この研究は、中国国家自然科学基金会(81973569、82274207、82104533)、成都中産大学の興林奨学生研究促進プロジェクト(XKTD2022013)、寧夏回族自治区重点研究開発プログラム(2023BEG02012)の支援を受けました。

Rhodiolaのcrenulata はZhuodaの雪山、甘子県、甘子チベット族自治州、四川省、中国(N 31.44570°、E 99.96086°、4892 m)から収集されます。これらの植物は、成都中医薬大学民族医学院のYi Zhang教授によって本物であると認証されています。
1. Rhodiola crenulataのコレクション
<ol><li>Rhodiola crenulataの生息地マップを撮影します。</li>
	<li>Rhodiola crenulataの植物全体、葉、萼、根茎を撮影します。</li>
	<li>スペードを使用して、 Rhodiola crenulata から1m以内の雑草や壊れた石を取り除き、その後のスムーズな採掘を確保します。</li>
	<li>根圏全体が見えるまで鍬で土を掘り起こし、直根を集めます。 注:薬用部分に使用されている イワベンケイ の根と根茎は、花茎が枯れる秋に収集する必要があります。</li>
</ol>2. 特性の同定
<ol><li>円筒形と短い主根と根茎、茶色の表面、ピンクのパターンを持つ膜状の黄色の表皮、オレンジレッドまたはバーガンディのスライスなど、 Rodiolaクレヌラータ の外観特性を肉眼で観察します。</li>
	<li>匂いで識別する:鼻の近くで香ばしい匂いを放ちます。</li>
	<li>味で識別する:根の小片を口に入れ、最初に一口飲んでから噛むと、少し苦い味がし、次に甘い味がします。</li>
</ol>3. 薬用粉末中のデンプン顆粒の顕微鏡的同定
<ol><li>イワベンケイの表面の土をブラシで取り除き、40°Cのオーブンに入れ、24時間ごとにハーブを回します。 注:薬用材料の破砕のしやすさは、水分を乾燥させるための基準と考えられています。</li>
	<li>乾燥した薬用材料を粉末機で粉末化し、薬用3号のふるいで薬用粉末を濾過する( 材料表参照)。</li>
	<li>きれいなスライドを取り( 材料表を参照)、解剖針で粉末を掘り( 材料表を参照)、スライドの3分の1に2mm以内に均等に配置します。</li>
	<li>ガラススポイト( 材料表を参照)を使用して、粉末に脱イオン水を一滴加えます。ピンセット( 材料表を参照)を使用してカバーガラスの一端を保持し( 材料表を参照)、液面にすばやく触れて粉末を覆います。 注:解剖学的針を使用して、水と薬用粉末を穏やかに混合し、サンプルが均一に混合されるようにします。スライド、パウダー、カバーガラスの間に気泡があってはなりません。</li>
	<li>顕微鏡を開き( 材料表を参照)、手順3.4のスライドをプラットフォームに置いて固定します。光源と粗い焦点のスパイラルを調整して、パウダーを確認します。組織がはっきりと見えるまで、細かい傍焦点スパイラルを調整します。40倍対物レンズに切り替えて、でんぷん顆粒を観察します。 注:でんぷん粒は単一または複数の粒として表示され、臍帯はヘリンボーンまたは亀裂の形に見えます。</li>
</ol>4. 薬用粉末中のカテーテル、コルク細胞、繊維、木質実質細胞、色素塊の顕微鏡的同定
<ol><li>きれいなスライドを取り( 材料表を参照)、解剖針で粉末を掘り( 材料表を参照)、スライドの3分の1に置きます。</li>
	<li>ガラススポイト( 材料表を参照)を使用して、粉末に抱水クロラール( 材料表を参照)を滴下します。ピンセットでスライドを取り( 材料表を参照)、アルコールランプで3回、毎回1秒間加熱します。 注意: 加熱中は気泡を避けてください。液体は流れていないままであり、浸透が完了していることを示しています。</li>
	<li>ガラススポイトを使用して、グリセリンを一滴加えます( 材料表を参照)。ピンセットを使用してカバーガラスの一方の端を持ち、液面にすばやく触れます。</li>
	<li>顕微鏡を開き、スライドをプラットフォームに置いて固定します。光源と粗い焦点のスパイラルを調整して、パウダーを確認します。組織がはっきりと見えるまで、細かい傍焦点スパイラルを調整します。カテーテル、コルク細胞、繊維、木実質細胞、色素ブロックを40倍の対物レンズに切り替えて観察します。 注:コルク細胞の多角形または長い多角形、明らかならせん構造を持つらせん状の容器、シュウ酸カルシウム砂の結晶を含む木部実質、および赤または茶色がかった赤色の顔料ブロック。</li>
</ol>5. Rodiolaのcrenulataおよびその参照の薄層クロマトグラフィー(TLC)サンプルの調製 
<ol><li>計量紙を天びんに置き( 材料表を参照)、 ロディオラ・クレヌラータの粉末3gを計量します。</li>
	<li>粉末を100 mLのコニカルボトル( 材料表を参照)に入れ、大きな腹部のピペットで25 mLのメタノールを加えます( 材料表を参照)。円錐形のボトルを超音波装置に入れます。電力を250 W、周波数を40 kHz、時間を30分に設定し( 材料表を参照)、機器の電源を入れます。 注:超音波装置の目的は、 Rodiolaクレヌラータ の粉末がその後の薄層クロマトグラフ実験の結果に影響を与えることなく完全に溶解することを確認することです。</li>
	<li>円錐形のボトルを取り外し、外側のボトルを流水で室温(RT)ですすいでください。</li>
	<li>ステップ5.3で調製した液体800μLを1mLのシリンジで吸引する。0.22 μmの微多孔性フィルターメンブレン( 材料表を参照)でろ過し、クロマトグラフィーサンプルボトルに Rodiola crenulata の400 μLの中間サンプル溶液を回収します。</li>
	<li>2 mgのサリドロシド、チロソール、没食子酸( 材料表を参照)をそれぞれ3本の別々の100 mLコニカルボトルに計量して加えます。25 mLのメタノールを大きな腹のピペットですべてのコニカルボトルに加えます。</li>
	<li>円錐形のボトルを超音波装置に入れ、出力を250 W、周波数を40 kHz、時間を30分に設定し、手順5.3を繰り返します( 材料表を参照)。</li>
	<li>ステップ5.6で調製した液体800μLを1mLのシリンジで吸引する。0.22 μm 微多孔性フィルターメンブレン( 材料表を参照)でろ過し、400 μL のサリドロシド、チロソール、没食子酸標準液を対応するクロマトグラフィーサンプルボトルに回収します。</li>
</ol>6. TLCの識別
<ol><li>ピペットトリクロロメタン(5 mL)、酢酸エチル(4 mL)、メタノール(2 mL)、ギ酸(0.5 mL)( 材料表を参照)。ダブルタンククロマトグラフィーシリンダーの片側( 材料表を参照)に加え、振とうし、均一に混合します。上部シリンダーヘッドを覆います。</li>
	<li>没食子酸、サリドロシド、チロソール標準液、 およびロディオラクレヌラータ 溶液をサンプルラックの位置A1〜A4に置きます。</li>
	<li>5 cm x 10 cmのシリコンシート( 材料表を参照)をサンプリングテーブルに置きます。自動サンプリング機( 材料表参照)を始動し、エアコントロールバルブを開きます。</li>
	<li>visionCATSソフトウェアを開きます( 材料表を参照)。[ 新規] > [新しいフォルダー] ( 名前は Rhodiola crenulata sample test) をクリックして [OK] >。 [New Method (name Rhodiola crenulata sample test)] > [ OK ] > ATS 4 をクリックします。</li>
	<li>「 ステップ定義の終了」をクリックします。[ Track Assignment ] をクリックして、説明 (没食子酸、サリドロシド、チロソール、 および Rhodiola crenulata サンプル) を編集します。</li>
	<li>「 HPTLC ステップ」をクリックします。薄層のパラメータ(5 cm x 10 cm)を設定します。 アプリケーションの種類 (バンド)を選択し、パラメータを設定し(表1)、 OK ボタンをクリックします。</li>
	<li>充填/すすぎ品質を開きます。Fill only Programmed Volumeにチェックを入れます。バイアルの底面レベル(mm)を0.5に設定し、[OK]ボタンをクリックします。</li>
	<li>「 メソッドの実行」をクリックします。</li>
	<li>[ Track Assignment ]ボタンをクリックし、[ Center]をオンにして、パラメータを設定します(表2)。</li>
	<li>[ 続行 ] ボタンをクリックすると、自動サンプリングが行われます。</li>
	<li>自動サンプリング機とエアバルブの電源を切ります。自動サンプリング機からシリコーンシートをはがします。</li>
	<li>ステップ6.1でシリコーンシートをダブルタンククロマトグラフィーシリンダーの反対側に入れ、上部シリンダーヘッドを覆い、20分間予飽和させます。</li>
	<li>薄層プレートの上端をピンセットで静かにクランプし、シリコンシートを現像剤にすばやく入れて、上部シリンダーヘッドを覆います。 注意: 展開する前端が薄層プレートの上端から0.5〜1.0cm離れているときに、シリコンシートを取り出します。</li>
	<li>有機溶媒が蒸発した後、発色溶液を室温でシリコーンシートの表面に噴霧して発色結果を得る。 注:発色溶液は、2%FeCl3 と1%K3 [Fe(CN)6]を含む水溶液です。</li>
</ol>

Rhodiola crenulateの顕微鏡的特性評価とTLCベースの同定

<ol>
	<li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+herbal+medicine+and+nanomedicine:+Converging+disciplines+to+improve+therapeutic+efficacy+and+human+health.">Traditional herbal medicine and nanomedicine: Converging disciplines to improve therapeutic efficacy and human health.</a> Advanced Drug Delivery Reviews. 178, 113964 (2021).</li><li>Yin, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+positive+role+and+mechanism+of+herbal+medicine+in+Parkinson's+Disease.">The positive role and mechanism of herbal medicine in Parkinson's Disease.</a> Oxidative Medicine and Cellular Longevity. 2021, 9923331 (2021).</li><li>Li, F. S., Weng, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demystifying+traditional+herbal+medicine+with+modern+approach.">Demystifying traditional herbal medicine with modern approach.</a> Nature Plants. 3, 17109 (2017).</li><li>da Fonseca, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herbal+medicinal+products+from+Passiflora+for+anxiety:+An+unexploited+potential.">Herbal medicinal products from Passiflora for anxiety: An unexploited potential.</a> The Scientific World Journal. 2020, 6598434 (2020).</li><li>Aziato, L., Antwi, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facilitators+and+barriers+of+herbal+medicine+use+in+Accra,+Ghana:+an+inductive+exploratory+study.">Facilitators and barriers of herbal medicine use in Accra, Ghana: an inductive exploratory study.</a> BMC Complementary and Alternative Medicine. 16, 142 (2016).</li><li>Tao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhodiola+species:+A+comprehensive+review+of+traditional+use,+phytochemistry,+pharmacology,+toxicity,+and+clinical+study.">Rhodiola species: A comprehensive review of traditional use, phytochemistry, pharmacology, toxicity, and clinical study.</a> Medicinal Research Reviews. 39 (5), 1779-1850 (2019).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dietary+supplement+Rhodiola+crenulata+extract+alleviates+dextran+sulfate+sodium-induced+colitis+in+mice+through+anti-inflammation,+mediating+gut+barrier+integrity+and+reshaping+the+gut+microbiome.">The dietary supplement Rhodiola crenulata extract alleviates dextran sulfate sodium-induced colitis in mice through anti-inflammation, mediating gut barrier integrity and reshaping the gut microbiome.</a> Food &amp; Function. 12 (7), 3142-3158 (2021).</li><li>Xie, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhodiola+crenulate+alleviates+hypobaric+hypoxia-induced+brain+injury+via+adjusting+NF-&#954;B/NLRP3-mediated+inflammation.">Rhodiola crenulate alleviates hypobaric hypoxia-induced brain injury via adjusting NF-&#954;B/NLRP3-mediated inflammation.</a> Phytomedicine. 103, 154240 (2022).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salidroside,+a+phenyl+ethanol+glycoside+from+Rhodiola+crenulata,+orchestrates+hypoxic+mitochondrial+dynamics+homeostasis+by+stimulating+Sirt1/p53/Drp1+signaling.">Salidroside, a phenyl ethanol glycoside from Rhodiola crenulata, orchestrates hypoxic mitochondrial dynamics homeostasis by stimulating Sirt1/p53/Drp1 signaling.</a> Journal of Ethnopharmacology. 293, 115278 (2022).</li><li>Ma, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+characteristics+of+Rhodiola+Crenulata+and+its+mechanism+in+acute+mountain+sickness+using+UHPLC-Q-TOF-MS/MS+combined+with+network+pharmacology+analysis.">Chemical characteristics of Rhodiola Crenulata and its mechanism in acute mountain sickness using UHPLC-Q-TOF-MS/MS combined with network pharmacology analysis.</a> Journal of Ethnopharmacology. 294, 115345 (2022).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhodiola+crenulata+attenuates+apoptosis+and+mitochondrial+energy+metabolism+disorder+in+rats+with+hypobaric+hypoxia-induced+brain+injury+by+regulating+the+HIF-1&#945;/microRNA+210/ISCU1/2(COX10)+signaling+pathway.">Rhodiola crenulata attenuates apoptosis and mitochondrial energy metabolism disorder in rats with hypobaric hypoxia-induced brain injury by regulating the HIF-1&#945;/microRNA 210/ISCU1/2(COX10) signaling pathway.</a> Journal of Ethnopharmacology. 241, 111801 (2019).</li><li>d'Avigdor, E., Wohlmuth, H., Asfaw, Z., Awas, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+current+status+of+knowledge+of+herbal+medicine+and+medicinal+plants+in+Fiche,+Ethiopia.">The current status of knowledge of herbal medicine and medicinal plants in Fiche, Ethiopia.</a> Journal of Ethnobiology and Ethnomedicine. 10, 38 (2014).</li><li>S&#225;nchez, M., Gonz&#225;lez-Burgos, E., Divakar, P. K., G&#243;mez-Serranillos, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-based+authentication+and+metabolomics+analysis+of+medicinal+plants+samples+by+DNA+barcoding+and+ultra-high-performance+liquid+chromatography/triple+quadrupole+mass+spectrometry+(UHPLC-MS).">DNA-based authentication and metabolomics analysis of medicinal plants samples by DNA barcoding and ultra-high-performance liquid chromatography/triple quadrupole mass spectrometry (UHPLC-MS).</a> Plants (Basel, Switzerland). 9 (11), 1601 (2020).</li><li>. Rhodiola renulata (Hook.fil &amp; Thomson) H.Ohba Available from: <a target="_blank" href="https://www.gbif.org/species/4200529">https://www.gbif.org/species/4200529</a> (2023)</li><li>. Plant + data center Available from: <a target="_blank" href="https://www.iplant.cn/">https://www.iplant.cn/</a> (2023)</li><li>. Chinese virtual herbarium Available from: <a target="_blank" href="https://www.cvh.ac.cn/">https://www.cvh.ac.cn/</a> (2023)</li><li>Cunningham, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=There+&amp;#34;ain't+no+mountain+high+enough&amp;#34;?:+The+drivers,+diversity+and+sustainability+of+China's+Rhodiola+trade.">There &amp;#34;ain't no mountain high enough&amp;#34;?: The drivers, diversity and sustainability of China's Rhodiola trade.</a> Journal of Ethnopharmacology. 252, 112379 (2020).</li><li>Wang, Q., Ruan, X., Jin, Z. H., Yan, Q. C., Tu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Rhodiola+species+by+using+RP-HPLC.">Identification of Rhodiola species by using RP-HPLC.</a> Journal of Zhejiang University. Science. B. 6 (6), 477-482 (2005).</li><li>Dong, X., Guo, Y., Xiong, C., Sun, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+two+major+Rhodiola+species+and+the+systemic+changing+characteristics+of+metabolites+of+Rhodiola+crenulata+in+different+altitudes+by+chemical+methods+combined+with+UPLC-QqQ-MS-based+metabolomics.">Evaluation of two major Rhodiola species and the systemic changing characteristics of metabolites of Rhodiola crenulata in different altitudes by chemical methods combined with UPLC-QqQ-MS-based metabolomics.</a> Molecules. 25 (18), 4062 (2020).</li><li>Zhao, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPLC+fingerprint+differentiation+between+natural+and+ex+situ+populations+of+Rhodiola+sachalinensis+from+Changbai+Mountain,+China.">HPLC fingerprint differentiation between natural and ex situ populations of Rhodiola sachalinensis from Changbai Mountain, China.</a> PloS One. 9 (11), 112869 (2014).</li><li>Yu, Y. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+evaluation+of+Tibetan+medicine+Rhodiolae+crenulatae+radix+et+rhizome+based+on+sensory+recognition+and+near+infrared+spectrum+analysis.">Quality evaluation of Tibetan medicine Rhodiolae crenulatae radix et rhizome based on sensory recognition and near infrared spectrum analysis.</a> Chengdu University of Traditional Chinese Medicine. , (2020).</li><li>Lv, X. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+determination+of+5+chemical+components+in+3+kinds+of+Rhodiola+rosea+by+HPLC.">Simultaneous determination of 5 chemical components in 3 kinds of Rhodiola rosea by HPLC.</a> China Pharmacy. 29 (18), 2515-2519 (2018).</li><li>Dong, T., Sha, Y., Liu, H., Sun, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altitudinal+variation+of+metabolites,+mineral+elements+and+antioxidant+activities+of+Rhodiola+crenulata+(Hook.f.+&amp;+Thomson)+H.Ohba.">Altitudinal variation of metabolites, mineral elements and antioxidant activities of Rhodiola crenulata (Hook.f. &amp; Thomson) H.Ohba.</a> Molecules. 26 (23), 7383 (2021).</li><li>Ma, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+UHPLC+fingerprints+combined+with+chemical+pattern+recognition+analysis+in+the+differentiation+of+six+Rhodiola+species.">Application of UHPLC fingerprints combined with chemical pattern recognition analysis in the differentiation of six Rhodiola species.</a> Molecules. 26 (22), 6855 (2021).</li><li>Li, X. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+discrimination+of+different+Rhodiola+species+using+1H-NMR+and+GEP+combinational+chemometrics.">Metabolic discrimination of different Rhodiola species using 1H-NMR and GEP combinational chemometrics.</a> Chemical &amp; Pharmaceutical Bulletin. 67 (2), 81-87 (2019).</li><li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Hippophae+species+(Shaji)+through+DNA+barcodes.">Identification of Hippophae species (Shaji) through DNA barcodes.</a> Chinese Medicine. 10, 28 (2015).</li></ol>

著者は何も開示していません。

世界には90種以上のイワベンケイの植物があり、すべての種の60%以上が中国で発見され、イワベンケイ、イワベンケイ、イワベンケイのアカリネンシス、イワベンケイなどの一般的なものが見られます17。中国薬局方(2020年)の最初の部分に記録されたRhodiola crenulataは、高地で栽培されたチベットの伝統薬です。Riolaのcrenulataのための市場の需?...

漢方薬は中国で長い歴史と豊富な応用経験があり、神農の漢方古典1の最初の体系的な記録でした。マラリアに応用されたアルテミシニンの発見は、漢方薬の開発を新たなステージ1に進めました。漢方薬の正確なメカニズムを明らかにするための現代の科学技術の使用は、漢方薬の利用率と需要を高め、それの新しい国際市場を開きます2,3,4。しかし、これは一連の悪影響につながっています。非専門家は漢方薬の特徴を漠然と理解しているため、漢方薬の使用は大きな安全上のリスクに直面しています5。
イワベンケイ属の植物の一つであるイワベンケイは、主にチベット、雲南省北西部、中国四川省西部に分布している(図1)6,7。Rhodiola crenulataは、「気を活性化し、血液循環を促進し、脈拍をきれいにし、喘息を落ち着かせる」機能を通じて、低酸素関連疾患の治療のためのサリドロシド、チロソール、没食子酸、およびその他の化合物で構成されています8,9,10,11。現地調査の結果、イワベンケイは標高4,000〜5,600mの高山距骨帯、峡谷斜面、岩の割れ目に見られることが分かりました。その生育環境は寒く、日光と強烈な放射線に満ちており、高山の牧草地の生態系に属しています。Rhodiola crenulataは、成長地形に応じて層状および点状の集団に分布することができ、他家受粉によって遺伝子流動を行うことができます。
イワベンケイ属の花粉流産、違法性の発掘、および退化した生態環境により、イワベンケイは絶滅危惧種となっています6,12。イワベンケイの薬効が高いことから、模倣品が市場に流入することが予想されます。この記事では、Rhodiola crenulataの生息地といくつかの便利な実験室での識別方法を紹介します。まず、イワベンケイの生育環境とその薬効を観察しました。第二に、薬用粉末の微細構造を顕微鏡で観察しました。最後のステップが重要なポイントです。Rhodiola crenulataの代表的な成分は、特定の物質におけるこれらの成分の異なる吸着または溶解特性に従って分離および同定されました。薬用植物のDNAベースの認証またはメタボロミクス分析法は複雑で高価です13。これらの基本的で便利で経済的な方法は、ロディオラ・クレヌラータを迅速に識別できます。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#956;m millipore filter</td>
			<td>Millipore</td>
			<td>SLGP033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic sampling machine</td>
			<td>CAMAG</td>
			<td>ATS 4</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloral hydrate</td>
			<td>Fuzhou Brunei Technology Co., Ltd</td>
			<td>ST1002</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromatographic&#160;sample&#160;bottles</td>
			<td>Zhejiang ALWSCI Technology Co., Ltd</td>
			<td>C0000008</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical flask</td>
			<td>Sichuan Shubo Co., Ltd</td>
			<td>1121</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Citotest Labware Manufacturing Co., Ltd</td>
			<td>10211818c</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting needle</td>
			<td>Shanghai Bingyu Fluid Technology Co., Ltd</td>
			<td>BY-5026</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>SHIMADZU</td>
			<td>ATX124</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl acetate</td>
			<td>Chengdu Kelong Chemical Co., Ltd</td>
			<td>2022120901</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Chengdu Kelong Chemical Co., Ltd</td>
			<td>2021110801</td>
			<td></td>
		</tr>
		<tr>
			<td>Gallic acid</td>
			<td>Chengdu Herbpurify Co., Ltd</td>
			<td>M-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10010618</td>
			<td></td>
		</tr>
		<tr>
			<td>High speed&#160; crusher</td>
			<td>Beijing Zhongxingweiye Instrument Co., Ltd</td>
			<td>FW-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Chengdu Kelong Chemical Co., Ltd</td>
			<td>20230108</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Chongqing Oprec Nistrument Co.,&#160; Ltd</td>
			<td>B203</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Citotest Labware Manufacturing Co., Ltd</td>
			<td>7105P-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Shanghai Yuejin Medical Equipment Co., Ltd</td>
			<td>DHG-8145</td>
			<td></td>
		</tr>
		<tr>
			<td>Pharmacopoeia sieve</td>
			<td>Hangzhou Xingrun sieve factory</td>
			<td>572423281330</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Changde BKMAM Biotechnology Co., Ltd</td>
			<td>120302008</td>
			<td></td>
		</tr>
		<tr>
			<td>Salidroside</td>
			<td>Chengdu Herbpurify Co., Ltd</td>
			<td>H-040</td>
			<td></td>
		</tr>
		<tr>
			<td>Saturate tank&#160;</td>
			<td>Yancheng Liegu Technology Co., Ltd</td>
			<td>10*20 P-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica gel plate</td>
			<td>Yantai Jiangyou Silica Gel Development&#160; Co., Ltd</td>
			<td>HSG20211227</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloromethane</td>
			<td>Chengdu Kelong Chemical Co., Ltd</td>
			<td>20221013-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer&#160;</td>
			<td>Changde BKMAM Biotechnology Co., Ltd</td>
			<td>130302027</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyrosol</td>
			<td>Chengdu Herbpurify Co., Ltd</td>
			<td>L-042</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound equipment</td>
			<td>Ningbo Xinyi Ultrasonic Equipment Co., Ltd</td>
			<td>SB-8200DTS</td>
			<td></td>
		</tr>
		<tr>
			<td>Volumetric pipet</td>
			<td>Changde BKMAM Biotechnology Co., Ltd</td>
			<td>120301006</td>
			<td></td>
		</tr>
	</tbody>
</table>

field collection and laboratory routine identification of rhodiola crenulata

医薬品の同定は、医薬品の安全性の前提であり、保証です。科学研究者の大多数は、ハーブの単純、迅速、効果的、そして安価な同定プロセスを支持するに違いありません。 イワベン ケイは、主に中国のチベット、雲南省、四川省の地域に分布する高地で栽培されている伝統的なチベット薬です。 Rhodiolaのcrenulateは 炎症抑制、抗低酸素症および酸化防止特性のような多数のbioactivityを、所有し、開発のための大きい潜在性があります。市場の需要の増加と資源含有量の急速な減少に伴い、 イワベン ケイの多数の混同された製品が人々を悩ませています。従って、この議定書はルーチンの実験室試験と結合される分野の Rodiolaのcrenulata の同一証明のための標準的なプロセスをもたらす。生息地、顕微鏡的特徴、および薄層クロマトグラフィーの組み合わせにより、 間違いなくロディオラ・クレヌラータ を迅速、効率的、経済的に同定し、チベット医学の継続的な発展と薬用材料の品質管理に貢献します。

この実験プロトコルは、現場で のロディオラのクレヌレート の識別と収集について説明しています。 イワベンケイ は、高山の距骨帯、峡谷の斜面、および高地の岩の裂け目に住む傾向があります。 イワベン ケイの生息地、全植物、花、葉を 図2に示すことができます。 イワベン ケイは赤褐色の根茎を持っています(図3A</stro...

Watch this Scientific Journal Video about Field Collection and Laboratory Routine Identification of Rhodiola crenulata at JoVE.com

著者スポットライト: Rhodiola crenulata の同定法の開発とその資源分布と薬理学的効果の調査

ここでは、生息地、植物形態、薬効成分、微視的特徴、および薄層クロマトグラフィーからの イワベンケイ の同定について説明します。

Rodiolaのcrenulataの野外収集そして実験室のルーチン同定

The identification of medicinal materials is the premise and guarantee of drug safety. The majority of scientific researchers are bound to favor the ...

私たちの研究の範囲は、民族医学のリソース調査の実施を中心としています。具体的には、この研究は、Rhodiola crenulataの同定のための迅速で便利な方法を開発することを目的としています。私たちが提供するプロトコルは、さまざまな薬用ハーブ材料を特定するための費用対効果が高く、簡単で迅速な方法を提示します。当研究室では、今後の研究活動として、ロディオラ・クレヌラータに関連する研究の分布と、本種の薬理学的影響という2つの主要な分野の研究に取り組んでいきます。

field-collection-laboratory-routine-identification-rhodiola

Research

JoVE Journal

Medicine

Field Collection and Laboratory Routine Identification of Rhodiola crenulata

482 ビュー.  Chengdu University of Traditional Chinese Medicine. 私たちの研究の範囲は、民族医学のリソース調査の実施を中心としています。具体的には、この研究は、Rhodiola crenulataの同定のための迅速で便利な方法を開発することを目的としています。私たちが提供するプロトコルは、さまざまな薬用ハーブ材料を特定するための費用対効果が高く、簡単で迅速な方法を提示します。当研究室では、今後の研究活動として、ロ?...

ビデオ: 著者スポットライト: Rhodiola crenulata の同定法の開発とその資源分布と薬理学的効果の調査

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

ヒト膵臓の収集プロトコル

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

医学

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

パーキンソン病の分野におけるミトコンドリアの表現型を監視するための主要ヒト線維芽細胞の使用

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Localization, Identification, and Excision of Murine Adipose Depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.

マウス脂肪デポのローカライズ、識別、および切除

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

Methodology for Sputum Induction and Laboratory Processing

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which is interesting in various respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), chronic cough, or idiopathic pulmonary fibrosis. This technique is well tolerated, safe and non-invasive, but is currently limited to research services and specialized centers in clinical practice because it is technically demanding, time-consuming, and requires trained staff. The success rate of sputum induction and analysis is about 80%.
Here, we describe the induction and laboratory processing of sputum samples. Sputum is induced by inhalation of hypertonic or isotonic saline with salbutamol. For the processing, we use the whole sputum technique. Dithiothreitol (DTT) is used to allow mucolysis of sputum samples. The primary aim of sputum processing is to obtain a differential cell count to study the cell types present in the airway lumen. Additional analyses may also be performed on sputum supernatant and sputum cells, which may allow further investigation into inflammatory processes and immune mechanisms. Examples include studying mediators in sputum supernatant and performing a large spectrum of analysis on sputum cells such as flow cytometry, genomics, or proteomics.
Finally, representative results of sputum analysis in healthy controls, asthmatics, and COPD patients are presented.

Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis.

喀痰誘発および実験室の処理のための方法論

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...

Routine Screening Method for Microparticles in Platelet Transfusions

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood banks. Cells fragment off microparticles (MP) when they are stressed. Blood and blood components may contain cellular fragments from a variety of cells, most notably from activated platelets. When performing their roles as innate immune cells and major players in coagulation and hemostasis, platelets change shape and generate microparticles. With dynamic light scattering (DLS)-based microparticle detection, it is possible to differentiate activated (high microparticle) from non-activated (low microparticle) platelets in transfusions, and optimize the use of this scarce blood product. Previous research suggests that providing non-activated platelets for prophylactic use in hematology-oncology patients could reduce their risk of becoming refractory and improve patient care. The goal of this screening method is to routinely differentiate activated from non-activated platelets. The method described here outlines the steps to be performed for routine platelet inventory management in a hospital blood bank: obtaining a sample from a platelet transfusion, loading the sample into the capillary for DLS measurement, performing the DLS test to identify microparticles, and using the reported microparticle content to identify activated platelets.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative in hospital blood banks. The goal is to differentiate activated from non-activated platelets to optimize the use of platelets. Providing non-activated platelets to hematology-oncology patients might reduce their high risk to become refractory.

血小板輸血で微粒子のスクリーニング法のルーチン

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood ...

Remote Laboratory Management: Respiratory Virus Diagnostics

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a multitude of issues ranging from transportation, access, facilities, equipment, and communication to provider training. To address this need, we have developed an innovative, scalable, logistics-enhanced, mobile, laboratory facility for emergencies and epidemics in resource-constrained global settings. Utilizing a background in clinical operations as an academic medical center, we designed a rapidly-deployable, modular BSL-2 and BSL-3 facility with user-friendly software for tracking and management of drugs and supplies in remote regions during epidemics and outbreaks. Here, we present our intermodal, mobile, expandable shipping-container laboratory units. The design of the laboratory facilitates off-grid usage by minimizing power consumption and allowing alternate water sources. The unit's information communication technology (ICT) platform provides (i) user-friendly tablet-based documentation, (ii) enhanced tracking of patients and supplies, and (iii) integrated communication onsite with built-in telehealth capabilities. To ensure quality in remote environments, we have developed a checklist for a basic laboratory workflow and a protocol for respiratory viral diagnosis using reverse-transcription polymerase chain reaction (RT-PCR). As described, this innovative and comprehensive approach allows for the provision of laboratory capability in resource-limited global environments.

A rapidly-deployable, off-grid laboratory has been designed and built for remote, resource-constrained global settings. The features and critical aspects of the logistically-enhanced, expandable, multifunctional laboratory modules are explored. A checklist for a basic laboratory workflow and a protocol for a respiratory viral diagnostic test are developed and presented.

遠隔実験システム管理: 呼吸器ウイルス診断

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a ...

Learning Modern Laryngeal Surgery in a Dissection Laboratory

Surgery for laryngeal malignancies requires millimetric accuracy from the different endoscopic and open techniques available. Practice of this surgery is almost completely reserved to a few referral centers that deal with a large proportion of this pathology. Practice on human specimens is not always possible for ethical, economic, or availability reasons. The aim of this study is to provide a reproducible method for the organization of a laryngeal laboratory on ex vivo animal models where it is possible to approach, learn, and refine laryngeal techniques. Porcine and ovine larynges are ideal, affordable, models to simulate laryngeal surgery given their similarity to the human larynx in their anatomical layout and tissue composition. Herein, the surgical steps of transoral laser surgery, open partial horizontal laryngectomy, and total laryngectomy are reported. The merging of endoscopic and exoscopic views guarantees an inside-out perspective, which is vital for the comprehension of the complex laryngeal anatomy. The method was successfully adopted during three sessions of a dissection course &#34;Lary-Gym&#34;. Further perspectives on robotic surgical training are described.

The purpose of this paper is to illustrate how to organize a reproducible laboratory for laryngeal surgery on affordable and closely similar animal laryngeal models in order to improve anatomical and surgical knowledge and skills.

解剖学室での現代喉頭手術の学習

Surgery for laryngeal malignancies requires millimetric accuracy from the different endoscopic and open techniques available. Practice of this surgery is ...

An In Vitro Dissolution Determination of Multi-Index Components in Tibetan Medicine Rhodiola Granules

The composition of the Tibetan medicine Rhodiola granules (RG) is complex, and the overall quality of RG is difficult to determine. Therefore, establishing a method to determine the multi-component in vitro dissolution of RG is of great significance for quality control. This study uses the second paddle method of the fourth general rule 0931 from the Chinese Pharmacopoeia (2020 edition), compliant with apparatus 2 of the United States Pharmacopeia (USP). The dissolution apparatus was set to a rotation speed of 100 rpm with ultrapure water as the dissolution medium. A sample volume of 1 mL was collected at each timepoint. Furthermore, the cumulative dissolution of gallic acid, salidroside, and ethyl gallic acid in RG at different time points was determined by high-performance liquid chromatography (HPLC). Finally, the dissolution curves were drawn, and the curves were fitted to the GompertzMod, the Gompertz, the Logistic, and the Weibull equations. The results showed that the cumulative dissolution of gallic acid in RG was over 80% at 1 min, the cumulative dissolution of salidroside and ethyl gallic acid was over 65% at 5 min, and the cumulative dissolution of each index component decreased after 30 min. The curve fitting demonstrated that the GompertzMod equation was the best-fitting model for each index component of RG. In conclusion, the dissolution test method described in this protocol is simple, accurate, and reliable. It can characterize the dissolution behavior of the index components in RG in vitro, which provides a methodological reference for quality control of RG and quality evaluation of other ethnic compounds.

An In Vitro Dissolution Determination of Multi-Index Components in Tibetan Medicine Rhodiola Granules

Here, we test the dissolution of Rhodiola granules (RG) in vitro, draw dissolution curves of salidroside, gallic acid, and ethyl gallate in ultrapure water, and fit the curves to different mathematical models. This protocol provides information and guidance for in vivo bioequivalence and in vivo-in vitro correlation studies of RG.

チベット医学ロディオラ顆粒におけるマルチインデックス成分の in vitro 溶解測定

The composition of the Tibetan medicine Rhodiola granules (RG) is complex, and the overall quality of RG is difficult to determine. Therefore, ...

Ferric Chloride-Induced Arterial Thrombosis and Sample Collection for 3D Electron Microscopy Analysis

Cardiovascular diseases are a leading cause of mortality and morbidity worldwide. Aberrant thrombosis is a common feature of systemic conditions like diabetes and obesity, and chronic inflammatory diseases like atherosclerosis, cancer, and autoimmune diseases. Upon vascular injury, usually the coagulation system, platelets, and endothelium act in an orchestrated manner to prevent bleeding by forming a clot at the site of the injury. Abnormalities in this process lead to either excessive bleeding or uncontrolled thrombosis/insufficient antithrombotic activity, which translates into vessel occlusion and its sequelae. The FeCl3-induced carotid injury model is a valuable tool in probing how thrombosis initiates and progresses in vivo. This model involves endothelial damage/denudation and subsequent clot formation at the injured site. It provides a highly sensitive, quantitative assay to monitor vascular damage and clot formation in response to different degrees of vascular damage. Once optimized, this standard technique can be used to study the molecular mechanisms underlying thrombosis, as well as the ultrastructural changes in platelets in a growing thrombus. This assay is also useful to study the efficacy of antithrombotic and antiplatelet agents. This article explains how to initiate and monitor FeCl3-induced arterial thrombosis and how to collect samples for analysis by electron microscopy.

The present protocol describes how to use a FeCl3-mediated injury to induce arterial thrombosis, and how to collect and prepare arterial injury samples at various stages of thrombosis for electron microscopy analysis.

塩化第二鉄誘発動脈血栓症および3D電子顕微鏡分析のためのサンプル収集

Cardiovascular diseases are a leading cause of mortality and morbidity worldwide. Aberrant thrombosis is a common feature of systemic conditions like ...

ヒト辺縁ニッチ細胞を分離、培養、および同定するためのプロトコール

Isolation and Identification of Limbal Niche Cells

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used for LNCs isolation. The tissue was divided into 12 pieces under aseptic conditions and digested for 18 h at 37 &#176;C in the cell culture incubator using collagenase A to obtain cell clusters with LNCs and limbal&#160;epithelial progenitor cells. The cell clusters were further digested for 15 min at 37 &#176;C using 0.25% trypsin-EDTA to obtain single cells and then cultured in modified embryonic stem cell medium (MESCM) on a plastic surface coated with 5% Matrigel. Cells were passaged upon 70% confluence, and LNCs were identified using immunofluorescence, real-time quantitative PCR (qPCR), and flow cytometry. Primary LNCs were isolated and passaged more than 12 times. The proliferation activity of LNCs from P4 to P6 was the highest. LNCs expressed higher stem cell markers than BMMSCs (SCF, Nestin, Rex1, SSEA4, CD73, CD90, MSX1, P75NTR, and PDGFR&#946;). Furthermore, results showed that P4 LNCs uniformly expressed VIM, CD90, CD105, and PDGFR&#946;, but not Pan-CK, which could be used as a marker for the identification of LNCs. Flow cytometric analysis showed that approximately 95%, 97%, 92%, and 11% of LNCs expressed CD73, CD90, CD105, and SCF respectively, while they were&#160;68%, 99%, 20%, and 3% in&#160;BMMSCs. The standard process for LNC&#160;isolation and identification could provide&#160;a reliable laboratory basis for the widespread use of LNCs.

著者スポットライト:広範なLNC研究をサポートするためのLimbal Niche Cell(LNC)の単離と特性評価の標準化(英語) 

Here, we present a protocol to isolate and identify the human limbal niche cells.

辺縁ニッチ細胞の単離と同定

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used ...