本研究は、NCI（R01-94160とR01-155086）、IUサイモンがんセンターと小児研究ウェルズセンターによってサポートされていました。

この研究は、医学インディアナ大学でIACUC委員会によって承認されています。 1。正しい遺伝子型を持つマウスを作製<ol><li>のBCCのマウスモデルを確立します。メイトK14-クレエマウスR26-SmoM2 YFPマウス （ジャクソン研究所ストック番号005130）（18）と（ジャクソン実験室ストック番号005107）K14-クレエ+とR26-SmoM2 YFP +マウスを生成する。 </li><li>ジェノタイピングを行います。 1 mg / mlのプロテ​​イナーゼKとdirectPCRテール溶解液に浸し尾検体（0.5センチ）55℃で振とうしながら一晩。翌日、マイクロ遠心機（ジェノタイピングのために上清を維持するため）で最高速度で遠心分離することにより、組織の破片を除去。ベンダが推奨するプライマー及び条件で各PCR反応のために上清の1μlを使用する。 FO右遺伝子を有するマウスを選択してください（3-6週齢で、0.05〜0.1 mg / kg体重でブプレノルフィンは2〜3日ごとに8-12時間尾中略後SQを介して適用されます）rは、手順2。 </li></ol> 2。皮膚のケラチノサイトにおけるSmoM2の発現を誘導する給餌針（20ゲージを湾曲）と強制経口投与を経由して5日間連続してタモキシフェン（各給餌における植物油50ml中5μgの/ kg体重）の経口投与によるケラチノサイトにおけるSmoM2発現を誘導する。 3。皮膚標本の準備一般的には、表現型はK14creER/R26SmoM2 GFPマウスの耳としっぽでより深刻である。細胞分析の準備をするために、我々は最初の機関承認手続（CO 2プラス頸椎脱臼）と動物の犠牲の後にマウスの皮膚を収穫。 4。表皮を分離<ol><li>承認手続き（ステップ3を参照）犠牲マウスの後、トリマーを使用してファーを取り外します。 37℃でディスパーゼ溶液（10％FBSを含むDMEMで5 mg / ml）で1〜2時間（未満8週齢と2時間マウスで1時間Cに浸し皮膚標本）8週より齢のマウスのために。 </li><li>以下のディスパーゼ処理、鉗子によって真皮から分離表皮。 </li></ol> 5。単一細胞を作製<ol><li> 37℃で1〜2時間、コラゲナーゼIV溶液（10％FBSを含むDMEM中で1 mg / mlの）に浸した後、単一の細胞集団を得るはさみによって表皮をミンチとする°〜50 mlチューブでC。優しく渦管の細胞解離を助けるために20分ごとにしてみてください。 </li><li>顕微鏡下で細胞解離を点検。単一細胞はコラゲナーゼ媒体に検出することができる場合には、細胞収率を高めるためにさらに30分間試料をインキュベートする。一般的には、マウスを一から表皮は、10 7個まで得ることができる。 </li><li>ベンチトップ遠心分離によって1,500 rpmで、セルストレーナー（細孔径70μm）を経由して、組織の混合、スピン細胞をダウンフィルタリングし、PBSで1回時間を洗う。 </li></ol> 6。ラベルの細胞およびフローサイトメトリー解析を実行<ol><li> 10％FBSで細胞を再懸濁しPBSで非特異的結合をブロックするために2.5×10 6細胞/ ml（30分間氷上で）少なくとも</li><li>特定の抗体標識用の1.5ミリリットルマイクロチューブのそれぞれに細胞のアリコートを取る。 2μgの/ mlの最終濃度で各チューブに異なる抗体を加える。数秒間穏やか頂点を経由して細胞と抗体を混合し、1mlのPBSで洗浄後、30分間氷上にそれらを残す。 （詳細については、試薬を参照）ビメンチン-FITC（線維芽細胞）のCD11b-PEプラスGr1と-APC（骨髄細胞や骨髄由来のサプレッサー細胞）：BCC標本のために、我々は次のバイオマーカーのための抗体を使用していました。 </li><li>細胞内染色のために、我々はキットを使用します（詳細については試薬を参照）ベンダーの説明書に従ってください。我々は、細胞表面マーカーを分析するために新たなセルを使用。表面マーカー染色後、細胞を洗浄し、PBSに再懸濁し。 1μgの/ mlの最終濃度で細胞混合物にDAPIを追加する。 DAPIはさらに分析からゲーテッド外となり死んだ細胞を染色します。 </li><li>納入仕様を使用してデータを分析するCソフトウェア。我々は最初のFSC対SSCプロットに基づいて単一のセルを選択して、さらに分析するためにDAPI陰性人口（生細胞）を使用します。 </li></ol>

<ol>
	<li>Rogers, H. 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=Incidence+estimate+of+nonmelanoma+skin+cancer+in+the+United+States.">Incidence estimate of nonmelanoma skin cancer in the United States.</a> Arch. Dermatol. 146, 283-287 (2006).</li><li>Epstein, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basal+cell+carcinomas:+attack+of+the+hedgehog.">Basal cell carcinomas: attack of the hedgehog.</a> Nat. Rev. Cancer. 8, 743-754 (2008).</li><li>Yang, L., Xie, G., Fan, Q., Xie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+hedgehog-signaling+pathway+in+human+cancer+and+the+clinical+implications.">Activation of the hedgehog-signaling pathway in human cancer and the clinical implications.</a> Oncogene. 29, 469-481 (2010).</li><li>Johnson, R. L., 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=Human+homolog+of+patched,+a+candidate+gene+for+the+basal+cell+nevus+syndrome.">Human homolog of patched, a candidate gene for the basal cell nevus syndrome.</a> Science. 272, 1668-1671 (1996).</li><li>Hahn, 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=A+mammalian+patched+homolog+is+expressed+in+target+tissues+of+sonic+hedgehog+and+maps+to+a+region+associated+with+developmental+abnormalities.">A mammalian patched homolog is expressed in target tissues of sonic hedgehog and maps to a region associated with developmental abnormalities.</a> J. Biol. Chem. 271, 12125-12128 (1996).</li><li>Hahn, 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=Mutations+of+the+human+homolog+of+Drosophila+patched+in+the+nevoid+basal+cell+carcinoma+syndrome.">Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome.</a> Cell. 85, 841-851 (1996).</li><li>Xie, 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=Activating+Smoothened+mutations+in+sporadic+basal-cell+carcinoma.">Activating Smoothened mutations in sporadic basal-cell carcinoma.</a> Nature. 391, 90-92 (1998).</li><li>Reifenberger, 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=Missense+mutations+in+SMOH+in+sporadic+basal+cell+carcinomas+of+the+skin+and+primitive+neuroectodermal+tumors+of+the+central+nervous+system.">Missense mutations in SMOH in sporadic basal cell carcinomas of the skin and primitive neuroectodermal tumors of the central nervous system.</a> Cancer Res. 58, 1798-1803 (1998).</li><li>Lam, C. 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=A+frequent+activated+smoothened+mutation+in+sporadic+basal+cell+carcinomas.">A frequent activated smoothened mutation in sporadic basal cell carcinomas.</a> Oncogene. 18, 833-836 (1999).</li><li>Nilsson, M., 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=Induction+of+basal+cell+carcinomas+and+trichoepitheliomas+in+mice+overexpressing+GLI-1.">Induction of basal cell carcinomas and trichoepitheliomas in mice overexpressing GLI-1.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 3438-3443 (2000).</li><li>Sheng, 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=Dissecting+the+oncogenic+potential+of+Gli2:+deletion+of+an+NH(2)-terminal+fragment+alters+skin+tumor+phenotype.">Dissecting the oncogenic potential of Gli2: deletion of an NH(2)-terminal fragment alters skin tumor phenotype.</a> Cancer Res. 62 (2), 5308-5316 (2002).</li><li>Reifenberger, 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=Somatic+mutations+in+the+PTCH,+SMOH,+SUFUH+and+TP53+genes+in+sporadic+basal+cell+carcinomas.">Somatic mutations in the PTCH, SMOH, SUFUH and TP53 genes in sporadic basal cell carcinomas.</a> Br. J. Dermatol. 152, 43-51 (2005).</li><li>Guha, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+inhibitor+gets+landmark+skin+cancer+approval,+but+questions+remain+for+wider+potential.">Hedgehog inhibitor gets landmark skin cancer approval, but questions remain for wider potential.</a> Nat. Rev. Drug Discov. 11, 257-258 (2012).</li><li>Sekulic, A., 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=Efficacy+and+safety+of+vismodegib+in+advanced+basal-cell+carcinoma.">Efficacy and safety of vismodegib in advanced basal-cell carcinoma.</a> N. Engl. J. Med. 366, 2171-2179 (2012).</li><li>Tang, J. 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=Inhibiting+the+hedgehog+pathway+in+patients+with+the+basal-cell+nevus+syndrome.">Inhibiting the hedgehog pathway in patients with the basal-cell nevus syndrome.</a> N. Engl. J. Med. 366, 2180-2188 (2012).</li><li>Aszterbaum, M., 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=Ultraviolet+and+ionizing+radiation+enhance+the+growth+of+BCCs+and+trichoblastomas+in+patched+heterozygous+knockout+mice.">Ultraviolet and ionizing radiation enhance the growth of BCCs and trichoblastomas in patched heterozygous knockout mice.</a> Nat. Med. 5, 1285-1291 (1999).</li><li>Pazzaglia, S., 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=Modulation+of+patched-associated+susceptibility+to+radiation+induced+tumorigenesis+by+genetic+background.">Modulation of patched-associated susceptibility to radiation induced tumorigenesis by genetic background.</a> Cancer Res. 64, 3798-3806 (2004).</li><li>Athar, M., 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=Inhibition+of+smoothened+signaling+prevents+ultraviolet+B-induced+basal+cell+carcinomas+through+regulation+of+Fas+expression+and+apoptosis.">Inhibition of smoothened signaling prevents ultraviolet B-induced basal cell carcinomas through regulation of Fas expression and apoptosis.</a> Cancer Res. 64, 7545-7552 (2004).</li><li>Mancuso, M., 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=Basal+cell+carcinoma+and+its+development:+insights+from+radiation-induced+tumors+in+Ptch1-deficient+mice.">Basal cell carcinoma and its development: insights from radiation-induced tumors in Ptch1-deficient mice.</a> Cancer Res. 64, 934-941 (2004).</li><li>Ellis, T., 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=Patched+1+conditional+null+allele+in+mice.">Patched 1 conditional null allele in mice.</a> Genesis. 36, 158-161 (2003).</li><li>Mao, 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=A+novel+somatic+mouse+model+to+survey+tumorigenic+potential+applied+to+the+Hedgehog+pathway.">A novel somatic mouse model to survey tumorigenic potential applied to the Hedgehog pathway.</a> Cancer Res. 66, 10171-10178 (2006).</li><li>Wang, G. Y., Wang, J., Mancianti, M. L., Epstein, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basal+cell+carcinomas+arise+from+hair+follicle+stem+cells+in+Ptch1(+/-)+mice.">Basal cell carcinomas arise from hair follicle stem cells in Ptch1(+/-) mice.</a> Cancer Cell. 19, 114-124 (2011).</li><li>Grachtchouk, M., 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=Basal+cell+carcinomas+in+mice+arise+from+hair+follicle+stem+cells+and+multiple+epithelial+progenitor+populations.">Basal cell carcinomas in mice arise from hair follicle stem cells and multiple epithelial progenitor populations.</a> J. Clin. Invest. 121, 1768-1781 (2011).</li><li>Kasper, M., 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=Wounding+enhances+epidermal+tumorigenesis+by+recruiting+hair+follicle+keratinocytes.">Wounding enhances epidermal tumorigenesis by recruiting hair follicle keratinocytes.</a> Proc. Natl. Acad. Sci. U.S.A. 108, 4099-4104 (2011).</li><li>Youssef, K. K., 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+the+cell+lineage+at+the+origin+of+basal+cell+carcinoma.">Identification of the cell lineage at the origin of basal cell carcinoma.</a> Nat Cell Biol. 12, 299-305 (2010).</li><li>Yauch, R. L., 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=Smoothened+mutation+confers+resistance+to+a+Hedgehog+pathway+inhibitor+in+medulloblastoma.">Smoothened mutation confers resistance to a Hedgehog pathway inhibitor in medulloblastoma.</a> Science. 326, 572-574 (2009).</li><li>Buonamici, S., 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=Interfering+with+resistance+to+smoothened+antagonists+by+inhibition+of+the+PI3K+pathway+in+medulloblastoma.">Interfering with resistance to smoothened antagonists by inhibition of the PI3K pathway in medulloblastoma.</a> Sci. Transl. Med. 2, 51ra70 (2010).</li><li>Dijkgraaf, G. 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=Small+molecule+inhibition+of+GDC-0449+refractory+smoothened+mutants+and+downstream+mechanisms+of+drug+resistance.">Small molecule inhibition of GDC-0449 refractory smoothened mutants and downstream mechanisms of drug resistance.</a> Cancer Res. 71, 435-444 (2011).</li><li>Guo, Z., Draheim, K., Lyle, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+adult+epithelial+stem+cells+from+human+skin.">Isolation and culture of adult epithelial stem cells from human skin.</a> J. Vis. Exp. (49), e2561 (2011).</li><li>Li, L., Fukunaga-Kalabis, M., Herlyn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+three-dimensional+human+skin+reconstruct+model:+a+tool+to+study+normal+skin+and+melanoma+progression.">The three-dimensional human skin reconstruct model: a tool to study normal skin and melanoma progression.</a> J. Vis. Exp. (54), e2937 (2011).</li><li>Anacker, D., Moody, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+organotypic+raft+cultures+from+primary+human+keratinocytes.">Generation of organotypic raft cultures from primary human keratinocytes.</a> J. Vis. Exp. (60), e3668 (2012).</li></ol>

著者は、彼らが競合する経済的利益を持っていないことを宣言します。

我々は、BCCの内の細胞集団を分析するための方法を記載している。腫瘍組織は、多くの細胞型の一貫しており、各細胞型は、in vitro条件下で最高の下でも取り戻すことは非常に困難である、発がん性のある時点で動作が異なります。このプロトコルを用いて、基底細胞癌の開発が表皮幹細胞の増殖、なら​​びに骨髄細胞の動員を伴っていることが示された。種々の細胞型に特異的な抗体が利用可能であるので、免疫組織化学または免疫蛍光解析と比較して、細胞集団の分析は、細胞集団の変化をより定量的な評価を与える。 遺伝子から誘導表現型の変化は時間を要するので、このプロトコルのために、10週間の研究を完了するために必要とされる。これは、事前に実験を計画することが重要である。細胞単離および分析のために、丸2日が必要とされてもよい。生きた細胞をanalysに使用されるためES、それは前もってFACSキャリバーを確保することが重要です。このプロトコルを使用して作業する場合、我々はまた、（下記参照）一般的な問題を記載しております。 我々の経験では、以下は、我々が遭遇した共通の問題である： <ol><li>表皮の分離不良：これは不完全なディスパーゼ処理（diapase処理の時間を増加してください）​​または不十分なディスパーゼ溶液（皮膚が完全にディスパーゼ溶液に浸漬する必要がある）によって引き起こされる可能性がある。我々は1マウス用ディスパーゼ溶液を少なくとも5ミリリットルを使用しています。ボリュームは、皮膚のサイズに基づいて異なる場合があります。 </li><li>細胞数の低収率：これはコラゲナーゼIVと不完全消化のためかもしれない。最初のチェックコラゲナーゼ濃度や有効期限をお願いします。これは、使用される表皮の不十分な量によって引き起こされ得る。さらに、酵素消化を°C（消化を実行する前に温度を調節してください）​​振とうしながら37で実行されるべきである。 </li><li>細胞塊：これは（皮膚細胞に対してピペット細胞は非常に一般的ですストレーナー（遠心分離前）、又はMg 2 +の存在と+溶液中（細胞洗浄のための Mg 2 +およびCa 2 +フリーPBSを使用してください）のCa 2を通過した後に数回。 </li><li>あまりにも多くの死細胞：これは（新鮮な組織を使用してください）​​劣化した組織の結果であるかもしれない、または過剰消化（ディスパーゼとコラゲナーゼIVと拡張インキュベーションを避けるため）。 </li></ol>このプロトコルは、骨髄移植または細胞起源を調べるために組織移植と組み合わせることができる。例えば、致死的に放射線照射したマウスに骨髄細胞をGFP発現の移植は、私たちは、腫瘍関連線維芽細胞と骨髄細胞の骨髄細胞の寄与を調べることができるようになります。同様に、皮膚腫瘍は、腫瘍 - 宿主相互作用を調べるために新しいマウス宿主に移植することができます。 発癌中に細胞集団の変化を調べることに加えて、このプロトコルはまた、研究者が調べることを可能にする腫瘍環境における細胞への薬物治療の効果。このプロトコルは発癌皮膚中に細胞集団の分析のために使用するように設計されているが、わずかな変更は、他の癌のタイプのために行うことができる。 皮膚癌のマウスモデルのサマリー、細胞集団の解析では悪性形質転換、変換プロセスのさまざまな細胞事象における細胞生物学のよりよい理解につながる、発癌の間に私達に動的な細胞変化を与えることができます。

ヒト癌の最も一般的なタイプとしては、基底細胞癌（BCC）は、約2万人のアメリカ人が年間1に影響を与えます。ヘッジホッグ（HH）シグナル伝達の異常な活性化を示す証拠を蓄積すると、BCCの開発を（2,3の投稿）の基礎となる原動力となっています。 BCCのにシグナリングのHhの変更は不活PTCH1 4-6の突然変異、SMO 7-9の機能獲得型変異、およびHh経路転写因子GLI1 10およびGLI2 11または負の調節因子蘇（フーの珍しい不活化変異の異常発現を含む）12。すべてのこれらの及び他の研究を通じ、連邦医薬品局（FDA）は最近、転移性および局所進行BCCの13-15を治療するためのHhシグナル伝達阻害剤Vismodegibの使用を承認した。 これらすべての成果を16にもかかわらず、我々はまだHhシグナル伝達がCARCを駆動する分子や細胞のメカニズムを理解していないinogenesis。 Hhシグナル伝達の組織特異的な活性化を用いた動物モデルを確立することは、これらの研究のために、薬剤耐性の我々の理解のためには重要です。マウスでは、野生型マウスでも、発がん性紫外線や電離放射線の重い線量下のBCCを発症しない。対照的に、PTCH1 + / -マウスは、BCC現像17,18の影響を受けやすい。通常の状態で保たれている場合、マウスめったに完全に成長したのBCCを開発しない- PTCH1におけるBCC開発の浸透度+ / - + /はPTCH1ものの、 マウスは 50％以上である18,19。 - / - PTCH1の胚致死により、PTCH1の組織特異的ノックアウトは、一般に、調査20のために使用される。また、発癌SmoM2 YFP（Krt14-クレエ：R26-SmoM2 YFPまたはKrt14-CRE：R26-SmoM2 YFP）の条件皮膚特異的発現は、のための簡単な遺伝的アッセイを提供する、非常に早い年齢で多数の微視的のBCCの形成につながるhhはないシグナリングSMO 21 wnstream。 BCC生物学の知識で三大問題があります。まず、のBCCの細胞起源は完全に明確ではありません。いくつかの研究は、幹細胞サイト22-24として毛包のびくともをサポートしていますが、ユセフら 25は、毛包に、間濾胞表皮領域内になるように皮膚HH-主導腫瘍の起源のマウス細胞ではなくローカライズ、ROSA26主導ジェニック変異SMOの発現を活性化する細胞特異的Creをを使用します。第二に、BCC開発中の細胞間相互作用はよく理解されていない。これは、活性化されたHhシグナル伝達とケラチノサイトはBCCの​​の形成をもたらすことが知られているが、他の細胞の変化は、よく理解されていない。さらに、マウスでSmoの拮抗薬の治療は、薬剤耐性26-28につながることができます。 BCCのためこのように新たな目標を大幅に必要とされている。これら3つの問題を理解することはcarcinog中にマウスの細胞の変化の分析を必要とするenesis。いくつかの方法がケラチノサイト培養、フローサイトメトリーおよび皮膚幹細胞の単離29-31で公開されているが、マウスのBCCにおける細胞分析のための包括的な手順は現在存在しない。のBCCのマウスモデル、表皮の分離、組織および細胞分析からの単一細胞の生成のための方法を組み合わせることにより、我々は、BCC開発中に細胞のシグナル伝達、細胞および分子間相互作用を研究するために一連の手順を読者に提供する。私たちは、このプロトコルは、読者がそれぞれの手続きがどのように達成されるか見ることができると信じています。また、結果がどのように見えるべきかを説明するためにいくつかのデータを提供され、読者はこれらの手順を実行する間、困難を克服するのに役立つトラブルシューティング。

<table border="1">
 <tbody>
 <tr>
 <td>Reagents</td>
 <td> Company</td>
 <td> Cat#</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>directPCR lysis reagent (tail) </td>
 <td>Viagen Biotech </td>
 <td>102-T </td>
 <td>store @ 4 &deg;C</td>
 </tr>
 <tr>
 <td>proteinase K </td>
 <td> Sigma </td>
 <td>P2308 </td>
 <td>store @ -20 &deg;C</td>
 </tr>
 <tr>
 <td>ApliTag 360 taq polymerase </td>
 <td>Applied biosystems </td>
 <td>N8080155 </td>
 <td> store @ -20 &deg;C</td>
 </tr>
 <tr>
 <td>Tamoxifen </td>
 <td>Sigma </td>
 <td>T5648 </td>
 <td> store @ 4 &deg;C</td>
 </tr>
 <tr>
 <td>Feeding needle (20 gauge) </td>
 <td>Fisher scientific </td>
 <td>01-290-9B</td>
 <td></td>
 </tr>
 <tr>
 <td>Dispase </td>
 <td>Roche Diagnostic </td>
 <td>04942078001</td>
 <td>store @ 4 &deg;C</td>
 </tr>
 <tr>
 <td>Collagenase IV </td>
 <td>Worthington </td>
 <td>LS004189</td>
 <td>store @ -20 &deg;C</td>
 </tr>
 <tr>
 <td>Cell strainer </td>
 <td>Fisher scientific </td>
 <td>08-771-2</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-CD11b-PE </td>
 <td>eBioscience </td>
 <td> 12-0112-81</td>
 <td>store @ 4 &deg;C</td>
 </tr>
 <tr>
 <td>Anti-Gr1-APC </td>
 <td>eBioscience </td>
 <td> 17-5931-81</td>
 <td>store @ 4 &deg;C</td>
 </tr>
 <tr>
 <td>Anti-vimentin-FITC </td>
 <td>eBioscience </td>
 <td>11-9897-80 </td>
 <td>store @ 4 &deg;C</td>
 </tr>
 </tbody>
</table>

cell population analyses during skin carcinogenesis

癌の開発は、癌の前駆細胞、免疫細胞、線維芽細胞および内皮細胞を含む多くの細胞型を含む多段階プロセスである。セルの各タイプは、発癌の間のシグナリングおよび機能的変化を受ける。多くの癌の研究者の現在の課題は、 インビボでの多段階プロセス中に各細胞型の変化を分析することである。ここ数年では、著者はK14creER/R26-SmoM2 YFPマウスを用いた皮膚癌の開発中の異なる細胞集団を分離するための手順のセットを開発しました。研究（このプロトコルでK14creER +とR26-SmoM2 YFP +マウス）に適したマウスを生成する1）、2）マウス皮膚における誘導SmoM2 YFP発現 、3）マウスの皮膚生検の準備、4）を単離：手順は、6部に分かれています皮膚の表皮; 5）表皮から単一細胞を調製する工程と、6）フローサイトメトリー分析のための単一の細胞集団を標識する。右の遺伝子型を持つマウスに十分な数の生成は、2ヶ月かかることがあり、このプロトコルでの律速段階です。手順の残りの部分は数日に数時間かかる。このプロトコルの中では、我々はまた、トラブルシューティングのためのセクションが含まれています。我々は、皮膚癌に焦点を当てているが、このプロトコルは、ヒトの疾患の他の動物モデルに適用するように変更することができる。

これは、マウスがヘッジホッグシグナル伝達活性化を除き、基底細胞癌を発症しないことが知られている。 図1は、発癌性の誘導発現平滑化、SmoM2 YFP後の皮膚の表現型を示している。 図2は、正常および腫瘍担持マウスにおける骨髄細胞の分析を示す。のCD11b + Gr1とは+細胞骨髄細胞に由来しており、そのうちのいくつかを導出サプレッサー細胞を骨髄されています。通常の状況では、のCD11b + Gr1と+細胞集団はほとんど検出されたが、炎症や腫瘍の発達に応じて増加します。我々の、CD11b + Gr1と+細胞の増加で皮膚結果にSmoM2YFPと誘導発現を示した。現在、BCCののこの変化の根底にある分子メカニズムは明らかではない。 <img alt="図1" src="/files/ftp_upload/50311/50311fig1.jpg" /> 図1。 Morphologicaマウス皮膚におけるSmoM2YFP誘導後のLに変更されます。トップは、マウスの遺伝子型の図を示します。中央のパネル：タモキシフェンで治療K14creER +とR26-SmoM2 YFP +マウスはH＆Eセクション（左）にひどくお肌に異常（右）と同様に、皮膚腫瘍を示した。下のパネル：マウスSmoM2YFP誘導せず、正常な肌（右）とH＆Eセクション（左）のない異常な形態を示した。 <img alt="図2" src="/files/ftp_upload/50311/50311fig2.jpg" /> 図2のCD11b + Gr1と+細胞のフロー分析。皮膚癌の開発中のCD11b + Gr1と+細胞の変化を調べるためには、我々の、CD11bおよびGr1とする蛍光標識抗体を用いた単一細胞で染色を単離した。 FlowJoと分析の後、我々は、タモキシフェンで処置したマウスから皮膚組織におけるこの細胞集団の増加に気づい。

Watch this Scientific Journal Video about Cell Population Analyses During Skin Carcinogenesis at JoVE.com

Cell Population Analyses During Skin Carcinogenesis

発癌は、癌細胞とがん微小環境との相互作用を伴うプロセスである。分子事象を分析するためには、癌の開発中の異なる段階で異なる細胞集団を単離する必要がある。基底細胞癌のマウスモデルを用いて、発癌の間に、細胞分析のためのプロトコールを述べる。

細胞集団分析スキン発がん中

Cancer development is  a multiple-step process involving many cell types including cancer precursor  cells, immune cells, fibroblasts and endothelial ...

cell-population-analyses-during-skin-carcinogenesis

Research

JoVE Journal

Medicine

12.3K ビュー.  Indiana University. 発癌は、癌細胞とがん微小環境との相互作用を伴うプロセスである。分子事象を分析するためには、癌の開発中の異なる段階で異なる細胞集団を単離する必要がある。基底細胞癌のマウスモデルを用いて、発癌の間に、細胞分析のためのプロトコールを述べる。

ビデオ: Cell Population Analyses During Skin Carcinogenesis

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

全血刺激のためのアッセイを用いて炎症性サイトカインIL -1βの正確かつ簡単に測定

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

医学

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

前立腺発がんおよび良性前立腺過形成の評価のための腎皮膜異種移植および皮下ペレット移植

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

Dried Blood Spot Collection of Health Biomarkers to Maximize Participation in Population Studies

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spots meet this need, and can be collected in the field with high response rates. These elements are particularly important in longitudinal study designs including interventions where attrition is critical to avoid, and high response rates improve the interpretation of results. Dried Blood Spot sample collection is simple, quick, relatively painless, less invasive then venipuncture, and requires minimal field storage requirements (i.e.&#160;samples do not need to be immediately frozen and can be stored for a long period of time in a stable freezer environment before assay). The samples can be analyzed for a variety of different analytes, including cholesterol, C-reactive protein, glycosylated hemoglobin, numerous cytokines, and other analytes, as well as provide genetic material. DBS collection is depicted as employed in several recent studies.

Biomarkers are directly-measured biological indicators of disease or health. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spot (DBS) collection meets this need, can be collected in the field with high response rates&#160;and analyzed for a variety of biomarkers.

集団研究への参加を最大化するために健康バイオマーカーの血液スポットコレクション乾燥させ

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

シーケンシャル<em&gt;インビボ</em&gt;骨折修復中に、骨形成幹細胞/前駆細胞のイメージング

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

マウス内視鏡検査のため<em&gt;インビボ</em&gt;発がんと腸の評価のマルチモーダルイメージング創傷治癒および炎症

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

瘢痕創傷修復のマウス胎児皮膚モデル

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).

Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).

前立腺癌臨床組織におけるmiRNA発現解析

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, ...

A Method of Trigonometric Modelling of Seasonal Variation Demonstrated with Multiple Sclerosis Relapse Data

This report describes a novel Stata-based application of trigonometric regression modelling to 55 years of multiple sclerosis relapse data from 46 clinical centers across 20 countries located in both hemispheres. Central to the success of this method was the strategic use of plot analysis to guide and corroborate the statistical regression modelling. Initial plot analysis was necessary for establishing realistic hypotheses regarding the presence and structural form of seasonal and latitudinal influences on relapse probability and then testing the performance of the resultant models. Trigonometric regression was then necessary to quantify these relationships, adjust for important confounders and provide a measure of certainty as to how plausible these associations were. Synchronization of graphing techniques with regression modelling permitted a systematic refinement of models until best-fit convergence was achieved, enabling novel inferences to be made regarding the independent influence of both season and latitude in predicting relapse onset timing in MS. These methods have the potential for application across other complex disease and epidemiological phenomena suspected or known to vary systematically with season and/or geographic location.

Combining plot analysis with trigonometric regression is a robust method for exploring complex, cyclical phenomena such as relapse onset timing in multiple sclerosis (MS). This method enabled unbiased characterisation of seasonal trends in relapse onset permitting novel inferences around the influence of seasonal variation, ultraviolet radiation (UVR) and latitude.

多発性硬化症の再発のデータを用いて実証季節変動の三角モデリングの方法

This report describes a novel Stata-based application of trigonometric regression modelling to  55 years of multiple sclerosis relapse data from 46 ...

Cutaneous Surgical Denervation:  A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies have suggested that nerves may also modulate skin disorders including atopic dermatitis, psoriasis and cancer. Here, we describe protocols for testing the requirement for nerves in maintaining a cutaneous mechanosensory organ, the touch dome (TD). Specifically, we discuss methods for genetically labeling, harvesting and visualizing TDs by whole-mount staining, and for performing unilateral surgical denervation on mouse dorsal back skin. Together, these approaches can be used to directly compare TD morphology and gene expression in denervated as well as sham-operated skin from the same animal. These methods can also be readily adapted to examine the requirement for nerves in mouse models of skin pathology. Finally, the ability to repeatedly sample the skin provides an opportunity to monitor disease progression at different stages and times after initiation.

This article includes detailed protocols for genetic labeling of mouse skin, surgical denervation, skin biopsy and visualizing labeled epithelia by whole-mount &#946;-galactosidase staining. These methods can be used to test the requirement for nerves in mouse models of normal and pathological skin.

皮膚外科神経切除：皮膚病のマウスモデルにおける神経のための要件をテストする方法

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies ...

Assessment of Child Anthropometry in a Large Epidemiologic Study

A high proportion of children have overweight and obesity in the United States and other countries. Accurate assessment of anthropometry is essential to understand health effects of child growth and adiposity. Gold standard methods of measuring adiposity, such as dual X-ray absorptiometry (DXA), may not be feasible in large field studies. Research staff can, however, complete anthropometric measurements, such as body circumferences and skinfold measurements, using inexpensive portable equipment. In this protocol we detail how to obtain manual anthropometric measurements from children, including standing and sitting height, weight, waist circumference, hip circumference, mid-upper arm circumference, triceps skinfold thickness, and subscapular skinfold thickness, and procedures to assess the quality of these measurements. To demonstrate accuracy of these measurements, among 1,110 school-aged children in the pre-birth cohort Project Viva we calculated Spearman correlation coefficients comparing manual anthropometric measurements with a gold standard measure of body fat, DXA fat mass1. To address reliability, we evaluate intra-rater technical error of measurement at a quality control session conducted on adult female volunteers.

In epidemiologic studies of children, well-trained research staff can accurately and precisely assess weight, height, sitting height, skinfold thicknesses, and body circumferences.

大規模な疫学研究における児童身体計測の評価

A high proportion of children have overweight and obesity in the United States and other countries. Accurate assessment of anthropometry is essential to ...