Name: a murine pancreatic islet cell-based screening for diabetogenic environmental chemicals
Uploaded: 2018-06-25T12:30:00.000+00:00
Duration: 7 min 39 s
Description: Watch this Scientific Journal Video about A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals at JoVE.com

この作品は、CREH センター主催 NIEHS、国家自然科学基金の中国 (第 31572626) からパイロット プロジェクトの助成金によって支えられました。

すべての動物実験は、すべての関連するガイドライン、規制、規制機関に準拠して実行されました。例示されているプロトコルは、指導と承認機関動物ケアおよび使用委員会 (IACUC)、テキサス A & M 研究所のゲノム医学の下で行った。
1. ソリューションの準備
<ol><li>ダブル蒸留 H2O, 1 x 10 x ハンクのバランスの取れた塩溶液を希釈し、4 ° C で保存</li>
	<li>HEPES (10 mM)、MgCl2 (1 mM)、グルコース (5 mM) を追加して、分離ソリューションを準備します。PH 7.4 に調整して氷の上。</li>
	<li>コラゲナーゼ タイプ 4 からコラゲナーゼの解決を準備します。 注:具体的には、コラゲナーゼ タイプ 4 には、乾燥重量の mg 当たり 160 U が含まれています。1 グラム コラゲナーゼ タイプ 4 は 5 mL 二重蒸留 H2O 200 Mg/ml の濃度で希釈しました。30 mL の氷冷分離ソリューションに希薄のコラゲナーゼの 2 mL が追加され、コラゲナーゼ溶液 6 mL が各マウスの 50 mL チューブに転送されます。</li>
	<li>1 mM CaCl2を分離ソリューションに追加することで洗浄液を準備します。</li>
	<li>浄化液の冷たい polysucrose/ナトリウム diatrizoate (1.1119 g/mL、5 mL) を準備します。</li>
	<li>RPMI 培地への L-グルタミン (20 mM)、(100 U/mL) ペニシリン、ストレプトマイシン (100 μ g/mL)、ウシ胎児血清 (FBS) (10%) の追加によって膵島培養液 500 mL を準備します。</li>
	<li>蒸留水 200 μ M で、-20 ° C で店で粉体を溶解することにより DCFH DA 原液を準備します。 注:染色液ロスたて、DCFH DA RPMI で 5 μ M の最終的な集中に原液を希釈することによって行われます。この試薬は、光から保護する必要があります。</li>
</ol>2. 手術の準備
<ol><li>腹腔内注入 (i. p.) タヴェルタン (2.5% タヴェルタン、0.4 mL/20 g) 付きのマウス。マウス麻酔が完全に応答しなくなった後ハインドに足をつまみ、マウスを安楽死させるし、生物のフードに移動します。</li>
	<li>腹部側を上に euthanized のマウスを置き、殺菌のための 70% のエタノールで皮膚をスプレーします。</li>
	<li>腹部に 1 cm U 切開で腹腔内を開き、腹腔内を公開する胸の上の吻側方向に皮膚を撤回します。</li>
</ol>3. 膵灌流と除去
<ol><li>図 1に示すように、乳頭部の場所を見つけます。オディ括約筋の位置の近くに十二指腸をクランプするのにカーブタイプ鉗子のペアを使用します。十二指腸に胆汁経路をブロックするのに十二指腸の壁をクランプするのにカーブタイプ鉗子の別のペアを使用します。 注:この手順では、乳頭が挿入されていません。この手順は、十二指腸で胆汁の経路をブロックするものです。</li>
	<li>頭の近位と遠位研究者に関しての足を使って、マウスの位置を変更します。</li>
	<li>総胆管を見つけるし、30 G 針、5 mL 注射器でゆっくりとコラゲナーゼ溶液 3 mL を注入します。総胆管と膵臓注入後膨張していることを確認します。</li>
	<li>下降結腸、腸、胃と脾臓から分離するカーブタイプ鉗子と罰金のはさみを使用して膨張した膵臓を削除します。コラゲナーゼ溶液 3 mL を含む 50 mL チューブに膵臓を置く氷の上保管してください。</li>
</ol>4. 膵消化
<ol><li>37 ° C の水浴に各 50 mL チューブを配置します。15-20 分間インキュベート (ひずみとマウスの年齢時間異なります)。</li>
	<li>渦を使用して管を振動させます。</li>
	<li>4-5 大型滅菌シンク ストレーナーの消化膵臓をプール、スクープを使用して、徹底的にバラバラ膵臓。強制的に画面オフ消化細胞をピペットに 23 G 針、10 mL シリンジを含む洗浄液を使用します。50 mL チューブの洗浄ソリューションを収集します。</li>
	<li>4 ° c 1 分 97 x g でチューブを遠心し、上清をデカントします。</li>
	<li>チューブに洗浄溶液 25 mL を追加し、再穏やかなボルテックスによって組織を中断します。4 ° c 1 分 97 x g で組織をスピンし、上清をデカントします。3 回繰り返します。ペレットの組織が外れないように注意してください。</li>
</ol>5. 島の精製
<ol><li>洗浄組織ペレットに浄化溶液 10 mL を追加し、軽く内容を再懸濁します。</li>
	<li>優しく浄化ソリューションに分離溶液 5 mL をオーバーレイします。2 つのソリューション間の鋭い液体インターフェイスを保つように注意します。</li>
	<li>低速の加速と 15 分のための 4 ° C で g x 560 でないブレーキ管を遠心します。インターフェイスにピペットを挿入し、新しい 50 mL チューブに小島層を収集します。</li>
	<li>3.5 の手順で説明されているように島を洗います。</li>
</ol>6. メッキと膵島細胞の治療
<ol><li>膵島培養液の 10 mL で小島を再懸濁します、ゆっくり、混ぜると、96 ウェル プレートにマルチ チャンネル ピペットを 100 μ L/ウェル。まあ 96 あたり約 5 小島を配置します。</li>
	<li>12 h の組織文化のインキュベーターでプレートを配置します。</li>
	<li>遠心分離機 (112 x g) の 96 ウェル プレート、培養培地を取り外します。</li>
	<li>膵島培養液中の環境汚染物質を希釈します。プレートの別の井戸に化学の希釈剤のそれぞれの 100 μ L を追加します。各ウェル中の小島を再懸濁します、37 ° C (異なる環境化学物質の濃度変化) で 12 時間孵化させなさい。</li>
</ol>7. ROS 染色と測定
<ol><li>1x PBS で洗浄小島治療後 (各 96 ウェルの 200 μ L)。 注:化学物質の濃度は以下の通りです: AFB1: 3 μ M、アトラジン: 100 μ M、BaP: 10 μ M、BPA: 100 μ M, PCB126 β エストロゲン: 10 μ M、ノニルフェノール、ビスフェノール A: 100 μ M。</li>
	<li>活性酸素を抑制するのに抗酸化物質として N アセチルのシステイン (NAC) を使用します。ROS 誘起化学希釈剤に NAC の 5 mM を追加し、37 ° C で 12 時間インキュベート</li>
	<li>112 x g で 96 ウェル プレートを遠心し、37 ° C, 60 分で蛍光プローブ (H2 DCFH DA RPMI メディアで解散) の 5 μ M 溶液で孵化させなさい。</li>
	<li>PBS で 3 回処理した膵島細胞を洗浄し、488 のマイクロ プレート リーダーで蛍光を測定 nm。 注:530 で蛍光性の増加によって測定されるセルの DCF の蓄積サンプルは 488 で興奮すると nm nm。</li>
	<li>活性酸素を誘発する異物にグルコース刺激によるインスリン分泌 (GSIS) 試金9を実行膵島の生理機能上異物の影響を測定する未処理の島と島を扱われます。</li>
</ol>

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著者は、彼らは競合する金銭的な利益があることを宣言します。

環境化学物質への曝露を示唆している証拠の蓄積 2 型糖尿病の開発の重要な役割を果たしています。化学物質による活性酸素は、2 型糖尿病の発展に貢献する潜在的な病因因子として認識されています。人間は、生体異物の化学薬品の広い範囲にさらされている、膵毒性物質を効果的に識別し、膵臓の細胞に固有の毒性のメカニズムを調査する新しい研究技術のための大きい必要があります?...

2 型糖尿病の有病率の増加は、近年公衆衛生1に深刻な脅威に地球規模の健康危機をなっています。多くの要因が見つかっている間、2 型糖尿病の開発にリンク作因的に定期的な示唆これらの要因の 1 つの一般的な収束点である過剰な活性酸素2の世代につながる酸化ストレス誘導,3。
Pcb、ダイオキシン、BaP など環境化学物質の広いスペクトルは、膵 β 細胞の機能を損なうし、インスリン抵抗性と 2 型糖尿病4につながる可能性のある酸化ストレスを誘導するために発見されています。活性酸素の生理学的なレベルでは、細胞機能に重要な役割を果たしている、抗酸化システムの容量を超える ROS への暴露細胞/組織に損傷の結果、5疾患に 。膵 β 細胞抗酸化酵素の低レベルを表現、従って酸化ストレスを介した損傷6、7のための敏感なターゲット。ストレスによる膵細胞機能不全5が発生する活性酸素の高レベルへの慢性露出が示されている8の肝臓や脂肪組織でのインスリン抵抗性と同様。
このプロジェクトの全体的な目標は、膵臓の細胞内活性酸素の誘導に基づく彼らの糖尿病誘発電位のスクリーン化学物質に細胞ベースのアッセイを開発することです。膵臓は、代謝解毒を欠いているし、異物による損傷6,7の重要なターゲットであります。したがって、膵臓細胞で生成された ROS を直接測定することにより、この試金は化学的に誘導膵損傷の直接近似を提供しなければなりません。このメソッドを開発するには、マウス膵ランゲルハンス島の分離、化学物質、細胞培養条件下で分離膵島を培養し、読み出しとして化学誘導活性酸素生成を利用します。この手順はシンプルで孤立した小島に ROS を誘発する化学物質を識別するに効果的膵臓体外に固有の毒性発現機構の解明にはさらに開発できます。

<table><tbody><tr><td>10&amp;times;Hank&amp;rsquo;s balanced salt solution&amp;nbsp;</td><td>GIBCO</td><td>14185-052</td><td></td></tr><tr><td>Collagenase Type 4</td><td>Worthington Biochemical Corporation</td><td>CLS-4</td><td></td></tr><tr><td>polysucrose/sodium diatrizoate solution&amp;nbsp;</td><td>Sigma</td><td>10771</td><td></td></tr><tr><td>2&amp;rsquo;,7&amp;rsquo;-dichlorofluorescein (DCFH-DA)</td><td>Sigma</td><td>D6883-50MG</td><td></td></tr><tr><td>fluorescence microplate reader&amp;nbsp;</td><td>Biotek</td><td></td><td></td></tr><tr><td>L-glutamine</td><td>Sigma</td><td>G8540-25G</td><td></td></tr><tr><td>streptomycin</td><td>GIBCO</td><td>15140148</td><td></td></tr><tr><td>FBS</td><td>GIBCO</td><td>26140079</td><td></td></tr><tr><td>RPMI 1640</td><td>GIBCO</td><td>11875-085</td><td></td></tr><tr><td>avertin</td><td>Sigma</td><td>T48402-25G</td><td></td></tr><tr><td>Rat/Mouse Insulin ELISA Kit</td><td>Millipore</td><td>EZRMI-13K</td><td></td></tr><tr><td>Centrifuge</td><td>Sorval</td><td>Sorval RT7</td><td>for 96-well plate centrifuge</td></tr><tr><td>Microplate reader</td><td>Biotek</td><td>Epoch 2</td><td>for fluorescence reading</td></tr></tbody></table>

a murine pancreatic islet cell-based screening for diabetogenic environmental chemicals

人間や動物の特定の環境化学物質への暴露は、2 型糖尿病 (2 型糖尿病) の開発につながる膵臓 β 細胞の細胞損傷を引き起こす発見されています。化学的に誘導 β 細胞障害のメカニズムが明白でなく、複雑になる可能性があります、1 つの定期的な発見は、これらの化学物質への損傷を引き起こす過剰な活性酸素種 (ROS) の生成につながる酸化ストレスを誘発します。β 細胞。C57bl/6 マウスの膵島細胞および 96 ウェルの細胞培養プレートに培養膵島細胞は、潜在的な糖尿病誘発環境化学物質を識別するに分離されました。膵島細胞が化学物質を投与し、2', 7'-ジクロルフルオレッセン (DCFH-DA) 蛍光色素による活性酸素生成が検出されました。このメソッドを使用すると、我々 は [] ピレン (BaP) やポリ塩化ビフェニル (Pcb) そのビスフェノール A (BPA)、ベンゾを発見、彼らは潜在的膵島細胞の損傷を引き起こす可能性があります示唆 ROS の高レベルを引き起こすことができます。このメソッドは、糖尿病誘発物質をスクリーニングするために役立ちます。さらに、培養された膵島細胞膵臓細胞に毒性を化学的に誘導の in vitro解析の適応もあります。

健全な分離膵島の顕微鏡写真に示す図 2小島が (ただし、サイズ均一性ひずみに異なります) サイズを比較的均一に円形または楕円形をしています。次に膵島を分離し、文化島のインスリン分泌を刺激することでの in vitroアッセイにおける膵島機能を調べた。3.3 〜 16.7 mM グルコース9による C57BL/6 マウス孤立島から情報科?...

Watch this Scientific Journal Video about A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals at JoVE.com

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

ここで潜在的な糖尿病誘発異物化学物質を識別するために、化学物質によって ROS の帰納法をスクリーニングするためのマウスの膵島細胞を分離するためのプロトコルを提案する.

マウスの膵島細胞ベースの糖尿病誘発環境化学物質のスクリーニング

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to ...

a-murine-pancreatic-islet-cell-based-screening-for-diabetogenic

Research

JoVE Journal

Immunology and Infection

7.2K ビュー.  Hunan Agriculture University. ここで潜在的な糖尿病誘発異物化学物質を識別するために、化学物質によって ROS の帰納法をスクリーニングするためのマウスの膵島細胞を分離するためのプロトコルを提案する.

ビデオ: A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Murine Pancreatic Islet Isolation

Hi, I am Greg Zott. I'm with the UCSF Diabetes Center in the Bluestone Laboratory, and today I'm gonna show you the process for isolating mouse eyelets. The procedure involves the injection of a collagenase solution into the pancreas of a C3 H donor mouse, where we will then remove that pancreas, digest it away, purify the eyelets on a fial gradient, and then handpick them for ultimate transplant into the kidney capsule of a diabetic NOD mouse, Which will be our recipient.Before we start this procedure, we diluted our ATE solution in a modified Hanks buffer and we have adjusted the concentration specific for the strain of mouse that we're using the C3 H.We found digests better at a 0.8 mgs per mil collagenase solution, and we will use that to inject into the common bile duct to distend the pancreas.Performing. The procedure today Will be Pavo codre of the diabetes center. He will inject, open up the mouse, expose the the intestines, and then inject into the common bile duct.We will spray down the mouse with ethanol to keep the hair down, and then we will also verify that the mouse has been sacrificed properly with a toe pinch. Pavo will now open up the mouse and exposing the intestine and the pancreas with a midline incision. The diaphragm will also be cut to verify that the mouse will be sacrificed.Before we get started, I'd like to point out some of the important structures. The stomach is lying right here with the pancreas attached underneath into the duodenum and above. This is the liver where we'll find the gallbladder and the common bile duct, which you can see right here.So what Pavo will do next is he will isolate the small intestine, find where the common bile duct enters the small intestine. Once the intestine is exposed, we clamp on either side of the common bile duct with mosquito clamps. We find that this allows more collagenates to enter the pancreas while other groups try to direct the clamp on top of the common bile duct.We find that this is a better technique in allowing enzyme to enter the pancreas. This is a gentle procedure. You don't want to tear the bowel.You wanna allow the clamps to lie gently on either side of the common bile duct. Now we will find a gallbladder, and once the gallbladder's exposed, we will use that as a guide to injecting the collagenase into the common bile duct. Powell's using a 30 gauge needle with a five cc syringe containing three mills of collagenase solution.As he's entering the common bile duct, he's distending that duct with collagenase solution and then he travels down the common bile duct till he reaches near the intestine. And as he's going down, the enzyme solutions being pumped into the injected into the pancreas. Now that the pancreas is inflated with collagenase solution, Powell's gonna remove the hemostats and then remove the pancreas from the the mouse carefully tearing it away from the intestine.He removes it from the stomach. Okay, and then using the spleen as a handle, he lifts up the pancreas and then slowly snips it away in the common bile duct, and that's it. Pancreas is laid out and then the spleen is removed, and there he's removing the meso enteric tissue, and then the pancreas is placed in a two mill collagenase solution and a sterilized glass vial and it's placed back on Ice.The isolated pancreas that we put into the glass vials will now be placed into a 37 degree water bath for approximately 17 minutes. We placed them into a 50 mil conical rack so that they don't float up. Once during the bath, the timer starts and the digestion begins.Once the digestion time is completed, vials are removed, and then they are shaken and disrupted gently. And once the tissue starts falling apart, the vial is then placed on ice. A sterile screen is placed over a 400 mil beaker that contains washing buffer.The pancreas digest is then carefully poured over the screen into the wash buffer. For demonstration purposes, the hood has been turned off and the glass of the hood has been raised so we could see what we're doing. Once the pancreas digest is poured into the beaker, the vials are rinsed with the same washing buffer and the wash buffer placed over the screen.We have a syringe filled with washing buffer. We will use that to forcefully wash the tissue through the screen. The idea is to release any loose eyelets that are unattached to the matrix, but leaving the membranes and the undigested tissue on the screen.And when you're finished washing a tissue, this is what you usually See at the end of the procedure. The Pancreas tissue that's in the bottom of the tube will now be transferred to two 50 mil conical, and the tissue will be washed, the tissue's mixed evenly, and then distributed evenly so that we have a equal pellet volume in each tube. The beaker is now rinsed with more washing buffer.The sides are rinse so that you get all tissue that is entered the beaker again. Then it's equally distributed between each two, both 50 mil. Conical then are topped off with wash buffer.The tissue is inverted several times and then placed into a centrifuge. This is a quick spin. The centrifuge is allowed to reach a thousand RPM and then is turned off.The tissue at this time is very S eyelets are very sensitive, and you don't want to over compact them. The tubes are removed and we will do this wash step three more times. This is the end of the third wash.The components of the 50 mil conical tube are transferred to the 15 conical tube and centrifuge for a thousand RP M and then stopped. The supernat will be removed and the first part of the FI Call will be started. We are at the step where we Have our pelleted di pancreas digest.We'll resuspend that tissue so it's nice and loose to the pellets. We'll add the first density gradient, which is 1 1 0 8. We'll add five mils to each tube.The pellet is then mixed thoroughly in each of the gradients. It's important that you get good tissue distribution in your heaviest gradient. Now we will layer two mils of 1 0 9 6 density.We will then on top of that, layer two mils of 1 0 6 9 density, and then on top of that 1 0 3 7 density. It's important while you're layering the densities that you don't mix. Then all the layers have been added to the tube.And you can see this is the top of 1 1 0 8, the top of 9 1 0 9 6, the top of 1 0 6 9 and the top of 1 0 3 7. And you could see the tissue is already starting to move to its proper density even without centri fusion. The fial gradients are brought up to speed at 1800 RPM for 15 minutes, and remember to keep the brake off after the tubes are removed from the centrifuge.You can see between the 0 0 9 6 and 0 0 7 layer, there's a nice band of eyelet tissue Pablo's. Now removing the 0 3 7 layer to remove any tissue debris and DNA that might have been released during the processing. Using a sterile plastic pasture pipette, he will now remove the islet layer and transfer them to a 50 mil conical containing 25 mils of wash buffer.You want do this step quickly because fol is toxic to eyelets. We actually removed the entire 0 6 9 and 0 9 6 layer. Pavo cuts the top of the plastic pasture pipette and rinses it with wash buffer.The eyelets are then centrifuge like we centrifuge previously. Centrifuge is brought up to a Thousand RPM and then we stop it and We aspirate off. The supernatant eyelets will be washed three times with wash media.So we just completed the eyelid Isolation process we took from the digest to the fol gradient to the purified eyelets after our third wash with wash buffer, we resuspended the eyelets in our RPMI media and we played it in a tissue culture plate. These eyelets are now ready for in vitro assays or in vivo preps like eyelet Transplant.

マウス膵島単離

生物学

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

マウス膵臓内分泌細胞の単一細胞トランスクリプトーム解析

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

発生生物学

Analysis of Non-Human Primate Pancreatic Islet Oxygen Consumption

The measurement of oxygen consumption in spheroid clusters of cells, such as ex vivo pancreatic islets, has historically been challenging. We demonstrate the measurement of islet oxygen consumption using a 96-well microplate designed for the measurement of oxygen consumption in spheroids. In this assay, spheroid microplates are coated with a cell and tissue adhesive on the day prior to the assay. We utilize a small volume of adhesive solution to encourage islet adherence to only the bottom of the well. On the day of the assay, 15 islets are loaded directly into the base of each well using a technique that ensures optimal positioning of islets and accurate measurement of oxygen consumption. Various aspects of mitochondrial respiration are probed pharmacologically in non-human primate islets, including ATP-dependent respiration, maximal respiration, and proton leak. This method allows for consistent, reproducible results using only a small number of islets per well. It can theoretically be applied to any cultured spheroids of similar size.

This protocol demonstrates the accurate and reproducible measurement of oxygen consumption in non-human primate pancreatic islets. The islet loading techniques and coating of the microplate provide a framework for efficient measurement of respiration in other types of cultured spheroids.

非ヒト霊長類膵島酸素消費量の分析

The measurement of oxygen consumption in spheroid clusters of cells, such as ex vivo pancreatic islets, has historically been challenging. We demonstrate ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

LEPR<sup&gt; DB</sup&gt; 2型糖尿病のマウスモデル：無傷の膵島の膵島単離およびライブセル2光子イメージング

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

医学

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

ハイコンテンツ<em&gt;インビトロ</em&gt;膵島β細胞の複製ディスカバリー・プラットフォーム

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

An In Ovo Model for Testing Insulin-mimetic Compounds

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and &#946;-cell failure. Various strategies, including the injection of insulin or the usage of insulin-sensitizing drugs, were pursued to treat T2DM or at least reduce the symptoms. In addition, the application of herbal compounds has attracted increasing attention. Thus, it is necessary to find efficient test systems to identify and characterize insulin-mimetic compounds. Here we developed a modified chick embryo model, which enables testing of synthetic compounds and herbal extracts with insulin-mimetic properties. Using a fluorescence microscopy-based primary screen, which quantifies the translocation of Glucose transporter 4 (Glut4) to the plasma membrane, we were able to identify compounds, mainly herbal extracts, which lead to an increase of intracellular glucose concentrations in adipocytes. However, the efficacy of these substances requires further verification in a living organism. Thus, we used an in-ovo approach to identify their blood glucose-reducing properties. The approval by an ethics committee is not needed since the use of chicken embryos during the first two-thirds of embryonic development is not considered an animal experiment. Here, the application of this model is described in detail.

An In Ovo Model for Testing Insulin-mimetic Compounds

In this study, we describe an in-ovo system as a promising tool to test insulin-mimetic compounds in a living organism. The main advantages include adequate throughput rates and acceptable costs, which enable the identification of phytochemicals with insulin-mimetic properties.

インスリン模倣化合物のテスト用のOvo でモデル

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and ...

神経科学

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

マウス モデルにおける膵島の門脈内移植

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

マウス膵島の単離と細胞内cAMP測定法

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

マウスからの膵島分離のための簡単な高効率プロトコル

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

マウス膵臓β細胞におけるマイトファジーの定量解析

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic &#946;-Cells

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient insulin release. Advanced quantitative assessments of mitophagy often require the use of genetic reporters. The mt-Keima mouse model, which expresses a mitochondria-targeted pH-sensitive dual-excitation ratiometric probe for quantifying mitophagy via flow cytometry, has been optimized in &#946;-cells. The ratio of acidic-to-neutral mt-Keima wavelength emissions can be used to robustly quantify mitophagy. However, using genetic mitophagy reporters can be challenging when working with complex genetic mouse models or difficult-to-transfect cells, such as primary human islets. This protocol describes a novel complementary dye-based method to quantify &#946;-cell mitophagy in primary islets using MtPhagy. MtPhagy is a pH-sensitive, cell-permeable dye that accumulates in the mitochondria and increases its fluorescence intensity when mitochondria are in low pH environments, such as lysosomes during mitophagy. By combining the MtPhagy dye with Fluozin-3-AM, a Zn2+ indicator that selects for &#946;-cells, and Tetramethylrhodamine, ethyl ester (TMRE) to assess mitochondrial membrane potential, mitophagy flux can be quantified specifically in &#946;-cells via flow cytometry. These two approaches are highly complementary, allowing for flexibility and precision in assessing mitochondrial quality control in numerous &#946;-cell models.

著者スポットライト:効果的で堅牢な補完的アプローチを使用した膵臓β細胞のマイトファジーフラックスの評価 

This protocol outlines two methods for the quantitative analysis of mitophagy in pancreatic &#946;-cells: first, a combination of cell-permeable mitochondria-specific dyes, and second, a genetically encoded mitophagy reporter. These two techniques are complementary and can be deployed based on specific needs, allowing for flexibility and precision in quantitatively addressing mitochondrial quality control.

膵β細胞におけるマイトファジーフラックスを調べるための補完的アプローチ

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient ...

マウスの膵島細胞ベースの糖尿病誘発環境化学物質のスクリーニング

要約

さらに動画を探す

この動画の章

マウス膵島単離

マウス膵臓内分泌細胞の単一細胞トランスクリプトーム解析

非ヒト霊長類膵島酸素消費量の分析

LEPR

ハイコンテンツ

インスリン模倣化合物のテスト用のOvo でモデル

マウスモデルにおける膵島の門脈内移植

マウス膵島の単離と細胞内cAMP測定法

マウスからの膵島分離のための簡単な高効率プロトコル

膵β細胞におけるマイトファジーフラックスを調べるための補完的アプローチ

マウスの膵島細胞ベースの糖尿病誘発環境化学物質のスクリーニング

要約

さらに動画を探す

この動画の章

マウス膵島単離

マウス膵臓内分泌細胞の単一細胞トランスクリプトーム解析

非ヒト霊長類膵島酸素消費量の分析

LEPR

ハイコンテンツ

インスリン模倣化合物のテスト用のOvo でモデル

マウス モデルにおける膵島の門脈内移植

マウス膵島の単離と細胞内cAMP測定法

マウスからの膵島分離のための簡単な高効率プロトコル

膵β細胞におけるマイトファジーフラックスを調べるための補完的アプローチ

マウスモデルにおける膵島の門脈内移植