이 작품은 NIEHS 및 국가 자연 과학 재단의 중국 (No. 31572626) 후원 CREH 센터에서 파일럿 프로젝트 교부 금에 의해 지원 되었다.

모든 동물 실험은 모든 관련 지침, 규정 및 규제 기관에 따라 실행 했다. 지도 및 게놈 의학 기관 동물 관리 및 사용 위원회 (IACUC) 텍사스 A & M 연구소의 승인 아래 프로토콜 입증 되 고 수행 되었다.
1. 솔루션 준비
<ol><li>두 배 증류수 H2O, 1 x 10 x 행 크의 균형된 소금 솔루션을 희석 하 고 4 ° c.에 저장</li>
	<li>HEPES (10 m m), MgCl2 (1 m m), 포도 (5 m m)를 추가 하 여 격리 솔루션을 준비 합니다. PH 7.4에 조정 하 고 얼음에 계속.</li>
	<li>유형 4 콜라에서 콜라 솔루션을 준비 합니다. 참고: 특히, 콜라 유형 4 건조 중량의 밀리 그램 당 160을 U 포함 되어 있습니다. 콜라 유형 4의 1 g 5 mL 두 배 증류수 H2O 200 mg/mL의 농도로 희석입니다. 희석된 콜라의 2 mL 30 mL 얼음 차가운 격리 솔루션에 추가 되 고 콜라 솔루션의 6 mL 50 mL 튜브 각 마우스에 대 한 전송 됩니다.</li>
	<li>1mm CaCl2 격리 솔루션에 추가 하 여 세척 솔루션을 준비 합니다.</li>
	<li>찬 polysucrose/나트륨 diatrizoate 솔루션 (1.1119 g/mL, 5 mL) 정화 솔루션을 준비 합니다.</li>
	<li>RPMI 매체에 L-글루타민 (20 m m), 페니실린 (100 U/mL), 스 (100 µ g/mL), 및 소 태아 혈 청 (FBS) (10%)를 추가 하 여 섬 문화 매체의 500 mL를 준비 합니다.</li>
	<li>200 µ M에서 증류수에-20 ° c.에 게 분말을 용 해 하 여 DCFH-다 재고 솔루션을 준비 참고: 솔루션 얼룩 선생님 갓 RPMI에 5 µ M의 최종 농도에 DCFH-다 재고 솔루션을 diluting 하 여 이루어집니다. 이 시 약은 빛 으로부터 보호 되어야 한다.</li>
</ol>2. 수술 준비
<ol><li>Intraperitoneally 주사 (I.P.) avertin (2.5 %avertin, 0.4 mL/20 g)와 마우스. 마우스 완전히 마 취 후 더 이상 응답 pinches 발 뒷 다리를 하 고 마우스를 안락사 한 생물 후드로 이동.</li>
	<li>복 면이 위로 향하도록 안락사 마우스를 배치 하 고 피부 살 균에 대 한 70% 에탄올 스프레이.</li>
	<li>상단 복 부에 1cm U-절 개 된 복 부 구멍을 열고 복 부 구멍을 노출 가슴 위에 rostral 방향으로 피부를 제거 합니다.</li>
</ol>3. 췌 관류 및 제거
<ol><li>그림 1에서 볼 수 있듯이 ampulla의 위치를 찾아. 곡선된 집게의 쌍을 사용 하 여 십이지 장 Oddi의 괄약근의 위치에 가까운 클램프. 또 한 쌍의 곡선된 겸 자 사용 하 여 클램프 십이지 장 벽에는 십이지 장 담 즙 통로 차단. 참고: ampulla이이 단계에서 삽입 되지 않습니다 있다. 이 단계에서 십이지 장 담 즙 통로 차단 하도록 의미 된다.</li>
	<li>근 위 머리와 연구원에 관해서는 원심 발 마우스 위치를 변경할.</li>
	<li>일반적인 담 관을 찾아서 30 G 바늘과 5 mL 주사기로 콜라 솔루션의 3 mL를 천천히 주입. 일반적인 담 관 및 췌 주입 후 증가 다는 것을 확인 하십시오.</li>
	<li>팽창된 췌 내림차순 콜론, 창 자, 복 부, 및 비장에서 분리 곡선된 겸 자 및 좋은 위를 사용 하 여 제거 합니다. 콜라 솔루션의 3 mL를 포함 하는 50 mL 튜브에 췌를 놓고 얼음에 그것을 유지 합니다.</li>
</ol>4. 췌 장 소화
<ol><li>37 ° C 물 목욕으로 각 50 mL 튜브를 놓습니다. 15-20 분 동안 품 어 (시간에 따라 다릅니다 긴장과 쥐의 나이).</li>
	<li>소용돌이 사용 하 여 튜브를 진동.</li>
	<li>대형 메 마른 싱크대 여과기에 4-5 소화 췌 풀 하 고 특 종 췌 철저 하 게 붕괴를 사용 하 여. 세척 솔루션을 포함 하는 23 G 바늘과 10 mL 주사기를 사용 하 여 강제로 화면에서 소화 셀 플라스틱. 세척 솔루션 50 mL 튜브에 수집 합니다.</li>
	<li>1 분에 4 ° C에서 97 x g에서 튜브 원심 고는 상쾌한 가만히 따르다.</li>
	<li>튜브를 세척 솔루션의 25 mL를 추가 하 고 다시 조직 부드러운 vortexing에 의해 일시 중단. 1 분에 4 ° C에서 97 x g에서 조직을 아래로 회전 시키십시오 그리고는 상쾌한 가만히 따르다. 3 번 반복 합니다. 수송과 조직을 꺼내 조심 해야.</li>
</ol>5입니다. 독도의 정화
<ol><li>씻어 조직 펠 릿을 정화 솔루션의 10 mL를 추가 하 고 내용을 부드럽게 resuspend.</li>
	<li>부드럽게 정화 솔루션에 격리 솔루션의 5 mL을 오버레이. 두 솔루션 간의 날카로운 액체 인터페이스를 유지 하려면 주의 해야 합니다.</li>
	<li>느린 가속 15 분 동안 4 ° C에서 g x 560 및 아무 브레이크 튜브 원심 인터페이스에는 피 펫을 삽입 하 고 새로운 50 mL 튜브에 독도 레이어를 수집 합니다.</li>
	<li>3.5 단계에서 설명한 대로 독도 씻어.</li>
</ol>6. 도금 및 작은 섬 세포의 치료
<ol><li>독도 문화 솔루션의 10 mL에는 독도 resuspend, 부드럽게 섞어 잘, 그리고 100 µ L/96 잘 접시에 멀티 채널 피 펫으로 잘 배치. 96 당 약 5 독도 잘 배치 합니다.</li>
	<li>12 h에 대 한 조직 문화 인큐베이터에 접시를 놓습니다.</li>
	<li>원심 (112 x g) 96 잘 접시와 문화 매체를 제거 합니다.</li>
	<li>독도 문화 매체에서 환경 화학 물질을 희석. 접시의 별도 우물에 각 화학 diluents 100 µ L를 추가 합니다. 각 잘에서 독도 resuspend 하 고 (다른 환경 화학 물질에 대 한 농도 다름) 37 ° C에 12 h에 대 한 품 어.</li>
</ol>7. 선생님 얼룩 및 측정
<ol><li>독도 치료 후 1 x PBS로 세척 (각 96 잘 200 µ L). 참고: 화학 물질의 농도 다음과 같습니다: AFB1: 3 µ M, 아트 가진: 100 µ M, BaP: 10 µ M, BPA: 100 µ M, β-에스트로겐, PCB126: 10 µ M, Nonylphenol, 및 BPA: 100 µ M.</li>
	<li>항 산화로 N-아 세 틸 시스테인 (NAC)를 사용 하 여 선생님을 끄다. NAC의 5mm 선생님 유발 화학 diluents을 추가 하 고 37 ° c.에 12 h에 대 한 품 어</li>
	<li>112 x g에서 96 잘 접시 원심 하 고 60 분 동안 37 ° C에서 5 µ M 솔루션 형광 프로브 (H2-DCFH-다 RPMI 미디어에 녹아 있는)의 품 어.</li>
	<li>PBS로 3 회 치료 작은 섬 세포를 세척 하 고 488에서 microplate 리더 형광 측정 nm. 참고: 셀에 DCF의 축적 증가 의해 형광 530에서 측정할 수 있습니다 때 샘플 488에 흥분은 nm nm.</li>
	<li>포도 당 자극 인슐린 분 비 (GSIS) 분석 결과9 선생님 유도 xenobiotic 수행 독도 췌 장 독도의 생리 기능에 xenobiotic 효과 측정 하기 위해 치료 독도 취급.</li>
</ol>

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저자 들은 아무 경쟁 금융 관심사 선언 합니다.

환경 화학 물질에 노출을 제안 증거를 축적 T2DM의 개발에 중요 한 역할. Xenobiotics 유도 선생님 T2DM의 발전에 기여 하는 잠재적인 etiological 요인으로 인식 하고있다. 인간은 다양 한 xenobiotic 화학 물질에 노출 되는 그리고 췌 장 이용과 효과적으로 식별 하 고 췌 장 세포에 독성의 기계 장치를 조사 하는 새로운 연구 기법에 대 한 큰 필요.
14, 게시 된 절차 따라 ?...

T2DM의 보급에 있는 증가 최근 몇 년 동안 공중 보건1에 심각한 위협이 포즈 세계 보건 위기 되고있다. 많은 요인이 발견 되었습니다 원인이 되는, T2DM의 개발에 연결 될 반복 결과 것이 좋습니다 이러한 요소에 대 한 하나의 일반적인 집중 포인트의 과도 한 선생님2 세대에 이르게 산화 스트레스의 유도 , 3.
환경 화학 물질 PCBs, 다이옥신, BaP 등의 다양 한 스펙트럼 유발 산화 스트레스, 췌 장 β 세포의 기능을 손상 하 고 인슐린 저항 및 T2DM4이어질 수 있습니다 발견 되었습니다. 선생님의 생리 적 수준 세포 기능에 중요 한 역할을, 비록 항 산화 시스템의 용량을 초과 하는 선생님에 노출 세포/조직에 손상 귀착되 고 질병5리드. 췌 장 β 세포 항 산화 효소의 낮은 수준의 표현 되며 따라서 산화 스트레스 중재 손상6,7에 대 한 중요 한 목표. 선생님의 상부에 만성 노출은 췌 장 세포 스트레스 유발 장애5 원인을 보여줘 되었습니다8간 및 지방 조직 인슐린 저항 뿐만 아니라.
이 프로젝트의 전반적인 목표는 췌 장 세포에 선생님의 그들의 유도에 따라 그들의 diabetogenic 잠재력에 대 한 화학 물질 화면에 세포 기반 분석 결과 개발 하는 것입니다. 췌 대사 해독 부족 고 xenobiotic 손상6,7에 대 한 중요 한 대상 이다. 따라서, 췌 장 세포에서 생성 하는 선생님을 직접 측정 하 여이 분석 결과 췌에 화학 유도 부상의 직접적인 근사를 제공 해야 합니다. 이 방법을 개발, 우리는 절연 마우스 췌 장 독도 교양 화학 물질, 셀 문화 조건 하에서 고립 된 섬 하 고 판독으로 유도 하는 화학 선생님 세대를 활용. 이 절차는 간단 하 고 효과적인 격리 된 섬; 선생님 유도 하는 화학 물질 식별 그것은 췌 체 외에 관련 된 독성의 메커니즘의 조사에 대 한 추가 개발할 수 있습니다.

<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

인간과 동물에서 특정 환경 화학 물질에 노출은 타입-2 당뇨병 mellitus (T2DM)의 개발으로 이어질 것 이다 췌 장 β 세포의 세포 손상을 발견 되었습니다. 화학 유도 β 세포 손상에 대 한 메커니즘은 불분명 하 고 복잡 한 것 같다, 비록 한 되풀이 발견은 이러한 화학 물질에 손상을 유발합니다 과도 한 반응성 산소 종 (선생님)의 생성을 선도 하는 산화 스트레스 유발 β 세포입니다. 우리 C57BL/6 마우스의 췌 장 섬 세포과 교양된 섬 셀 96 잘 세포 배양 배지;에 있는 고립 된 잠재적인 diabetogenic 환경 화학 물질 식별, 그리고, 작은 섬 세포는 화학 물질과 복용 했다 선생님 세대 2', dichlorofluorescein (DCFH-다)-7' 형광 염료에 의해 발견 되었다. 이 방법을 사용 하 여, 우리는 bisphenol A (BPA), Benzo [a] pyrene (BaP), 그리고 비페닐 (PCBs), 그들은 잠재적으로 작은 섬 세포에 손상을 유발 수 있습니다 제안 하는 선생님의 상부 유도 수 있습니다. 이 메서드는 diabetogenic xenobiotics를 심사 하는 데 유용 해야 합니다. 또한, 경작된 작은 섬 세포 췌 장 세포에서 화학 유도 된 독성의 생체 외에서 분석에 맞게 수도 있습니다.

건강 한 고립 된 섬의 현미경 사진 그림 2, 독도 원형 또는 타원형 모양으로 비교적 균일 한 크기 (비록 크기 균일성 스트레인 스트레인에서 다를 수 있습니다)를가지고 있는에 표시 됩니다. 우리 다음 조사 생체 외에서 분석 결과에서 췌 장 섬 기능 분리는 섬 하 고 문화 독도에서 인슐린 분 비를 자극. 그림 3 GSIS 분석 결?...

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

여기 우리는 잠재적인 diabetogenic xenobiotic 화학 물질 식별는 xenobiotics에 의해 선생님 inductions을 심사에 대 한 마우스 췌 장 섬 세포 격리 프로토콜을 제시.

Diabetogenic 환경 화학 물질에 대 한 Murine 췌 장 섬 세포 기반 검사

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

7.3K 조회수.  Hunan Agriculture University. 여기 우리는 잠재적인 diabetogenic xenobiotic 화학 물질 식별는 xenobiotics에 의해 선생님 inductions을 심사에 대 한 마우스 췌 장 섬 세포 격리 프로토콜을 제시.

비디오: 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.

Murine 췌장 아일릿 격리

연구

생물학

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

당뇨병 쥐의 부고환 지방 세포에 이식 된 지지체 지지대 지지대

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

생체공학

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 형 당뇨병의 마우스 모델 : 본래 독도 췌장 섬 세포의 분리 및 라이브 셀 2 광자 이미징

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, ...

의학

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.

Intraportal 이식 마우스 모델에서 췌 장 독도의

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.

마우스 췌장 섬의 분리 및 세포 내 cAMP를 결정하는 방법

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

Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices

Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared from live human and mouse pancreatic tissue embedded in agarose and cut using a vibratome. This method allows for the tissue to maintain viability and function in addition to preserving underlying pathologies such as type 1 (T1D) and type 2 diabetes (T2D). The slice method enables new directions in the study of the pancreas through the maintenance of the complex structures and various intercellular interactions that comprise the endocrine and exocrine tissues of the pancreas. This protocol demonstrates how to perform staining and time-lapse microscopy of live endogenous immune cells within pancreatic slices along with assessments of islet physiology. Further, this approach can be refined to discern immune cell populations specific for islet cell antigens using major histocompatibility complex-multimer reagents.

This study presents the application of live pancreatic tissue slices to the study of islet physiology and islet-immune cell interactions.

살아있는 췌장 조직 조각에서 아일렛 기능 및 아슬레 면역 세포 상호 작용을 관찰

Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared ...

면역학 및 감염병학

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

근접 결찰 분석분석서를 활용한 인간 췌장 베타 세포에서 내인성 미토파기 복합체의 시각화

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for islet transplantation in rodent models. However, the tight kidney capsule limits the transplantation of sufficient islets in large animals and humans. The inguinal subcutaneous white adipose tissue (ISWAT), a new subcutaneous space, was found to be a potentially valuable site for islet transplantation. This site has better blood supply than other subcutaneous spaces. Moreover, the ISWAT accommodates a larger islet mass than the kidney capsule, and transplantation into it is simple. This manuscript describes the procedure of mouse islet isolation and transplantation in the ISWAT site of syngeneic diabetic mouse recipients. Using this protocol, murine pancreatic islets were isolated by standard collagenase digestion and a basement membrane matrix hydrogel was used for fixing the purified islets in the ISWAT site. The blood glucose levels of the recipient mice were monitored for more than 100 days. Islet grafts were retrieved at day 100 after transplantation for histological analysis. The protocol for islet transplantation in the ISWAT site described in this manuscript is simple and effective.

Inguinal Subcutaneous White Adipose Tissue ISWAT Transplantation Model of Murine Islets

In this protocol, a method of murine islet isolation and transplantation into the inguinal subcutaneous white adipose tissue is described. Isolated syngeneic murine islets are transplanted into a murine recipient using a basement membrane hydrogel. The blood glucose level of the recipients is monitored, and histology analysis of the islet grafts is performed.

뮤린 아일렛의 인과 피하 백색 지방 조직 (ISWAT) 이식 모형

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for ...

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.

마우스에서 췌장 췌도 격리를 위한 간단한 고효율 프로토콜

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

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

마우스 췌도 세포의 하위 집단에 있는 화상 진찰 칼슘 역학

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...