伦蒂病毒介导基因在低附着板中制备的人类伪性沉默
Summary
提出了一种从分散的人类胰岛细胞中产生基因修饰的人类伪胰岛的函数,这种细胞由携带短发核糖核酸(shRNA)的慢病毒转导。该协议利用现成的酶和培养容器,易于执行,并产生适合功能和形态学研究的转基因人类假岛。
Abstract
各种基因工具可用于调节啮齿动物胰腺胰岛的基因,以解剖胰岛基因的功能,用于糖尿病研究。然而,由于物种之间在胰岛结构和功能方面众所周知的差异,从啮齿动物胰岛获得的数据往往不能完全复制或适用于人类胰岛。目前,可用于操纵人类胰岛基因表达的技术非常有限。腺病毒、质粒和寡核苷酸将转基因引入完整胰岛,往往效率低、毒性高。低效率在完整小岛的基因调控研究中尤其成问题,需要高效率。众所周知,酶分散的胰岛细胞在培养中重新聚合,形成称为伪胰岛的球状体。大小控制的人类胰岛细胞的再聚合产生伪胰岛,在长期培养后维持动态第一阶段胰岛素分泌,并提供一个窗口,以有效引入低毒性的慢病毒短发针RNA(shRNA)。这里,描述了使用两个市售的多孔板在慢病毒转导后创建人类伪小岛的详细方案。该协议可以很容易地执行,并允许有效地降低基因和评估使用人类小岛细胞的胰岛素分泌活力。因此,具有慢病毒介导基因调制的人类伪胰岛为评估人类胰岛细胞内的基因功能提供了一个强大而通用的模型。
Introduction
功能β细胞质量的丧失是1型和2型糖尿病1的中心病理学。虽然β细胞是胰腺胰岛胰岛素的产生者,但β细胞和非β细胞之间的通信在调节胰岛素分泌2方面起着至关重要的作用。此外,胰高血糖素分泌不良导致糖尿病高血糖3。因此,对胰腺胰岛内细胞的基因表达进行调节,以解决糖尿病胰岛功能障碍发展背后的机制有着浓厚的兴趣。包括转基因小鼠在内的各种方法可用于调节小鼠胰岛的基因表达。然而,人类和小鼠胰岛表现出明显的内向、细胞分布、β与α细胞的比例以及对分泌细胞4的响应。因此,直接评估人类胰岛的基因功能对于理解人类胰腺胰岛的病理生理学具有极其重要的作用。
腺病毒载体是体外转胰腺胰岛应用最广泛的病毒载体,因为非分裂细胞的转导效率很高。然而,腺病毒不能有效地渗透到胰岛的核心,特别是在人类胰岛5,并且在高剂量6细胞毒性。相比之下,慢病毒载体的细胞毒性较小,将外源基因永久地输送到后线细胞的染色体中,使其成为基因治疗7的广泛测试载体。然而,扁豆病毒穿透完整人类胰岛核心的能力也有限,因此需要通过酶消化进行部分分散,以提高转导效率8。完整人类胰岛分散的注意事项是细胞-细胞和细胞基质通信的中断,这损害了胰岛素分泌的动态调节,这对维持人类葡萄糖平衡至关重要9。因此,在人类胰岛模型中评估基因调制对胰岛功能动态调节的影响一直具有挑战性。
众所周知,人类和啮齿类小岛的分散胰岛细胞可自主地重新聚合成称为”伪胰岛”的胰岛状结构。伪胰岛显示β和非β细胞分布类似于原生小岛10,11。此外,经过长期培养后,原生胰岛逐渐失去强大的第一期胰岛素分泌5,10,11,12。然而,与同一文化期5之后的本地小岛相比,伪胰岛在葡萄糖反应下,第一阶段胰岛素分泌的保存效果更好。除了更好地保存胰岛素分泌外,在低附着板11中控制人体小岛细胞的重新聚集,为在将扁病毒载体重新聚集到其中提供了一个机会之窗伪小岛。几项研究已经证明了伪胰岛结合扁病毒介导转导的效用。Caton等人13日报告说,与未感染对照相比,引入表达慢病毒的绿色荧光蛋白(GFP)对胰岛素分泌作用甚微,同时实现大鼠伪胰岛体内GFP的同质表达。他们还通过慢病毒13过度表达康奈辛32、36和43,证明了不同康奈辛对胰岛素分泌的具体作用。用市售的96孔超低附着板制备的人类伪胰岛证明,经慢病毒介导的转录因子SIX3的过度表达可改善静态孵育评估的胰岛素分泌。最近,用96孔超低附着板制备的人类伪胰岛通过慢病毒短发针RNA(shRNA)降低调节葡萄糖素酶,以证明其原理,表明葡萄糖刺激的胰岛素分泌减少,同时KCl刺激胰岛素分泌保存5。研究还表明,人类伪胰岛在基因表达和分泌谱方面与原生胰岛相似,进一步支持了人类伪胰岛对胰岛功能5的调节的效用。虽然没有进行围产,但最近上市的生物工程微孔培养板也报告与慢病毒转导相容,并产生了具有优秀胰岛素的人类假小岛移植后体外和体内分泌11。总体而言,人类假岛的形成与慢病毒转导相结合是研究人类胰岛病理生理学的一种简单而有效的方法,为在人类胰岛中进行机械研究提供了宝贵的工具。
在本报告中,提出了一种利用两个市售平台——96孔超低附着板和微孔培养板——与慢病毒转导的人类伪小岛形成方案。两者都实现了基因表达的有效调制,并创造了与下游评估(包括静态孵育和围气又液)相容的人类伪岛。
Protocol
Representative Results
Discussion
这里给出了一种使用96孔超低附着板或微孔培养板生成由慢病毒转导的人类伪小岛的详细方案。据报道,伪小岛已显示出形态和分泌功能类似于本地人类胰岛,可以在体外进行长时间的体外培养。与在大小上表现出巨大差异的本地人类小岛不同,伪小岛在大小上相对均匀,减少了捐赠者和实验复制体5,11之间的变异。</sup…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国国家卫生研究院(R01-DK090490)和美国糖尿病协会对Y.I.(1-17-IBS-132)的财政支持。J.A.和Y.I.由鹰糖尿病研究中心兄弟会支持。A.B. 得到国家卫生研究院培训补助金(T32NS45549)的支持。作者利用了由NIDDK资助的希望之城综合胰岛分配方案(IIDP)提供的人类胰腺胰岛(2UC4DK098085)。
Materials
| Anti-adherence rinsing solution | Stemcell technologies | 7919 | |
| Biological safety cabinet | Thermo Scientific | 1300 Series Type A2 | |
| cell strainer, 40 micrometer | Corning | 431750 | |
| CMRL-1066 | ThermoFisher | 11530037 | |
| CO2 incubator | Thermo Scientific | Heracell VIOS 160i | |
| conical centrifuge tube, 15 mL | VWR | 89039-666 | |
| conical centrifuge tube, 50 mL | VWR | 89039-658 | |
| fetal bovine serum | ThermoFisher | 26140079 | |
| guanidinium thiocyanate RNA extraction reagent | ThermoFisher | 15596026 | Trizol |
| glutamine | ThermoFisher | 25030164 | |
| Hemocytometer | Marien Feld | Neubauer-Improved Bright line | |
| Human serum albumin | Sigma | A1653 | |
| inverted microscope | Fisher brand | 11-350-119 | |
| microcentrifuge | Beckman Coulter | Microfuge 20 | |
| microcentrifuge tube, 1.5 mL | USA Scientific | 1615-5500 | |
| microwell culture plate | Stemcell technologies | 34411 | Aggrewell 400, 24 well |
| motor-driven pestle | GAMUT | #399X644 | |
| non-tissue culture treated dish, 10 cm | Fisher Scientific | FB0875713 | |
| PBS | ThermoFisher | 14190250 | |
| Penicillin-streptomycin | ThermoFisher | 10378016 | |
| Petri dish, 35 mm | Celltreat | 229638 | |
| pipette, 5 mL | DOT Scientific, | 667205B | |
| pipette, 8-channel | VWR | #613-5253 | |
| pipette, 10 mL | VWR | 667210B | |
| pipette, P10 | Denville | UEZ-P-10 | |
| pipette, P200 | Denville | UEZ-P-200 | |
| pipette, P1000 | Denville | UEZ-P-1000 | |
| proteolytic and collagenolytic enzyme mixture | Sigma | A6965 | Accutase |
| reagent reservoir, 50 mL | VWR | 89094-680 | |
| reversible strainer, 37 micrometer | Stemcell technologies | 27251 | |
| swing bucket plate centrifuge | Beckman Coulter | Allegra X-14R | |
| swing bucket rotor | Beckman Coulter | SX4750A | |
| tuberculin syringe, 1 mL | BD | 309659 | |
| ultra low attachment microplate, 96 well | Corning | 4515 |
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