CRISPR-dCas9多重激活体内肝细胞内源基因直接重编程为胰岛β细胞及机制研究

Dysfunction and/or decrease in pancreatic β-cell function are the major causes of diabetes. The direct reprogramming of different kinds of adult cells to islet beta cells is an effective way to potentially address the lack of islet beta cells. It has been reported in the literature and our study found that exogenous expression of key transcription factors for pancreatic development can directly reprogram live hepatocytes to islet beta cells. However, since the chromatin of the endogenous transcription factor of the liver cells is in an inhibitory state, the exogenously introduced transcription factor is transiently expressed, resulting in inefficient and poor maturation of the directly reprogrammed beta cells. In order to solve this problem, we constructed a CRISPR/dCas9 based new Casilio system that targeted Tgif2, Pdx1, Ngn3 and Mafa, and continuously activated these four endogenous specific transcription factors in different periods. Therefore, we intends to use this Casilio system to directly reprogram hepatocytes to islet beta cells in vivo and observe the effects of treating diabetes. The detection of histone dynamics at specific sites by RNA-Seq, ChIP-Seq will elucidate the mechanism of gene editing in the chromatin remodeling of endogenous gene activation and direct reprogramming of hepatocytes into islet β cells. This will provide new technical solutions and theoretical basis for direct reprogramming and treatment of diabetes.

胰岛β细胞功能损伤和/或数量减少是导致糖尿病的主要原因。把不同种类的成体细胞直接重编程为胰岛β细胞是潜在解决胰岛β细胞缺乏的有效方法。文献报道和我们研究发现外源表达胰腺发育关键转录因子可直接重编程活体肝细胞为胰岛β细胞。但由于肝脏细胞内源转录因子的染色质处于抑制状态，而外源导入的转录因子仅瞬时表达，造成直接重编程的β细胞效率低和成熟度差。为解决此问题，课题组构建了可靶向激活Tgif2、Pdx1、Ngn3和Mafa的CRISPR/dCas9的Casilio体系，可分时段持续激活细胞内特定转录因子。因此，本项目拟利用该Casilio体系对活体内肝细胞直接重编程为胰岛β细胞并观察治疗糖尿病的效果。运用RNA-Seq，ChIP-Seq等技术检测特定位点的组蛋白动态修饰，阐明基因编辑在内源基因活化及肝细胞直接重编程为胰岛β细胞中的染色质重塑机制。为细胞直接重编程治疗糖尿病提供新的技术方案和理论依据。

胰岛β细胞功能损伤和/或数量减少是导致糖尿病的主要原因。利用直接重编程技术，可将一种终末分化的细胞直接转变成另一种终末分化的细胞，不需要经过多能干细胞阶段，避免了操作的复杂性，免疫排斥及伦理学问题。目前通过导入外源转录因子或通过CRISPR/dCas9基因编辑技术可实现肝细胞直接重编程为胰岛β细胞。但由于肝脏细胞内源转录因子的染色质处于抑制状态，而外源导入的转录因子仅瞬时表达，造成直接重编程的β细胞效率低和成熟度差。本研究构建了可同时靶向激活内源Pdx1、Ngn3和MafA的CRISPR/dCas9的Casilio体系，在两种肝细胞系中筛选到了特异性高，激活效率高的候选sgRNA系统，并且导入4条gRNA的激活效率相对于单独导入1条gRNA有叠加效应，表明Casilio系统实现内源靶基因的特异性激活调控。本研究利用该体系，结合PiggyBac转座子实现了dCas9、P65-HSF1片段在HepG2肝细胞系基因组的整合，并初步将HepG2细胞直接重编程为胰岛素分泌细胞。同样地，利用激活PNM的Casilio体系也在体外实现了THLE2细胞向胰岛素分泌细胞的重编程。体内动物实验结果显示，通过超声微泡增强的水流动力学注射在活体肝脏内过表达Pdx1、Ngn3、MafA基因可以实现肝细胞向胰岛素分泌细胞的重编程，并可改善糖尿病小鼠的高血糖症状，初步验证了活体肝细胞直接重编程为胰岛素分泌细胞治疗糖尿病的安全性与有效性。本研究利用基于CRISPR/dCas9的内源基因激活体系，建立了肝脏细胞直接重编程为胰岛素分泌细胞的初步方案，为胰岛细胞再生提供了一种新的研究思路和技术手段，为糖尿病的临床治疗奠定了实验基础，并具有一定的临床应用潜力。