纳米粒介导的人源诱导多能干细胞定向肝脏细胞的分化研究

发布时间：2018-06-30 19:22

本文选题：非病毒载体 + 重编程　；参考：《江苏大学》2017年硕士论文

【摘要】：自2006年Yamanaka首次利用“四因子”组合(p SOX2、p OCT4、p C-MYC、p KLF4)成功诱导生成i PSCs后,细胞重编程技术始终是研究热点之一。由于i PSCs来源于患者自身体细胞,并具有自我更新和多向分化能力,因此,将i PSCs作为一种替代胚胎干细胞(ESCs)的细胞应用于临床中,能够有效避免很多伦理及法律问题,且i PSCs源于患者,能够实现个体化治疗。原位肝移植是目前唯一能有效治疗肝癌的方法。然而,由于器官短缺,近年来因为肝癌死亡的人数不断增加;此外,世界范围内的肝癌发病率也在不断上升。肝细胞移植的临床试验带来振奋人心的成果。但作为替代肝脏移植的肝细胞移植仍然需要解决肝细胞来源的问题。目前,很多研究都致力于寻找一种能够产生稳定的功能性肝细胞的可行性方法。其中,可能会成为肝细胞不竭来源的细胞就是人源诱导多能干细胞(hi PSCs)。hi PSCs具有自我更新和多向分化能力,与人源胚胎干细胞(h ESCs)相比,hi PSCs涉及到的伦理问题较少,且完全不受来源限制。因此,hi PSCs来源肝细胞(hi PSC-HEPs)在药物筛选、细胞治疗以及体外疾病模型等方面具有重要的意义。本研究课题是以磷酸钙纳米粒作为基因载体,通过携带两种不同因子组合将人脐带间充质干细胞(h UMSCs)重编程为hi PSCs,继而进行肝细胞定向诱导分化,并筛选优化出最佳诱导分化培养基,采用最佳诱导方案对其进行培养,使其分化为肝细胞,为肝细胞移植提供理论基础,从而更好地解决肝细胞短缺问题。第一章重编程法制备人源肝脏细胞及其应用研究进展通过查阅大量资料,对人源肝细胞重编程方法进行了综述,总结并分析人源肝细胞重编程的影响因素;阐述了人源肝细胞重编程技术的研究现状及其在再生医学、药物筛选及体外疾病模型等方面的应用,并指出人源肝细胞应用于临床实践的局限性。为本论文实验工作的顺利开展和进行提供了理论依据。第二章非病毒纳米基因传递系统的hi PSCs重编程研究采用磷酸钙纳米粒作为基因载体,对比两种不同的因子组合,即p SOCK(p SOX2、p OCT4、p C-MYC、p KLF4)与p OS+mi R(p OCT4、p SOX2、mi R302b-367),制备得到磷酸钙纳米粒C-p SOCK与C-p OS+mi R,通过琼脂糖凝胶电泳、透射电镜观察、毒性评价、粒径以及Zeta电位测定等一系列检测进行表征;以人脐带间充质干细胞(h UMSCs)为源细胞,进行重编程研究。结果显示:两种因子组合所制备得到的纳米粒均呈现形态规整、分散均匀、表面带正电荷的球形或者椭球型;细胞毒性实验表明两种不同因子组合制备得到的磷酸钙纳米粒无毒或低毒;通过碱性磷酸酶染色,统计阳性克隆数,从而考察两种纳米粒的重编程效率;免疫荧光、Western blot均说明纳米粒C-p OS+mi R诱导生成的hi PSCs能够表达多能性标记蛋白,且能进行体内分化(内、中、外)。第三章hi PSCs的肝细胞定向诱导分化研究设置4种不同肝细胞诱导分化培养基(MediumⅠ、Ⅱ、Ⅲ、Ⅳ),对重编程生成的hi PSCs进行肝细胞定向诱导分化,采用酶联免疫检测法(ELISA)分别在诱导分化第0,3,7,11,15,19,23,27,32天检测AFP和ALB表达情况,从而筛选出最佳诱导方案;光学显微镜下观察细胞形态变化;用免疫荧光法、Western blot检测肝脏特征性蛋白(AFP、ALB、CK8、CK18、SOX17)的表达。结果显示:诱导培养基MediumⅡ、诱导方案“三步法”的肝细胞诱导效果较好;显微镜下观察细胞形态发生明显变化,从“克隆团”形态逐渐变化为“铺路石”样形态,诱导分化结束后,细胞形态基本一致,为多角多边形或者类圆形,且细胞核较明显,部分细胞表现出典型肝细胞形态:出现双核甚至多核现象,且诱导分化的肝细胞能够表达肝脏肝脏特征性蛋白(AFP、ALB、CK8、CK18、SOX17)。第四章二维培养体系中肝细胞药物代谢酶表达及细胞活性研究在二维培养体系下,选择Ⅰ相代谢酶细胞色素P450(CYP450)、Ⅱ相代谢酶葡萄糖醛酸转移酶(UGT)与谷胱甘肽硫转移酶(GST),用Western blot测定诱导后细胞蛋白表达,以人胚胎干细胞来源肝细胞(h ESCs-HEPs)为阳性对照;考察在诱导分化不同时间药物代谢酶的动态变化,同时比较hi PSCs诱导形成的肝细胞与胚胎干细胞来源肝细胞(h ESCs-HEPs)药物代谢酶的表达差异;不同时间取样,检测谷草转氨酶(AST)、谷丙转氨酶(ALT)和乳酸脱氢酶(LDH)的质量浓度;检测白蛋白分泌、尿素合成及葡萄糖消耗含量,分别采用溴甲酚绿法、二乙酰-肟法及葡萄糖氧化酶-过氧化物酶法。结果显示:hi PSCs诱导形成的肝细胞(hi PSCs-HEPs)能够较好表达CYP450、UGT与GST,且与h ESCs-HEPs表达无明显差异;而诱导前hi PSCs很少表达甚至不表达CYP450、UGT与GST;hi PSCs的肝细胞诱导分化过程中ALT、AST、LDH变化趋势与h ESCs基本一致;hi PSCs-HEPs与h ESCs-HEPs测定的白蛋白分泌水平、尿素合成浓度以及葡萄糖消耗量基本一致。第五章三维培养体系中hi PSCs定向肝细胞诱导分化在三维培养体系下,对重编程生成的hi PSCs进行肝细胞定向诱导分化。以Ⅰ型胶原和壳聚糖作为支架材料,采用冷冻干燥-热固化法制备胶原/壳聚糖三维支架,考察不同质量比与不同固化时间对支架性能的影响,优化三维支架制备工艺,通过形态观察、吸水率与保水率测定等对制备得到的支架进行表征;将纳米粒C-p OS+mi R与空白胶原/壳聚糖支架相结合,得到三维载基因纳米粒-胶原/壳聚糖支架;人脐带间充质干细胞(h UMSCs)在三维载基因纳米粒-胶原/壳聚糖支架中进行重编程后,形成边界清晰、形态规整的克隆团;对三维支架上重编程生成的hi PSCs进行肝细胞定向诱导分化,用ELISA法对比二维与三维培养体系中肝细胞功能;分别用溴甲酚绿法、二乙酰-肟显色法、葡萄糖氧化酶-过氧化物酶法比较二维与三维培养体系中肝细胞代谢活性。结果显示:在三维培养体系诱导21天后,细胞形态呈现明显的肝细胞样细胞形态;在三维培养体系下,诱导形成的肝细胞功能没有“损伤”,仍处于正常肝功能状态。
[Abstract]:After the successful induction of I PSCs by the "four factor" combination (P SOX2, P OCT4, P C-MYC, P KLF4) in 2006, cell reprogramming has always been one of the hotspots of research. Because I originates from the body and has self renewal and multi differentiation energy, it is used as a substitute for embryonic stem cells. The use of cells in the clinic can effectively avoid many ethical and legal problems, and I PSCs is derived from patients and can achieve individualized treatment. Orthotopic liver transplantation is the only effective treatment for liver cancer. However, the number of deaths due to liver cancer has increased in recent years due to the shortage of organs; in addition, the incidence of liver cancer in the world The rate is also rising. The clinical trials of hepatocyte transplantation have brought exciting results. But as a substitute for liver transplantation, liver cell transplantation still needs to solve the problem of hepatocyte origin. Many studies are currently working to find a feasible way to produce stable functional liver cells. The cell inexhaustible source is the ability of human induced pluripotent stem cells (HI PSCs).Hi PSCs to have self-renewal and multidirectional differentiation. Compared with human embryonic stem cells (H ESCs), the ethical problems involved in hi PSCs are less and are completely unrestricted from sources. Therefore, hi PSCs comes from liver cells (HI PSC-HEPs) in drug screening, cell therapy, and body. The model of external disease is of great significance. This research focuses on the use of calcium phosphate nanoparticles as a gene carrier to reprogram human umbilical cord mesenchymal stem cells (H UMSCs) into hi PSCs by carrying two different combinations of factors. The induction program can be cultured to differentiate into liver cells, provide a theoretical basis for hepatocyte transplantation, and thus better solve the problem of liver cell shortage. Chapter 1 reprogramming a human source liver cell and its application research progress through consulting a large number of data, summarizing and analyzing human liver cell heavy cell reprogramming methods, summarizing and analyzing people. The influence factors of reprogramming of source hepatocyte, the current status of human hepatocyte reprogramming and its application in regenerative medicine, drug screening and in vitro disease model were introduced, and the limitations of the application of human hepatocytes in clinical practice were pointed out. The theoretical basis for the successful development and development of this paper was provided. Second The hi PSCs reprogramming of non viral nanoscale gene transfer system uses calcium phosphate nanoparticles as the gene carrier, and compares two different combinations of factors, namely, P SOCK (P SOX2, P OCT4, P C-MYC, P KLF4) and the agarose gel electrophoresis. A series of tests, such as transmission electron microscopy, toxicity evaluation, particle size and Zeta potential measurement, were characterized by a series of tests. Human umbilical cord mesenchymal stem cells (H UMSCs) were used as the source cells for reprogramming. The results showed that the nanoparticles prepared by the two factors were uniformly distributed, dispersed evenly, with a spherical or ellipsoid with positive charges on the surface. The cytotoxicity test showed that the calcium phosphate nanoparticles prepared by two different combinations were non-toxic or low toxic; the reprogramming efficiency of the two nanoparticles was investigated by the alkaline phosphatase staining and the number of positive clones, and the immunofluorescence and Western blot showed that the hi PSCs induced by the nanoparticles C-p OS+mi R could express the multienergy standard. Third hi PSCs induced differentiation of hepatocytes and differentiation medium (Medium I, II, III, IV) were set up in 4 different hepatocytes (Medium, II, III, IV). The induced differentiation of liver cells was induced by reprogrammed hi PSCs, and the differentiation of 0,3,7,11 was induced by enzyme immunoassay (ELISA), respectively. The expression of AFP and ALB was detected by 15,19,23,27,32 days, and the optimal induction scheme was screened. The morphological changes of cells were observed under the optical microscope, and the expression of liver characteristic proteins (AFP, ALB, CK8, CK18, SOX17) was detected by immunofluorescence and Western blot. The results showed that the inducible medium Medium II was induced by the induction program "three steps" of hepatocyte induction. The morphological changes were observed under the microscope, and the morphology of the cells changed from "cloned group" to "pave" form. After the induction of differentiation, the cell morphology was basically the same, it was polygonal polygon or round, and the nucleus was more obvious. Some cells showed typical liver cell morphology: Double nucleus and even multinuclear appearance appeared. AFP, ALB, CK8, CK18, SOX17 in liver liver cells can be expressed in the differentiated hepatocytes. Fourth in two dimensional culture system, the expression of drug metabolizing enzymes and cell activity of hepatocytes in the two dimensional culture system, the selection of cytochrome P450 (CYP450), phase II metabolic enzyme glucuronotransferase (UGT) and Valley in the two-dimensional culture system Cystamine thiotransferase (GST) was used to determine the expression of cell protein after induction of Western blot, and the positive control was taken from human embryonic stem cell derived hepatocytes (H ESCs-HEPs). The dynamic changes of drug metabolizing enzymes at different time of induction of differentiation were investigated, and the drug derived from hi PSCs induced liver cells and embryonic stem cells derived from liver cells (H ESCs-HEPs) were compared. The difference in the expression of metabolic enzymes; sampling at different time, detecting the mass concentration of AST, ALT and LDH, detecting albumin secretion, urea synthesis and glucose consumption, using bromo methyl phenol green method, two acetyl oxime method and glucose oxidase peroxidase method respectively. The results showed: Hi PSCs The induced liver cells (HI PSCs-HEPs) can express CYP450, UGT and GST, and there is no obvious difference from H ESCs-HEPs, but before induction, hi PSCs seldom expresses or even expresses CYP450, UGT and GST. The protein secretion level, the urea synthesis concentration and the glucose consumption are basically the same. In the fifth chapter, in the fifth chapter three dimensional culture system, the induced differentiation of liver cells was induced and differentiated under the three-dimensional culture system. The reprogrammed hi PSCs was directed and differentiated to the liver cells. The type I collagen and chitosan were used as the scaffold material, and the freeze drying heat curing was used. A three-dimensional collagen / chitosan scaffold was prepared by the method. The effects of different mass ratio and curing time on the performance of the scaffold were investigated. The three-dimensional scaffold preparation process was optimized. The prepared scaffolds were characterized by morphological observation, water absorption and water retention ratio. The C-p OS+mi R of the nanoparticles was combined with blank collagen / chitosan scaffold. Gene loaded nanoparticles collagen / chitosan scaffold; human umbilical cord mesenchymal stem cells (H UMSCs) were reprogrammed in three dimensional gene loaded nanoparticles collagen / chitosan scaffold to form a well-defined and orderly cloned cloned group. The hi PSCs generated on the three dimensional stent was directed to induce differentiation of liver cells, and the two dimension was compared with the ELISA method. The liver cell function in the three-dimensional culture system was compared with bromocresol green method, two acetyl oxime colorimetric method and glucose oxidase peroxidase method to compare the metabolic activity of liver cells in two-dimensional and three-dimensional culture systems. The results showed that the morphology of the cells showed obvious liver cell like cells in the three dimensional culture system after the induction of the three-dimensional culture system for 21 days. Under the guidance of the system, the function of induced hepatocytes is not "damaged" and is still in normal liver function.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R329.2

【参考文献】