人羊膜上皮细胞横向分化为肝细胞样细胞及脾内移植的初步研究

发布时间：2018-01-07 17:25

本文关键词：人羊膜上皮细胞横向分化为肝细胞样细胞及脾内移植的初步研究　出处：《中南大学》2008年博士论文　论文类型：学位论文

【摘要】： 在胚胎发育过程中,肝脏的整个发育过程是一个涉及多基因、多环节、多途径的网络调控过程。1965年Farber的研究揭开了对于肝细胞研究的新篇章,他首次提出在肝内存在小上皮细胞,即卵圆细胞。他的发现推动了肝再生及肝细胞肝癌发病机制的深入研究。目前认为肝干细胞(hepaticstem cell,HSC)来源于前肠内胚层,在胚胎发育过程中以未成熟的肝细胞的形式存在,其形态与胆管上皮细胞相似,其生化特征类似于胚胎干细胞。20世纪末,大量研究表明,成体干细胞可以跨越胚层限制分化为肝细胞。美国科学家近年宣布,他们在羊水中发现了和胚胎干细胞一样有效的干细胞,并在实验室中使这种干细胞分化成肌肉、骨头、脂肪、血管、神经和肝脏细胞。和胚胎干细胞相比,这种干细胞更容易分化成各种组织,且不会形成一种名为畸胎瘤的良性肿瘤。来源于人废弃胎盘的羊膜上皮细胞(human amnioticepithelial cells,hAECs)是这样一类极具研究潜力的细胞。目前国内外这方面的研究尚处于起步阶段。本课题对人羊膜上皮细胞完成了体外检测和鉴定,对其向肝细胞样细胞体外分化的条件进行了摸索和优化,在成功诱导生成肝细胞样细胞的基础上,将诱导获得的肝细胞样细胞移植入裸小鼠体内,检测了诱导获得的肝细胞样细胞是否具有生物学功能。本研究分为3个部目的:建立人羊膜上皮细胞的体外分离方法及培养体系,检测人羊膜上皮细胞的生物学特性,检测人羊膜上皮细胞的AAV-EGFP感染效率。方法:使用胰蛋白酶从人羊膜上消化人羊膜上皮细胞进行培养,细胞免疫荧光检测胚胎干细胞表面SSEA-1、SSEA-3、SSEA-4和TRA-1-60、TRA-1-81标记;RT-PCR检测全能性相关基因TERF、THY1、LEFTYA、Rex-1、Sox-2、Oct-4和nanog等的表达;腺相关病毒感染检测人羊膜上皮细胞的AAV-EGFP感染效率。结果:人羊膜上皮细胞表达SSEA3、SSEA4、TRA-1-60、TRA-1-81等胚胎干细胞的特异性标记;表达TERF1、THY、EFTYA、Rex-1、SOX2、Oct-4和Nanog等全能性相关基因;人羊膜上皮细胞的AAV-GFP感染效率在病毒浓度为10~(-3)pfu/ml时可达58.2%±1.3%。结论:建立了hAECs的细胞分离方法及适合hAECs生长和增殖的体外培养体系;hAECs具干细胞特征;体外培养的hAECs细胞可以被AAV-GFP感染,并表达GFP,且表达效率达50%以上。目的:研究Dex,HGF,IGF等细胞因子联合使用的诱导体系对hAECs向肝细胞样(hepatocyte-like)细胞诱导分化的影响。方法:采用Dex,HGF,IGF等细胞因子联合使用方法诱导培养hAECs向肝细胞样细胞分化,诱导周期为两周。诱导过程中采用RT-PCR鉴定细胞ALB、CYP1A1、CYP1A2、IGFR、c-met等肝细胞相关关键功能基因的表达和HNF3、HNF4和C/EBPa三种转录因子的表达。流式细胞术分析集落细胞表面标记ALB、AFP和CK18的时程变化;为了优化诱导体系,检测了Dex、HGF、IGF等细胞因子的剂量依赖性;比较不同代数的hAECs在诱导向肝细胞样细胞分化的ALB基因表达情况。结果:在诱导两周后的hAECs中可以检测到ALB、CYP1A1、CYP1A2、IGFR、c-met等肝细胞相关关键功能基因的表达,且这些功能基因的表达呈现逐渐递增的趋势。提示hAECs可能已经被成功诱导为肝细胞样细胞。诱导前的hAECs表达HNF1(一种肝细胞转录因子),不表达HNF3、HNF4和C/EBPa;而诱导后的hAECs不仅可以表达HNF1,还可以表达HNF3,HNF4和C/EBPa,并且HNF1的表达在诱导前后的hAECs中也有所不同,诱导后的hAECs中HNF1的表达水平明显高于没有经过诱导的hAECs。细胞表面标记检测发现:诱导6天时,hAECs主要表达AFP+,约为15.1±2.1%;随着诱导时间的延长,10天时hAECs表达AFP+/ALB+,约为6.5±1.4%;而诱导14天时,hAECs基本上只表达ALB+,为13.9±2.3%。诱导10天时的hAECs开始表达ALB+/CKl8+;约为2.5±1.4%;诱导14天时,细胞依然表达ALB+/CK18+,且这种双阳性的细胞量有明显增加,为18.9±3.1%;同时CK18+细胞数并没有明显减少,诱导10天时,CK18+细胞数为16.1±1.2%;诱导14天时,CK18+细胞数为21.3±4.6%。提示,诱导过程中的hAECs随着诱导时间的延长有一个逐渐成熟的过程。剂量依赖性实验结果显示,IGF、HGF均存在剂量依赖性,而Dex的剂量依赖性实验结果不具有统计学意义,认为不存在剂量依赖性。对不同代数的hAECs在诱导向肝细胞样细胞分化的ALB基因表达情况进行比较,结果显示,传代三代的hAECs诱导后均能表达ALB基因,且在表达强度上没有明显区别结论:hAECs确实可以在体外向肝细胞样细胞进行诱导分化,并能被成功诱导分化为有功能的肝细胞样细胞;hAECs向肝细胞样细胞的诱导过程中,对于HGF和IGF具有剂量依赖性,而对于Dex的这种剂量依赖性并不明显;传代三代内的hAECs诱导后,在肝细胞样细胞特异性功能基因ALB的表达上没有明显区别。目的:观察人羊膜上皮细胞(hAECs)在肝脏受损裸鼠脾内移植后是否具有肝细胞样功能的表达并探讨其治疗肝功能损伤的可行性。方法: 40只裸小鼠,随机分为三组,A组:20只,行2/3肝叶切除后,自脾下极移植5×10~6绿色荧光蛋白标记的hAECs;B组:10只,不行肝叶切除,自脾下极移植5×10~6绿色荧光蛋白标记的hAECs;C组:10只,行2/3肝叶切除后,自脾下极注射生理盐水0.2 mL。荧光显微镜hAECs体内示踪并观察其是否表达肝细胞特异性蛋白;术后2周A、B组各10只裸小鼠采血定量检测裸鼠血中人白蛋白及肝功能,术后4周A组另10只及C组10只裸小鼠做以上相同检测。结果: A组镜下肝脏和脾脏组织中均发现绿色荧光信号的hAECs,大部分表达人白蛋白;B、C组镜下均未见绿色荧光细胞。A组血中可检测到人白蛋白且术后4周明显高于术后2(3.9±1.34vs0.37±0.14,P0.01);术后4周A组肝功能(ALB、ALT、AST)明显优于C组(p0.05),A组肝功能术后4周亦优于术后2周(p＜0.05)。结论:肝损伤可以有效诱导hAECs向肝迁移增殖,大部分hAECs在肝细胞损伤的微环境下有肝细胞样功能表达,hAECs移植可以在一定程度上修复受损肝功能。
[Abstract]:In the process of embryonic development, the development of the whole process of the liver is a multi gene, multi link, network control process.1965 Farber multi way opened a new chapter for stem cell research, he first proposed the existence of small epithelial cells in hepatic oval cells. Namely, promote the research of his discovery the pathogenesis of liver regeneration and hepatic cell carcinoma. It is believed that liver stem cells (hepaticstem cell, HSC) from the foregut endoderm, in immature liver cells form during embryonic development, the morphology and the bile duct epithelial cells like, its biochemical characteristics similar to embryonic stem cells.20 at the end of the century, a large number of studies show that that adult stem cells can differentiate into liver cells across the ectoderm limits. American scientists announced in recent years, they found the same and embryonic stem cells and effective stem cells in amniotic fluid, and that this in lab The differentiation of stem cells into muscle, bone, fat, blood vessel, nerve and liver cells. Compared with embryonic stem cells, the stem cells differentiate into various organizations more easily, and does not form a benign teratoma. From the abandoned placenta amniotic epithelial cells (human amnioticepithelial cells, hAECs) is such a kind of great research potential of cells. At present domestic and international research in this area is still in its infancy.
This subject has completed the in vitro detection and identification of human amniotic epithelial cells, has carried on the exploration and optimization into hepatocyte like cells in vitro differentiation conditions, based on the generation of hepatocyte like cells induced by success, will be obtained by the hepatocyte like cells transplanted into nude mice, detected the induced hepatocyte like cells whether it has obtained biological function. This research is divided into 3 parts
Objective: to establish an in vitro isolation method and culture system for human amniotic epithelial cells, to detect the biological characteristics of human amniotic epithelial cells, and to detect the AAV-EGFP infection efficiency of human amniotic epithelial cells.
Methods: using trypsin digestion of human amniotic epithelial cells were cultured from human amniotic cells, immunofluorescence of embryonic stem cell surface SSEA-1, SSEA-3, SSEA-4 and TRA-1-60, TRA-1-81; RT-PCR detection of pluripotency related genes TERF, THY1, LEFTYA, Rex-1, Sox-2, Oct-4 and Nanog expression; adeno-associated virus infection detection human amniotic epithelial cells AAV-EGFP infection efficiency.
Results: the expression of SSEA3 in human amniotic epithelial cells, SSEA4, TRA-1-60, TRA-1-81 and other specific markers of embryonic stem cells; the expression of TERF1, THY, EFTYA, Rex-1, SOX2, Oct-4 and Nanog of pluripotency related genes; human amniotic epithelial cells infected by AAV-GFP virus in the efficiency of concentration of 10~ (-3) pfu/ml up to 58.2%. 1.3%.
Conclusion: a cell isolation method for hAECs and an in vitro culture system suitable for hAECs growth and proliferation were established. HAECs has the characteristics of stem cells, and hAECs cells cultured in vitro can be infected by AAV-GFP and express GFP, and the expression efficiency is over 50%.
Objective: To study the effect of the combined use of Dex, HGF, IGF and other cytokines on the induction of differentiation of hAECs to hepatocyte like (hepatocyte-like) cells.
Methods: Dex, HGF, IGF and other cytokines induced by cultured hAECs into hepatocyte like cell differentiation, induction period of two weeks. The identification of RT-PCR ALB cells during induction of CYP1A1, CYP1A2, IGFR, HNF3 and the expression of related key functions such as c-met gene, the expression of HNF4 and C/EBPa three transcription factor. Flow cytometry analysis of colony cell surface markers ALB, AFP and CK18 of the time course of changes; in order to optimize the induction system, detection of Dex, HGF, IGF and other cytokines dose dependent; ALB gene expression comparison of different passages of hAECs cells into hepatocyte like cells in differentiation.
Results: at two weeks after the induction of hAECs was detected in ALB, CYP1A1, CYP1A2, IGFR, c-Met and other related key functional expression of liver cell gene, and the expression of these genes showed a gradual increasing trend. Indicating that hAECs may have been successfully induced into hepatocyte like cells. The expression of HNF1 (hAECs before induction a liver cell transcription factor), the expression of HNF3, HNF4 and C/EBPa; and after the induction of hAECs can not only express HNF1 can also express HNF3, HNF4 and C/EBPa, and the expression of HNF1 in hAECs before and after induction in different levels of HNF1 expression induced by hAECs was significantly higher than that without hAECs. cells surface markers detected by discovery: after 6 days induction, the expression of hAECs AFP+, about 15.1 + 2.1%; with the induction time, the expression of hAECs AFP+/ALB+ in 10 days, about 6.5 + 1.4%; and after 14 days, basically only the expression of ALB+ hAECs, 13.9 + 2.3%. induced 10 days when hAECs began to express ALB+/CKl8+; about 2.5 + 1.4%; after 14 days, cells still express ALB+/CK18+, and the double positive cells were significantly increased, 18.9 + 3.1%; at the same time, the number of CK18+ cells was not significantly reduced, by 10 days, the cell number was CK18+ 16.1 + 1.2%; after 14 days, the number of CK18+ cells was 21.3 + 4.6%., induced by the process of hAECs with the induction time is a mature process. Dose dependent experimental results showed that IGF dose dependently there were HGF, Dex and the dose dependence of experimental results was not statistically meaning that there is no dose dependence. The expression of ALB gene of different generations of the hAECs cells into hepatocyte like cells in differentiation were compared, the results showed that three generations after the induction of hAECs could express ALB gene, and the expression intensity in no obvious area other
Conclusion: hAECs can differentiate into hepatocyte like cells in vitro, and can be induced successfully for functional hepatocyte like cells; hAECs to induction of hepatocyte like cells, in a dose dependent manner for HGF and IGF, and the dose dependence of Dex is not obvious after three; the generation of the hAECs after the induction, there were no significant differences in the expression of hepatocyte like cells specific gene ALB.
Objective: To observe whether human amniotic epithelial cells (hAECs) have hepatocyte like function after liver transplantation in nude mice, and to explore the feasibility of treatment of liver function damage.
Methods: 40 mice were randomly divided into three groups, group A: 20, 2/3 after liver resection, with splenic transplantation 5 x 10~6 green fluorescent protein tagged hAECs; group B: 10, did not receive hepatectomy, with splenic transplantation 5 x 10~6 green fluorescent protein hAECs; group C: 10, 2/3 after liver resection, with splenic injection of saline 0.2 mL. fluorescence microscope hAECs tracing in vivo and to observe the expression of hepatocyte specific protein; after 2 weeks, A, albumin and liver function in B group of 10 mice were detected in blood of nude mice people, after 4 weeks, the other group A 10 and group C 10 nude mice to do more of the same.
Results: the green fluorescence signals of hAECs were found in A group with liver and spleen tissues, most expression of human albumin; B, C group under the microscope showed no green fluorescent cells in group.A blood can be detected in human albumin and after 4 weeks after transplantation was significantly higher than 2 (3.9 + 1.34vs0.37 + 0.14, P0.01); the 4 week A group of postoperative liver function (ALB, ALT, AST) was significantly higher than that of C group (P0.05), after 2 weeks of liver function in A group 4 weeks after surgery was superior to surgery (P < 0.05).
Conclusion: liver injury can effectively induce hAECs to migrate to liver, and most of hAECs has hepatocyte like expression in the microenvironment of liver injury. HAECs transplantation can repair damaged liver function to some extent.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329

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