间充质干细胞肝向分化特异性microRNA表达谱的鉴定及功能学研究

发布时间：2018-05-01 16:40

本文选题：microRNA + 肝细胞　；参考：《第四军医大学》2013年博士论文

【摘要】：【背景】终末期肝病在我国的发病率呈逐年增高趋势，年死亡率达30-50%。原位肝移植是目前治疗终末期肝病的唯一有效手段。但是供肝缺乏为原位肝移植治疗的瓶颈。此外原位肝移植治疗费用昂贵、术后需长期使用免疫抑制剂、围手术期风险大等问题，极大限制了原位肝移植的广泛应用。成体干细胞在体内外均可以突破胚层的限制，分化为具有功能的肝细胞。因此，成体干细胞移植技术成为一种新兴的终末期肝病治疗技术。但是其向肝细胞分化的机制仍不清楚。传统的诱导手段复杂、诱导周期长、分化效率低，限制了干细胞治疗技术在临床的广泛推广与应用。 microRNA是一类短的内源性非编码RNA，通过与靶基因mRNA的3’非编码区的结合位点结合，参与基因转录后调控。以往研究认为，microRNA调控基因表达并未起到开关的作用，而是发挥可变电阻器的作用。近年来研究发现microRNA的时空特异性分布规律使得microRNA在机体发育和细胞分化过程中发挥了重要的调控作用。利用特定的microRNA组合可以介导细胞从一种终末分化状态转化为另一种终末分化状态。尽管已经有文献陆续报道了microRNA在肝脏发育过程的重要性，但其在干细胞肝向分化过程中的作用尚不清楚。【目的】本研究的目的是筛选成体干细胞肝向分化过程特异性的microRNA表达谱，并探讨关键microRNA分子在肝向分化中的作用，为干细胞肝向分化机制的阐明提供理论支持。研究内容主要包括三部分：①建立成体干细胞向肝细胞分化的体外模型；②筛选并鉴定成体干细胞肝向分化过程特异的microRNA表达谱；③阐明关键microRNA分子在肝向分化过程中的作用。【方法】 1．通过免疫磁珠方法纯化外周血造血干细胞，通过贴壁方法分选脐带间充质干细胞（mesenchymal stem cell, MSC）。应用电子显微镜观察细胞形态、流式细胞技术检测细胞免疫表型，诱导成骨、成脂分化潜能方面检测分离的干细胞纯度及特性。在成功分离得到干细胞后，给予肝向分化诱导培养液，利用qRT-PCR方法和细胞免疫荧光方法分别从RNA、蛋白质水平检测MSC经肝向诱导后肝细胞特异性基因的表达变化，利用PAS染色方法、尿素氮检测试剂盒以及LDL摄取实验检测肝细胞特异性功能的水平，从而验证是否成功建立干细胞肝向分化的体外模型。 2．选取干细胞肝向分化模型7个不同时间点的细胞，提取RNA，进行microRNA芯片检测。依据差异倍数、表达量、以及变化规律筛选microRNA分子进行qRT-PCR检测以验证芯片结果。将qRT-PCR与芯片结果一致的差异表达的microRNA谱与其在成骨分化过程中的表达变化及其在肝癌细胞和正常肝细胞系的表达变化做比较，以明确该microRNA表达谱的肝向分化特异性。 3.分别构建包含miR-1246、miR-1290、miR-148a、mR-30a、miR-424、miR-542-5p干扰序列的慢病毒颗粒，感染入MSC后，给予肝向分化诱导培养液，观察分别抑制microRNA活性后对肝向分化过程的影响。另外分别合成以上microRNA的模拟物，并以肝脏富集的miR-122为对照，单一转染或共同转染后，利用qRT-PCR、细胞免疫荧光、PAS染色、尿素氮检测以及LDL摄取实验等方法检测外源性增加microRNA表达后能否启动MSC的肝向分化并将其转化为成熟的有功能的肝细胞。最后将诱导的肝细胞移植入肝损伤小鼠体内，通过检测小鼠肝功能的变化、肝组织形态学的变化以及人源性肝细胞和干细胞在肝脏的定植最终明确microRNA诱导的肝细胞能否在小鼠体内发挥肝细胞的功能。【结果】 1.成功建立了成体干细胞肝向分化体外细胞模型从外周血分离CD34+细胞后经流式细胞仪分析，，99%以上的细胞为CD34+细胞，细胞呈圆形不贴壁生长。经四种不同培养体系培养与扩增后，发现CD34+细胞在SFM34+IL3+SCF+GM-SCF培养液中扩增效率最高，可以扩增6倍。但是，扩增后的细胞失去了CD34的免疫表型，即SFM34+IL3+SCF+GM-SCF培养液虽然可以高效的扩增CD34细胞，但是，扩增的CD34细胞失去了造血干细胞的特征。从脐带间质分离的MSC具有典型的长索状形态，呈旋涡状生长。流式细胞分析结果显示，分离的细胞88.7%可以表达MSC标志分子CD105，不表达内皮细胞标志分子CD31和造血干细胞标志分子CD34。分离的细胞经成骨诱导培养液诱导14天后，具有钙盐沉积。即我们成功地从脐带间质分离到MSC。 qRT-PCR结果显示肝向诱导1周后，肝细胞特异性基因HNF4α、ALB和CK18的mRNA表达水平上调，于26天时达到高峰。免疫细胞荧光染色结果显示MSC经肝向诱导14天后可以检测到肝细胞特异性基因AFP、ALB、CK18的蛋白质的表达。PAS染色结果显示，MSC经肝向诱导14天后可以检测到紫红色的糖原颗粒。LDL摄取实验结果显示，MSC经肝向诱导14天时，大多数细胞可以摄取DIL标记的LDL。尿素氮检测结果显示，MSC肝向诱导6天后其合成尿素的能力逐渐提高，于22天时达到高峰。即我们成功地建立了脐带间质来源MSC肝向分化的体外细胞模型。 2．肝向分化特异性microRNA表达谱的筛选与鉴定本研究中共检测了1205个人类已知的和144个人类病毒相关microRNA分子在肝向分化7个不同时间点的表达变化情况。共61个microRNA分子在肝向分化前后变化倍数大于2倍，且在7个时间点中呈规律性表达。在此基础上，依据差异倍数和表达含量共筛选了7个差异倍数大于4倍的上调的microRNA、6个表达含量最高的上调的microRNA以及6个差异倍数大于4倍的下调的microRNA、10个表达含量最高的下调的microRNA进行了qRT-PCR验证。qRT-PCR结果显示，上调microRNA中miR-542-5p、miR-148a、miR-1290、miR-424、miR-30a和miR-1246的表达模式与芯片结果一致。下调microRNA中，除miR-3646外，其他下调microRNA均呈下调表达。但除miR-146a外，其他microRNA的表达模式与芯片结果吻合度较差。 qRT-PCR和芯片结果一致的6个上调的microRNA与8个下调的microRNA在成骨分化过程中的表达结果显示，部分在肝向分化过程中上调的microRNA在成骨分化过程中却呈下降趋势，部分在肝向分化过程中下调的microRNA在成骨分化过程中却呈上升趋势。同样经肝向诱导后部分表达上调（下调）的microRNA在L02和HepG2中的表达水平低于（高于）其在MSC中的表达。说明在肝向分化过程中差异表达的microRNA表达谱不同于其在成骨分化过程中的变化也并非肝癌细胞或肝细胞所特异性的，而是肝向分化过程所特异性的。 3.关键microRNA分子在MSC肝向分化中的作用未感染病毒的MSC以及感染miR-nc-lev的MSC在肝向诱导6天后白蛋白的表达水平上调，而感染了miR-1246-RNAi-lev、miR-1290-RNAi-lev、miR-148a-RNAi-lev、miR-30a-RNAi-lev、miR-424-RNAi-lev、miR-542-5p-RNAi-lev的MSC经过肝向诱导后，其白蛋白的水平并未上调。肝向诱导12天后，miR-nc-lev组无论是否成功感染慢病毒均可以摄取LDL。而miR-1246-RNAi-lev、 miR-1290-RNAi-lev、miR-148a-RNAi-lev、miR-30a-RNAi-lev、miR-424-RNAi-lev、miR-542-5p-RNAi-lev组，只有在未成功感染慢病毒的细胞中出现了LDL的摄取。此外，这些慢病毒颗粒可以抑制HepG2细胞肝细胞特异性白蛋白和G6P的表达。将miR-122、miR-1246、miR-1290、miR-148a、miR-30a、 miR-424和miR-542-5p模拟物分别转染入MSC后，可以提高相应microRNA的水平，但并不能促使MSC高表达白蛋白。7种microRNA模拟物共同转染入MSC后，不仅可以同时增加上述7种microRNA的含量，而且可以促使MSC高表达肝脏特异性早、中、晚期标志分子，不表达肝祖细胞、胰胆上皮标志分子。使MSC的细胞形态从间质样向上皮样转化，并具有肝细胞特异性的功能。移植入肝损伤小鼠体内后，不仅可以发挥肝细胞的功能即改善小鼠肝功能，还可以发挥部分MSC的功能，修复小鼠肝组织。【结论】我们成功掌握了分离纯化外周血造血干细胞和脐带MSC技术；在成功建立MSC肝向分化体外细胞模型基础上，通过芯片和qRT-PCR技术筛选并鉴定了一组MSC肝向分化过程所特异性的microRNA表达谱。在6个上调的microRNA中，抑制任意一个的活性均可以抑制MSC向肝细胞分化，而单纯过表达某一种microRNA并不能启动MSC向肝细胞分化。同时过表达时，可以促使MSC转化为在体内外均具有功能的肝细胞。本研究首次系统筛选了肝向分化特异性的microRNA谱并详细探讨了其在肝向分化中的作用。首次证实7-microRNA组合可以使MSC直接转化为有功能的肝细胞。为肝向分化机制理解提供了一定的理论支持。
[Abstract]:[background]
The incidence of end-stage liver disease in China is increasing year by year. The annual mortality rate is 30-50%. in situ liver transplantation is the only effective means to treat end-stage liver disease. However, the lack of donor liver is the bottleneck of orthotopic liver transplantation. In addition, the orthotopic liver transplantation is expensive, the long-term use of immunosuppressive agents should be used after operation, and the perioperative risk is great. These problems greatly restrict the wide application of orthotopic liver transplantation.
Adult stem cells can break through the restriction of the germ layer and differentiate into functional hepatocytes in vivo and in vivo. Therefore, adult stem cell transplantation has become a new technique for the treatment of end-stage liver disease. However, the mechanism of its differentiation to hepatocytes is still unclear. The traditional induction method is complex, the induction cycle is long, the differentiation efficiency is low, and the dry fine is limited. Cell therapy is widely popularized and applied in clinical practice.
MicroRNA is a short endogenous non coding RNA, which is involved in post transcriptional regulation by binding to the binding site of the 3 'non coding region of the target gene mRNA. Previous studies suggest that microRNA regulation gene expression does not play the role of switching, but plays the role of variable resistors. In recent years, the spatial and temporal distribution of microRNA has been discovered. Rules make microRNA play an important role in the development of body and cell differentiation. The use of specific microRNA combinations can mediate cells from a terminal differentiation to another terminal differentiation state. Although the literature has reported the importance of microRNA in the development of the liver, it is in stem cells. The role of liver differentiation in the process of liver differentiation is not clear.
[Objective]
The purpose of this study is to screen the specific microRNA expression profiles of adult stem cell liver differentiation process, and to explore the role of key microRNA molecules in liver differentiation, and provide theoretical support for the clarifying mechanism of stem cell differentiation. The main contents of this study include three parts: (1) establishing an in vitro model for differentiation of adult stem cells into hepatocytes. (2) to screen and identify the specific microRNA expression profiles of adult stem cells in the process of liver differentiation; (3) clarify the role of key microRNA molecules in the process of liver differentiation.
[method]
1. the peripheral blood stem cells (mesenchymal stem cell, MSC) were purified by the immunomagnetic bead method. The cell morphology was observed by the electron microscope, the cell immunophenotype was detected by flow cytometry, and the purity and characteristics of the separated stem cells were detected by the induction of osteogenesis and the potential of adipogenic differentiation. After the stem cells were successfully isolated, the hepatocyte differentiation induction culture was given. The expression of specific genes in the hepatocyte was detected by qRT-PCR and cell immunofluorescence methods from the RNA and protein levels, respectively. The specificity of the liver cell specific gene was detected by the PAS staining method, the urea nitrogen detection kit and the LDL uptake test for the detection of the liver cell specificity of MSC. The level of function, thereby verifying whether successful establishment of stem cell hepatocyte differentiation model in vitro.
2. the cells of 7 different time points of stem cell liver differentiation model were selected, RNA was extracted and microRNA chip was detected. According to the difference multiplier, the expression and the change rule, the microRNA molecules were screened by qRT-PCR to verify the results of the chip. The microRNA spectrum of differential expression of qRT-PCR and the result of the chip was consistent with the process of osteogenesis differentiation. The expression changes and their expression changes in hepatoma cells and normal hepatocyte lines were compared to clarify the liver differentiation specificity of the microRNA expression profile.
3. the lentivirus particles including miR-1246, miR-1290, miR-148a, mR-30a, miR-424, miR-542-5p interference sequences were constructed respectively. After infection into MSC, the liver differentiation induction culture was given, and the effects of inhibiting the activity of microRNA on the liver differentiation process were observed respectively. After single transfection or co transfection, qRT-PCR, cell immunofluorescence, PAS staining, urea nitrogen detection and LDL uptake test were used to detect the liver differentiation of MSC and convert it into mature and functional liver cells after exogenous microRNA expression, and then the induced hepatocytes were transplanted into the liver injury mice. By detecting the changes in the liver function of mice, the changes of liver histomorphology and the colonization of human hepatocytes and stem cells in the liver, the liver cells of microRNA induced liver cells can be played in mice.
[results]
1. successfully established adult stem cell hepatocyte differentiation model in vitro.
After the separation of CD34+ cells from peripheral blood, more than 99% of the cells were CD34+ cells, and the cells were round and non wall growth. After four different cultures and amplification, it was found that the amplification efficiency of CD34+ cells in the SFM34+IL3+SCF+GM-SCF culture solution was the highest, which could be expanded by 6 times. But the expanded cells lost the CD34 immunity. Although the SFM34+IL3+SCF+GM-SCF culture medium can amplify CD34 cells efficiently, the expanded CD34 cells lose the characteristics of hematopoietic stem cells.
The MSC separated from the umbilical cord was a typical long cable shaped form and was vortexed. Flow cytometry showed that the isolated cell 88.7% could express the MSC marker molecule CD105, and the cells that did not express the endothelial cell marker molecule CD31 and the hematopoietic stem cell marker CD34. were induced for 14 days after the induction of the osteogenic induction culture, with calcium salts. Deposition. That is, we succeeded in separating MSC. from umbilical cord stroma.
QRT-PCR results showed that after 1 weeks of liver induction, the mRNA expression level of hepatocyte specific gene HNF4 a, ALB and CK18 increased and reached the peak at 26 days. The results of immunofluorescence staining showed that the hepatocyte specific gene AFP, ALB, CK18 protein expression of MSC showed that MSC via the liver to the liver after 14 days of liver induction. 14 days after induction, the.LDL uptake test of purple red glycogen granules showed that when MSC was induced 14 days after liver induction, most cells could take the LDL. urea nitrogen test of DIL markers. The ability of MSC liver to synthesize urea gradually increased after 6 days of induction, and reached the peak at 22 days. That is, we successfully established the umbilical cord between the umbilical cord. A cell model of MSC liver differentiation in vitro.
Screening and identification of 2. liver specific microRNA expression profiles
In this study, 1205 human and 144 human virus related microRNA molecules were detected at 7 different time points of liver differentiation. A total of 61 microRNA molecules were more than 2 times more than 2 times before and after the liver differentiation, and were regularly expressed in 7 time points. On this basis, the difference multiplier and expression content were based on this. A total of 7 up-regulated microRNA, 6 up-regulated microRNA with the highest expression level, and 6 down-regulation of 4 times more than 4 times, were screened. The 10 down-regulated microRNA with the highest expression content showed that.QRT-PCR results showed that microRNA was up to miR-542-5p, miR-148a, miR-1290, miR-424, miR-3. The expression patterns of 0A and miR-1246 are in accordance with the results of the chip. In the down regulation of microRNA, all the other down-regulated microRNA is down down expression, except for miR-3646, but the expression pattern of other microRNA is not consistent with the chip results except miR-146a.
The expression of 6 up-regulated microRNA and 8 downregulated microRNA in the process of osteogenic differentiation showed that the up regulation of microRNA in the process of liver differentiation showed a downward trend in the process of osteogenesis, and the microRNA in the process of liver differentiation was rising in the process of osteogenesis differentiation. The expression level of microRNA in L02 and HepG2 is lower than (higher) in MSC. It shows that the differential expression of microRNA expressed in the process of hepatic differentiation is different from that in the process of osteogenesis, and not in the liver cells or liver cells, but the liver cells, but the liver. Specific to the process of differentiation.
3. the role of key microRNA molecules in MSC liver differentiation
The MSC of uninfected virus and MSC infected with miR-nc-lev increased in the expression level of albumin for 6 days after liver induction, while miR-1246-RNAi-lev, miR-1290-RNAi-lev, miR-148a-RNAi-lev, miR-30a-RNAi-lev, miR-424-RNAi-lev, miR-542-5p-RNAi-lev MSC were induced by liver direction, and the level of albumin was not up. The liver was induced to 12. Day after, group miR-nc-lev, whether or not successfully infected with lentiviruses, can take LDL. and miR-1246-RNAi-lev, miR-1290-RNAi-lev, miR-148a-RNAi-lev, miR-30a-RNAi-lev, miR-424-RNAi-lev, miR-542-5p-RNAi-lev, and LDL uptake only in cells that have not successfully infected the lentivirus. In addition, these lentivirus particles can inhibit HepG2. The expression of specific albumin and G6P in cell hepatocytes.
MiR-122, miR-1246, miR-1290, miR-148a, miR-30a, miR-424 and miR-542-5p can be transfected into MSC respectively, which can improve the level of the corresponding microRNA, but it can not promote the co transfection of MSC high expression albumin.7 microRNA analogue, which can not only increase the content of these 7 kinds, but also promote high expression. The liver specific early, middle and late marker molecules, which do not express liver progenitor cells and pancreatic bile duct epithelial markers, make the cell morphology of MSC transform from interstitial like to epithelioid and have the function of hepatocyte specific. After transplanted into the liver injury mice, it can not only improve the function of liver cells, but also improve the liver function of mice, and can also play a part of MSC. The function of repairing the liver tissue of mice.
[Conclusion]
We successfully obtained the isolation and purification of peripheral blood hematopoietic stem cells and umbilical cord MSC. On the basis of the successful establishment of MSC hepatocyte differentiation in vitro cell model, the microRNA expression profiles of a group of MSC liver differentiation processes were screened and identified by chip and qRT-PCR technology. In the 6 above adjusted microRNA, the inhibitory activity of any one was inhibited. It can inhibit the differentiation of MSC into hepatocytes, and simply overexpression of a certain kind of microRNA can not initiate MSC to differentiate into liver cells. At the same time, overexpression can induce the transformation of MSC into functional liver cells both in vivo and in vivo. This study first systematically screened the specific microRNA spectrum of liver differentiation and discussed in detail its liver differentiation. For the first time, it is confirmed that 7-microRNA combination can directly transform MSC into functional hepatocytes, providing some theoretical support for understanding the mechanism of liver differentiation.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R575.3

【共引文献】