microRNA介导人脐带间充质干细胞转分化为肝细胞样细胞的组合优化与验证

发布时间：2018-05-21 03:09

本文选题：间充质干细胞 + miRNA　；参考：《第四军医大学》2016年硕士论文

【摘要】：【背景】近年来,由病毒、酒精、毒物或代谢等因素引起的肝脏衰竭呈逐年上升趋势。特别是急性、亚急性及慢加急性肝功能衰竭,由于病因多,并发症多和重要器官损伤多等特点,成为高发病率、高死亡率和治疗非常棘手的疾病。肝移植是治疗肝衰竭最有效的手段,但供肝的严重缺乏、治疗费用昂贵及移植后的免疫排斥反应等限制了它的广泛应用。人工肝支持系统可部分地清除体内的毒性代谢产物,给患者自身肝脏再生创造条件。但由于其没有肝脏的合成及代谢等功能,其治疗疗效有限。因此,急需开发具有一定肝脏功能的生物型人工肝支持系统。近年来,生物型人工肝支持系统逐渐成为治疗急性肝衰竭最有效和最具潜力的治疗方式。生物型人工肝支持系统主要由细胞成分,生物反应器及辅助器材组成,其中细胞成分是生物型人工肝的核心问题。目前,种子细胞来源受到很多方面的限制,诸如自体肝细胞体外培养、增殖和保存技术有限,异种来源肝细胞存在免疫排斥效应等。这些因素限制了生物型人工肝支持系统应用于肝功能衰竭的治疗。因此,我们需要寻找有功能的人源肝细胞,并可大规模生产。而近年来干细胞的发展为解决肝细胞来源提供了新的思路。我们前期研究通过对间充质干细胞诱导分化得到的肝细胞样细胞HLC与间充质干细胞的micro RNA表达水平进行芯片分析,以及RT-PCR验证比对等获得一组表达量显著上调的micro RNA组合:mi R-148a,mi R-424,mi R-1290,mi R-542-5p,mi R-1246和mi R-30a。在此基础上,我们加入了肝脏特异性的mi R-122,作为一组micro RNA即7个mi RNA组合转染进人脐带间充质干细胞内,成功实现了干细胞转分化为肝细胞样细胞(hepatocyte-like cells 7,HLC-7),并进行了一系列肝细胞样细胞的功能验证。我们证实这组由mi RNA组合诱导得到的细胞不仅表达肝特异基因,还具有LDL摄取,糖原沉积,尿素合成等一系列肝细胞功能。进一步,我们通过每组减少一种mi RNA获得不同组合方式的方法继续筛选对于人脐带间充质干细胞转分化为肝细胞样细胞的关键mi RNA或者组合。我们通过删减mi R-30a和mi R-1290后的5种mi RNA(mi R-122,mi R-148a,mi R-424,mi R-542-5p和mi R-1246)转染进脐带MSC中,成功将其诱导分化为肝细胞样细胞(HLC-5),能够表达肝脏特异性基因ALB,AFP,HNF4A和TF等,PAS染色和LDL摄取试验均为阳性,证实了诱导分化的成功。本研究将继续采用n-1方法进一步删减mi RNA组合,并验证其体外的肝细胞功能。运用四氯化碳(Carbon tetrachloride,CCl4)建立裸鼠肝损伤和急性肝衰竭模型,并对肝损伤小鼠进行HLC移植治疗,观察对动物模型的修复作用。【目的】1.明确mi RNA介导间充质干细胞转分化为肝细胞样细胞的最优组合;2.阐明HLCs在裸鼠肝损伤和肝衰竭中的修复作用。【方法】1.贴壁培养MSC,倒置显微镜观察细胞形态,流式细胞仪检测表面标志,进行成脂和成骨诱导分化,明确MSC的纯度和分化能力。2.筛检mi RNA组合:验证5种mi RNA(mi R-122,148a,424,542-5p和1246)的转染效率以及诱导间充质干细胞转分化为肝细胞样细胞的能力。在此基础上,逐渐减少mi RNA的个数,观察转染后的效应:主要通过RT-PCR检测肝特异性基因表达水平,LDL摄取实验、ICG摄取实验、PAS糖原染色和尿素氮合成等试验检测HLC的肝细胞特异性功能。3.CCl4诱导裸鼠肝损伤,并随机分为生理盐水阴性对照组,HLC-7阳性对照和最优组合诱导的HLC-N组,将生理盐水和诱导细胞经裸鼠尾静脉移植(细胞数量1x106/只)。通过血清学检测AST、ALT、ALB水平,HE染色和天狼星红染色观察造模损伤程度和细胞移植后的修复情况。4.高浓度CCl4诱导裸鼠急性肝衰竭,随机分为生理盐水阴性对照组,HLC-7阳性对照和最优组合诱导的HLC-N组,并经裸鼠尾静脉进行移植。一周内观察裸鼠的生存情况。【结果】1.贴壁培养的MSC呈类成纤维样的梭形形态,流式细胞仪检测MSC表达99.8%的标志分子CD105,而造血干细胞标志CD34以及内皮细胞标志CD31阳性率仅为0.2%,0.2%,表明MSC细胞纯度较高。另外,茜素红和油红染色结果显示MSC可以在诱导液作用下分化为骨细胞和脂肪细胞,具备多向分化的潜能。2.5种mi RNA转染后的结果显示:在转染7天后,5种mi RNA均在转染细胞中成功过表达;与NC组相比,HLC-5的肝特异性基因ALB,HNF4A,AFP等表达水平显著升高,与HLC-7类似;而不表达其他非特异的PDX1,Ep CAM和CK7。肝特异性功能检测结果显示:LDL和ICG摄取实验表明大部分的HLC-5可以摄取LDL,约40%可以摄取ICG;PAS染色结果提示大约60%的HLC-5都可以合成糖原;尿素合成实验表明HLC-5具备合成尿素的能力,与对照组相比,具有统计学意义。3.4种mi RNA转染后的结果显示:转染7天后,各组的细胞形态并无明显改变;RT-PCR的结果显示各组间ALB,HNF4A,AFP,CYP3A4和TF的m RNA水平与对照组相比并无明显差异;LDL摄取实验也表明4种mi RNA诱导的细胞不能成功摄取LDL。4.急性肝损伤模型结果显示:CCl4处理后血清ALB明显下降,AST和ALT升高,HE染色可见肝脏结构破坏和炎细胞浸润,提示急性肝损伤模型建立成功。与生理盐水处理组相比,HLC-5和HLC-7移植后白蛋白水平升高,转氨酶水平下降,血清结果明显改善;HE染色提示肝脏组织结构改善,炎症减轻。5.急性肝衰竭模型结果表明:高浓度CCl4处理1天后,HE染色结果显示肝脏组织结构受损严重,炎性细胞浸润。观察一周内的生存情况发现,与生理盐水对照组相比,HLC-5和HLC-7移植后可显著改善裸鼠的生存时间。并且HLC-5和HLC-7两组间并无明显差异,说明在体内HLC-5具有与HLC-7相似的功能。【结论】本研究表明,5种mi RNA(mi R-122,148a,424,542-5p和1246)是诱导脐带间充质干细胞转分化为肝细胞样细胞的最优组合。诱导得到的HLC-5不仅在体外具备肝细胞的功能,在动物模型体内也可发挥与HLC-7相似的损伤修复功能,并可显著改善肝衰竭裸鼠的生存情况。这为肝细胞样细胞在生物性人工肝的应用提供了实验基础。而mi RNA调控转分化的具体机制和HLC-5的细胞特点尚需要进一步探索。
[Abstract]:[background] in recent years, liver failure caused by virus, alcohol, poison or metabolism has been increasing year by year. Especially acute, subacute and slow and acute liver failure, due to many causes, many complications and many important organ damage, it has become a high incidence, high mortality and very difficult treatment. Liver transplantation is a serious disease. The most effective means for the treatment of liver failure, but the severe deficiency of the donor liver, the expensive treatment and the immune rejection after the transplantation limit its wide application. The artificial liver support system can partially remove the toxic metabolites in the body and create conditions for the patient's own liver regeneration. However, it has no function of liver synthesis and metabolism. In recent years, biological artificial liver support system has gradually become the most effective and potential treatment for acute liver failure. Biological artificial liver support system is mainly composed of cell components, bioreactors and auxiliary equipment groups. The cell composition is the core problem of the biotype artificial liver. At present, the source of seed cells is limited by many aspects, such as the culture of autologous liver cells in vitro, the limited proliferation and preservation techniques, and the immune rejection of xenogenic liver cells. These factors restrict the application of biological artificial liver support system to liver failure. Therefore, we need to find functional human derived hepatocytes and can be produced on a large scale. In recent years, the development of stem cells provides a new way of thinking for the solution of hepatocyte origin. We have studied the level of micro RNA expression of HLC and mesenchymal stem cells derived from mesenchymal stem cells. Chip analysis, and RT-PCR validation is a micro RNA combination that is significantly up - up of a set of expressions than peer to peer: Mi R-148a, MI R-424, MI R-1290, MI R-542-5p. The stem cells were transformed into hepatocyte like cells (hepatocyte-like cells 7, HLC-7), and the function of a series of hepatocyte like cells was verified. We confirmed that this group of cells induced by Mi RNA combination not only expressed liver specific genes, but also had a series of hepatocyte functions, such as LDL uptake, glycogen deposition, urea synthesis, and so on. We continue screening the key mi RNA or combination of human umbilical cord mesenchymal stem cells transdifferentiated into hepatocyte like cells by reducing one kind of MI RNA in each group. We transfect the 5 mi RNA of MI R-30a and MI R-12 90 (MI R-122) into umbilical cord It is successfully induced to differentiate into hepatocyte like cells (HLC-5), which can express liver specific genes ALB, AFP, HNF4A and TF. Both PAS staining and LDL uptake test are positive, which confirm the success of induced differentiation. This study will continue to use n-1 method to further reduce the MI RNA combination and verify the function of liver cells in vitro. Using carbon tetrachloride in vitro. (Carbon tetrachloride, CCl4) to establish a model of liver injury and acute liver failure in nude mice, and to treat the mice with liver injury by HLC transplantation and observe the repair effect of the animal model. [Objective] 1. to clarify the optimal combination of MI RNA mediated mesenchymal stem cells to turn into hepatocyte like cells; and 2. clarify HLCs in nude mice liver injury and liver failure. Repair effect. [Methods] 1. adherent culture MSC, inverted microscope to observe cell morphology, flow cytometry to detect surface markers, lipid and osteogenesis induced differentiation, clear MSC purity and differentiation ability.2. screening mi RNA combination: verify the transfection efficiency of 5 mi RNA (MI R-122148a, 424542-5p and 1246) and induce mesenchymal stem cells to transfer. The ability to differentiate into hepatocyte like cells. On this basis, the number of MI RNA was gradually reduced and the effect after transfection was observed: the detection of liver specific gene expression by RT-PCR, LDL uptake experiment, ICG uptake experiment, PAS glycogen staining and urea nitrogen synthesis test to detect the liver cell specific function.3.CCl4 of HLC to induce liver injury in nude mice And randomly divided into the normal saline negative control group, the HLC-7 positive control and the optimal combination induced HLC-N group, the physiological saline and the induced cells were transplanted in the tail vein of nude mice (the number of cells 1x106/ only). By serological detection of AST, ALT, ALB level, HE staining and Sirius red staining, the damage degree of the model and the restoration of the cells after the cell transplantation were observed. The high concentration of CCl4 induced acute liver failure in nude mice, randomly divided into the normal saline negative control group, the HLC-7 positive control and the optimal combination induced HLC-N group, and transplantable by the tail vein of nude mice. In a week, the survival of nude mice was observed. [results] the MSC of 1. adherent culture was like the spindle shape of the fibrinolytic type, and the flow cytometry was used to detect the MSC expression. 99.8% of the marker molecule CD105, and the hematopoietic stem cell marker CD34 and the endothelial cell marker CD31 positive rate is only 0.2%, 0.2%, indicating that the MSC cell purity is high. In addition, alizarin red and oil red staining results show that MSC can differentiate into bone and fat cells under the action of inducer, with the potential of multidirectional differentiation potential.2.5 species after MI RNA transfected. The results showed that 7 days after transfection, 5 kinds of MI RNA were overexpressed in transfected cells. Compared with group NC, the expression level of HLC-5 specific gene ALB, HNF4A, AFP was significantly higher, similar to HLC-7, but not other non specific PDX1, Ep CAM, and CK7. liver specific function detection results showed that most of the experimental results showed that the majority of them were found. 5 can take LDL, about 40% can absorb ICG; PAS staining results suggest that about 60% of HLC-5 can synthesize glycogen. The urea synthesis experiment shows that HLC-5 has the ability to synthesize urea. Compared with the control group, the results of.3.4 after MI RNA are statistically significant: the cell morphology of each group is not obviously changed after 7 days of transfection; RT-PCR The results showed that the m RNA levels of ALB, HNF4A, AFP, CYP3A4 and TF were not significantly different from those of the control group. LDL uptake experiments also showed that 4 mi RNA induced cells failed to successfully absorb the LDL.4. acute liver damage model. The acute liver injury model was successfully established. Compared with the saline treatment group, the level of albumin increased after HLC-5 and HLC-7 transplantation, the level of transaminase decreased and the serum results were obviously improved; HE staining suggested the improvement of liver tissue structure. The results of inflammation alleviating.5. acute liver failure model showed that high concentration CCl4 treatment was 1 days after HE staining results. The liver tissue structure was severely damaged and inflammatory cells infiltrated. Observation of survival within a week was found, compared with the saline control group, HLC-5 and HLC-7 could significantly improve the survival time of nude mice. There was no significant difference between the HLC-5 and the HLC-7 two groups, indicating that HLC-5 had a similar function to HLC-7 in the body. [Conclusion] this study The results show that 5 kinds of MI RNA (MI R-122148a, 424542-5p and 1246) are the best combinations to induce the differentiation of umbilical cord mesenchymal stem cells into hepatocyte like cells. The induced HLC-5 not only has the function of hepatocyte in vitro, but also can play a similar damage repair function with HLC-7 in the animal model, and can significantly improve the liver failure in nude mice. This provides an experimental basis for the application of hepatocyte like cells in biological artificial liver. The specific mechanism of MI RNA regulation of transdifferentiation and the cell characteristics of HLC-5 need to be further explored.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R575.3

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