c-met基因修饰的骨髓间充质干细胞靶向治疗急性肝衰竭大鼠的实验研究

发布时间：2018-03-21 03:41

本文选题：c-met　切入点：骨髓间充质干细胞　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景与目的急性肝衰竭是由多种原因引起的肝细胞亚大片或大片坏死,致使肝脏功能严重受损一种临床症候群。该病病情凶险,进展迅速,病死率高,是严重威胁人类健康的疾病之一。目前,肝移植是该病最有效的治疗方法,但因肝脏供者的缺乏和手术价格昂贵而受到限制。近些年来,许多研究发现通过移植骨髓间充质干细胞(BMSCs)来治疗急性肝衰竭已成为一种很有前途的治疗方式。但其疗效有限。主要原因是移植细胞后归巢于损伤肝脏的细胞数目过少,以及归巢的细胞分化再生能力差。因此,目前迫切需要提高骨髓间充质干细胞移植后的归巢能力,从而改善其治疗急性肝衰竭的疗效。目前,已有研究指出急性肝衰竭患者的肝脏内肝细胞生长因子(HGF)的浓度较正常肝脏明显升高。且研究发现HGF/c-met信号通路在诱导骨髓间充质干细胞向损伤肝脏的归巢过程中发挥着重要的作用。在本研究中,我们通过构建稳定表达c-met基因的大鼠骨髓间充质干细胞细胞株(c-met-BMSCs),旨在证明损伤肝脏内高浓度的HGF可诱导c-met-BMSCs向急性肝衰竭大鼠损伤肝脏定向归巢,以提高骨髓间充质干细胞治疗急性肝衰竭的疗效。方法1.构建c-met慢病毒表达载体,并将此慢病毒稳定转染大鼠骨髓间充质干细胞,从而建立稳定表达c-met基因的骨髓间充质干细胞细胞株(c-met-BMSCs)。接着采用transwell系统体外观察不同浓度的肝细胞生长因子(HGF)诱导c-met-BMSCs迁移的能力。2.取36只SD大鼠随机分为3组(移植c-met-BMSCs治疗组、移植BMSCs治疗组和对照组),每组12只。采用D-氨基半乳糖(D-Gal N)和脂多糖(LPS)联合腹腔注射的方式制造大鼠急性肝衰竭模型,造模完成24h后,c-met-BMSCs组和BMSCs组急性肝衰竭大鼠分别经尾静脉注射c-met-BMSCs和BMSCs(注射细胞数为1.0×107/kg溶于1ml生理盐水中),对照组急性肝衰竭大鼠尾静脉注射1ml生理盐水。然后每天观察并记录三组大鼠的生存情况,并收集三组大鼠造模后不同时间点(0,24,48,72h)静脉血行肝功能检测及造模后24、48和72h的肝组织标本,采用HE染色后观察肝组织病理变化并进行HAI评分。3.采用染料DIR分别染色标记c-met-BMSCs和BMSCs,将标记的细胞以相同的细胞数(1×106)通过尾静脉分别输入到急性肝衰竭大鼠体内。24h后采用活体成像系统观察两组细胞向大鼠损伤肝脏的迁移情况及记录肝脏处的辐射效率。结果1.经PCR鉴定及测序结果显示c-met慢病毒表达载体构建成功,慢病毒滴度为2×108TU/ml。将此慢病毒转染大鼠骨髓间充质干细胞,经嘌呤霉素筛药后,荧光显微镜下检测荧光阳性率高于99%,且Western blot证实该细胞株过表达c-met蛋白,即成功构建了稳定表达c-met基因的骨髓间充质干细胞细胞株(c-met-BMSCs)。此外,体外transwell迁移实验表明肝细胞生长因子(HGF)具有诱导c-met-BMSCs定向迁移的能力。2.制造大鼠急性肝衰竭模型,在造模24、48小时后其肝脏内HGF浓度明显升高。治疗急性肝衰竭大鼠,移植c-met-BMSCs治疗组、移植BMSCs治疗组和对照组三组急性肝衰竭大鼠的生存率分别为83.3%、50%和0%。与移植BMSCs治疗组和对照组相比,移植c-met-BMSCs治疗组可显著改善大鼠肝功能及降低损伤肝组织HAI评分。3.活体成像系统显示移植c-met-BMSCs治疗组的大鼠肝脏处的辐射效率是移植BMSCs治疗组的20倍,表明移植c-met-BMSCs可明显提高归巢于损伤肝脏的细胞数。结论成功构建了c-met慢病毒表达载体,并建立了稳定表达c-met基因的大鼠骨髓间充质干细胞细胞株(c-met-BMSCs)。且该细胞株治疗急性肝衰竭大鼠时可靶向归巢于损伤肝脏,从而加快了急性肝衰竭的修复过程。
[Abstract]:Background and objective acute liver failure is caused by a variety of reasons of hepatocyte necrosis or large, causing the liver function severely damaged a clinical syndrome. The disease is a serious disease, rapid progression and high mortality, is a serious threat to human health. Currently, liver transplantation is the most effective treatment for the disease but, because of the lack of donor liver surgery and the price is expensive and limited. In recent years, many studies have found that the transplantation of bone marrow mesenchymal stem cells (BMSCs) for the treatment of acute liver failure has become a promising treatment. But the effect is limited. The main reason is that the number of cells homing after transplantation. In liver injury and regeneration capacity is too small, cell differentiation homing difference. Therefore, there is an urgent need to improve the transplantation of bone marrow mesenchymal stem cells after homing ability, so as to improve the treatment of acute liver failure treatment Effect. At present, researches have pointed out that the hepatocyte growth factor in patients with acute liver failure (HGF) concentration in the liver increased significantly. Compared with normal liver and found that HGF/c-met signaling pathway in the induction of bone marrow mesenchymal stem cells homing to liver damage plays an important role. In this study, we to construct stable c-met gene expression of rat bone marrow mesenchymal stem cells (c-met-BMSCs), aims to prove that high concentration of HGF in liver injury to acute liver failure in rats liver injury induced by c-met-BMSCs homing, in order to improve the curative effect of bone marrow mesenchymal stem cells in the treatment of acute liver failure. 1. methods to construct the c-met lentiviral vector the expression vector, and the lentiviral stable transfection of rat bone marrow mesenchymal stem cells, so as to establish a stable c-met gene expression of bone marrow mesenchymal stem cells (c-met-BMSCs). Then using Transwell In vitro effects of different concentration of hepatocyte growth factor (HGF) induced c-met-BMSCs migration ability of.2. 36 SD rats were randomly divided into 3 groups (c-met-BMSCs transplantation treatment group, BMSCs transplantation group and control group), 12 rats in each group. D- galactosamine (D-Gal N) and lipid polysaccharide (LPS the way of manufacturing) and intraperitoneal injection of rats with acute liver failure model, model 24h, c-met-BMSCs group and BMSCs group of rats with acute liver failure were treated with c-met-BMSCs and BMSCs tail intravenous injection (injection of cell number was 1 * 107/kg in normal saline 1ml), control group of acute liver failure rat tail vein injection of 1ml saline. Then observed and recorded daily survival of the three groups of rats, and collected three groups of rats at different time points (0,24,48,72h) detection of venous blood and liver function after modeling 24,48 and 72h liver tissues, liver tissues were observed by HE staining The pathological changes and HAI score by.3. dye staining c-met-BMSCs and BMSCs DIR respectively, the labeled cells with the same number of cells (1 * 106) through the tail vein were input to.24h in rats with acute liver failure after using in vivo imaging system observes two group of cells to radiation damage efficiency in rat liver and migration record the liver. Results 1. was identified by PCR and sequencing results showed that c-met expression vector was successfully constructed. The virus titer was 2 * 108TU/ml. the lentiviral transfection of rat bone marrow mesenchymal stem cells by puromycin drug screening, fluorescence microscope detection fluorescence positive rate higher than 99%, and Western blot the cell line overexpressing c-met protein, the c-met gene was successfully constructed by bone marrow mesenchymal stem cell line with stable expression of (c-met-BMSCs). In addition, the migration of Transwell in vitro experiments showed that HGF Sub (HGF) with c-met-BMSCs induced directional migration of manufacturing ability of.2. in acute liver failure rats model, significantly increased the concentration of HGF in the liver in the rats after 24,48 hours. In the treatment of acute liver failure rats transplanted c-met-BMSCs treatment group, BMSCs transplantation group and control group three groups of rats with acute liver failure survival rate as of 83.3%, 50% and 0%. and BMSCs transplantation group compared with the control group, c-met-BMSCs transplantation treatment group can significantly improve the liver function of rats and reduced liver tissue injury HAI score.3. in vivo imaging system the radiation efficiency of rat liver transplantation at the c-met-BMSCs treatment group was 20 times of transplantation in the BMSCs group, showed that the number of transplanted cells c-met-BMSCs can significantly improve the homing to liver injury. Conclusion the successful construction of c-met lentiviral expression vector, and a stable expression of c-met gene in rat bone marrow mesenchymal stem cells (c-met- cell line BMSCs). And the cell line can be targeted to the injured liver in the treatment of acute liver failure rats, thus accelerating the repair process of acute liver failure.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.3

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