發(fā)布時(shí)間：2018-03-21 03:41

  本文選題：c-met　切入點(diǎn)：骨髓間充質(zhì)干細(xì)胞　出處：《安徽醫(yī)科大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：研究背景與目的急性肝衰竭是由多種原因引起的肝細(xì)胞亞大片或大片壞死,致使肝臟功能?chē)?yán)重受損一種臨床癥候群。該病病情兇險(xiǎn),進(jìn)展迅速,病死率高,是嚴(yán)重威脅人類(lèi)健康的疾病之一。目前,肝移植是該病最有效的治療方法,但因肝臟供者的缺乏和手術(shù)價(jià)格昂貴而受到限制。近些年來(lái),許多研究發(fā)現(xiàn)通過(guò)移植骨髓間充質(zhì)干細(xì)胞(BMSCs)來(lái)治療急性肝衰竭已成為一種很有前途的治療方式。但其療效有限。主要原因是移植細(xì)胞后歸巢于損傷肝臟的細(xì)胞數(shù)目過(guò)少,以及歸巢的細(xì)胞分化再生能力差。因此,目前迫切需要提高骨髓間充質(zhì)干細(xì)胞移植后的歸巢能力,從而改善其治療急性肝衰竭的療效。目前,已有研究指出急性肝衰竭患者的肝臟內(nèi)肝細(xì)胞生長(zhǎng)因子(HGF)的濃度較正常肝臟明顯升高。且研究發(fā)現(xiàn)HGF/c-met信號(hào)通路在誘導(dǎo)骨髓間充質(zhì)干細(xì)胞向損傷肝臟的歸巢過(guò)程中發(fā)揮著重要的作用。在本研究中,我們通過(guò)構(gòu)建穩(wěn)定表達(dá)c-met基因的大鼠骨髓間充質(zhì)干細(xì)胞細(xì)胞株(c-met-BMSCs),旨在證明損傷肝臟內(nèi)高濃度的HGF可誘導(dǎo)c-met-BMSCs向急性肝衰竭大鼠損傷肝臟定向歸巢,以提高骨髓間充質(zhì)干細(xì)胞治療急性肝衰竭的療效。方法1.構(gòu)建c-met慢病毒表達(dá)載體,并將此慢病毒穩(wěn)定轉(zhuǎn)染大鼠骨髓間充質(zhì)干細(xì)胞,從而建立穩(wěn)定表達(dá)c-met基因的骨髓間充質(zhì)干細(xì)胞細(xì)胞株(c-met-BMSCs)。接著采用transwell系統(tǒng)體外觀察不同濃度的肝細(xì)胞生長(zhǎng)因子(HGF)誘導(dǎo)c-met-BMSCs遷移的能力。2.取36只SD大鼠隨機(jī)分為3組(移植c-met-BMSCs治療組、移植BMSCs治療組和對(duì)照組),每組12只。采用D-氨基半乳糖(D-Gal N)和脂多糖(LPS)聯(lián)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組和BMSCs組急性肝衰竭大鼠分別經(jīng)尾靜脈注射c-met-BMSCs和BMSCs(注射細(xì)胞數(shù)為1.0×107/kg溶于1ml生理鹽水中),對(duì)照組急性肝衰竭大鼠尾靜脈注射1ml生理鹽水。然后每天觀察并記錄三組大鼠的生存情況,并收集三組大鼠造模后不同時(shí)間點(diǎn)(0,24,48,72h)靜脈血行肝功能檢測(cè)及造模后24、48和72h的肝組織標(biāo)本,采用HE染色后觀察肝組織病理變化并進(jìn)行HAI評(píng)分。3.采用染料DIR分別染色標(biāo)記c-met-BMSCs和BMSCs,將標(biāo)記的細(xì)胞以相同的細(xì)胞數(shù)(1×106)通過(guò)尾靜脈分別輸入到急性肝衰竭大鼠體內(nèi)。24h后采用活體成像系統(tǒng)觀察兩組細(xì)胞向大鼠損傷肝臟的遷移情況及記錄肝臟處的輻射效率。結(jié)果1.經(jīng)PCR鑒定及測(cè)序結(jié)果顯示c-met慢病毒表達(dá)載體構(gòu)建成功,慢病毒滴度為2×108TU/ml。將此慢病毒轉(zhuǎn)染大鼠骨髓間充質(zhì)干細(xì)胞,經(jīng)嘌呤霉素篩藥后,熒光顯微鏡下檢測(cè)熒光陽(yáng)性率高于99%,且Western blot證實(shí)該細(xì)胞株過(guò)表達(dá)c-met蛋白,即成功構(gòu)建了穩(wěn)定表達(dá)c-met基因的骨髓間充質(zhì)干細(xì)胞細(xì)胞株(c-met-BMSCs)。此外,體外transwell遷移實(shí)驗(yàn)表明肝細(xì)胞生長(zhǎng)因子(HGF)具有誘導(dǎo)c-met-BMSCs定向遷移的能力。2.制造大鼠急性肝衰竭模型,在造模24、48小時(shí)后其肝臟內(nèi)HGF濃度明顯升高。治療急性肝衰竭大鼠,移植c-met-BMSCs治療組、移植BMSCs治療組和對(duì)照組三組急性肝衰竭大鼠的生存率分別為83.3%、50%和0%。與移植BMSCs治療組和對(duì)照組相比,移植c-met-BMSCs治療組可顯著改善大鼠肝功能及降低損傷肝組織HAI評(píng)分。3.活體成像系統(tǒng)顯示移植c-met-BMSCs治療組的大鼠肝臟處的輻射效率是移植BMSCs治療組的20倍,表明移植c-met-BMSCs可明顯提高歸巢于損傷肝臟的細(xì)胞數(shù)。結(jié)論成功構(gòu)建了c-met慢病毒表達(dá)載體,并建立了穩(wěn)定表達(dá)c-met基因的大鼠骨髓間充質(zhì)干細(xì)胞細(xì)胞株(c-met-BMSCs)。且該細(xì)胞株治療急性肝衰竭大鼠時(shí)可靶向歸巢于損傷肝臟,從而加快了急性肝衰竭的修復(fù)過(guò)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.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R575.3

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