碱性成纤维细胞生长因子在骨髓间充质干细胞治疗小鼠肝硬化中的作用

发布时间：2018-05-22 13:13

本文选题：肝硬化 + 骨髓间充质干细胞　；参考：《兰州大学》2017年硕士论文

【摘要】：背景:干细胞移植治疗肝硬化在动物实验、临床研究已被广泛研究,碱性成纤维细胞生长因子体外条件下可以促进干细胞增殖,利用bFGF培养后的干细胞治疗肝硬化的体内疗效目前尚不确定。目的:探索bFGF对BMSC生长和增殖的影响。加入0.1%bFGF培养后的骨髓间充质干细胞通过小鼠尾静脉注射后肝功能ALT、AST、ALB水平、组织病理学改变及胶原沉积变化情况。方法:全骨髓贴壁法提取雄性SD大鼠骨髓间充质干细胞;流式细胞检测BMSC。雌性昆明小鼠46只随机分为分为5组:A组(CCl4+PBS)、B组(CCl4+bFGF)、C组(CCl4+bFGF-BMSC)、D组(CCl4+bFGF+BMSC)、E组(CCl4+bFGF+BMSC3次移植)。小鼠CCl4造模10周后分别尾静脉注射bFGF+或bFGF-培养的BMSC,酶联免疫法检测肝功能,HE染色检测组织病理学改变情况;Masson三色染色法检测肝组织胶原沉积情况。结果:(1)BMSC生长情况:观察各组传代细胞的生长情况,bFGF-、bFGF+组细胞24h完全贴壁,贴壁细胞初期呈梭形或不规则形,细胞形态无明显异常,主要以梭形为主,bFGF+组细胞增殖较快,于第4天铺满瓶底。bFGF-组较bFGF+细胞生长相对缓慢,第6-7天时基本完全铺满细胞培养瓶。(2)细胞生长:10ng/ml bFGF体外培养条件下,BMSC生长速率显著高于无bFGF培养。(3)流式检测:CD29、CD44阳性表达,CD45呈阴性表达。(4)化学法检测肝脏功能:ALT水平在模型对照组(A组)与B组比较无显著性差异(P0.05),C组、D组、E组ALT水平与A组、B组比较均有下降(两两比较具有显著性差异,P0.05);ALT水平在D组、E组均较C组下降(P0.05);E组较D组下降(P0.05)。AST水平在A组与B组间无显著性差异(P0.05);C组、D组、E组两两之间无显著性差异(P0.05);而C组、D组、E组较A组或B组具有明显下降(P0.05)。(5)HE染色结果:A组、B组HE染色见肝细胞明显水肿、脂肪变性、桥接坏死,肝索排列紊乱,假小叶广泛形成。C组肝细胞水肿、脂肪变性,炎性细胞浸润,但程度较模型组有所减轻,汇管区纤维沉积,可见假小叶结构;D组、E组肝细胞水肿情况、炎性细胞浸润程度较BMSC组明显改善,假小叶结构可见,汇管区可见纤维沉积。(6)Masson结果显示,A组、B组肝门脉区大量绿染纤维沉积,肝实质内绿色纤维浸润,纤细的纤维间隔从汇管区延伸至肝实质,分隔肝小叶形成假小叶。A组纤维面积较B组无显著性差异(P0.05);与A组、B组相比,C组、D组、E组纤维量减少,较A组、B组明显改善,差异有统计学意义(p值均0.05);C组、D组汇管区、肝实质有可见纤维沉积,但两组纤维面积无显著性差异(P0.05);E组较C组、D组面积均有显著降低(P0.05)。结论:体外培养加入bFGF后BMSC生长速度较无bFGF时显著提高,细胞形态大致相同,流式检测细胞表面标志物CD29、CD44、CD45表达与无bFGF时相同。BMSC移植能够改善肝硬化小鼠肝功能、肝硬化程度、胶原纤维沉积,培养中加入bFGF的BMSC更为显著地改善肝损伤程度和纤维化程度,重复细胞移植后肝纤维化程度改善更为显著。
[Abstract]:Background: stem cell transplantation in the treatment of liver cirrhosis in animal experiments, clinical research has been widely studied, basic fibroblast growth factor can promote stem cell proliferation in vitro. The in vivo efficacy of stem cells cultured with bFGF in the treatment of liver cirrhosis is uncertain. Objective: to explore the effect of bFGF on the growth and proliferation of BMSC. Bone marrow mesenchymal stem cells (BMSCs) cultured with 0.1GF were injected through caudal vein in mice. The level of ALB, histopathological changes and collagen deposition in liver function were observed. Methods: bone marrow mesenchymal stem cells from male SD rats were extracted by whole bone marrow adherent method, and BMSCs were detected by flow cytometry. 46 female Kunming mice were randomly divided into 5 groups: group B: CCl4 bFGF group B group CCl4 bFGF group B group (group C): CCl4 bFGF group B group (group D): CCl4 bFGF group B group (group E): CCl4 bFGF BMSC3 transplants. After 10 weeks of mouse CCl4 model, bFGF or bFGF- cultured BMSCs were injected into tail vein respectively. Liver function was detected by enzyme-linked immunosorbent assay (Elisa) to detect histopathological changes. Masson trichrome staining was used to detect collagen deposition in liver tissue. Results the growth of BMSC: the growth of BMSCs in each group was observed. The cells in the bFGF-bFGF group were completely adhered to the wall for 24 hours. The adherent cells were fusiform or irregular at the initial stage, and the morphology of the cells was not obviously abnormal. The proliferation of the cells in the main fusiform group was faster than that in the bFGF group. On the 4th day, the growth of bFGF cells was slower than that of BFGF- group. After 6-7 days, the cell growth rate of bFGF was significantly higher than that of bFGF free culture.) flow cytometry showed negative expression of CD44 + CD44 and negative expression of CD45. 4) the liver function was detected by chemical method. There was no significant difference between group A and group B (P 0.05). The level of ALT in group D was significantly lower than that in group A (P 0.05) and the level of alt in group B (P 0.05) was significantly lower than that in group C (P 0.05). There was no significant difference between group A and group B in the level of P0.05A. There was no significant difference between group D and group E, while group C had a significant decrease in P0.05. 5HE staining results showed that the liver cells were edema in group A and group B compared with those in group A or group B, while in group C, there was no significant difference between group A and group B in the level of P0.05A, and there was no significant difference between group D and group B in the levels of P0.05A and P0.05.The results showed that there was no significant difference between group D and group B. Fatty degeneration, bridging necrosis, disordered arrangement of hepatic cord, edema of hepatocytes, steatosis and infiltration of inflammatory cells were widely formed in group .C, but the degree was less than that in the model group. The edema of hepatocytes, the degree of inflammatory cell infiltration and the structure of pseudolobules were obviously improved in group D and group E, and the fibrous deposition was observed in portal area of group A and B. The results showed that a large number of green stained fibers were deposited in portal vein of group A and B. Green fiber infiltration in hepatic parenchyma, fine fibrous septum extending from catchment area to hepatic parenchyma, no significant difference in fiber area between group A and group B in the formation of pseudolobules, and decrease in fiber content in group C and group D in comparison with group A and group B. Compared with group A, group B, the difference was statistically significant (P < 0.05). There was significant difference between group C and group C (P < 0.05), but there was no significant difference in fiber area between group A and group C (P 0.05), but the area of fiber in group E was significantly lower than that in group C (P 0.05), but there was no significant difference in fiber area between group C and group C (P 0.05). Conclusion: the growth rate of BMSC in vitro was significantly higher than that without bFGF, and the cell morphology was approximately the same. The expression of CD29, CD4, CD4, CD45, a marker on cell surface, could improve liver function and degree of liver cirrhosis in cirrhotic mice as compared with those without bFGF. The degree of liver injury and fibrosis was significantly improved by adding bFGF to BMSC and the degree of liver fibrosis was improved after repeated cell transplantation.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.2

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