血管内皮祖细胞移植对大鼠急性胰腺炎胰腺微循环改善作用的实验研究

发布时间：2019-03-26 12:49

【摘要】：目的：本实验通过检测血管内皮祖细胞（Endothelial progenitorcells，EPCs）移植到牛磺胆酸钠诱导的大鼠急性胰腺炎模型的CD133、CD34免疫组化表达和HE染色血管密度的变化，研究血管内皮细胞对大鼠急性胰腺炎微循环的改善作用。方法：1．细胞分离培养和鉴定：选取成年健康SD大鼠，取大鼠股骨、胫骨，PBS液冲洗骨髓腔，收集全部骨髓细胞，制成单核细胞悬液，加入到淋巴细胞分离液中，用密度梯度离心法离心，取中间白膜层的有核细胞，离心洗涤三次。利用差速贴壁法，取培养48h后未贴壁细胞，用含10％的胎牛血清、5ng／ml bFGF、20ng／ml VEGF的M199培养液培养于6孔板中。培养10d后,免疫组化法和荧光双染法鉴定细胞。待细胞增殖到移植所需的数量时，收集细胞，无血清培养基洗涤两次，用无血清培养基制成单细胞悬液，以备移植。2、大鼠急性胰腺炎模型的建立和EPCs的移植：将72只健康成年雄性大鼠随机分为正常对照组（A组，n=24），急性胰腺炎模型组（B组,n=24）,急性胰腺炎内皮祖细胞移植组（C组，n=24）。将SD大鼠用1%戊巴比妥钠0.6ml/100g腹腔注射，于胰腺被膜下注射1%牛磺胆酸钠1ml(正常对照组被膜下注射同等量生理盐水)，同时行空肠造瘘，术后均给予肠外营养。C组通过大鼠颈外静脉置管注射经体外培养的内皮祖细胞。各组于C组移植后第3d、7d、14d三个时间点各取6只大鼠处死并收集胰腺标本甲醛固定，胰腺组织HE染色后光镜病理学检查，观察胰腺病理形态变化，记录血管密度情况；免疫组织化学法(SP法)检测胰腺组织中CD133、CD34的累积光密度值（IOD值）。以上所收集的数据采用均数±标准差（x±s）表示，采用SPSS17.0统计软件进行统计学处理；样本均数的比较采用单因素方差分析；两者的相关性采用直线相关和直线回归分析，，P0.05，则差异有统计学意义。结果：（1）B组（模型组）和C组（移植组）的病理评分在3d、7d、14d各时间点高于A（对照组）组（P0.05)。B组的病理评分在7d、14d点高于C组(P0.05)。(2)C组胰腺组织HE染色随机选取镜下微血管数在7d、14d高于A组(P0.05)，C组微血管数7d、14d高于B组(P0.05)。（3）C组胰腺组织CD133的IOD值在各时间点均高于A、B组(P0.05)。(4)C组胰腺组织CD34的IOD值在各时间点均高于A、B组(P0.05)。（5）胰腺组织病理评分与CD133的IOD值呈直线相关(r=0.814，P=0.000)。结论：（1）通过密度梯度离心结合差速贴壁法能够从大鼠骨髓分离并培养得到的EPCs可以用于细胞移植。（2）培养的血管内皮祖细胞可通过成功移植到急性胰腺炎的大鼠胰腺组织中。（3）移植的内皮祖细胞能增加急性胰腺炎胰腺微血管的生成，从而使胰腺微循环障碍得到改善。
[Abstract]:Objective: to investigate the expression of CD133,CD34 and the changes of vascular density by HE staining in rats with acute pancreatitis induced by sodium taurocholate transplantation of vascular endothelial progenitor cells (Endothelial progenitorcells,EPCs). To study the effect of vascular endothelial cells on microcirculation of acute pancreatitis in rats. Methods: 1. Cell isolation, culture and identification: the adult healthy SD rats were selected. The femur, tibia and PBS solution were used to wash the bone marrow cavity, collect all the bone marrow cells, make monocyte suspension, and add it into the lymphocyte separation solution. The nucleated cells in the middle white membrane were centrifuged by density gradient centrifugation and washed for three times. Non-adherent cells were cultured in M199 medium containing 10% fetal bovine serum and 5ng/ml bFGF,20ng/ml VEGF in 6-well plate after 48 h culture. After 10 days of culture, the cells were identified by immunohistochemistry and fluorescence double staining. When cells proliferate to the amount required for transplantation, collect cells, wash them twice in serum-free medium, and use serum-free medium to make a single cell suspension for transplantation. 2, Establishment of acute pancreatitis model in rats and transplantation of EPCs: 72 healthy adult male rats were randomly divided into normal control group (group A, n = 24), model group of acute pancreatitis (group B, n = 24) and group C of endothelial progenitor cell transplantation (group C). (24). SD rats were injected intraperitoneally with 1% pentobarbital sodium 0.6ml/100g, 1% sodium taurocholate 1ml (the same amount of saline was injected under the capsule of normal control group) under the pancreatic capsule, and enterostomy was performed at the same time. Group C was injected with cultured endothelial progenitor cells (EPCs) through external jugular vein. On the 3rd, 7th and 14th day after transplantation, 6 rats in each group were killed and fixed with formaldehyde. After HE staining, the pathological changes of pancreas were observed and the density of blood vessels was recorded. Immunohistochemical method (SP) was used to detect the cumulative optical density (IOD) of CD133,CD34 in pancreatic tissue. The data collected above were expressed by the mean 卤standard deviation (x 卤s), and the SPSS17.0 software was used for statistical processing, and the comparison of the sample averages was analyzed by one-way ANOVA. The correlation between the two was statistically significant by linear correlation and linear regression analysis, P 0.05. Results: (1) the pathological scores of group B (model group) and group C (transplantation group) were higher than those of group A (control group) at 3 d, 7 d, 14 d (P0.05). B group). The number of microvessels in pancreatic tissue of group C was higher than that of group C (P0.05). (2) at 14 days, and the number of microvessels in pancreatic tissue of group C was significantly higher than that of group A (P 0.05). (2) at 7 days and 14 days respectively (P 0.05), C group). The IOD value of pancreatic tissue CD133 in group C was higher than that in group B (P0.05). (3) and the value of CD34 in group B (P0.05). (4) was higher than that in group A at each time point, and the IOD value of pancreatic tissue in group C was higher than that in group A at each time point. In group B (P0.05). (5), there was a linear correlation between the pathological score of pancreas and the iod value of CD133 (r = 0.814, P < 0.001). Conclusion: (1) EPCs can be isolated and cultured from rat bone marrow by density gradient centrifugation combined with differential adhesion method. (2) cultured vascular endothelial progenitor cells can be successfully transplanted to acute pancreas. (3) Transplantation of endothelial progenitor cells (EPCs) can increase the formation of pancreatic microvessels in patients with acute pancreatitis. Thus the pancreatic microcirculation disorder was improved.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R657.51

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