【摘要】：目的：本實(shí)驗(yàn)通過檢測血管內(nèi)皮祖細(xì)胞（Endothelial progenitorcells，EPCs）移植到�；悄懰徕c誘導(dǎo)的大鼠急性胰腺炎模型的CD133、CD34免疫組化表達(dá)和HE染色血管密度的變化，研究血管內(nèi)皮細(xì)胞對大鼠急性胰腺炎微循環(huán)的改善作用。方法：1．細(xì)胞分離培養(yǎng)和鑒定：選取成年健康SD大鼠，取大鼠股骨、脛骨，PBS液沖洗骨髓腔，收集全部骨髓細(xì)胞，制成單核細(xì)胞懸液，加入到淋巴細(xì)胞分離液中，用密度梯度離心法離心，取中間白膜層的有核細(xì)胞，離心洗滌三次。利用差速貼壁法，取培養(yǎng)48h后未貼壁細(xì)胞，用含10％的胎牛血清、5ng／ml bFGF、20ng／ml VEGF的M199培養(yǎng)液培養(yǎng)于6孔板中。培養(yǎng)10d后,免疫組化法和熒光雙染法鑒定細(xì)胞。待細(xì)胞增殖到移植所需的數(shù)量時，收集細(xì)胞，無血清培養(yǎng)基洗滌兩次，用無血清培養(yǎng)基制成單細(xì)胞懸液，以備移植。2、大鼠急性胰腺炎模型的建立和EPCs的移植：將72只健康成年雄性大鼠隨機(jī)分為正常對照組（A組，n=24），急性胰腺炎模型組（B組,n=24）,急性胰腺炎內(nèi)皮祖細(xì)胞移植組（C組，n=24）。將SD大鼠用1%戊巴比妥鈉0.6ml/100g腹腔注射，于胰腺被膜下注射1%牛磺膽酸鈉1ml(正常對照組被膜下注射同等量生理鹽水)，同時行空腸造瘺，術(shù)后均給予腸外營養(yǎng)。C組通過大鼠頸外靜脈置管注射經(jīng)體外培養(yǎng)的內(nèi)皮祖細(xì)胞。各組于C組移植后第3d、7d、14d三個時間點(diǎn)各取6只大鼠處死并收集胰腺標(biāo)本甲醛固定，胰腺組織HE染色后光鏡病理學(xué)檢查，觀察胰腺病理形態(tài)變化，記錄血管密度情況；免疫組織化學(xué)法(SP法)檢測胰腺組織中CD133、CD34的累積光密度值（IOD值）。以上所收集的數(shù)據(jù)采用均數(shù)±標(biāo)準(zhǔn)差（x±s）表示，采用SPSS17.0統(tǒng)計軟件進(jìn)行統(tǒng)計學(xué)處理；樣本均數(shù)的比較采用單因素方差分析；兩者的相關(guān)性采用直線相關(guān)和直線回歸分析，，P0.05，則差異有統(tǒng)計學(xué)意義。結(jié)果：（1）B組（模型組）和C組（移植組）的病理評分在3d、7d、14d各時間點(diǎn)高于A（對照組）組（P0.05)。B組的病理評分在7d、14d點(diǎn)高于C組(P0.05)。(2)C組胰腺組織HE染色隨機(jī)選取鏡下微血管數(shù)在7d、14d高于A組(P0.05)，C組微血管數(shù)7d、14d高于B組(P0.05)。（3）C組胰腺組織CD133的IOD值在各時間點(diǎn)均高于A、B組(P0.05)。(4)C組胰腺組織CD34的IOD值在各時間點(diǎn)均高于A、B組(P0.05)。（5）胰腺組織病理評分與CD133的IOD值呈直線相關(guān)(r=0.814，P=0.000)。結(jié)論：（1）通過密度梯度離心結(jié)合差速貼壁法能夠從大鼠骨髓分離并培養(yǎng)得到的EPCs可以用于細(xì)胞移植。（2）培養(yǎng)的血管內(nèi)皮祖細(xì)胞可通過成功移植到急性胰腺炎的大鼠胰腺組織中。（3）移植的內(nèi)皮祖細(xì)胞能增加急性胰腺炎胰腺微血管的生成，從而使胰腺微循環(huán)障礙得到改善。
[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 鹵standard deviation (x 鹵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.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R657.51

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