大麻素WIN55，212-2对体外培养牛眼小梁细胞MMP-3，MMP-9及TIMP-1表达的影响

发布时间：2018-03-16 01:00

  本文选题：大麻素WIN55　切入点：212-2　出处：《山西医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：研究大麻素WIN55,212-2对体外培养的牛眼小梁细胞MMP-3、MMP-9及TIMP-1表达的影响，从而探讨其降眼压的机制。 方法：1.牛眼小梁细胞的培养和鉴定：应用组织块培养法对牛眼小梁细胞进行原代培养，传代培养，采用免疫组织化学方法对第3代细胞（波形蛋白，NSE，VIII因子相关抗原染色）进行鉴定，应用透射电镜对细胞进行生长特性及形态学的观察,确定所得细胞大部分为小梁细胞组织来源；2.免疫组化SP染色法测定MMP-3,MMP-9的量：将经过消化离心后的传3代的小梁细胞接种于孔底铺有盖玻片的6孔培养板（密度：5×104个/mL），当细胞铺满大部分孔底时，改换培养液（不含FBS）进行饥饿培养（48h），后随机分为6个组（1孔/组）。1~5组施加含终浓度0（对照组）、1、10、20、40μmol/L大麻素WIN55,212-2的无血清培养液，第6组为阴性对照组（以PBS液为一抗）。继续培养48h后对各组细胞爬片进行免疫细胞化学染色（MMP-3及MMP-9），重复该实验共4次。结果进行计算机图像分析并进行统计学检验。3.提取细胞上清液用ELASA法检测随浓度的不同TIMP-1量的变化：将经消化离心后的传3代小梁细胞接种于96孔板（密度：5×104个/mL），当细胞铺满大部分孔底时，改换培养液（不含FBS）进行饥饿培养（48h），，以使所有细胞同步。随后将其随机分为5组（15孔/组），施加含终浓度0（对照组）、1、10、20、40μmol/L大麻素WIN55,212-2的无血清培养液，继续培养48h。后分组吸出各孔上清液置于EP管中，-20℃冰箱保存，应用ELASA法检测随浓度的不同TIMP-1量的变化情况。 结果：体外培养的牛眼小梁细胞表达MMP-3及MMP-9；含大麻素WIN55,212-2终浓度为1、10、20、40μmol/L的无血清培养液可促进牛眼小梁细胞MMP-3及MMP-9的表达，且与浓度的呈正相关性，并抑制TIMP-1的表达，与浓度呈负相关性。 结论：一定剂量的大麻素可以促进牛眼小梁细胞MMP-3及MMP-9的表达（P0.05），并抑制TIMP-1的表达（P0.01），从而达到降低眼压的目的。
[Abstract]:Aim: to investigate the effect of cannabinoid WIN55-212-2 on the expression of MMP-3, MMP-9 and TIMP-1 in cultured bovine trabecular meshwork cells, and to explore the mechanism of lowering intraocular pressure (IOP). Methods 1. Culture and identification of bovine trabecular cells: primary culture and subculture of bovine trabecular cells were carried out by tissue mass culture. The third generation of cells (vimentin NSEG factor VIII related antigen staining) were identified by immunohistochemical method. The growth characteristics and morphology of the cells were observed by transmission electron microscope (TEM). Immunohistochemical SP staining method was used to determine the quantity of MMP-3 and MMP-9: after digestion and centrifugation, the third generation trabecular cells were inoculated into a 6-well culture plate with covered glass on the bottom of the hole (density: 5 脳 104). When the cells are covered with the bottom of most of the holes, After 48 hours of starvation culture, the rats were randomly divided into 6 groups (n = 6) and treated with a serum-free medium containing a final concentration of 0 (control group) (control group, n = 10, n = 10, 2040 渭 mol/L) and cannabinoid (WIN55N, 212-2, n = 6). The sixth group was a negative control group (using PBS solution as the first anti). After 48 hours of culture, the cells in each group were stained with MMP-3 and MMP-9 for 4 times. The results were analyzed by computer image and analyzed statistically. ELASA method was used to detect the changes of TIMP-1 with different concentrations: trabecular meshwork cells were inoculated with 96-well plate (density: 5 脳 104 / mL) after digestion and centrifugation, when the cells were covered with the bottom of most of the holes. Culture medium (not containing FBS) was changed for 48h to synchronize all cells. Then, the cells were randomly divided into 5 groups with 15 holes per cell / group and serum-free medium containing 0 (control group: 1 10 mol/L) and cannabinoid 212-2 (WIN55H212-2). After 48 hours of culture, the supernatant of each hole was sucked out and stored in EP tube at -20 鈩

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