發(fā)布時(shí)間：2018-03-16 01:00

  本文選題：大麻素WIN55　切入點(diǎn)：212-2　出處：《山西醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：研究大麻素WIN55,212-2對(duì)體外培養(yǎng)的牛眼小梁細(xì)胞MMP-3、MMP-9及TIMP-1表達(dá)的影響，從而探討其降眼壓的機(jī)制。 方法：1.牛眼小梁細(xì)胞的培養(yǎng)和鑒定：應(yīng)用組織塊培養(yǎng)法對(duì)牛眼小梁細(xì)胞進(jìn)行原代培養(yǎng)，傳代培養(yǎng)，采用免疫組織化學(xué)方法對(duì)第3代細(xì)胞（波形蛋白，NSE，VIII因子相關(guān)抗原染色）進(jìn)行鑒定，應(yīng)用透射電鏡對(duì)細(xì)胞進(jìn)行生長(zhǎng)特性及形態(tài)學(xué)的觀察,確定所得細(xì)胞大部分為小梁細(xì)胞組織來源；2.免疫組化SP染色法測(cè)定MMP-3,MMP-9的量：將經(jīng)過消化離心后的傳3代的小梁細(xì)胞接種于孔底鋪有蓋玻片的6孔培養(yǎng)板（密度：5×104個(gè)/mL），當(dāng)細(xì)胞鋪滿大部分孔底時(shí)，改換培養(yǎng)液（不含F(xiàn)BS）進(jìn)行饑餓培養(yǎng)（48h），后隨機(jī)分為6個(gè)組（1孔/組）。1~5組施加含終濃度0（對(duì)照組）、1、10、20、40μmol/L大麻素WIN55,212-2的無血清培養(yǎng)液，第6組為陰性對(duì)照組（以PBS液為一抗）。繼續(xù)培養(yǎng)48h后對(duì)各組細(xì)胞爬片進(jìn)行免疫細(xì)胞化學(xué)染色（MMP-3及MMP-9），重復(fù)該實(shí)驗(yàn)共4次。結(jié)果進(jìn)行計(jì)算機(jī)圖像分析并進(jìn)行統(tǒng)計(jì)學(xué)檢驗(yàn)。3.提取細(xì)胞上清液用ELASA法檢測(cè)隨濃度的不同TIMP-1量的變化：將經(jīng)消化離心后的傳3代小梁細(xì)胞接種于96孔板（密度：5×104個(gè)/mL），當(dāng)細(xì)胞鋪滿大部分孔底時(shí)，改換培養(yǎng)液（不含F(xiàn)BS）進(jìn)行饑餓培養(yǎng)（48h），，以使所有細(xì)胞同步。隨后將其隨機(jī)分為5組（15孔/組），施加含終濃度0（對(duì)照組）、1、10、20、40μmol/L大麻素WIN55,212-2的無血清培養(yǎng)液，繼續(xù)培養(yǎng)48h。后分組吸出各孔上清液置于EP管中，-20℃冰箱保存，應(yīng)用ELASA法檢測(cè)隨濃度的不同TIMP-1量的變化情況。 結(jié)果：體外培養(yǎng)的牛眼小梁細(xì)胞表達(dá)MMP-3及MMP-9；含大麻素WIN55,212-2終濃度為1、10、20、40μmol/L的無血清培養(yǎng)液可促進(jìn)牛眼小梁細(xì)胞MMP-3及MMP-9的表達(dá)，且與濃度的呈正相關(guān)性，并抑制TIMP-1的表達(dá)，與濃度呈負(fù)相關(guān)性。 結(jié)論：一定劑量的大麻素可以促進(jìn)牛眼小梁細(xì)胞MMP-3及MMP-9的表達(dá)（P0.05），并抑制TIMP-1的表達(dá)（P0.01），從而達(dá)到降低眼壓的目的。
[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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