牛眼小梁细胞体外高压培养模型的建立及中药滴明眼液有效部位配比研究

发布时间：2018-09-03 08:30

【摘要】：目的：(1)建立牛眼小梁细胞体外高压培养模型并观察压力对小梁细胞的影响。(2)利用体外培养细胞系，初步探讨将中药有效部位成分进行配比筛选的可行性。(3)观察中药滴明眼液有效部位配比(GD)对体外培养小梁细胞的影响。方法：(1)对牛眼小梁细胞(bovine trabecular meshwork cells，BTM cells)进行常压体外培养。(2)将装有BTM细胞的培养瓶(瓶内放置载玻片)密闭，分别以20mmHg、40mmHg、60mmHg、80mmHg加压24小时，再以80mmHg加压12、24、48小时，取出爬有细胞的载玻片，免疫组化法对肌动蛋白(actin)、微管蛋白(tubulin)染色，测其平均光密度值(AO值)。(3)BTM细胞密闭加压至80mmHg，24小时后收集细胞，透射电镜观察其超微结构。(4)在BTM细胞中加入不同浓度的GD和PBS液，继续培养24小时，用噻唑兰比色法(MTT法)，测定每组的光密度值(OD值)。(5)在BTM细胞中加入G4D6，其中一组密闭加压至80mmHg，另一组不加压，24小时后取出载玻片，行actin、tubulin染色，测其平均光密度值。(6)在BTM细胞中加入乳胶微粒及G4D6，其中一组密闭并加压至80mmHg，另一组不加压，，24小时后取出载玻片，测其吞噬微粒数。结果：(1)压力为40mmHg、60mmHg、80mmHg加压24小时，BTM细胞的actin、tubulin表达均有减弱；压力为80mmHg加压24、48小时后actin的表达减弱，12、24、48小时后tubulin的表达减弱。(2)80mmHg加压24小时，BTM细胞的超微结构受损。(3)在常压培养下，加入0.00016mg／ml、0.000032mg／ml的G8D2，0.5mg／ml的G7D3，0.5mg／ml的G5D5，2.5mg／ml、0.5mg／ml、0.1mg／ml、0.004mg／ml、0.0008mg／ml、0.00016mg／ml、0.000032mg／ml的G4D6，0.5mg／ml的G3D7，0.5mg／ml的G2D8，0.5mg／ml的G1D9对BTM细胞的存活有促进作用。(4)在BTM细胞以80mmHg培养24h时，浓度为0.5mg／ml的G4D6对其actin、tubulin表达较单纯加压组均有增强。(5)浓度为0.5mg／ml的G4D6对常压及加压下的BTM细胞吞噬乳胶微粒数无影响。结论：(1)BTM细胞能承受一定的压力，当压力继续升高时，可减弱其actin、tubulin的表达，并与压力的升高和加压时间的延长成正相关。(2)80mmHg加压24小时后超微结构明显受损。(3)利用体外培养细胞系，将中药有效部位成分进行
[Abstract]:Objective: (1) to establish a high pressure culture model of bovine trabecular meshwork cells in vitro and to observe the effect of pressure on trabecular meshwork cells. To explore the feasibility of screening the effective components of traditional Chinese medicine (TCM). (3) to observe the effect of (GD) on trabecular cells cultured in vitro. Methods: (1) bovine trabecular meshwork cells (bovine trabecular meshwork cells,BTM cells) were cultured under normal pressure. (2) the culture bottle containing BTM cells (glass slide was placed in the bottle) was sealed. The culture bottle was pressurized with 20mm HgG 40mmHg 60mmHg 80mmHg for 24 hours, and then 80mmHg was used to pressurize 12448 hours. The actin (actin), tubulin (tubulin) was stained by immunohistochemical method. The mean optical density (AO) of BTM cells (AO value). (3) was measured. The cells were collected after airtight compression to 80mm HgG for 24 hours. The ultrastructure of BTM cells was observed by transmission electron microscope. (4) GD and PBS solution of different concentrations were added to BTM cells. After 24 hours of culture, the optical density (OD). (5) of each group was measured by MTT method. G4D6 was added to BTM cells. One group was pressurized to 80mm Hg, the other group took out the slide after 24 hours without pressure, and was stained with actin,tubulin. (6) Emulsion particles and G _ 4D _ 6 were added to BTM cells. One group was sealed and pressurized to 80 mm Hg, the other group took out the slide after 24 hours without pressure, and measured the number of phagocytic particles. Results: (1) the expression of actin,tubulin in BTM cells was decreased at the pressure of 40mm HgG 60mmHg for 24 hours, the expression of actin decreased after 24h of 80mmHg pressure for 48 hours. (2) the ultrastructure of BTM cells was damaged after 24 hours of 80mmHg pressure. (3) the ultrastructure of BTM cells was damaged under normal pressure. Add 0.00016 mg / ml, 0.000032 mg / ml, G8D2, 0.5mg/ ml, G5D52.5mgpml, G5D52.5mg / ml, 0.5mg / ml, 0.1 mg / ml, 0.0008 mg / ml, 0.00016mggrml, 0.000032mgrml, G4D6O0.5 mg / ml, G2D80.5mgrml, G1D9 can promote the survival of BTM cells. (4) when BTM cells are incubated with 80mmHg for 24 hours, G4D6O0.5 mg / ml G2D80.5mgrml can promote the survival of BTM cells. (4) when BTM cells are cultured in 80mmHg for 24 hours, G4D6O 0.5 mg / ml G2D80.5 mg / ml can promote the survival of BTM cells. (4) when BTM cells are cultured in 80mmHg for 24 hours, (5) G4D6 with 0.5mg/ml concentration had no effect on the number of phagocytic particles of BTM cells under normal pressure and pressure. Conclusion: (1) BTM cells can withstand certain pressure, and when the pressure continues to increase, it can reduce the expression of actin,tubulin. There was a positive correlation between the increase of pressure and the prolongation of pressure time. (2) the ultrastructure of 80mmHg was obviously damaged after 24 hours of compression. (3) the active components of traditional Chinese medicine were obtained by culture of cell line in vitro.
【学位授予单位】：成都中医药大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R-332

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