【摘要】：目的：(1)建立牛眼小梁細(xì)胞體外高壓培養(yǎng)模型并觀察壓力對(duì)小梁細(xì)胞的影響。(2)利用體外培養(yǎng)細(xì)胞系，初步探討將中藥有效部位成分進(jìn)行配比篩選的可行性。(3)觀察中藥滴明眼液有效部位配比(GD)對(duì)體外培養(yǎng)小梁細(xì)胞的影響。 方法：(1)對(duì)牛眼小梁細(xì)胞(bovine trabecular meshwork cells，BTM cells)進(jìn)行常壓體外培養(yǎng)。(2)將裝有BTM細(xì)胞的培養(yǎng)瓶(瓶?jī)?nèi)放置載玻片)密閉，分別以20mmHg、40mmHg、60mmHg、80mmHg加壓24小時(shí)，再以80mmHg加壓12、24、48小時(shí)，取出爬有細(xì)胞的載玻片，免疫組化法對(duì)肌動(dòng)蛋白(actin)、微管蛋白(tubulin)染色，測(cè)其平均光密度值(AO值)。(3)BTM細(xì)胞密閉加壓至80mmHg，24小時(shí)后收集細(xì)胞，透射電鏡觀察其超微結(jié)構(gòu)。(4)在BTM細(xì)胞中加入不同濃度的GD和PBS液，繼續(xù)培養(yǎng)24小時(shí)，用噻唑蘭比色法(MTT法)，測(cè)定每組的光密度值(OD值)。(5)在BTM細(xì)胞中加入G4D6，其中一組密閉加壓至80mmHg，另一組不加壓，24小時(shí)后取出載玻片，行actin、tubulin染色，測(cè)其平均光密度值。(6)在BTM細(xì)胞中加入乳膠微粒及G4D6，其中一組密閉并加壓至80mmHg，另一組不加壓，，24小時(shí)后取出載玻片，測(cè)其吞噬微粒數(shù)。 結(jié)果：(1)壓力為40mmHg、60mmHg、80mmHg加壓24小時(shí)，BTM細(xì)胞的actin、tubulin表達(dá)均有減弱；壓力為80mmHg加壓24、48小時(shí)后actin的表達(dá)減弱，12、24、48小時(shí)后tubulin的表達(dá)減弱。(2)80mmHg加壓24小時(shí)，BTM細(xì)胞的超微結(jié)構(gòu)受損。(3)在常壓培養(yǎng)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對(duì)BTM細(xì)胞的存活有促進(jìn)作用。(4)在BTM細(xì)胞以80mmHg培養(yǎng)24h時(shí)，濃度為0.5mg／ml的G4D6對(duì)其actin、tubulin表達(dá)較單純加壓組均有增強(qiáng)。(5)濃度為0.5mg／ml的G4D6對(duì)常壓及加壓下的BTM細(xì)胞吞噬乳膠微粒數(shù)無(wú)影響。 結(jié)論：(1)BTM細(xì)胞能承受一定的壓力，當(dāng)壓力繼續(xù)升高時(shí)，可減弱其actin、tubulin的表達(dá)，并與壓力的升高和加壓時(shí)間的延長(zhǎng)成正相關(guān)。(2)80mmHg加壓24小時(shí)后超微結(jié)構(gòu)明顯受損。(3)利用體外培養(yǎng)細(xì)胞系，將中藥有效部位成分進(jìn)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.
【學(xué)位授予單位】：成都中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R-332

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