【摘要】：目的：(1)建立牛眼小梁細胞體外高壓培養(yǎng)模型并觀察壓力對小梁細胞的影響。(2)利用體外培養(yǎng)細胞系，初步探討將中藥有效部位成分進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)對體外培養(yǎng)小梁細胞的影響。 方法：(1)對牛眼小梁細胞(bovine trabecular meshwork cells，BTM cells)進行常壓體外培養(yǎng)。(2)將裝有BTM細胞的培養(yǎng)瓶(瓶內(nèi)放置載玻片)密閉，分別以20mmHg、40mmHg、60mmHg、80mmHg加壓24小時，再以80mmHg加壓12、24、48小時，取出爬有細胞的載玻片，免疫組化法對肌動蛋白(actin)、微管蛋白(tubulin)染色，測其平均光密度值(AO值)。(3)BTM細胞密閉加壓至80mmHg，24小時后收集細胞，透射電鏡觀察其超微結(jié)構(gòu)。(4)在BTM細胞中加入不同濃度的GD和PBS液，繼續(xù)培養(yǎng)24小時，用噻唑蘭比色法(MTT法)，測定每組的光密度值(OD值)。(5)在BTM細胞中加入G4D6，其中一組密閉加壓至80mmHg，另一組不加壓，24小時后取出載玻片，行actin、tubulin染色，測其平均光密度值。(6)在BTM細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小時后取出載玻片，測其吞噬微粒數(shù)。 結(jié)果：(1)壓力為40mmHg、60mmHg、80mmHg加壓24小時，BTM細胞的actin、tubulin表達均有減弱；壓力為80mmHg加壓24、48小時后actin的表達減弱，12、24、48小時后tubulin的表達減弱。(2)80mmHg加壓24小時，BTM細胞的超微結(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BTM細胞的存活有促進作用。(4)在BTM細胞以80mmHg培養(yǎng)24h時，濃度為0.5mg／ml的G4D6對其actin、tubulin表達較單純加壓組均有增強。(5)濃度為0.5mg／ml的G4D6對常壓及加壓下的BTM細胞吞噬乳膠微粒數(shù)無影響。 結(jié)論：(1)BTM細胞能承受一定的壓力，當壓力繼續(xù)升高時，可減弱其actin、tubulin的表達，并與壓力的升高和加壓時間的延長成正相關(guān)。(2)80mmHg加壓24小時后超微結(jié)構(gòu)明顯受損。(3)利用體外培養(yǎng)細胞系，將中藥有效部位成分進行
[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.
【學位授予單位】：成都中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R-332

【參考文獻】

相關(guān)期刊論文 前10條

1 彭清華，曾自明，李偉力;活血利水治療慢性單純性青光眼31例[J];遼寧中醫(yī)雜志;1995年04期

2 黃慶山，朱應文，馬緒風，周光，蘇伯平;益氣活血法治療慢性單純性青光眼臨床研究[J];中國中醫(yī)眼科雜志;1994年02期

3 賀義恒，唐由之，高健生;青光眼四號對原發(fā)性開角型青光眼降眼壓的臨床研究[J];中國中醫(yī)眼科雜志;1994年01期

4 賀義恒,唐由之,高健生,陳惠民;青光眼四號對原發(fā)性開角型青光眼視功能影響的臨床研究[J];中國中醫(yī)眼科雜志;2000年02期

5 徐新榮,蔡豐英;葛根素對慢性高眼壓兔眼視神經(jīng)軸漿流影響的實驗研究[J];中國中醫(yī)眼科雜志;1997年01期

6 張文強,楊新光,李養(yǎng)軍;消化法牛眼小梁細胞的體外培養(yǎng)[J];眼科研究;2003年04期

7 林明楷,葛堅;青光眼住院病人的構(gòu)成比變化特點[J];眼科學報;1997年02期

8 沈鳳梅,馮學峰,張德秀,劉濤,劉菲;體外培養(yǎng)的牛眼小梁細胞及微管微絲的形態(tài)學觀察[J];眼視光學雜志;2002年02期

9 劉耀輝;疏通散煎劑治療原發(fā)性開角型青光眼[J];眼視光學雜志;1998年03期

10 薛蔚!430030武漢,杜蜀華!430030武漢,李勇,黃愷;壓力對牛眼小梁細胞誘導型一氧化氮合酶mRNA的表達及蛋白質(zhì)合成的影響[J];中華眼科雜志;2000年04期

相關(guān)碩士學位論文 前2條

1 張文強;壓力及BDNF對體外培養(yǎng)的牛眼小梁細胞的影響[D];中國人民解放軍第四軍醫(yī)大學;2003年

2 曾潔萍;DSX及其成分對體外培養(yǎng)鼠視網(wǎng)膜神經(jīng)節(jié)細胞影響的實驗研究[D];成都中醫(yī)藥大學;2003年

本文編號：2219441