本文選題：細(xì)胞培養(yǎng) + 小梁網(wǎng)細(xì)胞��； 參考：《福建醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的在體外培養(yǎng)人眼小梁網(wǎng)細(xì)胞的基礎(chǔ)上，研究myocilin蛋白對(duì)人眼小梁網(wǎng)細(xì)胞纖維連接蛋白(fibronectin，F(xiàn)N)表達(dá)以及對(duì)小梁網(wǎng)細(xì)胞遷移、粘附功能的影響，探索myocilin蛋白與原發(fā)性開(kāi)角型青光眼發(fā)病的關(guān)系。 方法采用組織塊培養(yǎng)法體外培養(yǎng)正常人眼小梁網(wǎng)細(xì)胞，并運(yùn)用倒置顯微鏡觀察、透射電鏡和免疫細(xì)胞化學(xué)等方法對(duì)細(xì)胞進(jìn)行鑒定。取傳三代的小梁網(wǎng)細(xì)胞分別加入含重組myocilin蛋白終濃度為0ug/m（l對(duì)照組）、0.5ug/ml、1ug/ml、1.5ug/ml、2ug/ml、2.5ug/ml的無(wú)血清培養(yǎng)基，繼續(xù)培養(yǎng)24小時(shí)后，運(yùn)用Western blot、Elisa法分別檢測(cè)小梁網(wǎng)細(xì)胞內(nèi)及細(xì)胞培養(yǎng)液中FN的表達(dá)水平；運(yùn)用Transwell小室法、CCK-8法檢測(cè)myocilin蛋白對(duì)小梁網(wǎng)細(xì)胞遷移、粘附的影響。 結(jié)果1.成功建立人眼小梁網(wǎng)細(xì)胞體外培養(yǎng)體系，經(jīng)鑒定確定為人眼小梁網(wǎng)細(xì)胞。 2. Western blot檢測(cè)結(jié)果顯示，在一定范圍內(nèi)，隨myocilin蛋白濃度的增高小梁網(wǎng)細(xì)胞內(nèi)FN表達(dá)呈下降趨勢(shì)。3. Elisa法檢測(cè)經(jīng)濃度為0ug/ml、0.5ug/ml、1ug/ml、1.5ug/ml、2ug/ml、2.5ug/ml myocilin蛋白干預(yù)的各組細(xì)胞培養(yǎng)液中FN濃度為：0.5817±0.0959ug/ml、0.5572±0.0443ug/ml、0.5868±0.0735ug/ml、0.5015±0.0472ug/ml、0.4263±0.0426ug/ml、0.2838±0.0972ug/ml。1.5ug/ml、2ug/ml、2.5ug/ml myocilin蛋白組與對(duì)照組的差異有統(tǒng)計(jì)學(xué)意義(P0.05)，，且此三組實(shí)驗(yàn)組組間比較存在顯著性差異。4.Transwell小室法檢測(cè)結(jié)果顯示，實(shí)驗(yàn)組細(xì)胞遷移數(shù)較對(duì)照組少，且實(shí)驗(yàn)組與對(duì)照組的差異有統(tǒng)計(jì)學(xué)意義(P0.05）。5.運(yùn)用CCK-8法檢測(cè)經(jīng)濃度為0ug/ml、0.5ug/ml、1ug/ml、1.5ug/ml、2ug/ml、2.5ug/mlmyocilin蛋白處理的各組細(xì)胞吸光度值的均值分別為0.1614±0.0061、0.1529±0.0139、0.1273±0.0172、0.1271±0.0230、0.1165±0.0084、0.1103±0.0045。隨myocilin蛋白濃度的增高各組細(xì)胞吸光度值呈現(xiàn)下降趨勢(shì)，各實(shí)驗(yàn)組與對(duì)照組相比差異有統(tǒng)計(jì)學(xué)意義（P0.05）。 結(jié)論1.myocilin蛋白能抑制小梁網(wǎng)細(xì)胞纖維連接蛋白的表達(dá)，并呈現(xiàn)一定的劑量依耐性；2.myocilin蛋白可降低小梁網(wǎng)細(xì)胞的遷移和粘附能力。
[Abstract]:Objective to study the effects of myocilin protein on the expression of fibronectin FN and the migration and adhesion of human trabecular meshwork cells in vitro. To explore the relationship between myocilin protein and primary open angle glaucoma. Methods normal human trabecular meshwork cells were cultured in vitro by tissue mass culture, and the cells were identified by inverted microscope, transmission electron microscope and immunocytochemistry. The third generation trabecular meshwork cells were added to the serum-free medium containing the final concentration of recombinant myocilin protein as the 0ug/m(l control group (0ug/m(l control group). After 24 hours of culture, the expression of FN in trabecular meshwork cells and cell culture medium was detected by Western blottimerassay. The effect of myocilin protein on the migration and adhesion of trabecular meshwork cells was detected by Transwell chamber assay and CCK-8 method. Result 1. The in vitro culture system of human trabecular meshwork cells was successfully established and identified as human trabecular meshwork cells. 2. The results of Western blot showed that the expression of FN in trabecular meshwork cells decreased with the increase of myocilin protein concentration in a certain range. Elisa娉曟嫻嬬粡嫻撳害涓

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