本文選題：增生性玻璃體視網(wǎng)膜病變 + 視網(wǎng)膜色素上皮細胞��； 參考：《吉林大學(xué)》2012年碩士論文

【摘要】：研究表明,視網(wǎng)膜色素上皮(retinal pigment epithelium, RPE)細胞是增生性玻璃體視網(wǎng)膜病變(proliferation vitreoretinopathy, PVR)的增殖膜中的主要細胞來源,因此增殖性玻璃體視網(wǎng)膜病變的研究和防治多圍繞探討RPE細胞的異常行為和抑制RPE細胞及其他相關(guān)細胞的增殖。雷帕霉素(rapamycin, RAPA)是一種新型的大環(huán)內(nèi)酯類免疫抑制劑,眼科方面已有研究應(yīng)用于動物及體外細胞培養(yǎng)藥物實驗。 目的： 本研究是在體外培養(yǎng)人視網(wǎng)膜色素上皮(human retinal pigment epithelium, hRPE)細胞的基礎(chǔ)之上,探討一定范圍濃度的RAPA對體外培養(yǎng)的hRPE細胞形態(tài)、增殖等生物學(xué)活性的影響。 方法： 復(fù)蘇并體外培養(yǎng)hRPE細胞并分為不同濃度雷帕霉素實驗組(Ong/ml,5ng/ml,10ng/ml,20ng/ml,40ng/ml,80ng/ml)。顯微鏡下觀察各組細胞及藥物不同作用時間下,細胞的增殖狀態(tài)、倍增時間、細胞活力、生長曲線及四唑鹽(MTT)比色法檢RAPA對hRPE細胞增殖的影響。 結(jié)果： 復(fù)蘇后細胞活力良好的hRPE細胞株,雷帕霉素在實驗范圍內(nèi)抑制hRPE細胞的增殖。鏡下可見隨雷帕霉素濃度增加,細胞數(shù)目逐漸減少。經(jīng)80ng/ml雷帕霉素作用72小時后部分細胞變形崩解,偶見細胞浮起。對照組細胞分裂時間為1.83天,隨RAPA藥物濃度增加細胞分裂時間隨之延長。生長曲線顯示隨著雷帕霉素濃度的增加,作用時間的延長,hRPE細胞的生長有明顯的抑制作用,并呈劑量時間效應(yīng)關(guān)系。通過生長曲線可見對照組細胞增殖旺盛,各項生長指標均高于用藥組。臺盼蘭細胞活力測定結(jié)果為不同濃度雷帕霉素(Ong/ml,5ng/ml,1Ong/ml,20ng/ml,40ng/ml,80ng/ml)細胞活力分別為92.3%、89.3%、88.7%、87.7%、86.0%、82.6%。MTT比色法觀察細胞生長基本規(guī)律結(jié)果示：各濃度藥物組作用24小時后與對照組比較計算得抑制率,分別為8.70%、22.06%、40.69%、42.31%、43.12%；72小時時分別為12.57%、26.35%、42.23%44.15%、47.12%。 結(jié)論： 1雷帕霉素對體外培養(yǎng)的hRPE細胞增殖具有有效抑制作用,其效應(yīng)在一定范圍內(nèi)呈濃度/時間依賴性。10～80ng/ml濃度范圍內(nèi),RAPA明顯抑制hRPE細胞增殖(P0.01)；但在20～80ng/ml濃度范圍內(nèi),各組間抑制作用無顯著性差異(P0.05)。 2雷帕霉素延長hRPE細胞倍增時間,降低體外培養(yǎng)的hRPE細胞活性。 3雷帕霉素在80ng/ml時細胞抑制率近50%。 4雷帕霉素可能為臨床預(yù)防和治療PVR提供新思路,提升其在眼科的應(yīng)用價值。
[Abstract]:The results show that retinal pigment epithelium (RPE) cells are the main cell sources in proliferative membrane of proliferative vitreoretinopathy (PVR) of proliferative vitreoretinopathy (PVR). Therefore, the study and prevention of proliferative vitreoretinopathy focus on the study of abnormal behavior of RPE cells and inhibition of proliferation of RPE cells and other related cells. Rapamycin (rapamycin) is a new type of macrolide immunosuppressant, which has been used in animal and in vitro cell culture experiments in ophthalmology. Objective: Based on the culture of human retinal pigment epithelium (RPE) retinal pigment epithelium, hRPE) cells in vitro, the effects of certain concentration of RAPA on the morphology and proliferation of hRPE cells in vitro were investigated. Methods: HRPE cells were resuscitated and cultured in vitro and divided into different concentrations of rapamycin experimental group: 5 ng / ml 10 ng / ml 10 ng / ml 20 ng / ml 40 ng / ml / ml 80 ng / ml / ml. The effects of RAPA on the proliferation of hRPE cells were observed under microscope. The proliferation state, doubling time, cell viability, growth curve and tetrazolium trioxide (MTT) colorimetric assay were used to detect the effect of RAPA on the proliferation of hRPE cells. Results: After resuscitation, rapamycin inhibited the proliferation of hRPE cells. Microscopically, the number of cells decreased with the increase of rapamycin concentration. After 72 hours of treatment with 80ng/ml rapamycin, some of the cells were deformed and disintegrated, and occasionally the cells floated. The cell division time of the control group was 1.83 days, and the cell division time was prolonged with the increase of RAPA concentration. The growth curve showed that with the increase of rapamycin concentration, the growth of hRPE cells was significantly inhibited with the prolongation of the action time, and showed a dose-time effect. The growth curve showed that the growth index of the control group was higher than that of the drug group. Trypan blue cell viability assay showed that the cell viability of different concentrations of rapamycin Ong / ml 5 ng / ml 1 Ong / ml + 20 ng / ml 20 ng / ml + 20 ng / ml + 40ng / ml + 80ng / ml) was 92.3% and 89.3% respectively. The cell growth was observed by MTT colorimetric method. The results showed that the inhibition rate was calculated after 24 hours of treatment in each concentration group as compared with the control group. 42.31 and 43.12 for 72 hours, 12.57 and 26.35, 42.23D.15 and 47.12. respectively, with 8.70 and 22.06 and 40.69, and 42.31 and 43.12, respectively, with 12.57 and 26.35, and 42.23D.15 and 47.12, respectively. Conclusion: 1 rapamycin had an effective inhibitory effect on the proliferation of hRPE cells cultured in vitro, and its effect was in a dose-dependent / time-dependent manner. Rapa significantly inhibited the proliferation of hRPE cells in a concentration range of 80 ng / ml, but in the range of 20~80ng/ml concentration, rapamycin inhibited the proliferation of hRPE cells in a dose-dependent manner, but in the range of 20~80ng/ml concentration, rapamycin inhibited the proliferation of hRPE cells in a dose-dependent manner. There was no significant difference in the inhibitory effect among the three groups (P 0.05). 2 rapamycin prolonged the doubling time of hRPE cells and decreased the activity of hRPE cells cultured in vitro. 3 the inhibitory rate of rapamycin on 80ng/ml was nearly 50%. Rapamycin may provide a new idea for clinical prevention and treatment of PVR, and enhance its application value in ophthalmology.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R774.1

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