【摘要】：目的：通過腺相關(guān)病毒.(AAV)載體介導(dǎo)人β-神經(jīng)生長因子(β-NGF)基因和人血小板源性生長因子基因B(PDGF-B)聯(lián)合轉(zhuǎn)染體外培養(yǎng)的貓角膜內(nèi)皮細(xì)胞,探討其對該細(xì)胞生物學(xué)效應(yīng)的影響。 方法：體外培養(yǎng)的貓角膜內(nèi)皮細(xì)胞,通過細(xì)胞形態(tài)學(xué)觀察、神經(jīng)特異烯醇化酶(NSE)單克隆抗體免疫熒光染色鑒定細(xì)胞的種類及純度。構(gòu)建AAV載體,利用AAV載體介導(dǎo)綠色熒光蛋白(GFP)基因轉(zhuǎn)染貓角膜內(nèi)皮細(xì)胞,根據(jù)其陽性表達(dá)率測定轉(zhuǎn)染效率。AAV-β-NGF和AAV-PDGF-B分別和聯(lián)合轉(zhuǎn)染貓角膜內(nèi)皮細(xì)胞后,Real-Time PCR及Western blot分別在mRNA和蛋白水平檢測轉(zhuǎn)染24h、48h、72h后β-NGF和PDGF-B的表達(dá)變化。AAV-β-NGF和AAV-PDGF-B分別和聯(lián)合轉(zhuǎn)染貓角膜內(nèi)皮細(xì)胞72小時后,通過MTT檢測細(xì)胞增殖能力的改變,流式細(xì)胞儀測定處于G1期的細(xì)胞比例,檢測外源基因?qū)?xì)胞周期的影響。劃痕實驗觀察AAV-β-NGF和AAV-PDGF-B對細(xì)胞遷移能力的影響。 結(jié)果：貓角膜內(nèi)皮細(xì)胞體外培養(yǎng)可形成完整的細(xì)胞單層,經(jīng)形態(tài)學(xué)觀察、NSE免疫熒光染色證明為純凈的貓角膜內(nèi)皮細(xì)胞。AAV介導(dǎo)的綠色熒光蛋白基因轉(zhuǎn)染貓角膜內(nèi)皮細(xì)胞72h后,熒光顯微鏡下觀察可見細(xì)胞內(nèi)清晰的綠色熒光,測得轉(zhuǎn)染效率可達(dá)67.8%。AAV-β-NGF和AAV-PDGF-B分別和聯(lián)合轉(zhuǎn)染貓內(nèi)皮細(xì)胞后,Real-Time PCR和Western blot結(jié)果顯示,轉(zhuǎn)染后各時間點貓角膜內(nèi)皮細(xì)胞中β-NGF和PDGF-B的mRNA和蛋白表達(dá)隨時間延長不斷增高,與對照組相比差異有統(tǒng)計學(xué)意義(P0.05)。聯(lián)合轉(zhuǎn)染組中β-NGF和PDGF-B在不同時間點的表達(dá)與單獨轉(zhuǎn)染組差別無統(tǒng)計學(xué)意義(P0.05)。MTT結(jié)果顯示,AAV-β-NGF和AAV-PDGF-B分別和聯(lián)合轉(zhuǎn)染貓角膜內(nèi)皮細(xì)胞后增殖能力較對照組增強,聯(lián)合轉(zhuǎn)染組更為明顯,差別均有統(tǒng)計學(xué)意義(P0.05)。流式細(xì)胞儀檢測AAV-β-NGF和AAV-PDGF-B轉(zhuǎn)染組細(xì)胞周期中G1期細(xì)胞比例增加,聯(lián)合轉(zhuǎn)染組增加更為明顯。劃痕實驗表明,AAV-β-NGF和AAV-PDGF-B轉(zhuǎn)染后可以增強細(xì)胞遷徙能力,聯(lián)合轉(zhuǎn)染組效果更為明顯,差異具有統(tǒng)計學(xué)意義(P0.05) 結(jié)論：AAV可有效介導(dǎo)人β-NGF基因和PDGF-B基因轉(zhuǎn)染體外培養(yǎng)的貓角膜內(nèi)皮細(xì)胞,并在貓角膜內(nèi)皮細(xì)胞中持續(xù)穩(wěn)定表達(dá)。AAV-β-NGF和AAV-PDGF-B分別和聯(lián)合轉(zhuǎn)染貓角膜內(nèi)皮細(xì)胞后可以增強細(xì)胞的增殖和遷移能力,聯(lián)合轉(zhuǎn)染的效果更加明顯。
[Abstract]:Objective: to co-transfect human 尾-nerve growth factor (尾-NGF) gene and human platelet-derived growth factor B (PDGF-B) gene into cat corneal endothelial cells by adeno-associated virus. (AAV) vector. To investigate the biological effects of these cells. Methods: cat corneal endothelial cells were cultured in vitro. The types and purity of cultured cat corneal endothelial cells were identified by morphological observation and immunofluorescence staining with monoclonal antibody against neuro-specific enolase (NSE). AAV vector was constructed and transfected into cat corneal endothelial cells by AAV vector mediated green fluorescent protein (GFP) gene. The transfection efficiency was measured according to its positive expression rate. AAV-尾-NGF and AAV-PDGF-B were transfected into cat corneal endothelial cells respectively and co-transfected with AAV-尾-NGF and AAV-PDGF-B. The expression of 尾-NGF and PDGF-B were detected at mRNA and protein levels for 24 h, 48 h and 72 h after transfection of Real-Time PCR and Western blot, respectively. AAV-尾-NGF and AAV-PDGF-B were transfected into cat corneal endothelial cells for 72 hours, respectively. The cell proliferation ability was detected by MTT, the proportion of cells in G1 phase was measured by flow cytometry, and the effect of exogenous genes on cell cycle was detected. The effects of AAV- 尾-NGF and AAV-PDGF-B on cell migration were observed by scratch assay. Results: cat corneal endothelial cells were cultured in vitro to form intact monolayers. Morphological observation and NSE immunofluorescence staining showed that they were pure cat corneal endothelial cells. AAV-mediated green fluorescent protein gene was transfected into cat corneal endothelial cells for 72 hours. A clear green fluorescence was observed under fluorescence microscope, and the transfection efficiency was 67.8%. After AAV-尾-NGF and AAV-PDGF-B were co-transfected with cat endothelial cells, the results of Real-Time PCR and Western blot showed that the transfection efficiency was 67.8%. The expression of mRNA and protein of 尾-NGF and PDGF-B in cat corneal endothelial cells increased with time after transfection, and the difference was statistically significant compared with the control group (P0.05). There was no significant difference in the expression of 尾-NGF and PDGF-B between the combined transfection group and the single transfection group at different time points (P0.05). The proliferation of cat corneal endothelial cells transfected with AAV- 尾-NGF and AAV-PDGF-B was significantly higher than that of the control group, and the difference was statistically significant (P0.05). The proportion of G1 phase cells in cell cycle of AAV- 尾-NGF and AAV-PDGF-B transfected group was increased by flow cytometry, especially in combined transfection group. Scratch test showed that AAV- 尾-NGF and AAV-PDGF-B could enhance the ability of cell migration after transfection, and the effect of combined transfection group was more obvious. The difference was statistically significant (P0.05) conclusion: AAV can effectively mediate the transfection of human 尾-NGF gene and PDGF-B gene into cat corneal endothelial cells in vitro. AAV-尾-NGF and AAV-PDGF-B could enhance the proliferation and migration of cat corneal endothelial cells, and the co-transfection of AAV-尾-AAV and AAV-尾-AAV could enhance the proliferation and migration of cat corneal endothelial cells.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R77

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