本文關(guān)鍵詞： 豬 血管內(nèi)皮細(xì)胞 細(xì)胞鑒定 細(xì)胞體外培養(yǎng) 端粒酶 PPARγ　出處：《湖南農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：對(duì)豬臍靜脈血管內(nèi)皮細(xì)胞進(jìn)行體外培養(yǎng)并使其永生化,進(jìn)而為研究過氧化物酶體增殖物激活受體PPARγ對(duì)豬胎盤及胎盤血管發(fā)生的影響提供良好的細(xì)胞模型。方法：采用膠原酶對(duì)新鮮仔豬臍靜脈血管內(nèi)皮細(xì)胞進(jìn)行消化分離,獲得原代細(xì)胞并在CO2培養(yǎng)箱條件下進(jìn)行體外培養(yǎng)；顯微鏡觀察細(xì)胞生長(zhǎng)的形態(tài)學(xué)變化,免疫組化法進(jìn)行細(xì)胞鑒定；利用ICELLigence細(xì)胞功能分析儀監(jiān)測(cè)體外培養(yǎng)細(xì)胞的生長(zhǎng)曲線變化,并采用流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期和凋亡；采用端粒酶轉(zhuǎn)入的方法進(jìn)行體外培養(yǎng)豬臍靜脈血管內(nèi)皮細(xì)胞的永生化處理,并用G418藥物篩選陽(yáng)性單克隆細(xì)胞；利用細(xì)胞功能分析儀監(jiān)測(cè)激動(dòng)劑和抑制劑對(duì)該細(xì)胞增殖能力的影響。結(jié)果：(1)原代細(xì)胞生長(zhǎng)狀態(tài)良好,呈小三角形、橢圓形、梭形、多邊形單層生長(zhǎng),界限清晰,融合后呈鋪路石狀排列,細(xì)胞增殖能力強(qiáng)。多次傳代后細(xì)胞增殖能力減弱,細(xì)胞活力差,凋亡細(xì)胞增多。兔抗人第八因子相關(guān)抗原(FⅧAg)抗體和兔抗人CD31多克隆抗體鑒定均呈陽(yáng)性。(2)原代細(xì)胞轉(zhuǎn)入端粒酶后,轉(zhuǎn)染效檢證明端粒酶成功轉(zhuǎn)入細(xì)胞內(nèi)；轉(zhuǎn)染細(xì)胞藥篩14d后獲得單克隆細(xì)胞,并能夠增殖傳代,細(xì)胞狀態(tài)良好。(3)當(dāng)激動(dòng)劑濃度為5μMol/ml時(shí),可以促進(jìn)PPARγ蛋白的表達(dá),加速原代細(xì)胞和單克隆細(xì)胞的生長(zhǎng)增殖,增強(qiáng)細(xì)胞活力；當(dāng)抑制劑濃度為20μMol/ml時(shí),明顯抑制PPARγ蛋白的表達(dá),從而抑制細(xì)胞的生長(zhǎng)增殖,降低細(xì)胞活力。結(jié)論：本研究由豬臍靜脈血管分離獲得的原代培養(yǎng)細(xì)胞,經(jīng)體外培養(yǎng)觀察和細(xì)胞特征性標(biāo)志物檢測(cè),符合血管內(nèi)皮細(xì)胞的基本特征；原代培養(yǎng)細(xì)胞經(jīng)端粒酶轉(zhuǎn)染和藥篩處理獲得轉(zhuǎn)染陽(yáng)性單克隆細(xì)胞,并表現(xiàn)出良好的生長(zhǎng)增殖能力；使用PPARγ激動(dòng)劑或抑制劑藥物干預(yù),原代培養(yǎng)細(xì)胞和轉(zhuǎn)染的單克隆細(xì)胞均表現(xiàn)出類似的促進(jìn)或抑制細(xì)胞生長(zhǎng)的反應(yīng)。
[Abstract]:Objective: to culture porcine umbilical vein endothelial cells in vitro and immortalize them. So as to provide a good cell model to study the effect of peroxisome proliferator activated receptor (PPAR 緯) on placenta and placental angiogenesis. Methods: fresh umbilical vein endothelial cells were digested and separated by collagenase. Primary cells were obtained and cultured in vitro in CO2 incubator; morphological changes of cell growth were observed under microscope and identified by immunohistochemical method; growth curves of cultured cells were monitored by ICELLigence cell function analyzer. Cell cycle and apoptosis were detected by flow cytometry, immortalization of porcine umbilical vein endothelial cells in vitro was treated by telomerase transfer, and positive monoclonal cells were screened by G418. The effects of agonists and inhibitors on the proliferation of the cells were monitored by cell function analyzer. Results: the primary cells were in good growth state, small triangle, ellipse, fusiform, polygonal monolayer growth, and the boundary was clear. After fusion, the cells were arranged in paving stone, the ability of cell proliferation was strong. After repeated passages, the ability of cell proliferation was weakened, and the activity of cells was poor. The number of apoptotic cells was increased. Both rabbit anti-human factor 8th antigen F 鈪，

本文編號(hào)：1529229