本文關(guān)鍵詞：改良大鼠肺微血管內(nèi)皮細(xì)胞原代培養(yǎng)技術(shù)及細(xì)胞鑒定　出處：《中華損傷與修復(fù)雜志(電子版)》2014年01期 　論文類型：期刊論文

  更多相關(guān)文章： 內(nèi)皮細(xì)胞 微血管 肺 細(xì)胞 培養(yǎng)的 細(xì)胞鑒定 免疫組織化學(xué)

【摘要】：目的改進(jìn)大鼠肺微血管內(nèi)皮細(xì)胞的原代培養(yǎng)方法,并對(duì)所培養(yǎng)的原代細(xì)胞進(jìn)行鑒定。方法應(yīng)用SD大鼠的外周肺組織,進(jìn)行大鼠肺微血管內(nèi)皮細(xì)胞的原代培養(yǎng)。從動(dòng)物選取、取材、肺灌注、植塊貼壁、培養(yǎng)基及添加物的選擇等方面對(duì)肺微血管內(nèi)皮細(xì)胞原代培養(yǎng)技術(shù)方法進(jìn)行改進(jìn)。從細(xì)胞形態(tài)學(xué)特征,免疫組織化學(xué)檢測(cè)血管內(nèi)皮細(xì)胞表面標(biāo)志物(CD31抗原和Ⅷ因子相關(guān)抗原的表達(dá)),熒光顯微鏡觀察異硫氰酸熒光標(biāo)記植物凝集素與肺微血管內(nèi)皮細(xì)胞特異性結(jié)合情況等方面鑒定所培養(yǎng)的肺微血管內(nèi)皮細(xì)胞。流式細(xì)胞儀檢測(cè)細(xì)胞純度,噻唑藍(lán)測(cè)量細(xì)胞生長(zhǎng)曲線。結(jié)果原代培養(yǎng)肺微血管內(nèi)皮細(xì)胞成多角形,融合為單層后呈典型的鵝卵石或鋪路石樣生長(zhǎng),傳代培養(yǎng)后可變?yōu)樗笮纬输鰷u樣生長(zhǎng)或聚集生長(zhǎng)。免疫組織化學(xué)顯示細(xì)胞CD31和Ⅷ因子相關(guān)抗原的表達(dá)陽(yáng)性;異硫氰酸熒光標(biāo)記植物凝集素結(jié)合試驗(yàn)陽(yáng)性。細(xì)胞生長(zhǎng)狀態(tài)良好、細(xì)胞純度達(dá)93.2%。結(jié)論改進(jìn)后的原代培養(yǎng)方法簡(jiǎn)便、可靠,獲得的肺微血管內(nèi)皮細(xì)胞污染少、純度高,狀態(tài)良好,保持了內(nèi)皮細(xì)胞的結(jié)構(gòu)和特性。
[Abstract]:Objective to improve the primary culture of rat pulmonary microvascular endothelial cells and to identify the cultured primary cells. Methods the peripheral lung tissue of SD rats was used to carry out primary culture of rat lung microvascular endothelial cells. The primary culture technology of pulmonary microvascular endothelial cells was improved from animal selection, sampling, perfusion, explant, culture medium and additives. From the cell morphological features, immunohistochemical detection of vascular endothelial cell surface markers (expression of CD31 antigen and factor VIII related antigen), fluorescein isothiocyanate labeled lectin and pulmonary microvascular endothelial cell specific binding and identification of cultured pulmonary microvascular endothelial cells were observed by fluorescence microscope. The cell purity was detected by flow cytometry, and the growth curve of the cell was measured by thiazolium. Results the primary culture of pulmonary microvascular endothelial cells was polygonal, and was monolayer, which was typical cobblestone or paving stone growth. After culture, it could be changed into spindle like growth or aggregation growth. Immunohistochemistry showed that the expression of CD31 and von Willebrand factor antigen; FITC labeled lectin binding test positive. The cell growth was good and the purity of the cells was 93.2%. Conclusion the improved primary culture method is simple and reliable, and the obtained pulmonary microvascular endothelial cells have less pollution, high purity and good state, which maintain the structure and characteristics of endothelial cells.
【作者單位】： 中山大學(xué)附屬第一醫(yī)院重癥醫(yī)學(xué)科SICU;
【基金】：國(guó)家自然科學(xué)基金(81071536),國(guó)家自然科學(xué)基金青年基金(81201452) 廣東省教育廳育苗基金(2012LYM_0006)
【分類號(hào)】：R329.2
【正文快照】： 血管內(nèi)皮細(xì)胞不僅具有屏障功能,同時(shí)還具有調(diào)節(jié)血管張力,調(diào)節(jié)抗凝、凝血和纖溶,表達(dá)黏附分子,參與血管活性物質(zhì)的代謝等多種功能,被認(rèn)為是一個(gè)“代謝和內(nèi)分泌器官”[1]。肺的血管內(nèi)皮細(xì)胞尤其是肺微血管內(nèi)皮細(xì)胞(pulmonary microvascularendothelial cells,PMVECs)廣泛涉及多

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