【作者】 卢玮；

【导师】 杜惠扬； 敖启林；

【作者基本信息】 华中科技大学 ， 病理学与病理生理学， 2006， 硕士

【摘要】 目的研究低氧条件下肺动脉内皮细胞（PAEC）向平滑肌样细胞的转分化及转化生长因子β1（TGFβ1）在此转分化过程中的作用。方法1.胰酶消化法获取猪肺动脉内皮细胞,并用免疫磁珠法（以PECAM-1/CD31阳性表达作为分选标记）对原代肺动脉内皮细胞进行纯化;2.形态学观察结合免疫组化检测Ⅷ因子和CD31对内皮细胞进行鉴定;3.实验分组:将纯化后的PAEC分成四组:（1）常氧组（N）（含21%O2,5%CO₂和74%N₂）;（2）低氧组（H）（含1%O₂,5%CO₂,94%N₂）;（3）常氧+TGFβ1抑制组（N+TGFβ1 Neutralizing Abs）;（4）低氧+TGFβ1抑制组（H+TGFβ1 Neutralizing Abs）。各组分别培养1、4、7天;4.平滑肌样细胞的鉴定:形态学观察结合免疫组化检测平滑肌标志性蛋白α-SM- actin,判定有无平滑肌样细胞的转分化;5.用逆转录-聚合酶链式反应法（RT-PCR）检测常氧和低氧组细胞TGFβ-1mRNA.;6.免疫印迹（Western-blot）检测常氧和低氧组细胞TGFβ-1蛋白的表达。结果1.免疫磁珠纯化可剔除内皮细胞中的杂细胞,获得高纯度的内皮细胞。纯化后的内皮细胞相差显微镜下观察呈典型的铺路石样外观,免疫组化检测Ⅷ因子（FⅧ）和CD31呈阳性表达。2.平滑肌样细胞的鉴定:形态学观察转化细胞呈梭形,免疫组化检测α-SM- actin呈阳性表达。3.各组细胞转化率比较:第1天各组均未出现平滑肌样细胞的转化（图7、8）;第4天和7天N组未出现转化细胞,H组出现转化细胞,且第7天H组转化率明显高于第4天H组转化率（P < 0.05）;阻断TGFβ1的作用后,第4天和第7天H+ TGFβ1 Neu Abs组的转化率分别低于其对应的H组（P < 0.05）,其余各组均未见转化细胞。4.低氧可增加肺动脉内皮细胞TGFβ1 mRNA和蛋白的表达（P<0.05）。结论1.磁珠分选法是一种有效的细胞纯化方法,可高效快捷的分离纯化出高纯度的血管内皮细胞。2.肺动脉内皮细胞中存在具有转分化为平滑肌样细胞潜能的细胞;3.低氧可明显促进内皮-平滑肌转分化;4. TGFβ1在此转分化过程中起促进作用。更多还原

【Abstract】 Objective:To investigate the transdifferentiation of pulmonary artery endothelium cells to smooth muscle-like cells under hypoxia and the role of transforming growth factor 1 ( TGFβ1) in this progress.Methods1. Endothelial cells from pulmonary artery of pig were purified by immunomagnetic (Dynabeads) purification techniques(based on PECAM-1/CD31 positive expression for sorting marker). The obtained pulmonary artery endothelial cells(PAECs)were primary cultured and subcultured.2. Using the immunocytochemistry to detect the expression of factorⅧand CD31 together with morphological observation through inverted phase contrast microscope to determined the purified cells were PAECs.3. The purified endothelial cells were divided into four groups :①normoxia group (cell culture box containing 21% O2, 5%CO2 and 74% N2)②hypoxia group (cell culture box containing 1%O2,5%CO2,94%N2 ).③normoxia culturing using TGFβ1neutralizing Abs to block the expression of TGFβ1,④hypoxia culturing using TGFβ1neutralizing Abs to block the role of TGFβ1. four groups were cultured 1day, 4days and 7 days respectively.4. Using the immunocytochemistry to detect the expression of smooth muscle cells specific markerα-SM- actin together with morphological observation to determined whether the endothelial cells transdifferentiated to the smooth muscle-like cells or not.5. Using reverse transcription- polymerase chain reaction (RT-PCR) analysis the expression of TGFβ1 mRNA in normoxia group and hypoxia group6 The expression of TGFβ1 protein were analyzed by Western blot. Results:1. The MACS (magnetic cell separation system) can highly purify endothelial cells. The cells which were purified by immunomagnetic (Dynabeads) purification techniques formed typical monolayer of cobblestone appearance and identifed by factorⅧand CD31 positive immunohistochemical stain.2. Identification of smooth muscle-like cells: These cells which were spindle-shaped and expressed smooth muscle cells specific markerα-SM- actin were identified SMCs.3. With the hypoxic time prolongation, the ratio of transdifferentiated smooth muscle-like cells from pulmonary aterial endothelial cells was gradually increased (P<0.05); blocking the role of TGFβ1, however, could significantly decrease the ration of transdifferentiation (P<0.05).4. The expression level of TGFβ1 mRNA and protein was higher in hypoxia groups than that in nomoxia groups (P<0.05).Conclusions:1. The MACS is efficiency in endothelial cell isolation and purification and can obtain highly purified endothelial cell fast and easily.2. Present study shows that pulmonary arterial endothelial cells colony contains those cells which possess the transdifferentiation potential to smooth muscle-like cells.3.Hypoxia is greatly attributed to the transdifferentiation.4.TGFβ1 plays a important role in this process.更多还原