大鼠主动脉血管内皮细胞原代培养方法改良及氧化损伤模型构建  

Improvement of primary culture method of rat aortic vascular endothelial cells and construction of oxidative damage model

作　　者：马露 杨雷 晏凡晨 刘晓丹[1] 丁煌 谭维 李菀榆 唐映红 张伟[1] 邓常清[1] MA Lu;YANG Lei;YAN Fan-chen;LIU Xiao-dan;DING Huang;TAN Wei;LI Wan-yu;TANG Ying-hong;ZHANG Wei;DENG Chang-qing(School of Integrative Medicine,Hunan University of Traditional Chinese Medicine,Key Laboratory of Hunan Province for Integrated Traditional Chinese and Western Medicine on Prevention and Treatment of Cardio-Cerebral Diseases,Hunan 410208,China)

机构地区：[1]湖南中医药大学中西医结合学院,湖南中医药大学中西医结合心脑疾病防治湖南省重点实验室,湖南长沙410208

出　　处：《中国药理学通报》2023年第11期2192-2198,共7页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 82174218,81774032);湖南省研究生科研创新项目(No CX20220786)。

摘　　要：目的改良大鼠原代主动脉血管内皮细胞(vascular endothelial cells,VECs)的提取和培养方法,并构建稳定的AVECs氧化损伤模型。方法采用六孔板植块联合胶原酶消化改良法建立大鼠原代AVECs提取和培养方法,倒置显微镜下观察细胞形态,免疫荧光染色法检测vW因子(vWF)进行细胞鉴定。选用2~4代的AVECs,用不同浓度的POVPC干预24 h,检测细胞增殖、细胞骨架、成管能力、细胞内活性氧(reactive oxygen species,ROS),构建POVPC诱导的AVECs氧化损伤模型。结果植块联合胶原酶消化法提取培养的内皮细胞具有典型的“铺路石”样特性,生长状态良好,vWF免疫荧光染色阳性,阳性率占0.95,鉴定为内皮细胞。该法可缩短细胞传代时间。采用4.375~70μmol·L^(-1)的POVPC干预培养AVECs,POVPC在17.5~70μmol·L^(-1)时可明显抑制AVECs增殖(P<0.05);POVPC在8.75~70μmol·L^(-1)时,可导致细胞骨架交叉断裂堆积、解聚、结构模糊,且细胞成管能力明显降低(P<0.01),细胞内ROS水平明显升高(P<0.05)。以上改变在POVPC浓度为35μmol·L^(-1)时尤为明显。结论采用六孔板植块联合胶原酶消化改良法可获得较多的内皮细胞,细胞生长状态良好,缩短了细胞的生长周期,是一种简便、可行的AVECs培养方法。17.5~705μmol·L^(-1)的POVPC可构建稳定AVECs氧化损伤模型。Aim To improve the extraction and culture method of rat primary aortic vascular endothelial cells(AVECs)and to construct a stable oxidative injury model of AVECs.Methods A modified method of extraction and culture of rat primary AVECs was established by using a six-well plate implantation block combined with collagenase digestion,cell morphology was observed under an inverted microscope,and vW factor(vWF)was detected by immunofluorescence staining for cell identification.AVECs of 2-4 generations were selected and intervened with different concentrations of 1-palmitoyl-2-(5-oxoacryloyl)-sn-glycero-3-phosphocholine(POVPC)for 24 h.Cell proliferation,cytoskeleton,tube-for ming ability and intracellular reactive oxygen species(ROS)were examined to construct POVPC-induced oxidative damage in AVECs model.Results The results showed that the endothelial cells extracted and cultured by the block and collagenase digestion method had typical"pavement stone"characteristics and were in good growth condition,and the positive rate of vWF immunofluorescence staining was 0.95.This method could shorten the cell passaging time.POVPC at 4.375-70μmol·L^(-1) significantly inhibited the proliferation of AVECs(P<0.05);POVPC at 8.75-70μmol·L^(-1) resulted in cytoskeletal cross breaks,accumulation,depolymerization,blurring of structure,and a significant decrease in cellular tube formation ability(P<0.01)and a significant increase in intracellular ROS levels(P<0.05).The above changes were especially obvious at the POVPC concentration of 35μmol·L^(-1).Conclusions The modified method of using six-well plate implants combined with collagenase digestion can obtain more endothelial cells with good cell growth status and shorten the cell growth cycle,which is a simple and feasible method for culturing AVECs.17.5-70μmol·L^(-1) POVPC can construct a stable AVECs oxidative injury model.

关 键 词：血管内皮细胞 主动脉 POVPC 模型 活性氧 氧化损伤 

分 类 号：R-332[医药卫生] R322.121R329.24R340.5R916.3