低浓度胰蛋白酶原代培养大鼠血管内皮细胞实验方法研究

Abstract

目的：探索改进一种简单、经济和高纯度的大鼠血管内皮细胞分离培养方法。方法：采用2%戊巴比妥钠麻醉Wistar大鼠，在无菌条件下截取主动脉约3~4 cm，再将主动脉血管外翻使内膜暴露，用丝线结扎动脉两端，以0.125%胰蛋白酶消化18~20 min，在倒置相差显微镜下直接观察细胞生长和细胞形态特征，分别应用免疫组化和免疫荧光方法检测第VIII因子相关抗原抗体结合情况，对培养传代的血管内皮细胞进行鉴定。结果：采用0.125%胰蛋白酶直接消化可获得高纯度的大鼠主动脉血管内皮细胞。经过3~4天培养，细胞呈集落样散在分布于六孔培养板底，到10~12天时，细胞贴壁增长至覆盖大部分培养板底，约85%的细胞汇合成单层，呈现典型的“鹅卵石”样内皮细胞特征；此外，免疫组化和免疫荧光方法检测第VIII因子相关抗原抗体结合情况，发现大多数细胞胞浆呈现棕黄色和绿色荧光的阳性反应，显示分离培养的细胞主要是血管内皮细胞。结论：本改良的细胞培养方法操作简便，只需浓度低至0.125%胰蛋白酶即可获取较高纯度的血管内皮细胞，不需要较昂贵的胶原酶及内皮细胞生长因子，与现有的其他培养方法相比更为经济实用。 Objective: To explore a simple, economic and highly purified modified method to isolate and cul-ture the endothelial cells of rat thoracic aorta. Methods: Under an aseptic condition, the thoracic aorta was harvested in a male adult Wistar rat anesthetized with 2% pentobarbital sodium. After having removed the peripheral connective tissue and fat, the thoracic aorta was turned inside out to expose its intima. Then, the thoracic aorta ends were ligated with the sterile silk thread, and thoracic aorta intima was digested with 0.125% trypsin for 18 min - 20 min in centrifugal tube. Cell growth and cell morphology was directly observed under inverted phase contrast microscope; immunohistochemistry and immunofluorescence method is applied to detect and identify if the primary culture rat aortic endothelia cells (RAECs) are the vascular endothelial cells by their combining reactivity with VIII factor related antigen antibody. Results: After 3 - 4 days, the cultured rat aortic endothelial cells were distributed on the basement of six-well plates in a cluster. 10 - 12 days later, the adherent cells spread to cover most bottom of culture plate with about 85% of the adherent cells in a confluent monolayer, and show a typical cobblestone appearance which is the characteristic of the vascular endothelial cells. In addition, immunohistochemical staining and mmunofluorescence staining with monoclonal anti-Factor-VIII antigen antibody in culturing RAECs were shown by the tan and green fluorescence positive reactive particulate matter found in most of the cell cytoplasm, suggesting that the primary culture RAECs are the vascular endothelial cells. Conclusions: Just using the concentrations as low as 0.125% trypsin, our modified cultural method of vascular endothelial cells is a convenient, economical and applicable one with a higher cell activity and purity, no needing the expensive collagen enzyme and endothelial cell growth factor.