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Isolation and Identification of Primary Human Umbilical Vein Endothelial Cells

【摘要】 目的 通過比較兩種原代人臍靜脈內皮細胞的分離培養方法并對細胞特異性抗原進行鑒定,探索提高原代內皮細胞體外培養存活率及純化率的方法。 方法 采用一次性無菌注射器向人臍靜脈灌注消化液,消化液的濃度和消化時間分別025%(質量體積比)胰蛋白酶,10 min和01%(質量體積比)膠原酶Ⅱ,15 min。通過在倒置顯微鏡下觀察細胞的形態特點和用免疫熒光染色的方法對細胞進行鑒定,比較兩種消化方法的優劣。 結果 01%膠原酶Ⅱ,15 min的消化方法較025%胰蛋白酶,10 min對原代人臍靜脈內皮細胞有更好的分離效果,活細胞數量多且細胞純度較高。免疫熒光染色結果表明細胞內有Ⅷ因子相關抗原表達。結論 膠原酶Ⅱ可以有效分離臍靜脈內皮細胞,最佳消化條件是01%膠原酶Ⅱ,37℃,15 min。【Abstract】 Objective To explore the optimal method for primary culture of human umbilical vein endothelial cells (HUVECs). Methods HUVECs were prepared from human umbilical cords by 01% collagenase Ⅱ digestion for 15 minutes and 025 trypsinase digestion for 10 minutes,respectively. HUVECs were observed under inverted microscope and identified by immunofluorescence.The two methods of digestion were compared. Results More HUVECs were harvested through the method of 01% collagenase Ⅱ for 15 minutes,which expressed Ⅷ related antigen. Conclusion The method of 0.1% collagenase Ⅱ digestion for 15 minutes is a better choice to isolate HUVECs.