低氧增强小鼠脑微血管内皮细胞CD73表达

目的：探讨低氧对小鼠脑微血管内皮细胞系bEnd.3细胞CD73的表达及活性的影响。 方法： ①构建bEnd.3细胞低氧模型。②用乳酸脱氢酶检测试剂盒测定细胞上清液中乳酸脱氢酶的含量。③高压液相层析法检测细胞 CD73的活性。④半定量RT-PCR法检测细胞CD73的mRNA表达。⑤生物素化bEnd.3细胞表面蛋白，继而用免疫沉淀及Western blot方法检测CD73蛋白表达。 结果： ①低氧24 h之后bEnd.3细胞乳酸脱氢酶漏出显著高于正常对照组(P＜0.01)。②低氧能刺激bEnd.3细胞表面CD73的酶活性增高，并呈时间依赖性(P＜0.05)。③低氧4 h、8 h，bEnd.3细胞CD73 mRNA表达增加（P＜0.05）。④低氧12 h、24 h，bEnd.3细胞表面CD73蛋白表达增加（P＜0.05）。 结论： 低氧刺激小鼠脑微血管内皮细胞bEnd.3 CD73 mRNA、蛋白水平及酶活性的提高。

Hypoxia upregulates CD73 expression in mouse microvascular endothelial cells

AIM: To study the effect of hypoxia on CD73 expression in mouse microvascular endothelial cell line bEnd.3. METHODS: ① bEnd.3 cells were exposed to different periods of hypoxia. ② Concentration of LDH released by bEnd.3 cells into the culture medium was detected. ③ Surface CD73 activity in bEnd.3 cells was measured by HPLC according to the conversion of E-AMP to E-ADO. ④ CD73 mRNA expression were analyzed by semiquantitative RT-PCR. ⑤ Cell surface proteins were biotinylated and CD73 was detected by avidin blots of immunoprecipitation with mAb TY23. RESULTS: ① bEnd.3 cells exposed to hypoxia for 24 h demonstrated a significant increase in LDH release (P<0.01). ②Hypoxia induced CD73 activity in bEnd.3 cells in a time-dependent manner. ③ CD73 mRNA expression increased markedly in hypoxia for 4 h and 8 h (P<0.05). ④ bEnd.3 cells exposured to hypoxia induced a time-dependent increase in expression of CD73 (P<0.05). CONCLUSION: Hypoxia induces CD73 mRNA, protein expression and increases CD73 activity in mouse microvascular endothelial cells.