摘要
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Westernblot方法检测CD73蛋白表达。结果:①低氧24h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4h、8h,bEnd.3细胞CD73mRNA表达增加(P<0.05)。④低氧12h、24h,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.3CD73mRNA、蛋白水平及酶活性的提高。
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.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2005年第7期1255-1259,共5页
Chinese Journal of Pathophysiology
基金
教育部回国人员科研基金资助项目
关键词
低氧
5'-核苷酸酶
腺苷
内皮
血管
Hypoxia
5’-nucleotidase
Adenosine
Endothelium, vascular
