摘要
研究溶血卵磷脂(lysophosphatidylcholine,LPC)及其受体GPR4(G protein-coupled receptor4,GPR4)相互作用后对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)增殖的影响及其凋亡的诱导。方法:采用脂质体2000将装有GPR4受体基因的pEFneo真核表达载体转染HUVEC,并通过G418(800μg/ml)筛选获得GPR4基因稳定表达细胞株;RT-PCR法检测转染前后GPR4受体的mRNA水平表达情况;MTT法检测细胞的生长活力;AO染色法观察LPC对HUVEC造成损伤后形态的改变;Western blot法检测LPC与其受体相互作用后对caspase-3前体表达的影响。结果:成功获得了GPR4基因稳定表达细胞株;随着LPC浓度的增加(0．5,1,2,4,8μmol/1),抑制率分别增加了3．1%,4．4%,9．3%,17．0%,25．7%;但caspase-3前体的表达却随着浓度的增加先增加后减少。结论:LPC及其受体GPR4相互作用后可抑制HUVEC的增殖,并对HUVEC造成损伤进而诱导凋亡,使caspase-3前体的表达先增加后减少。
Objective：To explore the expression of pro-caspase 3 and the proliferation of HUVEC （human umbilical vein endothelial cells,HUVEC）influcnccd by lysophosphaddylchollnc through its receptor GPR4. Methods：HUVEC cells were transfected with the expression plasmids pEFneo-GPR4（containing the GPR4 receptor gene） by the LipofectamineTM2000, and the neomydn-resitant（G418 sulfate at 800μg/ml for HUVEC cells ）stable cell lines were selected. The expression of GPR4 receptor was detected by RT-PCR. HUVEC cells growth inhibition was measured by MTT assay. Impairement and configuration changes of the HUVEC caused by lysophosphaddylcholine were observed by AO dyes. Expression of pro-caspase 3 influenced by LPC through GPR4 receptor was detected by western blot. Results：Stable HUVEC cell lines, which can express the GPR4 receptor stably, were obtained. After treatment with （0.5, 1, 2, 4, 8）μmol/l LPC for 16 h, result of MTT assay showed that the HUVEC cells growth inhibition compared with the control group increased 3.1%,4.4%,9.3%,17.0%, 25.7%, respectively. The expression of the pro-caspase 3 protein was promoted by LPC at the lower concentration of 0.5μmol/1, but inhibited at the higher concentration. Conclusions：LPC could induce the HUVEC apoptosis,inhibit the HUVEC proliferation and influence the expression of procaspase 3 protein through GPR4 receptors.
出处
《现代生物医学进展》
CAS
2007年第4期552-556,共5页
Progress in Modern Biomedicine
基金
湖南省教育厅重点基金(No 03A040)
国家教育部重点基金(No 204095)
