【作者】 张海强；

【导师】 严奉祥； 李凯；

【作者基本信息】 南华大学 ， 药理学， 2007， 硕士

【摘要】 目的研究GPR4 ( G protein-coupled receptor 4 , GPR4 )配体溶血卵磷脂(lysophosphatidylcholine,LPC)及鞘氨醇磷脂酰胆碱(sphingosylphosphorylcholine,SPC)对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)的生长效应及有关信号通路。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法鉴定稳定转染并检测转染前后GPR4受体mRNA的表达情况;MTT法检测细胞的生长活力;AO染色法观察LPC及SPC对HUVEC细胞造成的形态改变; Western blot法检测LPC及SPC与其受体相互作用后对caspase-3前体、p-JNK、p-Akt及MMP-2蛋白表达的影响。结果成功获得了GPR4基因稳定表达细胞株;随着LPC(0.5,1,2,4,8μmol/l)及SPC(0.25,0.5,1.0μmol/l)浓度的增加, LPC使HUVEC细胞活力下降, 570nm OD值依次为:1.50±0.136;1.45±0.050;1.42±0.208;1.36±0.191;1.32±0.122;1.08±0.189。SPC使细胞活力依次增强,570nm OD值依次为:1.08±0.089;1.15±0.097;1.21±0.096;1.30±0.11。LPC对细胞造成一定的损伤,随着浓度的增加,有些细胞可见致密浓染的黄绿色,而SPC却对细胞形态没有造成明显的改变。LPC使caspase-3前体蛋白的表达先增加后减少,MMP-2蛋白的表达下调,活化JNK激酶,使p-JNK蛋白的表达先增加后减少。SPC使p-Akt及MMP-2的表达先增加后减少,抑制caspase-3前体蛋白的表达。结论1. LPC与受体GPR4作用后使HUVEC细胞活性下降,该作用可能与调节pro-caspase 3、p-JNK及MMP-2蛋白的表达有关;2. SPC与受体GPR4作用后则能够提高HUVEC细胞活力并有一定的促增殖作用,可能与促进p-Akt及MMP-2蛋白的表达有关。更多还原

【Abstract】 Objective To explore the effect on HUVEC proliferation and the related cell signaling pathway caused by LPC and SPC through the G protein-coupled receptor4.Methods HUVEC cells were transfected with the expression plasmids pEFneo-GPR4 (containing the GPR4 receptor gene) by the lipofectamineTM 2000, and the neomycin-resitant(G418 sulfate at 800μg/ml for HUVEC cells )stable cell lines were established. The expression of GPR4 receptor was detected by RT-PCR. The cell viability was measured by MTT assay. The structure changes of the HUVEC caused by lysophosphatidylcholine or sphingosylphosphorylcholine were observed by AO dyes. The expression of pro-caspase 3、p-JNK、p-Akt and MMP-2 influenced by lysophosphatidylcholine or sphingosylphosphorylcholine through GPR4 receptor was detected by western blot.Results The HUVEC cell line stably expressing the GPR4 receptor was established. After treatment with 0.5-8μmol/l LPC for 16 h, MTT assay showed that LPC decreased the HUVEC cell viability, and the OD value (570nm) was: 1.50±0.136; 1.45±0.050;1.42±0.208;1.36±0.191;1.32±0.122;1.08±0.189 respectively. On the contrary, SPC (0.25, 0.5,1.0μmol/l) increased the cell viability, and the OD value (570nm) was: 1.08±0.089;1.15±0.097;1.21±0.096;1.30±0.11 respectively. LPC caused some impairment to the HUVEC with some morphological changes. However, the SPC did not cause evident morphological changes .The expression of the pro-caspase 3 protein was promoted by LPC at the lower concentration 0.5μmol/l, but the expression was inhibited at the higher concentrations. LPC activated the JNK kinase and inhibited the expression of the MMP-2 protein. The expression of the p-Akt and MMP-2 protein were promoted by SPC at the lower concentration 0.25-0.5μmol/l, but the expression was inhibited and restored the basal level almostly at the higher concentrations. SPC inhibited the expression of the pro-caspase 3 protein .Conclusion 1. LPC decreased the cell viability at the HUVEC. LPC decreased the expression of the pro-caspase 3 protein , activated the JNK kinase and inhibited the expression of the MMP-2 ; 2. SPC increased the HUVEC viability, promoted the expression of the p-Akt and MMP-2 protein at the lower concentration, but the expression was inhibited and restored the basal level almostly at the higher concentration. SPC inhibited the expression of the pro-caspase 3 protein .更多还原