【作者】 赵珅；

【导师】 谢露；

【作者基本信息】 广西医科大学 ， 生理学， 2008， 硕士

【摘要】 目的:利用人脐静脉内皮细胞（HUVEC）体外培养模型,探讨海带海带多糖L01对内毒素刺激下,人脐静脉内皮细胞表达和分泌iNOS、eNOS、PGI₂的影响,以及对内皮的细胞超微结构的影响,为研究和开发新型抗血栓药物提供理论和实验室依据。方法:（1）体外培养人脐静脉内皮细胞（HUVEC）,随机分成7组,空白对照组（10%FCS）,L-L01对照组（10mg/L L01）、LMWH对照组（50U/mlLMWH）、模型组（40mg/L LPS）、L-L01低剂量组（40mg/L LPS+10mg/LL01）、H-L01高剂量组（40mg/L LPS+100mg/L L01）、LMWH组（40mg/LLPS+50U/ml LMWH）,培养48h,酶联免疫吸附分析（ELISA）法测定HUVEC培养上清夜中的iNOS、eNOS、6-Keto-PGF1α水平。（2）逆转录多聚酶链反应（RT-PCR）检测各组HUVEC内iNOSmRNA、eNOSmRNA、PGISmRNA的表达,其扩增产物电泳后进行密度扫描及半定量分析。（3）电镜下观察各组细胞的超微结构。结果:40mg/L的内毒素培养48h,可致HUVEC培养液中iNOS水平显著升高,eNOS、6-K-PGF1α水平显著降低（P＜0.01,P＜0.05）;HUVEC中eNOSmRNA、PGISmRNA表达水平显著下降,iNOSmRNA表达水平显著升高;HUVEC细胞核不规则,细胞间微绒毛减少、消失,线粒体（Mi）肿胀,嵴溶解消失,核旁溶酶体增多。而LMWH及不同浓度的L01可拮抗内毒素所致的各种变化。结论:海带多糖L01可拮抗LPS对内皮细胞的作用,增多eNOS、6-K-PGF1α的分泌,降低iNOS的分泌;上调eNOSmRNA、PGISmRNA的表达,下调iNOSmRNA的表达,从而升高eNOS/β-actine、PGIS/β-actine的比值,降低iNOS/β-actine的比值;从超微结构上改善LPS对内皮细胞的损伤。因此海带多糖L01可以从功能和形态两方面保护内皮细胞。更多还原

【Abstract】 Objective:To study the effect of Polysaccharide L01 from Laminaria on the expression and secretion of inducible nitric oxide synthase（iNOS）,endothelial nitric oxide synthase（eNOS）,prostaglandin I₂（prostacyclin）（PGI₂）stimulated by Lipopolysaccharide in cultured human umbilical vein endothelial cell （HUVEC）,thus providing an experimental basis for developing a new drug for antithrombosisMethods:（1）The human umbilical vein endothelial cell was divided into 7 group randomly as blank control group,L01 control group,LMWH control group,model group.L-L01 treatment group,H-L01 treatment group,LMWH treatment group.With 40mg/L Lipopolysaccharide added,human umbilical vein endothelial cell was exposed to LMWH and different concentration of Polysacchatide of Laminaria（L01）and cultured for 48 hours.The level of iNOS, eNOS and 6-Keto-PGF1αin cultured supernatants was measured by enzyme-linked immunosorbent assay（ELISA）.（2）Reverse transcriptase chain reaction（RT-PCR）was used to detect the expression of iNOS,eNOS and PGIS messenger RNA（mRNA）in HUVEC in all groups and the related products were examined by densityscan and half-quantiative analysis after electrophoresis to reflect the variety of the transcription level.（3）The ultrastructure of samples was observed by transmission electron microscope. Result:（1）Stimulated by 40mg/L LPS for 48h,the level of eNOS and 6-Keto-PGF1αdecreased significantly（P＜0.01,P＜0.05）and iNOS increased significantly（P＜0.01,P＜0.05）in cultured supernants.Besides the expression of eNOSrnRNA and PGISmRNA were down-regulated and iNOSmRNA was up-regulated in the HUVEC.After observed by transmission electron microscope,we found that the irregularity cellular nucleus,microvilli decreased and disaapeared,engorgement of mitochondrium,cristae dissolved and disaapeared,cytolysosome increased.But the effect of LPS was reduced by HMWH and different concentration of polysaccharide L01 from Laminaria after cultured.Conclusion:L01 not only enhanced the LPS-stimulated secretion of eNOS and 6-Keto-PGF1α,reduced the secretion of iNOS in cultured supernants,but also increased the LPS-stimulated expression of eNOSmRNA and PGISmRNA, decreased the expression of iNOSmRNA in the HUVEC,so increase the ratio of eNOS/β-actine、PGIS/β-actine,decrease the ratio of iNOS/β-actine.Except that,L01 also reduce the injury of HUVEC in ultrastructure.So Polysaccharide L01 from Laminaria can protect HUVEC in function and morphology.更多还原