文献类型：学位论文

中文题名：人参皂苷Rb3对血管内皮细胞的类雌激素样保护效应研究

英文题名：The Estrogen-like Protective Effects of Ginsenoside Rb3on Vascular Endothelial Cells

作者：潘玉婷[1];

第一作者：潘玉婷

机构：[1]甘肃中医学院;

第一机构：甘肃中医药大学

导师：殷惠军;甘肃中医学院|李应东;甘肃中医学院|郭春雨;甘肃中医学院

授予学位：硕士

语种：中文

中文关键词：人参皂苷Rb3;氧化低密度脂蛋白;人脐静脉内皮细胞;氧化应激;细胞凋亡

外文关键词：ginsenoside Rb3;oxidized low density lipoprotein /(ox-LDL/);human umbilical vein endothelial cells/(HUVECs/);oxidative stress;apoptosis

年份：2014

摘要：目的：体外培养人脐静脉内皮细胞，探索人参皂苷Rb3对ox-LDL所致内皮细胞损伤的保护机制，为具有益气养阴作用的西洋参茎叶总皂苷的临床应用提供实验依据。 方法：取健康产妇分娩的新生儿脐带，用0.1%胶原酶Ⅰ消化取脐静脉内皮细胞；分别应用含20%FBS、10ng/ml EGF的DMEM培养液和含8%FBS、2%LSGS的M199培养液培养人脐静脉内皮细胞（HUVECs），观察其生长情况；0.25%猪胰蛋白酶溶液消化细胞传代培养；Ⅷ因子相关抗原间接免疫染色方法对细胞进行鉴定。以体外培养的HUVECs为研究对象，建立ox-LDL刺激内皮细胞损伤模型。实验共分为8个组：正常对照组（NOR）、模型组（MOD）、雌二醇组（E2，0.01μM）、雌二醇+雌激素受体抑制剂ICI182780组（E2I，E2：0.01μM，ICI：1μM）、人参皂苷Rb3低剂量组（RL，1μM）、人参皂苷Rb3中剂量组（RM，10μM）、人参皂苷Rb3高剂量组（RH，100μM）、人参皂苷Rb3中剂量组+雌激素受体抑制剂ICI182780组（RMI，RM：10μM，ICI：1μM），各给药组，在ox-LDL刺激前，药物预处理1小时，加予ICI182780组，在药物处理前1h给予ICI182780，然后共同作用24h后进行检测。取细胞培养上清液检测NO、ET-1、sE-selectins、sICAM-1含量，取细胞裂解液检测胞内NOS、SOD、MDA水平，提取细胞总RNA应用RT-PCR检测iNOS和eNOS基因表达水平，收集细胞采用流式细胞仪结合AnnexinV-FITC/PI双染色法测定HUVECs凋亡率，Western blot法检测细胞Akt、p-Akt、ERK1/2、p-ERK1/2、p38MAPK及p-p38MAPK蛋白水平。 结果： ①0.1%胶原酶Ⅰ消化脐静脉所得原代内皮细胞在接种后约4h开始贴壁生长，光镜下贴壁生长的内皮细胞可汇合成典型的铺路石状，用含8%FBS、2%LSGS的M199培养液培养的内皮细胞可稳定传代至6～7代。Ⅷ因子相关抗原间接免疫染色法鉴定可见细胞胞质中有棕黄色颗粒，即所培养细胞表达Ⅷ因子相关抗原，为内皮细胞。 ②氧化损伤的检测：与NOR相比，MOD组HUVECs SOD活力明显降低（P0.01），MDA含量明显增多（P0.01）；与MOD组比较，E2、RL、RM、RH组HUVECs SOD活力明显升高（P0.01或P0.05），MDA含量明显降低（P0.01）；运用ICI182780预处理后，与对应药物组相比， HUVECs SOD活力明显降低（P0.05），MDA合成明显增多（P0.01）。 ③致炎因子的检测：与NOR相比，MOD组HUVECs sE-selectin、sICAM-1含量均明显增多（P0.01）；与MOD组比较，E2、RM、RH组HUVECs sE-selectin含量均明显降低（P0.01），RL组无统计学差异；与MOD组比较，E2、RL、RM、RH组HUVECssICAM-1含量均明显降低（P0.01）；运用ICI182780预处理后，与对应药物组相比，HUVECs sE-selectin、sICAM-1含量均明显增多（P0.01）；另外，与RL组相比，RH组HUVECs sICAM-1含量进一步降低（P0.05）。 ④内皮细胞功能的检测：与NOR相比，MOD组HUVECs NOS、NO、ET-1含量及iNOSmRNA表达均显著升高（P0.01），eNOS mRNA表达显著降低（P<0.01）；与MOD组比较，E2、RL、RM、RH组HUVECs NOS、NO、ET-1含量及iNOS mRNA表达均明显降低（P0.01），eNOS mRNA表达显著升高（P<0.01）；运用ICI182780预处理后，与对应药物组相比，HUVECs NOS、NO、ET-1含量及iNOS mRNA表达均显著升高（P0.01或P0.05），eNOS mRNA表达显著降低（P0.01或P0.05）。 ⑤细胞凋亡的检测：与NOR相比，MOD组HUVECs早期凋亡细胞(Annexin V阳性/PI阴性)及晚期凋亡和坏死细胞(AnnexinV阳性/PI阳性)比率明显增加(P<0.01)；与MOD组比较，E2、RL、RM、RH组HUVECs早期凋亡细胞比率明显降低(P<0.01)，E2、RM、RH组HUVECs晚期凋亡和坏死细胞比率明显降低(P<0.01)，RL组无统计学差异；运用ICI182780预处理后，与对应药物组相比，HUVECs早期凋亡细胞及晚期凋亡和坏死细胞比率增加(P<0.01)。 ⑥雌激素相关信号通路蛋白的检测：与NOR组相比，MOD组HUVECs细胞p-Akt、p-ERK1/2蛋白表达明显增高（P0.01），p-p38MAPK蛋白表达明显降低（P0.01）；与MOD组比较，E2、RH、RM组HUVECs细胞p-Akt蛋白表达均明显减少（P0.05），RL组HUVECs细胞p-Akt蛋白表达也呈降低趋势，但无统计学意义；与MOD组比较，E2、RH组HUVECs细胞p-ERK1/2蛋白表达均明显减少（P0.01），RM、RL组HUVECs细胞p-ERK1/2蛋白表达无统计学差异；与MOD组比较，E2、RH、RM、RL组HUVECs细胞p-p38MAPK蛋白表达均明显增高（P0.01）；运用ICI182780预处理后，与对应药物组相比，HUVECs细胞p-Akt、p-ERK1/2蛋白表达明显增高（P0.01或P0.05），且E2I组细胞p-p38MAPK蛋白表达明显降低(P0.05)；但与RM组相比，RMI组HUVECs细胞p-p38MAPK蛋白表达无统计学差异。 结论： ①成功分离培养人脐静脉内皮细胞，为体外研究血管内皮细胞损伤及药物保护机制提供保障。 ②人参皂苷Rb3可通过与雌激素受体结合实现抑制内皮细胞氧化应激、炎症反应激活、ET-1和NO过度释放、细胞凋亡及降低Akt、ERK1/2蛋白磷酸化水平等类雌激素样血管保护作用，其作用效应较雌激素弱。 ③人参皂苷Rb3可通过影响p38MAPK介导内皮细胞保护作用，说明人参皂苷Rb3还可通过其它途径发挥血管内皮细胞保护作用。 ④气阴两虚是雌激素下降的主要病机，益气养阴有一定雌激素效应。
Objective: Human umbilical vein endothelial cells /(HUVECs/) were separated, incubatedand identified to provide solid foundation for the futher research in vitro. To explorethe protective mechanism of ginsenoside Rb3on impaired endothelial cells and compare theeffects with estrogen to provide experimental basis for the clinical application of Panaxquinquefolius saponin of stem and leaf /(PQS/) of replenishing Qi and nourishing Yin. Method: Newborn umbilical cord were taken from healthy parturient, then0.1/%collagenaseⅠwere used to digest the endothelia cells from umbilical vein. HUVECs werecultured in DMEM culture containing20/%fetal bovine serum（FBS）and10ng//ml EGFor in M199culture containing8/%FBS and2/%LSGS, observing the growth of HUVECsin the two medium. HUVECs was passaged by0.25/%trypsin solution. The method ofimmunocytochemistry of Ⅷ factor were used to identify the HUVECs. HUVECs werecultured in vitro for the study, ox-LDL stimulated endothelial cell injury model is established.There are eight groups: normal control group /(NOR/), model group /(MOD/),17-β estrodiolgroup /(E2,0.01μM/),17-β estrodiol+ICI182780group /(E2I, E2:0.01μM, ICI:1μM/),ginsenoside Rb3low-dose group /(RL,1μM/), ginsenoside Rb3middle-dose group /(RM,10μM/), ginsenoside Rb3high-dose group /(RH,100μM/), ginsenoside Rb3+ICI182780group/(RMI, RM:10μM, ICI:1μM/). According to the different groups, HUVECs werepretreated for1hour with ICI182780, and followed by treatment with E2or ginsenoside Rb3for1hour before the24hour exposure to ox-LDL. The NO, ET-1, sE-selectin and sICAM-1concentrations in the cell culture supernatant were measured by ELISA method. The activitiesof SOD, NOS and the contents of MDA in the cell lysate were examined by enzyme methodor spectrophotometry. HUVECs apoptosis rate was measured by flow cytometry usingAnnexinV-FITC//PI staining. HUVECs iNOS and eNOS expression were measured byRT-PCR, while p-Akt, p-ERK1//2, p-p38MAPK expression were measured by Western-blot. Results: ①The culture of HUVEC: The primary cultured endothelia cells/(ECs/) began toadhere to the flask in4h after inoculation, the ECs arrayed like pitching stone under light microscopy. Cells incubated in M199culture containing8/%fetalbovine serum and2/%LSGS can passage to6~7generation. The cells exhibited brownishyellow granules in the cytoplasm after factor VIII-related antigen indirectimmunocytochemistry staining, thus the cells were identified as HUVECs. ②Cellular oxidative damage: Compared with NOR group, MOD group’s SODactivity was obviously lower /(P<0.01/), and MDA content increased significantly /(P<0.01/);compared with MOD group, E2, RL, RM, RH group’s HUVECs SOD activity was obviouslyincreased /(P<0.01or P<0.05/), and MDA content decreased obviously /(P<0.01/). Pretreatedwith ICI182780，compared with the corresponding medication group, HUVECs SOD activitydecreased obviously /(P<0.05/), MDA content significantly increased /(P<0.01/). ③Proinflammatory cytokines: In comparison with NOR group, the level ofsE-selectin and sICAM in MOD groups were all significantly increased/(P0.01/). Incomparison with MOD group, E2, RM and RH group were all significantly decreased（P0.01）, the level of sICAM in RL group were also decreased/(P0.01/). Pretreated withICI182780，compared with the corresponding medication group, the level of sE-selectin andsICAM were all significantly increased/(P0.01/). In addition, compared with RL group, thelevel of sICAM-1in RH group is further decreased/(P<0.05/). ④Endothelial function: Compared with NOR group, the level of NOS, NO, ET-1, andiNOSmRNA expression in MOD group were significantly increased /(P<0.01/), eNOSmRNAexpression in MOD group were decreased significantly /(P<0.01/). Compared with MODgroup, these were all significantly increased or decreased respectively in E2, RL, RM and RHgroup/(P<0.01/). Pretreated with ICI18278，compared with the corresponding medication group,the level of NOS, NO, ET-1, and iNOS mRNA expression were significantly increased/(P<0.01or P<0.05/), and eNOS mRNA expression were decreased significantly /(P<0.01orP<0.05/). ⑤Apoptosis: In comparison with NOR group, the rate of early apoptosis /(AnnexinVpositive//PI negative/), later apoptotic and necrosis cells /(AnnexinV positive//PI positive/)increased significantly in MOD group/(P<0.01/). Compared with MOD group, the rate of earlyapoptotic cells was significantly decreased in E2, RL, RM and RH group /(P<0.01/), and the rate of later apoptotic and necrosis cells was significantly decreased in E2, RM and RHgroup/(P<0.01/), but the RL group had no statistical difference. Pretreated with ICI182780,compared with the corresponding medication group, the rate of early apoptosis, laterapoptotic and necrosis cells increased significantly /(P<0.01/). ⑥Related proteins expression: In comparison with NOR group，the phosphorylationlevels of Akt, ERK1//2in MOD groups were all increased significantly/(P0.01/), meanwhilethe phosphorylation levels of p38MAPK were decreased significantly /(P<0.01/). Comparedwith MOD group, the phosphorylation levels of Akt in E2, RH and RM groups were decreasedsignificantly /(P0.05/), the phosphorylation levels of ERK1//2in E2and RH groups weredecreased significantly /(P0.01/), and the phosphorylation levels of p38MAPK in E2, RH, RMand RL groups were increased significantly /(P0.01/). Pretreated with ICI182780，comparedwith the corresponding medication group, the phosphorylation levels of Akt, ERK1//2were allincreased significantly/(P0.01or P<0.05/), and the phosphorylation levels of p38MAPK weredecreased in E2I group significantly /(P<0.05/); but compared with RM group, thephosphorylation levels of p38MAPK in RMI groups had no statistical difference. Conclusions: ①HUVECs are isolated and cultured successfully which provides solid foundation forthe research on vascular endothelial cell injury and protection mechanism of the medicines. ②Ginsenoside Rb3has estrogen-like vascular protective effects of alleviatingoxidative stress and inflammatory reaction, reducing ET-1,NO excessive release, decreasingthe apoptosis and inhibiting Akt, ERK1//2protein phosphorylation by binding to estrogenreceptors and these effects are relatively weaker than estradiol. ③The effect that Ginsenoside Rb3increases the phosphorylation level of p38MAPKdemonstrates ginsenosides Rb3also play a protective role through other pathways. ④Both deficiency of Qi and Yin syndrome is the pathogenesis of the falling levelof estrogen, and there is estrogen-like effects of replenishing Qi and Nourishing Yin.