黄芪甲苷配伍三七总皂苷对脑缺血大鼠BMSCs移植后血管新生的影响  

Effect of AstragalosideⅣCombined with Notoginseng Total Saponins on Angiogenesis After Transplantation of BMSCs in Rats with Cerebral Ischemia

作　　者：李艳玲[1] 丁煌[1] 杨芙蓉 陆展辉 李煜雯 刘晓丹[1] 邓常清[1] LI Yan-ling;DING Huang;YANG Fu-rong;LU Zhan-hui;LI Yu-wen;LIU Xiao-dan;DENG Chang-qing(Hunan University of Chinese Medicine,Hunan Key Laboratory of Cell Biology and Molecular Technology,Hunan Key Laboratory of Integrated Chinese and Western Medicine for Prevention and Treatment of Heart and Brain Diseases,Changsha 410208,China)

机构地区：[1]湖南中医药大学中西医结合心脑疾病防治湖南省重点实验室,细胞生物学与分子技术湖南省高校重点实验室,长沙410208

出　　处：《中国实验方剂学杂志》2021年第21期73-79,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81904181);湖南中医药大学研究生创新课题(2020CX63);湖南省大学生创新创业训练计划项目(S202010541017)。

摘　　要：目的:探讨黄芪甲苷(ASTⅣ)与三七总皂苷(NTS)配伍联合骨髓间充质干细胞移植对脑缺血大鼠神经修复和血管新生的影响。方法:大鼠随机分为假手术组,模型组,ASTⅣ配伍NTS低、高剂量组,骨髓间充质干细胞(BMSCs)输注组,BMSCs输注联合黄芪甲苷和三七总皂苷低(10,25 mg·kg^(-1)),高(20,50 mg·kg^(-1))剂量组。全骨髓贴壁法分离、纯化BMSCs,流式细胞术检测BMSCs表面标志物CD29,CD90,CD34,CD45阳性表达率。采用大脑中动脉阻塞建立局灶性脑缺血模型。BMSCs输注组尾静脉注射PKH26标记的BMSCs(1次/d);联用组尾静脉注射BMSCs(1次/d)并灌胃给药(2次/d);其他组灌胃给药,2次/d;假手术组、模型组灌胃等体积生理盐水。Longa法测定神经功能缺损症状,红四氮唑(TTC)染色测定脑梗死率、免疫荧光法观察BMSCs移植后的存活及向血管的分化[PKH26/血管内皮生长因子(VEGF)的双阳性表达],蛋白免疫印迹法(Western blot)检测血管生成素1(Ang1)和转化生长因子-β_(1)(TGF-β_(1))蛋白的表达。结果:成功分离培养BMSCs,表面标志物CD29,CD90,CD34,CD45鉴定符合BMSCs特征。脑缺血后出现神经功能缺损症状,模型组神经功能缺损评分显著升高(P<0.01),脑梗死率显著增高(P<0.01),各药物及细胞移植均能不同程度减轻上述病理改变,其中效应最强为BMSCs输注联合高剂量ASTⅣ+NTS组(P<0.01),优于单用药物和单用BMSCs输注。脑缺血后脑血管损伤,输注BMSCs后,细胞可在缺血侧脑组织存活并促进血管新生,药物联合可增强其效应。脑缺血后,Ang1和TGF-β_(1)的表达增加,效应最强为BMSCs输注联合ASTⅣ+NTS组(P<0.01),优于单用药物和单用BMSCs输注。结论:ASTⅣ配伍NTS能促进骨髓间充质干细胞移植的存活,靶向修复脑缺血后受损血管促进血管新生,机制可能与改善脑缺血后脑内局部微环境,促进移植干细胞的存活和分化有关。Objective:To investigate the effect of astragalosideⅣ(ASTⅣ)and Notoginseng total saponins(NTS)combined with bone marrow mesenchymal stem cell(BMSC)transplantation on neural repair and angiogenesis in rats with cerebral ischemia.Method:The rats were randomly divided into a sham operation group,a model group,low-and high-dose ASTⅣ+NTS groups,a BMSC infusion group,and low-and highdose BMSC infusion+ASTⅣ(10 and 20 mg·kg^(-1))+NTS group(25,50 mg·kg^(-1)).BMSCs were isolated and purified by whole bone marrow adherent culture.The positive expression of surface markers of BMSCs(CD29,CD90,CD34,and CD45)was detected by flow cytometry.The focal cerebral ischemia model was established by middle cerebral artery occlusion(MCAO).The PKH26-labeled BMSCs were injected into the tail vein of rats in the BMSC infusion group,once a day.The rats in the combination groups received BMSC injection once a day and intragastric administration of drugs twice a day.Other groups were administered twice a day by gavage.The sham operation group and the model group received the same amount of normal saline.Symptoms and signs of neurological deficits were assessed by the Longa method and the cerebral infarction rate was determined by TTC staining.The survival and vascularization[double positive expression of PKH26/vascular endothelial growth factor(VEGF)]after transplantation of BMSCs were observed by the immunofluorescence method.The protein expression of Ang1 and TGF-β_(1) was measured by Western blot.Result:BMSCs were properly isolated and cultured.The identification of surface markers CD29,CD90,CD34,and CD45 was consistent with the characteristics of BMSCs.The neurological deficit score and cerebral infarction rate of the model group were significantly increased(P<0.01).All drugs and cell transplantation could alleviate the above pathological changes in varying degrees.The strongest effect was observed in high-dose BMSC infusion+ASTⅣ+NTS group(P<0.01),which was superior to those in the ASTⅣ+NTS groups or the BMSC infusion group.

关 键 词：黄芪甲苷 三七总皂苷 骨髓间充质干细胞 脑缺血 血管新生 

分 类 号：R2-0[医药卫生—中医学] R289