目的:探讨人参皂苷Rg1对高糖诱导损伤的内皮祖细胞(EPCs)生物学功能和分泌血管生成相关因子的影响.方法:体外分离和培养人脐带血EPCs,通过观察细胞形态、双荧光染色法对培养的EPCs进行鉴定.将鉴定成功的EPCs用30 mmol/L的葡萄糖预处理120 h后,分别在不同浓度梯度人参皂苷Rg1(0 mg/L、5 mg/L、10 mg/L、20 mg/L、40 mg/L、80 mg/L)条件下干预培养,确定人参皂苷Rg1促高糖受损EPCs增殖的最佳浓度.将高糖诱导损伤的EPCs随机分为实验组(最佳浓度人参皂苷Rg1干预)和模型组[磷酸缓冲盐溶液(PBS)干预],同时设置正常EPCs对照组(PBS干预).采用细胞计数试剂(CCK-8)、黏附能力测定试验、Matrigel体外成管试验、划痕实验及酶联免疫吸附试验(ELISA)法检测EPCs增殖、黏附、成管、迁移能力及EPCs分泌血管内皮生长因子(VEGF)、基质细胞衍生因子-1α(SDF-1α)、血管生成素-1(Ang-1)、基质金属蛋白酶-2(MMP-2)的水平.结果:人参皂苷Rg1促高糖受损EPCs增殖的最佳浓度为40 mg/L;与正常组比较,高糖受损EPCs增殖、黏附及成管能力明显降低(P<0.01);采用人参皂苷Rg1干预后,高糖受损EPCs增殖、黏附、成管及迁移能力明显增强(P<0.01).ELISA检测发现,与正常组比较,模型组EPCs分泌VEGF、SDF-1α、Ang-1明显减少,而MMP-2分泌增加,差异均有统计学意义(P<0.05);实验组在40 mg/L人参皂苷Rg1干预后,EPCs分泌VEGF、SDF-1α、Ang-1明显增加,而MMP-2分泌减少,差异有统计学意义(P<0.05).结论:人参皂苷Rg1处理能改善高糖受损EPCs的生物学功能及促进EPCs分泌血管生成相关生长因子,具有促血管新生的潜能.

Professional identity of medical students is not only the starting point of individual career, but also the source of the power of career development and success. To investigate the current situation and influencing factors of the professional identity of students majoring in Traditional Chinese and Western clinical medicine, and to explore ways to enhance their professional identity, the study does questionnaire survey on 492 undergraduates and postgraduates of traditional Chinese and western clinical medicine in a university in Hunan, and statistically analyzes the investigation results with SPSS 26.0. There are statistically significant differences in professional identity scores of students of Traditional Chinese and Western clinical medicine with different characteristics. Grades, whether they are student cadres, and different ways of choosing the clinical medicine major have the main impact on the professional identity of students of Traditional Chinese and Western clinical medicine. The professional identity of students majoring in Chinese and Western medicine can be enhanced by improving the professional education and professional identity education of Traditional Chinese and Western clinical medicine, positively developing career planning education, and building harmonious medical environment, etc.

目的 探讨黄芪甲苷(AstragalosideⅣ,AS-Ⅳ)干预的人内皮祖细胞外泌体(Endothelial progenitor cells derived exosomes,EPC-Exos)对高糖诱导损伤人脐血间充质干细胞(Human umbilical cord blood mesenchymal stem cells,hUCBMSCs)向内皮分化的影响.方法 体外分离和培养人内皮祖细胞(Endothelial Progenitor Cells,EPCs),同时体外分离和培养hUCBMSCs,取P4代细胞分别进行成骨、成软骨和成脂诱导分化鉴定.将鉴定成功的EPCs分别用100mg·L-1的黄芪甲苷和等量的PBS干预,培养24 h后收集两组细胞上清液中外泌体.采用透射电子显微镜观察EPC-Exos的形态,利用纳米粒子追踪分析(Nanoparticle Tracking Analysis,NTA)技术检测EPC-Exos的粒径,Western blot技术进行外泌体特征性标志物CD9、CD63和TSG101的检测.将hUCBMSCs用30 mmol·L-1的葡萄糖预处理120h后,随机分为实验组和对照组,同时设置正常组.三组细胞培养24 h后,通过Matrigel体外成管实验研究EPC-Exos对hUCBMSCs成管分化的影响.免疫荧光检测hUCBMSCs表达CD31、vWF等内皮细胞特异标志物的情况.结果 光镜下hUCBMSCs边缘清楚,形态均一,排列呈漩涡状,3系细胞分化为典型图像.透射电镜观察分离及提纯后黄芪甲苷干预EPC-Exos为包膜完整的圆形或椭圆形微囊泡结构;NTA技术检测97.6％人EPC-Exos为直径在81.4-142.1nm之间的微囊泡;EPC-Exos表面特异标志物CD9、CD63、TSG101呈阳性,以上实验证实成功提取EPC-Exos.Matrigel成管实验显示,与对照组相比,实验组MSCs细胞体外成管能力显著增强,差异显著(P<0.001).免疫荧光检测显示,与对照组相比,实验组MSCs表达内皮细胞特异性标志物CD31、vWF能力显著增强,差异显著(P均<0.01).结论 黄芪甲苷干预的EPC-Exos可有效恢复高糖受损人间充质干细胞的成管功能且显著改善其向内皮分化能力.

目的 探讨黄芪甲苷(AS-Ⅳ)对生信分析筛选出的人脐带血间充质干细胞(hUCBMSCs)外泌体(Exos)中与血管生成相关微小RNAs(miRNAs)表达的影响.方法 通过生信分析筛选hUCBMSCs-Exos中与血管生成高度相关的miRNAs.将hUCBMSCs随机分为实验组和对照组.实验组给予含300 mg·L-1 AS-Ⅳ的DMEM/F12培养基培养24 h,对照组加入等量磷酸盐缓冲溶液培养24 h.用二辛丁酸法检测Exos浓度,用实时荧光聚合酶链反应法检测miRNAs的表达水平.结果 实验组和对照组的外泌体浓度分别为(1.22±0.02)和(0.88±0.03)μg·μL-1,差异有统计学意义(P<0.01).miRNA-126-3p、miRNA-21、miRNA-214-5p、miRNA-126-5p、miRNA-145、miRNA-16-5p和miRNA-195-5p在实验组的表达量分别为6.69±0.57,1.06±0.16,1.06±0.21,0.68±0.05,0.51±0.17,0.48±0.14和0.46±0.10,在对照组中的表达量分别为1.04±0.14,0.74±0.10,0.48±0.14,1.04±0.14,1.02±0.17,1.09±0.15和1.18±0.20,差异均有统计学意义(均P<0.05).结论 AS-Ⅳ可改善hUCBMSCs分泌Exos的能力,且所分泌的Exos负载有与血管生成相关的miRNAs,具有强大的诱导血管新生的潜能.

目的:探讨黄芪甲苷(AS-IV)对高糖诱导损伤的人间充质干细胞(MSCs)分泌与创伤修复相关生长因子的影响.方法:无菌条件下取正常剖宫产胎盘中的脐带血,经密度梯度离心分离出人脐带血间充质干细胞(hUCBMSCs),分别进行成骨、成软骨和成脂诱导分化鉴定.将鉴定成功的hUCBMSCs用含30 mmol/L葡萄糖的DMEM/F12培养基培养120 h后随机分为实验组和模型组,实验组用300 mg/L的AS-IV进行干预,模型组用等体积的磷酸盐缓冲液(PBS)处理;同时设置正常组,用等体积PBS干预.各组干预48 h后收集细胞上清液,通过酶联免疫吸附实验检测表皮生长因子(EGF)、成纤维细胞生长因子(FGF)、胰岛素样生长因子-1(IGF-1)、神经生长因子(NGF)、血小板源性生长因子(PDGF)、干细胞因子(SCF)的吸光度.结果:与正常组相比,模型组中高糖受损hUCBMSCs所分泌的EGF、FGF、PDGF、SCF明显降低(P＜0.05);与模型组相比,实验组中高糖受损hUCBMSCs所分泌的EGF、FGF、IGF-1、NGF、PDGF、SCF含量明显升高(P＜0.05).结论:黄芪甲苷可促进高糖诱导损伤人MSCs分泌与创伤修复相关生长因子,具有较高临床应用价值.

目的 探讨黄芪甲苷(AS-Ⅳ)介导的内皮祖细胞外泌体(EPCs-Exos)对高糖受损内皮细胞(ECs)增殖和凋亡的影响.方法 从足月健康新生儿脐带血中分离、培养单个核细胞,用CD31抗体联合4,6-二脒基-2-苯基吲哚(DAPI)核染鉴定,获得的EPCs用100 mg·L-1 AS-Ⅳ干预24 h.用超速离心法结合超滤法提取细胞上清液中囊泡结构,用透射电子显微镜进行形态观察,用蛋白质印迹(WB)法检测外泌体表面特征性标志蛋白CD9、CD63、CD81.体外分离、培养人脐静脉内皮细胞(HUVECs),用vWF因子联合DAPI核染鉴定,获得的HUVECs用30 mmol·L-1葡萄糖干预120 h,随机分为实验组、对照组并设置正常组(用正常EGM-2培养基预培养120 h).实验组用AS-IV介导下的EPCs-Exos处理24 h,对照组和正常组用等容量PBS处理24 h,用CCK-8细胞增殖检测试剂盒,用流式细胞术分别检测细胞增殖和凋亡情况.结果 正常组、对照组和实验组OD值分别为0.73±0.00,0.45±0.00和0.74±0.01,凋亡率分别为(6.44±0.27)％,(24.41±1.12)％和(9.54±0.20)％.各组细胞凋亡率比较,差异均有统计学意义(均P<0.05).结论 高糖环境下ECs增殖能力抑制明显、凋亡率上升,而AS-IV介导的EPCs-Exos可显著促进高糖受损ECs增殖并抑制其凋亡,对ECs有明确的保护作用.

目的 观察人参皂苷Rg1对高糖环境下人脐带血间充质干细胞(hUCBMSCs)分泌炎症因子的影响.方法 无菌条件下取正常剖宫产胎盘中的脐带血,采用密度梯度离心法分离人脐带血间充质干细胞(hUCBMSCs)并鉴定.将鉴定成功的hUCBMSCs随机分为正常组、模型组、干预组.模型组和干预组加入含30 mmol/L葡萄糖的DMEM/F12培养基建立高糖受损细胞模型,正常组加入DMEM/F12培养基;培养5 d后,干预组加入40 mg/L的人参皂苷Rg1,正常组和模型组加入等量PBS,继续培养48 h.收集三组细胞上清液,采用酶联免疫吸附法检测炎症因子白细胞介素(IL)-1、IL-6、IL-10、巨噬细胞集落刺激因子(M-CSF)、单核细胞趋化蛋白1(MCP-1)水平.结果 与正常组相比,模型组细胞上清液中IL-1、IL-6、MCP-1升高(P均<0.05);与模型组相比,干预组细胞上清液中IL-1、IL-6降低,IL-10、M-CSF升高(P均<0.05).结论 高糖环境下,人参皂苷Rg1可抑制hUCBMSCs中的促炎因子释放,促进抑炎因子分泌,从而发挥抑制炎症反应的作用.

Objective:To investigate the effects of mesenchymal stem cells-derived exosomes (MSC-Exos) secreted by mesenchymal stem cells (MSCs) induced by astragaloside IV (AS-IV) on the biological function and pyroptosis of human umbilical vein endothelial cells (HUVECs) injured by high glucose.Methods:After human umbilical cord blood mesenchymal stem cells (hUCBMSCs) were intervened with 400 mg/L of AS-IV, exosomes were extracted, and then the morphology and specific markers of exosomes were identified. Human umbilical vein endothelial cells (HUVECs) were cultured in a medium with a glucose concentration of 30 mmol/L to prepare a high glucose-impaired HUVECs model. High glucose-impaired HUVECs were randomly divided into experimental and model groups, with the experimental group intervened with 100 μg/ml of MSC-Exos and the model group intervened with an equal volume of PBS solution, while a blank control group was also set up. Cell counting Kit-8 (CCK-8) cell proliferation assay, adhesion assay, matrigel tube formation assay and scratch assay were used to detect the effects of AS-IV-mediated MSC-Exos on the proliferation, adhesion, tube formation and migrationability of HUVECs; Western blot and real time fluorescence quantitative polymerase chain reaction (qRT-PCR) were used to detect the protein and mRNA expression of scorch death-related molecules, such as Caspase-1, GSDMD (Gasdermin D) and NLRP3 in each group.Results:The proliferation, adhesion number, tube number and migration width of HUVECs cells were significantly lower than those in the blank group ( P<0.05); The expression of Caspase-1, GSDMD, NLRP3 protein and their mRNA increased significantly ( P<0.001); Under the intervention of MSC-Exos mediated by AS-IV, the cell proliferation, adhesion number, tube number and migration width of HUVECs were significantly higher than those in the model group ( P<0.05); The expression of Caspase-1, GSDMD, NLRP3 protein and their mRNA decreased, with statistically significant difference ( P<0.05). Conclusions:AS-IV mediated MSC-Exos can significantly improve the biological function of high glucose-impaired endothelial cells, and its mechanism may be related to anti-pyroptosis.