摘要
目的:观察胰岛再生源蛋白Ⅲγ(RegⅢγ)修饰人脐带间充质干细胞(HuMSCs)对柯萨奇B3病毒(CVB3)诱导的小鼠心肌炎炎性细胞浸润及心脏功能的影响。方法:从新鲜脐带组织中提取HuMSCs,倒置显微镜观察形态,流式细胞仪检测细胞表面抗原表达;采用含过表达RegⅢγ的慢病毒感染HuMSCs,荧光显微镜观察绿色荧光蛋白表达,实时荧光定量聚合酶链式反应(qRT-PCR)检测RegⅢγ mRNA表达水平。将40只小鼠按照随机数字表法分为对照组、模型组、HuMSCs组和RegⅢγ-HuMSCs组,每组10只。模型组、HuMSCs组和RegⅢγ-HuMSCs组采用腹腔注射CVB3建立心肌炎模型,HuMSCs组和RegⅢγ-HuMSCs组再分别尾静脉注射HuMSCs、RegⅢγ-HuMSCs。14 d后,采用高分辨率小动物超声成像系统记录小鼠心脏超声指标;酶联免疫吸附法(ELISA)检测各组血清白细胞介素(IL)-2、IL-4、IL-6、IL-10和IL-23含量;苏木精-伊红(HE)染色观察心肌组织病理损伤情况,末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法(TUNEL)检测心肌组织细胞凋亡水平,免疫组织化学染色测定心肌组织T淋巴细胞表面抗原(CD4、CD8)和巨噬细胞特异性抗体(CD68)的表达。结果:分离的细胞呈典型纺锤形,细胞表面抗原CD44、CD73、CD90呈高表达,CD34、CD45、人类白细胞抗原-DR蛋白(HLA-DR)呈低表达,说明成功分离到HuMSCs。经过慢病毒感染的HuMSCs有明显绿色荧光蛋白表达,RegⅢγ mRNA表达水平高于未感染的细胞(P<0.05),说明成功得到RegⅢγ修饰的HuMSCs。与对照组比较,模型组小鼠射血分数(EF)、缩短分数(FS)降低,左室舒张末期容积(LVEDV)和左室收缩末期容积(LVESV)升高,血清IL-2、IL-6和IL-23含量增加,IL-4和IL-10含量减少,心肌组织内炎性细胞浸润明显,TUNEL阳性率增加,CD4、CD8及CD68蛋白表达均呈强阳性(P<0.05);与模型组比较,HuMSCs组和RegⅢγ-HuMSCs组小鼠EF和FS升高,LVEDV和LVESV降低,血清IL-2、IL-6和IL-23含量
Objective:To investigate the effects of islet regeneratingⅢγ(RegⅢγ)modified human mesenchymal stem cells(HuMSCs)on mice myocarditis induced by Coxsackievirus B3(CVB3).Methods:HuMSCs were extracted from fresh umbilical cord tissue,morphology was observed by inverted microscopy,and the expressions of cell surface antigen was detected by flow cytometry.HuMSCs were infected with lentivirus containing overexpressed RegⅢγ,the expressions of green fluorescent protein were observed by fluorescence microscopy,and the expression of RegⅢγmRNA was detected by quantitative real-time fluorescence polymerase chain reaction(qRT-PCR).Forty mice were randomly divided into control group,model group,HuMSCs group,and RegⅢγ-HuMSCs group,with 10 mice in each group.The model group,HuMSCs group and RegⅢγ-HuMSCs group were intraperitoneally injected with CVB3 to establish myocarditis models,and HuMSCs group and RegⅢγ-HuMSCs group were injected with HuMSCs and RegⅢγ-HuMSCs through tail vein,respectively.After 14 d,the cardiac ultrasound indexes of mice were recorded by high-resolution small animal ultrasound imaging system,serum interleukin(IL)-2,IL-4,IL-6,IL-10 and IL-23 of each group were measured by enzyme-linked immunosorbent assay(ELISA).The myocardial histopathological damage was observed by Hematoxylin-eosin(HE)staining,terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay(TUNEL)was used to detect apoptosis levels in myocardial tissue,and immunohistochemical staining was used to determine the expression of T-lymphocyte surface antigens(CD4,CD8)and macrophage-specific antibodies(CD68)in myocardial tissue.Results:The isolated cells showed a typical spindle shape with high expression of cell surface antigens CD44,CD73 and CD90,and low expression of CD34,CD45 and human leukocyte antigen-DR protein(HLA-DR),indicating successful isolation of HuMSCs.Lentivirally infected HuMSCs Showed significant green fluorescent protein expression,RegⅢγmRNA expression was increased than that of uninfected
作者
蹇强
李丹
程玮
孙敏
JIAN Qiang;LI Dan;CHENG Wei;SUN Min(Xi′an Children′s Hospital,Xi′an 710003,Shaanxi,China)
出处
《中西医结合心脑血管病杂志》
2023年第8期1415-1422,共8页
Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease
基金
陕西省重点研发计划项目(No.2020SF-103)。
