经尿道灌注SCL和GJA1基因慢病毒对糖尿病膀胱病变中CD117和Cx43蛋白及其mRNA表达的影响  

Effects of SCL and GJA1 gene lentivirus injected into urethra on CD117 and Cx43 protein and mRNA expression in diabetic bladder lesions

作　　者：孙鹏 王勤章[1] SUN Peng;WANG Qin-zhang(Department of Urology Surgery,the First Affiliated Hospital of the Medical College,Shihezi University,Shihezi 832000,China)

机构地区：[1]石河子大学医学院第一附属医院泌尿外科,新疆石河子832000

出　　处：《吉林医学》2023年第4期869-874,共6页Jilin Medical Journal

基　　金：国家自然科学基金资助项目[项目编号:81960147]。

摘　　要：目的:探索灌注干细胞白血病SCL和GJA1基因慢病毒对糖尿病膀胱病变中CD117(c-kit)和Cx43蛋白及其mRNA表达的影响。方法:选取70只健康成年雄性豚鼠,称量体重在300~400 g之间,一次性腹腔注射链脲佐菌素(STZ)200 mg/kg,浓度1%,构建豚鼠糖尿病膀胱模型,经尿流动力学筛选出26只成模型豚鼠。随机将24只糖尿病膀胱模型豚鼠分为4组,每组6只:阴性对照组(n=6)、Cx43组(n=6)、CD117组(n=6)、CD117+Cx43组(n=6)。阴性对照组将未携带基因的慢病毒+PBS液0.2 ml单次经尿道灌注,CD117组单次经尿道灌注SCL基因重组慢病毒药物(滴度2×10^(7)U)0.2 ml,Cx43组单次经尿道灌注GJA1基因重组慢病毒药物(滴度2×10^(7)U)0.2 ml,CD117+Cx43组单次经尿道灌注SCL+GJA1基因重组慢病毒药物(滴度2×10^(7)U)0.2 ml。灌注结束后迅速结扎尿管至2 h后解除尿管。各组的糖尿病豚鼠膀胱在结束灌注后第14天时于无菌操作和全身麻醉下取出,将组织迅速冻存于-80℃冰箱,ICC表面CD117蛋白和缝隙连接蛋白Cx43的表达通过Western印迹检测,CD117和Cx43mRNA表达情况通过实时荧光定量聚合酶链反应(qRT-PCR)法检测,采用免疫组织化学染色(1HC)法观察Cx43蛋白和ICC表面CD117蛋白在豚鼠膀胱中的分布和染色强度。结果:Western印迹显示CD117+Cx43组、Cx43组和CD117组的CD117蛋白及Cx43蛋白表达均高于阴性对照组,差异有统计学意义(P<0.05);CD117+Cx43组的CD117蛋白和Cx43蛋白表达均高于Cx43组和CD117组,差异有统计学意义(P<0.05);qRT-PCR检测发现CD117+Cx43组、Cx43组和CD117组的CD117和Cx43mRNA表达均高于阴性对照组,差异有统计学意义(P<0.05);CD117+Cx43组的CD117和Cx43mRNA表达均高于CD117组和Cx43组,差异有统计学意义;1HC结果显示CD117+Cx43组的CD117蛋白和Cx43蛋白的免疫染色强度均高于CD117组、Cx43组和阴性对照组。结论:SCL和GJA1基因重组慢病毒均能在糖尿病豚鼠膀胱中成功转染,两种慢病毒联合灌注�Objective To explore the effects of SCL and GJA1 lentivirus injected into stem cell leukemia on the expression of CD117(c-kit)and Cx43 protein and mRNA in diabetic bladder lesions.Method A total of 70 healthy adult male guinea pigs were selected and weighed between 300~400g.Intraperitoneal injection of streptozotocin(STZ)200mg/kg was performed at a concentration of 1%.A guinea pig diabetic bladder model was constructed,and 26 guinea pigs were screened by urine flow dynamics.A total of 24 diabetic guinea pig models were divided into 4 groups by random number table method,6 rats in each group:negative control group(n=6),group Cx43(n=6),CD117 group(n=6)and CD117+Cx43 group(n=6).The negative control group received a single transurethral infusion of 0.2ml lentivirus+PBS solution without gene,and the CD117 group received a single transurethral infusion of SCL gene recombinant lentivirus drug(titer 2×10^(7)u)0.2ml,Cx43 group,single transurethral infusion of GJA1 gene recombinant lentivirus drug(titer 2×10^(7)u)0.2ml,CD117+Cx43 group,single transurethral infusion of SCL+GJA1 gene recombinant lentivirus drug(titer 2×10^(7)U)0.2ml.After the perfusion,the urinary catheter was ligated rapidly and released 2 hours later.In each group,the bladder of diabetic guinea pigs was taken on the fourteenth day after the end of perfusion.The tissues were cryopreserved in the refrigerator at-80℃at the time of asepsis and general anesthesia.The expression of CD117 protein and connexin Cx43 on ICC surface were detected by Western blot.The expression of CD117 and Cx43mRNA was detected by qRT-PCR method,and the distribution and staining intensity of Cx43 protein and ICC surface protein in guinea pig bladder were observed by immunohistochemical staining.Results Western blot showed that the expressions of CD117 protein and Cx43 protein in CD117+Cx43 group,Cx43 group and CD117 group were significantly higher than those in negative control group(P<0.05);The expressions of CD117 protein and Cx43 protein in CD117+Cx43 group were significantly

关 键 词：Cx43 CD117蛋白 基因慢病毒 糖尿病膀胱 缝隙连接 

分 类 号：R587.2[医药卫生—内分泌] R694[医药卫生—内科学]