观察在体灌注SCL基因重组慢病毒对豚鼠糖尿病膀胱中ICC功能的影响  被引量:6

To observe the effect of recombinant lentiviral recombination on ICC function in DCP bladder of guinea pigs

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作  者:徐浩[1] 钱彪[1] 杨立[2] 张思源[1] 沈志远 邵润芃 王勤章[1] Xu Hao;Qian Biao;Yang li;Zhang Siyuan;Shen Zhiyuan;Shao Runpeng;Wang Qinzhang(The First Affiliated Hospital,School of Medicine,Shihezi University,Shihezi,Xinjiang 832002,China;The 474 Hospital of the People's Liberation Army,Shihezi,Xinjiang 830000 China)

机构地区:[1]石河子大学医学院第一附属医院,新疆石河子832000 [2]中国人民解放军第四七四医院,新疆乌鲁木齐830000

出  处:《石河子大学学报(自然科学版)》2018年第4期486-491,共6页Journal of Shihezi University(Natural Science)

基  金:国家自然科学基金项目(81360120)

摘  要:目的探究豚鼠糖尿病膀胱病(DCP)在经尿道灌注干细胞白血病(SCL)基因重组慢病毒后所引发的Cajal样间质细胞(ICC)电位活动的改变。方法购买健康雄性豚鼠90只,按照200 mg/kg单次腹腔注射1%的链脲佐菌素(STZ),正常喂养12周后,筛选出符合DCP模型标准的的豚鼠并将其随机分为3组:实验组、阳性对照组、空白对照组。实验组经尿道灌注0.2 m L滴度为2×107IU的SCL基因重组慢病毒,阳性对照组经尿道灌注0.2 mL空慢病毒,空白对照组经尿道灌注0.2 mL PBS液。每组在转染后2、7、14、28 d这4个时间节点每次处死3只豚鼠,迅速摘取膀胱,选择胶原酶V消融法分离提取初代ICC并继续培养。72 h后运用膜片钳技术检测ICC自发及ATP诱发电位活动。结果体外通过胶原酶消融法可分离培养DCP豚鼠膀胱中ICC,运用细胞膜片钳技术观察到在无刺激条件下检测不到ICC的内向电流,而在ATP(100μmol/L)条件激发下可检测到ICC的内向电流。实验组2 d时与其他两组内向电流无明显变化,实验组SCL慢病毒转染DCP膀胱7、14、28 d时可检测到ICC内向电流比其他两组高(P<0.05),14 d达到峰值350.0±29.9pA,随后降低。阳性对照组与空白对照组随着DCP膀胱持续受损,内向电流逐步降低(P<0.05)。结论 SCL基因重组慢病毒经尿道灌注可成功转染豚鼠DCP膀胱,使受损的膀胱ICC电流幅度逐步增高,说明SCL基因对DCP膀胱中受损ICC功能有部分改善作用。Objective To investigate the change of potential activity of interstitial cells of Cajal (ICC) induced by guinea pigs with diabetic urinary bladder disease (DCP) after transfection of stem cell leukemia (SCL) gene recombinant lentiviral. Methods A total of 90 healthy male guinea pigs were purchased and injected with 1% of streptozotocin (STZ) intraperitoneally at 200 mg/kg.After 12 weeks of normal feeding,guinea pigs that met the DCP model criteria were selected and randomly divided into three groups: experimental group,positive control group,blank control group.The experimental group was transfused through the urethra with 0.2 mL of recombinant SCL gene lentivirus with a titer of 2×107 IU.The experimental group was transfused through the urethra with 0.2 mL of recombinant SCL gene lentivirus with a titer of 2×107 IU.The positive control group was perfused with 0.2 mL of empty lentivirus through the urethra,and the control group was perfused with 0.2 mL of PBS via the urethra.In each group,3 guinea pigs were sacrificed at each of the four time points of 2,7,14,28 d after transfection.The bladder was quickly removed,and the collagenase V ablation method was chosen to separate and extract the primary ICC and continue the culture.Seventy-two hours later,patch clamp techniques were used to detect ICC spontaneous and ATP evoked potentials. Results The ICC of DCP guinea pig bladder was isolated and cultured in vitro by collagenase ablation method.It was observed by cell patch clamp technique that the inward current of ICC was not detected under the condition of no stimulation,and the inward current of ICC could be detected under the excitation of ATP (100 μmol/L).At day 2,there was no significant change in the inward current between the experimental group and the other two groups.The inward current of ICC was higher in the experimental group than in the other two groups at 7,14,28 d after transfection of the SCL lentiviruses in the experimental group (P〈0.05),and the peak value of 14d rea

关 键 词:干细胞白血病基因 糖尿病膀胱病变 重组慢病毒 CAJAL样间质细胞 

分 类 号:R694.5[医药卫生—泌尿科学]

 

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