中国组织工程研究

中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (8): 1161-1165.doi: 10.12307/2022.1017

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

Gja1基因重组慢病毒对糖尿病豚鼠膀胱病变模型中缝隙连接蛋白43蛋白及mRNA表达的影响

张永强1，潘  凤2，3，孙  鹏1，谭明辉1，轩留明1，王勤章1   

  1. 石河子大学医学院第一附属医院，1泌尿外科，2病理科，新疆维吾尔自治区石河子市  832000；3石河子大学医学院病理系，新疆维吾尔自治区石河子市  832000
  • 收稿日期:2021-12-27 接受日期:2022-02-15 出版日期:2023-03-18 发布日期:2022-07-27
  • 通讯作者: 王勤章，博士，教授，主任医师，石河子大学医学院第一附属医院泌尿外科，新疆维吾尔自治区石河子市 832000
  • 作者简介:张永强，男，1990年生，安徽省阜阳市人，汉族，石河子大学在读硕士，主要从事泌尿外科尿动力学的研究。
  • 基金资助:
    国家自然科学基金项目(8196030314)；项目负责人：王勤章

Effect of Gja1 gene recombinant lentivirus on Cx43 protein and mRNA expression in a diabetic guinea pig bladder model

Zhang Yongqiang1, Pan Feng2, 3, Sun Peng1, Tan Minghui1, Xuan Liuming1, Wang Qinzhang1   

  1. 1Department of Urology, 2Department of Pathology, The First Affiliated Hospital, Shihezi University School of Medicine, Shihezi 832000, Xinjiang Uygur Autonomous Region, China; 3Department of Pathology, Shihezi University School of Medicine, Shihezi 832000, Xinjiang Uygur Autonomous Region, China
  • Received:2021-12-27 Accepted:2022-02-15 Online:2023-03-18 Published:2022-07-27
  • Contact: Wang Qinzhang, MD, Professor, Chief physician, Department of Urology, The First Affiliated Hospital, Shihezi University School of Medicine, Shihezi 832000, Xinjiang Uygur Autonomous Region, China
  • About author:Zhang Yongqiang, Master candidate, Department of Urology, The First Affiliated Hospital, Shihezi University School of Medicine, Shihezi 832000, Xinjiang Uygur Autonomous Region, China
  • Supported by:
    the National Natural Science Foundation of China, No. 8196030314 (to WQZ)

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摘要：

文题释义：
糖尿病膀胱：糖尿病是一组以血糖升高为特征的慢性系统性代谢疾病，糖尿病膀胱是糖尿病常见慢性并发症之一，可导致膀胱感觉受损、容量增加等病理生理变化，临床常见为尿频、尿急、尿潴留等，最终引起尿毒症等严重不良后果，严重影响患者生活质量。
连接子：6个连接蛋白亚单位在细胞膜上形成六聚体，称为连接子或半通道，可以作为独立的通道，或与同位膜中的半通道对接，形成完整的缝隙连接通道，允许小分子物质自由通过，从而形成间隙连接的细胞间通讯功能，促进细胞间的物质信息和能量交换。

背景：缝隙连接蛋白43又被称为Gja1蛋白，Gja1基因重组慢病毒对糖尿病膀胱引起的收缩功能障碍可能有改善作用。
目的：构建豚鼠糖尿病膀胱病变模型，研究经尿道灌注Gja1基因重组慢病毒药物的方式，观察对受损膀胱逼尿肌细胞表面缝隙连接蛋白43蛋白和 mRNA表达的影响。
方法：选取80只鼠龄及体质量相近的健康豚鼠，随机原则挑选15只豚鼠常规饲养，设为正常组；剩余65只高糖高脂饮食喂养4周后给予腹腔注射1%链脲佐菌素200 mg/kg建立糖尿病豚鼠膀胱模型；尿动力学筛选出符合糖尿病膀胱病变豚鼠模型，依据随机分组原则分为3组：空白组(n=12)、对照组(n=12)、实验组(n=12)。空白组经尿道灌注0.2 mL磷酸缓冲盐溶液；对照组经尿道灌注0.2 mL空载基因重组慢病毒；实验组经尿道灌注0.2 mL Gja1基因重组慢病毒。每组在转染后第2，7，14，28天分别处死3只豚鼠，通过免疫组织化学染色法观察豚鼠膀胱组织缝隙连接蛋白43蛋白的表达及分布，分别采用Western-Blot和qRT-PCR检测缝隙连接蛋白43蛋白和 mRNA 的表达水平。
结果与结论：①经尿道灌注Gja1基因重组慢病毒后，实验组较空白组及对照组的缝隙连接蛋白43蛋白和 mRNA表达水平均明显升高(P < 0.01，P < 0.01)，且基因重组慢病毒在转染后第14天缝隙连接蛋白43蛋白和 mRNA表达最佳；空白组较对照组缝隙连接蛋白43蛋白和 mRNA表达水平差异无显著性意义(P > 0.01)；②结论：经尿道灌注Gja1基因可以稳定表达于膀胱组织中，并能上调缝隙连接蛋白43蛋白和mRNA的表达，有利于修复细胞间受损的信号转导及物质交换通路。
缩略语：缝隙连接蛋白43：Connexin43，Cx43

https://orcid.org/0000-0003-3211-3301(张永强)

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: Gja1基因, 重组慢病毒, 糖尿病膀胱, Cx43蛋白

Abstract: BACKGROUND: Connexin 43 is also known as Gja1 protein. Gja1 recombinant lentivirus may improve bladder contractile dysfunction in diabetes.
OBJECTIVE: To establish a guinea pig model of diabetic cystopathy, study the way of transurethral infusion of Gja1 recombinant lentiviral drugs, and observe the effect on the expression of connexin 43 protein and mRNA on the surface of detrusor muscle cells in the damaged bladder. 
METHODS: Eighty healthy guinea pigs of similar age and body mass were selected, 15 of which were randomly selected and conventionally fed and the remaining 65 were given intraperitoneal injection of 1% streptozotocin (200 mg/kg) to establish a guinea pig model of diabetic cystopathy. Urodynamics screening was performed to identify guinea pigs with diabetic cystopathy, which were further randomized into three groups: blank group (n=12), control group (n=12) and experimental group (n=12). The blank group was perfused with 0.2 mL of phosphate buffered saline, the control group was perfused with 0.2 mL of empty recombinant lentivirus, and the experimental group was perfused with 0.2 mL of Gja1 recombinant lentivirus. Three guinea pigs from each group were killed at each observation time point (2, 7, 14, and 28 days) after transfection. Expression and distribution of connexin 43 protein in the bladder were observed by immunohistochemical staining, and the protein and mRNA levels of connexin 43 were detected by western blot and qRT-PCR assays, respectively, 
RESULTS AND CONCLUSION: After transurethral infusion of Gja1 recombinant lentivirus, the expression levels of connexin 43 protein and mRNA were significantly higher in the experimental group than the blank and control groups (P < 0.01, P < 0.01), and the best expression levels of connexin 43 protein and mRNA were found at 14 days after transfection with Gja1 recombinant lentivirus. There were no significant differences in the expression levels of connexin 43 protein and mRNA between the blank and control groups (P > 0.01). To conclude, Gja1 gene via transurethral perfusion can stably express in bladder tissue and up-regulate the expression of connexin 43 at protein and mRNA levels, which may be beneficial to repair damaged signal transduction and exchange pathways between cells.

Key words: Gja1 gene, recombinant lentivirus, diabetic cystopathy, connexin 43

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