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目的构建、鉴定自身互补双链DNA的腺相关病毒(scAAV)重组载体,使其分泌表达Exendin-4,检测其转导效率并观察在糖尿病大鼠模型中的治疗作用。方法应用基因工程方法改建穿梭质粒pSSHG-CMV,插入外源性基因Exendin-4,构建重组scAAV载体,感染HEK293细胞,ELISA检测转染NIH3T3细胞上清Exendin-4滴度,链佐霉素诱导20只6周龄体质量180~220 g SD成年雄性大鼠为糖尿病鼠模型,逆向注射重组scAAV于糖尿病大鼠颌下腺,检测其血糖及胰岛素分泌水平。结果重组scpSSHG/exn4可有效包装和复制,病毒滴度为2.5×1011pfu/mL,转染细胞上清分泌Exendin-4浓度可达到4.53 ng/mL,scAAV治疗组血糖浓度在2、4周及8周均低于对照组[分别为(639.17±27.89)vs(396.00±34.00),(657.02±39.87)vs(315.62±42.56),(215.6±24.7)vs(458.6±19.7)mg/dL],胰岛素浓度均高于对照组[分别为(156.8±24.5)vs(535.9±35.6),(236.5±12.3)vs(495.3±18.6),(620.43±46.90)vs(381.56±21.78)pg/mL],二者比较有显著统计学差异(P<0.05)。结论成功构建重组双链腺伴病毒scAAV-Ex-4,具有高效转导能力,对糖尿病大鼠模型具有控制血糖及增加胰岛素分泌作用。
Objective To construct and identify a recombinant adeno-associated virus (scAAV) vector with its own complementary double-stranded DNA to secrete and express Exendin-4. The transduction efficiency and the therapeutic effect in diabetic rat model were observed. Methods The shuttle plasmid pSSHG-CMV was constructed by gene engineering method. The exogenous gene Exendin-4 was inserted into HEK293 cells. The expression of Exendin-4 in supernatant of NIH3T3 cells was detected by ELISA. Adult male rats with body weight 180-220 g at 6 weeks of age were all diabetic rats. Reverse scAAV was injected into the submandibular gland of diabetic rats to detect their blood glucose and insulin secretion. Results Recombinant scpSSHG / exn4 could be effectively packaged and replicated. The titer of virus was 2.5 × 10 11 pfu / mL. The concentration of Exendin-4 secreted by transfected cells was 4.53 ng / mL. The serum concentration of scpSSHG / Weekly lower than the control group [(639.17 ± 27.89 vs 396.00 ± 34.00 vs 657.02 ± 39.87 vs 315.62 ± 42.56 vs 215.6 ± 24.7 vs 458.6 ± 19.7 mg / dL, respectively) (156.8 ± 24.5 vs 535.9 ± 35.6 vs 236.5 ± 12.3 vs 495.3 ± 18.6 vs 620.43 ± 46.90 vs 381.56 ± 21.78 pg / mL, respectively) There was significant statistical difference (P <0.05). Conclusion Recombinant double-stranded adeno-associated virus scAAV-Ex-4 is successfully constructed and has high transduction ability. It can control blood glucose and increase insulin secretion in diabetic rats.