rAAV9介导的siRNA靶向USP11调控2型糖尿病大鼠血糖及视网膜NF-κB表达的机制研究

Study on mechanism of rAAV9-mediated siRNA targeting USP11 in regulating blood glucose and retinal NF-κB expression in rats with type 2 diabetes mellitus

目的 探讨9型重组腺相关病毒介导的去泛素化酶siRNA基因敲除对2型糖尿病大鼠血糖及视网膜核因子κB表达的影响。方法 将48只大鼠按照随机数字表法分为对照组、2型糖尿病组、9型重组腺相关病毒组和9型重组腺相关病毒+去泛素化酶敲低组。2型糖尿病组、9型重组腺相关病毒组和9型重组腺相关病毒+去泛素化酶敲低组大鼠采用链脲佐菌素腹腔注射诱导2型糖尿病大鼠模型,对照组大鼠仅注射等剂量生理盐水,造模成功后尾静脉分别注射生理盐水、9型重组腺相关病毒慢病毒载体和9型重组腺相关病毒+去泛素化酶敲低慢病毒载体。造模4周及8周末检测各组大鼠体质量和血糖、血脂水平,收集视网膜切片,采用免疫组织化学法和免疫印记法检测核因子κB的表达。结果 造模前后4组大鼠体质量比较差异有统计学意义(P<0.01),造模4周及8周末4组间比较差异有统计学意义(P<0.01),对照组最高,2型糖尿病组最低。造模前后4组大鼠血糖水平比较差异有统计学意义(P<0.05或0.01),造模4周及8周末4组间比较差异有统计学意义(P<0.01),2型糖尿病组最高,对照组最低。造模前后4组大鼠血清甘油三酯水平比较差异有统计学意义(P<0.05或0.01),造模4周及8周末4组间比较差异有统计学意义(P<0.01),2型糖尿病组最高,对照组最低。2型糖尿病组大鼠视网膜核因子κB阳性水平最高,其次为9型重组腺相关病毒组、9型重组腺相关病毒+去泛素化酶敲低组,对照组阳性水平最低。2型糖尿病组大鼠视网膜核因子κB蛋白表达最高,其次为9型重组腺相关病毒组、9型重组腺相关病毒+去泛素化酶敲低组,对照组最低。结论 9型重组腺相关病毒介导的去泛素化酶siRNA基因敲除可显著降低2型糖尿病大鼠血糖、血脂水平,并抑制视网膜核因子κB的表达,发挥保护视网膜的作用。

Objective To investigate the effect of ubiquitin-specific protease11(USP11) siRNA gene knockout mediated by recombinant adeno-associated virus 9(rAAV9) on blood glucose and retinal nuclear factor kappa-B(NF-κB) expression in rats with type 2 diabetes mellitus(T2DM). Methods Forty-eight rats were divided into control group, T2DM group, rAAV9 group and rAAV9+si-USP11 group. Rats in the T2 DM group, rAAV9 group and rAAV9+si-USP11 group were injected with streptozotocin(STZ) intraperitoneally to induce rat models of T2DM, and rats in the control group were injected with the same dose of normal saline. After successful modeling, normal saline, RAAV9 lentivirus vector and rAAV9+si-USP11 lentivirus vector were injected into the tail vein respectively. At the end of 4 weeks and 8 weeks of modeling, the body mass, blood glucose and blood lipids of rats in each group were measured, and retinal sections were collected. Immunohistochemistry and Western blotting were used to detect the expression of NF-κB.Results There was a statistical significance in the body mass in the four groups before and after modeling(P<0.01),and there was also a statistical difference among the four groups at the end of 4 weeks and 8 weeks of modeling(P<0.01),and the body mass was the highest in control group and the lowest in T2DM group.The difference in blood glucose in the four groups was statistically significant before and after modeling(P<0.05 or 0.01),and there was a statistical significance among the four groups at the end of 4 weeks and 8 weeks of modeling(P<0.01),and the level was the highest in T2DM group and the lowest in control group.Serum triglyceride level in the four groups showed a statistical difference before and after modeling(P<0.05 or 0.01),and the level also showed a statistical significance at the end of 4 weeks and 8 weeks of modeling among the four groups(P<0.01),and the level was the highest in T2DM group and the lowest in control group.The positive level of retinal NF-κB in T2DM group was the highest,followed by rAAV9group and rAAV9+siUSP11group,and the lowest in control group.The NF-κB protein expression was the highest in T2DM group,followed by rAAV9group and rAAV9+si-USP11group,and the lowest in control group.Conclusions rAAV9-mediated USP11siRNA gene knockout can significantly reduce blood glucose and blood lipid and inhibit the expression of retinal NF-κB in rats with T2DM,and it plays a protective role in the retina.