PI3-k/Akt shRNA对大鼠Thy-1肾炎增生病变的影响

【作者】 张艳；

【导师】 王迎伟；

【作者基本信息】 南京医科大学 ， 免疫学， 2008， 硕士

【摘要】 第一部分PI3-k/Akt shRNA真核表达质粒的构建及鉴定目的:构建并鉴定针对PI3-k/Akt基因的特异性短发卡RNA（shRNA）真核表达载体,探讨沉默大鼠肾组织内PI3-k/Akt基因对Thy-1肾炎（Thy-1 N）增生病变的影响。方法:用DNA重组技术将针对大鼠PI3-k/Akt基因不同位点所设计的8对shRNA序列克隆到真核表达质粒pGenesil-1中。在酶切分析及序列测定后,用脂质体转染至大鼠GMCs中,Western blotting检测蛋白的相对表达量来筛选最佳沉默效率的shRNA。结果:限制性酶切及核酸序列分析表明,重组质粒构建正确。Western blotting证实,在多种重组质粒中,以shPik3c3-2、shAkt1-4具有最佳的沉默效率。结论:成功构建了PI3-k/Akt shRNA的真核表达质粒,该结果为进一步体内研究沉默PI3-k/Akt信号通路基因对Thy-1 N病变的抑制作用奠定了实验基础。第二部分沉默PI3-k/Akt信号通路对Thy-1 N增生病变的影响目的:探讨用发夹状小干涉RNA（shRNA）沉默大鼠肾组织内PI3-k/Akt基因对Thy-1N增生病变的影响。方法:用水流动力学注射法将shPik3c3-2、shAkt1-4导入大鼠Thy-1 N肾组织,以Real-time PCR检测大鼠肾皮质中血小板反应蛋白-1（Thrombospondin-1,Tsp-1）、转化生长因子-beta 1（Transforming growth factor-β₁,TGF-β₁）、细胞周期蛋白（cyclinD₂）和纤维连接蛋白（Fibronectin,FN）mRNA丰度,Western blotting和免疫组化检测P-Akt、T-Akt、Tsp-1、TGF-β₁、cyclin D₂和FN蛋白表达水平。此外,实验还观察了大鼠肾脏组织病理学及24h尿蛋白的变化。结果:将shPik3c3-2、shAkt1-4导入Thy-1 N大鼠肾组织后,发现大鼠肾皮质中P-Akt和T-Akt蛋白表达水平均明显减低,同时,Tsp-1、TGF-β₁、cyclin D₂和FN mRNA和蛋白表达水平也显著下调。大鼠肾皮质的组织病理学检查显示,肾小球内系膜细胞增生及细胞外基质过度积聚现象显著减轻。24h尿蛋白分泌也明显降低。结论:沉默Thy-1 N大鼠肾组织内PI3-k/Akt基因,能够抑制Tsp-1、TGF-β₁的合成,减轻Thy-1 N增生病变。提示,PI3-k/Akt信号分子有望成为今后治疗人类系膜增生性肾炎的一个靶点。更多还原

【Abstract】 PartⅠConstruction and identification of eukaryotic vectors expressing shRNA targetting PI3-K/Akt geneObjective:To construct PI3-k/Akt shRNA expression vectors for observing the effect of silencing PI3-k/Akt signal pathway on preventing renal cell proliferation of rats with Thy-1 nephritis（Thy-1 N）. Methods:Eight 19～21bp reverse repeated motifs targeting of PI3-k/Akt gene were synthesized and cloned into eukaryotic expression plasmid pGenesil-1.After being screened and sequencing confirmed,the recombinant plasmids were transfected into rat glomerular mesangial cells （GMCs）,then the levels of P-Akt and T-Akt protein in rat GMCs were measured using Western blotting to select the optimal shRNA.Results:It was verified by partial nucleotide sequencing and restriction endonuclease digestion that the constructed eukaryotic vector expressing shRNA of PI3-K/Akt were correct.Western blotting results showed that the optimal shRNA plasmids which could effectively silence the target genes were shPik3c3-2 and shAkt1-4 respectively.Conclusion:The eukaryotic vectors expressing shRNA of PI3-k/Akt were constructed successfully.The results of the study lay the foundation for further studying on biological functions of PI3-k/Akt signal pathway in Thy-1 N. PartⅡSuppression of mesangial cell proliferation and extracelluar matrix production in Thy-1 nephritis rats by knockdown of PI3-k/Akt with shRNAObjective:To explore the effect of silencing PI3-k/Akt signal pathway on suppressing of mesangial cell proliferation and extracelluar matrix production in Thy-1 nephritis rats.Methods:shPik3c3-2 or/and shAkt1-4 plasmids were transferred into the kideny of Thy-1 nephritis rats by usinghydrodynamic injection method.The expressions of Tsp-1,TGF-β₁, cyclin D₂ and FN mRNA were assessed by Real-time PCR and the protein levels of P-Akt,T-Akt,Tsp-1,TGF-β₁ cyclind₂ and FN weredetected by Western blotting or Immunohisto- chemistry.In addition,histopathology and Urine protein were both observed.Results:In the renal cortex of Thy-1 nephritis rats transferred with shPik3c3-2,shAkt1-4, shPik3c3-2+shAkt1-4,the expressions of P-Akt and T-Akt were successfully reduced.And the expressions of Tsp-1,TGF-β₁,cyclin D₂ and FN were also inhibited.Meanwhile,the glomerular cell proliferation and extracellular matrix accumulation were markedly suppressed in concomitant with downregulation excretion of Urinary protein.Conclusion: The amelioration of the pathological findings of Thy-1 nephritis by silencing PI3-k/Akt signal pathway suggests that PI3-k/Akt may serve as a potential therapeutic target for mesangial proliferation nephritis in the future.更多还原