PI3-k/Akt shRNA对大鼠Thy-1肾炎增生病变的影响
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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-β_1,TGF-β_1)、细胞周期蛋白(cyclinD_2)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-β_1、cyclin D_2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-β_1、cyclin D_2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-β_1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。
PartⅠConstruction and identification of eukaryotic vectors expressing shRNA targetting PI3-K/Akt gene
Objective: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 shRNA
Objective: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-β_1, cyclin D_2 and FN mRNA were assessed by Real-time PCR and the protein levels of P-Akt,T-Akt,Tsp-1,TGF-β_1 cyclind_2 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-β_1,cyclin D_2 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.
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-β_1,TGF-β_1)、细胞周期蛋白(cyclinD_2)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-β_1、cyclin D_2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-β_1、cyclin D_2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-β_1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。
PartⅠConstruction and identification of eukaryotic vectors expressing shRNA targetting PI3-K/Akt gene
Objective: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 shRNA
Objective: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-β_1, cyclin D_2 and FN mRNA were assessed by Real-time PCR and the protein levels of P-Akt,T-Akt,Tsp-1,TGF-β_1 cyclind_2 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-β_1,cyclin D_2 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.
引文
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15. Shtivelman E, Sussman J, Stokoe D. A role for PI 3-kinase and PKB activity in the G2/M phase of the cell cycle[J].Curr Biol.2002;12:919-24.
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26. Barber DF, Bartolome A, Hernandez C, et al. PDKgamma inhibition blocks glomerulonephritis and extends lifespan in a mouse model of systemic lupus [J]. Nat Med. 2005; 11: 933-5.
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28. Thirone AC, Scarlett JA, Gasparetti AL, et al. Modulation of growth hormone signal transduction in kidneys of streptozotocin-induced diabetic animals: effect of a growth hormone receptor antagonist [J].Diabetes. 2002; 51: 2270-81.
29. Dobrzynski E, Montanari D, Agata J, Zhu J, Chao J, Chao L.Adrenomedullin improves cardiac function and prevents renal damage in streptozotocin-induced diabetic rats [J]. Am J Physiol Endocrinol Metab. 2002; 283: E1291-8.
30. Dai C, Yang J, Liu Y. Single injection of naked plasmid encoding hepatocyte growth factor prevents cell death and ameliorates acute renal failure in mice [J]. J Am Soc Nephrol. 2002; 13:411-22.
31. H Kuwana, Y Terada, T Kobayashi, et al. The phosphoinositide-3 kinase-Akt pathway mediates renal tubular injury in cisplatin nephrotoxicity [J]. Kidney International. 2008; 73: 430-445.
32. Rodriguez-Pena AB, Grande MT, Eleno N, et al. Activation of Erk1/2 and Akt following unilateral ureteral obstruction [J]. Kidney Int. 2008 Apr 30.
33. Sourbier C, Lindner V, Lang H, et al. The phosphoinositide 3-kinase/Akt pathway: a new target in human renal cell carcinoma therapy [J]. Cancer Res. 2006; 66: 5130-42.
34. Horiguchi A, Oya M, Uchida A, Marumo K, Murai M. Elevated Akt activation and its impact on clinicopathological features of renal cell carcinoma [J]. J Urol. 2003; 169: 710-713.
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