PTEN在血管重建早期的作用

1、Role of the Phosphatase PTEN in Early Vascular Remodeling磷酸酶PTEN在早期血管重建中的作用发表日期：2013年4月 杂志：PLOS ONE IF：3.534研究背景及目的 磷酸酶PTEN是PI3K（磷脂酰肌醇-3-羟激酶）蛋白激酶B（Akt）信号通路的一种重要的生理学抑制剂，在过去的研究中，我们致力于研究在体内和体外情况下PTEN在血管急性损伤后VSMC凋亡方面的作用。然而，PTEN在血管成形术诱导的血管损伤的早期阶段的作用尚不明了。 为了明确该作用机制，我们设计该实验以探究磷酸酶PTEN在早期血管重塑中的作用。技术路线1.体内(颈动

2、脉损伤模型构建)2.体外：成年Wistar大鼠（300g)颈总动脉分离未处理（对照）球囊损伤12h后PTEN表达量检测 取材（HcASMC）细胞培养至第六代干预24h转染共转染PTEN/活化的Akt突变体血清不同生长因子周期性拉伸装置H2O2WT-PTEN质粒SiRNA敲出PTEN基因凋亡VSMC数量检测、Akt磷酸化检测VSMC凋亡数量检测3.汇总数据、统计分析、得出结论PTEN表达量检测实验结果报告：图1A-D 为了探究血管受损后PTEN的表达及细胞凋亡情况，研究人员对实验大鼠颈动脉进行球囊损伤，12小时后，免疫组化检测各项指标如上图：PTEN (green), apoptotic smo

3、oth muscle cells (TUNEL staining,red) and nuclei (DAPI, blue） 由图可知：球扩后损伤较严重的区域PTEN表达量较多，正常细胞数量少、凋亡细胞数量密集（图上半部分）实验结果报告：图1E-Fwestern blot：using a specific PTEN antibody微管蛋白 不同时间点PTEN相对表达量 densitometric analysis of immunoblots 对经/未经球扩后的标本裂解后通过免疫沉淀反应进行PTEN活性检测： Immunoprecipitations employing an IgG iso-

4、antibody and without addition of any antibodies served as controls由图可知：在大鼠颈动脉受损后12H内，PTEN的表达具有时间依赖性（递增） 与对照组相比，其活性明显高于未受损的大鼠颈动脉标本。时间依赖性？实验结果报告：图2A-EA:Lysates of cells exposed to mechanical forces using a stretching devicewere analyzed by western blotting using specific antibodies；B:Lysates of SMC ex

5、posed to growth medium (FCS). Cdk4（周期素依赖性激酶） served as loading control.C:Lysates of SMC exposed to oxidative stress using 500 mM H2O2. p53 and Cdk4 served as apoptotic marker and loading control,respectively.D：PTEN相对表达量的检测： quantifiedby densitometric analysis of immunoblots E：PTEN的活性检测： phosphatase

6、activity assay of immunoprecip-itated protein from lysatesof HcASMC注：PTEN（抗PTEN抗体） Bpv(一种PTEN的特异性抑制剂)由图可知：PTEN受H2O2的氧化应激刺激后表达显著增加，且上调的PTEN活性可被Bpv抑制。 为了探究哪些因素可以诱导PTEN的表达设计如下实验：实验结果报告：图3A-CA:HcASMC transfected with a plasmid carrying WT-PTEN or a control (empty) vector. PTEN protein-expression levels

7、weredetermined by immunoblotting HcASMC were transfected as follows: non transfected (NT); transfected with an emptyplasmid without PTEN-cDNA (pControl); transfected with a plasmid coding for PTEN (pPTEN).B: SMC were incubated in basalmedium .C：SMC were incubated in basal medium supplemented with H2

8、O2 and therelative number of apoptotic cells was evaluated following TUNELstaining由图可知:转染了WT-PTEN质粒的HcASMC其PTEN表达明显增多，凋亡的细胞数显著增多且在基础培养基中加入H2O2之后更明显。为了进一步探究PTEN的上调与SMC凋亡的关系进行如下实验：实验结果报告：图4A-CA:Protein expression was determined bywestern blotting. Detection of Cdk4 served as a loading control. SMCtran

9、sfected with siRNA targeting PTEN or a scrambled control wereincubated in basal medium in the absence (B) or presence of H2O2 (C).SMC were transfected as follows: non transfected(NT); mock transfected (without siRNA, but with lipid carrier);transfected with a non-targeting (scrambled) siRNA (Control

10、 siRNA);transfected with a targeting siRNA against PTEN (PTEN siRNA). 由图可知：与对照组相比，经siRNA敲出PTEN基因之后，VSMC的PTEN表达量明显减少，凋亡的SMC数量亦显著减少。敲出PTEN基因后PTEN的表达及凋亡细胞数量变化实验结果报告：图5A-CA：Akt磷酸化检测（western blot）An antibody against total Akt （Pan Akt）wasused as control. B: PI3-K-inhibitor Ly294002 (50 nM) was supplement

11、ed to the medium for not-transfected cells. HcASMC weretransfected as follows: non transfected (NT); transfected with a plasmidcoding for GFP (pGFP); transfected with an empty plasmid withoutPTEN-cDNA (pControl); transfected with a plasmid coding for PTEN(pPTEN); mock trans-fected (without plasmid,

12、but with transfection reagent). PTEN- and Akt co-overexpression reverses PTENs proapoptotic effect (C). 注：注： GFP（绿色荧光蛋白）（绿色荧光蛋白）由图可知：与对照组相比，转染了PTEN质粒的HcASMC其Akt磷酸化明显减弱；在未转染组中加入PI3K抑制剂（Ly294002）之后Akt磷酸化也明显减弱；当共转染了PTEN质粒和活化的Akt之后可以部分翻转SMC凋亡。为了探究PTEN与Akt磷酸化及细胞凋亡的关系，进行如下实验：实验结果报告：图6A-BA：生长培养基（FCS）培养条件下

13、SMC增殖测定：PTEN overexpression attenuates serum Induced HcASMC proliferation (A). HcASMC transfected with a plasmidcarrying WT-PTEN or a control vector carrying GFP alone were incubated either in basal medium or growth medium in thepresence of BrdU. B:基础/生长培养基培养条件下 SMC增殖测定：HcASMC were transfected as following: transfected with a non-targeting (scrambled) siRNA (Control siRNA); transfected with a targeting siRNA against PTEN (PTEN siRNA).由图可知：与对照组相比，转染了WT-PTEN质粒后SMC增殖明显减少，敲出P