PTEN在血管重建早期的作用学习教案

1、会计学1PTEN在血管重建早期在血管重建早期(zoq)的作用的作用第一页，共13页。第1页/共12页第二页，共13页。1.体内(颈动脉损伤(snshng)模型构建)2.体外：成年(chngnin)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表达量检测第2页/共12页第三页，共13页。实验(

2、shyn)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 smooth 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(mj)（图上半部分）第3页/共12页第四页，共13页。实验(shyn)结果报告：图1E-Fwestern blot：using a specific PTEN antibody微

3、管(wi un)蛋白 不同时间(shjin)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-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？第4页/共12页第五页，共13页。

4、实验(shyn)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 exposed to growth medium (FCS). Cdk4（周期素依赖性激酶(jmi)） served as loading control.C:Lysates of SMC exposed to oxidative stress using 500

5、 mM H2O2. p53 and Cdk4 served as apoptotic marker and loading control,respectively.D：PTEN相对(xingdu)表达量的检测： quantifiedby densitometric analysis of immunoblots E：PTEN的活性检测： phosphatase 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

6、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：第5页/共12页第六页，共13页。实验(shyn)结果报告：图3A-CA:HcASMC transfected with a plasmid carrying WT-PTEN or a control (empty) vector. PTEN protein-expression levels weredetermined by immunoblotting HcASMC were transfected as follows: non transfected (NT); tra

7、nsfected 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 H2O2 and therelative number of apoptotic cells was evaluated following TUNELstaining由图可知:转染了WT-P

8、TEN质粒的HcASMC其PTEN表达明显增多(zn du)，凋亡的细胞数显著增多(zn du)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(shn dio)与SMC凋亡的关系进行如下实验：第6页/共12页第七页，共13页。实验(shyn)结果报告：图4A-CA:Protein expression was determined bywestern blotting. Detection of Cdk4 served as a loading control. SMCtransfected with siRNA targeting PTEN or a scrambled

9、 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 siRNA);transfected with a targeting siRNA again

10、st PTEN (PTEN siRNA). 由图可知(k zh)：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(jyn)后PTEN的表达及凋亡细胞数量变化第7页/共12页第八页，共13页。实验结果(ji gu)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 supplemented to the med

11、ium 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, but with tran

12、sfection reagent). PTEN- and Akt co-overexpression reverses PTENs proapoptotic effect (C). 注： GFP（绿色荧光(ynggung)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(b fen)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：第8页/共12页第九页，共13页。实验结果(ji gu)报告：图6A-BA

13、：生长培养基（FCS）培养条件下 SMC增殖(zngzh)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(zngzh)测

14、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PTEN基因后其增殖显著增多且在生长(shngzhng)培养基上表现更显著。探究PTEN对SMC增殖的影响：第9页/共12页第十页，共13页。实验(shyn)结论 通过上述实验结果得出如下结论： 通过对依赖PI3-K/Akt的生存信号的干扰， 氧化应激诱导的PTEN上调在损伤的颈动脉VSMC中能增强血管平滑肌细胞对在损伤后早期阶段细胞凋亡刺激的敏感度，进而增加细胞的凋亡和细胞缺失。 简言之：血管损伤早期 PTEN上调 Akt磷酸化被抑制(yzh) 细胞凋亡增多（增殖受抑）影响血管重塑第10页/共12页第十一页，共13页。第11页/