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發(fā)表日期：2013年4月 雜志：PLOS ONE IF：3.534研究背景及目的 磷酸酶PTEN是PI3K（磷脂酰肌醇-3-羥激酶）蛋白激酶B（Akt）信號通路的一種重要的生理學(xué)抑制劑，在過去的研究中，我們致力于研究在體內(nèi)和體外情況下PTEN在血管急性損傷后VSMC凋亡方面的作用。然而，PTEN在血管成形術(shù)誘導(dǎo)的血管損傷的早期階段的作用尚不明了。 為了明確該作用機(jī)制，我們設(shè)計(jì)該實(shí)驗(yàn)以探究磷酸酶PTEN在早期血管重塑中的作用。技術(shù)路線1.體內(nèi)(頸動(dòng)

2、脈損傷模型構(gòu)建)2.體外：成年Wistar大鼠（300g)頸總動(dòng)脈分離未處理（對照）球囊損傷12h后PTEN表達(dá)量檢測 取材（HcASMC）細(xì)胞培養(yǎng)至第六代干預(yù)24h轉(zhuǎn)染共轉(zhuǎn)染PTEN/活化的Akt突變體血清不同生長因子周期性拉伸裝置H2O2WT-PTEN質(zhì)粒SiRNA敲出PTEN基因凋亡VSMC數(shù)量檢測、Akt磷酸化檢測VSMC凋亡數(shù)量檢測3.匯總數(shù)據(jù)、統(tǒng)計(jì)分析、得出結(jié)論P(yáng)TEN表達(dá)量檢測實(shí)驗(yàn)結(jié)果報(bào)告：圖1A-D 為了探究血管受損后PTEN的表達(dá)及細(xì)胞凋亡情況，研究人員對實(shí)驗(yàn)大鼠頸動(dòng)脈進(jìn)行球囊損傷，12小時(shí)后，免疫組化檢測各項(xiàng)指標(biāo)如上圖：PTEN (green), apoptotic smo

3、oth muscle cells (TUNEL staining,red) and nuclei (DAPI, blue） 由圖可知：球擴(kuò)后損傷較嚴(yán)重的區(qū)域PTEN表達(dá)量較多，正常細(xì)胞數(shù)量少、凋亡細(xì)胞數(shù)量密集（圖上半部分）實(shí)驗(yàn)結(jié)果報(bào)告：圖1E-Fwestern blot：using a specific PTEN antibody微管蛋白 不同時(shí)間點(diǎn)PTEN相對表達(dá)量 densitometric analysis of immunoblots 對經(jīng)/未經(jīng)球擴(kuò)后的標(biāo)本裂解后通過免疫沉淀反應(yīng)進(jìn)行PTEN活性檢測： Immunoprecipitations employing an IgG iso-

4、antibody and without addition of any antibodies served as controls由圖可知：在大鼠頸動(dòng)脈受損后12H內(nèi)，PTEN的表達(dá)具有時(shí)間依賴性（遞增） 與對照組相比，其活性明顯高于未受損的大鼠頸動(dòng)脈標(biāo)本。時(shí)間依賴性？實(shí)驗(yàn)結(jié)果報(bào)告：圖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（周期素依賴性激酶） 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相對表達(dá)量的檢測： quantifiedby densitometric analysis of immunoblots E：PTEN的活性檢測： phosphatase

6、activity assay of immunoprecip-itated protein from lysatesof HcASMC注：PTEN（抗PTEN抗體） Bpv(一種PTEN的特異性抑制劑)由圖可知：PTEN受H2O2的氧化應(yīng)激刺激后表達(dá)顯著增加，且上調(diào)的PTEN活性可被Bpv抑制。 為了探究哪些因素可以誘導(dǎo)PTEN的表達(dá)設(shè)計(jì)如下實(shí)驗(yàn)：實(shí)驗(yàn)結(jié)果報(bào)告：圖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由圖可知:轉(zhuǎn)染了WT-PTEN質(zhì)粒的HcASMC其PTEN表達(dá)明顯增多，凋亡的細(xì)胞數(shù)顯著增多且在基礎(chǔ)培養(yǎng)基中加入H2O2之后更明顯。為了進(jìn)一步探究PTEN的上調(diào)與SMC凋亡的關(guān)系進(jìn)行如下實(shí)驗(yàn)：實(shí)驗(yàn)結(jié)果報(bào)告：圖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). 由圖可知：與對照組相比，經(jīng)siRNA敲出PTEN基因之后，VSMC的PTEN表達(dá)量明顯減少，凋亡的SMC數(shù)量亦顯著減少。敲出PTEN基因后PTEN的表達(dá)及凋亡細(xì)胞數(shù)量變化實(shí)驗(yàn)結(jié)果報(bào)告：圖5A-CA：Akt磷酸化檢測（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（綠色熒光蛋白）（綠色熒光蛋白）由圖可知：與對照組相比，轉(zhuǎn)染了PTEN質(zhì)粒的HcASMC其Akt磷酸化明顯減弱；在未轉(zhuǎn)染組中加入PI3K抑制劑（Ly294002）之后Akt磷酸化也明顯減弱；當(dāng)共轉(zhuǎn)染了PTEN質(zhì)粒和活化的Akt之后可以部分翻轉(zhuǎn)SMC凋亡。為了探究PTEN與Akt磷酸化及細(xì)胞凋亡的關(guān)系，進(jìn)行如下實(shí)驗(yàn)：實(shí)驗(yàn)結(jié)果報(bào)告：圖6A-BA：生長培養(yǎng)基（FCS）培養(yǎng)條件下

13、SMC增殖測定：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:基礎(chǔ)/生長培養(yǎng)基培養(yǎng)條件下 SMC增殖測定：HcASMC were transfected as following: transfected with a non-targeting (scrambled) siRNA (Control siRNA); transfected with a targeting siRNA against PTEN (PTEN siRNA).由圖可知：與對照組相比，轉(zhuǎn)染了WT-PTEN質(zhì)粒后SMC增殖明顯減少，敲出P