血小板源性生長(zhǎng)因子和血小板源性內(nèi)皮細(xì)胞生長(zhǎng)因子在內(nèi)皮細(xì)胞和血管平滑肌細(xì)胞中的作用研究

朱晉坤，毛 華，尹揚(yáng)光，董 文，杜 峰，魯玉明，熊宗華，鄧夢(mèng)揚(yáng)

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·論著·

血小板源性生長(zhǎng)因子和血小板源性內(nèi)皮細(xì)胞生長(zhǎng)因子在內(nèi)皮細(xì)胞和血管平滑肌細(xì)胞中的作用研究

朱晉坤，毛 華，尹揚(yáng)光，董 文，杜 峰，魯玉明，熊宗華，鄧夢(mèng)揚(yáng)

目的 通過體外轉(zhuǎn)染血小板源性生長(zhǎng)因子(PDGF)和血小板源性內(nèi)皮細(xì)胞生長(zhǎng)因子(PD-ECGF)質(zhì)粒，觀察兩種生長(zhǎng)因子對(duì)人臍靜脈內(nèi)皮細(xì)胞EAHY926和主動(dòng)脈血管平滑肌細(xì)胞T/G HA-VSMC的影響，評(píng)估兩種生長(zhǎng)因子對(duì)血管損傷治療的可行性。方法 構(gòu)建人pcDNA 3.1(+)空載(空載組)、pcDNA 3.1(+)-PDGF-透明質(zhì)酸(HA) (PPH組) 和pcDNA 3.1(+)-PD-ECGF-HA (PPEH組)質(zhì)粒，分別轉(zhuǎn)染入EAHY926和T/G HA-VSMC中，四甲基偶氮唑鹽微量酶反應(yīng)比色法(MTT法)檢測(cè)內(nèi)源性PD-ECGF和外源性PD-ECGF對(duì)EAHY926和T/G HA-VSMC細(xì)胞增殖的影響；細(xì)胞傷痕實(shí)驗(yàn)觀察內(nèi)源性PD-ECGF和外源性PD-ECGF對(duì)EAHY926和T/G HA-VSMC細(xì)胞遷移速度的影響。結(jié)果 MTT法檢測(cè)3組EAHY926吸光度值比較，差異有統(tǒng)計(jì)學(xué)意義(F=235.18，P<0.001)，其中PPH組高于空載組和PPEH組(P<0.05)；空載組與PPEH組比較，差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。MTT法檢測(cè)3組T/G HA-VSMC吸光度值比較，差異有統(tǒng)計(jì)學(xué)意義(F=82.89，P<0.001)，其中PPH組高于空載組(P<0.05)；PPEH組低于空載組(P<0.05)。MTT法檢測(cè)轉(zhuǎn)染PPEH質(zhì)粒的培養(yǎng)基(L-PPEH)對(duì)PPH誘導(dǎo)的EAHY926和T/G HA-VSMC增殖的影響，空載組、PPH組、PPH+L-PPEH組EAHY926吸光度值比較，差異無統(tǒng)計(jì)學(xué)意義(F=512.89,P=0.183)?？蛰d組、PPH組、PPH+L-PPEH組T/G HA-VSMC吸光度值比較，差異有統(tǒng)計(jì)學(xué)意義(F=317.40,P<0.001)；其中PPH組高于空載組和PPH+L-PPEH組(P<0.05)。細(xì)胞傷痕實(shí)驗(yàn)證實(shí)，轉(zhuǎn)染PPEH質(zhì)粒的EAHY926、T/G HA-VSMC細(xì)胞遷移能力增強(qiáng)；PPEH組EAHY926平均細(xì)胞遷移距離百分比(56.1%±2.2%)較空載組(27.8%±3.4%)升高(t=-15.08,P<0.001)；PPEH組T/G HA-VSMC平均細(xì)胞遷移距離百分比(69.1%±2.3%)較空載組(43.6%±5.8%)升高(t=-33.64,P<0.001)。用轉(zhuǎn)染PPEH質(zhì)粒的培養(yǎng)基培養(yǎng)EAHY926、T/G HA-VSMC發(fā)現(xiàn)，外源性PD-ECGF處理的EAHY926、T/G HA-VSMC細(xì)胞遷移能力亦明顯增強(qiáng)。結(jié)論 通過轉(zhuǎn)染PDGF質(zhì)粒，模擬血管損傷后內(nèi)皮細(xì)胞和平滑肌細(xì)胞受到PDGF和/或PD-ECGF刺激，發(fā)現(xiàn) PDGF能明顯上調(diào)內(nèi)皮細(xì)胞和血管平滑肌的增殖和遷移。PD-ECGF對(duì)血管內(nèi)皮細(xì)胞增殖的效果不明顯，但能上調(diào)血管內(nèi)皮細(xì)胞和血管平滑肌的遷移速度；同時(shí)PD-ECGF能抑制血管內(nèi)皮細(xì)胞的增殖。兩種因子同時(shí)使用能抑制平滑肌細(xì)胞的過度增殖，促進(jìn)內(nèi)皮細(xì)胞和平滑肌細(xì)胞的遷移。兩者聯(lián)合使用可能成為促進(jìn)經(jīng)皮冠狀動(dòng)脈介入術(shù)(PCI)后損傷血管恢復(fù)及預(yù)防血管再狹窄的新策略。

血小板源性生長(zhǎng)因子；血小板源性內(nèi)皮細(xì)胞生成因子；血管成形術(shù)，氣囊，冠狀動(dòng)脈；肌細(xì)胞,平滑??；內(nèi)皮細(xì)胞；細(xì)胞增殖

朱晉坤，毛華，尹揚(yáng)光，等.血小板源性生長(zhǎng)因子和血小板源性內(nèi)皮細(xì)胞生長(zhǎng)因子在內(nèi)皮細(xì)胞和血管平滑肌細(xì)胞中的作用研究[J].中國(guó)全科醫(yī)學(xué)，2015，18(9)：1023-1028.[www.chinagp.net]

Zhu JK,Mao H,Yin YG,et al.Role of PDGF and PD-ECGF in endothelial cells and vascular smooth muscle cells[J].Chinese General Practice，2015，18(9)：1023-1028.

經(jīng)皮冠狀動(dòng)脈介入術(shù)(percutaneous coronary intervention，PCI)后靶血管存在血栓形成及血管舒縮功能喪失現(xiàn)象，至今仍是臨床應(yīng)用面臨的重大難題。損傷局部血管再內(nèi)皮化不良是上述過程發(fā)生的機(jī)制之一。在血管損傷時(shí)，血管內(nèi)皮細(xì)胞起著血管修復(fù)以及新生血管的作用[1-2]。血小板源性生長(zhǎng)因子(PDGF)有助于內(nèi)皮細(xì)胞的增殖[3]，但同時(shí)PDGF也能加速平滑肌細(xì)胞增殖和遷移[4-5]，這就意味著PDGF很可能導(dǎo)致PCI后靶血管再狹窄。而血小板源性內(nèi)皮細(xì)胞生成因子(PD-ECGF)也被稱為胸苷磷酸化酶(TP)，能夠加速損傷局部血管內(nèi)皮生成和遷移，但與PDGF相反的是，PD-ECGF能抑制局部平滑肌細(xì)胞過度增殖[6]。PDGF最初是在血小板細(xì)胞中發(fā)現(xiàn)并由血小板分泌用于創(chuàng)傷修復(fù)的生長(zhǎng)因子，PDGF是創(chuàng)傷愈合過程中較早出現(xiàn)的生長(zhǎng)因子之一,在創(chuàng)面愈合的全過程中起重要作用。在正常情況下，PDGF在血管內(nèi)皮細(xì)胞僅有少量表達(dá)，在血管內(nèi)皮受損如缺血、創(chuàng)傷及炎癥等病理?xiàng)l件下，其在血管內(nèi)皮的表達(dá)水平明顯升高。本研究基于PDGF和PD-ECGF的特點(diǎn)，在人臍靜脈血管內(nèi)皮細(xì)胞EAHY926及主動(dòng)脈血管平滑肌細(xì)胞T/G HA-VSMC內(nèi)表達(dá)PDGF，借以模擬血管損傷后內(nèi)皮細(xì)胞和平滑肌細(xì)胞受到PDGF和/或PD-ECGF刺激后兩種細(xì)胞的增殖遷移變化，旨在研究?jī)烧呗?lián)合用于加速損傷血管重塑、預(yù)防PCI后靶血管再狹窄的可行性。

1 材料與方法

1.1 質(zhì)粒構(gòu)建 pcDNA 3.1(+)-透明質(zhì)酸(HA)由第三軍醫(yī)大學(xué)公共實(shí)驗(yàn)平臺(tái)留存，本載體在多克隆位點(diǎn)后引入了HA標(biāo)簽(氨基酸序列為：YPYDVPDYA)，可以與目的蛋白融合表達(dá)以標(biāo)記目的蛋白。合成PDGF和PD-ECGF引物。PDGF上游引物:5′-CCGGAATTCATGAATCGCTGCTGGGCGC-3′,下游引物:5′-ACCCAAGCTTGGCTCCAAGGGTCTCCTT-3′；PD-ECGF上游引物:5′-CCGGAATTCATGGCAGCCTTGATGAC-3′，下游引物:5′-ACCCAAGCTTTTGCTGCGGCGGCAGAACG-3′。采用聚合酶鏈?zhǔn)椒磻?yīng)(PCR)從cDNA中富集PDGF和PD-ECGF全長(zhǎng)。電泳純化后采用限制性內(nèi)切酶HindⅢ和EcoRⅠ進(jìn)行酶切，以表達(dá)載體pcDNA 3.1(+)為骨架，重組連接為pcDNA 3.1(+)-PDGF-HA(PPH)和pcDNA 3.1(+)-PD-ECGF-HA(PPEH)質(zhì)粒。

1.2 細(xì)胞培養(yǎng) EAHY926和T/G HA-VSMC購(gòu)自中國(guó)科學(xué)院典型培養(yǎng)物保藏委員會(huì)細(xì)胞庫(kù)，由本實(shí)驗(yàn)室凍存。兩種細(xì)胞均接種于50 ml細(xì)胞培養(yǎng)瓶中，在含10%胎牛血清(USA)、100 U/ml青霉素和100 μg/ml鏈霉素(hyclone，USA)的RPMI 1640完全培養(yǎng)基中(Life Technology，USA)中，于37 ℃環(huán)境中5%二氧化碳(CO2)孵箱中培養(yǎng)。

1.3 質(zhì)粒轉(zhuǎn)染 按照轉(zhuǎn)染試劑說明書，將2.5 μg (6孔板)或100 ng(96孔板) pcDNA 3.1(+)空載，PPH或PPEH質(zhì)粒與10 μl(6孔板)或0.5 μl (96孔板)轉(zhuǎn)染試劑(lipofectamin 2000，Life Technology)分別稀釋于無血清的240 μl培養(yǎng)基中，5 min后將兩種稀釋液1∶1混合，20 min后加入事先用無血清和抗生素培養(yǎng)基培養(yǎng)的細(xì)胞中，4～6 h后換成RPMI 1640完全培養(yǎng)基繼續(xù)培養(yǎng)。

1.4 免疫印跡法 收集分別轉(zhuǎn)染了pcDNA 3.1(+)空載(空載組)、PPH(PPH組)或PPEH(PPEH組)質(zhì)粒72 h后的EAHY926和T/G HA-VSMC的蛋白，用BCA蛋白濃度檢測(cè)試劑盒和分光光度計(jì)測(cè)定蛋白濃度，并加入上樣緩沖液備用。將總蛋白50 μg于預(yù)先配制好的10%或15%聚丙烯酰胺凝膠上樣孔中進(jìn)行分離，每個(gè)樣本至少用2塊平行膠進(jìn)行分離。通過“三明治”濕法轉(zhuǎn)印于預(yù)先活化好的聚偏氟乙烯(PVDF)膜(merck-millipore，USA)上。轉(zhuǎn)印完成后的PVDF膜用5%脫脂奶粉封閉過夜。一抗孵育2～4 h，磷酸鹽吐溫緩沖液(PBST)洗滌3次，15 min/次；二抗孵育1～2 h，PBST洗滌3次，15 min/次，其后顯色觀察。

1.5 四甲基偶氮唑鹽微量酶反應(yīng)比色法(MTT法) 細(xì)胞接種在96孔板中，每孔6 000個(gè)細(xì)胞，在培養(yǎng)基中培養(yǎng)約24 h后轉(zhuǎn)染PPH或PPEH質(zhì)粒；每組4個(gè)重復(fù)孔；繼續(xù)培養(yǎng)，72 h后，5 mg/ml MTT溶液加入到各孔中，100 μl/孔，于37 ℃溫育2～4 h，除去MTT后每孔

本研究創(chuàng)新點(diǎn)：

損傷血管再內(nèi)皮化及重塑是經(jīng)皮冠狀動(dòng)脈介入術(shù)(PCI)預(yù)后的關(guān)鍵。既往研究證實(shí)血小板源性生長(zhǎng)因子(PDGF)只在損傷血管的內(nèi)皮細(xì)胞中高表達(dá)，以促進(jìn)平滑肌細(xì)胞和內(nèi)皮細(xì)胞的遷移和增殖，但平滑肌細(xì)胞的過度增殖常引起血管的再狹窄，影響PCI效果。同時(shí)，有研究表明，正常情況下，血小板源性內(nèi)皮細(xì)胞生長(zhǎng)因子(PD-ECGF)可能抑制平滑肌細(xì)胞的增殖，但促進(jìn)平滑肌細(xì)胞和內(nèi)皮細(xì)胞的遷移。但對(duì)PD-ECGF在血管損傷、PDGF高表達(dá)的情況下，是否能拮抗PDGF的促增殖效果，抑制平滑肌細(xì)胞增殖，目前為止并沒有明確的報(bào)道。本研究提出假設(shè)，認(rèn)為在損傷血管細(xì)胞中，高表達(dá)PDGF的內(nèi)皮細(xì)胞或平滑肌細(xì)胞的增殖和遷移可能受到外源性PD-ECGF的影響，且PD-ECGF和PDGF的聯(lián)合使用在促進(jìn)血管內(nèi)皮細(xì)胞遷移增殖/加速血管重塑的同時(shí)也可抑制平滑肌細(xì)胞過度增殖。本研究通過高表達(dá)PDGF模擬血管損傷狀態(tài)下的內(nèi)皮細(xì)胞和平滑肌細(xì)胞，證實(shí)其促進(jìn)了平滑肌和內(nèi)皮細(xì)胞的遷移和增殖；在此基礎(chǔ)上，發(fā)現(xiàn)PD-ECGF可以抑制PDGF引起的平滑肌細(xì)胞增殖，但不影響PDGF引起的內(nèi)皮細(xì)胞增殖，且進(jìn)一步促進(jìn)平滑肌細(xì)胞及內(nèi)皮細(xì)胞的遷移。研究結(jié)果為改善PCI預(yù)后、加速損傷血管重塑、防止血管再狹窄提供了潛在的干預(yù)靶標(biāo)。

加入100 μl二甲基亞砜(DMSO)，搖床上緩慢搖動(dòng)30 min。在酶標(biāo)儀中490 nm處讀取吸光度值。

1.6 細(xì)胞傷痕實(shí)驗(yàn) 用標(biāo)記筆在6孔板底部均勻劃上3～4條橫線，將適量EAHY926或T/G HA-VSMC種植于6孔板內(nèi)〔(1～2)×105個(gè)/孔〕，貼壁轉(zhuǎn)染24 h或未轉(zhuǎn)染48 h后用藍(lán)色無菌1 ml槍尖在6孔板內(nèi)傷痕；PBS清洗，除去漂浮細(xì)胞后，加入低濃度血清培養(yǎng)基(含2% FBS的培養(yǎng)基)。以橫線為參照物，取3個(gè)點(diǎn)的平均值，細(xì)胞遷移距離百分比=(0 h時(shí)細(xì)胞平均間距-24 h后細(xì)胞平均間距)/0 h時(shí)細(xì)胞平均間距。

2 結(jié)果

2.1 PPH和PPEH對(duì)EAHY926增殖的影響 3組EAHY926吸光度值比較，差異有統(tǒng)計(jì)學(xué)意義(F=235.18，P<0.001)；其中PPH組高于空載組和PPEH組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；空載組與PPEH組比較，差異無統(tǒng)計(jì)學(xué)意義(P>0.05，見圖1)。

注：PPH=pcDNA 3.1(+)-血小板源性生長(zhǎng)因子-透明質(zhì)酸，PPEH=pcDNA 3.1(+)-血小板源性內(nèi)皮細(xì)胞生長(zhǎng)因子-透明質(zhì)酸；與PPH組比較，*P<0.05

圖1 MTT法檢測(cè)3組EAHY926吸光度值

Figure 1 Absorbance values of EAHY926 among three groups detected by MTT method

2.2 PPH和PPEH對(duì)T/G HA-VSMC增殖的影響 3組T/G HA-VSMC吸光度值比較，差異有統(tǒng)計(jì)學(xué)意義(F=82.89，P<0.001)；其中PPH組高于空載組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；PPEH組低于空載組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05，見圖2)。

注：與空載組比較，*P<0.05

圖2 MTT法檢測(cè)3組T/G HA-VSMC吸光度值

Figure 2 Absorbance values of T/G HA-VSMC among three groups detected by MTT method

2.3 外源性PD-ECGF對(duì)EAHY926和T/G HA-VSMC增殖的影響 MTT法檢測(cè)轉(zhuǎn)染PPEH質(zhì)粒的培養(yǎng)基(L-PPEH)對(duì)PPH誘導(dǎo)的EAHY926和T/G HA-VSMC增殖的影響，空載組、PPH組、PPH+L-PPEH組EAHY926吸光度值比較，差異無統(tǒng)計(jì)學(xué)意義(F=512.89,P=0.183)。空載組、PPH組、PPH+L-PPEH組T/G HA-VSMC吸光度值比較，差異有統(tǒng)計(jì)學(xué)意義(F=317.40,P<0.001)；其中PPH組高于空載組和PPH+L-PPEH組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05，見圖3)。

2.4 PD-ECGF對(duì)EAHY926遷移的影響 細(xì)胞傷痕實(shí)驗(yàn)證實(shí)，轉(zhuǎn)染PPEH質(zhì)粒的EAHY926細(xì)胞遷移能力增強(qiáng)(見圖4A)；PPEH組EAHY926平均細(xì)胞遷移距離百分比(56.1%±2.2%)較空載組(27.8%±3.4%)升高，差異有統(tǒng)計(jì)學(xué)意義(t=-15.08,P<0.001)。

用轉(zhuǎn)染PPEH質(zhì)粒的培養(yǎng)基培養(yǎng)轉(zhuǎn)染PPH質(zhì)粒的EAHY926，發(fā)現(xiàn)外源性PD-ECGF轉(zhuǎn)染PPEH質(zhì)粒后EAHY926細(xì)胞遷移能力亦明顯增強(qiáng)(見圖4B)。

注：A為EAHY926，B為T/G HA-VSMC；與PPH組比較，*P<0.05

圖3 MTT法檢測(cè)轉(zhuǎn)染PPEH質(zhì)粒的培養(yǎng)基對(duì)PPH誘導(dǎo)的EAHY926和T/G HA-VSMC增殖的影響

Figure 3 MTT method detecting the influence of medium transfected with PPEH on the proliferation of EAHY926 and T/G HA-VSMC induced by PPH

圖4 PD-ECGF對(duì)EAHY926細(xì)胞遷移能力的影響

2.5 PD-ECGF對(duì)T/G HA-VSMC遷移的影響 細(xì)胞傷痕實(shí)驗(yàn)證實(shí)，轉(zhuǎn)染PPEH質(zhì)粒的T/G HA-VSMC細(xì)胞遷移能力增強(qiáng)(見圖5A)；PPEH組T/G HA-VSMC平均細(xì)胞遷移距離百分比(69.1%±2.3%)較空載組(43.6%±5.8%)升高，差異有統(tǒng)計(jì)學(xué)意義(t=-33.64,P<0.001)。

用轉(zhuǎn)染PPEH質(zhì)粒的培養(yǎng)基培養(yǎng)轉(zhuǎn)染PPH質(zhì)粒的HA-VSMC，發(fā)現(xiàn)T/G HA-VSMC細(xì)胞遷移能力亦明顯增強(qiáng)(見圖5B)。

圖5 PD-ECGF對(duì)T/G HA-VSMC細(xì)胞遷移能力的影響

Figure 5 Influence of PD-ECGF on the migration of T/G HA-VSMC

3 討論

PDGF最初在血清和血小板中發(fā)現(xiàn)，能在體外誘導(dǎo)平滑肌細(xì)胞和成纖維細(xì)胞的分裂，因此被認(rèn)為是一種分裂素[7-8]，而且在促進(jìn)細(xì)胞增殖、遷移以及血管外周細(xì)胞重建中起著重要作用[9]。本研究發(fā)現(xiàn)，PDGF能明顯誘導(dǎo)血管內(nèi)皮細(xì)胞和平滑肌細(xì)胞的增殖，這與既往研究結(jié)果[10]一致。內(nèi)皮細(xì)胞和平滑肌細(xì)胞的遷移和增殖是血管受損后的正常生理反應(yīng)，目的是修復(fù)損傷的血管，PDGF在其中起到關(guān)鍵作用。但PDGF可以引起平滑肌細(xì)胞過度增殖，后者被認(rèn)為在血管再狹窄過程中扮演重要角色[11]。正常血管內(nèi)皮細(xì)胞幾乎不表達(dá)或者很少表達(dá)PDGF，而損傷的血管內(nèi)皮細(xì)胞則高表達(dá)PDGF。于是本研究通過高表達(dá)PDGF來模擬受損的內(nèi)皮細(xì)胞，同時(shí)采用由內(nèi)皮細(xì)胞表達(dá)的PD-ECGF來培養(yǎng)內(nèi)皮細(xì)胞和血管平滑肌細(xì)胞，外源PD-ECGF培養(yǎng)高表達(dá)PDGF的內(nèi)皮細(xì)胞和平滑肌細(xì)胞，觀察兩種細(xì)胞的增殖遷移情況。PD-ECGF能明顯抑制平滑肌細(xì)胞增殖，誘導(dǎo)內(nèi)皮細(xì)胞遷移，是理想的抑制平滑肌細(xì)胞過度增殖的細(xì)胞因子[12-13]。本研究結(jié)果還發(fā)現(xiàn)，在PDGF過表達(dá)的環(huán)境中內(nèi)皮細(xì)胞和平滑肌細(xì)胞的增殖雖然明顯上調(diào)，但PD-ECGF卻能拮抗并抑制平滑肌細(xì)胞的增殖，而對(duì)PDGF過表達(dá)引起的內(nèi)皮細(xì)胞增殖并無明顯抑制。PD-ECGF能誘導(dǎo)內(nèi)皮細(xì)胞增殖[14]，但是在與PDGF合用時(shí)卻沒有表現(xiàn)出拮抗作用，引起該結(jié)果的原因可能是內(nèi)皮細(xì)胞增殖的速度已經(jīng)趨于飽和。由上述結(jié)果，本研究假設(shè)PDGF可增殖血管內(nèi)皮細(xì)胞和平滑肌細(xì)胞，而PD-ECGF卻能抑制平滑肌細(xì)胞的過度增殖，兩者在血管重鑄的過程中共同作用加速傷口愈合防止血管狹窄。

PD-ECGF/TP是嘧啶核苷合成和分解過程中的一個(gè)重要酶,主要分布于血小板、淋巴細(xì)胞、網(wǎng)狀細(xì)胞核胎盤基質(zhì)中，其一個(gè)重要產(chǎn)物磷酸脫氧核糖(dRP)是血管生成和血管內(nèi)皮細(xì)胞增殖遷移的必須因子。同時(shí)，PD-ECGF在體內(nèi)血管生成中起著重要作用，能趨化內(nèi)皮細(xì)胞，抑制低氧環(huán)境下的細(xì)胞凋亡[15]。近年來，PD-ECGF作為血管內(nèi)皮生長(zhǎng)因子在血管重塑和血管生成中受到了廣泛關(guān)注。國(guó)內(nèi)外一些實(shí)驗(yàn)室證實(shí)，PD-ECGF能誘導(dǎo)腫瘤細(xì)胞模型中血管內(nèi)皮細(xì)胞的遷移卻不增強(qiáng)腫瘤的侵襲[16]。而最近關(guān)于PD-ECGF與血管平滑肌細(xì)胞的文章則認(rèn)為，PD-ECGF通過激活STAT3來抑制平滑肌細(xì)胞的增殖[17]。本研究結(jié)果則顯示，PDGF和PD-ECGF均能增強(qiáng)內(nèi)皮細(xì)胞和平滑肌細(xì)胞的遷移。內(nèi)源性PD-ECGF可以明顯促進(jìn)內(nèi)皮細(xì)胞和平滑肌細(xì)胞的遷移；外源性PD-ECGF可以進(jìn)一步促進(jìn)PDGF過表達(dá)狀態(tài)下內(nèi)皮細(xì)胞和平滑肌細(xì)胞的遷移，但卻能抑制過表達(dá)PDGF狀態(tài)下平滑肌細(xì)胞的增殖。兩者聯(lián)合在加速內(nèi)皮細(xì)胞增殖的同時(shí)，在抑制平滑肌細(xì)胞過度增殖上起重要作用。兩種生長(zhǎng)因子在損傷內(nèi)皮細(xì)胞和平滑肌細(xì)胞的定位功能上也可能有相互聯(lián)系，后續(xù)的具體機(jī)制還需進(jìn)一步在體內(nèi)進(jìn)行研究或驗(yàn)證。

總之，本研究提示，利用PDGF對(duì)內(nèi)皮細(xì)胞和平滑肌細(xì)胞的增殖作用，誘導(dǎo)兩種細(xì)胞增殖并遷移至受損處，同時(shí)利用PD-ECGF抑制平滑肌細(xì)胞過度增殖、趨化內(nèi)皮細(xì)胞的特點(diǎn)，將有助于PCI后損傷血管的修復(fù)，降低PCI后血管再狹窄風(fēng)險(xiǎn)，具有潛在的臨床應(yīng)用價(jià)值。

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(本文編輯：陳素芳)

Role of PDGF and PD-ECGF in Endothelial Cells and Vascular Smooth Muscle Cells

ZHUJin-kun,MAOHua,YINYang-guang,etal.

DepartmentofVasculocardiology,theFirstPeople′sHospitalofGuiyang,Guiyang550002,China

Objective To investigate the influence of platelet derived growth factor (PDGF) and platelet-derived endothelial cell growth factor (PD-ECGF) on human umbilical endothelial cells EAHY926 and human-aorta vascular smooth muscle cells T/G HA-VSMC by transfecting the plasmids of PDGF and PD-ECGF in vitro and to evaluate the possibility of treating vascular injury by the two kinds of plasmids.Methods pcDNA 3.1(+)no-load (no-load group)，pcDNA 3.1(+)-PDGF-HA (PPH group) and pcDNA 3.1 (+)-PD-ECGF-HA (PPEH group) plasmids were constructed and transfected into EAHY926 and T/G HA-VSMC.MTT method was used to evaluate the influence of endogenous and exogenous PD-ECGF on the proliferation of EAHY926 and T/G HA-VSMC before and after transfection.Cell wound healing assay was used to evaluate the influence of endogenous and exogenous PD-ECGF on the migration speed of EAHY926 and T/G HA-VSMC after transfection.Results MTT method was used to detect the absorbance value of EAHY926 among the three groups,and the differences were statistically significant (F=235.18,P<0.001),with the PPH group significantly higher than the no-load group and PPEH group (P<0.05).The no-load group and PPEH group showed no statistically significant differences (P>0.05).MTT method was also used to detect the absorbance value of T/G HA-VSMC among the three groups,and the differences were statistically significant (F=82.89,P<0.001),with the PPH group significantly higher than the no-load group(P<0.05),and the PPEH group significantly lower than the no-load group(P<0.05).MTT method detecting the influence of medium transfected with PPEH on the proliferation of EAHY926 and T/G HA-VSMC induced by PPH.There was no significant difference in the absorbance value of EAHY926 among no-load group，PPH group and PPH+L-PPEH group(F=512.89,P=0.183).There was significant difference in the absorbance value of T/G HA-VSMC among no-load group,PPH group and PPH+L-PPEH group (F=317.40,P<0.001),with the PPH group significantly higher than the no-load group and PPH+L-PPEH group(P<0.05).Cell wound healing assay demonstrated that EAHY926 and T/G HA-VSMC transfected with PPEH plasmid had a higher migration ability;compared with the no-load group,the average percentage of cell migration distance of EAHY926 in PPEH group was significantly increased〔(56.1%±2.2%) vs.(27.8%±3.4%)〕 (t=-15.08,P<0.001);compared with the no-load group,the average percentage of cell migration distance of T/G HA-VSMC in PPEH group was significantly increased〔(69.1%±2.3%) vs.(43.6%±5.8%)〕(t=-33.64,P<0.001).EAHY926 and T/G HA-VSMC cultured in medium transfected with PPEH showed that the migration ability of EAHY926 and T/G HA-VSMC transfected with PPEH processed by exogenous PD-ECGF also increased significantly.Conclusion After transfection of PDGF plasmid,the stimulation of PDGF and/or PD-ECGF on endothelial cells and smooth muscle cells is simulated.It is found that PDGF can significantly up-regulate the proliferation and migration of endothelial cells and smooth muscle cells.PD-ECGF has limited influence on the proliferation of vascular endothelial cells,but can up-regulate the migration speed of vascular endothelial cells and vascular smooth muscle cells.Meanwhile,PD-ECGF can inhibit the proliferation of vascular endothelial cells.The combined application of the two factors can inhibit over-proliferation of smooth muscle cells and promote migration of endothelial cells and smooth muscle cells.The combination may become a new strategy for the recovery of vascular damage and prevention of vascular restenosis after PCI.

Platelet-derived growth factor；Platelet-derived endothelial cell growth factor；Angioplasty,balloon,coronary；Myocytes,smooth muscle；Endothelial cells；Cell proliferation

貴州省科技廳社會(huì)發(fā)展項(xiàng)目(黔科合SY[2010]3087號(hào))

550002貴州省貴陽(yáng)市第一人民醫(yī)院心血管內(nèi)科(朱晉坤，毛華，董文，杜峰，魯玉明，熊宗華)；第三軍醫(yī)大學(xué)附屬新橋醫(yī)院心血管內(nèi)科(尹揚(yáng)光，鄧夢(mèng)揚(yáng))

朱晉坤，550002貴州省貴陽(yáng)市第一人民醫(yī)院心血管內(nèi)科；E-mail：jkz2001@163.com

R 349.51

A

10.3969/j.issn.1007-9572.2015.09.010

2014-07-21；

2015-01-02)