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      溴隱亭對(duì)垂體泌乳素腺瘤血管形成的影響及其分子作用機(jī)制研究

      2016-05-09 02:21:48陳宏謀鄭捷敏閆憲磊肖振勇陳家康
      實(shí)用心腦肺血管病雜志 2016年3期

      陳宏謀,鄭捷敏,閆憲磊,劉 全,黎 耀,肖振勇,陳家康

      545005廣西柳州市,廣西醫(yī)科大學(xué)第四附屬醫(yī)院神經(jīng)外科

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      ·論著·

      溴隱亭對(duì)垂體泌乳素腺瘤血管形成的影響及其分子作用機(jī)制研究

      陳宏謀,鄭捷敏,閆憲磊,劉 全,黎 耀,肖振勇,陳家康

      545005廣西柳州市,廣西醫(yī)科大學(xué)第四附屬醫(yī)院神經(jīng)外科

      【摘要】目的探討溴隱亭對(duì)垂體泌乳素腺瘤血管形成的影響及其分子作用機(jī)制。方法2014年1月—2015年6月,體外培養(yǎng)MMQ細(xì)胞株并進(jìn)行MTT比色實(shí)驗(yàn),共設(shè)置6個(gè)實(shí)驗(yàn)組、1個(gè)對(duì)照組和1個(gè)空白組,實(shí)驗(yàn)組在培養(yǎng)基和細(xì)胞懸液中添加不同濃度(0.125、0.250、0.500、1.000、2.000、4.000 μg/ml)溴隱亭,分別記為0.125 μg/ml組、0.250 μg/ml組、0.500 μg/ml組、1.000 μg/ml組、2.000 μg/ml組、4.000 μg/ml組,對(duì)照組加入等體積的培養(yǎng)基及細(xì)胞懸液,空白組僅加入等體積的培養(yǎng)基;根據(jù)半數(shù)抑制濃度(IC50)篩選最適濃度溴隱亭進(jìn)行后續(xù)試驗(yàn)。再采用最適濃度的溴隱亭處理MMQ細(xì)胞48 h,制備條件培養(yǎng)液(CM),各實(shí)驗(yàn)組在培養(yǎng)基和細(xì)胞懸液基礎(chǔ)上分別加入最適溶度的溴隱亭和CM,對(duì)照組加入等體積的培養(yǎng)基和細(xì)胞懸液,采用酶聯(lián)免疫吸附試驗(yàn)(ELISA)檢測(cè)泌乳素(PRL)濃度及其變化率。用攜帶GFP基因的慢病毒(LV-GFP)感染人臍靜脈血管內(nèi)皮細(xì)胞(HUVEC)制備HUVEC/LV-GFP,用CM孵育HUVEC/LV-GFP細(xì)胞,各實(shí)驗(yàn)組在培養(yǎng)基和細(xì)胞懸液基礎(chǔ)上分別加入最適濃度的溴隱亭和CM,CM組在培養(yǎng)基和細(xì)胞懸液基礎(chǔ)上僅加入CM,1.000 μg/ml組在培養(yǎng)基和細(xì)胞懸液基礎(chǔ)上僅加入1.000 μg/ml溴隱亭,對(duì)照組加入等體積培養(yǎng)基和細(xì)胞懸液;24 h后在熒光顯微鏡下觀察血管樣結(jié)構(gòu)形成情況并計(jì)數(shù)。采用Western blotting法檢測(cè)對(duì)照組與最適濃度溴隱亭組垂體瘤轉(zhuǎn)化基因(PTTG)和血管內(nèi)皮生長(zhǎng)因子(VEGF)表達(dá)情況。結(jié)果6個(gè)實(shí)驗(yàn)組MMQ細(xì)胞增殖抑制率時(shí)間與組間存在交互作用(P<0.05);培養(yǎng)48 h、72 h 0.125 μg/ml組、0.250 μg/ml組、0.500 μg/ml組、1.000 μg/ml組、2.000 μg/ml組、4.000 μg/ml組MMQ細(xì)胞增殖抑制率高于培養(yǎng)24 h,培養(yǎng)72 h 0.125 μg/ml組、0.250 μg/ml組、0.500 μg/ml組、1.000 μg/ml組、2.000 μg/ml組MMQ細(xì)胞增殖抑制率高于培養(yǎng)48 h(P<0.05);而培養(yǎng)48 h與培養(yǎng)72 h 4.000 μg/ml組MMQ細(xì)胞增殖抑制率比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。通過(guò)軟件計(jì)算抑制MMQ細(xì)胞增殖的IC50接近0.500 μg/ml,遂選用0.250、0.500、1.000 μg/ml溴隱亭進(jìn)行后續(xù)實(shí)驗(yàn)。1.000 μg/ml+CM組PRL濃度及變化率均低于0.500 μg/ml+CM組、0.250 μg/ml+CM組和對(duì)照組;0.500 μg/ml+CM組PRL濃度及變化率均低于0.250 μg/ml+CM組和對(duì)照組;0.250 μg/ml+CM組PRL濃度及變化率均低于對(duì)照組(P<0.05)。CM組MMQ細(xì)胞外血管樣結(jié)構(gòu)計(jì)數(shù)多于0.250 μg/ml+CM組、0.500 μg/ml+CM組、1.000 μg/ml+CM組、1.000 μg/ml組、對(duì)照組;0.250 μg/ml+CM組MMQ細(xì)胞外血管樣結(jié)構(gòu)計(jì)數(shù)多于0.500 μg/ml+CM組、1.000 μg/ml+CM組、1.000 μg/ml組、對(duì)照組;0.500 μg/ml+CM組MMQ細(xì)胞外血管樣結(jié)構(gòu)計(jì)數(shù)多于1.000 μg/ml+CM組、1.000 μg/ml組、對(duì)照組;1.000 μg/ml+CM組MMQ細(xì)胞外血管樣結(jié)構(gòu)計(jì)數(shù)多于1.000 μg/ml組、對(duì)照組;1.000 μg/ml組與對(duì)照組MMQ細(xì)胞外血管樣結(jié)構(gòu)計(jì)數(shù)比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。對(duì)照組PTTG、VEGF表達(dá)水平高于0.250 μg/ml組、0.500 μg/ml組、1.000 μg/ml組,0.250 μg/ml組PTTG、VEGF表達(dá)水平高于0.500 μg/ml組、1.000 μg/ml組,0.500 μg/ml組PTTG、VEGF表達(dá)水平高于1.000 μg/ml組(P<0.05)。結(jié)論溴隱亭可通過(guò)抑制垂體泌乳素腺瘤的血管形成而抑制其生長(zhǎng)與侵襲,而這種抑制作用與下調(diào)PTTG/VEGF信號(hào)通路有關(guān)。

      【關(guān)鍵詞】催乳素瘤;溴隱亭;血管內(nèi)皮生長(zhǎng)因子類

      陳宏謀,鄭捷敏,閆憲磊,等.溴隱亭對(duì)垂體泌乳素腺瘤血管形成的影響及其分子作用機(jī)制研究[J].實(shí)用心腦肺血管病雜志,2016,24(3):43-48.[www.syxnf.net]

      Chen HM,Zheng JM,Yan XL,et al.Impact of bromocriptine on vascularization of prolactinoma and the molecular mechanism[J].Practical Journal of Cardiac Cerebral Pneumal and Vascular Disease,2016,24(3):43-48.

      垂體瘤是顱內(nèi)常見(jiàn)疾病之一,占顱內(nèi)腫瘤的10%~15%,其中垂體泌乳素腺瘤(prolactinoma)占30%左右[1]。外科手術(shù)是治療垂體泌乳素腺瘤的首選方案,而溴隱亭是不能耐受手術(shù)或拒絕手術(shù)治療患者的最佳選擇。溴隱亭單獨(dú)使用或結(jié)合外科手術(shù)治療垂體泌乳素腺瘤已取得較好的臨床療效[2]。垂體泌乳素腺瘤的生長(zhǎng)和轉(zhuǎn)移依賴新生血管的形成,而腫瘤血管的形成極為復(fù)雜,包括正常靜脈血管系統(tǒng)激活、新生血管出芽以及腫瘤細(xì)胞、內(nèi)皮細(xì)胞、外周細(xì)胞間相互作用等多種因素共同作用。血管內(nèi)皮生長(zhǎng)因子(VEGF)是迄今公認(rèn)的最重要的血管形成因子,其可在多種生理、病理狀態(tài)下促進(jìn)周圍血管的形成。垂體瘤轉(zhuǎn)化基因(PTTG)在正常垂體中不表達(dá),而在垂體瘤中高表達(dá)。PTTG、VEGF對(duì)腫瘤血管的形成起重要作用。有研究表明,上調(diào)PTTG和VEGF表達(dá)可促進(jìn)垂體瘤周圍血管的形成,其表達(dá)情況可作為判斷垂體瘤侵襲性的一項(xiàng)生物學(xué)指標(biāo),對(duì)判斷患者預(yù)后也有一定的臨床意義[3]。

      本研究旨在分析溴隱亭對(duì)大鼠垂體泌乳素腺瘤MMQ細(xì)胞泌乳素(prolactin,PRL)分泌、血管形成及PTTG、VEGF表達(dá)的影響,初步探討溴隱亭對(duì)垂體泌乳素腺瘤的治療作用是否與通過(guò)調(diào)節(jié)PTTG/VEGF通路而抑制血管形成有關(guān),為臨床上應(yīng)用溴隱亭治療垂體泌乳素腺瘤提供參考。

      1材料與方法

      1.1實(shí)驗(yàn)材料

      1.1.1細(xì)胞株與病毒大鼠垂體泌乳素腺瘤細(xì)胞株MMQ及人臍靜脈血管內(nèi)皮細(xì)胞(HUVEC)均購(gòu)自American Type Culture Collection(ATCC)公司,攜帶GFP基因的慢病毒(LV-GFP)由Gene Chem公司制備。

      1.1.2主要試劑及試劑盒溴隱亭(Gedeon Richter Plc.),噻唑藍(lán)(MTT,Sigma),F(xiàn)12培養(yǎng)基(Hyclone),胎牛血清(Hyclone),馬血清(Gibco),0.25%胰蛋白酶消化液(Hyclone),二甲基亞砜(DMSO,Amresco),Polybrene(Sigma);Matrigel(Sigma),鼠源性抗VEGF、PTTG單克隆抗體(Santa Cruz),鼠源性抗GAPDH單克隆抗體(Santa Cruz),化學(xué)發(fā)光試劑ECL(Pierce),辣根過(guò)氧化物酶標(biāo)記的二抗羊抗鼠IgG(Santa Cruz);酶聯(lián)免疫吸附試驗(yàn)(ELISA)試劑盒(BOSTER),BCA蛋白定量試劑盒(Pierce)。

      1.2實(shí)驗(yàn)方法

      1.2.1MTT比色法2014年1月—2015年6月,將MMQ細(xì)胞復(fù)蘇后在含10%胎牛血清和5%馬血清的F12培養(yǎng)基中培養(yǎng),置于37 ℃、5% CO2培養(yǎng)箱中常規(guī)傳代培養(yǎng),觀察細(xì)胞狀態(tài),待生長(zhǎng)良好即進(jìn)行下一步實(shí)驗(yàn)。取處于對(duì)數(shù)生長(zhǎng)期的MMQ細(xì)胞,制成1×105個(gè)/ml細(xì)胞懸液,加入96孔板中,100 μl/孔(邊緣孔用無(wú)菌的PBS填充),37 ℃、5% CO2培養(yǎng)箱孵育過(guò)夜。共設(shè)置6個(gè)實(shí)驗(yàn)組、1個(gè)對(duì)照組和1個(gè)空白組,實(shí)驗(yàn)組加入培養(yǎng)基、細(xì)胞懸液和不同濃度(0.125、0.250、0.500、1.000、2.000、4.000 μg/ml)溴隱亭,分別記為0.125 μg/ml組、0.250 μg/ml組、0.500 μg/ml組、1.000 μg/ml組、2.000 μg/ml組、4.000 μg/ml組,對(duì)照組加入等體積的培養(yǎng)基及細(xì)胞懸液,空白組僅加入等體積的培養(yǎng)基,每組設(shè)6個(gè)復(fù)孔。置于培養(yǎng)箱中培養(yǎng)24、48、72 h后,每孔加入5 mg/ml MTT溶液20 μl,繼續(xù)培養(yǎng)4 h后,每孔加入150 μl DMSO;振蕩10 min后,用酶標(biāo)儀在570 nm波長(zhǎng)處測(cè)量吸光度(OD),并計(jì)算MMQ細(xì)胞增殖抑制率。實(shí)驗(yàn)獨(dú)立重復(fù)3次。MMQ細(xì)胞增殖抑制率=〔1-(實(shí)驗(yàn)組OD值-空白組OD值)/(對(duì)照組OD值-空白組OD值)〕×100%。并根據(jù)各組MMQ細(xì)胞增殖的半數(shù)抑制濃度(IC50)選取最適濃度的溴隱亭進(jìn)行后續(xù)試驗(yàn)。

      1.2.2制備條件培養(yǎng)液(CM)和提取總蛋白取對(duì)數(shù)生長(zhǎng)期的MMQ細(xì)胞,制成2×105個(gè)/ml的細(xì)胞懸液,加入6孔板中,1 ml/孔,置于培養(yǎng)箱中過(guò)夜。在孔板中加入最適濃度的溴隱亭培養(yǎng)48 h后,離心取上清液即為CM(4 ℃保存);6孔板中加入細(xì)胞裂解液,提取總蛋白(70 ℃保存)。

      1.3ELISA檢測(cè)CM中PRL濃度各實(shí)驗(yàn)組在培養(yǎng)基和細(xì)胞懸液基礎(chǔ)上分別加入最適溶度的溴隱亭和CM,對(duì)照組加入等體積的培養(yǎng)基和細(xì)胞懸液:(1)將100 μl的上述CM和標(biāo)準(zhǔn)品(標(biāo)準(zhǔn)品按試劑盒說(shuō)明稀釋)加入相應(yīng)的反應(yīng)孔中,37 ℃孵育2 h,洗板5次;(2)每孔加100 μl一抗,37 ℃孵育2 h,洗板5次;(3)每孔加100 μl酶標(biāo)二抗,37 ℃孵育30 min,洗板5次;(4)每孔加100 μl底物工作液37 ℃避光孵育15 min,洗板5次;(5)每孔加100 μl終止液混勻;(6)用酶標(biāo)儀在450 nm波長(zhǎng)處測(cè)量OD值;(7)根據(jù)標(biāo)準(zhǔn)曲線計(jì)算樣本OD值對(duì)應(yīng)的PRL濃度,并計(jì)算變化率,變化率(%)=(實(shí)驗(yàn)組PRL濃度/對(duì)照組PRL濃度)×100%。實(shí)驗(yàn)獨(dú)立重復(fù)3次取平均值。

      1.4體外血管形成

      1.4.1慢病毒感染制備HUVEC/LV-GFP細(xì)胞將HUVEC細(xì)胞接種于24孔板中培養(yǎng),24 h后用LV-GFP感染,根據(jù)細(xì)胞的增殖特性調(diào)整細(xì)胞密度,使感染時(shí)細(xì)胞匯合度為30%~40%。將60 μl LV-GFP(滴度為1×108 TU/ml)與435 μl培養(yǎng)液及5 μg/ml Polybrene 5 μl混勻制成感染混合體系,吸去細(xì)胞培養(yǎng)液,加入感染混合體系,37 ℃下孵育24 h,然后更換為常規(guī)培養(yǎng)液,72 h后用1 μg/ml嘌呤霉素篩選穩(wěn)定表達(dá)GFP的細(xì)胞,即HUVEC/LV-GFP細(xì)胞。

      1.4.2體外血管形成實(shí)驗(yàn)在96孔板加入Matrigel,50 μl/孔,37 ℃孵育1 h;將HUVEC/LV-GFP細(xì)胞懸液加入Matrigel上層,5×103個(gè)/孔;用CM孵育HUVEC/LV-GFP細(xì)胞。各實(shí)驗(yàn)組在培養(yǎng)基和細(xì)胞懸液基礎(chǔ)上分別加入最適濃度的溴隱亭和CM,CM組在培養(yǎng)基和細(xì)胞懸液基礎(chǔ)上僅加入CM,1.000 μg/ml組在培養(yǎng)基和細(xì)胞懸液基礎(chǔ)上僅加入1.000 μg/ml溴隱亭,對(duì)照組加入等體積的培養(yǎng)基和細(xì)胞懸液。24 h后,在熒光顯微鏡下觀察血管樣結(jié)構(gòu)的形成情況,任意取5個(gè)視野,計(jì)數(shù)血管樣結(jié)構(gòu)。實(shí)驗(yàn)獨(dú)立重復(fù)3次取平均值。

      1.5Western blotting法(1)用BCA蛋白定量試劑盒進(jìn)行蛋白定量,取一定體積總蛋白進(jìn)行聚丙烯酰胺凝膠(SDS-PAGE)電泳。(2)100 V恒壓電泳至溴酚藍(lán)遷移到分離膠底部0.5 cm處,300 mA恒流轉(zhuǎn)膜(PVDF膜)90 min。(3)用含5%脫脂奶粉的封閉液封閉PVDF膜;加入適當(dāng)稀釋的一抗,封于塑料膜中4 ℃過(guò)夜;用TBST洗滌,加入適當(dāng)稀釋的二抗,室溫孵育1 h;用TBST洗滌。(4)用增敏化學(xué)發(fā)光底物試劑(ECL)處理后在顯影儀中曝光成像,并用Image J軟件進(jìn)行灰度分析,以GAPDH作為內(nèi)參照。實(shí)驗(yàn)獨(dú)立重復(fù)3次取平均值。

      2結(jié)果

      2.1各組MMQ細(xì)胞增殖抑制率比較6個(gè)實(shí)驗(yàn)組MMQ細(xì)胞增殖抑制率時(shí)間與組間存在交互作用(P<0.05);培養(yǎng)24h、48h、72h6組MMQ細(xì)胞增殖抑制率比較,差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。其中培養(yǎng)48h、72h0.125μg/ml組、0.250μg/ml組、0.500μg/ml組、1.000μg/ml組、2.000μg/ml組、4.000μg/ml組MMQ細(xì)胞增殖抑制率高于培養(yǎng)24h,培養(yǎng)72h0.125μg/ml組、0.250μg/ml組、0.500μg/ml組、1.000μg/ml組、2.000μg/ml組MMQ細(xì)胞增殖抑制率高于培養(yǎng)48h,差異有統(tǒng)計(jì)學(xué)意義(P<0.05);而培養(yǎng)48h與培養(yǎng)72h4.000μg/ml組MMQ細(xì)胞增殖抑制率比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05,見(jiàn)表1)。通過(guò)軟件計(jì)算抑制MMQ細(xì)胞增殖的IC50接近0.500μg/ml,遂選用0.250、0.500、1.000μg/ml溴隱亭進(jìn)行后續(xù)實(shí)驗(yàn)。

      Table1ComparisonofinhibitionratioofMMQcellproliferationamongthesixgroupswithdifferentconcentrationofbromocriptine

      組別培養(yǎng)24h培養(yǎng)48h培養(yǎng)72h0.125μg/ml組27.12±2.2432.89±3.45a43.57±3.89ab0.250μg/ml組36.33±2.2641.44±3.22a55.59±3.12ab0.500μg/ml組50.48±1.5558.88±1.67a75.68±1.22ab1.000μg/ml組63.22±3.4574.24±3.89a86.22±2.24ab2.000μg/ml組75.65±3.2278.56±3.12a92.01±2.26ab4.000μg/ml組90.34±1.6796.44±1.22a96.67±1.55aF值F組間=103.32,F時(shí)間=44.58,F交互=115.07P值P組間=0.00,P時(shí)間=0.00,P交互=0.00

      注:與培養(yǎng)24 h比較,aP<0.05;與培養(yǎng)48 h比較,bP<0.05

      2.2對(duì)照組與最適濃度溴隱亭組PRL濃度及變化率比較4組PRL濃度及變化率比較,差異有統(tǒng)計(jì)學(xué)意義(P<0.05);其中1.000 μg/ml+CM組PRL濃度及變化率均低于0.500 μg/ml+CM組、0.250 μg/ml+CM組和對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P<0.05);0.500 μg/ml+CM組PRL濃度及變化率均低于0.250 μg/ml+CM組和對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P<0.05);0.250 μg/ml+CM組PRL濃度及變化率均低于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P<0.05,見(jiàn)表2)。

      表2對(duì)照組與最適濃度溴隱亭組PRL濃度及變化率比較

      Table2ComparisonofPRLconcentrationandthechangerateamongcontrolgroupandtestgroupswithbestconcentrationofbromocriptineandCM

      組別PRL(μU/ml)變化率(%)對(duì)照組85.66±1.34100.00±1.230.250μg/ml+CM組55.21±2.82a64.45±2.22a0.500μg/ml+CM組40.19±3.11ab46.92±1.89ab1.000μg/ml+CM組30.33±2.67abc35.41±33.11abcF值103.3379.54P值0.0060.000

      注:PRL=泌乳素;與對(duì)照組比較,aP<0.05;與0.250 μg/ml+CM組比較,bP<0.05;與0.500 μg/ml+CM組比較,cP<0.05

      2.3對(duì)照組、1.000 μg/ml組、CM組與最適濃度溴隱亭組MMQ細(xì)胞外血管樣結(jié)構(gòu)計(jì)數(shù)比較6組MMQ細(xì)胞外血管樣結(jié)構(gòu)計(jì)數(shù)比較,差異有統(tǒng)計(jì)學(xué)意義(P<0.05);其中CM組MMQ細(xì)胞外血管樣結(jié)構(gòu)計(jì)數(shù)多于0.250 μg/ml+CM組、0.500 μg/ml+CM組、1.000 μg/ml+CM組、1.000 μg/ml組、對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P<0.05);0.250 μg/ml+CM組MMQ細(xì)胞外血管樣結(jié)構(gòu)計(jì)數(shù)多于0.500 μg/ml+CM組、1.000 μg/ml+CM組、1.000 μg/ml組、對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P<0.05);0.500 μg/ml+CM組MMQ細(xì)胞外血管樣結(jié)構(gòu)計(jì)數(shù)多于1.000 μg/ml+CM組、1.000 μg/ml組、對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P<0.05);1.000 μg/ml+CM組MMQ細(xì)胞外血管樣結(jié)構(gòu)計(jì)數(shù)多于1.000 μg/ml組、對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P<0.05);1.000 μg/ml組與對(duì)照組MMQ細(xì)胞外血管樣結(jié)構(gòu)計(jì)數(shù)比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05,見(jiàn)表3)。

      2.4對(duì)照組與最適濃度溴隱亭組PTTG、VEGF表達(dá)水平比較4組PTTG、VEGF表達(dá)水平比較,差異有統(tǒng)計(jì)學(xué)意義(P<0.05);其中對(duì)照組PTTG、VEGF表達(dá)水平高于0.250 μg/ml組、0.500 μg/ml組、1.000 μg/ml組,差異有統(tǒng)計(jì)學(xué)意義(P<0.05);0.250 μg/ml組PTTG、VEGF表達(dá)水平高于0.500 μg/ml組、1.000 μg/ml組,差異有統(tǒng)計(jì)學(xué)意義(P<0.05);0.500 μg/ml組PTTG、VEGF表達(dá)水平高于1.000 μg/ml組,差異有統(tǒng)計(jì)學(xué)意義(P<0.05,見(jiàn)表4)。不同溴隱亭濃度組PTTG、VEGF表達(dá)水平電泳圖見(jiàn)圖1。

      Table 3Comparison of capillary structure count of MMQ cells among control group and test groups with best concentration of bromocriptine,with/without CM

      組別血管樣結(jié)構(gòu)計(jì)數(shù)對(duì)照組10.33±0.10abcd1.000μg/ml組9.3±0.05abcd1.000μg/ml+CM組20.5±0.90abc0.500μg/ml+CM組40.33±1.10ab0.250μg/ml+CM組55.23±1.90aCM組90.22±1.40F值119.43P值0.000

      注:與CM組比較,aP<0.05;與0.250 μg/ml+CM組比較,bP<0.05;與0.500 μg/ml+CM組比較,cP<0.05;與1.000 μg/ml+CM組比較,dP<0.05

      表4對(duì)照組和最適濃度溴隱亭組PTTG、VEGF表達(dá)水平比較

      Table4ComparisonofexpressionsofPTTGandVEGFamongcontrolgroupandtestgroupswithbestconcentrationofbromocriptine

      組別PTTGVEGF對(duì)照組1.00±0.011.00±0.010.250μg/ml組0.59±0.02a0.69±0.02a0.500μg/ml組0.29±0.01ab0.58±0.01ab1.000μg/ml組0.15±0.02abc0.36±0.02abcF值109.32105.02P值0.0070.009

      注:PTTG=垂體瘤轉(zhuǎn)化基因,VEGF=血管內(nèi)皮生長(zhǎng)因子;與對(duì)照組比較,aP<0.05;與0.250 μg/ml組比較,bP<0.05;與0.500 μg/ml組比較,cP<0.05

      注:PTTG=垂體瘤轉(zhuǎn)化基因,VEGF=血管內(nèi)皮生長(zhǎng)因子

      圖1對(duì)照組與最適濃度溴隱亭組PTTG、VEGF表達(dá)水平的電泳圖

      Figure 1Electrophoretogram for expressions of PTTG and VEG of control group and test groups with best concentration of bromocriptine

      3討論

      溴隱亭作為臨床最常用的一種D2受體激動(dòng)劑,在侵襲性垂體泌乳素腺瘤的臨床治療中已被證明安全有效。溴隱亭可使80%~90%患者的血清PRL水平恢復(fù)至參考范圍,使60%~75%垂體泌乳素大腺瘤患者腫瘤縮小,使80%~90%垂體泌乳素微腺瘤和60%~75%大腺瘤患者恢復(fù)性腺功能[4-6]。19世紀(jì)70年代,F(xiàn)olkman[7]提出腫瘤的生長(zhǎng)和轉(zhuǎn)移依賴于血管形成的假說(shuō)。垂體泌乳素腺瘤的生長(zhǎng)和侵襲亦依賴于腫瘤血管的形成,而腫瘤血管的形成過(guò)程極為復(fù)雜,與腫瘤細(xì)胞分泌的許多生長(zhǎng)因子有關(guān),目前已有文獻(xiàn)指出參與調(diào)控腫瘤血管形成的生長(zhǎng)因子有30多種,而在眾多調(diào)控因子中VEGF是最重要的促進(jìn)血管生成因子之一[8-9]。VEGF是目前公認(rèn)的作用最強(qiáng)、特異性最高的腫瘤血管形成的關(guān)鍵調(diào)控因子,其由多種正常細(xì)胞和腫瘤細(xì)胞分泌,在多種腫瘤組織的血管形成中起關(guān)鍵作用[10]。在腫瘤的生長(zhǎng)和侵襲過(guò)程中,當(dāng)腫瘤細(xì)胞與內(nèi)皮細(xì)胞通過(guò)縫隙連結(jié)相互作用后,VEGF分泌增加,基質(zhì)金屬蛋白酶1(MMP-1)明顯升高,基質(zhì)金屬蛋白酶原2(Pro-MMP-2)活化,胞膜型基質(zhì)金屬蛋白酶1(MT1-MMP)、尿激酶型纖溶酶原激活劑(uPA)、纖溶酶原激活物抑制劑(PAI-1)表達(dá)增高,從而促進(jìn)腫瘤侵襲和腫瘤血管的形成。多種因子可通過(guò)調(diào)節(jié)VEGF水平或血管內(nèi)皮生長(zhǎng)因子受體(VEGFR)活性而參與血管形成和調(diào)節(jié),如在缺血缺氧環(huán)境下,腫瘤血管形成可通過(guò)誘導(dǎo)缺氧誘導(dǎo)因子-1α(HIF-1α)增多而實(shí)現(xiàn)。VEGF是腫瘤血管形成過(guò)程中最重要的刺激因子,同時(shí)又是缺氧誘導(dǎo)因子1(HIF-1)重要的靶基因,機(jī)體缺氧時(shí)HIF-1能調(diào)節(jié)VEGFR表達(dá)上調(diào),促進(jìn)血管內(nèi)皮細(xì)胞增生并形成新的血管,以滿足自身營(yíng)養(yǎng)供應(yīng)[11]。PTTG是1997年從鼠垂體腫瘤細(xì)胞分離并確定的垂體洐生轉(zhuǎn)化基因,與腫瘤血管形成關(guān)系密切[12]。PTTG可通過(guò)正反饋機(jī)制促進(jìn)堿性成纖維細(xì)胞生長(zhǎng)因子(bFGF)的表達(dá),參與腫瘤血管的形成;且PTTG與基質(zhì)金屬蛋白酶2(MMP-2)成正相關(guān),可抑制腫瘤血管生成[13];PTTG也可能通過(guò)p53抑制血小板反應(yīng)蛋白(TSP-1)的表達(dá),從而促進(jìn)腫瘤血管生成??傊?,PTTG可通過(guò)多條途徑誘導(dǎo)腫瘤血管生成。而PTTG與VEGF之間是否存在相關(guān)性,目前尚不明確。有學(xué)者認(rèn)為,PTTG可上調(diào)VEGF和血管內(nèi)皮生長(zhǎng)因子受體(KDR)的表達(dá),因此提出了PTTG/VEGF/KDR自分泌信號(hào)通路[14-15]。也有學(xué)者認(rèn)為,盡管PTTG確實(shí)存在誘導(dǎo)血管形成的作用,但未必是通過(guò)調(diào)節(jié)VEGF表達(dá)而實(shí)現(xiàn),兩種基因表達(dá)不存在關(guān)聯(lián)性[16]。因此,在腫瘤血管形成方面,PTTG與VEGF的關(guān)系尚需進(jìn)一步探究。

      本研究中MTT實(shí)驗(yàn)和ELISA結(jié)果顯示,培養(yǎng)24 h、48 h、72 h,0.125 μg/ml組、0.250 μg/ml組、0.500 μg/ml組、1.000 μg/ml組、2.000 μg/ml組、4.000 μg/ml組MMQ細(xì)胞增殖抑制率間有差異;其中培養(yǎng)48 h、72 h 0.125 μg/ml組、0.250 μg/ml組、0.500 μg/ml組、1.000 μg/ml組、2.000 μg/ml組、4.000 μg/ml組MMQ細(xì)胞增殖抑制率高于培養(yǎng)24 h,培養(yǎng)72 h 0.125 μg/ml組、0.250 μg/ml組、0.500 μg/ml組、1.000 μg/ml組、2.000 μg/ml組MMQ細(xì)胞增殖抑制率高于培養(yǎng)48 h。1.000 μg/ml+CM組PRL濃度及變化率均低于0.500 μg/ml+CM組、0.250 μg/ml+CM組和對(duì)照組;0.500 μg/ml+CM組PRL濃度及變化率均低于0.250 μg/ml+CM組和對(duì)照組;0.250 μg/ml+CM組PRL濃度及變化率均低于對(duì)照組;表明溴隱亭可抑制MMQ細(xì)胞增殖和PRL的分泌。繼而構(gòu)建能穩(wěn)定表達(dá)GFP的HUVEC/LV-GFP,在添加CM的培養(yǎng)基中培養(yǎng),通過(guò)熒光顯微鏡下觀察血管樣結(jié)構(gòu)形成情況發(fā)現(xiàn),1.000 μg/ml組與對(duì)照組MMQ細(xì)胞外血管樣結(jié)構(gòu)計(jì)數(shù)間無(wú)差異,CM組MMQ細(xì)胞外血管樣結(jié)構(gòu)計(jì)數(shù)多于0.250 μg/ml+CM組、0.500 μg/ml+CM組、1.000 μg/ml+CM組、1.000 μg/ml組、對(duì)照組,0.250 μg/ml+CM組MMQ細(xì)胞外血管樣結(jié)構(gòu)計(jì)數(shù)多于0.500 μg/ml+CM組、1.000 μg/ml+CM組、1.000 μg/ml組、對(duì)照組,0.500 μg/ml+CM組MMQ細(xì)胞外血管樣結(jié)構(gòu)計(jì)數(shù)多于1.000 μg/ml+CM組、1.000 μg/ml組、對(duì)照組,1.000 μg/ml+CM組MMQ細(xì)胞外血管樣結(jié)構(gòu)計(jì)數(shù)多于1.000 μg/ml組、對(duì)照組;表明MMQ細(xì)胞培養(yǎng)48 h后分泌了能促進(jìn)HUVEC/LV-GFP細(xì)胞形成血管的因子,而這種因子在HUVEC/LV-GFP細(xì)胞自身合成中含量較低,溴隱亭處理MMQ細(xì)胞48 h后明顯抑制了血管的形成,且隨著溴隱亭濃度的增加,其血管形成抑制能力逐漸加強(qiáng)。為探索其相關(guān)機(jī)制,采用Western blotting實(shí)驗(yàn)分析MMQ細(xì)胞總蛋白中PTTG和VEGF表達(dá)情況,結(jié)果顯示,對(duì)照組PTTG、VEGF表達(dá)水平高于0.250 μg/ml組、0.500 μg/ml組、1.000 μg/ml組,0.250 μg/ml組PTTG、VEGF表達(dá)水平高于0.500 μg/ml組、1.000 μg/ml組,0.500 μg/ml組PTTG、VEGF表達(dá)水平高于1.000 μg/ml組;表明PTTG和VEGF表達(dá)受溴隱亭濃度的影響。分析原因?yàn)镸MQ細(xì)胞合成并分泌了PTTG和VEGF,即PTTG/VEGF信號(hào)通路,以促進(jìn)HUVEC/LV-GFP細(xì)胞形成血管樣結(jié)構(gòu),而溴隱亭可抑制PTTG/VEGF信號(hào)通路。

      綜上所述,溴隱亭可通過(guò)抑制垂體泌乳素腺瘤血管形成而抑制其生長(zhǎng)與侵襲,而這種抑制作用可能與下調(diào)PTTG/VEGF信號(hào)通路有關(guān)。

      作者貢獻(xiàn):陳宏謀進(jìn)行資料收集整理、撰寫論文、成文并對(duì)文章負(fù)責(zé);鄭捷敏進(jìn)行實(shí)驗(yàn)設(shè)計(jì)并組織實(shí)施;閆憲磊、劉全、黎耀、肖振勇進(jìn)行實(shí)驗(yàn)實(shí)施、評(píng)估、資料收集;陳家康進(jìn)行質(zhì)量控制及審校。

      本文無(wú)利益沖突。

      參考文獻(xiàn)

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      (本文編輯:毛亞敏)

      Impact of Bromocriptine on Vascularization of Prolactinoma and the Molecular Mechanism

      CHENHong-mou,ZHENGJie-min,YANXian-lei,etal.DepartmentofNeurosurgery,theFourthAffiliatedHospitalofGuangxiMedicalUniversity,Liuzhou545005,China

      【Abstract】ObjectiveTo investigate the impact of bromocriptine on vascularization of prolactinoma and the molecular mechanism.MethodsFrom January 2014 to June 2015,MMQ cell strains were cultured in vitro,MTT colorimetric assay was used to find the best concentration of bromocriptine.A group added 0.125 μg/ml of bromocriptine,B group added 0.250 μg/ml of bromocriptine,C group added 0.500 μg/ml of bromocriptine,D group added 1.000 μg/ml of bromocriptine,E group added 2.000 μg/ml of bromocriptine,F(xiàn) group added 4.000 μg/ml of bromocriptine,control group added isovolumetric culture medium and cell suspension,blank control group added isovolumetric culture medium.IC50 was calculated to find the best concentration of bromocriptine.After that,MMQ cell strains were cultured by the best concentration of bromocriptine for 48 hours,and conditioned medium(CM)was prepared at the same time,then B group added with CM served as B1 group,C group added with CM served as C1 group,D group added with CM served as D1 group,control group added isovolumetric culture medium and cell suspension as before,and ELISA method was used to detect the PRL concentration and the change rate.Lentivirus with GFP gene was used to infect the HUVEC and prepared for HUVEC/LV-GFP cells,then B group hatched by CM served as B2 group,C group hatched by CM served as C2 group,D group hatched by CM served as D2 group,control group add with CM served as control-CM group;after 24 hours of culture,fluorescence microscope was used to observe the formation and counts of capillary structure.Western blotting method was used to detect the expressions of PTTG and VEGF of control group,of B group,of C group,of D group.ResultsThere was interaction of inhibition ratio of cell multiplication between time and group among A group,B group,C group,D group,E group and F group(P<0.05);after 48 hours,72 hours of culture,inhibition ratio of cell multiplication of A group,of B group,of C group,of D group,of E group,of F group was statistically significantly higher than that after 24 hours of culture,respectively;after 72 hours of culture,inhibition ratio of cell multiplication of A group,of B group,of C group,of D group,of E group was statistically significantly higher than that after 48 hours of culture,respectively(P<0.05),while no statistically significant differences of inhibition ratio of cell multiplication of F group was found compared to that after 48 hours of culture(P>0.05).The calculation showed that,IC50 of inhibition of cell multiplication was close to 0.500 μg/ml.The PRL concentration and the change rate of D1 group were statistically significantly lower those of C1 group,of B1 group,of control group,PRL concentration and the change rate of C1 group were statistically significantly lower than those of B1 group,of control group,PRL concentration and the change rate of B1 group were statistically significantly lower than those of control group(P<0.05).Capillary structure count of control-CM group was statistically significant more than that of B2 group,of C2 group,of D2 group,of D group,of control group,respectively;capillary structure count of B2 group was statistically significant more than that of C2 group,of D2 group,of D group,of control group,respectively;capillary structure count of C2 group was statistically significant more than that of D2 group,of D group,of control group,respectively;capillary structure count of D2 group was statistically significant more than that of D group,of control group,respectively(P<0.05);while no statistically significant differences of capillary structure count was found between D group and control group(P>0.05).Expressions of PTTG and VEGF of control group were statistically significantly higher than those of B group,of C group,of D group;expressions of PTTG and VEGF of B group were statistically significantly higher than those of C group,of D group;expressions of PTTG and VEGF of C group were statistically significantly higher than those of D group(P<0.05).ConclusionBromocriptine can inhibit development and invasion of prolactinoma by inhibiting the vascularization,the molecular mechanism is possibly correlated with the down-regulation PTTG/VEGF signaling pathway.

      【Key words】Prolactinoma;Bromocriptine;Vascular endothelial growth factors

      (收稿日期:2015-11-19;修回日期:2016-02-23)

      【中圖分類號(hào)】R 736

      【文獻(xiàn)標(biāo)識(shí)碼】A

      doi:10.3969/j.issn.1008-5971.2016.03.012

      通信作者:陳家康,545005廣西柳州市,廣西醫(yī)科大學(xué)第四附屬醫(yī)院神經(jīng)外科;E-mail:lgsjwk45@163.com

      基金項(xiàng)目:廣西壯族自治區(qū)衛(wèi)生廳自籌經(jīng)費(fèi)科研課題(z2013624);廣西壯族自治區(qū)臨床重點(diǎn)??平ㄔO(shè)項(xiàng)目

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