潘俊 趙曉勇 張曉麗

(1海南省儋州市人民醫(yī)院神經(jīng)外科,海南 儋州 571700；2南方醫(yī)科大學(xué)附屬花都醫(yī)院神經(jīng)外科,廣東 廣州 510800)

·論著·

低氧條件下BAVM動(dòng)物模型VSMC中TGF-β表達(dá)及細(xì)胞功能改變

潘俊1趙曉勇2*張曉麗2

(1海南省儋州市人民醫(yī)院神經(jīng)外科,海南 儋州 571700；2南方醫(yī)科大學(xué)附屬花都醫(yī)院神經(jīng)外科,廣東 廣州 510800)

目的探討分析缺氧對(duì)腦動(dòng)靜脈畸形(BAVM)血管內(nèi)膜平滑肌細(xì)胞(VSMC)中轉(zhuǎn)化生長(zhǎng)因子-β(TGF-β)表達(dá)及VSMC細(xì)胞功能改變。方法建立穩(wěn)定的BAVM實(shí)驗(yàn)用豬動(dòng)物模型，分離腦底微血管網(wǎng)(RM)的VSMC后行原代培養(yǎng)。分組：①對(duì)照組采用正常實(shí)驗(yàn)用豬VSMC：(A組)21%O2濃度；②實(shí)驗(yàn)組采用BAVM模型實(shí)驗(yàn)用豬VSMC：(B組)21%O2濃度、(C組)1%O2濃度。細(xì)胞免疫熒光檢測(cè)VSMC密度、實(shí)時(shí)定量-多聚酶鏈反應(yīng)(RT-PCR)和蛋白印記(Western blot)法驗(yàn)證VSMC中TGF-β mRNA及蛋白差異、末端脫氧核苷酸轉(zhuǎn)移酶介導(dǎo)的生物素脫氧尿嘧啶核苷酸缺口末端標(biāo)記(TUNEL)法檢測(cè)VSMC的調(diào)亡、Transwell測(cè)定VSMC的侵襲能力。結(jié)果VSMC對(duì)缺氧敏感，實(shí)驗(yàn)組與對(duì)照組比較及實(shí)驗(yàn)組組間比較，TGF-β的mRNA及蛋白差異有統(tǒng)計(jì)學(xué)意義(Plt;0.01)、72 h VSMC凋亡及細(xì)胞侵襲數(shù)目差異有統(tǒng)計(jì)學(xué)意義(Plt;0.01)。結(jié)論缺氧對(duì)動(dòng)物模型VSMC中TGF-β表達(dá)有顯著的影響且促使VSMC凋亡及侵襲能力增加，加速畸形血管團(tuán)的形成。在細(xì)胞分子生物學(xué)水平分析能為BAVM臨床治療提供一種有益的思路。

缺氧; 豬模; 血管內(nèi)膜平滑肌細(xì)胞; 血管內(nèi)皮生長(zhǎng)因子; 細(xì)胞功能

腦動(dòng)靜脈畸形(brain arteriovenous malformations,BAVM)是中樞神經(jīng)系統(tǒng)最為常見(jiàn)的先天性腦血管異常[1]。豬的腦底微血管網(wǎng)(rete mirabile,RM)結(jié)構(gòu)與人BAVM畸形血管團(tuán)高度相似[2]，本研究是先建立穩(wěn)定型實(shí)驗(yàn)用豬BAVM模型基礎(chǔ)上，用缺氧因素干預(yù)分離培養(yǎng)好RM的原代血管內(nèi)膜平滑肌細(xì)胞(vessels smooth muscle cells,VSMC)，分析缺氧因素對(duì)于VSMC中轉(zhuǎn)化生長(zhǎng)因子-β(transforming growth factor-β,TGF-β)的影響和VSMC的細(xì)胞功能作用，為BAVM畸形血管團(tuán)的加速形成機(jī)制提供可能的基礎(chǔ)實(shí)驗(yàn)研究理論。

材料與方法

一、實(shí)驗(yàn)組建立穩(wěn)定的BAVM動(dòng)物模型

實(shí)驗(yàn)小型豬60頭，由南方醫(yī)科大學(xué)動(dòng)物實(shí)驗(yàn)中心提供，3～4個(gè)月齡，體重20～25 kg。實(shí)驗(yàn)組豬模(B組，n=20；C組，n=20)用2.5%硫賁妥鈉10～15 mg/kg靜脈誘導(dǎo)后氣管插管，機(jī)控呼吸，芬太尼分次靜脈注射維持麻醉[3]。再行暴露和分離左頸總動(dòng)脈、左、右頸外動(dòng)脈、左咽升動(dòng)脈和左頸外靜脈。結(jié)扎左頸外動(dòng)脈、左頸總動(dòng)脈近心端和左頸外靜脈遠(yuǎn)心端的殘端，將左咽升動(dòng)脈與左頸外靜脈端端斜切口吻合，結(jié)扎左咽升動(dòng)脈降支和枕動(dòng)脈降支。

二、實(shí)驗(yàn)方法

1.29 d行手術(shù)切除對(duì)照組(A組，n=20)和實(shí)驗(yàn)組豬模的RM，分別置入含5×105μ/L青、鏈霉素的D-hank's溶液中，4℃ 30 min。取內(nèi)膜內(nèi)皮細(xì)胞液氮保存后勻漿，消化液(0.25 g胰酶+0.1 g膠原酶Ⅲ+100 ml 磷酸緩沖鹽溶液(phosphate buffered solution,PBS)，用0.22 μm的過(guò)濾器除菌消化約10 min，將終濃度為1×105/ml細(xì)胞接種于培養(yǎng)瓶，抗細(xì)胞角蛋白和抗珠蛋白抗體鑒定[4]。

2.培養(yǎng)基為10%胎牛血清(10% fetal balf serum,FBS)，條件為37℃、5%CO2。A組：正常氧壓培養(yǎng)箱中培養(yǎng)(21% O2)；B組：正常氧壓培養(yǎng)箱中培養(yǎng)(21% O2)；C組:低氧培養(yǎng)箱中培養(yǎng)(1% O2)。

3.在滅菌的培養(yǎng)皿中鋪上3塊細(xì)胞爬片(4%多聚賴氨酸處理的圓片)，取細(xì)胞懸液分別滴到圓片上，4%多聚甲醛(paraformaldehyde,PFA)固定處理30 min，0.2% TxitonX-100室溫處理5 min、PBS漂洗5 min，3次。室溫封閉:置于試劑盒中保持濕潤(rùn)，37℃，30 min；滴加1∶200 PBS稀釋的綠色熒光一抗(CD34為1∶200)，陰性對(duì)照用PBS代替一抗，室溫反應(yīng)30～40 min，80%甘油封片，置顯微鏡下觀察拍片。

4.實(shí)時(shí)PCR和Western blot法檢測(cè)VSMC中TGF-β mRNA及蛋白：3組VSMC總RNA提取后逆轉(zhuǎn)錄合成cDNA，均加入0.3 ml氯仿，4℃ 13 000 rpm離心15 min。棄上清，清洗、振蕩后加入30～50 μl的焦碳酸二乙酯(diethy pyrocarbonate,DEPC)水溶解RNA。置于實(shí)時(shí)PCR儀(C1000型，美國(guó)Bio Rad公司)上進(jìn)行PCR反應(yīng)，β-actin和目的基因同時(shí)擴(kuò)增，擴(kuò)增反應(yīng)條件如下：①預(yù)變性Reps：95℃ 30 s；②PCR反應(yīng)Reps：95℃ 5 s、60℃ 34 s；③β-actin：85℃收集熒光，TGF-β：84.3℃收集熒光；④95℃，5 min，35個(gè)PCR循環(huán)，建立熔解曲線和擴(kuò)增曲線，引物設(shè)計(jì)結(jié)果列表(表1)。

表1 設(shè)計(jì)實(shí)時(shí)PCR的基因引物
Tab 1 Designed real-time PCR gene primers

GeneNameTwo?wayprimersequences(5＇?3＇)Annealingtemperature(℃)Productlength(bp) β?actinF：CCTGTACGCCAACACAGTGC57211 R：ATACTCCTGCTTGCTGATCC TGF?βF：CCTGAAGCTGACCCAGGTAG58133 R：TTCCAAACTGCATCAATGAAT

Note:F:upstream primer sequences,R:downstream primer sequences

Western blot蛋白質(zhì)印跡法；3組均加入裂解液(radio immunoprecipitation assay,RIPA)200 μl勻漿，于冰上加苯甲基磺酰氟(phenylmethanesulfonyl fluoride,PMSF) 2 μl，RIPA:PMSF=100∶1，4℃,12 000 rpm離心1 h，取上清后Bradford法測(cè)定蛋白含量。行聚丙烯酰胺凝膠(polyacrylamide gelelectrophoresis,SDS-PAGE)電泳，將一抗TGF-β(1∶3 000,Santa Cruz)和β-actin (1∶1 000,Santa Cruz)加入到封閉液中混均行二抗反應(yīng)。用封閉緩沖液稀釋的二抗雜交溶液，置于振蕩培養(yǎng)箱中進(jìn)行雜交(26℃,80 rpm/min,l h)；棄二抗溶液,用緩沖鹽溶液(TBST)洗膜3次(置于振蕩培養(yǎng)箱中,26℃,80 rpm/min,10 min/次)；化學(xué)發(fā)光試劑(electrochemiluminescence,ECL,購(gòu)自上海普飛生物公司)檢測(cè)，利用BIO-RAD2000凝膠成像系統(tǒng)及QUANTITY ONE分析系統(tǒng)測(cè)定條帶的積光光密度值。

5.末端脫氧核苷酸轉(zhuǎn)移酶介導(dǎo)的生物素脫氧尿嘧啶核苷酸缺口末端標(biāo)記(terminal deoxynucleotidyl transferase mediated biotinylated deoxyuridine triphosphate,TUNEL)法檢測(cè)VSMC調(diào)亡：培養(yǎng)皿中鋪上3塊細(xì)胞爬片，貼片30 min左右；室溫孵育10 min。用10 mM Tris溶液稀釋2 mg/ml的蛋白酶K溶液，滅活內(nèi)源過(guò)氧化物酶活性。在光學(xué)顯微鏡下觀察呈現(xiàn)深棕色染色的細(xì)胞為凋亡細(xì)胞，每張玻片隨機(jī)選擇5個(gè)高倍鏡視野，5個(gè)視野的平均數(shù)代表調(diào)亡細(xì)胞的數(shù)目[5]。

6.侵襲小室(Transwell)測(cè)定VSMC的侵襲能力

均勻的鋪在Transwell侵襲小室(美國(guó)CORNING公司24孔板，孔徑為8 mm，膜直徑為6.4 mm)上室的纖維聚碳酸酷膜上，每孔50 ul，放置于37℃孵育Transwell板2 h。

將3組的侵襲小室分別放入21% O2及1% O2(37℃,5%CO2)中繼續(xù)孵育48 h和72 h，可見(jiàn)VSMC貼附浸潤(rùn)膜的下室繼續(xù)生長(zhǎng)。

三、統(tǒng)計(jì)學(xué)分析

數(shù)據(jù)采用SPSS 20.0軟件分析計(jì)量資料以(x±SD)表示，多組比較采用單因素方差分析；組間比較用LSD檢驗(yàn)。當(dāng)Plt;0.05時(shí)，認(rèn)為差異具有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

一、AVM穩(wěn)定實(shí)驗(yàn)豬模型的建立

端端吻合后引流靜脈見(jiàn)圖1A、B。均建成由五支供血?jiǎng)用}-畸形血管團(tuán)-單支引流靜脈的穩(wěn)定實(shí)驗(yàn)豬模型，見(jiàn)圖1C、D。

二、VSMC的鑒定

VSMC用胰蛋白酶消化后呈圓形，見(jiàn)圖2A(倒置顯微鏡NF-2000 Nikon)。在透射電鏡(transmission electron microscope,TEM)見(jiàn)圖2B。

三、不同O2條件下細(xì)胞免疫熒光檢測(cè)VSMC密度比較，見(jiàn)圖3(熒光抗體染色×200)。

四、各組VSMC中TGF-β mRNA及蛋白比較

各組經(jīng)內(nèi)參校正后得出2-△△Ct。實(shí)驗(yàn)B、C組中TGF-β mRNA和蛋白均高于A組，差異有顯著性(Plt;0.01)。B組和C組相比較，差異有統(tǒng)計(jì)學(xué)意義(Plt;0.01，圖4，表2、3)。

圖1 吻合后引流靜脈及造影
Fig 1 Drainage vein and angiography after anastomosis
A:L-APA and L-EJV end to end anastomosis;B:L-APA and L-EJV end to end anastomosis after vascular filling;C:Immediately after the procedure (the arrow showed the L-APA-EJV anastomosis);D:Post-operative 28 d angiography (arrow showed L-APA-EJV anastomosis,drainage vein thickening.

圖2 VSMC的鑒定
Fig 2 Identification of VSMC
A:Primary culture of VSMC (×100);B:VSMC cells under transmission electron microscope (×l0000)

圖3 不同O2條件下細(xì)胞免疫熒光檢測(cè)VSMC密度比較
Fig 3 Comparison of VSMC density of cells under different O2conditions
A:Cultured VSMC by 21% O2(arrow showed CD34);B:Cultured VSMC by 1% O2(arrow showed CD34)

GroupnTGF?ββ?actinTGF?β/β?actin GroupA201．93±0．77a1．92±0．74a1．01±1．04a GroupB202．87±1．94b2．05±0．81b1．40±3．03b GroupC203．51±1．25b2．03±0．88b1．73±1．42b

aPlt;0.01,vsGroup B,C；bPlt;0.01,vsGroup A.

圖4 Western Blot法檢測(cè)各組蛋白TGF-β結(jié)果
Fig 4 VSMC TGF-β protein by Western Blot

GroupnTGF?ββ?actinTGF?β/β?actin GroupA203．83±0．63a5．47±1．13a0．70±0．56a GroupB204．99±1．77b5．69±1．41b0．88±1．26b GroupC207．02±1．23b5．50±1．35b1．28±0．91b

aPlt;0.01,vsGroup B,C;bPlt;0.01,vsGroup A.

五、TUNEL法檢測(cè)VSMC調(diào)亡和Transwell侵襲結(jié)果(表4，圖5)

24、48 h三組VSMC調(diào)亡和侵襲數(shù)目差異無(wú)統(tǒng)計(jì)學(xué)意義(Pgt;0.05)。72 h B、C組細(xì)胞調(diào)亡數(shù)目和侵襲顯著增多，且C組顯著多于B組，差異有統(tǒng)計(jì)學(xué)意義(Plt;0.01)。

圖5 不同O2條件下VSMC凋亡和侵襲下室結(jié)果(結(jié)晶紫染色,×200)
Fig 5 VSMC apoptosis and room numbers under attacking under different O2conditions (crystal violet staining,×200)
A:21% O2VSMC apoptosis at 72 h;B:1% O2VSMC apoptosis at 72 h (arrows indicated apoptotic cells);C:21% O2VSMC hit at 72 h (arrows indicated invading cells);D:1% O2VSMC hit at 72 h.

TimeGroupAapoptosishitGroupBapoptosishitGroupCapoptosishit 24h5．53±1．02a1．97±0．54a5．84±0．97a2．03±0．18a5．77±1．01a2．01±0．20a 48h6．87±1．55b2．25±0．66b6．96±1．14b2．38±0．51b6．91±1．09b2．32±0．55b 72h6．64±1．37b2．22±0．43b15．81±3．93b10．05±1．92b20．18±3．47b18．65±4．17b

aPlt;0.01,vsGroup B,C;bPlt;0.01,vsGroup A.

討 論

VSMC是構(gòu)成血管壁的最主要細(xì)胞。正常情況下，血管中膜的VSMC是保證血管壁的張力及收縮運(yùn)動(dòng)的結(jié)構(gòu)基礎(chǔ)[6]。近年研究認(rèn)為[7]，BAVM血管壁中的VSMC受損，收縮能力下降，管壁變薄弱,管壁擴(kuò)張[8]。TGF-β與BAVM密切相關(guān)，其信號(hào)蛋白功能的低失可導(dǎo)致明顯血管結(jié)構(gòu)異常[9]。

在本研究中，首先建立穩(wěn)定的BAVM動(dòng)物模型，然后分離培養(yǎng)實(shí)驗(yàn)組和對(duì)照組的VSMC，采用原代細(xì)胞進(jìn)行不同氧濃度條件下培養(yǎng)。免疫綠色熒光檢測(cè)在不同氧濃度培養(yǎng)后VSMC的分布密度，缺氧的條件可以導(dǎo)致VSMC的密度明顯減少，這說(shuō)明VSMC對(duì)缺氧敏感，缺氧可以引起血管內(nèi)膜構(gòu)筑的改變，使其薄化。Real-time PCR和Western Blot驗(yàn)證TGF-β mRNA及蛋白差異，對(duì)照組與實(shí)驗(yàn)組比較(Plt;0.01)；實(shí)驗(yàn)組組間比較 (Plt;0.01)；考慮缺氧因素可以引起TGF-β的表達(dá)水平顯著上調(diào)，這對(duì)于畸形血管團(tuán)的形成有促進(jìn)作用。TUNEL檢測(cè)VSMC調(diào)亡和Transwell的侵襲結(jié)果，24 h、48 h三組VSMC調(diào)亡和侵襲數(shù)目差異無(wú)統(tǒng)計(jì)學(xué)意義(Pgt;0.05)。72 h B、C組細(xì)胞調(diào)亡數(shù)目和侵襲顯著增多，C組顯著于B組，差異有統(tǒng)計(jì)學(xué)意義(Plt;0.01)。說(shuō)明在不同氧濃度條件下，早期VSMC能夠耐受輕度的缺氧，但隨著VSMC缺氧程度加重，VSMC中線粒體功能失調(diào)，導(dǎo)致VSMC調(diào)亡數(shù)目和侵襲顯著增多。BAVM的畸形血管團(tuán)結(jié)構(gòu)已經(jīng)重鑄[10]，致使VSMC分布及密度變化，在缺氧因素作用下加速了VSMC凋亡。

綜上所述，缺氧因素對(duì)BAVM豬模VSMC中TGF-β表達(dá)有顯著的作用，加速了畸形血管團(tuán)的形成，并且促使VSMC凋亡及侵襲能力增加。在細(xì)胞分子生物學(xué)水平分析缺氧因素對(duì)于VSMC中TGF-β的影響和VSMC的細(xì)胞功能作用，為BAVM畸形血管團(tuán)的加速形成機(jī)制提供可能的基礎(chǔ)實(shí)驗(yàn)研究理論。

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ChangesofTGF-βexpressionandcellularfunctioninBAVManimalmodelsunderlowoxygencondition

PANJun1,ZHAOXiaoyong2,ZHANGXiaoli2

1DepartmentofNeurosurgery,People'sHospitalofDanzhouCity,Danzhou571700;2DepartmentofNeurosurgery,HuaduHospitalAffiliatedtoSouthMedicalUniversity,Guangzhou510800,China

ObjectiveThe effect of hypoxia on endothelial growth factor (TGF-β) expression and functions of VSMC cells in brain arteriovenous malformations (BAVM) vascular intimal smooth muscle cells (VSMC) were studied.MethodsThe BAVM experimental pigs were well established and the separation of cerebral microvascular network (RM) was cultured by VSMC. There were three groups:the control group with normal experimental porcine VSMC (Group A) under 21%O2concentration;the experimental group using porcine VSMC (Group B) under 21%O2concentration,and 1%O2concentration (Group C). VSMC density was detected by immunofluorescence,TGF-β mRNA and protein of VSMC were detected by quantitative real-time polymerase chain reaction and Western blot,respectively. Terminal deoxyribonucleic transferase mediated dUTP nick labeling (TUNEL) method was used to detect the difference of apoptosis of VSMC,and Transwell was used for the determination of VSMC invasion.ResultsVSMC was sensitive to hypoxia. There were statistically significant differences in mRNA and protein of TGF-β between control and experimental groups and among the experiments (Plt;0.01). There was statistically significant difference in VSMC apoptosis and cell invasion number at 72 h (Plt;0.01).ConclusionHypoxia has a significant effect on expression of TGF-β in VSMC of animal model,and it improves the apoptosis and invasion of VSMC and accelerates the formation of the vascular malformation. The analysis of molecular biology can provide a useful idea for the clinical treatment of BAVM.

Hypoxia; Pig model; Vascular smooth muscle cell; Vascular endothelial growth factor; Cell function

1671-2897(2016)15-501-05

R 651.1

A

潘俊，主治醫(yī)師，博士，E-mail:ace15171517@sina.com

*通訊作者：趙曉勇，副教授、副主任醫(yī)師，E-mail:ace0829@sina.com

2015-10-12；修回時(shí)間：2016-01-20)