李周儒，滕道輝，董國(guó)凱，殷文江，蔡紅星

(1.徐州醫(yī)學(xué)院法醫(yī)學(xué)教研室，江蘇徐州 221002；2.江蘇省徐州市銅山區(qū)公安局刑警大隊(duì) 221000)

論著·基礎(chǔ)研究

GDNF通過(guò)AKT/β-catenin調(diào)節(jié)人膠質(zhì)瘤細(xì)胞增殖的研究*

李周儒1，滕道輝2，董國(guó)凱1，殷文江1，蔡紅星1

(1.徐州醫(yī)學(xué)院法醫(yī)學(xué)教研室，江蘇徐州 221002；2.江蘇省徐州市銅山區(qū)公安局刑警大隊(duì) 221000)

目的 研究膠質(zhì)細(xì)胞系源性神經(jīng)營(yíng)養(yǎng)因子(GDNF)促進(jìn)人膠質(zhì)瘤細(xì)胞增殖的機(jī)制。方法將膠質(zhì)瘤樣本分為低級(jí)別膠質(zhì)瘤組和高級(jí)別膠質(zhì)瘤組，腦挫裂傷患者樣本作為正常對(duì)照組，每組各12例；將膠質(zhì)瘤細(xì)胞系C6細(xì)胞分為細(xì)胞對(duì)照組、牛血清蛋白(BSA)組和GDNF組。CCK-8實(shí)驗(yàn)檢測(cè)細(xì)胞增殖率，蛋白免疫印跡(Western blot)法檢測(cè)各組AKT、p-AKT、β-catenin和p-β-catenin的表達(dá)。結(jié)果與正常對(duì)照組相比，膠質(zhì)瘤組AKT、p-AKT、β-catenin和p-β-catenin的蛋白水平顯著增加(P<0.05)，且高級(jí)別膠質(zhì)瘤組的蛋白水平明顯高于低級(jí)別膠質(zhì)瘤組(P<0.05)。CCK-8檢測(cè)C6膠質(zhì)瘤細(xì)胞實(shí)驗(yàn)中，與細(xì)胞對(duì)照組相比，GDNF組細(xì)胞增殖率明顯增高(P<0.05)，Akt表達(dá)水平?jīng)]有明顯變化，而p-Akt、β-catenin和p-β-catenin的蛋白表達(dá)均顯著增加(P<0.05)。結(jié)論GDNF可能通過(guò)上調(diào)膠質(zhì)瘤細(xì)胞p-AKT、β-catenin和p-β-catenin來(lái)促進(jìn)膠質(zhì)瘤細(xì)胞的增殖。

神經(jīng)膠質(zhì)瘤;膠質(zhì)細(xì)胞源性神經(jīng)營(yíng)養(yǎng)因子;AKT;β-catenin

膠質(zhì)瘤是人中樞神經(jīng)系統(tǒng)常見(jiàn)病、多發(fā)病，因發(fā)病機(jī)制未明，故臨床尚無(wú)根治膠質(zhì)瘤的有效辦法。膠質(zhì)瘤細(xì)胞內(nèi)一系列信號(hào)轉(zhuǎn)導(dǎo)的機(jī)制研究是近年來(lái)神經(jīng)外科學(xué)研究的熱點(diǎn)。普遍認(rèn)為，膠質(zhì)瘤細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)通路中信號(hào)傳導(dǎo)因子引發(fā)膠質(zhì)瘤一系列生物學(xué)效應(yīng)。有文獻(xiàn)報(bào)道膠質(zhì)瘤細(xì)胞AKT與β-catenin表達(dá)水平上調(diào)，且兩者之間存在著多種交叉聯(lián)系，都能夠促進(jìn)細(xì)胞增殖，然而這兩者相互作用的機(jī)制目前仍不甚清楚[1-3]。同時(shí)有研究證實(shí)膠質(zhì)細(xì)胞系源性神經(jīng)營(yíng)養(yǎng)因子(GDNF)能夠促進(jìn)C6膠質(zhì)瘤細(xì)胞增殖，GDNF下游通路因子對(duì)AKT/β-catenin信號(hào)通路存在調(diào)節(jié)作用，其可能參與GDNF促C6膠質(zhì)瘤細(xì)胞增殖的機(jī)制[4-7]。本試驗(yàn)首先通過(guò)GDNF促進(jìn)膠質(zhì)瘤細(xì)胞增殖，然后通過(guò)蛋白免疫印跡(Western blot)方法、CCK-8檢測(cè)細(xì)胞增殖率等方法，觀察在促進(jìn)膠質(zhì)瘤細(xì)胞增殖過(guò)程中GDNF是否通過(guò)調(diào)節(jié)AKT/β-catenin信號(hào)通路來(lái)實(shí)現(xiàn)的這一生物學(xué)作用。

1 材料與方法

1.1 材料 臨床膠質(zhì)瘤患者的術(shù)中所取膠質(zhì)瘤樣本,膠質(zhì)瘤細(xì)胞系C6細(xì)胞購(gòu)自中科院細(xì)胞庫(kù)。Akt(ab66138)、 p-Akt(ab8805)、β-catenin(ab32572)、p-β-catenin(ab24925)一抗購(gòu)自Abcam公司。羊抗小鼠熒光二抗(#610632-002)購(gòu)自Rockland公司。羊抗兔熒光二抗(#35568)購(gòu)自Thermo公司。牛血清白蛋白(BSA，A2153)購(gòu)自Sigma公司。

1.2 方法

1.2.1 組織樣本 膠質(zhì)瘤組織樣本24例，按照WHO診斷標(biāo)準(zhǔn)將膠質(zhì)瘤樣本分成低級(jí)別腦膠質(zhì)瘤組、高級(jí)別腦膠質(zhì)瘤組，每組12例。所有樣本均取自徐州醫(yī)學(xué)院附屬醫(yī)院神經(jīng)外科手術(shù)切除并經(jīng)過(guò)病理證實(shí)的人腦膠質(zhì)瘤樣本，其中男15例，女9例，年齡10～82歲。另取12例因腦挫裂傷行內(nèi)減壓術(shù)患者的非腫瘤腦組織設(shè)為正常對(duì)照組。Western blot方法檢測(cè)3組中Akt、p-Akt、β-catenin和p-β-catenin表達(dá)。

1.2.2 細(xì)胞培養(yǎng) C6膠質(zhì)瘤細(xì)胞購(gòu)自中科院上海細(xì)胞庫(kù)，依照其培養(yǎng)說(shuō)明書(shū)進(jìn)行培養(yǎng)。每3～4天，細(xì)胞處于對(duì)數(shù)生長(zhǎng)期，鋪滿培養(yǎng)瓶底約70%時(shí)傳代1次(主要保證細(xì)胞的活性)。按1∶3或1∶4的細(xì)胞比例傳代。將傳代所得細(xì)胞分為細(xì)胞對(duì)照組，BSA組和GDNF組。細(xì)胞對(duì)照組不進(jìn)行任何處理，BSA組加入BSA(60 μg/L)處理，GDNF組加入GDNF藥物(60 μg/L)，BSA組和GDNF組處理時(shí)間均為15 min。處理結(jié)束后細(xì)胞收集入EP管中，以備細(xì)胞提取。然后進(jìn)行樣品總蛋白的提取及蛋白含量的測(cè)定，分裝并置-80 ℃冰箱待用。

1.2.3 CCK-8試驗(yàn)檢測(cè)C6神經(jīng)膠質(zhì)瘤細(xì)胞的增殖 取對(duì)數(shù)生長(zhǎng)期的C6膠質(zhì)瘤細(xì)胞接種于96 孔培養(yǎng)板(每孔約5×103個(gè)細(xì)胞)，24 h后細(xì)胞生長(zhǎng)達(dá)約80% 融合時(shí),以脂質(zhì)體與DNA比例為2.2 μL∶1.0 μg轉(zhuǎn)染細(xì)胞。將轉(zhuǎn)染所得細(xì)胞分為細(xì)胞對(duì)照組，BSA組和GDNF藥物組。每組設(shè)4個(gè)復(fù)孔，在轉(zhuǎn)染46 h后每孔加CCK-8試劑10 μL，繼續(xù)孵育2 h，終止培養(yǎng)，小心吸取孔內(nèi)的液體，移入另外1個(gè)對(duì)應(yīng)的96孔板內(nèi)。在全波長(zhǎng)酶標(biāo)儀上測(cè)定波長(zhǎng)為450 nm的吸光光度值,試驗(yàn)重復(fù)3次，取均值。

1.2.4 Western blot檢測(cè) 等量蛋白樣品經(jīng)7.5% SDS聚丙烯凝膠電泳分離后，以濕轉(zhuǎn)法轉(zhuǎn)移至聚偏氟乙烯膜上，3% BSA封閉后加入一抗4 ℃過(guò)夜。用洗滌液除去多余一抗，然后加入相應(yīng)的二抗，37 ℃反應(yīng)1 h，洗膜。用熒光掃描儀掃描圖像，蛋白表達(dá)水平以目的蛋白/Actin蛋白的比值來(lái)表示。

2 結(jié) 果

2.1 組織樣本中Akt、p-Akt、β-catenin和p-β-catenin的表達(dá) Western blot檢測(cè)正常對(duì)照組、低級(jí)別膠質(zhì)瘤組和高級(jí)別膠質(zhì)瘤組樣品，結(jié)果顯示與正常對(duì)照組相比，低級(jí)別膠質(zhì)瘤組和高級(jí)別膠質(zhì)瘤組中Akt、p-Akt、β-catenin和p-β-catenin的蛋白水平顯著升高(P<0.05)，且高級(jí)別膠質(zhì)瘤組Akt、p-Akt、β-catenin和p-β-catenin的表達(dá)水平高于低級(jí)別膠質(zhì)瘤組(P<0.05)，見(jiàn)圖1。

N組：正常對(duì)照組；L組：低級(jí)別膠質(zhì)瘤組；H組：高級(jí)別膠質(zhì)瘤組。光密度比值：樣本光密度/Actin光密度。*：P<0.05，與N組比較；#：P<0.05，與L組比較。

圖1 3組膠質(zhì)瘤組織Akt、p-Akt、β-catenin和p-β-catenin蛋白表達(dá)

2.2 GDNF對(duì)C6膠質(zhì)瘤細(xì)胞系增殖的影響 CCK-8實(shí)驗(yàn)檢測(cè)GDNF誘導(dǎo)C6膠質(zhì)瘤細(xì)胞增殖，結(jié)果顯示與細(xì)胞對(duì)照組細(xì)胞增殖率相比，GDNF組細(xì)胞增殖率增大(P<0.05)，而B(niǎo)SA組細(xì)胞增值率與細(xì)胞對(duì)照組之間差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)，見(jiàn)圖2。

*：P<0.05，與細(xì)胞對(duì)照組比較。

圖2 CCK-8實(shí)驗(yàn)檢測(cè)GDNF作用對(duì)C6細(xì)胞增殖的影響

2.3 GDNF對(duì)C6膠質(zhì)瘤細(xì)胞系A(chǔ)KT、p-AKT、β-catenin和p-β-catenin表達(dá)的影響 Western blot檢測(cè)結(jié)果顯示與細(xì)胞對(duì)照組相比，GDNF組中的p-AKT、β-catenin和p-β-catenin的蛋白水平顯著升高(P<0.05)，而AKT沒(méi)有明顯變化，BSA組中的AKT、p-AKT、β-catenin和p-β-catenin的表達(dá)水平則無(wú)明顯變化，見(jiàn)圖3。

*：P<0.05，與細(xì)胞對(duì)照組比較。

圖3 GDNF對(duì)C6膠質(zhì)瘤細(xì)胞Akt、p-Akt、β-catenin和p-β-catenin蛋白表達(dá)的影響

3 討 論

腦膠質(zhì)瘤是顱內(nèi)最常見(jiàn)的惡性腫瘤，占原發(fā)性中樞神經(jīng)系統(tǒng)惡性腫瘤的50%～60%[8]。目前對(duì)其治療策略是在手術(shù)切除腫瘤主體的基礎(chǔ)上，采用多種手段殺滅殘存瘤細(xì)胞或抑制其增殖，但是手術(shù)加放化療的平均生存期僅為8～11個(gè)月。研究人員發(fā)現(xiàn)，腦膠質(zhì)瘤中GDNF及其受體人膠質(zhì)細(xì)胞系來(lái)源神經(jīng)營(yíng)養(yǎng)因子受體α1(GFRα1)的表達(dá)量顯著高于正常腦組織中的含量[9]，其中GDNF的含量大約是正常成年腦組織中含量的5倍。這種表達(dá)異常與腫瘤的哪些生物學(xué)活性有關(guān)，目前其具體的機(jī)制仍不明確。

GDNF是糖基化二硫鍵結(jié)合的同源二聚體蛋白質(zhì)，為轉(zhuǎn)化生長(zhǎng)因子(TGF)超家族的成員[10]。它是一種神經(jīng)營(yíng)養(yǎng)因子，對(duì)多種神經(jīng)元如多巴胺能神經(jīng)元、運(yùn)動(dòng)神經(jīng)元、交感神經(jīng)元等有明顯營(yíng)養(yǎng)作用[11-13]。有文獻(xiàn)報(bào)道GDNF能夠促使膠質(zhì)瘤細(xì)胞增殖[7]。有研究指出，GDNF通過(guò)激活A(yù)KT來(lái)發(fā)揮其對(duì)腫瘤細(xì)胞的促增殖作用[14]。AKT、JNK信號(hào)通路與許多信號(hào)途徑交叉，形成復(fù)雜的信號(hào)網(wǎng)絡(luò)。AKT信號(hào)通路在特定的條件下，可以被細(xì)胞因子、生長(zhǎng)因子和應(yīng)激等多種因素激活，參與細(xì)胞的增殖、分化、凋亡和惡變等多種生物學(xué)反應(yīng)，與腫瘤、糖尿病等多種疾病的發(fā)生、發(fā)展密切相關(guān)。

本研究發(fā)現(xiàn)，膠質(zhì)瘤組織中AKT、p-AKT、β-catenin和p-β-catenin均表達(dá)增高，而GDNF細(xì)胞因子處理后膠質(zhì)瘤細(xì)胞系p-AKT、β-catenin和p-β-catenin蛋白表達(dá)增高，但AKT表達(dá)無(wú)變化。這些蛋白表達(dá)的變化可能潛在地促進(jìn)膠質(zhì)瘤細(xì)胞的增殖。因此推測(cè)GDNF很可能通過(guò)誘導(dǎo)p-AKT、β-catenin和p-β-catenin的表達(dá)，促進(jìn)AKT和β-catenin向其活化形式的轉(zhuǎn)變，從而促進(jìn)膠質(zhì)瘤細(xì)胞的增殖。

有關(guān)GDNF表達(dá)與膠質(zhì)瘤惡性程度之間關(guān)系研究報(bào)道較少，根據(jù)本試驗(yàn)結(jié)果認(rèn)為GDNF作為重要的因子可能通過(guò)AKT磷酸化以及上調(diào)β-catenin和p-β-catenin來(lái)促進(jìn)膠質(zhì)瘤洗標(biāo)增殖。如果能夠闡明GDNF促進(jìn)膠質(zhì)瘤細(xì)胞增殖的機(jī)制，將為臨床研發(fā)抑制膠質(zhì)瘤的藥物提供良好的理論基礎(chǔ)。

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GDNF regulates the proliferation of glioma cells through AKT/β-catenin signaling pathway*

LiZhouru1,TengDaohui2,DongGuokai1,YinWenjiang1,CaiHongxing1

(1.DepartmentofForensicMedicine,XuzhouMedicalCollege,Xuzhou,Jiangsu221002,China;2.CriminalPoliceBrigadeofTongshanDistrict,PublicSecurityBureauofXuzhou,Xuzhou,Jiangsu221000,China)

Objective To study the mechanism that glial cell line-derived neurotrophic factor (GDNF) promotes human glioma cells proliferation.MethodsWe divided glioma samples into two groups,including low-grade glioma group and high-grade glioma group,while cerebral contusion patients were treated as the control group,12 cases in each group.C6 glioma cell lines were divided into three groups,such as GDNF group,BSA(bovine serum albumin) group and control group.CCK-8(cell counting kit-8) was used to detect the cell proliferation,while Western blot was used to detect the expression of AKT,p-AKT,β-catenin and p-β-catenin in each group.ResultsComparing with the control group,the expression levels of AKT,p-AKT,β-catenin and p-β-catenin in glioma group had a significantly increased (P<0.05).Meanwhile,the high-grade gliomas group also had a significant increase in those more than low-grade gliomas group (P<0.05).CCK-8 test showed that the cell proliferation in GDNF group was significantly higher than the control group (P<0.05),and the expression levels of p-AKT,β-catenin and p-β-catenin proteins all had a significant increase (P<0.05).However,the expression level of AKT had no obvious difference.ConclusionGDNF might promote the proliferation of glioma cells by up-regulating the expression of p-AKT,β-catenin and p-β-catenin.

glioma;glial cell line-derived neurotrophic factor;AKT;β-catenin

10.3969/j.issn.1671-8348.2015.22.003

國(guó)家自然科學(xué)基金項(xiàng)目(81101899)。

李周儒(1980-)，講師，碩士，主要從事腦損傷機(jī)制研究。

R739.4

A

1671-8348(2015)22-3034-03

2015-02-28

2015-07-09)