125I粒子短時低劑量率照射對胰腺癌Capan-2細(xì)胞神經(jīng)浸潤的影響

司佩任 路箏 劉巖 馬建霞 吳洪玉 李兆申

·論著·

125I粒子短時低劑量率照射對胰腺癌Capan-2細(xì)胞神經(jīng)浸潤的影響

司佩任 路箏 劉巖 馬建霞 吳洪玉 李兆申

目的觀察125I粒子短時低劑量率照射對胰腺癌Capan-2細(xì)胞神經(jīng)浸潤的影響，并探討其分子機(jī)制。方法建立胰腺癌Capan-2細(xì)胞和大鼠背根神經(jīng)節(jié)(DRG)共培養(yǎng)及Capan-2或DRG單培養(yǎng)模型。通過125I粒子低劑量率照射平板對3種模型進(jìn)行照射，以相應(yīng)未照射模型作為對照。倒置顯微鏡下觀察癌細(xì)胞、DRG的生長，圖像分析軟件計(jì)算神經(jīng)突和癌細(xì)胞集落占據(jù)的表面積，ELISA法檢測細(xì)胞培養(yǎng)上清液和基質(zhì)膠溶解液中神經(jīng)生長因子(NGF)及轉(zhuǎn)化生長因子α(TGF-α)濃度， RT-PCR法檢測胰腺癌Capan-2細(xì)胞神經(jīng)營養(yǎng)因子-3 (NT-3)mRNA表達(dá)。結(jié)果共培養(yǎng)模型中DRG發(fā)出的神經(jīng)突向癌細(xì)胞定向、集中生長，而癌細(xì)胞沿神經(jīng)突的方向生長。經(jīng)125I粒子照射后這種定向、集中和互逆的生長受到一定程度的抑制。共培養(yǎng)組第5天所增加的神經(jīng)突表面積為290.15±12.08，較DRG單培養(yǎng)組的124.83±6.96顯著增加(P<0.01)，經(jīng)照射后的神經(jīng)突表面積減少到201.53±12.20(P<0.01)；所增加的Capan-2細(xì)胞表面積為300.47±12.99，較Capan-2細(xì)胞單培養(yǎng)組的199.30±8.60顯著增加(P<0.01)，經(jīng)照射后的Capan-2細(xì)胞表面積減少到202.35±7.97(P<0.01)。共培養(yǎng)組不表達(dá)NT-3 mRNA，經(jīng)照射后NT-3mRNA表達(dá)量為0.68±0.04(P<0.05)。共培養(yǎng)組培養(yǎng)上清液中NGF及TGF-α濃度分別為(27.56±13.73)、(40.86±20.73)ng/ml，經(jīng)照射后分別升高到(94.98±33.80)、(157.54±83.76)ng/ml，差異有統(tǒng)計(jì)學(xué)意義(P<0.05或<0.01)。共培養(yǎng)組基質(zhì)膠溶解液中NGF及TGF-α濃度分別為(60.42±33.03)、(64.39±21.52)ng/ml，經(jīng)照射后分別升高到(132.52±53.01)、(138.38±83.58)ng/ml，其中NGF的差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。結(jié)論125I粒子短時低劑量率照射可以抑制胰腺癌和神經(jīng)的交互作用，其機(jī)制可能與癌細(xì)胞促神經(jīng)浸潤介質(zhì)NGF、TGF-α和NT-3等表達(dá)上調(diào)有關(guān)。

胰腺腫瘤； 碘同位素； 小劑量照射； 腫瘤浸潤

胰腺癌遠(yuǎn)處轉(zhuǎn)移擴(kuò)散的途徑包括血管、淋巴管和神經(jīng)浸潤，其中周圍神經(jīng)浸潤(perineural invasion, PNI)是被廣泛接受的特殊擴(kuò)散途徑[1]。極高頻率的PNI(90%，甚至100%)[1-3]是胰腺癌的特征。最初的胰腺內(nèi)PNI可能引起胰腺外神經(jīng)叢浸潤，是持續(xù)胰腺外播散和術(shù)后復(fù)發(fā)的主要原因[4]。最近的研究[5-7]證實(shí)，PNI可能涉及腫瘤細(xì)胞和神經(jīng)元之間相互的趨向性和旁分泌的交互作用。

胰腺癌內(nèi)放療治療是通過置入瘤內(nèi)的放射性粒子持續(xù)釋放射線來達(dá)到最大限度地殺傷腫瘤細(xì)胞的作用。目前文獻(xiàn)報(bào)道[8-9]，采用內(nèi)鏡超聲(EUS)引導(dǎo)下穿刺等方式植入放射性粒子治療不能手術(shù)的中晚期胰腺癌是安全有效的，可明顯改善患者生活質(zhì)量，尤其可以緩解患者的疼痛反應(yīng)。胰腺癌疼痛的發(fā)生多與其嗜神經(jīng)特性有關(guān)。為此，本研究用高神經(jīng)轉(zhuǎn)移的胰腺癌細(xì)胞Capan-2和大鼠背根神經(jīng)節(jié)(dorsal root ganglion,DRG)構(gòu)建共培養(yǎng)體，觀察125I粒子短時低劑量照射對胰腺癌細(xì)胞、DRG生長及培養(yǎng)上清中PNI相關(guān)細(xì)胞因子含量的影響，探討胰腺癌細(xì)胞PNI的分子機(jī)制。

材料和方法

一、胰腺癌細(xì)胞神經(jīng)浸潤的體外模型建立

4只SD雄性大鼠由第二軍醫(yī)大學(xué)實(shí)驗(yàn)中心提供，動物許可證號SCXK(滬)2012-0003，清潔級，體質(zhì)量80 g左右。參照文獻(xiàn)[5]用CO2將大鼠安樂死，75%乙醇消毒。在無菌條件下切除胸和腰段的DRGs，置RPMI培養(yǎng)液中備用。

胰腺癌細(xì)胞株Capan-2購自ATCC(American type cultrue collection)，常規(guī)培養(yǎng)、傳代。參照文獻(xiàn)[5]把100 μl EHS基質(zhì)膠(BD Biosciences公司)加進(jìn)置于冰上(為保持基質(zhì)膠的流動性)的直徑3.5 cm的細(xì)胞培養(yǎng)皿中央，放入1枚DRG，再種植105個Capan-2細(xì)胞，置細(xì)胞培養(yǎng)箱中加溫到37℃，使細(xì)胞和DRG固化在基質(zhì)膠中。同時制作單獨(dú)種植Capan-2或DRG的培養(yǎng)皿。然后在培養(yǎng)皿中加入含有10%熱滅活的胎牛血清(GIBCO公司)、100 U/ml青霉素、100 μg/ml鏈霉素的RPMI-1640培養(yǎng)液(GIBCO公司)。

二、125I粒子短時低劑量率照射模型建立

參照文獻(xiàn)[10]設(shè)計(jì)。用5 cm直徑的細(xì)胞培養(yǎng)皿灌注石蠟，晾干后制作照射平板。在3 cm直徑的圓周線找出8個等距離的點(diǎn)，在這8個點(diǎn)和圓心處刻出直徑6 mm的凹槽。把9顆1.0 mCi的125I粒子分別置入9個凹槽內(nèi)，制成照射平板。將上述制作的3種體外模型培養(yǎng)皿分別放在照射裝置的中央，置入專用的細(xì)胞培養(yǎng)箱中常規(guī)培養(yǎng)，總照射劑量為2 Gy。為避免各培養(yǎng)皿放射線的互相干擾，每個125I粒子照射裝置用金屬隔板隔開。以上述制作的3種體外模型培養(yǎng)皿放入普通培養(yǎng)箱內(nèi)培養(yǎng)作為相應(yīng)對照組。培養(yǎng)第3、5天在倒置顯微鏡下觀察各組Capan-2細(xì)胞、DRGs的形態(tài)并攝像，應(yīng)用圖像分析軟件Image pro-plus 5.0計(jì)算DRG發(fā)出的神經(jīng)突和選定區(qū)域的Capan-2細(xì)胞集落占據(jù)的表面積。照射第5天收集培養(yǎng)液上清，應(yīng)用基質(zhì)膠溶解劑(BD Biosciences公司)試劑盒將基質(zhì)膠溶解并收集基質(zhì)膠溶解液，收集從基質(zhì)膠溶解液中分離出的Capan-2細(xì)胞和DRGs。實(shí)驗(yàn)期間避免放射污染，凡是涉及接觸放射源的步驟均用鉛衣、鉛眼鏡、鉛圍脖和鉛手套防護(hù)。

三、人神經(jīng)生長因子(NGF)、轉(zhuǎn)化生長因子α(TGF-α)檢測

應(yīng)用NGF、TGF-α的ELISA試劑盒(Westang公司)檢測收集的細(xì)胞培養(yǎng)上清液和基質(zhì)膠溶解液內(nèi)NGF、TGF-α含量，按試劑盒說明書操作。通過試劑盒攜帶的NGF或TGF-α標(biāo)準(zhǔn)品繪制標(biāo)準(zhǔn)曲線，計(jì)算樣本中NGF、TGF-α濃度。

四、Capan-2細(xì)胞神經(jīng)營養(yǎng)因子3(NT-3)mRNA表達(dá)的檢測

收集各組用基質(zhì)溶解劑后分離出來的Capan-2細(xì)胞，用預(yù)冷的PBS洗滌3次，應(yīng)用Trizol 試劑(Invitrogen公司)提取細(xì)胞總RNA。采用逆轉(zhuǎn)錄試劑盒(Takara公司)合成cDNA。RT反應(yīng)條件：37℃ 15 min，85℃ 5 s。采用RT-PCP法檢測NT-3 mRNA的表達(dá)。引物通過Primer Premier 5.0軟件設(shè)計(jì)，NT-3引物上游為5′-AAGTCATCGGCCATCGACA-3′， 下游為5′-TCAGTGCTCGGACGTAGGTT-3′，擴(kuò)增片段200 bp；內(nèi)參GAPDH引物上游為5′-GCACCGTCAAGGCTGAGAAC-3′，下游為5′-ATGGTGGTGAAGACGCCAGT-3′，擴(kuò)增片段142 bp。引物均由上海英俊生物工程技術(shù)有限公司合成。PCR反應(yīng)條件： 94℃ 4 min，94℃ 45 s、58℃ 45 s、72℃ 45 s，35個循環(huán)，最后72℃延伸10 min。PCR擴(kuò)增產(chǎn)物經(jīng)瓊脂糖凝膠電泳分離，ImageJ軟件掃描，以目的條帶與內(nèi)參條帶的灰度比值表示mRNA的表達(dá)量。每組實(shí)驗(yàn)重復(fù)3次，取均值。

五、統(tǒng)計(jì)學(xué)分析

結(jié) 果

一、各組Capan-2細(xì)胞和神經(jīng)突的生長狀況

在共培養(yǎng)模型可以觀察到DRG與Capan-2細(xì)胞之間定向、集中和互逆的生長，即DRG發(fā)出的神經(jīng)突向癌細(xì)胞定向、集中生長，而癌細(xì)胞沿神經(jīng)突的方向生長。經(jīng)125I粒子持續(xù)低劑量率照射后這種定向、集中和互逆的生長雖仍存在，但其趨勢受到一定程度的抑制(圖1)。

圖1共培養(yǎng)照射組(上)和未照射組(下)在第1(a)、3(b)、5(c)天的細(xì)胞和神經(jīng)突生長

DRG和Capan-2共培養(yǎng)組在培養(yǎng)第3、5天所增加的神經(jīng)突表面積分別為182.15±10.05、290.15±12.08，而DRG單培養(yǎng)組所增加的神經(jīng)突表面積分別為82.08±3.48、124.83±6.96，共培養(yǎng)組顯著大于相應(yīng)時間的DRG單培養(yǎng)組(t值分別為23.044、29.056，P值均<0.01)。經(jīng)125I粒子照射后，共培養(yǎng)組所增加的神經(jīng)突表面積分別為128.30±10.02、201.53±12.20，均顯著低于相應(yīng)時間的共培養(yǎng)未照射組(t值分別為9.294、12.644，P值均<0.01)。

DRG和Capan-2共培養(yǎng)組在培養(yǎng)第3、5天所增加的Capan-2細(xì)胞表面積分別為162.93±6.96、300.47±12.99，而Capan-2細(xì)胞單培養(yǎng)組所增加的Capan-2細(xì)胞表面積分別為112.08±10.55、199.30±8.60，共培養(yǎng)組顯著大于相應(yīng)時間的Capan-2細(xì)胞單培養(yǎng)組(t值分別為90.855、15.901，P值均<0.01)。經(jīng)125I粒子照射后，共培養(yǎng)組所增加的Capan-2細(xì)胞表面積分別為95.12±4.83、202.35±7.97，均顯著低于相應(yīng)時間的共培養(yǎng)未照射組(t值分別為19.610,16.116，P值均<0.01)。

二、各組Capan-2細(xì)胞NT-3 mRNA表達(dá)的變化

DRG和Capan-2共培養(yǎng)照射組NT-3 mRNA表達(dá)量為0.68±0.04，而共培養(yǎng)未照射組不表達(dá)NT-3 mRNA，差異有統(tǒng)計(jì)學(xué)意義(t=5.66，P<0.05)。Capan-2細(xì)胞單培養(yǎng)照射組NT-3 mRNA表達(dá)量為0.71±0.04，較單培養(yǎng)未照射組的0.57±0.03明顯上調(diào)，差異有統(tǒng)計(jì)學(xué)意義(Z=2.882，P<0.05，圖2)。

圖2Capan-2細(xì)胞單培養(yǎng)未照射組(1)和照射組(2)及共培養(yǎng)未照射組(3)和照射組(4) NT-3 mRNA的表達(dá)

三、各組Capan-2細(xì)胞培養(yǎng)上清和基質(zhì)膠溶解液中NGF濃度的變化

DRG和Capan-2共培養(yǎng)照射組的培養(yǎng)上清中NGF濃度為(94.98±33.80)ng/ml，較共培養(yǎng)未照射組的(27.56±13.73)ng/ml顯著升高，差異有統(tǒng)計(jì)學(xué)意義(t=4.132，P<0.01)。Capan-2細(xì)胞單培養(yǎng)照射組的培養(yǎng)上清中NGF濃度為(24.62±10.10)ng/ml，單培養(yǎng)未照射組為(38.89±10.60)ng/ml，差異無統(tǒng)計(jì)學(xué)意義(t=2.182，P=0.06)。

DRG和Capan-2共培養(yǎng)照射組的基質(zhì)膠溶解液中NGF濃度為(132.52±53.01)ng/ml，較共培養(yǎng)未照射組的(60.42±33.03)ng/ml顯著升高，差異有統(tǒng)計(jì)學(xué)意義(t=2.581，P<0.05)。Capan-2細(xì)胞單培養(yǎng)照射組的基質(zhì)膠溶解液中NGF濃度為(56.59±8.15)ng/ml，單培養(yǎng)未照射組為(57.81±3.12)ng/ml，差異無統(tǒng)計(jì)學(xué)意義(t=0.410，P=0.890)。

四、各組Capan-2細(xì)胞培養(yǎng)上清和基質(zhì)膠溶解液中TGF-α濃度的變化

DRG和Capan-2共培養(yǎng)照射組的培養(yǎng)上清中TGF-α濃度為(157.54±83.76)ng/ml，較共培養(yǎng)未照射組的(40.86±20.73)ng/ml顯著升高，差異有統(tǒng)計(jì)學(xué)意義(t=2.704，P<0.05)。Capan-2細(xì)胞單培養(yǎng)照射組培養(yǎng)上清中TGF-α濃度為(54.80±34.71)ng/ml，單培養(yǎng)未照射組為(105.21±21.97)ng/ml，差異無統(tǒng)計(jì)學(xué)意義(t=2.454，P=0.05)。

DRG和Capan-2共培養(yǎng)照射組基質(zhì)膠溶解液中TGF-α濃度為(138.38±83.58)ng/ml，共培養(yǎng)未照射組為(64.39±21.52)ng/ml，差異無統(tǒng)計(jì)學(xué)意義(t=1.917，P=0.09)。Capan-2細(xì)胞單培養(yǎng)照射組基質(zhì)膠溶解液中TGF-α濃度為(157.71±39.57)ng/ml，較單培養(yǎng)未照射組的(83.39±26.69)ng/ml顯著升高，差異有統(tǒng)計(jì)學(xué)意義(t=3.359，P<0.05)。

討 論

以前的研究多用前列腺癌細(xì)胞與DRG共培養(yǎng)證實(shí)腫瘤細(xì)胞沿神經(jīng)節(jié)起源的神經(jīng)突移行，神經(jīng)突集中、定向性向腫瘤細(xì)胞集落外生長[5]。同樣，胰腺癌細(xì)胞與DRG共培養(yǎng)模型中，腫瘤細(xì)胞在移行前經(jīng)歷早期形態(tài)學(xué)改變和神經(jīng)細(xì)胞的神經(jīng)突定向朝向癌細(xì)胞生長，最終導(dǎo)致移行細(xì)胞成簇圍繞在神經(jīng)節(jié)周圍[6-7]。這些發(fā)現(xiàn)提示神經(jīng)元與癌細(xì)胞之間存在互惠的交互作用，這種交互作用有助于腫瘤細(xì)胞的增殖和抑制凋亡，并有利于神經(jīng)生長。

神經(jīng)營養(yǎng)因子家族(NTs)包括NGF、NT-3等，在胰腺癌的發(fā)生和演進(jìn)過程中發(fā)揮重要作用，包括刺激趨化性、腫瘤侵襲性、克隆增生、各種腫瘤細(xì)胞的形態(tài)學(xué)改變等[4]。

NGF在胰腺癌細(xì)胞系中過表達(dá)，其受體trkA在周圍神經(jīng)的神經(jīng)束膜上強(qiáng)表達(dá)，NGF與trkA相互作用調(diào)節(jié)神經(jīng)浸潤[6]。NT-3在胰腺癌標(biāo)本中過表達(dá)，刺激腫瘤細(xì)胞浸潤[11]。表皮生長因子受體(EGFR)和TGF-α在有胰腺癌細(xì)胞的組織中大量表達(dá)，神經(jīng)也產(chǎn)生TGF-α，TGF-α和EGFR相互作用觸發(fā)一系列增強(qiáng)細(xì)胞增殖的事件，與胰腺癌的神經(jīng)浸潤有關(guān)[12]。

針對胰腺癌細(xì)胞與神經(jīng)節(jié)之間互惠的交互作用，以及這些與胰腺癌神經(jīng)浸潤有關(guān)的因子，本研究結(jié)合文獻(xiàn)資料[5,10]構(gòu)建了一個新的125I籽源短時間低劑量率照射干預(yù)胰腺癌神經(jīng)浸潤的體外模型，用125I籽源短時間低劑量率持續(xù)照射(總照射劑量為2 Gy)的方法干預(yù)胰腺癌細(xì)胞與神經(jīng)的交互作用，觀察癌細(xì)胞與神經(jīng)突的生長和形態(tài)變化， 以及NGF、NT-3和TGF-α表達(dá)的改變。結(jié)果發(fā)現(xiàn)，125I籽源短時間低劑量率持續(xù)照射后在形態(tài)學(xué)和生物學(xué)行為上抑制胰腺癌細(xì)胞和神經(jīng)之間的交互作用，使癌細(xì)胞向神經(jīng)突的生長和移行減緩。但在共培養(yǎng)模型培養(yǎng)上清液和基質(zhì)膠溶解液中，照射組的NGF和TGF-α濃度高于未照射對照組，且照射組胰腺癌細(xì)胞NT-3 mRNA的表達(dá)顯著上調(diào)，提示125I籽源短時間低劑量率持續(xù)照射可能增強(qiáng)胰腺癌細(xì)胞的侵襲性，與Ohuchid等[13]的研究一致。他們把照射過的成纖維細(xì)胞與胰腺癌細(xì)胞共培養(yǎng)，可刺激成纖維細(xì)胞分泌MMP-2，增強(qiáng)腫瘤細(xì)胞的侵襲力，但使胰腺癌細(xì)胞的移行能力減弱，這也可以解釋本研究在體外模型中看到的現(xiàn)象，即神經(jīng)節(jié)與癌細(xì)胞的互逆生長明顯減弱。

以前的研究認(rèn)為，放療可作為很多惡性腫瘤主要的一種輔助治療。應(yīng)用于胰腺癌患者放療的原理是基于放射能抑制體外細(xì)胞增殖或使凋亡的細(xì)胞死亡，并抑制體內(nèi)腫瘤的生長[14]。然而，近來很多證據(jù)顯示放射通過激活與腫瘤浸潤和轉(zhuǎn)移有關(guān)的眾多途徑促進(jìn)癌細(xì)胞的惡性行為[15-17]。此現(xiàn)象可能由于多為體外實(shí)驗(yàn)，與體內(nèi)腫瘤生長的微環(huán)境不同，導(dǎo)致對照射的反應(yīng)不同。其次實(shí)驗(yàn)多為短時低劑量率照射，照射時間及劑量是否充分也是值得考慮的問題。因此深入研究125I粒子短時低劑量率照射干預(yù)胰腺癌的分子機(jī)制顯得尤為重要。

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EffectofIodine125seedsshorttimelowdoserateirradiationonperineuralinvasioninpancreaticcancerCapan-2cells

SIPei-ren,LUZheng,LIUYan,MAJian-xia,WUHong-yu,LIZhao-shen.

DepartmentofGastroenterology,ChanghaiHospital,SecondMilitaryMedicalUniversity,Shanghai200433,China

LIZhao-shen,Email：zhsli@81890.net

ObjectiveTo investigate the effect of Iodine 125 seeds short time low dose rate irradiation on perineural invasion (PNI) in pancreatic cancer Capan-2 cells, and explore its molecular mechanism.MethodsThe co-culture model was established by co-culturing the dorsal root ganglion (DRG) of SD rat and Capzn-2 cells line , while Capan-2 culture model and DRG culture model was also established. Iodine 125 seeds short time low dose rate irradiation tablet was used for the 3 models, and the model without irradiation was used as control. Cancer cell and DRG growth was observed under inverted microscopy, surface of neurite and cell colony growth was determined by image analysis software. The concentration of nerve growth factor (NGF), transforming growth factor-α (TGF-α) in cell culture supernatant and matrigel solution was tested by ELISA, and the expression of neurotrophin-3 (NT-3) mRNA was detected by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR).ResultsIn the co-culture model, neurite of DRG showed a direction to cancer cells and had a concentrated growth towards cancer cells. And Capan-2 cells formed more colonies towards neurite. However, in irradiation groups, the symbiotic phenomenon was inhibited to some degree. Increased surface of neurite in co-culture model at 5th day was 290.15±12.08, which was significantly higher than that in DRG group (124.83±6.96,P<0.01), but the surface of neurite was decreased to 201.53±12.20 after irradiation (P<0.01). Increased surface of Capan-2 cell was 300.47±12.99, which was significantly higher than that in Capan-2 group (199.30±8.60,P<0.01 ), but the surface of Capan-2 was decreased to 202.35±7.97 after irradiation (P<0.01). NT-3 mRNA was seldom or not expressed in supernatant of co-culture model, but it was strongly expressed (0.68±0.04) after irradiation (P<0.05). The concentration of NGF and TGF-α in supernatant of co-culture model were (27.56±13.73), (40.86±20.73)ng/ml, after irradiation they were increased to (94.98±33.80), (157.54±83.76)ng/ml, and the difference between the two groups was statistically significant (P<0.05 or <0.01). The concentration of NGF and TGF-α in matrigel lysate of co-culture model were (60.42±33.03), (64.39±21.52)ng/ml, after irradiation they were increased to (132.52±53.01), (138.38±83.58)ng/ml, and the difference of NGF concentration between the two groups was statistically significant (P<0.05).ConclusionsIodine-125 seeds short-time low-dose rate irradiation could inhibit interactions between nerve and Capan-2 cells, and the mechanism may be related to up-regulation of cancer cells perineural invasion promoter NGF, TGF-α and NT-3.

Pancreatic neoplasms; Iodine isotopes; Low-level irradiation; Neoplasm invasiveness

2013-07-10)

(本文編輯：屠振興)

10.3760/cma.j.issn.1674-1935.2013.06.001

國家自然科學(xué)青年基金(30801362)

264002 山東煙臺，解放軍107醫(yī)院(司佩任)；第二軍醫(yī)大學(xué)長海醫(yī)院消化內(nèi)科(路箏、劉巖、馬建霞、吳洪玉、李兆申)

李兆申，Email：zhsli@81890.net

共同第一作者：吳洪玉