高親和性神經(jīng)生長(zhǎng)因子受體對(duì)神經(jīng)生長(zhǎng)因子誘導(dǎo)神經(jīng)母細(xì)胞瘤細(xì)胞分化的影響

1、    高親和性神經(jīng)生長(zhǎng)因子受體對(duì)神經(jīng)生長(zhǎng)因子 誘導(dǎo)神經(jīng)母細(xì)胞瘤細(xì)胞分化的影響        【摘要】目的觀察人神經(jīng)母細(xì)胞瘤細(xì)胞系IMR-32細(xì)胞在恢復(fù)高親和性神經(jīng)生長(zhǎng)因子受體基因trkA表達(dá)后對(duì)神經(jīng)生長(zhǎng)因子(NGF)誘導(dǎo)分化的反應(yīng)。方法應(yīng)用基因重組技術(shù)構(gòu)建含外源trkA cDNA的逆轉(zhuǎn)錄病毒載體，經(jīng)PA317細(xì)胞包裝后感染靶細(xì)胞系IMR-32細(xì)胞，經(jīng)Southern blot雜交、逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-PCR)證實(shí)轉(zhuǎn)化細(xì)胞系中含有外源基因的整合及穩(wěn)定表達(dá)后，

2、進(jìn)行神經(jīng)生長(zhǎng)因子誘導(dǎo)分化實(shí)驗(yàn)。結(jié)果轉(zhuǎn)化細(xì)胞系未經(jīng)NGF誘導(dǎo)前與母系細(xì)胞在細(xì)胞形態(tài)及生長(zhǎng)狀態(tài)方面差異均無(wú)顯著性意義，而經(jīng)NGF誘導(dǎo)后細(xì)胞出現(xiàn)明顯神經(jīng)元樣分化，生長(zhǎng)及代謝速率減慢(四甲基偶氮唑鹽法測(cè)定原細(xì)胞系IMR-32和空載體轉(zhuǎn)化細(xì)胞系IMR-32/pIRV分別為0.258±0.017,0.237±0.011；而trkA基因轉(zhuǎn)化的細(xì)胞系則僅為0.028±0.003)，撤去NGF后分化細(xì)胞仍然維持分化狀態(tài)。轉(zhuǎn)化細(xì)胞在軟瓊脂內(nèi)很少形成集落，集落形成率僅為0.6；遠(yuǎn)低于原細(xì)胞系IMR-32和空載體轉(zhuǎn)化細(xì)胞系IMR-32/pIRV 的32.3 和33.6，在裸鼠體內(nèi)未見腫瘤

3、形成。結(jié)論高親和性神經(jīng)生長(zhǎng)因子受體基因的表達(dá)可以使轉(zhuǎn)化細(xì)胞系對(duì)NGF產(chǎn)生不可逆的誘導(dǎo)分化反應(yīng)，使腫瘤細(xì)胞惡性表型得到逆轉(zhuǎn)?！娟P(guān)鍵詞】神經(jīng)母細(xì)胞瘤；受體，神經(jīng)生長(zhǎng)因子；基因轉(zhuǎn)移；細(xì)胞分化Effects of high-affinity nerve growth factor (NGF) receptor gene on NGF-induced differentiation of neuroblastoma cell lineZHE Xiaoning, CHEN Jie(Email:chenj)*, LIU Tonghua, GAO Jie.*Department of Pathology, P

4、eking Union Medical College Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100730，China【Abstract】ObjectiveTo study the effects of transfection of high-affinity nerve growth factor receptor gene (trkA) on NGF-induced differentiation of human neuroblastoma cell li

5、ne IMR-32. MethodsThe recombinant retrovirus vector containing exogeneous trkA gene was constructed and packed by PA317 packaging cell line. The neuroblastoma cell line IMR-32 was transfected by virus containing supernatant. The transformant cell line was confirmed by Southern blot and RT-PCR techni

6、ques. The NGF was used to induce cellular differentiation of the transformant cells. ResultsThe trkA gene was successfully transferred and expressed in the neuroblastoma cells. After NGF treatment, the transformant cells displayed apparent neuron-like differentiation morphologically, and a slower ra

7、te of cell growth (MTT value 0.028±0.003) compared with original cell line (0.258±0.017) and empty virus transformed cell line (0.237±0.011). The cells remained in differentiated status after withdrawing the NGF from the medium. The transformant tumor cells rarely formed colonies in s

8、oft agar and failed to form tumor in nude mice. ConclusionRestoration of high-affinity NGF receptor (trkA) expression in neuroblastoma cells could induce non-reversal differentiation. The trkA might be the important factor during NGF-induced differentiation of neuroblastoma cell. 【Key words】Neurobla

9、stoma；Receptors, nerve growth factor；Gene transfer；Cell differentiation神經(jīng)母細(xì)胞瘤是兒童期常見的惡性腫瘤之一。我們實(shí)驗(yàn)室以往在對(duì)多種人神經(jīng)母細(xì)胞瘤細(xì)胞系及其他神經(jīng)外胚層腫瘤細(xì)胞系的研究中發(fā)現(xiàn)，缺乏神經(jīng)生長(zhǎng)因子受體（NGFR）表達(dá)的細(xì)胞系對(duì)神經(jīng)生長(zhǎng)因子(NGF)不產(chǎn)生誘導(dǎo)分化反應(yīng)；即使恢復(fù)了低親和性NGFR在人神經(jīng)母細(xì)胞瘤細(xì)胞系IMR-32中的表達(dá)，轉(zhuǎn)化細(xì)胞經(jīng)NGF誘導(dǎo)仍然不能發(fā)生明顯的分化反應(yīng)。近年來(lái)越來(lái)越多的實(shí)驗(yàn)證據(jù)表明，高親和性NGFR所介導(dǎo)的信號(hào)傳導(dǎo)通路在NGF誘導(dǎo)包括神經(jīng)母細(xì)胞瘤在內(nèi)的多種神經(jīng)源性腫瘤體外培養(yǎng)細(xì)胞系

10、分化過(guò)程中起到較為重要的作用。我們的研究目的即為觀察高親和性NGFR是否能介導(dǎo)神經(jīng)母細(xì)胞瘤細(xì)胞對(duì)NGF產(chǎn)生誘導(dǎo)分化反應(yīng)。材料與方法一、材料含trkA cDNA的質(zhì)粒pIRV-CMV-trkA及人神經(jīng)母細(xì)胞瘤細(xì)胞系IMR-32由美國(guó)伍斯特實(shí)驗(yàn)生物學(xué)研究所Dr.Ross惠贈(zèng)；小鼠纖維母細(xì)胞NIH 3T3、雙向性包裝細(xì)胞系PA317由中國(guó)醫(yī)學(xué)科學(xué)院醫(yī)學(xué)分子生物學(xué)重點(diǎn)實(shí)驗(yàn)室提供。TrkA cDNA擴(kuò)增引物5-CTGGGCGGAGTGCCTGAA-3及5-GGCTGCCGGCTC-CAGGAA-3,擴(kuò)增片段長(zhǎng)度為523 bp1；擴(kuò)增內(nèi)參照GAPDH引物：5-TCCCTCAAGATTGTCAGCAA-3及

11、5-AGATCCACAACGGATACATT-3,擴(kuò)增片段長(zhǎng)度為309 bp2。以上引物均由北京賽百盛(Cybersyn)公司合成。隨機(jī)引物標(biāo)記試劑盒為Gibcol BRL公司產(chǎn)品,逆轉(zhuǎn)錄試劑盒為Promega公司產(chǎn)品,-32P-dCTP購(gòu)自亞輝醫(yī)學(xué)生物工程公司，1.11×105 GBq/mmol, 370 MBq/ml。Balb/c裸鼠及飼料購(gòu)自中國(guó)醫(yī)學(xué)科學(xué)院腫瘤醫(yī)院動(dòng)物室，裸鼠動(dòng)物號(hào)：01-1004。二、方法1.質(zhì)粒的構(gòu)建：含trkA cDNA的逆轉(zhuǎn)錄病毒載體經(jīng)限制性內(nèi)切酶EcoR酶解，回收線狀空病毒載體，經(jīng)T4DNA連接酶體系連接，恢復(fù)其閉環(huán)結(jié)構(gòu)，即為pIRV-neo-CMV空

12、載體。2.細(xì)胞培養(yǎng)及轉(zhuǎn)化細(xì)胞系的建立：IMR-32細(xì)胞用含10%胎牛血清的IMDM培養(yǎng)基培養(yǎng)，PA317細(xì)胞用含10%胎牛血清的DMEM培養(yǎng)基培養(yǎng)；將經(jīng)純化的上述2種質(zhì)粒經(jīng)脂質(zhì)體法3轉(zhuǎn)染逆轉(zhuǎn)錄病毒雙向包裝細(xì)胞系PA317，經(jīng)G418篩選獲得陽(yáng)性克隆，擴(kuò)大培養(yǎng)后收集上清，測(cè)定病毒滴度4，用高滴度病毒上清轉(zhuǎn)染IMR-32細(xì)胞系，經(jīng)G418篩選獲得抗性克隆，擴(kuò)大培養(yǎng)，轉(zhuǎn)化細(xì)胞系分別命名為IMR-32/trkA及IMR-32/pIRV。3.轉(zhuǎn)化細(xì)胞中外源基因整合與表達(dá)的鑒定：分別提取2種轉(zhuǎn)化細(xì)胞系及IMR-32母系基因組DNA及總RNA，應(yīng)用-32P-dCTP標(biāo)記的neo cDNA探針行Souther

13、n雜交5，應(yīng)用逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-PCR)技術(shù)進(jìn)行目的片段的擴(kuò)增，實(shí)驗(yàn)按試劑盒說(shuō)明進(jìn)行操作。4.NGF誘導(dǎo)分化實(shí)驗(yàn)：NGF以200 ng/ml作為終濃度，持續(xù)給藥2周，觀察細(xì)胞生長(zhǎng)分化狀況，測(cè)定細(xì)胞生長(zhǎng)曲線，四甲基偶氮唑鹽(MTT)法檢測(cè)細(xì)胞增殖代謝率6，常規(guī)進(jìn)行軟瓊脂集落形成實(shí)驗(yàn)7，即每組用2個(gè)鋪雙層瓊脂的平皿，接種2×103細(xì)胞，培養(yǎng)2周后，分別計(jì)數(shù)集落數(shù)，用2個(gè)平皿的平均數(shù)除以接種細(xì)胞數(shù)得出集落形成率；應(yīng)用Northern 雜交檢測(cè)NGF作用后細(xì)胞中神經(jīng)絲蛋白mRNA的表達(dá)情況。所有實(shí)驗(yàn)均以不加NGF，同原細(xì)胞系作對(duì)照。5.裸鼠體內(nèi)致瘤性8: 選無(wú)致病原雌性4周齡Bal

14、b/c裸鼠，以原細(xì)胞系細(xì)胞做對(duì)照組，空病毒轉(zhuǎn)化細(xì)胞IMR-32/pIRV和trkA 轉(zhuǎn)化細(xì)胞IMR-32/trkA為實(shí)驗(yàn)組。每組34只，無(wú)菌條件下飼養(yǎng)，每周換飼料及墊料1次，分別于裸鼠背部皮下接種1×107細(xì)胞，4周后開始觀察，每周觀察1次，并記錄腫瘤體積，觀察36個(gè)月。計(jì)算方法如下：V=0.523 6(/6)×a×b2,其中a和b分別為腫瘤在體測(cè)量時(shí)的最大和最小徑線值9。結(jié)果1質(zhì)粒的酶切鑒定結(jié)果如1顯示，含外源cDNA的質(zhì)粒經(jīng)EcoR酶切可得到2.7 kb的cDNA片段及6.5 kb的載體，而空載體酶切后僅出現(xiàn)6.5 kb條帶。1：DNA/Hind，2：pIRV

15、-CMV-trkA/EcoR，3：pIRV-CMV/EcoR1pIRV-CMV-trkA及相應(yīng)空載體酶切鑒定結(jié)果1：IMR-32，2：IMR-32/pIRV，3：IMR-32/trkA2Southern雜交鑒定轉(zhuǎn)化細(xì)胞系中外源基因整合情況2Southern雜交結(jié)果如2顯示，在轉(zhuǎn)化細(xì)胞中可見前病毒整合，其中空病毒轉(zhuǎn)化細(xì)胞雜交帶為3.8 kb，為完整前病毒的長(zhǎng)度；而trkA轉(zhuǎn)化細(xì)胞中由于trkA cDNA 5端距起始300 bp處含有Sac位點(diǎn)，酶切后帶neo基因的片段長(zhǎng)度約剩3.3 kb，所以雜交帶較空載體細(xì)胞前移；3結(jié)果顯示IMR-32/trkA中可擴(kuò)增出長(zhǎng)523 bp的片段，而空載體轉(zhuǎn)化細(xì)胞

16、及母系對(duì)照中均未見擴(kuò)增帶，各細(xì)胞系GAPDH表達(dá)能力差別不大，說(shuō)明RNA總量基本一致。3轉(zhuǎn)化細(xì)胞對(duì)NGF誘導(dǎo)分化的反應(yīng)：NGF誘導(dǎo)之前，IMR-32/trkA的細(xì)胞生物學(xué)行為與空載體轉(zhuǎn)化細(xì)胞系及母系無(wú)明顯差別；在NGF(200 ng/ml)存在的條件下，IMR-32/trkA產(chǎn)生明顯分化，包括神經(jīng)突起生長(zhǎng)，胞體聚集(46)，分化反應(yīng)在給NGF后35 d開始出現(xiàn)，911 d達(dá)到高峰，分化細(xì)胞經(jīng)Northern雜交檢測(cè)發(fā)現(xiàn)神經(jīng)絲蛋白表達(dá)上調(diào)(7)，MTT法檢測(cè)細(xì)胞增殖代謝率明顯降低(表1)。軟瓊脂集落形成實(shí)驗(yàn)結(jié)果表明，經(jīng)NGF誘導(dǎo)分化的IMR-32/trkA細(xì)胞集落形成能力顯著下降(表2)；加藥2

17、周后撤去NGF，分化細(xì)胞依然維持分化狀態(tài)，觀察2周亦未見細(xì)胞恢復(fù)到分化前的幼稚狀態(tài)，相比之下，空載體轉(zhuǎn)化細(xì)胞及母系在NGF作用下未見明顯的分化反應(yīng)，僅有輕微的生長(zhǎng)加快，撤去NGF之后繼續(xù)維持原有的生長(zhǎng)狀態(tài)；轉(zhuǎn)化細(xì)胞在裸鼠體內(nèi)連續(xù)觀察6個(gè)月未見腫瘤形成，而其母系IMR-32和空病毒對(duì)照系均在接種的4周時(shí)可見明顯的腫瘤形成。M：pBR322/Hinf，1：IMR-32，2：IMR-32/pIRV， 3：IMR-32/trkA3RT-PCR檢測(cè)轉(zhuǎn)化細(xì)胞系中外源基因表達(dá)情況討論神經(jīng)母細(xì)胞瘤的發(fā)生是胚胎發(fā)育過(guò)程中神經(jīng)嵴來(lái)源的母細(xì)胞在某些階段分化受阻的結(jié)果，而在此過(guò)程中，神經(jīng)營(yíng)養(yǎng)因子及其相關(guān)受體的改變可能

18、不同程度地參與了該腫瘤的發(fā)生與發(fā)展過(guò)程。我們的主要研究?jī)?nèi)容是基于課題組以往工作成果之上的繼續(xù)與深入，實(shí)驗(yàn)思路以既往研究中兩項(xiàng)主要結(jié)論為出發(fā)點(diǎn)，即（1）NGF不能誘導(dǎo)包括IMR-32細(xì)胞系在內(nèi)的無(wú)NGFR表達(dá)的神經(jīng)母細(xì)胞瘤細(xì)胞分化，尤其是在MYCN基因擴(kuò)增的情況下10；（2）通過(guò)基因重組技術(shù)在上述細(xì)胞系中恢復(fù)低親和性神經(jīng)營(yíng)養(yǎng)因子受體表達(dá)之后，轉(zhuǎn)化細(xì)胞系仍不能在NGF作用之下出現(xiàn)形態(tài)學(xué)上的明顯分化11，提示低親和性NGFR所介導(dǎo)的信號(hào)傳導(dǎo)體系不足以引導(dǎo)神經(jīng)母細(xì)胞瘤細(xì)胞發(fā)生徹底的終末分化反應(yīng)，因此，對(duì)神經(jīng)母細(xì)胞瘤細(xì)胞誘導(dǎo)分化的研究可能通過(guò)更有效的途徑深入下去。近年來(lái)的研究表明，trkA基因的表達(dá)產(chǎn)物

19、即高親和性NGFR所介導(dǎo)的信號(hào)傳導(dǎo)通路在NGF誘導(dǎo)包括神經(jīng)母細(xì)胞瘤在內(nèi)的多種神經(jīng)源性體外培養(yǎng)細(xì)胞分化過(guò)程中起到較為決定性的作用12，13。因此，觀察高親和性NGFR是否能介導(dǎo)神經(jīng)母細(xì)胞瘤細(xì)胞對(duì)NGF產(chǎn)生明顯的誘導(dǎo)分化反應(yīng)即成為本研究的目的。46轉(zhuǎn)化細(xì)胞系在NGF誘導(dǎo)前后的形態(tài)特征，4為IMR-32母系的細(xì)胞形態(tài)，5為TrkA基因轉(zhuǎn)化后未加NGF的形態(tài)，6為TrkA基因轉(zhuǎn)化后的細(xì)胞系IMR-32/trkA在NGF處理5 d后，出現(xiàn)明顯的形態(tài)分化，細(xì)胞突起明顯延長(zhǎng)，胞體變小變圓7Northern 雜交檢測(cè)轉(zhuǎn)化細(xì)胞系在NGF誘導(dǎo)前后神經(jīng)絲蛋白mRNA的表達(dá)情況表1MTT法測(cè)定各細(xì)胞系經(jīng)NGF誘導(dǎo)2周

20、后增殖代謝能力細(xì)胞系A(chǔ)550 nm(±s)抑制率(%)IMR-320.258±0.0170.00IMR-32/pIRV0.237±0.0110.08IMR-32/trkA0.028±0.00389.10表2各細(xì)胞系經(jīng)NGF誘導(dǎo)2周后軟瓊脂集落形成能力檢測(cè)細(xì)胞系集落數(shù)集落形成率(%)IMR-3265832.3637IMR-32/pIRV68333.6669IMR-32/trkA80.615我們首先成功獲得了表達(dá)外源基因的轉(zhuǎn)化細(xì)胞系，在進(jìn)一步的NGF誘導(dǎo)分化實(shí)驗(yàn)中發(fā)現(xiàn)，含有trkA外源基因表達(dá)的轉(zhuǎn)化細(xì)胞系在NGF的作用之下出現(xiàn)了明顯的誘導(dǎo)分化反應(yīng)，而作為平行

21、對(duì)照的IMR-32母系以及空病毒載體轉(zhuǎn)化細(xì)胞系均未見分化反應(yīng)；另外，我們?cè)俅纹叫袑?duì)照了低親和性NGFR基因轉(zhuǎn)化細(xì)胞系在同一給藥條件下對(duì)NGF的反應(yīng)，結(jié)果該細(xì)胞系僅表現(xiàn)出輕微的分化反應(yīng)。通過(guò)對(duì)以上兩種分別表達(dá)高和低親和性NGFR的轉(zhuǎn)化細(xì)胞系的誘導(dǎo)分化過(guò)程的觀察，我們發(fā)現(xiàn)高親和性NGFR對(duì)NGF誘導(dǎo)神經(jīng)母細(xì)胞瘤細(xì)胞分化起到極為重要的作用，這種作用還特別表現(xiàn)在它所介導(dǎo)的分化反應(yīng)的不可逆性，即誘導(dǎo)神經(jīng)母細(xì)胞瘤細(xì)胞發(fā)生明顯分化后再撤去NGF，已分化的細(xì)胞依然能夠維持既有的生存狀態(tài)，在23周的觀察期內(nèi)，細(xì)胞不再恢復(fù)到原來(lái)的幼稚狀態(tài)，而穩(wěn)定維持在一種低代謝率的分化狀態(tài)。轉(zhuǎn)化細(xì)胞在軟瓊脂內(nèi)集落形成能力明顯下降

22、，在裸鼠體內(nèi)亦不能形成腫瘤。從腫瘤細(xì)胞惡性表型得到較為完全的逆轉(zhuǎn)這個(gè)角度出發(fā)，我們的實(shí)驗(yàn)結(jié)果具有相當(dāng)重要的意義，因?yàn)檫@是實(shí)現(xiàn)對(duì)該腫瘤進(jìn)行實(shí)驗(yàn)性基因治療的重要和可靠的指標(biāo)。基金項(xiàng)目：國(guó)家杰出青年基金資助項(xiàng)目(39625012)；國(guó)家教委跨世紀(jì)優(yōu)秀人才計(jì)劃基金資助項(xiàng)目作者單位：折曉寧(100730 北京，中國(guó)醫(yī)學(xué)科學(xué)院 中國(guó)協(xié)和醫(yī)科大學(xué) 北京協(xié)和醫(yī)院病理科)陳杰(100730 北京，中國(guó)醫(yī)學(xué)科學(xué)院 中國(guó)協(xié)和醫(yī)科大學(xué) 北京協(xié)和醫(yī)院病理科)通訊作者：陳杰(Email:chenj)參考文獻(xiàn)1 Martin-Zanca D, Oskam R, Mitra G, et al. Molecular and b

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