【作者】 严林海；

【导师】 肖强；

【作者基本信息】 广西医科大学 ， 胃肠腺体外科， 2014， 博士

【摘要】 1背景与目的迄今为止，化疗仍是胃癌的主要治疗方法之一，但是在反复接触某种抗癌药物或化合物后，肿瘤细胞可以获得对该药物以及其他结构类似药物（作用机制不尽相同）的耐受性，即多药耐药性(MDR)。MDR是一个多基因参与、多因素影响和多步骤演变的复杂过程，各作用因素之间既相互依赖也相互制约。胃癌细胞对化疗药物的耐药性是目前化疗最为棘手的问题，所以，深入的研究肿瘤细胞耐药性的分子基础，已成为肿瘤学家们研究肿瘤治疗的重要切入点。在药理学方面，目前，许多药物可以用来暂时性的逆转肿瘤细胞的MDR，但不能从根本上解决问题，只是起到了暂时性治疗的效果，远期或者总体治疗效果欠佳，而且具有相当大的毒副作用。与传统的药物治疗相比，基因治疗具有作用靶点精准、治疗效果彻底、毒副作用低等诸多优点，在MDR逆转中有广阔的发展前景，随着基因组计划的完成，科学家们发现并证实了许多胃癌多药耐药相关基因，如：MDR1、MRP、mTOR等。某些基因治疗的研究成果有可能在未来胃癌治疗中发挥有效的治疗作用，但是目前基因治疗仍有明显的不足：1、目的基因表达时间不长。2、肿瘤特异性基因寻找困难。3、没有根本上解码耐药基因的整个通路。4、整个基因系统是存在正负反馈联合调节机制的，很难依靠单一靶基因的定位治疗达到理想的治疗效果。以上各种原因限制了基因治疗的临床使用，这也提示我们，还有未知因素在耐药的发生和发展中扮演重要角色。为此，我们有必要进一步寻找治疗胃癌多药耐药的有效的目的基因。尾型同源基因CDX2作为尾型同源基因家族（Caudal-related homeobox，CDX）的一员，对消化道特别是结肠和小肠上皮的发育起着关键的作用，同时在胃肠道的发育过程中也扮演着重要的角色，大量研究证实CDX2基因在肠道内大量表达，参与肠型细胞的维护，而正常的胃粘膜中则不表达这种基因。在远端结肠中，CDX2对肿瘤的进展起了肿瘤抑制基因的作用，显示CDX2基因不同于其在消化道发展过程中的稳定功能，提示了CDX2基因新的作用途径。近日，有日本学者发现CDX2是肿瘤多药耐药性重要控制基因MDR1的直接上游基因，直接揭示了CDX2在肿瘤细胞多药耐药性方面的作用。我们在前期的研究中亦发现，CDX2在胃癌细胞的多药耐药性调节中扮演重要的角色，其调节作用十分确切，通过构建CDX2沉默的慢病毒载体转染胃癌耐顺铂细胞株SGC7901/DDP，我们发现转染后的SGC7901/DDP细胞株对顺铂、5-氟尿嘧啶、阿霉素这三种常用胃癌化疗药物的敏感性显著增加，将药物泵出细胞的效率明显降低，SGC7901/DDP细胞株的凋亡明显增加，并促使SGC7901/DDP细胞停滞于M期，此外我们的动物实验模型也支持CDX2低表达时，胃癌细胞耐药性逆转。但是这还不能够完全揭示CDX2在胃癌多药耐药性中起到的调节作用，因为很多基因的调节作用是受正负反馈同时调节的，简单的说就是一些基因只能在一个相对窄的表达区间，不管沉默还是过表达其都会发挥同一方向的作用，这类基因并不十分合适作为调节生物行为的靶基因，因为我们现有生物技术还不能很好的控制他们相对狭窄的表达区间。为了进一步明确CDX2沉默和过表达之后生物行为是否会相反。本课题构建CDX2基因过表达慢病毒载体，观察CDX2过表达后耐药细胞株在体外的功能变化，并运用CDX2过表达的慢病毒载体进行了初步的动物实验，同时，qRT-PCR、Western blot技术初步探讨其发生机制。2方法2.1应用基因重组技术将CDX2基因克隆到慢病毒载体GV287中，进行酶切鉴定。CDX2过表达重组体(PGC-FU-CDX2-EGFP)和慢病毒空白载体(PGC-FU-NC-EGFP)分别感染包装细胞293T，Western blot检测转染效果，收集富含慢病毒颗粒的细胞上清液，对其浓缩后得到高滴度的慢病毒浓缩液，在293T细胞中测定并标定病毒滴度。2.2将实验组PGC-FU-CDX2-EGFP和阴性对照组PGC-FU-NC-EGFP转染入人胃癌耐药细胞株SGC7901/DDP，分别命名为：SGC7901/DDP+CDX2组和SGC7901/DDP+NC组，同时设空白对照组，转染后72小时，分别应用蛋白印迹（western blotting）和实时半定量PCR (Real-time PCR)检测各组CDX2蛋白及mRNA的表达；CCK-8检测三组胃癌多药耐药细胞株对于顺铂、阿霉素、5-氟尿嘧啶（5-fu）的半数凋亡浓度（IC50）的变化；流式细胞仪检测三组胃癌多药耐药细胞株对阿霉素泵出和蓄积的变化；流式细胞仪检测三组胃癌多药耐药细胞株细胞周期阻滞和凋亡率的变化，激光共聚焦显微镜观察三组胃癌多药耐药细胞株凋亡形态的变化。2.3提取SGC7901/DDP+CDX2组和SGC7901/DDP+NC组的细胞的RNA及总蛋白，运用qRT-PCR和Western blot检测胃癌细胞内凋亡关键因子Caspase-3、Caspase-9、 PTEN,和耐药重要通路MDR1、MRP、mTOR、HIF-1α的mRNA和蛋白的表达。2.4将人胃癌多药耐药细胞株SGC7901/DDP接种于裸鼠（去胸腺，免疫力缺失小鼠）腋下，建立人胃癌多药耐药细胞株裸鼠皮下瘤模型，将成功接种移植瘤的45只裸鼠随机分为：实验组（SGC7901/DDP+CDX2组）、阴性对照组（SGC7901/DDP+NC组）和空白对照组（PBS组），每组15只。隔日分别向3组动物模型皮下肿瘤内注射重组慢病毒颗粒PGC-FU-CDX2-EGFP、空载体病毒颗粒PGC-FU-NC-EGFP或PBS(Phosphate buffer saline)，并按裸鼠体重腹腔注射化疗药顺铂DDP（30mg/kg），共10次；同时测量各组肿瘤的长径和短径，以计算肿瘤的体积大小；35天后颈椎脱臼处死裸鼠，取瘤体行HE染色后显微镜下观察移植瘤组织形态变化，脱氧核苷酸末端转移酶介导的缺口末端标记(TUNEL)检测肿瘤细胞凋亡指数。3结果3.1双酶切证实所设计的CDX2及阴性对照组NC正确插入慢病毒载体，DNA测序证实插入的序列正确，293T细胞成功包装CDX2的重组慢病毒载体及其阴性对照组的重组慢病毒载体，收集的细胞培养上清液中，PGC-FU-CDX2-EGFP病毒的滴度为5×109TU/ml，PGC-FU-NC-EGFP病毒的滴度为3×109TU/ml。3.2PGC-FU-CDX2-EGFP和PGC-FU-NC-EGFP成功转染胃癌多药耐药细胞株SGC7901/DDP后，PGC-FU-CDX2-EGFP有效增加了SGC7901/DDP细胞CDX2mRNA的表达，比阴性对照组和正常对照组分别增加了3.8倍和4.0倍以上(P <0.05)，PGC-FU-CDX2-EGFP明显增加了SGC7901/DDP细胞株CDX2蛋白的表达，与阴性对照组和正常对照组相比分别升高了约4.0倍和3.9倍以上(P <0.05)， PGC-FU-CDX2-EGFP明显降低了SGC7901/DDP细胞株对顺铂、阿霉素、5-氟尿嘧啶的敏感性(P<0.05)，PGC-FU-CDX2-EGFP转染后增加了SGC7901/DDP细胞株对阿霉素的泵出率(P <0.05)；PGC-FU-CDX2-EGFP使更多的SGC7901/DDP细胞DNA聚集于S期，SGC7901/DDP+CDX2组处于S期的DNA含量比例为（40.37%±4.52%）高于阴性对照组的（25.61%±2.02%）和正常对照组的（21.83%±4.32%），差异有统计学意义(P <0.05)。转染后，SGC7901/DDP+CDX2组凋亡率为（7.60%±0.28%），低于阴性对照组的（11.05%±1.50%）和正常对照组的（10.80%±1.64%），差异具有统计学意义(P <0.05)。3.3体外实验中，qRT-PCR结果显示：与空白对照组和SGC7901/DDP+NC组比较，SGC7901/DDP+CDX2组的Caspase-3、Caspase-9、PTEN mRNA表达明显下调，MDR1、 MRP、 mTOR、HIF-1α mRNA表达明显上调（P<0.05），空白对照组和SGC7901/DDP+NC组比较差异无统计学意义（P>0.05）；Western-blot结果显示：SGC7901/DDP+CDX2组Caspase-3、Caspase-9、PTEN的蛋白表达量显著低于空白对照组和SGC7901/DDP+NC组（P <0.05），MDR1、 MRP、 mTOR、HIF-1α的蛋白表达量显著高于空白对照组和SGC7901/DDP+NC组（P <0.05），空白对照组和SGC7901/DDP+NC组比较差异无统计学意义（P>0.05）。3.4成功构建人胃癌多药耐药细胞株裸鼠移植瘤模型，三组裸鼠成瘤情况为SGC7901/DDP+CDX2组15只、SGC7901/DDP+NC组14只、空白对照（PBS）组15只，各组皮下移植瘤生长曲线表明：与PBS组和SGC7901/DDP+NC组比较，SGC7901/DDP+CDX2组肿瘤生长速度明显增快（P <0.05），PBS组和SGC7901/DDP+NC组比较差异无统计学意义（P <0.05），移植瘤质量称量结果显示：SGC7901/DDP+CDX2组肿瘤重量（1.1g±0.3g）显著高于SGC7901/DDP+NC组（0.5g±0.3g）和PBS组（0.4g±0.2g），（P <0.05），PBS组和SGC7901/DDP+NC组比较差异无统计学意义（P>0.05）。HE染色后显微镜下确认切取组织为人胃癌皮下移植瘤组织，半定量RT-PCR和Western blot结果均显示SGC7901/DDP+CDX2组CDX2mRNA和蛋白表达较SGC7901/DDP+NC组和PBS组上调（P <0.05），PBS组和SGC7901/DDP+NC组比较差异无统计学意义（P>0.05），TUNEL染色结果显示：SGC7901/DDP+CDX2组的凋亡指数（7.34%±0.36%）明显低于SGC7901/DDP+NC组（16.21%±0.71%）和PBS组（17.25%±0.33%）（P <0.05），PBS组和SGC7901/DDP+NC组比较差异无统计学意义（P>0.05）。4结论4.1成功构建人CDX2基因过表达慢病毒载体，为探讨CDX2过表达对于人胃癌细胞耐药的体内实验、体外实验提供了前期准备，为开展靶向CDX2基因的胃癌耐药性基因治疗研究提供了必须的实验工具。4.2CDX2基因过表达慢病毒载体能明显增加CDX2mRNA和蛋白的表达，降低细胞对顺铂、阿霉素、5-fu的敏感性，增加细胞对阿霉素的泵出率，使G2/M期细胞DNA含量下降并阻滞在S期，同时抑制人胃癌多药耐药细胞的凋亡。4.3体内实验中，移植瘤内注射靶向过表达CDX2基因慢病毒颗粒PGC-FU-CDX2-EGFP可显著加快人胃癌多药耐药细胞SGC7901/DDP皮下移植瘤的生长速度，增加肿瘤重量，抑制多药耐药细胞在动物模型中的凋亡。4.4体外实验中，PGC-FU-CDX2-EGFP增加耐药细胞中CDX2的mRNA和蛋白的表达后，可引起Caspase-9、Caspase-3、PTEN表达降低，MDR1、MRP、 mTOR、HIF-1α表达升高。提示CDX2过表达有可能直接或间接通过PTEN/Caspase-3/Caspase-9凋亡信号传导通路和MDR1/MRP/mTOR/HIF-1α耐药信号传导通路加强人胃癌多药耐药细胞的多药耐药性。创新点1目前，对于利用基因重组技术使CDX2基因过表达于目标细胞后，探讨其对人胃癌多药耐药性进展过程的影响的相关研究国内外均未见报道。本课题从体内和体外实验及CDX2的蛋白间相互作用出发，初步明确CDX2基因过表达后对人胃癌多药耐药性进展的影响，结合前期CDX2沉默在胃癌多药耐药性方面的实验，全面阐明CDX2基因在胃癌多药耐药性中的作用及其功能机制，为CDX2作为胃癌多药耐药性逆转的一个治疗性靶基因提供实验依据。2本研究在机制方面，不仅关注耐药性本身的关键信号通路，同时考虑了凋亡信号通路可能多多药耐药产生的影响，并寻找可能相关的基因，有助于全面地阐明其作用的分子机制。更多还原

【Abstract】 1. Background and ObjectiveDespite the decline in its incidence in the past few decades, gastric cancerremains the second most common cause of cancer-related deaths worldwide, andabout two-thirds of gastric cancer cases occur in developing countries, and42%in China alone. Most deaths of gastric cancer are due to resistance tochemotherapies. Generally, the molecular genetic basis of resistance to cancertherapeutics is complex, involving multiple processes, such as drug transport,drug metabolism, DNA repair and apoptosis. However, the factors that regulatethe chemo-resistance of gastric cancer remain poorly understood.CDX2is a member of the caudal-related homeobox gene family. Severalinvestigators have reported that strong and robust expression of CDX2is foundin>80%of colorectal cancer and non-small cell lung cancer, CDX2enhancesproliferation and has tumorigenic potential in human colon cancer cell linesLoVo and SW48. In addition, CDX2transgenic mice have been shown to haveintestinal metaplasia and a high incidence of gastric carcinoma. Furthermore, Takakura et al. has been reported that inhibition of CDX2decreases endogenousMDR1expression. MDR1was originally identified as an overexpressed andamplified gene in multidrug-resistant cells. Its product, P-glycoprotein (P-gp),appears to play a critical role in drug resistance. In addition, in our previousstudy, we have found that reversal of multidrug resistance in gastric cancer cellsby CDX2downregulation. Although these evidence implicated that CDX2isassociated with carcinogenesis and development of multidrug resistance (MDR),the role of CDX2in multidrug resistant gastric cancer largely remainsunexplored.To define the effects of up-regulation CDX2in multidrug resistant gastriccancer, we constructed CDX2-overexpression lentiviral vector (LV-CDX2-GFP),transfected them into a cisplatin-resistant gastric cancer cell line SGC7901/DDP,selected stable transfectants, and explored changes in IC50, rate of doxorubicinefflux, cell cycle, and apoptosis. We also observed the effects of up-regulationCDX2on the expression of genes associated with apoptosis and multidrugresistant, including Caspase-3, Caspase-9, PTEN, MDR1, MRP, mTOR, andHIF-1α. Moreover, we investigated the effects of CDX2up-regulation on thegrowth and apoptosis of SGC7901/DDP cells in vivo.2Methods2.1CDX2was cloned through gene recombination technique, and inserted intothe retroviral expression vector GV287.The recombinant retroviral expressionvector LV-CDX2-GFP was identified by restriction endonuclease digestion andRT-PCR, then the recombinant vector and the empty vector were infected intothe packaging cell line293T.Transfect the recombinant retroviral expressionvector LV-CDX2-GFP and LV-NC-GFP into the cisplatin-resistant gastric cancer cell line (SGC7901/DDP). Western bolt and RT-PCR were used to detect theexpression of the mRNA and protein of CDX2gene.2.2LV-CDX2-GFP (SGC7901/DDP+CDX2group) and LV-NC-GFP(SGC7901/DDP+NC group) were transfected into gastric cancer resistance cellline SGC7901/DDP. After72hours, the expression of protein and CDX2mRNAin resistance gastric cancer cell line SGC7901/DDP were detected by westernblotting and Real-time quantitative PCR methods respectively; MTT assay isused to investgate the effect of DDP, ADR and5-FU on the cell growth ofhuman gastric cancer resistance cell line SGC7901/DDP; Flow cytometry assayis used to investgate the effect of LV-CDX2-GFP on ADR accumulation ofgastric cancer resistance cell line SGC-7901/DDP; The cell cycle arrest andapoptosis rate were detected by flow cytometry assay.2.3In our previous studies, we found that MDR1expression was decreasedwhen the CDX2was downregulated in SGC7901/DDP cells. Therefore, toinvestigate the mechanisms of overexpression of CDX2in gastric cancer cellspromotes the development of multidrug resistance. We observed the expressionof some apoptosis-associated genes (PTEN, caspase-3and caspase-9), and somemultidrug resistance-associated genes (MDR1, MRP, mTOR and HIF-1α) bysemi-quantitative RT-PCR and western blot in this research.2.4BALB/C nude mice were inocμlated subcutaneously with chemoresistantcells SGC7901/DDP to establish the subcutaneous tumor model of gastriccarcinoma. Until the tumors reached0.5cm in diameter, the mice were randomlydivided into SGC7901/DDP+CDX2group, SGC7901/DDP+NC group and PBSgroup. LV-CDX2-GFP, LV-NC-GFP or PBS (0.15ml per time) was injectedindividually every two days. BALB/C nude mice received an intraperitoneal injection of cisplatin (20mg/kg) every two days. The tumor growth-curve wasdrawn, and the weight of tumor was measured. The volume of tumor wasmeasured and after HE staining, histopathologieal characteristics ofsubcutaneous tumor were observed by microscope; the expression of CDX2wasdetected by semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) and Western Blot. The apoptosis of tumor xerographs was measuredby in situ end labeling technique (TUNEL).3Results3.1restriction endonuclease digestions revealed that the CDX2gene was clonedinto the retroviral expression vector pLXSN successfully, and the sequenceswere identified by DNA sequencing. The CDX2gene of the recombinantlentiviral vector was successfully packed into293T cells. The recombinantlentivirus was harvested from293T cells, and the titer of the virus ofSGC7901/DDP+CDX2and SGC7901/DDP+NC was5×109TU/ml and3×109TU/ml respectively.3.2After the experimental group and vector group were transfected successfully,Densitometry analysis showed that CDX2mRNA and protein in SGC7901/DDP+CDX2cells were about5-and4-fold higher, respectively, than those inSGC7901/DDP+NC cells and SGC7901/DDP cells (P <0.05). There were nodifferences between SGC7901/DDP+NC cells and SGC7901/DDP cells. Thedrug sensitivity of cell line SGC-7901/DDP was obviously decreased about DDP,ADR and5-FU(P <0.05); SGC7901/DDP+CDX2have decreased the ADRaccumulation of gastric cancer resistance cell line SGC7901/DDP (P <0.05);more mean DNA content of SGC7901/DDP cells accumulated at S phase ofcell cycle, the mean DNA content at G2/M phase of SGC7901/DDP+CDX2group was (40.37%±4.52%), the difference have compered with SGC7901/DDP+NC groups (25.61%±2.02%) and control group（21.83%±4.32%）(P <0.05); After transfection, the apoptosis rate of SGC7901/DDP cellswas（7.60%±0.28%）, the difference have compered with vector groups（11.05%±1.50%） and control group （10.80%±1.64%）(P <0.05).3.3To investigate the mechanisms of overexpression of CDX2in gastric cancercells promotes the development of multidrug resistance. We usedsemi-quantitative RT-PCR and western blot to examine the mRNA and proteinexpression of Caspase-3, Caspase-9, PTEN, MDR1, MRP, Mtor and HIF-1α.Densitometry showed that Caspase-3, Caspase-9and PTEN mRNA expressionin the SGC7901/DDP+CDX2group was lower while CDX2was higher thanthat of the SGC7901/DDP+NC and control groups (P <0.05), and no differencewas found between SGC7901/DDP+NC and control groups (P>0.05).3.4The model of human gastric cancer in nude mice was successfullyestablished by subcutaneous transplantation. The formation of transplantedtumour in SGC7901/DDP+CDX2, SGC7901/DDP+NC and PBS group was14,13and15respectively. The tumor growth-curve showed that the tumor growthrate in the SGC7901/DDP+CDX2treatment group was faster than that in theSGC7901/DDP+NC and PBS group (P <0.05). The weight of transplant tumorin SGC7901/DDP+CDX2group (1.1±0.3g) was lighter thanSGC7901/DDP+NC (0.5±0.3g) and PBS group (0.4±0.2g). The tissues gotfrom the nude mice haven been diagnosed that was malignant tumor undermicroscope after HE stain. the expression level of CDX2mRNA and proteinwere increased in the SGC7901/DDP+CDX2group (P <0.05). The apoptoticrate in the SGC7901/DDP+CDX2treatment group was7.34%±3.6%,significantly higher than that in the other two groups (16.21%±0.71%forSGC7901/DDP+NC and17.25%±0.33%for PBS, P <0.05). There was no difference to find between the SGC7901/DDP+NC and PBS group in all theexperiment mention above (P>0.05).4Conclusions4.1Successfully construct the recombinant lentiviral vector (LV-CDX2-GFP).The recombinant lentiviral vector LV-CDX2-GFP was successfully infectedSGC7901/DDP cells.4.2The expression of protion and mRNA were obviously up-regulated byLV-CDX2-GFP, and the drug sensitivity of SGC7901/DDP cells about DDP,ADR and5-FU was decresaed, decrease the accumulation of ADR, and theLV-CDX2-GFP made the mean DNA content of M down and accumulate at Sphase, the apoptosis rate of SGC7901/DDP cells was decreased aftertransfection.4.3LV-CDX2-GFP could decrease Caspase-3, Caspase-9and PTEN expressionwhile MDR1, MRP, mTOR and HIF-1α expression was increased. This resultindicates that CDX2overexpression probably upregulates MDR1, MRP, mTORand HIF-1α expression directly or indirectly and promotes the development ofmultidrug resistance in gastric cancer cells in vitro.4.4Intratumor injection of LV-CDX2-GFP has significantly promoting effect onthe growth of human gastric subcutaneous tumor of Nude Mice. This lentiviralexpression vetor would be an effective tool for gastric cancer treatment.Innovation1. This study is believed to be the first to demonstrate that up-regulation ofCDX2causes cell cycle arrest in the S phase, and suppresses apoptosis.Furthermore, up-regulation of CDX2in SGC7901/DDP cells inhibits thesensitivity of SGC7901/DDP cells to cisplatin,5-fluorouracil (P-gp-non-relateddrug), and doxorubicin (P-gp-related drug). The ability to pump doxorubicin was enhanced significantly, moreover, a strong promotion effect of thedevelopment of multidrug resistance in vivo was observed, as tumor growth wasstrengthened and tumor apoptosis was decreased in nude mice when CDX2mRNA and protein were up-regulated.2. The mechanism of MDR remains obscure. Besides regulation ofmTOR/HIF-1a/P-gp and MRP1signaling pathway, apoptosis is also a commonpathway that finally mediates the killing effects of anticancer drugs, which is animportant cause of MDR, in our result, we study both MDR signaling pathwayand apoptosis signaling pathway. It helped clarify the role of CDX2in thedevelopment of multidrug resistance更多还原