【作者】 孙梯业；

【导师】 余佩武；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 目的:胃癌的腹膜种植转移是影响胃癌患者预后的重要原因之一。文献报道在胃癌术后复发的类型中,腹膜种植约占50%,目前其发生机制尚不明确,且缺乏行之有效的防治手段。趋化因子受体CXCR4(CXC chemokine receptor4,CXCR4)是组织表达最为广泛的趋化因子受体之一。研究发现CXCR4的异常活化与乳腺癌、前列腺癌以及肺癌等恶性肿瘤发生发展密切相关,肿瘤细胞可能通过活化的CXCR4信号影响肿瘤的发生发展进程,但CXCR4与胃癌侵袭转移的关系如何,经系统文献检索至今尚未见报道,鉴于此,本课题首先观察胃癌不同组织中CXCR4的表达及其与胃癌临床病理参数之间的关系,随后构建CXCR4-siRNA慢病毒载体转染胃癌细胞MKN-45,以特异抑制胃癌细胞MKN-45中CXCR4的表达,观察其阻抑效应;再建立裸鼠胃癌腹膜种植转移模型,观察胃癌细胞腹膜种植转移的变化情况,由此揭示CXCR4在胃癌腹膜种植转移中的作用及可能机理,为胃癌腹膜种植转移防治提供新思路。方法:1.收集90例外科手术切除胃癌标本和30例正常胃组织,应用免疫组组织化学方法检测CXCR4、SDF-1和VEGF-C的表达,系统分析它们与临床病理参数之间的关系,研究三者在胃癌不同组织中的表达与胃癌侵袭转移的关系。2.构建CXCR4-siRNA慢病毒载体并直接感染胃癌细胞株MKN-45。实验分成6组:正常对照组(SDF-1α阴性)、正常对照组(SDF-1α阳性)、空病毒载体组(SDF-1α阴性)、空病毒载体组(SDF-1α阳性)、CXCR4-siRNA载体转染组(SDF-1α阴性)、CXCR4-siRNA载体转染组(SDF-1α阳性)。采用MTT试验、流式细胞、趋化实验(Transwell小室法)、RT-PCR、Western blot等技术,比较研究了CXCR4-siRNA慢病毒载体转染前后及SDF-1作用前后细胞增殖、细胞凋亡、细胞趋化的变化和对胃癌细胞株MKN-45胞浆VEGF-C、NRP-1表达的受抑程度。3.建立人胃癌细胞裸鼠腹腔种植转移模型,观察CXCR4-siRNA慢病毒载体对胃癌细胞裸鼠腹腔种植转移的影响。结果:1. CXCR4阳性表达主要在胃癌细胞的细胞膜和/或细胞质中。在90例胃癌原发灶、71例胃癌转移的淋巴结和9例腹膜转移灶中表达率分别为83.33%(75/90)、90.14% (64/71)和100%(9/9),30例正常胃组织中未见CXCR4表达。VEGF-C阳性表达主要在癌细胞的细胞质,正常胃组织中VEGF-C表达阳性率为6.67%(2/30);胃癌组织中VEGF-C表达阳性率80.00%(72/90),胃癌转移淋巴结中VEGF-C阳性率为91.55% (65/71)。而胃癌腹膜转移中VEGF-C阳性率为100.00%(9/9),与前四组相比,具有显著性差异(P<0.01或P<0.05)。SDF-1阳性表达主要位于癌细胞胞质和胞浆内。SDF-1在正常胃组织中的表达呈强阳性,阳性率为93.33%(28/30);SDF-1在胃癌组织中的阳性表达率明显降低,胃癌组织中SDF-1阳性表达率68.89%(62/90),胃癌淋巴结转移中SDF-1阳性表达率42.25%(30/71),胃癌腹膜转移组织中SDF-1阳性表达率11.11%(1/9),与正常胃组织相比,差异均具有显著性(P<0.01或P<0.05)。CXCR4、SDF-1和VEGF-C在胃癌原发灶中的表达与肿瘤的分期、浸润深度、分化程度及淋巴结转移相关(r=0.337,P<0.05),与病人的年龄、性别、肿瘤的部位及大小无相关性(P>0.05)。结果表明肿瘤的浸润深度、肿瘤的大小和原发灶中CXCR4、SDF-1的表达构成了胃癌淋巴结和腹膜转移的危险因素。2.成功构建了CXCR4-siRNA慢病毒载体。CXCR4-siRNA慢病毒载体直接感染胃癌细胞MKN-45后,细胞生长受到明显抑制,抑制作用呈时间依赖性;转染后细胞周期分布中,G0/G1期比例增高,而S期和G2/M期比例降低,细胞的增殖指数亦明显降低(P<0.05),细胞的凋亡指数明显增加(P<0.01),显著抑制胃癌细胞MKN-45的趋化运动能力(P<0.01)和VEGF-C、NRP-1的表达(P<0.01)。3.建立各转染组胃癌细胞裸鼠腹腔种植转移模型,CXCR4-siRNA慢病毒载体转染组与对照组、空病毒组裸鼠比较,腹水产生时间、腹水量、腹膜种植瘤最大直径、最大重量、种植瘤数目、生存时间、存活率、生命延长率相比均有显著性差异(P<0.01或P<0.05);CXCR4-siRNA慢病毒载体组肿瘤抑制率为59.58%;种植瘤细胞周期分布情况:CXCR4-siRNA慢病毒载体组与对照组、空病毒组细胞比较,G0/G1期比例增高,而S期和G2/M期比例降低,细胞的增殖指数降低,凋亡显著增加(P<0.05或P<0.01)。而对照组和空病毒组细胞周期相比,无明显变化(P>0.05)。结论:1.研究表明CXCR4和VEGF-C在胃癌组织中特异性高表达,与胃癌的浸润深度、淋巴结转移、临床分期及肿瘤转移,尤其腹膜种植转移呈显著相关。随病变进展,CXCR4和VEGF-C在胃癌组织中的表达呈现逐渐上调的趋势,其中腹膜转移灶中表达最强,二者在胃癌组织中的表达具有显著相关性,胃癌腹膜种植转移可能是胃癌细胞分泌的CXCR4促进VEGF-C的活性升高所引起。2.胃癌细胞MKN-45特异性高表达CXCR4 mRNA与蛋白,与胃癌侵袭转移显著相关;CXCR4-siRNA慢病毒载体能显著抑制MKN-45细胞中CXCR4 mRNA及蛋白的表达,显著降低胃癌细胞MKN-45的增殖和侵袭能力,升高凋亡率,并显著下调VEGF-C、NRP-1的表达,结果表明CXCR4-siRNA慢病毒载体体外显著抑制胃癌细胞的侵袭转移,体内动物实验显著抑制胃癌腹腔种植转移。CXCR4促进VEGF-C活性升高可能是导致胃癌腹膜种植转移重要的分子机理之一。3. CXCR4-siRNA慢病毒载体可显著抑制裸鼠胃癌腹腔种植瘤的形成和腹水产生,显著延长裸鼠生存时间,降低其死亡率。同时可显著增加种植瘤细胞G0/G1期细胞比率(P<0.01)和凋亡率(P<0.01);显著降低G2/M期细胞比率和增殖PI值(P<0.05);并且随CXCR4-siRNA作用时间延长,胃癌细胞凋亡率和G0/G1期细胞比率呈上升趋势,而G2/M期细胞比率和PI值呈下降趋势,结果表明CXCR4-siRNA可能通过阻断胃癌细胞进入增殖周期,抑制其增殖,诱导其凋亡升高可能是CXCR4-siRNA慢病毒载体抑制胃癌腹腔侵袭转移的作用机制之一。更多还原

【Abstract】 Objective: The peritoneal seeding metastasis after gastric cancer radical dissection is one of the important causes of death in patients of gastric cancer. In advanced gastric cancer, peritoneal metastasis is the most frequent (50%) type of recurrence after curative resection, as well as the death cause of most patients. The mechanism of peritoneal seeding of gastric cancer has not been identified, so effective prevent and cure methods are not available. CXC chemokine receptor4(CXCR4) is one of the most common chemokine receptors in tissues and plays important roles in malignant tumors growth, migration and metastasis. The abnormal activation of CXCR4 had been observed in a variety of human malignancies development, including breast cancers, prostatic carcinomas, lung cancers and so on, but whether the activated CXCR4 could affect peritoneal seeding and metastasis of gastric cancer or not, and what downstream genes regulated by CXCR4 could induce the appearance of cancer cells invasive phenotype are not known. So, clinical samples of gastric cancer patients were collected. The expressions of CXCR4 in those samples were detected, and the relationships between the expression level and pathologic parameters were observed firstly. Secondly, the CXCR4-siRNA Lentivirus expression vectors were transfected into gastric cancer cells MKN-45 to block the activated CXCR4 protein especially, and the repressive effects were observed. Finally, after the gastric cancer animal model of peritoneal implantation had been established in nude mice, changes of implantation capacity and the expressions of relative regulative factors in gastric cancer cells were observed in order to reveal the mechanism of CXCR4 in the course of gastric cancer peritoneal seeding and provide new strategy for tumor prevention and treatment.Methods:1. 90 clinical samples of tumor patients and 30 adjacent noncancerous specimens of human gastric cancer were collected. Peritoneal lavage cytology was used to exam the exfoliated cancer cells, and nested Immuohistochemistry(SP)was employed to detect the expression of CXCR4, SDF-1, and VEGF-C expression and relative clinical pathological parameters were observed.2. CXCR4-siRNA Lentivirus expression vectors were constructed and transfected into MKN-45 cells. There were 6 groups in our study, normal control group (SDF-1- negative), normal control group(SDF-1-positive), keno-viral vector group (SDF-1- negative), keno-viral vector group(SDF-1-positive), CXCR4-siRNA experimental group (SDF-1- negative), CXCR4-siRNA experimental group (SDF-1- positive). The changes of cell proliferation, apoptosis, cell cycle and chemotactic activity,and expressive changes of VEGF-C、NRP-1 were observed by MTT assay, flow cytometry, chemotaxis assay, RT-PCR and Western blot respectively. The molecular mechanisms of CXCR4-siRNA transfection on inhibiting peritoneal invasion and metastasis were investigated.3. The tumor animal model of peritoneal implantation was established in nude mice. The capability of cancer cell’s peritoneal seeding in nude mice was observed, and changes of cellular proliferation activity, cellular affinity and the invasive and metastatic potentials were observed in vivo, after CXCR4-siRNA Lentivirus expression vectors were transfected.Results:1. Immunohistochemistry illustrated that the expression of CXCR4 was on the membrane or in the cytoplasm of gastric cancer cell. There was no CXCR4 expression in normal gastric mucosa cell .The expression rate of CXCR4 in primary tumor, lymph mode and peritoneal metastatic tumor was 83.33%, 90.14% and 100% respectively(P<0.01 or P<0.05). The expression of VEGF-C was mainly in the cytoplasm of gastric cancer cell. The expression rate of VEGF-C in normal gastric mucosa cell, primary tumor, lymph mode and peritoneal metastatic tumor was 6.67%, 80.00%, 91.55% and 100% respectively (P <0.01 or P< 0.05). The expression of SDF-1was on the membrane and in the cytoplasm of gastric cancer cell. SDF-1 could be expressed in normal gastric mucosa cell(93.33%). The expression rate of SDF-1 in primary tumor, lymph mode and peritoneal metastatic tumor was 68.89%, 42.25% and 11.11% respectively (P<0.01 or P<0.05). The expressions of CXCR4, SDF-1, and VEGF-C in primary tumor were correlated with tumor stage, depth of tumor invasion, pathological differentiation and lymph node metastasis(r=0.337, P<0.05), but not with age of patients, gender, location of tumor and tumor size(P>0.05).2. CXCR4-siRNA Lentivirus expression vectors were constructed successfully. After the vector was transfected into gastric cancer cells, tumor cells` proliferation was inhibited in a time-dependent manner. Among the distribution of cell cycle, the ratio of G0/G1 increased, and the S stage and ratio of G2/M decreased. Meanwhile, the proliferation index decreased(P<0.05) and apoptotic index increased(P < 0.05), with significantly decreased expressions of VEGF-C and NRP-1(P<0.05). 3. The gastric cancer model of peritoneal seeding was established. There were significant differences in ascitic volume, tumor max diameter, max weight, quantity of tumor, survival time, survival rate, prolonged survival ratio between the CXCR4-siRNA group, the keno-viral vector group and the control group(P<0.05, P<0.01). Among the distribution of cell cycle, the rate of G0/G1 increased ,and the S stage and rate of G2/M decreased. The proliferation index decreased(P<0.05), and apoptotic index also increased obviously(P<0.05). The migrative and invasive abilities of transfected MKN45 cells in vivo decreased in some degree (P<0.05 or P<0.01), but the distribution of cell cycle was no difference between the control group and the keno-viral vector group(P>0.05).Conclusions:1. As close correlations existed between the expression of CXCR4 and the peritoneal implantation of gastric cancer, it could act as a standard for judging the invasive and metastatic states of gastric cancer and forecasting the prognostic of patients. Laparoscopic radical gastrectomy does not facilitate the peritoneal implantation of gastric cancer cells.2. Positive correlations existed between the expressive intensity of CXCR4 and the implantation and metastatic potentials of gastric cancer cells.3. Restraint of CXCR4 effects in nucleus could directly result in the impairment of implantation and metastatic potentials of gastric cancer cells both in vitro and in vivo.更多还原