【作者】 张琍；

【导师】 苏琦；

【作者基本信息】 南华大学 ， 病理学与病理生理学， 2011， 博士

【摘要】 研究背景胃癌是严重危害人类健康的恶性肿瘤,其治疗主要以手术、化疗、生物治疗等综合治疗方法。由于胃癌常发生侵袭转移,从而手术治疗复发率高,并且化疗药物毒副作用大、容易产生耐药。侵袭转移、多药耐药是导致胃癌化疗失败的重要原因,开发抗侵袭转移、逆转胃癌多药耐药和毒副作用小的新药具有重要意义。研究发现PPARγ（proliferateration-activated receptor gamma）配体参与脂肪细胞分化、调节糖代谢、调控细胞周期、诱导肿瘤细胞分化和凋亡等作用,发挥抗肿瘤效应。研究表明,PPARγ配体曲格列酮通过抑制NF-kB表达抑制结肠癌细胞的生长;罗格列酮（Rosiglitazone,ROS）能增强5-Fu抑制人肝癌细胞生长的作用。目前,罗格列酮对胃癌细胞的增殖、侵袭转移、多药耐药的影响及其机制的报道甚少。【罗格列酮抑制人胃癌细胞增殖与诱导凋亡的作用及其机制】目的:通过体内外实验研究ROS对人胃癌细胞增殖及凋亡的影响及其分子机制。方法与结果:ROS能明显抑制MGC803细胞的增殖,12.5μmol.L^-1以上浓度的ROS可呈浓度和时间依赖性抑制MGC803细胞增殖;AO染色和流式细胞术检测结果显示ROS能明显诱导MGC803细胞凋亡,使细胞周期呈浓度依赖性停滞于G1期;免疫细胞化学染色法结果表明人胃癌MGC803细胞表达PPARγ,ROS作用MGC803细胞后能诱导bax、PTEN表达上调、bcl-2表达下调（P<0.05或P<0.01）;ROS对MGC803细胞小鼠肾囊膜下移植瘤有显著的生长抑制作用（P<0.05）,呈剂量依赖关系;18mg.kg^-1、30mg.kg^-1、50mg.kg^-1的ROS抑瘤率分别为35.2%、45.8%、62.9%; HE染色光镜下可见瘤细胞变性和部分瘤细胞凋亡,宿主肾皮质边界完整,30mg.kg^-1 ROS的抑制率（45.8%）与2mg.kg^-1丝裂霉素的抑瘤率（45.5%）相当（P>0.05）;且罗格列酮(50mg.kg^-1)对荷瘤小鼠血糖、肝肾功能及结构无明显影响。结论:罗格列酮可抑制人胃癌MGC803细胞增殖及其小鼠肾囊膜下移植瘤的生长,其机理可能通过上调bax、PTEN,下调bcl-2蛋白在MGC803细胞中的表达,诱导细胞凋亡有关。罗格列酮(50mg.kg^-1)对荷瘤小鼠血糖、肝肾功能及结构无明显影响,为罗格列酮临床应用的可行性提供了理论依据。【罗格列酮抑制人胃癌细胞迁移与侵袭的作用及机制】目的:研究罗格列酮（ROS）对人胃癌SGC7901与SGC7901/VCR细胞的迁移侵袭能力的影响,并初步探讨其可能机制。方法与结果:划痕实验和Transwell小室侵袭实验检测结果发现, 40μmol.L^-1的ROS处理24h后,SGC7901与SGC7901/VCR细胞迁移与侵袭能力明显降低（P<0.05）;RT-PCR、Western-blot检测结果发现40μmol.L^-1的ROS处理24h后,SGC7901与SGC7901/VCR细胞p-ERK1/2、Fra^-1蛋白表达和uPA与MMP-9 mRNA及蛋白表达明显下调,TIMP-3 mRNA及蛋白表达上调（P<0.05）,uPAR mRNA及其蛋白表达无明显差异（P>0.05）;而Rac1-LIMK1-cofilin1通路相关的LIMK1、Rac1、Rock1、Pak1、cofilin1 mRNA及其编码蛋白在此两株细胞中的表达均下调（P<0.05）。结论:ROS能抑制人胃癌SGC7901与SGC7901/VCR细胞迁移、侵袭能力,其机制不但与罗格列酮能通过ERK/Fra^-1/uPA通路,下调p-ERK1/2、Fra^-1及uPA、MMP-9表达,上调TIMP-3表达有关,也与罗格列酮能通过阻断Rac1-Rock-Pak1通路,下调LIMK的表达,进而抑制cofilin的激活有关。【罗格列酮对人胃癌祼鼠移植瘤血管生成拟态、淋巴管生成的影响及其机理】目的:研究罗格列酮对人胃癌SGC7901细胞裸鼠移植瘤血管生成拟态及淋巴管生成的影响并初步探讨其分子机制。方法与结果:采用PAS-CD31双染计数血管生成拟态;免疫组化法检测D2-40的表达,计算微淋巴管密度（LVD）差异,结果发现罗格列酮可抑制人胃癌祼鼠移植瘤血管生成拟态和淋巴管的生成;逆转录-聚合酶链式反应（RT-PCR）、Western-blot检测结果表明罗格列酮可下调MMP-2、MMP-9、VEGF-C及VEGFR-3,上调TIMP-2的在人胃癌裸鼠移植瘤组织中的表达,进而抑制人胃癌祼鼠移植瘤的生长。结论:罗格列酮可能是通过下调MMP-2、MMP-9,上调TIMP-2在人胃癌裸鼠移植瘤组织中的表达,抑制血管生成拟态生成;通过下调VEGF-C及VEGFR-3在人胃癌祼鼠移植瘤组织中的表达,阻断VEGF-C与受体VEGFR-3结合的途径抑制肿瘤淋巴管的生成。【罗格列酮逆转丝裂霉素对人胃癌SGC7901/VCR细胞多药耐药及机制】目的:在研究发现罗格列酮能在体内外抑制胃癌细胞增殖和侵袭转移的基础上,本项目进一步通过体内外实验观察罗格列酮能否逆转人胃癌SGC7901/VCR细胞对丝裂霉素C耐药性,并探讨其可能的分子机制。方法与结果:通过细胞生长曲线和MMT方法研究表明罗格列酮能抑制SGC7901细胞、SGC7901/VCR细胞的增殖及SGC7901/VCR细胞祼鼠移植瘤的生长,呈浓度依赖性（P<0.05）;应用流式细胞术、AO-EB荧光染色、DNA梯度法检测表明罗格列酮联合MMC与其它各组比较在体内外能增强诱导SGC7901/VCR细胞的凋亡（均P<0.05）;罗格列酮能部分逆转SGC7901/VCR细胞及其祼鼠移植瘤对MMC的耐药;通过Real time PCR和Western blot检测发现罗格列酮下调MDR1、p-Akt、Bcl-2基因及P-gp和上调Bax基因及其蛋白在SGC7901/VCR细胞及其祼鼠移植瘤组织中的表达。结论:罗格列酮能部分逆转SGC7901/VCR细胞及其祼鼠移植瘤对MMC的耐药,其机制与罗格列酮抑制MDR1基因/P-gp途径和通过PI3K/Akt信号通路下调p-Akt、Bcl-2和上调Bax在移植瘤组织中的表达,诱导凋亡有关。从而为临床应用罗格列酮抗胃癌治疗提供了理论基础,为胃癌治疗开辟新途径提供了思路。更多还原

【Abstract】 Background Gastric cancer is a malignant tumor, which threat seriously to the health of human being. The treatments of gastric cancer have surgical therapy, radiation therapy, chemotherapy and biological therapy. Because invasion and metastasis were occur often in gastric cancer, thereby the recurrence is higher after surgical therapy, and the commonly used chemotherapy drugs have side effects and prone to resistance. Invasion, metastasis and multidrug resistances are the significant failure causes of gastric cancer chemotherapy. There is important significance to look for new effective anticancer drugs that can resist to invasion, metastasis, multi-drug resistance to treat gastric cancer. Peroxisome proliferateration-activated receptor gamma （PPARγ） ligand involves in adipocyte differentiation, regulating sugar metabolism, cell cycles, inducing tumor cell differentiation and apoptosis that have antitumor effects. It had been found that PPARγligand troglitazone inhibit the growth of colon cancer cells via inhibiting the expression of NF-kB; Rosiglitazone （ROS） can enhance the effects of 5-Fu inhibiting human liver cancer cells growth. Up to now, effects of ROS on gastric cancer proliferation, invasion and metastasis, multidrug resistance and its mechanism are still unknown.[The role and mechanisms of inhibiting proliferation and induceing apoptosis of human gastric cancer cells]Objective To investigate the effects of ROS on the proliferation and apoptosis and explore the molecule mechanism of apoptosis induced by ROS in human gastric cancer MGC803 cells in vivo and in vitro. Methods and Results The results showed that 12.5μmol/L ROS could inhibit the proliferation of MGC803 cells in a concentration-dependent and time-dependent manner detected by MTT assay and clone formation method. Acridine orange staining and FCM indicated that the apoptosis of MGC803 cells was induced and cell cycle was arrested at G1 stage by ROS in a concentration-dependent manner. Immunocyte chemistry indicated PPARγwas expressed in MGC803 cells. The expression of Bax, PTEN protein increased and bcl-2 protein decreased in MGC803 cells treated with ROS （P<0.05）. The MGC803 cells transplanted into subrenal-capsule tumor of mice were inhibited by ROS in a dose-dependent manner. The tumor inhibitory rate was 35.2% with ROS at the dose of 18mg.kg^-1. The tumor inhibitory rates were respectively 45.8%, 62.9% with ROS at the dose of 30mg.kg^-1 and 50mg/kg, and HE stain indicated tumor cells were metamorphic and parts of tumor cells were apoptosis, and the borderline of the host renal cortex was clean. There were the same tumor inhibition rates at the dose of 30mg.kg^-1of ROS （45.8%） and 2mg/kg of MMC （45.5%） in mices. ROS (50mg.kg^-1) had no effects on the blood sugar, liver function, renal function. Conlusions ROS could inhibit the proliferation of MGC803 cell in vitro and the growth of MGC803 cells transplanted into subrenal- capsule tumor of Kunming mice in vivo. The mechanism of cell apoptosis was connected with increasing the expression of bax and PTEN protein and decreasing bcl-2 protein in MGC803 cells. ROS had no effects on the blood sugar, liver function, renal function, and their structure of Kunming mice that were transplanted MGC803 cells into subrenal-capsule. Based on the results, the theoretical basis was provided to the feasibility of applying ROS to clinical treatment for gastric cancer.[Rosiglitazone inhibit migration and invasion in gastric cancer cells and its mechanisms]Objects To explore the effects and its molecular mechanisms of ROS on migration and invasion in human gastric cancer SGC7901 and SGC7901/VCR cells. Methods and Results The results showed that the cellular migration and invasion was evidently inhibited after the cells were treated with 40μmol L^-1 ROS for 24h by wound healing assay and invasion assay in vitro, which extenuated the expression of MMP-9, uPA, p-ERK1/2, Fra^-1 protein and LIMK1, Rac1, Rock, Pak1, cofilin1 protein related to ERK/Fra^-1/uPA and Rac1-LIMK1-cofilin1 signal transduction pathway respectively, and increased the expression of TIMP-3 mRNA and protein （P<0.05）, and which has no effects on the expression of uPAR mRNA and protein （P>0.05） in SGC7901 and SGC7901/ VCR cells detected by RT-PCR and Western-blot. Conclusions ROS can inhibit significantly migration and invasion in human gastric cancer SGC7901 and SGC7901/VCR cells, the possible mechanism involved in downregulating the expression of p-ERK1/2, Fra^-1, uPA and MMP-9 and upregulating TIMP-3 via ERK/Fra^-1/uPA pathway, and simultaneously downregulating the expression of Rac1, Rock, Pak1, LIMK1 and p-cofilin1 via Rac1-LIMK1-cofilin1 signal transduction pathway.[Effects of ROS on Vasculogenic Mimicry and Lymphangiogenesis in Nude Mice Human Gastric Cancer Xenograft and its Mechanisms]Objective To investigate the effects of ROS on vascularization mimicry and lymphangiogenesis in nude mice transplanted tumor bearing SGC7901 cells of human gastric cancer, and preliminarily probe into the mechanism of anti- vascularization mimicry and lymphangiogenesis. Methods and Results Results showed that the average weight of Nude Mice in control group and experimental groups was not statistically different before executed （P>0.05）. The volume of transplanted tumor was statistical significant lower in the experimental groups than that in the control group （P<0.05）, with the elevation of ROS concentration, the rate of inhibitory rate is gradually increasing. The number of vascularization mimicry and lymph vessel in transplanted tumor was significantly decreased in the treatment group compared with control group detected by PAS-CD31 double staining and D2-40 protein staining （P<0.05）. The expression of MMP-2, MMP-9, VEGF-C and VEGFR-3 was downregulated and TIMP-2 was upregulated in the treatment group compared with control group by RT PCR and Western blot （P<0.05）. Conclusions Rosiglitazone inhibits the growth of transplanted tumor and the formation of vascularization mimicry, lymphangiogenesis in transplanted tumor, and Rosiglitazone affects the formation of vascularization mimicry through down-regulating the expression of MMP-2, MMP-9, up-regulating the expression of TIMP-2, and ROS inhibits tumor lymphangiogenesis through down-regulating the expression of VEGF-C and VEGFR-3, blocking the binding of VEGF-C and its receptor VEGFR-3 in transplanted tumor.[Reversing of Multidrug resistance and the correlative mechanism in SGC7901/VCR cell lines by Rosiglitazone]Objective Based on the results above, we explore that whether Rosiglitazone （ROS） can reverse the resistance to Mitomycin （MMC） of human gastric cancer SGC7901/VCR cell line which has multidrug resistant phenotype in vitro and in vivo, and further explore the possible mechanism of ROS reversing multidrug resistance. Methods and Results The results showed that ROS at 40μM and above had inhibitory effects on the growths of human gastric cancer SGC7901/VCR cell line and its parental generation SGC7901 cell line （P<0.05）, and ROS can reverse the resistance to MMC of the human gastric cancer SGC7901/VCR cell line, expressing an obvious dose-dependent relationship by the analysis of cell growth curve and MMT assay. The AO-EB fluorescent staining, flow cytometry and DNA ladder indicated that ROS combined MMC can enhance the apoptosis of SGC7901/VCR cell line in vivo and in vitro. Rosiglitazone could down-regulate the expression of MDR1, p-Akt, Bcl-2 gene and encoding protein P-gp of MDR1 in SGC7901/VCR cell line, and up-regulate Bax gene and its encoding protein in the xenograft tumor by Real time PCR and Western blot. Conclusions To inhibit MDR1/ P-gp pathway and induce apoptosis through the PI3K/Akt signaling pathway maybe the important mechanism of ROS partly reversing the resistance to Mitomycin of human gastric cancer SGC7901/VCR cell line. It can further provide theoretical basis of applying ROS to clinical treatment for gastric cancer, and open a new avenues for gastric cancer treatment.更多还原