Hh信号通路论文：Hedgehog信号通路在胃癌发生发展中的作用及机制探讨.doc

Hh信号通路论文：Hedgehog信号通路在胃癌发生发展中的作用及机制探讨【中文摘要】胃癌是全世界发病率和死亡率均非常高的恶性肿瘤,全球近半数胃癌发生在我国,并且近年来发病呈年轻化的趋势。目前胃癌的总体治疗效果仍然非常差,其发病机制尚不清楚,也缺乏特异性治疗药物。国内外研究表明针对细胞信号转导通路的某些分子靶点研制靶向治疗药物是提高恶性肿瘤整体疗效的重要措施之一。Hedgehog(Hh)信号通路在胚胎发育、维持细胞增殖与凋亡动态平衡、组织损伤与修复以及多种恶性肿瘤的发生、发展中发挥重要作用。研究表明Sonic Hedgehog(SHH)信号通路的异常激活可能与胃癌有关,但其具体的分子机制目前尚不清楚,且在慢性浅表性胃炎萎缩性胃炎肠上皮化生异型增生胃癌这一经典的胃粘膜癌变过程中的表形也未见系统的研究报道。为此,本课题旨在通过临床病理标本及体外研究探讨Hh信号通路在胃粘膜癌变过程中的变化规律及其可能的分子作用机制,为胃癌分子靶向治疗研究提供新思路。研究1、研究Hh信号通路中的关键因子：Shh、Smo、SuFu、Glil、Gli2和Gli3以及细胞周期调控蛋白CyclinD1在慢性浅表性胃炎萎缩性胃炎肠上皮化生异型增生胃癌这一胃粘膜癌变过程中的表达特征,探讨其临床意义。2、研究Hh信号通路与CyclinD1的表达、胃粘膜上皮细胞增殖、凋亡及胃癌临床病理参数之间的关系,探讨Hh信号通路在胃癌发生发展中的分子作用机制。3、研究Hh信号通路阻断剂Cyclopamine对胃癌细胞增殖及Glil、CyclinD1蛋白表达的影响,进一步探讨Hh信号通路在胃癌中的作用机制。研究方法：一、临床病理标本研究部分选取南昌大学第一附属医院2007年1月至2009年9月胃镜活检或外科手术病理标本共186例,其中慢性浅表性胃炎(Chronic superficial gastritis, CSG)44例,萎缩性胃炎(化生性萎缩性胃炎,Metaphastic atrophy gastritis, MAG) 44例,异型增生(Dysplasia, Dys)48例,胃癌(Gastric cancer, GC)50例为研究对象,同时收集临床病理资料。通过HE染色确定病理诊断,采用免疫组织化学二步法检测Hh信号通路中的关键因子：Shh、Smo、SuFu、Gli1、Gli2和Gli3以及细胞周期调控蛋白CyclinD1的表达,同时检测增殖细胞核抗原PCNA的表达。采用脱氧核糖核酸末端转移酶介导的缺口末端标记(TUNEL)技术研究细胞调亡。二、体外研究部分通过不同浓度的Cyclopamine作用于胃癌细胞MKN28不同时间(24h、48h及72h),用倒置显微镜观察细胞形态,四唑蓝(MTT)比色试验检测细胞的生长抑制率,蛋白印迹(Western Blot,WB)方法检测阻断Hh信号通路后对Gli1及CyclinD1蛋白表达的影响。研究结果：一、临床病理标本研究部分(1)Shh在Dys组表达最高,MAG组最低：MAG组显著低于CSG组(P=0.0030.05),但显著低于MAG组及Dys组(P0.05)；细胞核染色阳性率GC组(32.00%)显著高于CSG组(4.55%)、MAG组(6.82%)及Dys组(8.33%)。Glil的表达与胃癌分化程度、浸润深度、TNM分期及淋巴结转移密切相关(P0.05)。(6)GC组Gli3的表达显著低于其他3组(P0.05)；(8)PCNA在GC组表达显著高于CSG及MAG组(P0.05)；(9)细胞凋亡指数GC组(4.081.41)及Dys组(6.141.56)均显著低于CSG组(12.544.23)及MAG组(21.376.27)(P0.01)。(10)在胃粘膜癌变过程中Gli1的表达与CyclinD1的表达呈显著正相关(R=0.214,P=0.0030.01);与PCNA的表达也呈正相关(R=0.145,P=0.0480.05)；与细胞凋亡呈显著负相关性(R=0.278,P=0.0020.01)。二、体外研究部分(1) Cyclopamine阻断Hh信号通路后,胃癌细胞MKN28出现类似凋亡样的形态改变,增殖受到显著抑制,并呈浓度及时间依赖性。(4)阻断Hh信号通路后,显著下调CyclinD1的表达,Gli1表达总量却未见明显下调。结论：1、胃粘膜癌变过程中Hh信号通路发生异常活化。2、Hh信号通路的异常活化与胃癌分化程度、浸润深度、TNM分期及淋巴结转移密切相关。3、阻断Hh信号通路可下调CyclinD1的表达,抑制MKN28细胞增殖。【英文摘要】Background:Gastric cancer is a public healthy problem in the whole world with high incidence and mortality. Nearly one half of gastric cancer patients in global occurred in our country. More and more youth are being suffered from it in recent years. Unclear pathogenesis and lack of specific targeted drugs result in poor therapeutic effect. The exact molecular mechanism of the signaling pathways involved in gastric cancer is one of important measures for improving therapeutic effect and developing targeted therapy drugs.Hedgehog signaling pathway plays an important role in embryonic development, maintenance of the dynamic balance of cell proliferation and apoptosis, process of tissue regeneration and tumorigenesis. Many studies showed that the abnormal activation of hedgehog signaling pathway may relate to gastric cancer, but the detailed mechanism is so far unclear. There is no systematic report about how Hh signaling changes in the classic process of gastric tumorigenesis, which includes chronic superficial gastritis, atrophy gastritis, intestinal metaplasia, dysplasia and gastric cancer. Therefore, we try to investigate the variation of expression and molecular mechanism of Hh signaling in the gastric tumorigenesis in vivo and vitro.:1. To investigate the significance of the core components of Hh signaling pathway, including:Shh, Smo, SuFu, Gli1, Gli2, Gli3 and downstream CyclinDl expression in gastric carcinoma and precancerous lesion in the gastric tumorigenesis process.2. To identify the relationships between Hh signaling and CyclinD1 expression, gastric mucosa cell proliferation and apoptosis, clinical stage of gastric cancer, and the possible mechanism of Hh signaling in gastric cancer.3. To evaluate the effects of Hh signaling pathway inhibitor cyclopamine on proliferation and expression of Glil and CyclinDl in gastric cancer cell, and further study the molecular mechanism of Hh signaling in the gastric tumorigenesis in vitro.MethodsClinical specimens A total of 186 cases of gastric mucosa lesions with clinical data retrieved from endoscopic biopsy and surgery were available for the study in the First Affiliated Hospital of Nanchang University from Jan.2007 to Sep.2009. The gastric mucosa lesions included chronic superficial gastritis(CSG) 44 cases, metaphastic atrophy gastritis (MAG) 44 cases, dysplasia (Dys) 48 cases and gastric cancer (GC) 50 cases, which were verified by HE dye. Expression of the core components of Hh signaling pathway, including:Shh, Smo, SuFu, Gli1, Gli2, Gli3 and CyclinD1 were assayed by immunohistochemistry, besides the expression of PCNA. TdT-mediated dUTP nick end labeling (TUNEL) was performed to detect cell apoptosis.In vitroCell morphology of gastric cancer cell lines MKN28 was observed under inverted microscope, the inhibition rates of cell growth were detected by MTT assay, and the expression of GLI1 and CyclinDl were determined by western blot after treated with cyclopamine for 24h,48h and 72h.Results:Clinical specimens(1) The expression of Shh in Dys group was the highest, which was the lowest in MAG group, Shh expression in MAG group was significantly lower than in CSG group (P=0.0030.05), but much lower in GC group than MAG and Dys group (P0.05).The nuclear staining positive rates of Glil expression in CSG, MAG, Dys and GC group were 4.5%,6.8%,8.3%and 32.0% (P0.05).(6) Expression of Gli3 protein in GC group was lower compared with the other three groups (P0.05).(8) PCNA expressed higher in GC group than that in CSG and MAG group (P0.05).(9)Apoptosis index was extremely lower in GC(4.081.41) and Dys group(6.141.56) than that in CSG (12.544.23) and MAG group(21.376.27) (P0.01).(10) Positive correlation was found between expression of Gli1 and CyclinD1 (R=0.214, P=0.0030.01), so did PCNA (R=0.145, P=0.048), whereas negative correlation between Glil expression and cell apoptosis(R=0.278, P=0.0020.01).In vitro(1) The proliferation of MKN28 was dose-and time-dependently inhibited by cyclopamine.(2) The expression of CyclinDl decreased significantly while inhibiting Hh signaling pathway, but the total expression of Glil was not significantly down-regulated.Conclusions:1. Hh signaling pathway was activated aberrantly in the process of gastric mucosal malignant transformation.2. Aberrant activation of Hh signaling had a closely correlation with differentiation degree, infiltration depth, TNM staging and node metastasis in gastric cancer.3. CyclinDl was down-regulated and the proliferation of MKN28 was inhibited by cyclopamine.【关键词】Hh信号通路 胃癌 癌前病变 Cyclopamine【英文关键词】Hh signaling pathway gastric cancer precancerous lesion cyclopamine【目录】Hedgehog信号通路在胃癌发生发展中的作用及机制探讨摘要3-6ABSTRACT6-9第1章 引言14-19第2章 Hh信号通路与胃癌发生发展的关系19-462.1 实验材料和方法19-252.1.1 研究对象1