Hh信號通路論文：Hedgehog信號通路在胃癌發(fā)生發(fā)展中的作用及機制探討.doc

Hh信號通路論文：Hedgehog信號通路在胃癌發(fā)生發(fā)展中的作用及機制探討【中文摘要】胃癌是全世界發(fā)病率和死亡率均非常高的惡性腫瘤,全球近半數(shù)胃癌發(fā)生在我國,并且近年來發(fā)病呈年輕化的趨勢。目前胃癌的總體治療效果仍然非常差,其發(fā)病機制尚不清楚,也缺乏特異性治療藥物。國內(nèi)外研究表明針對細胞信號轉導通路的某些分子靶點研制靶向治療藥物是提高惡性腫瘤整體療效的重要措施之一。Hedgehog(Hh)信號通路在胚胎發(fā)育、維持細胞增殖與凋亡動態(tài)平衡、組織損傷與修復以及多種惡性腫瘤的發(fā)生、發(fā)展中發(fā)揮重要作用。研究表明Sonic Hedgehog(SHH)信號通路的異常激活可能與胃癌有關,但其具體的分子機制目前尚不清楚,且在慢性淺表性胃炎萎縮性胃炎腸上皮化生異型增生胃癌這一經(jīng)典的胃粘膜癌變過程中的表形也未見系統(tǒng)的研究報道。為此,本課題旨在通過臨床病理標本及體外研究探討Hh信號通路在胃粘膜癌變過程中的變化規(guī)律及其可能的分子作用機制,為胃癌分子靶向治療研究提供新思路。研究1、研究Hh信號通路中的關鍵因子：Shh、Smo、SuFu、Glil、Gli2和Gli3以及細胞周期調(diào)控蛋白CyclinD1在慢性淺表性胃炎萎縮性胃炎腸上皮化生異型增生胃癌這一胃粘膜癌變過程中的表達特征,探討其臨床意義。2、研究Hh信號通路與CyclinD1的表達、胃粘膜上皮細胞增殖、凋亡及胃癌臨床病理參數(shù)之間的關系,探討Hh信號通路在胃癌發(fā)生發(fā)展中的分子作用機制。3、研究Hh信號通路阻斷劑Cyclopamine對胃癌細胞增殖及Glil、CyclinD1蛋白表達的影響,進一步探討Hh信號通路在胃癌中的作用機制。研究方法：一、臨床病理標本研究部分選取南昌大學第一附屬醫(yī)院2007年1月至2009年9月胃鏡活檢或外科手術病理標本共186例,其中慢性淺表性胃炎(Chronic superficial gastritis, CSG)44例,萎縮性胃炎(化生性萎縮性胃炎,Metaphastic atrophy gastritis, MAG) 44例,異型增生(Dysplasia, Dys)48例,胃癌(Gastric cancer, GC)50例為研究對象,同時收集臨床病理資料。通過HE染色確定病理診斷,采用免疫組織化學二步法檢測Hh信號通路中的關鍵因子：Shh、Smo、SuFu、Gli1、Gli2和Gli3以及細胞周期調(diào)控蛋白CyclinD1的表達,同時檢測增殖細胞核抗原PCNA的表達。采用脫氧核糖核酸末端轉移酶介導的缺口末端標記(TUNEL)技術研究細胞調(diào)亡。二、體外研究部分通過不同濃度的Cyclopamine作用于胃癌細胞MKN28不同時間(24h、48h及72h),用倒置顯微鏡觀察細胞形態(tài),四唑藍(MTT)比色試驗檢測細胞的生長抑制率,蛋白印跡(Western Blot,WB)方法檢測阻斷Hh信號通路后對Gli1及CyclinD1蛋白表達的影響。研究結果：一、臨床病理標本研究部分(1)Shh在Dys組表達最高,MAG組最低：MAG組顯著低于CSG組(P=0.0030.05),但顯著低于MAG組及Dys組(P0.05)；細胞核染色陽性率GC組(32.00%)顯著高于CSG組(4.55%)、MAG組(6.82%)及Dys組(8.33%)。Glil的表達與胃癌分化程度、浸潤深度、TNM分期及淋巴結轉移密切相關(P0.05)。(6)GC組Gli3的表達顯著低于其他3組(P0.05)；(8)PCNA在GC組表達顯著高于CSG及MAG組(P0.05)；(9)細胞凋亡指數(shù)GC組(4.081.41)及Dys組(6.141.56)均顯著低于CSG組(12.544.23)及MAG組(21.376.27)(P0.01)。(10)在胃粘膜癌變過程中Gli1的表達與CyclinD1的表達呈顯著正相關(R=0.214,P=0.0030.01);與PCNA的表達也呈正相關(R=0.145,P=0.0480.05)；與細胞凋亡呈顯著負相關性(R=0.278,P=0.0020.01)。二、體外研究部分(1) Cyclopamine阻斷Hh信號通路后,胃癌細胞MKN28出現(xiàn)類似凋亡樣的形態(tài)改變,增殖受到顯著抑制,并呈濃度及時間依賴性。(4)阻斷Hh信號通路后,顯著下調(diào)CyclinD1的表達,Gli1表達總量卻未見明顯下調(diào)。結論：1、胃粘膜癌變過程中Hh信號通路發(fā)生異?；罨?。2、Hh信號通路的異?；罨c胃癌分化程度、浸潤深度、TNM分期及淋巴結轉移密切相關。3、阻斷Hh信號通路可下調(diào)CyclinD1的表達,抑制MKN28細胞增殖?！居⑽恼緽ackground: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.【關鍵詞】Hh信號通路 胃癌 癌前病變 Cyclopamine【英文關鍵詞】Hh signaling pathway gastric cancer precancerous lesion cyclopamine【目錄】Hedgehog信號通路在胃癌發(fā)生發(fā)展中的作用及機制探討摘要3-6ABSTRACT6-9第1章 引言14-19第2章 Hh信號通路與胃癌發(fā)生發(fā)展的關系19-462.1 實驗材料和方法19-252.1.1 研究對象1