胃肠道间质瘤中Shh信号通路作用的实验研究

发布时间：2018-05-02 22:52

本文选题：胃肠道间质瘤 + Shh信号通路　；参考：《安徽医科大学》2015年博士论文

【摘要】：胃肠道间质瘤(gastrointestinal stromal tumors, GIST)是消化道最常见的间叶源性肿瘤,c-kit或血小板源性生长因子受体α (platelet-derived growth factor receptor a, PDGFRA)受体酪氨酸激酶(receptor tyrosine kinase, RTKs)的异常激活是其发生的关键因素。目前治疗以手术为主,对于中高危及复发的患者靶向药物甲磺酸伊马替尼(Imatinib Mesylate-Glivec)是一线治疗药物,但大部分患者仍会发生耐药。因此,寻找具有共性的c-kit/PDGFRA下游关键信号通路或者c-kit/PDGFRA信号通路以外有与之交叉的信号通路上的相关分子,可能为GIST靶向治疗提供靶点。Sonic hedgehog (Shh)信号转导通路在肿瘤的发生、发展过程中起到重要作用。阻断肿瘤细胞中的Shh信号转导通路某个节点,有可能为肿瘤的靶向治疗提供一个新的途径。GIST组织中Shh信号通路的表达已有报道,但Shh与GIST临床病理因素及预后关系、Shh与GIST细胞增殖、凋亡的关系以及Shh在GIST中具体的作用机制及与其他信号通路的交叉调控的尚未见报道。因此,本研究将集中阐述上述三个方面的内容。为探讨Shh信号通路在GIST发生和发展过程中的作用,验证其作用的可能机制是在GIST中与PI3K和MAPK信号通路存在交叉联系,本课题采用免疫组化检测GIST标本中Shh信号通路中重要蛋白(Shh, Ptch, Smo和Gli-1)的表达,分析其与GIST病理因素及复发预后等因素的相互联系,Western blot检测GIST组织中Shh通路、PI3K通路和MAPK通路的表达情况,分析三条信号通路表达的关联性；体外培养GIST-H1细胞株,用相应的信号刺激因子(EGF、N-Shh)刺激上述三条信号通路,分别或联合用靶向抑制剂(cyclopamine、wortmannin和PD98059)对三条信号通路进行抑制,检测抑制后信号通路的关键因子(Gli-1、 p-AKT及p-ERK)表达变化,验证上述三条信号通路在GIST中是否存在相互之间的交叉调控,以及在GIST抑制增殖和促进凋亡过程中起作用。初步探讨Shh信号通路在GIST中的作用机制及联合抑制Shh和PI3K信号通路、MAPK通路对肿瘤凋亡和增殖的作用,为靶向治疗奠定基础。第一部分Shh信号通路分子在胃肠道间质瘤组织中的表达及其与临床病理因素的关系目的：探讨GIST标本组织中Shh信号通路重要蛋白的表达及其与临床病理因素之间的关系。方法：收集安徽医科大学第一附属医院2001年1月至2008年12月间行手术治疗,资料完整的GIST患者125例。免疫组化检测Shh信号通路蛋白Shh, Ptch, Smo和Gli-1的表达情况,分析上述蛋白的表达与GIST的肿瘤部位、直径、核分裂像、NIH分级、复发及预后的关系。结果：GIST组织中Shh,Ptch, Smo和Gli-14种蛋白的表达率分别为90.5%%,83.3%,85.7%,85.7%；Shh表达水平与Smo、Gli-1表达水平均成正相关,Ptch表达水平与Smo表达水平呈正相关；Smo表达水平与Gli-1表达水平成正相关,Shh表达水平与Ptch表达水平无相关性；Ptch表达水平与Gli-1表达水平无相关性；Shh, Ptch, Smo和Gli-1四种蛋白的阳性表达水平与患者性别、年龄均无相关性,与肿瘤大小、核分裂像数和危险度分级有关。Smo和Gli-1的阳性表达还与原发肿瘤的部位、组织学分类有关；NIH分级为中、高危组的GIST中Shh, Ptch, Smo和Gli-1四种蛋白的阳性表达率明显高于NIH分级为极低、低危组的阳性表达率,差异有显著性。Shh, Ptch, Smo和Gli-1在复发GIST中的阳性率明显高于无复发GIST,差异有显著性。Shh, Ptch, Smo和Gli-1四种蛋白的表达为阳性组的1、3、5年生存率要显著低于表达为阴性组者。进一步的多因素分析发现,Gli-1的阳性表达是患者预后的独立危险因素。结论：GIST组织中,有Shh信号通路的存在并激活,Shh信号通路的重要蛋白在GIST中表达具有相关性,可能是通过配体依赖型的激活方式参与GIST的发生与发展。Shh信号通路蛋白的表达与GIST预后及复发有关,其中Gli-1蛋白的表达可能作为判断复发的重要指标之一。第二部分胃肠道间质瘤组织中Gli-1、p-AKT、 p-ERK的表达及意义目的：分析Shh、PI3K和MAPK信号通路中的关键因子Gli-1、p-AKT和p-ERK在GIST组织中的表达情况,探讨上述三条信号通路表达之间的相关性。方法：收集安徽医科大学第一附属医院在2013年1月至2013年12月间手术切除、资料完整的GIST石蜡组织标本145例,Western blot检测GIST组织中Gli-1、p-AKT及p-ERK的表达水平,分析上述三种蛋白的表达及其相关性。结果：Gli-1、p-AKT和p-ERK在不同危险度分级的GIST中均有表达,且其表达水平与肿瘤的危险度分级密切相关,表达具有一致性。结论：Shh、PI3K和MAPK信号通路的关键因子Gli-1、p-AKT和p-ERK,广泛表达于GIST中,上述信号通路参与了GIST的发生发展过程。Shh信号通路在GIST中所起作用可能是通过与PI3K通路和MAPK通路之间的交叉调控机制实现的。第三部分胃肠道间质瘤中Shh与PI3K、MAPK信号通路的交叉调控及对肿瘤细胞增殖和凋亡的影响目的：探讨GIST中Shh, PI3K和MAPK三条信号通路之间是否存在交叉调控及其对细胞增殖和凋亡的影响。方法：采用N-Shh和EGF分别诱导激活GIST-H1细胞株,分别使用各个信号通路的特异性抑制剂阻滞相应的信号通路。Western blot方法检测各个通路的关键因子Gli-1、p-AKT和p-ERK在的表达情况。分析单独使用或联合使用Shh、PI3K/AKT和MAPK通路的抑制剂对于未激活及激活状态下的各信号通路活性的影响；分析使用上述通路抑制剂阻断对于GIST-H1细胞增殖、凋亡的影响。结果：GIST-H1细胞株中,p-AKT, p-ERK和Gli-1的表达水平能分别被EGF和N-Shh刺激上调；EGF诱导GIST-H1细胞株p-AKT或p-ERK表达效应可分别被wortmannin或PD98059所抑制,同时这种效应也能够被cyclopamine抑制,联合使用两种抑制剂(cyclopamine+wortmannin,或cyclopamine +PD98059)能更进一步抑制上述通路的活性；N-shh诱导GIST-H1细胞株Gli-1表达效应可被cyclopamine抑制,同时这种效应也分别能被wortmannin或PD98059所抑制,联合使用两种抑制剂(cyclopamine+wortmannin,或cyclopamine+PD98059)能更进一步抑制Shh通路Gli-1的活性；在EGF处理下,GIST-H1细胞增殖速度显著加快,并且该促进能被PD98059和cyclopamine-KAAD预处理部分抑制,在N-Shh处理下,GIST-H1细胞的增殖速度显著加快,并且该效应能被PD98059和cyclopamine-KAAD预处理所部分抑制；Shh、PI3K和MAPK1信号通路特异的抑制剂可以诱导GIST-H1细胞凋亡。结论：GIST细胞中,Shh、PI3K;和MAPK信号转导是存在的,且存在相互之间的交叉联系。联合使用上述信号通路的抑制剂可以协同抑制细胞增殖、促进细胞凋亡,可作为GIST靶向治疗的新选择。
[Abstract]:Gastrointestinal stromal tumors (GIST) is the most common mesenchymal tumor in the digestive tract, and the abnormal activation of c-kit or platelet derived growth factor receptor alpha (platelet-derived growth factor receptor A, PDGFRA) receptor tyrosine kinase (receptor) is the key factor for its occurrence. The treatment is based on surgery, and the targeted drug Imatinib Mesylate-Glivec, a targeted drug for middle and high risk and recurrence, is a first-line treatment, but most patients still have resistance. Therefore, looking for a common signal of the c-kit/PDGFRA downstream key signaling pathway or the c-kit/PDGFRA signaling pathway is a cross signal. The related molecules on the pathway may provide a target.Sonic hedgehog (Shh) signal transduction pathway for GIST targeting therapy, which plays an important role in the development of tumor. Blocking a node in the Shh signal transduction pathway in tumor cells may provide a new pathway for the targeting therapy of cancer, Shh signaling pathway in.GIST tissue. The expression of Shh has been reported, but the relationship between Shh and GIST clinicopathological factors and prognosis, the relationship between Shh and GIST cell proliferation, apoptosis, the specific mechanism of Shh in GIST and the cross regulation with other signaling pathways have not yet been reported. Therefore, this study will focus on the contents of the above three aspects. In order to explore the Shh signaling pathway in GIST The possible mechanism in the process of birth and development verifies that the possible mechanism of its role is the cross connection with the PI3K and MAPK signaling pathways in GIST. This subject uses immunohistochemistry to detect the expression of important proteins in the Shh signaling pathway (Shh, Ptch, Smo and Gli-1) in the Shh signaling pathway in GIST specimens, and analyzes the correlation with the pathological factors of GIST and the recurrence prognosis and other factors. We Stern blot detected the expression of Shh pathway, PI3K pathway and MAPK pathway in GIST tissues, analyzed the correlation between the expression of three signal pathways, cultured the GIST-H1 cell lines in vitro, stimulated the three signal pathways with corresponding signal stimulating factors (EGF, N-Shh), or combined with target inhibitors (cyclopamine, wortmannin and PD98059) to three, respectively. The signal pathway is suppressed and the key factors (Gli-1, p-AKT and p-ERK) of the signal pathway after inhibition are detected, and the cross regulation between the three signaling pathways in GIST and the role of the GIST in inhibiting proliferation and promoting apoptosis are verified. The mechanism of Shh signaling pathway in GIST is preliminarily discussed and the mechanism of Shh signaling pathway is preliminarily discussed. Combined inhibition of Shh and PI3K signaling pathways and the role of MAPK pathway on tumor apoptosis and proliferation, laying the foundation for targeted therapy. Part 1 expression of Shh signaling molecules in gastrointestinal stromal tumors and its relationship with clinicopathological factors: To explore the expression of the important protein in the Shh signaling pathway and its presence in the tissue of GIST. Methods: the relationship between bed pathology factors. Methods: the First Affiliated Hospital of Medical University Of Anhui was collected from January 2001 to December 2008. 125 patients with complete data were collected. The expression of Shh signaling pathway protein Shh, Ptch, Smo and Gli-1 were detected by immunohistochemistry. The expression of the protein was analyzed with the tumor location, diameter, and nuclear score of the GIST. Results: the expression rates of Shh, Ptch, Smo and Gli-14 in GIST tissues were 90.5%%, 83.3%, 85.7%, 85.7%; Shh expression level was positively correlated with Smo, Gli-1 expression level, Ptch expression level was positively correlated with Smo expression level, and the level of expression was positively correlated with the level of expression. There was no correlation between the expression level and the expression level of Ptch, and no correlation between the expression level of Ptch and the expression level of Gli-1; the positive expression level of four proteins of Shh, Ptch, Smo and Gli-1 had no correlation with the sex and age of the patients. The positive expression of.Smo and Gli-1 related to the size of the tumor, the number of nuclear mitotic figures and the risk degree were also related to the site of the primary tumor. The positive rate of four proteins in GIST, Ptch, Smo and Gli-1 in high risk group was significantly higher than that of NIH grade, and the positive rate of the low risk group was significantly higher than that of the low risk group. The positive rate of Ptch, Smo and Gli-1 in the recurrent GIST was significantly higher than that in the recurrent GIST. The positive rates of Ptch, Smo and Gli-1 in the recurrent GIST were significantly higher than those in the recurrent GIST. The positive rates of Ptch, Smo and Gli-1 in the recurrent GIST were significantly higher than those in the recurrent GIST. The positive rates of the four kinds of proteins in the recurrent GIST were significantly higher than those in the recurrence GIST. The 1,3,5 year survival rate in the positive group and the Gli-1 four proteins was significantly lower than that in the negative group. Further multivariate analysis found that the positive expression of Gli-1 was an independent risk factor for the prognosis of the patients. Conclusion: the existence and activation of the Shh signaling pathway in the GIST tissue is expressed and the important protein in the Shh signaling pathway is expressed in GIST. It is possible to participate in the occurrence and development of GIST by ligand dependent activation. The expression of.Shh signaling protein is related to the prognosis and recurrence of GIST. The expression of Gli-1 protein may be one of the important indicators to judge the recurrence. The expression and significance of Gli-1, p-AKT, p-ERK in the second part of the gastrointestinal stromal tumor group Analysis of the key factors in the Shh, PI3K and MAPK signaling pathways Gli-1, p-AKT and p-ERK in the GIST tissue, and explore the correlation between the above three signaling pathways. Methods: 145 cases of complete GIST paraffin tissue specimens were collected from the First Affiliated Hospital of Medical University Of Anhui from January 2013 to December 2013. Western blot was used to detect the expression level of Gli-1, p-AKT and p-ERK in GIST tissues. The expression and correlation of the above three proteins were analyzed. Results: Gli-1, p-AKT and p-ERK were expressed in GIST of different risk grades, and the expression level was closely related to the risk classification of the tumor, and the expression was consistent. Conclusion: Shh, PI3K and letter are in agreement. Key factors, Gli-1, p-AKT and p-ERK, are widely expressed in GIST, the signaling pathway involved in the development of GIST, the role of.Shh signaling pathway in GIST may be achieved through the cross regulation mechanism between PI3K and MAPK pathways. Third part of the gastrointestinal stromal tumor, Shh and PI3K, MAPK signal pathway Cross regulation and effect on proliferation and apoptosis of tumor cells: To investigate whether there is a cross regulation between three signaling pathways of Shh, PI3K and MAPK in GIST and its effect on cell proliferation and apoptosis. Methods: N-Shh and EGF are used to induce activation of GIST-H1 cell lines, respectively, to block the specific inhibitors of each signal pathway. The corresponding signal pathway.Western blot method is used to detect the expression of key factors of each pathway, Gli-1, p-AKT and p-ERK. The effects of the inhibitors of Shh, PI3K/AKT, and MAPK pathway on the activity of each signal pathway in the state of activation and activation in the state of the Shh, PI3K/AKT and MAPK pathways are analyzed. The effect of -H1 cell proliferation and apoptosis. Results: the expression level of p-AKT, p-ERK and Gli-1 in the GIST-H1 cell lines can be up-regulated by EGF and N-Shh, respectively. EGF induced p-AKT or p-ERK expression of GIST-H1 cell lines can be suppressed respectively by wortmannin or N-Shh, and this effect can also be suppressed by two kinds of inhibition. The agent (cyclopamine+wortmannin, or cyclopamine +PD98059) can further inhibit the activity of the above pathway; N-shh induced Gli-1 expression effect in GIST-H1 cell line can be suppressed by cyclopamine, and this effect can also be inhibited by wortmannin or PD98059, combined with the use of two inhibitors (cyclopamine+wortmannin, or cyclopamine+PD9805). 9) can further inhibit the activity of Gli-1 in the Shh pathway; under the EGF treatment, the proliferation rate of GIST-H1 cells is significantly accelerated, and the promotion can be inhibited by PD98059 and cyclopamine-KAAD preconditioning, and the proliferation rate of GIST-H1 cells is significantly accelerated under N-Shh treatment, and this effect should be partially suppressed by PD98059 and cyclopamine-KAAD preconditioning. Shh, PI3K and MAPK1 signaling pathway specific inhibitors can induce apoptosis of GIST-H1 cells. Conclusion: Shh, PI3K, and MAPK signal transduction are existing in GIST cells, and there are cross links between each other. The combined use of these inhibitors can inhibit cell proliferation and promote cell apoptosis, which can be used as a GIST targeting treatment. A new choice of treatment.

【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735

【共引文献】