本文選題：胃腸道間質(zhì)瘤 + Shh信號通路　； 參考：《安徽醫(yī)科大學(xué)》2015年博士論文

【摘要】：胃腸道間質(zhì)瘤(gastrointestinal stromal tumors, GIST)是消化道最常見的間葉源性腫瘤,c-kit或血小板源性生長因子受體α (platelet-derived growth factor receptor a, PDGFRA)受體酪氨酸激酶(receptor tyrosine kinase, RTKs)的異常激活是其發(fā)生的關(guān)鍵因素。目前治療以手術(shù)為主,對于中高危及復(fù)發(fā)的患者靶向藥物甲磺酸伊馬替尼(Imatinib Mesylate-Glivec)是一線治療藥物,但大部分患者仍會發(fā)生耐藥。因此,尋找具有共性的c-kit/PDGFRA下游關(guān)鍵信號通路或者c-kit/PDGFRA信號通路以外有與之交叉的信號通路上的相關(guān)分子,可能為GIST靶向治療提供靶點。Sonic hedgehog (Shh)信號轉(zhuǎn)導(dǎo)通路在腫瘤的發(fā)生、發(fā)展過程中起到重要作用。阻斷腫瘤細(xì)胞中的Shh信號轉(zhuǎn)導(dǎo)通路某個節(jié)點,有可能為腫瘤的靶向治療提供一個新的途徑。GIST組織中Shh信號通路的表達(dá)已有報道,但Shh與GIST臨床病理因素及預(yù)后關(guān)系、Shh與GIST細(xì)胞增殖、凋亡的關(guān)系以及Shh在GIST中具體的作用機制及與其他信號通路的交叉調(diào)控的尚未見報道。因此,本研究將集中闡述上述三個方面的內(nèi)容。為探討Shh信號通路在GIST發(fā)生和發(fā)展過程中的作用,驗證其作用的可能機制是在GIST中與PI3K和MAPK信號通路存在交叉聯(lián)系,本課題采用免疫組化檢測GIST標(biāo)本中Shh信號通路中重要蛋白(Shh, Ptch, Smo和Gli-1)的表達(dá),分析其與GIST病理因素及復(fù)發(fā)預(yù)后等因素的相互聯(lián)系,Western blot檢測GIST組織中Shh通路、PI3K通路和MAPK通路的表達(dá)情況,分析三條信號通路表達(dá)的關(guān)聯(lián)性；體外培養(yǎng)GIST-H1細(xì)胞株,用相應(yīng)的信號刺激因子(EGF、N-Shh)刺激上述三條信號通路,分別或聯(lián)合用靶向抑制劑(cyclopamine、wortmannin和PD98059)對三條信號通路進(jìn)行抑制,檢測抑制后信號通路的關(guān)鍵因子(Gli-1、 p-AKT及p-ERK)表達(dá)變化,驗證上述三條信號通路在GIST中是否存在相互之間的交叉調(diào)控,以及在GIST抑制增殖和促進(jìn)凋亡過程中起作用。初步探討Shh信號通路在GIST中的作用機制及聯(lián)合抑制Shh和PI3K信號通路、MAPK通路對腫瘤凋亡和增殖的作用,為靶向治療奠定基礎(chǔ)。第一部分Shh信號通路分子在胃腸道間質(zhì)瘤組織中的表達(dá)及其與臨床病理因素的關(guān)系目的：探討GIST標(biāo)本組織中Shh信號通路重要蛋白的表達(dá)及其與臨床病理因素之間的關(guān)系。方法：收集安徽醫(yī)科大學(xué)第一附屬醫(yī)院2001年1月至2008年12月間行手術(shù)治療,資料完整的GIST患者125例。免疫組化檢測Shh信號通路蛋白Shh, Ptch, Smo和Gli-1的表達(dá)情況,分析上述蛋白的表達(dá)與GIST的腫瘤部位、直徑、核分裂像、NIH分級、復(fù)發(fā)及預(yù)后的關(guān)系。結(jié)果：GIST組織中Shh,Ptch, Smo和Gli-14種蛋白的表達(dá)率分別為90.5%%,83.3%,85.7%,85.7%；Shh表達(dá)水平與Smo、Gli-1表達(dá)水平均成正相關(guān),Ptch表達(dá)水平與Smo表達(dá)水平呈正相關(guān)；Smo表達(dá)水平與Gli-1表達(dá)水平成正相關(guān),Shh表達(dá)水平與Ptch表達(dá)水平無相關(guān)性；Ptch表達(dá)水平與Gli-1表達(dá)水平無相關(guān)性；Shh, Ptch, Smo和Gli-1四種蛋白的陽性表達(dá)水平與患者性別、年齡均無相關(guān)性,與腫瘤大小、核分裂像數(shù)和危險度分級有關(guān)。Smo和Gli-1的陽性表達(dá)還與原發(fā)腫瘤的部位、組織學(xué)分類有關(guān)；NIH分級為中、高危組的GIST中Shh, Ptch, Smo和Gli-1四種蛋白的陽性表達(dá)率明顯高于NIH分級為極低、低危組的陽性表達(dá)率,差異有顯著性。Shh, Ptch, Smo和Gli-1在復(fù)發(fā)GIST中的陽性率明顯高于無復(fù)發(fā)GIST,差異有顯著性。Shh, Ptch, Smo和Gli-1四種蛋白的表達(dá)為陽性組的1、3、5年生存率要顯著低于表達(dá)為陰性組者。進(jìn)一步的多因素分析發(fā)現(xiàn),Gli-1的陽性表達(dá)是患者預(yù)后的獨立危險因素。結(jié)論：GIST組織中,有Shh信號通路的存在并激活,Shh信號通路的重要蛋白在GIST中表達(dá)具有相關(guān)性,可能是通過配體依賴型的激活方式參與GIST的發(fā)生與發(fā)展。Shh信號通路蛋白的表達(dá)與GIST預(yù)后及復(fù)發(fā)有關(guān),其中Gli-1蛋白的表達(dá)可能作為判斷復(fù)發(fā)的重要指標(biāo)之一。第二部分胃腸道間質(zhì)瘤組織中Gli-1、p-AKT、 p-ERK的表達(dá)及意義目的：分析Shh、PI3K和MAPK信號通路中的關(guān)鍵因子Gli-1、p-AKT和p-ERK在GIST組織中的表達(dá)情況,探討上述三條信號通路表達(dá)之間的相關(guān)性。方法：收集安徽醫(yī)科大學(xué)第一附屬醫(yī)院在2013年1月至2013年12月間手術(shù)切除、資料完整的GIST石蠟組織標(biāo)本145例,Western blot檢測GIST組織中Gli-1、p-AKT及p-ERK的表達(dá)水平,分析上述三種蛋白的表達(dá)及其相關(guān)性。結(jié)果：Gli-1、p-AKT和p-ERK在不同危險度分級的GIST中均有表達(dá),且其表達(dá)水平與腫瘤的危險度分級密切相關(guān),表達(dá)具有一致性。結(jié)論：Shh、PI3K和MAPK信號通路的關(guān)鍵因子Gli-1、p-AKT和p-ERK,廣泛表達(dá)于GIST中,上述信號通路參與了GIST的發(fā)生發(fā)展過程。Shh信號通路在GIST中所起作用可能是通過與PI3K通路和MAPK通路之間的交叉調(diào)控機制實現(xiàn)的。第三部分胃腸道間質(zhì)瘤中Shh與PI3K、MAPK信號通路的交叉調(diào)控及對腫瘤細(xì)胞增殖和凋亡的影響目的：探討GIST中Shh, PI3K和MAPK三條信號通路之間是否存在交叉調(diào)控及其對細(xì)胞增殖和凋亡的影響。方法：采用N-Shh和EGF分別誘導(dǎo)激活GIST-H1細(xì)胞株,分別使用各個信號通路的特異性抑制劑阻滯相應(yīng)的信號通路。Western blot方法檢測各個通路的關(guān)鍵因子Gli-1、p-AKT和p-ERK在的表達(dá)情況。分析單獨使用或聯(lián)合使用Shh、PI3K/AKT和MAPK通路的抑制劑對于未激活及激活狀態(tài)下的各信號通路活性的影響；分析使用上述通路抑制劑阻斷對于GIST-H1細(xì)胞增殖、凋亡的影響。結(jié)果：GIST-H1細(xì)胞株中,p-AKT, p-ERK和Gli-1的表達(dá)水平能分別被EGF和N-Shh刺激上調(diào)；EGF誘導(dǎo)GIST-H1細(xì)胞株p-AKT或p-ERK表達(dá)效應(yīng)可分別被wortmannin或PD98059所抑制,同時這種效應(yīng)也能夠被cyclopamine抑制,聯(lián)合使用兩種抑制劑(cyclopamine+wortmannin,或cyclopamine +PD98059)能更進(jìn)一步抑制上述通路的活性；N-shh誘導(dǎo)GIST-H1細(xì)胞株Gli-1表達(dá)效應(yīng)可被cyclopamine抑制,同時這種效應(yīng)也分別能被wortmannin或PD98059所抑制,聯(lián)合使用兩種抑制劑(cyclopamine+wortmannin,或cyclopamine+PD98059)能更進(jìn)一步抑制Shh通路Gli-1的活性；在EGF處理下,GIST-H1細(xì)胞增殖速度顯著加快,并且該促進(jìn)能被PD98059和cyclopamine-KAAD預(yù)處理部分抑制,在N-Shh處理下,GIST-H1細(xì)胞的增殖速度顯著加快,并且該效應(yīng)能被PD98059和cyclopamine-KAAD預(yù)處理所部分抑制；Shh、PI3K和MAPK1信號通路特異的抑制劑可以誘導(dǎo)GIST-H1細(xì)胞凋亡。結(jié)論：GIST細(xì)胞中,Shh、PI3K;和MAPK信號轉(zhuǎn)導(dǎo)是存在的,且存在相互之間的交叉聯(lián)系。聯(lián)合使用上述信號通路的抑制劑可以協(xié)同抑制細(xì)胞增殖、促進(jìn)細(xì)胞凋亡,可作為GIST靶向治療的新選擇。
[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.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R735

【共引文獻(xiàn)】

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