本文選題：IL-23 + 黑色素瘤��； 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：【研究目的】雙向分化惡性腫瘤是指可以同時(shí)向間充質(zhì)樣組織和上皮樣組織分化的惡性腫瘤,而上皮樣分化和間充質(zhì)樣分化不同之處在于分化后的腫瘤細(xì)胞的形態(tài)以及組織排列兩個(gè)方面。惡性黑色素瘤就是一種常見的雙向分化惡性腫瘤[1]。血管生成擬態(tài)(vasculogenic mimicry,VM)是與傳統(tǒng)內(nèi)皮依賴性血管不同的腫瘤血供形式之一,其構(gòu)成不依賴于內(nèi)皮細(xì)胞,而是在特定的微環(huán)境條件下腫瘤細(xì)胞自身通過(guò)塑形以及與細(xì)胞外基質(zhì)相互作用而形成的血管壁類似結(jié)構(gòu),是可為腫瘤輸送血液的管道,VM與腫瘤的生長(zhǎng)、浸潤(rùn)和轉(zhuǎn)移有著密切的關(guān)系[2,3]。由炎癥細(xì)胞和炎癥因子參與構(gòu)成的腫瘤微環(huán)境,對(duì)腫瘤的增殖、生存和轉(zhuǎn)移都有著重要影響,是腫瘤形成不可缺少的一個(gè)方面[4-10]。而白細(xì)胞介素-23(IL-23)作為腫瘤微環(huán)境中的一個(gè)重要炎性細(xì)胞因子,通常是結(jié)合細(xì)胞表面膜受體復(fù)合物,激活下游STAT3、STAT4和NF-κβ等信號(hào)通路,從而發(fā)揮其生物學(xué)功能[11,12]。“腫瘤可能起源于慢性炎癥”的假說(shuō)Virchow早在1863年就已提出[13],這引起了后續(xù)一系列的有關(guān)炎癥和腫瘤之間關(guān)系的研究,但I(xiàn)L-23在黑色素瘤雙向分化及血管生成擬態(tài)形成過(guò)程中所起的作用尚未見報(bào)道。本研究旨在檢測(cè)IL-23在黑色素瘤中的表達(dá)并探討IL-23在黑色素瘤雙向分化及血管生成擬態(tài)(VM)形成過(guò)程中的作用,進(jìn)而研究其對(duì)黑色素瘤細(xì)胞遷移侵襲能力的影響。【研究方法】1.根據(jù)IL-23在黑色素瘤細(xì)胞系A(chǔ)375,A875,MUM-2B,MUM-2C中的表達(dá)水平,將IL-23表達(dá)質(zhì)粒轉(zhuǎn)染至黑色素瘤細(xì)胞系A(chǔ)375,MUM-2B,MUM-2C中,使高表達(dá)的A375,MUM-2B中IL-23外源性降表達(dá),低表達(dá)的MUM-2C中誘導(dǎo)IL-23外源性過(guò)表達(dá)。利用Western blot及細(xì)胞免疫熒光實(shí)驗(yàn)檢測(cè)IL-23的轉(zhuǎn)染效果。2.在倒置顯微鏡下觀察IL-23轉(zhuǎn)染后A375,MUM-2B,MUM-2C的細(xì)胞形態(tài)學(xué)改變。3.通過(guò)MTT增殖實(shí)驗(yàn)檢測(cè)IL-23轉(zhuǎn)染后A375,MUM-2B,MUM-2C細(xì)胞增殖能力的變化情況。4.利用細(xì)胞劃痕實(shí)驗(yàn)檢測(cè)IL-23轉(zhuǎn)染后A375,MUM-2B,MUM-2C細(xì)胞遷移運(yùn)動(dòng)能力的變化情況。5.通過(guò)Transwell侵襲實(shí)驗(yàn)檢測(cè)IL-23轉(zhuǎn)染后A375,MUM-2B,MUM-2C細(xì)胞侵襲能力的變化情況。6.利用三維培養(yǎng)實(shí)驗(yàn)檢測(cè)IL-23轉(zhuǎn)染后A375,MUM-2B,MUM-2C細(xì)胞成管能力的變化情況。7.利用Western blot及免疫熒光實(shí)驗(yàn)檢測(cè)IL-23轉(zhuǎn)染后A375,MUM-2B,MUM-2C細(xì)胞中相關(guān)蛋白(NF-κβ、N-cadherin、Vimentin、Snail、MMP-2和VE-cadherin)表達(dá)變化情況。8.利用明膠酶譜實(shí)驗(yàn)檢測(cè)IL-23轉(zhuǎn)染后A375,MUM-2B細(xì)胞中MMP-2和MMP-9表達(dá)活性的變化情況。9.將轉(zhuǎn)染的降表達(dá)A375和過(guò)表達(dá)MUM-2C黑色素瘤細(xì)胞接種至裸鼠體內(nèi),觀察腫瘤成瘤及生長(zhǎng)情況。10.包埋制成組織切片,運(yùn)用Endomucin/PAS雙染和免疫組化檢測(cè)移植瘤組織內(nèi)VM和相關(guān)蛋白(Vimentin、Snail和VE-cadherin)表達(dá)情況,分析其表達(dá)差異�！狙芯拷Y(jié)果】1.Western blot及細(xì)胞免疫熒光實(shí)驗(yàn)結(jié)果顯示經(jīng)慢病毒質(zhì)粒轉(zhuǎn)染并藥篩得到穩(wěn)定的IL-23外源性降表達(dá)的A375和MUM-2B細(xì)胞,IL-23外源性過(guò)表達(dá)的MUM-2C細(xì)胞。2.在倒置顯微鏡下觀察IL-23轉(zhuǎn)染后A375,MUM-2B,MUM-2C的細(xì)胞形態(tài)發(fā)生明顯改變。IL-23降表達(dá)的A375和MUM-2B細(xì)胞變大變圓,偽足減少,而IL-23過(guò)表達(dá)的MUM-2C細(xì)胞變小,呈梭型,偽足增多。3.MTT增殖實(shí)驗(yàn)顯示IL-23降表達(dá)的A375和MUM-2B細(xì)胞增殖能力顯著降低,而IL-23過(guò)表達(dá)的MUM-2C細(xì)胞增殖能力升高。4.細(xì)胞劃痕實(shí)驗(yàn)結(jié)果顯示IL-23降表達(dá)的A375和MUM-2B細(xì)胞遷移運(yùn)動(dòng)能力顯著降低,而IL-23過(guò)表達(dá)的MUM-2C細(xì)胞遷移運(yùn)動(dòng)能力明顯提高。5.Transwell侵襲實(shí)驗(yàn)顯示IL-23降表達(dá)的A375和MUM-2B細(xì)胞侵襲能力顯著降低,而IL-23過(guò)表達(dá)的MUM-2C細(xì)胞侵襲能力明顯提高。6.三維培養(yǎng)實(shí)驗(yàn)結(jié)果顯示IL-23降表達(dá)的A375和MUM-2B細(xì)胞形成管狀結(jié)構(gòu)的能力都顯著降低,而IL-23過(guò)表達(dá)的MUM-2C細(xì)胞形成管狀結(jié)構(gòu)的能力明顯提高。7.Western blot及免疫熒光實(shí)驗(yàn)結(jié)果顯示IL-23降表達(dá)的A375和MUM-2B細(xì)胞間充質(zhì)表型蛋白N-cad和Vimentin表達(dá)降低,VM相關(guān)蛋白MMP-2和VE-cadherin表達(dá)降低,相關(guān)調(diào)節(jié)蛋白NF-κβ、Snail表達(dá)也顯著降低,而IL-23過(guò)表達(dá)的MUM-2C細(xì)胞間充質(zhì)表型蛋白N-cad和Vimentin表達(dá)升高,VM相關(guān)蛋白MMP-2和VE-cadherin表達(dá)升高,相關(guān)調(diào)節(jié)蛋白NF-κβ、Snail表達(dá)也顯著升高。8.明膠酶譜實(shí)驗(yàn)結(jié)果顯示IL-23降表達(dá)的A375和MUM-2B細(xì)胞中MMP-2和MMP-9表達(dá)活性都顯著降低。9.接種IL-23降表達(dá)A375和過(guò)表達(dá)MUM-2C黑色素瘤細(xì)胞的裸鼠移植瘤成瘤率及大小均明顯小于對(duì)照組移植瘤。10.Endomucin/PAS雙染和免疫組化結(jié)果顯示接種IL-23降表達(dá)A375移植瘤組織內(nèi)VM和相關(guān)蛋白(Vimentin、Snail和VE-cadherin)表達(dá)降低,接種IL-23過(guò)表達(dá)MUM-2C移植瘤組織內(nèi)未形成VM而相關(guān)蛋白(Vimentin、Snail和VE-cadherin)表達(dá)均升高�！狙芯拷Y(jié)論】1.IL-23可促進(jìn)黑色素瘤細(xì)胞向間充質(zhì)表型分化,促進(jìn)黑色素瘤的增殖、遷移和侵襲,并促進(jìn)黑色素瘤血管生成擬態(tài)(VM)的形成。2.IL-23可能是通過(guò)NF-κβ調(diào)節(jié)Snail的表達(dá)進(jìn)而調(diào)節(jié)N-cadherin和Vimentin的表達(dá)促進(jìn)黑色素瘤細(xì)胞向間充質(zhì)表型分化。3.IL-23通過(guò)調(diào)節(jié)間充質(zhì)表型蛋白N-cadherin和Vimentin促進(jìn)黑色素瘤細(xì)胞向間充質(zhì)表型分化改變細(xì)胞形態(tài)使其更易于遷移和侵襲,調(diào)節(jié)MMP-2和MMP-9表達(dá)活性提供空間結(jié)構(gòu)基礎(chǔ),并同時(shí)調(diào)節(jié)MMP-2和VE-cadherin的表達(dá),進(jìn)而進(jìn)一步促進(jìn)血管生成擬態(tài)(VM)的形成。
[Abstract]:[Objective] bi-directional differentiation of malignant tumors is a malignant tumor that can differentiate into mesenchymal like tissue and epithelioid tissue at the same time. The differentiation of epithelioid and mesenchymal like differentiation lies in two aspects of the morphology and tissue arrangement of the differentiated tumor cells. Malignant melanoma is a common bidirectional differentiation malignant tumor. The tumor [1]. vasculogenic mimicry (VM) is one of the form of blood supply different from the traditional endothelium dependent blood vessel, and its composition is not dependent on the endothelial cells, but in the specific microenvironment, the tumor cell itself is formed by the shape of the tumor cells themselves and the interaction of the extracellular matrix. VM has a close relationship with the growth, infiltration and metastasis of tumor, which is closely related to the tumor microenvironment composed of inflammatory cells and inflammatory factors. It has an important influence on the proliferation, survival and metastasis of the tumor. It is an indispensable aspect of tumor formation, [4-10]. and IL-23 as a tumor. An important inflammatory cytokine in microenvironment, usually combined with cell surface membrane receptor complexes, activates the downstream STAT3, STAT4, and NF- kappa beta signaling pathways, thus giving play to the hypothesis that the biological function [11,12]. "tumor may originate in chronic inflammation", Virchow has been proposed in 1863, which has caused a series of follow-up. The study of the relationship between inflammation and tumor, but the role of IL-23 in the bi-directional differentiation of melanoma and angiogenesis has not yet been reported. The purpose of this study was to detect the expression of IL-23 in melanoma and to explore the role of IL-23 in the formation of melanoma bi-directional differentiation and angiogenesis (VM). The effect on the migration and invasion of melanoma cells. [method] 1. according to the expression level of IL-23 in the melanoma cell line A375, A875, MUM-2B, MUM-2C, the IL-23 expression plasmid was transfected into the melanoma cell line A375, MUM-2B, and MUM-2C to induce the expression of the high expression A375, MUM-2B IL-23, and low expression. Western blot and cell immunofluorescence test were used to detect the transfection effect of IL-23.2. under inverted microscope to observe the cell morphological changes of A375, MUM-2B and MUM-2C after IL-23 transfection..3. through MTT proliferation test was used to detect the proliferation of IL-23 cells. Changes in the mobility of A375, MUM-2B, and MUM-2C cells after IL-23 transfection were detected by the scratch test..5. was used to detect the changes of A375, MUM-2B, MUM-2C cell invasiveness after IL-23 transfection by Transwell invasion experiment. RN blot and immunofluorescence test detected the changes in the expression of related proteins (NF- kappa, N-cadherin, Vimentin, Snail, MMP-2 and VE-cadherin) in A375, MUM-2B, MUM-2C cells after IL-23 transfection. MUM-2C melanoma cells were inoculated into nude mice, and the tumor growth and tumor growth were observed by.10. embedding into tissue sections. The expression of VM and related proteins (Vimentin, Snail and VE-cadherin) in the transplanted tumor tissues were detected by Endomucin/PAS double staining and immunohistochemical staining, and the difference of expression was analyzed. [results] 1.Western blot and The results of cell immunofluorescence test showed that A375 and MUM-2B cells were stabilized by IL-23 transfection and drug sieves, and the IL-23 exogenous MUM-2C cell.2. was observed under inverted microscope for A375 in IL-23 transfection, and the cell morphology of MUM-2B and MUM-2C changed obviously to A375 and refinement of.IL-23 descending expression. The cells changed greatly and the pseudo foot decreased, while the MUM-2C cells overexpressed by IL-23 became smaller and showed shuttle type. The proliferation test of.3.MTT in.3.MTT showed that the proliferation ability of A375 and MUM-2B cells decreased significantly, while the proliferation ability of MUM-2C cells expressed by IL-23 was higher than that of.4. cells in.4. cells. The A375 and MUM-2B cell migration of IL-23 decreased expression. The migration ability of the MUM-2C cells expressed by IL-23 was significantly increased and the.5.Transwell invasion experiment showed that the invasion ability of A375 and MUM-2B cells decreased significantly, while the invasion ability of MUM-2C cells expressed by IL-23 was obviously enhanced by.6. three-dimensional culture experiment, which showed A375 and MUM-2 with IL-23 reduced expression. The ability of B cells to form tubular structure decreased significantly, while the ability of IL-23 over expressed MUM-2C cells to form tubular structures significantly increased.7.Western blot and immunofluorescence test results showed that IL-23 reduced expression of A375 and MUM-2B cells decreased in N-cad and Vimentin expressions of MUM-2B cells, VM related proteins MMP-2 and expression decreased. The expression of NF- kappa beta and Snail decreased significantly, while the expression of N-cad and Vimentin in IL-23 overexpressed MUM-2C cells increased, the expression of MMP-2 and VE-cadherin in VM related proteins increased, and the expression of NF- kappa beta and Snail expression of the related regulatory proteins increased significantly. The expression of MMP-2 and MMP-9 in B cells significantly decreased the tumor formation rate and size of nude mice transplanted with IL-23 descending A375 and overexpression of MUM-2C melanoma cells. The results showed that.10.Endomucin/PAS double staining and immunohistochemical results showed that IL-23 descending expression of A375 was VM and related proteins in A375 transplanted tumor tissues (Vimenti). The expression of N, Snail and VE-cadherin decreased, and the expression of VM and related proteins (Vimentin, Snail and VE-cadherin) were not formed in the MUM-2C transplanted tumor tissues of IL-23 over expression. [Conclusion] 1.IL-23 can promote the differentiation of melanoma cells into the mesenchymal phenotype, promote the proliferation, migration and invasion of melanoma, and promote the blood vessels of melanoma. The formation of.2.IL-23 (VM) may be mediated by the expression of Snail by NF- kappa beta and then regulating the expression of N-cadherin and Vimentin to promote the differentiation of melanoma cells into mesenchymal phenotypic differentiation.3.IL-23 by regulating mesenchymal phenotypic protein N-cadherin and Vimentin to promote melanoma cells to differentiate into mesenchymal phenotype and change cell morphology. It is easier to migrate and invasiveness, to regulate the spatial structure of MMP-2 and MMP-9 expression activity, and to regulate the expression of MMP-2 and VE-cadherin, and further promote the formation of angiogenic mimicry (VM).
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.5

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