本文選題：肝細(xì)胞癌 + 腫瘤微環(huán)境　； 參考：《廣西醫(yī)科大學(xué)》2017年博士論文

【摘要】：第一部分腫瘤相關(guān)巨噬細(xì)胞的誘導(dǎo)、鑒定及其對(duì)肝癌細(xì)胞的影響肝癌是經(jīng)典的炎癥相關(guān)性腫瘤,肝癌細(xì)胞所在微環(huán)境內(nèi)浸潤(rùn)著大量腫瘤相關(guān)巨噬細(xì)胞,并以M2型腫瘤相關(guān)巨噬細(xì)胞為主。本部分研究旨在通過(guò)體外誘導(dǎo)獲得不同活化狀態(tài)巨噬細(xì)胞并明確其對(duì)肝癌細(xì)胞生物學(xué)行為的影響。我們首先通過(guò)人單核白血病細(xì)胞THP-1經(jīng)佛波酯誘導(dǎo)為未分化巨噬細(xì)胞(UM0),再分別經(jīng)脂多糖,白介素4和13誘導(dǎo)獲得經(jīng)典活化的巨噬細(xì)胞M1及選擇性激活的巨噬細(xì)胞M2;然后通過(guò)形態(tài)學(xué)、熒光定量PCR和ELISA對(duì)其表型進(jìn)行鑒定;最后從增殖、遷移及侵襲實(shí)驗(yàn)探索不同活化狀態(tài)巨噬細(xì)胞條件培養(yǎng)基(conditioned medium,CM)對(duì)肝癌細(xì)胞生物學(xué)行為的影響,并檢測(cè)了肝癌細(xì)胞中上皮-間質(zhì)化轉(zhuǎn)換(EMT)標(biāo)志物的變化。結(jié)果顯示:(1)與UM0相比,M1呈長(zhǎng)梭形或多邊形,TNF-α和CCL3 m RNA的表達(dá)量及IL-12的分泌均明顯升高;M2偽足細(xì)長(zhǎng)而雜亂、呈抱團(tuán)式生長(zhǎng),AMAC-1、CCL22和Arg-1 m RNA的表達(dá)量及IL-10的分泌均顯著增加。該檢測(cè)結(jié)果與文獻(xiàn)報(bào)道的結(jié)果一致,說(shuō)明已成功誘導(dǎo)獲得不同活化狀態(tài)的巨噬細(xì)胞;(2)生物學(xué)功能實(shí)驗(yàn)顯示經(jīng)M2CM處理后的肝癌細(xì)胞與對(duì)照組相比,其增殖、遷移和侵襲能力均顯著增強(qiáng),而M1CM處理的肝癌細(xì)胞則無(wú)明顯變化,說(shuō)明M2型腫瘤相關(guān)巨噬細(xì)胞能顯著促進(jìn)肝癌細(xì)胞惡性變;(3)經(jīng)M2CM處理后的肝癌細(xì)胞上皮標(biāo)志物E-cadherin的表達(dá)明顯下降,伴隨間質(zhì)細(xì)胞標(biāo)志物N-cadherin、Vimentin和α-SMA顯著上升,經(jīng)M1CM處理的肝癌細(xì)胞各標(biāo)志物的表達(dá)量均無(wú)明顯變化,說(shuō)明僅有經(jīng)M2CM處理后的肝癌細(xì)胞發(fā)生了EMT。第二部分腫瘤相關(guān)巨噬細(xì)胞和肝癌細(xì)胞三維共培養(yǎng)體系中分泌蛋白的差異體內(nèi)狀態(tài)的腫瘤細(xì)胞生存于立體、復(fù)雜而開放的微環(huán)境中,分泌蛋白是該微環(huán)境中腫瘤細(xì)胞與TAMs間相互作用的重要橋梁。在本部分研究中,我們利用低熔點(diǎn)瓊脂糖作為支架建立腫瘤相關(guān)巨噬細(xì)胞與肝癌細(xì)胞的三維立體共培養(yǎng)體系,通過(guò)i TRAQ標(biāo)記為基礎(chǔ)的定量蛋白質(zhì)組學(xué)技術(shù)對(duì)三維培養(yǎng)體系中的分泌蛋白進(jìn)行比較分析和篩選,并應(yīng)用Western-Blot對(duì)部分差異蛋白的鑒定結(jié)果進(jìn)行驗(yàn)證。結(jié)果顯示:(1)不同三維立體培養(yǎng)體系中的條件培養(yǎng)基經(jīng)i TRAQ標(biāo)記、質(zhì)譜分析及數(shù)據(jù)庫(kù)搜索,一共鑒定到了26677條肽段及1640個(gè)蛋白(99%可信度)。將鑒定到的蛋白按比值1.3定義為上調(diào),比值0.7定義為下調(diào)的準(zhǔn)則進(jìn)行篩選,發(fā)現(xiàn)在與M2比較時(shí),有333個(gè)分泌蛋白差異性表達(dá)在M2+SMMC7721共培養(yǎng)體系;在與SMMC7721比較時(shí),有357個(gè)分泌蛋白差異性表達(dá)在M2+SMMC7721共培養(yǎng)體系;同時(shí)與M2、SMMC7721單一立體培養(yǎng)體系比較時(shí),有159個(gè)分泌蛋白差異性表達(dá)在M2+SMMC7721共培養(yǎng)體系中,其中上調(diào)的差異性分泌蛋白為63個(gè),下調(diào)的為96個(gè)。(2)Western-Blot的結(jié)果顯示IL-8、IL-1β、HB-EGF和IGFBP3的變化趨勢(shì)與質(zhì)譜結(jié)果完全一致,而MMP3與CXCL2,雖發(fā)現(xiàn)其在M2CM中的表達(dá)高于SMMC7721CM與質(zhì)譜結(jié)果相反,但兩者均在共培養(yǎng)條件培養(yǎng)基中升高,該趨勢(shì)與質(zhì)譜結(jié)果一致。第三部分差異分泌蛋白CXCL2對(duì)肝癌細(xì)胞轉(zhuǎn)移功能的影響及機(jī)制研究CXCL2在本研究中被發(fā)現(xiàn)是共培養(yǎng)條件培養(yǎng)基里明顯上升的差異蛋白,且在M2CM中的表達(dá)高于SMMC7721CM,說(shuō)明其主要源于M2。其對(duì)肝癌轉(zhuǎn)移能力的影響及作用機(jī)制未明。本部分通過(guò)采用不同濃度的外源性重組CXCL2蛋白刺激肝癌細(xì)胞,以觀察其對(duì)肝癌細(xì)胞遷移、侵襲、粘附等生物學(xué)行為的影響,并通過(guò)信號(hào)通路芯片及通路抑制劑等方法探索其可能的作用機(jī)制。結(jié)果顯示:(1)CXCL2在10例肝癌患者肝癌組織中的表達(dá)比相應(yīng)癌旁組織明顯增高;(2)不同濃度CXCL2刺激均可顯著增強(qiáng)肝癌細(xì)胞的遷移能力,1ng/m L刺激組的肝癌細(xì)胞侵襲能力明顯增加,10ng/m L及100ng/m L CXCL2刺激組的肝癌細(xì)胞粘附能力顯著降低;(3)中和共培養(yǎng)條件培養(yǎng)基中的CXCL2后其促進(jìn)肝癌細(xì)胞遷移及侵襲能力的作用均明顯下降;(4)通過(guò)特異性抑制劑SB225002抑制肝癌細(xì)胞表達(dá)的CXCR2后,CXCL2對(duì)肝癌細(xì)胞遷移、侵襲能力的促進(jìn)作用及對(duì)其粘附能力的抑制作用均消失,說(shuō)明CXCL2通過(guò)與其受體CXCR2相結(jié)合發(fā)揮作用;(5)CXCL2過(guò)表達(dá)后,通過(guò)信號(hào)通路芯片檢測(cè)發(fā)現(xiàn)肝癌細(xì)胞中HSP27、PRAS40、bad,Chk1、Ik Bα(Ser32/36)的磷酸化水平均顯著上調(diào),Ik Bα(Total)也明顯升高;Western blot的結(jié)果證實(shí)過(guò)表達(dá)CXCL2后肝癌細(xì)胞中p-Ik Bα的磷酸化水平明顯增高,說(shuō)明NF-k B通路在CXCL2過(guò)表達(dá)的肝癌細(xì)胞中被激活;(6)進(jìn)一步通過(guò)NF-k B特異性抑制劑BAY117082阻斷肝癌細(xì)胞的NF-k B通路,發(fā)現(xiàn)CXCL2促進(jìn)肝癌細(xì)胞細(xì)胞遷移及侵襲的能力減弱。上述結(jié)果表明腫瘤相關(guān)巨噬細(xì)胞源性的CXCL2主要通過(guò)激活NF-k B信號(hào)通路促進(jìn)肝癌細(xì)胞的遷移及侵襲。結(jié)論1.在體外成功誘導(dǎo)獲得具有典型表型特征的不同活化狀態(tài)巨噬細(xì)胞M1和M2;并發(fā)現(xiàn)M2條件培養(yǎng)基可促進(jìn)肝癌細(xì)胞增殖、遷移及侵襲并發(fā)生EMT。2.成功應(yīng)用低熔點(diǎn)瓊脂糖建立肝癌細(xì)胞及M2型腫瘤相關(guān)巨噬細(xì)胞的三維立體共培養(yǎng)體系,通過(guò)i TRAQ標(biāo)記的定量蛋白質(zhì)組學(xué)技術(shù)建立了不同三維立體培養(yǎng)體系中的分泌蛋白表達(dá)譜,并從共培養(yǎng)體系中鑒定到了159個(gè)差異分泌蛋白。3.CXCL2是共培養(yǎng)體系中顯著升高的蛋白,可促進(jìn)肝癌細(xì)胞的遷移、侵襲能力,并抑制其粘附能力;其作用的發(fā)揮是通過(guò)與肝癌細(xì)胞表達(dá)的CXCR2受體結(jié)合,從而激活NF-k B信號(hào)通路進(jìn)行。
[Abstract]:The first part is the induction of tumor related macrophages, identification and its effect on hepatoma cells. Liver cancer is a classic inflammatory tumor. A large number of tumor related macrophages are infiltrated by a large number of tumor related macrophages in the microring of the hepatoma cells, and M2 type tumor related macrophages are the main factors. This part of the study is aimed at obtaining different activation states through in vitro induction. Macrophage and its effect on the biological behavior of hepatoma cells. First of all, we induced undifferentiated macrophages (UM0) through human mononuclear cells THP-1 via phorbol ester, and then induced by lipopolysaccharide, interleukin 4 and 13 to obtain the classic activated macrophage M1 and selectively activated macrophage M2; and then through morphology, fluoro The phenotypes were identified by light quantitative PCR and ELISA. Finally, the effects of conditioned medium (CM) on the biological behavior of hepatoma cells were investigated from the proliferation, migration and invasion experiments, and the changes in the epithelial mesenchymal transition (EMT) markers in the hepatoma cells were detected. The results showed: (1) M, compared with UM0, M. 1 long spindle shape or polygon, the expression of TNF- alpha and CCL3 m RNA and the secretion of IL-12 were significantly increased; M2 pseudo foot was elongated and messy and cluttered. The expression of AMAC-1, CCL22 and Arg-1 m RNA and IL-10 secretion were all increased significantly. The results of this detection were consistent with the results reported in the literature, indicating that different activation states have been successfully induced. (2) (2) biological function experiments showed that the proliferation, migration and invasion of HCC cells treated by M2CM were significantly enhanced, while the M1CM treated hepatoma cells had no significant changes, indicating that the macrophages of type M2 tumor related macrophages could significantly promote the malignant transformation of liver cancer cells; (3) the epithelial cells of liver cancer treated by M2CM The expression of the marker E-cadherin was significantly decreased, with the interstitial cell marker N-cadherin, Vimentin and alpha -SMA significantly increased, and the expression of all the hepatoma cells treated by M1CM had no significant changes, indicating that the only M2CM treated hepatoma cells had a three dimensional co culture of the tumor related macrophages and hepatoma cells of the EMT. second parts. Tumor cells secreting proteins in the nutrient system exist in a three-dimensional, complex and open microenvironment. Secretory proteins are an important bridge between the interaction of tumor cells and TAMs in this microenvironment. In this part, we use low melting point agarose as a support to establish tumor related macrophages and hepatoma cells. A three-dimensional co culture system was used to compare and screen the secretory proteins in the three-dimensional culture system by quantitative proteomics based on I TRAQ markers. The results of identification of partial differential proteins were verified by Western-Blot. The results showed that: (1) the conditioned medium in different three-dimensional culture systems was treated with I TRAQ Markers, mass spectrometry and database search, 26677 peptides and 1640 proteins (99% reliability) were identified. The identified protein was up to up by ratio 1.3, and the ratio 0.7 was defined as down regulation. It was found that when compared with M2, 333 secretory proteins were expressed differentially in the M2+SMMC7721 co culture system; and in SMMC772 1 in comparison, 357 secretory proteins were expressed differently in the M2+SMMC7721 co culture system; at the same time, when compared with M2, SMMC7721 single stereoscopic culture system, 159 secretory proteins were expressed differently in the M2+SMMC7721 co culture system, among which 63 differential secretory proteins were up-regulated and 96 were downregulated. (2) the results of Western-Blot showed IL-8, I The changes in L-1 beta, HB-EGF and IGFBP3 were in full agreement with the mass spectrum, while MMP3 and CXCL2 showed that their expression in M2CM was higher than that of SMMC7721CM and mass spectrometry, but both of them were increased in co culture medium, and the trend was in accordance with the mass spectrum. Third differential secretory protein CXCL2 has a shadow on the metastasis function of liver cancer cells. In this study, CXCL2 was found to be a distinct rising protein in the co culture medium, and the expression in M2CM was higher than that of SMMC7721CM. It was mainly attributed to the effect of M2. on the metastasis ability of liver cancer and the mechanism of action was not clear. This part was used to stimulate liver cancer by using different concentrations of exogenous recombinant CXCL2 protein. Cell, to observe the effects of its biological behavior on the migration, invasion and adhesion of hepatoma cells, and to explore its possible mechanism by means of signal pathway chip and pathway inhibitor. The results showed: (1) the expression of CXCL2 in the liver cancer tissues of 10 patients with liver cancer was significantly higher than that of the corresponding para cancerous tissues; (2) different concentrations of CXCL2 stimulation could be used. Significantly enhanced migration of hepatoma cells, the invasion ability of hepatoma cells in 1ng/m L stimulation group increased significantly, and the adhesion ability of hepatoma cells in 10ng/m L and 100ng/m L CXCL2 stimulation group decreased significantly; (3) after neutralizing CXCL2 in the co culture medium, the effect of promoting the migration and invasion of hepatoma cells decreased obviously; (4) through special treatment. After the inhibition of the heterosexual inhibitor SB225002 to inhibit the expression of CXCR2 in the hepatoma cells, CXCL2 plays a role in promoting the migration of hepatoma cells, the promoting effect of invasion and the inhibition effect on its adhesion ability. It shows that CXCL2 is combined with its receptor CXCR2. (5) after CXCL2 overexpression, the detection of HSP27, PRAS40 in hepatoma cells through the signal pathway chip is found. The phosphorylation level of bad, Chk1, Ik B alpha (Ser32/36) was significantly up-regulated, and Ik B a (Total) also increased obviously. The results of Western blot confirmed that the phosphorylation level of p-Ik alpha in hepatoma cells was significantly higher after the overexpression CXCL2, indicating that the pathway was activated in the hepatocellular carcinoma cells, which were overexpressed. (6) further through the specific inhibitors 82 blocking the NF-k B pathway of hepatoma cells, and finding that the ability of CXCL2 to promote the migration and invasion of hepatoma cells weakened. The results showed that the CXCL2 of tumor related macrophage derived mainly through the activation of NF-k B signaling pathway to promote the migration and invasion of hepatoma cells. Conclusion 1. in vitro induction to obtain the typical phenotypic characteristics of the difference. Activated state macrophages M1 and M2, and M2 conditioned medium can promote the proliferation, migration and invasion of hepatoma cells, and EMT.2. successfully applied low melting point agarose to establish a three-dimensional co culture system of hepatoma cells and M2 type tumor related macrophages, and established different three-dimensional erect by quantitative proteomics technology of I TRAQ markers. The secretory protein expression profiles in the body culture system and 159 differentially secreted protein.3.CXCL2 are identified in the co culture system as a significant increase in the co culture system, which can promote the migration, invasiveness and inhibition of the adhesion of hepatoma cells, and its role is to combine with the CXCR2 receptor expressed in hepatoma cells. Activation of the NF-k B signal pathway is performed.

【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.7

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 吳孟超;;肝癌外科治療的近期進(jìn)展[J];中國(guó)普外基礎(chǔ)與臨床雜志;2006年02期

2 張志敏;;肝癌會(huì)遺傳嗎?[J];肝博士;2006年03期

3 徐連根;周晶;雷偉;沈志祥;;瘦素及其受體在肝癌組織中的表達(dá)及意義[J];胃腸病學(xué)和肝病學(xué)雜志;2006年05期

4 魏麗珍;李勝棉;張楠;溫登瑰;周燁;王士杰;;688例肝癌患者發(fā)病特點(diǎn)及診療狀況分析[J];肝臟;2010年02期

5 ;我國(guó)科研人員發(fā)現(xiàn)促進(jìn)肝癌生長(zhǎng)和轉(zhuǎn)移的相關(guān)基因[J];臨床薈萃;2010年10期

6 吳生;;盤點(diǎn)誘發(fā)肝癌的7大元兇[J];肝博士;2012年01期

7 鄧慶華;一家四例肝癌報(bào)道[J];中國(guó)廠礦醫(yī)學(xué);1995年06期

8 王定珠;;觥籌交錯(cuò) 過(guò)度疲勞 肝癌乘機(jī)而入[J];健康博覽;2005年11期

9 笑山,吳蓉蓉;喝咖啡可以減少肝癌發(fā)生率[J];健康之路;2005年04期

10 ;遠(yuǎn)離肝癌 預(yù)防在先[J];農(nóng)民文摘;2006年02期

相關(guān)會(huì)議論文 前10條

1 俞順章;穆麗娜;;飲水中藻類毒素與肝癌關(guān)系和控制的研究(摘要)[A];全國(guó)腫瘤流行病學(xué)和腫瘤病因?qū)W學(xué)術(shù)會(huì)議論文集[C];2007年

2 楊秉輝;;肝癌預(yù)防的新話題-非酒精性脂肪性肝病與肝癌[A];第十二屆全國(guó)肝癌學(xué)術(shù)會(huì)議論文匯編[C];2009年

3 丁光偉;楊玉秀;徐秋霞;徐存拴;;肝癌多基因表達(dá)異常初步研究[A];第五屆全國(guó)肝臟疾病臨床暨中華肝臟病雜志成立十周年學(xué)術(shù)會(huì)議論文匯編[C];2006年

4 吳孟超;;肝癌外科治療的近期進(jìn)展[A];第三屆全國(guó)重型肝病及人工肝血液凈化學(xué)術(shù)年會(huì)論文集（上冊(cè)）[C];2007年

5 劉耕陶;孫華北;方亞南;;肝癌發(fā)生的化學(xué)預(yù)防[A];第十次中國(guó)生物物理學(xué)術(shù)大會(huì)論文摘要集[C];2006年

6 阮秀花;田蔥;張效本;張喜梅;;肝癌患者乙型肝炎病毒感染血清流行病學(xué)分析[A];第五屆全國(guó)肝臟疾病臨床暨中華肝臟病雜志成立十周年學(xué)術(shù)會(huì)議論文匯編[C];2006年

7 阮秀花;田蔥;張效本;張喜梅;;肝癌患者乙型肝炎病毒感染血清流行病學(xué)分析[A];第五屆全國(guó)肝臟疾病臨床暨中華肝臟病雜志成立十周年學(xué)術(shù)會(huì)議論文匯編[C];2006年

8 呂巖;韓學(xué)鋒;;肝癌患者需求的評(píng)估[A];中華醫(yī)學(xué)會(huì)醫(yī)學(xué)工程學(xué)分會(huì)第八次學(xué)術(shù)年會(huì)暨《醫(yī)療設(shè)備信息》創(chuàng)刊20周年慶祝會(huì)論文集[C];2006年

9 孫華;魏懷玲;劉耕陶;;雙環(huán)醇對(duì)化學(xué)毒物促發(fā)肝癌的防治作用及作用機(jī)制研究[A];2009醫(yī)學(xué)前沿論壇暨第十一屆全國(guó)腫瘤藥理與化療學(xué)術(shù)會(huì)議論文集[C];2009年

10 謝靜媛;戴煒;涂愛民;趙文高;符花;胡應(yīng)龍;;深圳地區(qū)53例肝癌發(fā)生的相關(guān)因素探討[A];全國(guó)第2屆中西醫(yī)結(jié)合傳染病學(xué)術(shù)會(huì)議暨國(guó)家中醫(yī)藥管理局第1屆傳染病協(xié)作組會(huì)議論文匯編[C];2008年

相關(guān)重要報(bào)紙文章 前10條

1 湖北宜昌 胡獻(xiàn)國(guó);吸煙與肝癌[N];上海中醫(yī)藥報(bào);2013年

2 四川成都 孫清廉;遠(yuǎn)離肝癌 謹(jǐn)防四大危險(xiǎn)因素[N];上海中醫(yī)藥報(bào);2013年

3 特約撰稿 北京朝陽(yáng)醫(yī)院京西院區(qū)肝膽外科主任 孫文兵;警惕肝癌的過(guò)度治療[N];醫(yī)藥導(dǎo)報(bào);2007年

4 健康時(shí)報(bào)特約記者 孫理;三招不得肝癌[N];健康時(shí)報(bào);2008年

5 葉建平;專家提醒肝癌患者保護(hù)生命 重在“三早”防治[N];中國(guó)中醫(yī)藥報(bào);2008年

6 通訊員 韋夏 本報(bào)記者 鄧宏鷹 鐘少鴻;不良飲食習(xí)慣易誘發(fā)肝癌[N];中國(guó)食品報(bào);2009年

7 記者 顧泳;我國(guó)肝癌患者占全球55%[N];解放日?qǐng)?bào);2010年

8 解放軍302醫(yī)院 劉士敬;四成肝癌酒精泡出來(lái)的[N];健康時(shí)報(bào);2010年

9 記者 顏維琦 曹繼軍;我科學(xué)家揭示肝癌發(fā)生早期分子機(jī)制[N];光明日?qǐng)?bào);2012年

10 記者 胡德榮;肝癌發(fā)生早期階段分子機(jī)制被揭示[N];健康報(bào);2012年

相關(guān)博士學(xué)位論文 前10條

1 陳放;TOPK蛋白和肝癌發(fā)生以及相關(guān)調(diào)控機(jī)制的研究[D];復(fù)旦大學(xué);2011年

2 張怡安;中樞神經(jīng)特異性RNA結(jié)合蛋白NOVA1在肝癌中的表達(dá)及其促進(jìn)腫瘤發(fā)展的潛在分子機(jī)制探究[D];復(fù)旦大學(xué);2014年

3 殷杰;E3泛素連接酶Prp19對(duì)肝癌侵襲的影響及其機(jī)制研究[D];復(fù)旦大學(xué);2014年

4 周宇紅;分化抑制因子ID1在調(diào)控化療誘導(dǎo)的肝癌細(xì)胞“干性”改變中的作用研究[D];復(fù)旦大學(xué);2014年

5 王盛;FOXA1基因變異和調(diào)控異常促，

本文編號(hào)：1819839