本文選題：骨髓間質(zhì)干細(xì)胞 + 胃癌間質(zhì)干細(xì)胞　； 參考：《江蘇大學(xué)》2015年博士論文

【摘要】：目的：腫瘤微環(huán)境在腫瘤發(fā)生、發(fā)展中發(fā)揮著重要的調(diào)控作用,非編碼微小RNA(microRNAs/miRNAs)參與介導(dǎo)微環(huán)境對腫瘤細(xì)胞的作用及微環(huán)境重塑。癌相關(guān)間質(zhì)干細(xì)胞(mesenchymal stem cell,MSC)作為一種重要的腫瘤微環(huán)境細(xì)胞被相繼分離與鑒定,但是目前有關(guān)癌相關(guān)MSC的來源及其異常表達(dá)的miRNAs在其活化過程中的作用與分子機(jī)制尚不清楚。骨髓MSC是微環(huán)境細(xì)胞的重要來源,研究發(fā)現(xiàn)胃癌MSC在表面標(biāo)記和多向分化潛能與骨髓MSC相似,但較骨髓MSC具有較強(qiáng)的增殖能力和促胃癌作用。本研究旨在建立胃癌細(xì)胞誘導(dǎo)骨髓MSC轉(zhuǎn)分化模型,篩選胃癌MSC異常表達(dá)的miRNAs并以此為切入點(diǎn),明確調(diào)控骨髓MSC向胃癌MSC轉(zhuǎn)分化的關(guān)鍵miRNAs,闡明miRNAs再編程骨髓MSC的分子機(jī)制,方法：分離培養(yǎng)胃癌MSC、癌旁MSC、人骨髓MSC和615小鼠骨髓MSC。采用胃癌細(xì)胞上清處理骨髓MSC,建立體外胃癌細(xì)胞誘導(dǎo)骨髓MSC轉(zhuǎn)分化模型。采用Agilent human miRNA芯片篩選檢測胃癌MSC和癌旁MSC差異表達(dá)miRNAs、定量PCR檢測驗(yàn)證、結(jié)合體外胃癌細(xì)胞上清誘導(dǎo)前后骨髓MSC中miRNAs的檢測分析,確定胃癌MSC差異表達(dá)的miRNAs。模擬胃癌MSC中miRNAs低表達(dá),采用miRNAs inhibitor轉(zhuǎn)染骨髓MSC抑制miRNA的表達(dá)。采用免疫熒光染色檢測α平滑肌肌動蛋白(a-smooth muscle actin,α-SMA)和成纖維細(xì)胞活化蛋白(Fibroblast Activation Protein, FAP)的表達(dá),luminex檢測細(xì)胞培養(yǎng)上清中細(xì)胞因子的分泌量進(jìn)行表型分析。收集骨髓MSC的培養(yǎng)上清并作用胃癌細(xì)胞,細(xì)胞克隆形成試驗(yàn)、Transwell細(xì)胞遷移和侵襲試驗(yàn)、體內(nèi)裸鼠致瘤分析轉(zhuǎn)染后骨髓MSC對胃癌細(xì)胞的功能。采用miRNAs mimics轉(zhuǎn)染胃癌MSC過表達(dá)miRNAs,檢測轉(zhuǎn)染前后胃癌MSC的表型和功能,分析miRNAs對胃癌MSC逆分化作用。采用靶基因預(yù)測、3’UTR報(bào)告基因載體構(gòu)建和western blot檢測并確定miRNAs調(diào)控的靶基因。在此基礎(chǔ)之上,干預(yù)胃癌細(xì)胞上清誘導(dǎo)骨髓MSC、miRNAs轉(zhuǎn)染骨髓MSC轉(zhuǎn)分化模型和胃癌MSC中靶基因,檢測處理前后MSC的表型、功能及相關(guān)信號通路活化情況,分析靶基因在轉(zhuǎn)分化過程中的作用。結(jié)果：miRNAs芯片篩選檢測胃癌MSC中差異倍數(shù)大于2且P0.01的miRNA共有61個,其中高表達(dá)miRNAs有38個,低表達(dá)miRNAs有23個。隨機(jī)選擇部分表達(dá)差異顯著且與腫瘤調(diào)控密切相關(guān)的miRNAs,進(jìn)一步在5對胃癌MSC和癌旁MSC進(jìn)行檢測驗(yàn)證,結(jié)果顯示miR-99a-5p在胃癌MSC表達(dá)顯著低于癌旁MSC。體外成功建立胃癌細(xì)胞上清誘導(dǎo)骨髓MSC向胃癌MSC樣細(xì)胞轉(zhuǎn)分化模型,定量PCR檢測發(fā)現(xiàn)誘導(dǎo)后骨髓MSC中miR-99a-5p顯著低表達(dá)。抑制骨髓MSC中miR-99a-5p的表達(dá),可顯著促進(jìn)骨髓MSC中a-SMA和FAP的表達(dá),luminex檢測顯示轉(zhuǎn)染后骨髓MSC細(xì)胞培養(yǎng)上清中IL-6、IL-8、MCP-1、RANTES等細(xì)胞因子含量增加。將轉(zhuǎn)染后骨髓MSC細(xì)胞上清作用胃癌細(xì)胞,可顯著促進(jìn)胃癌細(xì)胞克隆形成、遷移、侵襲和裸鼠體內(nèi)致瘤力。過表達(dá)miR-99a-5p可顯著阻斷胃癌細(xì)胞上清對骨髓MSC的誘導(dǎo)作用。將胃癌MSC中miR-99a-5p高表達(dá),可顯著抑制其a-SMA和FAP的表達(dá)、細(xì)胞因子的分泌、阻斷其對胃癌細(xì)胞增殖、遷移和侵襲的促進(jìn)作用。靶基因預(yù)測和報(bào)告基因的檢測分析確定FGFR3和IGF1R為miR-99a-5p調(diào)控的靶點(diǎn)。Western blot檢測結(jié)果顯示FGFR3和IGF1R的蛋白及其下游信號通路分子AKT和ERK在胃癌細(xì)胞上清誘導(dǎo)后的骨髓MSC、低表達(dá)miR-99a-5p的骨髓MSC和胃癌MSC中表達(dá)顯著增加和活化。采用FGFR3抑制劑AZD4547口IGF1R抑制劑OSI-906分別預(yù)處理骨髓MSC,檢測結(jié)果顯示AZD4547可抑制胃癌細(xì)胞上清或miRNAs inhibitor對骨髓MSC中a-SMA和FAP的表達(dá)和下游信號通路活化的促進(jìn)作用,阻斷作用后的骨髓MSC對胃癌細(xì)胞的促進(jìn)作用。AZD4547處理胃癌MSC可直接抑制其a-SMA和FAP的表達(dá),阻斷其對胃癌細(xì)胞遷移的促進(jìn)作用。OSI-906的預(yù)處理不影響胃癌細(xì)胞上清和低表達(dá)的miR-99a-5p對骨髓MSC的轉(zhuǎn)分化作用。結(jié)論：體外成功建立胃癌細(xì)胞上清誘導(dǎo)骨髓MSC向胃癌MSC轉(zhuǎn)分化模型,確定胃癌MSC低表達(dá)miR-99a-5p。miR-99a-5p低表達(dá)可促進(jìn)骨髓MSC向胃癌MSC轉(zhuǎn)分化。高表達(dá)miR-99a-5p可促進(jìn)胃癌MSC向骨髓MSC樣細(xì)胞逆分化。FGFR3是介導(dǎo)胃癌細(xì)胞上清和miR-99a-5p再編程骨髓MSC的關(guān)鍵分子。本研究工作的完成將闡明骨髓MSC向胃癌MSC轉(zhuǎn)分化作用及分子機(jī)制,為胃癌微環(huán)境細(xì)胞胃癌MSC的來源與重塑提供新的試驗(yàn)依據(jù),為胃癌的治療提供靶向微環(huán)境新的靶點(diǎn)和治療策略,具有重要的科學(xué)研究意義和臨床應(yīng)用前景。
[Abstract]:Objective: tumor microenvironment plays an important role in the development of tumor, and it plays an important role in the development of tumor cells. Non coding micro RNA (microRNAs/miRNAs) is involved in mediating the effect of microenvironment on tumor cells and the remodeling of microenvironment. Cancer related mesenchymal stem cells (mesenchymal stem cell, MSC) are isolated and identified as an important tumor microenvironment cell. However, the function and molecular mechanism of the origin of cancer related MSC and its abnormal expression of miRNAs in its activation process is not clear. Bone marrow MSC is an important source of microenvironmental cells. The study found that the surface marker and multidirectional differentiation potential of gastric cancer MSC are similar to that of bone marrow MSC, but it has stronger proliferation and gastric cancer than bone marrow MSC. The purpose of this study is to establish a MSC transdifferentiation model of bone marrow induced by gastric cancer cells, to screen the miRNAs of abnormal expression of MSC in gastric cancer and take this as a breakthrough point, to clearly regulate the key miRNAs to MSC transdifferentiation of bone marrow MSC to gastric cancer, and to clarify the molecular mechanism of miRNAs reprogramming of bone marrow MSC, methods: the separation and cultivation of gastric cancer MSC, paracancerous MSC, human bone marrow MSC and 615 mice Bone marrow MSC. was treated with gastric cancer cell supernatant to treat bone marrow MSC, and to establish MSC transdifferentiation model of bone marrow induced by gastric cancer cells in vitro. Agilent human miRNA chip was used to screen and detect the differential expression of gastric cancer MSC and paracancerous MSC, and the quantitative PCR test was verified. SC differentially expressed miRNAs. simulated low expression of miRNAs in gastric cancer MSC, and miRNAs inhibitor was used to transfect bone marrow MSC to inhibit the expression of miRNA. Immunofluorescence staining was used to detect the expression of alpha smooth muscle actin (a-Smooth muscle actin, alpha -SMA) and fibroblast activation protein. The secretory quantity of cytokine in the supernatant was analyzed by phenotypic analysis. The culture supernatant of bone marrow MSC was collected and the gastric cancer cell, cell clone formation test, Transwell cell migration and invasion test, the function of bone marrow MSC on the gastric cancer cells after tumor analysis in nude mice. MiRNAs mimics transfection of gastric cancer MSC over expression miRNAs was used to detect the transfection. The phenotype and function of gastric cancer MSC were analyzed by miRNAs, and the target gene was predicted by target gene, 3 'UTR reporter gene vector construction and Western blot detection and miRNAs regulation target genes. On this basis, intervention of bone marrow MSC in gastric carcinoma cell supernatant, miRNAs transfection of bone marrow MSC transdifferentiation model and target base of gastric carcinoma MSC on this basis. Because of the phenotype, function and activation of the related signal pathway of MSC before and after detection, the role of target gene in the process of transdifferentiation was analyzed. Results: miRNAs chip screening detection of gastric cancer MSC was more than 2 and P0.01 miRNA had 61, of which there were 38 high expression miRNAs, 23 low expression miRNAs, and random selection of partial expression differences. MiRNAs, which was closely related to tumor regulation, was further tested in 5 gastric cancer MSC and paracancerous MSC. The results showed that the expression of miR-99a-5p in gastric cancer was significantly lower than that of MSC. in the side of the carcinoma, and the transdifferentiation of the bone marrow MSC to the MSC like cells was induced by the supernatant of gastric cancer cells, and the quantitative PCR detection was found in the miR-9 of the bone marrow MSC. The expression of miR-99a-5p in bone marrow MSC could significantly promote the expression of a-SMA and FAP in bone marrow MSC. Luminex detection showed that the content of IL-6, IL-8, MCP-1, RANTES and other cytokines increased in the culture supernatant of bone marrow MSC cells after transfection. The gastric cancer cells could be significantly promoted by the clearance of gastric cancer cells on the transfected bone marrow cells after transfection. Formation, migration, invasion and tumorigenicity of nude mice. Overexpression of miR-99a-5p can significantly block the induction of MSC in gastric cancer cell supernatant. High expression of miR-99a-5p in gastric cancer MSC can significantly inhibit the expression of a-SMA and FAP, the secretion of cytokines, and block the promotion of cell proliferation, migration and invasion of gastric cancer. The detection and analysis of the detected and reported genes identified the target of FGFR3 and IGF1R as the target of miR-99a-5p regulation and.Western blot detection results showed that the protein of FGFR3 and IGF1R and the downstream signal pathway molecules AKT and ERK were expressed in the bone marrow MSC after the induction of the supernatant of gastric cancer cells, and the expression of the low expression miR-99a-5p bone marrow MSC and gastric cancer was significantly increased and activated. 3 inhibitor AZD4547 IGF1R inhibitor OSI-906 pretreated bone marrow MSC respectively. The results showed that AZD4547 could inhibit the expression of a-SMA and FAP in the bone marrow MSC and the activation of the downstream signal pathway in the bone marrow MSC, and the promoting effect of the bone marrow MSC on the gastric cancer cells after the blocking action.AZD4547 treated gastric cancer Direct inhibition of the expression of a-SMA and FAP, blocking the promotion of gastric cancer cell migration,.OSI-906 preconditioning does not affect the transdifferentiation of gastric cancer cell supernatant and low expression miR-99a-5p on bone marrow MSC. Conclusion: in vitro successfully established gastric carcinoma cell supernatant induced MSC transdifferentiation model of bone marrow MSC to gastric cancer, and determined the low expression of miR in gastric cancer. The low expression of -99a-5p.miR-99a-5p can promote the MSC transdifferentiation of bone marrow MSC to gastric cancer. High expression of miR-99a-5p can promote the reverse differentiation of MSC to bone marrow MSC like cells, which is the key molecule to mediate gastric cancer cell supernatant and miR-99a-5p reprogramming bone marrow MSC. The completion of this work will clarify the MSC transdifferentiation and molecular mechanism of bone marrow MSC to gastric cancer. It provides new experimental basis for the origin and reshaping of gastric cancer microenvironment cell gastric cancer MSC, and provides new target and treatment strategy for the target microenvironment for the treatment of gastric cancer. It has important scientific research significance and clinical application prospect.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R735.2

【相似文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 顧紅兵;miR-99a-5p調(diào)控骨髓MSC向胃癌MSC轉(zhuǎn)分化作用及機(jī)制[D];江蘇大學(xué);2015年

，

本文編號：2108802