本文選題：exosome　切入點(diǎn)：miR-4669　出處：《江蘇大學(xué)》2017年碩士論文

【摘要】：目的:探討胃癌細(xì)胞旁分泌外泌體(exosome)促進(jìn)骨髓間質(zhì)干細(xì)胞(bone marrow mesenchymal stem cell,BMMSC)轉(zhuǎn)分化的重要作用和途徑,闡明其誘導(dǎo)BMMSC轉(zhuǎn)分化的分子機(jī)制,為胃癌微環(huán)境細(xì)胞重塑提供新的實(shí)驗(yàn)依據(jù)。方法:采用Nanosight檢測(cè)分析exosome直徑大小、數(shù)量并驗(yàn)證Rab27a-siRNA干擾組腫瘤細(xì)胞培養(yǎng)上清中exosome的量;透射電鏡檢測(cè)exosome形態(tài)和直徑大小;Western blot檢測(cè)exosome特異性標(biāo)記CD81、CD9蛋白的表達(dá)。免疫熒光檢測(cè)誘導(dǎo)前后BMMSC中α-SMA的表達(dá),Transwell遷移實(shí)驗(yàn)檢測(cè)胃癌細(xì)胞SGC-7901、HGC-27遷移能力的改變,定量PCR檢測(cè)比較誘導(dǎo)前后BMMSC中相關(guān)因子的表達(dá)和mi R-4669表達(dá)的差異。采用Rab27a-siRNA抑制exosome分泌,免疫熒光和Transwell遷移實(shí)驗(yàn)檢測(cè)Rab27a干擾的胃癌細(xì)胞上清誘導(dǎo)后BMMSC表型和功能的改變,評(píng)估exosome的誘導(dǎo)作用。運(yùn)用mi R-4669 micmis在胃癌細(xì)胞中上調(diào)mi R-4669的表達(dá),分析轉(zhuǎn)染mi R-4669 micmis的SGC-7901和HGC-27上清及exosome分別作用BMMSC后其表型、功能的變化,確定mi R-4669對(duì)BMMSC向胃癌間質(zhì)干細(xì)胞(gastric cancer tissue-derived MSC,GC-MSC)轉(zhuǎn)分化的調(diào)控作用。體外血管生成試驗(yàn)評(píng)價(jià)miR-4669過表達(dá)組BMMSC對(duì)血管形成能力的影響。定量PCR檢測(cè)轉(zhuǎn)染mi R-4669mimics的胃癌細(xì)胞上清或exosome誘導(dǎo)后BMMSC相關(guān)基因表達(dá)變化,分析胃癌細(xì)胞來源的exosome通過miR-4669重塑BMMSC向GC-MSC轉(zhuǎn)化的分子機(jī)制。結(jié)果:免疫熒光和Transwell遷移試驗(yàn)結(jié)果顯示,胃癌細(xì)胞來源的exosome可再現(xiàn)胃癌細(xì)胞上清的作用,誘導(dǎo)BMMSC使其獲得GC-MSC樣表型和功能,α-SMA表達(dá)顯著增強(qiáng),且誘導(dǎo)后BMMSC具有更強(qiáng)的促進(jìn)胃癌細(xì)胞遷移的能力。通過轉(zhuǎn)染Rab27a-siRNA抑制胃癌細(xì)胞exosome的分泌,與對(duì)照組相比,干擾組誘導(dǎo)后BMMSC的基質(zhì)細(xì)胞活化表型和促進(jìn)胃癌細(xì)胞遷移的能力均顯著減弱。qRT-PCR結(jié)果顯示,胃癌細(xì)胞來源exosome作用BMMSC后miR-4669表達(dá)水平較對(duì)照組顯著上調(diào)。與對(duì)照組相比,轉(zhuǎn)染mi R-4669 mimics的胃癌細(xì)胞上清和exosome誘導(dǎo)后的BMMSC顯示出增強(qiáng)的基質(zhì)細(xì)胞活化表型和促進(jìn)胃癌細(xì)胞遷移的能力,且轉(zhuǎn)染mi R-4669 mimics的胃癌細(xì)胞上清誘導(dǎo)后的BMMSC具有更強(qiáng)的促進(jìn)血管生成能力。轉(zhuǎn)染mi R-4669mimics的胃癌細(xì)胞上清或exosome誘導(dǎo)后BMMSC相關(guān)基因檢測(cè)結(jié)果顯示,miR-4669的靶基因RXRA以及pri-4669、pre-4669的表達(dá)并無顯著變化,而成熟的miR-4669表達(dá)上調(diào),且相關(guān)炎性因子CCL2、IL-6、IL-8表達(dá)上調(diào)。結(jié)論:胃癌細(xì)胞分泌的exosome是胃癌細(xì)胞培養(yǎng)上清中誘導(dǎo)BMMSC向GC-MSC轉(zhuǎn)化的重要組分,其富含的mi R-4669可被直接遞送至BMMSC中,進(jìn)而促使其獲得GC-MSC樣表型和功能,是介導(dǎo)胃癌細(xì)胞旁分泌作用BMMSC的關(guān)鍵信號(hào)分子。為MSC在胃癌微環(huán)境中的改造與重塑提供了新的機(jī)制。
[Abstract]:Objective: to investigate the important role and pathway of paracrine exosome-exosome (exosome) in promoting the transdifferentiation of bone marrow mesenchymal stem cells (BMSCs), bone marrow mesenchymal stem cell (BMMSCC), and to elucidate its molecular mechanism of inducing BMMSC transdifferentiation. Methods: the diameter and quantity of exosome were analyzed by Nanosight and the amount of exosome in the supernatant of tumor cell culture in Rab27a-siRNA interference group was verified. Transmission electron microscopy (TEM) was used to detect the morphology and diameter of exosome. Western blot was used to detect the expression of CD81G + CD9 protein specifically labeled by exosome. The expression of 偽 -SMA in BMMSC before and after induction was detected and the transwell migration assay was used to detect the migration ability of gastric cancer cell line SGC-7901HGC-27. Quantitative PCR was used to detect the expression of related factors and the expression of miR-4669 in BMMSC before and after induction. Rab27a-siRNA was used to inhibit the secretion of exosome, immunofluorescence and Transwell migration assay were used to detect the changes of BMMSC phenotype and function in the supernatant of gastric cancer cells induced by Rab27a interference. To evaluate the induction effect of exosome, the expression of mi R-4669 was up-regulated by mi R-4669 micmis in gastric cancer cells. The phenotypic and functional changes of the SGC-7901 and HGC-27 supernatants transfected with mi R-4669 micmis and the BMMSC treated with exosome were analyzed. To determine the regulatory effect of mi R-4669 on the transdifferentiation of BMMSC into gastric cancer cancer tissue-derived MSC. In vitro angiogenesis test was used to evaluate the effect of BMMSC on angiogenesis in miR-4669 overexpression group. Quantitative PCR was used to detect the supernatant of gastric cancer cells transfected with mi R-4669mimics. Or exosome induced changes in the expression of BMMSC related genes, To analyze the molecular mechanism of the transformation of exosome from gastric cancer cells to GC-MSC through miR-4669 remodeling of BMMSC. Results: the results of immunofluorescence and Transwell migration test showed that exosome derived from gastric cancer cells could reproduce the role of supernatant of gastric cancer cells. BMMSC was induced to obtain GC-MSC like phenotype and function, 偽 -SMA expression was significantly increased, and the induced BMMSC had a stronger ability to promote the migration of gastric cancer cells. Transfection of Rab27a-siRNA inhibited the secretion of exosome in gastric cancer cells, compared with the control group. The activation phenotype of stromal cells and the ability to promote the migration of gastric cancer cells in BMMSC induced by interference group were significantly decreased. The results of qRT-PCR showed that the expression of miR-4669 in gastric cancer cells derived from exosome was significantly up-regulated than that in the control group, compared with the control group. The supernatant of gastric cancer cells transfected with mi R-4669 mimics and BMMSC induced by exosome showed enhanced phenotype of stromal cell activation and enhanced migration of gastric cancer cells. The BMMSC induced by the supernatant of the gastric cancer cells transfected with mi R-4669 mimics has stronger ability of promoting angiogenesis. The detection of BMMSC related genes in the supernatant of gastric cancer cells transfected with mi R-4669mimics or induced by exosome showed that the target gene RXRA of miR-4669 and the surface of pri-4669 gene pre-4669 were detected. Da has not changed significantly. The expression of mature miR-4669 was up-regulated, and the related inflammatory factor CCL2, IL-6, IL-8 was up-regulated. Conclusion: exosome secreted by gastric cancer cells is an important component in inducing the transformation of BMMSC to GC-MSC in the supernatant of gastric cancer cell culture, and its rich mi R-4669 can be directly delivered to BMMSC. It is a key signal molecule that mediates paracrine action of gastric cancer cells to BMMSC and provides a new mechanism for the transformation and remodeling of MSC in gastric cancer microenvironment.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Shalini Vellasamy;Pratheep Sandrasaigaran;Sharmili Vidyadaran;Elizabeth George;Rajesh Ramasamy;;Isolation and characterisation of mesenchymal stem cells derived from human placenta tissue[J];World Journal of Stem Cells;2012年06期

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本文編號(hào)：1687516