本文選題：YAP + 間質(zhì)干細(xì)胞　； 參考：《江蘇大學(xué)》2017年碩士論文

【摘要】：目的:間質(zhì)干細(xì)胞(mesenchymal stem cell,MSC)作為腫瘤微環(huán)境中的重要組成成分調(diào)節(jié)著腫瘤的生物學(xué)行為。在過去的研究中我們成功從胃癌組織中分離得到胃癌間質(zhì)干細(xì)胞(gastric cancer-derived MSC,GC-MSC)并發(fā)現(xiàn)GC-MSC促進(jìn)胃癌的發(fā)展。但GC-MSC中YAP分子作用仍不清楚。本研究旨在闡明GC-MSC中YAP分子在胃癌發(fā)展過程中的作用。方法:Western blot檢測(cè)比較骨髓間質(zhì)干細(xì)胞(bone marrow mesenchymal stem cell,BM-MSC)與GC-MSC中YAP分子的表達(dá)差異。通過慢病毒YAP-ShRNA對(duì)GC-MSC中YAP分子進(jìn)行敲減,應(yīng)用qRT-PCR和Western blot檢測(cè)YAP的敲減效率。平板克隆實(shí)驗(yàn)、Western blot、免疫熒光實(shí)驗(yàn)、Transwell遷移實(shí)驗(yàn)和侵襲實(shí)驗(yàn)檢測(cè)敲減后的GC-MSC細(xì)胞增殖、遷移、侵襲、上皮間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition,EMT)以及干性能力的改變。平板克隆實(shí)驗(yàn),免疫熒光實(shí)驗(yàn)、流式細(xì)胞術(shù)、qRT-PCR和Western blot分別檢測(cè)Blank組SGC-7901細(xì)胞(Blank SGC-7901),Control組GC-MSC培養(yǎng)上清(conditioned medium,CM)處理的SGC-7901細(xì)胞[GC-MSC(Control)-CM SGC-7901]以及ShYAP組GC-MSC培養(yǎng)上清處理的SGC-7901細(xì)胞[GC-MSC(ShYAP)-CM SGC-7901]的增殖、凋亡、遷移、侵襲能力及EMT指標(biāo)的變化。qRT-PCR檢測(cè)3種不同培養(yǎng)上清處理的SGC-7901細(xì)胞中促血管形成相關(guān)基因IL-6、PDGF和VEGF的變化。采用血管形成試驗(yàn)和遷移實(shí)驗(yàn)檢測(cè)敲減YAP的GC-MSC培養(yǎng)上清處理SGC-7901細(xì)胞72h后SGC-7901上清促血管形成能力的改變。qRT-PCR和Western blot檢測(cè)三種不同處理的SGC-7901中β-catenin及其下游靶基因CD44和CyclinD的表達(dá)變化。分別將3種不同培養(yǎng)上清處理的SGC-7901細(xì)胞接種裸鼠,構(gòu)建裸鼠5致瘤模型,測(cè)定3組模型致瘤瘤體大小和重量。免疫組化和免疫熒光檢測(cè)瘤組織中Ki67、β-catenin、E-cadherin、Vimentin及CD31的表達(dá)。結(jié)果:與BM-MSC相比,YAP在GC-MSC中高表達(dá)。YAP敲減可抑制GC-MSC中增殖相關(guān)蛋白PCNA及Ki67的表達(dá),EMT相關(guān)蛋白N-cadherin、Vimentin的下調(diào)和E-cadherin的上調(diào),干性蛋白CD44、Sall4和Nanog的下調(diào)。同時(shí),GC-MSC增殖、遷移和侵襲能力也明顯下降。GC-MSC(ShYAP)-CM SGC-7901中PCNA及Ki67的表達(dá)降低,EMT相關(guān)蛋白N-cadherin、Vimentin的下調(diào)和E-cadherin的上調(diào),血管形成相關(guān)基因IL-8、PDGF和VEGF表達(dá)下調(diào),凋亡相關(guān)蛋白Bcl2和Bax沒有明顯改變。其增殖、遷移、侵襲能力及促血管形成能力也明顯下降。研究顯示YAP敲減的GC-MSC培養(yǎng)上清可以抑制SGC-7901中對(duì)胃癌發(fā)展有重要作用的β-catenin及其下游靶基因CD44和CyclinD的表達(dá)。裸鼠皮下致瘤實(shí)驗(yàn)表明,YAP敲減的GC-MSC培養(yǎng)上清處理的胃癌細(xì)胞體內(nèi)致瘤能力明顯下降。免疫組化、免疫熒光結(jié)果顯示GC-MSC(ShYAP)-CM SGC-7901形成的瘤組織中E-cadherin表達(dá)升高,Ki67、β-catenin、Vimentin及CD31表達(dá)明顯下降。結(jié)論:YAP敲減不僅可以抑制GC-MSC增殖、遷移、侵襲、EMT及干性分子的表達(dá),還能抑制體外胃癌細(xì)胞的增殖,遷移,侵襲及促血管形成能力,抑制體內(nèi)胃癌的發(fā)展。YAP敲減的GC-MSC培養(yǎng)上清可能通過調(diào)控胃癌細(xì)胞中β-catenin的表達(dá)來影響胃癌的發(fā)展。
[Abstract]:Aim: mesenchymal stem cells (MSCs), as an important component of tumor microenvironment, regulate the biological behavior of tumor. In previous studies, we successfully isolated gastric cancer mesenchymal stem cells from gastric cancer tissues and found that GC-MSC promoted the development of gastric cancer. However, the interaction of YAP molecules in GC-MSC is still unclear. The purpose of this study was to elucidate the role of YAP molecules in the development of gastric cancer. Methods the expression of YAP in bone marrow mesenchymal stem cells (BM-MSC) and GC-MSC was detected by Western blot. Yap molecules in GC-MSC were knocked down by lentivirus YAP-ShRNA, and the knockout efficiency of YAP was detected by qRT-PCR and Western blot. Western blot, Transwell migration assay and invasion assay were used to detect the proliferation, migration, invasion, epithelial-mesenchymal transitionof epithelial mesenchymal transition (EMTT) and the change of dry ability. The proliferation, apoptosis and migration of SGC-7901 cells treated with SGC-7901 cell line Blank SGC-7901 and SGC-7901 treated with GC-MSC culture supernatant were detected by plate cloning assay, immunofluorescence assay, flow cytometry qRT-PCR and Western blot, respectively, and the proliferation, apoptosis and migration of SGC-7901 cells treated with SGC-7901 and ShYAP GC-MSC culture supernatant were detected. The changes of invasion ability and EMT index. QRT-PCR was used to detect the changes of angiogenic genes IL-6, PDGF and VEGF in SGC-7901 cells treated with three different supernatants. Angiogenesis test and migration assay were used to detect the expression of 尾 -catenin and its downstream target genes CD44 and CyclinD in SGC-7901 cells treated with SC-MSC culture supernatant of knockdown Yap for 72 h. The expression of 尾 -catenin and its downstream target genes CD44 and CyclinD in SGC-7901 were detected by qRT-PCR and Western blot. SGC-7901 cells treated with three different culture supernatants were inoculated into nude mice, and the tumorigenic model of nude mice was established. The tumor size and weight of the three groups were measured. The expression of Ki67, 尾 -cateninine E-cadherinn and CD31 were detected by immunohistochemistry and immunofluorescence. Results: compared with BM-MSC, the expression of proliferation-associated protein PCNA and Ki67 in GC-MSC was inhibited by high expression of YAP. YAP knockout inhibited the down-regulation of N-cadherin and E-cadherin, and down-regulation of CD44-Sall4 and Nanog in GC-MSC. At the same time, the proliferation, migration and invasion ability of GC-MSC were also significantly decreased. The expression of PCNA and Ki67 in GC-MSCC ShYAP-CM SGC-7901 was significantly decreased, the expression of N-cadherin Vimentin and E-cadherin was down-regulated, and the expression of IL-8, PDGF and VEGF was down-regulated, while the apoptosis-related proteins Bcl2 and Bax were not significantly changed. Proliferation, migration, invasion and angiogenesis were also significantly decreased. The results showed that the supernatant of GC-MSC with YAP knockout could inhibit the expression of 尾 -catenin and its downstream target genes CD44 and CyclinD in SGC-7901. The results of subcutaneous tumorigenesis in nude mice showed that the tumorigenic ability of gastric cancer cells treated with the supernatant of GC-MSC by YAP knockout was significantly decreased. Immunohistochemistry and immunofluorescence showed that the expression of E-cadherin increased, 尾 -catenin Vimentin and CD31 decreased significantly in the tumor tissue formed by GC-MSC-ShYAP-CM SGC-7901. Conclusion WYAP knockout can not only inhibit the proliferation, migration, invasion of EMT and the expression of dry molecules in GC-MSC, but also inhibit the proliferation, migration, invasion and angiogenesis of gastric cancer cells in vitro. Inhibiting the development of gastric cancer in vivo. The culture supernatant of GC-MSC with YAP knockout may affect the development of gastric cancer by regulating the expression of 尾 -catenin in gastric cancer cells.
【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)：2023313