【摘要】：目的:外泌體是多泡小體產(chǎn)生并與細(xì)胞膜融合,之后釋放到細(xì)胞外的小囊泡,直徑在30-150nm,其含有micro RNA,可以作為細(xì)胞間信息交流的載體。本實(shí)驗(yàn)的目的在于研究CAF分泌的外泌體對(duì)于抗雄治療的CD LNCAP細(xì)胞系增殖及凋亡的影響,并驗(yàn)證CAF來源的外泌體中是否含有與增殖凋亡相關(guān)的mi RNAs。方法:運(yùn)用超速離心的方法提取CAF細(xì)胞中的外泌體。應(yīng)用透射電鏡,納米顆粒追蹤分析及蛋白印跡法檢測(cè)外泌體特異性蛋白CD63鑒定外泌體。運(yùn)用MTT,細(xì)胞周期檢測(cè),細(xì)胞計(jì)數(shù)及流式細(xì)胞學(xué)檢測(cè)細(xì)胞凋亡分析CAF細(xì)胞外泌體對(duì)CD LNCAP細(xì)胞增殖凋亡的影響。從數(shù)據(jù)庫(kù)及文獻(xiàn)庫(kù)篩選出mi RNA,并運(yùn)用q PCR及PCR技術(shù)驗(yàn)證其在CAF細(xì)胞和NF細(xì)胞及其外泌體中該mi RNA的情況。結(jié)果:超速離心提取的外泌體電鏡下可見圓盤狀,杯口狀外泌體。從納米顆粒追蹤分析的結(jié)果可以看出外泌體直徑大小約100nm左右,大部分集中在30-150nm之間。從蛋白印跡法檢測(cè)外泌體特異蛋白CD 63可以看到外泌體的CD 63拖尾影。由MTT、細(xì)胞周期及細(xì)胞計(jì)數(shù)結(jié)果可以看出,經(jīng)過CAF細(xì)胞條件培養(yǎng)基及外泌體處理過的CD LNCAP細(xì)胞增殖更加明顯(P0.05),而去除外泌體之后的上清液及對(duì)照組的結(jié)果陰性。由流式細(xì)胞學(xué)檢測(cè)結(jié)果可以看出,CD LNCAP細(xì)胞經(jīng)過CAF細(xì)胞條件培養(yǎng)基及外泌體處理過的CD LNCAP細(xì)胞凋亡更少(P0.05)。通過對(duì)數(shù)據(jù)庫(kù)及文獻(xiàn)庫(kù)的篩查得到mi R-21,通過q PCR及PCR技術(shù)初步確定,CAF細(xì)胞中mi R-21表達(dá)量明顯高于NF細(xì)胞中mi R-21,同時(shí)在CAF細(xì)胞外泌體中也存在miR-21。結(jié)論:CAF細(xì)胞分泌一定量的外泌體,直徑在30-150nm之間,其表面表達(dá)CD 63分子。CAF細(xì)胞來源的外泌體能夠促進(jìn)去雄前列腺癌細(xì)胞系CD LNCAP的增殖并抑制其凋亡。CAF細(xì)胞來源的外泌體內(nèi)含有促進(jìn)前列腺癌生長(zhǎng)的mi R-21,并且可能上述增殖由此介導(dǎo)。
[Abstract]:Objective: exocrine bodies are multivesicular bodies produced and fused with cell membrane, and then released into extracellular vesicles, which have a diameter of 30-150 nm. They contain micro RNAs and can be used as carriers for intercellular information exchange. The purpose of this study was to investigate the effects of exosomes secreted by CAF on the proliferation and apoptosis of CD LNCAP cell lines after anti-androgenic therapy, and to verify whether the exocrine derived from CAF contained mi RNAs related to proliferation and apoptosis. Methods: exocrine bodies were extracted from CAF cells by ultracentrifugation. Transmissive electron microscopy (TEM), nanoparticles tracing analysis and Western blotting were used to detect the exocrine specific protein CD63. The effects of exosome of CAF on the proliferation and apoptosis of CD LNCAP cells were analyzed by MTT, cell cycle detection, cell count and flow cytometry. The miRNAs were screened from the database and the library, and their miRNAs in CAF cells, NF cells and their exosomes were confirmed by Q PCR and PCR techniques. Results: the exocrine bodies extracted by ultracentrifugation were disc-shaped and cup-shaped under electron microscope. The size of the exocrine is about 100nm, most of which are between 30-150nm and 30-150nm. CD63 tail shadow of exocrine specific protein CD63 can be seen by Western blotting. From the results of MTT, cell cycle and cell count, the proliferation of CD LNCAP cells treated with CAF cell conditioned medium and exocrine was more obvious (P0.05), but the results of supernatant and control group were negative. The results of flow cytometry showed that the apoptosis of CD LNCAP cells treated with CAF cell conditioned medium and exocrine was less (P0.05). The expression of miR-21 in caf cells was significantly higher than that in NF cells by Q PCR and PCR techniques, and miR-21 was also found in exosomes of CAF cells. ConclusionThe exocrine bodies secreted by the cell line were between the diameters of 30-150nm. The exocrine derived from CD63 molecule. Caf cells can promote the proliferation of androgenic prostate cancer cell line CD LNCAP and inhibit its apoptosis. The exocrine derived from caf cells contains mi R-21, which can promote the growth of prostate cancer. The proliferation is thus mediated.
【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)】：R737.25

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 高旭;;中國(guó)前列腺癌早期診斷專家共識(shí)[J];中華泌尿外科雜志;2015年08期

，

本文編號(hào)：2139430