CAF细胞外泌体影响CD LNCAP细胞增殖及凋亡
发布时间：2018-07-23 12:35
【摘要】：目的:外泌体是多泡小体产生并与细胞膜融合,之后释放到细胞外的小囊泡,直径在30-150nm,其含有micro RNA,可以作为细胞间信息交流的载体。本实验的目的在于研究CAF分泌的外泌体对于抗雄治疗的CD LNCAP细胞系增殖及凋亡的影响,并验证CAF来源的外泌体中是否含有与增殖凋亡相关的mi RNAs。方法:运用超速离心的方法提取CAF细胞中的外泌体。应用透射电镜,纳米颗粒追踪分析及蛋白印迹法检测外泌体特异性蛋白CD63鉴定外泌体。运用MTT,细胞周期检测,细胞计数及流式细胞学检测细胞凋亡分析CAF细胞外泌体对CD LNCAP细胞增殖凋亡的影响。从数据库及文献库筛选出mi RNA,并运用q PCR及PCR技术验证其在CAF细胞和NF细胞及其外泌体中该mi RNA的情况。结果:超速离心提取的外泌体电镜下可见圆盘状,杯口状外泌体。从纳米颗粒追踪分析的结果可以看出外泌体直径大小约100nm左右,大部分集中在30-150nm之间。从蛋白印迹法检测外泌体特异蛋白CD 63可以看到外泌体的CD 63拖尾影。由MTT、细胞周期及细胞计数结果可以看出,经过CAF细胞条件培养基及外泌体处理过的CD LNCAP细胞增殖更加明显(P0.05),而去除外泌体之后的上清液及对照组的结果阴性。由流式细胞学检测结果可以看出,CD LNCAP细胞经过CAF细胞条件培养基及外泌体处理过的CD LNCAP细胞凋亡更少(P0.05)。通过对数据库及文献库的筛查得到mi R-21,通过q PCR及PCR技术初步确定,CAF细胞中mi R-21表达量明显高于NF细胞中mi R-21,同时在CAF细胞外泌体中也存在miR-21。结论:CAF细胞分泌一定量的外泌体,直径在30-150nm之间,其表面表达CD 63分子。CAF细胞来源的外泌体能够促进去雄前列腺癌细胞系CD LNCAP的增殖并抑制其凋亡。CAF细胞来源的外泌体内含有促进前列腺癌生长的mi R-21,并且可能上述增殖由此介导。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.25
本文编号：2139430
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.25
【参考文献】
相关期刊论文 前1条
1 高旭;;中国前列腺癌早期诊断专家共识[J];中华泌尿外科杂志;2015年08期
，本文编号：2139430
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