癌源外泌体的制备及其对胃癌细胞STK15表达的影响
|
摘要
目的探讨胃癌来源外泌体对胃癌细胞的增殖作用及其对丝氨酸/苏氨酸激酶15(STK15)表达的影响。方法采用超速梯度离心法从胃癌MGC803细胞培养上清液中提取外泌体,通过透射电镜技术对其超微结构进行鉴定,然后用不同浓度的外泌体(0.05、0.10、0.20、0.50μg/μL)处理胃癌细胞,利用甲基偶氮唑蓝(MTT)法检测其对细胞增殖活性的影响。利用免疫细胞化学方法分析外泌体对胃癌细胞中心体扩增激酶STK15表达的影响。结果胃癌MGC803细胞来源的外泌体具有特征性的盘状结构,由双层膜构成,直径为50～130nm。外泌体具有促进胃癌细胞增殖活性的作用(P<0.05)。随着时间的延长增殖活性也增强,12h细胞增殖活性从0.213上升到0.534(F=44.21,P=0.000),24h细胞增殖活性从0.251上升到0.998(F=66.06,P=0.000),36h细胞增殖活性从0.273上升到1.057(F=435.37,P=0.000),48h细胞增殖活性从0.342上升到1.414(F=195.30,P=0.000),外泌体以剂量依赖性-时间依赖性促进胃癌细胞增殖,其可能通过负载抗原上调STK15表达来影响细胞增殖进程。结论胃癌来源外泌体可能通过调节细胞STK15表达促进胃癌细胞增殖。
Objective To discuss the effect of exosomes on proliferation of gastric cancer cells and expression of serine/threonine kinase 15(STK15).Methods Exosomes were extracted from the culture supernatant of MGC803 gastric cancer cells by ultracentrifugal gradient centrifugation,and their ultrastructure was identified by transmission electron microscopy.Gastric cancer cells were then treated with exosomes at different concentrations(0.05,0.10,0.20,0.50μg/μL)The effect of exosomes on the proliferation of gastric cancer cells was examined by MTT assay.Immunohistochemical method was used to test the expression of serine/threonine kinase 15(STK15)in gastric cancer cells.Results The exosomes derived from gastric cancer MGC803 cells had a characteristic plate-like structure consisting of a bilayer membrane with the diameter of about 50-130 nm.Exosoms had the effect of promoting the proliferation acitivity of gastric cancer cells(P<0.05).The cell proliferation activity increased from 0.213 to 0.534 when treated 12 h(F=44.21,P=0.000),from 0.251 to 0.998 when treated 24 h(F=66.06,P=0.000),from 0.273 to 1.057 when treated 36 h(F=435.37,P=0.000),from 0.342 to 1.414 when treated 48 h(F=195.30,P=0.000).Exosomes promoted the proliferation of MGC803 cells in a dose-dependent and time-dependent manner,which might affect the cell proliferation process by regulating the expression of STK15.Conclusion Exosomes can accelerate the proliferation of gastric cancer cells by regulating STK15 expression.
Objective To discuss the effect of exosomes on proliferation of gastric cancer cells and expression of serine/threonine kinase 15(STK15).Methods Exosomes were extracted from the culture supernatant of MGC803 gastric cancer cells by ultracentrifugal gradient centrifugation,and their ultrastructure was identified by transmission electron microscopy.Gastric cancer cells were then treated with exosomes at different concentrations(0.05,0.10,0.20,0.50μg/μL)The effect of exosomes on the proliferation of gastric cancer cells was examined by MTT assay.Immunohistochemical method was used to test the expression of serine/threonine kinase 15(STK15)in gastric cancer cells.Results The exosomes derived from gastric cancer MGC803 cells had a characteristic plate-like structure consisting of a bilayer membrane with the diameter of about 50-130 nm.Exosoms had the effect of promoting the proliferation acitivity of gastric cancer cells(P<0.05).The cell proliferation activity increased from 0.213 to 0.534 when treated 12 h(F=44.21,P=0.000),from 0.251 to 0.998 when treated 24 h(F=66.06,P=0.000),from 0.273 to 1.057 when treated 36 h(F=435.37,P=0.000),from 0.342 to 1.414 when treated 48 h(F=195.30,P=0.000).Exosomes promoted the proliferation of MGC803 cells in a dose-dependent and time-dependent manner,which might affect the cell proliferation process by regulating the expression of STK15.Conclusion Exosomes can accelerate the proliferation of gastric cancer cells by regulating STK15 expression.
引文
[1]KURYWCHAK P,KALLURI R.An evolving function of DNA-containing exosomes in chemotherapy-induced immune response[J].Cell Res,2017,27(6):722-723.
[2]MILANE L,SINGH A,MATTHEOLABAKIS G,et al.Exosome mediated communication within the tumor microenvironment[J].J Control Release,2015,219(2):278-294.
[3]PARK J,CHOI Y.Exosome identification for personalized diagnosis and therapy[J].Biomed Eng Lett,2014,4(3):258-268.
[4]HAO S,MOYANA T,XIANG J.Cancer immunotherapy by exosome-based vaccines[J].Cancer Biother Radiopham,2007,22(5):692-703.
[5]HONG B S,CHO J H,KIM H,et al.Colorectal cancer cell-derived microvesicles are enriched in cell cycle-related m RNAs that promote proliferation of endothelial cells[J].BMC Genomics,2009,10(1):556.
[6]TAVERNA S,FLUGY A,SAIEVA L,et al.Role of exosomes released by chronic myelogenous leukemia cells in angiogenesis[J].Int J Cancer,2012,130(9):2033-2043.
[7]YAN Y M,FU G Z,YE Y F,et al.Exosomes participate in the carcinogenesis and the malignant behavior of gastric cancer[J].Scand J Gastroenterol,2017,52(5):499-504.
[8]骆阳,孟民杰,刁志宏.Liquid biopsies in cancer[J/CD].转化医学电子杂志,2017,4(9):48-54.
[9]ZHANG H G,GRIZZLE W E.Exosomes:a novel pathway of local and distant intercellular communication that facilitates the growth and metastasis of neoplastic lesions[J].Am J Pathol,2014,184(1):28-41.
[10]WHITESIDE T L.Tumor-deried exosoems and their role in cancer progression[J].Adv Clin Chem,2016,74(1):103-141.
[11]SUNG B H,WEAVER A M.Exosome secretion promotes chemotaxis of cancer cells[J].Cell Adh Migr,2017,11(2):187-195.
[12]卜宁,孙秉中,李奇灵,等.Exosomes对L1210肿瘤细胞攻击的免疫保护作用研究[J].临床血液学杂志,2006(6):358-361.
[13]王东关,孙善珍,王振光,等.肿瘤exosome诱导的细胞毒性T细胞的特异性杀伤作用[J].山东大学学报(医学版),2006,44(2):213-216.
[14]GREENING D W,GOPAL S K,XU R,et al.Exosomes and their roles in immune regulation and cancer[J].Semin Cell Dev Biol,2015,40(1):72-81.
[15]MAHMOODZADEH HOSSEINI H,HALABIAN R,AMINM,et al.Texosome-based drug delivery system for cancer therapy:from past to present[J].Cancer Biol Med,2015,12(3):150-162.
[16]AL-HAZMI N,ALHAZZAZI T,WILLIAMS G,et al.DNAreplication licensing factor MCM2,geminin,and Ki67define proliferative state and are linked with survival in oral squamous cell carcinoma[J].Eur J Oral Sci,2018,126(3):186-196.
[17]VALENTINO A,RECLUSA P,SIRERA R,et al.Exosomal microRNAs in liquid biopsies:future biomarkers for prostate cancer[J].Clin Transl Oncol,2017,19(6):651-657.
[18]朱磊,李响,刘朋飞.Exosomes在肿瘤免疫方面的研究进展[J].中国生物制品学杂志,2010,23(4):441-444.
[2]MILANE L,SINGH A,MATTHEOLABAKIS G,et al.Exosome mediated communication within the tumor microenvironment[J].J Control Release,2015,219(2):278-294.
[3]PARK J,CHOI Y.Exosome identification for personalized diagnosis and therapy[J].Biomed Eng Lett,2014,4(3):258-268.
[4]HAO S,MOYANA T,XIANG J.Cancer immunotherapy by exosome-based vaccines[J].Cancer Biother Radiopham,2007,22(5):692-703.
[5]HONG B S,CHO J H,KIM H,et al.Colorectal cancer cell-derived microvesicles are enriched in cell cycle-related m RNAs that promote proliferation of endothelial cells[J].BMC Genomics,2009,10(1):556.
[6]TAVERNA S,FLUGY A,SAIEVA L,et al.Role of exosomes released by chronic myelogenous leukemia cells in angiogenesis[J].Int J Cancer,2012,130(9):2033-2043.
[7]YAN Y M,FU G Z,YE Y F,et al.Exosomes participate in the carcinogenesis and the malignant behavior of gastric cancer[J].Scand J Gastroenterol,2017,52(5):499-504.
[8]骆阳,孟民杰,刁志宏.Liquid biopsies in cancer[J/CD].转化医学电子杂志,2017,4(9):48-54.
[9]ZHANG H G,GRIZZLE W E.Exosomes:a novel pathway of local and distant intercellular communication that facilitates the growth and metastasis of neoplastic lesions[J].Am J Pathol,2014,184(1):28-41.
[10]WHITESIDE T L.Tumor-deried exosoems and their role in cancer progression[J].Adv Clin Chem,2016,74(1):103-141.
[11]SUNG B H,WEAVER A M.Exosome secretion promotes chemotaxis of cancer cells[J].Cell Adh Migr,2017,11(2):187-195.
[12]卜宁,孙秉中,李奇灵,等.Exosomes对L1210肿瘤细胞攻击的免疫保护作用研究[J].临床血液学杂志,2006(6):358-361.
[13]王东关,孙善珍,王振光,等.肿瘤exosome诱导的细胞毒性T细胞的特异性杀伤作用[J].山东大学学报(医学版),2006,44(2):213-216.
[14]GREENING D W,GOPAL S K,XU R,et al.Exosomes and their roles in immune regulation and cancer[J].Semin Cell Dev Biol,2015,40(1):72-81.
[15]MAHMOODZADEH HOSSEINI H,HALABIAN R,AMINM,et al.Texosome-based drug delivery system for cancer therapy:from past to present[J].Cancer Biol Med,2015,12(3):150-162.
[16]AL-HAZMI N,ALHAZZAZI T,WILLIAMS G,et al.DNAreplication licensing factor MCM2,geminin,and Ki67define proliferative state and are linked with survival in oral squamous cell carcinoma[J].Eur J Oral Sci,2018,126(3):186-196.
[17]VALENTINO A,RECLUSA P,SIRERA R,et al.Exosomal microRNAs in liquid biopsies:future biomarkers for prostate cancer[J].Clin Transl Oncol,2017,19(6):651-657.
[18]朱磊,李响,刘朋飞.Exosomes在肿瘤免疫方面的研究进展[J].中国生物制品学杂志,2010,23(4):441-444.