外泌体在促进非小细胞肺癌脑转移中的作用
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摘要
目的探讨外泌体(Exosomes)促进非小细胞肺癌脑转移的作用,为今后的临床诊疗工作提供有价值的参考依据。方法采用电镜观察和Western blot检测鉴定收集的培养非小细胞肺癌上清中含有的源性Exosomes;利用Exosomes荧光染色与受体NHA细胞共培养,明确Exosomes对受体NHA细胞的作用形式及对NHA细胞活性的影响,并建立体外血脑屏障模型验证外泌体是否参与了非小细胞肺癌穿过血脑屏障转移入脑的过程。结果分离获得的crExo样品在电镜下呈现为圆形,直径30～1000 nm。Western blot结果显示,crExo样品表达Exosomes标志物蛋白CD63、CD9,对albumin为轻微表达,对内质网蛋白标志物calnexin则不表达。非小细胞肺癌细胞株crExoh浓度检测结果显示,晚期crExo蛋白浓度显著高于早期,差异有统计学意义(P<0.05)。染色观察细胞迁移率,结果显示,Exosomes参与了非小细胞肺癌穿过血脑屏障的过程。结论 Exosomes对肿瘤的增殖、侵袭和转移等生物学行为具有重要的调控作用,非小细胞肺癌发生脑转移也受Exosomes的影响。
Objective To explore the role of Exosomes in promoting brain metastasis of non-small cell lung cancer, so as to provide the valuable reference for future clinical diagnosis and treatment work. Methods The exogenous Exosomes contained in the cultured non-small cell lung cancer supernatant were observed by electron microscopy and identified by Western blot. Exosomes fluorescent staining and co-culture of receptor NHA cells wee used to determine the role form of Exosomes on the receptor NHA cells and the effect on NHA cell activity, and establish an in vitro blood-brain barrier model to verify whether Exosomes are involved in non-small cell lung cancer in the process of transferring the blood-brain barrier into the brain. Results The crExo sample obtained by the separation was circular under the electron microscope and had a diameter of 30 to 1000 nm. Western blot results showed that the excretion marker proteins CD63 and CD9 were expressed in the crExo sample, which was slightly expressed in albumin and not expressed in the endoplasmic reticulum protein marker calnexin. The detection of crExoh concentration in non-small cell lung cancer cell lines showed that the late crExo protein concentration was significantly higher than that in the early stage, and the difference was statistically significant(P<0.05). Cell migration was observed by staining, and the results showed that Exosomes was involved in the process of non-small cell lung cancer crossing the blood-brain barrier. Conclusion Exosomes plays an important role in the regulation of tumor growth, invasion and metastasis. The brain metastasis of non-small cell lung cancer is also affected by Exosomes.
Objective To explore the role of Exosomes in promoting brain metastasis of non-small cell lung cancer, so as to provide the valuable reference for future clinical diagnosis and treatment work. Methods The exogenous Exosomes contained in the cultured non-small cell lung cancer supernatant were observed by electron microscopy and identified by Western blot. Exosomes fluorescent staining and co-culture of receptor NHA cells wee used to determine the role form of Exosomes on the receptor NHA cells and the effect on NHA cell activity, and establish an in vitro blood-brain barrier model to verify whether Exosomes are involved in non-small cell lung cancer in the process of transferring the blood-brain barrier into the brain. Results The crExo sample obtained by the separation was circular under the electron microscope and had a diameter of 30 to 1000 nm. Western blot results showed that the excretion marker proteins CD63 and CD9 were expressed in the crExo sample, which was slightly expressed in albumin and not expressed in the endoplasmic reticulum protein marker calnexin. The detection of crExoh concentration in non-small cell lung cancer cell lines showed that the late crExo protein concentration was significantly higher than that in the early stage, and the difference was statistically significant(P<0.05). Cell migration was observed by staining, and the results showed that Exosomes was involved in the process of non-small cell lung cancer crossing the blood-brain barrier. Conclusion Exosomes plays an important role in the regulation of tumor growth, invasion and metastasis. The brain metastasis of non-small cell lung cancer is also affected by Exosomes.
引文
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[13]张干,刘彦龙,杨艳梅,等.肿瘤衍生外泌体传递的微RNA在肿瘤进展中作用的研究进展[J].肿瘤,2016,34(12):1161.
[14]金福军,曾川,张献全,等.全脑放疗联合靶向药物与单独全脑放疗治疗非小细胞肺癌脑转移疗效及安全性的Meta分析[J].中华肺部疾病杂志,2016,9(4):366-371.
[15]路长红,马莉,孙增峰,等.非小细胞肺癌脑转移手术治疗预后相关因素分析[J].中华肿瘤防治杂志,2016,23(10):677-681.
[16]刘芳.血清肿瘤标志物联合检测在非小细胞肺癌脑转移诊疗中的价值[J].实用癌症杂志,2017,32(4):578-581.
[17]Ou SH,Ahn JS,De Petris L,et al.Alectinib in crizotinibrefractory ALK-rearranged non-small-cell lung cancer:a phaseⅡglobal study[J].J Clin Oncol,2016,34(7):661-668.
[18]陈芳,张雯,宋淑霞,等.肿瘤及肿瘤基质细胞释放的外泌体对肿瘤耐药产生的影响[J].中国肿瘤生物治疗杂志,2016,23(3):432-436.
[19]施卫忠,卢仁泉,郭林.恶性肿瘤患者外泌体检测的研究进展[J].中国肿瘤,2015,24(10):849-854.
[20]刘锐,巴一.外泌体传输microRNA在肿瘤研究中的进展[J].中国肿瘤临床,2016,43(10):442-445.
[21]张肖肖,毕大鹏.非小细胞肺癌脑转移放化疗联合的疗效观察[J].临床肺科杂志,2015,18(3):562-563.
[22]刘长红,武明花,李桂源.肿瘤细胞来源的外泌体与恶性肿瘤的进展及化疗[J].中国生物化学与分子生物学报,2014,30(6):526-532.
[23]李孟娇,王浩,李庆伟.外泌体在肿瘤转移及诊疗中的研究进展[J].中国细胞生物学学报,2016,38(5):608-615.
[2]聂欢,袁帅,刘庆云,等.GPC5基因在肺腺癌细胞系中的功能研究[J].第三军医大学学报,2015,37(7):600-605.
[3]陈芳,张雯,宋淑霞,等.肿瘤及肿瘤基质细胞释放的外泌体对肿瘤耐药产生的影响[J].中国肿瘤生物治疗杂志,2016,23(3):432-436.
[4]Yang Q,Diamond MP,Al-Hendy A.The emerging role of extracellular vesicle-derived miRNAs:implication in cancer progression and stem cell related diseases[J].J Clin Epigenet,2016,2(1):13.
[5]孟祥宽.血清外泌体膜蛋白GPC1在非小细胞肺癌筛查及预后评估中的应用价值[D].长春:吉林大学,2016.
[6]石远凯,孙燕,于金明,等.中国肺癌脑转移诊治专家共识(2017年版)[J].中国肺癌杂志,2017,20(1):1-13.
[7]臧淑月,张振成,叶伟民,等.血清HE4与CYFRA21-1,NSE联合检测在肺癌诊断中的价值[J].检验医学与临床,2016,13(1):84-87.
[8]Ji Z,Bi N,Wang J,et al.Risk factors for brain metastases in locally advanced non-small cell lung cancer with definitive chest radiation[J].Int J Radiat Oncol Biol Phys,2014,89(2):330-337.
[9]李志坚,李哲,卢强,等.CA19-9、CA125、CEA及Ferritin联合检测诊断非小细胞肺癌脑、骨转移的临床价值研[J].现代生物医学进展,2014,14(19):3729-3732.
[10]王彩,魏素菊,洪雷,等.319例晚期肺腺癌患者临床预后因素分析[J].肿瘤基础与临床,2016,29(3):240-246.
[11]李思迪,侯信,亓洪昭,等.外泌体:为高效药物投递策略提供天然的内源性纳米载体[J].化学进展,2016,28(2):353-362.
[12]梅芬,卢忠心.肺癌脑转移与血清肿瘤标志物水平的关系[J].临床与实验病理学杂志,2016,32(1):96-98.
[13]张干,刘彦龙,杨艳梅,等.肿瘤衍生外泌体传递的微RNA在肿瘤进展中作用的研究进展[J].肿瘤,2016,34(12):1161.
[14]金福军,曾川,张献全,等.全脑放疗联合靶向药物与单独全脑放疗治疗非小细胞肺癌脑转移疗效及安全性的Meta分析[J].中华肺部疾病杂志,2016,9(4):366-371.
[15]路长红,马莉,孙增峰,等.非小细胞肺癌脑转移手术治疗预后相关因素分析[J].中华肿瘤防治杂志,2016,23(10):677-681.
[16]刘芳.血清肿瘤标志物联合检测在非小细胞肺癌脑转移诊疗中的价值[J].实用癌症杂志,2017,32(4):578-581.
[17]Ou SH,Ahn JS,De Petris L,et al.Alectinib in crizotinibrefractory ALK-rearranged non-small-cell lung cancer:a phaseⅡglobal study[J].J Clin Oncol,2016,34(7):661-668.
[18]陈芳,张雯,宋淑霞,等.肿瘤及肿瘤基质细胞释放的外泌体对肿瘤耐药产生的影响[J].中国肿瘤生物治疗杂志,2016,23(3):432-436.
[19]施卫忠,卢仁泉,郭林.恶性肿瘤患者外泌体检测的研究进展[J].中国肿瘤,2015,24(10):849-854.
[20]刘锐,巴一.外泌体传输microRNA在肿瘤研究中的进展[J].中国肿瘤临床,2016,43(10):442-445.
[21]张肖肖,毕大鹏.非小细胞肺癌脑转移放化疗联合的疗效观察[J].临床肺科杂志,2015,18(3):562-563.
[22]刘长红,武明花,李桂源.肿瘤细胞来源的外泌体与恶性肿瘤的进展及化疗[J].中国生物化学与分子生物学报,2014,30(6):526-532.
[23]李孟娇,王浩,李庆伟.外泌体在肿瘤转移及诊疗中的研究进展[J].中国细胞生物学学报,2016,38(5):608-615.