[1]王梦丽,韩利红,麻佑锋,等.肿瘤相关成纤维细胞外泌体对肺腺癌细胞增殖、迁移、侵袭的影响及机制[J].新乡医学院学报,2023,40(11):1016-1023.[doi:10.7683/xxyxyxb.2023.11.003]
　WANG Mengli,HAN Lihong,MA Youfeng,et al.Effect and mechanism of cancer-associated fibroblasts exosomes on proliferation,migration and invasion of lung adenocarcinoma cells[J].Journal of Xinxiang Medical University,2023,40(11):1016-1023.[doi:10.7683/xxyxyxb.2023.11.003]
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肿瘤相关成纤维细胞外泌体对肺腺癌细胞增殖、迁移、侵袭的影响及机制
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
40卷
期数:
2023年11
页码:
1016-1023
栏目:
基础研究
出版日期:
2023-11-05

文章信息/Info

Title:
Effect and mechanism of cancer-associated fibroblasts exosomes on proliferation,migration and invasion of lung adenocarcinoma cells
作者:
王梦丽1; 2; 韩利红2; 麻佑锋2; 郑其帆2; 3; 王若熹2
（1.新乡医学院， 河南　新乡　453003；2.洛阳市中心医院呼吸与危重症科， 河南　洛阳　471009；3.郑州大学，河南　郑州　450001）
Author(s):
WANG Mengli1; 2; HAN Lihong2; MA Youfeng2; ZHENG Qifan2; 3; WANG Ruoxi2
（1.Xinxiang Medical University,Xinxiang 453003,Henan Province,China;2.Department of Respiratory and Critical Care Medicine,Luoyang Central Hospital,Luoyang 471000,Henan Province,China;3.Zhengzhou University，Zhengzhou 450001,Henan Province,China)
关键词:
肿瘤相关成纤维细胞; 外泌体; 肺腺癌
Keywords:
cancer-associated fibroblasts; exosome; lung adenocarcinoma
分类号:
R734.2
DOI:
10.7683/xxyxyxb.2023.11.003
文献标志码:
A
摘要:
目的　探讨肿瘤相关成纤维细胞（CAFs）外泌体对肺腺癌细胞A549增殖、迁移、侵袭的影响及机制。
方法　取洛阳市中心医院胸外科收治的肺腺癌患者的癌组织和癌旁组织标本，并分离培养原代CAFs和正常成纤维细胞(NFs)；收集CAFs和NFs细胞培养上清液,使用差速离心法提取CAFs外泌体和NFs外泌体。取对数生长期的A549细胞，随机分为对照组、NFs外泌体组、CAFs外泌体组，NFs外泌体组、CAFs外泌体组细胞分别加入NFs外泌体、CAFs外泌体孵育，对照组细胞常规培养。采用细胞计数试剂盒-8（CCK-8）实验检测细胞增殖能力，细胞划痕实验检测细胞迁移能力，Transwell小室实验检测细胞侵袭能力，Western blot法检测细胞中磷酸酯酶与张力蛋白同源物（PTEN）/磷脂酰肌醇3激酶(PI3K)/ 蛋白激酶B（Akt）通路相关蛋白PTEN、PI3K、磷酸化磷脂酰肌醇3激酶（p-PI3K）、Akt、磷酸化蛋白激酶B （p-Akt）及上皮-间质转化（EMT）相关蛋白上皮钙黏素(E-cad)、神经钙黏素(N-cad)、波形蛋白(VIM)表达。采用随机数字表法将15只裸鼠分为A549裸鼠组、NFs外泌体裸鼠组、CAFs外泌体裸鼠组，每组5只，将对照组、NFs外泌体组、CAFs外泌体组细胞分别移植到A549裸鼠组、NFs外泌体裸鼠组、CAFs外泌体裸鼠组小鼠左侧腋下，移植6周内每周测量小鼠肿瘤体积。
结果　培养24 h时，3组细胞的增殖能力比较差异无统计学意义 (P>0.05)；培养48、72 h时，CAFs外泌体组细胞的增殖能力显著高于NFs外泌体组和对照组 (P<0.05)；培养48、72 h时，对照组与NFs外泌体组细胞的增殖能力比较差异无统计学意义（P>0.05）。培养24、48 h时，CAFs外泌体组细胞的迁移能力显著高于NFs外泌体组和对照组（P<0.05）；培养24、48 h时，NFs外泌体组与对照组细胞的迁移能力比较差异无统计学意义（P>0.05）。CAFs外泌体组侵袭细胞数显著多于NFs外泌体组和对照组(P<0.05)；NFs外泌体组与对照组的侵袭细胞数比较差异无统计学意义(P>0.05)。CAFs外泌体组细胞中N-cad、VIM蛋白相对表达量显著高于对照组和NFs外泌体组（P<0.05)，E-cad蛋白相对表达量显著低于对照组和NFs外泌体组（P<0.05)；NFs外泌体组与对照组细胞中N-cad、VIM、E-cad蛋白相对表达量比较差异无统计学意义（P>0.05）。CAFs外泌体组细胞中PTEN蛋白相对表达量显著低于对照组和NFs外泌体组（P<0.05)，p-PI3K /PI3K、p-Akt/Akt值显著高于对照组和NFs外泌体组（P<0.05)；NFs外泌体组与对照组细胞中PTEN蛋白相对表达量及p-PI3K/PI3K、p-Akt /Akt值比较差异无统计学意义（P>0.05）。移植第1周，3组小鼠的肿瘤体积比较差异无统计学意义（P>0.05）；移植第2～6周，CAFs外泌体裸鼠组小鼠的肿瘤体积显著大于A549裸鼠组和NFs外泌体裸鼠组（P<0.05）；移植第2～6周，NFs外泌体裸鼠组与A549裸鼠组小鼠的肿瘤体积比较差异无统计学意义（P>0.05）。
结论　CAFs外泌体可促进肺腺癌细胞的增殖、迁移和侵袭，在小鼠体内促进肺腺癌的生长，其机制可能与EMT和PTEN/PI3K/Akt信号通路有关。
Abstract:
Objective　To explore the effect and mechanism of cancer-associated fibroblasts (CAFs) exosomes on the proliferation,migration and invasion of lung adenocarcinoma cells A549.
Methods　The cancer tissue and adjacent tissue samples from lung adenocarcinoma patients admitted to the Department of Respiratory and Critical Care Medicine,Luoyang Central Hospital were collected,and the primary CAFs and normal fibroblasts (NFs) were isolated and cultured;the supernatant of cell culture of CAFs and NFs was collected,and the CAFs exosomes and NFs exosomes were extracted by differential centrifugation method.The A549 cells in the logarithmic growth phase were randomly divided into the control group,NFs exosomes group and CAFs exosomes group.The cells in the NFs exosomes group and CAFs exosomes group were incubated with NFs exosomes and CAFs exosomes,while the cells in the control group were cultured routinely.The cell proliferation ability was detected by cell counting kit-8 (CCK-8) assay,the cell migration ability was detected by cell scratch assay,the cell invasion ability was detected by Transwell cell assay,and the phosphatase and tension protein homolog (PTEN)/ phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway related proteins PTEN,PI3K,phosphorylated phosphoinositide 3-kinase (p-PI3K),phosphorylated protein kinase B (p-Akt) and epithelial mesenchymal transition (EMT) related proteins E-cadherin (E-cad),N-cadherin(N-cad) and vimentin (VIM) were detected by Western blot.A total of 15 nude mice were divided into A549 nude mice group,NFs exosomes nude mice group and CAFs exosomes nude mice group by random number table method,with five mice in each group.The cells in the control group,NFs exosomes group and CAFs exosomes group were transplanted to the left armpit of A549 nude mice group,NFs exosomes nude mice group and CAFs exosomes nude mice group,respectively;the tumor volume of the nude mice was measured weekly within 6 weeks of transplantation.
Results　At 24 hours of cultivation,there was no significant difference in the proliferation ability of cells among the three groups (P<0.05);at 48 and 72 hours of cultivation,the proliferation ability of cells in the CAFs exosomes group was significantly higher than that in the NFs exosomes group and control group (P<0.05);at 48 and 72 hours of cultivation,there was no significant difference in the proliferation ability of cells between the control group and the NFs exosomes group (P>0.05).At 24 and 48 hours of cultivation,the migration ability of cells in the CAFs exosomes group was significantly higher than that in the NFs exosomes group and control group (P<0.05);at 24 and 48 hours of cultivation,there was no significant difference in the migration ability of cells between the NFs exosomes group and the control group (P>0.05).The number of invasive cells in the CAFs exosomes group was significantly higher than that in the NFs exosomes group and control group (P<0.05);there was no significant difference in the number of invasive cells between the NFs exosomes group and the control group (P>0.05).The relative expression levels of N-cad and VIM protein in the CAFs exosomes group were significantly higher than those in the control group and the NFs exosomes group (P<0.05),the relative expression level of E-cad protein was significantly lower than that in the control group and NFs exosomes group (P<0.05);there was no significant difference in the relative expression levels of N-cad,VIM and E-cad protein between the NFs exosomes group and the control group (P>0.05).The relative expression level of PTEN protein in the CAFs exosomes group was significantly lower than that in the control group and NFs exosomes group (P<0.05),p-PI3K/PI3K,p-Akt/Akt values were significantly higher than those in the control group and NFs exosomes group (P<0.05);there was no significant difference in the relative expression level of PTEN protein and p-PI3K/PI3K,p-Akt/Akt values between the NFs exosomes group and the control group (P>0.05).At the first week of transplantation,there was no significant difference in the tumor tissue volume of mice among the three groups (P>0.05);at 2-6 weeks of transplantation,the tumor volume of mice in the CAFs exosomes nude mice group was significantly larger than that in the A549 nude mice group and the NFs exosomes nude mice group (P<0.05);at 2-6 weeks of transplantation,there was no significant difference in tumor volume of mice between the NFs exosomes nude mice group and the A549 nude mice group (P>0.05).
Conclusion　CAFs exosomes can promote the proliferation,migration and invasion ability of lung adenocarcinoma cells,and promote the growth of lung adenocarcinoma in mice;the mechanism may be related to the EMT and PTEN/PI3K/Akt signaling pathways.

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更新日期/Last Update: 2023-11-05