CXCL1对肝癌内质网应激作用的相关性研究

发布时间：2018-07-09 21:00

  本文选题：趋化因子CXCL1 + 肿瘤微环境　； 参考：《广西医科大学》2016年硕士论文

【摘要】：目的：目前研究认为肿瘤的许多生物特性与肿瘤微环境有着密切的关系。肿瘤微环境中的许多细胞因子可以促进肿瘤发展,也因其组织间质压力高、供血不足、营养相对缺乏,易引起肿瘤细胞发生内质网应激,激活相关通路,促进细胞因子表达以及肿瘤发展。许多报道认为趋化因子CXCL1与肿瘤发生、发展、迁移、侵袭具有相关性,如在一些黑色素瘤、肝癌、胃癌、卵巢癌等均有高表达,且高表达的肿瘤其恶性程度也增加。然而趋化因子CXCL1为什么在肿瘤微环境中高表达,又是如何影响肝癌发生、发展、迁移、侵袭报道很少。因此,本研究从肝癌微环境的角度研究CXCL1对肿瘤作用。为进一步研究CXCL1提供基础及肝癌治疗提供新的思路。方法：首先用不同浓度的衣霉素(TM)刺激HepG2细胞,引起细胞发生内质网应激(endoplasmic reticulum stress ER Stress),提取总RNA后反转录,再利用Q-PCR技术检测ELR+家族趋化因子的mRNA表达水平,同时利用ELISA方法检测培养基中CXCL1的水平。用衣霉素刺激HepG2细胞12h,引起细胞发生内质网应激后换成新鲜培养基,培养24h,收集培养基用于刺激HepG2细胞及诱导成熟的THP-1细胞,提取总RNA后反转录,再利用Q-PCR技术检测其内质网应激相关基因ATF-6、PERK、IRE1的mRNA表达水平。合成SiRNA,瞬时转染到HepG2细胞中,敲低其CXCL1表达,再用衣霉素刺激HepG2细胞12h后,换成新鲜培养基,培养24h,收集培养基用于刺激HepG2细胞及诱导成熟的THP-1细胞,提取mRNA,反转录后利用Q-PCR技术检测其内质网应激相关基因ATF-6、PERK、IRE1的mRNA表达水平。最后通过基因重组技术构建CXCL1载体,确认其在真核细胞表达后,瞬时转染到HepG2细胞内,使CXCL1过表达,再提取mRNA,反转录后利用Q-PCR技术检测内质网应激相关基因ATF-6、PERK、IRE1的mRNA水平。以上所得数据均用SPSS 16.0软件分析,采用单因素方差分析对结果进行比较,P0.05认为具有统计学意义。结果：HepG2在不同浓度的衣霉素刺激下,CXCL1、CXCL2、CXCL3的mRNA表达水平与对照组相比均升高(P0.05),CXCL8的mRNA水平降低,而CXCL5、CXCL6、CXCL7、CXCLR1、CXCLR2的mRNA未检测出；培养基中的CXCL1水平升高。用TM处理过的培养基刺激HepG2与对照组相比内质网应激相关基因ATF-6、PERK、IRE1的mRNA表达水平均升高(P0.05)；当CXCL1被敲低时,内质网应激相关基因ATF-6、PERK、IRE1的mRNA表达水平均降低(P0.05)；CXCL1载体构建成功并在真核细胞中表达,当CXCL1在HepG2细胞过表达时,HepG2细胞的内质网应激相关基因ATF-6、PERK、IRE1的mRNA水平均升高(P0.05)。结论：在肝癌微环境中,当细胞发生内质网应激时,可促进CXCL1、CXCI2、 CXCL3的表达,CXCL8降低,而CXCL1通过自分泌或者旁分泌又引起细胞发生内质网,两者相互促进。
[Abstract]:Objective: many biological characteristics of tumor are closely related to tumor microenvironment. Many cytokines in tumor microenvironment can promote the development of tumor. Because of the high interstitial pressure, insufficient blood supply and relative lack of nutrition, tumor cells are prone to endoplasmic reticulum stress and activation of related pathways. Promote cytokine expression and tumor development. Many reports suggest that chemokine CXCL1 is associated with tumorigenesis, development, migration and invasion, such as high expression of CXCL1 in some melanoma, liver cancer, gastric cancer, ovarian cancer and so on. However, there are few reports on why the chemokine CXCL1 is overexpressed in tumor microenvironment and how it affects the occurrence, development, migration and invasion of HCC. Therefore, the effect of CXCL1 on tumor was studied from the point of view of microenvironment of liver cancer. It provides a new idea for the further study of CXCL1 and the treatment of liver cancer. Methods: HepG2 cells were stimulated with different concentrations of chlortetracycline (TM) to induce endoplasmic reticulum stress (ER stress), and reverse transcription was performed after total RNAs were extracted. The expression of chemokines in the ELR family was detected by Q-PCR. At the same time, the level of CXCL1 in culture medium was detected by Elisa. HepG2 cells were stimulated by itriamycin for 12 hours, and then changed into fresh medium after endoplasmic reticulum stress. The culture medium was used to stimulate HepG2 cells and induce mature THP-1 cells for 24 hours. The total RNA was extracted and reverse transcripted. Q-PCR technique was used to detect the mRNA expression level of ER stress-related gene ATF-6 PERKN IRE1. SiRNAs were synthesized and transfected into HepG2 cells, and the expression of CXCL1 in HepG2 cells was reduced. After 12 h of stimulation with itamycin, HepG2 cells were transferred to fresh culture medium for 24 h. The culture medium was used to stimulate HepG2 cells and induce mature THP-1 cells. After mRNAs were extracted, Q-PCR technique was used to detect the mRNA expression level of ER stress-related gene ATF-6 and PERKN IRE1. Finally, CXCL1 vector was constructed by gene recombination technique. After eukaryotic expression, CXCL1 was transiently transfected into HepG2 cells to overexpression CXCL1, then mRNAs were extracted. Q-PCR technique was used to detect the mRNA level of ER stress-related gene ATF-6PERKN IRE1. All the above data were analyzed by SPSS 16.0 software, and the results were compared by single factor variance analysis (P0.05). Results the mRNA expression level of CXCL1, CXCL2, CXCL2, CXCL8, CXCL8, CXCL8, CXCL8, and CXCL1 in CXCL1, CXCL1, CXCL7, CXCL6, CXCL6, CXCL7, CXCL1, and CXCL1 in culture medium of HepG2 were significantly higher than those in control group (P0.05), while the mRNA expression of CXCL1, CXCL8, CXCL8, CXCL8, CXCL8 and CXCL1 in culture medium were not detected. Compared with the control group, the mRNA expression of ER stress-related gene ATF-6PERKnIRE1 was increased in HepG2 treated with TM (P0.05), and the mRNA expression level of ER stress-related gene ATF-6 / PERKN IRE1 was decreased when CXCL1 was knocked down (P0.05). CXCL1 vector was successfully constructed and expressed in eukaryotic cells. When CXCL1 was overexpressed in HepG2 cells, the mRNA level of ER stress-related gene ATF-6 / PERK-1 IRE1 was increased in HepG2 cells (P0.05). Conclusion: in the microenvironment of liver cancer, the expression of CXCL1, CXCI2 and CXCL3 can be decreased when the endoplasmic reticulum stress occurs, while CXCL1 can induce the endoplasmic reticulum formation through autocrine or paracrine.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7

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