XAF-1与TNF-α共表达腺病毒抑制肝癌细胞生长的作用研究

发布时间：2018-05-04 01:24

本文选题：腺病毒 + 肝细胞癌　；参考：《重庆医科大学》2016年博士论文

【摘要】：目的:本研究探讨共表达XAF-1和TFN-α对HCC细胞生长的作用,并分析其分子机制。方法:在本研究中,检测了肝细胞癌癌组织样本中XAF-1的mRNA和蛋白水平,并在肝癌细胞系中证实了共表达XAF-1和TFN-α的腺病毒对肝癌细胞的显著的抑制作用。建立HepG2 M人肝细胞癌97H细胞裸鼠移植瘤模型,并运用重组共表达XAF-1和TNF-α腺病毒及Ad-con对裸鼠移植瘤模型进行瘤内注射,对照组每次注射同等体积的生理盐水作为对照。观察裸鼠移植瘤的生长情况,并通过比较肿瘤体积的变化和对肿瘤的抑制率来评价其体内治疗的效果。结果:肝细胞癌癌组织样本中XAF-1的表达量明显低于肝细胞癌瘤旁样本,说明其降低与肿瘤恶性程度相关。同时也在肝细胞癌细胞系,如MHCC 97L,HepG2和MHCC 97H中证实了XAF-1表达量的降低。本研究用一段2A肽编码序列,构建重组的腺病毒Ad-XAF-1TNF-α,有效的共表达了XAF-1和TFN-α。当Ad-XAF-1和TNF-α转染MHCC 97L细胞后,XAF-1和TFN-α的mRNA和蛋白水平明显升高。此外,确认了被Ad-XAF-1TNF-α转染的MHCC 97L细胞,细胞增值率明显低于单独用Ad-XAF-1或Ad-TNF-α病毒感染的细胞。与PBS对照组注射的裸小鼠瘤体积相比较,,Ad-XAF-1TNF-α组及Ad-con组裸小鼠的瘤体积增长,差异有统计学意义(P0.05);Ad-XAF-1TNF-α治疗组裸小鼠瘤体积增长最为缓慢,与Ad-con组裸小鼠的瘤体积相比较,差异有统计学意义(P0.05)。Ad-XAF-1TNF-α注射后的第7天,抑瘤率最高,达到65.87%,随后抑瘤率呈下降趋势。在注射后第28天,抑瘤率为60.78%.免疫组化染色以及RT-PCR实验结果表明,与Ad-con组及PBS组裸鼠瘤组织XAF-1、TNF-α的阳性表达率相比(6.11%±1.73,6.42%±2.59),Ad-XAF-1TNF-α治疗组裸鼠瘤组织XAF-1、TNF-α的阳性率表达增高(16.39%±1.39),差异有统计学意义(P0.05)。Ad-XAF-1TNF-α治疗组裸鼠瘤组织中XAF-1、TNF-α基因的mRNA表达水平均较Ad-con组、PBS组裸鼠瘤组织中XAF-1、TNF-α基因的mRNA表达水平增高,差异有统计学意义(P0.05)。结论:Ad-XAF-1TNF-α共表达病毒可以较单表达XAF-1、TNF-α的腺病毒更有效的抑制肝细胞癌细胞增殖,介导XAF-1、TNF-α可在裸小鼠移植瘤模型内进行有效的表达,减小瘤体积从而发挥抑制移植瘤生长的作用,同时还能促进XAF-1、TNF-α在移植瘤中的表达及mRNA水平的表达,其有望成为一种有效抵抗肝细胞癌的抗肿瘤因子。
[Abstract]:Aim: to investigate the effect of coexpression of XAF-1 and TFN- 伪 on the growth of HCC cells and to analyze its molecular mechanism. Methods: in this study, the levels of mRNA and protein of XAF-1 in hepatocellular carcinoma (HCC) tissues were detected, and the inhibitory effect of adenovirus co-expressing XAF-1 and TFN- 伪 on HCC cells was confirmed. The transplanted tumor model of HepG2 M human hepatocellular carcinoma 97H cell in nude mice was established. The nude mice transplanted tumor model was injected with recombinant co-expression of XAF-1, TNF- 伪 adenovirus and Ad-con. The control group was injected with normal saline of the same volume each time. The growth of xenografts in nude mice was observed and the effect of treatment in vivo was evaluated by comparing the changes of tumor volume and tumor inhibition rate. Results: the expression of XAF-1 in hepatocellular carcinoma tissues was significantly lower than that in the adjacent samples, indicating that the decrease of XAF-1 expression was related to the malignancy of the tumor. The decrease of XAF-1 expression was also confirmed in hepatocellular carcinoma cell lines such as MHCC 97L HepG2 and MHCC 97H. In this study, the recombinant adenovirus Ad-XAF-1TNF- 伪 was constructed with a fragment of 2A peptide encoding sequence. The recombinant adenovirus Ad-XAF-1TNF- 伪 was effectively coexpressed with XAF-1 and TFN- 伪. When Ad-XAF-1 and TNF- 伪 were transfected into MHCC 97L cells, the mRNA and protein levels of XAF-1 and TFN- 伪 increased significantly. In addition, the proliferation rate of MHCC 97L cells transfected with Ad-XAF-1TNF- 伪 was significantly lower than that of cells infected with Ad-XAF-1 or Ad-TNF- 伪 alone. Compared with the nude mice injected with PBS, the tumor volume of Ad-XAF-1TNF- 伪 group and Ad-con group increased significantly (P 0.05). The tumor volume of nude mice treated with Ad-XAF-1TNF- 伪 was the slowest, and the tumor volume of Ad-con group was higher than that of Ad-con group. On the 7th day after injection, the tumor inhibition rate was the highest, reaching 65.87, and then decreased. On the 28th day after injection, the tumor inhibition rate was 60.78%. Immunohistochemical staining and RT-PCR assay showed that, Compared with Ad-con group and PBS group, the positive rate of XAF-1TNF- 伪 in tumor tissue of nude mice was increased by 6.11% 卤1.73 卤6.42% 卤2.59 ~ XAF-1TNF- 伪 treatment group. The positive rate of XAF-1TNF- 伪 expression in tumor tissue of nude mice in Ad-con group and PBS group was 16.39% 卤1.39% higher than that in Ad-con group. There was significant difference in the expression of XAF-1TNF- 伪 gene in tumor tissue of nude mice treated with P0.05. Ad-XAF-1TNF- 伪. The mRNA expression level of XAF-1TNF- 伪 gene was higher than that of Ad-con group. The mRNA expression level of XAF-1TNF- 伪 gene was increased in nude mice in PBS group. The difference was statistically significant (P 0.05). Conclusion compared with the adenovirus expressing XAF-1TNF- 伪 alone, the adenovirus can inhibit the proliferation of hepatocellular carcinoma more effectively, and mediate the expression of XAF-1TNF- 伪 in nude mice transplanted tumor model, and reduce the volume of tumor and thus play a role in inhibiting the growth of transplanted tumor. It can also promote the expression of XAF-1TNF- 伪 and the expression of mRNA in transplanted tumors, which is expected to be an effective anti-tumor factor against hepatocellular carcinoma.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

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