稳转LRAT、RDH12基因的人宫颈癌细胞在裸鼠中成瘤情况及机制分析

发布时间：2018-06-13 11:09

本文选题：宫颈癌 + LRAT基因　；参考：《北京协和医学院》2017年博士论文

【摘要】：背景与目的宫颈癌是妇科最常见恶性肿瘤之一,除HPV感染以外,被感染细胞的基因出现异常表达才能最终导致宫颈癌的发生发展。前期研究首次通过RNA-Seq技术,检测宫颈癌与癌旁组织的差异基因,发现视黄醇代谢相关基因LRAT和RDH12在宫颈癌组织和细胞中的表达明显低于癌旁组织。已有的体外实验证实:构建LRAT及RDH12过表达载体并转染宫颈癌细胞系,瞬时转染LRAT和RDH12后,能够抑制宫颈癌细胞的增殖、侵袭和迁移,阻滞宫颈癌细胞周期,增加凋亡率。这一现象与抑制MAPK及NF-kB信号通路有关。当LRAT及RDH12基因的表达升高时,宫颈癌细胞中MAPK及NF-kB通路中相关蛋白的表达水平有所下降。本课题拟在组织层面上,通过动物体内实验,进一步验证LRAT、RDH12基因与MAPK及NF-kB信号通路的关系。通过建立宫颈癌细胞的裸鼠移植瘤模型,分析稳定转染LRAT和RDH12基因的两种Hela细胞,在裸鼠中的成瘤情况。并对移植瘤组织的病理进行分析,探讨LRAT/RDH12与宫颈癌的临床病理因素的关系,旨在阐明LRAT/RDH12基因在宫颈癌进展中的作用及机制,为宫颈癌的防治提供新的理论支持和分子指标。方法一、培养稳定转染的细胞株:利用重组质粒,培养并获得稳定转染LRAT及RDH12基因的两种Hela细胞株。二、构建Hela细胞的裸鼠移植瘤模型:利用未做处理的对照组Hela细胞,探索裸鼠中最佳的Hela细胞成瘤浓度。将Hela细胞配置成5×106/mL、1×107/mL、5×107/mL和1×108/mL的单细胞悬液,4个浓度梯度下进行成瘤浓度探索。三、观察并记录裸鼠中Hela细胞的成瘤情况,记录肿瘤体积大小,绘制肿瘤生长曲线。四、对裸鼠皮下移植瘤进行取材,进行免疫组化切片。分析病理组织中,MAPK通路中p38及NF-kB通路中p50、p65的表达情况。结果培养并获得稳定转染LRAT、RDH12基因的两种Hela细胞系。经过qPCR验证,稳转的LRAT基因过表达倍数是空白对照的29360倍,稳转的RDH12基因过表达倍数是空白对照的39631倍。对培养获得的稳转细胞进行实验,发现稳转LRAT、RDH12基因的两种Hela细胞中,P38、P50、P65蛋白的表达水平均下调。裸鼠成瘤模型建立的实验中,成功找出Hela细胞在裸鼠中的最佳成瘤浓度为5×107/mL,并顺利构建Hela细胞的裸鼠移植瘤模型。观察肿瘤生长变化情况,对肿瘤大小进行比对分析,稳转LRAT、RDH12基因的两组Hela细胞,在裸鼠模型中的成瘤速度及肿瘤生长速度均慢于对照组,且LRAT组受抑制程度较RDH12组明显。对3组同周数的瘤体组织分别进行免疫组化染色实验及Western实验,表明在LRAT、RDH12两组实验组中,P38、P50、P65蛋白的表达水平均下降。LRAT、RDH12的过表达,会抑制MAPK、NF-kB通路的活性。结论LRAT与RDH12基因的过表达,能够有效抑制宫颈癌组织的生长,这一现象与抑制MAPK、NF-kB信号上的相关蛋白表达水平有关,过表达LRAT、RDH12基因可以抑制宫颈癌的发展,这两个基因有望成为宫颈癌治疗的新靶点。
[Abstract]:Background & objective Cervical cancer is one of the most common malignant tumors in gynecology. In addition to HPV infection, abnormal gene expression in infected cells can eventually lead to the occurrence and development of cervical cancer. For the first time, RNA-Seq technique was used to detect the differentially expressed genes of retinol metabolism related genes LRAT and RDH12 in cervical cancer tissues and adjacent tissues, and the expression of LRAT and RDH12 genes in cervical cancer tissues and cells was significantly lower than that in adjacent tissues. It has been confirmed in vitro that LRAT and RDH12 overexpression vectors were constructed and transfected into cervical cancer cell lines. After transient transfection of LRAT and RDH12, LRAT and RDH12 could inhibit the proliferation, invasion and migration of cervical cancer cells, block the cell cycle and increase the apoptosis rate. This phenomenon is related to the inhibition of MAPK and NF-kB signaling pathways. When the expression of LRAT and RDH12 gene increased, the expression of MAPK and NF-kB related proteins in cervical cancer cells decreased. At the tissue level, the relationship between LRATA RDH12 gene and MAPK and NF-kB signaling pathway was further verified by animal experiments in vivo. The tumor formation of two Hela cells stably transfected with LRAT and RDH12 gene in nude mice was analyzed by establishing the tumor model of nude mice. The relationship between LRATR / RDH12 and the clinicopathological factors of cervical cancer was discussed in order to elucidate the role and mechanism of LRATR RDH12 gene in the progression of cervical cancer and to provide new theoretical support and molecular index for the prevention and treatment of cervical cancer. Methods 1. Stable transfected cell lines: two Hela cell lines transfected stably with LRAT and RDH12 gene were obtained by using recombinant plasmid. Secondly, the model of Hela cell transplantation in nude mice was established. The optimal concentration of Hela cells in nude mice was explored by using untreated Hela cells. Hela cells were configured into 5 脳 10 6 / mL mLN 1 脳 10 7 / mL and 1 脳 10 8 / mL single cell suspensions. The tumor concentration was investigated under four concentration gradients. Third, the tumorigenesis of Hela cells in nude mice was observed and recorded, the tumor volume was recorded and the tumor growth curve was drawn. Fourthly, the subcutaneous transplanted tumor of nude mice was selected and immunohistochemical sections were made. The expression of p38 and NF-kB p50 p65 in MAPK pathway was analyzed. Results two Hela cell lines stably transfected with LRAT-RDH12 gene were obtained. The results of qPCR showed that the overexpression of LRAT gene was 29360 times higher than that of the control, and the overexpression of RDH12 gene was 39631 times higher than that of the control. The results showed that the expression level of P38-P50 / P65 protein was down-regulated in the two Hela cells transfected with LRAT-RDH12 gene. The optimal concentration of Hela cells in nude mice was found to be 5 脳 10 7 / mL, and the model of Hela cells was successfully constructed in nude mice. The tumor growth rate and tumor growth rate of two groups of Hela cells with stable transformation of LRATA RDH12 gene in nude mice were slower than those in control group, and the inhibition degree of LRAT group was significantly higher than that of RDH12 group. Immunohistochemical staining and Western blot analysis showed that the expression level of P38-P50 p65 protein decreased and the overexpression of RDH12 in LRAT-RDH12 group inhibited the activity of MAPK-NF-kB pathway. Conclusion the overexpression of LRAT and RDH12 gene can effectively inhibit the growth of cervical cancer. This phenomenon is related to the inhibition of the expression of related proteins in MAPK- NF-kB signal. Overexpression of LRAT-RDH12 gene can inhibit the development of cervical cancer. These two genes are expected to become a new target for cervical cancer treatment.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.33

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