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RDHl2 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RDHl2 过表达载体并转染宫颈癌细胞系， 瞬时转染LRAT 和RDHl2 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、RDHl2 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/RDHl2 与宫颈癌的临床病理因素的关系， 旨在阐明LRAT/RDHl2 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。

BACKGROUND AND PURPOSE: Cervical cancer is one of the most common cancers in females. It has been demonstrated that HPV infection alone is insufficient while the abnormal expression of related genes is critical in development of cervical cancer. Our studies have screened the differential expressed genes between cervical cancer tissues and matched adjacent non-·tumor tissues by RNA-Seq technology. We discovered that the expression of the retinol metabolism related genes (LRAT and RDH12) were lower in cervical cancer tissue. In our experiments in vitro, we found that overexpression of LRAT、RDH12 helps inhibiting the cell proliferation, transferation and invation. In the cervical cancer cells with overexpression of LRAT/RDH12, the expression of proteins involved in MAPK/NF-kB signal pathway changed accordingly. The expression of LRAT/RDH12 increased, the expression of P38 MAPK/NF-kB decreased. In this study, we are trying to carry on experiments in vivo, verify the relation between LRAT/RDH12 gene and the MAPK/NF-kB signal pathway. By establishing the nude mouse model, this project aims to analyze the tumor growth curve in nude mice injected with Hela cell of LRAT/RDH12 overexpression. In order to clarify the effect and mechanism of retinol metabolism related genes LRAT/RDH12 in the progress of cervical cancer, the study will also draw the tumor tissues from nude mice to make immunohistochemistry analysis and western blot experiments. In this way, this study can provide important theoretical supports and molecular targets for cervical cancer. METHODS: 1. Growing the stably transfected cell: using recombinant plasmid to culture two kinds of Hela cell lines stabled transfected by LRAT and RDH12 gene. 2. Establishing the nude-mouse transplanted tumor model: using original Hela cell in control group to explore the best concentration of Hela cell to form tumor tissues. We make different concentration of Hela cell suspension: 5×106/mL、1×107/mL、5× 107/mL 和1×108/mL. 3. Observing the tumor growth condition. Recording the volume size of tumor tissues and drawing the tumor growth curve. 6 4. Drawing the tumor tissues from nude mice to make immunohistochemistry analysis and western blot experiments. Verifying the relation between LRAT/RDH12 gene and expression of P38, P50 and P65 in the MAPK/NF-kB signal pathway. RESULTS: Culturing and acquiring the Hela cell lines stabled transfected by LRAT and RDH12 gene. By qPCR technique, we verify that the overexpression of LRAT gene in stably transfected Hela cell is over 20 thousand times than blank control group, while the overexpression of RDH12 gene is over 30 thousand times. Putting two kinds of Hela cell above into western blot experiment, we get the expected results: in the two kinds Hela cells stabled transfected by LRAT and RDH12 gene, the expression of P38, P50 and P65 in the MAPK/NF-kB signal pathway are all decreasing. In establishing nude-mouse transplanted tumor model, We get the 5×107/mL as the best concentration of Hela cell suspension to form tumor tissues. The nude-mouse transplanted tumor model is built successfully. By observing and recording the tumor growth condition, we discovered the nude mice in LRAT and RDH12 group has slower tumor growth rate than control group of original Hela cell. The inhibiting effect of LRAT gene is stronger than RDH12 gene. Drawing the tumor tissue of 3 weeks from nude mice in control group and two experimental group, carrying on immunohistochemistry analysis and western blot experiments in three groups. The results show the overexpression of LRAT/RDH12 gene can reduce the expression of P38, P50 and P65. In this way, the activity of MAPK/NF-kB signal pathway can be suppressed. CONCLUSION: The overexpression of LRAT/RDH12 gene can effectively inhibit the development of cervical cancer. This phenomenon is related with suppressing the protein in MAPK/NFkB signal pathway. LRAT and RDH12 gene have the potential to be the new targets for cervical cancer treatment in the future.