【作者】 吴姗；

【导师】 徐恩萍；

【作者基本信息】 浙江大学 ， 病理与病理生理学， 2011， 硕士

【摘要】 大肠癌是严重危害人类健康的恶性肿瘤之一。随着人类基因组计划的完成和基因技术的不断进步,基因治疗已成为大肠癌治疗的新热点,并从理论走向实践,其有效性已在细胞学和动物模型上得到验证。1998年RNA干扰(RNA interference,RNAi)的发现,特别是在哺乳动物细胞应用小干扰RNA成功介导RNAi,为肿瘤的治疗提供了强有力的工具。对于RNAi在大肠癌基因治疗中的应用来讲,靶点的选择是最重要的。SOX9基因是我们实验室运用生物信息学方法在结直肠癌正常粘膜和其配对癌组织中首次筛选到的一个在大肠癌中表达上调的蛋白,并经半定量RT-PCR、实时荧光定量PCR、免疫印迹、免疫组织化学反馈性验证。同时免疫组织化学结果表明SOX9高表达是一个独立的预后不良指标。另外已有研究支持SOX9是Wnt信号转导通路的下游基因,其可能通过抑制细胞分化参与结肠癌的发生、发展。因此我们推测SOX9很可能是大肠癌发生、发展中一个新的促癌基因。SOX是一类SRY(sex determination region of Y chromosome)相关基因构成的控制发育的.基因家族,编码一系列SOX家族的转录因子,其所有成员的共同特点是含有一个高度保守的HMG-box(high mobility group box)DNA结合域。SOX9可能是目前SOX家族中研究最为广泛的转录因子之一,是人体早期胚胎发育重要的相关基因,该基因的突变或重复可导致性别反转或CD综合症。近年来研究发现SOX9在脊椎动物的发育过程中起着重要作用,尤其在骨骼、生殖腺、神经胶质以及心脏的发育过程中发挥重要功能,同时SOX9的异常表达可能与人类多种肿瘤有关,研究显示SOX9在大肠癌、软骨肉瘤、前列腺癌、乳腺癌、卵巢癌、肺癌、基底细胞癌、膀胱癌及黑色素瘤等肿瘤组织中表达异常,并与肿瘤细胞的增殖分化、侵袭转移、凋亡等密切相关。SOX9在结直肠癌组织中的表达相对于正常组织来说较高,并且结直肠癌细胞系中SOX9的表达也较高,这个现象可能预示着SOX9表达升高可能与肿瘤的发生相关。这个现象给了我们一个思路：将高表达SOX9的结直肠癌细胞通过RNAi方法使得SOX9的表达降低后,这些细胞会产生怎样的变化?我们选用了具备不同遗传学背景的SW480和RKO细胞作为我们的细胞模型。本实验通过构建SOX9干扰质粒载体、转染细胞及稳筛的方法沉默结直肠癌细胞中SOX9的表达,并分别对SW480和RKO的空白对照组(B)、空质粒组(E)及转染组(S)的生物学行为进行了检测分析,包括细胞增殖实验、凋亡相关检测、迁移实验,并建立裸鼠人大肠癌移植模型,体内评价SOX9基因沉默对大肠癌实体瘤的靶向抑瘤效果,为SOX9作为临床基因治疗新靶点提供理论依据。MTT实验结果分析显示：转染了SOX9干扰质粒的SW480和RKO细胞的生长速度要明显慢于空白对照组和转染空载体对照组。并且通过实时定量仪检测细胞增殖速度,同样得到RKOS和SW480S组细胞相对于对照组增殖速度减慢。提示细胞中SOX9表达上调后可能通过加快细胞的增殖速率促进肿瘤的形成。PI/Annexin V双染的流式细胞术检测发现：转染了SOX9干扰质粒的SW480和RKO细胞凋亡率都明显增高,提示SOX9的沉默在结直肠癌细胞中可以诱导细胞的凋亡。细胞迁移实验表明：SOX9对结直肠癌SW480的迁移能力没有影响。裸鼠成瘤实验结果显示：将SW480B、SW480E和SW480S细胞分别注射入裸鼠体内,SW480S组的成瘤率相对于SW480B、SW480E组减少,但瘤体积并未有差异。通过以上关于SOX9沉默对结直肠癌细胞系生物学功能影响的初步研究,我们得出以下结论：SOX9沉默对结直肠癌细胞具有抑制生长、诱导凋亡的作用,表明其本身在结直肠癌中可能扮演着促癌基因的角色。更多还原

【Abstract】 Colorectal cancer (CRC) is one of the most common malignancies which threaten our health. Since the completion of the Human Genome Project and the development of gene-tech, gene therapy has become the focus of medical research, whose effectiveness has been verified in cytology and animal models. In 1998, the discovery of RNA interference provided a strong tool for tumor treatment.Selecting a target is the most important in the gene therapy of colorectal cancer. Our laboratory analyzed the expression of SOX9 gene in CRC by the methods of bioinformatics, showing that it was upregulated in most colorectal cancers. By reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining, we confirmed the overexpression of SOX9 in CRC tissues, compared to the paired normal mucosa. Also, we found the high expression of SOX9 in CRC cell lines. Meanwhile the results showed that high expression of SOX9 was an independent adverse prognosticator. Other studies supported that SOX9, a downstream gene of Wnt signalling pathways, might participate in the occurrence and development of colorectal cancer by inhibiting cell differentiation. So we speculated that SOX9 was probably a new cancer-promoting gene in colorectal cancer. SOX family is a kind of SRY related genes, encoding a series of transcription factors. All of its members contain a highly conservative HMG-box DNA-binding domain. SOX9 might be the most widely researched of the rapidly growing family. SOX9 was also expressed during organ development. SOX9 was shown to play an important role in both chondrogenesis and vertebrate sex determination. Meanwhile the abnormal expression of SOX9 might relate to multiple tumors.Reseaches showed that-SOX9 expressed abnormally in colorectal cancer, chondrosarcomas, prostate cancer, breast cancer, and bladder cancer.Also, it is closely related with cell proliferation and differentiation, invasion, metastasis, and apoptosis.Relative to the normal tissues, the expression of SOX9 is higher in colorectal cancer tissues. In addition, SOX9 was found in higher levels in many colorectal cancer cell lines. This phenomenon may indicate that an increasing expression is probably associated with tumorigenesis. The phenomenon provides us a way to study SOX9: What will the cancer cells change with SOX9 gene knockdown? We choose SW480 and RKO as our cell models. In this experiment, we constructed a small interfering (siRNA) SOX9 expression plasmid and transferred it into SW480 and RKO colorectal cancer cells. We detected the expression of SOX9 with the methods of Realtime-PCR and Westernblot. On this basis, we studied the changes of tumor cell growth, apoptosis, and other biological behaviours in SOX9 gene silencing cells.Reasults:1. There was significant difference in the proliferation ability of the cells. It was suggested that SOX9 gene silencing inhabited cell growth.2. Interference of SOX9 could induce apoptosis in SW480 and RKO cell lines.3. Downregulation of SOX9 had little effect on migration ability.4. In vivo, interference of SOX9 in SW480 could suppress tumorigenicity. Conclusions:SOX9 gene silencing can inhibit cell growth and induce apoptosis, indicating SOX9 maybe play a role as a cancer-promoting gene in colorectal cancer.更多还原