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学科主题: 骨科学
题名:
SOX2对尤文肉瘤的作用与机制研究
作者: 任翀旻
答辩日期: 2016-05-11
导师: 郭卫
专业: 骨科学
授予单位: 北京大学
授予地点: 北京大学第二临床医学院
学位: 博士
关键词: 尤文肉瘤 ; SOX2 ; 细胞周期 ; 凋亡 ; PI3K/Akt信号通路
其他题名: The Effect and Mechanism of SOX2 on Ewing's sarcoma progression
分类号: R738.6
摘要:

研究背景

 

尤文肉瘤是一种具有高度侵袭性的骨与软组织恶性肿瘤。由于其恶性程度高、 预后差等因素,用于靶向干预的新型肿瘤标志物位点亟待研究和发现,从而改善 和提高尤文肉瘤的治疗。EWS/FLI1融合基因是尤文肉瘤最具标志性的肿瘤生物 学特征,与尤文肉瘤的肿瘤起源密切相关。作为干细胞转录因子,SOX2在间充 质干细胞中被EWS/FLI1诱导表达并受其调控。SOX2能够影响许多类型肿瘤的进 展,如维持肿瘤干细胞特性、促进肿瘤细胞增殖和迁移。在肿瘤细胞增殖的调控 中,SOX2以促进细胞周期进展和抗凋亡为主要作用方式,同时涉及了多种信号 通路调控。然而,SOX2在尤文肉瘤中的具体作用调节机制目前仍有待研究。

研究方法

系统性从体内和体外实验层面,检测SOX2在尤文肉瘤细胞系、组织标本及 肿瘤移植模型内的表达,研究了SOX2影响尤文肉瘤进展的作用机制。

SOX2表达情况采用Western Blot和免疫组织化学技术在尤文肉瘤细胞系和 组织标本中检测。尤文肉瘤细胞系选用A673和RD-ES作为实验对象。利用siRNA 对EWS/FLI1和SOX2基因进行基因敲减,并采用实时定量PCR和Western Blot进行 RNA和蛋白层面的效率验证。SOX2对细胞周期和细胞凋亡的作用现象采用流式 细胞术和TUNEL实验进行检测,并且检测了相关调控蛋白以明确调节机制。Akt 过表达试验中利用了质粒转染技术。在具体调节蛋白的表达水平变化检测上,采 用Western Blot实验进行分析。SOX2体内实验在裸鼠模型上进行,利用siRNA瘤 内注射干扰SOX2基因表达。异体肿瘤生长变化情况在体内实验过程中测量,相关蛋白表达变化在模型处死后利用免疫组织化学与Western Blot进行检测分析。

研究结果

实验分四部分进行,分别研究了SOX2与尤文肉瘤的相关性、SOX2对尤文肉 瘤细胞增殖和迁移的作用、SOX2调控尤文肉瘤细胞周期和细胞凋亡的具体机制 和体内相关作用与机制。

首先验证得到SOX2是EWS/FLI1融合基因的下游靶点,并且在尤文肉瘤细胞 和组织内高表达。生物学作用方面,敲减SOX2能够显著抑制尤文肉瘤细胞的增 殖和迁移能力。在细胞周期层面,SOX2通过调节p21、p27和cyclin-E促进尤文肉 瘤细胞由G0/G1期向S期的转化;在细胞凋亡层面,SOX2通过外源性和内源性凋 亡通路,即通过Fas、caspase-8和Bad、Bcl-2、XIAP、caspase-9等凋亡因子,调 控caspase-3的活性,影响PARP对细胞凋亡的作用。因此,抑制尤文肉瘤细胞SOX2 表达能诱导细胞周期阻滞与细胞凋亡的发生。此外,SOX2对细胞周期和凋亡的 调控有PI3K/Akt信号通路进行参与。最后,SOX2对尤文肉瘤细胞的生存维持和 生长促进的作用机制在体内实验也得到了验证。

结论

实验证明了SOX2在尤文肉瘤细胞增殖、迁移、细胞周期和凋亡调控中的关 键作用,在体内和体外实验中阐明了相关的分子调节机制。研究结果表明,SOX2 可以作为一个潜在的生物标记靶点用于尤文肉瘤的靶向干预治疗,同时为更进一 步的研究提供了详实的实验基础。

英文摘要:

Background

Ewing’s sarcoma is an aggressive bone and soft tissue tumor with a high incidence in children and adolescents. Due to its high malignancy and poor prognosis, identification of novel biomarkers for intervention therapies is necessary to improve outcome. The EWS/FLI1 fusion gene is a acteristic of Ewing’s sarcoma in most cases. Sex determining region Y-box 2 (SOX2) is a primary target of EWS/FLI1. It has been identified as an oncogene and linked to apoptotic resistance in several types of cancer. However, its role and regulatory mechanisms in Ewing’s sarcoma are largely unknown.

Methods

We systematically investigated the role of SOX2 in Ewing’s sarcoma cell lines, human tissue samples and xenograft models.
The expression of SOX2 was detected in Ewing’s sarcoma samples by WB and IHC . siRNAs were used to knockdown EWS/FLI1 and SOX2 in A673 and RD-ES cell lines with the efficiencies tested by qRT-PCR and WB. The effect of SOX2 on cell cycle and apoptosis was determined by Flow cytometric assay and TUNEL. Akt overexpression was performed with plas. The protein expression of corresponding factors were examined by WB analysis. The SOX2 inhibition in vivo was performed by siRNA against SOX2 in xenograft models, and the protein expression of the regulators testified in vitro was examined in xenograft tumors by IHC and WB.

Results

The results confirmed that SOX2 was highly expressed in Ewing’s sarcoma and was the target of EWS/FLI1. SOX2 advanced Ewing’s sarcoma cell proliferation and migration. SOX2 facilitated G1/S phase transition of Ewing’s sarcoma cells by regulating p21, p27 and cyclin-E, and restrained cell apoptosis by mediating caspase-3, PA !E and caspase-8) and intrinsic (caspase-9, Bad, Bcl-2 and XIAP) apoptotic pathways. Inhibition of SOX2 induced G1/S arrest and cell apoptotsis of Ewing’s sarcoma cells. Additionally, SOX2 regulated the cell-cycle progression and apoptosis via activation of the PI3K/Akt signaling pathway. The mechanism was both proved in vitro and in vivo.

Conclusions

Our investigations demonstrate that SOX2 played a central role in promoting Ewing’s sarcoma cell proliferation with the specific mechanisms proved both in vitro and in vivo. These findings suggest that SOX2 may serve as a potential biomarker for targeted intervention in Ewing’s sarcoma and have provided an experimental basis for future investigations.

语种: 中文
相关网址: 查看原文
内容类型: 学位论文
URI标识: http://ir.bjmu.edu.cn/handle/400002259/124604
Appears in Collections:北京大学第二临床医学院_学位论文

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作者单位: 北京大学第二临床医学院

Recommended Citation:
任翀旻. SOX2对尤文肉瘤的作用与机制研究[D]. 北京大学第二临床医学院. 北京大学. 2016.
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