摘要
目的：探讨非催化区域酪氨酸激酶衔接蛋白1（Nck1）在宫颈鳞状细胞癌（CSCC）的血管生成中的作用及其分子机制。
方法：采用免疫组织化学和免疫印迹法分别检测CSCC标本和癌细胞中蛋白的表达。用CD34内皮标记结合Weinner计数法检测癌组织微血管密度（MVD）。用实时定量PCR检测癌细胞mRNA水平。分别通过表达质粒pCMV2-Nck1和Nck1-siRNA转染子宫颈鳞癌细胞获得Nck1基因过表达（SiHa-Nck1+）和基因沉默（SiHa-Nck1-）的细胞株。利用ELISA检测细胞上清液蛋白质水平。分别通过CCK-8细胞活力测定，transwell小室细胞迁移实验和体外Matrigel管腔实验分别检测人脐静脉内皮细胞（HUVECs）的增殖、迁移和管腔形成能力。
结果：Nck1的表达水平在正常宫颈上皮组织和低级别宫颈上皮内瘤变至高级别宫颈上皮内瘤变中逐渐升高。在宫颈鳞癌组织中Nck1的表达水平显著高于高级别上皮内瘤变，且Nck1的表达与宫颈鳞癌微血管密度正相关。与正常SiHa 上清液处理后的HUVECs相比，SiHa-Nck1+上清液处理后的内皮细胞的增殖能力、迁移能力和管腔形成能力显着增强，而SiHa-Nck1-上清液处理后的HUVECs 分别表现出显著降低的增殖、迁移和管腔形成能力。Nck1基因转染和siRNA分别导致SiHa中Rac1-GTP、p-PAK1和MMP2表达水平的上调和下降，而利用Rac1抑制剂（NSC23766）预处理降低SiHa-Nck1+中Rac1-GTP的同时，p-
柳月弯弯
PAK1和MMP2的水平也显著降低。并且在SiHa-Nck1+中利用PAK1抑制剂抑制PAK1的活化时，MMP2的水平也显著降低，而Rac1-GTP的水平却没有明显改变。此外，当SiHa细胞的Rac1或PAK1的活性受到抑制时，其上清液处理下的HUVECs 也相应地表现出较低的增殖、迁移和管腔形成能力。
蛋黄有什么营养结论：Nck1高表达于宫颈鳞癌，促进宫颈鳞癌的微血管生成，其分子机制与Nck1激活Rac1/PAK1/MMP2信号通路有关。
关键词：Nck1；微血管生成；MMP2；Rac1；PAK1；宫颈鳞癌
ABSTRACT
Objectives:The study was to explore the roles of Nck1 in the angiogenesis of cervical squamous cell carcinoma (CSCC) and the related moleclular mechanism.
Methods:The protein expression in CSCC samples and cancer cells was evaluated with immunohistochemisty and western blotting, respectively. The cancer microvesl density (MVD) was assayed with CD34 endothelial labeling and counted according to Weinner’s standard.Protein mRNA level was detected with real time quantitative PCR. Nck1 gene up-regulation(SiHa-Nck1+) and knockdown (SiHa-Nck1-) in cancer cells was achieved by gene transfection and siRNA, respectively.
简单汉字
Cellular supernatant protein level was measured with ELISA. Proliferation, migration and tube formation of the Human Umbilical Vein Endothelial cells (HUVECs) was evaluated by CCK-8 cell viability assay, transwell chamber assay and in vitro Matrigel tubulation assay, respectively.
Results: Nck1 showed gradually incread level from normal cervical epithelia and low-grade cervical intraepithelial neoplasia(LGCIN) to high-grade cervical intraepithelial neoplasia(HGCIN). The expression level of Nck1 in CSCC was significantly higher than that of HGCIN and was associated with cancer MVD. The HUVECs treated with supernatant of SiHa-Nck1+ showed significantly incread ability of proliferation, migration and tube formation than the HUVECs treated with normal SiHa supernatant, respectively. Compared with the HUVECs treated with normal SiHa supernatant, the HUVECs treated with supernatant of SiHa-Nck1- showed significantly decread ability of proliferation, migration and tube formation, respectively. Nck1 gene transfection and siRNA led to increa and decrea of Rac1-GTP、p-PAK1 and MMP2 level in SiHa, respectively. Furthermore, pretreatment with the Rac1 inhibitor (NSC23766) significantly decread the level of Rac1-GTP, p-PAK1 and MMP2 in the SiHa-Nck1+. PAK1 activation inhibited in SiHa-Nck1+, the MMP2 level was notably decread. However, the level of Rac1-GTP was not significantly changed. The cancer cells with inhibition of Rac1 or PAK1 also showed an impaired ability of proliferation, migration and tube formation.
Conclusions: Nck1 is highly expresd in CSCC and promotes the angiogenesis-inducing capacity. The molecular mechanism may be related to Nck1-mediated activation of the Rac1/PAK1/MMP2 signaling pathway.
人物三视图Key words: Nck1; angiogenesis; MMP2; Rac1; PAK1; CSCC
目录
第1 章引言 (1)
1.1 肿瘤微血管生成 (1)
1.2 NCK1 (2)
1.3 NCK1可能是调节宫颈鳞癌微血管生成的关键分子 (2)
第2 章材料与方法 (4)
2.1材料 (4)
2.1.1组织标本 (4)
2.1.2 细胞株 (4)
2.1.3实验试剂 (4)
2.1.4实验设备 (6)
2.1.5 实验试剂配制方法 (7)
2.2 实验方法 (8)
2.2.1 实验分组 (8)
2.2.2免疫组化 (8)
2.2.3 免疫组化测定方法及MVD计数方法 (9)
2.2.4 细胞培养 (9)
2.2.5 质粒转染 (11)
2.2.6 实时荧光定量PCR (12)
2.2.7 Western blot (13)
2.2.8 ElISA 实验 (16)
2.2.9 CCK8实验 (17)
2.2.10 Transwell 小室实验 (17)
2.2.11管腔形成实验 (17)
2.2.12 统计学分析 (18)阿密特2
第3章实验结果 (19)
3.1 NCK1在正常宫颈上皮、宫颈上皮内瘤变和宫颈鳞癌组织中的表达情况
以及与MVD的关系 (19)
3.2 Nck1促进SiHa细胞的血管生成诱导能力 (23)
3.3 Nck1增强CSCC的血管诱生能力与MMP2的上调有关 (26)
3.4 NCK1通过活化Rac1-PAK1信号上调SiHa细胞中MMP2的表达 (27)军匠>关于雪的句子
3.5 Nck1对CSCC血管诱生能力的调节作用依赖于Rac1-PAK1信号传导的
激活 (30)
第4 章讨论 (31)
第5 章结论 (34)
致谢 (35)
淡竹叶的功效
参考文献 (36)
攻读学位期间的研究成果 (40)
综述 (41)
