【作者】 苏颖洁；

【导师】 黄杰安；

【作者基本信息】 广西医科大学 ， 消化内科， 2012， 硕士

【摘要】 目的研究鞘氨醇激酶1(SphK1)和黏着斑激酶(FAK)在结肠癌中的表达及其与临床病理特征的关系,并探讨其在结肠癌发生发展中的作用及其可能的分子机制。方法①采用免疫组织化学SP法检测66例结肠癌、对应癌旁及正常结肠黏膜组织中SphK1和FAK的表达；用统计学方法分析二者表达与临床病理特征的关系。②体外培养人结肠癌细胞株lovo,用终浓度为100nmol/L的佛波醇-12-豆蔻酸酯-13-乙酸酯(PMA)以及终浓度为50μmol/L的N,N-二甲基鞘胺醇(DMS)分别作用于人结肠癌lovo细胞,一定时间后,采用MTT法和克隆形成实验检测细胞的增殖能力,Tranwell小室模型观察细胞迁移和侵袭能力的变化,RT-PCR检测FAKmRNA的表达,Western blot检测FAK蛋白的表达。结果①组织免疫组化结果显示：Sphk1和FAK蛋白在结肠癌及其癌旁、正常组织中的表达阳性率分别为：72.7%(48/66)、51.5.%(34/66)、34.8%(23/66)；77.3%(51/66)、37.9%(25/66)、22.7%(15/66),Sphk1和FAK在结肠癌与癌旁及正常结肠黏膜组织的阳性率差异均有统计学意义(P值<0.05)。SphK1和FAK表达之间存在明显关联性(r=0.480,P=0.000)。SphK1和FAK在癌组织中的表达水平均与肿瘤浸润程度、组织分化程度、有无远处转移、淋巴结转移、临床分期有关(P值均<0.05)。②体外实验结果显示：PMA显著促进细胞的增殖、迁移和侵袭能力,DMS则显著抑制细胞的增殖、迁移和侵袭能力(对照组、PMA组和DMS组的细胞增殖活力分别为：0.71±0.03vs1.05±0.05vs0.46±0.04；克隆形成率分别为：1.32%±0.26%vs2.17%±0.17%vs0.73%±0.13%；迁移细胞数分别为：72.19±3.36vs98.46±6.25vs40.48±4.27；侵袭细胞数分别为：75.48±4.12vs143.36±5.73vs38.57±3.24；P值均<0.05vs对照组)。PMA显著促进黏着斑激酶(FAK)的活性和表达,相反DMS则抑制FAK的活性和表达(对照组、PMA组和DMS组FAK mRNA的表达强度：0.151±0.008vs0.212±0.014vs0.114±0.021；蛋白：0.332±0.022vs0.374±0.029vs0.296±0.018；磷酸化FAK(p-FAK Tyr397)蛋白：0.186±0.032vs0.234±0.017vs0.112±0.023；P值均<0.05vs对照组)。结论1.SphKl和FAK可能与结肠癌的发生、发展密切相关,并可能在结肠癌的浸润和转移中起重要作用。2. SphK1可促进lovo细胞的增殖、侵袭和迁移能力,其机制可能是通过激活FAK通路而发挥作用。更多还原

【Abstract】 Objective To study the expression of sphingosine kinase1(SphK1) and Focal Adhesion Kinase (FAK) in colon carcinoma tissues and to explore their correlation with clinicopathological features. Then further investigate their relationship with the cell adhesion, migration and invasion of human colon cancer cell line lovo and its associated mechanism through vitro examination.Methods①Sixty-six paraffin-embedded colon carcinoma samples were tested with immunohistochemistry method, stactistical analysis was then carried out to explore their expression in clinicopathological features.②Cultured lovo cells were divided into three groups:PMA group, DMS group and control group. Cells of PMA group were treated with100nmol/L Phorbol12-myristate13-acetate (PMA), the DMS group was treated with50μmol/L N, N-dimethylsphingosine (DMS), while the control group was treated with equal volume of culture medium. After treatment, cell proliferation was detected by MTT assay and colony formation assay, the migration and invasion capability of the cells were assessed in Transwell chambers. RT-PCR and Western blot were used to evaluate the mRNA and protein expression of FAK.Results①The immunohistochemistry results show:the positive rates of SphKl and FAK in66colon carcinoma samples were72.7%(48/66) and77.3%(51/66), which were higher than those in adjacent tissues[SphK1:51.5%(34/66); FAK37.9%(25/66)] and normal mucosa[SphK1:34.8%(23/66); FAK:22.7%(15/66)], showing a significant statistical difference(P<0.05). There was a close correlation between SphKl and FAK expression levels (r=0.480, P=0.000). Overexpression of SphKl and FAK in colon carcinoma were all related with depth of invasion, differentiation, distant and lymph node metastasis and Dukes’stages (P<0.05).②Vitro tests’results show that PMA significantly enhanced cells proliferation, invasion and migration, whereas DMS suppressed cells proliferation, migration and invasion (the cells viability, cloning rate, migration and invasion cell count of control group, PMA group and DMS group were as follow:cells viability:0.71±0.03vs1.05±0.05vs0.46±0.04; cloning rate:1.32%±0.26%vs2.17%±0.17%vs0.73%±0.13%; migration cell count:72.19±3.36vs98.46±6.25vs40.48±4.27; invasion cell count:75.48±4.12vs143.36±5.73vs38.57±3.24; all P<0.05vs control group). PMA significantly up-regulated the expression and activity of focal adhesion kinase (FAK), while DMS down-regulated the expression and activity of FAK (FAK mRNA:0.151±0.008vs0.212±0.014vs0.114±0.021; FAK protein:0.332±0.022vs0.374±0.029vs0.296±0.018; phosphor-FAK protein:0.186±0.032vs0.234±0.017vs0.112±0.023; P<0.05vs control group).Conclusions1. Overexpression of SphK1and NF-κB may be involved in the occurrence and development of colon carcinoma. Moreover, SphKl and NF-κB may be correlated with the invasion and metastasis of colon carcinoma.2. SphKl enhances cell proliferation, migration and invasion in human colon cancer cell line lovo possibly by activating FAK.更多还原