【作者】 田翀；

【导师】 高青；

【作者基本信息】 重庆医科大学 ， 内科学， 2010， 硕士

【摘要】 目的:探讨结肠癌血管生成拟态(VM)现象及其生成的相关机制。方法:体外三维培养结肠癌细胞株SW480,倒置显微镜下观察VM形成情况,透射电镜下观察其超微结构。采用MTT法检测磷脂酰肌醇-3激酶(PI-3K)信号通路抑制剂2-(4-吗啉基)-8-苯基-4氢-1-苯并吡喃-4-酮(LY294002 )对SW480细胞的增殖抑制率。10μmo1/L LY294002处理SW480细胞72 h,在光学显微镜下观察VM密度,并用逆转录聚合酶链技术(RT-PCR)检测骨桥蛋白(OPN)基因,基质金属蛋白酶2(MMP2)基因的水平。Western Blot法检测10μmo1/L LY294002处理SW480细胞24～72 h后蛋白激酶B (PKB),磷酸化蛋白激酶B (p-PKB)蛋白的表达。结果:结肠癌细胞株SW480在体外三维培养条件下能够形成VM,电镜观察结果进一步证实了VM的存在。LY294002作用后SW480细胞的生长明显减慢,抑制率为13.01%～67.55%( P<0.01)且呈良好的时间-剂量效应关系。SW480细胞经10μmo1/L LY294002干预72 h后,VM密度由12.50±3.27下降至2.60±1.07( P<0.01),opn,mmp2 mRNA的表达强度分别为0.31±0.01,0.20±0.04,低于未干预细胞的表达强度0.75±0.03,0.55±0.05 (P<0.01)。10μmo1/L LY294002处理SW480细胞24～72 h后,PKB蛋白表达下降( P<0.01), p-PKB蛋白表达水平无明显变化。结论:LY294002通过下调opn,mmp2 mRNA的表达,抑制结肠癌细胞VM的形成,此过程与抑制PI-3K/PKB通路有关。更多还原

【Abstract】 AIM: To explore whether vasculogenic mimicry (VM) exists in colon carcinoma and the possible mechanism.METHODS: Three-dimensional culture system of human colon carcinoma cell line SW480 was constructed to observe VM under inverted microscope, and the ultrastructure of VM was observed under trans-electron microscope. The proliferation inhibitory rates of SW480 cells induced by the specific inhibitor 2-(4-morpholinyl)-8-phenyl -4H-1-benzopyran-4-one (LY294002) of the signaling pathway phosphatidy1inosito1-3 kinase (PI-3K) were detected by Methyl thiazolyl tetrazolium (MTT) method. After SW480 cells were treated with 10μmo1/L LY294002 for 72 h, the density of VM was observed under light microscope. Moreover, the expressions of osteopontin (OPN), matrix metalloproteinase2 (MMP2) genes were assessed by reverse transcription-polymerase chain reaction (RT-PCR). After SW480 cells were treated with 10μmo1/L LY294002 for 24-72 h, the expressions of protein kinase B (PKB), phospho-protein kinase B (p-PKB) proteins were analyzed by Western Blot.RESULTS: During Three-dimensional culture, SW480 cells can form VM. The result of trans-electron microscope validated the presence of VM. After LY294002 treatment,the growth of SW480 cells was inhibited significantly in a time and dose dependent manner, and the inhibition rates were 13.01%-67.55% ( P< 0.01) .When treated for 72 h ,The density of VM was decreased from [( 12.50±3.27 ) to ( 2.60±1.07) ,P< 0.01] , the expressions of opn, mmp2 genes were lower in 10μmo1/L LY294002 groups than in control groups [ (0.31±0.01) vs (0.75±0 .03) , (0.20±0 .04 ) vs (0.55±0 .05 ), P<0.0 1 ]. The expression of p-PKB protein was down-regulated ( P<0.01), but it had no change on PKB.CONCLUSION: LY294002 can inhibit the formation of VM in colon carcinoma by reducing opn and mmp2 expression, which might be related to the inhibition of PI3K/PKB signaling pathway.更多还原