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/LLY294002处理SW480细胞72h,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)基因水平.WesternBlot检测LY29400210μmo1/L处理SW480细胞24～72h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LY29400210μmo1/L干预72h后,VM密度由(12.50±3.27)个下降至(2.60±1.07)个(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).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,mmp2mRNA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.
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 LY294002 10 μmo1/L 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 LY294002 10 μmo1/L 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 LY294002 10 μmo1/L groups than in control groups[（0.31±0.01）vs（0.75±0.03）,（0.20±0.04）vs（0.55±0.05）,P〈0.01].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.
出处
《第四军医大学学报》
北大核心
2009年第22期2568-2571,共4页
Journal of the Fourth Military Medical University
