摘要
目的研究低氧状态下乙酰肝素酶（heparanase，Hpa）反义寡核苷酸（antisenseoligodeoxynu—cleotide，AS—ODN）对胰腺癌高转移细胞株MIAPaCa-2细胞基质金属蛋白酶．2（matrixmetalloproteinase．2，MMP-2）和MMP-9的影响。方法以HpaAS—ODN预先阻断MIAPaCa-2细胞Hpa表达并低氧培养，观察MMP-2、MMP-9mRNA和蛋白表达的变化，明胶酶法检测MMP-2和MMP-9活性的变化，观察阻断Hpa对MMP-2和MMP-9表达及活性的影响。结果低氧培养条件下，MIAPaCa-2细胞MMP-2mRNA及蛋白表达略有上调，但是与常氧条件下培养对比，在6、12和24h差异均无统计学意义（P值均〉0．05）。以ASODN阻断Hpa后，MMP-2mRNA及蛋白表达均无明显变化。低氧后6hMMP-9mRNA及蛋白表达开始升高，12h（P〈0．05）和24h（P〈0．01）更加明显。阻断Hpa表达后，MMP-9mRNA及蛋白表达在各时间段均有下降，而在24h下降明显，差异具有统计学意义（P〈0．01）。低氧后12h和24h，活化型的MMP-2和MMP-9均显著增强，当Hpa表达被抑制后，于12h（P〈0．01）和24h（P〈0．01）活化型的MMP-9活性受到明显抑制，而各时间内，活化型的MMP-2活性均不受HpaAS—ODN的影响（P〉0．05）。结论低氧可刺激MIAPaCa-2细胞MMP-9mRNA及蛋白的表达，增强酶活性。预先阻断Hpa表达在12h和24h可抑制MMP-9mRNA和蛋白的表达，降低MMP-9的活性，而对MMP-2则无明显影响。
Objective To investigate the expression of matrix metalloproteinase-2 （MMP-2）and matrix metalloproteinase-9 （MMP-9）in MIA PaCa-2 cells blocked by AS-ODN cultured in hypoxia. Methods Heparanase（Hpa） expression of MIA PaCa-2 cells was blocked by AS-ODN and cultured in hypoxia. The expression of MMP-2 and MMP-9 mRNA and proteins in cell lysate was evaluated by RT-PCR and Western blot respectively, and the enzymatic activities of MMP-2 and MMP-9 in supernatants were detected by gelatinase activity assay. Re suits Hypoxia stimulated mRNA and protein expression of MMP-9 in cultured MIA PaCa-2 cells and elevated at 6h, 12 h（P 〈0. 05）and 24 h（P 〈 0. 01 ）. When Hpa expression was inhibited by AS-ODN, the expression of MMP-9 mRNA and protein as well as the gelatinase activity in supernatant decreased dramatically at 12 h and 24 h, especially at 24h（P 〈 0. 01 ） , however, no significant difference of MMP-2 expression and gelatinase activity was observed after AS-ODN transfeetion. （ P 〉 0. 05 ）. Conclusion In hypoxia, MMP-9 expression, either mRNA or protein in cultured MIA PaCa-2 cells, increased gradually accompanied with elevated gelatinase activities. When the heparanase expression was inhibited, the MMP-9 mRNA and protein, as well as the gelatinase B activity in supernatant, were decreased dramatically at 12h and 24h, however, no significantly differences of MMP-2 expression and gelatinase A activity were observed after the AS-ODN transfection.
出处
《中华内分泌外科杂志》
CAS
2013年第6期473-477,共5页
Chinese Journal of Endocrine Surgery
基金
基金项目：山东省烟台市科学技术发展项目（2006131-15）
关键词
胰腺癌
乙酰肝素酶
基质金属蛋白酶
寡核苷酸类
反义
Pancreatic carcinoma
Heparanase
Matrix Metalloproteinase
Oligodeoxynucleotide
Antisense
