骨桥蛋白反义寡核苷酸抑制胃癌细胞MKN28粘附侵袭的实验研究

目的：骨桥蛋白（Osteopontin，OPN）是一种分泌型、粘附性的糖基化磷蛋白，许多肿瘤在发生发展过程中均伴有OPN的表达，而表达OPN的肿瘤也更易表现出侵袭、转移的倾向。本研究将观察OPN反义寡核苷酸（antisense oligonucleotide，ASODN）抑制高分化人胃癌细胞株MKN28粘附侵袭的作用。方法：用脂质体将骨桥蛋白ASODN转染胃癌细胞MKN28，实验分3组，对照组、随机序列寡核苷酸（random oligonucleotide，rODN）组和ASODN组。通过荧光定量RT—PCR、免疫组化方法、粘附细胞计数法、穿透小室聚碳酸酯膜侵袭实验分别测定骨桥蛋白mRNA和细胞表面骨桥蛋白的蛋白表达及癌细胞粘附力、侵袭力的变化。结果：转染后ASODN组骨桥蛋白mRNA的相对定量比值较对照组显著降低（1．83±0．10m3．32±0．16，P〈0．05）；免疫组化检测骨桥蛋白表达结果显示，转染后荧光强度明显降低，细胞表面骨桥蛋白表达下降（P〈0．01）；转染后ASODN组粘附力和侵袭力均较对照组显著下降（粘附细胞百分数为20．50±4．58vs41．50±8．40，穿透聚碳酸酯膜细胞数为21．80±6．90vs．42．00±9．40，P均小于0．01）。而rODN组上述各指标较对照组无明显变化。结论：骨桥蛋白的表达与粘附侵袭能力密切相关，反义寡核苷酸能有效抑制胃癌细胞株MKN28细胞骨桥蛋白基因的表达，从而抑制蛋白质的合成，降低其粘附侵袭能力。

Inhibitory effect of osteopontin antisense oligonucleotide on adhesion and invasion of gastric cancer cell line MKN28

Objective： Osteopontin （OPN） is a secretory adhesive glycosylated phosphoprotein. Many tumors express OPN during the development and progression period simultaneously, which is suggested be related with the tendency of invasion and metastasis. In this study, we observe the effect of OPN antisense oligonucleotide（ASODN） on the adhesion and invasion of highly differentiated human gastric cancer cell line MKN28. Methods：Three experimental groups were defined as ASODN group, random oligonucleotide （rODN） group and control group. ASODN was transfected into MKN28 cells by liposome. The expression of osteopontin mRNA and protein were determined by fluorescent quantitative RT-PCR and immunohistochemistry respectively. The abilities of adhesion and inva- sion were measured by counting adhesive cells and transwell polycarbonate filters invasive test. Results：Relative mRNA level of OPN was significantly lower in ASODN group than in control group（ 1.83±0. 10 vs. 3.32±0. 16, P 〈0. 05 ）. The mean fluorescent index of ASODN group detected by immunohistochemistry was evidently decreased than that of control group（ P 〈 0. 01 ）. The adhesive and invasive ability of ASODN group decreased more than control group（The ratio of adhesive cells was 20. 50% ± 4. 58% vs. 41.5% ± 8. 4% , and the number of cells that infiltrated the polycarbonate membrane was 21.8 ± 6. 9 vs. 42. 0 ± 9. 4, both of P 〈 0. 01 ）. However, no difference about above-mentioned indexes existed between rODN group and control group. Conclusion：The expression of OPN in MKN28 cells was related with adhesion and invasion. ASODN could effectively inhibit expression of osteopontin gene, inhibit protein synthesis, decrease ability of adhesion and invasion.