CAJ | 학술논문

目的研究原癌基因受体酪氨酸激酶(recepteur d'origine nantais,RON)对Burkitt淋巴瘤细胞株Raji侵袭性行为的影响及单克隆抗体Zt/f2(2F2)的靶向抑制效应.方法采用RON特异性配体巨噬细胞刺激蛋白(macrophage stimulating protein,MSP) (2.0 nmol/L)和Zt/f2 (2F2)(2.0 nmol/L)处理Raji细胞24和72 h,采用MTT比色法研究对Raji细胞增殖的作用；采用甲基纤维素半固体培养法研究对Raji细胞克隆形成的作用；通过Annexin Ⅴ/PI双染流式细胞术检测对Raji细胞凋亡和细胞周期的影响；采用Western blot法检测RON的表达和凋亡相关蛋白及周期蛋白的改变.结果与空白对照组细胞活性(1.00)和集落数(103.6±7.0)比较,MSP处理72 h后Raji细胞活性为1.35±0.20,集落数为133.7±10.4,而Zt/f2 (2F2)单抗处理组细胞活性为0.68±0.11,集落数为66.3±6.1,差异均有统计学意义(P值均＜ 0.05)；Zt/f2(2F2)单独处理组细胞凋亡率为(12.16±2.33)％,G0/G1期细胞所占比例为(54.96±3.70)％,与对照组细胞凋亡率[(2.89±1.03)％]、G0/G1期细胞所占比例[(39.10±2.30)％]比较差异均有统计学意义(P值均＜ 0.05)；Zt/f2 (2F2)可以显著降低MSP刺激引起的RON磷酸化水平以及β-catenin蛋白的表达,且使凋亡蛋白caspase-3、-8、-9和多聚ADP核糖聚合酶表达水平增高,抗凋亡基因MCL-1和凋亡抑制蛋白XIAP表达水平下降,而且G1期周期蛋白相应改变.结论 RON激活后促进Raji细胞恶性生物学行为；Zt/f2 (2F2)可通过抑制Raji细胞RON磷酸化水平改变其G1期周期蛋白和相关凋亡蛋白的表达水平,实现抑制细胞增殖和诱导凋亡的目的.
목적 연구원암기인수체락안산격매(recepteur d'origine nantais,RON)대Burkitt림파류세포주Raji침습성행위적영향급단극륭항체Zt/f2(2F2)적파향억제효응.방법 채용RON특이성배체거서세포자격단백(macrophage stimulating protein,MSP) (2.0 nmol/L)화Zt/f2 (2F2)(2.0 nmol/L)처리Raji세포24화72 h,채용MTT비색법연구대Raji세포증식적작용；채용갑기섬유소반고체배양법연구대Raji세포극륭형성적작용；통과Annexin Ⅴ/PI쌍염류식세포술검측대Raji세포조망화세포주기적영향；채용Western blot법검측RON적표체화조망상관단백급주기단백적개변.결과 여공백대조조세포활성(1.00)화집락수(103.6±7.0)비교,MSP처리72 h후Raji세포활성위1.35±0.20,집락수위133.7±10.4,이Zt/f2 (2F2)단항처리조세포활성위0.68±0.11,집락수위66.3±6.1,차이균유통계학의의(P치균＜ 0.05)；Zt/f2(2F2)단독처리조세포조망솔위(12.16±2.33)％,G0/G1기세포소점비례위(54.96±3.70)％,여대조조세포조망솔[(2.89±1.03)％]、G0/G1기세포소점비례[(39.10±2.30)％]비교차이균유통계학의의(P치균＜ 0.05)；Zt/f2 (2F2)가이현저강저MSP자격인기적RON린산화수평이급β-catenin단백적표체,차사조망단백caspase-3、-8、-9화다취ADP핵당취합매표체수평증고,항조망기인MCL-1화조망억제단백XIAP표체수평하강,이차G1기주기단백상응개변.결론 RON격활후촉진Raji세포악성생물학행위；Zt/f2 (2F2)가통과억제Raji세포RON린산화수평개변기G1기주기단백화상관조망단백적표체수평,실현억제세포증식화유도조망적목적.
Objective To study the proto-oncogene RON mediated aggression of Raji cells and the inhibitory effects by monoclonal antibody Zt/f2 (2f2).Methods The effects of RON ligand macrophage stimulating protein (MSP) (2.0 nmol/L) and inhibitory Zt/f2 (2F2) (2.0 nmol/L) antibody on proliferation of RON positive Raji cells after treatment for 24 and72 hours were detected by MTT method,colony formation units (CFU) of Raji cells by methylcellulose semi solid culture,Raji cells apoptosis and cell cycle analysis by Annexin Ⅴ/Pl double staining,expression of RON,apoptosis-related proteins,and cyclins by Western blot.Results ①Compared with the cell viability (1.0) and counts of CFU (103.6±7.0) in control group,Raji cells after MSP treatment had better viability (1.35±0.20) and CFU counts (133.7± 10.4) (P＜0.05),but worse viability (0.68±0.11) and CFU counts (66.3±6.1) after Zt/f2 (2F2) treatment (P＜0.05).②Percentage of Raji cells apoptosis after Zt/f2 (2F2) antibody treatment (12.16±2.33)％ was significantly increased than the control (2.89± 1.03)％ (P＜0.05).The percentage of Raji cells arrested in G0/G1 phase was increased after Zt/f2 (2F2) antibody treatment as compared to the control [(54.96± 3.70)％ vs (39.10±2.30)％,(P＜0.05)].③ High-level ofRON phosphorylation and β-catenin expression activated by MSP could be inhibited significantly by Zt/f2 (2F2),which also up-regulated the expression of caspase-3,caspase-8,caspase-9 and PARP and down-regulated anti-apoptotic MCL-1 gene and inhibitor of apoptosis protein XIAP expression,accompanied with G1 phase protein changes accordingly.Conclusion MSP could aggravate Raji cells proliferation.Inversely,Zt/f2 (2F2) could inhibit proliferation and induce apoptosis by inhibition of RON phosphorylation and up-regulation of apoptosis related proteins.