CAJ | 학술논문

目的探讨绿脓杆菌制剂(pseudomonas aeruginosa vaccine,PA)对有转移潜能的人肝癌MHCC97L细胞增殖和侵袭能力的影响.方法采用不同浓度的PA分别作用于MHCC97L细胞,观察细胞增殖、生长曲线、克隆形成率、流式细胞术分析(FCM)、侵袭、运动、迁移、VEGF和MMP2蛋白表达(ELISA法).结果 PA明显抑制MHCC97L细胞增殖和克隆形成,呈良好的剂量-效应关系.PA 48 h和72 h的IC50分别为3.1×108/ml和1. 9×108/ml.PA浓度为0.5×108/ml、1×108/ml和2×108/ml时,其细胞倍增时间依次增加,克隆形成率依次降低(P均＜0.01);FCM显示,G1期细胞比例随PA浓度增加而增加,S+G2期细胞比例随PA浓度增加而降低(P均＜0.01).PA浓度为1×108/ml时,穿过人工基底膜(侵袭实验)和上室底膜(运动实验)的细胞数(分别为4.8±1.3和8.8±2.2)明显低于对照组(8.6±2.1和15.6±1.2)(P均＜0.01);细胞经72 h培养后,对照组划痕逐渐愈合,1×108/ml的PA组细胞划痕依然明显.ELISA法检测发现,1×108/ml的PA组其VEGF蛋白和MMP2蛋白含量和对照组相比均无明显差异(P均＞0.05).结论在一定条件下,绿脓杆菌制剂可抑制人肝癌MHCC97L细胞增殖和克隆形成,其作用部分是通过使细胞周期阻滞在G1期实现的;绿脓杆菌制剂可以明显抑制MHCC97L细胞的侵袭、运动和迁移能力,其作用和VEGF、MMP2蛋白分泌关系不明显.
목적 탐토록농간균제제(pseudomonas aeruginosa vaccine,PA)대유전이잠능적인간암MHCC97L세포증식화침습능력적영향.방법 채용불동농도적PA분별작용우MHCC97L세포,관찰세포증식、생장곡선、극륭형성솔、류식세포술분석(FCM)、침습、운동、천이、VEGF화MMP2단백표체(ELISA법).결과 PA명현억제MHCC97L세포증식화극륭형성,정량호적제량-효응관계.PA 48 h화72 h적IC50분별위3.1×108/ml화1. 9×108/ml.PA농도위0.5×108/ml、1×108/ml화2×108/ml시,기세포배증시간의차증가,극륭형성솔의차강저(P균＜0.01);FCM현시,G1기세포비례수PA농도증가이증가,S+G2기세포비례수PA농도증가이강저(P균＜0.01).PA농도위1×108/ml시,천과인공기저막(침습실험)화상실저막(운동실험)적세포수(분별위4.8±1.3화8.8±2.2)명현저우대조조(8.6±2.1화15.6±1.2)(P균＜0.01);세포경72 h배양후,대조조화흔축점유합,1×108/ml적PA조세포화흔의연명현.ELISA법검측발현,1×108/ml적PA조기VEGF단백화MMP2단백함량화대조조상비균무명현차이(P균＞0.05).결론 재일정조건하,록농간균제제가억제인간암MHCC97L세포증식화극륭형성,기작용부분시통과사세포주기조체재G1기실현적;록농간균제제가이명현억제MHCC97L세포적침습、운동화천이능력,기작용화VEGF、MMP2단백분비관계불명현.
Objective To investigate the effects of Pseudomonas aeruginosa vaccine (PA) on proliferation and invasiveness of the hepatocellular carcinoma cell line MHCC97L with metastatic potential. Methods Proliferation, growth curve, plate efficiency, flow cytometry, transwell invasion assay, cell motility assay, scarification test, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP2) protein activity were evaluated after cells were treated with PA at various concentrations. Results PA can inhibit the proliferation and plate efficiency of MHCC97L cell markedly in a dose-dependent manner. The IC50 of cells treated with PA for 48 h and 72 h was 3.1 ×108/ml and 1.9 × 108/ml, respectively. The doubling time increased and plate efficiency decreased gradually when cells treated with 0.5 × 108/ml, 1 × 108/ml and 2 × 108/ml PA (P＜0.01). PA could induce cell cycle arrest at the G1 phase in a dose-dependent manner by flow cytometric analysis. The average amount of invading cell per field in cell invasion assay and motility assay were 4. 8 ± 1.3 and 8. 8±2.2 when cells treated with 1× 108/ml PA, which was significantly lower than that of control group (8. 6±2. 1 and 15. 6±1.2 ) (P＜0.01) Scarification test showed that the metastatic ability of cells treated with 1 × 108/ml PA significantly lower than that in the control group. Comparison between cells treated with 1 × 108/ml PA and control group, no remarkable difference was found regarding expression of VEGF and MMP2 in supernatant of cell culture. Conclusion PA can inhibit proliferation and plate efficiency of HCC cell line MHCC97L, which is in part mediated by the cell cycle arrest at the G1 phase. PA could inhibit invasiveness of HCC cell line MHCC97L, which is unrelated to the VEGF and MMP2 protein activity.