CAJ | 학술논문

目的探讨甘露糖敏感性绿脓杆菌制剂(PA-MSHA)对水浴加热后有转移潜能的人肝癌MHCC97L细胞增殖和侵袭能力的影响及其机制.方法 42℃水浴加热前2h加入1×1011/LPA-MSHA,加热组不加药,对照组不加热、不加药,其他处理相同并在同一时间点观察.在不同的时间观察细胞增殖、生长曲线、克隆形成率、流式细胞术分析细胞周期(FCM)、侵袭、运动、酶联免疫吸附试验(ELISA)法检测血管内皮生长因子(VEGF)和基质金属蛋白酶(MMP)-2蛋白表达.结果 PA-MSHA能够逆转加热对人肝癌MHCC97L细胞的增殖促进作用,和对照组比较,加热联合PA-MSHA能明显抑制人肝癌MHCC97L细胞的增殖(P＜0.01),其最大抑制效应为加热后48 h(42.3％),细胞倍增时间延长了1.68倍;加热联合PA-MSHA组其加热后48、96 h的细胞集落形成率均明显降低(P＜0.01);FCM显示,加热联合PA-MSHA能逆转加热对细胞周期的影响,和对照组比较,其48 h的G1期细胞比例明显升高,S+G2期细胞比例明显降低(P＜0.01),其96 h的G1期和S +G2期细胞比例差异无统计学意义(P＞0.05).加热联合PA-MSHA组其加热后48 h和96 h相同数量的细胞穿过人工基底膜到达Transwell小室膜背面的细胞平均数(侵袭实验)和穿过Transwell小室膜到达背面的MHCC97L细胞平均数(运动实验)均明显低于加热组(P＜0.01);ELISA法检测相同数量细胞的上清液发现,加热联合PA-MSHA组其加热后48、96h细胞VEGF和MMP-2蛋白的分泌量和加热组比较差异无统计学意义(P＞0.05).结论 PA-MSHA能抑制体外加热后肝癌细胞的增殖,其作用和抑制细胞进入DNA合成期和分裂期有关;进一步抑制其侵袭运动能力,其作用和MMP-2、VEGF表达无关.
목적 탐토감로당민감성록농간균제제(PA-MSHA)대수욕가열후유전이잠능적인간암MHCC97L세포증식화침습능력적영향급기궤제.방법 42℃수욕가열전2h가입1×1011/LPA-MSHA,가열조불가약,대조조불가열、불가약,기타처리상동병재동일시간점관찰.재불동적시간관찰세포증식、생장곡선、극륭형성솔、류식세포술분석세포주기(FCM)、침습、운동、매련면역흡부시험(ELISA)법검측혈관내피생장인자(VEGF)화기질금속단백매(MMP)-2단백표체.결과 PA-MSHA능구역전가열대인간암MHCC97L세포적증식촉진작용,화대조조비교,가열연합PA-MSHA능명현억제인간암MHCC97L세포적증식(P＜0.01),기최대억제효응위가열후48 h(42.3％),세포배증시간연장료1.68배;가열연합PA-MSHA조기가열후48、96 h적세포집락형성솔균명현강저(P＜0.01);FCM현시,가열연합PA-MSHA능역전가열대세포주기적영향,화대조조비교,기48 h적G1기세포비례명현승고,S+G2기세포비례명현강저(P＜0.01),기96 h적G1기화S +G2기세포비례차이무통계학의의(P＞0.05).가열연합PA-MSHA조기가열후48 h화96 h상동수량적세포천과인공기저막도체Transwell소실막배면적세포평균수(침습실험)화천과Transwell소실막도체배면적MHCC97L세포평균수(운동실험)균명현저우가열조(P＜0.01);ELISA법검측상동수량세포적상청액발현,가열연합PA-MSHA조기가열후48、96h세포VEGF화MMP-2단백적분비량화가열조비교차이무통계학의의(P＞0.05).결론 PA-MSHA능억제체외가열후간암세포적증식,기작용화억제세포진입DNA합성기화분렬기유관;진일보억제기침습운동능력,기작용화MMP-2、VEGF표체무관.
Objective To investigate the effects of thermotherapy combined pseudomonas aeruginosa mannose-sensitive hemagglutination pilus strain (PA-MSHA) on proliferation and invasion of hepatocellular carcinoma cell line MHCC97L with metastatic potential.Methods PA-MSHA (1 × 10n/L) was added 2 h before thermotherapy.Chemicals were not given in the thermotherapy group,and chemicals and thermotherapy were omitted in the control group.Proliferation,growth curve and plate efficiency were observed.Flow cytometry was used to examine the cell cycle.Transwell invasion assay and cell motility assay were used to assay the invasion and molity of MHCC97L cells.Vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) protein expression in MHCC97L cells after thermotherapy was detected.Results PA-MSHA reversed thermotherapy-augmented proliferation of MHCC97L cells.Compared to the control group,the proliferation of MHCC97L cells was significantly decreased after thermotherapy combined with PA-MSHA (P ＜ 0.01).The inhibition ratio reached the maximum at 48 h after thermotherapy up to 42.3％,and the doubling time increased by 1.68 times.The plate efficiency of the same number of cells at 48 h and 96 h after thermotherapy combined with PA-MSHA was decreased (P ＜ 0.01).Flow cytometric analysis revealed that the ratio of G1 phase was increased and that of S + G2 phase decreased 48 h after thermotherapy combined with PA-MSHA (P ＜ 0.01).The ratio of G1 phase S + G2 phase showed no obvious difference 96 h after thermotherapy combined with PA-MSHA (P ＞ 0.05).The average amount of invading cells per field in cell invasion assay and motility assay of the same number of cells at 48 h and 96 h after thermotherapy combined with PA-MSHA was significantly less than single thermotherapy group (P ＜ 0.01).Enzyme linked immunosorbent assay showed no remarkable difference was found regarding the expression of VEGF and MMP-2 in supematant of the same number of cells culture between thermotherapy combined with PA-MSHA group and single thermotherapy group (P ＞ 0.05).Conclusion PA-MSHA suppressed thermotherapy-augmented proliferation of MHCC97L cells,which was in part mediated by inhibiting DNA synthesis and mitosis of the cells; further inhibiting the invasion in vitro post-thermotherapy (42℃),which was unrelated to the VEGF and MMP-2 protein expression.