CAJ | 학술논문

目的：探讨沉默CD147基因对人乳腺癌MDA－MB－231细胞生长、增殖、运动能力的影响。方法构建CD147小干扰RNA（ siRNA）慢病毒载体并转染各实验组MDA－MB－231细胞，采用免疫细胞化学ABC法检测沉默前后MDA－MB－231细胞中CD147、基质金属蛋白酶（ MMP）－2、金属蛋白酶类组织抑制剂（ TIMP）－2蛋白的表达水平；采用即时定量PCR 和Western Blot 技术检测CD147、MMP－2、TIMP－2的mRNA和蛋白表达水平改变；采用CCK－8法和划痕实验分别检测细胞的增殖和迁移能力；建立裸鼠皮下移植瘤模型，观察CD147 siRNA对裸鼠瘤体质量和体积的影响。结果转染MDA－MB－231细胞后，CD147、MMP－2、TIMP－2蛋白呈弱阳性或阴性表达，明显低于对照组（ P＜0．01）。转染72 h时，CD147基因下调平均效率为96．03％±0．84％，MMP－2基因下调平均效率48．64％±0．35％，mRNA表达均明显下降（P＜0．05），而TIMP－2 mRNA表达水平与对照组相比差异无统计学意义（ P＞0．05）。转染第2天后， MDA－MB－231细胞生长明显受到抑制（ P ＜0．05）。转染24 h 后MDA－MB－231细胞平均迁移距离为（0．64±0．12） mm，而空白对照组细胞平均迁移距离为（4．69±0．85）mm，迁移速度减小。沉默CD147基因后，裸鼠瘤体体积和瘤体质量均下降（P＜0．05），平均瘤体质量为（1．85±0．98）g，移植瘤的生长受到抑制（P＜0．05）。结论 CD147可以影响MDA－MB－231细胞中的MMP－2／TIMP－2平衡，沉默CD147基因可以抑制MDA－MB－231细胞的增殖和迁移及裸鼠移植瘤的生长。
목적：탐토침묵CD147기인대인유선암MDA－MB－231세포생장、증식、운동능력적영향。방법구건CD147소간우RNA（ siRNA）만병독재체병전염각실험조MDA－MB－231세포，채용면역세포화학ABC법검측침묵전후MDA－MB－231세포중CD147、기질금속단백매（ MMP）－2、금속단백매류조직억제제（ TIMP）－2단백적표체수평；채용즉시정량PCR 화Western Blot 기술검측CD147、MMP－2、TIMP－2적mRNA화단백표체수평개변；채용CCK－8법화화흔실험분별검측세포적증식화천이능력；건립라서피하이식류모형，관찰CD147 siRNA대라서류체질량화체적적영향。결과전염MDA－MB－231세포후，CD147、MMP－2、TIMP－2단백정약양성혹음성표체，명현저우대조조（ P＜0．01）。전염72 h시，CD147기인하조평균효솔위96．03％±0．84％，MMP－2기인하조평균효솔48．64％±0．35％，mRNA표체균명현하강（P＜0．05），이TIMP－2 mRNA표체수평여대조조상비차이무통계학의의（ P＞0．05）。전염제2천후， MDA－MB－231세포생장명현수도억제（ P ＜0．05）。전염24 h 후MDA－MB－231세포평균천이거리위（0．64±0．12） mm，이공백대조조세포평균천이거리위（4．69±0．85）mm，천이속도감소。침묵CD147기인후，라서류체체적화류체질량균하강（P＜0．05），평균류체질량위（1．85±0．98）g，이식류적생장수도억제（P＜0．05）。결론 CD147가이영향MDA－MB－231세포중적MMP－2／TIMP－2평형，침묵CD147기인가이억제MDA－MB－231세포적증식화천이급라서이식류적생장。
Objective To investigate the influence of siRNA-mediated down-regulation of CD147 on growth, proliferation and movement of human breast cancer cell line MDA-MB-231.Methods The protein expression of CD147, MMP-2 and TIMP-2 of the MDA-MB-231 cells were analyzed by ABC.Lentiviral expression vector of CD 147 gene was constructed and transfected into MDA-MB-231 cells.RT-PCR and Western blot were used to detect the mRNA and protein level changes of CD 147 genes to identify the optimal time point , followed by detection of changes of mRNA and protein expression of MMP-2 and TIMP-2 genes. CCK-8 reagent method and cell scratch test were used to detect the proliferation and migration change of MDA-MB-231 cells.The nude mouse model of breast cancer by hypodermic injection with MDA-MB-231 cells was established to document the effect of CD 147 siRNA on the tumor transplants.Results After transfection of lentiviral expression vector of CD 147 gene, protein of CD147, MMP-2 and TIMP-2 were weakly or negative expressed , significantly weaker than those of control group ( P<0.01 ) .After 72 hours of transfection, average down-regulation rate of CD147 and MMP-2 were 96.03%±0.84% and 96.03%± 0.84%, respectively.Both CD147 mRNA and MMP-2 mRNA expression were down-regulated(P<0.05), while TIMP-2 mRNA expression showed no significant deference ( P >0.05 ) .No less than 2 days after transfection, cell growth of MDA-MB-231 cell line was found significantly inhibited ( P <0.05 ) .After 24 hours of tranfection, average migration distance of MDA-MB-231 cell line and control group were (0.64 ± 0.12)mm and (4.69 ±0.85)mm, respectively, which indicated a lower migrate speed.Down regulation of CD147 led to reduction of volume and mass of nude mouses .The growth of the carcinoma transplant was inhibited upon siRNA-mediated down-regulation of CD147 ( P <0.05 ) , with an average tumor mass of (1.85 ±0.98) g and both reduction of tumor size and tumor mass .Conclusions CD147 may alter the MMP-2/TIMP-2 balance in MDA-MB-231 cells. CD147 gene silencing inhibits the proliferation and migration of MDA-MB-231 cells and the growth of carcinoma transplants in nude mice .