乳移平含药血清对乳腺癌4T1细胞增殖和侵袭影响的实验研究

Effects of Ruyiping medicated serum on proliferation and invasion of mammary carcinoma 4T1 cells

目的研究乳移平含药血清对小鼠乳腺癌4T1细胞增殖与侵袭的影响,并探讨其作用机制。方法 (1)制备不同浓度的乳移平含药血清(10%、20%、30%、40%),并以10%、20%、30%、40%空白血清作对照,采用噻唑蓝(MTT)方法测定含药血清对4T1细胞存活率的影响,筛选最佳作用浓度和最佳作用时间。(2)设空白对照组、TGF-β1对照组、TGF-β1 10%乳移平含药血清组、TGF-β120%乳移平含药血清组;除空白对照组外,其他组细胞都用2 mg/L的TGF-β1预处理3 h,再分别选择空白血清或含药血清作用48 h。采用Transwell侵袭实验检测含药血清对TGF-β1预处理的4T1细胞侵袭率的影响;Western blotting及Real-time PCR检测乳移平含药血清对经TGF-β1预处理的4T1细胞Smad4和Smad7表达的影响。结果 (1)乳移平含药血清显著抑制4T1细胞的增殖活性;选择10%和20%作用浓度,48 h作用时间用于后续实验。(2)乳移平含药血清显著抑制TGF-β1诱导的4T1高侵袭能力,并且存在浓度依赖性;Western blotting和Real-time PCR结果显示,乳移平含药血清明显逆转了TGF-β1诱导的Smad4蛋白及mRNA的高表达,但对TGF-β1诱导的Smad7蛋白及mRNA表达量上调没有影响。结论乳移平含药血清能够抑制4T1的增殖和侵袭能力,其作用机制可能与调控TGF-β/Smads信号通路相关因子的表达有关。

Objective To study the effects of Ruyiping medicated serum on proliferation and invasion of mouse mammary carcinoma 4T1 cells, and discuss its mechanism. Methods ①The medicated serum of Ruyiping with different concentrations （ 10%, 20% ,30% and 40% ） were prepared, and the blank serum at same concentrations were taken as the control. The effect of medicated serum on the viability of 4T1 cells was detected by methylthiazolyldiphenyl-tetrazolium （MTr） assay to screen the optimum concentration and exposure time. ②The cells were divided into the blank control group, TGF-β1 control group, Ruyiping medicated serum groups with low（10% ）- and high（20% ）-concentration. Except the blank control group,the cens in the other groups were all given the pre- treatment with TGF-β1 at concentration of 2 mg/L for 3 hours. And then the ceils were treated with blank serum or medicated serum for 48 hours respectively. The effect of medicated serum on the invasion of TGF-β1 pre-treated 4T1 cells was detected TransweU test. The effects of medicated serum on the expressions of Smad4 and Smad7 in TGF-β1 pre-treated 4T1 cells were determined by Western blotting and Real-time PCR. Results ①The Ruylping medicated serum significantly inhibited the proliferation activity of 4T1 cells. 10% and 20% were taken as the optimum concentrations,and 48 hours was taken as the optimum exposure time for the experiment. ①The Ruyiping medicated serum significantly inhibited TGF-β1-induced high invasion of 4T1 cells,with a concentration-depandent manner. The results of Western blotting and Real-time PCR showed that, the Ruyiping medicated serum obviously reversed the high mRNA and protein expressions of Smad4 induced by TGF-β1 ,but no effects on the up-regulated mRNA and protein expressions of Smad7 were observed. Conclusion Ruyiping medicated serum could inhibit the proliferation and invasion Of 4T1 cells. Its mechanism may be associated with the regulation on the expressions of related factors in TGF-β/Smads signal pathway.