紫杉醇抑制大鼠血管平滑肌细胞增殖的实验研究

Inhibitory effect of paclitaxel on proliferation of vascular smooth muscle cell in rats

目的以体外培养的大鼠血管平滑肌细胞为研究对象,探讨紫杉醇对平滑肌细胞增殖的作用。方法SD大鼠腹腔注射麻醉后,解剖、分离其主动脉,仔细分离血管的平滑肌层并剪成小块置于培养瓶中培养。采用倒置像差显微镜下观察及α平滑肌肌动蛋白免疫荧光检测对传4代～6代的细胞进行鉴定。根据加入的干预药物将平滑肌细胞分为无血清M199培养基组(A组)、1nmol/L紫杉醇组(B组)、10nmol/L紫杉醇组(C组)及100nmol/L紫杉醇组(D组)共4组,每组均为5个样本。采用流式细胞仪检测不同干预组平滑肌细胞的生长周期,用增殖细胞核抗原(proliferating cell nuclearantigen,PCNA)检测方法检测细胞的PCNA阳性百分比。结果培养约2周后出现大量细胞生长,可见致密的细胞层。倒置像差显微镜下观察,细胞呈梭形;随着细胞融合度的增加,可以看到平滑肌细胞特有的"峰-谷"生长现象。培养的血管平滑肌细胞α平滑肌肌动蛋白免疫荧光检测呈阳性,总阳性率大于90%。随着紫杉醇用量的增加,细胞的PCNA阳性百分比呈逐渐减少的趋势,与对照组(A组)比较,10nmol/L(C组)和100nmol/L(D组)两种浓度下的紫杉醇引起的PCNA阳性率明显减低。流式细胞仪检测显示,随着紫杉醇用量的增加,细胞S期百分比呈逐渐减少的趋势。与对照组相比,10nmol/L(C组)和100nmol/L(D组)两种浓度下的紫杉醇引起的S期百分比明显减低。结论紫杉醇能够抑制大鼠血管平滑肌细胞的增殖。

Objectives To evaluate the inhibitory effect of paclitaxel on proliferation of vascular smooth muscle cell in rats. Methods Isolated the aortae of the rats, drawed off the smooth muscle and cut them into fragment, then put the fragment in the culture flask to cultivate. Verificated the cells by inverted aberratio microscope and α-smooth muscle-aetin immunofluoreseence detection after they have been passaged for 4-6 times. The smooth muscle cells were divided into 4 different groups according to the adding medicine： group A with non serum M199 medium; group B with 1 nmol/L paclitaxel, group C with 10 nmol/L paelitaxel and group D with 100 nmol/L paclitaxel. There were 5 samples in each group. Flow cytomery was used to detect the grow periodicity of the smooth muscle cells and vaseular smooth muscle cell proliferating cell nuclear antigen （ PCNA ） were also detected. Results A great quantity of cells growed after 2 weeks. The cells were observed fusiform shape and presented peak-valley grow phenomenon by inverted aberration microscope. The a-smooth muscle-actiu immunofluorescence detection of the culture smooth musele cells were positive and the positive rate was over 90%. The posititve rate of the smooth muscle cell PCNA presented a taper tendency, following the increase dosage of paclitaxel. Compared with group A, the posititve rate of the smooth muscle cell PCNA in group C and group D decreased significantly. The flow cytometry results indicated that the S period rate of the smooth muscle cell presented a taper tendency, following the increase dosage of paclitaxel. Compared with group A, the S period rate in group C and group D decreased significantly. Conclusions Paclitaxel can inhibit the proliferation of vascular smooth muscle cell in rats.