聚集蛋白聚糖和层黏连蛋白对PC12细胞突起生长及β-肌动蛋白表达的影响

摘要

目的 研究细胞外基质成分聚集蛋白聚糖与层黏连蛋白对PC12细胞突起生长形态的影响及细胞骨架中β-肌动蛋白表达的影响,为进一步探索细胞外基质对神经元轴突细胞骨架的影响机制奠定基础.方法 建立PC12细胞培养体系,分别在培养基中加入150 μg/ml聚集蛋白聚糖和50 μg/ml层黏连蛋白(分别为聚集蛋白聚糖组和层黏连蛋白组),设立空白培养基为对照组.体外培养5d后,通过细胞免疫荧光染色观察3组PC12细胞的形态并测量细胞突起的长度、宽度及面积.采用PCR和Western blot的方法测定PC12细胞骨架中β-肌动蛋白的RNA和蛋白表达情况. 结果 聚集蛋白聚糖组PC12细胞突起较为扁短,宽度为(3.65±0.35)μm,大于层黏连蛋白组[(1.00±0.65) μm] (P＜ 0.05),但略大于对照组[(2.26±0.49)μm] (P＞0.05),层黏连蛋白组的宽度小于对照组,但差异没有统计学意义(P＞0.05);层黏连蛋白组PC12细胞突起纤细而长,长度[(48.75±2.72)μm]大于聚集蛋白聚糖组[(17.21±2.75)μm]和对照组[(22.29±5.32)μm](P＜0.05);聚集蛋白聚糖组的长度小于对照组,但差异没有统计学意义(P＞0.05);聚集蛋白聚糖组、层黏连蛋白组和对照组PC12细胞突起面积分别为(39.71 ± 8.00) μm2、(60.42±8.51)μm2和(49.75 ± 8.20) μm2,组间差异没有统计学意义(P＞0.05).聚集蛋白聚糖组β-肌动蛋白RNA表达低于对照组(P＜0.01),层黏连蛋白组高于另外两组(P＜0.01).聚集蛋白聚糖组β-肌动蛋白的蛋白表达低于对照组(P＜0.01);层黏连蛋白组高于另外两组(P＜0.05). 结论 聚集蛋白聚糖对PC12细胞突起轴向生长和β-肌动蛋白的表达具有一定抑制作用,但不一定抑制其非轴向生长潜能;而层黏连蛋白对PC12细胞突起的轴向生长和β-肌动蛋白的表达具有促进作用.%Objective To study how those extracellular matrixes,aggrecan and laminin,influence to morphology of neurite of PC12 cells,and expression of β-actin in PC12 cells.And furthermore,it can lay a foundation for mechanism of investigating neuron axon cytoskeleton influenced by extracellular matrixes.Methods PC12 cells could divided to three groups,including laminin group (50 μg/ml laminin) and aggrecan group (150 μg/ml aggrecan),and blank medium as control group.Then those three groups PC12 cells cultured for 5 days to identify at what extend that laminin and aggrecan influence of PC12 cells neurite morphology and β-actin in cellular cytoskeleton,by using cell immunofluorescence and microscopy.RT-PCR was performed to evaluated RNA expression of PC12 β-actin in cellular cytoskeleton; Western-blot was performed to evaluated protein expression of PC12 β-actin in cellular cytoskeleton.Results Cell morphology observation:in the aggrecan group,the neurite growth of PC12 cells show short and flat,the width length and area of neurite growth of PC12 cells were 3.65 ± 0.35 μm,17.21 ± 2.75 μm,and 39.71 ± 8.00 μm2,respectively; the neurite growth of PC12 cells in laminin group show thin and long,the width length and area of neurite growth of PC12 cells were 1.00 ± 0.65 μm,48.75 ± 2.72 μm,and 60.42 ± 8.51 μm2,respectively; there were normal neurite growth of PC12 cells in control group,the width length and area of neurite growth of PC12 cells were 2.26 ± 0.49 μm,22.29 ± 5.32 μm,and 49.75 ± 8.20 μm2,respectively.In width,compared with laminin group,aggrecan group showed wider than laminin group (P ＜0.05),and aggrecan group showed more wider than control group,control group showed more wider than laminin group,but there were both with no difference.In length,compared with aggrecan and control groups,laminin group showed longer than other two groups (P ＜ 0.05); and there was no difference with aggrecan group and control group.In area,aggrecan group was smaller than other groups,laminin group was bigger than other groups,and control group was normal,but there were no differences as well.mRNA express of β-actin in PC12 cytoskeletal:The level of expression of β-actin mRNA of PC12 cells influenced by laminin groups higher than controlled treatment group(P ＜ 0.05),and the level of expression of β-actin mRNA of PC12 cells influenced by aggrecan lower than controlled treatment group (P ＜ 0.05).Protein express of β-actin in PC12 cytoskeletal:The level of expression of β-actin protein of PC12 cells influenced by laminin groups higher than controlled treatment group (P ＜0.05),and the level of expression of β-actin of PC12 cells influenced by aggrecan lower than controlled treatment group (P ＜ 0.05).Conclusion Aggrecan can inhibit growth of neurite of PC12 cells in length wise direction,and can decrease β-actin RNA and protein expression in PC12 cytoskeletal,but the growth potential of neurite of PC12 cells may not been depress.Laminin can promote growth of neurite of PC12 cells in length wise direction,and can increase β-actin RNA and protein expression in PC12 cytoskeletal.