摘要
目的 构建波形蛋白(vimentin)糖基化突变质粒并研究其对大鼠肾上腺嗜铬细胞(PC12细胞)分化的影响.方法 构建波形蛋白糖基化突变质粒,进行凝胶电泳并测序鉴定;采用空载质粒pcDNA3.1b(对照组)、波形蛋白质粒(Vim组)、波形蛋白糖基化突变质粒(Vim mut组)转染PC12细胞,分别在外源性寡糖化合物(Cyclo-ManN pro)处理与不处理条件下培养0、3 d,进行细胞分化记录与定量分析(细胞平均神经突长度及分化神经突细胞百分比).结果 成功构建含糖基化位点突变位点19(T-G)、97(A-G)、100(T-G)的波形蛋白基因片段(大小约为1404 bp)重组质粒.Cyclo-ManN pro处理前与处理后,Vim组的PC12细胞平均神经突长度与分化神经突细胞百分比均大于对照组和Vim mut组[(61.98±19.03)μm vs.(51.09±14.45)μm、(51.49±14.78)μm,(6.60±0.25)％vs.(4.27±0.18)％、(4.76±0.33)％;(78.01±18.31)μm vs.(69.98±12.85)μm、(68.45±13.84)μm,(10.62±0.25)％vs.(8.11±1.22)％、(5.89±0.60)％],差异有统计学意义(P<0.05).结论 波形蛋白糖基化修饰可作用PC12细胞分化.
Abstract
Objective To construct a vimentin glycosylated mutant plasmid,and to study its impact on rat adrenal chromaffin cell (PC12) cell differentiation.Methods The vimentin glycosylated mutant plasmid was constructed,and the gel electrophoretic was performed and the sequencing identification was performed. The empty plasmid pcDNA3.1b (control group),vimentin plasmid (Vim group) and vimentin glycosylated mutant plasmid (Vim mut group) were used to transfect PC12 cells,which were cultured for 0,3 d in the con-dictions of treatment and non-treatment of exogenous oligosaccharides (Cyclo-ManN pro) respectively,and the cell differentiation records and quantitative analysis (the cell average neurite length and differentiated neu-rite cells percentage) were performed.Results A recombinant plasmid containing a glycosylation site muta-tion site 19(T-G),97(A-G),100(T-G) was successfully constructed (about 1404 bp in size).Before and after Cyclo-ManN pro treatment,the average neurite length of PC12 cells and the percentage of differentiated neu-rite cells in the Vim group were greater than those in the control group and the Vim mut group[(61.98±19.03)μm vs.(51.09±14.45)μm,(51.49±14.78)μm,(6.60±0.25)％ vs. (4.27±0.18)％,(4.76±0.33)％;(78.01±18.31)μm vs. (69.98±12.85)μm,(68.45±13.84)μm,(10.62±0.25)％ vs. (8.11±1.22)％,(5.89±0.60)％],the difference was statistically significant (P<0.05).Conclusion The glycosyla-tion modification of vimentin could be used for PC12 cell differentiation.
基金项目
浙江省金华市公益性技术应用研究项目(2023-4-064)
维普
万方数据