摘要
为了研究热休克蛋白70(HSP70)基因对C57BL/6J-角膜混浊(B6-Co)小鼠眼睑成纤维细胞生物学功能的影响,本试验将前期研究并验证的siRNA-HSP70转染C57BL/6J(B6)小鼠眼睑成纤维细胞敲低HSP70基因,体外构建HSP70基因过表达载体转染B6-Co小鼠眼睑成纤维细胞过表达HSP70基因,通过实时荧光定量PCR和Western blot验证转染效率;CCK8法检测B6和B6-Co小鼠眼睑成纤维细胞的增殖水平;Annexin V-FITC法检测B6和B6-Co小鼠眼睑成纤维细胞的凋亡水平,Transwell试验检测B6-Co小鼠眼睑成纤维细胞的迁移能力。结果显示,B6-Co小鼠眼睑成纤维细胞转染HSP70基因过表达载体后,HSP70在mRNA和蛋白水平的表达均极显著提高(P<0.01);siRNA-HSP70组B6小鼠成纤维细胞增殖水平极显著低于siRNA-NC组(P<0.01),凋亡水平显著高于siRNA-NC组(P<0.05或P<0.01);pcDNA3.1-HSP70质粒转染组B6-Co小鼠成纤维细胞增殖水平和细胞迁移能力均极显著高于pcDNA3.1-NC组(P<0.01),凋亡水平极显著低于pcDNA3.1-NC组(P<0.01)。结果表明,B6-Co小鼠眼睑成纤维细胞转染过表达质粒后,HSP70基因表达显著提高,并显著促进细胞增殖和迁移,抑制细胞凋亡;B6小鼠眼睑成纤维细胞中敲低HSP70基因表达后,抑制细胞增殖,促进细胞凋亡;故HSP70表达水平显著影响成纤维细胞的生物学行为。
To investigate the effects of the heat shock protein 70(HSP70)gene on the biological functions of eyelid fibroblasts in C57BL/6J-corneal opacity(B6-Co)mice,this study utilized siRNA-HSP70 to knock down the HSP 70 gene in eyelid fibroblasts from C57BL/6J(B6)mice,an HSP 70 gene overexpression vector was constructed and transfected into B6-Co mouse eyelid fibroblasts to overexpress the HSP 70 gene,with transfection efficiency confirmed by real-time quantitative PCR and Western blot.The proliferation levels of B6 and B6-Co mouse eyelid fibroblasts were assessed using the CCK-8 assay;apoptosis levels were measured using Annexin V-FITC;and cell migration ability was evaluated by the Transwell assay.The results showed that after transfection with the HSP 70 overexpression vector,the expression of HSP70 at both mRNA and protein levels was significantly increased in B6-Co mouse eyelid fibroblasts(P<0.01).The proliferation levels of B6 mouse fibroblasts in the siRNA-HSP70 group were significantly lower than those in the siRNA-NC group(P<0.01),and the apoptosis levels were significantly higher(P<0.05 or P<0.01).In contrast,B6-Co mouse fibroblasts transfected with the pcDNA3.1-HSP70 plasmid showed significantly higher proliferation and migration abilities compared to the pcDNA3.1-NC group(P<0.01),with significantly lower apoptosis levels(P<0.01).These results indicate that HSP 70 gene overexpression in B6-Co mouse eyelid fibroblasts significantly enhances cell proliferation and migration while inhibiting apoptosis.Conversely,HSP 70 gene knockdown in B6 mouse fibroblasts inhibits cell proliferation and promotes apoptosis.Therefore,the expression level of HSP70 significantly affects the biological behaviors of fibroblasts.
作者
景瑾
邵义祥
Mice JING Jin;SHAO Yixiang(Laboratory Animal Center of Nantong University,Nantong 226001,China;Research Institute of Comparative Medicine of Nantong University,Nantong 226001,China)
出处
《中国兽医杂志》
CAS
北大核心
2024年第10期18-24,共7页
Chinese Journal of Veterinary Medicine
关键词
热休克蛋白70
成纤维细胞
过表达
细胞增殖
细胞凋亡
迁移能力
heat shock protein 70
fibroblasts
overexpression
cell proliferation
cell apoptosis
migration ability
