论文部分内容阅读
目的：针对人S100A10基因设计siRNA序列并构建shRNA表达载体；重组载体转染人关节软骨（HCs）细胞，观察细胞内S100A10基因表达及其对细胞内核转录因子κB（NF-κB）活性的影响。方法登陆美国国家生物技术信息中心（NCBI）数据库，根据人钙结合蛋白A10（S100A10）基因信息，设计3条针对其编码区（ CDS）区的小干扰RNA（ siRNA）序列。根据设计的siRNA序列，设计两条互补的shRNA序列，构建shRNA重组表达载体；阳离子脂质体法转染shRNA重组表达载体到HCs细胞，转染后48 h，收集细胞，提取细胞总 RNA及总蛋白，使用实时PCR（ RT-PCR）及蛋白质印迹法（ Western blot）的方法检测转染后细胞中S100A10 mRNA及蛋白相对含量；Western blot检测细胞内NF-κB的P65及P50亚基磷酸化。数据统计使用单因素方差分析。结果成功设计siRNA序列并成功构建了shRNA表达载体；mRNA及蛋白含量检测结果显示，三条siRNA序列对目的基因均有沉默效果，其中1号siRNA序列最为-有效，其转染后48 h，细胞内mRNA及蛋白含量相比较未转染组细胞，分别下降了74.2％和76.4％，差异均有统计学意义（t ＝5.21，P＜0.01）；HCs 细胞内S100A10基因沉默，能够明显抑制核转录因子NF-κB活性，与对照组比较，P65及P50亚基磷酸化明显减弱（ t＝3.02，P＜0.01）。结论 S100A10基因沉默可以有效抑制HCs细胞内NF-κB活性。“,”Objective To design siRNA sequences targeting human S100A10 gene, construct shRNA expression vectors with a plasmid expression vector and observe the effects on inhibition of NF-kappa B activity in human chondrocytes by transfecting with the recombinant vectors.Methods The human S100A10 gene sequence was obtained from National Center for Biotechnology Information ( NCBI) database.According to human S100A10 (NM_002966) gene information, three siRNA sequences were designed for the ( Sequence coding for amino acids in protein, CDS) of S100A10 gene.According to the designed siRNA sequences, two complementary DNA sequences were designed to construct recombinant shRNA expression vectors.Human articular chondrocytes ( HCs) were transfected with the recombinant shRNA expression vectors by cationic liposomes, and the transfection;48 h after the transfection, the cells were collected, the total RNA and total protein contents were extracted respectively.Relative S100A10 mRNA and protein levels in the transfected cells were detected by real-time PCR and Western blot, and NF-κB activity was detected by analysis of P65 and P50 subunit phosphorylation.One-way analysis of variance were used for data analysis.Results S100A10 gene information was found, siRNA sequences were designed and the shRNA expression vectors were constructed successfully.The results of the mRNA and protein showed that all three siRNA sequences had inhibitory effects on the target gene, and the 1st siRNA sequence was the most effective, which decreased mRNA and protein levels in the transfected groupby 74.2%and 76.4%, respectively;the difference compared with the untransfected cells was statistically significant (t=5.21, P<0.01).Compared with the control group, P65 and P50 subunit phosphorylation decreased obviously (t =3.02, P <0.01) in the gene silencing HCs, which meant S100A10 gene silence could obviously inhibit the transcription factor nuclear factor kappa B activity in HCs.Conclusion S100A10 gene silence can effectively reduce the NF-kappa B activity in HCs.