CRELD1基因表达量对心内膜垫发育中相关基因的影响

The effect of CRELD1 gene on the related genes in endocardial cushion development

目的探讨CRELD1基因表达量的改变对心内膜垫发育中相关基因的影响。方法通过慢病毒载体的构建实现CRELD1基因的过表达及沉默，然后用慢病毒载体感染人胚胎肺成纤维细胞（HFL-I），实验分为5组：空白对照组、干扰阴性对照（NC）组、干扰组、过表达NC组及过表达组。感染成功后，分别用实时荧光定量PCR（qPCR）和Western blot检测CRELD1、Sox9、Aggrecan和Scleraxis、Tenascin-C的mRNA和蛋白表达。结果通过测序表明干扰载体pLV3-shRNA-CRELD1和过表达载体pLV4-CRELD1构建成功。感染慢病毒的HFL-I在荧光倒置显微镜下观察，满视野呈现明显的绿色荧光，表明感染成功。qPCR和Western blot的检测结果一致：Sox9和Aggrecan的表达量在干扰组明显高于干扰NC组，在过表达组低于过表达NC组。而Scleraxis干扰组和过表达组均高于各自的NC组。Tenascin-C的表达量在干扰组明显低于干扰NC组，在过表达组高于过表达NC组。结论CRELD1基因对心内膜垫发育中的相关基因Sox9和Aggrecan的表达量有负调节作用，对Tenascin-C的表达量有正调节作用。这对于阐明其在房室间隔缺损中的作用提供一定的理论基础。

ObjectiveTo investigate the effect of alteration of CRELD1 gene expression on the related genes in the endocardial cushion development.MethodsOver-expression and silence of CRELD1 gene were realized by the construction of lentiviral vector.Afterwards, the lentiviral vectors were used to infect the human fetal lung fibroblasts（HFL）-I.All of the cells were divided into the following 5 groups： the blank control group, the negative control group of interference, the interference group, the negative control group of over-expression, and the over-expression group.Western blot and real-time fluorescent quantitative polymerase chain reaction were applied to examine the mRNA and protein expression of CRELD1, Sox9, Aggrecan, Scleraxis and Tenascin-C.ResultsThe DNA sequences of 2 recombinant plasmids pLV3-shRNA-CRELD1 and pLV4-CRELD1 matched very well with those which were designed according to the DNA sequence analysis.HFL-I was successfully infected with lentiviral vectors and displayed fluorescent green light under inverted fluorescence microscope.The results of real-time PCR detection and Western blot test were consistent： expressions of Sox9 and Aggrecan in the interference group were significantly higher than those in the negative control group of interference, while the expressions of the 2 genes in the over-expression group were significantly lower than those in the negative control group of over-expression.Expressions of Scleraxis increased in both the interference group and the over-expression group when compared with the negative control groups respectively.Compared to the corresponding negative control groups, Tenascin-C expression decreased markedly in the interference group, whereas it increased significantly in over-expression group.ConclusionsCRELD1 gene has negative effect on the expression of the related genes Sox9 and Aggrecan in the endocardial cushion development, whereas it has positive effect on the Tenascin-C expression.It serves as a theoretical framework to illustrate the effect of CRELD1 gene on the atrioventricular septal defect.