The Expression Of Shh And Wnt5a In Limb Buds Of NIPBL^+/- Mouse

Objective:Cornelia De Lange syndrome（Cdls）is a genetic defect disease.The mutation of NIPBL gene may change the transcription of related Shh and Wnt5a genes and affect the function of key factors in the Wnt signaling pathway,thus changing the direction of osteoblast differentiation and so on,and eventually leading to the abnormal development of long bones.Therefore,this research on mouse embryonic limb bud organization as the research object,research Shh,Wnt5a gene in mice limb bud NIPBL defective gene expression,revealing the Wnt signaling pathway related factors in NIPBL^+/- the role of gene knockout mice,to investigate the relationship between the two genes and Cdls syndrome,to provide the theoretical basis of pathogenesis Cdls syndrome.Methods:The experimental animals were nipbl-loxp mice and Cre mice of the background strain,57BL/6J,SPF grade,purchased from the animal experiment center of zhejiang university,and raised in the animal experiment center of school of pharmacy,shihezi university.Raised in the average temperature of25℃,humidity is about 40%.NIPBL^+/- mice,male weight 35g,female weight 30g,male and female mice were caged at 20:00 according to the ratio of 2:1,female mice were examined at 8:00 a.m.Fetal mice of the control group and the experimental group were collected at 10 days,11 days and 12 days respectively for experimental verification.In the experimental group,36 limb buds of NIPBL^+/- fetal limb bud at 10 days（n=12）,11 days（n=12）,12 days（n=12）were obtained after identification with rt-pcr reverse transcription kit.The control group,the use of rt-pcr retrovirus kit,can get 36 NIPBL^+/+fetal rat limb bud at 10 days（n=12）,11 days（n=12）,12 days（n=12）,ready to liquid nitrogen,the fetal rat limb bud organization into it,after 15-20 min,take out its limb bud tissue cryopreservation to-80℃refrigerator,set aside.Shh and Wnt5a gene transcription levels were detected by real-time PCR.The experimental data were normally distributed with mean standard deviation（s）,and were analyzed by SPSS 17.0 statistical software.One-way anova was used for the comparison of each group,and Pearson correlation analysis was performed among the data of each group.P<0.05 showed statistical difference,while P<0.01 showed significant difference.Results:NIPBL^+/- mouse model was obtained by nipbl-loxp mice and Cre mice,and then NIPBL+/-mice were hybridized,and the fetal mice in the experimental group and the control group were identified by rt-pcr.Real-time PCR was used to detect the transcription levels of Shh and Wnt5a genes.The expression levels of Shh and Wnt5a in the experimental group began to increase on the 10th day after surgery,reached the maximum on the 11th day,and decreased on the 12th day.Compared with the control group at each time point,the gene expression level in the experimental group was lower than that in the control group,and the difference was statistically significant（P<0.01）.Western blot was used to detect the expression levels of Shh and Wnt5a protein.The expression levels of Shh and Wnt5a in the experimental group began to increase on the 10th day after surgery,reached the maximum on the 11th day,and decreased on the 12th day.The expression levels of Shh and Wnt5a in the experimental group were lower than those in the control group（P<0.01）.Conclusion:By detecting the expression of Shh and Wnt5a genes in limb buds of NIPBL^+/- knockout mice,it was found that the expression of Shh and Wnt5a genes was inhibited on the 10th,11th and 12th days of gestation,indicating that NIPBL had an effect on the expression of Wnt5a and Shh genes,which is helpful for people to better understand the pathogenesis of CdLs syndrome bone deformity caused by NIPBL^+/- gene defects.