Observation Of The Phenomenon Of Short Stature Caused By Ihh Knockout In Mice After Birth And Related Mechanisms

Objective:A cartilage-specific Ihh knockout mouse model was established based on Cre/Lox P system,and the phenotypic changes were observed.The mechanism of abnormal ossification of mouse chondrocyte growth plate after conditional knockout of Ihh gene was discussed at the molecular level.The pathogenesis of diseases such as Jensen’s metaphyseal dysplasia,Laron syndrome,osteoporosis,and human Ihh gene mutations provide a new approach to treatment.Methods:(1)The newly-born mice were genetically identified by agarose gel electrophoresis,and suitable genotype mice were screened and Ihh conditional gene knockout was achieved by intraperitoneal injection of tamoxifen TM.The knockdown rate of the Ihh gene was verified by RT-q PCR.(2)The gross morphology of 8 weeks old mice was observed and recorded.The whole body and hind limb X-ray films were taken.The three-dimensional reconstruction of the knee joints was performed by Micro-CT,and the osteoporosis was analyzed.The selected 6d,8d,10 d,12d were selected.Mouse knee joints were treated with Safranin O staining and Von Kossa staining to observe the dynamic evolution of cartilage growth plates.(3)The rib cartilage cells of mice were cultured in vitro on the 6th day after birth.Flow cytometry was used to investigate the abnormal ossification of cartilage growth plate.RT-q PCR was used to detect the expression of signal factors related to Ihh-PTHr P signal axis and verified by cell rescue experiments.In order to resolve downstream related genes.Results:(1)RT-q PCR confirmed that the Ihh conditional gene knockout rate was 76.83%.(2)The general morphology of the 8-week-old mice showed short limb malformations,and the difference was statistically significant(P<0.01).There were significant osteoporosis in the experimental group(P<0.05),X-ray and Micro-CT three-dimensional reconstruction.The knockout mice showed obvious shortening of the femur and tibia and abnormal development of the epiphysis;Safranin O staining showed that Ihh conditional knockout was expressed in the specific region of the developing mouse tibia growth plate cartilage tissue.Von Kossa staining showed that the growth plate of Ihhd/d mice was ossified in advance and the callus was closed prematurely.(3)Apoptosis experiment showed that there was no significant difference between the two groups(P > 0.05).RT-q PCR showed that PTHr P expression was decreased(P<0.05)and BMP-6 expression was significantly increased(P<0.05)after conditional Ihh knockout,but runx-2 expression was not statistically significant(P>0.05).The results were also verified by the cell rescue experiment.Conclusion:Ihh gene plays an important role in maintaining the ordered and programmed differentiation of chondrocytes in the growth plate area of mice.After Ihh conditional gene knockout,the chondrocytes in the growth plate area of mice are disordered,partitioned,and the growth plate area.The chondrocytes were ossified in advance,and the epiphyseal growth plate was prematurely ossified,and the mice showed short limb deformities accompanied by obvious osteoporosis;Around the Ihh-PTHr P signal axis,Ihh knockdown inhibited the expression of the downstream gene PTHr P but promoted BMP-6 expression,and no correlation with Run X-2 signaling factor was found.