The Influence Of Postnatal Mice Growth Plate’s Early Ossification With Conditional Knockout Indian Hedgehog

Objective:1、Observe the conditional knockout Ihh effects on mouse femur, tibia length;2 、 Observe Ihh gene conditional knockout mice growth plate start day growth plate histomorphology changes before and after the ossification, in order to observe the four areas evolution process;3 、 Preliminary discussion Ihh’s influence on the growth plate early ossification mechanism;4 、 For abnormal related diseases, such as: BDA1, osteoarthritis and other ailments.caused by Ihh providing theoretical basis for the prevention and treatment;Methods:1、Taking genotype for Col2α1-Cre ERT2; Ihhfl/fl28 newborn mice were randomly divided into experimental group Ta group(6 days), control group Ca group(6 days),each group of 14. Then taking genotype for Col2α1-Cre ERT2;Ihhfl/fl14 newborn mice were randomly divided into experimental group Tb group(8 days), control group Cb group(8 days), six in each group. Intraperitoneal injection of the experimental group on the first day after birth in corn oil soluble TM(Tamoxifen)with 3 consecutive days.Intraperitoneal injection of the control group in the same amount of corn oil as a control.2、 RT-PCR detection experimental group Ta group, control group Ca group’s proximal tibial epiphyseal Ihh gene m RNA in the relative expression quantity, clear the knockout rate(n = 6).3、 Measurement the Ta, Ca, Tb, Cb group’s left femur, left tibia length, to observe the influence of the length of the femur, tibia(n = 6).4、Mice’s left the proximal tibia X irradiation and tissue slice Von kossa staining, clear the day of experimental proximal tibial growth plate began to ossification.5、The mouse left proximal tibial growth plate Safranine O stain, observe the proximal tibial growth plate morphology.6、RT-PCR detection experimental mice proximal tibial growth plate began the day before ossification, proximal tibial epiphyseal RUNX2 gene m RNA expression level(n = 6).Results:1、The experimental group Ta group Ihh gene expression quantity in the epiphyseal was obviously lower than control group Cb group(P < 0.05),Gene knock out at a rate of 65.17%.2、6 days and 8 days after birth, the experimental group femur length, tibia length significantly reduced than control group(P < 0.05).3、 The proximal tibia X-ray and growth plate biopsy Von Kossa staining were found in 6 days after birth, mice growth plate are not ossification, 8 days after birth, and the experimental group growth plate ossification is obvious.Confirm the conditional knockout Ihh growth plate began ossification the day before at after birth 6 days.4、The experimental group’s Safranine O stain red O staining showed that growth plate structure disorder, proliferating zone disappear, the arrangement of the standard type pillars cell are not found in the proliferating zone, growth plate was overcome with hypertrophy of chondrocytes proteoglycan secretion decreased, distribution of disorder.The control group of proximal tibial growth plate structure is normal.5、The experimental group started the day before ossification of proximal tibial epiphyseal RUNX2 gene expression rate is significantly higher than control group(P < 0.05).Conclusions:Conditional knockout Ihh with source of growth plate lead to mouse left tibia, left femur length is shorten, left proximal tibial growth plate structure disorder, plate ossification in advance, RUNX2 expressed in the growth plate in advance.Knocking out the source of growth plate Ihh, growth plate early ossification mechanism may be associated with the expression of RUNX2, For abnormal related diseases, such as: BDA1,osteoarthritis and other ailments caused by Ihh providing theoretical basis for the prevention and treatment.