Procedure Of Absence Of Growth Plate Introduced By Deletion Of Ihh

Objective:(1).To observe the change of gross structure of the growth plate,the morphological and distribution of the chondrocytes in the Ihh knockout mice;(2).To observe the change of mineralization in the growth plate of IHH knockout mice;(3).To preliminary observe the expression of PTHr P and BMP-6 in the growth plate of mice after IHH was knockout;Methods: Col2a1-Cre ERT2;Ihhfl/fl mice were bred and randomly divided into 2 groups.The first group were injected with TM(0.2ug)in P0 to delete Ihh.In another group was injected with vegetable oil at the same time.The postnatal mice were killed at the specific time point of P6/P8/P10/P12/P14.Safranin O–staining was used to observe the type and distribute of chondrocyte,and von Kossa–staining was used to observe the process of mineraliazation.Also,we use the mice of P6 conduct the immunocytochemistry to preliminary observe the the expression of PTHr P and BMP-6.Results: In P6,there was no significant divided zone and proliferation chondrocyte in the proximal tibia,and full of the hypertrophic chondrocyte in the deletion group showed by the Safranin O–staining;the control group showed normal structures.In P8,the chondrocyte became more hypertrophic in the deletion group,and emerged 55 little phenomenon of mineralization showed by the von Kossa–staining;and the control group can not find any mineralization.In P10,we find gradually expanded mineralization in the deletion group;we still can not find any mineralization in the control group,but began to form the second ossification of centre(SOC).In P12,the mineralization extend to most zone of the epiphysis in the deletion group;the structure of SOC formed almost entirely in the control group.In P14,the mineralization occupied almost zone of the epiphysis but the articular cartilage in the deletion group and filled to form the structure of growth plate;but the control group formed the significant divided structures.Also,we examined the change of the expression of paracrine factor parathyroid hormone related protein(PTHr P)induced by ihh and the paracrine factor Bone Morphogenetic Protein 6(BMP-6)inhibit by PTHr P.Staining of BMP-6 was localized within the hypertrophic chondrocytes of the growth plate and PTHr P was mainly observed in the proliferate chondrocytes in the control group.But the expression of PTHr P decreased obviously and the expression of BMP-6 increased significantly when ihh was knockout.