| Objective:To clarify the function and mechanism of Ihh in the hypertrophy chondrocyte of growth plate,we observed the changes of growth plate development and analyzed the expression level of cartilage related cytokines after knocking out the expression of Ihh in hypertrophy chondrocyte.The results would deepen the understanding of early period of the development of growth plate during the long bone growth.And may further provide a theoretical basis for the treatment of short deformities.Methods:1.The development of mice was observed by the photograph and X-ray.And the development of growth plate was observed by HE and Saf O&Fast green staining.Then,the different expressed genes were screened,the biomarkers and key regulated factors of hypertrophy chondrocyte were identified.After that,the IHC were performed to clarify the expression level、distribute and location of the type X collagen(the most important biomarker of hypertrophy chondrocyts)and Ihh(important chondrocyte regulator)during the formation of growth plate of newborn mice.2.To investigate the role of Ihh in chondrocytes of hypertrophic zone during growth plate development and its mechanism.Col10a1-CreERT2;Ihhfl/fl mice were constructed and bred,and Ihh expression in mouse X-type collagenpositive cells was knocked down by TM injection on the day of birth.After verifying the knockdown of Ihh,the overall and skeletal development of knockout mice was observed by gross photography and X-ray;the phenotypic alteration of knockout mouse growth plate was observed by HE and Saffaranin O&Fast green stanining.Furthermore,the expression of type Ⅱ collagen,Aggrecan,MMP13,PCNA,HDAC4,SOX9,PTHrP,Patchl,Smo.Gli1,OCN and ALP were evaluated by immunohistochemistry.3.Next,by breeding Col2al-CreERT2;Ihhfl/fl mice and knocking down the expression of Ihh in mice by the same way,the alterations of phenotypy were compared among control group,Col10a1-CreERT2;Ihhfl/fl group and Col2alCreERT2;Ihhfl/fl group mice.The knockdown of Ihh in Col2al-CreERT2;Ihhfl/fl mice was first evaluated using immunohistochemistry.Then the phenotypic changes in cartilage matrix synthesis,chondrocyte hypertrophy and ossification among three groups were observed by HE staining,Safaranin O&Fast green staining,toluidine blue staining,alisin blue staining,Masson trichrome staining,and von kossa staining.Then,the expression levels of type II collagen,Aggrecan,and MMP13 were examined by immunohistochemistry to investigate the differences between the three groups on cartilage matrix;Ki67 and PCNA were examined by to investigate the proliferation of chondrocytes in the three mice;the expression levels of type Ⅹ collagen,HDAC4,Runx2 and SOX9 were examined to investigate the differences between the three mice on chondrocyte hypertrophy;the expression levels of OC and ALP were detected and the differences of chondrocyte ossification among the three groups were investigated.Results:1.By observing the development of growth plate in normal mice after birth,we found that secondary ossification centers were gradually formed at 6-9 days of age after birth.And the development of hypertrophic zone and secondary ossification centers were important structure changes during the development of growth plate.After bioinformatics analysis,type Ⅹ collagen was screened as a biomarker of hypertrophic zone in the growth plate,and Ihh as an important regulator of chondrocyte hypertrophy.And IHC was used to further observed its expression level change,distribution pattern and tissue localization.2.Further by constructing and breeding Col10a1-CreERT2;Ihhfl/fl knockout mice,the expression of Ihh in hypertrophic chondrocytes was knocked out on the day of birth.The morphology of their growth plates and the expression level changes of important markers and regulatory factors of chondrocytes were further observed.The results showed that Col10a1-CreERT2;Ihhfl/fl knockout mice exhibited dwarfism,accelerated hypertrophy and ossification of chondrocytes at the lower end of their growth plate cartilage,along with delayed development of secondary ossification centers.Immunohistochemical results for important cartilage-related markers and regulators showed that knockout mouse growth plate chondrocytes exhibited significant impairment of chondrogenic matrix synthesis,accelerated chondrocyte hypertrophy,and secondary accelerated ossification.3.We further bred Col2al-CreERT2;Ihhfl/fl knockout mice,again injected with TM on the day of birth to knock out Ihh expression in this mouse.Next,the expression levels of growth plate morphology,chondrocyte-related important markers and regulatory factors were compared with those of Col2alCreERT2;Ihhfl/fl knockout mice and control mice.The results showed that with the knockout of Ihh,Col2a1-CreERT2;Ihhfl/fl knockout mice showed more intense chondrocyte hypertrophy and ossification at the lower end of the growth plate than Col10a1-CreERT2;Ihhfl/fl mice,but their secondary ossification centers did not develop and were replaced by upward invasion through the lower end of the growth plate where ossification was accelerated.Conclusions:1.during growth plate development,the prehypertrophic chondrocytes in the hypertrophic zone are the key to the morphofunctional transformation of chondrocytes.type X collagen is the most important marker of chondrocyte hypertrophy.Ihh is mainly localized in prehypertrophic chondrocytes,and it may have an important role in regulating the hypertrophy and ossification of chondrocytes in the secondary ossification centers and the lower part of growth plate.2.Knockdown of Ihh in chondrocytes in the hypertrophic region of the growth plate resulted in short body size,delayed development of secondary ossification centers,and accelerated hypertrophy and ossification of chondrocytes in the lower part of the growth plate.The mechanism is mainly due to the fact that the knockdown of Ihh impairs the synthesis of extracellular matrix in the cartilage of the growth plate,weakens the proliferation of chondrocytes and accelerates the hypertrophy of chondrocytes and their subsequent ossification.3.Ihh in prehypertrophic chondrocytes regulates the development of the growth plate by regulating chondrocyte proliferation and hypertrophy,while the development of secondary ossification centers in the growth plate requires the expression of Ihh in proliferating chondrocytes and its role in maintaining the proliferative capacity of chondrocytes... |
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