Expression Pattern Of NDRG2 And BMP4 In Limb Bud Of Mouse Embryo And Correlational Research

BACKGROUND: N-myc downstream regulated gene(NDRG)is a new gene family discovered in recent years,named for its up-regulated expression in N-myc knockout mouse embryonic tissues.NDRG2 is one of the four family members.The results of our and others’ studies on NDRG2 suggest that its functions are diverse,but the specific mechanism is not clear.At present,the research on the function of NDRG2 is mainly focused on its role as a candidate gene for tumor suppressor.However,the limited research information suggests that NDRG2 may be related to bone morphogenetic protein 4(BMP4),which is necessary for cartilage development.OBJECT: Taking the limb buds of mouse embryos and the rat osteoarthritis model as the research object,the expression patterns of NDRG2 and BMP4 were compared to explore the possible correlation between them.METHODS: The limb buds of mouse embryos at different gestational age were dissected,serial sections of tissues were prepared,and the limb bud development model of mouse embryos at different gestational age was established by hematoxylin eosin(H&E)and Saffron O-fast green stainings;The distribution patterns of NDRG2 and BMP4 proteins in limb buds of mouse embryos at different gestational ages were compared by immunohistochemistry;the expression trends of NDRG2,BMP2,BMP4,Sox9 and Runx2 m RNA in limb buds of mouse embryos at different gestational ages were detected by real time PCR;the expression trends of NDRG2 and BMP4 proteins in limb buds of mouse embryos at different gestationa ages were detected by Western blotting;the joint cavity of rat right knee was injected with 50 μ l of monosodium iodoacetate(MIA)solution(containing 2 mg MIA)to establish the osteoarthritis(OA)model of SD rats,and the normal saline(NS)was used as the control group;after the model was established,the animal behavior observation,the gross observation and score of articular cartilage,the histopathological observation and score by H & E and Saffron-O staining were used to evaluate the OA model of rats;Immunohistochemical method was used to detect the expression and distribution of NDRG2 and BMP4 protein in articular cartilage and subchondral tissue of normal adult rats and osteoarthritis(OA)rats.RESULTS: The main observation under anatomical microscope: the limb of mouse embryo appears in the form of limb bud on the side of the body trunk of embryo body.With the growth of fetal age,the limb grows rapidly along the long axis,and the web between toes disappears gradually,finally forming a complete and clear limb structure;After H & E and safranine o-fixation green staining,the development process of osteogenesis in limb cartilage can be clearly seen;The results of immunohistochemistry showed that the expression and distribution of NDRG2 and BMP4 protein in limb bud were similar.With the development of proliferative chondrocytes to hypertrophic chondrocytes,the expression of NDRG2 and BMP4 proteins increased gradually,especially in the cytoplasm of pre hypertrophic chondrocytes and hypertrophic chondrocytes.With the development of limb bone,the expression of NDRG2 protein in proliferative chondrocytes and pre hypertrophic chondrocytes was higher than that of BMP4 protein,while the expression of BMP4 protein in hypertrophic chondrocytes was higher than that of NDRG2 protein;The results of real time PCR showed that the expression level of NDRG2 and Runx2 m RNA was lower in E11.5 limb bud,while that of BMP4 and Sox9 was relatively higher.With the development of limb bud,the expression level of NDRG2,BMP2,BMP4,Sox9 and Runx2 genes showed an increasing trend,especially in NDRG2 and Runx2;Western blotting showed that the expression level of NDRG2 and BMP4 protein increased with the development of limb bud.After injecting MIA into the articular cavity,the rats were depressed in spirit,decreased in activity and avoided in touch;the appearance of the knee joint was swollen,the surface of the articular cartilage was rough,damaged and ulcerated with naked eyes,and the gross injury score of the articular cartilage was significantly higher than that of the NS control group(P < 0.01);The results of H&E and Safranine O-green stainings showed that there were cartilage exfoliation and defect in MIA model group,disordered fibers covering the damaged area of cartilage,the number of disordered chondrocytes significantly reduced,the area of Safranine O staining in cartilage matrix significantly reduced and uneven,and the damaged area seriously lost staining.The Mankin score of MIA group was significantly higher than that of NS control group(P < 0.01),indicating that the joint The OA model was successfully established after 4 weeks of single cavity injection of 2 mg MIA;The results of immunohistochemistry showed that: in normal adult rats,no obvious BMP4 protein positive staining was found in chondrocytes in articular cartilage or in chondrocytes in epiphyseal growth plate,while in subchondral bone,BMP4 protein positive staining was more significant.Different from BMP4 protein,NDRG2 protein positive staining was found in the cytoplasm of chondrocytes in the middle zone of articular cartilage The results showed that there was slight positive staining in subchondral bone tissue,and the same as BMP4 protein,there was no positive staining in chondrocytes of NDRG2 protein in epiphyseal growth plate;The results of immunohistochemistry in OA rats showed that the positive staining of BMP4 protein was more significant in the damaged articular cartilage area,especially in the articular surface layer of the damaged cartilage area and the fibrous tissue with disordered arrangement.On the contrary,the positive staining of NDRG2 protein became shallow or even disappeared in the damaged articular cartilage area.CONCLUSION: NDRG2 and BMP4 participate in the development of cartilage,the maintenance of articular cartilage homeostasis and the formation of articular cartilage injury.Theconfirmation of the correlation between NDRG2 and BMP4 in cartilage physiology and pathology not only adds new contents for understanding the regulation mechanism of cartilage development and cartilage homeostasis,but also provides new ideas for the repair of articular cartilage injury.