Study Of Cyclin Ⅰ And Matrilin 3 By Methods Of Immunohistochemistry And In Situ Hybridization In Broiler Tibial Dyschondroplasia

Tibial dyschondroplasia(TD),one of frequent leg diseases,is a skeletal abnormality characterized by an avascular plug of cartilage in the metaphysis of the long bones and an avascular jade white cartilage wedge by cartilage cellular proliferation of rapidly growing birds.The occurrence of TD owes to the factors of breeds,nutrition,environment and management,etc,it causes enormous economic losses on the poultry industry.Although TD has been researched gradually in recent years,its pathogenesis remains to be further verified.Cyclinâ… ,cell cycle regulatory factorâ… ,belongs to cell cycle regulatory protein family and plays an important role in cell division and proliferation.It can regulate cell cycle, promote cell growth,cell adhesion and cell migration,induced apoptosis,and control tumor growth,angiogenesis and endochondral ossification.Matrilin 3,is one of noncollagenous extracellular matrix protein family.It's involved in the formation of extracellular matrix fibers and the connection with collagen or non-collagen,regulates the interaction between the collagen fibers and other matrix such as proteoglycans.Its abnormal expression may lead to osteoepiphysis dysplasia,osteoarthritis and other diseases.We have found some differentially expressed genes,including Cyclinâ… and Matrilin3,by suppression subtractive hybridization(SSH) and gene chip in the pre-test.In order to exploer the different expression of Cyclinâ… and Matrilin 3 at different stages,we researched the sequential expression of these two genes in control and TD in the level of gene and protein by in situ hybridization and immunohistochemistry.The results are as follows:The cloning vectors pGEM-T-Cyclinâ… and pGEM-T-Matrilin 3 were constructed successfully.Then the plasmids of them were extracted to produce DIG-labeled cRNA probes for in situ hybridization.The result of in situ hybridization shows that the express level of Cyclinâ… in TD was upward or downward at different times:it's downward in 1^st and 4^th samples,but upward in 2^nd and 3^rd samples.The result of immunohistochemistry shows that,comparing with the control group, the Matrilin 3 gene expressed upward or downward at different times in the broilers with TD.It's as follows:Matrilin 3 gene expressed upward at 4^th sample,but downward at 1^st,2^nd,and 3^rd sample. The results of in situ hybridization on Matrilin 3 gene indicates that it expresses upward or downward in different period of the broilers with TD,too.It's reduced in 1^st and 2^nd samples of the broilers with TD,but enhanced in 3^rd and 4^th.This research demonstrates that CyclinI and Matrilin3 changed some extent at the level of gene and protein in broilers TD.It shows that Cyclinâ… and Matrilin 3 took part in the process of the development in broilers TD.And the analysis of these regularity and reason would provide some novel theoretical basis to continue to investigate TD in future.