Functional Study Of TGF-β1 Stimulating Rock Ⅰ-βActin Signaling Pathway In The Chondrycte Development Of Developmental Dislocation Of The Hip

[Background and Objective]Development dysplasia/dislocation of the hip (DDH) is one of the most frequently lower extremities deformities in children. The reported incidence rate of DDH throughout the world varies substantially between 0.87‰ and 10.5‰. The cause of DDH is still unknown, but some risk issues, including female, genetic, first born, and breech factors, confirm an increasing DDH prevalence. In Northern Europe and Northern China, DDH has a significantly higher incidence rate, which may be the result of swaddling in early-stage of life. Current explanations of bone morphology imply an important role of mechanical loading. The mechanical overload may act on controlled patterning systems and subsequent damage of joint structures. The clinic evidence showed that most of the children with DDH who received concentric closed reduction in early-stage may get a remodeling acetabulum close to normal hip, which might indicate the revisable potential of acetabulum morphology after changing the hip circumstance. However, the mechanism of acetabulum remodeling is undefined. Developmental dysplasia of the hip (DDH) is a dynamic disease with age. Acetabular chondrocyte has potentially capable of getting better or worse as the child develops. The development can be managed and can be optimized if diagnosed and treated early. Late treatment results in higher rates of failures and complications. It may play an important role in disabling osteoarthritis in adulthood. It is well acceptable that hip screen has shown effective way to avoid delay treatment in early stage of children. The earlier treatment the better result could be obtained. The acetabulars even grow normally in some cases. But it is unknown what the best time point/period of reversible development in DDH acetabular is and what the molecular mechanism is in this invertible process.In this study, we aimed to figure out the possible molecular mechanism of chondrocyte development in DDH animal model. It somewhat might mirror the optimized treatment time and indicate the treatment result in clinic.[Method]In newborn Wister rats, both lower extremities were banded on knees and hips extension position for DDH model at different time point, 1day,3days,5days,7days, 11 days for DDH animal model and non-bound age compared rats as control. The rats were sacrificed right at different time point, in order to observe the anatomic and histologic morphology. The expression of differenciation bio-marker in chondrocyte was detected by RT-PCR assay. Then, the expression and organization of Acin, one of the most important cytoskeleton proteins, was investigated by immunohistochemist, Western Blotting and qRT-PCR assays. In the mean time, the expression and secreation of TGF-β1 in chondrocyte was measured by qRT-PCR and ELISA assay. For the purpose of understanding the machenism of Rockl and Actin signaling patheway, Confocal Microscopy compared the expression of RockⅠ and Rock Ⅱ between DDH acetabular chondrocytes and normal acetabular chondrocytes, RT-PCR and Western Blot assay.[Result]The acetabular enlarged, the volume increased, and the epiphysis got narrow with age in normal SD rats. With bound duration increased, the morphologic changed dramatically. The labrum bent to inside, the volume of socket decrease, the upper part of acetabulum showed hypertrophy. The cells shape changed from round to spindle, disorganization. The cleft showed in the cartilage matrix, which may indicate the secretion of cells was immature. By using histochemisty, immunostaining nd Western Blotting assay, the dates showed that actin expression had not decreased significantly, but Actin located in cytosol in stead of under the membranes in dysplasia acetabular compared with normal acetabular. By using immunostaining and Western Blotting assay, the date was shown that G-actin expressed very faint. There was no significant difference in a-actin and F-actin expression and location. The result indicated that (3-Actin dislocation plays a role in dysplasia acetabular development. ROCK Ⅰ and ROCK Ⅱ expression increased in dysplasia chondrocyte which indicated that the Rho/ROCK pathway regulated Actin organization.[Conclusion]1. The chondrocyte morphologic change indicated that the chondrocytes shifted from differentiation to dedifferentiation.2. During the dedifferentiation process, increased expression and sectretion of TGF-β1 and disorganization of Actin in chondrocytes may play an important role.3. The pathway of TGF-β1 stimulating Rock I-PActin signaling may be one of the morlecular mechanism in DDH acetabulum development.