The Relationship Between The Expression Of Circulating MicroRNA-29 And The Injury Time After Fracture In Rats

Objective:To observe the histological changes of rat femoral fractures at different times,study the correlation between circulating microRNA-29 expression and injury time in different periods,and its applicability to the estimation of fracture injury time.Methods:45 Sprague-Dawley rats were randomly divided into blank control group?5 rats?,operation group?20 rats?and sham operation group?20 rats?.The operation group and the sham operation group were divided into 4 groups according to the injury time?1 day,4 days,7 days,14 days.There were 5 rats in each group respectively?.For the operation group,rats were received open osteotomy on the middle of left femur;For the sham operation group,only exposed the femoral tissue,and then the incision was sutured and disinfected.Rat femoral tissues were placed in 10%formalin neutral fixative,and tissue morphology was observed by Hematoxylin-Eosin?HE?staining.4.5 mL blood samples were obtained from abdominal aorta and extracted the microRNA from the plasma.the BestKeeper software was used to screen the internal reference genes,quantitative real-time PCR?qPCR?technology was used to detect the expression of microRNA-29,and the measured cyclic threshold?Ct?value was converted to 2-??ct.The SPSS software was used to carry out a single factor variance analysis,and the ? Ct values were analyzed by curve regression.Result:1.Microscopy results of HE Staining in Rat Femur:On the 1 st day after fracture,periosteum swelling,bleeding and inflammatory cell infiltration were found in rats.A large number of new capillaries and fibrous tissue hyperplasia were observed on the 4th day,transparent chondrocytes were formed and ossified on the 7th day,fibrous callus were gradually replaced by bony callus on the 14th day.The short-term repair of rat fractures had undergone three stages:hematoma formation,fibrous callus formation,and bony callus formation.2.U6 with the smallest standard deviation?SD?and coefficient of variation?CV?in plasma tissues was used as the optimal internal reference gene for this experiment.3.The expression of circulating microRNA-29a,microRNA-29b,microRNA-29c decreased within 1 to 14 days after fracture in rats?P<0.05?,the decrease was large within 1?7 d and gradually slowed.4.Taking fracture injury time as independent variable x,?Ct values of circulating microRNA-29a,microRNA-29b,microRNA-29c as dependent variables y,regression analysis was carried out,and the coefficient of cubic regression equation was the largest.(Regression equation:???_microRNA-29a=0.0278x³+0.1224x²+0.1721x+2.322,R²=0.454,P<0.05;???_microRNA-29b=0.0009X³-0.0249X²?0.242x+3.0335,R²=0.750,P<0.05;???_microRNA-29c=0.0059x³-0.0733x²+0.407x+2.0668,R²=0.577,P<0.05)Conclusion:1.The relative expressions of circulating microRNA-29a,microRNA-29b,and microRNA-29c were correlated with the time of femoral fracture injury in rats,and they showed a decreasing trend with time in the short term?P<0.05?.2.Detecting the relative expression levels of microRNA-29a,microRNA-29b,and microRNA-29c in plasma tissue can be used to estimate the fracture injury time,which has a greater value for estimating fracture time within 7 days.