| Objective:Appropriate inflammatory reaction is vital to fracture healing.Spleen is the largest immune organ in the body.It contains a large number of immune cells and inflammatory factors,which are closely related to injury repairs.As to the effects of splenectomy on fracture healing,there are very few reports in this area.Our clinical study found that fracture healing of patients with ruptured spleens were often delayed after splenectomy,but there are other mitigating factors in clinical cases.In order to eliminate these factors,this study intends to establish a splenectomy combined with femoral fracture model to clarify the related immune function after splenectomy and its influence on fracture healing.MicroRNA is a kind of short chain non-coding RNA,which can directly degrade the target gene mRNA by specific binding to the target gene mRNA in 3'non-translation regions.It plays an important role in physiology,development and pathology.In this study,microRNAs were selected by microRNA Microarrays(miRCURYTM LNA Array Exiqon)and their effects and mechanisms in regulating inflammatory response and fracture healing were studied.Methods:1.Effects of splenectomy on immuno-inflammatory response and fracture healing.(1)Sprague Dawley rats(male,230-270g)with SPF grade were divided into two groups according to random number table:femoral fracture plus pseudo-splenectomy group(F group),55 rats;femoral fracture plus splenectomy group(F+S group),60 rats.The models of femoral transverse fracture of the rats were established,which were intramedullary fixed firmly.Spleens were simultaneously excised in the F+S group.In the F group,spleens were dissociated,but the portal vessels were not cut off and the spleens were not excised.The histological examinations and molecular biology indexes were carried out by sampling the callus tissues and hematomas from the fracture sites and peripheral blood of rats at four different time points.(2)Detection of immuno-inflammatory indexes.The levels of IL-1 beta,IL-6,TNF-alpha and their mRNAs were detected by ELISA and Real-time PCR.F4/80/CD11b immunofluorescence stainings of the tissues from fracture sites were used to observe macrophage aggregation.(3)Detection of osteogenesis markers and histological examination.The activities of ALP in serum of each groups were detected by reagent box method;The levels of osteocalcin in serum weres detected by ELISA.The levels of ALP,osteocalcin mRNAs were detected by Real-time PCR.The expressions of OPG,RANKL,Collal and Col2al in fracture sites were detected by Western blot.HE stainings were used to observe the growth of calluses and their tissue morphologies.2.MicroRNA array screening for differential expressions of microRNAs in splenectomy plus fracture group.Three rats from femoral fracture plus pseudosplenectomy group and three rats from femoral fracture plus splenectomy group were screened on the third day after surgeries of model making.The differential expressions of microRNAs in peripheral blood were validated by Real-time PCR and bioinformatics analysis.3.Studys on the effects and mechanisms of miR-30d on the fracture healing after splenectomy.(1)In vitro experiment.1)To study the effects of serum after splenectomy on osteogenesis differentiation of BMSCs.In the process of osteogenesis differentiation of BMSCs,the serum from rats with fracture combined with pseudosplenectomy and fracture combined with splenectomy was added separately.ALP staining,alizarin red staining and ALP activities were used to analysis the osteogenesis differentiation of BMSCs.2)The effect of miR-30d on osteogenesis differentiation of BMSCs after splenectomy.The BMSCs cultured with serum from rats with fracture combined with splenectomy were transfected by antagomiR-30d and antagomiR-NC.Then the transfections were confirmed by real-time PCR.ALP staining,alizarin red staining,ALP activities and Western blot were used to analysis the osteogenesis differentiation of BMSCs.(2)In vivo experiment.There were 4 groups of SD rats:Group A:femur fracture plus pseudosplenectomy,Group B:femur fracture plus splenectomy,Group C:femur fracture with splenectomy plus antagomiR-30d,Group D:femur fracture with splenectomy plus antagomiR-30d,each group had 25 rats.In group C and group D,rats were given antagomiR-NC and antagomiR-30d respectively,1.5mg/kg,twice a week,until to the end of the experiment.The callus and haematoma on fracture sites and peripheral blood of rats in each groups were measured at four different time points after operations.1)The expressions of miR-30d and its target Fox03a mRNA at various time points in each groups were detected by real-time PCR.The target protein Fox03a in hematoma tissue of the fracture sites were detected by Western blot.2)Immuno-inflammatory indexs detection.IL-1 beta,IL-6,TNF-alpha levels of each groups were detected by ELISA.3)Detection of osteogenesis markers and histopathological observations.The ALP activitives and osteocalcin levels in serum of each groups were detected.The levels of ALP,osteocalcin mRNA were detected by Real-time PCR.HE stainings were used to observe the growth of calluses and their tissue morphologies.The expressions of OPG and RANKL were observed and analysised semi-quantitatively by immunohistochemical stainingsResults:1.Immunofluorescence double staining showed that F4/80/CD11b positive cells increased significantly on day 3 and decreased on day 30 after operation.But F4/80/CD1 lb positive cells decreased significantly in splenectomy group(P<0.01),indicating that the macrophages aggregation in fracture sites decreased after splenectomy compared with that of simple fracture group.2.The expressions of inflammatory factors TNF-alpha,IL-1 beta,IL-6 and their mRNAs in the serum and hematomas in fracture sites were significantly increased in simple fracture group and decreased by splenectomy on day 3 after operation(P<0.01),indicating that splenectomy inhibited the inflammatory reactions which were necessary for fracture healing.3.HE stainings showed that differentiation of fibroblasts,chondrocytes and fibrous callus were observed at the fracture sites on day 15 after surgery,which were significantly inhibited by splenectomy.4.The expressions of OPG increased after fracture happened.The expressions reached the peak point on day 15 after operation,and continued until day 30 after operation,detected by Western blot.While RANKL peaked on day 3 after fracture and continued to day 30 after fracture.The expression of these two indexes decreased significantly in splenectomy group(P<0.01),which indicated that splenectomy inhibited bone formation.The protein expression of Collal and Col2al increased significantly on day 3 after fracture.The expressions reached the peak at 15 days after fracture and lasted until day 30 after fracture.These datas showed that splenectomy inhibited the formation of bone matrix in fracture healing.5.The activities of ALP and the expressions of osteocalcin increased significantly from day 3 to day 30 after fracture and the expressions of these two indexes were significantly inhibited by splenectomy(P<0.01).6.Five different types of miRNAs were selected from two groups of samples(Fold>1.5,P-value<0.05),of which two miRNAs were increased:miR-30d-5p,miR-374-5p,and three miRNAs were decreased:miR-27a-3p,miR-183-5p and miR-196b-5p.The results of Real-time PCR and bioinformatics analysis showed that miR-30d was at the center of the damage regulatory genes network and was chosed for further research.7.The osteogenesis of BMSCs cells was significantly inhibited by serum of rats with fracture complicated with splenectomy.The numbers of calcified nodules in alizarin red staining,ALP activities and ALP staining results were lower in fracture complicated with splenectomy group than fracture complicated with pseudosplenectomy group.8.The transfection of antagomiR-30d in BMSCs may inhibit the increase of miR-30d caused by serums of splenectomy group,which improved the expressions of Fox03a at the gene and protein levels,improved the expressions of Runx2 protein,and promoted osteogenesis differentiation of BMSCs,thus reducd the inhibition of splenectomy serums on osteogenesis differentiation of BMSCs.9.On day 3,the expression of miR-30d was down regulated,the target gene Fox03a was elevated after implanting miR-30d inhibitor in the caudal vein of rats and miR-NC had no such effects.The expressions of ALP,osteocalcin,OPG,RANKL and inflammatory factors IL-1 beta,IL-6 and TNF-alpha were increased.HE staining and immunohistochemical staining showed that the number of chondrocytes in callus increased,the quality of callus inproved,and the expressions of OPG and RANKL in callus increased.Conclusion:Splenectomy significantly reduces the number of macrophages in fracture healing sites and inhibits the immune function of rats.Splenectomy significantly reduces the release of inflammatory cytokines during fracture healing and inhibits the inflammatory reactions which are necessary for fracture healing.Splenectomy inhibits the expression of bone formation markers during fracture healing,inhibits osteogenesis and bone matrix formation,and delays fracture healing in rats.MiRNAs are involved in the changes of immune function after splenectomy and affects fracture healing.MiR-30d is at the center of this regulatory network.MiR-30d and its target gene Fox03a play a regulatory role in immune system-mediated fracture healing. |
PDF Full Text Request