Identification And Function Study Of The Factors Related To Spontaneous Repair Of Cartilage And Subcartilaginous Bone In Yong Rabbit Knee Joint

Background: Joint injury repair is one of the difficulties of medical science. The main reason lies in that the joint injury can not build the permanent hyaline cartilage articular surface. Acording to the researchment, chondrocyte can change to chondroblast in the special conditions. So finding the factors and the mechanism to activate chondrocyte to dedifferentiate become the key to solve spontaneous cartilage repair. Acording to the researchment, after articular cartilage injury, the yong rabbit can rebuild the hyaline cartilage articular surface. In the repair tissue, the hyaline cartilage account is more than 80%, both the durability and the construction approach the normal articular cartilage. On the contrary, the old rabbit can only form fibrocartilage tissue in injury position. We conclude that the reason of the diffenrent repair result is the different gene expression in the joint injury. Therefore, to find the key factors related to the spontaneous repair by comparing the early gene expression of the yong rabbit's joint injury with the old rabbit will be very important and meaningful.Objective: To build the diferential expressed genes library and clone the aimed genes related to spontaneous cartilage and bone repair in yong rabbit knee joint after injury by suppression subtracted hybridization and rapid amplification cDNA ends methods. Analysis and screen the relative genes to lead spontaneous repair of the yong rabbit articular cartilage. Select the aim genes related to the repair of joint injury and clone the full length sequences of the target genes. Expres the proteins and obtain the bioactive proteins of the target genes. And then, identificate the functions of the target protein through observing the change of cell proliferation and shape of the mesenchymal stem cells cultured with the target proteins.Method: Selected three adult female New Zealand white rabbits (50 weeks) and their offsprings(8 weeks). Created an osteochondral defect on the medial femoral condyle in both knees. Used a bit to creat a defect, penetrating the subchondral bone plate, 3 mm in diameter and 3 mm in depth. Killed all the rabbits four weeks after operation with the method of aeroembolism. Immediately after death, separated the repaired tissue and stored in the liquid nitrogen. Extracted the totle RNA of repair tissue of the injury position after building the rabbit's injuried knee joint model.The total RNA of the yong and the adult rabbit's repaired tissue was prepared as the template of RT-PCR.The cDNA of the yong rabbit was the tester and the cDNA of adult rabbit was the driver. The both cDNA was digested into shorter with Rsa I. Aliquotted the tester cDNA into two parts. One was ligated with Adaptor 1 and another was ligated with Adaptor 2R.Then performed two hybridizations and two PCR amplifications.The PCR production was ligated with T vector and translated into competent E coli. Selected the positive colonies by blue-white experiment and constructed the differential library. Sequenced the positive colonies after identification by PCR. Obtained the ESTs of the differentially expressed genes with the software DNAStar 5.01 and the software Chromas. Identified the ESTs with the Reverse Northern blot.BLASTN the EST sequences in GENEBANK and selected the target gene fragments for further researchment.Clone the full gene sequences of the aimed genes by Rapid amplification cDNA ends and obtain the true full sequences through the contig splicing. Construct the prokaryotic expression system of aimed genes with pQE30 vector. Express the proteins and obtain the bioactive proteins of the target genes. Culture the mesenchymal stem cells by whole bone marrow culture. And then Observe the change of cell proliferation and shape of the mesenchymal stem cells cultured with the target proteins through the MTT assay and the Immunofluorescence staining.Research the role of VEGF in the differential expressed genes in the joint injury. Fourty-five adult New Zealand white rabbits were selected and divided into three groups randomly,gene transfer group, blank vector group and control group. The adenoviral vector comprising human VEGF (Ad-hVEGF) came from the central laboratory. The femoral head necrosis model was induced by injection with ethanol. The necrotic femoral head was transfected with a human adenoviral vector expressing VEGF. RT-PCT was performed to detect the express of VEGF in the three groups. Bone formation in the subchondral necrotic region was analysed using histology, by measuring the BMD value, and by observing bone trabecular morphology using image analysis.Results: One-step method combining plant RNAout kit could successfully extract the totle RNA with a high quality and the totle RNA was reversely transcripted into ds cDNA by LD-PCR. We constructed the diferential library of yong rabbit'repaired tissue and obtained 223 clones. Sixty-four of them were examined with PCR. All the clones were sent for sequence. Thereteen ESTs were obtained after got rid of duplicated sequences by DNASTAR5.01 software. Eleven of 13 ESTS were identified by homology search and 2 of them were unreported or unresearched. Dot blot testified that 12 of 13 ESTs were high expressed in the yong rabbit's repair tissue, which include 2 unreported gene fragments .VEGF in the differential expresse genes was significantly up-regulatied in the yong rabbit's repair tissue. The differential expressed gene fragments were all accepted by the GENEBANK..We obtain the full long sequence of the two unreported genes(A78 amd A80) by RACE method and electronic contig. According to the full long sequence, we designed the primers and amplified the physico-sequence by PCR. A78 had a high homology with the human?2 chain of type?collogen. We gave up the further researchment for type?collogen because Therer were a lot of repaortments about it. A80 have a homology with the S100A9 gene, which was not researched in the joint injury. So we constructed the prokaryotic expression system of the A80 gene by pQE30 vector, pQE30-12-IGI. We obtained 100 microgram aoluble protein of A80 gene through protein expression in the escherichia coli DH5?. We separated the mesenchymal stem cells(MSCs) by whole bone marrow culture and cultured the MSCs with the aimed protein. We observed that the aimed protein could accerated the cell proliferation of MSCs by MTT method and made MSCs hypertrophy by Immunofluorescence staining method.In the researchment of VEGF, bone formation rate, bone quality and quantity, and mineralization level in the subchondral necrotic region of the gene transfection group were significantly higher than the control groups. The control groups had more subchondral bone resorption compared with the gene transfection group.Conclusions:1. plant RNAout kit combining the one-step method has the advantages of speediness, simplification, cheapness and high-quality. This method fits for the extraction of totle RNA from the articular cartilage tissue with plenty of proteoglycan.2. The gene expression is significantly active in yong rabbit's joint injury. Reverse northern blot hybridization identify that 12 of 13 ESTs is high expressed, including three important known genes,VEGF, corticostatin-4(CS-4) and collagen type XI alpha 1, which relate with the repair of joint injury, and two unreported gene fragments. Further reaearchment for these gene are significant.3. We successfully clone the full gene sequences of two unreported genes(A78 and A80) by RACE. A78 had a high homology with the human?2 chain of type?collogen. A80 have a homology with the S100A9 gene, which was not researched in the joint injury. We obtained 100 microgram active protein of A80 gene. We observed that the A80 protein could accerated the cell proliferation of MSCs by MTT method and made MSCs hypertrophy by Immunofluorescence staining method.4. The differential expressed gene VEGF might promote bone formation and revascularization in the subchondral necrotic region of the femoral head, indirectly protecting the necrotic bone trabecula from absorption and avoiding a reduction in the mechanical function of the subchondral region.