Construction And Analysis Of Diferential Library Of The Factors Related To Spontaneous Cartilage Repair In Rabbit Knee Joint By Suppression Subtractive Hybridization

Background: Cartilage repair is one of the difficulties of medical science. The main reason of cartilage repair failure lies in that the permanent hyaline cartilage articular surface can not be rebuilded. 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 literature,the yong rabbit has the ability to rebuild the hyaline cartilage articular surface after articular cartilage injury. The hyaline cartilage account for more than 80% in the repair tissue, and the durability and the construction approximate the normal articular cartilage. But the old rabbit can only form a small quantity of fibrocartilage tissue in the injury position. We guess that the different gene expression lead to the diffenrent result in the repair tissue of yong rabbit. So we may find the key factor related to the spontaneous cartilage repair through comparing the early gene expression of the yong rabbit's articular cartilage after injury with the old rabbit. This is very important and meaningful.Objective: To construct the diferential library and clone differentially expressed genes related to spontaneous cartilage repair in yong rabbit knee joint after injury by suppression subtracted hybridization method. Analysis and screen the relative genes to lead spontaneous repair of the yong rabbit articular cartilage. Build a foundation for further cloning the full gene sequence and researching the function of the genes.Method: Isolate the differential expresed genes in the yong rabbit's injuried articular catilage in the course of repair with the suppression subtracted hybridization (SSH) technology and the Switching Mechanism At 5'end of the RNA Transcript (SMART) technology. Three adult female New Zealand white rabbits(aged 50 weeks) and their offsprings(aged 8 weeks) were selected. An osteochondral defect was created on the anterior weight-bearing area of the medial femoral condyle in both knees. A defect 3 mm in diameter and 3 mm in depth, penetrating the subchondral bone plate, was created on the medial femoral condyle as far to the rear as possible, using a bit. All the rabbits were killed four weeks after operation with the method of aeroembolism. Immediately after death, both hind limbs were disarticulated at the hip joint and the repaired tissue were separated and stored at the liquid nitrogen.The total RNA of the yong and the adult rabbit's repaired tissue was extracted and prepared as the template of RT-PCR.The cDNA prepared from the yong rabbit was used as the tester and the cDNA from adult rabbit as the driver. The ds tester and the driver cDNA was digested into shorter, blunt-ended ds cDNA fragments with Rsa I.The tester cDNA was aliquotted into two parts.One aliquot was ligated with Adaptor 1 and the second was ligated with Adaptor 2R.Then two hybridizations and two PCR amplifications were performed.The PCR production containing differentially expressed cDNAs was ligated with T vector and was translated into competent E coli. The positive colonies were selected by blue-white experiment and the differential library was constructed.The positive colonies were sequenced after identification by PCR. The ESTs of the differentially expressed genes were obtained with the software DNAStar 5.01 and the software Chromas and analysised by the Reverse Northern blot.BLASTN the EST sequences in GENEBANK and selected the aimed gene fragments as the further researching target.Results: The totle RNA extracted through one-step method combining plant RNAout kit had a high quality and was reversely transcripted into ds cDNA by LD-PCR. The diferential library of yong rabbit'repaired tissue was successfully constructed by suppression subtracted hybridization (SSH) technology and Switching Mechanism At 5'end of the RNA Transcript (SMART) technology.We obtained 223 individual cDNA clones,and 64 of them were examined by PCR. Twenty diffrential expressed gene fragments were obtained after sequencing and analysising with software, 13 of them were identified by homology search(including 1 vector sequence and 6 nonsense suquences such as mitochondrial gene, ribosomal gene etc.) and 6 of them were unreported. Dot blot testified that 18 of 20 were the differential expressed genes and 2 of 20 was excluded. In the testified 18 gene fragments, 9 are significantly up-regulatied, which include 3 unreported gene fragments, and 3 only express in the yong rabbit, which are all unreported gene fragments.The differential expressed gene fragments were all accepted by the GENEBANK. Conclusions:1. The one-step method combining plant RNAout kit has the advantages of simplification, speediness, cheapness and high-quality, and fits for the totle RNA extraction through the articular cartilage tissue with plenty of proteoglycan.2. The diferential library including 199 individual cDNA clones was successfully constructed by SSH technology and SMART technology.3. After BLASTNing the genes we screened from the library, we found the genes on the highest identities with them are involved in many biological processings.The identified differential expressed genes include calgranulin B, corticostatin-4(CS-4), collagen type I alpha 2, collagen type XI alpha 1 etc. which relate to metabolism and matrix synthesis of chondrocyte.4. Six unreported ESTs we submitted to Genbank were enrolled.5. The dot blot shew that 3 novel ESTs specially express in the injury articular cartilage of yong rabbit. They are maybe the factors related to spontaneous cartilage repair in the yong rabbit. It is important and significant to deeply research these genes.