Role Of CGI99 In Chondrogenesis Of Deer Antler

Cartilage which is a tissue composed of only one kind of cell has a strong ability to resist stress and strike. Since there are no blood vessels, cartilage almost loses the ability to repair itself. Study found that antler cartilage is a very unique cartilage tissue. It can not only repair under the natural condition, but also can regenerate in a fast rate each year. The characteristics of seasonal regeneration make antler become a potential medical model that studies for the regeneration process of organ and body. Previous studies have found CGI99 gene was highly-expressed in antler cartilage column(unpublished), but it hardly expressed in other parts of the antler, such as blood vessels. Therefore, we speculate that the gene may regulate chondrogenesis, and it may be involved in the molecular regulation of chondrogenesis. It is unclear that the mechanism of CGI99 in antler chondrogenesis. A study has found that the antler generation which is a stem cell-based process begins with antlerogenic periosteum(AP). We preliminarily discussed the role of CGI99 from AP cells in antler chondrogenesis in vitro.High score RNAi targeted sequence of CGI99 was designed and selected; a negative control sequence was designed; an oligo deoxynucleotide of shRNA was designed and synthesized, and the oligo DNA was ligated to pLVTHM to construct plasmid. The positive constructed plasmid was subsequently confirmed by PCR and sequencing, then transfected into 293 t cells with pSPAX2 and pMD2.G plasmids. AP cells were infected by the recombinant lentivirus and subsequently stable cell lines were obtained. The expression level of CGI99 mRNA in infected AP cells was detected by RT-PCR and Western-Blot. When CGI99 was in a suppression expression, its effect on AP cell proliferation was measured by MTT assay. Stable AP cell lines were used to cultivate micromass and paraffin sections with liveness micromass were made, then the differentiation tendency of cartilage was observed through these sections. After the extraction and inversed transcript of the total RNA of micromass, the expression level of CollagenⅡ mRNA was detected by RT-PCR to determine the microsomal cartilage differentiation tendency.The results showed that one shRNA aiming at CGI99 was successfully selected and verified by PCR and sequencing, and it was correctly inserted into pLVTHM to obtain the recombinant plasmid. Recombinant lentivirus particles were obtained through three plasmid transfection. The infected AP cells expressed green fluorescent protein and can be kept as stable genetic cell lines. RT-PCR results showed the expression level of CGI99 mRNA in AP cells that were infected by recombinant lentivirus was obviously decreased, and the interferential efficiency was about 70.8%. Western Blot results showed that the expression of CGI99 protein in infected AP cells decreased significantly. The MTT curves of three kinds of cells had similar tendency, which meant the suppression expression of CGI99 gene did not affect AP cell proliferation. Visible and dense micromass was obtained and its paraffin sections showed that compared with the negative control group and uninfected group, micromass cartilage differentiation of treatment group had a weak tendency, at the same time, the results of expression level of Collagen Ⅱ mRNA measured by RT-PCR also confirmed this conclusion.In this experiment, CGI99 in AP cells was taken as the research object. We find that CGI99 plays an inhibition role in chondrogenesis after its silencing. We preliminarily consider that the gene has some effect in the growth, development and differentiation of cartilage. CGI99 might be a key factor to regulate AP cells to differentiate into cartilage cells, but the mechanism of its influence in chondrogenesis still needs to study in the future.