| In recent years,stem cell therapy has entered a clinical trial phase for treating related diseases.But the phenomenon of immune rejection has always been a unavoid probelm,which is a stumbling block for stem cell therapy.GAL-1 is found to be highly expressed inmost mesenchymal stem cells,tumors and antler stem cells.These cells have appeared to show the phenomenon of immunosuppression.GAL-1,as a well-known immunomodulatory factor,is likely to be involved in the immune regulation of stem cells.This thesis studied the immunomodulatory effects of GAL-1 in several types of stem cells and hopes to provide some theoretical and technique data for stem cell therapy.The GAL-1gene in the deer antler stem cells was found to be highly expressed during antler generation and regeneration,indicating that it may be an important regulator for antler formation.To study the expression and possible biological function of GAL-1 in antler stem cells,the tissues within which antler stem ells reside were used as experimental materials.In this study,the GAL-1 gene coding sequence region of sika deer(Cervus nippon)was cloned and bioinformatics analysis of GAL-1gene was carried out using software or website methods.The relative expression level of GAL-1 in the antler stem cells was detected by using Western-Blot and Quantitative PCR Detecting System,Q-PCR technique.The results showed that the GAL-1 open reading frame was 408 bp in length and encoded 134 amino acids.The relative molecular mass was 14.69 KDa and the amino acid sequence was close to that of Bovine and Ovis aries.GAL-1 is mainly distributed in the interstitial substance,did not have transmembrane regions and glycosylation sites,and there may be two phosphorylation sites.The secondary structure of the GAL-1 proteinis was mainly β-sheets and random coil,and the tertiary structure was similar to that of the Bovine(Bos taurus).Expression level of GAL-1 was higher in the facial periosteum than in the antler stem cell tissues.In this study,we predicted the structural characteristics of GAL-1 protein,detected the expression level of GAL-1 protein in the antler stem cells and provided the basic data for the further study of antler regeneration.Antlerogenic Periosteal Stem Cells(APCs)have been classified as a kind of mesenchymal stem cells(MSCs),which was the basis for the initiation of antler growth.GAL-1 protein was abnormally expressed in APCs.Therefore,to explore the specific function of GAL-1 gene in the APCs,RNAi technique was used in this experiment and the interference efficiency was estimated to be about 62% by Q-PCR and Western-blot.In this study,PI and CCK8 were used to detect cell cycle and proliferation respectively.Q-PCR was used to detect IL-6 and C-MYC.The CFSE-stained peripheral blood lymphocytes were co-cultured with the APCs for 4 days and observed under a microscope and detected by flow cytometry.The percentage of S phase in the treated cells was increased,while percentage of G1 phase was decreased.The proliferation rate of the APCs was accelerated.IL-6 and C-MYC were up-regulated 1-fold and 5-fold,respectively.The cell division speed of peripheral blood lymphocytes was significantly accelerated.These results indicate that reduction of GAL-1 in cells leads to the majority of stem cells entering the dividing mode and the cell division speed was accelerated.Immune related factors such as IL-6 and C-MYC were upregulated,resulting in the activation and proliferation of immune cells.In summary,this study speculated that GAL-1 was the key factor in the proliferation and immune regulation for antler generation and regeneration. |
