Effect Of Pectin On T Cell Aggregation And Apoptosis Induced By Galectin-3

Galectin-3is a30—35KD protein with unique chimeric structure, which is a memberof Galectins family. Galectin-3is multifunctional both in vitro and in vivo, playing anindispensable role in the process of cell adhesion, cell growth, inflammation, immunemodulation, tumor aggression and metastasis, cell death, etc., the function among whichmodulating T cell activation and death is of great importance. Carbohydrate bindingdomain (CRD) on C terminal of Galectin-3recognize β-galactose, which endowsGalectin-3the ability to bind polysaccharide or glycoprotein.The immune modulating activity of pectin has become a hotspot in the pectin research;Galectin-3might be one of its target molecules, owing to numerous galactoses on pectinstructure which offer potential interaction with Galectin-3.In our research, WGPA and WGPA-E-1from ginseng, modified citrus pectin(MCP)and potato galactan were chosen as materials, Lactose was chosen as control, performingthe following trials: the best method of testing the growth and activity of suspension cellswas chose and optimized; the methods of Galectin-3inducing T cell aggregation andapoptosis were established successfully. Through these two methods the interactionbetween WGPA、WGPA-E-1、MCP、Galactan and Galectin-3was tested respectively,following which the effects of those polysaccharide inhibiting Galectin-3inducing T cellaggregation and apoptosis were tested. Besides，the method of Galectin-3suppressing Tcell activation was tried to construct in order to test the effect of pectin on T cell activation.The optimum dissolving Formazan solutions were chose from several common usedones, DMSO,20%SDS, SDS-DMF and SDS-isobutanol-HCl (triplex solution). The resultshowed that the bigger error could be caused by DMSO, certain limitation was found byusing triplex solution, thus both DMSO and triplex solution could not be used in testingsuspension cells. The optimum acid concentration in20%SDS and SDS-DMF was0.04M,both of which had higher sensitivity.20%SDS was chose to be the optimum formazansolution, owing its resolving Formazan more rapidly, much easier making, restoring for arelatively long time, etc..The interaction between polysaccharide and Galectin-3was tested by Hemagglutinationassay, the result indicated that WGPA-E-1and MCP had better interaction than Galactan, which was better than Lactose, the interaction between WGPA and Galectin-3was notobvious.The effect of saccharide inhibiting splenic cells aggregation induced by Galectin-3was tested through the method of Galectin-3inducing T cell aggregation, the result showedthat the effect was WGPA-E-1>MCP>Lactose, WGPA could not inhibit splenic cellsaggregation, for it had little interaction with Galectin-3. Galactan had the same effect asWGPA, owing to its steric hindrance and huge molecular weight.The saccharide inhibition effect on splenic cells apoptosis induced by Galectin-3wastested later; the result of MTT showed that MCP had potential effect, Galactan had littleeffect, Lactose had very good inhibition effect on inhibiting T cell apoptosis; the result ofAO/PI double staining method showed that WGPA could not inhibit T cell apoptosis, for ithad little interaction with Galectin-3; MCP and Galactan had little effect either, owing totheir steric hindrance, or their failure to inhibit the interaction between Galectin-3and Tcell death receptor. WGPA-E-1could inhibit T cell apoptosis obviously, the reason mightbecause it could inhibit T cell aggregation or competitively inhibit Galectin-3interactingwith death receptor, however, the inhibition rate (60%) is far less than Lactose(100%),which mainly due to the complex structure of WGPA-E-1.Method of Galectin-3suppressing T cell activation was tried to establish: usingdifferent kinds of T cells, Jurkat and splenic cells from mouse, and choosing ConA asactivator, tested the level of T cell activation by MTT. The results showed that ConA couldonly active splenic cells, thus three different splenic cells were chose, na ve T cells(fromnormal mouse),and antigen experienced T cells from mouse with ascitic tumor and withsolid tumor respectively. ConA, Lactose and Galectin-3was used to establish the method.The result showed that none of them has the ability to test Galectin-3suppressing T cellactivation, indicating that ConA was not suitable to be used in this method, and neededcontinuing work for further study.