Study On The Relationship Of N-GSLs Expression Of Immunocompetents Cells And MDR Tumor Cells & Tumor Immunology

ObjectiveGSLs are ubiquitous membrane constituents, which are embedded in the cell plasma membrane. GSLs that contain at least one monosaccharide and a sphingoid have been subdivided into neutral GSLs and acidic GSLs. Neutral GSLs may contain glucose, galactose, N-acetylgalactosamine, N-acetylglucosamine or fucose and can be formed by one or more saccharide residues. Acidic GSLs consist of two groups: the sialosyl-glycosylsphingolipids or gangliosides and the sulfoglycosylsphingolipids or sulfatides according to the saccharide core linked to sulfate or to one or more sialic acid residues. GSLs play essential roles in a variety of phenomena involving cell-cell recognition, cell adhesion, signal transduction, and cell growth and differentiation. Also, it is known that glycolipids are the receptors of some bacteria and viruses. Glycolipids have been identified as tumor or differentiation antigens. As cell markers, GSLs possess immunogenicity and play immunomodulatory roles in immunity system. Because of limited sensitivity of neutral GSLs determination, previous researchs were almost concentrated on gangliosides and lack of progress in these tissues such as lymphocytes which have a great small amounts of GSLs. To increase sensitivity of neutral GSLs determination, we have developed a microdetection system- autoradiography for N-GSL analysis. Then we detected N-GSLs expression of LAK cells and different tumor cells on different conditions via ARG. By means of the information, we primarialy explored the relationship of between N-GSLs and immunity functions.Method1. To develop a new neutral GSLs determination?ARG: (1) [14C(U)]-palmitic acid, which is a precursor of sphingolipid , was used as a tracer . Acurve was made that indicated the relation between the dosage of tracer and the amounts of labeled cells after detecting the dpm of labeled cells on the different dosage of tracer via liquid scintillation, detector. The optimum concentration wanted could be elected. (2) Labeled N-GSLs were developed in different concentrations with High-performance thin-layer chromato--graphy (HPTLC) , then X-ray film covering cosely HPTLC plate was exposed in days. The optimum time of exposure wanted could be obtained. (3) The sensitivity of both ARG and iodine visualization was compared.2. Study on the relationship of changes of morphology and functions & N-GSLs expression of LAK cells on different phases:(l) The morphological changes of PHA-LAK cells were observated during the whole period of culture. (2) PHA-LAK cells on both of the stages of LAK-I and LAK-M were labeled by [14C(U)]- palmitic acid respectively. The labeled N-GSLs of two stages were developed with HPTLC respectively, then X-ray film covering closely HPTLC plate was exposed and autoradiography.3. Study on the relationship of changes of morphology and functions & N-GSLs expression of LAK cells after co-incubation with KB, KBV20o, KBW: (1) Continous observation of the morphological changes after co-incubation. (2) MTT colorimetry were employed to evaluate the cytotoxic activity of LAK cells and NK cells against multidrug -resistant human oral carcinoma cell line-KBV2oo (before and after reversal of MDR) and parental drug-sensitive cell line KB. (3) LAK-M cells were labeled by [14C(U)]-palmitic acid and then incubated with KB, KBV200, KBW respectively. The labeled N-GSLs of LAK-M cells before and after incubation with 3 tumor cell lines were developed with HPTLC respectively, then X-ray film covering cosely HPTLC plate was exposed and autoradiography.4. Study on the relationship of changes of morphology and functions & N-GSLs expression of KB, KBV200, KBW in tumor immunity: (l)To observe the morphological changes of KB, KBV200, KBW respectively after 24h incubation with LAK cells. (2) MTT colorimetry were employed to evaluate the cytotoxic activity of LAK cells against KB, KBV200, KBW. (3)N-GSLs expression of KB, KBV200, KBW respectively before and after incubation with LAK cells were detected via ARG. KB, KBV20o, KBW were labeled by [14C(U)]- pal...