Effect Of Erythrocyte CD44s On The Immune Adherence To Gastric Cancer Cell

ObjectiveCD44 is a transmembrane glycoprotein of adhesion molecule family,and expresses on erythrocyte surface as a standard-type CD44(standard isoform of CD44, CD44s) in the largest numbers of immune molecules.CD44s is the major receptor for the macromolecular polysaccharide hyaluronic acid(HA).Hyaluronic acid expresses on tumor cells.Studies have shown that ability of erythrocyte immune-adhering cancer cells declined for cancer patients,and the expression of CD44s on erythrocyte surface was significantly lower.In view of this,we conclude that the combination between CD44s of erythrocyte as receptor and HA of tumor cell as ligand play a significant role in erythrocytes immune-adhering tumor cells.Our study aimed at the identification of CD44s on the plasma membrane of normocytes and the red blood cell of gastric cancer patient,and the expression of HA on the plasma membrane of gastric carcinoma cells. We blocked normocyte CD44s by mouse anti-human CD44s monoclonal antibody to observe the change of the ability of normocytes immune-adhering gastric carcinoma cells in order to explore the immune-adhering role of normocyte CD44s to gastric carcinoma cells.Materials and Methods1.Reagents and drugsMouse anti-human CD44s monoclonal antibody and PE mouse anti-human CD44s monoclonal antibody were purchased from BD company America;Biotinylated hyaluronic acid binding protein,Horseradish peroxidase labeled biotin-binding protein,DAB Color Kit were all purchased from senxiong biotech company,shanghai。2.Apparatuses and equipmentsBiological safety cabinet;CO₂ incubator;Inverted microscope;Flow cytometry 3.Preparation of erythrocytes and gastric cancer cellsHuman gastric cancer cell line MGC-803 was cultured and amplified,then cells was harvested after digestion by pancrease enzyme.1×10⁶/ml suspension of gastric cancer cells were cultured on coverslips for experiment.Normocytes were obtained from healthy donates and gastric cancer patient,then prepared 1×10⁸/ml and 1×10⁶/ml suspension for test.4.Experiment methodsThe expression of CD44s on the plasma membrane of erythrocyte was detected by using flow cytometry;The expression of HA in the plasma membrane of gastric carcinoma cell was detected by immunohistochemical staining;The immune adherent ability of normocytes to gastric carcinoma cells was detected by using rosette rate of normocytes immune-adhering tumor cells(R-T) rosette.ResultsThe mean fluorecence intensity of CD44s on normocyte plasma membrane was (308.694±8.643),and the expression of the erythrocyte of gastic cancer patient was significantly reduced compared with the normocye,the mean fluorecence intensity was(201.714±5.324).The mean IOD of HA on the gastric carcinoma cells was (4617±1775.363).The ability of normocyte immune-adhering gastric carcinoma cell was significantly lower after normocytes were treated by mouse anti-human CD44s monoclonal antibody.The R-T rosette was 20.6±9.232(%),which was less than the blank control group(P＜0.05)Conclusions1.There was HA expression on gastric carcinoma cells。2.The expression of the erythrocyte of gastic cancer patient was significantly reduced compared with the normocye.3.Erythrocyte played an important role in the immune adherence to tumor cells by combination of CD44s and HA.