A Study Of Assessment T Lymphocyte Subset Of OLP Patient And Sorting And Culture T Lymphocyte In Vitro

Objective:Assessment T lymphocyte subset of Olp patient peripheral blood,analysis patients immune condition.Immunomagnetic sorting T lymphocyte from Olp patient peripheral blood,culture Olp T lymphocyte in vitro.Sorting T lymphocyte from biopsy tissue,aim at know the procedure of sorting T lympho- cyte from local tissue.Methods:Assessment T lymphocyte subset of Olp patient peripheral blood:2 ml venous blood acquired from Olp patient,after antibody labeling and haemolysis test by FACS,control group deal with same operation.Immunomagnetic sorting T lymphocyte and culture in vitro:Obtain T lymphocyte from peripheral blood throught density gradient centrifugation and MACS.Tested by FACS after antibody labeling,to know the purity and apoptosis condition.Also draw the growth curve and undertake the morphologic evaluation.Sorting T lymphocyte from biopsy tissue:Biopsy tissue was dissociated by Dispase,after removal epithelium sheet,the biopsy tissue,with basal membrane revelation,countined operation. And got T lymphocyte proportion after antibody labeling by FACS.Results:1 Assessment T lymphocyte subset of Olp patient peripheral blood:Olp patients' CD8+T lymphocyte significant increased compare to control group(P＜0.001),CD4+T / CD8+T decreased significantly(P＜0.001),CD4+T decreased but no obviously different from control group(P＞0.05).2 Immunomagnetic sorting T lymphocyte and culture in vitro:The purity of T lymphocyte reached to 97.2%throught cell sorting,and increased to 97.7%after 3 days culture.Morphologic evaluation found T lymphocyte is oval-shap,hight transparent,and stay on buttom of dish.T lymphocyte proliferates and become cell clump with several hundreds cells,diameter of this clump variance from 30-100um.The proliferation capability of T lymphocyte declined later,and cell clump dissociated. Cellular membrane contracted obviously of dead cell.Cell growth curve like "s" shows the exponential phase of growth is 2-3d.Transfer of culture is 4-6 d.The T lymphocyte could be maintained in culture up to 3-4 generation.Three days after passage,apoptosis test shows,70.9%is healthy cell,19.4%is on the early stage of apoptosis,1.2%is early necrosis cell,8.5%is necrosis cell.Seven days after passage,apoptosis test shows,48.3%is healthy cell,31.2%is on the early stage of apoptosis,2.3%is early necrosis cell,18.2%is necrosis cell.3 Sorting T lymphocyte from biopsy tissue:The result is not very good,cause cell total amount is insufficiency.T lymphocyte is 6.48%of total suspension cell,and CD4+T is 3.3%,CD8+T is 3.18%.we can get 5~*10~4 cells from one biopsy,but only small part of it is T cell,more over there are many interstitial tissue cell.It makes problem for futher research.Conclusion:1 Assessment T lymphocyte subset of Olp patient peripheral blood indicates increase of CD8+T,means cytotoxicity T cell hyperfunction,cause decline of CD4+/CD8+ ratio.It shows disequilibrium of cell immunity in Olp patient.2 We have successfully sorting T lymphocyte and culture in vitro for 20 days.And we detect the decline of cell proliferation ability,while cell apoptosis increase.It suggests we should co-culture at early 3days.3 Sorting T lymphocyte from biopsy tissue still has not a good result.Because the size of oral biopsy is small,also T lymphocyte is infiltrate within the tissue which makes it diffculty to sort from tissue totally.On the whole there are much need to do on this subject.