Effects Of Artocarpus Lingnanensis Lectin On The Growth Of T Lymphocytes

Objective:Our Previous studies showed that Artocarpus lingnanensis lectin(ALL)may be an ideal new mitogen.In this research,in order to explore the effects of ALL on the growth of T lymphocytes,we treated T lymphocytes with ALL,and then detected the growth indices of T lymphocytes.These studies will lay a solid theoretical and experimental foundation not only on exploring the mechanisms of ALL in molecular level,but also its development and application in disease prevention and treatment.Method:(1)A convenient,sensitive and reliable protocol was established to isolated and purified ALL.[1-2].ALL was purified from the seeds of Artocarpus lingnanensis by ammonium sulfate fractional precipitation and affinity chromatography on Gal-Sepharose 6B eluted with 0.2mol/L Gal,then dislysis and ultrafiltration.After freeze drying,the hemagglutination was established to determine the activity of ALL.The purity of the protein was identified by discontinuous PAGE.After filtration sterilization,the concentration of ALL was determined by BCA kit.Different concentrations of ALL was prepared,which utend the cell culture experiment on the next step.(2)T lymphocytes were isolated from the health adult peripheral blood by Nylon Fiber Column T(L-Type).T cells were incubated with different concentrations of ALL,while laying PHA as the control group.After treatment at different time,①The proliferation of T lymphocytes was analyzed by MTT method and the change of cell morphology was observed by Inverted Microscope;②After synchronized by serum starvation,T lymphocytes were incubated with different concentrations of ALL,and cell cycle was analyzed by PI staining and flow cytometry assay(FCM);③ The subpopulations of CD3+CD4+ lymphocytes and CD3+CD8+ lymphocytes were evaluated by fluorescin-conjugated monoclonal antibody triple labeling technique and fluorescence-activated cell sorting(FACS);④The expression level of CD25 on the surface of T lymphocytes was evaluated by fluorescin-conjugated monoclonal antibody double labeling technique.(3)Jurkat T lymphocytes were incubated with different concentrations of ALL.①The proliferation and colony formation of Jurkat T lymphocytes were analyzed by using CCK-8 colorimetric assay and Inverted Microscope,respectively.②The expression level of CD25 on the surface of T lymphocytes was evaluated by fluorescin-conjugated monoclonal antibody double labeling technique.③ The human interleukin 2,interleukin 10 and Interferon γscreted by Jurkat T cells were analyzed by enzyme linked immune sorbent assay(ELISA).④The apoptosis of Jurkat T cells was analyzed by combining Annexin V-FITC with PI staining.⑤The effects of ALL on cell migration of Jurkat cells were investigate by Transwell chamber model assay.⑥Synchronized Jurkat T cells were incubated with different concentrations of ALL,and then cell cycle was analyzed by PI staining and FCM.⑦The expressions of MAPK signaling pathway proteins(p-ERKl/2,p-p38)and apoptosis caspase family members(p-caspase-3)was analyzed by Western Blotting.Result:The purified ALL agglutinates erythrocytes of the human’s O blood groups.It showed a single band of 59kDa on discontinuous PAGE.ALL was dissolved in phosphate buffer and the concentrations of ALL were 0.36μg/mL,1.8μg/mL,3.6μg/mL,18μg/mL,36μg/mL and 360μg/mL.On one hand,MTT showed that ALL significantly promoted the growth of T lymphocytes,showing dose-response relationship.The proliferation rate was 127.63%in ALL(360μg/ml)treatment group.It was also observed that T cells colony numbers increased with ALL concentration increase.After incubated with different concentrations of ALL for 24h,the proportion of T cells in GO/G1 phase decreased,but increased in S peak and G2/M,showing dose-response relationship.FACS results showed that ALL significantly promote the proportion of CD3+CD4+ subset cells.The proportion of CD3+CD4+ subset cells was increased to 80.59%by the dose of 36μg/ml.The CD25 expression on the surface of T lymphocytes was also enhanced significantly.The proportion of CD3+CD25+ cells was increased to 91.40%by the dose of 36μg/ml.On the other hand,ALL inhibited the growth of Jurkat T lymphocytes distinctly.ALL inhibited the proliferation of Jurkat cells in the concentration range of 0.36～360pg/ml,showing dose-response relationship.Inhibition rates were in the range of 1.82～59.17%(P<0.01),and its IC50 was 59.72μg/ml.It was also observed that Jurkat T cells colony numbers decreased with ALL concentration increase.ALL enhanced CD25 expression on the surface of Jurkat T lymphocytes significantly.The proportion of CD25+ Jurkat cells was increased to 60.02%by the dose of 1.8μg/ml,showing 9 times more than the control group.ALL enhanced IL-2 and IFN-γ secretion.Compared with the control group,the level of IL-2 and IFN-γ were 11.69 times and 21.29 times higher respectively in ALL(1.8μg/ml)treatment group.ALL induced apoptosis in Jurkat T lymphocytes in a time-and dose-dependent manner,and Western blot showed that caspase-3 was activated.Compared with control group,ALL strongly inhibited Jurkat T lymphocytes migration,and dose dependent inhibition was also observed.After synchronized Jurkat T lymphocytes were incubated with different concentrations of ALL for 24h,FACS showed that the cell cycle is predominantly blocked in S phase in a concentration-dependent manner.Western blot showed that ALL activated MAPK signaling pathway in Jurkat cells,with the up-regulation of p-ERK1/2 and p-p38.Conclusion:(1)ALL promotes the growth and cell cycle entry of peripheral blood T lymphocytes,especially the proliferation and activition of CD3+CD4+ subset cells.(2)ALL inhibits the proliferation of Jurkat T lymphocytes obviously,which is related to the S-phase cell cycle arrest and cell apoptosis.(3)ALL induces the activation of Jurkat T lymphocytes,and promotes IL-2 and IFN-γ secretion.(4)ALL inhibits the migration of Jurkat T lymphocytes significantly.(5)ALL activates the ERK1/2 and p3 8 MAPK signaling pathway in Jurkat cells,and enhances the activation of Caspase-3.