The Study Of Dendritic Cells Culture And Killing Effect By T-Lymphocytes After Activiated By Dendritic Cells Loaded With GC MGC-803 Cell With Over Expressed E2F-1

Objective: To study the methods of induction ,biological character of dendritic cells(DCs)which derived from the peripheral blood of health adult,and further to study the anti-tumor of kidney effect activated by DCs in vitro.It will provide the experimental foundation for clinical GC immunotherapy of DCs.Methods:1.The induction and biological character of DCs derived from human monocytes Monocytes isolated from the peripheral blood of health adult are cultured in medium.compare to the cultural effect with different cell factors and bolting optimal cultural method.at 37℃in humidified 5%CO2 in air in medium with rhGM-CSF,rhIL-4 and TNF-αfor 7-10 days,DCs from human monocytes are analyzed on morphology, cellar structure under optics and electronic microscope, Stronger allo-MLR is one of DCs characteristics,it is performed by using CCK8 assay to observe the ability of DCs stimulating lymphocytes proliferation.and then phenotype of DCs are detected rhIL-12 in medium supernate by Enzyme-linked immunohistochemistry assay. GC carcinoma cell MGC803 line are cultured with 1640 medium detected the double time is 52.2h by CCK8 assay and preferenced experiment with logarithmic growth phase cells.3.The ability of DCs loaded with tumor cell antigen stimulating lymphocytes proliferation in order to study the ability of DCs which loaded with antigen stimulating lymphocytes proliferation, monocytes isolated from the peripheral blood of health adult are cultured in medium with rhGM-CSF,rhIL-4 and TNF-α.At the same time,got the gc carcinoma cell antigen from MGC803 cell with over expressed E2F-1 line.Gain T lymphocytes by using cytokine IL-2 to stimulate antologous lymphocytes.Then cultured the tumor antigen, DCs and T lymphocytes all together,the proliferation of T lymphocytes in the 24h,48h and 72h are detected respectively by using cck8 method. calculate T lymphocytes stimulating index. 4.The killing effect of CTL by DCs loaded with GC freeze thawing antigen first use DCs loaded with tumor antigen induce the growth of CTL, detect the Absorbance of different group by MTT assay and calculate T lymphocytes stimulating index. then detect inf in DCs loaded with tumor antigen medium supernate by Enzyme-linked immunohis to chemistry assay. detect the killing effect by T-lymphocytes after activiated by Dendritic Cells loaded with GC MGC803 cell line freeze thawing antigen by MTT assay in vitro..Renal cell MGC803 line changes after killing action observe renal cell MGC803 line changes after killing by optics and electronic microscope,flow cytometry and wright's staining.Result:1.The identification of DCs and its biological character It can not obtain typical dendritic cells with purely rhM-CSF,rhIL-4 or TNF-α. Typical DCs can be induced when monocytes are cultured at 37℃in humidified 5%CO2 in medium with rhM-CSF,rhIL-4 and TNF-αfor 7-9 days. It shows typical morphology and cellular structure of dendritic cell under optics and electronic microscope. The detection of allo-MLR shows that DCs can stimulate strong T lymphocytes proliferation by cck8 assay. detected inf release in DCs medium supernaterh by Enzyme-linked immunohis to chemistry assay is up.2.GC carcinoma MGC803 cell line cultureed in vitro GC carcinoma MGC803 cell line cultureed and detected double time is good. selected the logarithmic growth phase cells to experimentize.3. the stimulating indexs show that DCs loaded with tumor cell antigen can stimulating strong antologous lymphocyte proliferation, it is more effective than control's(P<0.05).4.The killing effect of T-lymphocytes after activiated by Dendritic Cells loaded with GC MGC803 cell line freeze thawing antigen Detected INF in Dendritic Cells loaded with tumor antigen medium supernate by Enzyme-linked immunohistochemistry assay. DCs loaded with tumor cell antigen can generate strong specific CTL that kill the target mgc803 by DCs which cultured without antigen kill the target cells weakly.T lymphocytes stimulated by tumor antigen only generate non-specific immunoreaction. T-lymphocytes group after activiated by Dendritic Cells loaded with GC MGC803 cell line freeze thawing antigen compare with non-specific T lymphocytes(P<0.05).5.The renal carcinoma cell's change After T-lymphocytes activiated by Dendritic Cells loaded with GC antigen, the renal carcinoma cell volume are diminished and cell died under optics microscope; cell nucleus chromatin pycnosised under Wright's staining; cell apoptosis is found by electronic microscope; FCM result of Diclofenac sodium and Ketorolac tromethamine showed that the apoptosis cusp and rate increased.Conclusion:1.It is important of cell factor for Dendritic Cells culture.Culturing human monocytes in medium with rhGM-CSF,rhIL-4 and TNF-αcan induce typical DCs.It shows potent antigen presenting role to exogenous antigen.DCs from the peripheral blood of health adult with renal carcinoma cell antigen not only strongly stimulate autologous lymphocytes proliferation,but also play an important role in anti-tumor immunity.there have significant difference compare with control group.3.DCs loaded with tumor antigen could induce the growth of CTL. The cyotoxicity of obtained specific CTL against GC-803 was highly enhanced with a significant difference from that of non-specific CTL. The interleukin-12 release/secretion was increased by tumor antigen, which suggest an improved anti-tumor effect .It is suggested that DCs antitumor vaccines poses a clinically useful prospect with GC.4.It is convenient to use freeze thawing antigen loaded with DCs and avoid to identification of renal cell carcinoma special tumor antigen. specific CTL induced by DCs sensibilized by GC freeze thawing antigen exerts a remarkable killing activity on GC MGC803.It is suggested that DCs loaded with freeze thawing antigen antitumor vaccines poses a clinically useful prospect with GC.