| Objective:This experiment which based on NK cells in vitro to investigate the s MICA effects the occer of c GVHD through affecting the change of NKG2 D expression on the surface of NK cells in plants and IFN-γ secretion level after allo-HSCT. Methods:1. Purification of NK cells: high-purified NK cells were separated by Rosette Sep? NK cell enrichment kit in 20 ml of peripheral blood collected from healthy volunteers, detect the purification of cells before and after sorting by flow cytometry.2. Detection of NKG2 D expression level and IFN-γ secretion level: the purified NK cells were cultured in vitro. The change was observed in the number of cells before and after cultured. Using flow cytometry to detect NKG2 D expression levels on NK cells after co-culture with different concentrations of s MICA. ELISA detection training content of IFN-γ in a liquid. The supemant was collected to determine the concentration of IFN-γ by ELISA.3. Testing the killing activity of NK cells: determing the killing activity of NK cells which co-cultured with different concentrations of s MICA against K562 cells with the CCK-8 colorimetry when the E:T ratios was 10:1.4. The s MICA levels in serum which acquisited from the 100 th day after allo-HSCT was detected by ELISA. Results:1. Purity of NK cells: the purified NK cells was detected before and after sorting for(13.2 ± 2.1)% and(88.6±5.5)%.2. The rate of expression level of NKG2 D on NK cells: the expression of NKG2 D which co-cultured with s MICA0 and 100 ng/m L were(91.24±1.96)% and(80.11±2.21)% respectively, the difference was statistically significant(P<0.05).3. Determining the level of IFN-γ in the culture medium of each group: the concentration of IFN-γ in s MICA 0, 0.5 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml were(134.74±23.30) pg/ml,(190.43±15.41) pg/ml,(215.91±14.54) pg/ml,(128.35±18.75) pg/ml,(95.09±15.67) pg/ml, respectively. SMICA 0.5 ng/ml, 1 ng/ml group secreted IFN-γ levels were significantly higher than that of s MICA 0 ng/ml, the difference was statistically significant(P<0.05), while s MICA 10 ng/ml, 100 ng/ml group secreted IFN-γ levels was lower than s MICA concentration of 0 ng/ml group, the difference was statistically significant(P<0.05).4. Determing the killing activity of NK cells: K562 cells were target cells and E:T ratios was 10:1. The NK cell killing activity of s MICA 0 and 100 ng/ml group were(38.82±2.21)% and(12.71±1.48)%, respectively. SMICA 100 ng/ml group was significantly lower than that of the s MICA concentration of 0 group, the difference is statistically significant(P<0.05).5. The s MICA levels in serum was detected by ELISA: the patient with the number 6 s MICA levels was 386.08 pg/ml, significantly higher than other patients. Furthermore, he was the only one of the 12 patients with c GVHD. Conclusion:SMICA can downregulate the expression of NKG2 D receptor on NK cell surface, decrease the cytotoxicity of NK cells. It also has dose-dependent effect on the regulation of secreted IFN-γ levels. Low dose of s MICA(0.5 ng/ml and 1 ng/ml) increased the activation of NK cells secreted IFN-γ levels, while high doses of s MICA(10 ng/ml and 100 ng/ml) reduced activation of NK cells secreted IFN-γ levels. Level of s MICA in peripheral blood can be as a potential predictor for c GVHD, and become a new target for immunotherapy of potential weakening of c GVHD. |
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