| Part 1 Differential expression analysis of T cell function related mi RNAs and cytokines in patients of allogeneic hematopoietic transplantationObjective To investigate the different expression of 8 candidate mi RNAs and related cytokines in CD4+T cells of peripheral blood and plasma in patients with and without acute graft-versus-host disease(a GVHD) after allogeneic hematopoietic stem cell transplantation(allo-HSCT). Methods Differential expression analysis of 8 selected mi RNAs: choose healthy volunteers as control and patients without a GVHD and with II, III, IV a GVHD in 100 days after allo-HSCT according to the a GVHD score standard, analyze the difference expression of 8 candidate mi RNAs by fluorescence quantitative PCR in CD4+T lymphocytes of peripheral blood and plasma from different groups, analyze the expression of cytokines in different experimental groups using cytokine ELISA or CBA kit for the detection. Results 1. Compared with the control group, the a GVHD group has 4 mi RNAs, the expression of mi R-150 and mi R-181 a, which was significantly reduced(P<0.05), while the expression of mi R-155 and mi R-92 b increased significantly(P<0.05); and the different expression of mi R-17, mi R-92 a, mi R-146 a and mi R-146 b was not statistically significant(P>0.05); the expression of mi RNAs in plasma of patients is in coordinate with the expression levels in lymphocytes. In addition, the levels of IL-2, IFN-?, TNF, gamma and IL-17 A was significantly increased in patients with a GVHD(P<0.05), in which IL-2, IFN-? and TNF and a GVHD were positively correlated with the severity of a GVHD. The levels of IL-10 and IL-4 were significantly reduced(P<0.05), in which the levels of IL-10 was negative correlated with the severity of a GVHD while the secretion level of IL-6 was no obvious change(P>0.05); in non a GVHD group, levels of IL-2, IFN- ? and TNF were significantly reduced(P<0.05), while the levels of IL-10, IL-4 IL-17 A and IL-6 were not significantly changed(P>0.05). All cytokines detected had no significant changes before a GVHD happened. In patients with a GVHD, the expression of mi R-181 a and mi R-150 was significantly reduced(P<0.05), and was negatively correlated with the severity of a GVHD(P<0.05). The level of mi R-155 and mi R-92 b were significantly increased and were positively related to the severity of a GVHD(P<0.05). In addition, more than or equal to 4 days before a GVHD onset, the expression levels of mi R-181 a, mi R-150, mi R-155 and mi R-92 b had begun to change. Conclusion mi R-181 a, mi R-150, mi R-155 and mi R-92 b are differentially expressed in the pathogenesis of a GVHD; Th1 immune response is the leading effect of the mechanism of a GVHD; the expression of mi RNAs and cytokines are both correlated with the severity of a GVHD; differential expression of mi RNAs can be used to predict the occurrence of a GVHD.Part 2 Effect of mi R-181 a on CD4+T lymphocyte function, mechanism of action in vitro and targetgene screening, identificationObjective To predict, screen and identify the target genes of candidate mi RNA; to explore the effects of mi RNA on CD4+T lymphocytes and its possible mechanism. Methods Screen the potential target gene of mi RNA through bioinformatic analysis and determine the candidate target genes of mi RNAs by q RT-PCR, Western blot and luciferase reporter assay. Using lentiviral packaging by three plasmid systems, construct lentivirals expressing mi RNA precursors(LV-mi RNA) and empty vector(LV-Ctrl), infect normal human naive CD4+T cells, quantify the relative expression of mi RNA in CD4+T cells using real time quantitative PCR. Evaluate the proportion of Th subsets through detecting IFN-?, IL-4, IL-17 A and Foxp3 using flow cytometry. Detecting the proliferation and apoptosis using CCK-8 and Annexin V/7-AAD assays, quantify the levels of cytokines in cell culture supernatant by ELISA. In addition, through the preparation of LV-mi R-181 a, infect different amount of CD4+T cells with different titer of virus and evaluate the expression levels of IFN- ?.Results Through the bioinformatics analysis, 19 potential target genes of mi R-181 a were screened, in which 5 target genes including IFN-?, were significantly reduced. Luciferase reporter experiments showed that co-transfection of vector expressing the mi R-181 a with the vector of luciferase reporter containing the wide-type IFN-? 3'UTR decreased the luciferase activity significantly but no significant effect on the luciferase activity of the reporter contained the mutated 3'UTR of the IFN-?. After overexpression of mi R-181 a, the m RNA levels of IFN-? showed no significant changes in CD4+T cells compared with the blank control group while the protein levels were significantly decreased and the levels were negatively correlated with the levels of mi R-181a(P<0.05). Successfuliy construct the lentivirus vector expressing miR-181 a precursor and infect CD4+T cells, mi R-181 a expression increased about 4 times(P<0.05). The flow cytomery of T cell subgroup analysis showed that, compared with the control group, the percentage of Th1 cells in LV-181 a infected group was significantly reduced, Th2 and Treg cells were increased while no significant change was found in Th17. CCK-8 assay showed that the cell proliferation significantly decreased in group LV-mi R-181a(P<0.05), Annexin V/7-AAD analysis showed that after infected with LV-mi R-181 a, the percentage of apoptotic cells was significantly increased(P<0.05). ELISA showed that in LV-mi R-181 a group, IFN-? and IL-17 A secretion was decreased in cell culture supernatant(P<0.05), while IL-4 secretion was increased significantly(P<0.05). In addition, with the increase of mi R-181 a concentration, there was no difference change in the IFN-? m RNA level, but the protein level was found decreased and was negatively correlated with the concentration of mi R-181 a. Conclusion Mi R-181 a regulates the function of CD4+T lymphocytes by targeting IFN-?. The regulation of Mi R-181 a on CD4+T lymphocytes is in a concentration dependent manner.Part3 The effects of mi R-181 b on mouse model with acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantationObjective To investigate the function of mi RNA in mice with acute graft-versus-host disease. Methods q RT-PCR, Western blot and luciferase reporter assays were used to determine the mi RNAs on target genes in vivo. The C57BL/6 mice, tail-intravenous injection of LV-mi R-181 b, LV-Ctrl and RPMI 1640 culture medium, were used as donors. Aquire spleen lymphocytes and bone marrow cells from donor mice respectively and detect the expression of mi R-181 b in spleen and bone marrow cells by q RT-PCR. Use BALB/c mice as the recipients, and intravenously inject bone marrow and spleen mononuclear cells from donors into BALB/c mice to establish allo-HSCT mouse model. Observe mice living conditions, the changes of body weight and hematopoietic reconstitution after transplantation, according to the a GVHD clinical criteria of mice a GVHD score. Evaluate histopathological changes of liver and small intestine according to the a GVHD pathological score standard. Quantify plasma cytokines and Th lymphocyte subsets in spleen cells by flow cytometry and ELISA. Results Through the Gene Bank, et al database, we found that mi R-181 a in human and mouse is homologous which has the same mature sequence. The results of q RT-PCR showed that mi R-181 a for spleen mononuclear cells was increased about 2.48 times in donors intravenous injection of LV-mi R-181 a, compared with injection of LV-Ctrl and RPMI 1640 medium(P<0.05), while no significant different expression of mi R-181 a for bone marrow mononuclear cells. The levels of IFN-gamma m RNA gene and protein had no significant change in recipients that receive cells from tail-intravenous injection of mi R-181 a mice(LV-mi R-181 a group) after transplantation. The luciferase reporter gene assay further confirmed that mi R-181 a has no effect on IFN-gamma in mice. Go a further step, bio-informational analysis and luciferase reporter gene experiments confirmed that mi R-181 b and mi R-181 d, rather than mi R-181 a and mi R-181 c targeting IFN- Gamma 3 'UTR in mice. After over-expression of mi R-181 b in mice, the levels of IFN-? protein were detected significant decrease by Western blot, while the IFN-? gene expression had no changes detected by q RT-PCR. Which confirmed that mi R-181 b targeting IFN-? in mice. In the mice after transplantation, the counts of Th1 cell, as well as the levels of IFN-?, IL-2 and TNF increased significantly in the GVHD group, compared with the TBI group by detecting cytokines. In addition the level of mi R-181 b changed ahead of the occurrence of GVHD at the same time. Over-expression of mi R-181 b in mice could alleviate Th1 cell response effectively thus and prevent a GVHD: The results of q RT-PCR showed that mi R-181 b was increased about 4 times after over-expression of mi R-181 b in mice, and the mice of mi R-181 b group had reduced severity of a GVHD compared with the control group(P<0.05), while the total survival rates were higher in mi R-181 b group(P<0.05). Similarity, the histologic damage of liver, bowel and skin was markedly alleviated and the a GVHD pathological score was significantly lower in LV-mi R-181 b group(P<0.05). Conclusion mi R-181 b targeting IFN-? in mice. mi R-181 b can be a predictor in the occurrence of a GVHD in mice. mi R-181 b control Th1 cell response effectively thus alleviate a GVHD of mice. |
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