| We previously showed that miR-15a/16 was significantly down-regulated in hepatocellular carcinoma tissue compared with adjacent tissues by a microRNA chip assay. The miR-15a/16-1-/-mice was introduced to our lab for further study function of miR-15a/16. Here we aimed to analyze effects of miR-15a/16-1 deficiency on immune function of mice under physiological condition or of mice bearing transplanted tumors by using the miR-15a/16-1-/-mouse. This study would provide new ideas of immunotherapy against cancer.Our study can be divided into three parts.Part I. Breeding and identification of miR-15a/16-1-/-mice, and analysis of frequencies of cell subsets in spleen of mice of 8-10 weeks oldObjective:To analyze frequencies of cell subsets in spleen of miR-15a/16-1-/-mice of 8-10 weeks old by using the miR-15a/16-1-/- mice.Methods:Firstly, using conventional mating represented in mice and identification of miR-15a/16-1 gene knockout mice by PCR. Then, execute miR-15a/16-1-/-mice of 8-10 weeks old and mononuclear cells of spleen were isolated both from positive mice and control mice. Analysis of frequencies of cell subsets in spleen of miR-15a/16-1-/- mice of 8-10 weeks old by FCM. In addition, using animal blood analyzer to analyze the blood which was collected from the orbit of miR-153/16-1-/- mice of 8-10 weeks old and control mice.Results:The positive rate with miR-15a/16-1 gene of filial generation mice met Mendelian genetic law. PCR can be used to identify the genotype of the miR-15a/16-1 gene knockout mice, and to establish an animal experimental model to further study the important role of miR-15a/16-1. Compare to the wild-type mice, CD19+cells in spleen of miR-15a/16-1-/- mice of 8～10 weeks old were increased by FCM, although there were no significant changes on frequencies of other surface markers, such as CD4, CD8, Gr-1, NK1.1, CD11c, F4/80. Animal blood analyzer confirm the results that lymphocytes increased, the others have no change.Conclusions:Frequency of CD19+cells were increased in spleen of miR-15a/16-1-/- mice at 8-10 weeks old.Part II. Detection of phenotype and function of CD19+cells in spleen from miR-15a/16-1-/- mice of different agesObjective:To determine phenotype and function of CD19+cells in spleen of miR-15a/16-1-/-mice of different ages.Methods:Firstly, execute miR-15a/16-1-/-mice of 8-10 weeks and 8-12 months old and mononuclear cells of spleen were isolated both from positive mice and control mice, analysis of frequency of B220+CD 19+cells in spleen of miR-15a/16-1+mice by FCM. Selected CD19+cells through CD 19 magnetic, collecting the supernatant after LPS stimulation 0 h,24 h,48 h, detection the concentration of IL-10 in the supernatant by ELISA. Then, detection of IL-10 in splenic CD19+cells of the mononuclear cells of spleen which were isolated both from positive mice and control mice by FCM, and detection of CD19+Tim-1+, CD19hi FcyRlIbhi and CD19+ CD5+CD1dhi on lymphocytes in spleen by FCM, detection of IL-10 in splenic CD19+Tim-1+ cells of miR-15a/16-1-A mice of 8-12 month old. In addition, detection of CD19+Tim-1+on lymphocytes in spleen of miR-15a/16-1-’-mice of 8-10 weeks old after tumor bearing by FCM.Results:The frequency of B220+CD19+cells in spleen of miR-15a/16-1-/- mice of 8-10 weeks old were increased slightly, however, which was increased significantly of miR-15a/16-1-/-mice of 8-12 month old. ELISA results showed that compare to the wild-type mice, cytokine IL-10 lever, were increased significantly of miR-15 a/16-1’" mice of 8-12 month old which was stimulated by LPS. The CD19+IL-10+cells ratios in splenic cells were increased significantly of miR-15a/16-1-/- mice of 8-12 month old which was stimulated, comparing to the wild-type stimulated group by FCM. The frequency of CD19+Tim-1+on lymphocyte were increased in vcaR-15a/16-1-/- mice of 8-12 month old comparing to the wild-type which could secreted IL-10. The expression of CD19+Tim-1+on lymphocytes in spleen of miR-15a/16-1-/- mice of 8-10 weeks old after tumor bearing increased slightly.Conclusions:In miR-15a/16-1-/- mice of 8-12 months, frequency of CD19+IL-10+cells was significantly enhanced, its phenotype was CD19+Tim-1+.Part III. Analysis of T cell function and its anti-tumor activity in miR-15a/16-1-/- mice of 8-10 weeks oldObjective:To observe effects of miR-15a/16-1 on immune function against tumor in miR-15a/16-1-/- mice of 8～10 weeks old after tumor transplantation.Methods:Firstly, executemiR-15a/16-1-/- mice of 8～10 weeks old and mononuclear cells of spleen were isolated both from positive mice and control mice. Detection of T cell related surface receptors in spleen in physiological state by FCM. In addition, in vivo experiment was performed by injection of H22 transplantation tumor under the left armpit into miR-15a/16-1-/-(KO) mice of 8～10 weeks old and control mice.7 days after inoculation, the tumor could be touched by hand to the left side of the axillary fossa. The tumor was formed in seventh days, and the tumor was finished in 14 days, record the growth of tumor everyday. The size of transplanted tumor was measured to draw the tumor growth volume curves in different groups. Gross morphology of transplanted tumor in mice of each experimental group was observed by naked eye. The tumor was fixed by formalin and used to observe the morphology of tumor tissue of tumor bearing mice. Remaining part made into tumor tissue single cell suspension. At the same time, mononuclear cells of spleen were isolated both from positive mice and control mice, detection and analysis of CD8+, CD4+ T cell and CD8+ NKG2D+, CD4+ NKG2D+cell in spleen and tumor tissue of miR-15a/16-1-/-(KO) tumor bearing mice of 8-10 weeks old by FCM.Results:Under physiological condition, the results of T cell related surface receptors in spleen of miR-15a/16-1-/- mice of 8-10 weeks old comparing to the wild-type by FCM showed that the xpression of NKG2D on CD8+ cells were increased obviously, and were slightly increased on CD4+ cells. Growth of transplanted tumor was inhibited in miR-15a/16-1-/- mice of 8-10 weeks old. HE staining showed that there was no significant difference in pathological changes of the two tumor bearing mices. Under the condition of tumor, frequency of CD8+ and CD4+ cells and CD8+ NKG2D+ and CD4+ NKG2D+ cells of miR-15a/16-1-/- mice of 8-10 weeks old comparing to the wild-type by FCM showed that frequency of CD8+ and CD4+ cells and CD8+NKG2D+ and CD4+ NKG2D+ cells were no change in spleen, and CD4+ NKG2D+ cells were no change but CD8+ NKG2D+ cells was slightly higher in the tumor tissues.Conclusions:Growth of transplanted tumor was inhibited in miR-15a/16-1-/- mice of 8～10 weeks old. In addition, CD8+NKG2D+cells were higherly infiltrated in tumor tissues of miR-15a/16-1-/- mice, compared with those in wild-type mice. |
