小鼠NASP基因突变对脾脏淋巴细胞亚群的影响

Effects of mouse NASP gene mutation on spleen lymphocyte subsets

目的制备NASP基因突变小鼠,探讨NASP基因突变对小鼠脾脏淋巴细胞亚群的影响。方法通过ES打靶技术定点突变小鼠NASP基因,提取鼠尾DNA,通过PCR和DNA测序技术鉴定突变阳性小鼠B6-NASP^M,通过RT-PCR和Western blot检测NASP的mRNA和蛋白表达水平。取6月龄B6-WT小鼠和B6-NASP^M小鼠脾脏,计算脾脏指数并用流式细胞仪检测脾脏淋巴细胞亚群的比例和细胞周期。结果PCR和DNA测序检测显示,成功制备了B6-NASP^M小鼠;NASP基因突变后不影响NASP蛋白在皮肤、脾脏、胸腺的表达。NASP基因突变对小鼠脾脏指数无显著影响(P>0.05)。流式细胞术检测结果表明,B6-NASP^M小鼠与B6-WT小鼠相比,其脾脏CD3^+T细胞比例下降(P<0.01),CD19^+B细胞比例上升(P<0.01);CD3^+CD4^+T细胞比例下降(P<0.05),CD3^+CD8^+T细胞比例上升(P<0.01),CD4/CD8比值下降(P<0.01);NK细胞比例下降(P<0.05)。B6-NASP^M小鼠脾脏淋巴细胞S期细胞略高于B6-WT小鼠(P<0.05)。结论NASP基因突变不影响NASP蛋白的表达,但可以改变小鼠脾脏的淋巴细胞的比例,造成B细胞比例升高,T细胞比例降低,降低CD4/CD8比值,从而造成小鼠免疫功能紊乱。

This study was designed to prepare NASP gene mutant mice and investigate the effect of NASP gene mutation on the spleen lymphocyte subsets in mice.The NASP gene mutant mouse was spotted by ES targeting technique,and the tail genome was extracted.The mutant positive mouse B6-NASP^M was identified by PCR and DNA sequencing,and the expression level of NASP protein was detected by Western blot.Six months old B6-WT mice and B6-NASP^M mice were subjected to splenectomy,and the spleen index was calculated,while the proportion of the immune cell subsets and cell cycle in the spleen were detected by flow cytometry.PCR and DNA sequencing showed that B6-NASP^M mice were successfully prepared.The mutation of NASP gene did not affect the expression of NASP protein in skin,spleen and liver;NASP gene mutation had no significant effect on mouse spleen index (P> 0.05).Flow cytometry results showed that the proportion of CD3^+ T cells in spleen decreased in B6-NASP^M mice compared with B6-WT mice (P<0.01),while the proportion of CD19^+B cells increased (P<0.01),the proportion of CD3^+CD4^+ T cells decreased (P<0.05),the proportion of CD3^+CD8^+ T cells increased (P<0.01),the ratio of CD4/CD8 decreased (P<0.01),and the proportion of NK cells decreased (P<0.05).Furthermore,the S phase cells of spleen lymphocytes in B6-NASP^M mice were slightly higher than those in B6-WT mice (P<0.05).In conclusion,mutation of NASP gene does not affect the expression of NASP protein,but it can change the proportion of immune cells in mouse spleen,resulting in an increase in the proportion of B cells,a decrease in the proportion of T cells,and a decrease in the ratio of CD4/ CD8,thus lead to immune dysfunction in mice.