载脂蛋白C3 转基因小鼠脾脏免疫细胞表型及其活性分析

目的：分析载脂蛋白C3（Apo C3）转基因小鼠脾脏免疫细胞的表型及活性。方法：以12只ICR背景Apo C3转基因小鼠为研究模型，选取鼠龄/性别匹配的野生型小鼠作为对照，采集小鼠球后静脉丛血液，以酶反应显色比色法联合酶标仪测定血浆甘油三酯水平，选取血浆甘油三酯含量≥ 1 000 mg/dL （11.3 mmol/L）的小鼠用于后续实验。取小鼠脾脏制备成单细胞悬液，采用细胞膜表面分子、胞内分子及核内分子染色法，通过流式细胞术检测转基因小鼠和野生型小鼠脾脏免疫细胞频率和表型。再将转基因小鼠或野生型小鼠脾脏细胞与结肠癌细胞MC38分别以1：1、1：3、3：1效靶比共培养72 h后，通过流式细胞术检测CD8⁺ T细胞CD107a的表达。结果：Apo C3转基因小鼠血浆甘油三酯含量较野生型小鼠显著升高（P<0.01），而体质量差异无统计学意义（P>0.05）。与野生型小鼠相比，Apo C3转基因小鼠脾脏CD3⁺CD8⁺、CD3⁺CD8⁺NKG2D⁺细胞频率明显增多，髓系抑制性Gr-1⁺CD11B⁺细胞频率增加，脾脏NK1.1⁺NK、NK1.1⁺NKG2D⁺细胞的频率明显减少（P均>0.05）；脾脏CD3⁺CD4⁺NKG2D⁺、CD3⁺CD4⁺IL-17⁺细胞频率以及调节性T细胞，NKT细胞，γδT细胞，B细胞和巨噬细胞频率均无明显变化（P均>0.05）。Apo C3转基因小鼠CD8⁺CD62L^-CD44⁺、CD8⁺CD44⁺KLGR1⁺频率升高，以效应性T细胞为主，且CD8⁺ T细胞分泌IFN-γ活性增强；转基因小鼠中CD8⁺ T淋巴细胞CD107a的表达增加差异均具有统计学意义（P均<0.05）。结论：Apo C3转基因小鼠呈现高水平血浆甘油三酯，其免疫细胞表型及活性具有显著变化，其中CD8⁺ T细胞频率及生物活性上调，髓系抑制性细胞频率升高，NK细胞频率降低；并且转基因小鼠中CD8⁺T淋巴细胞对靶细胞杀伤能力增强。

Characterization of spleen immune cells in apolipoprotein C3 transgenic mice

OBJECTIVE:To analyze the suptype and activity of spleen immune cells in apolipoprotein CⅢ (Apo C3) transgenic mice. METHODS:Using ICR Apo C3 transgenic mice as a research model,weights of transgenic and wild-type mice were measured and blood samples were collected from the posterior venous plexus of mice. The triglyceride assay kit was used to determine plasma triglyceride levels by enzyme reaction colorimetric method in a microplate reader. Mice with a plasma triglyceride level of ≥ 1 000 mg/dL (11.3 mmol/L) were selected for supsequent experiments. The spleens of these mice were prepared into single cell suspensions. The frequencies and suptypes of various immune cells in the spleen of the transgenic and wild-type mice were detected by flow cytometry using cell membrane surface molecules, intracellular molecules and intranuclear molecular staining. Then,the spleen cells of transgenic and wild-type mice were co-cultured with colon cancer cell MC38 in 1:1,3:1,3:1 ratios for 72 h,and expressions of CD107a in CD8⁺ T cells were determined. RESULTS: Plasma triglyceride levels were elevated in the transgenic mice but there was no significant difference in body weight compared to wild-type mice. Furthermore,the frequencies of CD3⁺CD8⁺ and CD3⁺CD8⁺ NKG2D⁺ cells in spleen of the transgenic mice were increased significantly;of myeloid-derived suppressor cells MDSC) were increased;of spleen NK cells was,however,significantly reduced; of CD3⁺CD4⁺NKG2D⁺,CD3⁺ CD4⁺IL^-17⁺ cells in spleens and of regulatory T cells,NKT cells,γδT cells,B cells and macrophages did not changed significantly. The frequencies of CD8⁺CD62L^-CD44⁺ and CD8⁺CD44⁺KLGR1⁺ in Apo C3 transgenic mice were increased, effector T cells mainly, and the secretions of IFN-γ by CD8 ⁺ T cells were enhanced. The expressions of CD107a in CD8⁺ T cells were increased in transgenic mice. CONCLUSION:Plasma triglyceride levels were significantly increased in Apo C3 transgenic mice. Compared with wild-type mice,the frequencies and activities of CD8 + T cells were up-regulated in transgenic mice, the frequencies of myeloid suppressor cells were increased,and that of NK cells were decreased. Moreover,the ability of CD8⁺T lymphocytes to kill target cells in transgenic mice was enhanced.