摘要目的 探讨Npc1基因突变型(Npc1-/-)和野生型(Npc1+/+)小鼠免疫系统差异,从免疫学角度完善对C1型尼曼-匹克病(Niemann-Pick disease type C1,NPC1)发病机制的研究,为人NPC1疾病的治疗提供重要支持.方法 分别测量不同时间节点Npc1-/-与Npc1+/+小鼠体重、胸腺及脾脏重量,计算胸腺与脾脏指数;血常规检测小鼠外周血白细胞数量;qPCR检测小鼠外周血白细胞细胞因子表达;流式细胞术检测小鼠外周血与脾脏中CD4+、CD8+和CD19+淋巴细胞比例;免疫荧光及β-半乳糖苷酶染色检测小鼠脾脏的凋亡与衰老.结果 与野生型小鼠相比,Day14±2时Npc1-/-小鼠脾脏和胸腺重量差异无统计学意义,Day42±2时,Npc1-/-小鼠脾脏重量显著增加,而胸腺重量却显著降低,至Day63±2时,脾脏重量显著降低;其次,Day42±2时Npc1-/-小鼠中性粒细胞数量的显著增加,淋巴细胞绝对数量虽与野生型小鼠相比差异无统计学意义,但所占百分比显著降低;其次,Day42±2时Npc1-/-小鼠血液白细胞中细胞因子(IL-1、IL-2、IFN-γ、TNFα、IL-4、GA及GB)的基因表达与野生型小鼠相比具有明显异常;进一步,流式细胞术检测发现,Day42±2时Npc1-/-小鼠无论在血液还是脾脏中,T(CD4+和CD8+)淋巴细胞数量较野生型小鼠显著减少,而B(CD19+)淋巴细胞却显著增多;最终,通过对小鼠脾脏组织的凋亡和衰老染色发现,Day63±2时Npc1-/-小鼠脾脏细胞凋亡和衰老明显.结论 Npc1基因突变引起的脂质代谢异常,导致Npc1-/-小鼠在其生长过程中免疫系统及功能活性与野生型小鼠存在明显异常.该结果可为NPC1患者在临床上的用药与治疗提供借鉴,最大限度地改善患者生活质量并延长患者生存时间.

abstractsObjective To analyze the differences in immune system between Npc1 gene mutant (Npc1-/ -) and wild-type (Npc1+/ +) mice for better understanding the pathogenesis of Niemann-Pick disease type C1 (NPC1) from an immunological perspective and providing reference for NPC1 treatment in clinic.Methods Body, thymus and spleen weight of Npc1-/ -and Npc1+/ + mice aged (14±2) days, (42±2) days and (63±2) days (Day14±2 , Day42±2 and Day63±2 ) were recorded and the associated organ index were calcu-lated. White blood cell count in peripheral blood of mice aged Day42±2 was examined by routine blood test. Expression of cytokines at mRNA level in mouse peripheral blood was detected by qPCR. Percentages of CD4+, CD8+ and CD19+ lymphocytes in peripheral blood and spleen of mice aged Day42±2 were measured by flow cytometry. Apoptosis and senescence of spleen in mice aged Day63±2 were examined by immunofluores-cence and β-galactosidase staining. Results Compared with Npc1+/ + mice, there was no significant differ-ence in the weight of spleen and thymus in Npc1-/ - mice aged Day14±2; the weight of spleen in Npc1-/ - mice aged Day42±2 significantly increased, but the weight of thymus showed a significant decrease; furthermore, both the weight of spleen and thymus in Npc1-/ - mice aged Day63±2 significantly decreased; and the body weight of Npc1-/ - mice of each age group significantly decreased. Moreover, compared with Npc1+/ + mice, the absolute number of lymphocytes in the peripheral blood of Npc1-/ - mice aged Day42±2 showed no signifi-cant difference, but the percentage in whole white blood cells significantly decreased due to the significantly increased neutrophils. Expression of cytokines ( IL-1, IL-2, IFN-γ, TNF-α, IL-4, granzyme A and granzyme B) at mRNA level in the peripheral blood leukocytes of Npc1-/ - mice aged Day42±2 was abnormal as compared with that in Npc1+/ + mice. The number of T (CD4+ and CD8+) lymphocytes in Npc1-/ - mice aged Day42±2 significantly decreased, while the number of B (CD19+) lymphocytes increased significantly as com-pared with those in the Npc1+/ + mice. Compared with Npc1+/ + mice, apoptosis and senescence of the spleen in Npc1-/ - mice aged Day63±2 aggravated significantly. Conclusion The abnormal lipid metabolism triggered by Npc1 gene mutation causes severe immune dysfunction in Npc1-/ - mice. Therefore, immune dysfunction should be taken into full consideration when treating patients with NPC1, which might help improve the life quality and prolong the survival time.