摘要
目的建立造血干细胞特异性Hdac3条件性敲除小鼠模型,探讨Hdac3基因对小鼠造血发育的影响。方法 Hdac3^(fl/fl)小鼠与Mx1-Cre小鼠杂交获得Hdac3^(fl/fl)Mx1-Cre小鼠,PCR鉴定小鼠基因型。连续注射poly(I:C),诱导Cre表达,得到造血干细胞Hdac3敲除鼠Hdac3^(fl/fl)Mx1-Cre^+(CKO)及对照组小鼠Hdac3^(+/+)Mx1-Cre^+(WT),Q-PCR分析Hdac3 mRNA表达水平。流式细胞术检测小鼠外周血淋巴细胞数量及比例。HE染色观察小鼠胸腺、脾脏形态学改变。流式细胞术检测小鼠外周血CD3^+、B220^+细胞比例。克隆形成实验分析小鼠造血干/祖(LSK)细胞的分化能力。结果成功建立造血干细胞Hdac3^(fl/fl)Mx1-Cre^+(CKO)小鼠模型。与对照组相比,CKO小鼠外周血淋巴细胞绝对数量(P<0.001)及比例(P<0.01)均显著降低;细胞亚群分析显示,CKO小鼠外周血CD3^+淋巴细胞及B220^+淋巴细胞显著减少(P<0.01,P<0.001),CD11b^+髓系细胞比例显著增加(P<0.01)。HE染色结果显示,实验组鼠胸腺和脾淋巴细胞数减少。克隆形成实验显示,实验组小鼠粒系克隆数无差异,前B细胞样克隆缺失。结论造血干细胞中Hdac3基因促进造血干细胞向淋巴细胞分化。
Objective To investigate the role of histone deacetylase 3 gene(Hdac3) in hematopoiesis by developing a conditional Hdac3 knockout mice. Methods We crossed the Hdac3floxp/floxpmice with Mx1-Cre mice to generate Hdac3fl/flMx1-Cre mice, and then confirmed the mice genotype by PCR. After continuous injection of polyinosinic-polycytidylic acid [poly(I:C)], Cre was induced to express, and Hdac3fl/flMx1-Cre+ mice(CKO) and Hdac3+/+Mx1-Cre+ mice(WT) were generated. The Hdac3 expression in hematopoietic stem cells(HSCs) was validated by q-PCR. Flow cytometry was used to detect the number and proportion of lymphocytes and the frequencies of CD3+ cells and B220+ cells in the peripheral blood(PB). Hematoxylin and eosin staining(HE) was applied to observe the morphology of the thymus and spleen. In vitro colony-forming assay was conducted to determine differentiation capacity of the hematopoietic stem/progenitor(LSK) cells. Results The CKO mouse model was successfully constructed. Flow cytometry analysis of the peripheral blood showed a significant decreased number(P<0.001) and percentage(P<0.01) of the lymphocytes in the CKO mice as compared with the WT mice. Subpopulation analysis of the lymphocytes showed that the percentages of CD3+ lymphocytes(P<0.01) and B220+ lymphocytes(P<0.001) were significantly decreased in PB of CKO mice than of WT mice, while an obviously elevated frenquency of CD11 b+ myeloid cells was found in PB of CKO mice(P<0.01). HE staining showed the decrease of lymphogenesis in the thymus and spleen of CKO mice. The in vitro colony-forming assay showed Hdac3-deficient HSCs had deletion in the pre-B colony-forming units, but did not affect colony-forming units of myeloid precursors. Conclusion Hdac3 promotes the lymphogenesis of the HSCs in mice.
作者
吕文琼
舒逸
杨碧洁
张航
张佳
张虹洋
周永杰
石毓君
邹琳
张鹏辉
LYU Wenqiong;SHU Yi;YANG Bijie;ZHANG Hang;ZHANG Jia;ZHANG Hongyang;ZHOU Yongjie;SHI Yujun;ZOU Lin;ZHANG Penghui(Center for Laboratory Medicine,Center for Clinical Molecular Medicine,Key Laboratory of Child Development and Disorders of Ministry of Education,Chongqing Key Laboratory of Pediatrics,Chongqing International Science and Technology Cooperation Base for Child Development and Critical Disorders,Children’s Hospital of Chongqing Medical University,Chongqing,400014;Key Laboratory of Transplantation Engineering and Transplantation Immunity of National Health Commission,West China Hospital of Sichuan University,Chengdu,Sichuan Province,610041,China)
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2019年第9期844-850,共7页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(81570142
81870126)~~
