应用NB4细胞株建立SCID beige小鼠急性早幼粒细胞白血病病理模型

Establishment of the Human Acute Promyelocytic Leukemia Pathological Model in the SCID Beige Mice by Using NB4 Cell Line

目的:本研究旨在建立稳定、有效、重复性好的人急性早幼粒白血病重度联合免疫缺陷(SCID)小鼠移植瘤模型,观察模型的病程特点和生物学行为。方法:将3-5周龄SCID beige小鼠随机分为实验组和对照组;实验组鼠尾静脉注射对数生长期的NB4细胞5×106个/只,动态监测小鼠外周血白细胞数和外周血涂片中人早幼粒细胞阳性率;应用形态学和组织病理学方法确认白血病细胞浸润肝、脾、肺、肾、脑等器官情况,Western blot检测组织中PML-RARa融合蛋白表达情况。结果:接种NB4细胞2周内的实验组SCID小鼠外周血白细胞计数与对照组相比差异无统计学意义(P>0.05);同时,血涂片姬姆萨染色后镜检未见到NB4细胞;接种第21天和第28天实验组SCID小鼠外周血白细胞计数分别为(4.79±1.13)×109/L和(7.62±2.24)×109/L,显著高于对照组(P<0.05);血涂片吉姆萨染色后可见NB4细胞比例分别占(2.14±0.63)%和(6.6±2.76)%;形态学观察显示,接种21 d后实验组SCID小鼠体内出现单个或多个肿瘤实体包块;同时,组织切片HE染色表明,肝、脾、肺、肾、脑组织均有不同程度的瘤细胞浸润;细胞免疫荧光结果显示,实验组小鼠骨髓细胞CD33表达呈阳性(P<0.05);Western blot数据证实,肝、肾、脑组织中检测到不同水平的PML-RARa融合蛋白表达。结论:SCID小鼠鼠尾静脉注射NB4细胞可成功构建人急性早幼粒细胞白血病小鼠模型,该模型能模拟临床白血病累及骨髓和弥漫生长特点,是研究人类白血病发病机理及实验治疗的良好模型。

Objective： This study was to establish a stable, effective and reproducible human acute promyelocytic leukemia model in severe combined immunodeficient （SCID） mice by using NB4 cell line, and to investigate the disease course character and biological behaviors. Methods： Three-five-week-old SC1D beige mice were divided randomly into two groups ： experimental and control group. SCID mice of experimental group were transplanted by tail vein （iv） injection of 5 ×10^6 NB4 cells. The WBC cell count and the positive rate of promyelocytes in peripheral blood were dynamically monitored by using smears. Morphological examination and histopathological assay were employed to confirm NB4 cell infiltration in organs （liver, spleen, lung, kidney and brain）. The expression level of PML-RARa fusion protein was detected by Western blot. Results： Within two weeks there was no significant difference in peripheral blood WBC count between two groups （ P 〉 0.05 ）, meanwhile, NB4 cells were not found. At the day 21 and 28 after inoculation, the peripheral blood white blood cell count of experimental group reached to （4.79 ±1.13） × 10^9/L and （7.62 ±2.24） × 109/L respectively, which were significantly higher than that in control group （ P 〈 0.05 ） ; simultaneously, the positive rates of promyelocytes on smears were （2.14 ±0.63 ） % and （ 6.6 ± 2.76） %, respectively. Morphological observation showed single or multiple tumor lumps at day 21 after inoculation; HE staining of tissue biopsies demonstrated a large number of promyelocyte in the liver, spleen, lung, kidney and brain tissue. Cell immunofluorescence results showed that the CD33 expression of bone marrow cells in mice of experimental group was strongly positive （ P 〈 0. 05 ）. Western blot confirmed that the PML-RARoL fusion protein was expressed variously in liver, kidney and brain tissue.Conclusions： The human acute promyelocytic leukemia SCID mouse model is succesfuUy established by tail vein injection of NB4 cells. This model can mimic the characters of involved bone marrow and diffuse growth of cells. This model is a useful tool to explore the pathogenic mechanism and experimental treatment of human leukemia.