小鼠粒单细胞白血病模型的建立及相关白血病基因表达的实验研究

发布时间：2018-05-15 05:26

本文选题：小鼠 + 粒单细胞白血病　；参考：《中国人民解放军军事医学科学院》2009年硕士论文

【摘要】： 目的本实验利用WEHI-3粒单白血病细胞在正常BALB/c小鼠体内建立粒单细胞白血病动物模型并研究其生物学特性。然后通过对粒单细胞白血病小鼠骨髓中分化抑制因子nm23-M1基因以及原癌基因c-myc表达情况的检测,来分析这两个基因在小鼠粒单细胞白血病中可能发挥的作用。方法1.将体外培养的处在指数生长期的WEHI-3细胞通过尾静脉接种于BALB/c小鼠体内,再通过外周血计数涂片分类、骨髓甩片、流式细胞学、染色体、病理检测小鼠粒单细胞白血病的生物学特性。 2.将粒单细胞白血病小鼠处死后提取骨髓中单个核细胞总的RNA,逆转录成cDNA。查找nm23-M1基因和c-myc基因的序列,通过软件设计引物及其探针。用荧光定量PCR的方法来检测nm23-M1基因和c-myc基因的表达情况,经非参数检验分别比较这两个基因在粒单细胞白血病小鼠与正常小鼠中的表达情况,并分析其表达量与白血病的相关性。结果1. BALB/c小鼠尾静脉接种1×101～1×103个WEHI-3白血病细胞后均可长期存活,无一发病,而接种1×104～1×107个WEHI-3白血病细胞后100%发病,成功建立了全身播散的粒单白血病模型。静脉接种白血病细胞的小鼠存活时间与接种细胞数有相关性。BALB/c小鼠接种WEHI-3白血病细胞后白细胞逐渐上升,晚期可升高到正常的5～20倍,外周血中粒细胞比值逐渐上升,淋巴细胞比值逐渐下降,晚期外周血涂片分类可见5～20%左右的白血病细胞。 2.病理检查可见白血病细胞浸润肝、脾、脑、肺、骨髓等器官,尤其是肝脾肿大明显。 3.发病小鼠骨髓中的原始细胞流式细胞术检测高表达髓系分化抗原MAC-3。 4.白血病小鼠染色体检测发现多为超二倍体核型,数目在71～77之间。 5.在静脉接种106个WEHI-3白血病细胞后第3天,我们给予小鼠注射200mg/kg的CTX化疗,小鼠生存期延长,相当于小鼠体内大约还存在103～104范围内的白血病细胞,而这些残留的白血病细胞仍可以导致复发,可以作为白血病微小残留状态。 6.我们分别检测了20只粒单细胞白血病小鼠与20只正常小鼠骨髓中的nm23-M1基因的表达情况。经非参数检验结果示,与正常对照组相比,粒单细胞白血病小鼠组的nm23-M1基因表达差异有统计学意义(Z=-5.343,P0.05)。 7.我们将nm23-M1基因的表达与粒单细胞白血病小鼠的性别、年龄、外周血白细胞数、骨髓原始细胞数以及脏器白血病侵袭情况做相关性分析,结果显示nm23-M1基因与外周血白细胞数、骨髓原始细胞数以及脏器白血病侵袭情况相关,而与白血病小鼠的性别、年龄无关。 8.与正常小鼠骨髓中c-myc基因的表达情况相比,粒单细胞白血病小鼠骨髓中c-myc基因的表达差异无统计学意义(Z=-0.990,P0.05)。结论1.我们于正常BALB/c小鼠尾静脉接种WEHI-3白血病细胞,成功建立了全身播散的粒单细胞白血病模型,该模型较好地反映白血病在体内的演变过程,是进行新药疗效试验、生物导向治疗及基因治疗的理想工具。 2.白血病细胞的增殖失控、分化障碍以及凋亡异常都与基因的表达异常密切相关。在粒单细胞白血病小鼠体内,nm23-M1基因表达明显增高。nm23-M1基因在粒单细胞白血病小鼠中的表达情况与那些能反应白血病恶性程度、白血病侵袭情况的指标相关。因此,我们认为nm23-M1基因可以作为一个辅助诊断小鼠粒单细胞白血病的标志和衡量预后的参考指标。 3. c-myc基因的表达和粒单细胞白血病无明显的相关性。
[Abstract]:Objective to establish an animal model of granulocyte leukemia in normal BALB/c mice and study its biological characteristics by using WEHI-3 granulocyte monocyte, and then analyze the two genes by detecting the nm23-M1 gene and the expression of the proto oncogene c-myc in the bone marrow of granulocyte leukemia mice. The possible role of the mouse granulocyte leukemia.
Methods 1. WEHI-3 cells, cultured in vitro, were inoculated in the tail vein to BALB/c mice through the tail vein, and then the biological characteristics of the murine monocyte leukemia were detected by the classification of peripheral blood count smears, bone marrow flinging, flow cytology, chromosome and pathology.
2. the total RNA of mononuclear cells in bone marrow was extracted after the death of granulocyte leukemia mice. Reverse transcriptase into cDNA. to find the sequence of nm23-M1 and c-myc genes. The primers and probes were designed by software. The expression of nm23-M1 and c-myc genes were detected by the method of fluorescence quantitative PCR, and the two bases were compared by non parametric test. The expression of granulocytic leukemia in mice and normal mice was analyzed, and the correlation between the expression level and leukemia was analyzed.
Results 1. BALB/c mice were inoculated with 1 x 101~1 x 103 WEHI-3 leukemia cells for a long time, without one disease, and 1 x 104~1 x 107 WEHI-3 leukemia cells were inoculated 100%. The whole body disseminated granulocyte leukemia model was successfully established. The survival time of the mice inoculated with leukemic cells was related to the number of inoculated cells. When the.BALB/c mice were inoculated with WEHI-3 leukemia cells, the leukocytes gradually increased, and the late stage could rise to 5~20 times normal. The ratio of granulocytes in the peripheral blood gradually increased, the ratio of lymphocyte decreased gradually. The leukemia cells of 5 to 20% in the late peripheral blood smear classification could be seen.
2. pathological examination showed that leukemic cells infiltrated liver, spleen, brain, lung, bone marrow and other organs, especially hepatomegaly and splenomegaly.
3.. The myeloid differentiation antigen MAC-3. was detected by flow cytometry in the bone marrow of the onset mice.
4. chromosomal detection of leukemia mice showed that most of them were hyper diploid karyotypes, with a number of 71~77.
5. after third days of intravenous inoculation of 106 WEHI-3 leukemia cells, we gave 200mg/kg CTX chemotherapy to mice. The survival period of mice was prolonged, which was equivalent to about 103~104 leukemic cells in the mice, and these residual leukemic cells could still lead to relapse, which could be used as a small residual state of leukemia.
6. we detected the expression of nm23-M1 gene in the bone marrow of 20 granulocyte leukemia mice and 20 normal mice. The results of non parameter test showed that the difference of nm23-M1 gene expression in the mice with granulocyte leukemia was statistically significant (Z= -5.343, P0.05) compared with the normal control group.
7. we analyzed the correlation between the expression of nm23-M1 gene and the sex, age, the number of white blood cells in peripheral blood, the number of bone marrow cells and the invasion of visceral leukemia in mice with granulocyte leukemia. The results showed that the nm23-M1 gene was related to the number of white blood cells in the peripheral blood, the number of bone marrow cells and the invasion of visceral leukemia, and the white blood. The sex of the sick mice was not related to age.
8. compared with the expression of c-myc gene in the bone marrow of normal mice, there was no significant difference in the expression of c-myc gene in the bone marrow of granulocyte leukemia mice (Z=-0.990, P0.05).
Conclusion 1. we successfully inoculated WEHI-3 leukemia cells in the tail vein of normal BALB/c mice, and successfully established a systemic disseminated granulocyte leukemia model. This model is a good reflection of the evolution process of leukemia in the body. It is an ideal tool for new drug efficacy test, biological guided therapy and gene therapy.
2. the proliferation of leukemic cells is out of control, differentiation and apoptosis are closely related to the abnormal expression of genes. In mice with granulocytic leukemia, the expression of nm23-M1 gene increases obviously in the expression of.Nm23-M1 gene in mice with granulocytic leukemia and those that can respond to the malignancy of leukemia and the invasion of leukemia. Therefore, we believe that the nm23-M1 gene can be used as a marker for the diagnosis of granulocyte leukemia in mice and a prognostic indicator.
3. there was no significant correlation between c-myc gene expression and granulocyte leukemia.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R733.7;R-332

【引证文献】