本文選題：小鼠 + 粒單細(xì)胞白血病��； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2009年碩士論文

【摘要】： 目的本實(shí)驗(yàn)利用WEHI-3粒單白血病細(xì)胞在正常BALB/c小鼠體內(nèi)建立粒單細(xì)胞白血病動(dòng)物模型并研究其生物學(xué)特性。然后通過對粒單細(xì)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表達(dá)情況的檢測,來分析這兩個(gè)基因在小鼠粒單細(xì)胞白血病中可能發(fā)揮的作用。 方法1.將體外培養(yǎng)的處在指數(shù)生長期的WEHI-3細(xì)胞通過尾靜脈接種于BALB/c小鼠體內(nèi),再通過外周血計(jì)數(shù)涂片分類、骨髓甩片、流式細(xì)胞學(xué)、染色體、病理檢測小鼠粒單細(xì)胞白血病的生物學(xué)特性。 2.將粒單細(xì)胞白血病小鼠處死后提取骨髓中單個(gè)核細(xì)胞總的RNA,逆轉(zhuǎn)錄成cDNA。查找nm23-M1基因和c-myc基因的序列,通過軟件設(shè)計(jì)引物及其探針。用熒光定量PCR的方法來檢測nm23-M1基因和c-myc基因的表達(dá)情況,經(jīng)非參數(shù)檢驗(yàn)分別比較這兩個(gè)基因在粒單細(xì)胞白血病小鼠與正常小鼠中的表達(dá)情況,并分析其表達(dá)量與白血病的相關(guān)性。 結(jié)果1. BALB/c小鼠尾靜脈接種1×101～1×103個(gè)WEHI-3白血病細(xì)胞后均可長期存活,無一發(fā)病,而接種1×104～1×107個(gè)WEHI-3白血病細(xì)胞后100%發(fā)病,成功建立了全身播散的粒單白血病模型。靜脈接種白血病細(xì)胞的小鼠存活時(shí)間與接種細(xì)胞數(shù)有相關(guān)性。BALB/c小鼠接種WEHI-3白血病細(xì)胞后白細(xì)胞逐漸上升,晚期可升高到正常的5～20倍,外周血中粒細(xì)胞比值逐漸上升,淋巴細(xì)胞比值逐漸下降,晚期外周血涂片分類可見5～20%左右的白血病細(xì)胞。 2.病理檢查可見白血病細(xì)胞浸潤肝、脾、腦、肺、骨髓等器官,尤其是肝脾腫大明顯。 3.發(fā)病小鼠骨髓中的原始細(xì)胞流式細(xì)胞術(shù)檢測高表達(dá)髓系分化抗原MAC-3。 4.白血病小鼠染色體檢測發(fā)現(xiàn)多為超二倍體核型,數(shù)目在71～77之間。 5.在靜脈接種106個(gè)WEHI-3白血病細(xì)胞后第3天,我們給予小鼠注射200mg/kg的CTX化療,小鼠生存期延長,相當(dāng)于小鼠體內(nèi)大約還存在103～104范圍內(nèi)的白血病細(xì)胞,而這些殘留的白血病細(xì)胞仍可以導(dǎo)致復(fù)發(fā),可以作為白血病微小殘留狀態(tài)。 6.我們分別檢測了20只粒單細(xì)胞白血病小鼠與20只正常小鼠骨髓中的nm23-M1基因的表達(dá)情況。經(jīng)非參數(shù)檢驗(yàn)結(jié)果示,與正常對照組相比,粒單細(xì)胞白血病小鼠組的nm23-M1基因表達(dá)差異有統(tǒng)計(jì)學(xué)意義(Z=-5.343,P0.05)。 7.我們將nm23-M1基因的表達(dá)與粒單細(xì)胞白血病小鼠的性別、年齡、外周血白細(xì)胞數(shù)、骨髓原始細(xì)胞數(shù)以及臟器白血病侵襲情況做相關(guān)性分析,結(jié)果顯示nm23-M1基因與外周血白細(xì)胞數(shù)、骨髓原始細(xì)胞數(shù)以及臟器白血病侵襲情況相關(guān),而與白血病小鼠的性別、年齡無關(guān)。 8.與正常小鼠骨髓中c-myc基因的表達(dá)情況相比,粒單細(xì)胞白血病小鼠骨髓中c-myc基因的表達(dá)差異無統(tǒng)計(jì)學(xué)意義(Z=-0.990,P0.05)。 結(jié)論1.我們于正常BALB/c小鼠尾靜脈接種WEHI-3白血病細(xì)胞,成功建立了全身播散的粒單細(xì)胞白血病模型,該模型較好地反映白血病在體內(nèi)的演變過程,是進(jìn)行新藥療效試驗(yàn)、生物導(dǎo)向治療及基因治療的理想工具。 2.白血病細(xì)胞的增殖失控、分化障礙以及凋亡異常都與基因的表達(dá)異常密切相關(guān)。在粒單細(xì)胞白血病小鼠體內(nèi),nm23-M1基因表達(dá)明顯增高。nm23-M1基因在粒單細(xì)胞白血病小鼠中的表達(dá)情況與那些能反應(yīng)白血病惡性程度、白血病侵襲情況的指標(biāo)相關(guān)。因此,我們認(rèn)為nm23-M1基因可以作為一個(gè)輔助診斷小鼠粒單細(xì)胞白血病的標(biāo)志和衡量預(yù)后的參考指標(biāo)。 3. c-myc基因的表達(dá)和粒單細(xì)胞白血病無明顯的相關(guān)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.

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R733.7;R-332

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 彭恩蘭;基因芯片技術(shù)用于篩選小鼠粒單細(xì)胞白血病標(biāo)志基因的研究[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2010年

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本文編號：1891182