尾静脉注射HL60、HL60/ADR细胞建立SCID beige小鼠白血病动物模型的研究
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摘要
目的应用急性早幼粒细胞HL60及耐阿霉素(adriamycin,ADR)细胞HL60/ADR,构建SCID beige小鼠白血病模型。方法将15只4~5周龄的雌性SCID beige小鼠,随机分为对照组3只、四组模型12只(每组3只)。经2Gy X射线预处理,模型组分别经尾静脉接种对数生长期HL60细胞悬液1×10~7个/只(M1组)、5×10~6个/只(M2组),HL60/ADR细胞悬液1×10~7个/只(M3组)、5×10~6个/只(M4组),对照组鼠尾静脉注射等剂量生理盐水。观察一般情况,于照射前、造模第10、18、28天检测血常规、外周血白细胞分类、外周血CD33阳性细胞比例,第32天或濒死时处死取材,组织切片病理检查。结果各模型组于接种细胞第7天开始,出现竖毛、萎靡少动、脊背弓起、步态不稳、偏侧或转圈,M1、M3两组较M2、M4两组体重下降更明显(P<0.05),显著低于对照组(P<0.01)。至观察周期32 d,M1、M3两组生存率分别为33%、67%,M2、M4两组全部生存。各组小鼠经X线照射7 d内白细胞数迅速降低,随时间推移逐渐上升,第28天各模型组白细胞计数均显著高于对照组(P<0.01);且各模型组白血病细胞比例亦明显升高,以接种HL60/ADR的M3组最为显著(P<0.05);接种第28天时各模型组CD33阳性细胞比例显著高于对照组(P<0.01),然各模型组间差异无显著性。第32天或濒死前取材,病理组织切片,造模各组脾均可见弥漫性白血病细胞浸润,肝偶见白血病细胞浸润灶。结论 SCID beige小鼠经2Gy X射线预处理后,每只尾静脉注射HL60、HL60/ADR细胞1×10~7个或5×10~6个均可构建急性髓系白血病动物模型,符合急性髓系白血病的生物学特点,高浓度成瘤更快,中位生存期约29 d。
Objective To establish a stable,effective,and reproducible acute promyelocytic leukemia model in severe combined immunodeficient( SCID) beige mice using acute myelocyte HL60 and adriamycin( ADR)-resistant HL60/ADR cell lines. Methods Female SCID beige mice( 4 – 5 weeks old) were divided randomly into one control group and four model groups with three mice in each group. After X-ray irradiation,SCID mice received 1 × 10~7( M1 group) or 5 × 10~6( M2 group) HL60 cells,or 1 × 10~7( M3 group) or 5 × 10~6( M4 group) HL60/ADR cells via tail vein injection. The white blood cell( WBC) count and positive rate of promyelocytes in peripheral blood were dynamically monitored by detecting cells expressing CD33 using flow cytometry. Morphological examination and histopathological assays were employed to confirm promyelocyte infiltration into organs( liver,spleen,and kidneys). Results The four model groups displayed abnormal behaviors of tremors,retardation,and piloerection. The two high dose model groups experienced significant weight loss compared with the two low dose model groups( P < 0. 05). Moreover,the four model groups had significantly lower body weights than the control group( P < 0. 01). At day 32,survival rates of M1 and M3 groups were33% and 67%,respectively. All mice in M2 and M4 groups survived. The WBC count in peripheral blood declined after X-ray irradiation. At day 28 after inoculation,the peripheral blood WBC counts of the four model groups were significantly higher than that in the control group( P < 0. 01). At day 28,the leukemic cell percentage of the M3 group was the highest compared with the other three model groups( P < 0. 05). The rates of CD33-positive cells in flow cytometric analysis of the four model groups were higher than that in the control group at day 28,but there were no significant differences between the four model groups. Morphological examination and HE staining of tissue biopsies demonstrated a large number of promyelocytes in the spleen and liver. Conclusions The human acute leukemia SCID beige mouse model was successfully established by tail vein injection of 5 × 10~6 or 1 × 10~7 HL60 or HL60/ADR cells. This model mimics the biological characteristics of acute myeloid leukemia. The survival period of the high dose SCID beige mouse model was short with a median survival time of about 29 days.
Objective To establish a stable,effective,and reproducible acute promyelocytic leukemia model in severe combined immunodeficient( SCID) beige mice using acute myelocyte HL60 and adriamycin( ADR)-resistant HL60/ADR cell lines. Methods Female SCID beige mice( 4 – 5 weeks old) were divided randomly into one control group and four model groups with three mice in each group. After X-ray irradiation,SCID mice received 1 × 10~7( M1 group) or 5 × 10~6( M2 group) HL60 cells,or 1 × 10~7( M3 group) or 5 × 10~6( M4 group) HL60/ADR cells via tail vein injection. The white blood cell( WBC) count and positive rate of promyelocytes in peripheral blood were dynamically monitored by detecting cells expressing CD33 using flow cytometry. Morphological examination and histopathological assays were employed to confirm promyelocyte infiltration into organs( liver,spleen,and kidneys). Results The four model groups displayed abnormal behaviors of tremors,retardation,and piloerection. The two high dose model groups experienced significant weight loss compared with the two low dose model groups( P < 0. 05). Moreover,the four model groups had significantly lower body weights than the control group( P < 0. 01). At day 32,survival rates of M1 and M3 groups were33% and 67%,respectively. All mice in M2 and M4 groups survived. The WBC count in peripheral blood declined after X-ray irradiation. At day 28 after inoculation,the peripheral blood WBC counts of the four model groups were significantly higher than that in the control group( P < 0. 01). At day 28,the leukemic cell percentage of the M3 group was the highest compared with the other three model groups( P < 0. 05). The rates of CD33-positive cells in flow cytometric analysis of the four model groups were higher than that in the control group at day 28,but there were no significant differences between the four model groups. Morphological examination and HE staining of tissue biopsies demonstrated a large number of promyelocytes in the spleen and liver. Conclusions The human acute leukemia SCID beige mouse model was successfully established by tail vein injection of 5 × 10~6 or 1 × 10~7 HL60 or HL60/ADR cells. This model mimics the biological characteristics of acute myeloid leukemia. The survival period of the high dose SCID beige mouse model was short with a median survival time of about 29 days.
引文
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[14]Greenblatt S,Li L,Slape C,et al.Knock-in of a FLT3/ITDmutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model[J].Blood,2012,119(12):2883-2894.
[15]刘伟,季明春,李厚达.人慢性粒细胞白血病动物模型研究进展[J].中国比较医学杂志,2005,15(1):55-58.
[16]张佳,杨文华,杨向东,等.尾静脉注射K562细胞建立慢性髓系白血病小鼠模型及其鉴定[J].中国实验血液学杂志,2012,20(3):773-776.
[17]金字林,吴洁莹,陆琰,等.6.0Gy X射线辐照所致BALB/c小鼠造血系统损伤的动态检测[J].中国医药导报,2017,14(13):13-16.
[18]张晓红,江和碧,叶铁真,等.HL60/VCR耐药细胞荷瘤鼠模型的建立[J].2013,44(5):1-3.
[19]文良雪,刘鑫,李会,等.KCL22/NOD-SCID小鼠慢性粒细胞白血病移植瘤模型的建立及其鉴定[J].中国实验动物学报,2015,23(2):188-193.
[20]田晓琳,许亚梅,王珍珍,等.尾静脉注射及皮下接种方法建立小鼠高表达miR-17-92的L1210白血病模型的比较研究[J/CD].中华临床医师杂志(电子版),2013,7(8):3449-3453.
[2]Rocha Gda G,Oliveira RR,Kaplan MA,et al.3β-Acetyltormentic acid reverts MRP1/ABCC1 mediated cancer resistance through modulation of intracellular levels of GSH and inhibition of GST activity[J].Eur J Pharmacol,2014,741:140-149.
[3]Wang H,Wang H,Liang J,et al.Cell-penetrating apoptotic peptide/p53 DNA nanocomplex as adjuvant therapy for drugresistant breast cancer[J].Mol Pharm,2014,11(10):3352-3360.
[4]Sprouse AA,Herbert BS.Resveratrol augments paclitaxel treatment in MDA-MB-231 and paclitaxel-resistant MDA-MB-231breast cancer cells[J].Anticancer Res,2014,34(10):5363-5374.
[5]范磊,吴雨洁,张建富,等.伴有t(8;21)染色体异常急性髓系白血病的免疫表型特征[J].中国实验血液学杂志,2010,18(6):1410-1413.
[6]岳保红,张秋堂,秦东春,等.K562和HL60细胞分化抗原的表达[J].郑州大学学报(医学版),2006,41(3):467-470.
[7]Lin JJ,Hsu HY,Yang JS,et al.Molecular evidence of antileukemia activity of gypenosides on human myeloid leukemia HL-60 cells in vitro and in vivo using a HL-60 cells murine xenograft model[J].Phytomedicine,2011,18(12):1075-1085.
[8]王礼学.K562/BALB/c裸鼠白血病模型的建立及LDR、IM7对CML患者骨髓干/祖细胞集落形成能力影响的研究[D].徐州医学院,2008.
[9]于文俊,杨文华,史哲新,等.NOD/SCID小鼠模型在实验血液学研究中的应用[J].中国实验血液学杂志,2008,16(4):964-968.
[10]时彦胜,耿志贤,战大伟,等.SCID小鼠和BALB/c裸鼠对两株人体肿瘤细胞系移植敏感性的研究[J].中国实验动物学杂志,2000,10(1):5-10.
[11]Vasiliki E,Jennifer C,Shruti K,et al.Comparing the MRIappearance of the lymph nodes and spleen in wild-type and immuno-deftcient mouse strains[J].PLo S One,2011,6(11):e27508.
[12]Larbouret C,Gaborit N,Chardes T,et al.In pancreatic carcinoma,dual EGFR/HER2 targeting with cetuximab/trastuzumab is more effective than treatment with trastuzumab/erlotinib or lapatinib alone:implication of receptors downregulation and dimers disruption[J].Neoplasia,2012,14(2):121-130.
[13]Zunino SJ,Storms DH,Newman JW,et al.Resveratrol given intraperitoneally dose not inhibit the growth of high-risk t(4;11)acute lymphoblastic leukemia cells in a NOD/SCID mouse model[J].Int J Oncol,2012,40(4):1277-1284.
[14]Greenblatt S,Li L,Slape C,et al.Knock-in of a FLT3/ITDmutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model[J].Blood,2012,119(12):2883-2894.
[15]刘伟,季明春,李厚达.人慢性粒细胞白血病动物模型研究进展[J].中国比较医学杂志,2005,15(1):55-58.
[16]张佳,杨文华,杨向东,等.尾静脉注射K562细胞建立慢性髓系白血病小鼠模型及其鉴定[J].中国实验血液学杂志,2012,20(3):773-776.
[17]金字林,吴洁莹,陆琰,等.6.0Gy X射线辐照所致BALB/c小鼠造血系统损伤的动态检测[J].中国医药导报,2017,14(13):13-16.
[18]张晓红,江和碧,叶铁真,等.HL60/VCR耐药细胞荷瘤鼠模型的建立[J].2013,44(5):1-3.
[19]文良雪,刘鑫,李会,等.KCL22/NOD-SCID小鼠慢性粒细胞白血病移植瘤模型的建立及其鉴定[J].中国实验动物学报,2015,23(2):188-193.
[20]田晓琳,许亚梅,王珍珍,等.尾静脉注射及皮下接种方法建立小鼠高表达miR-17-92的L1210白血病模型的比较研究[J/CD].中华临床医师杂志(电子版),2013,7(8):3449-3453.