CN105670999B - 稳定表达绿色荧光蛋白的骨髓增生异常综合征转白细胞株 - Google Patents
稳定表达绿色荧光蛋白的骨髓增生异常综合征转白细胞株 Download PDFInfo
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Abstract
本发明属微生物动物细胞系领域。设计稳定表达绿色荧光蛋白的骨髓增生异常综合征转白细胞株及其建立方法和用途,本发明以人骨髓增生异常综合征转白细胞系SKM‑1为母系细胞,通过携带GFP基因的慢病毒进行转染,以有限稀释法获得GFP阳性的单细胞克隆,并将扩大培养后的细胞通过皮下注射的方式在小鼠体内筛选,致瘤后将瘤块分离,继续培养后,获得稳定表达绿色荧光蛋白的骨髓增生异常综合征转白细胞株SKM‑1/GFP。该细胞系形态和生长特性与母系细胞无差异,生长曲线与母系细胞无差异,可以稳定表达GFP,并且具有致瘤性。可进一步用于小鼠动物模型的建立以及为MDS微小残留病及其他临床前研究提供平。
Description
技术领域
本发明属生物技术、微生物动物细胞系领域,涉及骨髓增生异常综合征转白细胞株,具体涉及一种稳定表达绿色荧光蛋白的骨髓增生异常综合征转白细胞株及其制备方法和用途。
背景技术
现有技术公开了骨髓增生异常综合征(myelodysplastic syndeome,MDS)是一组克隆性细胞异常增生的疾病,以无效造血、病态造血及高风险向急性髓系白血病(AML)转化为特点,其中,约20%-30%的MDS患者向AML进展。据报道,在美国,MDS是最常见的髓系来源的肿瘤,男性发病率高于女性,平均每105个美国人中有3-4人患有MDS;随着年龄的增加,MDS的发病率逐渐增加;在年龄大于60岁的患者中,发病率高达7-35/10万,有研究显示先前行化疗及放疗治疗的患者是MDS发病的高危人群。有统计显示,我国MDS的发病率为1.45/10万,略低于欧美国家,随着我国老龄化进程逐渐加剧,MDS的发病率也呈持续上升状态。基于形态学、分子生物学、细胞遗传学和免疫表型,世界卫生组织(WHO)将MDS分为:难治性血细胞减少症伴单系病态造血(RCUD)、难治性血细胞减少伴多系病态造血(RCMD)、环形铁粒幼细胞性难治性贫血(RARS)、难治性贫血伴原始细胞增多1型(RAEB-1)、难治性贫血伴原始细胞增多2型(RAEB-2)、5q-综合征、不能分类的MDS(MDS-U)。
研究显示,在MDS的早期阶段,造血细胞过度凋亡导致一系或者多系细胞减少。在MDS中细胞遗传学的进一步改变导致造血细胞增殖加速、分化受阻,最终进展为AML。与初发AML(de novo AML)相比,MDS转化的AML(sAML)患者年龄偏大,治疗难度大,常常不能应用异基因造血干细胞移植(Allo-HSCT)和强烈化疗进行治疗,其预后不佳;研究还显示,随着地西他滨及阿扎胞苷等去甲基化药物的研发,治疗MDS的手段取得了突破性进展,但该些药物对高危MDS和sAML的治疗作用有限。由于临床实践缺乏有效的治疗手段,体内的白血病细胞难以清除,最终威胁患者的生命;然而,目前国内外缺乏有效可靠的体内外研究平台,严重制约了MDS的基础及临床前研究。
随着基因标记技术的发展,细胞示踪在肿瘤的研究中得到了越来越多的应用;绿色荧光蛋白(green fluorescent protein,GFP)可以在相应波长的紫外光激发下发出绿色荧光,与传统的新霉素耐药基因(NeoR)和编码大肠杆菌β-半乳糖苷酶的Lac-Z基因标记技术相比,GFP标记肿瘤细胞具有动态直观、操作简便、检测方便、灵敏度高的优点,可以灵敏地显示肿瘤细胞的位置,并进行定量研究,可应用于微小残留病等的研究,而且随着动物活体成像技术的发展,GFP也适合体内细胞的观察;同时,由于其分子量小,GFP可以与其他目的基因表达产物形成融合蛋白,不影响目的蛋白的空间构象和功能,用于目的蛋白的位置标记、功能和定量研究;基于以上几点,以GFP进行细胞示踪为相关的肿瘤的研究提供了新的平台。
微量残留白血病(minimal residual leukemia,MRL)是指白血病经诱导治疗获得完全缓解后体内残留少量白细胞的状态,其是白血病复发的根源;由于微小残留白血病分布隐蔽,通常临床不易检测,成为MRL研究的瓶颈。
基于现有技术的现状,本申请的发明人拟提供使GFP高效和稳定地在细胞中表达的技术方案,具体涉及提供一种稳定表达绿色荧光蛋白的骨髓增生异常综合征转白细胞株及其制备方法,进一步用于建立小鼠动物模型,利用荧光在成像上的优势,在动物体内长期观察,明确白血病细胞分布模式;为微小残留病的研究提供平台。
与本发明相关的现有技术有:
1.Tefferi A,Vardiman JW.Myelodysplastic syndromes.N Engl J Med 2009;361:1872-85.
2.Garcia-Manero G,Fenaux P.Hypomethylating agents and other novelstrategies in myelodysplastic syndromes.J Clin Oncol 2011;29:516-23.
3.Morel P,Hebbar M,Lai JL,Duhamel A,Preudhomme C,Wattel E,Bauters F,Fenaux P.Cytogenetic analysis has strong independent prognostic value in denovo myelodysplastic syndromes and can be incorporated in a new scoringsystem:a report on 408cases.Leukemia 1993;7:1315-23.
4.Lee JJ,Kim HJ,Chung IJ,Kim JS,Sohn SK,Kim BS,Lee KH,Kwak JY,ParkYH,Ahn JS and others.Comparisons of prognostic scoring systems formyelodysplastic syndromes:a Korean multicenter study.Leuk Res 1999;23:425-32.
5.Rollison DE,Howlader N,Smith MT,Strom SS,Merritt WD,Ries LA,EdwardsBK,List AF.Epidemiology of myelodysplastic syndromes and chronicmyeloproliferative disorders in the United States,2001-2004,using data fromthe NAACCR and SEER programs.Blood 2008;112:45-52.
6.Dan C,Chi J,Wang L.Molecular mechanisms of the progression ofmyelodysplastic syndrome to secondary acute myeloid leukaemia and implicationfor therapy.Annals of Medicine 2015:1-9.
7.Lubbert M,Suciu S,Baila L,Ruter BH,Platzbecker U,Giagounidis A,Selleslag D,Labar B,Germing U,Salih HR and others.Low-dose decitabine versusbest supportive care in elderly patients with intermediate-or high-riskmyelodysplastic syndrome(MDS)ineligible for intensive chemotherapy:finalresults of the randomized phase III study of the European Organisation forResearch and Treatment of Cancer Leukemia Group and the German MDS StudyGroup.J Clin Oncol 2011;29:1987-96.
8.Fili C,Malagola M,Follo MY,Finelli C,Iacobucci I,Martinelli G,Cattina F,Clissa C,Candoni A,Fanin R and others.Prospective phase II Study on5-days azacitidine for treatment of symptomatic and/or erythropoietinunresponsive patients with low/INT-1-risk myelodysplastic syndromes.ClinCancer Res 2013;19:3297-308.
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发明内容
本发明的目的是基于现有技术的现状,提供一种稳定表达绿色荧光蛋白的骨髓增生异常综合征转白细胞株及其制备方法,进一步用于建立小鼠动物模型,利用荧光在成像上的优势,在动物体内长期观察,明确白血病细胞分布模式;为微小残留病的研究提供平台。
本发明中,将绿色荧光蛋白(GFP)导入细胞,使GFP高效和稳定地在细胞中表达,建立稳定表达绿色荧光蛋白的骨髓增生异常综合征转白细胞株。
本发明中,以人骨髓增生异常综合征转白细胞系SKM-1为母系细胞,通过携带GFP基因的慢病毒进行转染,以有限稀释法获得GFP阳性的单细胞克隆,将扩大培养后的细胞通过皮下注射的方式在小鼠体内筛选,致瘤后将瘤块分离,继续进行培养,获得稳定表达绿色荧光蛋白的骨髓增生异常综合征转白细胞株SKM-1/GFP,该细胞系能稳定表达GFP。所述的细胞株已于2015年12月28日保藏,保藏单位:中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,100101,保藏编号:CGMCC No.11798,分类命名:人稳定表达绿色荧光蛋白的骨髓增生异常综合征转白细胞SKM-1/GFP。
本发明采用下述方法建立稳定表达绿色荧光蛋白的骨髓增生异常综合征转白细胞株SKM-1/GFP:采用以HIV-1(人类免疫缺陷I型病毒)为载体的慢病毒转染技术,携带GFP外源基因稳定地整合入宿主细胞基因组中,在细胞分裂、转移和分化时,GFP也可以稳定表达,而不出现荧光逐渐减低的情况;同时,在小鼠体内筛选前,对细胞进行单细胞克隆化培养,使GFP荧光均一,防止细胞在后续培养中出现GFP脱失的情况;所建立的SKM-1/GFP细胞株生长特性与母细胞基本保持一致,为后续的动物实验建立了良好的基础。
本发明中,慢病毒转染后72h,荧光显微镜下观察,多数细胞带有绿色荧光,流式细胞仪检测GFP阳性率在85%左右,以SKM-1母系细胞为对照,在普通显微镜下观察,转染前后细胞大小及形态未见明显变化(如图2所示)。
本发明中,采用有限稀释法将转染后的SKM-1细胞接种到96孔细胞培养板中,96孔细胞培养板中共接种32个单个细胞,在普通显微镜和荧光显微镜下观察,挑选出表达GFP的单个细胞20个,经完全培养基培养4周后得到1株可持续增殖的单细胞克隆,并继续扩增培养;小鼠体内筛选后,经流式细胞仪检测,单细胞克隆化培养的细胞GFP阳性率为100%(如图3所示),结果表明,经体内或体外培养后,SKM-1/GFP细胞荧光表达率稳定,无明显衰减。
本发明中,所述的SKM-1细胞系购自日本Health Science Research ResourcesBank(HSRRB),长期液氮保存和传代,采用10%胎牛血清及90%的RPMI 1640培养液配置成完全培养基,在37℃、5%CO2、饱和湿度条件下培养,每2-3天传代一次。
本发明采用细胞内稳定表达的GFP标记MDS转白细胞,可进一步建立小鼠动物模型,利用荧光成像的优势,在动物体内长期观察,明确白血病细胞分布模式;本发明的实施例中,在小鼠体内筛选前,对细胞进行单细胞克隆化培养,使GFP荧光均一,GFP表达效率在100%,防止细胞在后续培养中出现GFP脱失的情况,所建立的SKM-1/GFP细胞株生长特性与母细胞基本保持一致,为后续的动物实验建立了良好的基础。
本发明中,采用的动物为Nod/scid小鼠雄性,鼠龄为6周龄,体重23g,饲养于无特定病原体(SPF)级,单细胞克隆扩增培养后接种至Nod/scid小鼠皮下,接种14天时,小鼠注射细胞处皮下可见黄豆大小的瘤块,致瘤30天时,可肉眼观察到凸出皮肤的肿块,大小为14.75*21.75mm;
应用CCK-8法测定生长曲线,结果显示,SKM-1/GFP与SKM-1生长曲线无明显差异。
本发明的SKM-1/GFP细胞株具有以下生物学特性:
1.SKM-1/GFP细胞呈圆形,悬浮生长,形态和生长特性与SKM-1母系细胞无差异;
2.流式细胞仪检测SKM-1/GFP细胞的GFP阳性率为100%;
3.致瘤性:接种30天时,肉眼可观察到凸出皮肤的肿块,大小为14.75*21.75mm;
4.SKM-1/GFP细胞生长曲线与SKM-1母系细胞无差异;
5.小鼠致瘤后,小动物活体荧光显像可以显示瘤块。
本发明以人骨髓增生异常综合征转白细胞系SKM-1为母系细胞,通过携带GFP基因的慢病毒转染,GFP阳性的单细胞克隆及小鼠体内筛选等建立了稳定表达绿色荧光蛋白的骨髓增生异常综合征转白细胞株SKM-1/GFP,该细胞系形态和生长特性与母系细胞无差异,生长曲线与母系细胞无差异,可以稳定表达GFP,并且具有致瘤性。进一步可用于小鼠动物模型的建立,为MDS微小残留病及其他临床前研究提供平台,以及为药物筛选及MDS治疗的选择提供有意义的指导。
附图说明
图1为pCMV-dR8.91质粒图。
图2为SKM-1和SKM-1/GFP细胞光镜和荧光显微镜下细胞形态,其中,A为SKM-1细胞光镜下细胞形态;B为SKM-1细胞荧光显微镜下图像;C为SKM-1/GFP细胞光镜下细胞形态;D为SKM-1/GFP细胞荧光显微镜下图像。
图3,流式细胞仪检测单细胞克隆扩大培养后细胞的GFP阳性率。
图4是SKM-1/GFP细胞接种30天后,小鼠体内致瘤情况。
图5是SKM-1和SKM-1/GFP细胞生长曲线。
图6是小动物活体荧光显像。
图7 SKM-1/GFP组荷瘤小鼠瘤块行冰冻切片后,荧光显微镜下可见较为均一的GFP阳性肿瘤细胞浸润。
具体实施方式
实施例1
采用下述方法:以人骨髓增生异常综合征转白细胞系SKM-1为母系细胞,通过携带GFP基因的慢病毒进行转染,以有限稀释法获得GFP阳性的单细胞克隆,并将扩大培养后的细胞通过皮下注射的方式在小鼠体内筛选,致瘤后将瘤块分离,继续进行培养,获得SKM-1/GFP,该细胞系可以稳定表达GFP。
1)慢病毒包装及转染:
pCMV-dR8.91质粒购自上海斯丹赛生物技术有限公司,转染前24h将对数生长期的293T细胞接种于六孔板,在37℃、5%CO2条件下培养,当细胞密度到达80%-90%时,进行转染;将pCMV-dR8.91质粒以及辅助质粒pVSVG按照一定比例加入100μl无血清DMEM中,同时将10μl Lipofectamine 2000溶于100μl无血清DMEM中,静置5min;然后将二者混匀静置15min,加入无抗生素和无血清的DMEM培养基800μl至1ml,均匀加入到接种了293T细胞的六孔板中,放入培养箱,8h后换液;次日荧光显微镜观察转染效率;48h后收集培养液,3000rpm×10min离心,用0.45uM滤器过滤上清滤除细胞碎片后,分装到无菌的2mL细胞冻存管中,可直接感染或-80℃冻存;
实验前接种5×104个目的细胞于12孔培养板中,加入浓度为1×108TU/ml慢病毒20μl,用适量完全培养基调整总体积为500μl,同时向培养基中加入终浓度为5μg/ml的polybrene增强转染,轻轻混匀;8-12小时后观察细胞状态,离心,弃去细胞上清,用新鲜培养基重悬细胞,继续培养;72-96小时观察荧光表达情况;
2)单细胞克隆筛选:
调整细胞密度为0.5个/100μl,充分混匀,向96孔培养板中每孔加入100μl细胞悬液,分别在普通显微镜和荧光显微镜下挑选出表达GFP且为单个细胞的孔,进行标记,并进行扩大培养;
3)小鼠体内筛选:
在皮下注射肿瘤细胞前72小时、24小时腹腔注射环磷酰胺(CTX)120mg/kg,调整细胞浓度至5×107/ml,皮下致瘤按照0.1ml/10g注射液体量标准,在小鼠右侧胁下部位注射肿瘤细胞悬液,观察致瘤情况;30天时,常规处理小鼠,无菌分离小鼠皮下瘤块并剪碎,注射器针芯碾磨后200目滤网过滤,制备单细胞悬液,继续培养;
4)CCK-8法检测生长曲线
收集SKM-1及SKM-1/GFP细胞悬液,调整细胞浓度为105/ml,在96孔板中铺板,每孔加入100μl细胞悬液,分别在0、24、48、72、96小时加入CCK-8溶液10μl/孔,在培养箱中孵育4小时后,在酶标仪上读取OD值,检测波长为450nm,同时选用参考波长为630nm,每次每组样本设置3个平行样本,计算平均值,取3次实验数据进行统计;
5)冰冻切片:
取处理后小鼠新鲜的肿块组织,浸入30%的蔗糖溶液,沉底24h后,用OCT包埋,-70℃冰冻使其固化,制备连续冰冻切片,在荧光显微镜下475nm处观察各组织中的绿色荧光;
结果显示,建立的细胞系形态和生长特性与母系细胞无差异,生长曲线与母系细胞无差异,可以稳定表达GFP,并且具有致瘤性。
Claims (3)
1.一种稳定表达绿色荧光蛋白的骨髓增生异常综合征转白细胞株,其特征在于,所述细胞株于2015年12月保藏,保藏单位:中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏编号:CGMCC 11798 ,分类命名:稳定表达绿色荧光蛋白的骨髓增生异常综合征转白细胞株 SKM-1/GFP;
所述的细胞株 SKM-1/GFP具有以下生物学特性:
1)SKM-1/GFP细胞呈圆形,悬浮生长,形态和生长特性与SKM-1母系细胞无差异;
2)流式细胞仪检测SKM-1/GFP细胞的GFP阳性率为100%;
3) 致瘤性:接种30天时,肉眼可观察到凸出皮肤的肿块,大小为14.75*21.75mm;
4)SKM-1/GFP细胞生长曲线与SKM-1母系细胞无差异。
2.权利要求1所述的稳定表达绿色荧光蛋白的骨髓增生异常综合征转白细胞株在用于建立MDS微小残留病研究模型中的用途。
3.权利要求1所述的稳定表达绿色荧光蛋白的骨髓增生异常综合征转白细胞株在用于建立MDS微小残留病药物筛选模型中的用途。
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