CN115109755A - 敲除Dicer的单核细胞、组合物及其在制备治疗胶质母细胞瘤药物中的应用 - Google Patents
敲除Dicer的单核细胞、组合物及其在制备治疗胶质母细胞瘤药物中的应用 Download PDFInfo
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Abstract
本发明提供一种敲除Dicer基因的单核细胞、组合试剂及其在制备治疗胶质母细胞瘤药物中的应用。所述单核细胞来源于巨噬细胞条件性Dicer基因敲除小鼠的高表达Ly6C骨髓源性细胞,制备方法包括:A.提取巨噬细胞条件性Dicer基因敲除小鼠骨髓源性细胞并标记磁珠;B.单核细胞分选。所述组合试剂由独立包装的Dicer基因敲除的单核细胞注射液和替莫唑胺注射液组成。所述应用是将Dicer基因敲除的单核细胞注射液和替莫唑胺注射液先后依次注射入胶质母细胞瘤原位移植瘤小鼠体内。本发明向胶质母细胞瘤荷瘤鼠输入敲除了Dicer基因的单核细胞使得荷瘤鼠生存期显著延长,促进了巨噬细胞吞噬肿瘤细胞,且增强了替莫唑胺(TMZ)对胶质母细胞瘤的治疗效果。
Description
技术领域
本发明涉及药物制剂技术领域,具体涉及一种Dicer基因敲除的单核细胞、组合试剂及 其在制备治疗胶质母细胞瘤药物中的应用。
背景技术
胶质母细胞瘤是最常见的颅内原发性恶性肿瘤,复发率高、患者预后极差。胶质母细胞 瘤的生长依赖于特殊的肿瘤微环境。巨噬细胞是胶质母细胞瘤微环境中数量最多的免疫细胞, 因其表型和生物学功能异于正常巨噬细胞,故被称作胶质瘤相关巨噬细胞。研究表明,胶质 瘤相关巨噬细胞主要呈现促肿瘤表型,可通过促进胶质瘤细胞增殖、侵袭、血管生成和胶质 瘤干细胞自我更新等恶性生物学行为,进而促进胶质瘤生长和演进。在胶质瘤微环境中,胶 质瘤相关巨噬细胞的炎性活化与吞噬功能被显著抑制,深入研究胶质瘤相关巨噬细胞表型调 控的机制,找出调控其表型的方法或将为提高胶质母细胞瘤患者生存质量、延长罹患胶质母 细胞瘤患者的生存期带来新希望。
借助10×genomics单细胞转录组测序技术,科学家们发现胶质母细胞瘤内存在多个功能 各异的胶质瘤相关巨噬细胞亚群,分别为高表达Sepp1的巨噬细胞亚群、缺氧相关的巨噬细 胞亚群、具有增殖特性的巨噬细胞亚群、高表达I FN的巨噬细胞亚群与具有吞噬活性的巨噬 细胞亚群等。其中,高表达Sepp1的巨噬细胞亚群与缺氧相关的巨噬细胞亚群高表达Mrc1、 Arg1等抗炎细胞因子基因,可能具有促肿瘤效应;高表达I FN的巨噬细胞亚群与具有吞噬活 性的巨噬细胞亚群则具备炎性活化与吞噬潜能,可能具有抗肿瘤效应。该研究显示:提高具 有抗肿瘤效应潜能的巨噬细胞亚群比例,和/或降低具有促肿瘤效应潜能的巨噬细胞亚群比 例,可能作为靶向胶质母细胞瘤巨噬细胞的精准治疗手段。
Dicer是核糖核酸内切酶家族的一员,位于细胞质,能剪切pre-miRNAs进而促进绝大部 分miRNAs的成熟。miRNAs通过竞争性结合靶基因的3’-UTR端抑制基因转录,发挥调控各 种细胞生理活动的作用。在巨噬细胞内,Dicer显著影响miRNA-125、let-7b等miRNAs的表 达量,进一步影响巨噬细胞的炎性活化状态,从而影响慢性炎症等多种疾病的进展。因此, 敲除Dicer基因的单核细胞有望改善胶质瘤相关巨噬细胞炎性活化状态。
将敲除Dicer基因的单核细胞、组合试剂用于胶质母细胞瘤临产治疗,尚未有报道。
发明内容
有鉴于此,为了克服现有技术的不足,本发明提供一种敲除Dicer基因的单核细胞、组 合试剂及其在制备治疗胶质母细胞瘤药物中的应用。
本发明提供的一种敲除Dicer基因的单核细胞,所述单核细胞来源于巨噬细胞条件性 Dicer基因敲除小鼠的高表达Ly6C骨髓源性细胞。
上述Dicer基因敲除的单核细胞的制备方法,包括步骤:
A.提取巨噬细胞条件性Dicer基因敲除小鼠骨髓源性细胞并标记磁珠;
B.单核细胞分选。
进一步,所述A包括步骤:
1)使巨噬细胞条件性Dicer基因敲除小鼠的骨髓形成细胞悬液;
2)通过Fico l l分离法富集所述小鼠骨髓源性细胞悬液中的单个核细胞;
3)用含有单核细胞封闭液的PBS将细胞稀释为5×108个细胞/ml;
4)按体积比,细胞悬液:CD11 b磁珠分选抗体=9:1,在细胞悬液中加入磁珠抗体,将 细胞悬液充分混匀,于冰上孵育20min;
5)用PBS洗涤细胞后,用1ml PBS重悬备用。
进一步,所述B包括步骤:
6)将所需LS型细胞分选柱同时放于QuadroMACS磁铁上,在所述LS柱下方放置离心管;
7)将权利要求3获得的所述PBS细胞重悬液依次加在各LS型细胞分选柱上分选;
8)分选获得的细胞沉淀用体积比1:200的流式抗体Ly6C-APC/Cy7重悬,通过流式分 选Ly6C高表达的细胞;
9)离心后将细胞用PBS重悬,调整细胞浓度至2000个细胞/μl,得到所述Dicer基因敲除的单核细胞悬液。
本发明还提供上述Dicer基因敲除的单核细胞在制备治疗胶质母细胞瘤药物中的应用。
所述的应用是将Dicer基因敲除的单核细胞悬液从尾静脉注射入胶质母细胞瘤小鼠体 内,每0.015-0.02kg体重的小鼠注射2000个细胞/μl的细胞悬液80-120μl。
本发明还提供一种组合试剂,由独立包装的Dicer基因敲除的单核细胞注射液和替莫唑 胺注射液组成。
进一步,所述单核细胞注射液为2000个细胞/μl的PBS细胞悬液;所述替莫唑胺注射 液为4-6%DMSO、28-33%PEG和ddH2O,按顺序依次添加溶解获得。
本发明还提供上述组合试剂在制备治疗胶质母细胞瘤药物中的应用。
所述应用是将所述Dicer基因敲除的单核细胞注射液和替莫唑胺注射液先后依次注射入 胶质母细胞瘤小鼠体内;所述单核细胞注射液从尾静脉注射入胶质母细胞瘤小鼠体内,每 0.02kg小鼠注射2000个细胞/μl的细胞悬液80-120μl;所述替莫唑胺注射液腹腔注射入 胶质母细胞瘤小鼠体内,每小鼠注射替莫唑胺10mg/kg。
敲除巨噬细胞Dicer能促进巨噬细胞向经典活化表型(M1样)转分化,通过释放促炎细 胞因子I FNγ激活T细胞的免疫杀伤功能,从而发挥抑制肿瘤生长的作用。本发明首先利用 10×genomics单细胞转录组测序技术,探讨敲除巨噬细胞Dicer对巨噬细胞亚群的影响,发 现敲除巨噬细胞Dicer能上调高表达I FN的巨噬细胞亚群与具有吞噬活性的巨噬细胞亚群比 例,同时下调具有增殖活性的巨噬细胞亚群、高表达Sepp1的巨噬细胞亚群与缺氧相关的巨 噬细胞亚群比例;基于单细胞结果的发现,利用移植瘤模型进一步评估了敲除Dicer基因对 胶质瘤相关巨噬细胞增殖、吞噬、炎性活化等多种表型的影响,提示Dicer调控胶质瘤相关 巨噬细胞的多种功能,进而抑制胶质母细胞瘤移植瘤生长,延长荷瘤鼠生存期。该现象可作 为Dicer干预胶质瘤相关巨噬细胞表型的具体途径及抑制胶质瘤生长的潜在机制。
在促进巨噬细胞吞噬肿瘤细胞这一效应上,细胞治疗相比传统的抗体治疗具有更显著的 优势。抗体依赖的细胞吞噬效应(Ant ibody-dependent ce l l u l arphagocytos i s,ADCP)限 制部分抗体治疗的临床效果。2018年,Sh icheng Su等人发现,虽然抗Her2抗体曲妥珠单抗 能促进巨噬细胞吞噬乳腺癌细胞,然而,肿瘤细胞在被巨噬细胞吞噬后,其内部的DNA释放 到吞噬体内,能进一步通过Caspase-1途径诱导巨噬细胞产生PD-L1与I DO,进而抑制T细 胞与NK细胞活化,最终抑制肿瘤免疫。本发明发现,敲除巨噬细胞Dicer显著增强巨噬细胞 吞噬胶质母细胞瘤细胞的能力,且具有吞噬活性的巨噬细胞亚群依然能通过TNF与I L-2途径 活化NK细胞,通过TNF途径活化T细胞,避免了ADCP在治疗中出现的免疫抑制效应。
由于胶质母细胞瘤微环境中,促肿瘤样胶质瘤相关巨噬细胞亚群与抗肿瘤胶质瘤相关巨 噬细胞亚群同时存在,但单核细胞对不同胶质瘤相关巨噬细胞亚群分化的具体途径未知,因 此尚不能精准调控单核细胞向特定亚群胶质瘤相关巨噬细胞的分化,因此缺乏针对特定亚群 胶质瘤相关巨噬细胞的治疗手段。明确单核细胞向特定亚群胶质瘤相关巨噬细胞分化的途径 具有更为重要的意义。在此背景下,敲除巨噬细胞Dicer基因具有显著的优势,即敲除巨噬 细胞Dicer能上调促肿瘤胶质瘤相关巨噬细胞亚群比例,下调抗肿瘤胶质瘤相关巨噬细胞亚 群比例,若能将敲除巨噬细胞Dicer与针对不同胶质瘤相关巨噬细胞亚群的个性化治疗方案 相结合,可能得到更有效的基于巨噬细胞的胶质母细胞瘤治疗方案。这预示,未来将能充分 利用胶质瘤中庞大的巨噬细胞亚群,延长胶质瘤患者生存期,改善胶质瘤患者生存质量。
本发明通过将健康Dicer WT与Dicer KO小鼠骨髓及外周血中分离单核细胞注射给荷瘤 鼠,评估向荷瘤鼠注射敲除Dicer基因单核细胞的治疗学意义。通过F4/80免疫荧光染色观 察了巨噬细胞在肿瘤中的浸润情况。
与现有技术相比,本发明的有益效果在于:
1.本发明向胶质母细胞瘤荷瘤鼠注射敲除Dicer基因的健康小鼠骨髓单核细胞导致荷瘤 鼠生存期显著延长。通过对移植瘤切片F4/80免疫荧光染色观察到巨噬细胞吞噬肿瘤细胞这 一明显现象,且在肿瘤中心与肿瘤侵袭前缘均能观察到该现象。说明了敲除Dicer基因的巨 噬细胞对胶质母细胞瘤具有显著的治疗效果。提示提取患者单核细胞,构建敲除Dicer基因 的单核细胞并回输给患者这一细胞治疗手段有望成为新的胶质母细胞瘤治疗方式。
2.注射敲除Dicer基因的单核/巨噬细胞增强了替莫唑胺(TMZ)对胶质母细胞瘤的治疗 效果。
附图说明
图1注射敲除Dicer基因的单核/巨噬细胞对胶质母细胞瘤荷瘤鼠生存期及巨噬细胞吞 噬能力的影响;
其中:A)实验流程图;B)Kap l an-Meier生存曲线分析:向胶质母细胞瘤荷瘤鼠注射敲除 Dicer基因的单核细胞与来源于野生型小鼠的单核细胞对荷瘤小鼠生存期的影响对比;C-D) F4/80免疫荧光染色分析:向胶质母细胞瘤荷瘤鼠注射敲除Dicer基因的单核细胞与来源于 野生型小鼠的单核细胞对小鼠胶质瘤相关巨噬细胞吞噬胶质瘤细胞能力的影响对比。**, p<0.01
图2注射敲除Dicer基因的单核/巨噬细胞与注射TMZ联合使用对胶质母细胞瘤的治疗 效果;
A.GL261接种后第7天、第14天和第21天Dicer WT、Dicer WT+TMZ、Dicer cKO和Dicer cKO+TMZ组中肿瘤生长的体内生物发光图像;B.生物发光图像数据统计图;C.各组脑切片的H&E染色,比例尺,2mm;D.每组肿瘤面积统计图;E.各组小鼠Kaplan-Meier生存 曲线。(第1组和第2组n=10,第3组n=8,第4组n=9)。**p<0.01。
具体实施方式
下面结合具体实例对本发明的方法进行详细描述和说明。其内容是对本发明的解释而非 限定本发明的保护范围。
实施例1:转基因小鼠的繁殖及获取巨噬细胞条件性Dicer基因敲除小鼠
C57BL/6品系Dicer1 f lox/f lox转基因小鼠购自Jackson实验室(目录号:006366); C57BL/6品系LysM-Cre工具鼠购自Jackson实验室(目录号:004781)。
Dicer1 f lox/f lox转基因小鼠与LysM-Cre工具鼠均饲养于陆军军医大学全军免疫学研 究所无特定病原体(Specific Pathogen Free,SPF)动物房。动物房每日持续监测环境温度 与湿度,每日保持12小时光照与12小时黑暗时间,确保动物饲养环境的稳定。此外,每个 动物饲养笼的小鼠数量控制在5只以内(新生鼠除外)。小鼠所用鼠笼、垫料、饮用水及饲 料经严格高温、高压灭菌。新生鼠离乳后,将雌性鼠与雄性鼠分笼喂养。为了保证小鼠的营 养,对周龄大于6周的小鼠每周投喂5粒/只的灭菌向日葵籽。
设立LysM-Cre工具鼠与Dicer1 f lox/f lox转基因小鼠种鼠繁殖笼,将上述小鼠单独饲 养与繁殖。将Dicer1 f lox/f lox转基因小鼠与LysM-Cre工具鼠子代进行杂交,得到F1代 杂合子小鼠。将F1代杂合子小鼠互交,对经互交产生的适龄子代鼠进行鉴定,得到Dicer1 f lox/f lox;LysM-Cre纯合子小鼠,经功能鉴定后,即得到巨噬细胞条件性Dicer基 因敲除小鼠。
实施例2:转基因小鼠的鉴定
鉴定转基因鼠的步骤分为鼠尾DNA提取、聚合酶链式反应(Po lymerase ChainReact ion, PCR)与核酸电泳三个主要步骤。
1.鼠尾DNA提取。
(1)用无菌眼科剪减去小鼠的尾巴尖,长度为0.2cm,将其装入1.5ml EP管内,每管做好标记;
(2)用眼科剪将小鼠尾巴尖充分剪碎,在EP管内加入50μl Qu ick Extract So lut ion,并用1ml移液器充分吹打混匀;
(3)打开水浴锅,温度预热至65℃,将EP管置于水浴锅内,反应15min;
(4)涡旋混匀15s,95℃终止2min;
(5)1000rpm,5min进行离心,所得的液体即为小鼠基因组DNA溶液,保存于-20℃。
2.小鼠基因组DNA PCR扩增
以下操作在洁净室进行:
(1)在操作台上喷洒DNA/RNA酶去除剂;
(2)分别取出1.5ml EP管及PCR管备用;
(3)参照表1中PCR反应体系与表2中的引物,在1.5ml EP管中配制足够量的Taq酶、引物预混液;
(4)将Taq酶、引物预混液分别分装入对应的EP管中;
(5)在对应的EP管中加入DNA模板;
(6)执行表3的PCR反应条件,进行PCR扩增,针对LysM-Cre工具鼠的鉴定,需将Lyz2-3066与Lyz2-3067,Lyz2-3067与Lyz2-3068分开进行PCR扩增与核酸电泳。;
(7)所得PCR产物置于-20℃保存。
PCR反应体系如下:
表1鉴定Dicer1 f lox/f lox转基因鼠与LysM-Cre工具鼠的PCR反应体系
表2动物鉴定引物序列
表3.D icer1 f l ox/f l ox转基因鼠及LysM-Cre工具鼠PCR扩增条件
3.核酸电泳
(1)将1×TBE溶液配置1.2%或1.5%琼脂糖凝胶于制胶专用锥形瓶中,将微波炉调至“中 火”档,促进琼脂糖凝胶变为透明状;
(2)将锥形瓶置于室温冷却,使用制胶专用温度计实时监测凝胶温度,待凝胶温度降至 60℃以下,在凝胶中加入5μl Go ldview染料,轻轻摇晃锥形瓶,使染料充分溶解于凝胶内;
(3)准备制胶板,将梳子插入制胶板内;
(4)待凝胶温度降至60℃左右时,将凝胶倒入制胶板,等待凝胶冷却至室温;
(5)在电泳槽内倒入1×TBE缓冲液至最高刻度线,轻轻将凝胶放入电泳槽内;
(6)预先准备一张PCR封板膜,在封板膜上依次加入2μl DNA上样缓冲液,之后将8μl DNA PCR产物与其混匀,将混匀过后的PCR产物加入凝胶对应孔内,记录每个孔对应的小鼠编号;
(7)分别在每行的第一个凝胶孔或最后一个的凝胶孔内加入DNA marker;
(8)将电泳槽电压设置为100V,启动电泳,实时观察条带位置,适时终止电泳;
(9)取出凝胶,将凝胶置于显影仪中,拍照;
(10)根据电泳结果,逐一确认各小鼠基因型。
实施例3 BMDCs提取
1.BMDCs提取与磁珠标记
(1)提取小鼠骨髓源性细胞(Bone marrow-der ived ce l l,BMDC)
a向经基因型鉴定完毕的巨噬细胞条件性Dicer基因敲除小鼠注射0.1%戊巴比妥钠溶液 (剂量为50mg/kg),对小鼠实施麻醉;
b麻醉状态下,处死小鼠;
c将小鼠置于体积为250ml烧杯中,烧杯中添加200ml 75%乙醇溶液;
d对小鼠逆向刷毛,充分对小鼠进行灭菌;
e灭菌完毕,将小鼠置于生物安全柜中;
f用眼科剪将剪开小鼠腹股沟出皮肤,将切口扩大至小鼠跟腱处,钝性分离小鼠皮下筋 膜及肌肉组织,充分暴露小鼠股骨,并取出小鼠股骨与胫骨。
g去除小鼠股骨与胫骨肌肉、肌腱组织后,将小鼠股骨、胫骨置于含有双抗的预冷PBS 中;
h用预冷PBS反复冲洗3遍小鼠骨组织,之后将2.5ml注射器吸入PBS备用;
i分别用眼科剪剪开股骨、胫骨两端,之后将注射器针头插入2.5ml注射器中,适当推 动注射器冲出骨髓,重复操作1次;
j用1ml移液器反复吹打骨髓冲洗液至形成细胞悬液;
k将40μm细胞筛置于50ml离心管上,将上一步得到的细胞悬液吹打入细胞筛,丢弃细胞筛,离心管内余下液体即为BMDC悬液;
l离心,设置离心机条件为:300×g,常温,离心5min,升速8,降速8;
m离心完毕,弃上清,细胞沉淀备用。
(2)通过Fico l l富集小鼠骨髓源性细胞中的单个核细胞;
a取洁净15ml离心管,并做好标记,每个管子预填充3ml Fico l l;
b将骨髓源性细胞悬液吹打混匀后,缓慢加至Fico l l液面上;
c离心,设置离心条件为500×g,30min,温度为18℃,升速1,降速0;
d离心完毕后,用1ml移液器小心翼翼吸取中间白色细胞层至新的15ml离心管内;
e向离心管内加入PBS,使细胞悬液体积稀释为原体积的5-10倍;
f离心,设置离心条件为300×g,10min,温度为室温,升速8,降速8;
g离心完毕,弃上清,
(3)进行细胞计数,按照细胞个数,用含有单核细胞封闭液(稀释度1:20)的PBS将细胞浓度稀释为5×108个细胞/ml;
(4)15min后,按细胞悬液:CD11 b磁珠分选抗体=9:1,在细胞悬液中加入磁珠抗体;
(5)将细胞悬液充分混匀,于冰上孵育20min;
(6)用PBS洗涤细胞2次,离心条件为:300×g,4℃,10min,升速8,降速8;
(7)将细胞用1ml PBS重悬备用;
其中,CD11 b磁珠分选抗体购于德国美天旎公司,货号130-126-725。
2.单核细胞分选
(1)按照细胞总数计算所需LS柱的数量。1根LS柱最多可获得2×107个阳性细胞,最 多可通过4×107个细胞;
(2)将所需LS柱同时放于QuadroMACS磁铁上,用3ml PBS润湿LS柱;
(3)在LS柱下方放至一15ml离心管;
(4)将样品依次加在各LS柱上,待样本缓缓滴下;
(5)待LS柱不再滴下液体时,用3ml PBS润洗LS柱,重复3次;
(6)从磁铁中取下LS柱,将其放置于一15ml离心管上方,在柱内加入5ml PBS,推动活塞,将柱内液体快速注射入离心管内;
(7)重复上一步操作后离心,4℃,300×g,离心10min;弃上清
(8)将细胞沉淀用1:200Ly6C-APC/Cy7重悬,通过流式分选Ly6C高表达的细胞;
(9)离心300×g,室温,10min;
(10)将细胞用500μl PBS重悬,调整细胞浓度至2000个细胞/μl备用。
其中,流式分选(FCM分选)胶质瘤相关巨噬细胞的步骤为:
(1)将细胞沉淀用1:200Ly6C-APC/Cy7重悬,冰上孵育30min;
(2)向各EP管内加入1ml预冷PBS,离心,离心机条件不变;
(3)弃上清,重复上一步骤一次;
(4)将所有EP管中的细胞悬液转移至流式管内,将流式管置于冰上;
(5)流式上机检测,圈出Ly6C高表达一群,进行细胞分选,收集细胞于15ml离心管,使细胞充分混匀,抽取100μl细胞悬液作注射实验用。
实施例4建立小鼠GL261脑胶质瘤原位模型
一、小鼠GL261细胞的培养
1.GL261细胞的复苏
(1)打开水浴锅,温度设置为37℃;
(2)GL261细胞冻存管长期储存于液氮。从液氮罐中取出一支GL261细胞,并做好记录;
(3)将GL261细胞冻存管从液氮中取出后,立即将其置于水浴锅中,直至冻存管内液体 溶解;
(4)将冻存管内液体转移至15ml无菌、无酶离心管内,设置离心机参数为1000×rpm, 常温,离心5min,升速8,降速8;
(5)离心完毕,弃上清,将细胞沉淀用1ml DMEM完全培养基(含10%FBS)重悬,再次离心,设置离心机参数为1000×rpm,常温,离心5min,升速8,降速8;
(6)弃上清,将细胞沉淀用8ml DMEM完全培养基(含10%FBS)重悬,转移至10cm直径细胞培养皿中培养;
(7)将培养皿转移至培养箱中,设置培养箱参数为37℃,5%CO2。
(8)为保证培养箱清洁,每周对培养箱进行清扫、灭菌,每月对培养箱进行一次大循环 彻底灭菌。
二、使用GFP标记的GL261细胞,建立小鼠GL261原位移植瘤模型
(1)待GL261细胞生长至大于80%细胞密度,考虑开始建立移植瘤模型;
(2)将GL261细胞消化成单个细胞,用含10%FBS的DMEM培养基终止消化;
(3)1000×rpm,常温,离心5min,弃上清,用1ml PBS重悬细胞,将细胞转移至1.5ml EP管内,计数细胞,并调整细胞密度为4000个细胞/μl;
(4)将1.5ml离心管置于冰上,带至独立通风笼具(I nd ividua l ly vent i lated cage, IVC)动物房,放置于操作台上;
(5)向小鼠注射0.1%戊巴比妥钠溶液(剂量为50mg/kg),对小鼠实施麻醉,将小鼠眼内滴入50μl(一滴)玻璃酸钠滴眼液确保小鼠眼睛湿润;
(6)将麻醉好的小鼠固定于操作台中的小动物立体定位仪上,检查小动物麻醉机异氟烷 余量,将小鼠鼻腔连接至小动物麻醉机中,打开小动物麻醉机,设置异氟烷/空气流速为300 ml/min,对小鼠头皮进行表面消毒并脱毛,眼科剪沿颅骨中线剪开约0.5cm伤口,暴露颅骨;
(7)200μl移液器混匀细胞,汉密尔顿微量进样器吸5μl细胞悬液后垂直放置进样器;
(8)找到小鼠冠、矢状缝,小鼠前正中线与外眦连线交接点后右侧旁开0.5cm处即为进 针点,进针深度为0.3cm;
(9)将5μl细胞悬液在10分钟内缓慢注射入小鼠颅内(GL261细胞浓度为5000细胞/μl),注射时间1min,注射完毕,原位停留30秒后再缓慢拔针;
(10)利用持针钳、缝皮针与缝合线缝合小鼠头皮,对小鼠头皮进行表面消毒,将小鼠移 至37℃保温垫上,直至小鼠苏醒;
(11)将苏醒后的小鼠转移至普通动物房的鼠笼内饲养,每日观察小鼠情况。
实施例5单核细胞输入原位移植瘤模型小鼠
(1)将GL261原位移植瘤模型小鼠麻醉后,置于小鼠尾静脉注射显像仪内;
(2)打开光源,暴露小鼠尾静脉,用胰岛素注射器沿尾静脉注射100μl实施例3中通过流式分选获得的Ly6C高表达的巨噬细胞条件性Dicer基因敲除小鼠骨髓源性单核细胞(细 胞浓度至2000个细胞/μl);
(3)小鼠置于保温垫上复温,待小鼠苏醒后,将小鼠转移至动物笼内饲养。
实施例6输入单核细胞的原位移植瘤模型小鼠脑冰冻切片与F4/80免疫荧光染色
(1)对实施例5中输入单核细胞的实验小鼠实施灌注,注意在PBS灌注后,采用4%多聚 甲醛进一步灌注小鼠,每只小鼠约灌注200ml 4%多聚甲醛,期间观察到小鼠四肢及尾部显 著抽搐,灌注完毕,小鼠全身僵硬,关注过程中,操作人员作好防护。具体方法如下:
a向小鼠腹腔注射0.1%戊巴比妥钠溶液(剂量为50mg/kg),对小鼠实施麻醉;
b将小鼠固定于灌注器上,将灌注器置于灌注台内;
c用眼科剪剪开小鼠胸腔,暴露小鼠心包;
d向左心室注入0.1%EDTA抗凝剂200μl,行左心室主动脉插管,用血管钳固定灌注针,设置灌注液(预冷PBS)流速为90滴/min;
e剪开小鼠右心房,此时血液从小鼠右心房中流出,密切关注小鼠灌注状态;
f约5min后,降低灌注速度为60滴/min;
g约15min后,小鼠右心房中流出清亮且透明的液体,小鼠肝脏颜色显著变淡,四肢变 白,停止灌注;
h将小鼠从灌注台上取下,置于原代操作台中;
(2)将灌注后的小鼠脑放置于台面上,去掉嗅叶与小脑,之后将大脑置于30%蔗糖溶液 中脱水;
(3)待大脑接触蔗糖溶液底部,将大脑从中取出,使用冰冻切片仪对大脑进行20μm厚 切片;
(4)对小鼠大脑进行F4/80免疫荧光染色,由于蔗糖脱水后脑组织特别容易掉片,整个 过程需确保轻柔;
(5)染色完毕,用激光共聚焦显微镜观察切片,并拍照。
(6)统计学分析
对于免疫染色切片,每组选取至少5只老鼠来源的切片,每张切片选取至少10个随机视 野进行拍照,计算阳性细胞数及相应细胞比例;
对于两组数据之间的比较,采用Graphpad Pr i sm 6软件进行统计学分析。采用独立样本 的T检验对比两组数据之间的差异。误差线显示标准差。若总组别设置大于两组,即使比较 其中任意两组的数据,亦采用单因素方差分析方式进行。采用Kap l an-Meier生存分析评估 荷瘤鼠生存期。
(7)结论:注射敲除Dicer基因的单核/巨噬细胞对胶质母细胞瘤荷瘤鼠生存期及巨噬 细胞吞噬能力的影响。
为了初步探讨敲除巨噬细胞Dicer的治疗学意义,遂评估注射敲除Dicer基因的单核/ 巨噬细胞对胶质母细胞瘤荷瘤鼠生存期的影响(图1A),发现相较对照组及注射野生型单 核细胞组,注射敲除Dicer基因的单核/巨噬细胞显著延长荷瘤鼠生存期(图1B)。进一步对各组移植瘤样本进行F4/80免疫荧光染色,发现注射敲除Dicer基因的单核/巨噬细胞显著增加了巨噬细胞对GL261细胞的吞噬能力(图1C,D)。
实施例7:单核细胞输入与TMZ注射联用于原位移植瘤模型小鼠
替莫唑胺TMZ溶液:TMZ购自se l leck(S1237)。
替莫唑胺注射液为5%DMSO、30%PEG和ddH2O,按顺序依次添加溶解获得。
实施例4的小鼠GL261脑胶质瘤原位模型在注射GL261细胞(肿瘤种植)第8天开始,随机分成2组,第一组输入正常小鼠骨髓源性单核细胞;第二组同实施例5,输入Ly6C高表达的巨噬细胞条件性Dicer基因敲除小鼠骨髓源性单核细胞。
完成上述注射后,第一组再平分为2组:一组进行TMZ给药,10mg/kg(即图2中的WT+TMZ); 另一组注射等量TMZ溶解的溶剂做对照(即图2中的Dicer-WT);
第二组再平分为2组:一组进行TMZ给药,10mg/kg(即图2中的KO+TMZ);另一组注射等量TMZ溶解的溶剂做对照(即图2中的Dicer-KO)。
(其中,所述单核细胞注射液从尾静脉注射入胶质母细胞瘤小鼠体内,每0.02kg小鼠注 射2000个细胞/μl的细胞悬液100μl;所述替莫唑胺注射液(或TMZ溶解的溶剂)腹腔注射入胶质母细胞瘤小鼠体内,每小鼠注射替莫唑胺10mg/kg。)
在肿瘤种植后的第7、14、21天进行小动物活体成像,并对荧光值进行统计。
结论:TMZ是胶质母细胞瘤的一线治疗药物,本发明评估了注射敲除Dicer基因的单核/ 巨噬细胞与替莫唑胺(TMZ)联合使用对胶质母细胞瘤荷瘤鼠生存期的影响。与其他组相比, TMZ显著地抑制了注射敲除Dicer基因单核/巨噬细胞组胶质母细胞瘤的生长,极大地延长了 注射敲除Dicer基因的单核/巨噬细胞组小鼠的存活时间(图A-E)。该数据验证了注射敲除 Dicer基因的单核/巨噬细胞增强了替莫唑胺(TMZ)对胶质母细胞瘤的治疗效果。二者的联 合使用具有明显的协同效应。
最后说明的是,以上优选实施例仅用以说明本发明的技术方案而非限制,尽管通过上述 优选实施例已经对本发明进行了详细的描述,但本领域技术人员应当理解,可以在形式上和 细节上对其作出各种各样的改变,而不偏离本发明权利要求书所限定的范围。
Claims (10)
1.一种Dicer基因敲除的单核细胞,其特征在于,所述单核细胞来源于巨噬细胞条件性Dicer基因敲除小鼠的高表达Ly6C骨髓源性细胞。
2.根据权利要求1所述Dicer基因敲除的单核细胞的制备方法,其特征在于,包括步骤:
A.提取巨噬细胞条件性Dicer基因敲除小鼠骨髓源性细胞并标记磁珠;
B.单核细胞分选。
3.根据权利要求2所述的制备方法,其特征在于,所述A包括步骤:
1)使巨噬细胞条件性Dicer基因敲除小鼠的骨髓形成细胞悬液;
2)通过Ficoll分离法富集所述小鼠骨髓源性细胞悬液中的单个核细胞;
3)用含有单核细胞封闭液的PBS将细胞稀释为5×108个细胞/ml;
4)按体积比,细胞悬液:CD11b磁珠分选抗体=9:1,在细胞悬液中加入磁珠抗体,将细胞悬液充分混匀,于冰上孵育20min;
5)用PBS洗涤细胞后,用1ml PBS重悬备用。
4.根据权利要求2所述的制备方法,其特征在于,所述B包括步骤:
6)将所需LS型细胞分选柱同时放于QuadroMACS磁铁上,在所述LS柱下方放置离心管;
7)将权利要求3获得的所述PBS细胞重悬液依次加在各LS型细胞分选柱上分选;
8)分选获得的细胞沉淀用体积比1:200的流式抗体Ly6C-APC/Cy7重悬,通过流式分选Ly6C高表达的细胞;
9)离心后将细胞用PBS重悬,调整细胞浓度至2000个细胞/μl,得到所述Dicer基因敲除的单核细胞悬液。
5.根据权利要求1所述Dicer基因敲除的单核细胞在制备治疗胶质母细胞瘤药物中的应用。
6.根据权利要求5所述的应用,其特征在于,将Dicer基因敲除的单核细胞悬液从尾静脉注射入胶质母细胞瘤小鼠体内,每0.015-0.02kg体重的小鼠注射2000个细胞/μl的细胞悬液80-120μl。
7.一种组合试剂,由独立包装的Dicer基因敲除的单核细胞注射液和替莫唑胺注射液组成。
8.根据权利要求7所述的组合试剂,其特征在于,所述单核细胞注射液为2000个细胞/μl的PBS细胞悬液;所述替莫唑胺注射液为4-6%DMSO、28-33%PEG和ddH2O,按顺序依次添加溶解获得。
9.根据权利要求7所述的组合试剂在制备治疗胶质母细胞瘤药物中的应用。
10.根据权利要求9所述的应用,其特征在于,所述Dicer基因敲除的单核细胞注射液和替莫唑胺注射液先后依次注射入胶质母细胞瘤小鼠体内;所述单核细胞注射液从尾静脉注射入胶质母细胞瘤小鼠体内,每0.02kg小鼠注射2000个细胞/μl的细胞悬液80-120μl;所述替莫唑胺注射液腹腔注射入胶质母细胞瘤小鼠体内,每小鼠注射替莫唑胺10mg/kg。
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