CN114027263A - 一种获得性再生障碍性贫血模型小鼠的生产方法 - Google Patents
一种获得性再生障碍性贫血模型小鼠的生产方法 Download PDFInfo
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Abstract
本发明公开了一种获得性再生障碍性贫血模型小鼠的生产方法,包括对小鼠每日腹腔注射一次环磷酰胺和环孢霉素,所述环磷酰胺的用量为50mg/kg/d,所述环孢霉素的用量为5mg/kg/d,所述环磷酰胺和环孢霉素的注射间隔时间为3~5分钟,连续注射14天。本发明的方法生产的获得性再生障碍性贫血模型小鼠可用于研究获得性再生障碍性贫血。
Description
技术领域
本发明属于医药领域,特别是涉及一种获得性再生障碍性贫血动物模型的生产方法。
背景技术
再生障碍性贫血(aplastic anemia,AA)是一种以骨髓造血组织减少、骨髓造血功能下降、全血细胞减少为特征的恶性血液系统疾病。再生障碍性贫血可分为先天性和获得性两大类。先天性AA是一种遗传性疾病,主要由FA基因突变导致。获得性AA的发病机制包括造血干细胞缺陷、造血微环境损伤以及免疫功能异常。先天性再生障碍性贫血模型的建立成本高且技术难度大,一般实验室难以完成。获得性AA动物模型针对不同的发病机制采用不同方法建立,包括化学、物理、免疫介导、物理免疫介导法和物理化学结合法等。如采用化学试剂的造模方法,可能存在外周血项指标无统计学意义,维持时间短,一定时间内小鼠可自行修复,不符合获得性再障的临床诊断指标等缺点。物理射线、病毒感染、淋巴细胞输注等造模方法虽然可以成功制作符合AA病理特点的模型,但是存在着实验成本高、操作复杂、模型小鼠死亡率高、小鼠生存期短,不能满足实验药物疗效观察的等弊端。化学试剂联合物理射线法,特别要考虑小鼠射线照射的剂量,把控化学药物的计量,且操作复杂,造模成本较高,且改造模方法可造成造血干细胞或造血微环境损伤。
发明内容
本发明提供了一种获得性再生障碍性贫血模型小鼠的生产方法,包括对小鼠每日腹腔注射一次环磷酰胺和环孢霉素,所述环磷酰胺的用量为50mg/kg/d,所述环孢霉素的用量为5mg/kg/d,所述环磷酰胺和环孢霉素的注射间隔时间为3~5分钟,连续注射14天。
在一优选例中,所述环磷酰胺和环孢霉素的注射间隔时间为4分钟。
本发明采用腹腔注射环磷酰胺和环孢霉素制备获得性AA小鼠模型,造模药物易获取,造模方法操作简单,可替代物理射线、病毒感染等造模方法。经验证,当环磷酰胺联合环孢霉素腹腔连续注射14天后,小鼠外周血项变化、骨髓病理改变、脾脏免疫变化均达到获得性再生障碍性贫血的诊断标准,且该造模方法只需腹腔注射、操作简便、成功率高、维持时间较长,实验动物死亡率低,模型稳定持久,可作为获得性再生障碍性贫血发病机制及治疗研究的小鼠模型。
本发明各个方面的细节将在随后的章节中得以详尽描述。通过下文以及权利要求的描述,本发明的特点、目的和优势将更为明显。
附图说明
图1为实施例各组骨髓细胞形态a.对照组b.模型组
图2为实施例各组骨髓组织病理学检查a.对照组b.模型组
图3为各组小鼠脾脏组织病理学观察a.对照组b.模型组
具体实施方式
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中所使用的试剂和原料均可通过商业途径购买获得。下列实施例中未注明具体条件的实验方法,通常按照常规条件或按照制造厂商所建议的条件。除非另行定义,文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外,任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
本发明提到的上述特征,或实施例提到的特征可以任意组合。本专利说明书所揭示的所有特征可与任何组合物形式并用,说明书中所揭示的各个特征,可以任何可提供相同、均等或相似目的的替代性特征取代。因此除有特别说明,所揭示的特征仅为均等或相似特征的一般性例子。
实施例
实验动物及材料
(1)实验动物及分组
SPF级6月龄BALB/C雌性小鼠20只,体重20-25g,购于上海市计划生育科学研究所实验动物经营部。动物质量合格证号:SCXK(沪)2018-0006。饲养于上海医药工业研究院药理评价研究中心,室温24℃-26℃,每日采光8h-12h,环境湿度为55%左右。实验动物饲养设施合格证号:SYXK(沪)2019-0027。将20只SPF级6月龄BALB/C雌性小鼠20只随机分为为空白组和模型组,每组10只。
(2)试剂与仪器
环孢霉素(华北制药股份有限公司),环磷酰胺(上海源叶生物科技有限公司);BC-6800动物血细胞分析仪(迈瑞医疗国际有限公司),倒置光学显微镜(BX53,日本Olympus公司),荧光显微镜(BX53,Olmpus),组织包埋机(EG1160,LEICA)、水纯化系统(Milli-QDirect 8,MerckMillipore),石蜡切片机(Leica公司),恒温水浴箱(上海精宏实验设备有限公司),台式恒温振荡器(海门市其林贝尔仪器制造有限公司)。
实验方法
1、药物配制及注射
环孢霉素软胶囊剂量为25mg/粒,生理盐水稀释浓度为25mg/ml。环磷酰胺用生理盐水稀释浓度为250mg/ml。模型组每日腹腔注射一次环磷酰胺和环孢霉素,环磷酰胺的剂量为50mg/kg/d,环孢霉素的剂量为5mg/kg/d,环磷酰胺和环孢霉素的注射间隔时间为4分钟。空白组每日腹腔注射一次与上述两种药液同等体积的生理盐水。每组连续注射14天。
2、体征表现及存活期观察
观察每组小鼠一般体征表现及存活期,主要包括活动状况、饮食状况、体型、体重、皮毛等变化,并记录模型组小鼠死亡数量,比较每组小鼠生存情况。
3、外周血细胞计数
于第14天每组各取8只小鼠,经眼眶取血,每只取200μl,EDTA抗凝,稀释10倍后经迈瑞BC-6800全自动血细胞分析仪分析外周血白细胞数(WBC)、红细胞数(RBC)、血小板(PLT)、血红蛋白(HGB)等指标。
4、骨髓组织Wright染色观察
(1)于第14天每组各取8只小鼠处死,迅速剥离小鼠左侧股骨,剥除附于股骨的肌肉,用骨剪从膝关节向髋关节处纵剖,使骨髓暴露,记录骨髓颜色及状态;
(2)用尖头镊子将骨髓左右混合均匀,用镊子尖部取出骨髓约0.5mm放入装有1.5~2ml胎牛血清试管中;
(3)再用尖头玻璃滴管反复吹打30次左右,抽吸混有骨髓的血清,使骨髓均匀分散其中,注意尽量不要将血清吹打出气泡;
(4)将骨髓液用微量加样枪取30~50L(视所取骨髓量而定)加入细胞离心涂片机的小漏斗中,以600~800r/min经10min中速离心;
(5)取出载玻片,标本制作完成;
(6)甲醇固定,Wright染色;
(7)在显微镜下观察细胞形态,并进行骨髓有核细胞计数。
5、骨髓组织HE染色观察
(1)剥离小鼠右侧股骨,10%多聚甲醛固定24h;
(2)PBS浸泡清洗后,脱钙18天,每2-3天换脱钙液;
(3)PBS浸泡清洗后,组织脱水机中18h脱水;
(4)石蜡包埋机中浸蜡包埋,矢状位切片(4μm),捞片;
(5)载玻片平置于烤片机进行贴片5h;
(6)载玻片垂置于60℃烤箱流蜡;
(7)通风橱脱蜡至水(二甲苯2min×3→无水乙醇5min→95%乙醇2min→85%乙醇2min→75%乙醇2min→水洗×3);
(8)苏木素1.5min,水洗×3;
(9)2%盐酸乙醇5sec,水洗×3;
(10)0.5%氨水15sec,水洗×3;
(11)伊红1.5min,水洗×3;
(12)脱水透明(95%乙醇2min→无水乙醇5min→二甲苯5min×3);
(13)中性树胶封片;
(14)放置通风橱4h,光镜下观察骨髓病理改变,摄片观察骨髓造增生情况,并进行有核细胞、红细胞和巨核细胞计数。
6、脾脏组织HE染色观察
(1)将大鼠肾脏样品在10%中性缓冲福尔马林中固定液浸泡48小时后,流水冲洗0.5小时,常规脱水、石蜡包埋、4μm矢状位连续切片,常规脱蜡;
(2)通风橱脱蜡至水(二甲苯2min×3→无水乙醇5min→95%乙醇2min→85%乙醇2min→75%乙醇2min→水洗×3);
(3)苏木素1.5min,水洗×3;
(4)2%盐酸乙醇5sec,水洗×3;
(5)0.5%氨水15sec,水洗×3;
(6)伊红1.5min,水洗×3;
(7)脱水透明(95%乙醇2min→无水乙醇5min→二甲苯5min×3);
(8)中性树胶封片;
(9)放置通风橱4h,光镜下观察脾脏增生情况、淋巴结形态及脾窦、白髓、红髓等。
实验结果
1、模型组小鼠经过腹腔注射环磷酰胺和环孢霉素之后,出现皮毛光泽度降低、松弛蓬乱,部分小鼠有皮损及脱毛。消瘦、摄食减少、精神不佳,唇色眼睑苍白、活动减少,鼻部、足底出现瘀斑等症状,部分小鼠出现上呼吸道感染的症状。各组小鼠在实验期间,均未出现死亡。
2、模型组小鼠经环磷酰胺、环孢霉素腹腔注射后,外周血三系细胞数和血红蛋白浓度均明显低于空白组小鼠(P<0.05)(见表1)。
3、骨髓涂片Wright染色结果显示,空白组小鼠骨髓增生活跃,有核细胞密布,各系比例基本正常,成熟红细胞形态正常,巨核细胞多见,血小板多见,成簇分布;模型组小鼠骨髓增生障碍,呈全面衰竭,成熟红细胞大小一致,血小板减少,巨核细胞未可见(见图1)。各组小鼠骨髓涂片骨髓有核细胞镜下计数结果显示,模型组小鼠经腹腔注射环磷酰胺、环孢霉素后,骨髓有核细胞数量与空白组相比显著减少(P<0.05)(见表2)。
4、骨髓HE染色观察显示空白组骨髓造血细胞丰富;模型组骨髓中造血细胞减少、脂肪细胞等非造血细胞增多(见图2)。骨髓病理切片HE染色后镜下计数显示模型组有核细胞、红细胞及巨核细胞数量显著下降(P<0.05)(见表3)。
5、脾脏HE染色观察显示空白组小鼠脾脏淋巴结呈椭圆规则形态,红髓白髓分界清晰;模型组小鼠脾脏淋巴结形态不规则,红髓白髓分界不清,并且出现局灶性增生(见图3)。
*P<0.05,与对照组相比.
*P<0.05,与对照组相比.
*P<0.05,与对照组相比.
Claims (2)
1.一种获得性再生障碍性贫血模型小鼠的生产方法,包括对小鼠每日腹腔注射一次环磷酰胺和环孢霉素,所述环磷酰胺的用量为50mg/kg/d,所述环孢霉素的用量为5mg/kg/d,所述环磷酰胺和环孢霉素的注射间隔时间为3~5分钟,连续注射14天。
2.如权利要求1所述的生产方法,所述环磷酰胺和环孢霉素的注射间隔时间为4分钟。
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