CN111518835A - 一种原发性多发性骨髓瘤小鼠模型构建方法及其应用 - Google Patents
一种原发性多发性骨髓瘤小鼠模型构建方法及其应用 Download PDFInfo
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Abstract
本发明涉及一种原发性多发性骨髓瘤小鼠模型构建方法,包括筛选样品基因组序列,制备逆转病毒载录载体,逆转病毒载录及诱导转化等四个步骤。本发明一方面模型构建效率高,且生理特征稳定性好;另一方面可实现对模型中原发性多发性骨髓瘤基因序列全生命周期精确观测,且观测便捷、观测精度高,从而有效的提高对原发性多发性骨髓瘤疾病病情诊治、治疗及药物研发提供精确的依据。
Description
技术领域
本发明涉一种原发性多发性骨髓瘤小鼠模型构建方法及其应用,属多发性骨髓瘤研究技术领域。
背景技术
目前在进行多发性骨髓瘤研究过程中,主要是通过构建的多发性骨髓瘤小鼠模型为基础进行,但在实际研究工作中发现,当前所使用的多发性骨髓瘤小鼠模型一方面均不同程度存在制备效率低、制备成功率低以及多发性骨髓瘤小鼠模型的鼠体生理特征及活性受到影响,从而导致人原癌基因对鼠体造成的病变检测精度受到严重影响;另一方面也存在研究人员无法准确对多发性骨髓瘤小鼠模型体内原癌基因序列在多发性骨髓瘤小鼠模型体内的扩散、分布变化情况,从而无法直接获得原癌基因致病机理、病情发育及药物作用效果等相关的研究数据,从而对疾病治疗和药物开发造成了严重的影响,同时也对多发性骨髓瘤小鼠模型适用范围造成严重影响。
因此针对这一问题,迫切需要开发一种新的多发性骨髓瘤小鼠模型制备方法及应用方法,以满足实际科研工作的需要。
发明内容
为了解决现有分类技术上的一些不足,本发明提供一种原发性多发性骨髓瘤小鼠模型构建方法及其应用。
为了实现上面提到的效果,提出了一种原发性多发性骨髓瘤小鼠模型构建方法及其应用方法,包括以下步骤:
一种原发性多发性骨髓瘤小鼠模型构建方法,包括以下步骤:
S1,筛选样品基因组序列,首先随机选定从至少10名原发性多发性骨髓瘤患者组织样本,并分别放置在培养皿中培养3—72小时,然后对培养后的组织样本中分别进行DNA提取,最后对提取后的DNA样本进行基因序列测序,并筛选出个样本中的人原癌基因,并对筛选的各人原癌基因序列混合后进行存放备用;
S2,制备逆转病毒载录载体,在进行S1步骤同时,从选定的活体小鼠身上提取任意一种病毒,然后针对选定病毒制备并选用该类病毒为基础的逆转病毒载录载体;
S3,逆转病毒载录,完成S1和S2步骤后,将S1步骤制备得到人原癌基因序列同时与荧光分子标靶及一对引物混合,并通过聚合酶链式反应进行扩链,最后将扩链后的人原癌基因序列S2步骤制备的逆转病毒载录载体进行混合并,得到逆转病毒转录体,并对逆转病毒转录体进行荧光标靶检测,且含荧光标靶逆转病毒转录体量不小于逆转病毒转录体总量的98%后,即可进行后续作业;
S4,诱导转化,首先收集纯化的S2步骤同类活体小鼠IgM阳淋巴细胞,并利用化学诱导剂刺激分化小鼠IgM阳淋巴细胞,同时诱导Pre-B细胞凋亡;然后对小鼠IgM阳淋巴细胞进行培育生长分裂1—7天,然后将S3步骤制备得到逆转病毒转录体添加到小鼠IgM阳淋巴细胞进行培育基体中,并继续与小鼠IgM阳淋巴细胞进行培育1—10天;最后将S2步骤选定的活体小鼠进行γ射线辐照1—8小时,再将培育后的小鼠IgM阳淋巴细胞分别注射至活体小鼠的淋巴细胞及骨髓细胞中,并对活体小鼠在无菌环境下培育1—3天,即可得到原发性多发性骨髓瘤小鼠模型。
进一步的,所述的S2步骤中选定的活体小鼠鼠龄为1.5—3个月,且每组中均若干小鼠,且同组中个小鼠均来源同一母体同批次繁育所得。
进一步的,所述的S3步骤中,引物包括正向引物5'....CTTCGAAATTC3';反向引物5'....CCGATCGGGAGA3'。
一种原发性多发性骨髓瘤小鼠模型的应用,对原发性多发性骨髓瘤小鼠模型进行持续观测,通过荧光标靶对病毒在鼠体内扩散速度及路径进行持续观测并荧光标记。
一种原发性多发性骨髓瘤小鼠模型的应用,对原发性多发性骨髓瘤小鼠模型进行药物喂食,并通过荧光标靶对病毒在鼠体内扩散速度及路径进行持续观测并荧光标记,从而获得当前药物对原发性多发性骨髓瘤治愈效果评估。
一种原发性多发性骨髓瘤小鼠模型的应用,对原发性多发性骨髓瘤小鼠模型进行不同类型药物喂食,并通过荧光标靶对病毒在鼠体内扩散速度及路径进行持续观测并荧光标记,从而获得各类药物对对原发性多发性骨髓瘤治愈效果评估,为新药开发提供依据。
本发明一方面模型构建效率高,且生理特征稳定性好;另一方面可实现对模型中原发性多发性骨髓瘤基因序列全生命周期精确观测,且观测便捷、观测精度高,从而有效的提高对原发性多发性骨髓瘤疾病病情诊治、治疗及药物研发提供精确的依据。
附图说明
下面结合附图和具体实施方式来详细说明本发明;
图1为本发明方法流程图;
具体实施方式
为使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解,下面结合具体实施方式,进一步阐述本发明。
如图1所述的一种原发性多发性骨髓瘤小鼠模型构建方法,包括以下步骤:
S1,筛选样品基因组序列,首先随机选定从至少10名原发性多发性骨髓瘤患者组织样本,并分别放置在培养皿中培养3—72小时,然后对培养后的组织样本中分别进行DNA提取,最后对提取后的DNA样本进行基因序列测序,并筛选出个样本中的人原癌基因,并对筛选的各人原癌基因序列混合后进行存放备用;
S2,制备逆转病毒载录载体,在进行S1步骤同时,从选定的活体小鼠身上提取任意一种病毒,然后针对选定病毒制备并选用该类病毒为基础的逆转病毒载录载体;
S3,逆转病毒载录,完成S1和S2步骤后,将S1步骤制备得到人原癌基因序列同时与荧光分子标靶及一对引物混合,并通过聚合酶链式反应进行扩链,最后将扩链后的人原癌基因序列S2步骤制备的逆转病毒载录载体进行混合并,得到逆转病毒转录体,并对逆转病毒转录体进行荧光标靶检测,且含荧光标靶逆转病毒转录体量不小于逆转病毒转录体总量的98%后,即可进行后续作业;
S4,诱导转化,首先收集纯化的S2步骤同类活体小鼠IgM阳淋巴细胞,并利用化学诱导剂刺激分化小鼠IgM阳淋巴细胞,同时诱导Pre-B细胞凋亡;然后对小鼠IgM阳淋巴细胞进行培育生长分裂1—7天,然后将S3步骤制备得到逆转病毒转录体添加到小鼠IgM阳淋巴细胞进行培育基体中,并继续与小鼠IgM阳淋巴细胞进行培育1—10天;最后将S2步骤选定的活体小鼠进行γ射线辐照1—8小时,再将培育后的小鼠IgM阳淋巴细胞分别注射至活体小鼠的淋巴细胞及骨髓细胞中,并对活体小鼠在无菌环境下培育1—3天,即可得到原发性多发性骨髓瘤小鼠模型。
其中,所述的S2步骤中选定的活体小鼠鼠龄为1.5—3个月,且每组中均若干小鼠,且同组中个小鼠均来源同一母体同批次繁育所得。
同时,所述的S3步骤中,引物包括正向引物5'....CTTCGAAATTC3';反向引物5'....CCGATCGGGAGA3'。
一种原发性多发性骨髓瘤小鼠模型的应用,对原发性多发性骨髓瘤小鼠模型进行持续观测,通过荧光标靶对病毒在鼠体内扩散速度及路径进行持续观测并荧光标记。
一种原发性多发性骨髓瘤小鼠模型的应用,对原发性多发性骨髓瘤小鼠模型进行药物喂食,并通过荧光标靶对病毒在鼠体内扩散速度及路径进行持续观测并荧光标记,从而获得当前药物对原发性多发性骨髓瘤治愈效果评估。
一种原发性多发性骨髓瘤小鼠模型的应用,对原发性多发性骨髓瘤小鼠模型进行不同类型药物喂食,并通过荧光标靶对病毒在鼠体内扩散速度及路径进行持续观测并荧光标记,从而获得各类药物对对原发性多发性骨髓瘤治愈效果评估,为新药开发提供依据。
本发明一方面模型构建效率高,且生理特征稳定性好;另一方面可实现对模型中原发性多发性骨髓瘤基因序列全生命周期精确观测,且观测便捷、观测精度高,从而有效的提高对原发性多发性骨髓瘤疾病病情诊治、治疗及药物研发提供精确的依据。
以上显示和描述了本发明的基本原理和主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (6)
1.一种原发性多发性骨髓瘤小鼠模型构建方法,其特征在于:所述的一种原发性多发性骨髓瘤小鼠模型构建方法包括以下步骤:
S1,筛选样品基因组序列,首先随机选定从至少10名原发性多发性骨髓瘤患者组织样本,并分别放置在培养皿中培养3—72小时,然后对培养后的组织样本中分别进行DNA提取,最后对提取后的DNA样本进行基因序列测序,并筛选出个样本中的人原癌基因,并对筛选的各人原癌基因序列混合后进行存放备用;
S2,制备逆转病毒载录载体,在进行S1步骤同时,从选定的活体小鼠身上提取任意一种病毒,然后针对选定病毒制备并选用该类病毒为基础的逆转病毒载录载体;
S3,逆转病毒载录,完成S1和S2步骤后,将S1步骤制备得到人原癌基因序列同时与荧光分子标靶及一对引物混合,并通过聚合酶链式反应进行扩链,最后将扩链后的人原癌基因序列S2步骤制备的逆转病毒载录载体进行混合并,得到逆转病毒转录体,并对逆转病毒转录体进行荧光标靶检测,且含荧光标靶逆转病毒转录体量不小于逆转病毒转录体总量的98%后,即可进行后续作业;
S4,诱导转化,首先收集纯化的S2步骤同类活体小鼠IgM阳淋巴细胞,并利用化学诱导剂刺激分化小鼠IgM阳淋巴细胞,同时诱导Pre-B细胞凋亡;然后对小鼠IgM阳淋巴细胞进行培育生长分裂1—7天,然后将S3步骤制备得到逆转病毒转录体添加到小鼠IgM阳淋巴细胞进行培育基体中,并继续与小鼠IgM阳淋巴细胞进行培育1—10天;最后将S2步骤选定的活体小鼠进行γ射线辐照1—8小时,再将培育后的小鼠IgM阳淋巴细胞分别注射至活体小鼠的淋巴细胞及骨髓细胞中,并对活体小鼠在无菌环境下培育1—3天,即可得到原发性多发性骨髓瘤小鼠模型。
2.根据权利要求1所述的一种原发性多发性骨髓瘤小鼠模型构建方法,其特征在于:所述的S2步骤中选定的活体小鼠鼠龄为1.5—3个月,且每组中均若干小鼠,且同组中个小鼠均来源同一母体同批次繁育所得。
3.根据权利要求1所述的一种原发性多发性骨髓瘤小鼠模型构建方法,其特征在于:所述的S3步骤中,引物包括正向引物5'....CTTCGAAATTC3';反向引物5'....CCGATCGGGAGA3'。
4.根据权利要求1或3所述的一种原发性多发性骨髓瘤小鼠模型的应用,其特征在于:对原发性多发性骨髓瘤小鼠模型进行持续观测,通过荧光标靶对病毒在鼠体内扩散速度及路径进行持续观测并荧光标记。
5.根据权利要求1所述的一种原发性多发性骨髓瘤小鼠模型的应用,其特征在于:对原发性多发性骨髓瘤小鼠模型进行药物喂食,并通过荧光标靶对病毒在鼠体内扩散速度及路径进行持续观测并荧光标记,从而获得当前药物对原发性多发性骨髓瘤治愈效果评估。
6.根据权利要求1所述的一种原发性多发性骨髓瘤小鼠模型的应用,其特征在于:对原发性多发性骨髓瘤小鼠模型进行不同类型药物喂食,并通过荧光标靶对病毒在鼠体内扩散速度及路径进行持续观测并荧光标记,从而获得各类药物对对原发性多发性骨髓瘤治愈效果评估,为新药开发提供依据。
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