CN113549655B - Phf6△和jak3m511i双突变急性t淋巴细胞白血病小鼠模型构建方法和应用 - Google Patents
Phf6△和jak3m511i双突变急性t淋巴细胞白血病小鼠模型构建方法和应用 Download PDFInfo
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Abstract
本发明公开了一种PHF6△和JAK3M511I双突变急性T淋巴细胞白血病小鼠模型构建方法和应用。本发明利用Vav1‑Cre小鼠和Phf6fl/fl小鼠交配获得Vav1‑Cre；Phf6fl/Y(Phf6‑/Y)雄性小鼠；利用Mx1‑Cre小鼠和Phf6fl/fl交配获得Mx1‑Cre；Phf6fl/Y雄性小鼠，分别富集两种小鼠骨髓中Lin‑细胞，感染过表达GFP‑JAK3M511I的逆转录病毒，通过尾静脉注射，将GFP+细胞移植入致死剂量照射的受体小鼠，构建得到两种T‑ALL白血病小鼠模型。本申请模型可以快速在小鼠中传代，模拟PHF6基因和JAK3基因双突变的T‑ALL病人的白血病进程，用于靶向治疗药物筛选和疗效评价。
Description
技术领域
本发明涉及动物模型构建技术领域，具体涉及一种PHF6△和JAK3 M511I双突变急性T淋巴细胞白血病小鼠模型构建方法和应用。
背景技术
白血病(Leukemia)是一类造血干细胞恶性克隆性疾病，体现为造血干细胞的恶性转化及异常增殖并具有侵润其他组织的能力，以及造成骨髓中正常细胞的明显减少。急性T淋巴细胞白血病(T-ALL)作为白血病中的一大类型，起源于T淋巴细胞祖细胞。异常增生的原始淋巴细胞不仅在骨髓内聚集，同时可以浸润骨髓外组织，如脑膜、淋巴结、性腺、肝等。T-ALL 在儿童和成人白血病中都有较高的发生率，占儿童淋巴细胞白血病(ALL)的 10％–15％，占成人淋巴细胞白血病的25％。
经典的T淋巴细胞白血病致病机制可以概括为以下几个方面：1)持续的NOTCH1信号通路激活。2)调控细胞生长的重要分子发生激活型突变。例如，JAK分子突变导致的JAK-STAT信号通路异常活化。3)T细胞发育相关转录因子的异常表达。例如，TLX1，TLX3，TAL1，LMO2，HOXA 等。4)细胞周期调控异常。例如，CDKN2A基因缺失。5)表观遗传学修饰的重要调节分子发生突变，例如TET2突变导致的DNA甲基化修饰异常， PHF6突变导致的染色质结构改变等。目前，T淋巴细胞恶性转化的内在调控机制仍不清楚，针对T-ALL的经典信号通路的靶向药物，只能延缓T-ALL 进程。T-ALL患者5年生存率不足50％，小儿T-ALL患者死亡率达20％，而复发性成年T-ALL患者的死亡率高达90％。
在T-ALL发展研究中，应用小鼠动物模型作为主要研究对象揭示其内在机理是最常见的技术手段之一。常见的急性淋巴细胞白血病小鼠模型包括由NOTCH1诱导的单突变T-ALL小鼠模型，MYC诱导的T-ALL转基因小鼠模型，Ezh2缺失导致的ETP-ALL的小鼠模型以及由JAK3M511I引起的白血病小鼠模型等。但在临床病人中T-ALL的发生发展往往伴随着多种基因突变出现，并不是由单一的致病因素引起的。这类T-ALL患者其预后往往更差，对药物治疗更不敏感，由此可见，阐明T淋巴细胞恶性转化的内在分子机制对于为难治性T-ALL患者的精准治疗提供理论依据起着至关重要的作用。
PHF6基因位于X染色体Xq26–27，在脊椎动物中高度保守。PHF6 在血液系统恶性肿瘤中具有较高的突变频率，且常见于男性患者。在急性T 淋巴细胞白血病患者中，PHF6突变率高达30％，已有研究报道Phf6缺失 (PHF6△)虽然不是诱发白血病的独立因素，但可以加速NOTCH1/TLX3诱导的T-ALL发展。JAK-STAT信号通路主要参与细胞增殖、分化、凋亡以及免疫调节等重要的生物学过程，其过度激活引起正常造血细胞恶性转变，最终导致白血病发生。其中JAK3M511I型突变，在T-ALL中JAK3突变比例高达16.1％。通过多个独立中心的临床数据分析发现，PHF6与 JAK-STAT通路成员共突变事件在急性淋巴细胞白血病患者中高达2.65％-10.0％，其中PHF6和JAK3最常见的共突变基因，且这一部分病人生存期明显缩短。因此，研究JAK3和PHF6共突变所引起的T-ALL的内在致病机制，很可能为临床患者的治疗提供一定理论依据，提高临床患者的生活质量，达到更好的治疗效果。
发明内容
本发明为了解决上述技术问题，而提供一种PHF6△和JAK3 M511I双突变急性T淋巴细胞白血病小鼠模型构建方法和应用。
本发明是按照以下技术方案实现的：
一种PHF6△和JAK3 M511I双突变急性T淋巴细胞白血病小鼠模型的构建方法，包括以下步骤：
S1.使Vav1-Cre小鼠和Phf6fl/fl小鼠交配获得造血系统特异性敲除 Phf6的Vav1-Cre；Phf6fl/Y(Phf6-/Y)雄性小鼠；或使Mx1-Cre小鼠和Phf6fl/fl交配获得Mx1-Cre；Phf6fl/Y雄性小鼠；
S2.分别富集步骤S1获得的两种小鼠骨髓中Lin-细胞，并感染过表达 GFP-JAK3M511I的逆转录病毒，通过尾静脉注射，将两种GFP+细胞分别移植入半致死剂量照射的受体小鼠；
S3.移植后，每两周取小鼠外周血检测GFP+细胞占细胞群体的比例，检测GFP+细胞分化情况，在小鼠外周血白血病细胞比例达到80％-90％时，对小鼠进行相关表型数据检测，得到Vav1-Cre；Phf6fl/y+JAK3M511I(VC Phf6fl/y+JAK3M511I)小鼠和Mx1-Cre；Phf6fl/y+JAK3M511I(MCPhf6fl/y+JAK3M511I) 两种T-ALL白血病小鼠模型。
进一步的，步骤S2中，受体小鼠采用4.5Gy照射。
一种上述PHF6△和JAK3 M511I双突变急性T淋巴细胞白血病小鼠模型的构建方法构建得到的PHF6△和JAK3 M511I双突变急性T淋巴细胞白血病小鼠模型。
一种上述PHF6△和JAK3 M511I双突变急性T淋巴细胞白血病小鼠模型在靶向治疗药物筛选和疗效评价中的应用。
本发明具有的优点和有益效果是：
本发明弥补了现有小鼠模型的空缺，构建了两种模拟携带PHF6△和 JAK3M511I双突变T-ALL病人的急性淋巴细胞白血病小鼠模型，在构建的小鼠外周血及骨髓中可以检测到GFP+细胞明显成群且可稳定增殖，具备CD3 (T细胞标志)，CD8(T细胞标志)。本发明的小鼠构建模型方法，具有可连续传代，传代时间短，可模拟T-ALL患者等特点，可为T-ALL发生发展的内在调控机制研究及靶向治疗药物筛选和疗效评价，提供理论基础。
附图说明
图1是本发明VCPhf6fl/y和Mx1-Cre；Phf6fl/y小鼠构建图谱；
图2是本发明每隔2周连续检测构建得到的VCPhf6fl/y+JAK3M511I小鼠外周血GFP+细胞变化情况图(A.Vav1-Cre；Phf6fl/y+JAK3M511I小鼠构建图谱；B. VCPhf6fl/y+JAK3M511I小鼠生存期曲线；C.VCPhf6fl/y+JAK3M511I小鼠外周血 GFP+细胞变化情况)；
图3是本发明在小鼠发病末期检测构建得到的VCPhf6fl/y+JAK3M511I小鼠外周血、骨髓及脾脏等器官的GFP+细胞比例及分化情况图(A.Vav1-Cre； Phf6fl/y+JAK3M511I小鼠和Phf6 WT+JAK3M511I小鼠外周血血常规变化情况及血涂片；B-C.Vav1-Cre；Phf6fl/y+JAK3M511I小鼠和Phf6 WT+JAK3M511I小鼠各器官GFP+细胞比例及HE染色浸润情况；D-E.Vav1-Cre；Phf6fl/y+JAK3M511I小鼠和Phf6 WT+JAK3M511I小鼠骨髓中GFP+细胞比例及三系分化情况)；
图4是本发明在第21天确认构建得到的Mx1-Cre；Phf6fl/y+JAK3M511I小鼠发展成为T-ALL后，注射PIPC，在后期WB检测Phf6敲除情况图 (A.Mx1-Cre；Phf6fl/y+JAK3M511I小鼠构建图谱；B.MCPhf6fl/y+JAK3M511I小鼠和Phf6 WT+JAK3M511I小鼠21天检测外周血GFP+比例及分化情况；C.MC Phf6fl/y+JAK3M511I小鼠PIPC注射后骨髓细胞Western Blot检测phf6敲除情况及小鼠生存期曲线)；
图5是本发明在小鼠发病末期检测构建得到的 Mx1-Cre；Phf6fl/y+JAK3M511I小鼠外周血、骨髓及脾脏等器官的GFP+细胞比例及分化情况图(A.MCPhf6fl/y+JAK3M511I小鼠和Phf6WT+JAK3M511I小鼠外周血血常规变化情况；B.MCPhf6fl/y+JAK3M511I小鼠和Phf6 WT+JAK3M511I小鼠外周血和骨髓GFP+细胞比例；C.MCPhf6fl/y+JAK3M511I小鼠和Phf6 WT+JAK3M511I小鼠骨髓中GFP+细胞比例及三系分化情况；D-E.MC Phf6fl/y+JAK3M511I小鼠和Phf6 WT+JAK3M511I小鼠各器官GFP+细胞比例及重量)。
具体实施方式
下面结合附图及实施例对本发明进行详细的说明。
实施例1
构建VCPhf6fl/y和Mx1-Cre；Phf6fl/y小鼠
Cre-lox重组系统包括Cre重组酶和Lox位点两个要素:Cre重组酶能特异识别并催化两个Lox位点之间的片段进行重组。Lox位点是一段包含8个核心核苷酸片段、全长为34个核苷酸的序列。Lox位点的方向和位置决定了Cre 酶的功能效应，包括染色体DNA的敲除、倒位或易位。Cre重组酶基因和Lox 位点并不存在于小鼠的基因组中，因此通常需要利用转基因的方式将Cre基因以及Lox位点整合入小鼠胚胎干细胞中并繁育出相应的转基因小鼠。
Vav是Rho/Rac/CDC42家族的一员，作为GTP/GDP转换因子在调节G 蛋白方面发挥重要作用，同时它还是淋巴细胞抗原提呈必须的信号分子。内源性Vav在成体或胚胎中所有造血细胞各个生长阶段特异性表达，所以利用 Vav调控的转录起始序列启动内切酶Cre表达(Vav-Cre)常被用在特定基因对造血系统功能的研究中。
除组织特异的Cre转基因小鼠外，还有可对Cre进行诱导表达的Mx1-Cre 小鼠，Mx1-Cre小鼠在一般状态下不表达Cre重组酶，但可以通过干扰素-α、干扰素-β或人工合成的双链RNA类似物poly I:C(PIPC)的处理而诱导表达 Cre。利用Mx1-Cre虽然可用于在发育过程中的任意时间点诱导目的基因敲除小鼠，因此也被广泛的用于造血系统中，尤其是针对在造血系统中敲除引起胚胎致死的基因的研究中。综上所述，根据上述理论背景，构建了如下小鼠：
1.利用Vav1-Cre小鼠和Phf6fl/fl小鼠交配获得造血系统特异性敲除 Phf6的Vav1-Cre；Phf6fl/Y(Phf6-/Y)雄性小鼠；
将6-12周雌性纯合的Phf6fl/fl小鼠1-2只和1只6-12周的Vav1-Cre雄鼠合笼交配，通过PCR的方式鉴定子代鼠中loxp位点和Vav-1，确认小鼠基因型筛选得到Vav1-Cre；Phf6fl/Y(Phf6-/Y)雄性小鼠。因为子代鼠中血液细胞 Vav调控的转录起始序列启动内切酶Cre的表达，Cre识别loxp位点发挥作用，Phf6基因4-5号外显子被切割从而实现造血系统特异性敲除基因获得目的小鼠。
2.利用Mx1-Cre小鼠和Phf6fl/fl交配获得Mx1-Cre；Phf6fl/Y雄性小鼠。
将6-12周雌性纯合的Phf6fl/fl小鼠1-2只和1只6-12周的Mx1-Cre雄鼠合笼交配，通过PCR的方式鉴定子代鼠中loxp位点和Mx1，确认小鼠基因型筛选得到Mx1-Cre；Phf6fl/Y雄性小鼠。小分子PIPC注入小鼠体内，可以诱导Cre的表达，Cre识别loxp位点发挥作用，Phf6基因4-5号外显子被切割从而实现敲除Phf6基因获得目的小鼠。子代鼠未经PIPC诱导敲除，正常表达Phf6基因。
实验结果参见图1，在WT小鼠phf6基因的第四号和五号外显子两端插入两个同向的loxp位点和neo筛选基因获得第一代小鼠，将此代小鼠与全身表达flp的小鼠交配去掉抗性基因neo得到第二代小鼠，再将第二代小鼠与 Cre小鼠交配，cre发挥作用，将4/5号外显子以及前端loxp切割掉导致phf6 基因遭到破坏，不能够完整翻译，从而得到phf6敲除的目的小鼠VCPhf6fl/y。
实施例2
构建VCPhf6fl/y+JAK3M511I小鼠和Phf6 WT+JAK3M511I小鼠
处死phf6lox/Y和Vav1-Cre；phf6lox/Y小鼠后取股骨、胫骨和髂骨，去除骨髓周围的肌肉及脂肪组织，使用1ml注射器吸取PBE(含2％胎牛血清的磷酸盐缓冲液)冲取骨髓细胞，然后通过生物素磁珠富集小鼠的Lin-细胞，将其铺在用重组人纤维连接片断(Retronectin)处理过的24孔板中，加入细胞因子mSCF、TPO和Flt3预刺激8-12小时后，进一步感染由293T产生的 JAK3M511I的逆转录病毒，48小时后使用荧光显微镜观察，后收集感染的细胞用流式细胞仪检测GFP+细胞比例。将受体小鼠进行致死剂量照射(9Gy) 清除骨髓细胞，分两次照射，每次间隔4小时，在第二次照射四小时后将感染JAK3M511I病毒的Lin-细胞通过尾静脉注射移植入致死剂量照射的受体小鼠，移植后每间隔7天通过流式细胞仪检测一次受体小鼠外周血中GFP+细胞比例，诱发小鼠白血病，从而成功构建VCPhf6fl/y+JAK3M511I小鼠和Phf6 WT+JAK3M511I第一代白血病小鼠模型，后续用于Phf6缺失协同Jak3M511I促进T-ALL疾病发展的作用研究。通过获取发病小鼠骨髓中的的白血病起始细胞，并通过流式分选仪器分选GFP+细胞，按照3-5×105GFP+/只小鼠通过尾静脉注射的方式移植到半致死剂量照射(4.5Gy)的小鼠体内，连续移植2-3代，用于后续研究。
实验结果参见图2，分别是VC Phf6fl/y+JAK3M511I小鼠和Phf6 WT+JAK3M511I白血病构建流程示意图(A)，长周期检测白血病小鼠发病情况后所收集的生存期曲线(B)以及移植后两组小鼠每隔7天检测外周血GFP+细胞比例的增长曲线(C)。以上实验结果表明，在造血起始阶段，血液细胞中特异性敲除Phf6可以明显缩短JAK3M511诱导的急性淋巴细胞白血病小鼠的生存时间。
实施例3
测定VCPhf6fl/y+JAK3M511I小鼠外周血骨髓及各器官中GFP+细胞比例及分化情况
血细胞计数：尾静脉采集20μL外周血于加有180μL稀释液的肝素抗凝管内，利用血细胞计数仪器对VC Phf6fl/y+JAK3M511I小鼠和Phf6 WT+JAK3M511I小鼠外周血细胞进行分析。主要检测红细胞数，血红蛋白水平，红细胞压积，平均红细胞体积，平均红细胞血红蛋白浓度，白细胞和血小板数目。
形态学和组织学观察小鼠骨髓、脾和外周血变化：尾静脉采集小鼠外周血，制备血涂片，瑞氏-吉姆萨染色后，显微镜下观察血细胞的形态。每四周检测一次。小鼠处死当日取外周血和骨髓细胞制备血涂片，瑞氏-吉姆萨染色观察形态变化。对于各器官的病理检测，分别取2组小鼠的骨髓，脾脏，胸腺等，4％多聚甲醛固定，石蜡包埋后切片，分别利用苏木精-伊红(HE) 染色法对组织切片染色，显微镜下观察组织的形态变化和纤维化程度。
小鼠的生存情况的检测：根据Kaplan-Meier分析和log-rank test统计2 组小鼠的生存情况；同时观察疾病的发展过程，每个月分析，记录小鼠的表型，根据Bethesdaproposals标准判断其疾病的类型，绘制疾病与时间轴的模型图。
获取VCPhf6fl/y+JAK3M511I小鼠和Phf6 WT+JAK3M511I小鼠全骨髓细胞以及外周血、脾脏、肝脏、肺以及胸腺细胞，根据免疫表型(白血病细胞： GFP+白血病起始细胞LICs：GFP+CD4–CD8–CD25+CD127+)，运用流式分析技术分析细胞群体的比例。标记CD4,CD8，B220，Mac-1,Gr-1分析白细胞各类细胞比例。
实验结果参见图3，分别是两组小鼠外周血血常规(白细胞/中性粒细胞 /淋巴细胞)细胞比例及血细胞相应形态(A),两组白血病小鼠相应器官(脾脏/肝脏/胸腺)的GFP+细胞比例及各个器官病理检测的白血病细胞侵润情况 (B-C),以及两组小鼠骨髓细胞形态，GFP+细胞比例，TBM各系细胞的比例及流式检测分群情况(D-E)。以上实验结果表明，在造血起始阶段，血液细胞中特异性敲除Phf6可以加速JAK3M511诱导的急性淋巴细胞白血病小鼠的发病进展。
实施例4
构建MCPhf6fl/y+JAK3M511I小鼠和Phf6 WT+JAK3M511I小鼠
处死phf6lox/Y和MX1-Cre；phf6lox/Y小鼠骨髓细胞后取股骨、胫骨和髂骨，去除骨髓周围的肌肉及脂肪组织，使用1ml注射器吸取PBE(含2％胎牛血清的磷酸盐缓冲液)冲取骨髓细胞，然后通过生物素(Biotin)磁珠富集小鼠的Lin-细胞，将其铺在用重组人纤维连接片断(Retronectin)处理过的24 孔板中，加入细胞因子mSCF、TPO和Flt3预刺激8-12小时后,进一步感染由293T产生的JAK3M511I的逆转录病毒，48小时后使用荧光显微镜观察后收集感染的细胞用流式细胞仪检测GFP+细胞比例。将受体小鼠进行致死剂量照射(9Gy)清除骨髓细胞，分两次照射，每次间隔4小时，在第二次照射四小时后将感染JAK3M511I病毒的Lin-细胞通过尾静脉注射移植入致死剂量照射的受体小鼠，诱发小鼠白血病，构建MCPhf6fl/y+JAK3M511I小鼠和Phf6 WT+JAK3M511I白血病小鼠模型。在移植后21天检测小鼠外周血GFP+细胞变化情况，当GFP+比例达到20％左右并且三系分化偏向T细胞分化时，按照 10mg/kg小鼠体重注射PIPC，每间隔一天注射一次，连续注射三次，在注射后一个月通过WB检测小鼠骨髓细胞中Phf6的表达情况。
实验结果参见图4，分别是phf6lox/Y和MX1-Cre；phf6lox/Y白血病小鼠构建流程示意图(A),小鼠移植21天后外周血GFP+细胞及T细胞比例及流式分群情况(B)，注射PIPC一个月后小鼠骨髓细胞Western blot检测phf6 蛋白表达确认其敲除，以及长周期观察后两组小鼠生存曲线(C)。以上实验结果表明，T-ALL疾病发展中期敲除Phf6同样可以明显加速JAK3M511急性淋巴细胞白血病小鼠的疾病进程。
实施例5
测定MCPhf6fl/y+JAK3M511I小鼠外周血骨髓及各器官中GFP+细胞比例及分化情况
血细胞计数：尾静脉采集20μL外周血于加有180μL稀释液的肝素抗凝管内，利用血细胞计数仪器对MC Phf6fl/y+JAK3M511I小鼠和Phf6 WT+JAK3M511I小鼠外周血细胞进行分析。主要检测红细胞数，血红蛋白水平，红细胞压积，平均红细胞体积，平均红细胞血红蛋白浓度，白细胞和血小板数目。
形态学和组织学观察小鼠骨髓、脾和外周血变化：尾静脉采集小鼠外周血，制备血涂片，瑞氏-吉姆萨染色后，显微镜下观察血细胞的形态。每四周检测一次。小鼠处死当日取外周血和骨髓细胞制备血涂片，瑞氏-吉姆萨染色观察形态变化。对于各器官的病理检测，分别取2组小鼠的骨髓，脾脏，胸腺等，4％多聚甲醛固定，石蜡包埋后切片，分别利用苏木精-伊红(HE) 染色法对组织切片染色，显微镜下观察组织的形态变化和纤维化程度。
小鼠的生存情况的检测：根据Kaplan-Meier分析和log-rank test统计2 组小鼠的生存情况；同时观察疾病的发展过程，每个月分析，记录小鼠的表型，根据Bethesdaproposals标准判断其疾病的类型，绘制疾病与时间轴的模型图。
获取MCPhf6fl/y+JAK3M511I小鼠和Phf6 WT+JAK3M511I小鼠全骨髓细胞以及外周血、脾脏、肝脏、肺以及胸腺细胞，根据免疫表型(白血病细胞：GFP+白血病起始细胞LICs：GFP+CD4–CD8–CD25+CD127+)，运用流式分析技术分析细胞群体的比例。标记CD4,CD8，B220，Mac-1，Gr-1分析白细胞各类细胞比例。
实验结果参见图5，分别是MC Phf6fl/y+JAK3M511I小鼠和Phf6 WT+JAK3M511I小鼠外周血血常规(白细胞/淋巴细胞/血小板)细胞比例(A), 两组白血病小鼠外周血和骨髓的GFP+细胞比例(B),两组小鼠骨髓GFP+细胞和TBM三系细胞的流式检测分群图及细胞比例(C)，以及两组小鼠骨髓外周各器官(脾脏/肝脏/胸腺)的GFP+比例及重量(D-E)。以上实验结果表明，T-ALL疾病发展中期敲除Phf6加速JAK3M511诱导的急性淋巴细胞白血病小鼠的发病进展。
Claims (3)
1.一种PHF6 △ 和JAK3 M511I 双突变急性T淋巴细胞白血病小鼠模型的构建方法，其特征在于：包括以下步骤：
S1. 使Vav1-Cre 小鼠和 Phf6 fl/fl小鼠交配获得造血系统特异性敲除 Phf6 的Vav1- Cre; Phf6 fl/Y 雄性小鼠；或使Mx1-Cre小鼠和Phf6 fl/fl 交配获得Mx1-Cre; Phf6 fl/Y 雄性小鼠；
S2.富集步骤S1获得的小鼠骨髓中 Lin- 细胞，并感染过表达GFP-JAK3 M511I 的逆转录病毒，通过尾静脉注射，将GFP+细胞移植入半致死剂量照射的受体小鼠；
S3. 移植后，每两周取小鼠外周血检测GFP+细胞占细胞群体的比例，检测GFP+细胞分化情况，在小鼠外周血白血病细胞比例达到80%-90%时，对小鼠进行相关表型数据检测，得到PHF6 △ 和JAK3 M511I 双突变急性T淋巴细胞白血病小鼠模型。
2.根据权利要求1所述的一种PHF6 △ 和JAK3 M511I 双突变急性T淋巴细胞白血病小鼠模型的构建方法，其特征在于：步骤S2中，受体小鼠采用4.5Gy 照射。
3.一种权利要求1或2所述构建方法构建得到的PHF6 △ 和JAK3 M511I 双突变急性T淋巴细胞白血病小鼠模型在靶向治疗药物筛选中的应用。
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