CN106172212A - Ccm3基因敲除小鼠模型的建立方法及用途 - Google Patents
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Abstract
本发明公开了CCM3基因敲除小鼠模型的建立方法，包括：通过交配CCM3lox/lox小鼠与他莫昔芬诱导Cdh5CreERT2的敲除小鼠，获得他莫昔芬诱导的血管内皮细胞特异性的CCM3基因敲除小鼠模型。本发明的小鼠模型经过他莫昔芬诱导后具有人脑海绵状血管畸形的病理特点，非常适合用于人脑海绵状血管畸形发病机制研究和开发相应的治疗药物和治疗方法，也可用于药物的药效观察和药效评价。
Description
技术领域
本发明属于生物医药技术领域，尤其涉及CCM3基因敲除小鼠模型的建立方法及用途。
背景技术
脑海绵状血管畸形(cerebral carvernoμs malformation,CCM)是发生在中枢神经系统的一种血管错构畸形，是先天性脑血管畸形的一种,约占脑血管畸形的10％～20％,人群患病率约0.1％～4％。CCM，外观紫红色,剖面呈海绵状或蜂窝状,由不规则的、大小不等的血管腔隙聚集成海绵状异常血管团,管腔1～10mm不等,内壁为一层扁平内皮细胞,血管腔隙之间无神经组织,只有少量结缔组织。肉眼下CCM是一团堆积的薄壁血管腔,呈桑葚状,边界清楚,可见含铁血黄素沉着。镜下可见其由许多不规则,异常扩大的窦状血管间隙和玻璃样胶原基质构成,无平滑肌及弹力纤维,血管腔内面附有单层扁平内皮细胞,无基膜,血管间仅少量结缔组织,病灶内反复出血,血管腔机化血栓,钙化及血管再通,周围有含铁血黄素沉积及胶质增生。大多数CCM累及中枢神经系统,但也可见于视网膜、皮肤等其它器官。该病临床症状隐袭,主要表现为头痛、痫性发作、颅内出血和局部神经功能障碍。
CCM分为散发性和常染色体显性遗传,遗传性CCM与3个致病基因种系突变相关,包括CCM1(KRIT1,7q11.2-q21)、CCM2(MGC4607、7p15-p13)和CCM3(PDCD10、3q25.2-q27)。90％以上的CCM病人携带这3个致病基因的突变。近十几年来,3个CCM致病基因种系突变的研究报道非常多。由于CCM病灶组织内体细胞基因突变的发现提出了“二次打击”机制(two-hitmechanism)学说,认为其参与了脑海绵状血管瘤的发病机制,导致表达于脑海绵状血管瘤毛细血管腔内皮细胞的致病基因编码蛋白完全失去功能。这种学说最近已经在小鼠动物模型中被证实，虽然CCM发病机制尚不清楚,但分子遗传学研究为我们深入理解CCM的发病机制和临床治疗提供了重要依据。鉴于目前缺乏有效的治疗药物，手术切除是CCM的唯一治疗手段，因此，研究CCM的发病机制和寻找新的有效药物或治疗方法是当前的研究热点。
研究CCM的发病机制和探索有效的治疗药物或方法都需要建立一个合适的实验动物模型。CCM1，CCM2，CCM3是目前已发现的CCM致病基因，但是内皮细胞特异性敲除CCM1，CCM2，CCM3的小鼠由于血管发育异常往往导致胚胎致死，无法用于CCM发病机制和治疗方法或药物的研究。
发明内容
一方面，本发明的目的在于克服现有技术存在的不足之处而提供了CCM3基因敲除小鼠模型的建立方法，本发明的小鼠模型经过他莫昔芬诱导后具有人脑海绵状血管畸形的病理特点，非常适合用于人脑海绵状血管畸形发病机制研究和开发相应的治疗药物和治疗方法，也可用于药物的药效观察和药效评价。
本发明采用的技术方案为：一种CCM3基因敲除小鼠模型的建立方法，包括：通过交配CCM3lox/lox小鼠与他莫昔芬诱导Cdh5CreERT2的敲除小鼠，获得他莫昔芬诱导的血管内皮细胞特异性的CCM3基因敲除小鼠模型。
作为对上述技术方案的进一步改进，所述方法还包括以下步骤：所述他莫昔芬诱导的血管内皮细胞特异性的CCM3基因敲除小鼠出生后，给小鼠喂养他莫昔芬，该小鼠即发展为具有CCM病变表型的CCM3基因敲除小鼠模型(Ccm3lox/lox:Cdh5-CreERT2，简称为Ccm3-iecKO)。
作为对上述技术方案的进一步改进，他莫昔芬诱导的血管内皮细胞特异性的CCM3基因敲除小鼠出生后1至3天，给小鼠喂养他莫昔芬。
作为对上述技术方案的更进一步改进，按每克小鼠体重10μg的剂量给小鼠喂养他莫昔芬。选择此剂量是为了达到最好的敲除效果，且此剂量的它莫西芬本身对正常鼠的体重和发育等不造成影响。
作为对上述技术方案的更进一步改进，给小鼠喂养他莫昔芬采用以下步骤实施：用玉米油把它莫西芬配成浓度为10mg/mL的液体给小鼠喂养。
另一方面，本发明还提供了所述的CCM3基因敲除小鼠模型作为脑海绵状血管瘤发病机制、治疗方法或药物研究中的小鼠模型的用途。
相对于现有技术，本发明的有益效果为：
本发明建立的CCM3基因敲除小鼠模型采用他莫昔芬诱导的形式，克服了基因敲除CCM致病基因导致的胚胎致死问题；经过他莫昔芬诱导，本模型在出生后第五天即可在小脑观察到膨大的毛细血管，第10天可观察到明显的血管损伤，通过分析大脑组织神经血管单元、血管完整性和血脑屏障(BBB)功能的病理性改变，证实该小鼠模型与临床表型高度一致。因此，这个模型具有人脑海绵状血管畸形的病理特点，非常适合用于人脑海绵状血管畸形发病机制研究和开发相应的治疗药物和治疗方法，也可用于药物的药效观察和药效评价。
附图说明
图1显示了诱导型的血管内皮细胞特异性CCM3基因敲除小鼠快速出现脑和视网膜CCM病变表型；野生型(WT)和诱导型的血管内皮细胞特异性CCM3基因敲除(Ccm3-iecKO)小鼠在出生后1-3天喂他莫昔芬，在P2-P10天取脑组织进行检测；其中，A显示了P2，P5，P10天的WT和Ccm3-iecKO小鼠脑组织切片HE染色结果；B显示了P2，P5，P10天的WT和Ccm3-iecKO小鼠脑组织总CCM病灶数量统计结果；C显示了P2，P5，P10天Ccm3-iecKO小鼠脑组织CCM不同大小病灶数量统计结果。
图2显示了CCM病变部位血管渗漏；其中，A显示了P10天的WT和Ccm3-iecKO小鼠新鲜脑组织；B-D显示了P10天的小鼠用FITC-葡聚糖灌注后进行免疫荧光检测结果，B为脑组织荧光图，箭头指示血管渗漏；C为CD31染色图，箭头表示正常脑组织结构，星号*显示CCM病灶；D为伊文思蓝白蛋白检测评价小鼠血脑屏障渗透性。
图3显示了Ccm3-iecKO小鼠视网膜病变；对P10天的WT和Ccm3-iecKO小鼠视网膜组织的CD31和isolectin蛋白进行免疫荧光染色，放大倍数A为20X，B为80X，星号*显示CCM病变部位呈海绵状异常血管团，C显示了损害区域占比。
具体实施方式
为更好的说明本发明的目的、技术方案和优点，下面将结合具体实施例对本发明作进一步说明。实施例中未进行详细描述的方法均可通过常规试验操作方法实施。
它莫西芬(tamoxifen)，也即他莫昔芬；Ccm3与CCM3可替换使用；Cdh5-CreERT2转基因小鼠与他莫昔芬诱导Cdh5CreERT2的敲除小鼠可替换使用；Px天也即第x天。
1、材料与方法
1.1它莫西芬诱导的血管内皮细胞特异性的Ccm3基因敲除鼠(Ccm3-iecKO)模型
为了便于通过Cre-LoxP系统研究Ccm3在血管内皮细胞中的功能，我们使用Cdh5-CreERT2转基因小鼠。Cdh5-CreERT2转基因技术原理是，首先将雌激素受体(ER)的配体结合域(LBD)突变，进而使其与Cre重组酶融合产生一个嵌合重组酶(Cre-ER)，该酶活性依赖于它莫西芬的存在，而不受体内雌激素的影响。通过利用Cdh5基因的启动子驱动Cre重组酶基因在血管内皮细胞中的表达。该转基因小鼠若服用它莫西芬，Cre重组酶的活性被激活，即可导致Cre介导的重组在预定时间内和特定的组织细胞类型(这里是指血管内皮细胞)上发生。Cdh5-CreERT2转基因小鼠的获取也可参考已报道的文献(可参见：Wang,Y.,etal.Ephrin-B2controls VEGF-indμced angiogenesis and lymphangiogenesis.Natμre.2010May 27；465(7297):483-6.doi:10.1038/natμre09002.)。Ccm3fl/fl小鼠(也即CCM3lox/lox小鼠，其获取可参见：He Y,et al.Stabilization of VEGFR2signalingbycerebral cavernoμs malformation 3is critical for vascμlar development.Sci Signal.2010Apr 6；3(116):ra26.doi:10.1126/scisignal.2000722.)与Cdh5-CreERT2转基因小鼠交配，即得到可诱导的血管内皮细胞特异性的Ccm3基因敲除鼠(Ccm3-iecKO)。为了在小鼠体内用它莫西芬诱导Ccm3基因的敲除，我们用玉米油把它莫西芬(Sigma,T5648)配成浓度为10mg/mL的液体，给刚出生的Ccm3-iecKO和WT(Ccm3fl/fl)幼鼠从P1至P3每天喂10μg/g(体重)的它莫西芬一次，连续三天。
1.2异硫氰酸荧光素葡聚糖(FITC-dextran)灌注和伊文思蓝(Evan Blμe)染料渗
透试验
异硫氰酸荧光素葡聚糖(FD2000S；Sigma-Aldrich公司,St.Loμis,MO,美国)溶于无菌的生理盐水，离心10,000g 5分钟，调整浓度为50mg/mL，收集上清，避光。同胎出生的WT或Ccm3-iecKO小鼠经ANGPT2中和抗体处理或不经ANGPT2中和抗体处理均称重，并腹部注射麻醉((氯胺酮100mg/kg+甲苯噻嗪10mg/kg))。眼球后注射按照已发表的文献进行。注射部位选定为左眼角外眦。用3/10mL胰岛素注射器(31G针头)轻轻的沿45°角刺入小鼠的眼眶静脉窦。以10μl异硫氰酸荧光素葡聚糖对应1g体重来根据体重换算相应异硫氰酸荧光素葡聚糖注射量，并注射入小鼠眼眶静脉窦。5分钟后，收集脑和视网膜，并进行组织铺片或切片免疫染色处理。
伊文思蓝染料渗透性实验(Miles实验)。伊文思蓝染液(用0.9％NaCl溶液配成1％伊文思蓝溶液；Sigma-Aldrich)注射入已麻醉的WT和Ccm3-iecKO小鼠眼球后血管丛，注射量为10μl/g体重。注射30分钟后，杀死小鼠并往左心室灌注PBS，以清洗血管内的染料。收集小脑组织，并在60℃中干燥过夜，在伊文思蓝染料取出前称重，用1mL的甲酰胺55℃下孵育小脑组织16小时以取出伊文思蓝染料。用分光光度计(激发光波长为630nm)来检测伊文思蓝的含量。
1.3脑组织切片HE染色及总CCM病灶数量统计
收集小鼠脑组织，用4％多聚甲醛固定1天，常规梯度酒精脱水，二甲苯透明，浸蜡，脑组织包埋。矢状面50μm连续切片,60℃烤片2小时。将烤好的组织切片脱蜡至水，Harry’s苏木素染色8分钟，流水冲洗1分钟，1％盐酸酒精分化4秒钟，流水冲洗20分钟，伊红染色1分钟，流水冲洗，酒精脱水，干燥，二甲苯透明，封片，荧光显微镜的普通明场状态下观察、照相，并采用ImageJ软件计算总CCM病灶数量。
2.结论
诱导型的血管内皮细胞特异性Ccm3敲除的小鼠产生CCM损害
给刚出生的WT(Ccm3fl/fl)幼鼠和可诱导的血管内皮细胞特异性的Ccm3基因敲除(Ccm3-iecKO)小鼠从P1至P3每天喂他莫昔芬，在P2-P10取脑组织进行检测。结果发现，它莫西芬诱导的血管内皮细胞特异性Ccm3敲除的小鼠(Ccm3-iecKO)能在小脑快速产生CCM血管损害，即扩张膨胀伴出血的毛细血管，该病损在P5可见、P10急剧增大增多(如图1A -C所示)。没有Ccm3-iecKO能存活超过P15(n>50)。FITC-dextran小鼠体内循环灌注实验显示，FITC-dextran局限于WT小鼠脑和视网膜的毛细血管床内，但在Ccm3ECKO小鼠FITC-dextran弥散渗漏到小脑扩张的毛细血管床外的周边组织(如图2A –C所示)。Evans Blμe染料渗漏实验也证实了Ccm3-iecKO小鼠小脑增强的血管渗漏(如图2D所示)。CCM样静脉血管畸形也在Ccm3-iecKO小鼠视网膜周边血管丛被检测到(如图3A –C所示)。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
Claims (6)
1.一种CCM3基因敲除小鼠模型的建立方法，其特征在于：包括：通过交配CCM3lox/lox小鼠与他莫昔芬诱导Cdh5CreERT2的敲除小鼠，获得他莫昔芬诱导的血管内皮细胞特异性的CCM3基因敲除小鼠模型。
2.根据权利要求1所述的方法，其特征在于：所述方法还包括以下步骤：所述他莫昔芬诱导的血管内皮细胞特异性的CCM3基因敲除小鼠出生后，给小鼠喂养他莫昔芬，该小鼠即发展为具有CCM病变表型的CCM3基因敲除小鼠模型。
3.根据权利要求2所述的方法，其特征在于：所述他莫昔芬诱导的血管内皮细胞特异性的CCM3基因敲除小鼠出生后1至3天，给小鼠喂养他莫昔芬。
4.根据权利要求2所述的方法，其特征在于：按每克小鼠体重10μg的剂量给小鼠喂养他莫昔芬。
5.根据权利要求2所述的方法，其特征在于：给小鼠喂养他莫昔芬采用以下步骤实施：用玉米油把它莫西芬配成浓度为10mg/mL的液体给小鼠喂养。
6.根据权利要求1～5中任一项所述的CCM3基因敲除小鼠模型作为脑海绵状血管瘤发病机制、治疗方法或药物研究中的小鼠模型的用途。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110622921A (zh) * | 2019-09-29 | 2019-12-31 | 镇江杰胜瑞科技有限公司 | 阿尔茨海默病病变区FoxG1过表达小鼠模型的构建方法及应用 |
| CN110692594A (zh) * | 2019-10-12 | 2020-01-17 | 上海交通大学 | 一种人血管瘤动物模型及其高成功率构建方法 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011217667A (ja) * | 2010-04-09 | 2011-11-04 | Kanazawa Univ | 非ヒトノックアウト動物、並びにその用途およびその作製方法 |
| CN102492686A (zh) * | 2011-12-12 | 2012-06-13 | 湖南师范大学 | Kctd10基因剔除小鼠模型、构建方法及其应用 |
| WO2012149342A1 (en) * | 2011-04-27 | 2012-11-01 | Georgia Health Sciences University | Mouse model for avm |
| CN105296600A (zh) * | 2014-05-26 | 2016-02-03 | 中国科学院上海生命科学研究院 | Med23基因的用途及其相关药物 |
| CN105664179A (zh) * | 2016-01-12 | 2016-06-15 | 中国人民解放军第二军医大学 | Phf14基因敲除合并急性肾损伤及损伤后肾纤维化动物模型的建立方法 |
-
2016
- 2016-07-25 CN CN201610589393.3A patent/CN106172212A/zh not_active Withdrawn
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011217667A (ja) * | 2010-04-09 | 2011-11-04 | Kanazawa Univ | 非ヒトノックアウト動物、並びにその用途およびその作製方法 |
| WO2012149342A1 (en) * | 2011-04-27 | 2012-11-01 | Georgia Health Sciences University | Mouse model for avm |
| CN102492686A (zh) * | 2011-12-12 | 2012-06-13 | 湖南师范大学 | Kctd10基因剔除小鼠模型、构建方法及其应用 |
| CN105296600A (zh) * | 2014-05-26 | 2016-02-03 | 中国科学院上海生命科学研究院 | Med23基因的用途及其相关药物 |
| CN105664179A (zh) * | 2016-01-12 | 2016-06-15 | 中国人民解放军第二军医大学 | Phf14基因敲除合并急性肾损伤及损伤后肾纤维化动物模型的建立方法 |
Non-Patent Citations (1)
| Title |
|---|
| LUCA BRAVI 等: "Sulindac metabolites decrease cerebrovascular malformations in CCM3-knockout mice", 《PNAS》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110622921A (zh) * | 2019-09-29 | 2019-12-31 | 镇江杰胜瑞科技有限公司 | 阿尔茨海默病病变区FoxG1过表达小鼠模型的构建方法及应用 |
| CN110692594A (zh) * | 2019-10-12 | 2020-01-17 | 上海交通大学 | 一种人血管瘤动物模型及其高成功率构建方法 |
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