CRISPR/Cas9技术构建的ITGA2B c.2659C>T(p.Q887X)无义突变的血小板无力症小鼠模型对血小板功能的影响  

Effects of the ITGA2B Nonsense Mutation(c.2659C > T, p.Q887X) on Platelet Function in a Mouse Model of Glanzmann’s Thrombasthenia Generated with CRISPR/Cas9 Technology

作　　者：杨飞 江淼[2] 林赠华[1] 谢展利 马珍妮[2] 杨力[1] 刘红[1] 王兆钺[2] 周鹭[1] YANG Fei;JIANG Miao;LIN Zeng-Hua;XIE Zhan-Li;MA Zhen-Ni;YANG Li;LIU Hong;WANG Zhao-Yue;ZHOU Lu(Departmen of Hematology Affiliated Hospital of Nantong University,Naniong 226001 Jiangsu Province,China;Key Laboratory of Thrombosis&Hemostasis of Ministry of Health Jiangsu Institute of Hematology,The First Affiliated Hospital of Soochow University Suzhou 215006,Jiangsu Province,China;Institute of Clinical Medicine Research,Suzhou Science and Technology Town Hospital Affiliated to Nanjing Medical University uzhou 215001 Jiangsu Province,China)

机构地区：[1]南通大学附属医院血液内科,江苏南通226001 [2]卫生部血栓与止血重点实验室,江苏省血液研究所,苏州大学第一附属医院,江苏苏州215006 [3]临床医学研究中心,苏州科技城医院,江苏苏州215001

出　　处：《中国实验血液学杂志》2022年第2期559-564,共6页Journal of Experimental Hematology

基　　金：中国博士后基金(2019M661910);江苏省自然科学基金青年基金(BK20170361);南通市科技计划指令性项目(JC2019039)。

摘　　要：目的:用CRISPR/Cas9技术构建稳定表达的ITGA2B c.2659C>T (p.Q887X)无义突变的血小板无力症小鼠模型,并对其血小板膜表面糖蛋白αIIbβ3的功能做进一步研究。方法:根据ITGA2B基因信息设计并合成针对c.2659C位置的供体寡核苷酸和g RNA载体并进行体外转录。将g RNA和Cas9 m RNA与供体寡核苷酸共注射到受精卵,并送回到代孕鼠输卵管中。鼠出生后进行PCR基因分型和序列分析,获得阳性F0鼠。阳性F0鼠分别与野生型鼠配繁一代,获得经PCR和测序验证的F1代杂合型点突变GT小鼠,而后F1代小鼠通过彼此交配得到纯合型点突变GT小鼠以及对照组WT小鼠。通过检测小鼠鼠尾出血时间和隐静脉出血时间、血小板聚集功能、血小板表面αIIbβ3的表达和功能,对该小鼠模型的表型进一步验证。结果:小鼠鼠尾出血实验表明,GT小鼠割尾后的出血时间较WT小鼠明显延长(P<0.01)。小鼠隐静脉出血实验与鼠尾出血实验结果一致,即割断隐静脉后,GT小鼠的出血时间较WT小鼠明显延长(P<0.01)。在胶原、凝血酶、二磷酸腺苷诱导的血小板聚集实验中,GT小鼠血小板聚集率较WT小鼠有明显降低(P<0.01,P<0.01,P<0.05),表明GT小鼠血小板聚集功能受到明显抑制。流式结果显示,GT小鼠血小板表面αIIbβ3的表达量较WT小鼠明显下降(P<0.01),在凝血酶刺激后,诱导活化的血小板与纤维蛋白原的结合量较WT小鼠明显减少(P<0.01)。纤维蛋白原铺展实验结果显示,GT小鼠的血小板在纤维蛋白原上的铺展面积较WT小鼠显著缩小(P<0.05)。结论:应用CRISPR/Cas9技术成功建立了ITGA2B c.2659C>T (p.Q887X)无义突变的GT小鼠模型。该小鼠模型血小板聚集功能缺陷,符合血小板无力症的表现。Objective: To construct a mouse model of Glanzmann’s thrombasthenia(GT) with ITGA2 B c.2659 C>T(p.Q887 X) nonsense mutation by CRISPR/Cas9 technology, and then further explore the expression and function of glycoprotein αIIbβ3 on the surface of platelet membrane. Methods: The donor oligonucleotide and g RNA vector were designed and synthesized according to the ITGA2 B gene sequence. The g RNA and Cas9 m RNA were injected into fertilized eggs with donor oligonucleotide and then sent back to the oviduct of surrogate mouse. Positive F0 mice were confirmed by PCR genotyping and sequence analysis after birth. The F1 generation of heterozygous GT mice were obtained by PCR and sequencing from F0 bred with WT mice, and then homozygous GT mice and WT mice were obtained by mating with each other. The phenotype of the model was then further verified by detecting tail hemorrhage time, saphenous vein bleeding time, platelet aggregation, expression and function of αIIbβ3 on the surface of platelet. Results: The bleeding time of GT mice was significantly longer than that of WT mice(P<0.01). Induced by collagen, thrombin, and adenosine diphosphate(ADP), platelet aggregation in GT mice was significantly inhibited(P<0.01, P<0.01, P<0.05). Flow cytometry analysis showed that the expression of αIIbβ3 on the platelet surface of GT mice decreased significantly compared with WT mice(P<0.01), and binding amounts of activated platelets to fibrinogen were significantly reduced after thrombin stimulation(P<0.01). The spreading area of platelet on fibrinogen in GT mice was significantly smaller than that in WT mice(P<0.05). Conclusion: A GT mouse model with ITGA2 B c.2659 C>T(p.Q887 X) nonsense mutation has been established successfully by CRISPR/Cas9 technology. The aggregation function of platelet in this model is defective, which is consistent with GT performance.

关 键 词：小鼠模型 血小板无力症 CRISPR/Cas9 

分 类 号：R558[医药卫生—血液循环系统疾病]