建立一种新型凝血因子Ⅴ基因突变小鼠模型:突变小鼠无显著骨组织变化  

Establishment of a transgenic mouse model with coagulation factor V mutation, but with no obvious changes of bone tissue

作　　者：王宇英[1] 张琰 祁进伟 尹兆光 

机构地区：[1]杭州师范大学生命与环境科学学院,浙江省杭州市310036 [2]石家庄场站医院,河北省石家庄市050000 [3]石家庄市灵寿县医院泌尿外科,河北省石家庄市050500 [4]唐山市滦南县医院外一科,河北省唐山市063500

出　　处：《中国组织工程研究》2016年第46期6861-6867,共7页Chinese Journal of Tissue Engineering Research

基　　金：浙江省自然科学基金(LQ13H060003)~~

摘　　要：背景:研究显示,凝血因子Ⅴ基因突变在自发性非创伤性股骨头坏死的发生率比健康对照组和继发性非创伤性股骨头坏死高,血栓形成的发生率与此吻合。凝血因子Ⅴ的失活能够加速和促进凝血酶原的激活及凝血酶的生成。其重链上的3个位点arg-306,arg-506,arg-679的突变导致血栓倾向和高凝状态。文章将调查第506位和679位的同时突变R506Q/R679Q对骨坏死产生的功能和影响。目的:建立凝血因子Ⅴ第506位和679位的谷氨酰胺至精氨酸(Factor VR506Q/R679Q)点突变小鼠模型。方法:应用分子克隆技术构建Factor VR506Q/R679Q点突变打靶载体,将其线性化后电转到胚胎干细胞中,然后筛选G418抗性的胚胎干细胞克隆进行胚泡显微注射,将注射好的胚泡移植至同期发情的假孕受体母鼠输卵管内,获得携带双侧LoxP基因的Chimera小鼠。将突变鼠与CMV-cre转基因鼠杂交后,得到只含Factor VR506Q/R679Q的点突变鼠。PCR法进行基因型鉴定后,将突变小鼠和野生小鼠的骨苏木精-伊红染色和空骨陷窝率等进行比较,对其骨组织情况及骨量进行分析。结果与结论:(1)与野生鼠相比,Factor VR506Q/R679Q点突变鼠胚胎及生后的生长发育无明显异常,骨量及空骨陷窝率并无显著变化;(2)结果提示,成功建立了凝血因子Factor VR506Q/R679Q点突变小鼠,但突变小鼠并无显著骨组织变化。之后的研究应关注在外加因素刺激下突变小鼠的骨坏死诱发率。BACKGROUND： Blood coagulation factor V gene mutation in non-traumatic femoral head necrosis has been shown to have an higher incidence than that in healthy and secondary non-traumatic femoral head necrosis, and the incidence of thrombosis is positively related. Inactivated blood coagulation factor V can accelerate the activation of prothrombin and the generation of thrombin. Mutations at arg-306, arg-506 and arg-679 will result in the blood clots and hypercoagulable state. Here, this study is designed to investigate the influence of R506Q/R679 Q on osteonecrosis. OBJECTIVE： To establish the mouse model of mutations of Gln506 Arg and Gln679 Arg in coagulation Factor V（Factor VR506Q/R679Q）. METHODS： Factor VR506Q/R679 Q point mutation target vector was constructed by molecular cloning technology, the linearization vector was transfected into embryonic stem cells, and then G418-resistant cells were screened and used for microinjection. The target blastocysts were transplanted to the fallopian tube of estrus mice to obtain the Chimera mice carrying bilateral LoxP gene, followed by mated with CMV-cre transgenic mice, and then the mice with Factor VR506Q/R679 Q point mutations were obtained. After genotype identification by PCR, hematoxylin-eosin staining results and percentage of empty lacunae were compared between the mutant and wild-type mice, and rat bone tissue and bone mass were analyzed. RESULTS AND CONCLUSION： There were no obvious abnormalities in the embryonic and postnatal development, percentage of empty lacunae and bone mass of Factor VR506Q/R679 Q point mutation mice when compared with the wild-type mice. These results suggest that the mouse model with Factor VR506Q/R679 Q point mutation is established successfully, but there is no significant change in the bone tissue. The following research should focus on the effect of external stimulus on the incidence of osteonecrosis in a mutant mouse.

关 键 词：模型 动物 基因 血液凝固因子 组织工程 组织构建 骨组织工程 基因突变小鼠 凝血因子Ⅴ 显微注射 动物模型 浙江省自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]