中枢神经系统TauT基因敲除大鼠的构建及对线粒体DNA氧化损伤影响  

Construction of TauT gene knockout in the central nervous system of rats and its effect on oxidative damage of mitochondrial DNA

作　　者：夏一鸣 黄晓玲 仝慧慧 齐浩铭 莫丽冬[2] 王辰[2] 范维佳[2] 黄慧玲[2] XIA Yiming;HUANG Xiaoling;TONG Huihui;QI Haoming;MO Lidong;WANG Chen;FAN Weijia;HUANG Huiling(Tianjin Medical University,Tianjin 300070,China;Tianjin Huanhu Hospital,Neurosurgery Institute of Tianjin,Tianjin Key Laboratory of Cerebrovascular and Neurodegenerative Diseases,Tianjin 300350)

机构地区：[1]天津医科大学,天津300070 [2]天津市环湖医院,天津市神经外科研究所,天津市脑血管与神经变性重点实验室,天津300350

出　　处：《中国比较医学杂志》2021年第8期38-47,共10页Chinese Journal of Comparative Medicine

基　　金：天津市应用基础及前沿技术重点项目(16JCZDJC35500);国家自然科学基金面上项目(81571216)。

摘　　要：目的利用CRISPR/Cas9和Cre-loxP技术相结合构建牛磺酸转运体(taurine transporter,TauT)在中枢神经系统中条件性敲除大鼠,并对其脑组织线粒体mtDNA及线粒体呼吸链酶活性进行初步研究。方法利用CRISPR/Cas9和Cre-loxP技术获得TauT基因第5外显子两端含有loxP位点的杂合子大鼠(TauT^(loxP/WT))。将获得的TauT^(loxP/WT)大鼠与Nestin-Cre大鼠交配,经繁殖和鉴定最终获得神经特异性TauT基因敲除大鼠(TauT^(loxP/loxP/Cre+))。通过Real-time PCR、Westen blot、免疫组化对TauT^(loxP/loxP/Cre+)大鼠进行基因和蛋白表达检测,用HE染色观测其脑组织形态,并对脑组织mtDNA拷贝数和线粒体呼吸链酶(Ⅰ/Ⅱ/Ⅲ/Ⅳ/Ⅴ)活性进行检测。结果与野生型大鼠相比,TauT^(loxP/loxP/Cre+)大鼠脑组织中TauT基因和蛋白表达显著降低,TauT基因在中枢神经系统被成功敲除,模型构建成功;HE染色显示TauT^(loxP/loxP/Cre+)大鼠脑细胞数量和密度有所降低,而老年TauT^(loxP/loxP/Cre+)大鼠脑中细胞病变更加明显。此外,与野生型大鼠相比,TauT^(loxP/loxP/Cre+)大鼠脑组织线粒体呼吸链复合酶Ⅰ、Ⅲ、Ⅳ、Ⅴ酶活性显著降低,但mtDNA拷贝数却明显上升。结论利用CRISPR/Cas9和Cre-loxP技术成功了构建中枢神经系统TauT基因敲除大鼠模型,并初步验证了此模型对脑组织及其线粒体呼吸链酶和mtDNA的影响,为研究牛磺酸及TauT对脑组织的分子机制提供了新的模型平台。Objective CRISPR/Cas9 and Cre-loxP were used to construct the conditional knockout of taurine transporter(TauT)in the central nervous system of rats.The mitochondrial mtDNA and mitochondrial respiratory chain enzyme activities in brain tissues were studied.Methods CRISPR/Cas9 and Cre-loxP techniques were used to obtain heterozygous rats(TauT^(loxP/WT))with loxP at both ends of exon 5 of the TauT gene.The obtained TauT^(loxP/WT)rats were mated with Nestin-Cre rats.After breeding and identification,neural-specific TauT gene knockout rats(TauT^(loxP/loxP/Cre+))were obtained.The gene and protein expressions of TauT^(loxP/loxP/Cre+)rats were detected by Real-time PCR,Western blot,and immunohistochemistry.The morphology of brain tissue was observed by hematoxylin-eosin staining.The mtDNA copy number and mitochondrial respiratory chain enzyme(Ⅰ/Ⅱ/Ⅲ/Ⅳ/Ⅴ)activity in brain tissue were examined.Results Compared with wild-type rats,TauT gene and protein expressions in TauT^(loxP/loxP/Cre+)rat brain tissues were significantly decreased,and the TauT gene was successfully knocked out in the central nervous system indicating the knockout model was successfully constructed.Hematoxylin-eosin staining showed that the number and density of brain cells were decreased in TauT^(loxP/loxP/Cre+)rats and cytopathic changes more obvious in the brains of old TauT^(loxP/loxP/Cre+)rats.In addition,compared with wild-type rats,the activities of mitochondrial respiratory chain complex enzymesⅠ,Ⅲ,Ⅳ,andⅤwere significantly decreased,but the copy number of mtDNA was significantly increased.Conclusions The model of TauT gene knockout in the central nervous system of rats was successfully constructed using CRISPR/Cas9 and Cre-loxP technology.The effects of TauT knockout in the central nervous system on brain tissues,respiratory chain enzymes,and mtDNA of mitochondria were verified,providing a new model platform for the study of the molecular mechanisms of taurine and TauT in brain tissues.

关 键 词：TauT基因 条件敲除 CRISPR/Cas9 CRE-LOXP 大鼠 线粒体 

分 类 号：R-33[医药卫生]