人突变TDP-43转基因小鼠ALS模型的建立  

The establishment of human TDP-43 mutant transgenic mice of ALS

作　　者：王旭[1] 沈星辉[1] 孙瑞珍[1] 李兆瑗[1] 单智焱[1] 王振东[1] 雷蕾[1] 金连弘[1] 申景岭[1] 

机构地区：[1]哈尔滨医科大学组织学与胚胎学教研室哈尔滨医科大学胚胎与干细胞工程实验室,150081

出　　处：《国际遗传学杂志》2012年第3期139-145,共7页International Journal of Genetics

基　　金：国家自然科学基金（30900413、31000645）;中国博士后科学基金（20100481020）;中国博士后特别资助基金（201104421）

摘　　要：目的构建带有脊髓运动神经元特异性启动子HB9的人类TDP-43野生型和突变型的转基因小鼠模型，研究TDP-43突变导致肌萎缩侧索硬化症（amyotrophiclateralsclero—sis，ALs）的机制。方法构建pHB9-hTDP-43^Q331K,M337V-IRES-EGFP转基因的载体，通过受精卯原核注射的方法制备转基因小鼠。出生的后代利用PCR方法鉴定。免疫组织化学法观察转基因小鼠的脊髓运动神经元中TDP-43的定位及包涵体的形成。采用步态评估和悬挂实验观察转基因小鼠行为学的改变。结果获得人TDP．43突变转基因小鼠，其运动神经元胞质中TDP-43染色阳性，未见TDP-43包涵体。小鼠行为学检测显示，转基因小鼠的步距与同龄正常小鼠相比明显下降（Q331K型小鼠同Ntg相比前后肢步距t值分别为6．05和5．15，M337V型小鼠同Ntg相比前后肢步距t值分别为10．71和9．91；P〈0．01）；步态宽度升高（Q331K型小鼠同Ntg相比前后肢步态宽度i值分别为-11．35和-2．73；M337V型小鼠同Ntg相比后肢步态宽度t=2．49；P〈0．05）；平衡抓握力明显下降（Q331K型小鼠同Ntg相比落地时间t=47．43；M337V型小鼠同Ntg相比落地时间t=26．35，P〈0．01）。结论利用HB9启动子可以获得在脊髓运动神经元特异性表达人TDP-43突变的转基因小鼠，TDP-43突变转基因小鼠运动神经元发生退行性改变。转基因小鼠前后肢步距、步态宽度及平衡抓握能力发生改变，结果显示脊髓运动神经元中过表达人TDP-43突变对小鼠的运动能力产生影响。Objective To investigate the role of TDP-43 in the pathogenesis of Amyotrophic lat- eral sclerosis （ALS） , We generated several transgenic mouse lines expressing human wild-type TDP-43 and mutant-type TDP-43（ M337V and Q331K） under the control of a HB9 promoter （pHB9） which drives gene expression in motor neurons of spinal cord. Methods The transgenic plasmid was constructed by inserting the human TDP-43 mutator gene into the downstream of HB9 promoter. The linearized con- struct of the vectors was microinjected into fertilized B6D2F2 mouse eggs and reimplanted into pseudopregnant females mice. The genotype of the transgenic mice were detected by PCR. Furthermore, immuno- histochemichemistry was used to study the localization of TDP-43 and the formation of its inclusions in mo- tor neurons of spinal cord. Transgenic mice carrying mutation hTDP-43 gene were tested for behavioral phenotypes by gait assessment and hanging wire. Results We have created transgenic mice expressing human mutant TDP-43 in motor neurons of spinal cord. TDP-43 staining in motor neurons of spinal cord of transgenic mice is positive but negative in Ntg mice. Our results showed that gait width and length of forelimb and hindlimb were ＂decreased in TDP-43 transgenic mice than non-transgenic mice （P〈0.01） with that the t- test value of Q331 K- type transgenic mice were 6. 05 and 5. 15, and M337 V-type transgenic mice were 10.71 and 9.91. Length of forelimb and hindlimb were also decreased in TDP-43 transgenic mice compared with non-transgenic mice （ P 〈 0. 05 ） with that the t-test value of Q331 K- type transgenic mice were - 11.35 and - 2.73, and M337V-type transgenic mice were 2.49. The a- bility of balance and grip strength of transgenic mice （P 〈 0.01 ）were decreased with that the t-test val- ue of Q331K-type transgenic mice were 47. 43, and M337V-type transgenic mice were 26. 35. Conclusion The specific expression of mutant TDP-43 in motor neurons of spinal cord by HB9-depend- ent promotor may contribute to

关 键 词：肌萎缩侧索硬化症 TDP-43 转基因小鼠 

分 类 号：R-332[医药卫生]