活化型TGFβRI(T204D)转基因小鼠的建立及其对牙釉质发育影响的初步研究

Establishment of constitutively active TGFβRI (T204D) transgenic mice and preliminary study of its effects on enamel development

目的建立人活化型TGFβRI(T204D)的转基因小鼠,并利用转基因动物模型初步研究T204D对牙齿釉质发育的影响。方法克隆小鼠釉原蛋白(Amelogenin,Amelx)启动子及人T204D的全长基因,并利用缺失突变原理扩增T204D基因。运用Gateway技术将T204D基因插入Amelx启动子下游构建转基因表达载体,通过显微注射法建立T204D转基因C57BL/6小鼠。PCR鉴定转基因小鼠的基因型,RT-PCR检测外源性TGFβRI基因表达。体视显微镜及扫描电镜下观察转基因小鼠磨牙釉质的矿化程度和病理性磨损程度。结果构建了T204D转基因表达载体并制备转基因小鼠;转入的人T204D基因在小鼠颌骨组织中特异性表达;组织学分析显示:活化型TGFβRI转基因小鼠磨牙釉质矿化程度较野生型小鼠差。6个月龄时,与野生型小鼠相比,活化型TGFβRI转基因小鼠磨牙釉质有严重磨损。结论 TGFβRI在牙齿釉质发育过程中具有重要作用。

Objective To establish a transgenic mouse model of human activated TGFβRI（T204 D）,and to study the effect of T204 Don the development of dental enamel using a transgenic animal model.Methods The full length gene of murine amelogenin promoter and human T204 D was cloned and the T204 D gene was amplified by deletion mutation.The T204 Dgene was inserted into the downstream of Amelx promoter by Gateway technology to construct transgenic expression vector,and T204 D transgenic C57 BL/6 mice were established by microinjection method.The genotypes of transgenic mice were identified by PCR,and the expression of exogenous TGFβRI gene was detected by RT-PCR.The degree of mineralization and the degree of pathological wear of the molars enamel in the transgenic mice were observed under stereomicroscope and scanning electron microscope.Results The T204 Dtransgene expression vector was constructed and the transgenic mice were prepared.Transfection of human T204 Dgene was specifically expressed in mouse jaw tissues.Histological analysis showed that the activation of TGFβRI transgenic mouse molar enamel mineralization worse than wild-type mice.At 6 months of age,the activated enamel of TGFβRI transgenic mice had severe wear compared to wild-type mice.Conclusion TGFβRI plays an important role in tooth enamel development.