HPSE2基因功能缺失引发非神经源性膀胱的初步机制研究

Non-Neurogenic bladder (NNB) is one of serious bladder dysfunction belong to the lower urinary tract symptoms which may leading to the dysfunction of kidney, but the mechanism under NNB is not clear. The urofacial syndrome (UFS, OMIM #236730) is an autosomal recessive disease belong to NNB, with which the patients show a frown-like facial expression when try to smile. We recently screened one of the defect gene of UFS named HPSE2, we confirmed that deficient of HPSE2 in mice can cause low urinary tract dysfunction in mice model. Based on the common reorganization that NNB is caused by the combination of detrusor overactivity (DO) and detrusor-sphincter dyssynergia (DSD), and UFS patients show the special characters with multiple sphincter dyssynergia including the anal sphincter, orbicularis oculi muscle and orbicularis oris muscle. We hypothesized that the major function of HPSE2 is to coordinate the movement of sphincter with other muscles, this molecule may also regulate the non-specific contraction of detrusor smooth muscles. In this study we proposed to dissect the mechanism of HPSE2 through urodynamic analysis, electromyogram study in vivo to describe the pathogenic progress of NNB, through patch clamp and Immunofluorescence to study the ion channel on cells, density of presynaptic nerve teminal to dissect the cell mechanism of NNB, and through glycosidase treatment and DNA site mutation to check the enzyme activity of HPSE2 protein. to help understand how loss of HPSE2 function can cause Completion of our studies would provide novel information into NNB pathoetiology, including its major pathogenesis of DO and DSD, for the development of more effective therapeutic strategies against this bladder dysfunction.

非神经源性膀胱（NNB）是威胁患者肾功能的下尿路疾病，但其具体机制不明。Urofacial 综合征 (UFS)是具有面部表情异常及常染色体隐性遗传病特征的NNB分支。我们近期扫描到UFS的缺陷基因之一HPSE2，并证实该基因在小鼠中缺失可引发下尿路功能碍障。依据NNB现有的研究结果及UFS病人多种括约肌协同失调的特点，我们推测HPSE2主要参与协调括约肌与其它肌肉的动作，同时可能有抑制逼尿肌非特异性收缩的功能。申报者拟利用传统敲除及诱导敲除HPSE2基因小鼠，以尿动力学、肌电图等技术在活体动物水平阐明NNB发生的病理过程；以膜片钳、免疫荧光等技术检测离子通道、突触前神经末梢密度等，在细胞水平阐明NNB发生的机制；以定点突变及糖苷酶处理等技术，探讨HPSE2参与蛋白糖基化修饰的酶学基础，以期阐明HPSE2缺失引发逼尿肌过度活动、逼尿肌-括约肌协同失调的机制，进而为NNB的治疗提供新靶点。

人常染色体隐性遗传病Urofacial综合征是非神经源性膀胱（NNB）的一个分枝，我们在前期已经证实其缺陷基因是HPSE2，但对该基因的功能完全未知。在本项目中，我们建立了HPSE2条件性基因敲除及传统敲除小鼠，分别与全身、平滑肌、骨骼肌诱导表达Cre小鼠交配，获得诱导型全身敲除/平滑肌/骨骼肌特异性敲除HPSE2小鼠，检测其生理状态下的尿动力学参数；发现所有类型的HPSE2基因敲除小鼠均具有排尿量减少、排尿周期缩短及最高排尿压力增高等排尿异常。此外还发现，HPSE2敲除小鼠的眼睑闭合不全，这些都是典型的人Urofacial综合征症状。我们取小鼠膀胱组织行免疫荧光检测，结果显示HPSE2基因敲除小鼠的乙酰胆碱受体聚集增加，在体外实验中也证实，HPSE2高表达可以抑制乙酰胆碱受体（AchR）的聚集、减弱膀胱平滑肌的收缩，而敲减HPSE2的表达可以增强AchR聚集、增强膀胱平滑肌的收缩。对HPSE2基因敲除小鼠的膀胱胞外基质进行扫描电镜检测，发现其纹理明显稀疏，对胞外基质成份进行了质谱分析，结果表明细胞外基质中透明质酸（HA）的含量显著减少，而硫酸乙酰肝素和硫酸软骨素未见显著差异，该结果通过ELISA进行检测再次获得验证。这些研究结果显示我们建立的诱导型全身敲除/平滑肌/骨骼肌特异性敲除HPSE2小鼠可完全模拟人Urofacial综合征，具有典型的非神经源性膀胱症状。HPSE2敲除小鼠的膀胱逼尿肌更易在轻微的刺激下产生不自主的收缩，从而引发膀胱过度活动，其机制是HPSE2基因功能缺失降低HA的合成，改变胞外基质的组成，从而无法正常抑制膀胱的非自主性收缩。