老年期痴呆同病异证3个典型证候Tau蛋白磷酸化位点的特异性研究

Study on the specificity of Tau protein phosphorylation sites in three typical patterns of senile dementia based on the same disease with different patterns

目的观察3个老年期痴呆病证结合典型证候模型的大鼠Tau蛋白磷酸化位点的情况,分析总结其磷酸化的特异性。方法间隔1周结扎并剪断左、右颈总动脉,之后腹腔注射D-半乳糖,建立老年期痴呆病大鼠模型,最后在疾病模型的基础上分别进行房劳水劳刺激、投喂高脂饲乳和低温环境刺激,建立3种病证结合(肾虚精亏证、痰浊阻窍证和寒凝血瘀证)的老年期痴呆大鼠模型,通过测定血脂、血流变、血清睾酮水平检查验证各组模型是否成功。随后通过蛋白免疫印迹的方法观察Tau各磷酸化位点在各证候模型中的不同。结果在Tau蛋白脯氨酸富集区,与病理组相比,痰浊阻窍证组主要在205和231位磷酸化水平显著增强,肾虚精亏证组主要在181、205、231位磷酸化水平显著增强,寒凝血瘀证组磷酸化水平增强主要发生在205、231、262位;在C末端区域,与病理组相比,痰浊阻窍证组在404位磷酸化水平显著升高,肾虚精亏证组在396和404位磷酸化水平均显著升高,寒凝血瘀证组两者的升高幅度最高。结论老年期痴呆各典型证候在Tau蛋白磷酸化位点上有特异性,揭示了同病异证的科学内涵。

Objective To observe Tau protein phosphorylation in rats with three typical patterns of senile dementia, analyzing and summarizing the specificity of phosphorylation.Methods We ligated and dissected the left and right carotid arteries at one week＆#39;s interval with intraperitoneal injection of D-galactose to establish senile dementia model in rats.Those rats then received the intervention of excessive sexual and physical exertion, high-fat feeding milk and low temperature environment respectively to establish the rat models of senile dementia with three different patterns （group K： kidney deficiency and essence loss pattern;group P： retention of phlegm and turbid substance in orifices pattern;group C： congealing cold and blood stasis pattern）.Blood lipid, blood rheology and serum testosterone were measured to identify whether those model was built successfully.Different positions of Tau phosphorylation sites were measured with Western blot method.Results In the Tau protein proline-rich region, compared with the diseased groups, phosphorylation level increased significantly at the sites of 205 and 231 in group P.This increase was also present at sites of 181, 205, and 231 in group K and 205, 231 and 262 in group C.At the C-terminal region, compared with the diseased group, phosphorylation level of P group and K group were significantly increased at the sites of 404, and the increase of group C was the highest among those groups.Conclusion There is specificity at the Tau protein phosphorylation sites in each typical senile dementia pattern.This study may reveal the scientific rationale for the presence of different patterns in the same disease.