CDK5调节羊驼黑色素生成的作用研究

The proline-directed serine/threonine kinase cyclin-dependent kinase 5 (CDK5) is a member of Cdk family of serine/threonine kinases. But CDK5 is different from the other members and does not regulate the cell cycle. Now it has been verified that CDK5 is expressed in many tissues, where it plays roles in cell adhesion, migration and differentiation.Our previous research found that CDK5 was expressed in the outer root sheath and upper hair bulb of alpaca skin where melanocytes are localized. CDK5 was differentially expressed between white and brown coat color. Moreover, over-expression of CDK5 in the alpaca melanocytes led to a significant increase of TYR and MC1R mRNA and protein, two molecules that have well established function in the skin color. Those results suggested that CDK5 may regulate melanogenesis in alpaca melanocytes. To investigate whether CDK5 also plays a role in regulating melanogenesis in alpaca, Liposome transfection method will be used to make melanocyte transfected by CDK5 over-expression and shRNA down-expression, and then the expression of MC1R and TYR will be tested. The result of gain-of-function and loss-of-function will give evidence of the function that CDK5 has a novel function in regulating the melanogenisis in alpaca.

CDK5（Cyclin-Dependent Kinase 5）是丝氨酸/苏氨酸激酶，属细胞周期依赖激酶（CDK）家族成员，但与其他成员的功能不同，在细胞周期中不起作用。现已发现CDK5多个组织中表达，参与细胞粘附、迁移和分化。我们前期的研究发现CDK5在羊驼毛囊黑色素分布密集的毛乳头上方和外根鞘表达，在不同毛色的羊驼皮肤中CDK5的基因表达和蛋白表达均存在显著差异，且CDK5在体外培养的黑色素细胞中过表达会使毛色基因MC1R和TYR的表达量上升。该结果提示CDK5可能对黑色素细胞产生黑色素有调节作用。本项目主要采用脂质体转染法将CDK5基因及其shRNA转染羊驼黑色素细胞，检测转染后二者对GPCR路径上重要毛色基因的表达影响，以进行功能获得性和缺失性研究。该研究将揭示CDK5的新功能,即在黑色素细胞产生黑色素过程中起重要调控作用，为人为改造哺乳动物毛色提供理论依据和遗传资源。

摘要：CDK5是脯氨酸导向的丝氨酸／苏氨酸激酶（Cyclin-dependent kinase, CDK）家族成员之一，其功能与其他成员不同，不具有调节细胞周期的功能。但CDK5发挥其功能时与其他成员一样，与特异的调节子结合并激活后，通过磷酸化底物组织和细胞中发挥重要的作用。然而，CDK5在不同的组织中，其激活子不同，磷酸化底物也不同。本研究通过CDK5过表达和抵制表达，均发现色素形成基因MC1R、MITF、TYR、TYRP1和TYRP2 mRNA和蛋白表达，从而调控黑色素细胞产生黑色素颗粒；通过构建小鼠shRNA-CDK5敲减模型，分别产生小鼠毛色表型变化（黑色表型变为棕色）和小鼠表皮增厚的表型。通过筛选CDK5的激活子和磷酸化底物，发现在羊驼皮肤黑色素细胞中，CDK5的激活子是P35结合，二者结合后激活磷酸化底物MATP和K10，影响色素形成基因MC1R、MITF、TYR、TYRP1和TYRP2 mRNA和蛋白表达，从而调控黑色素颗粒的产生；通过对体外培养的黑色素细胞培养基中添加α－MSH和TGFβ3，发现α－MSH和TGFβ3均对CDK5在羊驼黑色素细胞中的表达受到调控，影响黑色素细胞中黑色素的产量；在黑色素细胞中转染lpa-miR-nov-66，对羊驼黑色素细胞中CDK5磷酸化产生影响，从而对黑色素的生成过程起调控作用。本项目为揭示CDK5的新功能以及为哺乳动物毛色形成的分子机制提供了理论依据。

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