UHRF1蛋白介导的Geminin泛素化参与VSMCs增殖周期调控机制的研究

Geminin plays an important role in regulating VSMCs proliferation.We found that Geminin interference facilitate VSMCs proliferation by promoting the transition the G0/G1 to S phase and it is degradated by ubiquitin - proteasome pathway which is regulated by the ubiquitin molecule in the nucleus. In addition,UHRF1 protein with both UBL domain and RING domain that has E3-ligase is abundantly expressed in the nucleus in the VSMCs proliferation late G1 and G2/M phase. Therefore, we speculate UHRF1 protein may be involved in the ubiquitination-like regulation of Geminin protein,thus it can regulate chromatin segregation in mitotic and play an important regulatory role in VSMCs proliferation cycle. Through FBS stimulates VSMCs proliferation, the relationship between the two proteins was analysised by CO-IP and FRET; whether the mode of action of the two protein were related to the ubiquitination degradation was detected by Mass Spectrometry and Ubiquitin fluorescent probes; chromatin segregation and proliferation process of VSMCs were studied by FCM and microscopic chromatin separation technology. This subject is to study the molecular regulation mechanism in the VSMCs proliferation. It will provide a new mentality for studying the feedback control mechanism in VSMCs proliferation and a new method for treating vascular proliferative diseases and preventing restenosis after angioplasty.

Geminin蛋白在VSMCs增殖中发挥了重要的调控作用。我们通过沉默Geminin发现可以通过促进VSMCs增殖周期的G0/G1期向S期的转化从而使其增殖速率明显增加。该蛋白在胞核内经泛素-蛋白酶体通路降解，受到泛素化分子调控；而具有UBL结构域及包含E3连接酶的RING结构域的UHRF1蛋白在VSMCs增殖的G1期末和G2/M期大量表达于胞核中，因此我们推UHRF1蛋白可能参与Geminin泛素化调控作用，影响分裂期染色质分离，从而对VSMCs增殖进程发挥重要作用。通过血清培养VSMCs，运用CO-IP、FRET等分析两蛋白间关系，质谱分析及泛素荧光探针检测两蛋白作用方式是否与泛素化降解相关，运用FCM及显微染色体分离技术研究VSMCs染色质分离及增殖周期进程。本课题旨在研究VSMCs增殖及分子调控机制，为探讨其自身调控机制提供一条新思路，从而为增生性血管疾病和PCI术后血管再狭窄的防治提供理论依据。

动脉粥样硬化性心血管病是危害人类健康最常见的一种疾病，具有高致残率及致死率。血管成形术是目前用于治疗动脉粥样硬化等血管增生性疾病最广泛且最有效的方法，术后再狭窄为影响其远期疗效的主要原因。血管平滑肌细胞（VSMCs）的增殖、移行以及合成细胞外基质是疾病的发病基础。因此，探索其分子机制具有极其重要的现实意义。Geminin蛋白在VSMCs增殖中发挥了重要的调控作用。该蛋白在胞核内经泛素-蛋白酶体通路降解，受到泛素化分子调控；而类泛素化蛋白UHRF1在VSMCs增殖的G1期末和G2/M期大量表达于胞核中，因此我们推测UHRF1蛋白可能参与Geminin泛素化调控作用，对VSMCs增殖进程发挥重要作用。本研究应用能稳定传代的血管平滑肌细胞珠A10，进行复苏、传代、培养，提取蛋白及RNA，通过PCR、 Western blot等方法进行实验，在基因水平及蛋白水平探讨UHRF1与Geminin 的关系，结果提示UHRF1通过调控Geminin蛋白参与细胞增殖过程。构建过表达UHRF1转染细胞珠，研究高表达UHRF1蛋白干扰Geminin的表达进而影响VSMCs的增殖及细胞周期的进程。基因测序分析了参与UHRF1调节Geminin蛋白表达的信号通路，提示UHRF1过表达影响了PI3K-AKt信号通路进而调控Geminin蛋白表达和VSMCs细胞周期进程。本研究阐明了 VSMCs 增殖的新机制，为临床上治疗血管增生性疾病提供新思路。同时我们研究发现交感神经递质对不同亚型的VSMCs的增殖起调节作用，神经肽Y能通过干扰Geminin从而对增殖型和收缩型VSMCs起不同的调节作用，从另一个角度证明了研究调节Geminin功能的因子对发现治疗干预动脉粥样硬化的发生发展有重要的意义。