肝素对KGF-2介导的NIH3T3增殖及信号转导机制研究

发布时间：2018-01-08 04:02

本文关键词：肝素对KGF-2介导的NIH3T3增殖及信号转导机制研究　出处：《吉林农业大学》2011年硕士论文　论文类型：学位论文

更多相关文章： KGF-2 肝素 NIH3T3细胞 信号通路 ERK

【摘要】：KGF-2是一种非常具有潜力的治疗分子,能够特异性促进细胞迁移、细胞增殖、细胞分化；在脊椎动物的多种组织和器官发育中起重要调节作用。KGF-2具有两种酪氨酸激酶受体FGFR1和FGFR2, FGFR1是其低亲和力受体,FGFR2是高亲和力受体。NIH3T3细胞是一种上皮细胞,属成纤维细胞一种,它的表面含有FGFR1,因此,KGF-2能够与其表面受体结合,使受体发生自身磷酸化,将信号传递到细胞内,实现信号传递。肝素/硫酸肝素类自身具有多种生物学功效,如抗凝、抗炎等,这些生物学功效需要与多种蛋白质相互作用来发挥,以往实验表明多糖是生长因子信号传递的必需分子。本实验中首先应用生物学活性检测的常规方法MTT法,研究KGF-2对NIH3T3细胞的促增殖作用,其次,探索了肝素对KGF-2发挥生物学活性起到的调控作用,证明肝素能够促进KGF-2与FGFR1的结合,促进NIH3T3细胞的增殖,最佳肝素浓度为200μg/ml,能够显著增强KGF-2对NIH3T3细胞增殖的促进作用。然后,建立应用NIH3T3测定KGF-2生物学活性的新方法,并优化了活性测定的条件,最优条件为：细胞接种密度为8000,基础培养基中血清浓度为0.5%,药物作用时间为48h,最佳药物浓度为100μg/m1,药物储存时尽量避免反复冻融,药物缓冲体系选择磷酸盐缓冲体系。最后,对ERK在KGF-2促进NIH3T3增殖时的作用进行研究,胞外信号调节激酶途径是涉及调节细胞生长信号网络的核心,对ERK磷酸化水平进行研究,结果表明KGF-2在促进NIH3T3增殖时p-ERK有表达,启动ERK途径,最佳磷酸化时间是KGF-2作用15min；然后加入ERK信号通路抑制剂PD98059,再检测p-ERK水平,结果显示加入抑制剂后ERK磷酸化水平被抑制,证明此增殖实验中所启动的信号通路为ERK途径。总之,本实验证明肝素能够调控KGF-2对NIH3T3细胞的增殖作用,建立了应用NIH3T3细胞测定KGF-2生物学活性的新方法,并优化了活性测定条件,对KGF-2发挥作用时的ERK信号通路进行研究,为KGF-2在临床治疗研究奠定了基础。
[Abstract]:KGF - 2 is a very potential therapeutic molecule capable of specifically promoting cell migration , cell proliferation , cell differentiation , and plays an important role in the development of various tissues and organs of vertebrates . KGF - 2 has two tyrosine kinase receptors FGFR1 and FGFR2 , FGFR1 is a low - affinity receptor , FGFR2 is a high - affinity receptor . The heparin / heparan sulfate class itself has a variety of biological effects , such as anticoagulation , anti - inflammation , etc . These biological effects need to interact with a variety of proteins , and previous experiments have shown that polysaccharides are essential molecules for signal transduction of growth factors . The effects of KGF - 2 on the proliferation of NIH 3T3 cells were studied by MTT assay . The effect of KGF - 2 on the proliferation of NIH 3T3 cells was investigated . The optimal conditions were as follows : cell inoculation density was 8000 , the optimal concentration of heparin was 200 渭g / ml , the optimal concentration of heparin was 100 渭g / ml . In conclusion , this experiment proves that heparin can regulate the proliferation of KGF - 2 to NIH 3T3 cells , establish a new method to measure the biological activity of KGF - 2 by using NIH 3T3 cells , optimize the activity assay condition , and study the ERK signal pathway when KGF - 2 plays a role , which lays a foundation for the clinical treatment of KGF - 2 .

【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

【引证文献】