β1整合素在涡虫neoblast增殖和迁移中的功能及分子机制研究

Planarians which are an ideal experimental model for studying the problems of stem cell and regeneration have a large number of adult stem cells called neoblasts. Following amputation of a planarian, neoblasts proliferated and were recruited to wounds to achieve regenaration. However, the mechanism by which wound stimulates neoblast proliferation and migration is not clear. A large amount of research showed that beta integrin is a key regulator of cell proliferation and migration. Our previous study revealed: planarians possessed one β1-integrin gene highly homologous with human beta 1 integrin. Further we confirmed that β1 integrin was predominantly expressed in the neoblasts; expression of β1 integrin in planarian was significantly increased response to regeneration following amputation. Based on this information, we speculated that upregulation of β1 integrin expression was closely related to neoblasts proliferation and migration, β1 integrin probably a key regulator of neoblast proliferation and migration. To verify our hypothesis, we will employ β1 integrin inhibitors, overexpression/silence of β1 integrin gene, tissue and cell transplantation to investigate the function and molecular mechanism of β1 integrin in the proliferation and migration of neoblast, and further reveal the molecular cell biology mechanism of proliferation, migration of planaria neoblast

涡虫作为理想的再生和干细胞研究模式生物，具有被称为neoblast的大量成体干细胞，机体受损时，neoblast增殖并迁移至受损位点完成再生。然而损伤诱导neoblast增殖和迁移的分子机制尚不清楚。研究表明β整合素是细胞增殖和迁移的关键调控者。我们前期研究发现：涡虫体内有一个与人高度同源的β1整合素，主要表达于涡虫neoblast；再生过程中β1整合素的表达显著上调。我们推测：β1整合素的表达上调与涡虫neoblasts增殖、迁移密切相关，可能是其主要的调控因子。本研究拟通过分析涡虫再生中β1整合素的时间和空间表达谱，研究其与干细胞增殖和迁移的关联；通过knockdown、过表达β1整合素，以及应用组织、细胞移植术研究β1整合素在涡虫neoblast增殖和迁移中的功能，初步揭示涡虫neoblast增殖和迁移的分子机制。

发现通过干扰β1-integrin的表达，诱导涡虫再生发现其再生异常，尾部不能正常再生，胚基较小且呈豁状。通过Real time PCR检测β1-integrin RNAi后涡虫不同再生阶段的PCNA表达水平，发现PCNA表达量大幅下调；同时利用涡虫有丝分裂Marker H3Ser10p抗体，检测β1-integrin RNAi后涡虫不同再生阶段的有丝分裂细胞数量，发现有丝分裂细胞明显下调。这些数据说明β1-integrin RNAi后涡虫neoblasts分裂增殖异常导致涡虫再生异常，当然不排除β1-integrin RNAi后neoblasts有丝分裂异常、同时迁移能力降低导致涡虫再生异常。.还发现RACK1也参与涡虫再生过程。RACK1 RNAi后涡虫不能再生出正常的头部和尾部。Rack1 RNAi后观察其平衡态，发现20d后从尾部开始自溶解。检测RACK1 RNAi后，涡虫不同再生阶段的有丝分裂细胞数量，发现实验组再生7天之后增殖细胞数量明显多于对照组但其涡虫Djprog1的表达又明显降低。说明RACK1通过影响细胞的分化，进而影响涡虫的再生过程。.同时，还发现RPS3、RPS20等蛋白也参与涡虫再生过程。RPS3或RPS20 RNAi后涡虫皆不能再生出正常的头部和尾部，并且RNAi后所有涡虫再生片段存活不能超过13d。进一步分析涡虫neoblasts增殖和分化，发现RPS3和RPS20也通过都参与neoblasts早期分化影响涡虫再生。