昆虫中肠干细胞离体诱导分化体系的建立及分化机制的研究

The insect midgut is responsible for digestion, absorption, secretion, and other physiological functions. The midgut epithelium is a natural barrier to pathogenic microorganisms and insecticidal toxin. The midgut is composed by three main cell types, columnar, goblet, and stem cells, the stem cells are critically important in that they are capable of massive proliferation and differentiation, and they are responsible for midgut growth and development, yet the differentiation processes and differentiation mechanisms of midgut stem cells are largely unknown up to now. Due to lack of midgut stem cell lines, there are no efficient in vitro induced differentiation system to study the differentiation of insect midgut stem cells. In this work, we will use several approaches and technologies in model organism Bomyx mori to study the: (1) Isolation and cloning of midgut stem cells from Bomyx mori midgut cell line Bm-Mg-1, established already, and establishment of midgut stem cell lines; (2) Screening of key cell factors and conditions related to midgut stem cell differentiation, and establishment of induced differentiation system of midgut stem cells in vitro; (3) Expression analysis of regulated genes related the differentiation signal pathway at the transcriptional level and protein level, and explanation of differentiation molecular mechanisms in vitro. Finally, we expect that this work can provide a new in vitro model system and scientific proofs to study the biology of insect midgut.

昆虫中肠具有消化、吸收和分泌等重要生理功能，是病原微生物和有害物质入侵的一道天然屏障。中肠细胞由柱状细胞、杯状细胞和干细胞组成，干细胞具有不断增殖和分化的特性，参与肠道的生理和一些病理过程，在中肠的生长发育过程中扮演着重要角色。目前昆虫中肠干细胞的研究处于起步阶段，对其分化机制的了解还不清楚，研究遇到的一个关键技术问题是缺少昆虫中肠干细胞系和有效的离体诱导分化体系。本项目将以模式昆虫－家蚕为材料，在已建立其中肠细胞系的基础上，通过细胞克隆和鉴定，建立中肠干细胞系；筛选最佳的细胞分化因子和诱导条件，建立中肠干细胞的高效离体诱导分化体系；应用基因芯片技术分析干细胞分化过程中基因表达谱的变化，确定分化调控目的基因；应用RT-PCR和蛋白免疫技术研究调控基因在转录水平和蛋白水平的表达，阐明中肠干细胞分化的分子机制。该项目将为昆虫中肠干细胞和中肠生物学功能的研究奠定理论基础以及提供离体的研究模型。

昆虫中肠具有消化、吸收和分泌等重要生理功能，同时也是病原微生物和有害物质入侵的一道天然屏障。昆虫中肠干细胞具有不断增殖和分化的特性，参与肠道的生理和一些病理过程，中肠干细胞分化机制的研究在昆虫生理学和病理学研究中具有重要意义。本项目围绕昆虫中肠干细胞分化的分子机制这一重大科学问题，以模式昆虫－家蚕为材料，从家蚕中肠细胞的培养、中肠干细胞的分离和增殖分化监测、中肠干细胞的诱导分化、中肠细胞分化基因表达谱分析以及相关信号通路基因的表达等方面开展系列研究。结果主要包括：通过对家蚕中肠细胞离体培养，明确了离体中肠细胞具有增殖和分化的特性；探索获得了Ficoll-Paque Plus梯度法分离家蚕中肠干细胞的方法和应用BrdU标记监测中肠干细胞增殖和分化的方法；通过筛选中肠细胞分化诱导因子和条件，建立了中肠干细胞离体诱导分化体系；通过中肠细胞转录组测序和分析，筛选获得多个可能与中肠细胞分化相关的信号通路基因；通过对家蚕离体和活体中肠细胞中相关信号通路基因的表达分析，推测Notch信号通路上Neurogenic locus protein delta和Neurogenic locus Notch protein基因的上调表达可能与中肠干细胞的分化密切相关。以上结果在理论上丰富了昆虫中肠干细胞分化机制的理论，将为昆虫中肠干细胞生物学的深入研究奠定理论基础。