利用iMIC细胞体外重建肠癌发生发展过程的实验研究

Intestinal epithelial stem cells(iSCs) located at the bottom of the intestinal crypts. Difficult to culture crypt cells in vitro is an obstacle hinder the related studies of iSCs. A recently work made a breakthrough: a three-dimensional culture system constitute of Matrigel and ENR medium which contained a variety of growth factors, in this culture system, iSC would form a structure called "Organoid" which similar to intestine "Villi - Crypt "unit, and the Organoids could maintain long-term survival. We improved this extremely expensive culture system, reduced 80% cost of ENR medium by preparing and using RSPO2 conditional medium instead commercial factors which is most expensive in all components. We also developed effective methods to deliver genes into Organoid through recombinant adenovirus, but it is still difficult to stably introduce genes into cultured crypt cells. To this end, after worked out two-dimensional culture conditions of primary intestinal crypt cells, we had created a stable mouse intestinal crypt cell line- iMIC by using piggyBac based immortalizing tool: pMPH86. iMICs were capable of long-term survival, only healthily grew in ENR medium and probably had multiple differentiation potential, more important, iMIC can also form a cavity-like Organoid when cultured in three-dimensional conditions. .Intestinal cancer originated from the normal crypt stem cells, whose development is a multi-stages process. A number of driver genes’ mutation are known to play an important role in the transition of stages’ shifting. This study attempt to prove iMICs possess main characteristics of intestinal stem cells /progenitors, then, by taking advantage of iMIC’s two-dimensional culture manner and piggyBac plasmid tools we constructed, well recognized gene mutations of early or later stage of colorectal cancer would introducing into iMICs, we hope to reconstitute the intestinal tumorigenesis and development process in vitro. Further on, we expect to set iMIC as a research platform for functional identification of cancer--related-gene candidates, and large-scale screening for new potential therapeutic targets..Intestinal cancer originated from the normal crypt stem cells, whose development is a multi-stage process. A number of driver genes’ mutation are known to play an important role in the transition of stages’ shifting. This study attempt to prove iMICs possess main characteristics of intestinal stem cells /progenitors, then, by taking advantage of iMIC’s two-dimensional culture manner and piggyBac plasmid tools we constructed, well recognized gene mutations of early or later stage of colorectal cancer were introducing into iMICs, we hope to reconstitute the intestinal tumorigenesis and development process in vitro. Further on, we expect to set iMIC as a research platform for functional identification of cancer--related-gene candidates, and large-scale screening for new potential therapeutic targets.

肠癌起源于肠隐窝正常干细胞，但从干细胞逐步恶化到癌的过程一直缺乏良好的体外研究模型。本研究在改进了肠上皮细胞昂贵的三维培养体系基础上，摸索出肠隐窝原代细胞二维培养条件，运用自行开发的基于piggyBac原理的永生化工具pMPH86，创建了小鼠肠隐窝细胞稳定系iMIC。试图通过i与原代肠隐窝细胞比对，谱系内多向分化潜能检测，三维条件下形成肠隐窝类器官Organoid的能力及对形成物的鉴定等，证明iMIC细胞具有肠上皮祖/干细胞的基本特性。借助二维培养的iMIC方便基因操作的优势，选择已知的在肠癌早，晚期阶段跃迁过程中扮演驱动者角色的重要基因突变体，运用前期开发的piggyBac载体工具，分别导入iMIC细胞中。利用二维、三维和移植等实验体外重现肠癌发生发展过程。研究结果将为肠癌相关基因的功能鉴定和新治疗靶点的大规模筛选提供研究平台。

肠癌是隐窝干/祖细胞经多阶段突变积累发展而来，如何寻找和甄别哪些基因在肠上皮细胞恶性转化过程中发挥作用并不容易。.近年来肠上皮隐窝细胞体外培养难题取得突破。为了更方便的对肠隐窝细胞进行基因水平的操作，我们在摸索出了肠隐窝细胞原代培养的方法之后，发现二维培养条件下原代细胞在传代后将很快出现分化。经多次尝试，针对刚贴壁的原代细胞，利用pMPH86成功建立了可连续传代的永生化的肠隐窝细胞系---iMIC。并通过对iMIC细胞进行肠上皮干细胞生物特性的鉴定，多向分化潜能的检测和形成Organoid的能力等证明iMIC细胞保持了肠上皮祖/干细胞的基本特性。通过在iMIC基础上导入已知公认的肿瘤进展不同阶段的分子事件，观察到细胞出现可直接观察的表型或生物学行为变化。实现通过基因操作，体外可控地重现肠隐窝细胞恶性转化的基本过程。.本研究不仅为明确某个特定的基因突变与肠癌发生发展之间的可能关系提供了新的研究手段，也为以后利用包括RNAi文库等在内的新的肠癌基因及相关药物靶点大规模筛选提供了节约资源，快速简便的策略。