中文摘要：
组织内稳态是一个动态的过程,需要新细胞的产生来补充那些衰老和损伤的细胞,保持良好平衡。肠道作为新陈代谢最快的组织之一,当肠道损伤时,肠道干细胞可以通过持续增殖来补充受损的上皮细胞,因此肠道干细胞在维持肠道稳态、肠道屏障功能方面的作用至关重要。果蝇消化道上皮是单层的,其在形态上分化为不同区域。其中中肠是最具代表性的,包含基于不同形态和组织学特性的亚区以及基因表达谱。多能肠干细胞(ISCs)显示中后肠(PMG)的增殖率较高,表达Notch配体Delta,并持续产生EBs。尽管ISCs和EBs都表达转录因子escargot(esg),但EB中的Notch受体激活导致抑制Su(H)的活化并分化成ECs,表达POU结构域的转录因子Pdm1。重要的是,一部分Su(H)~+EB分化成表达Prospero的肠内分泌细胞(EE)。在生理和应激条件下,ISC分裂和分化受到JNK,Egfr/Ras/MAPK,Notch,Wnt,JAK-STAT和m TOR等几种途径的控制。m TOR通路是一个著名的自噬调控器。果蝇的肠道结构及细胞组成与哺乳动物的肠道非常相似,果蝇的前胃前肠相当于哺乳动物的胃,用于贮存食物;果蝇的中肠相当于哺乳动物的小肠,消化食物吸收营养物质,果蝇的后肠相当于哺乳动物的大肠,用于排泄。哺乳动物的肠道干细胞在肠绒毛隐窝处,靠近基底膜,由隐窝向肠腔纵向进行分裂分化,ISC分裂产生中间过渡细胞(TA cell),随后分化EC,EE及潘氏细胞,靠近肠腔有一层粘膜;果蝇肠道表皮细胞组成与其相似,肠道干细胞也靠近基底膜,分裂产生EB细胞,进而分化为肠道上皮细胞(EC),具有毛刷状边缘及多倍体核,吸收营养物质,肠内分泌细胞(EE),分泌激素,外层是围食膜,粘膜。正因为果蝇与哺乳动物肠道结构组成的相似性及遗传学优势,果蝇肠道干细胞已经成为研究肠道干细胞的经典模型。近年来,人们发现lnc RNA在调控细胞正常发育和疾病发展过程中起着非常重要的调节剂的作用。它们参与组织和细胞特异性的表达模式,并潜在参与诸如细胞命运和分化等生物学过程。lnc RNA的作用通常在核中,在转录反应和调节染色质的结构和功能等过程中至关重要。目前,据我们所知,尚无参与果蝇ISC特异调控且必不可少的lnc RNA的相关报道。所以我们想知道lnc RNA是否也参与果蝇肠道干细胞增殖与分化的调控。我们通过DSS喂养果蝇后筛选发现CR43282-CR46040~(KO)果蝇中肠道干细胞的数目与对照相比明显减少,表明在果蝇肠道损伤应激的条件下,CR43282-CR46040影响肠道干细胞的增殖。之后我们通过PH3抗体染色发现CR43282-CR46040敲除果蝇株ISC/EB细胞数目的减少是由于ISC的增殖减少造成的,表明肠道再生时CR43282-CR46040参与ISCs的增殖过程。同时我们利用互补实验证实了正是因为CR43282-CR46040的缺失才导致了这一现象。我们通过MARCM克隆实验再次证实了CR43282-CR46040参与损伤后ISCs的增殖过程。接下来我们检测了损伤条件下相关通路的激活情况,我们发现CR43282-CR46040的缺失导致JAK-STAT通路激活明显减弱。有趣的是我们研究发现CR43282-CR46040并不参与ISCs的分化过程。lnc RNA对肠道干细胞功能的调控还未有过报道,本研究为进一步揭示lnc RNA对肠道干细胞增殖与分化的调控机理提供了帮助。
英文摘要：
Tissue homeostasis is a dynamic process that requires the production of new cells to replenish those that are aging and damaged,and to maintain a good balance.Stem cells play a key role in this process.Intestinal is one of the tissues with the fastest metabolism.When the intestinal is damaged,intestinal stem cells can continue to proliferate to renew damaged epithelial cells.Therefore,intestinal stem cells play a very important role in maintaining intestinal homeostasis and intestinal barrier function.Drosophila digestive tract epithelium is a pseudo-lamellar monolayer that is morphologically differentiated into different regions.The midgut is the most representative,including different subregions and gene expression profiles based on different morphological and histological characteristics.Intestinal stem cells(ISCs)show the highest proliferation rate in the midgut(PMG),express Notch ligand Delta(D1),and continue to produce doubleeffective intestinal cells(EBs).Although both ISCs and EBs express the transcription factor escargot(esg),activation of the Notch receptor in EB results in inhibition of Su(H)activation and differentiation into resorbable intestinal cells(EC)that express the POU domain transcription factor Pdm1.Importantly,a portion of Su(H)+ EB differentiates into type II enteroendocrine cells(EE)that express Prospero and specific neuropeptides.Under physiological and stress conditions,ISC division and differentiation are controlled by several pathways including JNK,Egfr / Ras / MAPK,Notch,Wnt,JAK-STAT and m TOR.The m TOR pathway is a well-known master regulator of autophagy,but its downstream effectors are unknown in the context of ISC division / differentiation.The structure and cell composition of the drosophila intestinal tract is similar to that of mammals.The midgut is the mammalian small intestine,which digests food and absorbs nutrients,and the hindgut is the mammalian large intestine.Mammalian intestinal stem cells divide and differentiate longitudinally from the crypts to the lumen near the basement membrane,and the ISC divides to produce the TA cell,followed by the differentiation of EC、EE and paneth cells,with a layer of mucosa near the lumen.The composition of the intestinal epidermal cells of the fruit fly is similar to that of the fruit fly.Intestinal stem cells are also close to the basement membrane and divide to produce EB cells,which then differentiate into intestinal epithelial cells(EC).They have bristles and polyploid nuclei,absorb nutrients,intestinal endocrine cells(EE)and secrete hormones.The drosophila gut has become a classic model for studying intestinal stem cells because of its structural similarities and genetic predominance in mammals.In recent years,lnc RNA has become an important regulator of cell processes in normal development and disease.They generally exhibit tissue and cell type-specific expression patterns and are potentially involved in biological processes such as cell fate designation and differentiation.Their role is usually in the nucleus,in the process of transcriptional responses and regulating the structure and function of chromatin.At present,to our knowledge,there are no reports of lnc RNAs specific and essential for Drosophila ISC.We wonder whether lnc RNA is also involved in regulating the proliferation and differentiation of intestinal stem cells.We found that the number of intestinal stem cells in knockout Drosophila CR43282-CR46040 knockout Drosophila was reduced compared to the control by DSS feeding on Drosophila screening,indicating that CR43282-CR46040 affects the proliferation of intestinal stem cells under the conditions of intestinal regeneration of Drosophila.After that,we found that the decrease in the number of CR43282-CR46040 knockout Drosophila strain ISC / EB cells was caused by the decrease of ISC proliferation by PH3 immunofluorescent antibody staining,indicating that CR43282-CR46040 participates in the proliferation of ISCs during intestinal regeneration.At the same time,we used complementary experiments to confirm that the absence of CR43282-CR46040 caused this phenomenon.We have confirmed through the MARCM cloning experiment that CR43282-CR46040 is involved in the proliferation of ISCs after injury.Then we examined the activation of related pathways under injury conditions,and we found that CR43282-CR46040 did indeed cause a significant reduction in JAK-STAT pathway activation.Interestingly,our research found that CR43282-CR46040 does not participate in the differentiation process of ISCs.Lnc RNA has not yet been reported on the regulation of intestinal stem cells,and this study provides further insights into the regulatory mechanism of intestinal stem cell proliferation and differentiation.
