Numb分子在肠粘膜屏障形成中的作用及其机制研究

发布时间：2018-02-16 13:40

本文关键词： Numb 肠粘膜屏障 Notch信号通路粘蛋白2 顶端连接复合体肌球蛋白调节性轻链微丝骨架　出处：《第三军医大学》2012年博士论文　论文类型：学位论文

【摘要】：背景＆目的肠黏膜屏障包括:机械屏障、免疫屏障、化学屏障和生物屏障。肠粘膜粘液屏障是上皮细胞表面覆盖的由粘蛋白形成的水化的凝胶样粘液，而肠黏膜上皮屏障是肠上皮本身及其紧密连接构成的在体内外环境之间形成一种组织学屏障，它们都具有机械性的保护作用。Numb分子因其能够不对称的分布到干细胞的两个子代细胞，并能够通过抑制Notch信号通路而使子代细胞具有不同的“命运”而引起人们的广泛关注。既往研究显示Numb表达在包括乳腺，肺，，睾丸，唾液腺等在内的大多数组织上，但Numb在肠上皮细胞的表达及其功能目前还没有报道。我们预实验发现Numb并非表达在肠隐窝底干细胞上，而是普遍的表达在沿着隐窝-绒毛轴的所有上皮细胞上。既往研究发现，Numb能够调节细胞的分化，调节细胞粘附，细胞迁移和细胞极性。在肠粘膜上皮细胞上，Numb是否能够通过抑制Notch通路促进肠上皮细胞粘蛋白Muc2的分泌及肠粘膜屏障的形成，以及Numb能否通过调节肠上皮细胞间顶端连接复合体(Apicaljunctional complex，AJC）的组装而维持肠粘膜上皮细胞屏障的完整性目前还不清楚。围绕以上问题，本研究揭示了Numb分子与Notch通路相关分子Hes1，Hath1的表达定位关系。通过干扰Numb分子的表达揭示了其在肠上皮细胞中对Notch通路的抑制作用，及其对肠上皮细胞LS174T细胞杯状细胞表型的影响。利用Caco-2单层细胞模型，揭示了Numb在肠上皮单层细胞屏障形成中的作用，并揭示了其通过抑制肌球蛋白轻链磷酸化及改变细胞微丝骨架的结构影响上皮细胞屏障通透性的作用机制。方法 1．采用RT-PCR和Western blot的方法检测Numb-PRRL和Numb-PRRS在肠上皮细胞系和肠粘膜细胞的表达，免疫组织化学检测Numb，Hes1，Hath1，Muc2在小鼠肠粘膜上皮细胞分布，揭示它们在肠粘膜上皮细胞的定位关系，并通过AB染色显示其与杯状细胞的定位关系。 2．构建干扰质粒pRNAT-U6.1-shRNA-Numb，转染LS174T细胞，筛选稳定转染细胞克隆，荧光素酶质粒pGa981-6检测干扰Numb表达对Notch活性的影响，及其下游靶基因Hes1，Hath1表达的影响。 3．MTT实验检测干扰Numb表达对细胞增殖的影响，RT-PCR，Western blot及免疫荧光染色检测Numb对Muc2表达的影响，PAS染色检测Numb对LS174T细胞粘液分泌的影响。 4．RT-PCR和Western blot的方法检测Numb在肠上皮细胞系Caco-2中的表达，免疫荧光染色检测其与粘附连接分子ZO-1，E-cadherin及Par3的表达定位关系，免疫共沉淀检测其与E-cadherin及Par3之间的相互作用。 5．干扰Numb分子的表达，TEER和FITC-Dextran渗透实验检测Numb分子在肠单层上皮细胞屏障通透性中的作用。钙转换实验和TNF-a/INF-γ刺激实验验证Numb在AJC组装和维持细胞连接完整中的作用。 6．免疫荧光染色检测抑制Numb表达对钙转换试验前后Caco-2细胞F-actin结构的影响，Western blot检测在生理和病理条件下，抑制Numb表达对细胞连接相关蛋白ZO-1，Occludin，E-cadhern，β-catenin，Claudin-1，Claudin-2的表达变化，并检测对Caco-2细胞肌球蛋白轻链磷酸化水平的影响。 7．DSS诱导小鼠溃疡性结肠炎动物模型，HE染色检测结肠组织形态变化，免疫荧光染色检测炎症条件下Numb在结肠上皮细胞的表达变化。结果 1．Numb-PRRL和Numb-PRRS亚型在肠上皮细胞系和肠粘膜上皮细胞内都有表达， Numb与Notch通路相关分子Hes1，Hath1在肠粘膜上皮细胞存在共定位关系，与Muc2和AB染色阳性的杯状细胞也有共定位关系。 2．序列测定证实pRNAT-U6.1-shRNA-Numb成功构建，转染并经G418筛选出稳定细胞克隆，荧光素酶质粒pGa981-6检测发现Numb能够抑制Notch通路活性，抑制Hes1表达，促进Hath1表达。 3．Numb抑制肠上皮细胞增殖；促进LS174T表达杯状细胞分子标志物Muc2的表达，PAS染色发现Numb促进LS174T细胞粘液的分泌。 4．肠上皮细胞系Caco-2表达Numb，Numb与ZO-1，E-cadherin及Par3分子共定位，IP检测发现Numb与E-cadherin和Par3之间存在相互作用。 5．抑制Numb分子的表达能够增加Caco-2上皮细胞屏障通透性。钙转换和TNF-a/INF-γ刺激实验证实Numb在AJC组装和维持细胞连接完整性中发挥作用。 6．抑制Numb表达能够改变钙转换试验前后Caco-2细胞F-actin的结构；Numb对正常和炎症条件下Caco-2细胞内ZO-1，Occludin，E-cadhern，β-catenin，Claudin-1的蛋白水平没有影响，但能够升高Claudin-2的水平；Numb能够抑制Caco-2细胞肌球蛋白轻链磷酸化。 7．HE染色发现DSS成功诱导小鼠结肠炎症反应，炎症条件下Numb在结肠上皮细胞的表达减少，且在细胞内分布紊乱。结论 Numb能够通过抑制Notch通路促进肠上皮细胞粘蛋白Muc2的表达及粘液的分泌，参与肠粘膜粘液屏障的形成；同时Numb通过抑制肌球蛋白轻链的磷酸化，调节肠上皮细胞间AJC的组装，维持肠粘膜上皮细胞屏障的完整性，参与肠粘膜机械屏障的形成。
[Abstract]:Background & purpose
The intestinal mucosal barrier includes: mechanical barrier, immune barrier, chemical barrier and biological barrier. Intestinal mucus barrier is formed by covering the surface of the epithelial mucin hydration gel like mucus, and intestinal epithelial barrier is the intestinal epithelial tight junction in itself and between the in vivo environment forming a tissue barrier and they all have the protective effect of.Numb for the distribution of the molecular machinery to asymmetric stem cell into two daughter cells, and through inhibition of the Notch signaling pathway to daughter cells with different "fate" and caused widespread concern. Previous studies showed that the expression of Numb in breast, lung and testis. Most of the salivary gland, tissue, but the expression of Numb in intestinal epithelial cells and its function has not been reported to date. We found that Numb is not expressed in the pre experimental intestinal crypt stem cells on the bottom But, generally expressed in all epithelial cells along the crypt villus axis. Previous studies have found that Numb can regulate cell differentiation, regulating cell adhesion, cell migration and cell polarity in epithelial cells of intestinal mucosa, whether Numb formation by secreting and intestinal mucosa barrier inhibiting Notch pathway to promote intestinal epithelial cell adhesion protein Muc2, and the Numb can pass through the apical junctional complex regulation between intestinal epithelial cells (Apicaljunctional complex, AJC) of the assembly and to maintain the integrity of the intestinal mucosal epithelial barrier is unclear.
Based on the above problems, this study reveals that Numb molecules associated with Notch pathway molecules Hes1, Hath1 expression and localization. By interfering expression of Numb molecules reveals its inhibitory effect on Notch pathway in intestinal epithelial cells, and its effect on the phenotype of intestinal epithelial cells in LS174T cells the number of goblet cells. The Caco-2 cell monolayer model, reveal the formation of Numb in the intestinal epithelial cell monolayer barrier function, and reveals its structure through inhibition of myosin light chain phosphorylation and actin cytoskeleton change effect of epithelial cell barrier permeability.
Method
1. with the methods of RT-PCR and Western blot to detect the expression of Numb-PRRL and Numb-PRRS in intestinal epithelial cells and intestinal mucosa cells, immunohistochemical detection of Numb, Hes1, Hath1, Muc2 in the distribution of epithelial cells in the intestinal mucosa of mice, reveal their relationship in the localization of intestinal epithelial cells, and through AB staining and goblet positioning the relationship between the cells.
2., we constructed interference plasmid pRNAT-U6.1-shRNA-Numb, transfected LS174T cells, and screened stable transfected cell clones. Luciferase plasmid pGa981-6 was used to detect the effect of Numb expression on Notch activity and the effect of downstream target gene Hes1 and Hath1 expression.
3.MTT assay was used to detect the effect of Numb interference on cell proliferation. RT-PCR, Western blot and immunofluorescence staining were used to detect the effect of Numb on Muc2 expression. PAS staining was used to detect the effect of Numb on mucus secretion of LS174T cells.
4.RT-PCR and Western blot were used to detect the expression of Numb in intestinal epithelial cell Caco-2. Immunofluorescence staining was used to detect the relationship between the expression of Numb and ZO-1, E-cadherin and Par3, and the interaction between E-cadherin and Par3 was detected by immunoprecipitation.
5., interfere with the expression of Numb, TEER and FITC-Dextran permeability test to detect the role of Numb molecules in barrier permeability of intestinal epithelial cells. Calcium transformation test and TNF-a/INF- gamma stimulation test verify the role of Numb in AJC assembly and maintenance of cell integrity.
6. immunofluorescence staining to inhibit the expression of Numb on calcium conversion of Caco-2 cells F-actin the influence of the structure before and after experiment, Western blot detection in physiological and pathological conditions and inhibition of Numb on the expression of cell junction proteins ZO-1, Occludin, E-cadhern, beta -catenin, Claudin-1, Claudin-2 expression, and detect the effect on Caco-2 cells of myosin light chain phosphorylation the level.
7.DSS induced animal model of ulcerative colitis in mice, HE coloring was used to detect colonic tissue morphology, and immunofluorescence staining was used to detect the expression of Numb in colonic epithelial cells under inflammatory condition.
Result
1.Numb-PRRL and Numb-PRRS subtypes are expressed in intestinal epithelial cells and intestinal epithelial cells. Numb and Notch pathway related molecules Hes1 and Hath1 have colocalization relationship in intestinal epithelial cells, and have co localization with Muc2 and AB stained goblet cells.
2. sequencing confirmed that pRNAT-U6.1-shRNA-Numb was successfully constructed, transfected and screened stable cell clone by G418. Luciferase plasmid pGa981-6 showed that Numb could inhibit Notch pathway activity, inhibit Hes1 expression and promote Hath1 expression.
3.Numb inhibited the proliferation of intestinal epithelial cells, and promoted LS174T expression of Muc2, a marker of goblet cell. PAS staining showed that Numb promoted mucus secretion in LS174T cells.
4. the intestinal epithelial cell line Caco-2 expressed Numb, and Numb was Co located with ZO-1, E-cadherin and Par3 molecules. IP detected the interaction between Numb and E-cadherin and Par3.
5. inhibition of Numb expression can increase the permeability of Caco-2 epithelial barrier. Calcium transformation and TNF-a/INF- gamma stimulation test confirm that Numb plays a role in AJC assembly and maintenance of cell integrity.
6. inhibition of Numb expression can change the structure of Caco-2 F-actin cell calcium conversion test before and after Numb; on ZO-1, Caco-2 cells in normal and inflammatory conditions, Occludin, E-cadhern, beta -catenin, did not affect the protein level of Claudin-1, but can increase the level of Claudin-2; Numb can inhibit Caco-2 cell myosin light chain phosphorylation.
7.HE staining showed that DSS successfully induced colonic inflammation in mice. The expression of Numb in colonic epithelial cells decreased in inflammatory conditions and was disordered in the cells.
conclusion
Numb can inhibit the Notch pathway to promote the secretion of intestinal epithelial cells and mucus expression of mucin Muc2, involved in the formation of intestinal mucus barrier; at the same time Numb by inhibiting the phosphorylation of myosin light chain assembly, regulation of intestinal epithelial cells AJC, to maintain the integrity of intestinal epithelial cell barrier, involved in the formation of intestinal mucosal mechanical barrier.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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