肿瘤坏死因子-α对肠上皮细胞通透性的影响及机制研究

发布时间：2018-05-11 21:45

本文选题：肿瘤坏死因子-α + Caco-2细胞　；参考：《中南大学》2010年硕士论文

【摘要】：研究背景与目的肠黏膜屏障是机体最重要的免疫防御屏障之一,它将机体与肠道内的外源性物质隔离开来,避免病原微生物侵袭和抗原分子的损伤。肠黏膜屏障包括肠上皮细胞屏障、免疫屏障和微生物屏障,而肠上皮细胞屏障是其最重要的一道屏障,是肠黏膜屏障具有选择性通透的基础。目前研究发现肠上皮细胞屏障通透性增高参与多种疾病的发生(如炎症性肠病、败血症、烧伤等),并且在这些情况下,血清肿瘤坏死因子-α(TNF-α)明显升高,与肠上皮细胞屏障的损害程度呈正相关,抗TNF-α抗体可以恢复受损的肠上皮细胞屏障。因此认为TNF-α是增加肠上皮细胞屏障通透性的重要因素。虽然目前已经形成共识TNF-α可以增加肠上皮细胞屏障的通透性,但其具体作用机制和方式还存在争议。早期认为与TNF-α引起的细胞凋亡密切相关,近年来则倾向于TNF-α引起肠上皮细胞屏障损伤是凋亡非依赖性的。另外TNF-α引起肠上皮细胞屏障通透性增高的具体调控环节也不十分清楚,它可能与蛋白激酶C(PKC)、核因子一κB(NF-κB).肌球蛋白轻链激酶(MLCK)、丝裂原活化的蛋白激酶(MAPK)等信号途径有关。因此本研究利用Caco-2细胞株建立体外肠上皮细胞屏障模型,观察TNF-α对其通透性的影响,并探讨TNF-α导致肠上皮细胞屏障通透增加的可能机制。研究方法 1体外肠上皮细胞屏障模型的建立应用Caco-2细胞株建立体外肠上皮细胞屏障模型,应用跨上皮细胞电阻(transepithelial electrical resistance,TEER)指标检测肠上皮细胞屏障的形成情况。 2质粒转染及分组待Caco-2细胞成紧密单层后无血清培养24h,根据有无转染NF-K B抑制质粒(Mu-IκB)将其分为Mu-IκB质粒转染组(M-TNF-α组)和Mu-IκB质粒未转染组(TNF-α组)。进一步根据TNF-α组预处理时间,两组分别分为M-TNF-α0、3、6、12、24h；TNF-α0、3、6、12、24h 5个亚组,各亚组于其相应的时间点在Transwell基底侧加入100μg/L TNF-α分别孵育0、3、6、12、24h,其中Oh亚组未予TNF-α刺激。 3 TNF-α对肠上皮细胞屏障通透性的影响加入100μg/L TNF-α作用不同时间(0、3、6、12、24h),观察各组Caco-2细胞屏障TEER的改变。 4 TNF-α对肠上皮细胞NF-κB活性的影响加入100μg/L TNF-α作用不同时间(0、3、6、12、24h),采用荧光素酶报告基因检测Caco-2细胞NF-κB活性。 5 TNF-α对肠上皮细胞肌球蛋白轻链(myosin light chain,MLC)磷酸化的影响加入100μg/L TNF-α作用不同时间(0、3、6、12、24h),采用蛋白质印迹法(western blot)检测MLC磷酸化蛋白的表达水平 6 TNF-α对肠上皮细胞F-actin的影响加入100μg/L TNF-α作用不同时间(0、3、6、12、24h),罗丹明-鬼笔环肽直接染色免疫荧光显微镜下观察Caco-2细胞间F-actin改变。研究结果 1 TNF-α致肠上皮细胞的通透性变化 TNF-α引起Caco-2细胞TEER降低,并具有时间依赖性。TNF-α作用3h后,TEER值下降,与TNF-α0h组比较差异有显著性降低(P0.05),TNF-α作用24h,TEER降至最低,是TNF-α0h组的68.7%。抑制NF-κB的活性(转染Mu-IκB质粒)后,TNF-α引起Caco-2细胞TEER降低,M-TNF-α各亚组与TNF-α相应各亚组相比,其TEER降幅均有显著减少(P0.05)。 2 TNF-α致肠上皮细胞NF-κB的活性变化 TNF-α3h、6h、12h组NF-κB活性增高,与TNF-α0h组比较有显著性差异(均P0.05),其中TNF-α12h亚组NF-κB活性最高,与TNF-α0h、3h、6h、24h亚组比较有显著性差异(均P0.05)。转染Mu-IκB质粒后TNF-α作用3h时NF-κB活性开始增高,与M-TNF-α0h组比较有显著性差异(P0.05),随作用时间延长,NF-κB活性进一步增高,12h达到最高,随后NF-κB活性下降。M-TNF-α各亚组与TNF-α相应各亚组相比,其NF-κB活性增高幅度均有显著性降低(P0.05)。 3 TNF-α致肠上皮细胞MLC磷酸化水平变化与TNF-αOh组比较,TNF-α作用6h,MLC磷酸化水平增加,转入Mu=IκB质粒后,TNF-α作用6h,MLC磷酸化水平无明显改变,作用12h才出现MLC磷酸化增加。显示降低NF-κB活性后TNF-α引起MLC磷酸化作用延迟。 4 TNF-α致肠上皮细胞间F-actin变化正常CaCo-2细胞,紧密连接结构完整,呈致密的带状结构。加入100μg/L TNF-α作用12h细胞外周致密带边缘变得毛糙不规整,细胞荧光染色减弱,作用24h细胞外周轮廓模糊难辨,逐渐出现锯齿样断裂,趋于变细崩解消散。转染Mu-IκB质粒后,M-TNF-α3、6、12h组Caco-2细胞的F-actin无明显改变,M-TNF-α24h组细胞外周致密带边缘开始变得毛糙不规整,细胞荧光染色减弱。研究结论肠上皮细胞MLC磷酸化及F-actin重组是TNF-α致其通透性增高的机制之一,此过程可能受NF-κB的调控。
[Abstract]:Research background and purpose
Intestinal mucosal barrier is one of the most important immune defense barriers in the body. It isolates the organism from the exogenous substances in the intestinal tract and avoids the invasion of pathogenic microorganisms and the damage of the antigen molecules. Intestinal mucosal barrier includes intestinal epithelial cell barrier, immune barrier and microbial barrier, and intestinal epithelial cell barrier is the most important barrier. It is the basis for selective permeability of intestinal mucosal barrier. It is found that the increase of barrier permeability of intestinal epithelial cells is involved in the occurrence of various diseases (such as inflammatory bowel disease, septicemia, and burn). In these cases, the serum tumor necrosis factor - alpha (TNF- alpha) is significantly elevated, and is positively related to the damage of the intestinal epithelial cell barrier, and is resistant to the damage of the intestinal epithelial cell barrier. TNF- alpha antibody can restore the damaged intestinal epithelial barrier. Therefore, it is considered that TNF- alpha is an important factor in increasing the permeability of intestinal epithelial barrier. Although the consensus that TNF- alpha can increase the permeability of the intestinal epithelial cell barrier, the specific mechanisms and ways of its action are still controversial. It was believed that the cell withering caused by TNF- alpha was early. It is closely related to death. In recent years, TNF- alpha induced intestinal epithelial barrier damage is not dependent on apoptosis. In addition, the specific regulatory link that TNF- alpha induces the increase of intestinal epithelial barrier permeability is not very clear. It may be associated with protein kinase C (PKC), nuclear factor kappa B (NF- kappa B), myosin light chain kinase (MLCK) and mitogen activated mitogen Protein kinase (MAPK) and other signaling pathways are related. Therefore, this study uses Caco-2 cell lines to build a stereoscopic intestinal epithelial cell barrier model, to observe the effect of TNF- alpha on its permeability, and to explore the possible mechanism of the increase of the permeability of the intestinal epithelial cell barrier by TNF- alpha.
research method
1 the establishment of an in vitro intestinal epithelial cell barrier model, the Caco-2 cell line was used to build a stereoscopic intestinal epithelial cell barrier model, and the formation of intestinal epithelial barrier was detected by the cross epithelial cell resistance (transepithelial electrical resistance, TEER).
2 plasmids transfection and grouping
After Caco-2 cells became compact monolayers, 24h was cultured without serum. According to the transfection of NF-K B suppressing plasmid (Mu-I kappa B), it was divided into Mu-I kappa B plasmid transfection group (M-TNF- Alpha Group) and Mu-I kappa B plasmid untransfected group (TNF- Alpha Group). Further, the two groups were divided into 5 subgroups according to the pretreatment time of alpha group. At the corresponding time point, the subgroup was incubated with 0,3,6,12,24h with 100 mu g/L TNF- alpha at the basal side of Transwell, and the Oh subgroup was not stimulated by TNF- alpha.
The effect of 3 TNF- alpha on the permeability of intestinal epithelial cell barrier
The changes of Caco-2 cell barrier TEER in each group were observed at different time (0,3,6,12,24h) after adding 100 g/L TNF- alpha.
Effect of 4 TNF- alpha on the activity of NF- kappa B in intestinal epithelial cells
The NF- kappa B activity of Caco-2 cells was detected by luciferase reporter gene at different time (0,3,6,12,24h) after adding 100 mu g/L TNF- alpha.
Effect of 5 TNF- alpha on phosphorylation of myosin light chain (MLC) in intestinal epithelial cells
The expression level of MLC phosphorylated protein was detected by Western blotting (Western blot) at 100 0,3,6,12,24h g/L TNF-.
The effect of 6 TNF- alpha on the F-actin of intestinal epithelial cells
After adding 100 mu g/L TNF- 0,3,6,12,24h for different time (0,3,6,12,24h), Luo Danming phallin cyclic peptide was directly stained with F-actin immunofluorescence microscope to observe the change of F-actin between cells.
Research results
Permeability change of 1 TNF- - alpha induced intestinal epithelial cells
TNF- alpha induces the decrease of TEER in Caco-2 cells, and the time dependent.TNF- alpha action 3h, the TEER value decreases, and the difference between the TNF- alpha 0h group and the TNF- alpha 0h group is significantly lower (P0.05). The TNF- alpha action is 24h and the TEER decreases to the lowest. Compared with the TNF- subgroups, the TEER decreased significantly (P0.05).
Changes of activity of NF- kappa B in 2 TNF- - alpha induced intestinal epithelial cells
The activity of NF- kappa B in the TNF- alpha 3h, 6h and 12h group was higher than that of the TNF- alpha 0h group (all P0.05), and the NF- kappa of TNF- alpha 12h subgroup had the highest activity. With the prolongation of action time, the activity of NF- kappa B increased further and the 12h reached the highest, then the NF- kappa B activity decreased in.M-TNF- A and the NF- kappa B activity increased significantly compared with the TNF- alpha subgroups (P0.05).
Changes of MLC phosphorylation in 3 TNF- alpha induced intestinal epithelial cells
Compared with the TNF- alpha Oh group, TNF- alpha acts on 6h, the phosphorylation level of MLC increases, and after the transfer of Mu=I kappa B plasmid, TNF- alpha acts on 6h, and the phosphorylation level of MLC does not change obviously, and MLC phosphorylation increases.
Changes of F-actin between 4 TNF- alpha induced intestinal epithelial cells
The normal CaCo-2 cells were tightly connected with a compact band structure. Adding 100 mu g/L TNF- alpha, the peripheral dense zone of 12h cells became rough and irregular, and the fluorescent staining of the cells weakened. The outer circumference of 24h cells was blurred, and the serrated fracture gradually appeared, and it tended to disintegrate and dissipate. After transfection of Mu-I kappa B plasmid, M-TNF- a 3, There was no significant change in the F-actin of Caco-2 cells in group 6,12h, and the margins of the peripheral dense zone of M-TNF- 24h group began to become rough and irregular, and the fluorescence staining of cells decreased.
research conclusion
Phosphorylation of MLC and recombination of F-actin in intestinal epithelial cells are one of the mechanisms by which TNF- alpha increases its permeability, which may be regulated by NF- B.

【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

【参考文献】