本文選題：腫瘤壞死因子-α + Caco-2細胞　； 參考：《中南大學(xué)》2010年碩士論文

【摘要】：研究背景與目的 腸黏膜屏障是機體最重要的免疫防御屏障之一,它將機體與腸道內(nèi)的外源性物質(zhì)隔離開來,避免病原微生物侵襲和抗原分子的損傷。腸黏膜屏障包括腸上皮細胞屏障、免疫屏障和微生物屏障,而腸上皮細胞屏障是其最重要的一道屏障,是腸黏膜屏障具有選擇性通透的基礎(chǔ)。目前研究發(fā)現(xiàn)腸上皮細胞屏障通透性增高參與多種疾病的發(fā)生(如炎癥性腸病、敗血癥、燒傷等),并且在這些情況下,血清腫瘤壞死因子-α(TNF-α)明顯升高,與腸上皮細胞屏障的損害程度呈正相關(guān),抗TNF-α抗體可以恢復(fù)受損的腸上皮細胞屏障。因此認為TNF-α是增加腸上皮細胞屏障通透性的重要因素。雖然目前已經(jīng)形成共識TNF-α可以增加腸上皮細胞屏障的通透性,但其具體作用機制和方式還存在爭議。早期認為與TNF-α引起的細胞凋亡密切相關(guān),近年來則傾向于TNF-α引起腸上皮細胞屏障損傷是凋亡非依賴性的。另外TNF-α引起腸上皮細胞屏障通透性增高的具體調(diào)控環(huán)節(jié)也不十分清楚,它可能與蛋白激酶C(PKC)、核因子一κB(NF-κB).肌球蛋白輕鏈激酶(MLCK)、絲裂原活化的蛋白激酶(MAPK)等信號途徑有關(guān)。因此本研究利用Caco-2細胞株建立體外腸上皮細胞屏障模型,觀察TNF-α對其通透性的影響,并探討TNF-α導(dǎo)致腸上皮細胞屏障通透增加的可能機制。 研究方法 1體外腸上皮細胞屏障模型的建立應(yīng)用Caco-2細胞株建立體外腸上皮細胞屏障模型,應(yīng)用跨上皮細胞電阻(transepithelial electrical resistance,TEER)指標檢測腸上皮細胞屏障的形成情況。 2質(zhì)粒轉(zhuǎn)染及分組 待Caco-2細胞成緊密單層后無血清培養(yǎng)24h,根據(jù)有無轉(zhuǎn)染NF-K B抑制質(zhì)粒(Mu-IκB)將其分為Mu-IκB質(zhì)粒轉(zhuǎn)染組(M-TNF-α組)和Mu-IκB質(zhì)粒未轉(zhuǎn)染組(TNF-α組)。進一步根據(jù)TNF-α組預(yù)處理時間,兩組分別分為M-TNF-α0、3、6、12、24h；TNF-α0、3、6、12、24h 5個亞組,各亞組于其相應(yīng)的時間點在Transwell基底側(cè)加入100μg/L TNF-α分別孵育0、3、6、12、24h,其中Oh亞組未予TNF-α刺激。 3 TNF-α對腸上皮細胞屏障通透性的影響 加入100μg/L TNF-α作用不同時間(0、3、6、12、24h),觀察各組Caco-2細胞屏障TEER的改變。 4 TNF-α對腸上皮細胞NF-κB活性的影響 加入100μg/L TNF-α作用不同時間(0、3、6、12、24h),采用熒光素酶報告基因檢測Caco-2細胞NF-κB活性。 5 TNF-α對腸上皮細胞肌球蛋白輕鏈(myosin light chain,MLC)磷酸化的影響 加入100μg/L TNF-α作用不同時間(0、3、6、12、24h),采用蛋白質(zhì)印跡法(western blot)檢測MLC磷酸化蛋白的表達水平 6 TNF-α對腸上皮細胞F-actin的影響 加入100μg/L TNF-α作用不同時間(0、3、6、12、24h),羅丹明-鬼筆環(huán)肽直接染色免疫熒光顯微鏡下觀察Caco-2細胞間F-actin改變。 研究結(jié)果 1 TNF-α致腸上皮細胞的通透性變化 TNF-α引起Caco-2細胞TEER降低,并具有時間依賴性。TNF-α作用3h后,TEER值下降,與TNF-α0h組比較差異有顯著性降低(P0.05),TNF-α作用24h,TEER降至最低,是TNF-α0h組的68.7%。抑制NF-κB的活性(轉(zhuǎn)染Mu-IκB質(zhì)粒)后,TNF-α引起Caco-2細胞TEER降低,M-TNF-α各亞組與TNF-α相應(yīng)各亞組相比,其TEER降幅均有顯著減少(P0.05)。 2 TNF-α致腸上皮細胞NF-κB的活性變化 TNF-α3h、6h、12h組NF-κB活性增高,與TNF-α0h組比較有顯著性差異(均P0.05),其中TNF-α12h亞組NF-κB活性最高,與TNF-α0h、3h、6h、24h亞組比較有顯著性差異(均P0.05)。轉(zhuǎn)染Mu-IκB質(zhì)粒后TNF-α作用3h時NF-κB活性開始增高,與M-TNF-α0h組比較有顯著性差異(P0.05),隨作用時間延長,NF-κB活性進一步增高,12h達到最高,隨后NF-κB活性下降。M-TNF-α各亞組與TNF-α相應(yīng)各亞組相比,其NF-κB活性增高幅度均有顯著性降低(P0.05)。 3 TNF-α致腸上皮細胞MLC磷酸化水平變化 與TNF-αOh組比較,TNF-α作用6h,MLC磷酸化水平增加,轉(zhuǎn)入Mu=IκB質(zhì)粒后,TNF-α作用6h,MLC磷酸化水平無明顯改變,作用12h才出現(xiàn)MLC磷酸化增加。顯示降低NF-κB活性后TNF-α引起MLC磷酸化作用延遲。 4 TNF-α致腸上皮細胞間F-actin變化 正常CaCo-2細胞,緊密連接結(jié)構(gòu)完整,呈致密的帶狀結(jié)構(gòu)。加入100μg/L TNF-α作用12h細胞外周致密帶邊緣變得毛糙不規(guī)整,細胞熒光染色減弱,作用24h細胞外周輪廓模糊難辨,逐漸出現(xiàn)鋸齒樣斷裂,趨于變細崩解消散。轉(zhuǎn)染Mu-IκB質(zhì)粒后,M-TNF-α3、6、12h組Caco-2細胞的F-actin無明顯改變,M-TNF-α24h組細胞外周致密帶邊緣開始變得毛糙不規(guī)整,細胞熒光染色減弱。 研究結(jié)論 腸上皮細胞MLC磷酸化及F-actin重組是TNF-α致其通透性增高的機制之一,此過程可能受NF-κB的調(diào)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.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R363

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