本文選題：骨髓間充質(zhì)干細(xì)胞 + CX3CR1��； 參考：《天津醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的:在體外成功分離、培養(yǎng)及鑒定大鼠骨髓間充質(zhì)干細(xì)胞(Bone marrow me senchymal stem cells,BM MSCs)及成功利用腫瘤壞死因子-α(tumor necrosis factor-α,TNF-α)刺激結(jié)腸腺癌細(xì)胞Caco-2建立體外腸黏膜上皮損傷模型的基礎(chǔ)上,探討趨化因子受體CX3CR1在BM MSCs修復(fù)受損腸上皮細(xì)胞間緊密連接中所發(fā)揮的作用。方法:1.體外貼壁篩選法分離培養(yǎng)大鼠BM MSCs至3代,觀察BM MSCs的細(xì)胞形態(tài)、流式檢測細(xì)胞表型、成脂成骨誘導(dǎo)分化方法檢測BM MSCs的分化能力。2.使用Caco-2模擬腸黏膜上皮細(xì)胞,免疫熒光、RT-PCR和Western blot技術(shù)檢測緊密連接蛋白ZO-1和Occludin來評(píng)價(jià)Caco-2的腸道屏障功能。將TNF-α以0 ng/m L、50 ng/m L、100 ng/m L和200 ng/m L四種不同濃度處理Caco-2,探索TNF-α作用Caco-2的最佳損傷濃度;選擇造模最佳濃度點(diǎn)作用Caco-2細(xì)胞,作用時(shí)間分別為0 h、12 h、24 h和48 h,篩選TNF-α作用Caco-2的最佳損傷時(shí)間,從而摸索出建立穩(wěn)定腸黏膜上皮損傷模型的最佳條件。3.將BM MSCs與受損Caco-2通過transwell小室間接共培養(yǎng),采用RT-PCR和Western blot技術(shù)檢測Caco-2細(xì)胞上ZO-1和Occludin以及BM MSCs上CX3CR1的含量變化。4.用CX3CR1抗體孵育封閉BM MSCs上CX3CR1受體,將anti-CX3CR1-BM MSCs與受損Caco-2通過Transw ell小室共培養(yǎng),RT-PCR和Western blot技術(shù)檢測Caco-2細(xì)胞上ZO-1和Occludin的含量變化,進(jìn)一步明確CX3CR1在BM MSCs修復(fù)受損腸上皮細(xì)胞間緊密連接中所發(fā)揮的作用。結(jié)果:1.體外成功提取培養(yǎng)BM MSCs。鏡下BM MSCs貼壁成長,呈長梭形,似漩渦或菊花狀排列,具備典型MSCs的形態(tài)特征。流式檢測三代BM MSCs,93.0%的細(xì)胞表達(dá)CD90而不表達(dá)CD45,97.5%的細(xì)胞表達(dá)CD29而不表達(dá)CD34,97.0%的細(xì)胞表達(dá)RT1A而不表達(dá)RT1B,這說明我們提取的原代細(xì)胞培養(yǎng)至三代可以獲得極純的BM MSCs,幾乎沒有造血干細(xì)胞等混雜。將三代BM MSCs使用成脂誘導(dǎo)培養(yǎng)液培養(yǎng)后,油紅O染色細(xì)胞質(zhì)內(nèi)出現(xiàn)橘紅色脂滴,BM MSCs使用成骨誘導(dǎo)培養(yǎng)液培養(yǎng)后,Von Kossa染色細(xì)胞內(nèi)出現(xiàn)黑色鈣鹽沉積。這些提示我們:采用貼壁篩選法提取的BM MSCs,正是我們需要的目的細(xì)胞。2.應(yīng)用不同濃度及不同作用時(shí)間的TNF-a處理Caco-2,免疫熒光檢測可見正常Caco-2中ZO-1連接呈蜘蛛網(wǎng)狀,隨著TNF-a濃度的增加、作用時(shí)間的延長,ZO-1網(wǎng)狀結(jié)構(gòu)遭到破壞、熒光強(qiáng)度減弱,RT-PCR和Western blot檢測結(jié)果顯示隨著TNF-a作用濃度的增大、作用時(shí)間的延長,ZO-1、Occludin的表達(dá)水平降低越來越明顯,呈濃度依賴和時(shí)間依賴,而100 ng/m L和200 ng/m L TNF-a對(duì)Caco-2中ZO-1、Occlud in的表達(dá)影響差異無統(tǒng)計(jì)學(xué)意義,最終證實(shí)本實(shí)驗(yàn)條件下TNF-α的最佳造模濃度為100 ng/m L,造模時(shí)間為48 h。3.BM MSCs與受損Caco-2共培養(yǎng)后,RT-PCR結(jié)果顯示:ZO-1、Occludin m RNA的表達(dá)水平較受損Caco-2組增加,同時(shí)BM MSCs上CX3CR1 m RNA的表達(dá)量較正常BM MSCs組增加,具有統(tǒng)計(jì)學(xué)意義;Western blot結(jié)果顯示:ZO-1、Occludin蛋白的表達(dá)水平較受損Caco-2組增加,同時(shí)BM MSCs上CX3CR1蛋白的表達(dá)量較正常BM MSCs組增加,具有統(tǒng)計(jì)學(xué)意義。m RNA水平與蛋白水平結(jié)果一致。提示:BM MSCs具有修復(fù)受損腸上皮細(xì)胞間緊密連接的作用。4.Anti-CX3CR1-BM MSCs與受損Caco-2共培養(yǎng)后,RTPCR結(jié)果顯示:ZO-1、Occludin m RNA的表達(dá)量較阻斷CX3CR1前減少,但高于受損Caco-2組的表達(dá)量。Western blot結(jié)果顯示:ZO-1、Occludin蛋白的表達(dá)量較阻斷CX3CR1前減少,但高于受損Caco-2組的表達(dá)量。揭示了BM MSCs上趨化因子受體CX3CR1參與其修復(fù)受損腸上皮細(xì)胞間緊密連接。結(jié)論:1.采用貼壁篩選法可以成功提取、培養(yǎng)獲得純度高、數(shù)量多的大鼠BM MSCs,該方法簡單易行。2.在體外環(huán)境下,使用100 ng/ml TNF-α作用48h刺激Caco-2細(xì)胞,可成功建立穩(wěn)定的腸黏膜上皮損傷模型。3.BM MSCs表面CX3C R1高表達(dá)促進(jìn)受損腸黏膜上皮間緊密連接蛋白及m RNA的表達(dá),發(fā)揮保護(hù)腸黏膜上皮細(xì)胞的作用。封閉BM MSCs上CX3CR1受體后,BM MSCs修復(fù)腸上皮緊密連接蛋白及m RNA表達(dá)的作用減弱,證實(shí)CX3CR1在BM MSCs修復(fù)受損腸黏膜上皮間緊密連接中發(fā)揮作用。
[Abstract]:Objective: to successfully isolate and cultivate rat bone marrow mesenchymal stem cells (Bone marrow me senchymal stem cells, BM MSCs) and to successfully use tumor necrosis factor alpha (tumor necrosis factor- alpha, TNF- a) to stimulate colon adenocarcinoma cells in vitro. 1 the role of BM MSCs in the repair of intercellular close connections between damaged intestinal epithelial cells. Methods: 1. the BM MSCs to 3 generations of rats were isolated and cultured in vitro, and the cell morphology of BM MSCs was observed, the phenotype of the cells was detected by flow cytometry, the differentiation method of lipid forming osteogenesis was used to detect the differentiation energy of BM MSCs by using Caco-2 to simulate intestinal mucosal epithelial cells and immunization. Fluorescence, RT-PCR and Western blot techniques were used to detect close connexin ZO-1 and Occludin to evaluate the intestinal barrier function of Caco-2. TNF- alpha was treated with 0 ng/m L, 50 ng/m L, 100 ng/m and 200 different concentrations. 0 h, 12 h, 24 h and 48 h respectively, screening the optimum damage time of TNF- alpha action Caco-2, thus finding out the best condition for establishing the model of the intestinal mucosal epithelium injury, which is the indirect co culture of BM MSCs and the damaged Caco-2 through the Transwell chamber. The content change of R1.4. was incubated with CX3CR1 antibody to seal the CX3CR1 receptor on BM MSCs. The anti-CX3CR1-BM MSCs and the damaged Caco-2 were co cultured in Transw ell chamber. Results: 1. the BM MSCs adherent growth under the BM MSCs. microscope was successfully extracted and cultured in vitro, with a long shuttle shape, like a whirlpool or Chrysanthemum like arrangement, with the morphological characteristics of a typical MSCs. Flow cytometry was used to detect three generation of BM MSCs, 93% of the cells expressed CD90 but did not express CD45,97.5% in the expression of CD29 but did not express CD34,97.0% cell expression RT1A but not Up to RT1B, which indicates that the primary cells extracted from the three generation can obtain extremely pure BM MSCs, almost no hybrid hematopoietic stem cells. After the use of the three generation BM MSCs in the culture medium of lipid induced culture, the orange red lipid droplets appear in the cytoplasm of the oil red O staining, and the BM MSCs is cultured in the bone induced culture medium, and the Von Kossa staining cells appear in the cells. Black calcium salt deposition. These suggest that the BM MSCs extracted by the adherent screening method is exactly what we need for the target cell.2. to treat Caco-2 with different concentrations and different time of action of TNF-a. The immunofluorescence detection shows that the ZO-1 connection in the normal Caco-2 is spider network, with the increase of TNF-a concentration, the prolongation of the action time, the ZO-1 reticular formation. The structure was destroyed and the fluorescence intensity was weakened. The results of RT-PCR and Western blot detection showed that as the concentration of TNF-a increased, the expression level of ZO-1 and Occludin decreased more and more obviously with the concentration dependence and time dependence, while the difference of the expression of 100 ng/m L and 200 ng/m L TNF-a was no statistical difference. The optimal model concentration of TNF- alpha was 100 ng/m L and the molding time was 48 h.3.BM MSCs and the damaged Caco-2 co culture. The RT-PCR results showed that the expression level of ZO-1 and Occludin m RNA was higher than that of the damaged Caco-2 group. The results of Western blot show that the expression level of ZO-1, Occludin protein is higher than that of the damaged Caco-2 group, and the expression of CX3CR1 protein on BM MSCs is higher than that of the normal BM MSCs group. After co culture of 3CR1-BM MSCs and damaged Caco-2, the results of RTPCR showed that the expression of ZO-1, Occludin m RNA decreased compared with that before blocking CX3CR1, but higher than that of the damaged Caco-2 group. The sub receptor CX3CR1 participates in the repair of the close intercellular connection between the damaged intestinal epithelial cells. Conclusion: 1. the adherent screening method can be successfully extracted and cultured to obtain BM MSCs with high purity and a large number of rats. This method is simple and easy to use.2. to stimulate Caco-2 cells in 48h by using the action of 100 ng/ml TNF- alpha in 48h, and a stable upper lesion of the intestinal mucosa can be successfully established. The high expression of CX3C R1 on the surface of the injury model.3.BM MSCs promotes the expression of interepithelial tight connexin and m RNA in the damaged intestinal mucosa, and plays a role in protecting intestinal epithelial cells. After blocking the CX3CR1 receptor on BM MSCs, BM MSCs repair the intestinal epithelial tight connexin and the role of M Expression. It plays a role in the interskin close connection.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R574

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