小鼠角膜基质细胞的间充质干细胞样表型和多向分化潜能以及抑制树突状细胞成熟的功能
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摘要
角膜基质细胞(corneal stroma cells, CSCs)是散在分布于角膜基质内神经嵴来源的细胞,对维持角膜透明性发挥着重要作用。在体CSCs数量稀少,所以在体外对细胞进行培养扩增是必经的研究路径。研究表明,培养于含胎牛血清(FBS)的完全培养基内的CSCs会丧失其原有的生物学特性。然而,当培养于无血清的基础培养基时,细胞虽能保持其特性不变,却无法进行有效增殖。因此,如何在保持细胞生物学特性不变的情况下高效扩增CSCs,是目前研究难点之一。
研究表明,出生后增殖性CSCs的细胞数量会迅速减少。当睑裂打开后,所有CSCs的细胞周期进入G0期。最近研究证实,CSCs表达众多干细胞标记物,并且具有多向分化潜能,与间充质干细胞的生物学特性十分相似。然而,目前尚缺乏小鼠CSCs是否具有间充质干细胞特性的研究。
树突状细胞(dendritic cells, DCs)是目前已知体内功能最强的抗原呈递细胞。成熟DCs可引发机体免疫反应,而未成熟DCs则会诱导机体免疫耐受。而且,角膜内的DCs广泛的参与了多种角膜相关疾病以及角膜移植免疫排斥反应,且以角膜内DCs为靶细胞的治疗方法已取得可喜的疗效。因此,对角膜内的DCs,尤其对DCs成熟状态的研究具有重要意义。
最近研究表明,位于角膜中央区的DCs完全处于未成熟状态,而位于角膜周边区的DCs则大多处于成熟状态。局部微环境对DCs的成熟状态发挥着重要的调节功能。所以,我们推测CSCs可能具有影响角膜内DCs成熟状态的功能,然而至今尚未见相关报道。
因此,本研究旨在探索如何在体外有效扩增小鼠CSCs以及对CSCs的间充质干细胞特性和抑制DCs成熟的功能进行探讨。如下:
第一部分小鼠角膜基质细胞的提取、鉴定以及培养扩增
目的:研究使用KSFM培养基能否获取具有增殖能力且保持生物学特性不变的小鼠CSCs。
方法:将中央区角膜置于EDTA液(20mmol/L)内孵育45min后,用手术显微镊小心剥离角膜上皮层以及内皮层,并将获取的角膜基质置于含300U/mL I型胶原酶的溶液中消化4h。离心后采用DMEM基础培养基、DMEM完全培养基(含10% FBS)以及KSFM培养基重悬细胞,接种于培养瓶内常规培养,并采用含1U/mL分散酶的EDTA液消化传代细胞。同时,观察细胞并绘制细胞生长曲线;采用逆转录聚合酶链式反应(RT-PCR)检测细胞角膜蛋白多糖(keratocan)、乙醛脱氢酶(ALDH)、细胞角蛋白12(CK12)和神经元特异性烯醇化酶(NSE)等基因的表达情况;采用细胞免疫荧光染色以及蛋白质印迹方法检测细胞keratocan蛋白的表达情况。
结果:通过胶原酶消化的方法可以从每只小鼠的角膜基质获取约1×104单个细胞。RT-PCR结果显示:原代细胞表达CSCs标记物keratocan和ALDH,不表达角膜上皮细胞标记物CK12以及角膜内皮细胞标记物NSE;免疫荧光染色和蛋白质印迹结果显示:原代细胞表达keratocan蛋白。因此,本实验获取的原代细胞为CSCs。培养于DMEM基础培养基内的原代CSCs无法增殖。培养于DMEM完全培养基内的CSCs可增殖,但第3代细胞不表达keratocan和ALDH基因以及keratocan蛋白。培养于KSFM培养基内的CSCs也可增殖,第3代细胞仍表达keratocan和ALDH基因以及keratocan蛋白,且与原代细胞相比,表达强度无统计学差异(P>0.05)。
结论:KSFM培养基不仅能维持小鼠CSCs的生物学特性不变,还能有效促进细胞增殖。
第二部分小鼠角膜基质细胞的间充质干细胞样表型以及多向分化潜能
目的:研究KSFM培养基培养扩增后的小鼠CSCs是否具有间充质干细胞样表型以及多向分化潜能。
方法:在去除角膜上皮层以及内皮层后,通过胶原酶消化的方法获取小鼠中央区角膜来源的CSCs,并采用KSFM培养基对其培养扩增。收集第2代CSCs,将细胞与造血干细胞标记物抗体(CD34-FITC、CD45-PE)以及间质细胞标记物抗体(CD105-PE、CD90-FITC、CD71-FITC、CD29-APC)共孵育30min后,应用流式细胞技术进行检测。当培养于KSFM培养基内的CSCs达细胞融合后,更换成骨细胞诱导培养基(含10%FBS、100nmol/L地塞米松、10mmol/Lβ-磷酸甘油、50mg/L维生素C的DMEM培养基)、脂肪细胞诱导培养基(含10% FBS、0.5μmol/L地塞米松、0.5mmol/L 3-异丁基-1-甲基黄嘌呤、10mg/L胰岛素的DMEM培养基)以及对照培养基(含10% FBS的DMEM培养基),进行常规培养,每3d更换一次培养基。21d后,对培养于成骨细胞诱导培养基以及对照培养基内的细胞进行2%茜素红S染色,并通过RT-PCR检测细胞碱性磷酸酶和骨钙素等基因的表达情况;对培养于脂肪细胞诱导培养基以及对照培养基内的细胞进行0.3%油红O染色,并通过RT-PCR检测细胞脂蛋白脂酶和过氧化物酶增殖物激活受体γ等基因的表达情况。
结果:应用流式细胞技术对第2代CSCs的表型特征进行分析,结果显示:细胞低表达CD34(3.68%±1.44%)以及CD45(9.56%±1.83%),高表达CD29(96.85%±1.91%)、CD90(93.62%±1.65%)、CD105(50.91%±2.56%)以及CD71(45.27%±3.56%)。在成骨诱导条件下,3d时,细胞形态仍然保持梭形,与对照组细胞无明显差别。7d时,细胞形态逐渐转变为多角形,胞浆内出现黑色颗粒。14d时,开始形成矿化结节,并逐渐增大,21d时,经茜素红S染色,结节呈现鲜红色。对照组细胞未显现出以上成骨细胞分化的形态学征象,且经茜素红S染色未见阳性结果。通过RT-PCR检测成骨细胞标记物基因的表达情况,结果显示:成骨诱导条件下细胞高表达碱性磷酸酶和骨钙素,而对照组细胞低表达碱性磷酸酶且不表达骨钙素。在脂肪诱导条件下,7d时,细胞形态逐渐由梭形转变为类圆形,胞浆内液滴也逐渐增多。14d时,细胞胞浆内满布液滴,经油红O染色,液滴被特异性染成橘红色。RT-PCR结果显示:脂肪诱导条件下细胞表达脂蛋白脂酶和过氧化物酶增殖物激活受体γ。而对照组细胞未显现出向脂肪细胞分化的任何征象。
结论:经KSFM培养基培养扩增的小鼠中央区角膜来源的CSCs具有与间充质干细胞相似的表型特征,以及向成骨细胞和脂肪细胞分化的能力。
第三部分小鼠角膜基质细胞培养上清液对树突状细胞成熟的抑制作用
目的:研究小鼠CSCs培养上清液是否具有抑制脂多糖诱导的DCs成熟的作用。
方法:通过尼龙毛柱法获取BALB/c小鼠脾脏来源的T细胞,并通过流式细胞技术检测细胞表面标记物CD3以测定T细胞纯度。原代小鼠CSCs(105/mL)培养于RPMI 1640基础培养基内,3d后半量换液,6d后收集培养上清液以备用。在裂解红细胞后,将由C57BL/6小鼠股骨获取的骨髓单核细胞培养于含10% FBS以及10ng/mL重组小鼠粒细胞-巨噬细胞集落刺激因子的RPMI 1640培养基内,2d后全量换液,4d后半量换液,6d后收集悬浮和半贴壁细胞,即为未成熟DCs。通过流式细胞技术检测细胞表面标记物CD11c以测定DCs纯度。向DCs培养液内加入脂多糖(1μg/mL),48h后未成熟DCs可被诱导成熟。为研究CSCs培养上清液对DCs成熟的作用,在DCs成熟过程中,不同浓度的培养上清液(25%、50%)被添加至DCs培养液中。而后,通过流式细胞技术检测DCs成熟状态标记物CD80、CD86和主要组织相容性抗原Ⅱ类分子(MHC-Ⅱ),以对DCs的表型成熟状态进行鉴定;通过混合淋巴细胞反应检测DCs刺激T细胞增殖能力以及通过FITC标记葡聚糖内吞实验检测抗原吞噬功能,以对DCs的功能成熟状态进行鉴定。
结果:小鼠脾脏细胞经红细胞裂解以及尼龙毛柱筛选提取后,可得到大量单个悬浮的小细胞。经流式细胞技术检测,细胞高表达T细胞标记物CD3(93.97%±3.06%)。小鼠骨髓单核细胞诱导培养6d后,细胞集落明显,呈悬浮或半贴壁生长。细胞表面可见长短不一的毛刺状突起,且高表达CD11c(78.61%±4.27%),低表达CD80、CD86和MHC-Ⅱ。细胞经脂多糖刺激48h后,CD80、CD86和MHC-Ⅱ的表达明显上调。在DCs成熟过程中,将不同浓度的CSCs培养上清液(25%、50%)添加至DCs培养液后,与对照组相比,DCs CD80、CD86和MHC-Ⅱ的表达均降低(P<0.01),CD11c的表达无明显差异(P>0.05);刺激T细胞增殖能力降低(P<0.05);抗原吞噬功能增强(P<0.01)。此外,CSCs培养上清液抑制DCs成熟的作用还呈现出剂量依赖性(25% vs. 50%, P<0.05)。
结论:小鼠CSCs培养上清液可以抑制脂多糖诱导的DCs表型以及功能成熟,且呈剂量依赖性。因此,我们推测CSCs可以通过分泌可溶性免疫调节因子抑制DCs成熟。
第四部分小鼠角膜基质细胞通过分泌转化生长因子β2以及前列腺素E2抑制树突状细胞成熟
目的:探索小鼠CSCs是否通过分泌转化生长因子β2(TGF-β2)、前列腺素E2(PGE2)、白介素10(IL-10)以及巨噬细胞集落刺激因子(M-CSF)抑制DCs成熟。
方法:采用RT-PCR检测原代小鼠CSCs TGF-β2、IL-10、M-CSF以及前列腺素内过氧化物合酶2(PTGS2)等基因的表达情况。据此,通过酶联免疫吸附实验(ELISA)测定CSCs培养上清液以及新鲜RPMI 1640培养基内PGE2和TGF-β2的含量。而后,通过应用TGF-β2中和抗体(15μg/mL)以及PGE2受体阻滞剂AH6809(100μmol/L),对CSCs是否通过分泌TGF-β2以及PGE2抑制DCs成熟作进一步鉴定。在DCs成熟过程中,分别作以下不同处理:1,LPS;2,LPS+50% CSCs培养上清液;3,LPS+50% CSCs培养上清液+AH6809;4,LPS+50% CSCs培养上清液+中和抗体;5,LPS+50% CSCs培养上清液+AH6809+中和抗体。然后,应用流式细胞技术检测DCs CD11c、CD80、CD86和MHC-Ⅱ的表达情况,通过混合淋巴细胞反应检测刺激T细胞增殖能力,以及通过FITC标记葡聚糖内吞实验检测抗原吞噬功能。
结果:RT-PCR结果表明:原代小鼠CSCs高表达TGF-β2和PTGS2,低表达M-CSF,不表达IL-10;ELISA数据显示:与新鲜RPMI 1640培养基相比,CSCs培养上清液内含有较高浓度的TGF-β2(1.46±0.38 ng/mL)和PGE2(21.27±0.94 ng/mL)。向CSCs培养上清液中加入TGF-β2中和抗体,可以不同程度的逆转CSCs培养上清液对DCs表型以及功能成熟的抑制作用(P<0.05或P<0.01)。使用AH6809预处理未成熟DCs同样可以不同程度的逆转CSCs培养上清液对DCs功能成熟的抑制作用(P<0.05),以及对CD86和MHC-Ⅱ表达的抑制作用(P<0.05或P<0.01),但不能逆转对CD80表达的抑制作用(P>0.05)。同时应用TGF-β2中和抗体以及AH6809,可以提高DCs MHC-Ⅱ的表达和刺激T细胞增殖能力,且存在交互作用(P<0.05);同时可以提高DCs CD80和CD86的表达以及降低DCs抗原吞噬功能,但不存在交互作用(P>0.05)。此外,同时应用TGF-β2中和抗体以及AH6809未完全逆转CSCs培养上清液对DCs成熟的抑制作用(P<0.05或P<0.01)。
结论:在体外,小鼠CSCs可以通过分泌TGF-β2以及PGE2抑制DCs成熟,且此两种细胞因子可发挥叠加效应。
Corneal stroma cells (CSCs), a unique population of neural crest-derived cells embedded in the corneal stroma, play a major role in maintaining corneal transparency. Since the cell number in vivo is scarce, CSCs must be expanded in vitro. Previous studies have indicated that when cultured in the complete medium (containing fetal bovine serum, FBS), CSCs readily lost their biological characteristic and transformed into some other cells. Unfortunately, CSCs cultured in the serum-free medium do not proliferate. Therefore, expanding CSCs while maintaining their normal biological characteristic in vitro is very desirable.
Recent studies have shown that between birth and eyelid opening, the number of proliferating CSCs decreases dramatically, and at the time of eyelid opening, CSCs have withdrawn from the cell cycle, remaining in G0 rather than undergoing complete terminal differentiation. Moreover, CSCs express stem cell markers and have transdifferentiation potency, which are similar to mesenchymal stem cells. However, the study on the mesenchymal stem cell characteristic of murine CSCs is rare.
Dendritic cells (DCs) are the most efficient antigen-presenting cells that initiate or control adaptive immune responses to invading pathogens, and are found in two distinct functional states. Immature DCs can uptake antigens and induce immunity tolerance; mature DCs are uniquely able to stimulate naive T cell responses efficiently. Since corneal DCs play a critical role in corneal transplantation and corneal disorders, and some approaches targeted DCs have been applied, further studies on regulation of DCs maturation should be required.
Recent studies have demonstrated that DCs are uniformly immature in the central cornea, but mainly mature in the peripheral region. And local microenvironment has been widely recognized as an important regulator for DCs maturation. Therefore, we deem that CSCs should have regulative effect on DCs maturation. However, to date, no systematic study has been performed.
Consequently, this study was carried out to explore how to expand murine CSCs, and to investigate the mesenchymal stem cell characteristic and the inhibitory effect on DCs maturation of murine CSCs. As follows: Part 1 Isolation, identification, cultivation, and expansion of murine corneal stroma cells
Objective: To investigate whether murine CSCs expanded in the KSFM medium still hold the original biological characteristic.
Methods: After incubated in EDTA solution (20mmol/L) for 45 minutes, the corneal epithelium and endothelium were carefully peeled away from the corneal stroma with fine forceps. And then, central corneal stromas were digested with collagenase I (300U/mL) for 4 hours. Following centrifugation, isolated single cells were harvested, and seeded on plastic in the DMEM basic medium (serum-free) or the DMEM complete medium (containing 10% FBS), or in the KSFM medium. The cells were cultured at 37℃in a 5% CO2 atmosphere, and subcultured with EDTA solution (containing 1U/mL dispase). Meanwhile, the cells were observed and further the cell growth curve was drawn; the gene expression of keratocan, aldehyde dehydrogenase (ALDH), cytokeratin 12 (CK12), and neuron-specific enolase (NSE) was examined by reverse transcription polymerase chain reaction (RT-PCR); the protein expression of keratocan was analyzed by immunofluorescence and Western Blot.
Results: After collagenase digestion, cell suspension obtained from two murine corneal stromas yielded about 1×10~4 single cells. The data of RT-PCR indicated that the primary cell exhibited positive expression of keratocan and ALDH, which are considered as hallmarks for keratocytes, and negative expression of CK12 and NSE, which are expressed in corneal epithelium and endothelium respectively; the data of immunofluorescence and Western Blot further showed that these cells expressed keratocan protein. And thus, the primary cells in this study were of stromal origin. In the DMEM basic medium, primary CSCs could not proliferate; in the DMEM complete medium, CSCs proliferated, but passage 3 cells lost the gene expression of ALDH and keratocan and the protein expression of keratocan; in the KSFM medium, CSCs also proliferated, and passage 3 cells still maintained the gene expression of ALDH and keratocan and the protein expression of keratocan, with no significant difference compared with primary CSCs (P>0.05).
Conclusion: KSFM medium can maintain the biological characteristic of murine CSCs while promoting cell proliferation.
Part 2 The mesenchymal stem cell-like phenotype and multilineage potential of murine corneal stroma cells
Objective: To investigate whether murine CSCs, cultured and expanded in the KSFM medium, share the same phenotype and multilineage potential with mesenchymal stem cell.
Methods: The central region of murine cornea was treated with collagenase digestion after the epithelium and endothelium were removed. Then the single cells were cultured and expanded in the KSFM medium. Passage 2 CSCs were harvested and incubated with hematopoietic marker antibodies (CD34-FITC, CD45-PE) and mesenchymal marker antibodies (CD105-PE, CD90-FITC, CD71-FITC, CD29-APC) for 30 minutes at 4℃in dark. Then the stained cells were analyzed on a flow cytometer. CSCs were maintained in the KSFM medium. At day 2 post-confluence, the medium was changed with the osteogenic differentiation medium (DMEM supplemented with 10% FBS, 100nmol/L dexamethasone, 10mmol/Lβ-glycerophosphate, and 50mg/L ascorbic acid), the adipogenic differentiation medium (DMEM supplemented with 10% FBS, 0.5μmol/L dexamethasone, 0.5mmol/L isobutylmethylxanthine, and 10mg/L insulin), or the control medium (DMEM supplemented with 10% FBS). The medium was changed every other day. After 21 days, the cells, cultured in the osteogenic differentiation medium and the control medium, were stained with 2% alizarin red S solution, and the gene expression of alkaline phosphatase and osteocalcin was examined by RT-PCR; and the cells, cultured in the adipogenic differentiation medium and the control medium, were stained with 0.3% oil red O solution, and the gene expression of lipoprotein lipase and peroxisome proliferator activated receptorγwas examined by RT-PCR.
Results: The phenotypic characterization of passage 2 CSCs was analyzed by flow cytometry. Data showed that cells were negative for CD34 (3.68%±1.44%) and CD45 (9.56%±1.83%); but positive for CD29 (96.85%±1.91%), CD90 (93.62%±1.65%), CD105 (50.91%±2.56%), and CD71 (45.27%±3.56%). Within 3 days after osteoblastic induction, cells continued to exhibit fibroblast-like morphology similar to cells maintained in the control medium. After 7 days, cells had transformed to a many-horned shape, with black particles appeared in cytoplasm. One week later, center of the colony increased gradually and finally formed mineralization nodules, which were stained by alizarin red S. Cells cultured in the control medium did not show any morphological sign of osteoblastic differentiation and were not stained by alizarin red S. Moreover, the expression of osteoblast-specific markers was analyzed by RT-PCR. Data showed that, cells under osteoblastic conditions exhibited positive expression of alkaline phosphatase and osteocalcin, whereas cells cultured in the control medium showed lower expression of alkaline phosphatase and negative expression of osteocalcin. After 7 days under adipogenic conditions, cells changed their shape from spindle to round, coincided with the accumulation of intracellular droplets. Two weeks after initial induction, the cytoplasm was completely filled with lipid rich vacuoles, which were stained positively by oil red O. Differentiation was demonstrated further by RT-PCR analysis. The lipoprotein lipase and peroxisome proliferator activated receptorγmRNA were both detected. These changes were not found in cells cultured in the control medium.
Conclusion: Similar to mesenchymal stem cells, the murine central cornea-derived CSCs, cultured and expanded in the KSFM medium, have special phenotypic marker expression profile and can differentiate into adipocytes and osteoblasts.
Part 3 The inhibitory effect of murine corneal stroma cells culture supernatant on dendritic cells maturation
Objective: To investigate whether murine CSCs culture supernatant can inhibit lipopolysaccharide-induced DCs maturation.
Methods: Splenic T cells from BALB/c mice were collected by nylon wool columns, and the purity of cultured T cells was determined by flow analysis of surface CD3 staining. Three days after primary murine CSCs (105/mL) were cultured in the serum-free RPMI 1640 medium, the medium was semi-changed. Three days later, the culture supernatant was harvested and tested. Bone marrow mononuclear cells were prepared from C57BL/6 mouse femur bone marrow suspension by depletion of red cells and then cultured in the RPMI 1640 medium supplemented with 10% FBS and 10ng/mL recombinant murine granulocyte macrophage colony stimulating factor. The medium was wholly changed on day 3 and semi-changed on day 5. On day 7, nonadherent and loosely adherent cells were harvested as immature DCs, the purity of which was tested by flow cytometry with anti-CD11c antibody staining. To induce DCs maturation, lipopolysaccharide (1μg/mL) was added for another 48 hours of culture. To explore the effect of CSCs culture supernatant on DCs maturation, various concentrations of culture supernatant (25%, 50%) was added into the culture medium during the DCs maturation stage. And then, to evaluate the phenotypic maturation of DCs, the cellular surface markers for maturation, including CD80, CD86 and major histocompatibility complex classⅡ(MHC-Ⅱ) were analyzed by flow cytometry. Furthermore, to evaluate the functional maturation of DCs, the capability of stimulating the proliferation of T lymphocytes was measured by allogeneic mixed lymphocyte reactions and the function of endocytosis was assessed by fluorescein isothiocyanate-dextran uptake.
Results: For T cells, following lysis of red cells, splenic cells were passed through nylon wool columns and nonadherent small cells were harvested. Phenotypic analysis by flow cytometry indicated that these cells were positive for CD3 (93.97%±3.06%), which is considered as a hallmark for T cells. For DCs, on day 7 of culture, bone marrow mononuclear cells generated many distinctive cell clusters with nonattachment and loose attachment to plate bottoms. These cells displayed different protruding veils, and expressed high level of CD11c (78.61%±4.27%), but low levels of the maturation markers CD80, CD86, and MHC-Ⅱ. An additional 48 hours of stimulation with lipopolysaccharide later, the levels of the maturation markers were increased. In the next step, the effect of CSCs culture supernatant on DCs maturation was explored. After adding the culture supernatant (25%, 50%) during the mature stage of DCs, compared with the control group, the expression of CD80, CD86, and MHC-Ⅱwas down-regulated (P<0.01), and the expression of CD11c was not altered (P>0.05); the capability of stimulating the proliferation of T lymphocytes was decreased (P<0.05); and the function of endocytosis was increased (P<0.01). Furthermore, the inhibitory effect seemed in a dose-dependent manner (25% vs. 50%, P<0.05). Conclusion: Murine CSCs culture supernatant can inhibit lipopolysaccharide-induced phenotypic and functional maturation of DCs dose-dependently. And thus, we speculate that CSCs could inhibit DCs maturation via secretion of soluble immunomodulatory cytokines.
Part 4 Murine corneal stroma cells inhibit dendritic cells maturation partially through transforming growth factorβ2 and prostaglandin E2-mediated mechanism in vitro
Objective: To explore which kind of immunomodulatory cytokines secreted by murine CSCs inhibits DCs maturation.
Methods: The gene expression of transforming growth factorβ2 (TGF-β2), macrophage colony stimulating factor (M-CSF), interleukin 10 (IL-10), and prostaglandin endoperoxide synthase 2 (PTGS2) in primary murine CSCs was examined by RT-PCR. After that, the levels of TGF-β2 and prostaglandin E2 (PGE2) in CSCs culture supernatant and the fresh RPMI 1640 medium were analyzed by enzyme linked immunosorbent assay (ELISA). Then, to further identify whether TGF-β2 and PGE2 are involved in the inhibitory effect on DCs maturation mediated by CSCs, the neutralizing TGF-β2 antibody and the EP2 receptor antagonist AH6809 were applied. During the DCs maturation stage, different treatments were executed: 1, LPS; 2, LPS + 50% CSCs culture supernatant; 3, LPS + 50% CSCs culture supernatant + AH6809; 4, LPS + 50% CSCs culture supernatant + antibody; 5, LPS + 50% CSCs culture supernatant + AH6809 + antibody. Subsequently, the cellular surface markers for DCs, including CD11c, CD80, CD86, and MHC-Ⅱ, were analyzed by flow cytometry; the capability of stimulating the proliferation of T lymphocytes was evaluated by allogeneic mixed lymphocyte reactions and the function of endocytosis was assessed by fluorescein isothiocyanate-dextran uptake.
Results: The data of RT-PCR indicated that primary murine CSCs exhibited high positive expression of TGF-β2 and PTGS2, low positive expression of M-CSF, and negative expression of IL-10; and the data of ELISA showed a higher concentration of TGF-β2 (1.46±0.38 ng/mL) and PGE2 (21.27±0.94 ng/mL) in murine CSCs culture supernatant than in the fresh RPMI 1640 medium. After adding neutralizing TGF-β2 antibody into CSCs culture supernatant, the phenotypic and functional modifications mediated by the supernatant were partially reversed (P<0.05 or P<0.01). After pretreating immature DCs with AH6809, the functional modification (P<0.05) and inhibition of CD86 and MHC-Ⅱexpression (P<0.05 or P<0.01) mediated by CSCs culture supernatant were also partially reversed, but the expression of CD80 was not altered (P>0.05). After applying AH6809 and neutralizing TGF-β2 antibody simultaneously, the expression of MHC-Ⅱand the capability of stimulating the proliferation of T lymphocytes were up-regulated, with statistical difference in interaction (P<0.05); the expression of CD86 and CD80 was elevated and the function of endocytosis were down-regulated, with no statistical difference in interaction (P>0.05). Furthermore, simultaneous application of AH6809 and neutralizing TGF-β2 antibody could not reversed the modification mediated by CSCs culture supernatant completely (P<0.05 or P<0.01).
Conclusion: TGF-β2 and PGE2 contribute to the inhibitory effect on DCs maturation mediated by murine CSCs in vitro, and further have additive effect on the immunosuppression of DCs.
研究表明,出生后增殖性CSCs的细胞数量会迅速减少。当睑裂打开后,所有CSCs的细胞周期进入G0期。最近研究证实,CSCs表达众多干细胞标记物,并且具有多向分化潜能,与间充质干细胞的生物学特性十分相似。然而,目前尚缺乏小鼠CSCs是否具有间充质干细胞特性的研究。
树突状细胞(dendritic cells, DCs)是目前已知体内功能最强的抗原呈递细胞。成熟DCs可引发机体免疫反应,而未成熟DCs则会诱导机体免疫耐受。而且,角膜内的DCs广泛的参与了多种角膜相关疾病以及角膜移植免疫排斥反应,且以角膜内DCs为靶细胞的治疗方法已取得可喜的疗效。因此,对角膜内的DCs,尤其对DCs成熟状态的研究具有重要意义。
最近研究表明,位于角膜中央区的DCs完全处于未成熟状态,而位于角膜周边区的DCs则大多处于成熟状态。局部微环境对DCs的成熟状态发挥着重要的调节功能。所以,我们推测CSCs可能具有影响角膜内DCs成熟状态的功能,然而至今尚未见相关报道。
因此,本研究旨在探索如何在体外有效扩增小鼠CSCs以及对CSCs的间充质干细胞特性和抑制DCs成熟的功能进行探讨。如下:
第一部分小鼠角膜基质细胞的提取、鉴定以及培养扩增
目的:研究使用KSFM培养基能否获取具有增殖能力且保持生物学特性不变的小鼠CSCs。
方法:将中央区角膜置于EDTA液(20mmol/L)内孵育45min后,用手术显微镊小心剥离角膜上皮层以及内皮层,并将获取的角膜基质置于含300U/mL I型胶原酶的溶液中消化4h。离心后采用DMEM基础培养基、DMEM完全培养基(含10% FBS)以及KSFM培养基重悬细胞,接种于培养瓶内常规培养,并采用含1U/mL分散酶的EDTA液消化传代细胞。同时,观察细胞并绘制细胞生长曲线;采用逆转录聚合酶链式反应(RT-PCR)检测细胞角膜蛋白多糖(keratocan)、乙醛脱氢酶(ALDH)、细胞角蛋白12(CK12)和神经元特异性烯醇化酶(NSE)等基因的表达情况;采用细胞免疫荧光染色以及蛋白质印迹方法检测细胞keratocan蛋白的表达情况。
结果:通过胶原酶消化的方法可以从每只小鼠的角膜基质获取约1×104单个细胞。RT-PCR结果显示:原代细胞表达CSCs标记物keratocan和ALDH,不表达角膜上皮细胞标记物CK12以及角膜内皮细胞标记物NSE;免疫荧光染色和蛋白质印迹结果显示:原代细胞表达keratocan蛋白。因此,本实验获取的原代细胞为CSCs。培养于DMEM基础培养基内的原代CSCs无法增殖。培养于DMEM完全培养基内的CSCs可增殖,但第3代细胞不表达keratocan和ALDH基因以及keratocan蛋白。培养于KSFM培养基内的CSCs也可增殖,第3代细胞仍表达keratocan和ALDH基因以及keratocan蛋白,且与原代细胞相比,表达强度无统计学差异(P>0.05)。
结论:KSFM培养基不仅能维持小鼠CSCs的生物学特性不变,还能有效促进细胞增殖。
第二部分小鼠角膜基质细胞的间充质干细胞样表型以及多向分化潜能
目的:研究KSFM培养基培养扩增后的小鼠CSCs是否具有间充质干细胞样表型以及多向分化潜能。
方法:在去除角膜上皮层以及内皮层后,通过胶原酶消化的方法获取小鼠中央区角膜来源的CSCs,并采用KSFM培养基对其培养扩增。收集第2代CSCs,将细胞与造血干细胞标记物抗体(CD34-FITC、CD45-PE)以及间质细胞标记物抗体(CD105-PE、CD90-FITC、CD71-FITC、CD29-APC)共孵育30min后,应用流式细胞技术进行检测。当培养于KSFM培养基内的CSCs达细胞融合后,更换成骨细胞诱导培养基(含10%FBS、100nmol/L地塞米松、10mmol/Lβ-磷酸甘油、50mg/L维生素C的DMEM培养基)、脂肪细胞诱导培养基(含10% FBS、0.5μmol/L地塞米松、0.5mmol/L 3-异丁基-1-甲基黄嘌呤、10mg/L胰岛素的DMEM培养基)以及对照培养基(含10% FBS的DMEM培养基),进行常规培养,每3d更换一次培养基。21d后,对培养于成骨细胞诱导培养基以及对照培养基内的细胞进行2%茜素红S染色,并通过RT-PCR检测细胞碱性磷酸酶和骨钙素等基因的表达情况;对培养于脂肪细胞诱导培养基以及对照培养基内的细胞进行0.3%油红O染色,并通过RT-PCR检测细胞脂蛋白脂酶和过氧化物酶增殖物激活受体γ等基因的表达情况。
结果:应用流式细胞技术对第2代CSCs的表型特征进行分析,结果显示:细胞低表达CD34(3.68%±1.44%)以及CD45(9.56%±1.83%),高表达CD29(96.85%±1.91%)、CD90(93.62%±1.65%)、CD105(50.91%±2.56%)以及CD71(45.27%±3.56%)。在成骨诱导条件下,3d时,细胞形态仍然保持梭形,与对照组细胞无明显差别。7d时,细胞形态逐渐转变为多角形,胞浆内出现黑色颗粒。14d时,开始形成矿化结节,并逐渐增大,21d时,经茜素红S染色,结节呈现鲜红色。对照组细胞未显现出以上成骨细胞分化的形态学征象,且经茜素红S染色未见阳性结果。通过RT-PCR检测成骨细胞标记物基因的表达情况,结果显示:成骨诱导条件下细胞高表达碱性磷酸酶和骨钙素,而对照组细胞低表达碱性磷酸酶且不表达骨钙素。在脂肪诱导条件下,7d时,细胞形态逐渐由梭形转变为类圆形,胞浆内液滴也逐渐增多。14d时,细胞胞浆内满布液滴,经油红O染色,液滴被特异性染成橘红色。RT-PCR结果显示:脂肪诱导条件下细胞表达脂蛋白脂酶和过氧化物酶增殖物激活受体γ。而对照组细胞未显现出向脂肪细胞分化的任何征象。
结论:经KSFM培养基培养扩增的小鼠中央区角膜来源的CSCs具有与间充质干细胞相似的表型特征,以及向成骨细胞和脂肪细胞分化的能力。
第三部分小鼠角膜基质细胞培养上清液对树突状细胞成熟的抑制作用
目的:研究小鼠CSCs培养上清液是否具有抑制脂多糖诱导的DCs成熟的作用。
方法:通过尼龙毛柱法获取BALB/c小鼠脾脏来源的T细胞,并通过流式细胞技术检测细胞表面标记物CD3以测定T细胞纯度。原代小鼠CSCs(105/mL)培养于RPMI 1640基础培养基内,3d后半量换液,6d后收集培养上清液以备用。在裂解红细胞后,将由C57BL/6小鼠股骨获取的骨髓单核细胞培养于含10% FBS以及10ng/mL重组小鼠粒细胞-巨噬细胞集落刺激因子的RPMI 1640培养基内,2d后全量换液,4d后半量换液,6d后收集悬浮和半贴壁细胞,即为未成熟DCs。通过流式细胞技术检测细胞表面标记物CD11c以测定DCs纯度。向DCs培养液内加入脂多糖(1μg/mL),48h后未成熟DCs可被诱导成熟。为研究CSCs培养上清液对DCs成熟的作用,在DCs成熟过程中,不同浓度的培养上清液(25%、50%)被添加至DCs培养液中。而后,通过流式细胞技术检测DCs成熟状态标记物CD80、CD86和主要组织相容性抗原Ⅱ类分子(MHC-Ⅱ),以对DCs的表型成熟状态进行鉴定;通过混合淋巴细胞反应检测DCs刺激T细胞增殖能力以及通过FITC标记葡聚糖内吞实验检测抗原吞噬功能,以对DCs的功能成熟状态进行鉴定。
结果:小鼠脾脏细胞经红细胞裂解以及尼龙毛柱筛选提取后,可得到大量单个悬浮的小细胞。经流式细胞技术检测,细胞高表达T细胞标记物CD3(93.97%±3.06%)。小鼠骨髓单核细胞诱导培养6d后,细胞集落明显,呈悬浮或半贴壁生长。细胞表面可见长短不一的毛刺状突起,且高表达CD11c(78.61%±4.27%),低表达CD80、CD86和MHC-Ⅱ。细胞经脂多糖刺激48h后,CD80、CD86和MHC-Ⅱ的表达明显上调。在DCs成熟过程中,将不同浓度的CSCs培养上清液(25%、50%)添加至DCs培养液后,与对照组相比,DCs CD80、CD86和MHC-Ⅱ的表达均降低(P<0.01),CD11c的表达无明显差异(P>0.05);刺激T细胞增殖能力降低(P<0.05);抗原吞噬功能增强(P<0.01)。此外,CSCs培养上清液抑制DCs成熟的作用还呈现出剂量依赖性(25% vs. 50%, P<0.05)。
结论:小鼠CSCs培养上清液可以抑制脂多糖诱导的DCs表型以及功能成熟,且呈剂量依赖性。因此,我们推测CSCs可以通过分泌可溶性免疫调节因子抑制DCs成熟。
第四部分小鼠角膜基质细胞通过分泌转化生长因子β2以及前列腺素E2抑制树突状细胞成熟
目的:探索小鼠CSCs是否通过分泌转化生长因子β2(TGF-β2)、前列腺素E2(PGE2)、白介素10(IL-10)以及巨噬细胞集落刺激因子(M-CSF)抑制DCs成熟。
方法:采用RT-PCR检测原代小鼠CSCs TGF-β2、IL-10、M-CSF以及前列腺素内过氧化物合酶2(PTGS2)等基因的表达情况。据此,通过酶联免疫吸附实验(ELISA)测定CSCs培养上清液以及新鲜RPMI 1640培养基内PGE2和TGF-β2的含量。而后,通过应用TGF-β2中和抗体(15μg/mL)以及PGE2受体阻滞剂AH6809(100μmol/L),对CSCs是否通过分泌TGF-β2以及PGE2抑制DCs成熟作进一步鉴定。在DCs成熟过程中,分别作以下不同处理:1,LPS;2,LPS+50% CSCs培养上清液;3,LPS+50% CSCs培养上清液+AH6809;4,LPS+50% CSCs培养上清液+中和抗体;5,LPS+50% CSCs培养上清液+AH6809+中和抗体。然后,应用流式细胞技术检测DCs CD11c、CD80、CD86和MHC-Ⅱ的表达情况,通过混合淋巴细胞反应检测刺激T细胞增殖能力,以及通过FITC标记葡聚糖内吞实验检测抗原吞噬功能。
结果:RT-PCR结果表明:原代小鼠CSCs高表达TGF-β2和PTGS2,低表达M-CSF,不表达IL-10;ELISA数据显示:与新鲜RPMI 1640培养基相比,CSCs培养上清液内含有较高浓度的TGF-β2(1.46±0.38 ng/mL)和PGE2(21.27±0.94 ng/mL)。向CSCs培养上清液中加入TGF-β2中和抗体,可以不同程度的逆转CSCs培养上清液对DCs表型以及功能成熟的抑制作用(P<0.05或P<0.01)。使用AH6809预处理未成熟DCs同样可以不同程度的逆转CSCs培养上清液对DCs功能成熟的抑制作用(P<0.05),以及对CD86和MHC-Ⅱ表达的抑制作用(P<0.05或P<0.01),但不能逆转对CD80表达的抑制作用(P>0.05)。同时应用TGF-β2中和抗体以及AH6809,可以提高DCs MHC-Ⅱ的表达和刺激T细胞增殖能力,且存在交互作用(P<0.05);同时可以提高DCs CD80和CD86的表达以及降低DCs抗原吞噬功能,但不存在交互作用(P>0.05)。此外,同时应用TGF-β2中和抗体以及AH6809未完全逆转CSCs培养上清液对DCs成熟的抑制作用(P<0.05或P<0.01)。
结论:在体外,小鼠CSCs可以通过分泌TGF-β2以及PGE2抑制DCs成熟,且此两种细胞因子可发挥叠加效应。
Corneal stroma cells (CSCs), a unique population of neural crest-derived cells embedded in the corneal stroma, play a major role in maintaining corneal transparency. Since the cell number in vivo is scarce, CSCs must be expanded in vitro. Previous studies have indicated that when cultured in the complete medium (containing fetal bovine serum, FBS), CSCs readily lost their biological characteristic and transformed into some other cells. Unfortunately, CSCs cultured in the serum-free medium do not proliferate. Therefore, expanding CSCs while maintaining their normal biological characteristic in vitro is very desirable.
Recent studies have shown that between birth and eyelid opening, the number of proliferating CSCs decreases dramatically, and at the time of eyelid opening, CSCs have withdrawn from the cell cycle, remaining in G0 rather than undergoing complete terminal differentiation. Moreover, CSCs express stem cell markers and have transdifferentiation potency, which are similar to mesenchymal stem cells. However, the study on the mesenchymal stem cell characteristic of murine CSCs is rare.
Dendritic cells (DCs) are the most efficient antigen-presenting cells that initiate or control adaptive immune responses to invading pathogens, and are found in two distinct functional states. Immature DCs can uptake antigens and induce immunity tolerance; mature DCs are uniquely able to stimulate naive T cell responses efficiently. Since corneal DCs play a critical role in corneal transplantation and corneal disorders, and some approaches targeted DCs have been applied, further studies on regulation of DCs maturation should be required.
Recent studies have demonstrated that DCs are uniformly immature in the central cornea, but mainly mature in the peripheral region. And local microenvironment has been widely recognized as an important regulator for DCs maturation. Therefore, we deem that CSCs should have regulative effect on DCs maturation. However, to date, no systematic study has been performed.
Consequently, this study was carried out to explore how to expand murine CSCs, and to investigate the mesenchymal stem cell characteristic and the inhibitory effect on DCs maturation of murine CSCs. As follows: Part 1 Isolation, identification, cultivation, and expansion of murine corneal stroma cells
Objective: To investigate whether murine CSCs expanded in the KSFM medium still hold the original biological characteristic.
Methods: After incubated in EDTA solution (20mmol/L) for 45 minutes, the corneal epithelium and endothelium were carefully peeled away from the corneal stroma with fine forceps. And then, central corneal stromas were digested with collagenase I (300U/mL) for 4 hours. Following centrifugation, isolated single cells were harvested, and seeded on plastic in the DMEM basic medium (serum-free) or the DMEM complete medium (containing 10% FBS), or in the KSFM medium. The cells were cultured at 37℃in a 5% CO2 atmosphere, and subcultured with EDTA solution (containing 1U/mL dispase). Meanwhile, the cells were observed and further the cell growth curve was drawn; the gene expression of keratocan, aldehyde dehydrogenase (ALDH), cytokeratin 12 (CK12), and neuron-specific enolase (NSE) was examined by reverse transcription polymerase chain reaction (RT-PCR); the protein expression of keratocan was analyzed by immunofluorescence and Western Blot.
Results: After collagenase digestion, cell suspension obtained from two murine corneal stromas yielded about 1×10~4 single cells. The data of RT-PCR indicated that the primary cell exhibited positive expression of keratocan and ALDH, which are considered as hallmarks for keratocytes, and negative expression of CK12 and NSE, which are expressed in corneal epithelium and endothelium respectively; the data of immunofluorescence and Western Blot further showed that these cells expressed keratocan protein. And thus, the primary cells in this study were of stromal origin. In the DMEM basic medium, primary CSCs could not proliferate; in the DMEM complete medium, CSCs proliferated, but passage 3 cells lost the gene expression of ALDH and keratocan and the protein expression of keratocan; in the KSFM medium, CSCs also proliferated, and passage 3 cells still maintained the gene expression of ALDH and keratocan and the protein expression of keratocan, with no significant difference compared with primary CSCs (P>0.05).
Conclusion: KSFM medium can maintain the biological characteristic of murine CSCs while promoting cell proliferation.
Part 2 The mesenchymal stem cell-like phenotype and multilineage potential of murine corneal stroma cells
Objective: To investigate whether murine CSCs, cultured and expanded in the KSFM medium, share the same phenotype and multilineage potential with mesenchymal stem cell.
Methods: The central region of murine cornea was treated with collagenase digestion after the epithelium and endothelium were removed. Then the single cells were cultured and expanded in the KSFM medium. Passage 2 CSCs were harvested and incubated with hematopoietic marker antibodies (CD34-FITC, CD45-PE) and mesenchymal marker antibodies (CD105-PE, CD90-FITC, CD71-FITC, CD29-APC) for 30 minutes at 4℃in dark. Then the stained cells were analyzed on a flow cytometer. CSCs were maintained in the KSFM medium. At day 2 post-confluence, the medium was changed with the osteogenic differentiation medium (DMEM supplemented with 10% FBS, 100nmol/L dexamethasone, 10mmol/Lβ-glycerophosphate, and 50mg/L ascorbic acid), the adipogenic differentiation medium (DMEM supplemented with 10% FBS, 0.5μmol/L dexamethasone, 0.5mmol/L isobutylmethylxanthine, and 10mg/L insulin), or the control medium (DMEM supplemented with 10% FBS). The medium was changed every other day. After 21 days, the cells, cultured in the osteogenic differentiation medium and the control medium, were stained with 2% alizarin red S solution, and the gene expression of alkaline phosphatase and osteocalcin was examined by RT-PCR; and the cells, cultured in the adipogenic differentiation medium and the control medium, were stained with 0.3% oil red O solution, and the gene expression of lipoprotein lipase and peroxisome proliferator activated receptorγwas examined by RT-PCR.
Results: The phenotypic characterization of passage 2 CSCs was analyzed by flow cytometry. Data showed that cells were negative for CD34 (3.68%±1.44%) and CD45 (9.56%±1.83%); but positive for CD29 (96.85%±1.91%), CD90 (93.62%±1.65%), CD105 (50.91%±2.56%), and CD71 (45.27%±3.56%). Within 3 days after osteoblastic induction, cells continued to exhibit fibroblast-like morphology similar to cells maintained in the control medium. After 7 days, cells had transformed to a many-horned shape, with black particles appeared in cytoplasm. One week later, center of the colony increased gradually and finally formed mineralization nodules, which were stained by alizarin red S. Cells cultured in the control medium did not show any morphological sign of osteoblastic differentiation and were not stained by alizarin red S. Moreover, the expression of osteoblast-specific markers was analyzed by RT-PCR. Data showed that, cells under osteoblastic conditions exhibited positive expression of alkaline phosphatase and osteocalcin, whereas cells cultured in the control medium showed lower expression of alkaline phosphatase and negative expression of osteocalcin. After 7 days under adipogenic conditions, cells changed their shape from spindle to round, coincided with the accumulation of intracellular droplets. Two weeks after initial induction, the cytoplasm was completely filled with lipid rich vacuoles, which were stained positively by oil red O. Differentiation was demonstrated further by RT-PCR analysis. The lipoprotein lipase and peroxisome proliferator activated receptorγmRNA were both detected. These changes were not found in cells cultured in the control medium.
Conclusion: Similar to mesenchymal stem cells, the murine central cornea-derived CSCs, cultured and expanded in the KSFM medium, have special phenotypic marker expression profile and can differentiate into adipocytes and osteoblasts.
Part 3 The inhibitory effect of murine corneal stroma cells culture supernatant on dendritic cells maturation
Objective: To investigate whether murine CSCs culture supernatant can inhibit lipopolysaccharide-induced DCs maturation.
Methods: Splenic T cells from BALB/c mice were collected by nylon wool columns, and the purity of cultured T cells was determined by flow analysis of surface CD3 staining. Three days after primary murine CSCs (105/mL) were cultured in the serum-free RPMI 1640 medium, the medium was semi-changed. Three days later, the culture supernatant was harvested and tested. Bone marrow mononuclear cells were prepared from C57BL/6 mouse femur bone marrow suspension by depletion of red cells and then cultured in the RPMI 1640 medium supplemented with 10% FBS and 10ng/mL recombinant murine granulocyte macrophage colony stimulating factor. The medium was wholly changed on day 3 and semi-changed on day 5. On day 7, nonadherent and loosely adherent cells were harvested as immature DCs, the purity of which was tested by flow cytometry with anti-CD11c antibody staining. To induce DCs maturation, lipopolysaccharide (1μg/mL) was added for another 48 hours of culture. To explore the effect of CSCs culture supernatant on DCs maturation, various concentrations of culture supernatant (25%, 50%) was added into the culture medium during the DCs maturation stage. And then, to evaluate the phenotypic maturation of DCs, the cellular surface markers for maturation, including CD80, CD86 and major histocompatibility complex classⅡ(MHC-Ⅱ) were analyzed by flow cytometry. Furthermore, to evaluate the functional maturation of DCs, the capability of stimulating the proliferation of T lymphocytes was measured by allogeneic mixed lymphocyte reactions and the function of endocytosis was assessed by fluorescein isothiocyanate-dextran uptake.
Results: For T cells, following lysis of red cells, splenic cells were passed through nylon wool columns and nonadherent small cells were harvested. Phenotypic analysis by flow cytometry indicated that these cells were positive for CD3 (93.97%±3.06%), which is considered as a hallmark for T cells. For DCs, on day 7 of culture, bone marrow mononuclear cells generated many distinctive cell clusters with nonattachment and loose attachment to plate bottoms. These cells displayed different protruding veils, and expressed high level of CD11c (78.61%±4.27%), but low levels of the maturation markers CD80, CD86, and MHC-Ⅱ. An additional 48 hours of stimulation with lipopolysaccharide later, the levels of the maturation markers were increased. In the next step, the effect of CSCs culture supernatant on DCs maturation was explored. After adding the culture supernatant (25%, 50%) during the mature stage of DCs, compared with the control group, the expression of CD80, CD86, and MHC-Ⅱwas down-regulated (P<0.01), and the expression of CD11c was not altered (P>0.05); the capability of stimulating the proliferation of T lymphocytes was decreased (P<0.05); and the function of endocytosis was increased (P<0.01). Furthermore, the inhibitory effect seemed in a dose-dependent manner (25% vs. 50%, P<0.05). Conclusion: Murine CSCs culture supernatant can inhibit lipopolysaccharide-induced phenotypic and functional maturation of DCs dose-dependently. And thus, we speculate that CSCs could inhibit DCs maturation via secretion of soluble immunomodulatory cytokines.
Part 4 Murine corneal stroma cells inhibit dendritic cells maturation partially through transforming growth factorβ2 and prostaglandin E2-mediated mechanism in vitro
Objective: To explore which kind of immunomodulatory cytokines secreted by murine CSCs inhibits DCs maturation.
Methods: The gene expression of transforming growth factorβ2 (TGF-β2), macrophage colony stimulating factor (M-CSF), interleukin 10 (IL-10), and prostaglandin endoperoxide synthase 2 (PTGS2) in primary murine CSCs was examined by RT-PCR. After that, the levels of TGF-β2 and prostaglandin E2 (PGE2) in CSCs culture supernatant and the fresh RPMI 1640 medium were analyzed by enzyme linked immunosorbent assay (ELISA). Then, to further identify whether TGF-β2 and PGE2 are involved in the inhibitory effect on DCs maturation mediated by CSCs, the neutralizing TGF-β2 antibody and the EP2 receptor antagonist AH6809 were applied. During the DCs maturation stage, different treatments were executed: 1, LPS; 2, LPS + 50% CSCs culture supernatant; 3, LPS + 50% CSCs culture supernatant + AH6809; 4, LPS + 50% CSCs culture supernatant + antibody; 5, LPS + 50% CSCs culture supernatant + AH6809 + antibody. Subsequently, the cellular surface markers for DCs, including CD11c, CD80, CD86, and MHC-Ⅱ, were analyzed by flow cytometry; the capability of stimulating the proliferation of T lymphocytes was evaluated by allogeneic mixed lymphocyte reactions and the function of endocytosis was assessed by fluorescein isothiocyanate-dextran uptake.
Results: The data of RT-PCR indicated that primary murine CSCs exhibited high positive expression of TGF-β2 and PTGS2, low positive expression of M-CSF, and negative expression of IL-10; and the data of ELISA showed a higher concentration of TGF-β2 (1.46±0.38 ng/mL) and PGE2 (21.27±0.94 ng/mL) in murine CSCs culture supernatant than in the fresh RPMI 1640 medium. After adding neutralizing TGF-β2 antibody into CSCs culture supernatant, the phenotypic and functional modifications mediated by the supernatant were partially reversed (P<0.05 or P<0.01). After pretreating immature DCs with AH6809, the functional modification (P<0.05) and inhibition of CD86 and MHC-Ⅱexpression (P<0.05 or P<0.01) mediated by CSCs culture supernatant were also partially reversed, but the expression of CD80 was not altered (P>0.05). After applying AH6809 and neutralizing TGF-β2 antibody simultaneously, the expression of MHC-Ⅱand the capability of stimulating the proliferation of T lymphocytes were up-regulated, with statistical difference in interaction (P<0.05); the expression of CD86 and CD80 was elevated and the function of endocytosis were down-regulated, with no statistical difference in interaction (P>0.05). Furthermore, simultaneous application of AH6809 and neutralizing TGF-β2 antibody could not reversed the modification mediated by CSCs culture supernatant completely (P<0.05 or P<0.01).
Conclusion: TGF-β2 and PGE2 contribute to the inhibitory effect on DCs maturation mediated by murine CSCs in vitro, and further have additive effect on the immunosuppression of DCs.
引文
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