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

发布时间：2018-07-21 18:49

【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(P0.05)。 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(P0.01),CD11c的表达无明显差异(P0.05);刺激T细胞增殖能力降低(P0.05);抗原吞噬功能增强(P0.01)。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%, P0.05)。 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(P0.05或P0.01)。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(P0.05),以及对CD86和MHC-Ⅱ表达的抑制作用(P0.05或P0.01),但不能逆转对CD80表达的抑制作用(P0.05)。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(P0.05);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(P0.05)。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(P0.05或P0.01)。 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。
[Abstract]:Corneal stroma cells (CSCs), which is scattered in the source of the neural crest scattered in the corneal stroma, plays an important role in maintaining corneal transparency. The number of CSCs in the body is scarce, so the culture and expansion of cells in vitro is a required study path. The study shows that the culture of fetal bovine serum (FBS) is completely cultured. CSCs can lose its original biological characteristics. However, when it is cultured on a serum-free basic medium, the cell can not be effectively proliferated, although it can keep its characteristics unchanged. Therefore, it is one of the difficulties at present how to effectively amplify CSCs under the condition of keeping the cell biological characteristics unchanged.
Studies have shown that the number of cells in the proliferative CSCs after birth is rapidly reduced. When the palpebral cleft is opened, the cell cycle of all CSCs enters the G0 phase. Recent studies have confirmed that CSCs expresses numerous stem cell markers and has multiple differentiation potential and is very similar to the biological specificity of mesenchymal stem cells. However, it is still lack of CSCs in mice. Studies of the characteristics of mesenchymal stem cells.
Dendritic cells (DCs) is now known as the most potent antigen presenting cell in the body. Mature DCs can induce immune response, while immature DCs induces immune tolerance. Moreover, the DCs in the cornea is widely involved in a variety of corneal related diseases and corneal transplantation immune rejection, and the DCs in the cornea is in the cornea. Target cell therapy has achieved gratifying results. Therefore, it is important to study DCs in the cornea, especially DCs.
Recent studies have shown that DCs in the central cornea of the cornea is completely in the immature state, while most of the DCs in the peripheral area of the cornea are in mature state. Local microenvironment plays an important role in the maturation of DCs. Therefore, we speculate that CSCs may have the function of affecting the maturity of DCs in the cornea. However, there has been no phase yet. Close the report.
Therefore, the purpose of this study is to explore the effective amplification of mouse CSCs in vitro and the characteristics of CSCs mesenchymal stem cells and the inhibition of DCs maturation.
The first part is the extraction, identification and culture amplification of mouse corneal stromal cells.
Objective: To study whether KSFM medium can obtain CSCs. with the ability to proliferate and maintain biological characteristics.
Methods: after incubating the central region cornea in EDTA liquid (20mmol/L) to incubate 45min, the corneal epithelium and the endothelium were carefully stripped by surgical microtweezers, and the acquired corneal stroma was placed in a solution containing 300U/mL I collagenase, and after 4h. centrifugation, DMEM basal medium was used, DMEM complete medium (containing 10% FBS) and KSFM culture medium suspended fine. The cells were inoculated in the culture bottle for routine culture, and the cells were digested with the EDTA solution containing 1U/mL dispersing enzyme. The cells were observed and the cell growth curves were observed. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the cell corneal proteoglycan (keratocan), acetaldehyde dehydrogenase (ALDH), cytokeratin 12 (CK12) and neuron specific enol The expression of NSE and other genes were detected, and the expression of keratocan protein was detected by cellular immunofluorescence staining and Western blotting.
Results: by collagenase digestion, approximately 1 * 104 single cell.RT-PCR results were obtained from the corneal stroma of each mouse. The CSCs markers keratocan and ALDH were expressed in the primary cells, and the corneal epithelial cell marker CK12 and the corneal endothelial cell marker NSE were not expressed; the immunofluorescence staining and Western blotting results showed that the original cells were original. Therefore, the primary cells obtained in this experiment can not proliferate in the primary cultured CSCs cultured in the basal culture base of DMEM. CSCs can proliferate in the complete culture base of DMEM, but the third generation cells do not express the keratocan and ALDH genes and keratocan protein in the third generation cells. CSCs also can proliferate in the culture base of KSFM, third. The keratocan and ALDH genes and keratocan protein were still expressed in the generation cells, and there was no significant difference in the expression intensity compared with the primary cells (P0.05).
Conclusion: KSFM medium can not only maintain the biological characteristics of CSCs in mice, but also effectively promote cell proliferation.
The second part is the mesenchymal stem cell like phenotype and multipotential differentiation of mouse corneal stromal cells.
Objective: To study whether KSFM CSCs culture medium has mesenchymal stem cell like phenotype and multipotential differentiation potential.
Methods: after removing the corneal epithelium and the endothelium, the CSCs of the cornea of the central region of the mice was obtained by collagenase digestion and was amplified by KSFM medium. Second generations of CSCs were collected, and the cell and hematopoietic stem cell marker antibody (CD34-FITC, CD45-PE) and the antibody of interstitial cell markers (CD105-PE, CD90-FITC, CD71-) were collected. FITC, CD29-APC) were incubated with 30min and detected by flow cytometry. After the fusion of CSCs cells cultured in the KSFM culture base, the culture medium was replaced by osteoblasts (including 10%FBS, 100nmol/L dexamethasone, 10mmol/L beta glycerophosphate, DMEM Pei Yang Ji of 50mg/L vitamin C), and the adipocyte inducible medium (including 10% FBS, 0.5 mu) Siamethasone, 0.5mmol/L 3- isobutyl -1- methyl xanthine, DMEM medium of 10mg/L insulin) and a control medium (10% FBS DMEM medium) were used for routine culture. After each 3D was replaced by a culture medium.21d, 2% alizarin red S staining was performed on the osteoblast induced medium and the cells in the control culture base, and through RT-PCR. The expression of alkaline phosphatase and osteocalcin and other genes were detected, and 0.3% oil red O staining was performed on the cells cultured in the adipocyte induced medium and in the control culture base, and the expression of cell lipoprotein lipase and peroxidase proliferator activated receptor gamma were detected by RT-PCR.
Results: the phenotypic characteristics of the second generation CSCs were analyzed by flow cytometry. The results showed that the cells were low expression of CD34 (3.68% + 1.44%) and CD45 (9.56% + 1.83%), high expression of CD29 (96.85% + 1.91%), CD90 (93.62% + 1.65%), CD105 (50.91% +) and CD71 (45.27% + 3.56%). Under the osteogenic induction condition, the cell morphology remained the shuttle. When there was no obvious difference between the cells of the control group and the control group.7d, the cell morphology gradually changed into polygon, and when the black granule.14d in the cytoplasm appeared, the mineralized nodules began to form and gradually increased. The nodules were bright red when 21d was dyed with alizarin red S. The cells of the control group did not show the morphological signs of the osteoblast differentiation, and were dyed by alizarin red S. No positive results were found. The expression of osteoblast marker gene was detected by RT-PCR. The results showed that the cells expressed high expression of alkaline phosphatase and osteocalcin under the induction of osteogenesis, while the cells in the control group had low expression of alkaline phosphatase and did not express osteocalcin. In the condition of fat induction, the cell morphology gradually changed from spindle shape to round like form when 7d was induced. When the intracellular droplets were gradually increased by.14d, the cells were filled with liquid droplets in cytoplasm, stained with oil red O, and the droplets were stained specifically to orange red.RT-PCR. The results showed that the cells expressed lipoprotein lipase and peroxidase proliferator activated receptor gamma under the condition of fat induction. The cells in the control group did not show any signs of differentiation to adipocytes.
Conclusion: the CSCs of the central region of the mouse cornea derived from KSFM culture medium has similar phenotypic characteristics with mesenchymal stem cells and the ability to differentiate into osteoblasts and adipocytes.
The third part is the inhibitory effect of mouse corneal stromal cell culture supernatant on the maturation of dendritic cells.
Objective: To study whether the supernatant of mouse CSCs can inhibit the maturation of DCs induced by lipopolysaccharide.
Methods: the T cells from the spleen of BALB/c mice were obtained by nylon hairy column method, and the cell surface marker CD3 was detected by flow cytometry to determine the purity of T cells. The primary mouse CSCs (105/mL) was cultured in the basal culture base of RPMI 1640, the second half of the 3D was changed, and the cultured supernatant was collected after 6D. After the lysis of red blood cells, it would be C57BL/6. The bone marrow mononuclear cells obtained from the mouse femur were cultured in the RPMI 1640 culture base containing 10% FBS and 10ng/mL recombinant mouse granulocyte macrophage colony stimulating factor. After 2D, the total amount of fluid was changed, the latter half of the 4D was changed, and the suspension and half adherent cells were collected after 6D, that is, the immature DCs. was used to detect the cell surface marker CD11c by flow cytometry. The purity of DCs was determined. Lipopolysaccharide (1 u g/mL) was added to DCs culture, and immature DCs could be induced to mature after 48h. In order to study the effect of CSCs culture supernatant on DCs maturation, the culture supernatant of different concentrations (25%, 50%) was added to the DCs medium during the maturation of DCs, and then the DCs mature marker CD8 was detected by flow cytometry. 0, CD86 and the main histocompatibility antigen class II molecule (MHC- II), to identify the phenotypic maturation of DCs, and to detect the proliferation of T cells stimulated by DCs by mixed lymphocyte reaction and to detect the phagocytic function of the antigen by the FITC labeled dextran endocytosis test in order to identify the functional maturity of DCs.
Results: after the splenic cells were lysed and the nylon wool column was screened and extracted, a large number of single suspended small cells were obtained. The cells were detected by flow cytometry, and the high expression of T cell marker CD3 (93.97% + 3.06%). After the mouse bone marrow mononuclear cells were induced and cultured for 6D, the cell colonies were obviously suspended or half adhered to the wall. The surface of the cells was available. The expression of CD11c (78.61% + 4.27%), low expression of CD80, CD86 and MHC- II. The expression of CD80, CD86 and MHC- II was obviously up-regulated after 48h was stimulated by lipopolysaccharide. In the process of DCs maturation, the CSCs culture supernatant (25%, 50%) of different concentrations was added to the DCs culture solution. The expression of - II was decreased (P0.01), the expression of CD11c was not significantly different (P0.05), the proliferation of T cells was reduced (P0.05), and the function of antigen phagocytosis was enhanced (P0.01). In addition, the effect of CSCs culture supernatant on the inhibition of DCs maturation was also dose-dependent (25% vs. 50%, P0.05).
Conclusion: the mouse CSCs culture supernatant can inhibit the DCs phenotype and function maturity induced by lipopolysaccharide, and it is dose-dependent. Therefore, we speculate that CSCs can inhibit the maturation of DCs by secreting soluble immunoregulatory factors.
The fourth part of mouse corneal stromal cells inhibit dendritic cell maturation by secreting transforming growth factor beta 2 and prostaglandin E2.
Objective: To explore whether mouse CSCs inhibits DCs maturation by secreting TGF beta 2 (TGF- beta 2), prostaglandin E2 (PGE2), interleukin 10 (IL-10) and macrophage colony stimulating factor (M-CSF).
Methods: the expression of CSCs TGF- beta 2, IL-10, M-CSF and prostaglandin synthase 2 (PTGS2) were detected by RT-PCR. According to this, the content of CSCs culture supernatant and fresh RPMI 1640 medium PGE2 and TGF- beta 2 were measured by enzyme linked immunosorbent assay (ELISA). Then, the TGF- beta 2 and antibody (1) were used. 5 mu g/mL) and PGE2 receptor blocker AH6809 (100 mu mol/L) for further identification of CSCs through the secretion of TGF- beta 2 and PGE2 to inhibit DCs maturation. In the process of DCs maturation, the following different treatments were made: 1, LPS; 2, LPS+50% CSCs culture supernatant; 3, 4, culture supernatant + neutralization antibody; 5, +50% CSCs culture supernatant +AH6809+ neutralization antibody. Then, flow cytometry was used to detect the expression of DCs CD11c, CD80, CD86 and MHC- II. The proliferation ability of T cells was detected by mixed lymphocyte reaction, and the function of antigen phagocytosis was detected by FITC labelled dextran endocytosis test.
Results: RT-PCR showed that CSCs expressed TGF- beta 2 and PTGS2 with low expression of M-CSF and did not express IL-10. ELISA data showed that the CSCs culture supernatant contained a higher concentration of TGF- beta 2 (1.46 + 0.38 ng/mL) and PGE2 (21.27 + 0.94), compared with the fresh RPMI 1640 medium, and the antibody was added to the culture supernatant. Different levels of CSCs supernatant were reversed to DCs phenotype and functional maturity.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R772.2

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