【作者】 刘飞；

【导师】 杨明珍；

【作者基本信息】 安徽医科大学 ， 内科学， 2012， 硕士

【摘要】 目的:本实验通过观察1，25-二羟维生素D3对树突状细胞（dendritic cells，DCs）的表型、功能以及对其TLR4、TLR7表达的影响，探讨TLR4、TLR7在1，25-二羟维生素D3诱导耐受性DCs中可能的作用。为以后研究三者在自身免疫性疾病中的关系奠定基础，同时也为治疗及预防自身免疫性疾病提供新的思路。方法:采用C57BL/6小鼠为供体，雌雄不拘，无菌取小鼠胫骨和股骨骨髓，用红细胞裂解液溶去红细胞后PBS洗2次，利用含rmGM-CSF （20ng/mL）, rmIL-4（10ng/mL）的RPMI1640培养基调整细胞浓度至1×10⁶/mL，置于6孔板中。分别根据加和不加10^-8mol/L1,25-（OH）₂D₃，将细胞分成加药组(10^-8mol/L1,25-（OH）₂D₃)和对照组（等体积RPMI1640），相同的温度、湿度及CO2浓度下培养。24h后轻轻吸弃上清液，去除悬浮细胞，及时补充含细胞因子浓度相同的完全培养基。分别于第3、5d换液，并于第5d加入1μg/ml的LPS刺激DCs成熟，继续培养48h,第7天收集细胞用于后续实验。1.采用倒置显微镜观察每日观察DCs形态的变化。2.使用流式细胞仪检测两组DCs表型变化：分别收集两组细胞，加入FITC-CD80,PE-cy5-CD86,PE-MHCⅡ单克隆抗体进行荧光标记，流式细胞仪进行表型分析。3.混合淋巴细胞反应：分别收集两组DC，作为刺激细胞。取正常C57BL/6小鼠脾脏，尼龙毛吸附法制备纯化T细胞，作为反应细胞。刺激细胞与反应细胞按1:10、1:50和1:100混合培养，采用CCK-8比色法检测1,25-（OH）₂D₃组及对照组DC对T淋巴细胞增殖的影响。4.利用RT-PCR半定量方法测定1,25-（OH）₂D₃组及对照组中DCs的TLR4、TLR7mRNA相对表达量。结果:1.显微镜下小鼠骨髓来源的细胞，在rmGM-CSF和rmIL-4作用1d后，多数细胞仍呈圆形贴壁生长，部分区域出现较少不规则形状的细胞。作用2-3d后，细胞明显增大，单个散在的细胞逐渐集聚成簇状细胞集落。5d以后，细胞集落增大，集落的周围有许多毛刺状突起，可见少量具有突起的悬浮细胞。加入LPS继续培养48h后，细胞集落较前减少，悬浮细胞明显增多，光镜下可见细胞形态不规则，表面有大量树枝状、伪足状、长短不一的突起，呈现典型的DCs形态2.流式细胞术（FCM）结果表明1,25-二羟维生素D3处理后，DCs表达CD86和MHC II的荧光强度均较对照组明显降低，差异有统计学意义（p<0.05）3.细胞生长抑制试验显示在相同浓度的情况下，1,25-（OH）₂D₃组中的DC较对照组可显著地抑制T淋巴细胞的增殖。4.半定量RT-PCR发现，1,25-（OH）₂D₃组中DCs其TLR7/β-actin的比值较对照组有显著降低（p<0.05），而TLR4差异不明显（p>0.05）。结论：（1）1,25-（OH）₂D₃可以抑制LPS刺激DCs成熟，诱导耐受性DCs；（2）1,25-（OH）₂D₃诱导的DCs低表达CD86、MHCⅡ，并且对T细胞的刺激能力也较成熟DCs弱；（3）1,25-（OH）₂D₃可能通过下调TLR7的表达，从而下调DC表面共刺激分子，降低其功能，抑制DCs成熟。故TLRs在诱导DCs的成熟及耐受性过程中起着重要作用。更多还原

【Abstract】 Objective: To investigate how Toll-like receptors (TLRs) may be involved in theinduction of tolerogenic dendritic cells (DCs) induced by1,25-dihydroxyvitamin D3weobserved the effects of1,25-dihydroxyvitamin D3on the phenotype and function ofDCs and their expression levels of TLR4and TLR7. The research will lay thefoundation for further exposition of the relationship between DCs, TLRs, and1,25-dihydroxyvitamin D3in autoimmune diseases, but also provide new ideas fortreatment and prevention of autoimmune diseases.Methods: C57BL/6mice either male or female were used as donor.The thighbone andshinbone were separated under sterile conditions.Cells were collected and washed twicewith PBS after removing the red cells using a red cell lytic buffer. Cells were adjustedconcentration to1×106/mL by using RPMI1640medium containing rmGM-CSF (20ng/mL) and rmIL-4(10ng/mL), placed in6well plates, respectively. According to withand without10-8mol/L1,25-dihydroxyvitamin D3,the cells were divided into dosinggroup and the control group, cultured under the same temperature, humidity and CO2concentration.The suspension cells were removed24h later, and the adherent cells weresupplemented with fresh complete medium. Medium was renewed on the3rd and5thday, and1μg/mL LPS was added to stimulate the maturation of DCs on the5th day.Cells were cultured for48h continuously and the cells were harvested on day7th forfurther experiments.1. We observed changes to the cytologic morphology under an inverted microscopedaily.2. Detection of two sets of DCs phenotypic changes by flow cytometry: Two groups of cells were collected by adding fluorescently labeled monoclonal antibody includingCD80, CD86and MHCII, and were assayed by flow cytometery.3. Two groups of cells were collected as stimulator cells, respectively. Take the normalof C57BL/6mice spleens, nylon wool adsorption preparation of purified T cells, as areaction cell. The DCs (stimulated cells) were mixed with the T cells (responder cells)at different proportions (1:10,1:50and1:100).The capability to stimulate theproliferation of lymphocytes were examined through CCK-8method.4. The effects of1,25-dihydroxyvitamin D3on the expression of TLR4and TLR7mRNA from DC was measured by RT-PCR.Results:1. Mouse bone marrow derived DCs treated for1day with rmGM-CSF andrmIL-4were observed under a microscope. Most cells appeared round in shape andadhered to the tissue culture plastic wall, but some had an anomalous appearance. After2–3days, the cells appeared markedly enlarged in shape and scattered single cellsgathered in fascicular cell colonies. Five days later the cell colonies grew and appearedmany stings around on edge, there were also some suspended cells beside cellcolonies.Following the addition of LPS, the cell colonies decreased in number whilethe suspended cells increased. The shape of the cells was irregular with many branchesor pseudopodia-like formations, and suggested a typical dendritic cell morphology bylight microscopy.2. After treatment with1,25-dihydroxyvitamin D3, the fluorescence intensity of CD86and MHC class II levels on DCs was significantly decreased (P <0.05).3. The stimulating capacity of1,25-dihydroxyvitamin D3-treated DCs to T cells wassignificantly reduced compared with the control group (P <0.05)in the case of the sameconcentration.4. In comparison with controls,1,25-dihydroxyvitamin D3treatment down–regulatedthe expression of TLR7(P <0.05), but not TLR4.Conclusion:(1)1,25-dihydroxyvitamin D3can inhibit LPS-stimulated DCs maturationand induce tolerogenic DCs. (2)1,25-Dihydroxyvitamin D3-induced DC had lower expression of CD80and MHCⅡ and DCs’ ability to stimulate T cells was significantly lower than the control.(3)1,25-dihydroxyvitamin D3may down-regulated the expression of TLR7, whichlowered the DC surface costimulatory molecules, and inhibit DCs maturation. Therefore,TLRs play an important role in the induction of DC maturation and tolerance process.更多还原