与创面愈合启动阶段相关的人皮肤αβT细胞和γδT细胞免疫激活机制的体外研究
发布时间：2018-08-08 18:56
【摘要】：目的：皮肤T细胞的激活在创面愈合的启动阶段有重要的作用，人皮肤T细胞无法免疫激活可能是慢性创面形成的主要原因之一。研究发现小鼠皮肤T细胞表面表达的连接粘附分子样蛋白（JAML）是创面愈合启动阶段中促进皮肤T细胞激活的重要共刺激分子。人体内也有JAML的表达，但其是否具有类似作用尚未得到验证。因此，我们提取了人皮肤αβT细胞和γδT细胞，用与JAML相关的多种激活剂和阻滞剂对其进行干扰，并测定其对干扰的不同反应，观察JAML是否具有作为人创面愈合启动阶段促进皮肤T细胞免疫激活的重要共刺激分子的能力，旨在进一步探索创面愈合过程中人皮肤T细胞的激活活化的细节，深化对创面愈合过程中免疫学机制的认识。 材料与方法：1.从组织中提取人皮肤T细胞，观察提取细胞的活性，检测皮肤T细胞的数量和表型。2.用100ng/ml的PMA和1000ng/ml的ionomycin满负荷刺激外周血T细胞及人皮肤T细胞，，检测不同T细胞合成IL-2和IGF-1的水平。3.分别对外周血T细胞及人皮肤T细胞进行培养扩增，观察不同T细胞的扩增能力。4.采用不同剂量的anti-CD3mAb（0.1μg/ml、0.5μg/ml、1μg/ml、2μg/ml、5μg/ml、10μg/ml）刺激人皮肤T细胞，检测其表面激活标志CD25、CD69的表达以及细胞分泌IL-2和IGF-1的水平。5.用4μg/ml重组柯萨奇-腺病毒受体蛋白（CAR）联合0.5μg/ml anti-CD3mAb或单独4μg/mlCAR刺激人皮肤T细胞，检测其表面激活标志CD25、CD69的表达以及细胞分泌IL-2和IGF-1的水平。6.将人皮肤αβT和γδT细胞分离，检测其表面JMAL和CD28的表达。7.从人皮肤T细胞中提取人皮肤γδT细胞，分别用4μg/ml CAR联合0.5μg/ml anti-CD3mAb，单独4μg/ml CAR或单独0.5μg/mlanti-CD3mAb刺激，检测其表面激活标志CD25、CD69和表面共刺激分子CD80、CD86、JAML的表达，以及细胞分泌IL-2和IGF-1的水平。8.从人皮肤T细胞中提取人皮肤αβT细胞，用4μg/ml CAR联合0.5μg/ml anti-CD3mAb刺激，检测其表面激活标志CD25、CD69和表面共刺激分子CD80、CD86、JAML的表达，以及细胞分泌IL-2和IGF-1的水平。再分别用4μg/ml CAR或0.5μg/ml anti-CD3mAb刺激，检测其表面共刺激分子CD28的表达。9.用激活的人皮肤γδT细胞和0.5μg/ml anti-CD3mAb刺激人皮肤αβT细胞，检测人皮肤αβT细胞表面激活标志CD25、CD69的表达。 结果：1.从组织中提取的人皮肤T细胞活性良好，平均每克表皮组织能提出T细胞（2.63±1.72）×107个，αβT细胞：γδT细胞为7.3±1.08；每克真皮组织能提出T细胞（1.95±1.31）×107个，αβT细胞：γδT细胞为6.7±1.31。两者在细胞数量及细胞亚群之间的比例上差异不明显。皮肤中的γδT细胞均为Vδ1+细胞，未见Vδ2+细胞。2.在PMA和ionomycin的满负荷刺激下，外周血T细胞及人皮肤T细胞的IL-2和IGF-1合成水平明显升高，不同T细胞之间的合成水平无明显差异。3.外周血αβT细胞和γδT细胞，及人皮肤αβT细胞被成功扩增，人皮肤γδT细胞扩增失败，外周血和人皮肤αβT细胞的扩增倍数明显高于外周血γδT细胞。4.高于2μg/ml的anti-CD3mAb可激活人皮肤T细胞，细胞表面CD25和CD69表达上调，分泌IL-2和IGF-1增多。5.4μg/ml CAR联合0.5μg/ml anti-CD3mAb可激活人皮肤T细胞，细胞表面CD25和CD69表达上调，分泌IL-2和IGF-1增多。单独4μg/ml CAR刺激不能激活人皮肤T细胞。6.人皮肤αβT细胞表面无JAML的表达，CD28的表达率为45.59±5.73%；人皮肤γδT细胞无CD28的表达，JAML的表达率为8.56±2.19%。7.4μg/ml CAR联合0.5μg/ml anti-CD3mAb可激活人皮肤γδT细胞，细胞表面CD25、CD69、CD80、CD86、JAML表达上调，分泌IL-2和IGF-1增多。单独4μg/ml CAR或单独0.5μg/mlanti-CD3mAb刺激不能激活人皮肤γδT细胞。但单独0.5μg/ml anti-CD3mAb刺激能使人皮肤γδT细胞表面JAML表达上调。8.4μg/ml CAR联合0.5μg/ml anti-CD3mAb不能激活人皮肤αβT细胞，但能使细胞表面CD28表达上调。单独0.5μg/ml anti-CD3mAb刺激也能使人皮肤αβT细胞表面CD28表达上调，单独4μg/ml CAR刺激对人皮肤αβT细胞表面CD28表达无影响。9.激活的人皮肤γδT细胞联合0.5μg/ml anti-CD3mAb可激活人皮肤αβT细胞，细胞表面CD25、CD69、表达上调。 结论：人皮肤T细胞分为αβT细胞和γδT细胞，JAML是协助全体人皮肤T细胞激活的重要共刺激分子，能为人皮肤γδT细胞的激活提供第二信号，协助其激活；而激活的人皮肤γδT细胞能为人皮肤αβT细胞提供激活所需的第二信号，协助其激活。提示创面中全部人皮肤T细胞的激活可能是通过先激活人皮肤γδT细胞，再由激活的人皮肤γδT细胞协助人皮肤αβT细胞的激活这个顺序进行的。
[Abstract]:Objective: the activation of skin T cells plays an important role in the initiation of wound healing. The non immune activation of human skin T cells may be one of the main reasons for the formation of chronic wounds. The study found that the adhesion molecule like protein (JAML) expressed on the surface of T cells in the skin of mice is the activation of T cell activation in the initiation stage of the wound healing. JAML is also expressed in the human body, but its similar effect has not been verified. Therefore, we have extracted human skin alpha beta T cells and gamma delta T cells, interfered with a variety of activators and blockers associated with JAML, and measured their different reactions to the interference, and observed whether JAML had a human wound surface. The ability of the important costimulators to promote the activation of T cells in the healing phase of the skin is designed to further explore the activation and activation of human skin T cells during wound healing, and to deepen the understanding of the immunological mechanism in the healing process of the wound.
Materials and methods: 1. the human skin T cells were extracted from the tissue, and the activity of the extracted cells was observed. The number of T cells in the skin and the phenotypic.2. were detected with the ionomycin full load of PMA and 1000ng/ml of 100ng/ml to stimulate the peripheral blood T cells and human T cells. The skin T cells were cultured and amplified to observe the amplification ability of different T cells,.4. using different doses of anti-CD3mAb (0.1 mu g/ml, 0.5 mu g/ml, 1 mu g/ml, 2 mu g/ml, 5 micron, 10 micron g/ml) to stimulate the human skin T cells, and to detect the surface activation marker CD25, the expression of the cell division and the level of 4 micron recombinant coxsackie adenovirus. The receptor protein (CAR) stimulates human skin T cells with 0.5 mu g/ml anti-CD3mAb or separate 4 g/mlCAR. The expression of the surface activation marker, the expression of CD69 and the level of the level.6. secreting the IL-2 and IGF-1 cells of the human skin are separated from the human skin alpha beta T and the gamma delta T cells. The surface activation markers CD25, CD69 and surface CO stimulator CD80, CD86, JAML, and human skin alpha beta cells were extracted by 4 mu g/ml CAR combined with 0.5 mu g/ml anti-CD3mAb, alone 4 mu g/ml CAR or alone 0.5 mu g/mlanti-CD3mAb, respectively. The surface activation markers, CD25, CD69 and surface costimulatory molecules CD80, CD86, JAML, and the level of IL-2 and IGF-1 were detected by g/ml anti-CD3mAb stimulation. The surface CO stimulatory molecules were detected by 4 micron g/ml CAR or 0.5 micron g/ml. CD3mAb stimulates human skin alpha beta T cells and detects the expression of CD25 and CD69 on the surface of human skin alpha beta T cells.
Results: 1. the activity of human skin T cells extracted from the tissue was good. The average per gram of epidermal tissue was able to propose T cells (2.63 + 1.72) x 107, alpha beta T cells: 7.3 + 1.08, and T cells (1.95 + 1.31) x 107 cells per gram of dermal tissue, and the size of gamma delta T was 6.7 + 1.31. between the number of cells and the cell subsets. There was no significant difference in the proportion of V Delta T cells in the skin. No V Delta 2+ cell.2. was stimulated by full load of PMA and ionomycin. The IL-2 and IGF-1 synthesis levels of T cells in peripheral blood and T cells of human skin were significantly increased, and there was no significant difference in the synthesis level between different cells. The amplification of the skin alpha beta T cells was successfully amplified, and the amplification of human skin gamma delta T cells failed. The amplification of peripheral blood and human skin alpha beta T cells was significantly higher than that in the peripheral blood and the.4. of the T cells of the peripheral blood was higher than the anti-CD3mAb of 2 Mu g/ml. The expression of CD25 and CD69 expression on the cell surface was up and the secretion IL-2 and IGF-1 increased together 0.5 Mu MAb activates human skin T cells, the expression of CD25 and CD69 is up-regulated, and the secretion of IL-2 and IGF-1 is increased. A single 4 uh CAR stimulation can not activate the expression of JAML in the surface of the human skin T cell.6. human skin alpha beta T cells. The expression rate is 45.59 + 5.73%, and the expression rate of human skin gamma delta cells is 8.56 +. CAR combined with 0.5 mu g/ml anti-CD3mAb activates human skin gamma delta T cells, the cell surface is CD25, CD69, CD80, CD86, JAML expression up, and IL-2 and IGF-1 increase. Individual 4 micron g/ml or 0.5 micron stimulus can not activate human skin gamma delta cells. The upregulation of.8.4 micron g/ml CAR combined with 0.5 u g/ml anti-CD3mAb can not activate human skin alpha beta T cells, but the expression of CD28 expression on the cell surface can be up-regulated. A single 0.5 u g/ml anti-CD3mAb stimulation can also increase the CD28 expression of the surface of human skin alpha beta T cells. T cells combined with 0.5 mu g/ml anti-CD3mAb can activate human skin alpha beta T cells, and the expression of CD25 and CD69 on the cell surface is upregulated.
Conclusion: human skin T cells are divided into alpha beta T cells and gamma delta T cells. JAML is an important co stimulator to assist the activation of all human skin T cells. It can provide second signals for the activation of human skin gamma delta T cells, and the activated human skin gamma delta T cells can provide second signals needed for activation of human skin alpha beta T cells and assist in their excitation. Activation of all human skin T cells in the wound may be activated by activating human skin gamma delta T cells and activating human skin gamma delta T cells to assist the activation of human skin alpha beta T cells.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R641
本文编号：2172709
[Abstract]:Objective: the activation of skin T cells plays an important role in the initiation of wound healing. The non immune activation of human skin T cells may be one of the main reasons for the formation of chronic wounds. The study found that the adhesion molecule like protein (JAML) expressed on the surface of T cells in the skin of mice is the activation of T cell activation in the initiation stage of the wound healing. JAML is also expressed in the human body, but its similar effect has not been verified. Therefore, we have extracted human skin alpha beta T cells and gamma delta T cells, interfered with a variety of activators and blockers associated with JAML, and measured their different reactions to the interference, and observed whether JAML had a human wound surface. The ability of the important costimulators to promote the activation of T cells in the healing phase of the skin is designed to further explore the activation and activation of human skin T cells during wound healing, and to deepen the understanding of the immunological mechanism in the healing process of the wound.
Materials and methods: 1. the human skin T cells were extracted from the tissue, and the activity of the extracted cells was observed. The number of T cells in the skin and the phenotypic.2. were detected with the ionomycin full load of PMA and 1000ng/ml of 100ng/ml to stimulate the peripheral blood T cells and human T cells. The skin T cells were cultured and amplified to observe the amplification ability of different T cells,.4. using different doses of anti-CD3mAb (0.1 mu g/ml, 0.5 mu g/ml, 1 mu g/ml, 2 mu g/ml, 5 micron, 10 micron g/ml) to stimulate the human skin T cells, and to detect the surface activation marker CD25, the expression of the cell division and the level of 4 micron recombinant coxsackie adenovirus. The receptor protein (CAR) stimulates human skin T cells with 0.5 mu g/ml anti-CD3mAb or separate 4 g/mlCAR. The expression of the surface activation marker, the expression of CD69 and the level of the level.6. secreting the IL-2 and IGF-1 cells of the human skin are separated from the human skin alpha beta T and the gamma delta T cells. The surface activation markers CD25, CD69 and surface CO stimulator CD80, CD86, JAML, and human skin alpha beta cells were extracted by 4 mu g/ml CAR combined with 0.5 mu g/ml anti-CD3mAb, alone 4 mu g/ml CAR or alone 0.5 mu g/mlanti-CD3mAb, respectively. The surface activation markers, CD25, CD69 and surface costimulatory molecules CD80, CD86, JAML, and the level of IL-2 and IGF-1 were detected by g/ml anti-CD3mAb stimulation. The surface CO stimulatory molecules were detected by 4 micron g/ml CAR or 0.5 micron g/ml. CD3mAb stimulates human skin alpha beta T cells and detects the expression of CD25 and CD69 on the surface of human skin alpha beta T cells.
Results: 1. the activity of human skin T cells extracted from the tissue was good. The average per gram of epidermal tissue was able to propose T cells (2.63 + 1.72) x 107, alpha beta T cells: 7.3 + 1.08, and T cells (1.95 + 1.31) x 107 cells per gram of dermal tissue, and the size of gamma delta T was 6.7 + 1.31. between the number of cells and the cell subsets. There was no significant difference in the proportion of V Delta T cells in the skin. No V Delta 2+ cell.2. was stimulated by full load of PMA and ionomycin. The IL-2 and IGF-1 synthesis levels of T cells in peripheral blood and T cells of human skin were significantly increased, and there was no significant difference in the synthesis level between different cells. The amplification of the skin alpha beta T cells was successfully amplified, and the amplification of human skin gamma delta T cells failed. The amplification of peripheral blood and human skin alpha beta T cells was significantly higher than that in the peripheral blood and the.4. of the T cells of the peripheral blood was higher than the anti-CD3mAb of 2 Mu g/ml. The expression of CD25 and CD69 expression on the cell surface was up and the secretion IL-2 and IGF-1 increased together 0.5 Mu MAb activates human skin T cells, the expression of CD25 and CD69 is up-regulated, and the secretion of IL-2 and IGF-1 is increased. A single 4 uh CAR stimulation can not activate the expression of JAML in the surface of the human skin T cell.6. human skin alpha beta T cells. The expression rate is 45.59 + 5.73%, and the expression rate of human skin gamma delta cells is 8.56 +. CAR combined with 0.5 mu g/ml anti-CD3mAb activates human skin gamma delta T cells, the cell surface is CD25, CD69, CD80, CD86, JAML expression up, and IL-2 and IGF-1 increase. Individual 4 micron g/ml or 0.5 micron stimulus can not activate human skin gamma delta cells. The upregulation of.8.4 micron g/ml CAR combined with 0.5 u g/ml anti-CD3mAb can not activate human skin alpha beta T cells, but the expression of CD28 expression on the cell surface can be up-regulated. A single 0.5 u g/ml anti-CD3mAb stimulation can also increase the CD28 expression of the surface of human skin alpha beta T cells. T cells combined with 0.5 mu g/ml anti-CD3mAb can activate human skin alpha beta T cells, and the expression of CD25 and CD69 on the cell surface is upregulated.
Conclusion: human skin T cells are divided into alpha beta T cells and gamma delta T cells. JAML is an important co stimulator to assist the activation of all human skin T cells. It can provide second signals for the activation of human skin gamma delta T cells, and the activated human skin gamma delta T cells can provide second signals needed for activation of human skin alpha beta T cells and assist in their excitation. Activation of all human skin T cells in the wound may be activated by activating human skin gamma delta T cells and activating human skin gamma delta T cells to assist the activation of human skin alpha beta T cells.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R641
【参考文献】
相关期刊论文 前6条
1 陈宏远;芮雯;花文峰;杜军;蔡绍晖;;鼠抗人TCR/CD3单克隆抗体的制备及其初步应用[J];广东药学院学报;2008年06期
2 陈国江,黎燕;抗CD3单克隆抗体的作用机制及其在临床上的应用[J];国外医学.药学分册;2005年01期
3 吕明;冯健男;黎燕;沈倍奋;;基因工程化抗CD3单克隆抗体及其诱导免疫耐受的研究进展[J];免疫学杂志;2006年S1期
4 付小兵,顾小曼,孙同柱,陈伟,杨银辉,孙晓庆,盛志勇;创面愈合组织三种生长相关因子表达及信号转导机制的实验研究[J];中华外科杂志;2001年09期
5 陈同辛,何振娟,吴圣楣,应大明,朱亚忠,王耀平;CD3抗体诱导的新生儿脐带血淋巴细胞信号传导的研究[J];中华微生物学和免疫学杂志;2003年01期
6 谭锦泉;肖威;王岚;何玉玲;;NKT细胞发育及功能研究进展[J];中国免疫学杂志;2010年06期
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