與創(chuàng)面愈合啟動階段相關的人皮膚αβT細胞和γδT細胞免疫激活機制的體外研究
發(fā)布時間：2018-08-08 18:56
【摘要】：目的：皮膚T細胞的激活在創(chuàng)面愈合的啟動階段有重要的作用，人皮膚T細胞無法免疫激活可能是慢性創(chuàng)面形成的主要原因之一。研究發(fā)現(xiàn)小鼠皮膚T細胞表面表達的連接粘附分子樣蛋白（JAML）是創(chuàng)面愈合啟動階段中促進皮膚T細胞激活的重要共刺激分子。人體內(nèi)也有JAML的表達，但其是否具有類似作用尚未得到驗證。因此，我們提取了人皮膚αβT細胞和γδT細胞，用與JAML相關的多種激活劑和阻滯劑對其進行干擾，并測定其對干擾的不同反應，觀察JAML是否具有作為人創(chuàng)面愈合啟動階段促進皮膚T細胞免疫激活的重要共刺激分子的能力，旨在進一步探索創(chuàng)面愈合過程中人皮膚T細胞的激活活化的細節(jié)，深化對創(chuàng)面愈合過程中免疫學機制的認識。 材料與方法：1.從組織中提取人皮膚T細胞，觀察提取細胞的活性，檢測皮膚T細胞的數(shù)量和表型。2.用100ng/ml的PMA和1000ng/ml的ionomycin滿負荷刺激外周血T細胞及人皮膚T細胞，，檢測不同T細胞合成IL-2和IGF-1的水平。3.分別對外周血T細胞及人皮膚T細胞進行培養(yǎng)擴增，觀察不同T細胞的擴增能力。4.采用不同劑量的anti-CD3mAb（0.1μg/ml、0.5μg/ml、1μg/ml、2μg/ml、5μg/ml、10μg/ml）刺激人皮膚T細胞，檢測其表面激活標志CD25、CD69的表達以及細胞分泌IL-2和IGF-1的水平。5.用4μg/ml重組柯薩奇-腺病毒受體蛋白（CAR）聯(lián)合0.5μg/ml anti-CD3mAb或單獨4μg/mlCAR刺激人皮膚T細胞，檢測其表面激活標志CD25、CD69的表達以及細胞分泌IL-2和IGF-1的水平。6.將人皮膚αβT和γδT細胞分離，檢測其表面JMAL和CD28的表達。7.從人皮膚T細胞中提取人皮膚γδT細胞，分別用4μg/ml CAR聯(lián)合0.5μg/ml anti-CD3mAb，單獨4μg/ml CAR或單獨0.5μg/mlanti-CD3mAb刺激，檢測其表面激活標志CD25、CD69和表面共刺激分子CD80、CD86、JAML的表達，以及細胞分泌IL-2和IGF-1的水平。8.從人皮膚T細胞中提取人皮膚αβT細胞，用4μg/ml CAR聯(lián)合0.5μg/ml anti-CD3mAb刺激，檢測其表面激活標志CD25、CD69和表面共刺激分子CD80、CD86、JAML的表達，以及細胞分泌IL-2和IGF-1的水平。再分別用4μg/ml CAR或0.5μg/ml anti-CD3mAb刺激，檢測其表面共刺激分子CD28的表達。9.用激活的人皮膚γδT細胞和0.5μg/ml anti-CD3mAb刺激人皮膚αβT細胞，檢測人皮膚αβT細胞表面激活標志CD25、CD69的表達。 結(jié)果：1.從組織中提取的人皮膚T細胞活性良好，平均每克表皮組織能提出T細胞（2.63±1.72）×107個，αβT細胞：γδT細胞為7.3±1.08；每克真皮組織能提出T細胞（1.95±1.31）×107個，αβT細胞：γδT細胞為6.7±1.31。兩者在細胞數(shù)量及細胞亞群之間的比例上差異不明顯。皮膚中的γδT細胞均為Vδ1+細胞，未見Vδ2+細胞。2.在PMA和ionomycin的滿負荷刺激下，外周血T細胞及人皮膚T細胞的IL-2和IGF-1合成水平明顯升高，不同T細胞之間的合成水平無明顯差異。3.外周血αβT細胞和γδT細胞，及人皮膚αβT細胞被成功擴增，人皮膚γδT細胞擴增失敗，外周血和人皮膚αβT細胞的擴增倍數(shù)明顯高于外周血γδT細胞。4.高于2μg/ml的anti-CD3mAb可激活人皮膚T細胞，細胞表面CD25和CD69表達上調(diào)，分泌IL-2和IGF-1增多。5.4μg/ml CAR聯(lián)合0.5μg/ml anti-CD3mAb可激活人皮膚T細胞，細胞表面CD25和CD69表達上調(diào)，分泌IL-2和IGF-1增多。單獨4μg/ml CAR刺激不能激活人皮膚T細胞。6.人皮膚αβT細胞表面無JAML的表達，CD28的表達率為45.59±5.73%；人皮膚γδT細胞無CD28的表達，JAML的表達率為8.56±2.19%。7.4μg/ml CAR聯(lián)合0.5μg/ml anti-CD3mAb可激活人皮膚γδT細胞，細胞表面CD25、CD69、CD80、CD86、JAML表達上調(diào)，分泌IL-2和IGF-1增多。單獨4μg/ml CAR或單獨0.5μg/mlanti-CD3mAb刺激不能激活人皮膚γδT細胞。但單獨0.5μg/ml anti-CD3mAb刺激能使人皮膚γδT細胞表面JAML表達上調(diào)。8.4μg/ml CAR聯(lián)合0.5μg/ml anti-CD3mAb不能激活人皮膚αβT細胞，但能使細胞表面CD28表達上調(diào)。單獨0.5μg/ml anti-CD3mAb刺激也能使人皮膚αβT細胞表面CD28表達上調(diào)，單獨4μg/ml CAR刺激對人皮膚αβT細胞表面CD28表達無影響。9.激活的人皮膚γδT細胞聯(lián)合0.5μg/ml anti-CD3mAb可激活人皮膚αβT細胞，細胞表面CD25、CD69、表達上調(diào)。 結(jié)論：人皮膚T細胞分為αβT細胞和γδT細胞，JAML是協(xié)助全體人皮膚T細胞激活的重要共刺激分子，能為人皮膚γδT細胞的激活提供第二信號，協(xié)助其激活；而激活的人皮膚γδT細胞能為人皮膚αβT細胞提供激活所需的第二信號，協(xié)助其激活。提示創(chuàng)面中全部人皮膚T細胞的激活可能是通過先激活人皮膚γδT細胞，再由激活的人皮膚γδT細胞協(xié)助人皮膚αβT細胞的激活這個順序進行的。
[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.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R641
本文編號：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.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R641
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