组织工程皮肤种子细胞免疫原性及组织工程皮肤移植SCID鼠的研究

发布时间：2018-04-10 07:50

本文选题：组织工程皮肤　切入点：免疫原性　出处：《第三军医大学》2008年博士论文

【摘要】： 组织工程皮肤是目前治疗难治性溃疡、大面积烧伤等皮肤缺损性疾病最有效的方法之一。组织工程皮肤是采用组织工程学方法,体外分离自体或异体细胞并长期培养和扩增后,与其他天然或合成材料共同构建而成。采用自体细胞虽然避免了免疫排斥反应,但为患者造成了新的创面,并限制了组织工程皮肤的商品化。异体细胞经体外长期培养后是否被受体免疫系统识别即免疫原性如何,仍然存在争论。目前构建组织工程皮肤主要使用角质形成细胞(keratinocyte,KC)、成纤维细胞(fibroblast,FB)作为种子细胞。一般认为这两种细胞正常情况下仅表达MHC-Ⅰ类抗原,不表达MHC-Ⅱ类抗原及共刺激分子,本身不具备递呈抗原能力,因此免疫原性较弱。但有实验显示在炎症因子如干扰素-γ(interferon-γ,IFN-γ)的作用下,上述细胞能诱导HLA-DR的表达。HLA-DR属于MHC-Ⅱ分子,是专职抗原递呈细胞(antigen presenting cell,APC)呈递抗原的重要结构之一。还有研究者认为KC可通过诱导表达B7分子来向T淋巴细胞传递协同刺激信号,从而激活T淋巴细胞。因此KC、FB能否成为APC,从而激活T细胞需要实验加以明确。混合淋巴细胞培养试验是以研究细胞免疫为主,一定程度上模拟体内免疫识别途径,广泛应用于研究免疫排斥反应的实验中。本实验将不同代KC、FB与异体淋巴细胞混合培养,观察淋巴细胞的增殖程度,反映异体KC、FB在体外培养的过程中免疫原性的变化情况。异体细胞移植后是否被受体免疫系统排斥,另一个关键之处在于其能否被受体的专职APC所识别。朗格汉斯细胞(langerhans cell, LC)是专职APC,长期定植于皮肤、呼吸道上皮及消化道粘膜上皮中,摄取抗原后向局部淋巴组织迁移,在那里最终分化成熟,激活初始T淋巴细胞,启动免疫反应或诱导免疫耐受。LC如何识别抗原,激活淋巴细胞的具体机制目前尚不完全清楚。随着LC的体外培养成功,对于LC能否识别异体KC、FB的问题能够进一步深入研究。本实验采用多种细胞因子联合诱导培养LC,将培养的LC与异体KC、FB混合培养后,观察LC的表型变化情况,反映LC对异体KC、FB的识别情况。组织工程皮肤支架材料也是影响其免疫原性的一个重要因素,组织工程皮肤采用的支架材料主要分为合成高分子材料及天然高分子材料,目前的研究主要集中于支架材料的生物毒性和降解性等,对其免疫原性的研究较少。动物胶原属于天然高分子材料,是由动物细胞合成,是细胞外间质的主要成分,因其易于获取,在组织工程皮肤支架材料中的应用最为广泛,虽然不同种类的动物分离出来的胶原极其相似,但毕竟属于异种蛋白,植入后是否具有免疫原性,有必要系统地研究。组织工程皮肤免疫原性的研究进展缓慢,其中一个主要原因就是移植于人体的临床研究受到伦理学等多方面的制约;另外长期缺乏一种良好的能够模拟人体免疫内环境的实验动物也影响了深入研究。重度联合免疫缺陷(severe combined immunodeficiency,SCID)鼠是细胞免疫、体液免疫均缺陷的动物,容易植入人的免疫细胞和组织,构建人的免疫系统。有实验显示植入SCID鼠的人淋巴细胞能够长期存活,仍然保持排斥异体移植物的特性。另有实验将人皮肤移植于SCID鼠后,移植的人皮肤能长期维持正常的皮肤结构,以上特点将使SCID鼠成为研究组织工程皮肤免疫原性的一个有力工具。本实验将组织工程皮肤、人皮肤和无细胞真皮支架移植于SCID鼠后,植入异体人脾淋巴细胞后观察组织工程皮肤三者的免疫排斥迹象,从组织水平反映组织工程皮肤的免疫原性。现将本实验方法、结果和结论分述如下: 方法: 1.采用组织工程的方法分离培养KC、FB,并不断传代。将不同代的KC、FB与异体淋巴细胞混合培养,观察淋巴细胞的增殖程度。CD1a标记LC,流式细胞仪测不同代数KC中混杂的LC的变化情况,以及KC在不同培养体系下细胞表面CD80、CD86的变化情况,从细胞水平观察组织工程皮肤种子细胞的免疫原性。 2.体外采用多种细胞因子联合诱导外周血单核细胞分化发育为LC,流式细胞仪测细胞表面CD1a、HLA-DR、CD80、CD86和CD83的表达情况,电镜鉴定培养的细胞为LC。将纯化的KC、FB分别与LC共培养后测LC的细胞表型的变化情况,从细胞水平观察LC对异体KC、FB的识别情况。 3.将体外分离的不同数量的人脾淋巴细胞注入SCID鼠腹腔内,观察SCID鼠的生长情况,流式细胞测鼠血中人淋巴细胞的分类情况,ELISA法测鼠血中人IgG的含量,摸索出一条稳定植入人脾淋巴细胞的实验方法。 4.将组织工程皮肤、人包皮、无细胞真皮支架移植于SCID鼠,随后植入异体人脾淋巴细胞后,用组织学和免疫组织化学的方法观察组织工程皮肤等的免疫排斥迹象,从组织水平反映组织工程皮肤整体的免疫原性。结果: 1.原代～4代KC、原代FB能显著刺激异体淋巴细胞增殖。流式细胞仪分析显示KC中混杂的LC逐渐减少,5代后无法测出LC的存在。KC刺激异体淋巴细胞增殖与其中混杂的LC明显相关。5代KC在无血清、有血清和PHA存在的培养体系均无法检测出细胞表面CD80、CD86的表达。 2.人外周血单核细胞在GM-CSF、IL-4、TGF-β1的联合诱导下,分化发育为LC,流式细胞仪显示细胞高表达CD1a,低表达HLA-DR、CD80、CD86,不表达CD83,电镜下观察到LC的特殊结构birbeck小体。与纯化的KC、FB共培养后LC的CD80、CD86和CD83无明显改变,也不能明显刺激同源淋巴细胞增殖。 3.将3×107人脾淋巴细胞植入SCID鼠后,动物无明显的移植物抗宿主反应。2周至10周鼠血中分离出的淋巴细胞均测得T细胞、B细胞、NK细胞。其中T、B细胞的百分比8周达到峰值,分别为18.2±0.4%和7.5±0.3%。鼠血中人IgG10周达到182.51±4.96 ug/ml。 4.组织工程皮肤、人包皮移植于SCID鼠后能维持其正常人皮肤组织结构,植入异体淋巴细胞后,组织工程皮肤未发生明显的免疫排斥反应,人包皮则发生了明显的免疫排斥反应。结论:本实验将组织工程皮肤种子细胞与异体淋巴细胞混合培养,纯化后种子细胞不能显著刺异体激淋巴细胞的增殖;纯化的种子细胞与体外诱导培养的不成熟LC混合培养后,也不能明显上调LC CD80、CD86和CD83的表达。将人脾淋巴细胞植入SCID鼠后,人淋巴细胞能继续增殖和分化,仍具有分泌人IgG等功能。组织工程皮肤移植于SCID鼠后植入异体淋巴细胞,未发生明显排斥反应。综上所述,本实验构建的组织工程皮肤免疫原性较弱,不易引起机体的免疫排斥反应。
[Abstract]:The skin tissue engineering is the treatment of refractory ulcers, burns and other methods of large area skin defect disease. One of the most effective skin tissue engineering is by tissue engineering method, in vitro autologous or allogeneic cells and long term culture and amplification, and other natural or synthetic materials constructed by autologous cells can avoid. Immune rejection, but caused a new wound for patients, and limit the commercialization of tissue engineered skin. Allogeneic cells after long-term in vitro cultivation is the immune system of receptor recognition is immunogenic, is still controversial. The main use of tissue engineering skin keratinocytes (keratinocyte, KC), a fiber cells (fibroblast, FB) as seed cells. Generally these two kinds of cells normally only the expression of MHC- class I antigen, the expression of MHC- class II antigens and costimulatory molecules That does not have antigen-presenting ability, so the weak immunogenicity. But some experiments showed that in inflammatory cytokines such as interferon gamma (interferon- y, IFN- y) under the effect of the expression of.HLA-DR cells can induce HLA-DR belongs to the MHC- II molecule is professional antigen presenting cells (antigen presenting cell, APC) important the structure of antigen presentation. And researchers believe that KC can also induce the expression of B7 molecules to T cells transmit costimulatory signal to activate T lymphocytes. So KC, FB can become APC, which activates T cells need to clear. The mixed lymphocyte culture test is to study the cell immune, immune to a certain extent in the way of recognition simulation, widely applied in the study of immune rejection in the experiment. In the experiment of different generations of KC, cultured with allogeneic lymphocytes in mixed FB lymphocytes proliferation, degree of observation, reflection The changes of immunogenicity during the culture of KC and FB in vitro.
Allogeneic cell transplantation is rejected by host immune system, another key lies in whether it can be a full-time receptor identified in the APC. Hans Lange (Langerhans cell, LC cells) is a full-time APC, long-term colonization in the skin, respiratory and digestive tract epithelial mucosa, migrate to the local lymph tissue uptake of antigen after the final where the initial differentiation, activation of T lymphocytes, immune response or immune tolerance induced by.LC antigen recognition, the specific mechanism of activated lymphocytes remains unclear. With the LC cultured in vitro successfully, for LC can identify allogeneic KC, FB problem can be further studied. This experiment adopts a variety of cytokines induced by the combination of culture LC, LC and KC in cultured allogenic FB, mixed culture, to observe the phenotype change of LC and LC reflect on allogeneic KC, identification of FB.
Scaffold materials of tissue engineering skin is also an important factor affecting the immunogenicity of the scaffold material of tissue engineering skin used consists of synthetic polymer materials and natural polymer materials, the present study focuses on the scaffold materials for biological toxicity and degradation, study on the immunogenicity of small animal collagen belongs to natural. Polymer materials are synthesized by animal cells, is the main component of extracellular matrix, because of its easy access and application in tissue engineering scaffold material in the skin is the most widely used, although different animal separated collagen is very similar, but after all, belongs to the heterogeneous protein is immunogenic after implantation, it is necessary to to study.
Research progress of tissue engineering skin immunogenicity is slow, clinical study of one of the main reasons is transplanted into the body is restricted by ethics and other aspects; another long-term lack of a good environment to mimic human immune animal also studied. Effects of severe combined immunodeficiency (severe combined, immunodeficiency, SCID) in cellular immunity, humoral immune defects in animal, easy implantation of human immune cells and tissues, construct the human immune system. Some experiments showed that human lymphocytes transplanted in SCID mice could still maintain long-term survival, graft rejection characteristics. The human skin transplanted to SCID mice after skin transplantation can the normal skin structure maintained for a long time, the above features make SCID mice become a powerful tool to study tissue engineering skin immunogenicity. This experiment will be organized Engineering skin, human skin and acellular dermal scaffold were transplanted to SCID rats, observe signs of rejection immune tissue engineering skin of the three implanted allogenic human splenic lymphocytes, reflect the immunogenicity of tissue engineering skin from the organizational level. The experimental methods, results and conclusions are as follows:
Method:
1. using tissue engineering methods for isolation of KC, FB, and continuously passaged. Different generation of KC culture, mixed with allogenic lymphocytes FB,.CD1a marker LC lymphocyte proliferation were observed, the changes of LC mixed flow cytometry test in different generations of KC, and KC cells in different culture systems under the surface of CD80 and the change of CD86, observe the immunogenicity of seed cells for skin tissue engineering from the cell level.
2. in vitro by cytokines induced by peripheral blood mononuclear cells and differentiation of LC cells was measured by flow cytometry on CD1a, HLA-DR, CD80, CD86 and CD83 expression of the cultured cells were identified by electron microscopy, LC. purified KC, FB changes respectively after cultured with LC LC fine test the cell phenotype, from the cell level to observe the effect of LC on allogeneic KC, identification of FB.
3. in vitro separation of different number of human spleen lymphocytes in SCID mice by intraperitoneal injection, to observe the growth of SCID rats, the classification of flow cytometric measurement of blood lymphocytes, determination of blood IgG ELISA method, and find out a stable implantation of human spleen lymphocytes experiment method.
4. of the tissue engineering skin, human foreskin acellular dermal scaffold were transplanted to SCID rats, then implanted in allogenic human splenic lymphocytes, using histological and immunohistochemical observation of immune rejection of the tissue engineering skin signs reflect the immunogenicity of tissue engineering skin tissue from the whole level.
Result:
The 1. primary to the 4 generation KC, primary FB could stimulate the proliferation of allogeneic lymphocytes. KC analysis showed that in mixed LC gradually decreased flow cytometry after 5 generations could not be detected in the presence of LC.KC to stimulate the proliferation of allogeneic lymphocytes and mixed with LC was significantly related to.5 generation KC in serum-free culture system, there exists the serum and PHA were unable to detect cell surface CD80, CD86 expression.
IL-4 2. in human peripheral blood mononuclear cells in GM-CSF, TGF-, and beta 1 induced differentiation, LC, flow cytometry showed that the cells with high expression of CD1a and low expression of HLA-DR, CD80, CD86, the expression of CD83 were observed by electron microscope LC Birbeck special structure bodies. With purified KC. After LC CD80 co culture FB, CD86 and CD83 had no obvious change, and could not stimulate homologous lymphocyte proliferation.
3. 3 x 107 human splenic lymphocytes transplanted in SCID mice after T cells were measured in isolated animal lymphocytes had no obvious graft-versus-host reaction of.2 to 10 weeks of blood B cells, NK cells. In T, the percentage of B cells reached the peak in 8 weeks, 0.4% and 7.5 respectively 18.2. 0.3%. + in the blood of mice IgG10 weeks to reach 182.51 + 4.96 ug/ml.
4., tissue engineered skin and human foreskin transplanted to SCID mice can maintain their normal skin tissue structure. After implantation of allogenic lymphocytes, no obvious immune rejection is found in tissue-engineered skin, and there is a significant immunological rejection in human foreskin.
Conclusion: the tissue engineering skin seed cells and allogeneic mixed lymphocyte culture, purified seed cells significantly stimulate allogenic lymphocytes proliferation; immature LC mixed culture cultured seed cells and purified in vitro after not significantly up-regulated LC expression of CD80, CD86 and CD83. The human spleen lymphocytes implanted SCID after, people can continue to lymphocyte proliferation and differentiation, still has secrete human IgG. Tissue engineering skin transplantation in SCID rats after implantation of allogeneic lymphocytes, no obvious rejection. In conclusion, this experiment is the primary tissue engineering skin immune building, is not easy to cause immune rejection.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392

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