再生家蚕丝素蛋白激发的T细胞应答

发布时间：2018-06-29 10:47

本文选题：再生 + 丝素蛋白　；参考：《苏州大学》2011年硕士论文

【摘要】：丝素蛋白是从蚕丝中提取的天然高分子纤维蛋白,具有良好的理化特性及生物相容性。再生丝素蛋白是由丝素蛋白经物理方式、环氧剂或SD交联而成的新型医用生物材料。近年来,研究者通过物理或化学方法将丝素蛋白制备了符合临床实际需要的再生多孔丝素膜,用于创面的修复。该材料具有良好的机械强度,不溶于水,膜的多孔结构使其富有较好通气性的同时,可阻挡病原微生物侵入创面。该材料经机体吸收后可在体内逐渐被降解代谢。本研究在已对家蚕丝素蛋白对B细胞应答影响的研究基础上,又深入探讨了不同性状的再生家蚕丝素蛋白在小动物体内及体表应用后对T细胞应答的影响,从而为丝素蛋白类材料临床应用后的免疫原性研究提供实验依据。目的:通过研究不同性状再生家蚕丝素蛋白材料对T细胞活化、增殖及分化的影响,分析丝素蛋白类材料的免疫原性。方法: 1.液状再生家蚕丝素蛋白体内运用对小鼠T细胞的激发作用将ICR小鼠随机分组,每组6只,实验组经腹腔注射1%的液状再生家蚕丝素蛋白0.5ml/只,以注射等量的无菌PBS为对照组。分别于第7d及第14d无菌取脾脏。制备细胞悬液作以下分析: 1.1小鼠脾脏细胞中CD3~+CD25~+、CD4~+CD25~+及CD4~+CD25~+ Foxp3~+T细胞的分析将上述分离的脾脏细胞置于流式分析管中(3×105细胞/管)。分别加入下列抗鼠直标抗体:第一组:CD3-FITC、CD25-APC;第二组:CD4-FITC、CD25-APC;第三组:CD4-FITC、CD25-APC及Foxp3-PE,反应后经FCM分析。 1.2经再生家蚕丝素蛋白处理的小鼠脾脏细胞对ConA的反应性将上述分离的小鼠脾脏细胞加入96孔培养板,2×105/100μL/孔,加入ConA,终浓度分别为0μg/ml、2.5μg/ml及5μg/ml。培养至72h,MTT检测细胞的增殖情况。同时采用FCM分析CD4~+CD25~+及CD4~+CD25~+Foxp3~+T的表达。 2、膜状再生家蚕丝素蛋白在大鼠创面运用后对T细胞的激活作用 2.1外科创伤面的建立将SD大鼠麻醉后经外科手术切除背部皮肤建立创面,面积为2 cm×2 cm。覆盖经预处理的膜状再生多孔丝素,将已切除皮肤的表皮盖在丝素膜上,进行缝合。以PVA海绵设为阳性对照,假手术组为阴性对照。 2.2移植处皮肤的病理学变化分别于术后第3d、14d、28d、56d及90d,在麻醉状态下取移植处皮肤,制作石蜡切片后行HE染色,分析炎性细胞的浸润及创面愈合情况。 2.3外周血、脾脏及胸腺中T细胞活化的动态变化于上述各时间点,取抗凝血分离PBMC,将大鼠处死,分离脾脏及胸腺细胞经FCM检测CD3~+CD25~+T的比率。同时行免疫组化分析。结果: 1.1液状再生家蚕丝素蛋白体内运用后对小鼠T细胞活化与分化的影响FCM分析的结果显示,腹腔注射液状再生丝素蛋白后第7d及第14d,脾脏细胞中CD3~+CD25~+T细胞的百分率分别为(7.32±1.02)%及(7.58±0.98)%;与对照组((7.262±1.31)%及(6.98±1.22)%)比较稍有升高,但无显著性差异(P㧐0.05)。CD4~+CD25~+T细胞的百分率分别为(7.22±0.92)%及(7.38±0.49)%;与对照组((7.19±0.58)%及(7.03±0.69)%)比较稍有升高,但无显著性差异(P㧐0.05)。CD4~+CD25~+Foxp3~+T百分率分别为(3.52±0.62)%及(3.78±0.56)%。与对照组((3.47±0.31)%及(3.44±0.65)%)比较稍有升高,但无显著性差异(P㧐0.05)。提示再生丝素蛋白可能对小鼠T细胞无明显的激发作用。 2、膜状再生家蚕多孔丝素蛋白对大鼠T细胞的激活作用 2.1移植处皮肤的病理学变化膜状再生家蚕丝素蛋白移植后第3d及第14d时,移植处可见少量炎性细胞,28d后炎症细胞已明显减少,第56d时已无炎症细胞。而阳性对照组大鼠术后均有炎症细胞浸润,至56d后才逐渐减少。 2.2外周血、脾脏及胸腺细胞中T细胞活化的动态变化于术后第3d、14d、28d、56d及90d各时间点,膜状再生家蚕丝素蛋白移植组大鼠PBMC、脾脏及胸腺中CD3~+CD25~+T细胞百分比率经FCM分析结果如表4所示:三者与阴性对照组比较均无显著性差异(P㧐0.05)。而与阳性对照组比较均有显著性差异(P0.05)。同时免疫组化的分析结果显示:实验组脾脏及胸腺组织中只有极少量的CD25~+T细胞,与FCM分析的结果一致。提示再生丝素蛋白对T细胞的激发作用较弱。结论: 体内外研究显示,再生家蚕丝素蛋白对机体T淋巴细胞的激发作用较小,引发机体细胞免疫应答的能力较弱,是一种低免疫原性的生物材料。
[Abstract]:Silk fibroin is a natural macromolecular fibrin extracted from silk and has good physicochemical properties and biocompatibility. Regenerative fibroin protein is a new medical biomaterial made of silk fibroin through physical way, epoxy agent or SD. In recent years, the researchers have prepared the silk fibroin by physical or chemical methods to meet the clinical value. The regenerated multi porous silk fibroin membrane is needed to repair the wound. The material has good mechanical strength and insoluble in water. The porous structure of the membrane makes it well ventilated and can prevent the pathogenic microorganism from invading the wound. This material can be gradually degraded and metabolize in the body after the body is absorbed. This study has been used in the family of silk fibroin. On the basis of the study on the effect of B cell response, the effect of the regenerative silk fibroin on the response of T cells after the application of the different characters in the small animals and the body surface was discussed, thus providing the experimental basis for the immunogenicity of the silk fibroin material after clinical application.
Objective: To study the immunogenicity of Silk Fibroin Materials by studying the effects of Silk Fibroin Materials from different characters on the activation, proliferation and differentiation of T cells.
Method:
1. ICR mice were randomly divided into 1. liquid regenerated silk fibroin proteins in mice, each group was randomly divided into 6 rats in each group. The experimental group was injected with 1% liquid regenerated silk fibroin 0.5ml/ only by intraperitoneal injection, and the same quantity of aseptic PBS was taken as the control group. The spleen was aseptic at 7d and 14d. The following analysis was made for the preparation of cell suspension.
Analysis of CD3~+CD25~+, CD4~+CD25~+ and CD4~+CD25~+ Foxp3~+T cells in spleen cells of 1.1 mice
The isolated splenic cells were placed in the flow analysis tube (3 x 105 cells / tubes). The following anti rat direct antibody antibodies were added: the first group: CD3-FITC, CD25-APC; second groups: CD4-FITC, CD25-APC; third groups: CD4-FITC, CD25-APC and Foxp3-PE. After the reaction, FCM analysis.
1.2 reactivity of mouse spleen cells treated with regenerated silk fibroin to ConA
The isolated mouse spleen cells were added to 96 hole culture plate, 2 x 105/100 mu L/ hole and ConA. The final concentration was 0 mu g/ml, 2.5 mu g/ml and 5 micron g/ml. were cultured to 72h. MTT was used to detect the proliferation of cells. FCM analysis of CD4~+CD25~+ and CD4~+CD25~+Foxp3~+T expression was used.
2, the activation of fibroblast protein from membrane regeneration on T cells after wound healing in rats.
2.1 establishment of surgical trauma surface
After the SD rats were anaesthetized, the back skin was excised by surgical operation. The area was 2 cm x 2 cm. covering the pretreated membranous regenerated multi hole silk fibroin. The skin covered by the skin was covered on the silk fibroin membrane and sutured with the PVA sponge. The sham operation group was negative.
Pathological changes of skin in 2.2 transplant areas
After 3D, 14d, 28d, 56d and 90d after the operation, the skin of the transplant was taken in the anesthetic state. After the paraffin section was made, HE staining was made to analyze the infiltration of inflammatory cells and the healing of the wound.
2.3 the dynamic changes of T cell activation in peripheral blood, spleen and thymus.
At each time point, PBMC was separated by anticoagulation. The rats were sacrificed and the ratio of spleen and thymocytes to FCM was detected by CD3~+CD25~+T. Immunohistochemical analysis was performed at the same time.
Result:
1.1 the effect of FCM analysis on the activation and differentiation of T cells in mice after the use of the liquid fibroin protein in vivo showed that the percentage of CD3~+CD25~+T cells in the spleen cells was (7.32 + 1.02)% and (7.58 + 0.98)%, respectively, and (7.262 + 1.31)% and (6.98 + 1.22)%) compared with the control group. The percentage of no significant difference (P? 0.05).CD4~+CD25~+T cells was (7.22 + 0.92)% and (7.38 + 0.49)%, respectively. Compared with the control group (7.19 + 0.58)% and (7.03 + 0.69)%), the percentage was slightly higher, but no significant difference (P? 0.05).CD4~+CD25~ +Foxp3~+T percentage was (3.52 + 0.62)% and (3.78 +%)% respectively. % and (3.44 + 0.65)% increased slightly, but there was no significant difference (P? 0.05), suggesting that regenerated silk fibroin might have no significant stimulating effect on T cells in mice.
2, membrane reactivation of Bombyx mori porous silk fibroin on rat T cells.
Pathological changes of skin in 2.1 transplant areas
A small amount of inflammatory cells were found at 3D and 14d after the transplantation of membrane regenerated silk fibroin. After 28d, inflammatory cells were obviously reduced and no inflammatory cells were found at 56d. The inflammatory cells were infiltrated in the positive control group after the operation, and the decrease gradually after 56d.
2.2 the dynamic changes of T cell activation in peripheral blood, spleen and thymocytes.
At the time points of 3D, 14d, 28d, 56d and 90d after operation, the percentage rate of CD3~+CD25~+T cells in the membrane like regenerated silk fibroin transplantation group rats PBMC, the percentage rate of CD3~+CD25~+T cells in the spleen and thymus, as shown in Table 4, as shown in Table 4: there was no significant difference between the three and the negative control groups (P? 0.05), but there were significant differences compared with those in the positive control group (P0.05). The results of immunohistochemical analysis showed that there were only a very small number of CD25~+T cells in the spleen and thymus tissue of the experimental group, which was in accordance with the results of FCM analysis, suggesting that the regenerated silk fibroin has a weak stimulating effect on T cells.
Conclusion:
In vivo and in vitro studies have shown that the regenerated silk fibroin protein has less stimulating effect on the body's T lymphocyte and is a low immunogenic biomaterial, which is weak in the immune response of the body.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R318.08;R392.1

【参考文献】