再生家蚕丝素蛋白激发的T细胞应答
|
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﹥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﹥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﹥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﹥0.05)。而与阳性对照组比较均有显著性差异(P<0.05)。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。
Silk fibroin extracted from the natural polymer fibrin, has a good physical and chemical properties and biocompatibility. Regenerated silk fibroin is a new type of medical biological materials by physical means, epoxy agents or SD crosslinking. In recent years, researchers prepared regenerated porous silk fibroin for clinical practice needs by physical or chemical methods, which can be used for wound repair. This material has good mechanical strength and do not dissolve in water. And this porous structure of membrane has better ventilation, which can stop the invasion of wound pathogens. This material can be gradually degraded after absorbing in the body. In this study, we aim to further study T cell response to the regeneration of the different characters of Silk Fibroin after application in small animals body surface on the basis of the study the B cell response to silk fibroin after implanting in animal body, so as to provide the immunology experimental evidence for fibroin-like material application in the clinical.
Objective: To investigate the immunogenicity of fibroin-like material by researching different characters silk fibroin material effect T cell activation, proliferation and differentiation
Methods:
1. The effect to T cells after injecting Liquid regenerated silk fibroin into vivo ICR mice were randomly divided into two groups, 6 mice each group. In experimental group every mouse was injected 1% liquid regenerated silk fibroin 0.5ml by intraperitoneal injection. In control group every mouse was injected 0.5ml sterile PBS . On the 7th and 14th day after injection, the spleen was taken sterilly. Analysis as following after isolating cells.
1.1 Analysis the ratio of CD3~+CD25~+, CD4~+CD25~+and CD4~+CD25~+T cell of spleen cells
The isolated spleen cells were added into flow tube, there were 3×105 cell/tube. Following the directly labeled anti-mouse antibody were added, there were there groups: the first one:CD3-FITC、CD25-APC;the second one: CD4-FITC、CD25-APC; the third one: CD4-FITC、CD25-APC and Foxp3-PE, analysis by FCM after reaction.
1.2 The reaction to ConA of spleen cell
The isolated spleen cells were added into 96 holes plate, there were 2×105 cell/hole. then ConA was added, the final concentration were 0μg/ml、2.5μg/ml and 5μg/ml. Cultured for 72h, the proliferation of T cell was analysed by MTT. And the expression of CD4~+CD25~+ and CD4~+CD25~+Foxp3~+T cell was analysed by FCM.
2. Study on the activation of T lymphocytes by regenerated porus silk fibrion film after local application on trauma
2.1 To establish the surgical wound
To randomly select the SD rats and create a wound with 2 cm×2 cm by removing its skin of back after anesthesia. Then we covered the wound surface with a piece of regenerated porous silk fibroin film and then lay the epidermis of the excisional skin on the regenerated porous silk fibroin film. The wound was sutured .
2.2 Histopathological examination
The rats were anaesthetized at the 3rdd, 14thd, 28thd, 56thd and 90thd after operation and the skins which at the site of transplantation were stained by hematoxylin–eosin (HE). Then we analysed the infiltration of inflammatory cells and the healing of wound.
2.3 Analyzing the ratio of CD3~+CD25~+T cell of PBMC, spleen and thymus
Five rats belonging to either group were sacrificed by an overdose of sodium pentobarbital at the same time points as before and then obtained PBMC, spleen and thymus which were detected the ratio of CD3~+CD25~+/CD3~+T cells with two-color immunofluorescence and flow cytometry technology. At the same time immunohistochemistry was performed to analysed the positive staining of CD3 and CD25.
Results:
1.1 The effect to T cells after injecting Liquid regenerated silk fibroin into vivo FCM analysis results showed that: at the 7th and 14th day after injecting liquid regenerated silk fibrion in vivo the expression of CD3~+CD25~+T of spleen cells was (7.32±1.02)% and (7.58±0.98)% respectively. It is slightly higher than control ((7.262±1.31)% and (6.98±1.22)%), but there was no significantly difference. The expression of CD4~+CD25~+T of spleen cells was (7.22±0.92)% and (7.38±0.49)% respectively. It is slightly higher than control ((7.19±0.58)% and (7.03±0.69)%), but there was no significantly difference.
The expression of CD4~+CD25~+Foxp3~+T of spleen cells was (3.52±1.03)% and (3.78±0.96)% respectively. It is slightly higher than control ((7.19±0.58)%及(7.03±0.69)%), but there was no significantly difference.
2. Study on the activation of T lymphocytes by regenerated Bombyx mori porus silk fibrion film after local application on trauma
2.1 Histopathological examination
Inflammatory cells were observed in site of implant on the 3rd and 14th day after surgery. The implanted silk fibroin film is clearly visible. The inflammatory cells reduced obviously after 28 days. There were no inflammatory cells and material after 56 and 90 days, and there were no difference from the normal skin. But there were always inflammatory cells of the skin of positive control. To 56d they decrease gradually.
2.2 Analyzing the ratio of CD3~+CD25~+T cell of PBMC, spleen and thymus
In the postoperative 3rdd, 14thd, 28thd, 56thd and 90thd, the percent of CD3~+CD25~+T of PBMC, spleen cell and thymus cell which were from experiment group was showed as following Table 4. There was no significant difference between experiment group and negative group(P﹥0.05). But there was significant difference between experiment group and positive group(P<0.05). Immunohistochemical analysis also showed that: the experimental group the spleen and thymus, only small amounts of CD25 positive staining. It was consistent with the results of FCM analysis.
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﹥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﹥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﹥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﹥0.05)。而与阳性对照组比较均有显著性差异(P<0.05)。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。
Silk fibroin extracted from the natural polymer fibrin, has a good physical and chemical properties and biocompatibility. Regenerated silk fibroin is a new type of medical biological materials by physical means, epoxy agents or SD crosslinking. In recent years, researchers prepared regenerated porous silk fibroin for clinical practice needs by physical or chemical methods, which can be used for wound repair. This material has good mechanical strength and do not dissolve in water. And this porous structure of membrane has better ventilation, which can stop the invasion of wound pathogens. This material can be gradually degraded after absorbing in the body. In this study, we aim to further study T cell response to the regeneration of the different characters of Silk Fibroin after application in small animals body surface on the basis of the study the B cell response to silk fibroin after implanting in animal body, so as to provide the immunology experimental evidence for fibroin-like material application in the clinical.
Objective: To investigate the immunogenicity of fibroin-like material by researching different characters silk fibroin material effect T cell activation, proliferation and differentiation
Methods:
1. The effect to T cells after injecting Liquid regenerated silk fibroin into vivo ICR mice were randomly divided into two groups, 6 mice each group. In experimental group every mouse was injected 1% liquid regenerated silk fibroin 0.5ml by intraperitoneal injection. In control group every mouse was injected 0.5ml sterile PBS . On the 7th and 14th day after injection, the spleen was taken sterilly. Analysis as following after isolating cells.
1.1 Analysis the ratio of CD3~+CD25~+, CD4~+CD25~+and CD4~+CD25~+T cell of spleen cells
The isolated spleen cells were added into flow tube, there were 3×105 cell/tube. Following the directly labeled anti-mouse antibody were added, there were there groups: the first one:CD3-FITC、CD25-APC;the second one: CD4-FITC、CD25-APC; the third one: CD4-FITC、CD25-APC and Foxp3-PE, analysis by FCM after reaction.
1.2 The reaction to ConA of spleen cell
The isolated spleen cells were added into 96 holes plate, there were 2×105 cell/hole. then ConA was added, the final concentration were 0μg/ml、2.5μg/ml and 5μg/ml. Cultured for 72h, the proliferation of T cell was analysed by MTT. And the expression of CD4~+CD25~+ and CD4~+CD25~+Foxp3~+T cell was analysed by FCM.
2. Study on the activation of T lymphocytes by regenerated porus silk fibrion film after local application on trauma
2.1 To establish the surgical wound
To randomly select the SD rats and create a wound with 2 cm×2 cm by removing its skin of back after anesthesia. Then we covered the wound surface with a piece of regenerated porous silk fibroin film and then lay the epidermis of the excisional skin on the regenerated porous silk fibroin film. The wound was sutured .
2.2 Histopathological examination
The rats were anaesthetized at the 3rdd, 14thd, 28thd, 56thd and 90thd after operation and the skins which at the site of transplantation were stained by hematoxylin–eosin (HE). Then we analysed the infiltration of inflammatory cells and the healing of wound.
2.3 Analyzing the ratio of CD3~+CD25~+T cell of PBMC, spleen and thymus
Five rats belonging to either group were sacrificed by an overdose of sodium pentobarbital at the same time points as before and then obtained PBMC, spleen and thymus which were detected the ratio of CD3~+CD25~+/CD3~+T cells with two-color immunofluorescence and flow cytometry technology. At the same time immunohistochemistry was performed to analysed the positive staining of CD3 and CD25.
Results:
1.1 The effect to T cells after injecting Liquid regenerated silk fibroin into vivo FCM analysis results showed that: at the 7th and 14th day after injecting liquid regenerated silk fibrion in vivo the expression of CD3~+CD25~+T of spleen cells was (7.32±1.02)% and (7.58±0.98)% respectively. It is slightly higher than control ((7.262±1.31)% and (6.98±1.22)%), but there was no significantly difference. The expression of CD4~+CD25~+T of spleen cells was (7.22±0.92)% and (7.38±0.49)% respectively. It is slightly higher than control ((7.19±0.58)% and (7.03±0.69)%), but there was no significantly difference.
The expression of CD4~+CD25~+Foxp3~+T of spleen cells was (3.52±1.03)% and (3.78±0.96)% respectively. It is slightly higher than control ((7.19±0.58)%及(7.03±0.69)%), but there was no significantly difference.
2. Study on the activation of T lymphocytes by regenerated Bombyx mori porus silk fibrion film after local application on trauma
2.1 Histopathological examination
Inflammatory cells were observed in site of implant on the 3rd and 14th day after surgery. The implanted silk fibroin film is clearly visible. The inflammatory cells reduced obviously after 28 days. There were no inflammatory cells and material after 56 and 90 days, and there were no difference from the normal skin. But there were always inflammatory cells of the skin of positive control. To 56d they decrease gradually.
2.2 Analyzing the ratio of CD3~+CD25~+T cell of PBMC, spleen and thymus
In the postoperative 3rdd, 14thd, 28thd, 56thd and 90thd, the percent of CD3~+CD25~+T of PBMC, spleen cell and thymus cell which were from experiment group was showed as following Table 4. There was no significant difference between experiment group and negative group(P﹥0.05). But there was significant difference between experiment group and positive group(P<0.05). Immunohistochemical analysis also showed that: the experimental group the spleen and thymus, only small amounts of CD25 positive staining. It was consistent with the results of FCM analysis.
引文
1. Thierry Lefèvrea, Fran?ois Paquet-Merciera, Stéphanie Lesagea, et al. Study by Raman spectromicroscopy of the effect of tensile deformation on the molecular structure of Bombyx mori silk. Recent progress in vibration spectroscopy. 2009, 51(1):136-141.
2. Ha, Sung-Won. Structural study of bombyx mori silk fibroin during processing for regeneration. J Biol Chem. 2005,66(1):302.
3.黄君霆,朱万民,夏建国,向仲怀主编.中国蚕丝大全,四川科学技术出版社,1995.
4. Inoue S, Tanaka K, Arisaka F, et al. Silk fibroin of bombyx mori is secreted, assembling a high moleculai mass elementary unit consisiting of H-chain, L-chain, and P25, with a 6:6:1 molar ration. J Biol Chem. 2000, 275(51): 40517-40528.
5. Cong-zhao Zhou, Joel janin. Silk fibroin: structural implication of a remarkable aminl acid squence. Proteins. 2001, 44: 119-122.
6. Mori K, Tanaka K, Kikuchi Y, et al. Production of a chimeric fibroin light-chain polypetide in a fibroin secreyion-deficient nakee pupa mutant of the silkworm bombyx mori. J Mol Biol. 1995, 251(2): 217-228.
7. Tanaka K, Inoue S, Mizuno S. Hydrophobic interaction of P25, containing asn-linked oligosaccharide chains, with the H-L complex of silk fibroin produced by bombyx mori. Insect Biochem Mol Biol. 1999, 29(3):269-276.
8. H Ishikawa, M Nagura. Structure and physical properties of silk fibroin. Sen-iGakkaishi 1983, 39(10): 353-363.
9. R E Marsh, R B Corey, L Pauling. Crystal structure of silk fibroin. Biochemical Biophysical Acta 1955, 16: 81-86.
10.许才定,杨建康,陈寿菊,等.蚕丝蛋白的开发利用[J].国外丝绸, 1996, (6):28-33.
11. Hollander DH. Interstitial cystitis and silk allergy. Med Hypotheses 1994, 43(3):155- 156.
12. Wen CM, Ye ST, Zhou LX, et al. Silk- induced asthma in children: A report of 64 cases. Ann Allergy. 1990, 65(5): 375- 378.
13. Kurosaki S, Otsuka H, KunitomoM, et al. Fibroin allergy: IgE mediated hypersensitivity to silk suture materials. Nippon Ika Daigaku Zasshi 1999, 66(1): 41- 44.
14. Dewair M, Baur X, Ziegler K. Use of immunoblot technique for detection of human IgE and IgG antibodies to individual silk proteins. J Allergy Clin Immun 1985, 76(4): 537- 542.
15. ZhaomingW, Codina R, Fernandez- Caldas E, et al. Partial characterization of the silk allergens in mulberry silk extract. J Investing Allergy Clin Immun 1996, 6(4):237-241.
16. Uff CR, Scott AD, Pockley AG, et al. Influence of soluble suture factors on in vitromacrophage function.Biomaterials, 1995,16(5): 355-360.
17. Panilaitis B, Altman GH, Chen J, et al. Macrophage responses to silk. Biomaterials, 2003, 24(18): 3079- 3085.
18. Kum JP, Hai HJ, Chang KH. Antigenotoxicity of peptides produced from silk fibroin. Process Biochem 2002, 38(3):411-418.
19. Biman BM, Subhas CK. Cell proliferation and migration in silk fibroin 3D scaffolds. Biomaterials 2009, 30(15):2956-2965.
20. Marcos GF, Anne JM, Monika H, et al. Silk fibroin/hyaluronan scaffolds for human mesenchymal stem cell culture in tissue engineering. Biomaterials 2009, 30(28):5068-5076.
21. Ronald EU, Michael W, Kirsten P, et al. Growth of human cells on a non-woven silk fibroin net : a potential for use in tissue engineering. Biomaterials, 2004,25(6):1069-1075.
22.吴海涛,钟翠平,顾云娣等.蚕丝在软骨细胞立体培养中的应用.中国修复重建外科杂志2000, 14(5):301-305.
23. Sofia S, McCarthy MB, Gronowicz G, et al. Functionalized silk-based biomaterials for bone formation. Biomed Mater Res 2001, 54(1):139-148.
24.游晓波,傅荣,屈树新.组织工程多孔生物材料的制备及表征.实用医院临床杂志, 2008, 5(1): 23-25.
25.李慕勤,王健平,赵莉.天然高分子/羟基磷灰石支架材料对成骨细胞增殖的影响.黑龙江医药科学2008, 31(5): 1-2.
26.苗宗宁,潘宇红,祝建中等.丝素蛋白支架材料复合骨髓间充质干细胞构建组织工程化软骨.中国组织工程研究与临床康复2008, 12(27): 5243-5247.
27.刘向阳,李明忠.丝素膜血液相容性研究.中国血液流变学杂志2002, 12(4): 266-269.
28.黄福华,郑军,孙立忠.一种新的人工血管涂层及其实验研究.北京生物医学工程2008, 8(4): 399-403
29.张幼珠,吴徽宇,杨晓马,等.中药丝素膜的研制及其性能.丝绸, 1999, 8: 29-30.
30.孙皎,宁丽,顾国珍,等.医用丝素蛋白皮肤再生膜的细胞相容性评价.生物医学工程学杂志2000, 17(4):393-395.
1. Otsuka H, Ikeya T, Okano T, et al. Activation of lymphocyte proliferation by boronate-containing polymer immobilised on substrate: the effect of boron content on lymphocyte proliferation. Eur Cell Mater, 2006, 12:36-43.
2. Hunt JA, Flanagan BF, McLaughlin PJ, et al. Effect of biomaterial surface charge on the in?ammatory response: evaluation of cellular infiltration and TNF-alpha production. Biomed Mater Res, 1996, 31(1):139-144.
3. McGettrick AF, O’Neill LA. Toll-like receptors: key activators of leucocytes and regulator of haematopoiesis. Br J Haematol, 2007, 139(2):185-193.
4. Kabelitz D. Expression and function of Toll-like receptors in T lymphocytes. Curr Opin Immunol, 2007, 19(1):39-45.
5. Kim S. Jones. Effects of biomaterial-induced in?ammation on fibrosis and rejection. Seminars in Immunology, 2008, 20(2):130-136.
6. JJ Zhong and T Yoshida. High-density cultivation of Perilla frutescens cell suspensions for anthocyanin production: Effects of sucrose concentration and inoculum size. Enzyme and Microbial Technology, 1995, 17(12):1073-1079.
7.赵洁,王曦鸣,李继祥等. MTT法检测淋巴细胞增殖能力的影响因素.畜牧业, 2008, (2):28-30.
8. Koh WS, Yoon SY, Kwon BM, et al. Cinnamaldehyde inhibits lymphocyte proliferation and modulates T-cell differentiation. Int J Immunopharmacol, 1998,20(11):643-660.
9.张成钧,张佳林,钟嘉明,等.肝癌患者外周血CD4+CD25highT淋巴细胞亚群变化及临床意义.中国医科大学学报, 2009, 38(7):539-541.
10.周茂华,黄蔚,张敏,等.老年消化道肿瘤患者外周血CD4+CD25high调节性T细胞的表达及其临床意义.实用医学杂志, 2009, 25(15):2570-2572.
11. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol, 2003, 4(4):330-6.
1. Li Mingzhong, Tao Wei, Lu Shenzhou, et al. Compliant film of regenerated Antheraea pernyi silk fibroin by chemical crosslinking. Int J Biol Macromol, 2003, 32:159-163.
2.卢神州,李明忠,康宁,等.环氧交联剂在丝素膜剂制备中的应用.丝绸, 2000, 3:7-9.
3. Seo YK, Yoon HH, Park YS, et al. Correlation between scaffold in vivo biocompatibility and in vitro cell compatibility using mesenchymal and mononuclear cell cultures. Cell Biol Toxicol, 2009, 25:5135-22.
4. Nogueira GM, Rodas AC, Leite CA, et al. Preparation and characterization of ethanol-treated silk fibroin dense membranes for biomaterials application using waste silk fibers as raw material. Bioresour Technol, 2010, 101:8446-8451.
5. Morgan AW, Roskov KE, Lin-Gibson S, et al. Characterization and optimization of RGD-containing silk blends to support osteoblastic differentiation[J]. Biomaterials, 2008, 29:2556-2563.
6. Kim HJ, Kim UJ, Kim HS, et al. Bone tissue engineering with premineralized silk scaffolds[J]. Bone, 2008, 42:1226-1234.
7. Wang G, Yang H, Li M, et al. The use of silk fibroin/hydroxyapatite composite co-cultured with rabbit bone-marrow stromal cells in the healing of a segmental bone defect. J Bone Joint Surg Br, 2010, 92:320-325.
8. Chiarini A, Petrini P, Bozzini S, et al. Silk fibroin /poly( carbonate) -urethane as a substrate for cell growth: In vivo interactions with human cells. Biomaterials, 2003, 24: 789-799.
9. Pra DI, Petrini P, Chiarini A, et al. Silk fibroin-coated three-dimensional polyurethane scaffolds for tissue engineering: Interactions with normal human fibroblasts. Tissue Eng, 2003, 9: 1113-1121.
10. Panilaitis B, Altman GH, Chen J, et a1. Macrophage responses to silk. Biomaterials, 2003, 24: 3079-3085.
11. Santin M, Motta A, Freddi G, et a1. In vitro evaluation of the inflammatory potential of the silk fibroin[J]. J Biomed Mater Res, 1999, 46: 382-389.
12. Mnasria K, Lagaraine C, Manaa J, et al. IL-2/IL-2R pathway in dendritic cell modulates both their cytokine synthesis profiles and their capacity to activate allogeneicCD4+ T lymphocytes. Pathol Biol. 2009, 15. [Epub ahead of print].
13. Wu D, Guo Z, Ren Z, et al. Green tea EGCG suppresses T cell proliferation through impairment of IL-2/IL-2 receptor signaling. Free Radic Biol Med, 2009, 1;47:636-643.
14. Wang J, Wicker LS, Santamaria P. IL-2 and its high-affinity receptor: genetic control of immunoregulation and autoimmunity. Semin Immunol, 2009, 21:363-371.
1 Ni Y, Zhao X, Zhou L, et al. Radiologic and histologic characterization of silk fibroin as scaffold coating for rabbit tracheal defect repair. Otolaryngol Head Neck Surg, 2008, 139(2): 256-261.
2 Inoue S, Tanaka K, Arisaka F, et a1. Silk Fibroin of Bombyx mori Is Secreted, Assembling a High Molecular Mass Elementary Unit Consisting of H-chain, L-chain, and P25, with a 6:6:1 Molar Ratio. J Biol Chem, 2000, 275(51): 40517–40528.
3 Zhang YQ. Applications of natural silk protein sericin in biomaterials. Biotechnol Adv, 2002, 20(2): 91-100.
4 Yongzhong Wang, Hyeon-Joo Kim, Gordana Vunjak-Novakovic, et al. Stem cell-based tissue engineering with silk biomaterials. Biomaterials, 2006, 27(36): 6064–60822.
5 Brian D, Lawrence, Jeffrey K, et al. Silk film biomaterials for cornea tissue engineering. Biomaterials, 2009, 30(7): 1299–1308.
6 Charu Veparia, David L. Kaplan. Silk as a biomaterial. prog. ploym. sci, 2007, 32: 911-1007.
7田莉,闵思佳.丝素蛋白在组织工程细胞支架方面的研究进展.生物医学工程学杂志,2006, 23 (6): 1375-1378.
8 Zhou CZ, Confalonieri F, Jacque M, et al. Silk fibroin: structural implications of a remarkable amino acid sequence. Proteins, 2001, 44(2): 119-122.
9 Liu L, Melissa K, Callahan D, et al. Chemokine Receptor CXCR3: An Unexpected Enigma. Curr Top Dev Biol, 2005, 68: 149-154.
10 Zhang YY, Yoneyama H, Wang Y, et al. Mobilization of dendritic cell precursors into circulation by administrating MIP-1αin mice. J. Natl. Cancer Inst, 2004, 96(3): 201-209.
11 Zhou ZH, Shi Q, Wang JF, et al. Sensitization of multiple myeloma and B lymphoma lines to dexamethasone andγ–radiation-induced apoptosis by CD40 activation. Apoptosis, 2005, 10(1): 123-134.
12 Hollander DH.Interstitial cystitis and silk allergy.Med Hypotheses, 1994, 43(3): 155-156.
13 Wen CM, Ye ST, Zhou LX, et a1.Silk-induced asthma in children:Areport of
64 cases. Ann Allergy, 1990, 65(5): 375-378.
14 Dewair M, Baur X, Ziegler K. Use of immunoblot technique for detection of human IgE and IgG antibodies to individual silk protein. J. Allergy Clin Immunol, 1985, 76(4): 537-542.
15 Zhaoming W, Codina R, Fernandez CE, et a1. Partial characterization of the silk allergens in mulberry silk extract. J. Investig Allergol Clin Immunol, 1996, 6(4): 237-241.
16 Uff CR, Scott AD, Pockley AG, et a1. Influence of soluble suture factors on in vitro macrophage function. Biomaterials, 1995, 16(5): 355-360.
17 Panilaitis B, Ahman GH, Chen J, et a1. Macrophage responses to silk. Biomaterials, 2003, 24(18): 3079-3085.
18 Meinel L, Hofmann S, Karageorgiou V, et a1. The inflammatory responses to silk film in vitro and in vivo. Biomaterials, 2005, 26(2): 147-155.
19 Pra HD, Freddi G, Minic J, et a1. De novo engineering of reticular tissue in vivo by silk fibroin non woven materials. Biomaterials, 2005, 26(14): 1987-1979.
20 Santin M, Motta A, Freddi G, et al. In vitro evaluation of the inflammatory potential of the silk fibroin. J Biomed Mater Res, 1999, 46(3): 382-389.
21栾希英,张学光.丝素蛋白的免疫学特性及组织相溶性研究进展.国际生物医学工程杂志, 2006, 29(5): 2 96-299.
22 Altman GH, Diaz F, Jakuba C, et al. Silk-based biomaterials. Biomaterials, 2003, 24 (3): 401-416.
23 Korayem AM, Hauling T, Lesch C, et al. Evidence for an immune function of lepidopteran silk proteins. Biochem Biophys Res Commun, 2007, 352(2): 317-322.
2. Ha, Sung-Won. Structural study of bombyx mori silk fibroin during processing for regeneration. J Biol Chem. 2005,66(1):302.
3.黄君霆,朱万民,夏建国,向仲怀主编.中国蚕丝大全,四川科学技术出版社,1995.
4. Inoue S, Tanaka K, Arisaka F, et al. Silk fibroin of bombyx mori is secreted, assembling a high moleculai mass elementary unit consisiting of H-chain, L-chain, and P25, with a 6:6:1 molar ration. J Biol Chem. 2000, 275(51): 40517-40528.
5. Cong-zhao Zhou, Joel janin. Silk fibroin: structural implication of a remarkable aminl acid squence. Proteins. 2001, 44: 119-122.
6. Mori K, Tanaka K, Kikuchi Y, et al. Production of a chimeric fibroin light-chain polypetide in a fibroin secreyion-deficient nakee pupa mutant of the silkworm bombyx mori. J Mol Biol. 1995, 251(2): 217-228.
7. Tanaka K, Inoue S, Mizuno S. Hydrophobic interaction of P25, containing asn-linked oligosaccharide chains, with the H-L complex of silk fibroin produced by bombyx mori. Insect Biochem Mol Biol. 1999, 29(3):269-276.
8. H Ishikawa, M Nagura. Structure and physical properties of silk fibroin. Sen-iGakkaishi 1983, 39(10): 353-363.
9. R E Marsh, R B Corey, L Pauling. Crystal structure of silk fibroin. Biochemical Biophysical Acta 1955, 16: 81-86.
10.许才定,杨建康,陈寿菊,等.蚕丝蛋白的开发利用[J].国外丝绸, 1996, (6):28-33.
11. Hollander DH. Interstitial cystitis and silk allergy. Med Hypotheses 1994, 43(3):155- 156.
12. Wen CM, Ye ST, Zhou LX, et al. Silk- induced asthma in children: A report of 64 cases. Ann Allergy. 1990, 65(5): 375- 378.
13. Kurosaki S, Otsuka H, KunitomoM, et al. Fibroin allergy: IgE mediated hypersensitivity to silk suture materials. Nippon Ika Daigaku Zasshi 1999, 66(1): 41- 44.
14. Dewair M, Baur X, Ziegler K. Use of immunoblot technique for detection of human IgE and IgG antibodies to individual silk proteins. J Allergy Clin Immun 1985, 76(4): 537- 542.
15. ZhaomingW, Codina R, Fernandez- Caldas E, et al. Partial characterization of the silk allergens in mulberry silk extract. J Investing Allergy Clin Immun 1996, 6(4):237-241.
16. Uff CR, Scott AD, Pockley AG, et al. Influence of soluble suture factors on in vitromacrophage function.Biomaterials, 1995,16(5): 355-360.
17. Panilaitis B, Altman GH, Chen J, et al. Macrophage responses to silk. Biomaterials, 2003, 24(18): 3079- 3085.
18. Kum JP, Hai HJ, Chang KH. Antigenotoxicity of peptides produced from silk fibroin. Process Biochem 2002, 38(3):411-418.
19. Biman BM, Subhas CK. Cell proliferation and migration in silk fibroin 3D scaffolds. Biomaterials 2009, 30(15):2956-2965.
20. Marcos GF, Anne JM, Monika H, et al. Silk fibroin/hyaluronan scaffolds for human mesenchymal stem cell culture in tissue engineering. Biomaterials 2009, 30(28):5068-5076.
21. Ronald EU, Michael W, Kirsten P, et al. Growth of human cells on a non-woven silk fibroin net : a potential for use in tissue engineering. Biomaterials, 2004,25(6):1069-1075.
22.吴海涛,钟翠平,顾云娣等.蚕丝在软骨细胞立体培养中的应用.中国修复重建外科杂志2000, 14(5):301-305.
23. Sofia S, McCarthy MB, Gronowicz G, et al. Functionalized silk-based biomaterials for bone formation. Biomed Mater Res 2001, 54(1):139-148.
24.游晓波,傅荣,屈树新.组织工程多孔生物材料的制备及表征.实用医院临床杂志, 2008, 5(1): 23-25.
25.李慕勤,王健平,赵莉.天然高分子/羟基磷灰石支架材料对成骨细胞增殖的影响.黑龙江医药科学2008, 31(5): 1-2.
26.苗宗宁,潘宇红,祝建中等.丝素蛋白支架材料复合骨髓间充质干细胞构建组织工程化软骨.中国组织工程研究与临床康复2008, 12(27): 5243-5247.
27.刘向阳,李明忠.丝素膜血液相容性研究.中国血液流变学杂志2002, 12(4): 266-269.
28.黄福华,郑军,孙立忠.一种新的人工血管涂层及其实验研究.北京生物医学工程2008, 8(4): 399-403
29.张幼珠,吴徽宇,杨晓马,等.中药丝素膜的研制及其性能.丝绸, 1999, 8: 29-30.
30.孙皎,宁丽,顾国珍,等.医用丝素蛋白皮肤再生膜的细胞相容性评价.生物医学工程学杂志2000, 17(4):393-395.
1. Otsuka H, Ikeya T, Okano T, et al. Activation of lymphocyte proliferation by boronate-containing polymer immobilised on substrate: the effect of boron content on lymphocyte proliferation. Eur Cell Mater, 2006, 12:36-43.
2. Hunt JA, Flanagan BF, McLaughlin PJ, et al. Effect of biomaterial surface charge on the in?ammatory response: evaluation of cellular infiltration and TNF-alpha production. Biomed Mater Res, 1996, 31(1):139-144.
3. McGettrick AF, O’Neill LA. Toll-like receptors: key activators of leucocytes and regulator of haematopoiesis. Br J Haematol, 2007, 139(2):185-193.
4. Kabelitz D. Expression and function of Toll-like receptors in T lymphocytes. Curr Opin Immunol, 2007, 19(1):39-45.
5. Kim S. Jones. Effects of biomaterial-induced in?ammation on fibrosis and rejection. Seminars in Immunology, 2008, 20(2):130-136.
6. JJ Zhong and T Yoshida. High-density cultivation of Perilla frutescens cell suspensions for anthocyanin production: Effects of sucrose concentration and inoculum size. Enzyme and Microbial Technology, 1995, 17(12):1073-1079.
7.赵洁,王曦鸣,李继祥等. MTT法检测淋巴细胞增殖能力的影响因素.畜牧业, 2008, (2):28-30.
8. Koh WS, Yoon SY, Kwon BM, et al. Cinnamaldehyde inhibits lymphocyte proliferation and modulates T-cell differentiation. Int J Immunopharmacol, 1998,20(11):643-660.
9.张成钧,张佳林,钟嘉明,等.肝癌患者外周血CD4+CD25highT淋巴细胞亚群变化及临床意义.中国医科大学学报, 2009, 38(7):539-541.
10.周茂华,黄蔚,张敏,等.老年消化道肿瘤患者外周血CD4+CD25high调节性T细胞的表达及其临床意义.实用医学杂志, 2009, 25(15):2570-2572.
11. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol, 2003, 4(4):330-6.
1. Li Mingzhong, Tao Wei, Lu Shenzhou, et al. Compliant film of regenerated Antheraea pernyi silk fibroin by chemical crosslinking. Int J Biol Macromol, 2003, 32:159-163.
2.卢神州,李明忠,康宁,等.环氧交联剂在丝素膜剂制备中的应用.丝绸, 2000, 3:7-9.
3. Seo YK, Yoon HH, Park YS, et al. Correlation between scaffold in vivo biocompatibility and in vitro cell compatibility using mesenchymal and mononuclear cell cultures. Cell Biol Toxicol, 2009, 25:5135-22.
4. Nogueira GM, Rodas AC, Leite CA, et al. Preparation and characterization of ethanol-treated silk fibroin dense membranes for biomaterials application using waste silk fibers as raw material. Bioresour Technol, 2010, 101:8446-8451.
5. Morgan AW, Roskov KE, Lin-Gibson S, et al. Characterization and optimization of RGD-containing silk blends to support osteoblastic differentiation[J]. Biomaterials, 2008, 29:2556-2563.
6. Kim HJ, Kim UJ, Kim HS, et al. Bone tissue engineering with premineralized silk scaffolds[J]. Bone, 2008, 42:1226-1234.
7. Wang G, Yang H, Li M, et al. The use of silk fibroin/hydroxyapatite composite co-cultured with rabbit bone-marrow stromal cells in the healing of a segmental bone defect. J Bone Joint Surg Br, 2010, 92:320-325.
8. Chiarini A, Petrini P, Bozzini S, et al. Silk fibroin /poly( carbonate) -urethane as a substrate for cell growth: In vivo interactions with human cells. Biomaterials, 2003, 24: 789-799.
9. Pra DI, Petrini P, Chiarini A, et al. Silk fibroin-coated three-dimensional polyurethane scaffolds for tissue engineering: Interactions with normal human fibroblasts. Tissue Eng, 2003, 9: 1113-1121.
10. Panilaitis B, Altman GH, Chen J, et a1. Macrophage responses to silk. Biomaterials, 2003, 24: 3079-3085.
11. Santin M, Motta A, Freddi G, et a1. In vitro evaluation of the inflammatory potential of the silk fibroin[J]. J Biomed Mater Res, 1999, 46: 382-389.
12. Mnasria K, Lagaraine C, Manaa J, et al. IL-2/IL-2R pathway in dendritic cell modulates both their cytokine synthesis profiles and their capacity to activate allogeneicCD4+ T lymphocytes. Pathol Biol. 2009, 15. [Epub ahead of print].
13. Wu D, Guo Z, Ren Z, et al. Green tea EGCG suppresses T cell proliferation through impairment of IL-2/IL-2 receptor signaling. Free Radic Biol Med, 2009, 1;47:636-643.
14. Wang J, Wicker LS, Santamaria P. IL-2 and its high-affinity receptor: genetic control of immunoregulation and autoimmunity. Semin Immunol, 2009, 21:363-371.
1 Ni Y, Zhao X, Zhou L, et al. Radiologic and histologic characterization of silk fibroin as scaffold coating for rabbit tracheal defect repair. Otolaryngol Head Neck Surg, 2008, 139(2): 256-261.
2 Inoue S, Tanaka K, Arisaka F, et a1. Silk Fibroin of Bombyx mori Is Secreted, Assembling a High Molecular Mass Elementary Unit Consisting of H-chain, L-chain, and P25, with a 6:6:1 Molar Ratio. J Biol Chem, 2000, 275(51): 40517–40528.
3 Zhang YQ. Applications of natural silk protein sericin in biomaterials. Biotechnol Adv, 2002, 20(2): 91-100.
4 Yongzhong Wang, Hyeon-Joo Kim, Gordana Vunjak-Novakovic, et al. Stem cell-based tissue engineering with silk biomaterials. Biomaterials, 2006, 27(36): 6064–60822.
5 Brian D, Lawrence, Jeffrey K, et al. Silk film biomaterials for cornea tissue engineering. Biomaterials, 2009, 30(7): 1299–1308.
6 Charu Veparia, David L. Kaplan. Silk as a biomaterial. prog. ploym. sci, 2007, 32: 911-1007.
7田莉,闵思佳.丝素蛋白在组织工程细胞支架方面的研究进展.生物医学工程学杂志,2006, 23 (6): 1375-1378.
8 Zhou CZ, Confalonieri F, Jacque M, et al. Silk fibroin: structural implications of a remarkable amino acid sequence. Proteins, 2001, 44(2): 119-122.
9 Liu L, Melissa K, Callahan D, et al. Chemokine Receptor CXCR3: An Unexpected Enigma. Curr Top Dev Biol, 2005, 68: 149-154.
10 Zhang YY, Yoneyama H, Wang Y, et al. Mobilization of dendritic cell precursors into circulation by administrating MIP-1αin mice. J. Natl. Cancer Inst, 2004, 96(3): 201-209.
11 Zhou ZH, Shi Q, Wang JF, et al. Sensitization of multiple myeloma and B lymphoma lines to dexamethasone andγ–radiation-induced apoptosis by CD40 activation. Apoptosis, 2005, 10(1): 123-134.
12 Hollander DH.Interstitial cystitis and silk allergy.Med Hypotheses, 1994, 43(3): 155-156.
13 Wen CM, Ye ST, Zhou LX, et a1.Silk-induced asthma in children:Areport of
64 cases. Ann Allergy, 1990, 65(5): 375-378.
14 Dewair M, Baur X, Ziegler K. Use of immunoblot technique for detection of human IgE and IgG antibodies to individual silk protein. J. Allergy Clin Immunol, 1985, 76(4): 537-542.
15 Zhaoming W, Codina R, Fernandez CE, et a1. Partial characterization of the silk allergens in mulberry silk extract. J. Investig Allergol Clin Immunol, 1996, 6(4): 237-241.
16 Uff CR, Scott AD, Pockley AG, et a1. Influence of soluble suture factors on in vitro macrophage function. Biomaterials, 1995, 16(5): 355-360.
17 Panilaitis B, Ahman GH, Chen J, et a1. Macrophage responses to silk. Biomaterials, 2003, 24(18): 3079-3085.
18 Meinel L, Hofmann S, Karageorgiou V, et a1. The inflammatory responses to silk film in vitro and in vivo. Biomaterials, 2005, 26(2): 147-155.
19 Pra HD, Freddi G, Minic J, et a1. De novo engineering of reticular tissue in vivo by silk fibroin non woven materials. Biomaterials, 2005, 26(14): 1987-1979.
20 Santin M, Motta A, Freddi G, et al. In vitro evaluation of the inflammatory potential of the silk fibroin. J Biomed Mater Res, 1999, 46(3): 382-389.
21栾希英,张学光.丝素蛋白的免疫学特性及组织相溶性研究进展.国际生物医学工程杂志, 2006, 29(5): 2 96-299.
22 Altman GH, Diaz F, Jakuba C, et al. Silk-based biomaterials. Biomaterials, 2003, 24 (3): 401-416.
23 Korayem AM, Hauling T, Lesch C, et al. Evidence for an immune function of lepidopteran silk proteins. Biochem Biophys Res Commun, 2007, 352(2): 317-322.