本文選題：再生 + 絲素蛋白��； 參考：《蘇州大學(xué)》2011年碩士論文

【摘要】：絲素蛋白是從蠶絲中提取的天然高分子纖維蛋白,具有良好的理化特性及生物相容性。再生絲素蛋白是由絲素蛋白經(jīng)物理方式、環(huán)氧劑或SD交聯(lián)而成的新型醫(yī)用生物材料。近年來(lái),研究者通過(guò)物理或化學(xué)方法將絲素蛋白制備了符合臨床實(shí)際需要的再生多孔絲素膜,用于創(chuàng)面的修復(fù)。該材料具有良好的機(jī)械強(qiáng)度,不溶于水,膜的多孔結(jié)構(gòu)使其富有較好通氣性的同時(shí),可阻擋病原微生物侵入創(chuàng)面。該材料經(jīng)機(jī)體吸收后可在體內(nèi)逐漸被降解代謝。本研究在已對(duì)家蠶絲素蛋白對(duì)B細(xì)胞應(yīng)答影響的研究基礎(chǔ)上,又深入探討了不同性狀的再生家蠶絲素蛋白在小動(dòng)物體內(nèi)及體表應(yīng)用后對(duì)T細(xì)胞應(yīng)答的影響,從而為絲素蛋白類材料臨床應(yīng)用后的免疫原性研究提供實(shí)驗(yàn)依據(jù)。 目的:通過(guò)研究不同性狀再生家蠶絲素蛋白材料對(duì)T細(xì)胞活化、增殖及分化的影響,分析絲素蛋白類材料的免疫原性。 方法: 1.液狀再生家蠶絲素蛋白體內(nèi)運(yùn)用對(duì)小鼠T細(xì)胞的激發(fā)作用將ICR小鼠隨機(jī)分組,每組6只,實(shí)驗(yàn)組經(jīng)腹腔注射1%的液狀再生家蠶絲素蛋白0.5ml/只,以注射等量的無(wú)菌PBS為對(duì)照組。分別于第7d及第14d無(wú)菌取脾臟。制備細(xì)胞懸液作以下分析: 1.1小鼠脾臟細(xì)胞中CD3~+CD25~+、CD4~+CD25~+及CD4~+CD25~+ Foxp3~+T細(xì)胞的分析 將上述分離的脾臟細(xì)胞置于流式分析管中(3×105細(xì)胞/管)。分別加入下列抗鼠直標(biāo)抗體:第一組:CD3-FITC、CD25-APC;第二組:CD4-FITC、CD25-APC;第三組:CD4-FITC、CD25-APC及Foxp3-PE,反應(yīng)后經(jīng)FCM分析。 1.2經(jīng)再生家蠶絲素蛋白處理的小鼠脾臟細(xì)胞對(duì)ConA的反應(yīng)性 將上述分離的小鼠脾臟細(xì)胞加入96孔培養(yǎng)板,2×105/100μL/孔,加入ConA,終濃度分別為0μg/ml、2.5μg/ml及5μg/ml。培養(yǎng)至72h,MTT檢測(cè)細(xì)胞的增殖情況。同時(shí)采用FCM分析CD4~+CD25~+及CD4~+CD25~+Foxp3~+T的表達(dá)。 2、膜狀再生家蠶絲素蛋白在大鼠創(chuàng)面運(yùn)用后對(duì)T細(xì)胞的激活作用 2.1外科創(chuàng)傷面的建立 將SD大鼠麻醉后經(jīng)外科手術(shù)切除背部皮膚建立創(chuàng)面,面積為2 cm×2 cm。覆蓋經(jīng)預(yù)處理的膜狀再生多孔絲素,將已切除皮膚的表皮蓋在絲素膜上,進(jìn)行縫合。以PVA海綿設(shè)為陽(yáng)性對(duì)照,假手術(shù)組為陰性對(duì)照。 2.2移植處皮膚的病理學(xué)變化 分別于術(shù)后第3d、14d、28d、56d及90d,在麻醉狀態(tài)下取移植處皮膚,制作石蠟切片后行HE染色,分析炎性細(xì)胞的浸潤(rùn)及創(chuàng)面愈合情況。 2.3外周血、脾臟及胸腺中T細(xì)胞活化的動(dòng)態(tài)變化 于上述各時(shí)間點(diǎn),取抗凝血分離PBMC,將大鼠處死,分離脾臟及胸腺細(xì)胞經(jīng)FCM檢測(cè)CD3~+CD25~+T的比率。同時(shí)行免疫組化分析。 結(jié)果: 1.1液狀再生家蠶絲素蛋白體內(nèi)運(yùn)用后對(duì)小鼠T細(xì)胞活化與分化的影響FCM分析的結(jié)果顯示,腹腔注射液狀再生絲素蛋白后第7d及第14d,脾臟細(xì)胞中CD3~+CD25~+T細(xì)胞的百分率分別為(7.32±1.02)%及(7.58±0.98)%;與對(duì)照組((7.262±1.31)%及(6.98±1.22)%)比較稍有升高,但無(wú)顯著性差異(P㧐0.05)。CD4~+CD25~+T細(xì)胞的百分率分別為(7.22±0.92)%及(7.38±0.49)%;與對(duì)照組((7.19±0.58)%及(7.03±0.69)%)比較稍有升高,但無(wú)顯著性差異(P㧐0.05)。CD4~+CD25~+Foxp3~+T百分率分別為(3.52±0.62)%及(3.78±0.56)%。與對(duì)照組((3.47±0.31)%及(3.44±0.65)%)比較稍有升高,但無(wú)顯著性差異(P㧐0.05)。提示再生絲素蛋白可能對(duì)小鼠T細(xì)胞無(wú)明顯的激發(fā)作用。 2、膜狀再生家蠶多孔絲素蛋白對(duì)大鼠T細(xì)胞的激活作用 2.1移植處皮膚的病理學(xué)變化 膜狀再生家蠶絲素蛋白移植后第3d及第14d時(shí),移植處可見(jiàn)少量炎性細(xì)胞,28d后炎癥細(xì)胞已明顯減少,第56d時(shí)已無(wú)炎癥細(xì)胞。而陽(yáng)性對(duì)照組大鼠術(shù)后均有炎癥細(xì)胞浸潤(rùn),至56d后才逐漸減少。 2.2外周血、脾臟及胸腺細(xì)胞中T細(xì)胞活化的動(dòng)態(tài)變化 于術(shù)后第3d、14d、28d、56d及90d各時(shí)間點(diǎn),膜狀再生家蠶絲素蛋白移植組大鼠PBMC、脾臟及胸腺中CD3~+CD25~+T細(xì)胞百分比率經(jīng)FCM分析結(jié)果如表4所示:三者與陰性對(duì)照組比較均無(wú)顯著性差異(P㧐0.05)。而與陽(yáng)性對(duì)照組比較均有顯著性差異(P0.05)。同時(shí)免疫組化的分析結(jié)果顯示:實(shí)驗(yàn)組脾臟及胸腺組織中只有極少量的CD25~+T細(xì)胞,與FCM分析的結(jié)果一致。提示再生絲素蛋白對(duì)T細(xì)胞的激發(fā)作用較弱。 結(jié)論: 體內(nèi)外研究顯示,再生家蠶絲素蛋白對(duì)機(jī)體T淋巴細(xì)胞的激發(fā)作用較小,引發(fā)機(jī)體細(xì)胞免疫應(yīng)答的能力較弱,是一種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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R318.08;R392.1

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