HSNGLPL多肽修饰聚氨酯的体内反应性研究

发布时间：2018-05-21 16:30

本文选题：聚氨酯 + HSNGLPL多肽　；参考：《南方医科大学》2017年硕士论文

【摘要】：第一章分析HSNGLPL多肽修饰聚氨酯(HSNGLPL-PUs)的体外细胞相容性[目的]通过分析材料浸提液对C2C12细胞增殖及毒性的影响,探讨HSNGLPL-PUs的细胞相容性。[方法]将合成的 L-P-PU(50mg HSNGLPL 修饰)、M-P-PU(100mg HSNGLPL 修饰)及H-P-PU(150mg HSNGLPL修饰)按试样表面积/浸泡液体积=6cm2/mL比率提取浸提液(DMEM,48h),并与C2C12细胞共培养48h,CCK-8检测细胞增殖活性。[结果]RGR细胞毒性评定表明,聚氨酯浸提液具有一定的细胞毒性(1级),但50-100mg浓度范围内HSNGLPL多肽修饰并未改变聚氨酯的细胞活性。[结论]HSNGLPL多肽成分的添加及处理过程不影响聚氨酯材料的细胞相容性。第二章HSNGLPL-PUs材料的体内炎症和免疫反应性研究[目的]分析植入肌内材料诱导的肌内炎性渗出及肌纤维的坏死和再生,明确HSNGLPL-PUs的体内反应性。[方法]将 HSNGLPL 多肽液、BDO-PU(无 HSNGLPL 修饰)、L-P-PU、M-P-PU和H-P-PU材料(0.2×0.4cm2)植入腓肠肌内,在14d、28d、42d和56d,摘取小鼠腓肠肌,冰冻切片、HE染色或免疫荧光染色并观察:i)单核/巨噬细胞、T细胞、树突状细胞(DCs)的渗出;ii)巨噬细胞凋亡;iii)植入物周边肌纤维的坏死和再生、再生肌纤维MHC-I表达;[结果]14d,BDO-PU炎性渗出最显著,随后逐渐消退,56d时炎症基本消失;L-P-PU、M-P-PU和H-P-PU诱发的炎性渗出在移植初期(7-14d)时低于BDO-PU,但炎症持续时间长,56d仍可见渗出的淋巴细胞;在14d、28d和42d,BDO-PU肌内巨噬细胞凋亡数显著高于L-P-PU、M-P-PU和H-P-PU组,但56d时L-P-PU、M-P-PU和H-P-PU组仍可见较多的凋亡细胞;BDO-PU组肌内罕见DCs细胞(CD11c+),但L-P-PU、M-P-PU和H-P-PU材料诱发更显著的DCs渗出,渗出细胞的数量与PU材料内的HSNGLPL多肽浓度呈正相关;28d和42d时,各组材料周围肌组织内都可见CD3+/CD4+T细胞,56d时,BDO-PU材料周围CD4+T细胞基本消失,L-P-PU、M-P-PU和H-P-PU组仍观察到CD4+T细胞;各组植入材料周围均可见肌纤维溃变,BDO-PU组于28d后可见新生肌纤维(Dystrophy+),L-P-PU、M-P-PU 和 H-P-PU 组则于植入早期(14d)出现肌纤维再生,HSNGLPL多肽浓度较高的H-P-PU组肌纤维的再生更为显著,42d时该组材料周围的损伤肌纤维基本完成修复,BDO-PU材料于56d时仍可见中央核肌纤维;各组PU材料均不能诱发再生肌纤维表达MHC-I分子。[结论]HSNGLPL-PUs肌内植入后,将迅速诱发肌内炎症反应及肌细胞坏死,渗出,以单核-巨噬细胞为主。PU材料内HSNGLPL肽成分可能抑制炎症细胞凋亡,从而导致炎症的持续。此外,体内的植入材料逐渐降解并释放HSNGLPL肽成分,可能有助于局部肌卫星细胞的激活、增殖与分化,于移植后期促进肌纤维再生。第三章分析体内、外条件下HSNGLPL-PUs对TGF-β的富集及释放[目的]明确合成的HSNGLPL-PUs对TGF-β的富集及体内释放。[方法]将各组HSNGLPL-PUs浸泡于含有500pg/mL的重组人TGF-β缓冲液中4℃C 1h,Elisa检测缓冲液中残余的TGF-β浓度;肌内植入各组HSNGLPL-PUs材料,分别于14d和28d摘取肌组织,匀浆,Elisa检测匀浆上清中TGF-β浓度。将预富集TGF-β的PU材料(HSNGLPL-PU-TGF-β)植入小鼠肌肉,分别于植入术后7,14,21,28d摘取肌肉,冰冻切片,HE及免疫荧光观察炎症渗出,masson染色观察局部纤维化程度,或提取肌组织RNA,qRT-PCR观察肌组织内质网应激相关分子水平改变。[结果]体外条件下,HSNGLPL-PUs浸泡缓冲液中TGF-β水平显著低于BDO-PU浸泡液,其降低程度与PU包含的HSNGLPL肽浓度呈正相关。14d时,包含H-P-PU材料的肌组织TGF-β浓度显著高于HSNGLPL多肽注射组及BDO-PU组;28d时各组肌组织TGF-β浓度无显著差异。较之BDO-PU和HSNGLPL-PU组,于移植7d和14d时,HSNGLPL-PU-TGF-β材料诱导更为剧烈的炎性渗出,但渗出范围相对局限,持续时间较短,28d时炎症基本消退。Masson染色可见,HSNGLPL-PU-TGF-β材料诱发的临近肌组织纤维化(蓝色)较其他材料组更加显著。ER Stress标志分子(ATF6、Bip、Calnexin、Spliced XBP1、CHOP 和 Cathepsin D)mRNA 表达水平可见,材料各组显著高于正常肌组织,随种植时间延长,上述分子水平逐渐下调。移植早期(7d),HSNGLPL-PU-TGF-β组的ER Stress分子mRNA水平显著高于BDO-PU和HSNGLPL-PU组。[结论]体内、外条件下,HSNGLPL多肽修饰的聚氨酯均可富集TGF-β分子。肌内植入后,预富集的TGF-β自HSNGLPL多肽-聚氨酯材料快速释放并可能干预肌内炎性反应和肌组织纤维化,并影响肌组织的ER Stress反应。
[Abstract]:In the first chapter, the cytocompatibility of HSNGLPL polypeptide modified polyurethane (HSNGLPL-PUs) in vitro was analyzed. [Objective] by analyzing the effect of material extract on the proliferation and toxicity of C2C12 cells, the cytocompatibility of HSNGLPL-PUs was discussed. [method] the synthesis of L-P-PU (50mg HSNGLPL modification), M-P-PU (100mg HSNGLPL modification) and H-P-PU (150mg modification) The extraction solution (DMEM, 48h) was extracted by the =6cm2/mL ratio of the sample surface area / soaking liquid volume, and 48h was co cultured with C2C12 cells and CCK-8 was used to detect the cell proliferation activity. [results the toxicity assessment of]RGR cells showed that the polyurethane extract had a certain cytotoxicity (1), but the HSNGLPL polypeptide modification in 50-100mg concentration did not change the cells of the polyurethane. [conclusion the addition and treatment of]HSNGLPL polypeptide components do not affect the cellular compatibility of polyurethane materials. Second chapter HSNGLPL-PUs in vivo inflammation and immunoreactivity study [Objective] to analyze the intramuscular inflammatory exudation induced by intramuscular materials and the necrosis and regeneration of muscle fibers, and to clarify the internal reactivity of HSNGLPL-PUs. ] HSNGLPL polypeptide liquid, BDO-PU (without HSNGLPL modification), L-P-PU, M-P-PU and H-P-PU materials (0.2 x 0.4cm2) were implanted into the gastrocnemius muscle. In 14d, 28d, 42d and 56d, the gastrocnemius, frozen section, HE staining or immunofluorescence staining and the infiltration of dendritic cells, dendritic cells and macrophages were implanted. The necrosis and regeneration of the muscle fibers around the peripheral muscle and regenerated muscle fiber MHC-I expression, [results]14d, BDO-PU inflammatory exudation is the most significant, then gradually subsided, and the inflammation basically disappeared at 56d; L-P-PU, M-P-PU and H-P-PU induced inflammatory exudation was lower than BDO-PU at the early stage of transplantation (7-14d), but the inflammation continued to be long, 56d still showed exudative lymphocytes; 14d, 28d. The number of apoptosis in 42d, BDO-PU intramuscular macrophages was significantly higher than that in L-P-PU, M-P-PU and H-P-PU groups, but there were still more apoptotic cells in the L-P-PU, M-P-PU and H-P-PU groups at 56d, and in BDO-PU group, the DCs cells (CD11c+) were rare in the BDO-PU muscle, but the number of exuded cells and the concentration of polypeptide in the material were more significant. At 28d and 42d, CD3+/CD4+T cells were found in all the muscles around each group. When 56d, the CD4+T cells around BDO-PU material disappeared basically. CD4+T cells were still observed in the L-P-PU, M-P-PU and H-P-PU groups. In group H-P-PU, muscle fiber regeneration was found at early stage of implantation (14d), and the regeneration of muscle fibers in group H-P-PU with high HSNGLPL polypeptide concentration was more significant. At 42d, the injured muscle fibers around the group basically completed the repair, and the BDO-PU material still showed the central nuclear muscle fiber in 56d. All PU materials could not induce the regenerated muscle fibers to express the MHC-I molecules. After]HSNGLPL-PUs intramuscular implantation, the intramuscular inflammation and myocyte necrosis and exudation will be quickly induced, and the HSNGLPL peptide in the mononuclear macrophage.PU material may inhibit the apoptosis of inflammatory cells and lead to the persistence of inflammation. In addition, the implanted materials in the body gradually degrade and release the HSNGLPL peptide components, which may contribute to the local muscle defense. The activation, proliferation and differentiation of the astrocytes and the regeneration of muscle fibers in the late stage of transplantation. The third chapter analyzes the enrichment and release of TGF- beta by HSNGLPL-PUs in the body and external conditions. [Objective] the concentration and release of TGF- beta by HSNGLPL-PUs is clearly synthesized. [method] HSNGLPL-PUs was soaked in each group of 500pg/mL in the recombinant TGF- beta buffer solution of C. 1H, Elisa detected the residual TGF- beta concentration in the buffer solution; intramuscular implantation of HSNGLPL-PUs materials in each group, 14d and 28d were taken to extract muscle tissue, homogenate, and Elisa to detect the concentration of TGF- beta in the homogenate supernatant. The PU materials preconcentration TGF- beta (HSNGLPL-PU-TGF- beta) were implanted in the muscle of mice. The inflammatory exudation was observed by the immunofluorescence, the degree of local fibrosis was observed by Masson staining, or the RNA of the muscle tissue was extracted, and the level of the endoplasmic reticulum stress related molecules in the muscle tissue was observed by qRT-PCR. [results] the level of TGF- beta in the soaked buffer of HSNGLPL-PUs was significantly lower than that of the BDO-PU soaking solution in vitro, and the degree of decrease was compared with the concentration of HSNGLPL peptide contained in PU, Cheng Zhengxiang. At.14d, the TGF- beta concentration of the muscle tissue containing H-P-PU was significantly higher than that of the HSNGLPL polypeptide injection group and the BDO-PU group, and there was no significant difference in the concentration of TGF- beta in the muscle tissues at 28d. The HSNGLPL-PU-TGF- beta material induced more acute inflammatory exudation than the BDO-PU and HSNGLPL-PU groups in the transplantation of 7D and 14d, but the scope of the exudation was relatively limited and the duration was relatively longer. Short, 28d inflammation basically subsided.Masson staining visible, HSNGLPL-PU-TGF- beta induced adjacent muscle tissue fibrosis (blue) more significant than the other materials group.ER Stress markers (ATF6, Bip, Calnexin, Spliced XBP1, CHOP and Cathepsin) expression level can be seen, the material is significantly higher than the normal muscle tissue, with the planting time extended. The level of these molecules is down gradually. Early transplantation (7D), the mRNA level of ER Stress molecules in HSNGLPL-PU-TGF- beta group is significantly higher than that in BDO-PU and HSNGLPL-PU groups. [Conclusion] in the body, the polyurethane modified by HSNGLPL polypeptide can enrich TGF- beta molecules. After intramuscular implantation, the TGF- beta self HSNGLPL polypeptide polyurethane material is released quickly. It may interfere in intramuscular inflammatory reaction and muscle tissue fibrosis, and affect the ER Stress reaction of muscle tissue.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.08

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