静电纺纤维载药系统动物体内应用的疗效研究

发布时间：2018-05-14 23:34

本文选题：酸性敏感 + 静电纺纤维　；参考：《上海交通大学》2015年博士论文

【摘要】：目的:静电纺纤维作为组织工程支架及药物控释载体已有广泛研究。本论文通过静电纺纤维内复合碳酸氢钠(NaHCO3)的方法构建具有酸度敏感的三维支架,并使其携载不同的药物,作为抗炎和抗肿瘤的药物载体应用于相应的动物模型中,以观察其生物相容性、安全性,并探讨其抗炎及抑制肿瘤的作用机制。与此同时,通过聚多巴胺(pDA)对载药静电纺纤维材料进行表面改性后,观察其抑瘤效果的改变。方法:抗炎型静电纺纤维的体外实验:不同组别的静电纺纤维支架及其浸提液分别与大鼠成肌细胞株(L6)共培养,MTT法评估细胞毒性、TUNEL染色法检测细胞凋亡。抗炎型静电纺纤维的体内实验,不同组别的静电纺纤维支架分别植入皮肤,肌肉受损的大鼠皮肤与骨骼肌内,在各时间点通过组织化学染色、免疫组织化学染色、PCR分析等技术手段检测其对皮肤、骨骼肌的修复情况。抗肿瘤型静电纺纤维的体外实验:不同组别的静电纺纤维支架浸提液与人肝细胞癌细胞(HuH-7)、人乳腺癌细胞(MDA-231)共培养,MTT法评估细胞毒性。抗肿瘤型静电纺纤维的体内实验,不同组别的静电纺纤维支架分别植入裸鼠皮下肝癌瘤体切除后的残癌内,在各时间点通过组织化学染色、免疫组织化学染色、ELISA检测、PCR分析等技术手段检测其对残癌的抑制情况。结果:1.抗炎型静电纺纤维的体外实验中,MTT分析不同组别的静电纺纤维支架及其浸提液对L6细胞无毒性;TUNEL染色显示不同组别的静电纺纤维支架浸提液对细胞凋亡无明显促进作用。抗炎型静电纺纤维支架的体内实验中,HE染色结果显示其可显著控制过度炎症反应,对大鼠骨骼肌急性损伤的愈合有明显促进作用;免疫组化与PCR结果显示抗炎型静电纺纤维支架可抑制促炎型细胞因子(IL-6和TNF-α)的表达,而对促增生型细胞因子(VEGF、b-FGF、TGF-1)的表达有促进作用。抗炎型静电纺纤维支架对大鼠皮肤急性损伤的愈合也有明显促进作用,HE染色显示其可显著控制过度炎症反应,减少皮肤胶原的沉积;免疫组化与Western blot结果显示抗炎型静电纺纤维支架对细胞因子b-FGF的表达有促进作用,而对a-SMA的表达有抑制作用;PCR结果显示collagen I/collagen III和TGF-β1/TGF-β3的比率降低,而MMP-1/TIMP-1的比率增高,提示抗炎型静电纺纤维支架可减少皮肤疤痕组织的形成,有利于皮肤无疤痕愈合。2.抗肿瘤型静电纺纤维的体外实验中,MTT分析不同组别的静电纺纤维支架浸提液对Huh-7、MDA-231肿瘤细胞增殖有明显抑制作用。抗肿瘤型静电纺纤维支架的体内实验表明其对残癌有明显抑制作用,HE染色结果显示其可引起肿瘤细胞凋亡;ELISA结果显示其可减少CRP的表达;免疫组化、ELISA、Western blot与PCR结果综合显示抗肿瘤型静电纺纤维支架早期可控制炎症反应,抑制炎症因子的表达,后期持续抑制肿瘤细胞增殖,下调IL-6,STAT3和NF-κB的表达,上调Caspase-3表达,并上调Bcl-2/Bax比值。经pDA表面改性后的载药静电纺PLLA纤维支架,亲水性增强,抑瘤效果显著提高。结论:抗炎型静电纺纤维支架对大鼠皮肤、骨骼肌急性损伤的愈合有明显促进作用,可显著控制过度炎症反应,加速愈合;并通过对疤痕形成相关的细胞因子的调节,实现皮肤无疤痕愈合。抗肿瘤型静电纺纤维支架对残癌有明显抑制作用,可实现早期对炎症因子的控制,后期长效抗肿瘤的作用。酸性敏感静电纺纤维支架可作为安全的药物载体对疾病进行有效的治疗;经pDA修饰的载药静电纺PLLA纤维支架可以作为有效的药物载体,用于对残癌的治疗,对其作用机制与应用范围尚需进一步研究。
[Abstract]:Objective: ESD fibers have been widely studied as tissue engineering scaffolds and drug controlled-release carriers. In this paper, an acidity sensitive three-dimensional scaffold was constructed by electrostenting fiber composite sodium bicarbonate (NaHCO3), and was carried with different drugs to be used as an anti-inflammatory and anti-tumor drug carrier in the corresponding animal model. To observe its biocompatibility, safety, and explore its anti-inflammatory and anti-tumor mechanism. At the same time, after the surface modification of the electrospun fiber material carried by polydopamine (pDA), the effect of its antitumor effect was observed. Method: in vitro experiment of anti-inflammatory electrostf fiber: electrostenting scaffold of different groups and its immersion The extracts were co cultured with rat myoblast cell line (L6), MTT method was used to evaluate cytotoxicity and TUNEL staining was used to detect cell apoptosis. In vivo experiments of anti-inflammatory electrospun fiber in vivo, electrostenting scaffolds of different groups were implanted in skin, muscle damaged rat skin and skeletal muscle, by histochemical staining and immuno tissue at all time points. Chemical staining, PCR analysis and other technical methods to detect the repair of skin and skeletal muscle. In vitro experiments of antitumor electrospun fibers: electrostenting extract of different groups and human hepatocarcinoma cell (HuH-7), human breast cancer cell (MDA-231) co culture, MTT method to evaluate cytotoxicity. The electrostenting scaffolds of different groups were implanted in the residual tumor after subcutaneously subcutaneously resected in nude mice. The inhibition of the residual cancer was detected by histochemical staining, immunohistochemical staining, ELISA detection and PCR analysis at all time points. Results: 1. in vitro, MTT analysis of the anti-inflammatory electrospun fiber in vitro. The electrostatic spun scaffolds of different groups and their extracts were nontoxic to L6 cells, and TUNEL staining showed that the electrostatic spun scaffolds of different groups had no obvious promoting effect on cell apoptosis. In vivo experiments of anti-inflammatory electrospun fiber scaffolds, HE staining results showed that it could significantly control excessive inflammatory reaction and acute skeletal muscle in rats. The healing of sexual injury has a significant effect. Immunohistochemical and PCR results show that anti-inflammatory electrostf scaffolds can inhibit the expression of pro-inflammatory cytokines (IL-6 and TNF- a), but promote the expression of hyperplastic cytokines (VEGF, b-FGF, TGF-1). HE staining showed that it could significantly control excessive inflammatory reaction and reduce the deposition of collagen in the skin. Immunohistochemical and Western blot results showed that the anti-inflammatory electrostf scaffold had a promoting effect on the expression of cytokine b-FGF, while the expression of a-SMA was inhibited, and PCR results showed collagen I/collagen III and TGF- beta 1. The ratio of /TGF- beta 3 decreased and the ratio of MMP-1/TIMP-1 increased, suggesting that the anti-inflammatory electrostf scaffold could reduce the formation of skin scar tissue, which was beneficial to the skin free scar healing.2. antitumor electrospun fiber in vitro. MTT analysis of different groups of electrostatic spun fibrous scaffold extracts had the proliferation of Huh-7, MDA-231 tumor cells. In vivo experiment of antitumor electrospun fiber scaffold showed that it had obvious inhibitory effect on the residual cancer. HE staining results showed that it could induce apoptosis of tumor cells; ELISA results showed that it could reduce the expression of CRP; immunohistochemical, ELISA, Western blot and PCR results showed that the antitumor electrospun fiber scaffold could be used in a comprehensive way. Control the inflammatory response, inhibit the expression of inflammatory factors, continue to inhibit the proliferation of tumor cells in the later period, down regulate the expression of IL-6, STAT3 and NF- kappa B, up regulation of Caspase-3 expression, and up regulation of Bcl-2/Bax ratio. The hydrophilic and anti tumor effect of the electrostatic spinning PLLA fiber stent after pDA surface modification is enhanced. Conclusion: anti-inflammatory electrostatic spun fiber branch The scaffold has an obvious promoting effect on the healing of acute injury of rat skin and skeletal muscle, which can significantly control excessive inflammatory reaction and accelerate healing, and achieve skin free scar healing by regulating the related cytokines of scar formation. The antitumor electrostun fiber scaffold has obvious inhibitory effect on the residual cancer and can achieve early inflammatory factors. The acid sensitive electrospun fiber scaffold can be used as a safe drug carrier for the effective treatment of the disease. The pDA modified electrostatically electrostatically spun PLLA fiber scaffold can be used as an effective drug carrier for the treatment of residual cancer, and the mechanism and application scope of it still need further study.

【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R965

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