虾青素抗肺纤维化作用中的促凋亡机制研究

发布时间：2018-05-25 03:26

  本文选题：虾青素 + 肺纤维化　； 参考：《滨州医学院》2013年硕士论文

【摘要】：目的：采用博莱霉素和TGF-pl诱导肺纤维化动物和细胞模型,探讨虾青素的抗纤维化作用,观察虾青素对线粒体分裂的影响,研究其对p53/PUMA/Drp-1凋亡信号通路的调控作用,探讨虾青素促进转分化细胞凋亡的分子机制。 方法：SD大鼠随机分为正常组,模型组,虾青素干预组和地塞米松组,BLM诱发大鼠IPF,造模3d后给药,21d,28d处死动物；取出部分肺组织液氮冷冻,同一部位小块组织进行戊二醛固定-环氧树脂包埋处理、常规固定-石蜡包埋处理；HE染色观察肺内炎症和纤维化的程度并进行分级,通过检测肺组织羟脯氨酸的含量、透射电子显微术以及masson染色检测虾青素对胶原表达的影响；免疫荧光法、western blot技术检测虾青素对转分化以及凋亡相关蛋白平滑肌肌动蛋白(a-SMA)、波形蛋白(vimentin)、E-钙粘蛋白(E-cadherin)、p53、Bcl-2表达的影响。采用SRB法检测虾青素的药物毒性及对细胞增殖的影响,分别应用免疫荧光法、流式细胞术和western blot技术检测虾青素对肺泡Ⅱ型上皮细胞和成纤维细胞转分化以及激活细胞凋亡的影响。采用线粒体荧光探针标记观察线粒体形态,免疫双标的方法检测虾青素对细胞凋亡的影响,对线粒体分裂相关蛋白Drp-1表达的影响,对uma, Bax和Drp-1定位的影响。Western blot检测虾青素对转分化后凋亡相关蛋白Puma, Bax和Drp-1表达的影响。 结果：结果表明,虾青素可以明显改善肺泡结构,减轻体内的胶原沉积。虾青素治疗组与模型组相比,能够下调α-SMA、羟脯氨酸、波形蛋白和Bcl-2的表达,上调E-钙粘蛋白和凋亡蛋白p53的表达。同时,虾青素能够抑制A549和MRC-5细胞的增殖,半数抑制率IC50值为分别40和30μM。虾青素能够促进体内外转分化的细胞凋亡,促进线粒体分裂,线粒体三维网状结构破坏,数目增多,管状线粒体变小变圆,有凝集现象。线粒体分裂相关蛋白Drp-1表达升高,线粒体分裂时由胞质转位到线粒体内。虾青素组与模型组相比,凋亡相关蛋白Bax, Puma的表达升高。 结论：虾青素可以缓解肺纤维化症状,阻止肺纤维化进展,可能是通过抑制转分化和细胞增殖,促进转分化的细胞凋亡实现的。其促进转分化的细胞凋亡可能是通过上调凋亡蛋白Bax, Puma的表达,影响Drp-1的表达和定位,打破线粒体的融合-分裂平衡促进凋亡。
[Abstract]:Aim: to investigate the antifibrotic effect of astaxanthin on mitochondrial division and the regulation of apoptosis signal pathway of p53/PUMA/Drp-1 by using bleomycin and TGF-pl to induce pulmonary fibrosis in animal and cell models. To explore the molecular mechanism of astaxanthin promoting apoptosis of transdifferentiation cells. Methods the rats were randomly divided into normal group, model group, astaxanthin intervention group and dexamethasone group. The degree of inflammation and fibrosis in lung was observed by HE staining and the content of hydroxyproline in lung tissue was detected by means of glutaraldehyde immobilization and epoxy resin entrapment in the same part of tissue, and HE staining of routine fixation-paraffin embedding treatment, and the content of hydroxyproline in lung tissue was determined by HE staining. The effects of astaxanthin on collagen expression were detected by transmission electron microscopy (TEM) and masson staining, and the effects of astaxanthin on transdifferentiation and apoptosis related protein smooth muscle actin (SMA) and vimentin E cadherin (E-cadherin) p53 Bcl 2 were detected by Western blot. The toxicity of astaxanthin and its effect on cell proliferation were detected by SRB assay. Immunofluorescence assay was used to detect the toxicity of astaxanthin. The effects of astaxanthin on the transdifferentiation and apoptosis of alveolar type 鈪，

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