本文選題：蝦青素 + 肺纖維化��； 參考：《濱州醫(yī)學(xué)院》2013年碩士論文

【摘要】：目的：采用博萊霉素和TGF-pl誘導(dǎo)肺纖維化動(dòng)物和細(xì)胞模型,探討蝦青素的抗纖維化作用,觀察蝦青素對(duì)線粒體分裂的影響,研究其對(duì)p53/PUMA/Drp-1凋亡信號(hào)通路的調(diào)控作用,探討蝦青素促進(jìn)轉(zhuǎn)分化細(xì)胞凋亡的分子機(jī)制。 方法：SD大鼠隨機(jī)分為正常組,模型組,蝦青素干預(yù)組和地塞米松組,BLM誘發(fā)大鼠IPF,造模3d后給藥,21d,28d處死動(dòng)物；取出部分肺組織液氮冷凍,同一部位小塊組織進(jìn)行戊二醛固定-環(huán)氧樹脂包埋處理、常規(guī)固定-石蠟包埋處理；HE染色觀察肺內(nèi)炎癥和纖維化的程度并進(jìn)行分級(jí),通過檢測(cè)肺組織羥脯氨酸的含量、透射電子顯微術(shù)以及masson染色檢測(cè)蝦青素對(duì)膠原表達(dá)的影響；免疫熒光法、western blot技術(shù)檢測(cè)蝦青素對(duì)轉(zhuǎn)分化以及凋亡相關(guān)蛋白平滑肌肌動(dòng)蛋白(a-SMA)、波形蛋白(vimentin)、E-鈣粘蛋白(E-cadherin)、p53、Bcl-2表達(dá)的影響。采用SRB法檢測(cè)蝦青素的藥物毒性及對(duì)細(xì)胞增殖的影響,分別應(yīng)用免疫熒光法、流式細(xì)胞術(shù)和western blot技術(shù)檢測(cè)蝦青素對(duì)肺泡Ⅱ型上皮細(xì)胞和成纖維細(xì)胞轉(zhuǎn)分化以及激活細(xì)胞凋亡的影響。采用線粒體熒光探針標(biāo)記觀察線粒體形態(tài),免疫雙標(biāo)的方法檢測(cè)蝦青素對(duì)細(xì)胞凋亡的影響,對(duì)線粒體分裂相關(guān)蛋白Drp-1表達(dá)的影響,對(duì)uma, Bax和Drp-1定位的影響。Western blot檢測(cè)蝦青素對(duì)轉(zhuǎn)分化后凋亡相關(guān)蛋白Puma, Bax和Drp-1表達(dá)的影響。 結(jié)果：結(jié)果表明,蝦青素可以明顯改善肺泡結(jié)構(gòu),減輕體內(nèi)的膠原沉積。蝦青素治療組與模型組相比,能夠下調(diào)α-SMA、羥脯氨酸、波形蛋白和Bcl-2的表達(dá),上調(diào)E-鈣粘蛋白和凋亡蛋白p53的表達(dá)。同時(shí),蝦青素能夠抑制A549和MRC-5細(xì)胞的增殖,半數(shù)抑制率IC50值為分別40和30μM。蝦青素能夠促進(jìn)體內(nèi)外轉(zhuǎn)分化的細(xì)胞凋亡,促進(jìn)線粒體分裂,線粒體三維網(wǎng)狀結(jié)構(gòu)破壞,數(shù)目增多,管狀線粒體變小變圓,有凝集現(xiàn)象。線粒體分裂相關(guān)蛋白Drp-1表達(dá)升高,線粒體分裂時(shí)由胞質(zhì)轉(zhuǎn)位到線粒體內(nèi)。蝦青素組與模型組相比,凋亡相關(guān)蛋白Bax, Puma的表達(dá)升高。 結(jié)論：蝦青素可以緩解肺纖維化癥狀,阻止肺纖維化進(jìn)展,可能是通過抑制轉(zhuǎn)分化和細(xì)胞增殖,促進(jìn)轉(zhuǎn)分化的細(xì)胞凋亡實(shí)現(xiàn)的。其促進(jìn)轉(zhuǎn)分化的細(xì)胞凋亡可能是通過上調(diào)凋亡蛋白Bax, Puma的表達(dá),影響Drp-1的表達(dá)和定位,打破線粒體的融合-分裂平衡促進(jìn)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，

本文編號(hào)：1931879