發(fā)布時(shí)間：2018-07-15 09:29

【摘要】：目的在改良已有方法基礎(chǔ)上,制備小鼠脫細(xì)胞心臟支架,探究其組成、成分及結(jié)構(gòu),建立“骨髓間充質(zhì)干細(xì)胞(Bone Mesenchymal Stem Cells,BMSCs)-脫細(xì)胞心臟支架”共培養(yǎng)體系,評(píng)價(jià)脫細(xì)胞心臟支架在細(xì)胞生長(zhǎng)、分化等方面的細(xì)胞生物學(xué)特性;探究脫細(xì)胞心臟支架及BMSCs在心臟組織工程研究中潛在應(yīng)用價(jià)值,為進(jìn)一步開展相關(guān)的組織工程研究及應(yīng)用,建立實(shí)驗(yàn)基礎(chǔ)。方法本實(shí)驗(yàn)聯(lián)合應(yīng)用胰蛋白酶、十二烷基硫酸鈉(SDS)、聚乙二醇辛基苯基醚(TritonX-100)對(duì)修剪后的大鼠心臟浸泡震蕩,以快速脫除細(xì)胞。以HE染色觀察支架內(nèi)細(xì)胞殘留;超微分光光度計(jì)檢測(cè)脫細(xì)胞支架的DNA殘留;掃描電鏡觀察支架的形態(tài)和纖維排列規(guī)律;Masson染色分析支架的成分;免疫組織化學(xué)染色檢測(cè)脫細(xì)胞支架Ⅰ型膠原蛋白、Ⅲ型膠原蛋白、肌紅蛋白、肌動(dòng)蛋白。選用BMSCs與脫細(xì)胞心臟支架體外構(gòu)建“細(xì)胞-支架復(fù)合物”共培養(yǎng)體系,選擇共培養(yǎng)7d、14d的“細(xì)胞-支架復(fù)合物”,HE染色觀察細(xì)胞在支架中的分布情況;掃描電鏡觀察“細(xì)胞-復(fù)合物”中細(xì)胞的粘附情況。以心肌組織裂解液進(jìn)行定向誘導(dǎo)培養(yǎng),實(shí)時(shí)熒光定量PCR檢測(cè)誘導(dǎo)后“細(xì)胞-支架復(fù)合物”中GATA-4和α-MHC的表達(dá)。結(jié)果1、脫細(xì)胞處理后的心臟支架呈半透明囊狀,HE染色觀察未見細(xì)胞核殘留,SEM可見脫細(xì)胞支架由大量纖維構(gòu)成,呈多孔網(wǎng)狀結(jié)構(gòu)。2、脫細(xì)胞心臟支架成分的檢測(cè),總DNA含量為43.75±0.03ng/μL;Masson染色可見脫細(xì)胞支架主要由網(wǎng)狀膠原纖維構(gòu)成;免疫組織化學(xué)染色可見,脫細(xì)胞心臟支架主要由Ⅰ型膠原蛋白及少量Ⅲ型膠原蛋白組成,未見細(xì)胞核與肌紅蛋白、肌動(dòng)蛋白表達(dá)。3、“細(xì)胞-支架復(fù)合物”HE染色可見,細(xì)胞分布在脫細(xì)胞支架內(nèi)部,比較共培養(yǎng)7d、14d的“細(xì)胞-支架復(fù)合物”,支架內(nèi)的細(xì)胞數(shù)量明顯增加;SEM觀察可見BMSCs緊密粘附在支架纖維表面;RT-PCR的結(jié)果顯示:定向誘導(dǎo)分化培養(yǎng)的“細(xì)胞-支架復(fù)合物”中,可檢測(cè)到α-MHC、GATA-4表達(dá),其表達(dá)量分別與“竹節(jié)樣”和“肌管樣”結(jié)構(gòu)階段相當(dāng)。結(jié)論本實(shí)驗(yàn)采取改良后的脫細(xì)胞方法可以有效去除小鼠心臟的細(xì)胞成分,保留了細(xì)胞外Ⅰ、Ⅲ型膠原蛋白成分;該脫細(xì)胞支架在保留心臟原有空間結(jié)構(gòu)的基礎(chǔ)上具有良好的細(xì)胞相容性,同時(shí)有助于BMSCs向心肌細(xì)胞分化。
[Abstract]:Objective to prepare the acellular heart scaffolds of mice based on the improved methods, and to investigate the composition, composition and structure of the scaffolds, and to establish a co-culture system of "bone mesenchymal stem cells (BMSCs) and acellular heart scaffolds". To evaluate the cellular biological characteristics of acellular cardiac scaffolds in cell growth and differentiation, and to explore the potential application value of acellular cardiac scaffolds and BMSCs in heart tissue engineering, so as to further develop the relevant tissue engineering research and application. Establish the experimental foundation. Methods in this experiment, trypsin, sodium dodecyl sulfate (SDS) and polyethylene glycol octyl phenyl ether (Triton X-100) were used for rapid removal of cells. Cell residues in scaffolds were observed by HE staining, DNA residues in acellular scaffolds were detected by ultramicro spectrophotometer, morphology and fiber arrangement of scaffolds were observed by scanning electron microscope (SEM), and components of scaffolds were analyzed by Masson staining. Immunohistochemical staining was used to detect type 鈪，

本文編號(hào)：2123606