【摘要】：心肌細(xì)胞外基質(zhì)(extracellular matrix,ECM)可由心肌經(jīng)脫細(xì)胞處理制得,被廣泛認(rèn)為是一種理想的制備工程心肌的生物支架材料。然而目前的脫細(xì)胞方法尚存在不足,本研究擬聯(lián)合使用經(jīng)典去垢劑改良脫細(xì)胞方法,制備性能更為優(yōu)良的心肌ECM薄片,以用于構(gòu)建工程心肌片。用振蕩切片機將包埋于低熔點瓊脂糖中的成年昆明小白鼠心室肌組織沿心臟橫軸切成300μm厚的薄片,隨機分為正常對照組、SDS脫細(xì)胞組(0.1%SDS處理)和改良脫細(xì)胞組(0.1%SDS和0.5%Triton X-100聯(lián)合處理)。通過總RNA和總蛋白質(zhì)含量分析、HE染色和免疫熒光染色等方法評估各組的脫細(xì)胞程度和ECM成分保留狀態(tài);將改良脫細(xì)胞組ECM與小鼠胚胎干細(xì)胞源心肌細(xì)胞(murine embryonic stem cell-derived cardiomyocytes,m ES-CMs)和小鼠胚胎成纖維細(xì)胞(murine embryonic fibroblasts,MEFs)共培養(yǎng)以檢測其生物相容性。結(jié)果顯示:SDS脫細(xì)胞組和改良脫細(xì)胞組ECM中殘留的總RNA及蛋白質(zhì)含量均低于對照組。HE染色結(jié)果顯示改良脫細(xì)胞組核質(zhì)去除較SDS脫細(xì)胞組更徹底。改良脫細(xì)胞組可見膠原蛋白IV和層粘連蛋白兩種ECM關(guān)鍵成分表達量和分布接近正常心肌組織,而SDS脫細(xì)胞組中這兩種蛋白明顯減少且分布紊亂。m ES-CMs和MEFs能存活于改良脫細(xì)胞組ECM表面12天以上并向內(nèi)遷移。綜上,SDS和Triton X-100聯(lián)合脫細(xì)胞法制備ECM薄片效果明顯,能更好地保留天然ECM成分和結(jié)構(gòu),具有良好的生物相容性。
[Abstract]:Myocardial extracellular matrix (extracellular matrix,ECM), which can be prepared by acellular treatment of myocardium, is widely regarded as an ideal biomaterial for preparing engineered myocardium. However, there are still some shortcomings in the present acellular method. In this study, we intend to use the classical scale remover to improve the acellular method to prepare the better performance of myocardial ECM thin slices for the construction of engineering myocardial slices. The ventricular muscle tissue of adult Kunming mice embedded in agarose at low melting point was cut into 300 渭 m thick slices along the transverse axis of heart by oscillatory slicing machine and randomly divided into normal control group. SDS acellular group (0.1%SDS treatment) and modified acellular group (0.1%SDS and 0.5%Triton Xa 100 combined treatment). Total RNA and total protein content analysis, HE staining and immunofluorescence staining were used to evaluate the acellular degree and the retention status of ECM components in each group. The modified acellular group ECM was co-cultured with mouse embryonic stem cell-derived cardiomyocytes (murine embryonic stem cell-derived cardiomyocytes,m ES-CMs) and mouse embryonic fibroblasts (murine embryonic fibroblasts,MEFs) to detect their biocompatibility. The results showed that the contents of total RNA and protein in ECM of SDS acellular group and modified acellular group were lower than those of control group. The results of HE staining showed that the nucleoplasmic removal of modified acellular group was more complete than that of SDS acellular group. In the modified acellular group, the expression and distribution of collagenin IV and laminin ECM were close to normal myocardial tissue. M-ES-CMs and MEFs could survive more than 12 days on the surface of ECM in the modified acellular group and migrate inward. In the SDS acellular group, the expression of these two proteins was significantly decreased and the distribution of the two proteins was disordered. In conclusion, the preparation of ECM slices by acellular method of SDS and Triton X + 100 has obvious effect, which can retain the natural ECM composition and structure better, and has good biocompatibility.
【作者單位】： 華中科技大學(xué)同濟醫(yī)學(xué)院基礎(chǔ)醫(yī)學(xué)院生理學(xué)系 中德干細(xì)胞中心 湖北省藥物靶向研究評價重點實驗室;
【基金】：supported by the National Natural Science Foundation of China(No.31100828) the Natural Science Foundation of Hubei Province,China(No.2011CDB363) the Scientific Research Foundation for the Returned Overseas Chinese Scholars,State Education Ministry,China,the Fundamental Research Funds(No.2013TS145) the Fundamental Research Funds for the Undergraduates(No.HUST:2011B142,2012B366)in Huazhong University of Science and Technology the National Undergraduate Training Programs for Innovation and Entrepreneurship(No.HUST:2012175) the Science and Technology Innovation Funds for the University or College Students,China(No.9)
【分類號】：R318.08

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本文編號：2451462