小鼠心脏脱细胞支架的制备及初步评价
发布时间：2018-07-15 09:29
【摘要】：目的在改良已有方法基础上,制备小鼠脱细胞心脏支架,探究其组成、成分及结构,建立“骨髓间充质干细胞(Bone Mesenchymal Stem Cells,BMSCs)-脱细胞心脏支架”共培养体系,评价脱细胞心脏支架在细胞生长、分化等方面的细胞生物学特性;探究脱细胞心脏支架及BMSCs在心脏组织工程研究中潜在应用价值,为进一步开展相关的组织工程研究及应用,建立实验基础。方法本实验联合应用胰蛋白酶、十二烷基硫酸钠(SDS)、聚乙二醇辛基苯基醚(TritonX-100)对修剪后的大鼠心脏浸泡震荡,以快速脱除细胞。以HE染色观察支架内细胞残留;超微分光光度计检测脱细胞支架的DNA残留;扫描电镜观察支架的形态和纤维排列规律;Masson染色分析支架的成分;免疫组织化学染色检测脱细胞支架Ⅰ型胶原蛋白、Ⅲ型胶原蛋白、肌红蛋白、肌动蛋白。选用BMSCs与脱细胞心脏支架体外构建“细胞-支架复合物”共培养体系,选择共培养7d、14d的“细胞-支架复合物”,HE染色观察细胞在支架中的分布情况;扫描电镜观察“细胞-复合物”中细胞的粘附情况。以心肌组织裂解液进行定向诱导培养,实时荧光定量PCR检测诱导后“细胞-支架复合物”中GATA-4和α-MHC的表达。结果1、脱细胞处理后的心脏支架呈半透明囊状,HE染色观察未见细胞核残留,SEM可见脱细胞支架由大量纤维构成,呈多孔网状结构。2、脱细胞心脏支架成分的检测,总DNA含量为43.75±0.03ng/μL;Masson染色可见脱细胞支架主要由网状胶原纤维构成;免疫组织化学染色可见,脱细胞心脏支架主要由Ⅰ型胶原蛋白及少量Ⅲ型胶原蛋白组成,未见细胞核与肌红蛋白、肌动蛋白表达。3、“细胞-支架复合物”HE染色可见,细胞分布在脱细胞支架内部,比较共培养7d、14d的“细胞-支架复合物”,支架内的细胞数量明显增加;SEM观察可见BMSCs紧密粘附在支架纤维表面;RT-PCR的结果显示:定向诱导分化培养的“细胞-支架复合物”中,可检测到α-MHC、GATA-4表达,其表达量分别与“竹节样”和“肌管样”结构阶段相当。结论本实验采取改良后的脱细胞方法可以有效去除小鼠心脏的细胞成分,保留了细胞外Ⅰ、Ⅲ型胶原蛋白成分;该脱细胞支架在保留心脏原有空间结构的基础上具有良好的细胞相容性,同时有助于BMSCs向心肌细胞分化。
[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，
本文编号：2123606
[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，
本文编号：2123606
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