鹿茸软骨组织脱细胞基质材料的制备及生物相容性研究
发布时间：2018-10-23 09:35
【摘要】：目的探讨鹿茸软骨制备脱细胞基质材料的可行性以及生物相容性,为软骨修复重建探索新材料。方法取梅花鹿鹿茸生长中心间充质层,进行由DNA酶、RNA酶、抑肽酶等介导的脱细胞处理;行组织学和DNA含量检测,评价脱细胞效果。取第2代鹿生茸区骨膜(antlerogenic periosteum,AP)细胞,行荧光干细胞标记明确其干细胞特性后,用PKH26荧光标记并与制备的间充质层脱细胞基质进行复合培养;7 d后取材行HE染色观察以及荧光显微镜下观察PKH26标记的AP细胞在基质表面生长情况。以上观测均以未复合AP细胞的脱细胞基质作为对照。将复合培养7 d的样本移植至裸鼠一侧腹股沟(实验组),取空白培养样本移植于另一侧(对照组)。于移植后7、21 d取材行HE染色,同时对组织进行冰冻切片并在荧光显微镜下观察PKH26标记成功的AP细胞在脱细胞基质表面及内部的生长情况,评价含AP细胞的脱细胞基质在裸鼠体内的组织相容性。结果HE和DAPI染色显示脱细胞处理后材料中无细胞残留,DNA含量为(19.367±5.254)ng/mg,较脱细胞处理前的(3 805.500±519.119)ng/mg显著降低(t=12.630,P=0.000),提示成功制备间充质层脱细胞基质。AP细胞与间充质层脱细胞基质复合培养7 d后,AP细胞主要黏附于材料表面,部分进入脱细胞基质内部。植入裸鼠体内后,随观察时间延长,接种AP细胞可以在脱细胞基质材料中增殖并逐渐进入材料内部,并诱导血管生成。结论实验成功制备鹿茸软骨脱细胞基质,该基质材料在离体和活体情况下适于种子细胞(AP细胞)的黏附和增殖,并具有刺激血管生成的功能,为其用于软骨组织修复提供理论依据。
[Abstract]:Objective to investigate the feasibility and biocompatibility of acellular matrix material from antler cartilage and explore new materials for cartilage repair and reconstruction. Methods the mesenchymal layer of deer antler growth center in sika deer was treated with DNA enzyme, RNA enzyme, aprotinin mediated acellular treatment, histology and DNA content were detected to evaluate the acellular effect. The second generation of antlerogenic periosteum,AP cells in the antler region of deer were taken, and the characteristics of the stem cells were determined by fluorescent stem cell labeling. The cells were labeled with PKH26 and co-cultured with the acellular matrix of mesenchymal layer, and the growth of AP cells labeled with PKH26 on the matrix was observed by HE staining and fluorescence microscope 7 days later. The acellular matrix of AP cells was used as control. The samples of co-culture for 7 days were transplanted into one side of groin of nude mice (experimental group), and blank culture samples were transplanted to the other side (control group). HE staining was performed on 7 days after transplantation and frozen sections of the tissue were made. The growth of AP cells labeled with PKH26 on the surface and inside of acellular matrix was observed under fluorescence microscope. To evaluate the histocompatibility of acellular matrix containing AP cells in nude mice. Results HE and DAPI staining showed that there was no residual cells in the acellular treated materials, and the DNA content of (19.367 卤5.254) ng/mg, was significantly lower than that of (3 805.500 卤519.119) ng/mg before acellular treatment. The results indicated that the acellular matrix of the mesenchymal layer was successfully prepared. The acellular matrix of AP cells and mesenchymal layer was successfully prepared. After 7 days of co-culture, AP cells were mainly adhered to the surface of the material. Part into the acellular matrix. After implanted into nude mice, AP cells could proliferate in acellular matrix material and gradually enter into the material, and induce angiogenesis with the prolongation of observation time. Conclusion the acellular matrix of velvet antler cartilage was successfully prepared. The matrix material was suitable for the adhesion and proliferation of seed cells (AP cells) in vitro and in vivo, and had the function of stimulating angiogenesis. It provides theoretical basis for cartilage tissue repair.
【作者单位】： 中国农业科学院特产研究所特种经济动物分子生物学国家重点实验室;吉林农业大学中药材学院;
【基金】：国家自然科学基金资助项目(31500792) 吉林省自然科学基金资助项目(20170101032JC)~~
【分类号】：R318.08;R68
本文编号：2288837
[Abstract]:Objective to investigate the feasibility and biocompatibility of acellular matrix material from antler cartilage and explore new materials for cartilage repair and reconstruction. Methods the mesenchymal layer of deer antler growth center in sika deer was treated with DNA enzyme, RNA enzyme, aprotinin mediated acellular treatment, histology and DNA content were detected to evaluate the acellular effect. The second generation of antlerogenic periosteum,AP cells in the antler region of deer were taken, and the characteristics of the stem cells were determined by fluorescent stem cell labeling. The cells were labeled with PKH26 and co-cultured with the acellular matrix of mesenchymal layer, and the growth of AP cells labeled with PKH26 on the matrix was observed by HE staining and fluorescence microscope 7 days later. The acellular matrix of AP cells was used as control. The samples of co-culture for 7 days were transplanted into one side of groin of nude mice (experimental group), and blank culture samples were transplanted to the other side (control group). HE staining was performed on 7 days after transplantation and frozen sections of the tissue were made. The growth of AP cells labeled with PKH26 on the surface and inside of acellular matrix was observed under fluorescence microscope. To evaluate the histocompatibility of acellular matrix containing AP cells in nude mice. Results HE and DAPI staining showed that there was no residual cells in the acellular treated materials, and the DNA content of (19.367 卤5.254) ng/mg, was significantly lower than that of (3 805.500 卤519.119) ng/mg before acellular treatment. The results indicated that the acellular matrix of the mesenchymal layer was successfully prepared. The acellular matrix of AP cells and mesenchymal layer was successfully prepared. After 7 days of co-culture, AP cells were mainly adhered to the surface of the material. Part into the acellular matrix. After implanted into nude mice, AP cells could proliferate in acellular matrix material and gradually enter into the material, and induce angiogenesis with the prolongation of observation time. Conclusion the acellular matrix of velvet antler cartilage was successfully prepared. The matrix material was suitable for the adhesion and proliferation of seed cells (AP cells) in vitro and in vivo, and had the function of stimulating angiogenesis. It provides theoretical basis for cartilage tissue repair.
【作者单位】： 中国农业科学院特产研究所特种经济动物分子生物学国家重点实验室;吉林农业大学中药材学院;
【基金】：国家自然科学基金资助项目(31500792) 吉林省自然科学基金资助项目(20170101032JC)~~
【分类号】：R318.08;R68
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