【摘要】：目的探討鹿茸軟骨制備脫細胞基質材料的可行性以及生物相容性,為軟骨修復重建探索新材料。方法取梅花鹿鹿茸生長中心間充質層,進行由DNA酶、RNA酶、抑肽酶等介導的脫細胞處理;行組織學和DNA含量檢測,評價脫細胞效果。取第2代鹿生茸區(qū)骨膜(antlerogenic periosteum,AP)細胞,行熒光干細胞標記明確其干細胞特性后,用PKH26熒光標記并與制備的間充質層脫細胞基質進行復合培養(yǎng);7 d后取材行HE染色觀察以及熒光顯微鏡下觀察PKH26標記的AP細胞在基質表面生長情況。以上觀測均以未復合AP細胞的脫細胞基質作為對照。將復合培養(yǎng)7 d的樣本移植至裸鼠一側腹股溝(實驗組),取空白培養(yǎng)樣本移植于另一側(對照組)。于移植后7、21 d取材行HE染色,同時對組織進行冰凍切片并在熒光顯微鏡下觀察PKH26標記成功的AP細胞在脫細胞基質表面及內(nèi)部的生長情況,評價含AP細胞的脫細胞基質在裸鼠體內(nèi)的組織相容性。結果HE和DAPI染色顯示脫細胞處理后材料中無細胞殘留,DNA含量為(19.367±5.254)ng/mg,較脫細胞處理前的(3 805.500±519.119)ng/mg顯著降低(t=12.630,P=0.000),提示成功制備間充質層脫細胞基質。AP細胞與間充質層脫細胞基質復合培養(yǎng)7 d后,AP細胞主要黏附于材料表面,部分進入脫細胞基質內(nèi)部。植入裸鼠體內(nèi)后,隨觀察時間延長,接種AP細胞可以在脫細胞基質材料中增殖并逐漸進入材料內(nèi)部,并誘導血管生成。結論實驗成功制備鹿茸軟骨脫細胞基質,該基質材料在離體和活體情況下適于種子細胞(AP細胞)的黏附和增殖,并具有刺激血管生成的功能,為其用于軟骨組織修復提供理論依據(jù)。
[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 鹵5.254) ng/mg, was significantly lower than that of (3 805.500 鹵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.
【作者單位】： 中國農(nóng)業(yè)科學院特產(chǎn)研究所特種經(jīng)濟動物分子生物學國家重點實驗室;吉林農(nóng)業(yè)大學中藥材學院;
【基金】：國家自然科學基金資助項目(31500792) 吉林省自然科學基金資助項目(20170101032JC)~~
【分類號】：R318.08;R68

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