酶联合消化法分离犬心房肌成纤维细胞及其生物学特性的研究

Combined enzymatic digestion method used on primary culture and biological characteristic identification of atrial fibroblasts of canine

目的探讨适用于犬心房肌成纤维细胞(CAF)体外分离培养及鉴定的技术方法。方法无菌手术取犬左心耳,应用木瓜蛋白酶、Ⅰ型胶原酶联合消化法分离心房肌成纤维细胞并进行体外培养。在倒置显微镜下观察原代心房肌成纤维细胞生长特点;苏木精-伊红(HE)染色观察细胞形态,并对细胞进行纤维连接蛋白、波形蛋白、盘状结构域受体2蛋白免疫荧光染色;通过绘制生长曲线法、2-(2-甲氧基-4-硝基苯基)-3-(4-硝基苯基)-5-(2,4-二磺基苯)-2-四唑单钠盐(WST-8)法观察细胞的增殖情况;流式细胞仪分析比较各代细胞DNA周期特点;观察不同培养体系对第3代(P3)CAF生长的影响。结果酶联合消化法分离培养的细胞1 d后,可见细胞贴壁呈长椭圆形生长,3 d后生长迅速呈长梭形,7 d后细胞排列成单层,连接紧密;HE染色显示细胞长梭形呈螺旋状排列;免疫荧光检测纤连蛋白、波形蛋白、盘状结构域受体2表达阳性;P1、P2、P3细胞生长曲线近似"S"形,WST-8法显示P1、P2、P3细胞生长于第3天至第5天光密度值变化较明显,增殖迅速;P1、P2、P3G0/G1的百分率分别为(56.83±1.72)%、(68.10±1.33)%、(68.23±1.33)%,各代之间G0/G1的百分率比较差异无统计学意义(P>0.05);P1、P2、P3(S+G2)/M的百分率分别为(43.17±1.72)%、(31.90±1.33)%、(31.77±1.33)%,各代之间(S+G2)/M的百分率比较差异无统计学意义(P>0.05);杜尔伯克改良伊格尔/F12培养基培养的P3细胞增殖力明显高于RPMI-1640培养基培养的细胞,差异有统计学意义(P<0.05)。结论酶联合消化法可以高效快速分离和稳定培养CAF,为研究犬心房纤维化提供了充足的种子细胞。

Objective To explore and set up the methodology of isolation,cuhivation and identification of canine atrial fibroblasts （CAF） in vitro. Methods The left aurcle of canine was del6ved from aseptic operation. Atrial fibroblasts were isola- ted using papain together with type I collagen enzyme and cultured in vitro. The growing states of primary atrial fibroblast were observed under the inverted phase-contrast microscope;cellular morphology was observed after hematoxylin-eosin staining; fi- broblasts were stained by immanofluorescence with fibronectin, vimentin and discoidin domain receptor 2. The CAF prolifera- tion profile was analyzed by curve of growth and WST-8 assay. Characteristic of DNA cell cycle was analyzed by flow eytome- try. The influences of different culture medium on the growth of P3 fibroblasts were observed. Results It was observed oblong cells grewing with adherence one day after the atrial fibroblasts isolated and cultured using enzyme digestion method, atrial fi- broblasts grew with long fusiform shape quickly after 3 clays and the fibroblasts remained monolayer and arranged closely after 7 days ; cells arranged a long spindle spiraling after HE staining. The expressions of fibronectin, vimentin and diseoidin domain receptor 2 were positive after immunofluorescence staining. The cell growth curves of P1, P2, P3 were like S-types, cells in the optical density value changed significantly from the third day to the fifth day. The rates of G0/G1 phase cells of P1 ,P2 ,P3 were （56.83 ± 1.72） %, （68.10 ± 1.33 ） %, （68.23 ± 1.33 ） % respectively and there was no variances in cells of PI, P2, P3 （P 〉 0. 05） ;the rates of（ S ＋ G2）/M phase cells of P1 ,P2 ,P3 were（43.17 ± 1.72） %, （31.90 ± 1.33）%, （31.77 ± 1.33 ） % re- spectively and there was no variances in cells of Pl, I〉2, P3 （ P 〉 0. 05 ）. The proliferation of P3 was higher in dulbecco modified eagle/Nutrient Mixture F-12 medium than that in RPMI-1640 medium（P 〈 0.05）. Conclusion CAF can be isolated quickly and cultured stably by enzyme digestion method which can supply plenty of seed cells for studying CAF.