成纤维细胞与脱细胞真皮基质复合培养的实验研究  

Study of fibrablast and acellular dermal matrix co-culturing in vitro

作　　者：车鹏程[1] 何洪彬 肖芳[1] 孙瑞军[1] 孙红[1] 

机构地区：[1]河北联合大学基础医学院,河北唐山063000 [2]秦皇岛市第一人民医院烧伤科,河北秦皇岛066000

出　　处：《中国现代医学杂志》2014年第27期29-33,共5页China Journal of Modern Medicine

基　　金：河北省自然科学基金资助项目(No:H2012401017)

摘　　要：目的本研究用酶消化法体外培养高纯度的人皮肤成纤维细胞(FB),将FB与高渗盐-氢氧化钠(NaCl-NaOH)消蚀法制作的人脱细胞真皮基质(ADM)复合培养并评价其生物相容性。方法中性蛋白酶-Ⅰ型胶原酶消化法体外培养人FB,通过组织学方法鉴定其纯度。携带绿色荧光蛋白基因的腺病毒(Ad-GFP)转染人FB,组织学方法观察荧光蛋白的表达情况,流式细胞仪检测转染比例。将FB和Ad-GFP转染的FB接种在NaCL-NaOH消蚀法制作的人ADM上,组织学方法观察细胞的生长和增殖情况;逆转录聚合酶链反应(RT-PCR)检测ADM内FB角质细胞生长因子(KGF)、碱性成纤维细胞生长因子(bFGF)mRNA的蛋白表达。结果体外培养的人FB形态为细长梭形,有较长的突起和明显的核分裂相,波形蛋白、Ⅰ型胶原蛋白、Ⅲ型胶原蛋白的表达均为阳性。Ad-GFP转染FB的比例为72%。复合培养显示人FB不仅黏附在ADM表面,还可以较为均匀地在ADM天然孔隙中生长和增殖;与正常FB比较,ADM内FB KGF、bFGF mRNA的蛋白表达无明显变化。结论中性蛋白酶-Ⅰ型胶原酶消化法体外可以培养出高纯度的人FB。Ad-GFP转染FB获得成功。NaCL-NaOH消蚀法制作的ADM蕴含丰富的生物信息,FB作为种子细胞与其生物相容性好,为进一步开展皮肤创伤修复的实验研究奠定了基础。【Objective】Fibrablasts（FBs） were digested in vitro by enzyme and cultured with acellular dermal matrix（ADM） made by hypertonic saline-sodium hydroxide（NaCl-NaOH） maceration to evaluate biocompatibility.【Methods】FBs were digested by Dispase Ⅱ-type Ⅰcollagenase and cultured in vitro.FBs were identified by immunohistochemistry. Adenovirus（Ad-GFP） transfected FBs, the green fluorescence protein were observed and the transfection ratio was detected by fluorescent protein expression by flow cytometry. FBs and Ad-GFP transfected FBs were cultured in the human ADM and the growth and proliferation of cells were observed histologically. The keratinocyte growth factor（KGF）/basic fibroblast growth factor（bFGF） mRNA expression in the FBs were examined by reverse transcription-polymerase chain reaction（RT-PCR）. 【Results】Fusiform FBs had long break out and clear caryokinesis and vimentin/type Ⅰ Collagen/type Ⅲ Collagen expression were positive. Transfection ratio was 72%.FB grew and proliferated well in the ADM. Compared with the one grown on the plate, there is no change in the KGF/ bFGF mRNA expression of FBs grown in the ADM. 【Conclusions】Wecould get highly purified FBs by Dispase Ⅱ-type Ⅰ collagenase digestion in vitro. Ad-GFP transfecting FBs achieved success. The ADM made by NaCl-NaOH maceration containing abundant bioinformation, FBs as seed cells had good biocompatibility with the ADM, these provided foundations to carry out the study of repairing skin wound.

关 键 词：成纤维细胞 高渗盐-氢氧化钠消蚀法 脱细胞真皮基质 生物相容性 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]