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.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
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)
