Comparison of Primary Keloid Fibroblast Cultivation Methods and the Characteristics of Fibroblasts Cultured from Keloids, Keloid-surrounding Tissues, and Normal Skin Tissues
Comparison of Primary Keloid Fibroblast Cultivation Methods and the Characteristics of Fibroblasts Cultured from Keloids, Keloid-surrounding Tissues, and Normal Skin Tissues
复制标题
DOI：
10.4067/s0717-95022021000100302
复制
发表时间：
2021-02
影响因子：
0.5
通讯作者：
Haiyan Qin;Rui-Qin Liu;Wen-Jia Nie;Mingxi Li;Liehao Yang;Chang-lin Zhou;Lianbo Zhang;Guang Zhang
中科院分区：
文献类型：
--
作者：
Haiyan Qin;Rui-Qin Liu;Wen-Jia Nie;Mingxi Li;Liehao Yang;Chang-lin Zhou;Lianbo Zhang;Guang Zhang
建立原代培养的瘢痕疙瘩成纤维细胞一直是研究瘢痕疙瘩发病机制的根本措施。原代细胞培养的质量可以直接影响进一步实验的结果。通过对S教授的研究，探讨了体外细胞培养模型的最佳生长条件，包括最佳的保存时间和胶原酶处理时间，以及不同组织中适合的表皮-真皮分离方法。采集患者的瘢痕疙瘩组织、瘢痕疙瘩周围组织和正常皮肤组织，进行原代成纤维细胞培养。采用组织块植入法和胶原酶消化法，并进行比较。用这两种方法检测瘢痕疙瘩相关基因α-SMA、COL1和COL3在细胞中的表达水平，以验证原代细胞的质量。对两种MET组织和三种不同组织进行了比较分析。样品经过处理后，细菌和脂肪污染立即降至最低。不同的组织需要不同的去表皮方法和不同的胶原酶消化时间才能获得最佳的效果。实时荧光定量聚合酶链式反应结果显示，瘢痕疙瘩相关基因在培养的成纤维细胞中的mRNA表达水平与其体内表达谱有关，这与其他研究报道的情况相同。本研究结果揭示了原代瘢痕疙瘩成纤维细胞培养的几个关键点，并证实了体内瘢痕疙瘩成纤维细胞与体外原代瘢痕疙瘩成纤维细胞基因表达的相关性。
The establishment of primary keloid fibroblast culture has always been a fundamental measure for studying mechanisms of keloid disease. The quality of the primary cell culture can directly affect the results of further experiments. T h s study was performed to investigate the optimal growth conditions, including the optimal storage time and collagenase treatment time, for in vitro cell culture models and the suitable methods for epidermis-dermis separation in different tissues. Keloid tissues, keloid-surro unding tissues, and normal skin tissues were collected from patients, for primary fibroblast culture. Two methods, tissue explant and col genase digestion, were deployed and compared. Expression levels of the keloid-related genes α-SMA, Col1, and Col3 were assessed in cells cultured using both methods, to verify the qualities of the primary cells. A comparative analysis was conducted between the two met ods and among the three different tissues used. Bacterial and lipid contamination was immediately minimized after the samples were processed. Different methods of epidermis removal and different durations of collagenase digestion were required in different tissues to g enerate optimal results. Real-time PCR results showed that the mRNA expression levels of keloid-related genes in cultured fibroblasts c or elated to their in vivo expression profile, as previously reported in other studies. The results of this study have revealed several k ey points in the culture of primary keloid fibroblasts and demonstrated the correlation in gene expression between in vivo keloid fibroblasts an d in vi ro primary keloid fibroblasts.