Establishment and identification of a human keloid fibroblasts cell line
10.3760/cma.j.cn114453-20240111-00018
- VernacularTitle:人原代瘢痕疙瘩成纤维细胞系的建立及鉴定
- Author:
Mengli XU
1
;
Qifei WANG
;
Jingyi WANG
;
Yuhao LU
;
Zelian QIN
Author Information
1. 北京大学第三医院成形外科，北京 100191
- Keywords:
Keloid;
Fibroblasts;
Immortalization;
Cell line;
Chromosome karyotype identification;
Gene
- From:
Chinese Journal of Plastic Surgery
2024;40(5):545-554
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish an immortalized human keloid fibroblasts(KFbs) cell line and identify its characteristics and functions.Methods:The specimen was obtained from a 32-year-old female patient who underwent surgical resection of an earlobe keloid at Peking University Third Hospital in November 2019. The keloid tissue obtained was removed from the subcutaneous fat and epidermis. It was then separated and cultured using the tissue sticking method to obtain primary KFbs, which were passaged using the trypsin digestion method. After the primary KFbs were infected with an SV40 lentivirus, purified by puromycin, and passaged, a human KFbs cell line was established. Chromosomal karyotype analysis, short tandem repeat (STR) profiling, and gender gene detection were conducted to identify the primary KFbs and the cell line. The CCK-8 method was used to assess the proliferation ability of the cells. Quantitative real-time PCR (qRT-PCR) and Western blotting were used to detect the mRNA and protein expression levels of specific genes (PGK1, ENO1, LDHA, GLUT1, TGF-β1, COL1, COL3, FN). The comparative analysis of relevant data between primary KFbs and the cell line was conducted using t-test, and P<0.05 indicated statistical significance. Results:The morphology of both the primary KFbs and the cell line was typically spindle-shaped. The cell line morphology was basically similar to that of the primary KFbs, which were continuously cultured and passaged for 20 generations. The gender gene(Amelogenin) detection showed both were females. The chromosome karyotyping of the primary KFbs and cell line was satisfactory, maintaining the fundamental characteristics of normal cells without undergoing malignant transformation. The STR identification results showed that no multiple alleles were found in the cell line, indicating a normal cell genotype. Furthermore, the cell line did not match any entries in known cell databases. After 24, 48, and 72 hours of culture, the proliferation ability of the cell line increased by 76.1%, 125.8%, and 60.3% compared to primary KFbs. The proliferation rates of the cell line were significantly faster than those of primary KFbs ( P<0.05). The mRNA and protein expression levels of the aforementioned genes in the cell line showed no significant changes compared to the primary KFbs ( P>0.05). Conclusion:An immortalized human KFbs cell line was successfully established, showing no significant changes in morphology, characterization, and function, while exhibiting a faster proliferation rate compared to that of primary KFbs.
