无血清培养不同年龄人表皮细胞(hECs)的扩增规律与原代表型的比较研究

Comparison of expansion capabilities and primary phenotypes of human epidermal cells between donors of different age in serum free culture

目的无血清培养条件下,研究不同年龄人表皮细胞(humanepidermalcells,hECs)的体外扩增规律,比较不同年龄hECs原代细胞表型。方法临床收集50例包皮环切术中丢弃的皮肤组织,包括中年组(A,35～52岁,11例)、青年组(Y,18～27岁,17例)、儿童组(C,2～12岁,22例)。以Dispase和胰酶(tripsin)-EDTA分离,收集hECs并进行体外培养。每个年龄组随机选取5例,各代细胞计数,计算最大扩增倍数,并统计分析。收集原代细胞测量平均直径,记录克隆形成率,流式细胞仪(FACS)检测细胞K19阳性百分率。结果中年、青年、儿童组包皮来源的表皮细胞最大扩增倍数分别为(719±65)、(730±47)、(729±56)倍,各组间无统计学差异(P>0.05)。全体随机标本平均可扩增(728±53)倍,达到最大扩增量所需平均时间为(31±6)d。中年、青年、儿童组,表皮细胞平均直径分别为(3.44±0.42)μm,(3.54±0.46)μm,(3.55±0.37)μm,各组间无统计学差异(P>0.05);克隆形成率分别为(36.5±5.3)%,(36.9±6.8)%,(36.4±5.1)%,各组间无统计学差异(P>0.05);FACS检测中K19阳性细胞百分率分别为,(42.55±4.61)%,(42.47±6.58)%,(41.77±4.73)%,各组间无统计学差异(P>0.05)。结论无血清培养hECs的体外扩增规律与年龄因素无关,不同年龄hECs最大扩增能力取决于原代细胞表型?

Objective To compare the expansion capabilities and primary phenotypes of human epidermal cells (hECs) between donors of different age in serum free culture. Methods The human skin samples obtained during surgery operations were classified into 3 groups according to the donor's age: adult group (A, 11 samples), young group (Y, 17 samples) and children group (C, 22 samples). The hECs were isolated by dispase and trypsin digestions, and then cultured in vitro. The average diameter of primary human epidermal cells was measured. Meanwhile cloning efficiency was also analyzed by Giemsa stain. The rate of K19 positive cells was counted by flow cytometer. The cells were subcultured when they became 3 quarters confluent. Results The maximum expansions of middle age group, young group and children group were (719±65), (730±47), (729±56) fold respectively(no statistically significant difference). Average cell diameters of the 3 groups were (3.44±0.42)μm, (3.54±0.46)μm, (3.55±0.37)μm (P >0.05). For the 3 groups respectively, clone forming efficiency was (36.5±5.3)%,(36.9±6.8)%,(36.4±5.1)% (P >0.05), and the percentage of keratin19 (K19) positive cells was (42.55±4.61)%,(42.47±6.58)%,(41.77±4.73)% (P >0.05). Conclusion The expansion capability of cells does not relate to the donor's age, but to the primary cellular phenotypes.