人角质形成细胞体外扩增与生物学特征变化

Proliferation and biological character changes of human keratinocytes cultured in vitro

目的 研究人角质形成细胞体外扩增过程中生物学特征的变化,观察其角蛋白19(K19)mRNA表达及克隆形成率与扩增受限的关系。方法 取2-3岁健康儿童包皮20例,通过2.4 U/mL dispase和0.05％胰酶(tripsin)分离,获得角质形成细胞体外培养。细胞计数仪计数描绘生长曲线。光镜观察细胞体外扩增过程中形态学改变,同时计数克隆形成率。RT-PCR检测角质形成细胞K19和Involucrin的mRNA表达情况。结果 儿童包皮角质形成细胞体外平均最大扩增能力为(700±37)倍。细胞平均获得率为(1.64±0.297)×106/cm2包皮。角质形成细胞原代(P0)、第2代(P2)、第4代(P4)、第5代(P5)克隆形成率分别为:(45±4)％,(37±4)％,(28±3)％,(9±2)％,第6代(P6)克隆形成率消失。RT-PCR检测发现P0、P2、P4、P5有K19 mRNA表达,P6未见表达;Involucrin各代均可见表达。结论 人角质形成细胞体外扩增能力随传代逐渐降低,扩增受限与角质形成细胞中K19 mRNA表达及克隆形成率消失相关。

Objective To investigate the biological character changes of keratinocyte proliferated in vitro and to investigate the relationship between growth arrest and keratin19 mRNA expression and clonal forming efficiency. Methods The human foreskin was obtained from 20 patients, and the keratinocytes were isolated by 2.4 U/mL dis-pase and 0.05% tripsin digestion, and then cultured in vitro. Growth curve was made from data of cell counting. Cell phenotype was observed during cell culture under light microscope. Meanwhile colonal analysis was also performed. The mRNA expression of keratin 19 and involucrin was detected by RT-PCR. Results The children foreskin keratinocyte could be expanded for 700 ±37 times. RT-PCR results showed that K19 mRNA expressed at passages 0,2,4,5 vanished at passage6 ; involucrin expressed at all passages. Clonal forming efficiency is (45±4) % , (37 ±4)%,(28±3)%,(9±2)% at passage 0,2,4,5 respectively and vanished at passage6. Conclusion During expansion of keratinocytes, the ability of proliferation reduces. Growth arrest may be relative to decreased clony forming efficiency and K19 mRNA expression.