Screening Differentially Expressed Genes In Cultured Epidermal Stem Cells Of The Different Development Stages Of Human Skin By CDNA Microarray Technique

Objective: The research screened differentially expressed genes in different developmental stages of human epidermal stem cells by cDNA microarray technique, analyzed the characteristics of human epidermal stem cell gene expression changes of different developmental stages. It can provide clues to further search for the proliferation and differentiation mechanisms of human epidermal stem cells.Method: Samples of human epidermal stem cells were obtained from 28-32 weeks of fetal, 4-12Y children and 35-55Y adult skins. Separation of the epidermis used trypsin digestion and EDTA. The typeâ…£collagen was used to isolate and purify the human epidermal stem cells. Theβ1 integrin and k19 were detected by the immunohistochemistry stain. 21,522 Oligo DNAs were put on a glass slide through chemical modification by Biochip Microarrayer. Total RNA were extracted from epidermal stem cells of two samples and detected by formaldehyde denaturing agarose gel electrophoresis. Through the reverse transcription method to make probe, Cy3-dCTP was used to label human control group DNA (the human genome as a common control), Cy5-dCTP was used to label be the experimental group (fetal group, adult group and children group) cDNA. Then, it hybridized with the chip. We used LuxScan 10KA dual-channel laser scanner to scan chips and used LuxScan 3.0 image analysis software to analyze the images. Then, made image signal into digital signal, screened the differentially expressed genes with the standard of 2 times differrence. Select the significant up-regulated genes or down-regulated genes, made the verification by real time RT pcr.Results: According to the hybridization results from oligonucleotide DNA microarray,gene expression patterns and functions were analyzed. Adult group compared to children group, there are differentially expressed genes in 1808, up-regulated genes have 1089, and down-regulated genes have 719. There are 1462 known genes (Sign in Gene Bank), and 346 unknown genes. Children group compared to fetal group, there are differentially expressed genes in 4534, up-regulated genes have 1783, and down-regulated genes have 2751. There are 3577 known genes (Sign in Gene Bank), and 957 unknown genes. 166 genes have a significant difference (Ratio> 4 or Ratio <0.25) in gene expression, up-regulated genes have 93 and down-regulated genes have 73. According to its expression of the protein function, the differentially expressed genes in adult group and children group include 128 categories, and 216 categories in children group and fetal group. A gene can have multiple functions, the total of them more than the sum of differential genes. 1104 genes were differentially expressed in continuing in fetal, children and adult group. They were divided into 32 categories on gene function. Different genes involved in protein translation, energy synthesis and DNA replication, they are closely related to the development process of epidermal stem cells. In the 32 categories, there were 94 continued up-regulated genes and 75 continued down-regulated genes. The continued up-regulated genes mainly are nucleotide-binding protein genes (for example, NM₀05381, NM₀24045), unfolded protein binding genes (for example, NM₀02157, NM₀06260), zinc ion binding and ATP binding genes. The continued down-regulated genes mainly are GTP binding genes, sequence-specific DNA binding genes (for example, NM₀05253,NM₀01421) and MAP kinase phosphatase activity genes (for example, NM₀02746,NM₀02754). Real time RT-PCR results showed that the fetus, children and adult samples TUBGCP3 continued to increase in the expression, and expression of FOSL2 continued to decrease.Real time RT-PCR results showed that TUBGCP3 was continued up-regulated in the expression in the fetus, children and adult samples, gene FOSL2 was continued down-regulated. It was consistent with the chip results. This shows that the results of gene chip are reliable.Conclusion: The gene expression is obviously different in fetal, children and adult epidermal stem cell in vitro. From fetal to children and adult, the genes of cells mature and cytoskeleton-related protein increases, and the genes of cell differentiation and proliferation-related down. This suggests that with the age grows, epidermal stem cells become more mature in maintaining cell shape, the internal structure of cells and the characteristics of the cells. However, their proliferation and differentiation may show a downward trend.