Dewu Liu

Expression of β-catenin and cyclin D1 in epidermal stem cells of diabetic rats

The healing of diabetic wounds represents a formidable clinical challenge, and the molecular mechanisms involved in diabetic wound healing are far from clear. In this study, we investigated the expression of β-catenin and cyclin D1 in the epidermal stem cells (ESCs) of diabetic rats, and explored whether the reduction of β-catenin and its downstream target in ESCs, cyclin D1, lead to poor wound healing in diabetes mellitus (DM). We found that, compared to the controls, the ESCs of diabetic rats were markedly reduced, the clone formation efficiency of the ESCs was markedly lower, and the mRNA and protein expression of β-catenin and cyclin D1 was significantly decreased. These findings suggest that the low expression of β-catenin and cyclin D1 may reduce the activity of ESCs from diabetic rats, which might be one of the important mechanisms of delayed wound healing in DM.

A Comparative Study on the Expression of Telomerase Reverse Transcriptase in Different Development Stages of Human Epidermal Stem Cells in vitro

Aim: The main reasons for limiting the replication and expansion ability of the adult stem cells in vitro are the loss of telomerase activity and the changes of related proliferation gene expression, in which telomerase reverse transcriptase(TERT) plays a key role. In this chapter, we focus on the isolation, culture and identification of the epidermal stem cells derived from human fetus, child and adult skins, and compare the TERT expression of the different developmental periods of the epidermal stem cells. Methods: The skin samples of child, adult and fetus were disposed with Trypsin-EDTA respectively. The epidermal stem cells were isolated and purified by means of the type IV collagen rapid adhering method and cultured in vitro using keratinocyte serum free medium contained epidermal growth factor and fetal bovine serum. The keratinocytes were used as the control groups. The colony forming efficiency were calculated. The β1 integrin, keratin19 and p63 transcription factor were detected by the immunocytochemical stain and the image quantitative analysis. Results: The cultured cells revealed colony growth, the colony forming efficiency of the epidermal stem cell is higher than the keratinocyte. The β1 integrin,k19 and p63 had positive expression in the epidermal stem cells but not the keratinocyte cells. The TERT expression level of the fetus epidermal stem cells was higher than that of the adult and the child. The detection of the average value of absorbance and positive area of each group exhibited that the value decreased with the age. Conclusion: The expression of TERT in the cultured human epidermal stem cells derived from human fetus, child and adult skins were all positive. And the expression intensity level was followed as the period of the fetus, the child and the adult. It showed that the induction and the reinforcement of the telomerase reverse transcriptase activity may have important influence in the maintenance of the self-renewal and proliferation ability of the epidermal stem cell.

Substance P combined with epidermal stem cells promotes wound healing and nerve regeneration in diabetes mellitus.

Exogenous substance P accelerates wound healing in diabetes, but the mechanism remains poorly understood. Here, we established a rat model by intraperitoneally injecting streptozotocin. Four wounds (1.8 cm diameter) were drilled using a self-made punch onto the back, bilateral to the vertebral column, and then treated using amniotic membrane with epidermal stem cells and/or substance P around and in the middle of the wounds. With the combined treatment the wound-healing rate was 100% at 14 days. With prolonged time, type I collagen content gradually increased, yet type III collagen content gradually diminished. Abundant protein gene product 9.5- and substance P-immunoreactive nerve fibers regenerated. Partial nerve fiber endings extended to the epidermis. The therapeutic effects of combined substance P and epidermal stem cells were better than with amniotic membrane and either factor alone. Our results suggest that the combination of substance P and epidermal stem cells effectively contributes to nerve regeneration and wound healing in diabetic rats.

Differential microRNA expression profile comparison between epidermal stem cells and differentiated keratinocytes.

The aim of the current study was to analyze the differential microRNA (miRNA) expression profiles of human epidermal stem cells (ESCs) and differentiated keratinocytes. Enzyme digestion was used in combination with rapid adhesion to collagen IV to isolate primary human ESCs and differentiated keratinocytes, from which total RNA was extracted. Fluorescence labeling, microarray hybridization and differential expression analyses were performed. Reverse transcription quantitative polymerase chain reaction (RT‑qPCR) was performed to validate the reliability of the microarray results and predict the target genes of the differentially expressed miRNAs. A total of 25 miRNAs, including hsa‑miR‑197‑5p, hsa‑miR‑125b‑5p and hsa‑miR‑376a‑3p, were upregulated, whereas 166 miRNAs, including hsa‑miR‑29b‑3p, hsa‑miR‑203 and hsa‑miR‑34a‑3p, were downregulated in the human ESCs compared with the expression in differentiated keratinocytes. RT‑qPCR results confirmed the upregulation of hsa‑miR‑197‑5p and the downregulation of hsa‑miR‑29b‑3p, which were consistent with the microarray results. miRNA target prediction indicated that the miRNA expression levels correlated with cell proliferation, differentiation, apoptosis and senescence. Expression levels of miRNAs significantly differed between human ESCs and differentiated keratinocytes. This finding may be attributed to their biological characteristics, such as proliferative behavior and differentiation abilities.

Expression of telomerase reverse transcriptase gene in human epidermal stem cells and its significance

It is well known that the telomerase is present at high levels in embryonic tissues, which plays an important role in replication and proliferation capacity of the stem cells. But the telomerase activity in cultured epidermal stem cells derived from human skins is not fully understood. In this chapter, we focus on the isolation, culture and identification of the epidermal stem cells derived from human fetal skins, and investigating the mRNA and protein expression of telomerase reverse transcriptase in cultured epidermal stem cells. The telomerase activity and the mRNA and protein expression of telomerase reverse transcriptase were determined by using TRAP-ELISA, western blot, immunocytochemical stain and RT-PCR techniques, respectively. The results showed that the cultured epidermal stem cells revealed colony growth. The telomerase activity and the mRNA and protein expression of telomerase reverse transcriptase were positive and in low level in cultured epidermal stem cells. It indicated that the expression of telomerase activity in human epidermal stem cells seems to be directly associated with their proliferation capacity and may play a critical role in overcoming growth limitations of seed cells in tissue engineering skins.

Detection of Differentially Expressed Genes in Cultured Epidermal Stem Cells Derived From Children and Adult Skins by cDNA Microarray Technique

Epidermal stem cells have the characteristic of the undifferentiated cells in the morphology, and are the biological source of the skin and its subsidiary in the development, repair and reconstruction. The proliferation and differentiation of epidermal stem cell are influenced by many factors, such as integrin, signal transduction pathway and cytokines. Current researches have not yet been able to find absolute recognized epidermal stem cells markers. And on certain characteristics of a single gene expression and its impact studies have made some progress, but the overall characteristics and gene expression changes and developments in the law was unclear. This research detected differentially expressed genes in cultured epidermal stem cells derived from children and adult skins. Samples were derived from 4-12 Y children and 35-55 Y adult skin. The human epidermal stem cells were isolated by trypsin digesting method and purified by collagen adhering method, respectively. Total RNA extracted from epidermal stem cells of two samples. Differentially expressed genes detected by cDNA microarray technique. According to the hybridization results from oligonucleotide DNA microarray, gene expression patterns and functions were analyzed. Adult group compared to childrens, there were differentially expressed genes in 1805, which mainly associated with protein translation, energy synthesis and DNA replication, and closely related to the development process of human epidermal stem cells. Down-regulated genes had 716, included GTP binding, sequence-specific DNA binding, MAP kinase phosphatase activity, they related to proliferation and differentiation of epidermal stem cell. Up-regulated genes had 1089, which mainly included cytoskeletal structural protein genes and associated with cell maturation. These data suggest that the gene expression is obviously different between children and adult epidermal stem cell in vitro. This may be closely related to different abilities of proliferation and differentiation of human epidermal stem cell and wound healing ability of skin with different developmental stages. It can provide clues to further search for new markers and the development of regulatory genes, and lay the foundation for further applications.

High‑throughput sequencing reveals differentially expressed lncRNAs and circRNAs, and their associated functional network, in human hypertrophic scars

Growing evidence suggests that long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) are involved in the occurrence and development of tumors and fibrotic diseases. However, the integrated analysis of lncRNA and circRNA expression, alongside associated co-expression and competing endogenous RNA (ceRNA) networks, has not yet been performed in human hypertrophic scars (HS). The present study compared the expression levels of lncRNAs, circRNAs and mRNAs in human HS and normal skin tissues by high-throughput RNA sequencing. Numerous differentially expressed lncRNAs, circRNAs and mRNAs were detected. Subsequently, five aberrantly expressed lncRNAs and mRNAs, and six circRNAs were measured to verify the RNA sequencing results by reverse transcription-quantitative polymerase chain reaction. Furthermore, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed for the dysregulated genes, in order to elucidate their principal functions. In addition, a coding-noncoding gene co-expression (CNC) network and ceRNA network were constructed for specific significantly altered genes. The CNC network analysis suggested that AC048380.1 and LINC00299 were associated with metastasis-related genes, including inhibin subunit βA (INHBA), SMAD family member 7 (SMAD7), collagen type I α1 chain (COL1A1), transforming growth factor β3 (TGFβ3) and MYC proto-oncogene, bHLH transcription factor (MYC). Inhibitor of DNA binding 2 was associated with the lncRNAs cancer susceptibility 11, TGFβ3-antisense RNA 1 (AS1), INHBA-AS1, AC048380.1, LINC00299 and LINC01969. Circ-Chr17:50187014_50195976_-, circ-Chr17:50189167_50194626_-, circ-Chr17:50189167_ 50198002_- and circ-Chr17:50189858_50195330_- were also associated with INHBA, SMAD7, COL1A1, TGFβ3 and MYC. COL1A1 and TGFβ3 were associated with circ-Chr9:125337017_125337591_+ and circ-Chr12:120782654_120784593_-. The ceRNA network indicated that INHBA-AS1 and circ-Chr9:125337017_125337591_+ were ceRNAs of microRNA-182-5p targeting potassium voltage-gated channel subfamily J member 6, ADAM metallopeptidase with thrombospondin type 1 motif 18, SRY-box 11, MAGE family member L2, matrix metallopeptidase 16, thrombospondin 2, phosphodiesterase 11A and collagen type V a1 chain. These findings suggested that lncRNAs and circRNAs may act as ceRNAs, which are implicated in the pathophysiology and development of human HS, and lay a foundation for further insight into the novel regulatory mechanism of lncRNAs and circRNAs in hypertrophic scarring.

Differential miRNA expression profiles in human keratinocytes in response to protein kinase C inhibitor

Aberrant expression of microRNAs (miRNAs) is widely accepted to be involved in keratinocyte differentiation and to be dependent on activation of the protein kinase C (PKC) pathway. However, the miRNA profiles and biological characteristics of keratinocytes induced by specific inhibitors of PKC have yet to be elucidated. The present study aimed to explore the differential miRNA expression profiles in keratinocytes treated with the PKC inhibitor GF109203X, by conducting a bioinformatics analysis. Parts of the GF109203X-induced keratinocytes formed distinct clones after 2 days of culture, and the expression of intergrin β1, cytokeratin (CK)19 and CK14 were positive, whereas CK10 expression was negative. A total of 79 miRNAs were differentially expressed in keratinocytes treated with GF109203X, among which 45 miRNAs were upregulated and 34 were downregulated. The significantly upregulated microRNAs includedhsa-miR-1-3p and miR-181c-5p, whereas hsa-miR-31-5p and hsa-let-7c-3p were significantly downregulated. In addition, the results of reverse transcription-quantitative polymerase chain reaction exhibited consistency with the microarray results. An enrichment analysis demonstrated that certain target genes of the differentially expressed miRNAs serve an important role in cell proliferation and differentiation, cell cycle progression and apoptosis, etc. These results revealed that GF109203X induced the differential expression of certain miRNAs when keratinocytes began showing the characteristics of epidermal-like stem cells, which may provide a novel approach for wound healing and regeneration of skin tissues.

Introduction of telomerase reverse transcriptase gene into epidermal stem cells derived from human skins

Telomerase extends the proliferation and prevent replicative senescence in most somatic cells. Whether it has similar function in human epidermal stem cells remains to be determined. In this study, the human telomerase reverse transcriptase (hTERT) cytalytic subunit was introduced into epidermal stem cells derived from human fetal skins. The expression of hTERT mRNA, protein and telomerase activity in the transduced cells was observed by real time PCR (RT-PCR) techniques, western blot analysis and telomeric repeat amplification protocol enzyme-linked immunosorbent assay (TRAP-ELISA) assay, respectively. The proliferation of the transduced cells was examined. The results showed the introduction of hTERT into the epidermal stem cells upregulated the expression of the hTERT gene and protein. And also the hTERT-transduced epidermal stem cells exhibited significantly elevated telomerase activity and proliferation. These works establish the base of further gene modification, monoclone screening and cell differentiation mechanism. The ability to maintain the biological characteristics and proliferation of epidermal stem cells could have important applications in wound healing and skin tissue engineering.

Comparative Study on Expression of Relevant Proteins of Proliferation and Differentiation of Human Epidermal Stem Cells between Diabetic Skin and Normal Skin

β-catenin plays an important role in the tissue repair. But its expression and meanings in the diabetic cutaneous wound remains to be determined. In this study, we investigated the expression of β-catenin and PCNA in epidermal stem cells(ESCs) of diabetic rat skin. The diabetes mellitus (DM) rats were induced by a single i.p. injection of streptozotocin. ESCs were extracted from the dorsal skin samples of rats by trypsin digesting method and further purified by collagen adhering methods. Expression of ss1-integrin and keratin 19 were identified as ESCs by immunocytochemical staining . The results showed the amount of β-catenin and PCNA were decreased in ESCs of DM rat skin, and the clone forming efficiency of ESCs in DM rat skin was significantly lower than that in normal skin. It reveals impaired wounds repair in DM is characterized by the protracted epithelialization which is due to a decreased proliferative capacity of epidermal stem cells.