Isolation and characterization of in vitro culture of hair follicle cells differentiated from umbilical cord blood mesenchymal stem cells.

The present investigation explored the in vitro culture, isolation and characterization of hair follicle cell differentiation from umbilical cord blood mesenchymal stem cells (MSCs). Flow cytometry was used to obtain MSCs from the isolation and purification of human umbilical cord blood MSCs. Culture suspension of hair follicle organ was centrifuged and the supernatant used in the culture medium of MSCs, and the entire process of induced differentiation was recorded by photomicroscopy. The expression level of surface marker CK15 of hair follicle cells obtained from induced differentiation was detected with immunofluorescence. RT-PCR method was used to further detect the difference in expression of CK15 between hair follicle cells and umbilical cord blood MSCs, and statistical analysis was carried out. CD44+CD29+ double-labeled cells accounted for 50.8% of all the samples of umbilical cord blood MSCs in this study. The diameter of hair follicle cells differentiated from umbilical cord blood stem cells reached 800×10-3 mm after 3 weeks of cell culture. Based on the detection and colocalization of CK15 expression in induced hair follicle cells, the overlap ratio between CK15 and nuclei reached 83% in hair follicle cells, which was obviously higher than that in umbilical cord blood stem cells. The difference had statistical significance (P<0.05). In conclusion, hair follicle cells can be successfully differentiated from umbilical cord blood stem cells by using the supernatant from hair follicle cells. This method can be used for high-speed induced differentiation with high purity, which is promising for clinical application.

Normalization of regulatory T cells, serum TGF-ß1, and LTN after 5-aminolevulinic acid-photodynamic therapy in patients with condyloma acuminate.

The purpose of this study was to investigate the changes in peripheral blood regulatory T (Treg) cells, serum transforming growth factor-ß1 (TGF-ß1), and lymphotactin (LTN) following treatment of patients with condyloma acuminata (CA) with 5-aminolevulinic acid-photodynamic. A total of 46 patients with CA were selected as the experimental group and 43 healthy individuals were included in the control group. Before the treatment, the CA patients had a higher number of CD4+CD25+Foxp3+ Treg cells and CD4+CD25+ Treg cells than the healthy group. CA patients also had lower levels of serum TGF-ß1 and LTN than the healthy controls. After the treatment, the number of CD4+CD25+Foxp3+ Treg cells and CD4+CD25+ Treg cells decreased significantly in the CA patients and normalized to the levels in the control group after 3 weeks. The treatment also elevated the levels of serum TGF-ß1 and LTN in the CA patients, which were close to the values in the control group after 3 weeks. The results showed that low levels of serum TGF-ß1 and LTN played important roles in the occurrence and development of CA and cellular immune functions were closely related to the occurrence and development of CA.

Overexpression of vascular endothelial growth factor (VEGF) receptors on keratinocytes in psoriasis: regulated by calcium independent of VEGF.

Psoriasis is a common chronic inflammatory disease of the skin characterized by epidermal hyperplasia and angiogenesis. Recently, vascular endothelial growth factor receptors (VEGFRs, including VEGFR-1, VEGFR-2 and VEGFR-3) were found to be expressed in normal human epidermis and associated with proliferation and migration of keratinocytes. The purpose of this study is to investigate the expression of VEGFRs on psoriatic keratinocytes and the roles of calcium and VEGF in regulating VEGFR expression. Skin samples from 17 patients with chronic plaque psoriasis and 11 normal controls were included. The expression of VEGFRs in psoriatic keratinocytes at mRNA and protein levels was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis. Localization of the VEGFRs in skin lesions was determined by immuno-fluorescent method. Since keratinocyte proliferation and differentiation rely on calcium concentrations, and VEGF is overexpressed in psoriatic epidermis, we further investigated the roles of calcium and VEGF in regulating the expression of VEGFRs. Overexpression of VEGFR-1, VEGFR-2 and VEGFR-3 in psoriatic epidermis was demonstrated both at mRNA and protein levels in vitro. VEGFRs were strongly labeled in non-lesional, perilesional and lesional psoriatic keratinocytes in all viable epidermal stratums in vivo. Furthermore, both exogenous VEGF165 and calcium enhanced the expression of VEGFRs. Calcium also enhanced the expression of VEGF in non-lesional psoriatic keratinocytes, while targeted blockade of VEGF activity by bevacizumab could not inhibit calcium-induced up-regulation of protein levels of VEGFRs. We conclude from these results that VEGFRs are overexpressed in lesional psoriatic epidermal keratinocytes. Both calcium and VEGF regulate VEGFRs expression in psoriatic epidermis. More importantly, calcium is a potential regulator for VEGFR independent of VEGF.

Expression of VEGFR-2 on HaCaT cells is regulated by VEGF and plays an active role in mediating VEGF induced effects.

Vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 play important roles in mitogenesis and chemotaxis of endothelial cells. In normal human skin, VEGF is expressed and secreted by epidermal keratinocytes. Emerging data suggest that keratinocyte-derived VEGF targets other cell types besides the dermal endothelial cells. We have recently showed that keratinocytes from human normal skin expressed all five known VEGF receptors and co-receptors (neuropilin 1 and 2). To define the functional significance of VEGFR-2 in epidermis, we examined its role in a keratinocyte cell line, HaCaT cells, in response to VEGF treatment. Expression of VEGFR-2 on HaCaT cells was confirmed at both RNA and protein levels and was regulated by VEGF165 treatment. Treatment of HaCaT cells with VEGF165 induced tyrosine-autophosphorylation of VEGFR-2 and phosphorylation of PLC-gamma and p44/42 MAPK in a time-dependent manner. Preincubation with a neutralizing antibody for VEGFR-2 (MAB3571) completely abrogated these phosphorylation effects. Furthermore, VEGF165 stimulated proliferation and migration of HaCaT cells, and this effect was significantly blocked by a pretreatment with MAB3571. Neutralizing VEGFR-2 in HaCaT cells increased cell adhesion during culture. Our results suggest that VEGFR-2 expressed on HaCaT cells plays a crucial role in VEGF-mediated regulation of cell activity.