ABSTRACT
Parathyroid hormone/parathyroid hormone-related peptides are polypeptide hormones. They have similar gene struc-ture and same membrane receptor .They play great roles in regulating the calcium and phosphorus metabolism in vivo.PTH/PTHrP and their receptors are expressed in tissues of tumor ,skin, hair follicle and other normal tissues .Because of their physiological action on the proliferation and differentiation of epidermis and hair growth ,they may be a potential therapeutic target for some skin diseases including psoriasis.
ABSTRACT
Objective To investigate the influence of recombinant human parathyroid hormone [rhPTH (1-34)]on the proliferation of HaCaT cells induced by tumor necrosis factor-α (TNF-α) in vitro.Methods Cultured HaCaT cells were treated with various concentrations of rhPTH (1-34) for different durations after incubation with recombinant human TNF-α of 10 g/L for 24 hours.MTT assay and flow cytometry were performed to detect the proliferation and cell cycle of HaCaT cells,respectively.Results As contrast phase microscopy showed,the growth of HaCaT cells was inhibited by rhPTH (1-34) along with a decrease in the growth speed.MTT assay showed a suppressed proliferation of HaCaT cells after being treated with rhPTH (1-34) of 0.05,0.2,0.8,3.2 and 12.8 pmol/L for 36 and 48 hours (P＜ 0.01 or 0.05).The percentage of cells at G1 phase in HaCaT cells markedly increased (all P ＜ 0.01 ),while that at S phase declined (all P ＜ 0.01 )after 48-hour treatment with rhPTH(1-34) of 0.2,0.8,3.2 and 12.8 μ mol/L.Conclusions rhPTH(1-34) has an obvious inhibitive effect on the proliferation of HaCaT cells induced by TNF-α in vitro,and the effect is in a dose-dependent manner.
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Objective To investigate the expressions and significance of IL-22 and related cytokines (IL-23pl9 and IL-6) in sera and PBMCs of patients with psoriasis. Methods Sera and PBMCs were obtained from the venous blood samples from 58 patients with psoriasis vulgaris and 20 normal human controls. The PBMCs were subjected to culture for 5 hours followed by the collection of cells and culture supernatant. Then,quantitative real-time RT-PCR was used to examine the mRNA expressions of IL-22, IL-23pl9 and IL-6 in PBMCs, enzyme-linked immunosorbent assay (ELJSA) to detect the level of IL-22 protein in the sera and culture supernatant of PBMCs. Results In the patients with psoriasis and controls, the relative expression level in PBMCs was 4.48 ± 2.64 and 2.35 ± 0.91 respectively for IL-22 mRNA, 6.07 ± 4.09 and 2.61 ± 1.46 respectively for IL-23pl9 mRNA, 3.87 ± 1.49 and 1.48 ± 0.62 respectively for IL-6 mRNA; significant differences were observed between the two groups in all the above parameters (all P < 0.01). ELISA revealed that the level of IL-22 protein in the patients and controls was (86.23 ± 25.58) ng/L and (43.67 ± 14.82) ng/L respectively in the sera (P< 0.01), (119.11 ± 21.51) ng/L and (57.70 ± 13.17) ng/L respectively in the culture supernatant of PBMCs (P< 0.01). Conclusion There is an overexpression of IL-22 in the PBMCs and sera of patients with psoriasis, implying that IL-22 is involved in the pathogenesis of psoriasis.
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Objective To construct an artificial skin model of melanoma by mixed culture of human keratinocytes and MV3 melanoma cells on de-epidermized dermis (DED) in order to study the effect of keratinocytes on melanoma invasion. Methods Epidermal cell suspension was obtained by a two-step digestion method from the circumcised foreskin of a child, keratinocyte serum-free medium was applied to the culture and passage of keratinocytes. MV3 melanoma cells were cultured and passaged in RPMI 1640 medium. Log-phase keratinocytes and MV3 cells were mixed with a ratio of 3:1 and seeded onto the surface of DED followed by a liquid culture and air-liquid culture for a total of 2 weeks. Thereafter, the artificial tissue model was assessed by HE staining and immunohistochemical staining for S-100 protein, HM64S and keratin. Results HE staining showed that MV3 cells formed band-like tumor masses or foci on the surface of DED, with keratinocytes intermingling among the tumor cells, but no typical epidermis-like structure was observed. Some tumor cells infiltrated into the surface of DED and showed a cluster distribution; some tumor cells invaded the lumen of the DED, and attached to the luminal wall in a ring shape; some tumor cells penetrated through the wall into the surrounding dermal tissue. On the bottom and lateral side of DED, tumor cells were infiltrating dispersedly.The tumor loci stained positive for S-100 protein and keratin, and weakly positive for HMB45. Conclusion Keratinocytes enhance the invasion of MV3 melanoma cells into the skin tissue model of melanoma.
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Objective To construct tissue-engineered skin via in vitro inoculation of epidermal stem cells(ESCS)onto de-epidermized dermis.Methods Skin tissue was obtained from the foreskin of a healthy 6-year-old child.and keratinocytes were isolated by two-step trypsinization method followed by the collection of ESCS via rapid adhesion by collagen Ⅳ.The ESCS were identified by morphological observation and immunohistochemical staining with K19 and integrin β1.To construct tissue-engineered skin,selected ESCS were seeded onto the surface of de-epidermized dermis followed by a one-week culture immersed in the medium and a subsequent 4-week culture at the air-medium interface.The tissue-engqneered skin was evaluated with haematoxylin & eosin(HE)staining as well as keratin immunohistochemistry.Results Micro scopically,cultured ESCs showed a paving stone-like appearance and grew into colonies.Immunohistochemistry revealed that the ESCs were positive for integrin-β1 and keratin 19.After 5 weeks of culture,3-6 layers of epidermal cell were observed on the dermis with the formation of stratum corneum.Keratin protein was observed in the artificial epidermal skin.Conclusion Tissue-engineered skin is successfully constructed with epidermal stem cells and de-epidermized dermis in vitro.