Expression of pituitary tumor-transforming gene-1 and its pathogenic role in systemic sclerosis / 南方医科大学学报

OBJECTIVE@#To investigate the expression of tumor-transforming gene-1 (PTTG1) in systemic sclerosis (SSc) and its role in fibrosis.@*METHODS@#Skin biopsy samples were collected from 21 patients with SSc and 22 patients with healthy skin for detecting the mRNA and protein expressions of PTTG1 using real-time PCR (RT-PCR) and immunohistochemistry, respectively. In cultured primary human dermal fibroblasts, PTTG1 expression was knocked down via RNA interference (siRNA), and the mRNA expression levels of PTTG1 and the fibrosis-related genes @*RESULTS@#Compared with those in normal skin samples, the mRNA and protein expressions of PTTG1 increased significantly in the skin tissue of patients with SSc (@*CONCLUSIONS@#PTTG1 is highly expressed in skin tissues of patients with SSc, and PTTG1 knockdown can reduce the activity of the dermal fibroblasts, suggesting a close correlation of PTTG1 with fibrosis in SSc.

Expression of calponin-1 and its pathogenic role in systemic sclerosis / 南方医科大学学报

OBJECTIVE@#To investigate the expression of calponin-1 (CNN1) in systemic sclerosis (SSc) and its pathogenic role in fibrosis.@*METHODS@#Skin biopsy samples were collected from 19 patients with SSc and 21 healthy subjects. Real-time PCR was used to detect the expression of and mRNAs in the samples, and the protein expression of CNN1 was detected using immunohistochemistry. In cultured primary human dermal fibroblasts, expression was knocked down RNA interference, and the mRNA expression levels of and the fibrosis-related genes , , , , and were detected using real-time PCR; the proliferation of the cells was assessed using a real-time cell proliferation detection system.@*RESULTS@#Compared with that in samples from normal subjects, the expression of mRNA was significantly increased in the skin tissue of patients with SSc ( < 0.05) with a positive correlation with α-SMA (=0.7219, < 0.0001); the protein expression of CNN1 was also significantly increased in the skin tissue of patients with SSc. In cultured primary skin fibroblasts, the expression of CNN1 mRNA was positively correlated with and mRNA expressions (=0.6547, < 0.05; =0.6438, < 0.05). knockdown in the fibroblasts significantly inhibited the cell proliferation, obviously lowered the expressions of fibrosis-related genes, and reduced the protein expression of collagen.@*CONCLUSIONS@#The expression of is increased in the skin tissues of patients with SSc, and knockdown can reduce the activity of dermal fibroblasts, suggesting the close correlation of CNN1 with fibrosis in SSc.

Human amniotic epithelial cells:isolation, identification and multi-directional differentiation / 中国组织工程研究

BACKGROUND: Human placenta is a stable source for human amniotic epithelial cells, which is becoming a cellsource in the regenerative medicine that attracts widespread attentions.OBJECTIVE: To establish the method of isolation, culture, and adipogenic, chondric and osteogenic differentiationof human amniotic epithelial cells. METHODS: Trypsin-EDTA digestion was used to isolate human amniotic epithelial cells from human amnion tissue,which were then cultured and identified in vitro. The growth curve of the cells was observed in 12 days. Passage 1human amniotic epithelial cells were induced to differentiate into adipocytes, chondrocytes and osteoblasts, andconventional cultured cells were used as controls. After 16 days induction, oil red O, Masson and alkaline phosphatesstaining methods were carried out, and adipogenic transcription factor, type Ⅱ collagen, osteopontin, alkalinephosphatase mRNA expressions were detected using real-time fluorescene quantitative PCR.RESULTS AND CONCLUSION: Human amniotic epithelial cells were successfully obtained from human amnion tissue.Immunofluorescence data showed the expression of epithelial cell surface marker CK19. Passage 1 cells had a strongability to divide and proliferate. Compared with passage 1 ones, passage 2 cells showed a slight decrease in proliferationability, and the proliferation ability of passage 3 cells was the worst. Red lipid droplets, brilliant blue cartilage matrix andreddish brown calcium nodes were detected by oil red O, Masson and alkaline phosphates staining after adipogenic,chondrogenic and osteogenic differentiation, respectively. With the time prolonged, the expressions of adipogenictranscription factor, type Ⅱ collagen, osteopontin and alkaline phosphatase mRNA were increased. These resultsdemonstrated that human amniotic epithelial cells could be isolated from human amniotic membrane by enzymedigestion method, and these amniotic epithelial cells could be induced to differentiate into differentiate into adipocytes,chondrocytes and osteoblasts.

Involvement of collagen-binding heat shock protein 47 in scleroderma-associated fibrosis

Uncontrolled fibrosis of skin and internal organs is the main characteristic of scleroderma, and collagen is a major extracellular matrix protein that deposits in the fibrotic organs. As the chaperone of collagen, heat shock protein 47 (HSP47) is closely related with the development of fibrosis. To explore the potential function of HSP47 in the pathogenesis of scleroderma, the clinical, in vivo and in vitro studies were performed. In clinical, the increased mRNA level of HSP47 was observed in the skin fibroblasts and PBMC from scleroderma patients, and the enhanced protein level of HSP47 was also detected in the skin biopsy and plasma of the above patients. Unexpectedly, the enhanced levels of HSP47 were positively correlated with the presence of anti-centromere antibody in scleroderma patients. Moreover, a high expression of HSP47 was found in the skin lesion of BLM-induced scleroderma mouse model. Further in vitro studies demonstrated that HSP47 knockdown could block the intracellular and extracellular collagen over-productions induced by exogenous TGF-β. Therefore, the results in this study provide direct evidence that HSP47 is involved in the pathogenesis of scleroderma. The high expression of HSP47 can be detected in the circulatory system of scleroderma patients, indicating that HSP47 may become a pathological marker to assess the progression of scleroderma, and also explain the systemic fibrosis of scleroderma. Meanwhile, collagen over-expression is blocked by HSP47 knockdown, suggesting the possibility that HSP47 can be a potential therapeutic target for scleroderma.

Differentiation and osteoprotegerin secretion of human osteoblasts:R-spondin 1 effect via Wnt/beta-catenin signal pathway / 中国组织工程研究

BACKGROUND:Studies have funded that reduced Wnt/β-catenin signaling is involved in the onset and/or progression of bone erosion in rheumatoid arthritis. It can lead to potential new treatment approaches of bone erosion by enhancing Wnt/β-catenin signaling pathway. R-spondin 1 may act as a Wnt agonist, but there is no study in human osteoblasts. OBJECTIVE:To verify the effect of R-spondin 1 on promoting differentiation and maturation of human osteoblasts by inhibiting DKK1. METHODS:S40-transfected human osteoblast lines, hFOB1.19, were treated with R-spondin 1, Wnt-3a and DKK1 to detecting the proliferation, alkaline phoshpatase activity and osteoprotegerin concentration. RESULTS AND CONCLUSION:R-spondin 1 had no effects on hFOB1.19 cel s. Wnt-3a upregulated the activity of alkaline phoshpatase, which could be enhanced by addition of R-spondin 1. R-spondin 1 could reduce the DKK1-mediated inhibition of alkaline phoshpatase activity in hFOB1.19 cel s. R-spondin 1 increased the concentration of osteoprotegerin, and moreover, the promotion of osteoprotegerin by R-spondin 1 alone was stronger than the inhibition by DKK1. These findings suggest that R-spondin 1 can inhibit DKK1 by Wnt/β-catenin signal pathway to promote the differential and maturation of human osteoblasts to excrete osteoprotegerin.