The influence of progesterone and prostaglandin F2α on decorin and the adhesion of caruncular epithelial cells of bovine placenta at early-mid pregnancy—Part II

One of the most important processes determining the proper course of gestation and its physiological termination in cows is the adhesion of epithelial cells allowing for direct contact of maternal and foetal parts of the placenta. Throughout pregnancy, placental cells are under strict hormonal control, which among others regulates the concentration and activity of specific proteins participating in the extracellular matrix remodelling of foetal membranes. The aim of the study was to evaluate the influence of progesterone and prostaglandin F_2α on the adhesion of epithelial cells at early-mid pregnancy in cows. Additionally, the impact of selected hormones on anti-adhesive properties of decorin was evaluated. Caruncular epithelial cells were isolated from healthy cows during pregnancy, immediately after slaughter. Primary cell cultures derived from the 2nd and 4th month of gestation were used in the experiments. The viability of cells was assessed by MTT assay. The adhesion of cells to fibronectin was measured spectrophotometrically. The activity of metalloproteinases was confirmed by the metalloproteinase assay. Progesterone (10^–5 and 10^–7 mol/L) and prostaglandin F_2α (10^–4_, 10^–5 and 10^–7 mol/L) increased the viability of bovine caruncular epithelial cells in the 2nd month of pregnancy. The treatment with prostaglandin F_2α significantly reduced the number of adherent cells from the 2nd month of gestation at the doses of 10^–4 and 10^–5 mol/L. Both progesterone and prostaglandin F_2α were shown to have an effect of decorin resulting in both a decrease in metalloproteinase activity and an increase in adhesion of cells to fibronectin.     

Inhibitory effect of decorin on human pterygium fibroblasts in vitro

AIM: To investigate the effect of decorin (DCN) on the proliferation and apoptosis of human pterygium fibroblasts (HPF) in vitro , and to compare the effect of mitomycin C (MMC) in order to search for a new method to prevent the recurrence after pterygium surgery. METHODS: Human pterygium fiborblasts were isolated from the caudomedial part of pterygium tissues in pterygium patients and then cultured in vitro using tissue inoculation method. The cells were treated with DCN and MMC at concentrations of 0.01, 0.1, 1, 5 and 10 mg/L. The morphological alterations of HPF were observed after 24 h, 48 h or 72 h of treatment. MTT method was used to assay the effects of the 2 drugs at different doses after 12 h, 24 h and 48 h on the proliferation of the cells. The expression of proliferating cell nuclear antigen (PCNA) in each group treated with different doses of DCN was detected by the method of immunohistochemistry after 48 h. The cell cycle distribution was determined by flow cytometry analysis. RESULTS: After administration of 10 mg/L DCN or 1 mg/L MMC for 12 h, the proliferation of HPF was significantly inhibited by both drugs in a dose- and time-dependent manner (P<0.05). After treated with 1~10 mg/L DCN for 48 h, the percentage of HPF in G₀/G₁ phase was increased, while the percentage of HPF in G₂/M phase and S phase (G₂/M%+S%) was decreased after treated with 5~10mg/L DCN for 48 h (P<0.05). The late-apoptotic cells were not found in DCN group and MMC group. DCN dose-dependently inhibited the expression of PCNA in HPF (P<0.05). CONCLUSION: Decorin significantly inhibits the proliferation of HPF, and blocks the cells in G₁ phase.     

Extracellular matrix proteoglycan decorin-mediated myogenic satellite cell responsiveness to transforming growth factor-beta1 during cell proliferation and differentiation Decorin and transforming growth factor-beta1 in satellite cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inhibitor of muscle cell proliferation and differentiation. Decorin, a small proteoglycan in the extracellular matrix, binds to TGF-beta1 and modulates the activity of TGF-beta1 during muscle cell growth and development. However, its interaction with TGF-beta1 and involvement in myogenesis is not well characterized. In the present study, chicken myogenic satellite cells, myogenic precursors for muscle growth and repair, were isolated from the pectoralis major muscle and used to investigate the biological function of TGF-beta1 and decorin during myogenesis. The over-expression of decorin in satellite cells significantly increased cell proliferation, compared to the control cells. Consistent with this result, reducing decorin expression decreased cell proliferation, which suggests a decorin-mediated mechanism is involved in the regulation of myogenic satellite cell proliferation. Satellite cells over-expressing decorin were less sensitive to TGF-beta1 during proliferation, which indicates that decorin may sequester TGF-beta1 leading to increased proliferation. During satellite cell differentiation, the over-expression of decorin induced differentiation by increasing the muscle specific creatine kinase concentration. However, the addition of TGF-beta1 diminished decorin-mediated cell responsiveness to TGF-beta1 during differentiation. Taken together, these results suggest that decorin induces myogenic satellite cell proliferation and differentiation by regulating cellular responsiveness to TGF-beta1. An alternative TGF-beta1-independent pathway may be involved in the regulation of satellite cells by decorin.     

Expression features of human decorin in E.coli DH5α

AIM: To study the expression features of human decorin in E. coli DH5α.METHODS: The pGEX-4T-1-decorin fusion clone was expressed in E. coli DH5α. Positively expressed clone was selected by SDS-PAGE. The optimized inducing time by 1 mmol/L IPTG was determined. The solubility of GST-decorin fusion protein was analyzed by ultrasonic crush method, and its quality was examined by Western blotting. RESULTS: With the induction of 1 mmol/L isopropy-β-D-thiogalactoside (IPTG), the fusion protein was expressed in E. coli DH5α. The optimized inducing time by 1 mmol/L IPTG was 4 hours. Most of fusion protein existed in the form of inclusion body. The expressed protein was GST fusion protein.CONCLUSION: It is suggested that the fusion protein of GST-decorin may be expressed in E.coli DH5α in a large amount in the form of inclusion body.     

鹿茸尖端组织核心蛋白多糖基因全长cDNA的克隆及其表达分析

为了进一步了解鹿源DCN基因的结构与功能,揭示该基因在鹿茸尖端不同组织层的表达规律,本研究从梅花鹿鹿茸尖端组织cDNA文库中首次克隆具有完整编码区的DCN基因全长cDNA序列,并结合生物信息学方法和实时荧光定量RT-PCR技术对该基因的氨基酸序列结构和表达特征进行分析.结果表明,梅花鹿DCN基因cDNA全长为1 831 bp,编码360个氨基酸;其编码蛋白具有N端信号肽,相对分子质量为39.9 ku,理论等电点为8.8,其一级结构中亮氨酸所占比例最高(12.5%);梅花鹿与绵羊DCN氨基酸序列的相似性最高(98%).实时荧光定量RT-PCR分析表明,DCN在间充质层的表达显著高于其他3层组织,提示DCN的抗纤维化活性可能是维持鹿茸间充质层快速生长的重要调节因素.     

Effect of decorin on proliferation and collagen type I synthesis of stiff knee joint synovial type B cells

AIM: To explore the effects of decorin on procollagen type I (PcI), mRNA expression,collagen type I synthesis and proliferation of synovial type B cells of stiff knee joint synovial membrane. METHODS: Type B cells of synovial membrane were isolated from the stiff knee joint synovial membrane and cultured in vitro. The cells were treated with decorin at concentrations of 0.1 mg/L, 5 mg/L and 10 mg/L. After cultured for 24 h, 48 h and 72 h, the cell proli-feration rates were measured by MTT colorimetric determination. Cell cycle distribution and apoptosis were analyzed by flow cytometry. The mRNA level of Pc I was detected by RT-PCR, while collagen type I was measured by Western blot. RESULTS: The proliferation of synovial type B cells was significantly inhibited, the percentage of synovial type B cells at G₁ phase was significantly increased by 5 mg/L and 10 mg/L decorin (P<0.05), and PcⅠmRNA expression and collagen type I synthesis were significantly decreased. The cells with late apoptosis were not found in control group and experimental groups. CONCLUSION: Recombinant human decorin inhibits synovial type B cell proliferation and decreases PcⅠmRNA expression and collagen type I synthesis in synovial type B cells of stiff knee joint synovial membrane in vitro, suggesting that decorin potentially contributes to the therapy of human knee stiffness.