[The Inhibitory Effect and Mechanism of Human Umbilical Cord Mesenchymal Stem Cells on Prostate Cancer Metastasis].

OBJECTIVE: To test whether umbilical cord mesenchymal stem cells (UMSCs) can inhibit the growth and metastasis of prostate cancer cells, and to investigate the mechanism. METHODS: The UMSCs from human umbilical cord tissue were isolate by explant technique. After being co-cultured the UMSCs with LNCaP and PC-3 cells for 24 h, 48 h and 72 h, LNCaP and PC-3 cells' proliferation were tested and the 72 h proliferation inhibitory rate (IR) was calculated. Transwell invasion assay was used to test the migration and invasion abilities of prostate cancer cells after being co-cultured with UMSCs for 48 h. The invasion IR were calculated. The expression of matrix metalloproteinases (MMP)-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in the 72 h co-culture supernatants were tested by MILLIPLEX®MAPmethod. RESULTS: The proliferation of prostate cancer cells was inhibited after being co-cultured with UMSCs. The proliferation rate of LNCaP was lower than control group at 72 h (P<0.05), and the proliferation IR was 37.21%. The proliferation rate of PC-3 was lower than control group at 48 h and 72 h (P<0.05), and the proliferation IR was 31.27% at 72 h. Transwell invasion assays showed that co-culturing 48 h with UMSCs inhibited the invasive abilities of LNCaP and PC-3, and the invasion IR were 48.35% (LNCaP) and 46.91% (PC-3). Co-culturing 72 h, the expression of MMP-2 and MMP-9 were down-regulated (P<0.05) and the secretion of TIMP-1 and TIMP-2 were up-regulated (P<0.05) compared with control group. CONCLUSION: UMSCs can inhibit the proliferation and invasion abilities of prostate cancer cells by secreting TIMPs, the antagonist of MMPs, which suppressed the overexpression of MMPs.

[Differentiation of human bone marrow mesenchymal stem cells into Leydig or steroidogenic cells in vivo].

OBJECTIVE: To study the differentiation of human bone marrow mesenchymal stem cells (BMSCs) into Leydig or steroidogenic cells in vivo and the immunoreaction related to transplantation into mouse testis. METHODS: After differentiation and cultivation, the 3rd-passage BMSCs were collected and labeled with Hoechest 33342, and joined the saline fluorouracil to form cell-suspending fluid. After injection of the etgane dimethane sulphonate (EDS), the mice received the transplanted cell-suspending fluid by testis net injection with a dose of each side testicular 0.05 mL. Since the first day prior to transplantation, mice were executed every 2 d (one mouse each time) and the testosterone concentrations were analyzed. The control group included 20 BALB/c mice without any treatment during the same period. The results were analyzed by microscopic observation, using 3beta-hydroxysteroid dehydrogenase (3beta-HSD) monoclonal antibody and mouse anti-human cell nucleus monoclonal antibody for immunofluorescence assay on the testis. For tracking the BMSCs, the cells which were positively stained with both 3beta-HSD and mouse anti-human cell nucleus monoclonal antibodies were retrieved. RESULTS: Certain killing effect of EDS to the mouse Leydig cells was observed. Transplantation of human BMSCs into the mouse testis by testis net injection was effective and feasible, no immunoreactions were detected. After transplantation, no positive cells of 3beta-HSD) and mouse anti-human cell nucleus monoclonal antibody were found. CONCLUSION: Transplantation of human BMSCs into the mouse testis by testis net injection was effective and feasible, no immunoreactions were detected. After transplantation, the human BMSCs failed to differentiate into Leydig cells or steroidogenic cells.

Constant pH molecular dynamics (CpHMD) and mutation studies: insights into AaegOBP1 pH-induced ligand releasing mechanism.

AaegOBP1, isolated from the male and female antenna of yellow fever mosquitoes, may serve as crucial molecular targets for the development of mosquitoes' attractants and for the control of mosquito populations. Nowadays crystal structures of AaegOBP1 in the neutral environment have been obtained, whereas little research is focused on the conformational change of AaegOBP1 in the acid solution. In our study, the conformational change and the ligand bound poses in different solution pH were investigated using constant pH molecular dynamics (CpHMD) as well as mutation studies. Results demonstrate that the protein changes dramatically in low pH solution and that the pH-sensing triad (Arg23-Tyr54-Ile125) plays an indispensable role in the structural stability and ligand binding. In addition, we have proved that the residue Arg23 is the most important one of the pH-sensing triad. This work could provide more penetrating understanding of the pH-induced ligand-releasing mechanism.

[Differentiation of human bone marrow mesenchymal stem cells into Leydig or steroidogenic cells in vitro].

OBJECTIVE: To investigate the possibility of differentiating mesenchymal stem cells (MSCs) into steroidogenic or testicular Leydig cells in vitro. METHODS: The 3rd-passage cells of MSCs were divided into 4 groups to be induced and cultured. The experimental groups were cultured with conditional medium which consisted of luteinizing hormone (LH), human chorionic gonadotrophin (hCG), platelet-derived growth factor (PDGF) and interlukin-1alpha (IL-1alpha) as follows. Group A: LH 0.75 U/mL, hCG 40 U/mL,PDGF 10 ng/mL, IL-1alpha 0.0005 ng/ mL; Group B: LH 0.375 U/mL, hCG 20 U/mL, PDGF 10 ng/mL, IL-1alpha 0.0005 ng/mL; Group C: LH 0.1875 U/ mL,hCG 10 U/mL, PDGF 10 ng/mL, IL-1alpha 0.0005 ng/mL. Meanwhile, the control group was cultured in basal medium with normal sodium. The results were analysed by microscopic observations, 3beta-hydroxysteroid dehydrogenase (3beta-HSD) immunocytochemistry and immunofluorescence on the 7th, 14th and 21st day of induction respectively. The function of the induced cells was characterized by testosterone ELISA. RESULTS: 10-14 days after induction, the induced cells with a typical reticular intercellular connection possessed the morphologic characteristic of Leydig cells. 3beta-HSD immunocytochemistry and immunofluorescence showed that Group A at 21th day had the most positive cells (P < 0.05). 21 days of induction revealed a higher testosterone production than others (P < 0.05). CONCLUSION: MSCs from human bone marrow are able to differentiate into steroidogenic or testicular Leydig cells in vitro. Human bone marrow-derived MSC may become important cells for studies of steroidogenic differentiation and offer a potential clinical stem cell source for diseases of steroidogenic organs.

Gonadotropins facilitate potential differentiation of human bone marrow mesenchymal stem cells into Leydig cells in vitro.

Infertility due to low testosterone levels has increased in recent years. This has impacted the social well-being of the patients. This study was undertaken to investigate the potential of gonadotropins in facilitating differentiation of human bone marrow mesenchymal stem cells (BMSCs) into Leydig cells in vitro. BMSCs were isolated, cultured, and their biological characteristics were observed. BMSCs were induced with gonadotropins in vitro and their ability to differentiate into Leydig cells was studied. The level of expression of 3-beta hydroxysteroid dehydrogenase (3ß-HSD) and secretion of testosterone were determined using flow cytometry and enzyme-linked immunosorbent assay, respectively, and the results were compared between the experimental and control groups. The cultured BMSCs showed a typical morphology of the fibroblast-like colony. The growth curve of cells formed an S-shape. After inducing the cells for 8-13 days, the cells in the experimental group increased in size and showed typical characteristics of Leydig cells, and the growth occurred in spindle or stellate shapes. Cells from the experimental group highly expressed 3ß-HSD, and there was a gradual increase in the number of Leydig cells. The control group did not express 3ß-HSD. The level of testosterone in the experimental group was higher than the control group (p < 0.05). Additionally, the cells in the experimental group secreted higher levels of testosterone with increased culture time. The expression of Leydig cell-specific markers in the experimental group was significantly higher (p < 0.05). With these findings, BMSCs can be considered a new approach for the treatment of patients with low androgen levels.

Fosfomycin induced structural change in fosfomycin resistance kinases FomA: molecular dynamics and molecular docking studies.

Fosfomycin resistance kinases FomA, one of the key enzymes responsible for bacterial resistances to fosfomycin, has gained much attention recently due to the raising public concern for multi-drug resistant bacteria. Using molecular docking followed by molecular dynamics simulations, our group illustrated the process of fosfomycin induced conformational change of FomA. The detailed roles of the catalytic residues (Lys18, His58 and Thr210) during the formation of the enzyme-substrate complex were shown in our research. The organization functions of Gly53, Gly54, Ile61 and Leu75 were also highlighted. Furthermore, the cation-π interaction between Arg62 and Trp207 was observed and speculated to play an auxiliary role in the conformation change process of the enzyme. This detailed molecular level illustration of the formation of FomA·ATP·Mg·Fosfomycin complex could provide insight for both anti-biotic discovery and improvement of fosfomycin in the future.

Constant pH molecular dynamics (CpHMD) and molecular docking studies of CquiOBP1 pH-induced ligand releasing mechanism.

The odorant binding protein of Culex quinquefasciatus (CquiOBP1), expressed on the insect antenna, is crucial for the investigation of trapping baited with oviposition semi-chemicals and controlling mosquito populations. The acidic titratable residues pKa prediction and the ligand binding poses investigation in two systems (pH 7 and pH 5) are studied by constant pH molecular dynamics (CpHMD) and molecular docking methods. Research results reveal that the change of the protonation states would disrupt some important H-bonds, such as Asp 66-Asp 70, Glu 105-Asn 102, etc. The cleavage of these H-bonds leads to the movement of the relative position of hydrophobic tunnel, N- and C- termini loops and pH-sensing triad (His23-Tyr54-Val125) in acid solution. Ligand MOP has lower affinity and shows different binding poses to protein CquiOBP1 at pH 5. This ligand may be released from another tunnel between helices α3 and α4 in acidic environment. However, it would bind to the protein with high affinity in neutral environment. This work could provide more penetrating understanding of the pH-induced ligand-releasing mechanism.