Objective: To investigate DBP's damages to female reproductive system with DBP-toxicated granulosa cell (GCs) models. Methods: DBP-toxicated ovarian GCs models were established in rats to simulate the concentration of DBP in vivo, which were verified after separation. The effects of DBP at different concentrations on the growth and proliferation of ovarian GCs and on the secretion of estradiol (E2) and progesterone (P) in ovarian GCs were investigated. The variation of P450 aromatase (P450arom), P450 side-chain cleavage (P450scc), IGF-1 and TNF-alpha was determined at both mRNA and protein levels when treated by different concentrations of DBP. Results: Prolonged DBP treatment at a low concentration could promote the growth and proliferation of rat ovarian GCs in vitro, while DBP at a high concentration showed obvious cytotoxicity, and the apoptosis rate of GCs had a dose-response relationship with DBP. Simultaneous detection of cell cycles showed that the proportion of cells in the S phase decreased. The treatment of Ovarian GCs with DBP for a long time could increase its secretion of E2 and P, while the expression of P450arom and P450scc in corresponding with ovarian GCs was increased at mRNA and protein levels. Conclusion: This research revealed that prolonged treatment of DBP can inhibit the growth and proliferation of ovarian GCs, and affect the secretion of E2 and P as well as the expression of P450arom and P450scc. The effect may be through the increased expression of DBP-induced IGF-1 and activated P450arom and P450scc.

Objective: Transplantation of mesenchymal stem cells (MSCs) may promote bone healing. Endothelial progenitor cells (EPCs) may enhance the osteogenic properties of MSCs by improving their microenvironment. We aimed to investigate whether EPCs can enhance the osteogenic properties of MSCs in vitro, and whether transplantation of EPC-MSC cell sheets could promote bone regeneration in a rat model of alveolar bone defect.Design: MSCs and EPCs were obtained from 2-week-old Sprague-Dawley rats. Cell sheets were prepared using MSCs and MSCs co-cultured with EPCs. Morphological characteristics of cell sheets were observed by H&E staining. Osteogenic differentiation capacities of the cell sheets were assessed by alkaline phosphatase (ALP) staining, Alizarin Red S staining and qRT-PCR. Cell sheets were transplanted into alveolar bone defects in 8-week-old rats. Six weeks later, bone formation was assessed by micro-CT.Results: EPC-MSC sheets exhibited faster osteogenesis than MSC sheets. Six weeks after implantation, alveolar bone defects transplanted with EPC-MSC sheets exhibited a better bone reconstruction. MSC sheets generated new bone that partially covered the defect areas, while EPC-MSC sheets exhibited more robust osteogenic activity, with continuous new bone that almost covered the entire defect area.Conclusions: Transplantation of cell sheets containing EPCs and MSCs promoted bone regeneration. (C) 2016 Elsevier Ltd. All rights reserved.

A hot issue in current research regarding stem cells for regenerative medicine is the retainment of the stemness and multipotency of stem cell. Endothelial progenitor cells (EPCs) are characterized by an angiogenic switch that induces angiogenesis and further ameliorates the local microenvironment in ischemic organs. This study investigated whether EPCs could modulate the multipotent and differential abilities of mesenchymal stem cells (MSCs) in vitro and in vivo. We established an EPC/MSC indirect Transwell coculture system and then examined the effects of EPCs on the regulation of MSC biological properties in vitro and bone formation in vivo. The in vitro studies showed that cocultured MSCs (coMSCs) display no overt changes in cell morphology but an enhanced MSC phenotype compared with monocultured MSCs (monoMSCs). Our studies regarding the cellular, molecular, and protein characteristics of coMSCs and monoMSCs demonstrated that EPCs greatly promote the proliferation and differentiation potentials of coMSCs under indirect coculture condition. The expression of the pluripotency factors OCT4, SOX2, Nanog, and Klf4 was also upregulated in coMSCs. Furthermore, coMSCs combined with fibrin glue showed improved bone regeneration when used to repair rat alveolar bone defects compared with monoMSC grafts in vivo. This study is the first to demonstrate that EPCs have dynamic roles in maintaining MSC stemness and regulating MSC differentiation potential.

The Forkhead box M1 (FOXM1), an important regulator of cell differentiation and proliferation, is overexpressed in a number of aggressive human carcinomas. The purpose of this study was to examine the expression levels of FOXM1 in epithelial ovarian cancer (EOC), to identify the relationship between FOXM1 expression and patient survival, and to investigate the role of FOXM1 in human ovarian cancer development.

OBJECTIVE To investigate the effect of endothelial progenitor cell (EPC) on regulating proliferation and apoptosis of bone marrow mesenchymal stem cell (BMSC) in the indirect co-culture system. METHODS BMSC and EPC were cultivated and identified in vitro.Using transwell inserts to establish EPC and BMSC indirect co-culture system.Experimental groups were prepared as follows: BMSC/EPC indirect co-culture: BMSC were co-cultured with EPC that were separated in transwell insert. CONTROL BMSC/BMSC were indirect co-cultured at the same cell counts with the experimental groups. Colony forming unit-fibroblast(CFU-F) assay was studied with three types of EPC/BMSC ratios (1: 1, 10: 1, 100: 1) to assess the capacity and efficiency for cell self-renewal.In addition, flow cytometry technique was used to detect the cell cycle at co-cultured 3 days and the cell apoptosis at co-cultured 3, 7, 10 d. RESULTS Under indirect co-culture condition,EPC could significantly promote cell cycle progress and enhance capacity of cell self-renewal. Co-cultured EPC resulted in an accumulation of BMSC at S phase [experimental group: (15.72 ± 2.93)%, control group: (2.02 ± 0.66)%, P < 0.01]. CFU-F assay showed that the self-renewal capacity of EPC: BMSC/10: 1 group [(50.98 ± 6.32)%] and 100:1 group[(57.87 ± 14.06)%] were significantly higher than that in control group [(33.07 ± 9.60)% and (30.06 ± 7.20)%](P < 0.01). However,EPC had a slight but non-significant down-regulated effect on BMSC apoptosis(P > 0.05). CONCLUSIONS Under indirect co-culture condition, EPC could enhance the proliferation of BMSC, but could not regulate cell apoptosis in vitro.

It is well‑known that estrogen-related receptor α (ERRα) affects numerous metabolic pathways and biological functions in the body, although the function of ERRα in the mandibular condylar chondrocytes (MCCs) of the temporomandibular joint remains unclear. The aim of the present study was to investigate the effect of ERRα on the biological characteristics of MCCs in female rats. Immunofluorescent staining was used to observe the expression level and distribution of ERRα in MCCs and tissues. Quantitative polymerase chain reaction (qPCR) was performed to detect the impact of estrogen intervention on the biological characteristics of female rat MCCs and ERRα expression levels. Liposome transfection and XCT‑790 were used to overexpress and inhibit ERRα expression, respectively, and then qPCR was performed to detect changes in the biological characteristics of MCCs. ERRα expression was detected in the nucleus and cytoplasm of rat MCCs. 17‑β estradiol (E2) (10‑8 M) increased the mRNA and protein expression levels of ERRα, Sox9, GDF‑5 and aromatase during in vitro MCC cultivation. In addition, E2 affected MCC proliferation through the regulation of ERRα expression levels. Overexpression of ERRα positively regulated the mRNA and protein expression levels of Sox9 and GDF‑5, but did not exhibit a significant effect on the mRNA and protein expression levels of aromatase and Col2a1. In conclusion, ERRα exhibited an important regulatory role in the proliferation and differentiation of female Sprague‑Dawley rat MCCs in vitro through regulating Sox9 and GDF-5.

Objective To analyze the effect of ginsenoside-Rd,a selective competitive Ca2+ receptor antagonist,on the prognosis of acute ischemic stroke.Methods We extracted the data of ginsenoside-Rd in patients with acute ischemic stroke of randomized,double-blind,placebo-controlled,multicenter,phase Ⅱ and Ⅲ studies,with using ginsenoside-Rd or not as the prognostic variable.The clinical outcome was assessed by using the modified Rankin Scale(mRS) and categorized as good(score 0-2) or poor(score 3-6).Univariate and multivariate logistic regression analyses were performed to explore predictors of ischemic stroke.Results A total of 428 patients were analyzed.Of them,344(56.2%) had good outcome and 84(43.8%) had poor outcome.The poor outcome was associated with old age(OR: 1.034;95% CI: 1.003-1.066),higher NIHSS total score(OR: 1.53;95% CI: 1.386-1.690) and the non-use of ginsenoside-Rd(OR: 2.024;95% CI: 1.120-3.663).Conclusion The results suggest that ginsenoside-Rd can improve the outcome of acute ischemic stroke.

Recently, it has been reported that the orphan nuclear receptor estrogen-related receptor α (ERRα) is involved in the osteogenic differentiation of mesenchymal stem cells (MSCs). Moreover, ERRα has been identified as a novel therapeutic target for treating osteoporosis and other bone diseases. Human periodontal ligament tissue-derived mesenchymal stem cells (hPDLSCs) have recently been used in stem cell-mediated therapies because of their multipotency, particularly toward osteogenic differentiation. However, it is still unclear whether ERRα can regulate the osteogenic differentiation of hPDLSCs. In the present study, we investigated the role of ERRα in the osteogenic differentiation of hPDLSCs in vitro. We isolated hPDLSCs and confirmed their capacity for multipotent differentiation. Furthermore, we examined ERRα expression in hPDLSCs by RT-PCR and immunocytochemistry. We found that the expression of ERRα mRNA was significantly increased during the late stage of osteogenic differentiation of hPDLSCs. Moreover, transfection of recombinant lentiviral-mediated miRNA targeting ERRα significantly suppressed ALP activity, mineralization capacity, and the mRNA expression of osteogenesis-related genes (ALP, OCN, RUNX2 and OPN) in hPDLSCs. Our results indicate that ERRα may promote the osteogenic differentiation of hPDLSCs in vitro.

Background Basigin, which has four isoforms, has been demonstrated to be involved in progression of various human cancers. The aim of this study was to examine the prognostic value of basigin-2 protein expression in epithelial ovarian cancer. Furthermore, the function of basigin-2 in ovarian cancer was further investigated in cell culture models. Methods Immunohistochemistry staining was performed to investigate basigin-2 expression in a total of 146 ovarian tissue specimens. Kaplan Meier analysis and Cox proportional hazards model were applied to assess the relationship between basigin-2 and progression-free survival (PFS) and overall survival (OS). Real-time PCR, RT-PCR and western blot were used to explore basigin-2, basigin-3 and basigin-4 expression in ovarian cancer cell lines and tissues. To evaluate possible contributions of basigin-2 to MMP secretion and cell migration and invasion, the overexpression vectors pcDNA3.1-basigin-2 and basigin-2 siRNA were transfected into HO-8910 and HO-8910 PM cells respectively. Results High basigin-2 expression was associated with lymph-vascular space involvement, lymph node metastasis and poor prognosis of epithelial ovarian cancer. Multivariate analyses indicated that basigin-2 positivity was an independent prognostic factor for PFS ( P = 0.006) and OS ( P = 0.019), respectively. Overexpression of basigin-2 increased the secretion of MMP-2/9 and cancer cell migration and invasion of HO-8910 cells, whereas knockdown of basigin-2 reduced active MMP-2/9 production, migration and invasion of HO-8910 PM cells. Conclusions The expression of basigin-2 might be an independent prognostic marker and basigin-2 inhibition would be a potential strategy for epithelial ovarian cancer patients, especially in inhibiting and preventing cancer cell invasion and metastasis.

Spinal cord injury (SCI) is conventionally considered as an irreversible condition with damaged gray matter and myelinated fiber tracts in white matter. This condition also results in limited functional recovery and life-threatening secondary complications. Regenerative medicine is a rapidly growing field that bears great promise to replace or repair the damaged neural system. A wide range of cell types have been investigated. Positive results have been demonstrated through multiple approaches, i.e., transplanting and/or migrating stem cells to the injury site, suppressing the activity of molecules that inhibit axon regrowth and remyelination, or a combination of the methods. However, there are still numerous issues to be addressed before establishing it as a clinical routine. In the current article, the recent technique advancements in regenerative medicine are reviewed with an emphasis on relevant patents awarded between 2007 and 2011. Keywords: Patent, regenerative medicine, spinal cord, stem cell, Stem Cell Therapy, SPINAL CORD INJURY, Glial Scar Associated Inhibitors, Myelin Associated Inhibitors, Embryonic Stem Cells, Neural Stem Cells, Induced Pluripotent Stem Cells

Background: The transcription factor Forkhead box M1 (FoxM1) is known to play an important role in the development and progression of many malignancies including lung cancer. However, the relationship of FoxM1 expression and the clinical response to chemotherapy and prognosis in non-small cell lung cancer (NSCLC) remains unknown.Methods: Total 162 NSCLC (stages IIIB and IV) patients who had tumor specimens available before treatment were assessed for FoxM1 expression using immunohistochemistry. Clinical significance was analyzed by Kaplan-Meier curves, log-rank test and multivariate Cox regression analysis. Sensitivities to cisplatin were detected by the MTT assay and drug-resistance related genes were analyzed by real-time PCR and western blot between DDP-sensitive A549 and the corresponding DDP-resistant cell subline (A549/DDP). Furthermore, small interfering RNA (siRNA) targeting FoxM1 was transfected into A549 and A549/DDP cell lines in vitro and migration and invasion were examined separately by Transwell chamber assay.Results: Patients with FoxM1 expression had a significantly lower response rate (P = 0.009) and poor progression-free survival (PFS, P = 0.002) and overall survival (OS, P = 0.007) than those without FoxM1 expression. Multivariate analyses indicated that FoxM1 positivity was an independent prognostic factor for PFS (P = 0.006) and OS (P = 0.021), respectively. Moreover, the expression of FoxM1 was significantly higher in A549/DDP cell subline than in A549 cells at both mRNA and protein levels. The FoxM1 inhibitor thiostrepton also showed efficacy in causing cell death and proliferative arrest in the cisplatin-resistant cells through the downregulation of FoxM1 expression. Knockdown of FoxM1 by siRNA suppressed cell migration and invasion in A549 and A549/DDP cells. Cisplatin resistance in A549/DDP cells could be partially reversed through siRNA-mediated FoxM1 inhibition.Conclusions: The expression of FoxM1 might be an independent prognostic marker for advanced NSCLC patients and FoxM1 inhibition would be a potential strategy for chemosensitization of NSCLC cells. (c) 2012 Elsevier Ireland Ltd. All rights reserved.

L-type voltage-dependent Ca(2+) channels (VDCC(L)) play an important role in the maintenance of intracellular calcium homeostasis, and influence multiple cellular processes. They have been confirmed to contribute to the functional activities of osteoblasts. Recently, VDCC(L) expression was reported in mesenchymal stem cells (MSCs), but the role of VDCC(L) in MSCs is still undetermined. The aim of this study was to determine whether VDCC(L) may be regarded as a new regulator in the proliferation and osteogenic differentiation of rat MSC (rMSCs). In this study, we examined functional Ca(2+) currents (I(Ca)) and mRNA expression of VDCC(L) in rMSCs, and then suppressed VDCC(L) using nifedipine (Nif), a VDCC(L) blocker, to investigate its role in rMSCs. The proliferation and osteogenic differentiation of MSCs were analyzed by MTT, flow cytometry, alkaline phosphatase (ALP), Alizarin Red S staining, RT-PCR, and real-time PCR assays. We found that Nif exerts antiproliferative and apoptosis-inducing effects on rMSCs. ALP activity and mineralized nodules were significantly decreased after Nif treatment. Moreover, the mRNA levels of the osteogenic markers, osteocalcin (OCN), bone sialoprotein (BSP), and runt-related transcription factor 2 (Runx2), were also down-regulated. In addition, we transfected α1C-siRNA into the cells to further confirm the role of VDCC(L) in rMSCs, and a similar effect on osteogenesis was found. These results suggest that VDCC(L) plays a crucial role in the proliferation and osteogenic differentiation of rMSCs.

Hormone deficiency has been recognized as a risk factor for periodontal disease in postmenopausal women. However, the anabolic effects of progesterone on human periodontal ligament cells (hPDLCs) are still unclear. Therefore, the objective of this study was to detect the expression of progesterone receptor (PgR) in hPDLCs and investigate the bone-sparing effects of progesterone. We detected PgR expression in hPDLCs by reverse transcriptase-polymerase chain reaction and immunocytochemistry. After progesterone stimulation, the percentage of hPDLCs entering the S + G2M phase of the cell cycle increased significantly, accompanied by an increased cell growth curve. In both basic culture medium and osteogenic medium, progesterone activated alkaline phosphatase-positive cells and alizarin red-positive nodules. Moreover, mineralization-related markers were up-regulated by progesterone in both time-dependent and dose-dependent manners. In contrast, these effects of progesterone were blocked by the PgR antagonist (RU486). Our results demonstrated that the PgR is expressed in hPDLCs at the gene and protein level, and that progesterone can stimulate the proliferation and differentiation of the hPDLCs. These findings suggest that progesterone may play a significant role in osteoblastic function of hPDLCs and may influence the maintenance of alveolar bone mass.

Objective:To investigate the feasibility of inducing the differentiation of rat bone marrow mesenchymal stem cells(BMSCs)under the influence of periodontal ligament fibroblasts(PDLFs)in vitro. Method:The non-contact co-culture system was established by cell culture inserts. The rat BMSCs were co-cultured with the rat PDLFs for 14 days. The expression of PDLFs protein markers(ALP,OCN,COL Ⅲ)were measured at different time courses. Result:The rat bone mesenchymal stem cells acquire the characteristics of the periodontal ligament fibroblasts .There are no differences of all markers between co-cultured BMSCs and PDLFs cultured individually,and observed changes between co-cultured BMSCs and BM-SCs cultured individually. Conclusion:The factors secreted by PDLFs may induce the rat BMSCs to obtain periodontal ligament-like marker expression.

Objective:To observe and reveal the effects of 17-β estradiol on modulating in vitro osteogenic differentiation and proliferation of adipose tissue-derived mesenchymal stem cells(ADSCs)isolated from 18-month-old female Sprague Dawleg rats.Methods:17-β estradiol was investigated for modulating in vitro osteogenic differentiation of ADSCs of the third generation isolated from a 18-month-old female rat.The capacity of 17-β estradiol on cell osteogenic differentiation was observed by alizarin red staining,RT-PCR and detection of morphological and molecular biology.Results:17-β estradiol could effectively enhance ADSCs' proliferation.After ADSCs' exposure to osteogenic differentiation medium supplemented with 17-β estradiol,the expression of osteogenic markers was significantly improved.Conclusion:17-β estradiol can modulate the differentiation and proliferation,and potentially improve the efficiency of ADSCs in stem cell-based tissue engineering and regeneration.However,further study is needed to verify the regulatory functions of 17-β estradiol on ADSCs' differentiation.

Objective: To construct a recombinant CypA eukaryocyte expression vector,and to establish a high expression CypA hepatocellular carcinoma(HCC) cell model for further studying the function and mechanism of CypA in HCC.Methods: The full gene fragment of human CypA was cloned,and subcloned into the eukaryotic gene expression vector pcDNA3.1(+).Then the recombinant vector was transfected into the FHCC98 cells,a hepatocellular carcinoma cell line,which were cultured selectively with 800 μg/ml G4l8 to get a stable CypA expression cell line.The expression of CypA was examined by immunobloting and the proliferation rate was determined by MTT.Results: The eukaryotic expression vector pcDNA3.1-CypA was examined by two restrictive endonuclease digesting and sequence measuring,and both results were correct.The immunobloting results showed that the stable transfected pcDNA3.1-CypA-FHCC98 cell expressed higher level of CypA than the stable transfected pcDNA3.1-FHCC98 cell or untransfected FHCC98 cell.Comparing to control,pcDNA3.1-CypA-FHCC98 cell had a higher proliferation rate.Conclusion: The recombinant eukaryocyte expression vector and its stable expression HCC cell line were established.Over expression of CypA in FHCC98 cell could stimulate its proliferation.

Objective To investigate the effects of L-type calcium channels on rat bone mesenchymal stem cells. Methods Marrow mesenchymal stem cells were separated and cultured in vitro. RT-PCR was used to detect the mRNA expression of alC,CCHL2a .The L-type calcium channel blocker,nifedipin,was used to block the L-type calcium channels. Methyl thiazol tetrazolium assay and flow cytometry were applied to measure the cellular proliferation and cellular cycle. Results A high expression of mRNA in alC was detected by RT-PCR.The cells in treatment group proliferated more slowly than those in the control group after blocking L-type calcium channels. There was significant difference of two groups after continuous culture for 3 days. After 72 hours' stimulation,the percentage of phase S,S+G2M was decreased,compared with the control group,and the percentage of phase GO/G1 increased,compared with the control group .There were significant differences between the two groups. Conclutions L-type calcium channels may be involved in the proliferation of marrow mesenchymal stem cells.