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Objective In order to meet the needs of maxillofacical bone defect repair, the aim of this study was to synthesize graphene oxide(GO) modified three-dimensional conneted nano- zirconia(ZrO2) bone tissue engineering scaffold and evaluate its surface morphology, compressive strength and cytocompatibility. Methods GO was synthesized by a modified Hummers method and then was testified by scanning electron microscope, transmission electron microscopy and fourier transform infrared spectroscopy. ZrO2 scaffold was modified by different concentrations(0.5,1.0,1.5mg/mL) of GO dispersion via a silane-mediated method. The composite scaffold with uniform GO coating was chosen for compressive strength test and co-cultured with human dental pulp stem cells(hDPSCs). Actin staining was used to observe the growth of the cells on the scaffold, and MTS was used to detect the cell activity. Results The characterization results showed that, under scanning electron microscope, the GO was flaky and the surface morphology of folds could be seen. Part of the GO layer folds up at the edge. Under transmission electron microscopy, the GO was clearly observed to have a gossylike, translucent and slightly wrinkled lamellar structure. The crystal structure in this area in the high-resolution filter image showed a six-member ring structure like graphite. Under high power electron microscope, the 1.0mg/ml GO-ZrO2 scaffold could be seen to deposit a thin layer of GO at the crack of the scaffold skeleton, connecting the two ends of the crack, and lamellar GO with folds could be observed on the surface of ceramic particles. The comparison of mechanical properties showed that the compression strength of GO-ZrO2 scaffold was sgnificantly increased compared with that of ZrO2 scaffold[（1.292±0.087）vs（1.031±0.076）, P<0.05]. Compared with the simple ZrO2 scaffold, the GO-ZrO2 scaffold showed more dense extension and adhesion to the surface of scaffolds, showing more active cell proliferation. The cell viability test showed that the viability of hDPSCs was significantly improved on GO-ZrO2 scaffold after 1, 3 and 5 days of proliferation compared with the simple ZrO2 scaffold(P<0.05). Conclusion The ZrO2 scaffold modified by GO improved compressive strength, promoted the early proliferation of hDPSCs with good cytocompatibility.
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Acute liver failure (ALF) is a severe clinical syndrome with rapid progression, poor prognosis and high mortality. Liver transplantation is the most effective option for the treatment of ALF, but only a few ALF patients can receive the donor liver in time due to the insufficient supply of the organ. Stem cell-related techniques, including stem cell transplantation, bioartificial liver, etc., with their roles of detoxication, synthesis, metastasis and secretion, have brought some hope for the solution of this problem. This article presents an overview of the three aspects as follows: mechanisms and clinical translation of stem cell transplantation for ALF, establishment of the coculture system for mesenchymal stem cells with porcine hepatocytes and clinical translation of the coculture-based bioartificial liver, and establishment of human-induced hepatocytes and clinical translation of bioartificial liver based on human-induced seed cells.
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Hormone therapy (androgen deprivation therapy, ADT) is the first line treatment for advanced prostate cancer. Gonadotropin-releasing hormone (GnRH) agonist is commonly used for ADT, with the risk of flare-up effect at the first dose and testosterone fluctuation, which needs combination of anti-androgen drugs to alleviate the deterioration of the clinical condition. GnRH antagonists directly bind to GnRH receptor, blocking its activity, and they can be used in prostate cancer treatment, without testosterone flare-up effect. This review focused on functional mechanisms of GnRH agonists and antagonists, and history and clinical application of GnRH antagonists, so as to evaluate the clinical safety and effectiveness of GnRH antagonists and to discuss their clinical value and future prospects.
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<p><b>BACKGROUND</b>Mesenchymal stem cells (MSCs) transplantation has been proven to have therapeutic potential for acute liver failure (ALF). However, the mechanism remains controversial. Recently, modulation of inflammation by MSCs has been regarded as a crucial mechanism. The aim of the present study was to explore the soluble cytokines secreted by MSCs and their therapeutic effects in ALF.</p><p><b>METHODS</b>MSCs isolated from Sprague-Dawley rats were identified by fluorescence-activated cell sorting analysis. Conditioned medium derived from MSCs (MSCs-CM) was collected and analyzed by a cytokine microarray. MSCs and MSCs-CM were transplanted into rats with D-galactosamine-induced ALF. Liver function, survival rate, histology, and inflammatory factors were determined. Exogenous recombinant rat interleukin (IL)-10, anti-rat IL-10 antibody, and AG490 (signal transducer and activator of transcription 3 [STAT3] signaling pathway inhibitor) were administered to explore the therapeutic mechanism of MSCs-CM. Statistical analysis was performed with SPSS version 19.0, and all data were analyzed by the independent-sample t-test.</p><p><b>RESULTS</b>There are statistical differences of the survival curve between ALF+MSCs group and ALF+Dulbecco's modified Eagle's medium (DMEM) group, as well as ALF+MSCs-CM group and ALF+DMEM group (all P < 0.05). Serum alanine aminotransferase (ALT) level in the ALF+MSCs and ALF+MSCs-CM groups was lower than that in the ALF+DMEM group (865.53±52.80 vs. 1709.75±372.12 U/L and 964.72±414.59 vs. 1709.75±372.12 U/L, respectively, all P < 0.05); meanwhile, serum aspartate aminotransferase (AST) level in the ALF+MSCs and ALF+MSCs-CM groups was lower than that in the ALF+DMEM group (2440.83±511.94 vs. 4234.35±807.30 U/L and 2739.83±587.33 vs. 4234.35±807.30 U/L, respectively, all P < 0.05). Furthermore, MSCs or MSCs-CM treatment significantly reduced serum interferon-γ (IFN-γ), IL-1β, IL-6 levels and increased serum IL-10 level compared with DMEM (all P < 0.05). Proteome profile analysis of MSCs-CM indicated the presence of anti-inflammatory factors and IL-10 was the most distinct. Blocking of IL-10 confirmed the therapeutic significance of this cytokine. Phosphorylated STAT3 was upregulated after IL-10 infusion and inhibition of STAT3 by AG490 reversed the therapeutic effect of IL-10.</p><p><b>CONCLUSIONS</b>The factors released by MSCs, especially IL-10, have the potential for therapeutic recovery of ALF, and the STAT3 signaling pathway may mediate the anti-inflammatory effect of IL-10.</p>
Subject(s)
Animals , Male , Rats , Interleukin-10 , Physiology , Liver , Pathology , Liver Failure, Acute , Pathology , Therapeutics , Mesenchymal Stem Cell Transplantation , Rats, Sprague-Dawley , STAT3 Transcription Factor , Physiology , Signal Transduction , PhysiologyABSTRACT
OBJECTIVE@#To explore the effect and molecular mechanism of SPHK1 in the invasion and metastasis process of non-small-cell lung cancer cells (A549).@*METHODS@#Recombinant retrovirus was used to mediate the production of A549/vector, A549/SPHK1, A549/scramble, and A549/SPHKl/RNAi that stably expressed or silenced SPHK1. The invasion and migration capacities of A549 cells overexpressing or silencing SPHK1 were determined using Transwell invasion assay and scratch wound repair experiment. The protein and mRNA expression levels of E-cadherin, fibronectin, vimentin in A549/vector, A549/SPHK1, A549/scramble, A549/SPHK1/RNAi were detected with Western blot (WB) and quantitative PCR (QPCR) methods, respectively.@*RESULTS@#Transwell invasion assay and scratch wound repair experiments showed that over-expression of SPHK1 obviously enhanced the invasion and migration capacities of A549 cells. WB and QPCR detection results showed that, the expression of E-cadherin (a molecular marker of epithelial cells) and fibronectin, vimentin (molecular markers of mesenchymal cells) in A549 cells was upregulated after overexpression of SPHK1; while SPHK1 silencing significantly reduced the invasion and metastasis capacities of A549 cells, upregulated the expression of molecular marker of epithelial cells, and downregulated the expression of molecular marker of mesenchymal cells.@*CONCLUSIONS@#SPHK1 promotes epithelial mesenchymal transition of non-small-cell lung cancer cells and affects the invasion and metastasis capacities of these cells.
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Objective: To explore the effect and molecular mechanism of SPHK1 in the invasion and metastasis process of non-small-cell lung cancer cells (A549). Methods: Recombinant retrovirus was used to mediate the production of A549/vector, A549/SPHK1, A549/scramble, and A549/SPHKl/RNAi that stably expressed or silenced SPHK1. The invasion and migration capacities of A549 cells overexpressing or silencing SPHK1 were determined using Transwell invasion assay and scratch wound repair experiment. The protein and mRNA expression levels of E-cadherin, fibronectin, vimentin in A549/vector, A549/SPHK1, A549/scramble, A549/SPHK1/RNAi were detected with Western blot (WB) and quantitative PCR (QPCR) methods, respectively. Results: Transwell invasion assay and scratch wound repair experiments showed that over-expression of SPHK1 obviously enhanced the invasion and migration capacities of A549 cells. WB and QPCR detection results showed that, the expression of E-cadherin (a molecular marker of epithelial cells) and fibronectin, vimentin (molecular markers of mesenchymal cells) in A549 cells was upregulated after overexpression of SPHK1; while SPHK1 silencing significantly reduced the invasion and metastasis capacities of A549 cells, upregulated the expression of molecular marker of epithelial cells, and downregulated the expression of molecular marker of mesenchymal cells. Conclusions: SPHK1 promotes epithelial mesenchymal transition of non-small-cell lung cancer cells and affects the invasion and metastasis capacities of these cells.
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<p><b>OBJECTIVE</b>To evaluate whether a combination therapy using allogeneic mesenchymal stem cell (MSC) transplantation and interleukin-1 receptor antagonist (IL-1Ra) chitosan nanoparticles is more robust than MSC transplantation alone for treating acute liver failure and to investigate the mechanisms of the improved therapeutic effect using a swine model system.</p><p><b>METHODS</b>IL-1Ra-loaded nanoparticles were made of lactosylated chitosan-FITC using the electrostatic spray method and analyzed by enzyme-linked immunosorbent assay. The active live targeting of these nanopaticles were investigated by fluorescence microscope and flow cytometer(FCM). The combined therapy was given and the Detailed Steps as follows: Chinese experimental mini swine were given D-galactosamine to build models of acute liver failure. Thirty-four pigs were randomly divided into five groups. In the control group(A),the normal saline was injected into liver via portal veins after 24 h; in IL-1Ra group(B), IL-1Ra was injected via ear veins 6 h before normal saline; In the MSCs transplantation group (C), 8 * 107 MSCs were injected into liver via portal veins after 24 h; IL-1Ra together with MSCs transplantation group(D) and nanopaticles group(E) as follows: on the one hand, 8 * 107 MSCs were injected into liver via portal veins after 24 h, on the other hand, rhIL-1Ra in group C or IL-1Ra chitosan nanopaticles in group D was injected via ear veins respectively at 6 h before. Liver function, serum inflammation and pathological changes were measured. The fate of MSCs was also observed.</p><p><b>RESULTS</b>The profiles in vitro shown that there was a steady-state release after a fast linear release; The live targeting of the lactosylated chitosan-based nanoparticles was achieved by ligand-receptor specificity; The biochemical assay, the serum inflammation lever and pathological changes of the nanopaticles group were all greatly different from the other groups, the hepatocytes grow rate was significantly improved after 1 week; The liver engraftment was very low in group C and D with significantly higher numbers found in nanopaticles group, but the differentiation of MSCs after 2 weeks relatively rare; western blot showed that there was more HGF and VEGF secreted in nanopaticles group.</p><p><b>CONCLUSION</b>IL-lRa-loaded lactosylated chitosan-based nanopaticles have significant liver targeting abilities and slow release characteristics in PBS solution. The combined therapy showed great synergistic effects through suppression of liver inflammation and paracrine.</p>
Subject(s)
Chitosan , Liver Failure, Acute , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Cell Biology , NanoparticlesABSTRACT
<p><b>OBJECTIVE</b>To investigate the potential transmissibility of porcine endogenous retrovirus (PERV) from a newly-developed porcine hepatocyte bioartificial liver (BAL) system prior to human clinical trial by using a live canine model.</p><p><b>METHODS</b>Five normal beagles were treated with the new BAL support system for six hours. Samples of plasma from the BAL system and whole blood from the beagles were collected at regular intervals over the six month study period. DNA and RNA were isolated from both the peripheral blood mononuclear cells (PBMCs) and plasma for evaluation by polymerase chain reaction (PCR) and reverse transcription (RT)-PCR, respectively, to detect PERV and the Sus scrofa cytochrome B normalization standard. In addition, RT activity and the in vitro infectivity of the plasma were detected in HEK293 cells.</p><p><b>RESULTS</b>All five beagles remained in stable physical health throughout the treatment and survived until the end of the study. PERV RNA-positivity and RT activity were only detected in the plasma samples from the 3rd BAL treatment cycle. All other samples, including PBMCs and plasma, were negative for PERV RNA, PERV DNA, and RT activity. In addition, none of the sera samples showed in vitro infectivity.</p><p><b>CONCLUSION</b>Application of our BAL system does not lead to PERV transmission.</p>
Subject(s)
Animals , Dogs , Humans , Cell Line , Endogenous Retroviruses , HEK293 Cells , Hepatocytes , Virology , Leukocytes, Mononuclear , Virology , Liver, Artificial , Models, Animal , SwineABSTRACT
<p><b>OBJECTIVE</b>To evaluate the therapeutic efficacy and safety of liver transplantation for patients with cholangiocarcinoma.</p><p><b>METHODS</b>According to the requirements of Cochrane systematic review, a thorough literature search was performed in Pubmed/Medline, Embase and Cochrane Central Register electronic databases ranged between 1995 and 2009 in terms of the key words "liver transplantation", and "cholangiocarcinoma" or "cholangiocellular carcinoma" or "bile duct cancer". And restricted the articles published in the English language. Two reviewers independently screened the studies for eligibility, evaluated the quality and extracted the data from the eligible studies with confirmation by cross-checking. Data were processed for a meta-analysis by Stata 10 software with 1-, 3-, 5-year survival rates and incidence of complications.</p><p><b>RESULTS</b>A total of 14 clinical trials containing 605 patients were finally enrolled in this study. The overall 1-, 3-, 5-year pooled survival rates were 73% (95%CI: 0.65 - 0.80), 42% (95%CI: 0.33 - 0.51) and 39% (95%CI: 0.28 - 0.51), respectively. Of note, preoperative adjuvant therapies (OLT-PAT group) rendered the transplanted individuals comparably favorable outcomes with 1-, 3-, 5-year pooled survival rates of 83% (95%CI: 0.57 - 0.98), 57% (95%CI: 0.18 - 0.92) and 65% (95%CI: 0.40 - 0.87), respectively. In addition, the overall pooled incidence of complications was 62% (95%CI: 0.44 - 0.78), among which that of OLT-PAT group (58%, 95%CI: 0.20 - 0.92) was relatively acceptable compared to those of liver transplantation alone (61%, 95%CI: 0.33 - 0.85) and liver transplantation with extended bile duct resection (78%, 95%CI: 0.55 - 0.94).</p><p><b>CONCLUSIONS</b>In comparison to curative resection of cholangiocarcinoma with the 5-year survival rate reported from 20% to 40%, the role of liver transplantation alone is so limited, but neoadjuvant radiochemotherapy combined with liver transplantation can bring better short- and long-term prognosis.</p>
Subject(s)
Humans , Bile Duct Neoplasms , General Surgery , Cholangiocarcinoma , General Surgery , Clinical Trials as Topic , Liver Transplantation , Treatment OutcomeABSTRACT
<p><b>OBJECTIVE</b>To evaluate the efficacy of newly developed multi-layer flat-plate bioartificial liver in treatment of canines with acute liver failure.</p><p><b>METHODS</b>Porcine hepatocytes and bone marrow mesenchymal stem cells were cocultured in newly developed multi-layer flat-plate bioreactor. Acute liver failure in canine models was induced by D-galactosamine administration.Sixteen canine models were divided into two groups: treatment group (n = 8) and control group (n = 8). Biochemical parameters were determined for 7 days after treatment and liver specimens were collected for histological analysis.</p><p><b>RESULTS</b>Hepatic encephalopathy and general conditions were significantly improved in the treatment group, but no changes in the control group. Alanine aminotransferase was significantly decreased from (1512 ± 183) U/L to (86 ± 25) U/L in the treatment group, aspartate aminotransferase was significantly decreased from (1472 ± 365) U/L to (46 ± 11) U/L, lactate dehydrogenase was significantly decreased from (463 ± 76) U/L to (312 ± 84) U/L, total bilirubin was significantly decreased from (28.8 ± 6.2) µmol/L to (12.5 ± 3.6) µmol/L, ammonia was significantly decreased from (56 ± 15) µmol/L to (34 ± 10) µmol/L, and prothrombin time were significantly decreased in the treatment group but increased in the control group, albumin was improved in the treatment group but decreased in the control group. There were 5 canines survived in the treatment group but only 3 in the control group. But there was no difference on survival rates between the two group (P = 0.294).</p><p><b>CONCLUSION</b>The application of newly developed multi-layer flat-plate bioartificial liver system was effective in the treatment of canines with acute liver failure.</p>
Subject(s)
Animals , Dogs , Bioreactors , Bone Marrow Cells , Cell Biology , Coculture Techniques , Disease Models, Animal , Hepatocytes , Cell Biology , Liver Failure, Acute , Therapeutics , Liver, ArtificialABSTRACT
<p><b>OBJECTIVE</b>To investigate the expression and distribution of extracellular matrix (ECM) in the co-culture of porcine primary hepatocytes and bone marrow mesenchymal stem cells (MSCs) in vitro.</p><p><b>METHODS</b>Mononuclear cells were isolated from bone marrow of swine by density gradient centrifugation. MSCs of passage 3 and primary hepatocytes harvested by a two-step in situ collagenase perfusion technique were co-cultured, and the morphological and functional changes of heterotypic interactions were characterized. Immunocytochemical analysis was performed to monitor the expression and distribution of ECM.</p><p><b>RESULTS</b>The purity of the third passage MSCs and primary hepatocytes was more than 90% and 99%, respectively. More than 95% of the hepatocytes were viable. Compared to hepatocytes culture, co-culture with MSCs significantly enhanced hepatic function: including albumin secretion and urea synthesis (P < 0.01). The best hepatic function level was achieved on day 2 and gradually decreased in the following co-culture days. Immunocytochemical staining suggested that higher amounts of naturally occurring ECM proteins including fibronectin, laminin, and several kinds of collagens were produced in co-culture group compared to hepatocyte homo-culture (P < 0.01). RNAi experiments verified that there was a correlation between ECM and hepatic functions.</p><p><b>CONCLUSION</b>ECM may indeed play a key role in the up-regulation of hepatocyte functions in MSC/hepatocytes co-culture.</p>
Subject(s)
Animals , Female , Albumins , Metabolism , Bone Marrow Cells , Cell Biology , Cell Separation , Methods , Cell Survival , Cells, Cultured , Coculture Techniques , Extracellular Matrix , Metabolism , Hepatocytes , Cell Biology , Metabolism , Immunohistochemistry , Mesenchymal Stem Cells , Cell Biology , Metabolism , Microscopy, Confocal , Microscopy, Electron, Scanning , Swine , Urea , MetabolismABSTRACT
Objective To test the hypothese that extreme low estrogen level will lead to elevation of blood pressure.Methods Thirty-two adult male SD rats were submitted to following approaches:control(n=8),orchi- ectomy(n=8),orchieetomy plus letrozole,an inhibitor of estrogen synthese [5 mg/(kg?d)by gavage,n=8], letrozole treatment in intact testis rats(n=8).Four weeks after treatment,SBP,serum testosterone(T),estradiol (E_2),nitric oxide(NO),endotheline(ET),thromboxane(TXB_2),prostacyelin(6-Keto-PGF_(1?)),atrial naturetic pep- tide(ANP),C type natriuretic peptide(CNP)were determined.Results SBP was increased significantly after or- chiectomy compared with control rats(orehiectomy:171?17 vs control:156?14 mmHg,P