Hepatopoietin Cn suppresses apoptosis of human hepatocellular carcinoma cells by up-regulating myeloid cell leukemia-1

AIM:To investigate the role of hepatopoietin Cn(HPPCn) in apoptosis of hepatocellular carcinoma(HCC)cells and its mechanism. METHODS:Two human HCC cell lines,SMMC7721 and HepG2,were used in this study.Immunostaining, Western blotting and enzyme linked immunosorbent assay were conducted to identify the expression of HPPCn and the existence of an autocrine loop of HPPCn/ HPPCn receptor in SMMC7721 and HepG2.Apoptotic cells were detected using fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide.RESULTS:The HPPCn was highly expressed in human HCC cells and secreted into culture medium(CM). FITC-labeled recombinant human protein(rhHPPCn) could specifically bind to its receptor on HepaG2 cells. Treatment with 400 ng/mL rhHPPCn dramatically increased the viability of HCC-derived cells from 48.1% and 36.9%to 85.6%and 88.4%,respectively(P< 0.05).HPPCn silenced by small-interfering RNA reduced the expression and secretion of HPPCn and increased the apoptosis induced by trichostatin A.Additionally, HPPCn could up-regulate the expression of myeloid cell leukemia-1(Mcl-1)in HCC cells via mitogen-activated protein kinase(MAPK)and sphingosine kinase-1. CONCLUSION:HPPCn is a novel hepatic growth factor that can be secreted to CM and suppresses apoptosis of HCC cells by up-regulating Mcl-1 expression.

Maternal inappropriate calcium intake aggravates dietary-induced obesity in male offspring by affecting the differentiation potential of mesenchymal stem cells

BACKGROUND The effects of inappropriate dietary calcium intake in early life on later obesity have not been fully elucidated.AIM To raise the mechanism of maternal calcium intake on the multi-differentiation potential of mesenchymal stem cells among their male offspring.METHODS Four-week-old female C57BL/6N mice were fed by deficient,low,normal and excessive calcium reproductive diets throughout pregnancy and lactation.Bone MSCs(BMSCs)were obtained from 7-day-old male offspring to measure the adipogenic differentiation potential by the Wnt/β-catenin signaling pathway.The other weaning male pups were fed a high-fat diet for 16 wk,along with normalfat diet as the control.Then the serum was collected for the measurement of biochemical indicators.Meanwhile,the adipose tissues were excised to analyze the adipocyte sizes and inflammatory infiltration.And the target gene expressions on the adipogenic differentiation and Wnt/β-catenin signaling pathway in the adipose tissues and BMSCs were determined by real-time reverse transcription polymerase chain reaction.RESULTS Compared with the control group,maternal deficient,low and excessive calcium intake during pregnancy and lactation aggravated dietary-induced obesity,with larger adipocytes,more serious inflammatory infiltration and higher serum metabolism indicators by interfering with higher expressions of adipogenic differentiation(PPARγ,C/EBPα,Fabp4,LPL,Adiponectin,Resistin and/or Leptin)among their male offspring(P<0.05).And there were significantly different expression of similar specific genes in the BMSCs to successfully polarize adipogenic differentiation and suppress osteogenic differentiation in vivo and in vitro,respectively(P<0.05).Meanwhile,it was accompanied by more significant disorders on the expressions of Wnt/β-catenin signaling pathway both in BMSCs and adulthood adipose tissues among the offspring from maternal inappropriate dietary calcium intake groups.CONCLUSION Early-life abnormal dietary calcium intake might program the adipogenic differentiation potential of BMSCs from male offspring,with significant expressions on the Wnt/β-catenin signaling pathway to aggravate high-fat-diet-induced obesity in adulthood.

Keratinocyte growth factor gene therapy ameliorates ulcerative colitis in rats

AIM:To investigate the effect of keratinocyte growth factor(KGF) gene therapy in acetic acid-induced ulcerative colitis in rat model.METHODS:The colitis of Sprague-Dawley rats was induced by intrarectal infusion of 1 mL 5%(v/v) acetic acid.Twenty-four hours after exposed to acetic acid,rats were divided into three experimental groups:control group,attenuated Salmonella typhimurium Ty21a strain(SP) group and SP strain carrying human KGF gene(SPK) group,and they were separately administered orally with 10% NaHCO3,SP or SPK.Animals were sacrificed and colonic tissues were harvested respectively on day 3,5,7 and 10 after administration.Weights of rats,colonic weight/length ratio and stool score were evaluated.Histological changes of colonic tissues were examined by hematoxylin and eosin(HE) staining method.The expression of KGF,KGF receptor(KGFR) and TNF-α were measured either by enzyme-linked immunosorbent assay or Western blotting.Immunohistochemistry was used to detect the cellular localization of KGFR and Ki67.In addition,superoxide dismutase(SOD) activity and malondialdehyde(MDA) contents in the homogenate were measured.RESULTS:Body weight and colonic weight/length ratio were declined in SPK group compared with SP and control groups(body weight:272.78 ± 17.92 g vs 243.72 ± 14.02 g and 240.68 ± 12.63 g,P < 0.01;colonic weight/length ratio:115.76 ± 7.47 vs 150.32 ± 5.99 and 153.67 ± 5.50 mg/cm,P < 0.01).Moreover,pathological changes of damaged colon were improved in SPK group as well.After administration of SPK strain,KGF expression increased markedly from the 3rd d,and remained at a high level till the 10th d.Furthermore,KGFR expression and Ki67 expression elevated,whereas TNF-α expression was inhibited in SPK group.In the group administered with SPK,SOD activity increased significantly(d 5:26.18 ± 5.84 vs 18.12 ± 3.30 and 18.79 ± 4.74 U/mg,P < 0.01;d 7:35.48 ± 3.35 vs 22.57 ± 3.44 and 21.69 ± 3.94 U/mg,P < 0.01;d 10:46.10 ± 6.23 vs 25.35 ± 4.76 and 27.82 ± 6.42 U/mg,P < 0.01) and MDA contents decreased accordingly(d 7:7.40 ± 0.88 vs 9.81 ± 1.21 and 10.45 ± 1.40 nmol/mg,P < 0.01;d 10:4.36 ± 0.62 vs 8.41 ± 0.92 and 8.71 ± 1.27 nmol/mg,P < 0.01),compared with SP and control groups.CONCLUSION:KGF gene therapy mediated by attenuated Salmonella ameliorates ulcerative colitis induced by acetic acids,and it may be a safe and effective treatment for ulcerative colitis.

HGF percutaneous endocardial injection induces cardiomyocyte proliferation and rescues cardiac function in pigs

Objective：To investigate the effect of cardiomyocyte proliferation induced by human hepatocyte growth factor（HGF）in pigs with chronic myocardial infarction（CMI）.Methods：A steerable,deflectable 7F catheter incorporating a 27-guage needle was advanced percutaneously to the left ventricular myocardium of 18 pigs with CMI.Pigs were randomized（1：1：1）to receive adenoviral vector HGF（total dose,1×10^10 genome copies）,which was administered as five injections into the infarcted myocardium（total,1.0 mL）,or saline,or Ad-null（control groups）.Injections were guided by Ensite NavX left ventricular electroanatomical mapping.HGF and cyclin proteins were detected by western blot and immunoprecipitation analysis.Histological and immunohistochemical analysis determined proliferating cardiomyocytes.Myocardial perfusion and cardiac function were estimated by Gated-Single Photon Emission Computed Tomography（G-SPECT）.Results：Western blot analyses showed that HGF were predominantly expressed in the infarct core and border in the myocardium of the infarcted heart.G-SPECT analysis indicated that the HGF group had better cardiac function and myocardial perfusion four weeks after the injection of Ad-HGF than before the injection of Ad-HGF.After treatment there were more proliferating cardiomyocytes in the HGF group compared to either of the control groups.Furthermore,the HGF group myocardial samples expressed higher levels of p-Akt,cyclin A,cyclin E,cyclin D1,cdk2,cdk4 than those in the control groups.Conclusion：The over-expression of HGF activates pro-survival pathways,induces cardiomyocyte proliferation,and improves the perfusion and function of the porcine CMI heart.

Easily-handled method to isolate mesenchymal stem cells from coagulated human bone marrow samples

AIM:To establish an easily-handled method to isolate mesenchymal stem cells(MSCs) from coagulated human bone marrow samples. METHODS: Thrombin was added to aliquots of seven heparinized human bone marrow samples to mimic marrow coagulation. The clots were untreated,treated with urokinase or mechanically cut into pieces before culture for MSCs. The un-coagulated samples and the clots were also stored at 4 ℃ for 8 or 16 h before the treatment. The numbers of colony-forming unit-fibroblast(CFU-F) in the different samples were determined. The adherent cells from different groups were passaged and their surface profile was analyzed with flow cytometry. Their capacities of in vitro osteogenesis and adipogenesis were observed after the cells were exposed to specific inductive agents.RESULTS: The average CFU-F number of urokinasetreated samples(16.85 ± 11.77/106) was comparable to that of un-coagulated control samples(20.22 ± 10.65/106,P = 0.293),which was significantly higher than those of mechanically-cut clots(6.5 ± 5.32/106,P < 0.01) and untreated clots(1.95 ± 1.86/106,P < 0.01). The CFU-F numbers decreased after samples were stored,but those of control and urokinase-treated clots remained higher than the other two groups. Consistently,the numbers of the attached cells at passage 0 were higher in control and urokinase-treated clots than those of mechanically-cut clots and untreated clots.The attached cells were fibroblast-like in morphology and homogenously positive for CD44,CD73 and CD90,and negative for CD31 and CD45. Also,they could be induced to differentiate into osteoblasts and adipocytes in vitro. CONCLUSION: Urokinase pretreatment is an optimal strategy to isolate MSCs from human bone marrow samples that are poorly aspirated and clotted.

Cross-Talk between Extracellular S1P/S1P2 and P42/44 MAPK in bcr/abl Positive Chronic Myeloid Leukemia Cells

Objective： BCR/ABL oncoprotein-expression is associated with uncontrolled cell growth. Sphingosine kinase 1 （SPK1） regulates the production of sphingosine 1-phosphate （S1P）, a key lipid signal molecular in cell proliferation and survival. The objective of this study was to elucidate the roles of S1P and its receptors in bcr/abl positive chronic myeloid leukemia （CML） cells. Methods： The expressions of SIP receptors： S1P1, S1P2 and S1P3 in CML cells were detected by RT-PCR. SPK1 expression, activity and extracellular S1P were determined in ECV304 and HL-60 cells which were transfected with bcr/abl gene. To elucidate the relationship between the BCR/ABL, ERK/MAPK （extracellular signal-regulated kinase/mitogen-activated protein kinase）, SPK/S 1P and S 1P/S 1 P2 signal pathways, bcr/abl positive CML cell line K562 was treated with STI571, PD98059, N,N-dimethyl sphingosine （DMS） and JTE-013. Results： Retrovirus-mediated overexpression of bcr/abl gene in ECV304 and HL-60 cells resulted in upregulation of the expression, activity of SPK1 and increase of the secretion of SIP, whereas treatment of STI571 and PD98059 decreased the BCR/ABL-induced S1P secretion. Treatment of DMS reduced S1P secretion and P42/44MAPK phosphorylation. S1P2-selective antagonist JTE-013 could also decrease P42/44MAPK phosphorylation. Conclusion： These results suggest that BCR/ABL up-regulates extracellular sphingosine 1-phosphate through sphingosine kinase 1 and there is cross-talk between SPK1/S1P/S1P2 and P42/44MAPK in bcr/abl positive CML cells.

Effect of human hepatocyte growth factor on promoting wound healing and preventing scar formation by adenovirus-mediated gene transfer

Objective To evaluate the effects of hepatocyte growth factor (HGF) on the prevention of scar formation and the promotion of wound healing by gene transfer Methods A total of 12 female New Zealand rabbits were used in this study Rabbits were anesthetized with an intravenous injection of sodium pentobarbital, and identical wounds were made over the ventral surface of each ear Five circular wounds, 7 mm in diameter, were created in each ear by excision through the skin to the underlying cartilage using sterile technique After the surgical procedures, 10 of the rabbits were randomly allocated to five groups, with 2 rabbits in each group: Ad HGF group 1, Ad HGF group 2, Ad HGF group 3, Ad GFP (a reporter gene) group and the solvent group Immediately after surgery, 6×10 7 pfu Ad HGF, 6×10 8 pfu Ad HGF, 6×10 9 pfu of Ad HGF, 6×10 9 pfu of Ad GFP, or same volume of solvent (PBS, pH 7.2) was applied once to each wound in groups 1 to 5, respectively One additional rabbit was used to evaluate the transfer efficiency of the adenovirus vector by transferring Ad GFP (6×10 9 pfu) into its wounds Ice slides of wounds from this animal were observed under fluorescence microscopy Another additional rabbit was used to evaluate the expression of HGF and TGFβ1 after transferring Ad HGF (6×10 9 pfu) into each of its wound Immunohistochemistry was used for detection Results The effect of HGF on reducing excessive dermal scarring was observed by adenovirus mediated gene transfer Transfection of the human HGF cDNA into skin wounds through an adenoviral vector suppressed the over expression of TGFβ1, which plays an essential role in the progression of dermal fibrogenesis Application of HGF to the wounds significantly enhanced wound healing and inhibited over scarring Conclusion HGF gene therapy could be a new approach for preventing excessive dermal scarring in wound healing

Human augmenter of liver regeneration: molecular cloning, biological activity and roles in liver regeneration

The complete amino acid sequence of human augmenter of liver regeneration (hALR) was reported by deduction from nucleotide sequence of its complementary DNA . The cDNA for hALR was isolated by screening a human fetal liver cDNA library and the sequencing of this insert revealed an open reading frame encoding a protein with 125aa and highly homologous (87% ) with rat ALR encoding sequence. The recombinant hALR expressed from its cDNA in transient expression experiments in cos-7 cells could stimulate DNA synthesis of HTC hepatoma cell in the dose-dependent and heat-resistant way. Northern blot analysis with rat ALR cDNA as probe confirmed that ALR mRNA was expressed in the normal rat liver at low level and that dramatically increased in the regenerating liver after partial hepatectomied rat. This size of hALR mRNA is 1.4 kb long and expressed in human fetal liver, kidney and testis. These findings indicated that liver itself may be the resource of ALR and suggested that ALR seems to be an im-portant paracrined regulator of liver regeneration.

Hydrogen peroxide preconditioning enhances the therapeutic efficacy of Wharton＇s Jelly mesenchymal stem cells after myocardial infarction

Background Exposure of cells to sublethal concentrations of hydrogen peroxide （H202） can alleviate subsequent oxidative stress-induced apoptosis. We assessed the effects of H202 preconditioning on the therapeutic potential of human umbilical cord Wharton＇s Jelly mesenchymal stem cells （WJ-MSCs） in a murine model of myocardial infarction. Methods WJ-MSCs were incubated in the media for 2 hours with or without 200 iJmol/L H202. Mice underwent left anterior descending coronary artery ligation, and received injection of phosphate buffered saline, 1×10^6 WJ-MSCs, or 1×10^6 H202 preconditioned WJ-MSCs 3 hours later via tail vein. Echocardiography was performed 0, 7, 14 and 28 days after surgery, and the mice were euthanized on day 28 for histological analysis. In vitro cytokine concentrations in the WJ-MSC cell supernatant were measured by enzyme-linked immunosorbent assay （ELISA）. The effect of WJ-MSC cell supernatant on the migration and proliferation of endothelial cells were observed by transwell migration and 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyl tetrazoliumbromide （MTT） assays. Results Echocardiographic measurements revealed a significant improvement in the left ventricular contractility of the WJ-MSCs-H2O2 group compared to the WJ-MSCs group. Histological analysis revealed increased neovascularization and reduced myocardial fibrosis in the WJ-MSCs-H2O2 group compared to the WJ-MSCs group. Pretreatment of WJ-MSCs with H2O2 increased the secretion of interleukin-6 （IL-6） into the cell culture supernatant by approximately 25-fold. The culture supernatant from WJ-MSCs-H2O2 significantly increased the migration and proliferation of endothelial cells; these effects could be blocked using an anti-IL-6 antibody. Conclusions This study demonstrates that H2O2 preconditioning significantly enhanced the therapeutic potential of WJ-MSCs, possibly by stimulating the production of IL-6 by WJ-MSCs, which may cause migration and proliferation of endothelial cells and increase neovascularization.

Human RBCs blood group conversion from A to O using a novel α-N-acetylgalactosaminidase of high specific activity

α-N-acetylgalactosaminidase (αNAGA) can convert group A human red blood cells (RBCs) to group O. One novel αNAGA gene was cloned by PCR from Elizabethkingia meningosepticum isolated from a domestic clinical sample. Pure recombinant αNAGA was obtained by genetic engineering and protein purification with a calculated molecule of 49.6 kD. αNAGA was selective for terminal α-N-acetylgalacto-samine residue with a high specific activity. αNAGA could completely remove A antigens of 1 U (about 100 mL) group A1 or A2 RBCs in 1 h at pH 6.8 and 25℃ with a consumption of 1.5 or 0.4 mg recombinant enzyme. Enzyme-converted group A RBCs did not agglutinate after being mixed with monoclonal anti-A or sera of groups A,B,AB and O. Other blood group antigens except ABO had no change. FCM analy-sis showed that A antigens and A1 antigens disappeared while H antigens increased. It indicated that αNAGA successfully converted human blood group A RBCs to universally transfusable group O RBCs without the risk of ABO-incompatible transfusion reactions. This αNAGA was suitable for producing universal RBCs to increase clinical transfusion safety,improve the RBCs supply,and to decrease transfusion cost and support transfusion service in case of emergency.

The Principle of Concerted Evolution Between Interacting Proteins—Darwinian Selection at Molecular Level

The neutral theory raised by Motoo Kimura in 1968 asserts that 'the great majority of evolutionary changes at the molecular level, as revealed by comparative studies of protein and DNA sequences. are caused not by Darwinian selection but by random drift of selectively neutral or nearly neutral mutants'.

The Principle of Modulated Evolution Between cDNA Coding Region and Nontranslated Region—Non-neutral Evolution at Molecular Level

A cDNA encoding a protein is composed of coding region and nontranslated region (NTR). There are two parts of NTR. i.e. 5’ NTR or 3’ NTR, upstream or downstream the coding region respectively. According to neutral theory of molecular evolution, the following expectations might be true at cDNA level: (i)evolutionary rate of NTR is higher than that of coding region; (ii) synonymous substitutions in coding region evolution are more than missense substitutions; (iii) transition substitutions are more than

Principle of Concerted Evolution of Cytokine-receptor and Verification of Its Corollary

Cytokines mediate their pleiotropic actions by binding to and activating theircell-surface receptors.So far,people have learned much about their biological characteristics,biochemical profile,and the interaction or modulation between cytokines and theirreceptors as well.However,people have no idea about what relationship of cytokines withtheir receptors in phylogeny exists.

A Correlation Study on Molecular Evolution of Hemopoietic Growth Factors and Development of Hemopoietic Cells

Development and evolution are two fundamental activities of life. In 1866 Ernst Hackell published the fundamental law of organ evolution, i. e. ontogeny is a recapitulation of phylogeny. So far, little progress has been achieved in the elucidation of its mechanism, particularly at molecular level. In mammalian tissues, the regulatory mechanism of cellular proliferation and differentiation of blood forming system has been studied in detail, and the regulatory

THE MECHANISM OF CLINICAL EFFECTIVENESS OF HUMAN FETAL LIVER CELL TRANSFUSION

During the development of human fetus,all organs consisting of various types of cells are in rapid growth.The significance of these cells and the chemical components involved,especially those of special functions in medical treatment of certain refractory diseases,have been noticed gradually.The questions as a step further are therefore raised,e.g.what is the mechanism underlying the clinical effectiveness and how to produce the active substances in large amount by biological engineering technique or chemical synthetic method,instead of using the crude preparation directly derived from fetus.