Fibroblast growth factor-4 and hepatocyte growth factor induce differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocytes

AIM: To investigate the differentiation of human umbilical cord blood (HUCB)-derived mesenchymal stem cells (MSCs) into hepatocytes by induction of fibroblast growth factor-4 (FGF-4) and hepatocyte growth factor (HGF), and to find a new source of cell types for therapies of hepatic diseases.METHODS: vSCs were isolated by combining gradient density centrifugation with plastic adherence. When HUCB-derived MSCs reached 70% confluence, they were cultured in Iscove modified Dulbecco medium (IMDM) supplemented with 10 mL/L FBS, 20 ng/mL HGF and 10 ng/mL FGF-4. The medium was changed every 4 d and stored for albumin, alpha-fetoprotein (AFP) and urea assay. Expression of CK-18 was detected by immunocytochemistry. Glycogen storage in hepatocytes was determined by PAS staining.RESULTS: By combining gradient density centrifugation with plastic adherence, we could isolate MSCs from 25.6% of human umbilical cord blood. When MSCs were cultured with FGF-4 and HGF, approximately 63.6% of cells became small, round and epithelioid on d 28 by morphology. Compared with the control, the level of AFP increased significantly from d 12 to 18.20±1.16 μg/L (t = 2.884, P＜0.05) in MSCs cultured with FGF-4 and HGF, and was higher (54.28±3.11 μg/L) on d 28 (t = 13.493, P＜0.01). Albumin increased significantly on d 16 (t = 6.68, P＜0.01) to 1.02±0.15 μg/mL, and to 3.63±0.30 μg/mL on d 28 (t = 11.748, P＜0.01). Urea(4.72±1.03 μmol/L) was detected on d 20 (t = 4.272,P＜0.01), and continued to increase to 10.28±1.06 μmol/L on d 28 (t = 9.276, P＜0.01). Cells expressed CK-18 on d 16. Glycogen storage was observed on d 24. CONCLUSION: HUCB-derived MSCs can differentiate into hepatocytes by induction of FGF-4 and HGF. HUCBderived MSCs are a new source of cell types for cell transplantation therapy of hepatic diseases.

In vitro effects of sodium hyaluronate on the proliferation and the apoptosis in chondrocytes from patients with Kashin-Beck disease and osteoarthritis

Objective：To identify the in vitro effects of sodium hyaluronate（HA） on the proliferation and the apoptosis of chondrocytes from patients with Kashin-Beck disease（KBD） and osteoarthritis（OA）. Methods：Samples of articular cartilages from KBD and OA patients, as well as healthy volunteers（6 subjects in each of the 3 groups） were dissected, digested with collagenase and the cells cultured in monolayers. Chondrocytes from each sample were assigned to an untreated group and two HA-treated groups： H0（no HA）, H100（HA, 0.1 g/L） and H500（HA, 0.5 g/L）. The first passage chondrocytes were used to observe proliferation using the MTT assay, and apoptosis by flow cytometry through Annexin V/PI staining. Results：HA promoted proliferation of chondrocytes in all the three groups, and.in KBD and OA groups, for cells cultured for 4 and 6 days, H500 significantly promoted the cell proliferation. The apoptotic rates of both KBD and OA group chondrocytes were in the order H500 〈 HA100 〈 H0. Conclusion：Sodium hyaluronate administration has a dosedependent in vitro effect to promote proliferation and inhibit apoptosis of chondrocytes from patients with KBD and OA.

Expression of p-PPARγ in the aging thoracic aorta of spontaneously hypertensive rat and inhibitory effect of rosiglitazone

Objective:To investigate the expression of phosphorylated peroxisome proliferators-activated receptor y(p-PPARY) in the aging thoracic aorta of spontaneously hypertensive rat(SHR) and the inhibitory effect of rosiglitazone on the phosphorylation of PPART.Methods:16,32 and 64 week-old Wistar-Kyoto rats(WKY) and SHR were randomly and respectively divided into WKY,SHR and SHR+rosiglitazone group(9 in each group).The rats in SHR+rosiglitazone group were treated with rosiglitazone(5 mg/kg,intragastrically) for 56 d,whereas normal saline was applied in WKY and SHR groups.Systolic blood pressure(SBP)of rats was measured by tail cuff method.Histopathological damage of thoracic aorta was analyzed using Hematoxylin-Eosin(HE) staining.Immunohistochemical staining and western blot were performed to test the level of p-PPARY protein in the thoracic aorta arising from each group.Results:The SBP in 16,32 and 64 week-old SHR were significantly higher as compared with those in matched WKY rats(P<0.05,respectively).HE staining showed increased content of smooth muscle cell,wrinkled lining endothelium and increased thickness of internal elastic lamina in the thoracic aorta of SHR.Immunohistochemical staining and western blot indicated that the levels of p-PPARY in the thoracic aorta arising from SHR were obviously higher than those in the thoracic aorta arising from WKY rats(P<0.05,respectively).Importantly,the high SBP,histopathological abnormalities of the thoracic aorta and elevated p-PPARY expression were prominently abrogated by rosiglitazone treatment in SHR(P<0.05,respectively).Furthermore,the SBP,histopathological abnormalities of the thoracic aorta and p-PPARY expression were positively correlated with age in SHR(P<0.05,respectively).Conclusions:The PPARY phosphorylation was observed in the thoracic aorta of SHR and its expression was increased by the increase of age.Furthermore,rosiglitazone inhibited the PPARY phosphorylation and suppressed vascular aging in SHR.

PROPERTIES OF PROLIFERATION AND DIFFERENTIATION OF NEONATAL RAT RETINAL PROGENITOR CELLS IN VITRO

Objective To investigate the properties of proliferation and differentiation of neonatal rat retinal progenitor cells (RPCs) in vitro. Methods RPCs were isolated from neonatal SD rats neural retina and cultured in DMEM/F12+N2 with EGF and bFGF (suspension medium )or 10%FBS without EGF and bFGF (differentiation medium). The cells grew as suspended spheres or adherent monolayers, depending on different culture conditions. The neural stem cells or retinal progenitors, neurons, astrocytes, retinal ganglion cells, rod photoreceptors and the proliferating cells were evaluated with immunofluorescence analysis by Nestin or Pax6, Map2, GFAP, Thy-1, Rhodopsin and BrdU antibodies respectively. Results RPCs could propagate and differentiate in suspension or differentiation medium and express the markers of Nestin (92.86%) or Pax6 (86.75%), Map2 (38.54%), GFAP (20.93%), Thy-1 (27.66%) and Rhodopsin(13.33%)in suspension medium; however, Nestin (60.27%), Pax6 (52%), Map2 (34.94%), GFAP (38.17%), Thy-1(30.84%) and Rhodopsin (34.67%) in differentiation medium. 96.4% of the population in the neurospheres was BrdU-positive cells. The cells could spontaneously adherent forming some subspheres and retinal specific cell types. Conclusion Neonatal rat RPCs possess the high degree of proliferation and can differentiate into neurons, astrocytes, retinal ganglion cells and rod photoreceptors in vitro. There are different proportions for RPCs to differentiate into specific cell types.

Construction and identification of Fas-targeting siRNA-expressing plasmid

Objective: To study the therapeutic potential of Fas inhibition in different diseases, a Fas-targeting siRNA (small interfering)-expressing plasmid was constructed. Methods: The U6 promoter cassette and siFas (small interfering RNA that inhibit Fas expression) template sequence were obtained by PCR method. They were cloned into modified pcDNA3.1. The resultant plasmid pU6-siFas was transfected into P815 cells with lipofectin2000 and selected under G-418-containing culture medium. Fas inhibition in stably transfected cells was detected by immunocytochemistry. Results: The plasmid pU6-siFas efficiently reduced the expression of Fas and conferred G-418 resistance in P815 cells. Conclusion: The successful construction of the siRNA expressing plasmid will facilitate the application of RNA interference technique and lay the foundation for further study of Fas inhibition in the treatment of different diseases such as aplastic anemia and acute liver failure.

ALLELE DISTRIBUTION OF FIVE X-CHROMOSOME SHORT TANDEM REPEAT LOCI IN EWENKE POPULATION OF NORTH CHINA

Objective To study the allele genetic polymorphism of five short tandem repeat(STR) loci on X-chromosome in Ewenke population of north China and to provide basic data for forensic identification. Methods Genomic DNA was extracted from EDTA-whole blood of Ewenke population by Chelex-100. The DNA samples were amplified by PCR and were analyzed by polyacrylamide gel electrophoresis and silver staining. The sequence length variations of DXS6799,DXS8378,DXS101,HPRTB,and DXS6789 loci on X-chromosome in 98 unrelated Ewenke individuals were investigated. Results All five loci analyzed showed high polymorphism and genetic stability. The data of the five X-chromosome STR loci in Ewenke ethnic group of China was in accordance with Hardy-Weinberg equilibrium by Chi-square test. Conclusion Allele polymorphism of five X-chromosome STR loci can be used as a genetic marker for forensic identification and population genetic research.

THE MORPHOLOGICAL CHANGES IN MUSCLE SPINDLES AND ALTERATIONS IN CELL ACTIVITY OF THE RATS' RED NUCLEUS AFTER 2 WEEKS' SIMULATED WEIGHTLESSNESS

Objective To study the morphological changes of soleus muscle spindle and electrical activity of neurons in Red Nucleus(RN) of the rat after 2 weeks' simulated weightlessness, and to reveal the interaction between proprioceptive inputs of muscle spindles and reciprocal alterations in RN under simulated weightlessness. Methods Twenty female rats were exposed to weightlessness simulated by tail-suspension for 14 days (SW-14d). Body weight(200-220g) matched female rats were control group(Con). The morphological changes in isolated muscle spindle of soleus muscle, the discharges of red nucleus neurons were observed after 14d tail-suspensions by silver staining and extracellular recording respectively. Results Compared with control group ,the nerve ending of muscle spindle in SW-14d was distorted, degenerated and dissolved; the diameters of intrafusal fibers and capsule in equatorial region of soleus muscle spindles were diminished(P<0.05). The spontaneous cell activity and discharge of RN neurons (spikes/s) induced by afferent firing from muscle spindles after injection of succinylcholine were reduced after 2 weeks' simulated weightlessness respectively (18.44±5.96 vs. 10.19±6.88, 32.50±8.08 vs. 16.86±5.97, P<0.01). Conclusion The degeneration of muscle spindle induced by simulated weightlessness may be one of the causes that led to alterations in discharges of RN.

Alterations of serum cholinesterase in patients with gastric cancer

AIM: To understand the correlation of serum cholinesterase (CHE) activity with gastric cancer and to assess their clinical significance.METHODS: The velocity method was adopted to detect the activity of serum CHE in patients with gastric cancer and in patients with non-malignant tumor as controls.RESULTS: The serum CHE activity in the treatment group was significantly lower than that in the control group with a very significant difference between the two groups (83.3:113.1,P = 0.0003). Age was significantly associated with the incidence of gastric caner.CONCLUSION: Serum CHE activity has a close relation with the incidence of gastric cancer.

Effects of moniliformin and selenium on human articular cartilage metabolism and their potential relationships to the pathogenesis of Kashin-Beck disease

Objective:To investigate the effects of mycotoxin moniliformin (MON) on the metabolism of aggrecan and type II collagen in human chondrocytes in vitro and the relationship between MON and Kashin-Beck disease (KBD).Methods:Human chondrocytes were isolated and cultured on bone matrix gelatin to form an artificial cartilage model in vitro with or without MON toxin.Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.The expression of aggrecan and type II collagen in the cartilage was determined using immunocytochemical staining.Results:MON toxin inhibited chondrocyte viability in dose-dependent and time-dependent manners.MON reduced aggrecan and type II collagen syntheses in the tissue-engineered cartilage.MON also increased the expression of matrix metalloproteinase-1 (MMP-1),MMP-13,BC4 epitopes,and CD44 in cartilages.However,the expression of 3B3(-) epitopes in cartilages was inhibited by MON.Selenium partially alleviated the damage of aggrecan induced by MON toxin.Conclusion:MON toxin promoted the catabolism of aggrecan and type II collagen in human chondrocytes.

Distributions of HLA-A and-B alleles and haplotypes in the Yi ethnic minority of Yunnan, China: relationship to other populations

Objective: To investigate the distributions of human leukocyte antigen (HLA)-A and-B alleles and HLA-A-B haplotypes in the Yi ethnic minority of the Yunnan Province, situated in southwestern China. Methods: DNA typing for HLA-A and-B loci was performed using the polymerase chain reaction-sequence-based typing (PCR-SBT) method on 114 randomly selected healthy individuals of the Yi population. The allelic frequencies of HLA-A and-B loci were calculated by direct counting and HLA-A-B haplotypes were estimated using the expectation maximization algorithm. Results: A total of 17 HLA-A and 38 HLA-B alleles were found in the Yi population. The most frequent alleles were A2402 (32.46%), A1101 (26.32%), and A0203 (10.09%) at the HLA-A locus and B4601 (12.28%), B1525 (10.09%), B4001 (8.77%), and B3802 (7.89%) at the HLA-B locus. The predominant HLA-A-B haplotypes were A2402-B1525 (7.86%) and A0203-B3802 (5.64%), followed by A1101-B4001 (4.69%). Phylogenetic analysis indicates that the Yi population in the Honghe, Yunnan Province of China basically belongs to groups of southeastern Asian origin, but shares some characteristics with northeastern Asian groups. Conclusion: The present study may add to the understanding of HLA polymorphism in the Yi ethnic group that was poorly defined previously, and provide useful information for bone marrow transplantation, anthropological research, and forensic sciences as well as for disease-association studies.

Serum resistin and adiponectin concentrations in patients with overweight and obesity

Objective:To explore the serum levels of resistin and adiponectin in patients with overweight and obesity.Methods:Fifty-eight cases with normal weight and 24 patients with overweight and obesity have taken fasting blood samples for measurements of plasma glucose,plasma lipids,insulin,C-peptide, thyroid hormones,C-response protein,interleukin-6,TNF-α,leptin,adiponectin and resistin.Results: The concentrations of resistin in cases with overweight and obesity were significant higher than those in the normal weight cases (16.01±8.60 vs 11.63±9.05ng/ml,P=0.047).Pearson relation analysis showed that serum resistin concentrations were positively correlated with age (r=0.476,P=0.019),but negatively correlated with C-peptide (r=-0.45,P=0.024),and adiponectin concentrations were posi- tively correlated with HDL-c (r=0.463,P=0.023) and systolic blood pressure (r=0.409,P=0.047)in overweight and obesity cases.Conclusion:The concentrations of resistin in cases with over-weight and obesity are higher,and there is no correlation between resistin and blood glucose,blood lipids and insulin, while the serum adiponectin concentrations positively correlated with HDL-c and systolic blood pressure.

Relationships between endothelial nitric oxide synthase gene polymorphisms and osteoporosis in postmenopausal women

Objective:To investigate the relationships between endothelial nitric oxide synthases(eNOS) G894T and 27 bpvariable number tandem repeat(VNTR) gene polymorphisms and osteoporosis in the postmenopausal women of Chinese Han nationality.Methods:In the present study,281 postmenopausal women from Xi'an urban area in West China were recruited,and divided into osteoporosis,osteopenia,and normal groups according to the diagnostic criteria of osteoporosis proposed by World Health Organization(WHO).The bone mineral density(BMD) values of lumbar vertebrae and left hips were determined by QDR-2000 dual energy X-ray absorptiometry.Blood samples were tested for plasma biochemical indicators including testosterone,estradiol,calcitonin,osteocalcin,and procollagen type I amino-terminal propeptide by enzyme-linked immunosorbent assay(ELISA),tartrate-resistant acid phosphatase by spectrophotometric method,and the content of nitric oxide by Griess method.Genome DNA was extracted from whole blood,and G894T polymorphism of eNOS gene was analyzed by using polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) method and 27 bp-VNTR polymorphism of eNOS gene was genotyped by PCR method.Then the relationships between genotypes and biochemical indicators,genotypes and osteoporosis,and haplotypes and osteoporosis were analyzed.Results:The average BMD values of the femoral neck,ward's triangle and lumbar vertebrae 1～4(L1～L4) in the subjects with T/T genotype in eNOS G894T locus were significantly higher than those in the subjects with G/T and G/G genotypes(P<0.05).The average BMD of the femoral neck in the subjects with a/a genotype of eNOS 27 bp-VNTR locus was evidently higher than that in the subjects with b/b genotype(P<0.05).The plasma testosterone and os-teocalcin concentrations in the subjects of eNOS G894T G/T genotype were evidently higher than those in the subjects of other genotypes(P<0.05);the plasma estradiol concentration in the subjects of eNOS 27 bp-VNTR a/a genotype was obviously higher than that in the subjects of b/b genotype(P<0.01).eNOS G/G homozygous frequencies in osteoporosis women,osteopenia women,and normal women were 85.37%,76.38%,and 83.87%,respectively(P>0.05).0% osteoporosis woman,0.79% osteopenia women,and 3.23% normal women were eNOS a/a homozygous(P<0.05).The frequencies of eNOS 27 bp-VNTR a allele were 5.33% in the osteoporosis group,10.24% in the osteopenia group,and 16.13% in the normal group(P<0.05,odds ratio(OR)=0.29,95% confidence interval(CI)=0.11～0.77),suggesting that a/a genotype and a allele might have protective effects on osteoporosis.The haplotype analysis showed that G-b was 87.7%(214/244) in the osteoporosis group(P<0.05,OR=2.48,95% CI=1.18～5.18).G-a was 5.3%(13/244) in the osteoporosis group(P<0.05,OR=0.29,95% CI=0.11～0.77).G-b was a risk factor for osteoporosis,and G-a a protective factor.Conclusion:eNOS G894T G/T genotype influenced the plasma testosterone and osteocalcin concentrations,and T/T genotype influenced BMD.eNOS 27 bp-VNTR a/a genotype increased plasma estradiol concentration to have a protective effect on osteoporosis.

Methotrexate ameliorates pristane-induced arthritis by decreasing IFN-γ and IL-17A expressions

Objective: This study was carried out to test the effects of methotrexate (MTX) and black seed oil (BSO) on pristane-induced arthritis (PIA) in rats.Methods: Inbred dark agouti (DA) rats were induced by a single subcutaneous injection of pristane,and then treated with MTX or BSO.Arthritis severity was evaluated macroscopically and microscopically.Plasma nitric oxide (NO) concentration was determined by the Griess method and cytokine mRNA expression in the spleen was detected by the real-time reverse transcription-polymerase chain reaction (RT-PCR).Results: The clinical arthritis severity was decreased after MTX treatment,while the BSO groups did not show significant changes compared with the disease group.The plasma NO level of the MTX group was significantly decreased compared with the disease group,but the BSO groups showed no difference from the disease group in plasma NO levels.The interferon-γ (IFN-γ) and interleukin-17A (IL-17A) mRNA expressions in the spleens were significantly decreased in the MTX group,but only showed a declining trend in the BSO groups compared with the disease group.Neither MTX nor BSO had an effect on the mRNA expressions of IL-4,transforming growth factor β (TGF-β),and tumor necrosis factor-α (TNF-α) in the spleen.Conclusions: MTX,but not BSO,can reduce the arthritis severity and decrease the mRNA expressions of IFN-γ and IL-17A in pristane-induced arthritis of rats.