BACKGROUND: Present studies have demonstrated that during neural development, differentiation of neural stem cells (NSCs) was affected by various regulatory factors from surrounding microenvironment. Sonic hedgehog (Shh) is a key induction signal during neural fetal development, and can be an effective inductor to regulate differentiation of neural cells. OBJECTIVE: To investigate the signal transduction pathway of SHH for differentiation of rhesus monkey bone marrow mesenchymal stem cells (BMSCs) into of neuron-like cells by sonic hedgehog factor. METHODS: Rhesus monkey BMSCs were isolated and cultured by conventional density gradient centrifugation. BMSCs in the induction group were treated with L-DMEM containing FGF2, B27 and fetal bovine serum for preinduction of 24 hours, and then with DMEM supplemented with 0.5 μmol/L retinoic acid or 400 μmol/L SHH for 8 days. Non-induced cells served as control group. Following labeling with neuron enolase, positive cells were screened by flow cytometry. RT-PCR and Western-blot were used to detect SHH- and retinoic acid-induced cell membrane receptor and intracellular signal protein changes. RESULTS AND CONCLUSION: SHH specific membrane receptor Ptc, retinoic acid specific receptor RARα, signal protein molecule ptch1 and Smad expressed in normal cells. Ptc expression upregulated in SHH-induced cells. High expression lasted for a long time with induction time, which was significantly stronger compared with the retinoic acid and control groups (P < 0.01). Intracellular ptch1 protein molecule expression showed similar tendency as this, but could not induce upregulation of RARα expression. During induction, retinoic acid-stimulated cells did not activate Ptc pathway. Four days following induction, RARα expression upregulated and lasted till 6 days, but there were no significant differences. No significant change in ptch1 expression was determined. SHH- and retinoic acid-induced cell Smad molecule expression upregulated, but no significant difference was determined. Results verified that SHH-induced scheme participated in cell induction and differentiation by persistently activating its specific receptors. However, there was no significant receptor pathway crossing between retinoic acid-induced and SHH-induced schemes.

BACKGROUND:Bone marrow mesenchymal stem cells(BMSCs)differentiating into neural cells is an effective way of cell therapy of nervous system disease.However,the methods used nowadays still need to be improved.OBJECTIVE:To induce the differentiation of rhesus monkey BMSCs into neuron-like cells by using sonic hedgehog factor.METHODS:Rhesus monkey BMSCs differentiating into neuron-like cells was induced by typical retinoic acid and sonic hedgehog factor.Rhesus monkey BMSCs were isolated and cultured by density gradient centrifugation method.Cell growth was observed under an inverted phase contrast microscope and cell growth curve was determined using MTT assay.Flow cytometry was performed to characterize the phenotype of BMSCs,and immunohistochemistry was utilized to assess differentiated cells.Ultra-structure of the differentiated cells was observed by transmission and scanning electron microscopes.RESULTS AND CONCLUSION:Rhesus monkey BMSCs cultured in vitro were identified by flow cytometry,with high homogenicity.Following sonic hedgehog factor disposal for 7 days,differentiated cells were mainly positive for neurone specific enolase,neurofilament protein,Tau and glial fibrillary acidic protein(GFAP).Image statistical analysis found that in sonic hedgehog factor scheme,neural stem cells marker Neetin positive rate was significantly higher compared with the rstinoic acid scheme(P＜0.01).GFAP-positive rate was greater in the retinoic acid scheme than in the sonic hedgehog factor scheme(P＜0.05).Results indicated that sonic hedgehog factor scheme is an effective pathway of rhesus monkey BMSC differentiation into neuron-like cells.

AIM:To investigate the effects of 17-AAG on apoptosis and cell cycle of HCT-15 cells and to clar-ify the related mechanisms .METHODS: MTT method was employed to evaluate the inhibitory effects of 17-AAG with Aifferent time and different doses on the proliferation of HCT-15 cells.The cells were stained with Annexin V-FITC/propid-iumiodide and measured by flow cytometry .The expression of STAT3, cyclin D1, Cyt C, caspase 9 and caspase 3 at mR-NA and protein levels was determined by RT-PCR and Western blotting .RESULTS:Treatment with 17-AAG at concentra-tion of 1.25~20 mg/L for 24 h and 48 h significantly inhibited the activity of HCT-15 cells at both time-and concentra-tion-dependent manners .Treatment with 17-AAG at concentrations of 0.425, 0.85 and 1.7 mg/L for 48 h significantly in-duced apoptosis and cell cycle arrest of HCT-15 cells.The exposure of 17-AAG at concentrations of 0.425, 0.85 and 1.7 mg/L for 48 h to the HCT-15 cells significantly down-regulated the expression of STAT 3 and cyclin D1 at mRNA and pro-tein levels, but up-regulated Cyt C, caspase 9 and caspase 3 mRNA and protein in a concentration-dependent manner . CONCLUSION:17-AAG inhibits the cell activity , induces apoptosis and G 1 arrest by down-regulating the expression of cyclin D1, and promoting the mitochondria apoptosis through STAT 3 pathway.

ObjectiveTo investigate the role of p38 and ERK1/2 during rhesus monkeys mesenchymal stem cells differentiated into neuron-like cells.MethodsTo induce the neuronal phenotype,rhesus monkeys mesenchymal stem cells were maintained in sub-confluent cultures in serum-contain medium supplement with Sonic hedgehog.Western blot analysis the change of p38 and ERK1/2 during rhesus monkeys mesenchymal stem cells differentiated into neuron-like cells.Under transmission and scanning electron microscope,ultra-structure of the differentiated cell were observed.ResultsDuring BMSCs differentiated into neuron-like cells by SHH,Mitogen-activated protein kinases (MAPK) involved in their signal transduction,p38 was activated and ERK1/2 was inhibited.P38 inhibitor SB203580 increased induced differentiation time compared with normal induced cells,and inhibited neurite outgrowth.ConclusionActivation of p38 and inhibition of ERK was impacted on differentiation into neuron-like cells from rhesus monkeys mesenchymal stem cells induced by Sonic hedgehog,which may has potential application on neuroprotection of stem cells in Nervous system diseases

This study aimed at analyzing the medication regularity based on differentiation in traditional Chinese medicine (TCM) for losing weight using text mining technique.All the references over losing weight were retrieved in CNKI,Wangfang Database,VIP Database and Pubmed.The drugs from the references were classified in accordance with drug property,drug flavor,channel tropism and drug efficacy.Frequency and constituent ratio of a single drug in TCM prescriptions for losing weight were put into analysis using chi square test and factor analysis to find out the medication regularity.It was found that the properties of TCM drugs in the prescriptions contained both cold and warm,while the flavors of the drugs involved pungent,sweet and light.The channel tropism of the drugs mainly belonged to spleen meridian,liver meridian,stomach meridian and lung meridian.They were mostly tonic,relieving,blood-activating,qi regulating,inhibiting-damp and antipyretic drugs.Through factor analysis we found that the common formula compatibilities were concluded as:cassia seed,lotus leaf,hawthorn,salvia miltiorrhiza,polygonum cuspidatum and radix polygonum multiflorum;capillary artemisia,epimedium herb,stephania tetrandra and ligusticum wallichii;dried tangerine peel,pinellia ternata and poria cocos;plantain seed,pericarpium arecae and selfheal;paeonia lactiflora,angelica sinensis,scutellaria baicalensis and ligusticum wallichii;poria cocos,cassia twig,atractylodes and glycyrrhiza;immature bitter orange and bark of magnolia;radix bupleuri,lycium chinensis and jujube;Chinese yam and coix seed;and astragalus,pueraria lobata and polygonatum.In conclusion,formula compatibility mainly combined syndrome differentiation with disease differentiation for the treatment of obesity in clinic,using the drugs belonging to liver meridian,spleen meridian,stomach meridian and lung meridian with the flavors of sweet,bitterness or pungent and the nature of both warm and cold.

The aim of the present study was to investigate the effects and mechanisms of lidocaine on lipopolysaccharide(LPS)-induced matrix metalloproteinase(MMP)-9 and MMP-2 activity in plasma, and the effects of lidocaine on LPS-induced acute lung injury(ALI). Mice were pretreated with lidocaine(2, 4, 8 mg/kg )for 30 minutes, and then treated with 10 mg/kg LPS(ip)for 12 h to induce ALI. The 7-day survival rate and lung wet/dry weight ratio of mice were monitored. Phosphorylation level of p38 was measured by western blot. The activity of MMP-9 and MMP-2 in plasma was evaluated by gelatin zymography. The results showed that pretreatment with lidocaine could significantly reduce the death rate of ALI mice as well as the lung wet/dry weight ratio in a dose-dependent manner and suppress the activity of MMP-9 and MMP-2 in plasma. Moreover, lidocaine also markedly inhibited LPS-induced upregulation of p-p38 in a dose-dependent manner. In conclusion, lidocaine alleviated LPS-induced acute lung injury by suppressing MMP-9 and MMP-2 activity.

We expressed and prepared the recombinant fusion protein sTNFRII-gAD consisted of soluble TNF receptor II and the globular domain of adiponectin by Adenovirus Vector System in mammalian BHK21c022 cells. First we used the adenovirus vector containing EGFP gene (rAd5-EGFP) to infect BHK21c022 cells at different MOI (from 0 to 1 000), and then evaluated their transduction efficiency and cytotoxicity. Similarly, we constructed the replication-deficient adenovirus type 5-sTNFRII-gAD (rAd5-sTNFRII-gAD). We collected the supernatants for Western blotting to determine the optimal MOI by comparing the expression levels of sTNFRII-gAD fusion protein, 48 h after the BHK21c022 cells were infected by rAd5-sTNFRII-gAD at different MOIs (from 0 to 1 000). Then, we chose rAd5-sTNFRII-gAD at MOI 100 to infect five bottles of BHK21c022 cells in 100 mL of serum-free chemically defined media 100 mL, harvested the supernatant every 48 h for 6 times, and condense and purify sTNFRII-gAD fusion protein by ammonium sulfate salt-out and size-exclusion chromatography, respectively. Finally, we analyzed anti-TNFalpha activity of sTNFRII-gAD fusion protein on L929 cells in vitro. The results showed that the number of BHK21c022 cells expressing EGFP protein was increased significantly with the increase of MOI. However, some cells died at MOI of 1 000 while there was no significant cytotoxicity at MOI from 0 to 100. Western blotting analysis showed that the more adenoviruses, the higher expression of sTNFRII-gAD fusion protein in the supernatant with the highest expression at MOI 1 000. We successfully obtained about 11 mg bioactive and purified sTNFRII-gAD fusion protein at last. The in vitro assay demonstrated that the sTNFRII-gAD fusion protein was potent to antagonize TNFalpha's cytotoxicity to L929 cells. Put together, we established a recombinant adenovirus vector/BHK21 cell expression system, characteristic of the efficient serum-free culture and easy scaling-up.
Adenoviridae ; genetics ; metabolism ; Adiponectin ; biosynthesis ; genetics ; Animals ; Cell Line ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Receptors, Tumor Necrosis Factor, Type II ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

Objective:To observe the effect of Gongyanping Capsule on genital tract infected by ureaplasma urealyticum and fertility of mice, and to explore its mechanism.Methods:One hundred female ICR mice were divided into normal control group, model control group, azithromycin group, the low and high-dose group of Gongyanping Capsule according to random number table method. Except for the normal control group, the other groups were infected with ureaplasma urealyticum to establish the reproductive tract inflammation model. The azithromycin group was given 40 mg/kg of azithromycin, the low and high-dose groups were given 50 and 100 mg/kg of Gongyanping Capsule respectively, the normal control group and model control group were given equal volume of normal saline, once a day, for 4 consecutive weeks. The fertility status of each group was recorded. HE staining was used to observe the pathological changes of the vaginal tissues of the mice in each group, and the content of monocyte chemoattractant protein 1 (MCP-1), IL-4, IL-12, TNF-α myeloperoxidase (MPO) and GSH-Px of the mice in each group were determined; RT-PCR and Western blot were used to determine the vagina level of mRNA and protein expressions of TLR4 and NF-κB.Results:Compared with the model control group, the birth time and the number of dead mice in the azithromycin group and the low and high dose groups of Gongyanping Capsule decreased ( P<0.05), and the number of born mice increased ( P<0.05). The level of MCP-1, MPO, IL-4, IL-12, TNF-α decreased ( P<0.05), the level of GSH-Px increased ( P<0.05), the expression of TLR4 mRNA (1.25±0.33, 2.97±0.92, 2.32±0.72 vs. 3.69±1.32), NF-κB mRNA (1.48±0.42, 2.91±0.99, 2.13±0.70 vs. 3.83±1.41) decreased ( P<0.05), the expression of TLR4 (0.63±0.13, 1.32 ± 0.34, 1.04 ± 0.33 vs. 1.63 ± 0.41), NF-κB (0.63 ± 0.14, 1.36 ± 0.32, 1.03 ± 0.30 vs. 1.94 ± 0.58) decreased ( P<0.05), and had a certain dose-dependence. Conclusion:Gongyanping Capsule has obvious therapeutic effect on genital tract mice infected by ureaplasma urealyticum, and can significantly improve the fertility of mice; the mechanism may be related to that Gongyanping Capsule could inhibit the vaginal TLR4/NF-κB pathway in mice.

Objective To study the effect of high mobility group box B1(HMGB1)gene knockout on alleviating a-cute lung injury and inhibiting toll-like receptor 4(TLR4)/nuclear factor-KB(NF-κB)pathway of sepsis mice.Methods Wild-type(WT)mice were divided into WT-Sham group and WT-model group,and HMGB1 knockout(KO)mice were divided into KO-sham group and KO-model group.Sepsis ALI model was established by cecal ligation and perforation in WT-model group and KO-model group.Sham operation was performed in WT-Sham group and KO-Sham group.24 h after modeling,the partial pressure of arterial oxygen(PaO2)was detected,oxy-genation index(OI)was calculated,pathological changes of lung tissue were detected and lung injury score was calculated,the concentrations of tumor necrosis factor-α(TNF-α),interleukin-1 β(IL-1 β),interleukin-6(IL-6),reactive oxygen species(ROS),malondialdehyde(MDA),superoxide dismutase(SOD),in serum and lung tissues and the expression of HMGB1,TLR4 and nuclear NF-κB in lung tissues were detected.Results The PaO2,OI and the concentration of SOD in serum and lung tissue of WT-model group were lower than those of WT-Sham group,the lung injury scores,the concentrations of TNF-α,IL-1 β,IL-6,ROS and MDA in serum and lung tissue,and the expression levels of HMGB1,TLR4 and nuclear NF-κB in lung tissue were higher than those in WT-Sham group(P＜0.05).HMGB1 was not expressed in lung tissue of KO-model group,and the concentrations of PaO2,OI and the concentration of SOD in serum and lung tissue of KO-model group were higher than those of WT-model group,the lung injury scores,the concentrations of TNF-α,IL-1β,IL-6,ROS and MDA in serum and lung tissue,and the expression levels of TLR4 and nuclear NF-κB in lung tissue were lower than those of the WT-model group(P＜0.05).Conclusion HMGB1 gene knockout alleviates acute lung injury of sepsis mice,the re-lated molecular mechanism may be the inhibition of TLR4/NF-κB pathway mediated inflammation and oxidative stress.

Dirofilaria immitis (heartworm) infections affect domestic dogs, cats, and various wild mammals with increasing incidence in temperate and tropical areas. More sensitive antibody detection methodologies are required to diagnose asymptomatic dirofilariasis with low worm burdens. Applying current transcriptomic technologies would be useful to discover potential diagnostic markers for D. immitis infection. A filarial homologue of the mammalian translationally controlled tumor protein (TCTP) was initially identified by screening the assembled transcriptome of D. immitis (DiTCTP). A BLAST analysis suggested that the DiTCTP gene shared the highest similarity with TCTP from Loa loa at protein level (97%). A histidine-tagged recombinant DiTCTP protein (rDiTCTP) of 40 kDa expressed in Escherichia coli BL21 (DE3) showed immunoreactivity with serum from a dog experimentally infected with heartworms. Localization studies illustrated the ubiquitous presence of rDiTCTP protein in the lateral hypodermal chords, dorsal hypodermal chord, muscle, intestine, and uterus in female adult worms. Further studies on D. immitis-derived TCTP are warranted to assess whether this filarial protein could be used for a diagnostic purpose.
Animal Structures/chemistry ; Animals ; Antibodies, Helminth/blood ; Antigens, Helminth/chemistry/*genetics/immunology/*isolation & purification ; Cloning, Molecular ; Dirofilaria immitis/chemistry/*genetics/immunology ; Disease Models, Animal ; Dogs ; Escherichia coli/genetics ; Gene Expression ; Molecular Sequence Data ; Molecular Weight ; Recombinant Fusion Proteins/chemistry/genetics/immunology/isolation & purification ; Sequence Analysis, DNA ; Tumor Markers, Biological/chemistry/*genetics/immunology/*isolation & purification