Erythropoietin-overexpressed umbilical cord mesenchymal stem cells inhibit neuroapoptosis in ischemic-hypoxic SH-SY5Y and its mechanism / 中国组织工程研究

BACKGROUND:Previous studies have successfully constructed erythropoietin-overexpressed umbilical cord mesenchymal stem cells.It was found that the apoptosis of ischemic and hypoxic human neuroblastoma cell line(SH-SY5Y)was significantly reduced by erythropoietin-overexpressed umbilical cord mesenchymal stem cells. OBJECTIVE:To explore the possible neuroprotective mechanisms of erythropoietin-overexpressed umbilical cord mesenchymal stem cells against ischemic-hypoxic SH-SY5Y and their associated epigenetic mechanisms. METHODS:Oxygen-glucose deprivation was applied to ischemia-hypoxia-induced SH-SY5Y cell injury,and multifactorial assays were applied to detect the expression levels of inflammatory factors in the cells before and after hypoxia and co-culture,respectively,with mesenchymal stem cells,as well as lentiviral-transfected null-loaded plasmids of the negative control mesenchymal stem cells and erythropoietin-overexpressed umbilical cord mesenchymal stem cells.The expression levels of supernatant inflammatory factors were detected by multifactor assay after co-culture.Proteomics was used to detect the differentially expressed proteins of negative control mesenchymal stem cells and erythropoietin-overexpressed umbilical cord mesenchymal stem cells.Cleavage under targets and tagmentation sequencing was applied to detect genomic H3K4me2 modification,and joint analysis was conducted with RNA-sequencing.Lentiviral vector infection was applied to construct the stable knockdown of REST in SH-SY5Y cells.qRT-PCR and western blot assay were performed to detect the expression level of REST.The apoptosis was detected by flow cytometry after co-culture of oxygen-glucose deprivation treatment with erythropoietin-overexpressed umbilical cord mesenchymal stem cells.The expression difference of H3K36me3 group proteins was detected by western blot assay,and transcriptome sequencing was performed to analyze the differentially expressed genes. RESULTS AND CONCLUSION:(1)Compared with the control group,monocyte chemotactic protein 1,interleukin-6,interleukin-18,and interleukin-1 beta,interferon α2,and interleukin-23 levels significantly increased in the cerebrospinal fluid supernatant of patients with ischemic-hypoxic encephalopathy(P<0.01).(2)After co-culturing SH-SY5Y cells with erythropoietin-overexpressed umbilical cord mesenchymal stem cells under ischemia and hypoxia,the expression levels of monocyte chemotactic protein 1 and interleukin-6 were significantly reduced.(3)Analysis of protein network interactions revealed significant downregulation of monocyte chemotactic protein 1,interleukin-6 related regulatory proteins CXCL1 and BGN.(4)Transcriptome sequencing analysis found that pro-inflammatory genes were down-regulated,and functional enrichment of histone modifications,and the expression of transcription factors REST and TET3 significantly up-regulated in the erythropoietin-overexpressed umbilical cord mesenchymal stem cell group compared with the negative control mesenchymal stem cell group.(5)Combined analysis of transcriptome sequencing and cleavage under targets and tagmentation revealed changes in epigenetic levels as well as significant activation of the promoter regions of transcription factors REST and TET3.(6)Stable knockdown REST in SH-SY5Y cells was successfully constructed;the transcript levels of REST mRNA and protein expression were both decreased.(7)After the REST knockdown SH-SY5Y cells were co-cultured with erythropoietin-overexpressed umbilical cord mesenchymal stem cells,apoptosis was significantly increased and H3K36me3 expression was significantly decreased.Transcriptome sequencing results showed that the expression of inflammation-related genes Aldh1l2 and Cth,as well as apoptosis-suppressor genes Mapk8ip1 and Sod2 was reduced at mRNA transcription level(P<0.01).(8)It is concluded that erythropoietin-overexpressed umbilical cord mesenchymal stem cells activated the expression of REST and TET3 by altering the kurtosis of H3K4me2 and upregulated the modification level of H3K36me3,which in turn regulated the expression of inflammation-related genes Aldh1l2 and Cth,as well as apoptosis-suppressor genes Mapk8ip1 and Sod2,and facilitated neuronal survival.

Melatonin promotes osteogenesis of bone marrow mesenchymal stem cells by improving the inflammatory state in ovariectomized rats / 中国修复重建外科杂志

OBJECTIVE@#To investigate the effects of melatonin (MT) on bone mass and serum inflammatory factors in rats received ovariectomy (OVX) and to investigate the effects of MT on the levels of inflammatory factors in culture medium and osteogenic ability of bone marrow mesenchymal stem cells (BMSCs) stimulated by lipopolysaccharide.@*METHODS@#Fifteen 12-week-old Sprague Dawley (SD) rats were randomly divided into 3 groups. The rats in Sham group only received bilateral lateral abdominal incision and suture, the rats in OVX group received bilateral OVX, and the rats in OVX+MT group received 100 mg/(kg·d) MT oral intervention after bilateral OVX. After 8 weeks, the levels of serum inflammatory factors [interleukin-1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α)] were detected using ELISA assay. Besides, the distal femurs were detected by Micro-CT to observe changes in bone mass and microstructure, and quantitatively measured bone volume fraction, trabecular thickness, and trabecular number. The BMSCs were extracted from the femurs of three 3-week-old SD rats using whole bone marrow culture method and passaged. The 3rd-5th passage BMSCs were cultured with different concentrations of MT (0, 1, 10, 100, 1 000 µmol/L), and the cell viability was then detected using cell counting kit 8 (CCK-8) to select the optimal concentration of MT for subsequent experiments. Cells were devided into osteogenic induction group (group A) and osteogenic induction+1/5/10 μg/mL lipopolysaccharide group (group B-D). The levels of inflammatory factors (IL-1β, IL-6 and TNF-α) in cell culture medium were detected using ELISA assay after corresponding intervention. According to the results of CCK-8 method and ELISA detection, the cells were intervened with the most significant concentration of lipopolysaccharide for stimulating inflammation and the optimal concentration of MT with osteogenic induction, defining as group E, and the cell culture medium was collected to detect the levels of inflammatory factors by ELISA assay. After that, alkaline phosphatase (ALP) staining and alizarin red staining were performed respectively in groups A, D, and E, and the expression levels of osteogenic related genes [collagen type Ⅰ alpha 1 chain (Col1a1) and RUNX family transcription factor 2 (Runx2)] were also detected by real time fluorescence quantitative PCR (RT-qPCR).@*RESULTS@#ELISA and Micro-CT assays showed that compared with Sham group, the bone mass of the rats in the OVX group significantly decreased, and the expression levels of serum inflammatory factors (IL-1β, IL-6, and TNF-α) in OVX group significantly increased (P<0.05). Significantly, the above indicators in OVX+MT group were all improved (P<0.05). Rat BMSCs were successfully extracted, and CCK-8 assay showed that 100 µmol/L was the maximum concentration of MT that did not cause a decrease in cell viability, and it was used in subsequent experiments. ELISA assays showed that compared with group A, the expression levels of inflammatory factors (IL-1β, IL-6, and TNF-α) in the cell culture medium of groups B-D were significantly increased after lipopolysaccharide stimulation (P<0.05), and in a concentration-dependent manner. Moreover, the expression levels of inflammatory factors in group D were significantly higher than those in groups B and C (P<0.05). After MT intervention, the expression levels of inflammatory factors in group E were significantly lower than those in group D (P<0.05). ALP staining, alizarin red staining, and RT-qPCR assays showed that compared with group A, the percentage of positive area of ALP and alizarin red and the relative mRNA expressions of Col1a1 and Runx2 in group D significantly decreased, while the above indicators in group E significantly improved after MT intervention (P<0.05).@*CONCLUSION@#MT may affect the bone mass of postmenopausal osteoporosis by reducing inflammation in rats; MT can reduce the inflammation of BMSCs stimulated by lipopolysaccharide and weaken its inhibition of osteogenic differentiation of BMSCs.

The role of B-cell scaffold protein with ankyrin repeats 1 in collagen-induced arthritis / 中华风湿病学杂志

Objective To analyze the roles of B-cell ccaffold protein with ankyrin repeats 1 (BANK1) in collagen-induced arthritis (CIA) murine model and the correlation with disease severity.Methods CIA murine model were established and evaluated.In different disease stages,the serum levels of anti-C Ⅱ auto-antibodies and BANK1 were detected by electrochemiluminescence immunoassay(ELISA).Moreover,the expression of BANK1 mRNA in peripheral blood cells were detected by real-time polymerase chain reaction (PCR) and its correlation with clinical scores was analyzed.Then the percentage of BANK1 expression in B cells in spleen and draining lymph nodes were detected by flow cytometry and the level of BANK1 protein in spleen was detected by Western blotting according to the results afore mentioned.The data was analyzed by Statistical Product and Service Solutions (SPSS) Stastistics 21.0 and figures were made with Graph Pad Prism 6.Repeated measure ANOVA was used to assess differences between the two groups.Correlations were analyzed by Spearman correlation analysis.Linear regression analysis was done when a correlation was identified.Results The incidence of CIA was over 90％.The clinical scores of arthritic mice was positively correlated with the serum levels of anti C Ⅱ total IgG antibody (r=0.717 5,P ＜0.01),anti C] IgG2a antibody (r=0.675 3,P＜0.01) and anti C Ⅱ IgG2b antibody (r=0.889 4,P＜0.01) respectively.The BANK1 level in the serum and the BANK1 mRNA expression were significantly decreased in different disease stages in CIA mice when compared with normal mice.The negative correlation between the BANK1 mRNA expression and clinical scores (r=-0.485 4,P＜0.01) was observed.The percentage of BANK1 +CD19+ cells in spleen and draining lymph nodes and the level of BANK1 protein in spleen were reduced in CIA as well.Conclusion Along with the disease progress in CIA,BNK1 expression is declined,which weakens the negative regulation of BANK1 on B cells.This change goes hand in hand with the severity of arthritis.

Interactions between induced T regulatory cells and B cells in inflammatory milieu in mice with collagen-induced arthritis / 中国免疫学杂志

Objective:To investigate the interactions between induced T regulatory cells ( iTregs ) and B cells in the inflammatory milieu in mice with collagen-induced arthritis(CIA). Methods: CD19+ cells were isolated from the spleen cells of normal DBA1/J(N-B) mice and CIA mice(CIA-B) on the 35th day after the first immunization with established arthritis. These B cells were used as antigen-presenting cells to observe their effects on the induction of Tregs. Tregs were induced with the classic method and co-cultured with CIA-B cells. CIA-B cell effects on iTreg proliferation and the expression of CTLA-4 on iTregs were explored. iTregs′ influence on the expressions of co-stimulators(CD80,CD86) and MHCⅡon B cells was studied and its mechanism was determined by the Transwell experiments. Results: CIA-B could induce more Treg production and proliferation. CIA-B could also promote the CTLA-4 expression on iTreg cell surface which worked through a cell-contact pathway, while iTregs could increase the expressions of co-stimulators(CD80,CD86) and MHCⅡby the same way. Conclusion: iTregs could show their immune suppressive function through the interactions with CIA-B cells in the inflammatory milieu in mice with CIA. These interactions work by a cell-contact pathway.

Gene clone, eukaryotic expression vector and tissue expression profile analysis of porcine BST-2 gene / 中国兽医学报

In order to study the biological function of pig BST-2 gene,the BST-2 gene was amplified with specific primers from porcine kidney tissue,and molecular characterization of BST-2 nuclectide and amino acid sequence were analyzed with bioinformatics tools and online server.Then the prokaryotic expression and tissue expression profile analysis was carried out.The results showed that the full length of pig BST-2 gene was 851 bp and contained 23 bp of 5'-UTR,294 bp of 3'-UTR and 534 bp of CDS and the gene encoded 177 aa.Amino acid sequence analysis of pig BST-2 protein showed 46.1％ identity with gorilla gorilla,41.7％ with cricetulus griseus,39.5％ with mus musculus,35.4％ with equus asinus,42.0％ with felis catus,40.5％ with bos mutus,44.4％ with macaca mulatta,38.7％ with ovis aries and 46.8％ with homo sapiens.BST-2 protein contained 2 transmembrane structure (27-49 aa and 154-176 aa),2 glycosylation sites and 14 potential phosphorylation sites including ATM,CK Ⅱ,PKA,PKC binding sites.The pig BST-2 protein was expressed in Vero cells after translated the recombinant plasmid FLAG-BST-2.Semiquantitative PCR results showed that BST-2 gene was expressed in all the tissues,especially in lymph nodes,thymus,tonsils,spleen,large intestine and small intestine.This study provide a foundation for further understanding the antiviral mechanism of pig BST-2 protein.

Clinical research of interleukin-34 in psoriatic arthritis patients / 中华风湿病学杂志

Objective To investigate the relationship between interleukin (IL)-34 and bone erosions in psoriatic arthritis (PsA) patients.Methods Forty PsA patients,20 psoriasis (Ps) patients and 20 healthy volunteers were recruited into this study.The levels of IL-34 and osteoclast related cytokines [including tumor necrosis factor (TNF)-α,receptor activator of nuclear factor-κB ligand (RANKL),osteoclast precursors (OPG)] were detected in the serum samples of all subjects.The correlations among IL-34,the number of osteoclast precursors (OCP),disease activity and imaging scores were analyzed.All data were analyzed by graphpad prism 6.Differences between groups was analyzed with One-way analysis of variance,q tests,and Spearman's correlation was used to explore the relation between disease activity/radiographic scores and laboratory results and followed by linear regressions.Results The serum level of IL-34 in patients with PsA [(328±476) pg/ml] was higher than that in Ps [(33±52) pg/ml,q =3.92,P＜0.01] and healthy controls [(32±32) pg/ml,q =3.93,P＜0.01],the erosive PsA group were higher than the non-erosive PsA group [(449±527) pg/ml and (47±24) pg/ml,q=4.04,P＜0.01].The levels of TNF-α,RANKL and OCP in patients with PsA [(125±79) pg/ml,(488± 475) pg/ml and (17.7±4.8) 5 sigh views] were higher than those in PS [(40±22) pg/ml,(26±3) pg/ml and (5.2± 0.8),q=7.32,6.14 and 2.94,P＜0.01] and healthy controls [(41±19) pg/ml,(65±8) pg/ml and (6.2±1.8),q=6.67,5.62 and 2.71,P＜0.01],whereas the OPG/RANKL ratio in PsA patients (0.5±0.4) was significantly lower than Ps patients (4.3±2.7,q=-3.30,P＜0.01) and healthy controls (1.8±0.6,q=-1.72,P＜0.01).IL-34,TNF-α and RANKL levels were all positively correlated with OCP (r=0.10,P＜0.05;r=0.12,P＜0.05;r=0.13,P＜0.(5,respectively).Conclusion The level of IL-34 is not only high in patients with PsA but also positively correlates with the number of OCP.In PsA,IL-34 is probably related to the OCP and osteoclast differentiation,and further participates in the process of bone destruction.Therefore,IL-34 is promising to become a new target or alternative choice for the treatment of PsA.

Effect of different types of nutrient solution on preoperative fasting time / 中华临床营养杂志

Objective To discuss the feasibility of preoperative diet by measuring gastric emptying time of carbohydrate and protein nutrient solutions in healthy volunteers.Methods A total of 20 healthy volunteers were collected from August 2013 to May 2014.On the morning of the trial,baseline gastric residual volume of each volunteer was measured with magnetic resonance imaging at 8 a.m.,then each of the 20 healthy volunteers took 12.5％ carbohydrate solution 400 ml (containing 40 g of maltodextrin and 10 g of sucrose) or 12.5％ whey protein solution (containing 50 g whey protein) in 5 minutes.Magnetic resonance imaging was conducted to measure the gastric residual volume every 25 minutes.The volunteers were shifted to the other nutrient solution after a 1-week interval.The gastric emptying time of both nutrient solutions was calculated to generate the curves illustrating the process of gastric emptying.Results The baseline gastric residual volume of the volunteers was (14.90 ± 9.39) ml.The total gastric emptying time of carbohydrate solution was (104.90 ± 27.98) min (95 ％ CI 98.64-111.16 min),while that of whey protein solution was (199.6 ± 34.17) min (95％ CI 184.47-214.73 min).There was a significant difference between these two types of nutrient solution in terms of gastric emptying time (P ＜ 0.000 1).Conclusions The induction of anesthesia could be performed 2 hours after carbohydrate administration,and at least 4 hours after whey protein administration.

Gastric emptying time for liquids in healthy volunteers: determination by magnetic resonance imaging / 中华麻醉学杂志

Objective To determine the gastric emptying time for liquids in the healthy volunteers by magnetic resonance imaging (MRI) and to provide a reference for reasonable preoperative fasting time.Methods Nineteen healthy volunteers of both sexes,of ASA physical status Ⅰ or Ⅱ,aged 20-60 yr,were enrolled in the study.The volunteers were fasted from the intake of liquids or solids starting from 22:00 the night before the trial,and 12.5％ carbohydrate solution 400 ml containing 40 g maltodextrin and 10 g sucrose was given orally.MRI was performed to measure the baseline gastric fluid volume at 8:00 on the day of the trial (T0).The gastric fluid volume was measured immediately after administration of the oral solution,and then measured every 25 min until the gastric fluid volume was returned to the baseline before administration of the oral solution or to ＜25 ml,and was recorded as T25,T50,T75,T100,et al.The gastric fluid volume was drawn using a computer to obtain the curve for gastric emptying.The gastric half and total emptying time was calculated using the curves.Results The gastric half emptying time was (32± 12) min,and the gastric total emptying time was (99±22) min in the volunteers.Compared with those at Tb,the gastric fluid volume was significantly increased at T25,T50,T75,T100,and no significant change was found in gastric fluid volume at T125,T150and T175.Conclusion After oral intake of liquids,the gastric emptying time is about 2 h,indicating that the preoperative fasting time for liquids can be shortened to 2 h before anesthesia in the healthy volunteers.

Inhibition of Nogo expression to promote repair after spinal cord injury / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>One of the reasons for poor neuroregeneration after central nervous system injury is the presence of inhibitory factors such as Nogo. Here, we tested the inhibition of Nogo by RNA interference both in vitro and in vivo, using recombinant adenovirus-mediated transfection of short hairpin RNAs, to explore a new method of treatment for spinal cord injury.</p><p><b>METHODS</b>We designed and cloned two Nogo-specific short hairpin RNAs and an unrelated short hairpin RNA, packaged the clones into adenovirus, and amplified the recombinant virus in 293 cells. We then tested the inhibition of Nogo expression both in vitro in adenovirus-transfected oligodendrocytes and in vivo in spinal cord tissue from adenovirus-transfected spinal cord injury model rats. We tested Nogo expression at the mRNA level by reverse-transcription PCR and at the protein level by Western blotting and immunohistochemistry.</p><p><b>RESULTS</b>In vitro, the two specific Nogo short hairpin RNAs decreased Nogo mRNA expression by 51% and 49%, respectively, compared with Nogo expression in cells transfected with the unrelated control small hairpin RNA (P < 0.005). Similarly, Nogo protein expression decreased by 50% and 48%, respectively (P < 0.005). In vivo, in spinal cord injury model rats, the two specific Nogo short hairpin RNAs decreased Nogo mRNA expression by 45% and 40%, respectively, compared with Nogo expression in spinal cord injury model rats transfected with the unrelated control short hairpin RNA (P < 0.005). The Nogo protein level was similarly decreased.</p><p><b>CONCLUSIONS</b>We were successful in specifically downregulating Nogo at the mRNA and protein levels by adenovirus-mediated delivery of short hairpin RNAs, both in vitro and in vivo. This confirms the effectiveness of RNA interference for the inhibition of Nogo gene expression and the efficiency of using adenovirus for delivery. Thus gene therapy may be an effective treatment for spinal cord injury.</p>

The association of BANK1 single nucleotide polymorphisms with rheumatoid arthritis in Chinese Han population / 中华风湿病学杂志

ObjectiveTo investigate the association of BANK1 single nucleotide polymorphisms (SNPs) with rheumatoid arthritis(RA) in Chinese Han. MethodsTwo hundreds and twenty-one RA patients and 310 healthy controls who were Chinses Han population from Huashan Hopital and Changzheng Hospital in Shanghai,China were included.DNAs were extracted from peripheral whole blood for study.Samples were genotyped for three variants rs10516487,rs17266594 and rs3733197 in BANK1 by unlabelled probe high resolution melting (HRM) assay.The genotype frequencies of the detected polymorphisms were analyzed in relation to RA and the production of autoantibodies in RA patients.ResultsThe Tr genotype frequency was much higher in RA patients than in healthy controls(X2=6.241,P=0.044).The frequencies of rs10516487 G allele,rs17266594 T allele and rs3733197 G allele were increased among RA patients compared with healthy controls,although they didn't reach statistical significance.The rs10516487 and rs17266594 were found in strong linkage disequilibrium(D'=0.993,r2=0.985).And also the major TGG haplotype of 3-SNP was significantly associated with RA patients[P=0.037,OR =1.345,95％CI (1.018-1.776)].ConclusionBANK1 rs17266594 polymorphism is susceptible to RA,while rs10516487 and rs17266594 are linked in Chinese Han population.BANK1 SNPs TGG haplotype may contribute to RA susceptibility,too.

Performance characteristic comparison of two commerical kits for anti-HCV confirmatory test / 中华检验医学杂志

Objective To detect anti-HCV in serum of hepatic disease patients by performing the confirmatory test, and further to confirm HCV infection. Methods Two recombinant immunoblot assays (CWT and CHIRON RIBA HCV 3.0 Strip Immunoblot Assay) were used respectively to detect anti-HCV in 477 human serum samples, which comprised 350 HCV-infected patients' specimens, 7 none-A none-E hepatitis specimens, 30 HBV-infected patients' specimens, 30 hepatitis E virus infected patients'specimens, and 60 specimens drawn from blood donors. The latter three groups served as controls. Results A total of 120 control non-HCV-infected patients' specimens were negative when tested by both assays. Among 350 HCV-infected patients, 341 were positive and 9 were indeterminated by CWT assay; 343 were positive and 7 were indeterminated by CHIRON RIBA HCV 3. 0 SIA. Seven none-A none-E hepatitis specimens tested by both assays turned out to be 2 positive, 4 negative and 1 indeterminate. The consistency rate of these two assays was 99. 16% (Kappa=0.98). Conclusion CWT assay is highly coherent with CHIRON RIBA HCV 3.0 SIA assay in the methodology of anti-HCV antibody detection, which can be applied in the determination of HCV infection among none-A none-E hepatitis patients.

Correlation analysis of bispectral index,spectral edge frequency of electroencephalogram with midazolam-induced sedation / 中华麻醉学杂志

Objective: To analyse correlation of bispectral index,spectral edge frequency of electroencephalogram with midazolam-induced sedation. Method: 30ASA grade Ⅰ-Ⅱ adult patients, undergoing elective surgery under regional anesthesia were randomly devided into three groups according to intravenous bolus doses of midazolam,i, e. group Ⅰ:0.05mg?kg~(-1),group Ⅱ:0.1mg?kg~(-1),group Ⅲ:0.2mg?kg~(-1). After an intravenous bolus dose of mida zolam was administered,both bispectral index (BIS), 95% spectral edge freguency (SEF) of electroencephalogram were monitored and their correlation with midazolam induced sedation was analysed. Result: Both BIS and 95% SEF-correlated with midazolam-induced sedation significantly (r= 0.86,0.73, P