Micro/nano topological modification of TiO2 nanotubes activates Thy-1 signaling to control osteogenic differentiation of stem cells.

Micro/nano topological modification is critical for improving the in vivo behaviors of bone implants, regulating multiple cellular functions. Titania (TiO2) nanotubes show the capacity of promoting osteoblast-related cell differentiation and induce effective osseointegration, serving as a model material for studying the effects of micro/nano-topological modifications on cells. However, the intracellular signaling pathways by which TiO2 nanotubes regulate the osteogenic differentiation of stem cells are not fully defined. Thy-1 (CD90), a cell surface glycoprotein anchored by glycosylphosphatidylinositol, has been considered a key molecule in osteoblast differentiation in recent years. Nevertheless, whether the micro/nano topology of the implant surface leads to changes in Thy-1 is unknown, as well as whether these changes promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Here, TiO2 nanotubes of various diameters were prepared by adjusting the anodizing voltage. qPCR and immunoblot were carried out to assess the mechanism by which TiO2 nanotubes regulate Thy-1. The results revealed Ti plates harboring TiO2 nanotubes ∼100-nm diameter (TNT-100) markedly upregulated Thy-1. Subsequently, upregulated Thy-1 promoted the activation of Fyn/RhoA/MLC Ⅱ/F-actin axis, which enhanced the nuclear translocation of YAP. After Thy-1 knockdown by siRNA, the Fyn/RhoA/MLC Ⅱ/F-actin axis was significantly inhibited and TiO2 nanotubes showed decreased effects on osteogenic differentiation. Therefore, Thy-1 upregulation might be a major mechanism by which micro/nano-topological modification of TiO2 nanotubes promotes osteogenic differentiation in BMSCs. This study provides novel insights into the molecular mechanism of TiO2 nanotubes, which may help design improved bone implants for clinical application.

Progress of synergistic factors of histone lysine specific demethylase 1 in colorectal cancer / 肿瘤研究与临床

The expression of histone lysine-specific demethylase 1 (LSD1) in colorectal cancer cells is increased, and LSD1 is closely related to its occurrence, development, proliferation, invasion and metastasis. LSD1 is a demethylase whose function depends on flavin adenine dinucleoside. It can specifically catalyze the demethylation reaction of histone lysine, and regulate the expression of target genes by reaction of demethyl and dimethyl (H3K4me, H3K4me2, H3K9me, and H3K9me2) at the 4th and 9th positions of lysine H3. Targeted inhibition of LSD1 has been proved to be able to exert significant anti-tumor effect, but since the tumors involve multiple centers and factors, later studies have found that single inhibition of LSD1 cannot completely and effectively kill tumor cells. Moreover, the specificity of the LSD1 catalytic substrate depends to a large extent on the synergistic factors that bind to it and form complexes. The double-target inhibitors based on LSD1 shows more remarkable effect in tumor inhibition. Therefore, finding the combined synergistic factors of LSD1 may provide the basis for the research of multi-target inhibitors.

Influences of interaction between dengue virus type 2-infected human umbilical vein endothelial cells and macrophages on major inflammatory cytokines / 中华微生物学和免疫学杂志

Objective To study the influences on the production of major inflammatory cytokines after co-culturing macrophages with human umbilical vein endothelial cells ( HUVECs) that were infected with dengue virus type 2 (DENV-2). Methods Density gradient centrifugation was used to isolate periph-eral blood mononuclear cells ( PBMC) from concentrated human leukocytes. Adherent monocytes in culture flasks were obtained and stimulated with macrophage colony-stimulating factor ( M-CSF) to prepare macro-phages. The purity of CD14+CD11b+ cells was measured by flow cytometry. Changes in the expression of NS1 at mRNA level in HUVECs were detected by real-time PCR following DENV-2 infection. DENV-2-in-fected HUVECs were co-culture with macrophages in Transwell chambers. A control group was set up by pre-treating HUVECs with sphingosine-1-phosphate (S1P) type 1 (S1P1)-specific receptor agonist CYM-5442 for 24 h to remove the drug before infection and then co-culturing the infected cells with macrophages. Real-time PCR was used to detect the expression at mRNA level of IL-6 and IL-8 in HUVECs and IL-6, IL-8, TNF-α and IL-1β in macrophages. A double-antibody sandwich ELISA was used to detect the expression of above cytokines in culture supernatants. Results After HUVECs were infected with DENV-2, expression of NS1 gene at mRNA level gradually increased to the peak at 24 h (2. 66±0. 53, P<0. 05) and then de-creased. The purity of macrophages detected by flow cytometry was (89. 16±2. 07) ％. Expression of IL-6 and IL-8 at mRNA level in DENV-2-infected HUVECs was up-regulated. The peak values reached at 24 h of IL-6 and IL-8 expression were 16. 10±0. 17 and 29. 76±0. 58, while the expression levels at 24 h in the un-infected group were 1. 46±0. 67 and 1. 60±0. 54, respectively. Expression of IL-6, IL-8, TNF-αand IL-1βat mRNA level in DENV-2-infected macrophages was increased significantly. The levels of IL-6, IL-8, TNF-αand IL-1β expression at 24 h were 45. 82±3. 72, 52. 34±1. 69 (12 h), 8. 94±1. 75 and 30. 96±1. 44 in the infected macrophages, and 1. 16±0. 22, 1. 15±0. 21, 1. 11±0. 09 and 1. 47±0. 31 in the uninfected group. Expression of these cytokines was decreased at every time points after co-culturing of DENV-2-infec-ted HUVECs with macrophages, but still significantly higher than that in the uninfected group. In the co-cul-ture group with DENV-2 infection, CYM-5442 pretreatment significantly decreased the expression at mRNA level of IL-6 and IL-8 in HUVECs (P<0. 01) and that of IL-6, IL-8, TNF-αand IL-1βin macrophages (P<0. 01). Conclusions DENV-2 could infect primary HUVECs, and then activate macrophages to promote the secretion of large amounts of IL-6, IL-8, TNF-αand IL-1β. Moreover, the activated macrophages could reduce the production of inflammatory cytokines in HUVECs to a certain extent.

Influences of interaction between DENV-2-infected HUVECs and regulatory T cells on major inflam-matory cytokines / 中华微生物学和免疫学杂志

Objective To investigate the influences on major inflammatory cytokines after co-cul-turing regulatory T cells (Treg) with human umbilical vein endothelial cells ( HUVECs) that were infected with dengue virus type 2 (DENV-2). Methods Peripheral blood mononuclear cells (PBMC) were extrac-ted from concentrated human leukocytes by density gradient centrifugation. Treg cells were sorted by immu-nomagnetic beads. Expression of CD4,CD25 and CD127 molecules on the membrane of Treg cells was detec-ted by flow cytometry to identify the purity of Treg cells. HUVECs pretreated with or without sphingosine-1-phosphate S1P type 1 (S1P1)-specific receptor agonist CYM-5442 for 24 h were first infected with DENV-2 and then co-cultured with Treg cells. Expression of IL-6,IL-8,TNF-α,IL-10 and TGF-β at mRNA level was detected by real-time RT-PCR. Levels of IL-6,IL-8,IL-10 and TGF-β in the culture supernatants were detec-ted by a double-antibody sandwich ELISA. Results The purity of Treg cells was (84. 3±0. 5)%. Expression of NS1 at mRNA level in DENV-2-infected HUVECs first gradually increased and then decreased after reac-hing the peak at 24 h (3. 03±0. 26, P<0. 01). Enhanced expression of IL-6,IL-8 and TNF-α at mRNA level in HUVECs was observed after DENV-2 infection ( P<0. 01). Expression of these cytokines at every time point was decreased after co-culturing DENV-2-infected HUVECs with Treg cells ( P<0. 05),but was still higher than that before infection. CYM-5442 pretreatment decreased the expression of IL-6,IL-8 and TNF-α at mRNA level in DENV-2-infected HUVECs and inhibited the secretion of IL-10 and TGF-β by Treg cells that were co-cultured with DENV-2-infected HUVECs. Conclusion Primary HUVECs infected by DENV-2 can enhance the secretion of IL-10 and TGF-β by Treg cells,and the suppressive cytokines produced by Treg cells can reduce the production of inflammatory cytokines by DENV-2-infected HUVECs.

Effects of interaction between dengue virus type 2-infected HUVECs and human CD4+T cells on the expression of adhesion molecules and immunosuppressive factors / 中华微生物学和免疫学杂志

Objective To investigate the effects of interaction between human umbilical vein endothelial cells (HUVECs) which were infected with dengue virus type 2 (DENV-2) and CD4+T cells on the expression of ICAM-1 (intercellular adhesion molecule 1),VCAM-1 (vascular cell adhesion molecule 1),IL-10 and TGF-β1 at mRNA level for further understanding the immunological mechanism of DENV infection.Methods HUVECs were treated with CYM-5442,a selective agonist for sphingosine-1-phosphate receptor 1 (S1P1),for 24 hours and then infected with 103 TCID50 (50% tissue culture infective dose) of DENV-2 before co-culturing with CD4+T cells.Changes in the expression of NS1 (DENV-2 nonstructural protein),SPHK1 (sphingosine kinase 1,phosphorylating sphingosine to S1P),ICAM-1,VCAM-1,IL-10 and TGF-β1 at mRNA level were detected by real-time PCR after 4,8,12,24,48 and 72 hours of co-culturing.Results There was a certain timeliness in the expression of NS1 at mRNA level after infecting HUVECs with DENV-2 and the expression reached a peak at 24 h.Treating HUVECs with or without CYM-5442 had no significant influence on the expression of DENV-2 NS1 at mRNA level.The expression of SPHK1 at mRNA level was significantly increased after treating HUVECs with CYM-5442 and DENV-2 (P<0.05).Compared with DENV-2-infected or untreated HUVECs,Co-culturing DENV-2-infected HUVECs with CD4+T cells increased the expression of ICAM-1 and VCAM-1 in HUVECs at mRNA level (P<0.01) as well as the expression of IL-10 in CD4+T cells at mRNA level (P<0.05),but had no significant influence on the expression of TGF-β1 in CD4+T cells at mRNA level.Conclusion This study shows that DENV-2 can replicate and proliferate in HUVECs,but CD4+T cells inhibit the replication and proliferation.CD4+T cells play an important role in promoting the expression of VCAM-1 and ICAM-1 in DENV-2-infected HUVECs at mRNA level,activating HUVECs and increasing inflammation,which may be associated with increased vascular permeability induced by DENV-2 infection.Co-culturing CD4+T cells with DENV-2-infected HUVECs promotes the expression of IL-10 in CD4+T cells at mRNA level,but has no significant effect on TGF-β1.

Prevalence of Hepatitis B Virus Infection in Systemic Lupus Erythematosus Patients / 现代检验医学杂志

Objective To investigate the prevalence of Hepatitis B virus(HBV)infection in systemic lupus erythematosus (SLE)patients.Methods HBV and HCV serological tests performed in the Gulou Hospital Affiliated to Medical College of Nanjing University from January 2010 to March 2015 were retrospectively investigated for analysis HBV and HCV infection rate.The clinical testing data of 866 SLE inpatients (SLE group)from January 2010 to March 2015 were retrospectively in-vestigated for analysis HBV and HCV infection rate.The serological tests performed in 1 795 health examination people (Control group)to estimate the HBV and HCV infection ratein general population using ELISA.Compare the difference of HBV/HCV infection rate between SLE group and Control group.Results In the SLE group,17 patients were HBsAg posi-tive,the positive rate was 1.96%.In the control group HBsAg postive 204 patients,the positive rate was 11.4%,there were significant differences between these two groups (χ2 =67.81,P <0.0001).The HBsAg positive rate was lower in male SLE patients compared with controls (5.26% VS 13.9%,χ2 =4.58,P <0.05).For the female SLE patients,the HBsAg positive rate was significantly lower than the control (1.64% VS 8.12%,χ2 = 35.65,P <0.0001).The HBsAg positive rate was lower in SLE group compared with control group among different age groups,and the difference was significant in 21~30, 31~40 and 41~50 age group (χ2 =21.86,22.78,20.36;all P <0.001).There had no statistical difference between SLE and control group for the HBsAb positive rate(Total,χ2 =0.50,P =0.48;Male,χ2 =0.12,P =0.73;Female,χ2 =2.00,P =0.16).Conclusion The prevalence of HBV infection in SLE patients was lower than general population.

Retrospective Analysis of Epstein Barr Virus or Cytomegalovirus Infection in Patients with Systemic Lupus Erythematosus / 现代检验医学杂志

Objective To analyze the epidemiological characteristics of Epstein Barr virus (EBV)or cytomegalovirus (CMV) infection in systemic lupus erythematosus (SLE)patients in order to provide reference for clinic.Methods The clinical data of 202 female cases in-patients diagnosed with SLE (SLE group)from January 2012 to May 2015 in Nanjing Drum Tower Hospital were analyzed retrospectively.Meanwhile,203 cases including female renal transplant donors,obstetrics and gyne-cology in-patients selected randomly were enrolled as control group.The infection rate between SLE and control groups was analyzed and compared based on the results of EBV-DNA and CMV-DNA in peripheral blood quantified by real time PCR. Results The positive rate of EBV-DNA in SLE group was 11.39% (23/202),with significantly statistical difference when comparing with the control group [3.45% (6/174)](χ2 = 8.28,P < 0.01).The positive rate of CMV-DNA [7.92% (16/202)]was significantly higher than that in control group [1.97% (4/203)](χ2 =7.64,P <0.05).In addition,the positive rate of EBV-DNA and CMV-DNA was also greatly higher in reproductive-age (20~45 years old)of SLE patients than those in control group,10.94%(14/128)vs 3.45%(4/116)(χ2 =4.99,P <0.05)and 7.75%(10/129)vs 2.22%(3/135)(χ2 =4.31,P <0.05),respectively.Conclusion SLE patients were more inclined to be accompanying infected by EBV or CMV, indicating the possible correlation between SLE and EBV or CMV infection;and physicians should pay more attention to the viral infection of SLE in clinical treatment.

Ribosomal protein S7 affects apoptosis of HeLa cervical cancer cells / 国际肿瘤学杂志

Objective To preliminarily investigate the effect of ribosomal protein S7 on apoptosis of HeLa cervical cancer cells.Methods The previously constructed recombinant plasmid pIRES2-EGFP-RPS7 was transfected into HeLa cells,the empty vector pIRES2-EGFP transfected cells as control.Enhanced green fluorescent protein(EGFP)expressing cells were quantified by flow cytometry,and RPS7 protein level was also determined by Western blotting.Cell apoptosis of both RPS7 over-expression cells and knockdown cells were evaluated by flow cytometry after staining using allophycocyanin labeled Annexin-V.Results Apoptotic cell level in the obtained RPS7 transient over-expression HeLa cells was significantly higher than that of vector con-trol cells [(1 0.00 ±0.60)% vs (5.73 ±0.61 )%],with a statistic difference (t =8.63,P =0.001 ). Moreover,the apoptotic level in RPS7 knockdown cells was lower than that in control cells [(3.08 ± 0.49)% vs (5.97 ±0.63)%],with a statistic difference (t =6.40,P =0.003).Conclusion Up-regula-tion of RPS7 may promote apoptosis,while down-regulation of RPS7 may inhibit apoptosis of HeLa cells,indi-cating that RPS7 may play roles in regulating cell apoptosis.

Genetic Variation of the VP1 Gene of the Virulent Duck Hepatitis A Virus Type 1 (DHAV-1) Isolates in Shandong Province of China / 中国病毒学·英文版

To investigate the relationship of the variation of virulence and the external capsid proteins of the pandemic duck hepatitis A virus type 1(DHAV-1) isolates,the virulence,cross neutralization assays and the complete sequence of the virion protein 1(VP1) gene of nine virulent DHAV-1 strains,which were isolated from infected ducklings with clinical symptoms in Shandong province of China in 2007-2008,were tested.The fifth generation duck embryo allantoic liquids of the 9 isolates were tested on 12-day-old duck embryos and on 7-day-old ducklings for the median embryonal lethal doses(ELD50s) and the median lethal doses(LD50s),respectively.The results showed that the ELD5s of embryonic duck eggs of the 9 DHAV-1 isolates were between 1.9 × 106/mL to 1.44 × 107/mL,while the LD50s were 2.39 × 105/mL to 6.15 × 106/mL.Cross-neutralization tests revealed that the 9 DHAV-1 isolates were completely neutralized by the standard serum and the hyperimmune sera against the 9 DHAV-1 isolates,respectively.Compared with other virulent,moderate virulent,attenuated vaccine and mild strains,the VP1 genes of the 9 strains shared 89.8％-99.7％ similarity at the nucleotide level and 92.4％-99.6％ at amino acid level with other DHAV-1 strains.There were three hypervariable regions at the C-terminus(as 158-160,180-193 and 205-219) and other variable points in VPI protein,but which didn't cause virulence of DHAV-1 change.

Gambogic acid induces Raji cell apoptosis in vitro and its mechanism / 白血病·淋巴瘤

Objective To study the inducing apoptosis effect of a traditional Chinese medicine gambogic acid (GA) on Raji cell line and its mechanism. Methods The effect of GA on the proliferation of human peripheral blood mononuclear cells induced by lipopolysaccharide (LPS) was analyzed. Raji cells were treated with GA at different concentrations and times, and the inhibitory effect was detected by MTT assay.Apoptosis induced by GA was observed by Annexin V/PI doubling staining and flow cytometry assay.Mitochondrial membrane potential was measured by JC-1 assay. Activated Caspase-3, Caspase-8 and Caspase-9 in living Raji cells were measured by caspGLOWTM fluorescein staining kit and quantificated by flow cytometry. Results After incubation with GA, the proliferation rates of both normal blood mononuclear cells and Raji cells were dramatically inhibited in a concentration dependent manner. GA induced Raji cells to undergo apoptosis. GA decreased the mitochondrial membrane potential of Raji cells. GA increased the level of activated caspase 3, caspase 8, caspase 9 for 0.37 %, 33.57 %, 18.27 % in 24 h and 28.2 %, 69.2 %,76.7 % in 48 h respectively. Conclusion GA have an inhibitory effect on Raji cells, and can trigger apoptosis of Raji cells through both intrinsic and extrinsic pathways.

Killing K562 cells by RNA interference compared with imatinib / 肿瘤研究与临床

Objective To compare RNA interference (RNAi) with imatinib in killing K562 cells. Methods Design effective shRNA sequences special for bcr-abl silencing and insert them into the eukaryotic expression vector for RNAi by gene engineering. The recombinant plasmi(ts were then transfected into K562 cells. 48 hours later, the efficiency of transfection was identified by fluorescent microscope, bcr-abl mRNA level was detected by RT-PCR. Another group of K562 cells were treated respectively by imatinib with different concentration. All groups of K562 cells were finally analyzed in apoptosis, cell proliferation and phosphotyrosine-containing proteins. Results Both RNAi and imatinib induced apoptosis, decreased proliferation and reduced phosphotyrosine-containing proteins. Conclusion BNAi can kill K562 cells successfully as imatinib, and it may be a promising way to treat CML patients in clinic, especially for those who fail in imatinib or other chemotherapy.

The influences of long-term cryopreservation on cell viability and lymphocyte phenotypes of peripheral blood HS/PC collections / 中国实用内科杂志

Objective To study the influences of long-term cryopreservation on nucleated cell viability,colony-form- ing units-granulocyte-macrophage(CFU-GM)-forming capability and Lymphocyte immune-phenotypes of pe- ripheral blood hematopoietic stem/progenitor cell(HS/PC)collections.Methods Fresh samples(n=12)were as control,frozen samples were divided into two groups:group 1(cryopreserved for 3-5 years,n=26)and group 2 (cryopreserved for 5-7 years,n=9).Flow cytometry(FCM)method based on 7 AAD or caspase-3staining was ap- plied to assess the degree of cell necrosis or apoptosis in cryopreserved and red-cell lytic collection samples.Lympho- cyte immune-phenotypes(CD3/CD4/CD8/CD19/CD16+56,CD4/CIM5RA/CIMSRO,CD8/CD28)were also de- tected by FCM.CFU-GM culture was done to study the hematopoietic activity.Results Percentages of necrosis and apoptosis cells of the control were both less than the groupl and group2(P

Expression of Th1/Th2 cytokines stimulated by CpG-ODN 2216 in lymphocytes of peripheral blood / 临床检验杂志

Objective To investigate the Th1/Th2 polarization of T lymphocytes in human peripheral blood stimulated by CpG-ODN2216,and the secretion of cytokines in supernatant of cultured PBMCs after stimulation of CpG-ODN2216.Methods Human PBMCs were isolated from blood of donors.The PBMCs were incubated with CpG-ODN2216 for 24 hours.Th1/Th2 subsets in the cultured PBMCs were examined by flow cytometry,and IFN-?,IL-2,IL-4 and IL-10 in the supernatant were assayed by ELISA.Results The percentage of Th1 and Tc1 increased significantly after stimulation of CpG-ODN2216 compared with control group (P0.05).Conclusion The Th1 cells and Tc1 cells in T lymphocytes of peripheral blood could be polarizated by CpG-ODN 2216.IFN-? secretion in PBMCs could be induced by CpG-ODN2216.

On exploration into the medicine of crititcal disease and disciplinary construction / 中华医院管理杂志

The medicine of critical disease is a clinical discipline that gradually sprang up in the late 1960s.Yet at present controversy still exists as to whether the discipline should be regarded as a separate one and it has not been ranked among the separate disciplines listed by the State Educational Committee. The authors, however, give an account of the customers and the cunent situation of disciplinary development of the discipline of critical disease from the petspective of a discipline. They also offer an account of the organizational structure and the form of staff of ICUs,pointing out that ICUs have 3 subjects structurally and are divided into CategoriesⅠ,Ⅱand Ⅲ. Except for ICUs in a few hospitals that are up to the level of Category Ⅱl, ICU s in most hospitals in China are lower than or just up to the level of Category Ⅰ. Then the authors give an introduction to the three forms of ICU management: open, semi-open and closed.

On exploration into the medicine of critcal disease and dsciplinary construction / 中华医院管理杂志

The medicine of critical disease is a clinical discipline that gradually sprang up in the late 1960s. Yet at present controversy still hats as to whether the discipline should be regarded as a separate one and it has not been ranked among the separate disciplines listed by the State Educational Committee. The authors, however, give an account of the customers and the current situation of disciplinary development of the discipline of critical disease ho the perspective of a discighne. They also offer an account of the organizational structure and the form of staff of ICUs, pointing out that ICUs have 3 subjects structurally and are divided into CategoriesⅠ,Ⅱ and Ⅲ. Except for ICUs in a few hospitals that are up to the level of Category n, ICUs in most hospitels in China are lower than or just up to the level of CategoryⅠ. ho the authorss give an introduction to the ho formsn of ICU management open, semi-opened-cpc ana closed.