Objective To explore the feasibility of establishment of NOD/SCID-mouse model with multiple myeloma by using plasma cells from myeloma patients.Methods The femurs and tibias were removed from the New Zealand white rabbits;the muscles,periosteum and cartilage tissues were cleared.Then each bone was cut into two pieces gently along its middle.The NOD/SCID mice weighing 25 - 30 g (4 - 6 weeks)were anesthetized by intraperitoneal injection;rabbit bone was inserted into the right side of the mouse back and engraftment of the bones was allowed to take place after 4 weeks.The 5000 000 purified plasma cells which expressed CD38 +/CD45 - were immunofluorescence labeled and then injected slowly into the implanted rabbit bone through the distal end.The mice were observed weekly;the plasma cells growth in mice was screened by the living-imaging system and the tumor from the mice was determined by biopsy.Results The implanted rabbit bone survived after 4 weeks.The tumor in mice was observed 2 weeks after the purified myeloma cells were injected into the rabbit bone,and it reached 100 mm3 after 8 weeks.Results of the living-imaging system showed that the myeloma cells had uptake in the rabbit bone after 2 weeks of injection and this phenomenon was more pronounced after 8 weeks of injection (2.4×10 4 vs .1.5× 10 5 ,P < 0.05 ).The tumor infiltrated with numerous plasma cells and osteoclasts increased upon the biopsy. Conclusion Rabbit bone marrow implanted into NOD/SCID mice can effectively support local injection of plasma cells of multiple myeloma patients,and the NOD/SCID-mouse model of myeloma has been established.This model can be used to study in vivo experiments related to myeloma and clinical therapeutic approaches for this disease.

Objective To investigate the effects and mechanism of Sufentanil on Hep3B cell viability in liver cancer.Methods Sufentanil of different concentrations (0.01,0.1,1,5,10,1 5 μmol/L)was used to treat Hep3B cells.The changes of cell cycle,apoptosis and protein expression were detected to explore the potential mechanisms by MTT method,flow cytometry and Western blot,respectively.Results Reduced viability of Hep3B cells,arrested cell cycle in the G0/G1 phase,decreased expression of survivin protein,and increased expression of caspase-3 protein were observed with the increase of Sufentanil concentration. Conclusion Sufentanil inhibited the viability of Hep3B cells through affecting cell cycle,promoting cell apoptosis and changing protein expressions of survivin and caspase-3.

Objective To investigate the adverse effects of nano-titanium dioxide (TiO2 )on kidney tissues in healthy rats and rats with oxidative stress (OS).Methods Thirty-six healthy male SD rats were randomly divided into control group, alloxan-treated group, nano-TiO2 treated (NM) group, and alloxan and nano-TiO2 dual treatment (OS-NM)group.Nano-TiO2 of three concentrations (0.5,5 and 50 mg/kg body weight)was injected intraperitoneally.The level of OS biochemical factors and blood urea nitrogen (BUN)and pathological changes of kidney were determined.Results Compared with those in NM and OS groups,the levels of O2 -· and superoxide dismutase (SOD)in OS-NM group were significantly increased after exposure to nano-TiO2 of 5 mg/kg body weight (P <0.05).Nano-TiO2 of 50 mg/kg body weight led to significant changes of O2 -·,SOD,and glutathione (GSH) levels in OS-NM group in comparison with OS and NM groups (P <0.01).The levels of O2 -· and GSH between OS group and NM group changed significantly (P < 0.05 ).Compared with healthy rats,OS rats showed significant increased BUN level (P <0.01),cell number and edema of renal tubular epithelial cells after nano-TiO2 exposure.A synergic effect between OS condition and nano-TiO2 level was shown on the increased level of O2 -·.Conclusion Nano-TiO2 induced more adverse effects on the kidney in OS rats,suggesting a synergistic effect between nano-TiO2 and OS.This result provides experimental evidence for patients’safe use of nano-TiO2 .

Objective To investigate the inhibitory effects of docosahexaenoic acid (DHA)and 5-fluorouracil (5-FU)in combination on human gastric cancer cell line AGS in vitro .Methods Human gastric cancer line AGS was treated with different concentrations of DHA and 5-FU alone or in combination.The inhibition of cell proliferation was evaluated by MTT assay.Dose of median (IC50 )of drugs (alone or in combination)and the combination index (CI)were calculated using the median-effect equation and the combination index equation of Chou-Talalay.Flow cytometry was used to detect the cell cycle distribution.The expression of mitochondrial respiratory membrane protein complex in AGS cells was analyzed with Western blot.Results DHA and 5-FU alone or in combination could markedly suppress the proliferation of AGS in significantly time-dependent and dose-dependent manners (P <0.05).IC50 values with DHA or 5-FU administered for 24 h and 48 h were 5 1.60 μg/mL (DHA:24 h),34.82 μg/mL (DHA:48 h),45.90 μg/mL (5-FU:24 h),and 1 6.86 μg/mL (5-FU:48 h), respectively.DHA remarkably strengthened the inhibitory effect of 5-FU and decreased IC50 of 5-FU by 3.56 -2.1 5 folds.The combination of DHA and 5-FU showed synergism.Flow cytometry showed that AGS cells treated with DHA and 5-FU were arrested in G0/G1 phase and the proportion of AGS cells in G0/G1 phase increased compared with that in the control group,DHA group and 5-FU group,while the proportion of the cells in S phase decreased significantly (P < 0.05 ).Western blot showed after treatment with DHA and 5-FU for 48 h,the expression of mitochondrial respiratory membrane protein complex was significantly decreased compared with control group,DHA group and 5-FU group (P <0.05).Conclusion DHA could act synergistically with 5-FU in inhibiting the growth of gastric carcinoma cells,and meanwhile decrease the dose of 5-FU.The mechanism may be associated with cell cycle arrest in G0/G1 phase and interference in the energy metabolism of AGS cells due to inhibition of the expression of mitochondrial oxidative respiratory chain complexes by the two compounds.

Objective To investigate the effect of recombinant human erythropoietin on immune function in mice with multiple myeloma.Methods Multiple myeloma model mice were divided randomly into control group and treatment groups (low,middle and high doses).The rats in treatment groups were injected with recombinant human erythropoietin 100 μL (2.5,5.0 and 10 mg/kg)via the tail vein for 30 days,and the rats in control group were injected isotonic saline solution of the same volume instead.After the experiment,the colorimetric MTT assay,neutral red method and ELISA kit were used to evaluate the proliferation of B and T lymphocytes,the phagocytic ability of macrophages and the content of serum TNF-α.Results Compared with those in control group,different doses of recombinant human erythropoietin could significantly increase the proliferation of lymphocytes,increase the phagocytosis of macrophages and improve the production of serum TNF-α(P < 0.05 ). Conclusion Recombinant human erythropoietin can significantly improve immunoregulation of mice with multiple myeloma.

Objective To investigate the functions of triple point-mutants of hypoxia-inducible factor 1α(HIF1α)in angiogenesis in bone defect region under normoxic conditions.Methods Triple point-mutations (the 402,564 and 803 amino acids)in HIF1α coding sequence (CDS)were induced.The triple mutant of HIF1α(402/564/803)was inserted into the adenovirus pAdEasy-1 system to complete viral packaging and titer measurements. The wild-type HIF1α gene and the empty adenovirus vector were packaged in the same way.For the in vitro experiment,the rabbit bone marrow mesenchymal stem cells (MSCs)were divided into four experimental groups:A,MSCs infected with viral solution containing mutant HIF1α;B,MSCs infected with viral solution containing wild-type HIF1α;C,MSCs infected with viral solution without any HIF1α;and D,MSCs without viral infection. The efficiency of infection was observed by the expression of human renilla reniformis green fluorescent protein (hrGFP).The expression levels of HIF1α mRNA and protein in infected cells in each experimental group were measured.For the in vivo experiment,the MSCs were divided into the same four groups and infected with the virus solutions from each group and cultured under normoxic conditions.At 72h after the infection,the MSCs were used as seed cells and transplanted into Apatite-wollastonite magnetic bioactive glass-ceramic (AW MGC)vector to construct artificial tissue-engineering scaffolds,and then the scaffolds were implanted into the in vivo rabbit radial bone defect model.The animals from each group were sacrificed 8 weeks after the surgery and the tissues from the implantation region were harvested for evaluation of angiogenesis.Results The 402,564 and 803 amino acids in CDS area were point mutated into alanine;three types of recombinant adenovirus were successfully constructed, packaged and characterized.The expression levels of HIF1αmRNA were significantly higher in Group A and Group B than in Group C and Group D (P <0.05 ).There was no significant difference between Group A and Group B group or between Group C and Group D (P >0.05 ).The HIF1α protein expression in Group A was significantly higher than that in the other three groups (P <0.05);however,there was no significant difference among groups B,C and D (P >0.05).There was prominent angiogenesis in bone defect region in Group A,but not in the other three groups.Conclusion ① Triple point-mutants of HIF1αefficiently expressed functional proteins under normoxic conditions.② Triple point-mutants of HIF1αeffectively promoted in vivo angiogenesis in bone defect region.

Objective To explore the expressions and correlation of RWD containing sumoylation enhancer (RSUME),small ubiquitin-like modifier (SUMO-1),hypoxia inducible factor-1α(HIF-1α)and vascular endothelial growth factor (VEGF)in gliomas of different pathologic grade.Methods We investigated the expression levels of RSUME mRNA,HIF-1α mRNA and VEGF mRNA with reverse transcription-polymerase chain reaction (RT-PCR),and investigated the immunohistochemical staining to determine the expressions of SUMO-1,HIF-1α and VEGF in 63 cases of human gliomas of different pathologic grade and 9 cases of normal brain tissues.We studied its correlation with the pathologic grade and the relationship between the expression of RSUME promoter sumoylation and HIF-1α/VEGF pathway in gliomas.Results There were significant differences (P <0.01)in the expressions of RSUME mRNA,HIF-1αmRNA and VEGF mRNA in glioma tissues.With the increasing degree of pathologic grade in tumor specimens,the expression levels of RSUME mRNA,HIF-1α mRNA and VEGF mRNA increased markedly (P <0.01 ).There was a positive correlation of the expression levels of RSUME mRNA with HIF-1αmRNA and VEGF mRNA.There were significant differences (P <0.01 )in the expressions of SUMO-1,HIF-1αand VEGF in glioma tissues by immunohistochemical staining.With the ascending of pathologic grade of tumor specimens,the expression levels of SUMO-1,HIF-1α and VEGF increased markedly (P < 0.01 ).There was a positive correlation between the expression level of SUMO-1 and HIF-1α(r =0.857,P <0.01).The Kaplan-Meier analysis and Log-rank test showed significant differences in progress free survival (PFS)between the RSUME high-expression and low-expression groups (χ2 =36.032,P <0.01).Conclusion RSUME may enhance HIF-1α/VEGF pathway through sumoylation in gliomas.It implicates that RSUME is related to angiogenesis in gliomas and can promote tumor invasion and progression,indicating that RSUME can be a novel target in gliomas treatment.

Objective To explore the therapeutic effects of combined application of survivin antisense nucleic acid and taxol in subcutaneous xenograft mouse model of Balb/c and to preliminarily investigate the mechanism of the anticancer effects.Methods The model of subcutaneous tumor was established by hypodermic injection of C26 cells into Bal b/c mice.The mice were then randomly divided into five groups through the internal tumor injection:the blank group (C),lipo2000 group (L),paclitaxel group (T),survivin antisense nucleic acid group (A),and survivin antisense nucleic acid combined with paclitaxel group (A+T).We observed tumor growth,determined cell apoptosis by TUNEL method,and detected the expression of survivin by Western blot.Results ① All treatment groups had T/C<60%,which was significantly different from that of group L (P <0.05);the intervention was proved effective in vivo .The tumor inhibition rate of mice tumor weight showed that there were significantly curative effects in groups T,A and A+ T compared with that in group C (P < 0.05 ).The antitumor activity of paclitaxel (tumor inhibition rate of 21.82%±0.84%)could be improved by more than 59% through combination therapy (tumor inhibition rate of 54.1 6% ± 0.32%)concerning inhibition of tumor weight growth.② TUNEL method detected apoptotic cells:The tumor cells hardly had apoptosis in the blank group while T group and A group had a certain number of apoptotic cells.The experiment results suggested that PTX could promote tumor cell apoptosis,and that not only A+T strengthened the effect in killing tumor cells,but also the synergy of both could influence tumor resistance and ultimately make the effect in promoting tumor cell apoptosis conspicuous.③ The expression of survivin protein:The results showed that the expression of survivin protein in group A + T was obviously decreased without the expression of β-actin affected;it did not change significantly in group C compared with group L.The ratio of the A-value in groups T,A and A+T was 0.895 ±0.01 1,0.704 ±0.121 and 0.345 ± 0.01 9,respectively.Analysis of variance t-test showed that the expression level in group A+T obviously differed from that in groups C,L,A and T (P <0.05).Conclusion The combined therapy of survivin antisense nucleic acid and taxol can promote tumor cell apoptosis by downregulating the expression of survivin protein,reduce the body’s resistance to drugs and create synergetic effects.

Objective To study the profiles and function of small RNA (sRNA)gene during chondrogenesis in rats so as to clarify the mechanisms of chondrocytes proliferation and differentiation.Methods All the sRNAs were identified from the female SD rats femoral head cartilages at three time points:at birth,ablactation and maturation,and three sRNA libraries were constructed.The Solexa sequencing and the bioinformatics analysis were employed to be blasted with the genomes of SD rats.Results The perfect match reads in the three libraries were screened out,which were correspondent to the 21 7 921 (41.23%),1 96 650 (38.74%)and 245 436 (41.54%)unique sRNA sequence,respectively.The percentages of 20-24 nt sRNA were 71.94% (d0),72.85% (d21),and 86.39%(d42).Half of clean sequences were 22 nt sRNA.The distribution characteristics of the reads were in line with the high-quality sRNA.More than 62% clean reads were from mature miRNA while the ratios in the three libraries were only 0.69%,0.78% and 0.63%.About 60% of the unique sRNA could not be matched with miRBase20.0 or Rfam9.1.Conclusion The distribution model of miRNA in the three libraries indicates that the miRNAs with different functions or from different sources are involved in the regulation of chondrocytes proliferation and differentiation in bone development and formation.

ABSTRACT:Objective To evaluate the clinical therapeutic effect and safety of tumor necrosis factor alpha (TNF-α)blockers in treating moderately to severely active ulcerative colitis (UC)by meta-analysis.Methods Such databases as the Cochrane Central Register of Controlled Trials,PubMed,OVID,Embase,ISI,CBM,CNKI, VIP,and WanFang Data were searched from establishment to June 2013.All randomized clinical trials (RCTs)on tumor necrosis factor alpha blockers in treating UC were collected,and then selected on the basis of the inclusion and exclusion criteria.We assessed the methodological quality,extracted the data from the included articles and performed the meta-analysis with Revman 5.1.Results A total of 13 RCTs involving 3334 patients were analyzed.TNF-αblockers group was superior to the control group in the short-term clinical response (OR =2.5 1, 95% CI 1.73,3.64),short-term clinical remission (OR =2.74,95% CI 1.80,4.1 6),long-term clinical response (OR =2.98,95% CI 1.98,4.47),1ong-term clinical remission (OR =2.64,95% CI 1.89,3.67),and mucosal healing (OR =1.89,95% CI 1.39,2.59)compared with control group.TNF-αblockers could also reduce the rate of colectomy (OR =0.61,95% CI 0.41,0.89)and improve inflammatory bowel disease questionnaire scores (MD=14.74,95% CI 1 1.43,18.06 ).There was no significant difference between the two groups in all reported adverse effects (OR =1.14,95% CI 0.97,1.34)and serious adverse effects (OR=0.78,95% CI 0.56,1.09).Conclusion Compared with conventional therapy or placebo,TNF-αblocking agents can improve the therapeutics effect on UC in clinical response,clinical remission and mucosal healing,and also can reduce the rate of colectomy. In patients with moderately to severely active UC treated with TNF-α blocking agents,it is easier to achieve the improvement of life quality.TNF-αblocking agents treatment is safe for UC.This conclusion should be verified with more large-scale and high-quality RCTs.