Acupuncture Therapy ; Adult ; Aged ; Combined Modality Therapy ; Female ; Humans ; Male ; Middle Aged ; Needles ; Periarthritis ; therapy ; Punctures ; Treatment Outcome

Objective To investigate the effects of standardized tertiary rehabilitation (STR) on limb motor function (LMF) after stroke.Methods Eighty-two patients were divided into a primary cerebral infarction group (PCI group) and a primary cerebral hemorrhage group (PCH group),and then randomly further divided into experi- mental and control sub-groups.All patients received routine internal medicine treatment,supplemented with stand- ardized tertiary" rehabilitation in the experimental groups.All patients were assessed with the simplified Fugl-Meyer motor function assessment (S-FMMFA) at enrollment,and 1,3 and 6 months after their stroke.Results The scores of the experimental groups were higher than those of the controls.The experimental groups scores were 26.10% of normal at the time of the enrollment,and improved to 42.52%,65.62% and 83.71% by the end of the 1st,3rd and 6th month,respectively.The control groups started at 18.51%,and progressed to 24.85% ,37.24% and 45.84% over the same interval.Conclusion STR was associated with improved LMF scores of stroke pa- tients.

OBJECTIVETo prepare the genetic engineering regulatory T cells (Treg) via the self-inactivating (SIN) lentiviral vectors carrying Foxp3 gene, and assay the phenotype and abilities of its proliferation and immunosuppression.

METHODSThe bicistronic SIN lentiviral transfer plasmid containing Foxp3 gene and internal ribosomal entry site-green fluorescent protein gene (IRES-GFP) was constructed. Human embryonic kidney 293T cells were co-transfected using liposome by lentiviral packing system, which included the packaging plasmid Delta NRF, the transfer plasmid and the envelope plasmid VSVG. The efficiency of gene transduction and the expressions of Foxp3, CD25, GITR, CTLA-4 of CD4(+)CD25(-) T cells, which were isolated by magnetic beads from the spleen, and then co-cultured with 293T cells, were detected by flow cytometry (FCM). The proliferative and suppressive capacities of transduced T cells were estimated by Cell Count Kit-8 (CCK-8) and the cytokine production was performed by ELISA.

RESULTSThe lentiviral transfer plasmid pXZ208-Foxp3-IRES-GFP was successfully constructed, the virus titers were above 10(6) IU/ml in the supernatant. pXZ208-IRES-GFP was used as control group. After cocultured, the CD4(+)CD25(-) T cells expressed significantly higher Foxp3, CD25, GITR and CTLA-4 in experimental group than in control group. Upon stimulation with anti-CD3 epsilon and APCs, the proliferative capacity of Foxp3-transduced T cells and the production of IL-2, IL-4, IL-10, IFN-gamma were significantly lower than those in control group (P < 0.01); Foxp3-transduced T cells also significantly inhibited the proliferation of CD4(+)CD25(-) T cells.

CONCLUSIONSThe genetic engineering Treg mediated by SIN lentiviral vectors are successfully constructed and their phenotype and function are similar to natural CD4(+)CD25(+) Treg.

Animals ; Cell Proliferation ; Cells, Cultured ; Forkhead Transcription Factors ; genetics ; metabolism ; Genetic Engineering ; Genetic Vectors ; HEK293 Cells ; Humans ; Lentivirus ; genetics ; Mice ; Mice, Inbred BALB C ; Phenotype ; T-Lymphocytes, Regulatory ; cytology ; immunology ; metabolism ; Transfection

OBJECTIVETo explore the influence of the lentiviral vector mediated mouse genetic engineering regulatory T cells (Treg) infusion on post-allogeneic bone marrow transplantation (allo-BMT) acute graft-versus-host disease (GVHD) in mice.

METHODSLentivirus-mediated Forkhead box P3 (Foxp3) gene was transduced into BALB/c mice CD4(+)CD25(-) T cells (Treg) to construct engineered Tregs in vitro. An allo-BMT model of BALB/c to C57BL/6 mice was established. After irradiation, the recipients were injected with donor cells plus the genetic engineering Tregs. Survival time, clinical GVHD score, histopathological findings, activation of donor T cells or serum levels of inflammatory cytokines were observed after allo-BMT.

RESULTSThe mean survival times for radiation alone group (Gp I), transplantation control group (Gp II), engineering Treg infusion group (Gp III) and empty vector control group (Gp IV) were (8.8 ± 0.6) d, (36.7 ± 2.5) d, (51.6 ± 4.0) d and (34.1 ± 2.3) d, respectively. The survival time was significantly longer in Gp III than in other groups (P < 0.05). Histopathological finding in several target organs (skin, liver and small intestine) confirmed the presence of severe GVHD in Gp II and Gp IV, while no histological signs of GVHD were observed in long survival recipients in Gp III, and clinical GVHD scores in Gp III were significantly lower than that in Gps II and IV. The numbers of donor T cells and the percentage of IFN-producing donor T cells in the spleen of recipients in Gp III were significant lower than those in Gps II and IV at days 3 and 4, and at day 3 after transplantation, respectively (P < 0.05). The serum levels of IFN-γ, IL-2 and TNF-α were increased at day 21 to 28 after transplantation in all groups. The peak concentrations of IFN-γ, IL-2 and TNF-α in Gp III were significantly lower than those in Gps II and IV control groups at day 21 (P < 0.05).

CONCLUSIONCo-injection of genetic engineering Treg can efficiently prevent recipients from lethal GVHD after allo-BMT in mice by inhibiting the early activation and expansion of donor T cells and reducing the serum levels of inflammatory cytokines.

Animals ; Bone Marrow Transplantation ; adverse effects ; Cytokines ; blood ; Female ; Forkhead Transcription Factors ; genetics ; Genetic Engineering ; Genetic Vectors ; Graft vs Host Disease ; immunology ; Lentivirus ; Lymphocyte Activation ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; T-Lymphocytes, Regulatory ; cytology ; immunology ; Transduction, Genetic ; Transplantation, Homologous

OBJECTIVETo evaluate the effectiveness of anterior versus posterior surgical treatments of thoracolumbar fractures.

METHODSRandomized controlled trials (RCTs) and clinical controlled trials (CCTs) were identified from MEDLINE (1966 - 2006.7), EMBASE (1966 - 2006.7), PubMed (1996 - 2006.7), Cochrane Library (Issue 2, 2006).We hand-searched Chinese Journal of Orthopedics (from establishment to May 2006) and Orthopaedic Journal of China (from establishment to May 2006). RCTs and CCTs were included. Data were extracted by two reviewers with designed extraction form. RevMan 4.2.8 software was used for data analysis.

RESULTSTwo RCTs and four prospective clinical trials were included. The combined results showed that compare with posterior surgical management, anterior approach in the treatment of thoracolumbar fractures proved the less incidence of complications; better neurologic recovery and corrected kyphosis angle; more complete and reliable decompression of the canal. However, there was not difference between the two groups in the general status outcomes.

CONCLUSIONSTo compare with posterior fixation system, anterior surgical managements in the thoracolumbar spinal trauma might be the optimal choices because the lower rates of complications and loss of corrected kyphosis angle; better neurologic recovery, also. Besides, due to the lack of Evidence-based guidelines for the treatment of thoracolumbar spinal injuries, the results which indicated above need further study.

Adult ; Female ; Fracture Fixation, Internal ; methods ; Humans ; Lumbar Vertebrae ; injuries ; Male ; Spinal Fractures ; surgery ; Thoracic Vertebrae ; injuries ; Treatment Outcome

Antimicrobial Cationic Peptides ; biosynthesis ; genetics ; DNA-Binding Proteins ; biosynthesis ; genetics ; Humans ; Microbial Sensitivity Tests ; Muramidase ; biosynthesis ; genetics ; Peptides, Cyclic ; biosynthesis ; genetics ; Pichia ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

OBJECTIVETo investigate the effect of AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) on proliferation, differentiation and apoptosis of U937 cells and explore its possible mechanism.

METHODSU937 cells were cultured with different concentrations of AICAR for 24 h and 48 h. Cell proliferation was evaluated. Cell growth curve was analyzed by CCK-8; cell apoptosis was analyzed by cell morphology, Annexin V/7-AAD double labeling. The differentiation of U937 cells was evaluated by expression of CD11b. The Bcl-xL, Bax, Bim, caspase-3 mRNA expressions of U937 cells were determined by real time PCR.

RESULTSAICAR significantly inhibited the growth of U937 cells in a time-and dose-dependent manner, with a 24 h IC50 value of 1.1 mmol/L and 48 h of 0.9 mmol/L. 1.0 mmol/L AICAR didn't induce differentiation of U937 cells with the increase of CD11b expression for 24 h (P > 0.05). The U937 cells apoptosis was confirmed by cell morphology and Annexin V/7-AAD labeling. AICAR induced apoptosis of U937 cells and the apoptosis rate was (6.81 ± 1.16)% at 1 mmol/L AICAR higher than control group (2.74 ± 0.32)% without AICAR for 24 h treatment (P < 0.05). The real time PCR assay revealed that as compared with control group, the expression of Bim and caspase-3 mRNA were increased, while Bcl-xL and Bax were unchanged on the AICAR treatment.

CONCLUSIONAICAR can effectively inhibit proliferation and induce apoptosis of U937 cells. However, it has no significant effect on differentiation of U937 cells. The mechanism may be related with up-regulating Bim and Caspase-3.

Aminoimidazole Carboxamide ; analogs & derivatives ; pharmacology ; Apoptosis ; drug effects ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Humans ; Ribonucleotides ; pharmacology ; U937 Cells

Recently, gene therapy has been become a promising approach to cure hemophilia A, a most common recessive bleeding disease. The aim of this study was to determine the perspective of lentiviral vector in hemophilia A gene therapy in vitro and in NOD/SCID mice. Lentivirus transfer vector pXZ9/BDDFVIII containing human B-domain-deleted Factor VIII-IRES-eGFP coding sequence and mock control pXZ9 were constructed. Lentivirus was prepared by co-transfecting 3 plasmids into 293FT cells. 293FT, HLF, human bone marrow mesenchymal stem cells and Chang-liver cells were transfected with the prepared virus. Coagulant activity of human FVIII, human FVIII antigen, human FVIII mRNA transcription and genomic integration were assayed by ELISA, one-step method, RT-PCR and PCR after infection. Lentiviral particles were concentrated by ultracentrifugation and NOD/SCID mice were transfected via portal vein injection. Human FVIII antigen in mouse blood plasma was analyzed by ELISA. eGFP expression was observed by fluorescent microscopy and human FVIII transcription in mouse liver was analyzed by RT-PCR at one month after transduction. The results showed that the high titer of recombinant virus was prepared and used to efficiently transduce the target cells in vitro. At 72 h after transfection, high levels of FVIII activity and FVIII antigen were detected. Human FVIII gene transcription could be detected in the liver of NOD/SCID mice received lentiviral particles carrying FVIII gene. Mouse hepatocytes were transfected with recombinant lentivirus efficiently in vivo. Human FVIII level in mouse blood plasma reached to (49 ± 6) mU, (54 ± 8) mU and (23 ± 4) mU at 72 h, one week and one month after transfection respectively. It is concluded that the lentiviral particles carrying BDDhFVIII gene can high efficiently transfect the target cells both in vitro and in vivo, and the transfected target cells can secrete hFVIII efficiently. The sustained expression of human FVIII in NOD/SCID mice is observed after lentivirus transfection via portal vein injection.
Animals ; Cell Line ; Factor VIII ; genetics ; Gene Expression ; Genetic Therapy ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Peptide Fragments ; genetics ; Plasmids

OBJECTIVETo explore gene expression of NANOG in T-cell acute lymphoblastic leukemia (T-ALL) cell lines and the effects of NANOG gene down-regulation on apoptosis of leukemia cells.

METHODSReal-time PCR (RT-PCR) and Western blot were used to detect the expression level of NANOG gene and protein in MOLT-4, CCRF-HSB2 and Jurkat cells. To test the efficiency of RNA interference, MOLT-4 cells were firstly infected by lentiviral vectors, which were successfully constructed with NANOG specific shRNA. NANOG expression levels were subsequently re-evaluated by RT-PCR and Western blot. The percentages of early apoptotic cells (Annexin V⁺/7-AAD⁻) and late apoptotic cells (Annexin V⁺/7-AAD⁺) were analyzed by flow cytometry. The expression of apoptosis-related genes was also detected.

RESULTSBoth NANOG gene and protein expression was positive in MOLT-4 and CCRF-HSB2 cells. The lentiviral vectors pLB-shNANOG-1, pLB-shNANOG-2, and pLB-sh control were successfully constructed, as evidenced by the viral titers (1.83-3.12)× 10⁸ IU/ml. The experimental data on infection of MOLT-4 cells with such lentiviral vectors revealed that both shRNA interfering sequences (shNANOG-1 and shNANOG-2) could stably down-regulate NANOG gene and protein expressions. The percentages of early apoptotic cells in groups of shNANOG-1[(8.06 ± 1.61)%]and shNANOG-2[(5.67 ± 1.59)%]were significantly increased as compared to that of MOLT-4 group[(1.13 ± 0.40)%]or sh-control [(1.15±0.49)%](P<0.05). However, no statistical difference among them was observed for late apoptotic cells (P>0.05). The gene expression of TP53, PMAIP1, and CASP9 of either shNANOG-1 or shNANOG-2 group was augmented as compared to that of MOLT-4 group or sh-control (P<0.05). Reversely, a significant down-regulation of Bcl-2 gene expression was observed (P<0.05).

CONCLUSIONNANOG can be expressed in various human T-ALL cell lines. Down-regulation of NANOG can trigger leukemia cellular apoptosis through mitochondria-dependent apoptosis pathway.

Apoptosis ; genetics ; Cell Line, Tumor ; Down-Regulation ; Gene Expression ; Genetic Vectors ; Homeodomain Proteins ; genetics ; Humans ; Nanog Homeobox Protein ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics

This study was aimed to construct the targeting AATF shRNA eukaryotic expression vector and establish the stably transfected U937 cell lines. The sequence of AATF mRNA was obtained from GenBank. After excluding homology, three plasmid expression vectors coding shRNA targeting 228 ∼ 249, 303 ∼ 324 and 443 ∼ 464 of AATF gene sequence were synthesized. Two terminals of shRNA carried BamHI and HindIII restriction sites. The selected nucleotides were cloned into the plasmid pSilencer 3.1-H1 neo respectively, and the resultant recombinant plasmids were named as pSA-1, pSA-2, pSA-3. The sequences of the recombinant plasmids were identified by DNA sequencing. The recombinant plasmids were transfected into the cell line U937 by electroporation with Neon(TM) Transfection System. The transfected cells were persistently screened under G418 (500 mg/L), and isolated with a limited dilution for 8 weeks. The inhibition of AATF mRNA and protein expression was respectively detected by RT-PCR and Western blot. The results indicated that RNAi eukaryotic expression vectors targeting AATF had correct reading frame and nucleotide sequence. Real-time PCR revealed that AATF shRNA effectively silenced mRNA expression of AATF. Western blot analysis found that AATF shRNA obviously suppressed protein expression of AATF (P < 0.05). It is concluded that the shRNA eukaryotic expression vector has been successfully constructed which can inhibit the expression of AATF, and the establishment of stably transfected U937 cell lines provide a original route for exploring the mechanism of AATF in human Leukemia further.
Apoptosis Regulatory Proteins ; genetics ; Gene Expression ; Genetic Vectors ; Humans ; Plasmids ; RNA Interference ; RNA, Messenger ; RNA, Small Interfering ; genetics ; Repressor Proteins ; genetics ; Transfection ; U937 Cells