Objective To demonstrate the clinical outcome of bilateral gluteus maximus musculocutaneous flap in conjunction with continuous postoperative negative pressure wound therapy in reconstruction of sacral soft tissue defects.Methods From January, 2008 to April, 2013, 18 patients (8 males and 10 females, aged from 34 to 78 years old) with full-thickness sacral soft tissue defects were treated.The size of the defects after initial debridement ranged from 3.0 cm × 2.0 cm to 18.0 cm × 14.0 cm, with the exposure of sacrum or ligament.Bilateral gluteus maximus musculocutaneous flap were applied in all the patients.Two drainage tubes were placed on each side of the flaps during the surgery and suck for 10 to 12 consecutive days after the operation.Results The size of the harvested flaps ranged from 12.0 cm × 8.0 cm to 18.0 cm × 12.0 cm, and all the donor sites of the flaps were closed with primary suture.Thirty-five flaps in 17 patients survived without any complication.Partial necrosis of one flap was found in 1 patient and managed successfully with conservative dressing change.Fourteen patients were followed-up ranged from 8 months to 2.5 years (mean follow-up was 18 months).Color and texture of the flaps were satisfactory and no recurrence of sacral defect was noted.Conclusion Bilateral gluteus maximus musculocutaneous flap in conjunction with continuous postoperative negative pressure wound therapy may serve as a useful option for fullthickness sacral soft tissue defects.

After half a century of self-innovation, the integrated traditional Chinese and Western medicine has witnessed the great progress in both clinical and basic research. However, the theoretical system of the integrative medicine does not break through the limitations of traditional Chinese medicine and Western medicine, which hinders its implication in experimental study and clinical work. In view of the current situation, to develop the integration of traditional Chinese and Western medicine further, efforts should be made in such aspects as educational system construction, talent personnel training, improving the level of clinical practice and corresponding basic research as well as the establishing the basic theoretical system.
Clinical Medicine ; History, 20th Century ; Humans ; Integrative Medicine ; history ; methods ; Medicine, Chinese Traditional ; history ; methods ; Research

OBJECTIVETo explore the killing effect of the mutant herpes simplex virus thymidine kinase (HSV-sr39tk) and its wild-type (HSV-tk) mediated by lentiviral vector on T lymphocytes in vitro and compare T cell survival rate after GCV or ACV treatment.

METHODSThe three-plasmid lentiviral vector system including packaging plasmid DeltaNRF, envelope plasmid VSV-G and vector plasmid (pTK151 + HSV-sr39tk or pTK151 + HSV-tk) were cotransfected into human embryonic kidney 293T cells using modified calcium phosphate precipitation methods. The packaged virus was harvested 72 h later. The survival of T cells expressing HSV-sr39tk or HSV-tk was measured by MTT assay after 4 day-culture against a gradient of GCV or ACV concentrations.

RESULTSThe three plasmids were effectively cotransfected and a high titre of lentivirus was obtained (2 x 10(6) IU/ml). 39tk(+) T cell survival rates declined promptly when the prodrug GCV/ACV concentrations increased from 0 micromol/L to 10 micromol/L. The T cell survival rates in GCV group declined from (96.04 +/- 3.23)% to (36.76 +/- 4.38)% while in ACV group from (97.31 +/- 4.61)% to (43.75 +/- 8.99)%. However, when GCV/ACV concentrations were more than 10 micromol/L, further decline of 39tk(+) T cell survival rates became unobvious. The growth rate of 39tk(+) T cell exposed to GCV or ACV was obviously lower than that in un-transfected T cells (P < 0.05). Tk(+) T cells were sensitive to GCV (P < 0.05), but not to ACV (P > 0.05). There was a significant difference in killing effects between 39tk(+) T cell + GCV group and tk(+) T cell + GCV group (P < 0.05).

CONCLUSIONThe lentiviral vectors containing HSV-sr39tk gene could infect T lymphocytes effectively and stably without affecting the proliferation of the transduced cell. In contrast to HSV-tk gene, T cells infected HSV-sr39tk were more sensitive not only to GCV but also to ACV.

Acyclovir ; pharmacology ; Animals ; Cell Survival ; drug effects ; Cells, Cultured ; Ganciclovir ; pharmacology ; Genetic Vectors ; Lentivirus ; genetics ; Mice ; Mice, Inbred C57BL ; Plasmids ; genetics ; T-Lymphocytes ; cytology ; drug effects ; Thymidine Kinase ; genetics ; Transfection

OBJECTIVETo explore the change of serious abdominal traumatic patients' cellular immunity and the effect of Astragalus Injection (AI) on it.

METHODSSixty-three serious abdominal traumatic patients were randomly assigned into two groups, the conventional group and the treated group, patients in the conventional group were given conventional treatment, while others in the treated group were given conventional treatment as the basis, with AI 20 ml was added into 250 ml of 5% glucose solution given through intravenous dripping, and then on the first day and 14th day, their T cell activated antigens as well as that of 10 healthy subjects were monitored.

RESULTSOn the first day, in the conventional group and treated group, the levels of CD(3)(+), CD(4)(+), CD(4)(+)/CD(8)(+), CD(16)(+), CD(69)(+) and CD(3)(+)/homologous leucocytic antigen-DR (HLA-DR(+)) were apparently lower than those in the healthy group (P < 0.05), while the CD(8)(+) was significantly higher than that in the healthy group (P < 0.05), and there was no significant difference between the conventional group and the treated group (P > 0.05); on the 14th days, the levels of CD(3)(+), CD(4)(+), CD(4)(+)/CD(8)(+), CD(16)(+), CD(69)(+) and CD(3)(+)/HLA-DR(+) of the treated group got closed to healthy subject value, and got even higher than those of conventional group (P < 0.05); CD(8)(+) got close to that of healthy subjects, while obviously lower than that of conventional group (P < 0.05).

CONCLUSIONAfter serious abdominal trauma, cellular immunity lowered, auxiliary use of AI was beneficial to the restoration of cellular immunity.

Abdominal Injuries ; immunology ; Adjuvants, Immunologic ; therapeutic use ; Adult ; Antigens, CD ; analysis ; Antigens, Differentiation, T-Lymphocyte ; analysis ; Astragalus membranaceus ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Humans ; Immunity, Cellular ; Lectins, C-Type ; Lymphocyte Activation ; Male ; Middle Aged ; Phytotherapy ; Receptors, IgG ; analysis ; T-Lymphocyte Subsets

OBJECTIVETo construct a recombinant lentiviral vector (pXZ208-BDDhFVIII) mediating B-domain-deleted human coagulation factor VIII (BDDhFVIII) gene and investigate its expression in HLF, Chang-Liver and MSC cells.

METHODSBDDhFVIII gene fragment was separated by endonuclease digestion and was cloned into the multiple cloning sites of pXZ208 to construct a recombinant lentiviral vector pXZ208-BDDhFVIII. Viral particles were prepared by means of three-plasmid cotransfection of 293T package cells by calcium phosphate precipitation. After infection, the coagulant activity of human FVIII in the culture medium of 293T, HLF, Chang-Liver and MSC cells was assayed by one-stage method. The gene transduction efficiency was assayed by flow cytometry (FCM). Furthermore, PCR was performed to test the integration of BDDhFVIII.

RESULTSThe infection rates of HLF, Chang-Liver and MSC were (74.52 +/- 7.57)%, (27.24 +/- 6.53)% and (42.34 +/- 5.84)% respectively. The activities of FVIII in supernatants of HLF, Chang-Liver and MSC were (54.1 +/- 5.6)%, (22.5 +/- 2.9)% and (12.5 +/- 2.7)% respectively. BDDhFVIII gene integration was detected in all the infected cells.

CONCLUSIONThe recombinant lentiviral vector pXZ208-BDDhFVIII was successfully constructed and efficiently integrated into target cells to express human FVIII activity in vitro.

Cell Line ; Factor VIII ; biosynthesis ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Plasmids ; Transfection

OBJECTIVETo construct a three plasmids lentiviral vector containing canine coagulation factor IX (cFIX) gene with ubiquinone promoter (PUB) and observe the expression of cFIX gene.

METHODSLentivirus was generated by transient three-plasmid transfection, namely, the VSV-G envelope expression cassette, the delta NRF packaging plasmid and the PTK 164 plasmid. Viral particles were used to infect the target cell, third passage mesenchymal stem cells (MSCs) and 293T cell respectively at MOI 3: 1. The cFIX activity was detected in cultured cells with one-stage clotting assay.

RESULTSThe MSCs were obtained in vitro. The lentivirus infected MSCs and 293T cells all expressed the active factor IX with the activity of (26.30 +/- 2.10)% and (19.70 +/- 1.53)%, respectively, which are significantly higher than that of control (1.00 +/- 0.05)%.

CONCLUSIONSThe lentiviral vector of three plasmids with ubiquinone promoter (PUB) was constructed and can transfect the MSCs and 293T cells.

Animals ; Bone Marrow Cells ; metabolism ; Cells, Cultured ; Dogs ; Factor IX ; genetics ; metabolism ; Genetic Vectors ; Hemophilia B ; genetics ; metabolism ; Humans ; Lentivirus ; genetics ; Plasmids ; genetics ; Transfection

OBJECTIVETo establish strategies for preventing graft versus host disease (GVHD) and reducing treatment associated morbidity while preserving graft versus leukemia (GVL) effect in nonmyeloablative allogeneic bone marrow transplantation (allo-BMT), with or without donor lymphocyte infusion (DLI) after BMT.

METHODS3 x 10(7) bone marrow cells mixed with 1 x 10(7) spleen cells from the same BALB/c mouse were transplanted into the nonablative irradiated inbred 615 mouse which received a single subcutaneous injection of 1 x 10(6) L615 leukemia cells three days before. The experiments were designed as follows (ten mice in each group): myeloablative BMT control group (group A), nonmyeloablative conditioning without BMT group (group B), nonmyeloablative BMT group (group C), and nonmyeloablative BMT + DLI group (group D). GVL effects were assessed by survival time, white blood cell count and L615 cells in peripheral blood and histologic changes. GVHD was assessed by signs of weight loss, ruffled fur, diarrhea and histologic changes of skin, liver and small intestines. Chimerism was detected by cytogenetic analysis and PCR technique.

RESULTSThe survival time of group A, B, C and D was (20.3 +/- 13.4), (15.9 +/- 1.1), (21.6 +/- 1.7) and (37.8 +/- 2.0) days, respectively, being no significant difference between group A and group C (P > 0.05). The survival time of group C was longer than that of group B (P < 0.01). And among group B, C and D, group D had the longest survival time (P < 0.01). GVHD signs and histologic changes were observed in 60% of control group mice at + 14 day, but none of group C and group D. 40% of mice in group A died of treatment associated morbidity within two weeks, but none in group C and group D. Allogeneic chimerism was kept in group A, but excluded gradually in group C.

CONCLUSIONGVL effect seems preserved in nonmyeloablative BMT mice, but weaker than that in myeloablative BMT mice. GVL effect seems to be enhanced by DLI after nonmyeloablative BMT. GVHD and transplantation associated morbidity seems to be reduced in nonmyeloablative BMT.

Animals ; Bone Marrow Transplantation ; immunology ; methods ; Combined Modality Therapy ; Female ; Graft vs Host Disease ; prevention & control ; Graft vs Leukemia Effect ; Leukemia, Experimental ; therapy ; Leukemia, Lymphoid ; therapy ; Lymphocyte Transfusion ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Inbred Strains ; Transplantation Conditioning ; methods ; Transplantation, Heterologous

BACKGROUNDThe B7/CD28 pathway provides critical costimulatory signals for complete T cell activation, and members of this pathway have served as useful targets for immunotherapeutic strategies. In this study, we investigated the RNA interference (RNAi) effect induced by small interfering RNA (siRNA) targeting CD28 mRNA on human lymphocytes and its specificity.

METHODSAccording to CD28 gene sequence, we designed and synthysized three different siRNAs (siRNA-1, siRNA-2, siRNA-3) containing 21 bases using Silencertrade mark siRNA construction kit. These siRNAs were transfected into freshly isolated human lymphocytes with Lipofectamine 2000 reagent. At 24-hour, 48-hour and 72-hour post transfection, these cells were collected and analyzed. The changes of surface expression of CD28 gene were detected by flow cytometry, and the changes of CD28 mRNA levels were determined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The cell viability of transfected lymphocytes was determined by methyl thiazolyl tetrazolium (MTT) assay and trypan blue dye exclusion assay.

RESULTSThree siRNAs (siRNA-1, siRNA-2, siRNA-3) specifically targeting CD28 mRNA were successfully designed and constructed. Flow cytometry analysis showed that a decrease in CD28 expression was detectable at 24-hour post transfection. Different siRNA showed different inhibition effects on CD28 expression. At 48-hour post transfection, the degrees of reduction with siRNA-1, siRNA-2 and siRNA-3 were 22.10% +/- 1.63%, 73.50% +/- 1.02% and 42.90% +/- 0.89% respectively compared with the control (P < 0.001). Neither of the groups transfected only with siRNA or lipo showed marked reduction in CD28 expression (3.15% +/- 0.75% and 4.55% +/- 0.80%) (P > 0.05). Moreover, lymphocytes treated with siRNA-co showed no marked reduction in CD28 expression (5.07% +/- 0.96%) (P > 0.05). The results of semi-quantitative RT-PCR assay indicated CD28 mRNA level was inhibited after transfection of specific siRNAs. At least 4-fold of reduction in siRNA-2 group occurred at 48-hour post transfection compared with the control (P < 0.001). MTT assay and trypan blue dye exclusion assay demonstrated that the viable cell rations of transfected lymphocytes were significantly reduced in siRNA-1, siRNA-2 and siRNA-3 groups at 48-hour post transfection (P < 0.01). The control groups showed no marked reduction in cell viability (P > 0.05).

CONCLUSIONSThree different siRNAs were synthesized and transfected into lymphocytes. They could reduce the expression of CD28 and the CD28 mRNA level. siRNA-2 was the most efficient. The cell viability reduced correspondingly. Therefore, the silencing effect on CD28 mRNA induced by siRNA may contribute to costimulatory blockade. This result show that siRNA may be useful for further study on graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT).

Adolescent ; Adult ; CD28 Antigens ; genetics ; Cell Survival ; Cells, Cultured ; Flow Cytometry ; Gene Silencing ; Humans ; Lymphocytes ; metabolism ; RNA, Small Interfering ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo analyze the relationship between the expression of FasL, Perforin and Granzyme B and the development of acute graft versus host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT).

METHODSThe peripheral blood mRNA expression of granzyme B, perforin, fasL from 17 patients after allo-HSCT was detected by competitive quantitative RT-PCR and the relationship between FasL, Granzyme B and Perforin expressions and clinical symptom of aGVHD was analyzed.

RESULTSThe expression level of Granzyme B, Perforin and FasL was 4.6 +/- 0.2, 4.5 +/- 0.1, 1.4 +/- 0.1 before aGVHD occurrence respectively, and was 98.7 +/- 2.5, 91.8 +/- 3.4, 61.5 +/- 2.2, after the occurrence in 14 patients (P < or = 0.05). Over expressions of Granzyme B, Perforin, and FasL during acute GVHD were detected in 13 of 14, 12 of 14, and 12 of 14 patients respectively. The upregulated expressions occurred prior to clinical symptom of aGVHD.

CONCLUSIONThe expressions of Granzyme B, Perforin, and FasL were significantly high in patients with acute aGVHD. Monitoring of the expressions, might predict the occurrence of clinical aGVHD and it severity and prognosis.

Acute Disease ; Adult ; Fas Ligand Protein ; genetics ; Female ; Gene Expression ; Graft vs Host Disease ; blood ; diagnosis ; etiology ; Granzymes ; genetics ; Hematopoietic Stem Cell Transplantation ; adverse effects ; methods ; Humans ; Male ; Perforin ; genetics ; Postoperative Complications ; blood ; diagnosis ; etiology ; Prognosis ; RNA, Messenger ; blood ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transplantation, Homologous

OBJECTIVETo assess the therapeutic effect of losartan on type 2 diabetes mellitus (DM2) with gas chromatography (GC)-based metabonomics.

METHODSDM2 patients were dosed with losartan (100 mg/d) and urines were collected at week 8 and 12. The biochemical criteria (blood pressure, urinary albumen, urinary 8-hydroxy-2'-deoxyguanosine and blood creatinine) were analyzed. Urine samples were derivatived and analyzed by GC. Multivariate metabonomics analysis was performed after peak alignment.

RESULTSAfter 8-12 weeks, losartan showed little curative effect and no remarked changes of biochemical criteria were observed. However, metabonomics analysis revealed that some biomarkers such as glucitol and inositol changed.

CONCLUSIONGC-based metabonomics analysis enables the rapid identification of metabolic differences and provides information concerning therapeutic effect of losartan.

Albuminuria ; urine ; Biomarkers ; blood ; chemistry ; urine ; Chromatography, Gas ; methods ; Creatinine ; blood ; Deoxyguanosine ; analogs & derivatives ; urine ; Diabetes Mellitus ; drug therapy ; Diabetes Mellitus, Type 2 ; drug therapy ; Drug Monitoring ; Humans ; Hypoglycemic Agents ; therapeutic use ; Inositol ; chemistry ; Losartan ; therapeutic use ; Metabolome ; drug effects ; Sorbitol ; chemistry