Objective To study the effect of the ECV-304 cells modified with LIF gene on the ex vivo culture of HSC/HPC in cord blood.Methods The ECV-304 cells were infected by Eukaryotic Expression plasmid pcDNA3.0LIF,and the positive ECV-304 cells were obtained by selected with G418.These cells were used to co-culture with CD34+ cells of cord blood.The phenotype of CD34+ and CD34+ CD54+、CD34+ CD62L+ primitive progenitors was detected by flow cytometry.Results The LIF gene can express in ECV-304 cells steadily.ECV-304 cells modified with LIF gene can improve expansion of CB CD34+、CD34+CD54+ and CD34+ CD62L+ cells while sustaining the expression of homing-related adhesion molecule.Conclusion The ECV-304 cells modified with LIF gene can not only significantly expand CB hematopoietic progenitor cells ex vivo,but the expanded CD34+ cells may well retain their homing ability.

Objective To summarize the clinical data of cardiac transplantation in 28 patients of our hospital,and study the effectiveness of operation and the methods of immunosuppressive regime.Methods From January 2000 to May 2005,in our center,28 patients were subjected to orthotopic cardiac transplantation.In early stage(before 2003) ciclosporin A and mycophenolate mofetil were used for immunosuppression.In late stage(after 2003) Daclizumab and mycophenolate mofetil were used for immunosuppression.Results Orthotopic cardiac transplantations were done successfully in 28 patients.There was no haemorrhage,twist and right heart insufficiency postoperatively.In the patients before 2003,the complications of hepatic-renal inadequacy to varying degrees occurred in 6 cases and infection in 3 cases respectively.In the patients after 2003,there were no complications of hepatic-renal inadequacy and infection.Conclusions Cardiac transplantion is an effective way for end stage cardiomyopathy.Immunosuppressive effect of Daclizumab and mycophenolate mofetil is satisfactory.

Objective To construct the human IL-24 recombined adenovirus(Ad-IL24)and to observe the effect of Ad-IL24 on ex vivo culture of HL-60 cells.Methods pAdTrack-CMV-IL24 was constructed by PCR with pcDNA3.0-IL24 recombined plasmid as a template,enzyme digestion and ligation.The pAdTrack-CMV-IL24 lineared by Pme Ⅰ was co-transformed into BJ5183 with pAdEasy-1.The pAdEasy-1-pTrack-CMV-IL24 recombined adenovirus vector was lineared with Pac Ⅰ and then transfected in to QBI-293A cells.The Ad-IL24 was obtained and used to infect HL-60 ceils,and the effect on HL-60 cells was tested by LSCM,Mrrr,FCM,ELISA and immunohistochemistry staining.Results The pAdEasy-1-pTrack-CMV-IL24 vector was constructed and the Ad-IL24 was successfully obtained.The effect of inhibiting growth of HL-60 cells and inducing cell apoptosis by IL-24 was proved by LSCM,MTT and FCM.IL-24 down-regulated the expression of anti-apoptosis factor Bcl-2 and increased the secretion of IFN-γ,TNF-α of HL-60 cells.Conclusion Ad-IL24 can inhibit the growth of HL-60 cells and induce cell apoptosis through down-regulating expression of anti-apoptosis factor and increasing the secretion of IFN-γ,TNF-α of HL-60 cells.The human Ad-IL24 may provide a basic study for the future target therapy to tumors.

Objective:To construct the cells which can express LIF protein steadily and study the effect of rhLIF and IL-24 on HL-60 cells.Methods:ECV-304 cells were infected by eukaryotic expression plasmid pcDNA3-LIF,then the positive cells were selected by G418.The positive cells were collected and their culture supernatant was stored for further use.Expression of LIF mRNA was detected by RT-PCR.The pAdeasy-1-pAdTrack-CMV-IL-24 was extracted from DH5a.The recombined adenovirus vector was lineared with PacI and transfected into 293 cells.The IL-24 recombined adenovirus(Ad-IL-24) was obtained and used to infect HL-60 cells.At the same time,the culture supernatant containing rhLIF was added into the HL-60 cells which was identified as positive expression of IL-24 by RT-PCR.Synergistic effect of the cytokines on HL-60 cells was tested by LSCM, FCM and immunohistochemistry stain assay.Results:The cells expressing LIF protein steadily were constructed successfully, the high titer of the recombined adenovirus(Ad-IL-24) was obtained. Expression of IL-24 in infected HL-60 cells was identified by RT-PCR.The apoptosis of HL-60 cells induced by rhLIF and IL-24 was proved by LSCM ,FCM and immunohistochemistry stain assay. They had synergistic effect.Conclusion:rhLIF and IL-24 can inhibit growth of HL-60 cells and induce apoptosis of the cells.They have synergistic effect.

Objective:To study the antitumor effect and mechanism of cocultured CIK cells modified with IL-24 gene and autologous DCs on HL-60 cells in vitro.Methods:DCs and CIK cells were prepared routinely from human peripheral blood mononuclear cells ( PBMC).IL-24 gene was transferred into CIK cells via electroporation.The cells obtained were named CIK-IL24.RT-PCR and ELISA were used to evaluate expression of IL-24 gene in transfected CIK cells.The phenotypic changes of CIK cells were identified by flowcytometry analysis.The concentration of IFN-γ and TNF-α in supernatant of CIK was determined by ELISA.FCM was used to determine the cytotoxicity of cocultured CIK cells modified with IL-24 gene and autologous DCs against HL-60 cells.Results:Eukaryotic expressing plasmid pcDNA3.0-IL24 was transferred into CIK cells successfully via electroporation.The expressing rate of CD3~+、CD3~+CD56~+ cells had no significant change in CIK cells.However,the rate of CD4~+CD25~+ cells was significantly decreased compared with that of the control group.Expression of adhesion molecules CD54,CXCR4 were significantly increased on CD3+CD56+ cells.CIK-IL24 cells produced markedly higher levels of IFN-γ and TNF-α as compared with the CIK cells.By comparison with non-transfected CIK cells co-cultured with DCs,transfected CIK cells co-cultured with DCs had a significantly higher lytic activity against HL-60 cells.Conclusion:IL-24 gene modification can enhance the anti-tumoral immunity of CIK cells,the mechanism of which might be related to the increased secretion of IFN-γ,TNF-α,up-regulation of adhesion molecule expression,and reduction of the rate of CD4~+CD25~+ cells in CIK cells.

Objective To study the antitumor effect and mechanism of co-cultured cytokine-induced killer(CIK) cells and autologous DC modified with IL-24 gene on A549 cells in vitro. Methods DC and CIK cells were prepared routinely from human peripheral blood mononuclear cells(PBMC). Recombinant adenovirus vector pAdEasy-1-pTrack-CMV-IL-24 was extracted from DH5α, it was lineared with Pac I and transfected into A293 cells, and then the IL-24 recombined adenovirus(Ad-IL-24) was obtained. Ad-IL-24 was used to infect DC. The cells obtained were named DC-IL-24. RT-PCR and ELISA were used to evaluate the expression of IL-24 gene in transfected DC. The phenotypes change of DC were identified by flow cytometry analysis, the concen-tration of IL-12 and TNF-α in supernatant of DC were determined by EIJSA. The ability of CIK producing per-forin was measured by homolysis method. FCM was used to determine the cytotoxicity of cocultured CIK cells and autologous DC modified with IL-24 gene to A549 cells. Results We obtained the high titre of Ad-IL-24.IL-24 gene was transfered into DC successfully via Ad-IL-24. The green fluorescence was observed on DC by fluorescence microscope. The expression rate of CD80, CD83, HI.A-DR, CD40, CXCR4 on DC-IL-24 was sig-nificantly increased compared with that of the control group. DC-IL-24 produced markedly higher levels of IL-12 and TNF-α as compared with DC. DC-IL-24 can enhance the ability of CIK cells producing perforin. On com-parison with non-transfected DC co-cultured with CIK cells, transfected DC co-cultured with CIK cells had a sig-nificantly higher lytic activity against A549 cells. Conclusion IL-24 gene modification can enhance the anti-tu-moral immunity of DC. The mechanism of which might be related to the increased secretion of IL-12 and TNF-α, up-regulation expression of co-stimulatory molecules and MHC Ⅱ class molecules on DC, promoting the acti-vation and maturation of DC, and then enhancing CIK cells to generate specific anti-tumoral immunity.

Objective To investigate the efficacy of intra-arterial thrombolysis with stenting for acute cerebral infarction. Methods Using a prospective case-control design, 24 patients with acute cerebral infarction who remained angiostegnosis ( ＞ 50%) after intra-arterial thrombolysis were randomly divided into stent treatment group and drug treatment group. They were treated with stenting + drug treatment and conventional drug treatment. The rates of vascular complete revascularization and residual stenosis, and the modified Rankin scale scores at 3 months in both groups were evaluated. Results The rate of complete revascularization in the stent treatment group was significantly higher than that in the drug treatment group (54. 5% vs.0%,χ2 =6.382, P ＜0. 001), and the rate of residual stenosis was significantly lower than that in the drug treatment group ([4.5 ±5.2]% vs. [82. 5 ±10. 5]%, t =7.464, P＜0.001). The rate of favorable clinical outcome in the stent treatment group was significantly higher than that in the drug treatment group (100% vs. 76. 9%,χ2 = 14. 263, P = 0.038). Conclusion The efficacy of intra-arterial thrombolysis with stenting in the treatment of acute cerebral infarction is superior to that in the drug treatment group, and it is safer.

BACKGROUND: Conventional means in treatment of distal tibiofibular syndesmosis disruption include plaster cast or splint immobilization as external fixation and internal fixation using screws, and bone bolts or plates;however, some disadvantages such as unstable fixation and uncertain fixation exist.OBJECTIVE: To observe the primary clinical curative effect of newly self-developed fixation, Hook-plate fixation (HPF), for distal tibiofibular syndesmosis disruption and its biochemical characteristics of HPF.DESIGN: Self-control observation.PARTICIPANTS: We recruited 23 patients with sustaining abruption of lower tibiofibular ligament union with fibular fracture who received treatment in the Department of Orthopedics, Affiliated Hospital of Yangzhou University Medical College, between October 2001 and March 2004. According to the Lauge-Hansen classification system, there were 11 cases of supination-lateral rotations, 7 cases of pronation-lateral rotations and 5cases of pronation-abductions. Among them 14 had bimalleolar fractures and 9 had trimalleolar fractures.METHODS: HPF was used on the 23 patients with distal tibiofibular syndesmosis disruption. Radiographs were taken routinely. Talocrural joint function exercise was recommended at week 1 and partial weight bearing was allowed 2 months later. Functions were evaluated according to modified Mazur's criteria (excellent, good, fair and poor).MAIN OUTCOME MEASURES: ①Healing time and function ev aluation after operation for distal tibiofibular syndesmosis disruption. ② Adverse events and side effectsRESULTS: Totally 23 patients entered the result analysis, with the mean of 11 months' follow-up. ①Results of healing time and function evaluation in the patients after operation for distal tibiofibular syndesmosis disruption: The healing time of fracture ranged from 12 to 18 weeks: 16 cases were excellent, 5 good, and 2 fair. ② Adverse events and side effects:There was no separation of the tibiofibular space, the distance between medial malleolus or lateral malleolus and anklebone was symmetrical.There was no mobilization or rupture of the internal fixation after operation.CONCLUSION: HPF is applied in treating distal tibiofibular syndesmosis disruption. It causes no postoperative complications, and can recover the ankle joint function with stable internal fixation and good biomechanical features.

Objective To screen the mutations of MYOC gene in a Chinese primary open angle glaucoma (POAG) family from Cbengqing and investigate the relationship between the mutations in MYOC/TIGR gene and POAG.Methods In a large 4-generation glaucoma family, myocilin gene (MYOC) was screened in 39 family members, 8 of which were confirmed patients. Normal controls included 100 normal Chinese subjects.The known mutations of MYOC gene ( including G34C, C136T, G144T, G227A, C624G,G736A, C1009G, A1036G, C1081T, G1099A, G1138A, A1139C, T1430A, C1441A and C1442T) were detected by single strand conformation polymorphism(SSCP) , po]ymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis and DNA sequencing.Results G227A mutation was detected in 2 POAG patients and 1 asymptomatic patient, but not in the controls.Cl009del mutation was identified in all patients of the pedigree and an offspring member but not in the controls. No other mutations were detected.Since the C1009del mutation was revealed for the first time, a new GenBank number FJ237047 correponding to ACI62293 was applied.Conclusions The G227A mutation is a known site and there is no relationship between G227A mutation and glaucoma. But C1009del may be related to glaucoma which suggests that morbidity could be higher in the relatives of POAG than the controls.

Objective To explore the mechanism of the role of mitomycin C(MMC)in regulating miR-200b expression and inducing fibroblasts apoptosis. Methods Fibroblasts cultured in vitro were treated with different concentrations of MMC for 5 min and continue culture for 24 h. The expression of miR-200b were analyzed by Real-time PCR. Cell apoptosis were observed using TUNEL staining. The expression of cleaved-PARP,Bax and Bcl-2 were detected by Western blot. The methylation level of miR-200b promoter were measured by BSP. Results After treated with MMC,The expression of miR-200b significantly downregulated.TUNEL Staining analysis demonstrated MMC could significantly induce human fibroblasts apoptosis. Western blot results showed cleaved-PARP,Bax increased and Bcl-2 decreased.The methylation ratio of miR-200b promotor increased and has a significant dose dependent. Conclusion MMC induced human fibroblasts apoptosis by promoting miR-200b promoter methylation.