AIM: To investigate the effectiveness of recombinanted retrovirus vector in gene therapy. METHODS: The retroviral vector pLXSN S was constructed and transferred into PA317 by means of electroporation, then HepG 2?RAW264.7 and EL 4 cells were infected with the pseudovirus produced from PA317, which highly expressed HBsAg . HBsAg expression was tested by RT PCR and ELISA. RESULTS: HBsAg was expressed variously in the eukaryotic cells mentioned above. HBsAg ( A value) of the cell supernatants (48 hours) were 0.92,0.53,0.42, respectively. CONCLUSION: The vector used in this study was an effective vector to carry genes of interest to target cells and macrophage, and high level HBsAg was expressed in antigen presenting cell such as macrophage, It indicated that plasmid immunity can induce the B lymphocyte and T lymphocyte response to hepatitis B virus surface antigen by stimulating macrophage. As a vector, it may be useful in the test for gene immunity and gene therapy.

Recently,study on cencer stem cells has been a focus of study.Cancer stem cell is a small population of cencer cells possessing the properties of stem cells:self-renewal,differentiation and proliferation.To date,the existence of cancer stem cells has been proven in acute and chronic myeloid leukemia,breast cancer,brain tumors,liver cancer and colon cancer,etc..In this article we reviews the current progress on cancer stem cells,including the defination,existing evidence,research methods, and challenges in clinical application.

Objective To study the effects of two stimulators of the innate immune system, polyinosine-polycytidylic acid [Poly (I:C)] and R848, on the activation of microglia in rat spinal cord. Methods Thirty male Lewis rats (6 to 8 weeks old) were divided into Poly(I:C) group (n=12), R848 group (n=15) and control group (n=3). According to the killing time, the Poly(I:C) group and R848 group were divided into 4 and 5 sub-groups, respectively, with 3 rats in each sub-group. The rats in the subgroups received intraperitoneal injection of a single bolus of Poly(I:C) (5 mg/kg) or R848 (1 mg/kg) accordingly, and in the control group, the same volume of phosphate-buffered saline (PBS) was administered. Activated microglia were observed using immunohistochemistry for ED-1, AIF-1, EMAP Ⅱ, OX6, and P2X4R, and BrdU staining was used to identify the proliferating cells. Results Compared with the control group, both Poly (I:C) and R848 groups showed a significant but transient increase of ED-1-positive spinal cord microglia 4 days after the injection, while no significant differences were found in the microglial markers AIF-1, EMAP Ⅱ, OX6, P2X4 receptor (P2X4R), indicating that the microglia were not fully activated. Tracing of the cell proliferation by BrdU revealed that only a small fraction of the proliferating cells were microglia (less than 5%). Conclusion Poly(I:C) and R848 have definite effects on the innate immune system of the spinal cord and modulate the immune activity in the spinal cord, suggesting the value of these agents in modulating local regenerative processes in injured spinal cord.

Objective To determine the difference of macrophage RAW264.7 apoptosis induced by Brucella melitensis virulent strain 16M and attenuated strain M5-90 and elucidate the regulatory role of caspases 3,8 and 9.Methods The best multiplicity of infection (MOI) was determined through kinetic analysis of Brucella melitensis strain 16M and M5-90 induced mouse macrophages apoptosis(bacterium ∶ cell =100 ∶ 1,50 ∶ 1,10 ∶1).The infection model was established using the best MOI =50 ∶ 1.The numbers of in vivo bacteria by colony formation units were calculated after macrophages were infected for different times,including 2,4,8,12,24 and 48 h,and the infected cells were collected.The ratios of apoptosis were detected and the regulation of caspases 3,8 and 9 in apoptosis pathway was elucidated by flow cytometry.Results The numbers of 16M in vivo bacteria were 105.4,104.8,105.8,106.5,108.0 and 109.0,respectively and of M5-90 were 106.1,106.2,106.4,106.3,106.1 and 105.0,respectively.The number of in vivo bacteria of 16M was significantly increased than that of M5-90 after infected for 24 h to 48 h.The ratios of apoptosis induced by 16M after infected for 2,4,8,12,24 and 48 h was (2.67 ± 0.09)％,(13.13 ± 0.30)％,(6.56 ± 0.42)％,(6.49 ± 0.28)％,(16.07 ± 0.86)％ and (24.23 ± 1.67)％,respectively,and by M5-90 was (3.62 ± 0.02)％,(32.01 ± 2.59)％,(17.58 ± 0.44)％,(16.09 ± 0.10)％,(62.53 ± 2.70)％ and (85.53 ± 0.15)％,respectively,and by control group was [(1.90 ± 0.20)％,(1.92 ±0.16)％,(1.99 ± 0.03)％,(2.48 ± 0.11)％,(3.56 ± 0.07)％,(5.26 ± 0.33)％].The differences were statistically between groups in same time.The Brucella melitensis vaccine strain M5-90 was more powerful than virulent strain 16M in respect of inducing macrophage apoptosis after infected for 24 to 48 h.Twenty-four hours after infection,the expression of caspases 3,8 and 9 was (1.47 ± 0.05)％,(1.52 ± 0.02)％ and (2.47 ± 0.12)％,respectively,in control group and the expression was (9.70 ± 0.46)％,(6.08 ± 0.56)％ and (35.08 ± 1.64)％,respectively,after infected for 24 h induced by M5-90.The expression of caspases 3,8 and 9 was significantly higher than that control group (P ＜ 0.01).Twenty-four hours after given caspases 3,8 and 9 inhibitor,apoptosis rate in control group was (66.72 ± 1.28)％,in M5-90 group was (22.58 ± 0.55)％,(53.15 ± 1.85)％ and (29.18 ± 0.23)％,respectively,and compared with control group,apoptosis rate of caspases 3,8 and 9 was significantly lower(P ＜ 0.01).Conclusions Apoptosis of macrophage can be induced by Brucella melitensis virulent vaccine strain 16M and attenuated strain M5-90.M5-90 is stronger than that of strain 16M.Caspases 3,8 and 9 can regulate macrophage apoptosis after M5-90 infection.

Adult ; Female ; Hepatitis B, Chronic ; complications ; drug therapy ; Humans ; Interferon-alpha ; adverse effects ; therapeutic use ; Nephrotic Syndrome ; etiology

ObjectiveTo analysis the clinical features of thoracic spine and spinal cord injury (SCI) and summarize the inclusive standard of cellular transplant clinical trial for SCI.MethodsThe data of 72 cases with thoracic spine and spinal cord injury from 1990 to 2005 were analyzed retrospectively.ResultsMean follow-up period was 20 months (6～48 months). There was no recovery in 12 spinal cord injury without radiographic abnormality (SCIWORA) patients, but improvement of urine function in 4 cases. 5 cases of 52 fracture-dislocation complete injury were improved to grade B (sense recovery), rate of recovery was 9.6%; recovery rate was 62.5% in incomplete injury. Sense recovery of all cases was better than motor recovery. Partial cases appeared spasm paralysis relief.ConclusionIncidence rate of complete injury is high and recovery is bad in thoracic spine and spinal cord injury. The inclusive standard of cellular transplant clinical trial for SCI is old complete thoracic spinal cord injury without residual compression.

Animals ; Animals, Newborn ; Astragalus Plant ; Doxorubicin ; adverse effects ; Drugs, Chinese Herbal ; pharmacology ; Mice ; Mice, Inbred BALB C ; Myocardium ; pathology ; ultrastructure

Biometry ; Clinical Protocols ; Cryoultramicrotomy ; methods ; Diagnostic Techniques and Procedures ; Frozen Sections ; utilization ; Humans ; Orthopedics ; methods

Objective To develop an ideal cultural method to amplify embryonic hepatic stem cells and inhibit their differentiation in vitro. Methods Suspension of ED 14 Fischer (F) 344 rat em-bryonic hepatic stem cells was prepared by collagenase digestion and mechanical disaggregation. Then cells were divided into two groups randomly. The cells in group 1 were seeded into type I collagen-coated plates by adherent culture while those in group 2 were seeded into soft agar medium by suspen-sion culture. After culture for 2 weeks, the morphology and ultrastructure of cells in both groups were observed and compared by inverted microscope and transmission electron microscope, respectivley.The expression of CD90. 1 and CD49F, the two specific stem cell surface markers, was tested by flow cytometry to manifest the establishment of embryonic hepatic stem cells. Alkaline phosphatase stai-ning was used to detect stem cell differentiation. Result Embryonic hepatic stem cells in group 2 were characterized by higher nucleus-cytoplasm ratio and less cell organelles, higher expression of CD90. 1 and CD49F, and stronger positive reaction for alkaline phosphatase staining compared with those in group 1. Moreover, the cells in group 1 showed significant differentiation features. Conclusion Em-bryonic hepatic stem cells cultured suspendedly in soft agar medium experience less differentiation than those adherently cultured in serum-added culture medium, and can proliferate and form clone ball with a specific stem cell feature.

ObjectiveTo compare the outcomes and indications between exchange nailing (EN) and augmentation plating (AP) with a nail left in situ for femoral shaft hypertrophic nonunion after femoral nailing. MethodsFrom April 1998 to June 2009, 20 patients with femoral shaft hypertrophic nonunions after femoral nailing were treated with EN (11 patients) and AP (9 patients) respectively. There were no significant differences between the two groups with respect to the patient's age, gender, associated injuries, anatomical location and type of femoral fracture. Patients were evaluated by imaging and clinical function at 1, 2, 3, 4,6, and 12 months after surgery, and then every year postoperatively, to observe the callus and the recovery condition of the affected limb function. Fisher exact test and t'-test were performed to compare the outcome and complications respectively. ResultsAn unpaired t'-test showed no significant differences with respect to follow-up time, operating time, intraoperative blood loss, postoperative drainage, length of stay, time to radiographic union, time to clinical union, and AAOS score between the two groups. The cost of hospitalizations in the EN group was higher than in the AP group(t'=16.4, P=0.013). Four nonunions in the EN group failed to achieve union, which 3 patients were subsequently treated with AP and simultaneous autogenous bone grafting and 1 patient was treated with nail dynamization. All 9 hypertrophic nonunions in the AP group obtained osseous union. Fisher exact test showed a higher nonunion rate of EN compared with AP (χ2=6.01 ,P=0.008). ConclusionEN has been an excellent choice for aseptic isthmal femoral nonunion without a large bone defect subsequent to intramedullary fracture fixation, and AP can be an effective solution in cases of nonisthmal femoral nonunion and bone defect and failed exchange nailing.