Objective To construct and identify recombinant expression plasmid of small interfering RNA (siRNA)targeting hepatitis B virus X protein(HBx), and observe its effect on mitoehondrial function in healthy liver cell line steadily expressed HBx gene (HL-7702/HBx). Methods Two siRNA sequences containing short hairpin structure, which target on the total length HBx gene, were synthesized and cloned into the vector psiRNA-Hh1GFPzeo to eonstruct recombinant expression plasmids pX1 and pX2. Non-specific recombinant pScr plasmid served as control. After siRNA transfected into HL-7702/HBx cells line by liposome, reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were performed to identify the suppressive effect on HBx expression. Levels of intraeellular reactive oxygen species (ROS) and mitochondrial membrane potential (△ m) were determined by flow cytometry. The experimental results were compared by analysis of variance. Results Successful constructions of pX1 and pX2 were confirmed by restriction enzyme digestion and sequencing. The expressions of HBx mRNA and protein after 48 h of transfection into HL-7702/HBx cells in control group were 0.65± 0.12 and 0.62± 0.09, respectively, which were both higher than those (0.33±0.10 and 0.19±0.08, respectively) in group pX1 (t=4.73, P<0.05; t=7.53, P<0.05) and those (0.48±0.10 and 0.37±0.11, respectively) in group pX2 (t=2.39, P<0.05;t=4.43,P<0.05). But the inhibition of group pX1 was stronger than that of pX2 (t=2.28,P<0.05). Levels of ROS and △ m after RNA interference were 5.00±0.38 and 33.86±0.50, respectively, while those in control group were 72. 10±0. 55 and 3. 57±0.26, respectively (ROS: t=276.22, P<0.05; △ m: t=107.15, P<0.05). Conclusions siRNA targeting HBx can efficiently and specifically suppress the HBx expression in HL-7702/HBx cells, and decrease the level of ROS and increase the level of △ m, thus relieve cellular oxidative stress.

In this article, the authors firstly summarized the number of applications submitted to and projects supported by the National Natural Science Foundation of China (NSFC) in the field of traditional Chinese medicine research in 2010. Then they described the district distribution, research direction layout and allotment of the approved projects in the three primary disciplines (traditional Chinese medicine, Chinese materia medica and integrated traditional Chinese and Western medicine) and their 43 subdisciplines. The targeting suggestions for improvement were given respectively by concluding the reason of disapproved projects from the point of view of applicants and supporting institution, and by stating the common problems existing in the review process from the perspectives of fund managers and evaluation experts. Lastly, the major funding fields in the near future were predicted in the hope of providing guidance for applicants.

Acupuncture Therapy ; Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Scalp ; Sleep Initiation and Maintenance Disorders ; therapy ; Treatment Outcome ; Young Adult

0.05).2.Two days after treatment,total protein(TP) and albumin(Alb) of hemotoplasma were significantly higher than before treatment(all P0.05).3.Toxic side effects: gastrointestinal reaction was seen in 20 cases ((52.63%)),reduction of WBC in 2 cases(5.26%),baldness in 38 cases(100%) and heart impairment in 1 case(2.63%).The sperm test and menstrual onset were normal in 18 cases,whose ages ≥ 8 years at beginning of treatment,who were clinically cured and whose ages ≥17 years at the time of last following up,7 of them who had married could have children.Conclusions Combining CTX(conti-)(nuous) stosstherapy with prednisone standard scheme treated the children in Zhuang nationality with NS,We could got a good curative effect.By using this treatment,not only the plasma TP and Alb level could be increased rapidly and the urine protein could be conversed to negative quickly.The accumulated amount of CTX was less than 80 mg/kg,but the treatment was safety in the recent term, and no impairment of sexual gland was seen at long-term following up.

Objective To investigate the tissue stimulation of copper ions to the smooth muscle of murine terminal rectum.Methods In this study copper needle used for electrochemical therapeutic equipment was punctured into submucosal smooth muscle tissue of terminal rectum above the dentate line in 65 male rats.Rats were then divided into 5 groups (10 rats in each group) at random and sacrificed at 1, 3, 4, 6, 8 weeks after respectively, samples were sent for observation of macroscopic and microscopic tissue reaction.Results Rats had no abnormal histological change in the puncture points grossly. Undor microscope, mild edema was detected in the submucosal layer in 1 week group. Remarkable infiltration of inflammatory cells and lymphocytes subsided 3 weeks after, and disappeared five weeks after.Smooth muscle was normal microscopically in all groups.In contrast, platinum and steel needles caused infiltration of plasma cells and neutrophils, ulcers and small abscess formation in around puncture points.Conclusion Inflammatory reaction was induced after puncturing of copper needle into the tissues and the tissue reaction disappeared after 5 Ws.In contrast with platinum and steel needles, copper needle was non-traumatic to the smooth muscle tissue.

Objective To analyze the clinical characteristics of adult patients with hemophagocytic lymphohistiocytosis (HLH) receiving haploidentical donor hematopoietic stem cell transplantation (HID HSCT).Method We retrospectively reviewed 20 adult patients with HLH from August 2009 to August 2014.The clinical features and outcome were analyzed.Results Conditioning regimens consisted of total body irradiation/etoposide/cyclophosphamide (TBI/VP-16/CTX) and busulfan (Bu)/VP-16/CTX in HLH with anti-thymocyte globulin (ATG) 8 mg/kg.The stem cells were mobilized from donors' peripheral blood.Median time to white blood cell engraftment was 13 (9-27) days.Median time to platelet engraftment was 14 (10-28) days.Mixed chimerism after transplantation developed in 4 patients and no patient presented graft failure.Eight patients developed grade Ⅱ to Ⅲ acute graft-versus-host disease (GVHD),while as chronic GVHD occurred in 9 patients.Among 12 patients with EB virus (EBV) reactivation,2 patients developed post-transplant lymphoproliferative disorder (PTLD),7 were suspected as PTLD and 3 were considered as relapse of primary disease.With a median follow-up of 20 months (range:0.5-108 months) after transplantation,the estimated 2-year overall survival (OS) rate was (60.0 ± 11.0) ％ in all patients.During the follow-up,12 patients survived,8 died including 5 within 100 days after HSCT.Among 5 non-remission patients before HSCT,4 patients died within 100 days after HCT.Conclusions HID HSCT is an effective treatment for adult patients with HLH to achieve remission and long-term survival.High proportion of mixed chimerism has been seen at early stage after transplantation.EBV reactivation and early transplant-related mortality are common.

[Objective] To detect the expression of CD137L mRNA and protein in colorectal cancer tissues and discuss its possible clinical significance.[Methods] To detect the expression of CD137L mRNA in colorectal cancer tissues and adjacent noncancerous tissues by real-time RT-PCR and immunohistochemistry and to analyze the relationship between CD137L expression and clinical features.[Results] The mean level of CD137L mRNA in colorectal cancer tissues is significantly higher than that of adjacent noncancerous tissues (0.035 ± 0.013 vs.0.008 ± 0.005,P ＜ 0.05 ).CD137L mRNA expression in colorectal cancer show no significant differences among different age,sex,tumor location,Dukes classification,histological differentiation,and serum levels of CEA.We selected 8 colorectal cancer tissues and 3 adjacent tissues randomly to analyze the expression of CD137L by immunohistochemistry.CD137L was expressed on tumor cells in colorectal cancer tissues.The expression of CD137L was observed only in colorectal cancer tissues (6/8),but not in adjacent noncancerous tissues (0/3).[Conclusions] The expression of CD137L Mrna in colorectal cancer tissues is higher than that of adjacent noncancerous tissues and CD137 is expressed on tumor cell surface,it might be of biological importance for the progression of colorectal cancer.

AIM: To discuss the relationship between prostate specific membrane antigen(PSMA) and prostate cancer and to seek a target for diagnosis and therapy of prostate cancer.METHODS: A pair of primers was designed according to the published PSMA mRNA sequence.Total RNA was extracted from prostate cancer tissues and was reversely transcribed into cDNA,which was used as a template for PCR to amplify the PSMA gene.The recombinant was sequenced and the result was analyzed by BLAST.The PSMA5 gene specific primers were designed to identify its expression in different cells and prostate tissues.RESULTS: A new alternatively spliced variant of PSMA named PSMA5 was discovered when sequencing the recombinant.PSMA5 showed well pathological tissue-specificity,and its expression rate in prostate cancer,benign prostatic hyperplasia of prostate,and normal prostate tissue were 92.6%,78.8% and 10.0%,respectively.It expressed specifically in Pca cell line LNCaP,not in cell lines of PC3,bladder carcinoma,renal carcinoma,or hepatoma.CONCLUSION: A new alternative spliced variant of PSMA named PSMA5 was discovered,which was well correlated with prostate cancer and benign prostatic hyperplasia.This finding may give a new clue to the evolution of prostate cancer and may provide a target for the diagnosis and therapy of prostate cancer.

AIM: To find out the gene structure and diversity of protate specific membrane antigen(PSMA) alternative spliced variants, and probe into the pathogenesis of prostate cancer.METHODS: 5'-RACE and 3'-RACE methods were used to amplify the 5' and 3'end of alternative spliced variant and then those viariants were sequenced for analyzing the gene stucture and diversity of PSMA alternative spliced variants of prostate cancer tissues.RESULTS: Four new alternative spliced variants of PSMA were discovered from prostate cancer tissues.Compared with reported PSMA alternative spliced variants,different insertions and deletions existed in different sites of those new variants.CONCLUSION: The discovery of the new variants confirms the diversity of PSMA spliced variants and provides the clues for seeking the target of diagnosis and therapy of prostate cancer.

Objective To construct a recombinant Bacillus Calmette-Guerin ( BCG ) vaccine strain, rBCG::Rv3478-pMV261, expressing the Rv3478 protein of Mycobacterium tuberculosis and to inves-tigate its immunogenicity.Methods The gene fragments encoding Rv3478 antigen were amplified by PCR and then respectively cloned into pMV261 and pET-28a vectors to construct the recombinant expression plas-mids (Rv3478-pMV261 and Rv3478-pET-28a).The Rv3478-pMV261 plasmids were transformed into the BCG cells to construct the rBCG vaccine strains, while the Rv3478-pET-28a plasmids were transformed into Escherichia coli BL21 strains for the expression of Rv3478 protein.Polyclonal antibodies were induced in mice upon the immunization with Rv3478 protein.The rBCG vaccine strains overexpressing Rv3478 protein were screened out with Western blot assay.The C57BL/6 mice were divided into four groups including the PBS treated group, BCG treated group, rBCG::pMV261 ( R0) treated group and rBCG::Rv3478-pMV261 ( R3) treated group.All mice were sacrificed in 4 or 12 weeks after immunization.Enzyme-linked immunos-pot assay ( ELISPOT) , ELISA and flow cytometry analysis were performed to evaluate the induced humoral and cell-mediated immune responses in mice.Results The Rv3478 protein was successfully expressed and could induce polyclonal antibodies in mice.High levels of IFN-γand TNF-αwere detected in mice treated with R3, indicating that the immunization with R3 enhanced the cellular immunity.Moreover, the ratios of CD4+to CD8+T cells and the percentages of CD44+CD62L+T cells were increased in mice upon the immuni-zation with R3.Conclusion The recombinant BCG vaccine strain overexpressing Rv3478 protein could in-duce stronger cell-mediated immune responses in mice.It might be have a great significance as a new tuber-culosis( TB) vaccine strain against TB infection in the future.