Objective To investigate the effect of modified Huangqi-Jianzhong decoction on healing quality of gastric ulcer in rats and to explore its mechanism. Methods 40 Wistar rats were randomly divided into a normal group, a model group, a modified Huangqi-Jianzhong decoction group and a omeprazole group (n=10) according to the random number table method. These rats were used to model of chronic gastric ulcer induced by acetic acid 100%. On third day after surgery, the rats in the modified Huangqi-Jianzhong decoction group were treated with 10% of the modified Huangqi-Jianzhong decoction according to the dose of 20 ml per kilogram of body weight. The rats in the omeprazole group were treated with a 3% omeprazole suspension according to the of 20 ml per kilogram of body weight. The rats in the normal group and the model group were treated with the same volume of saline. After treated with corresponding treatment for 16 days, the gastric mucosa pathology, ulcer index, regenerative mucosal thickness, mucosal surface mucus thickness, muscularis mucosal layer width of the defect, cystically dilated glands number and level of serum prostaglandin (6-K-PGF1α), epidermal growth factor (EGF), nitric oxide (NO) were observed. Results Compared with the model group, the rat ulcer index (8.95 ± 2.78 mm2 vs. 20.82 ± 5.12 mm2), mucosal muscularis defect width (123.56 ± 32.89 μm vs. 229.32 ± 49.69 μm), cystic glandular expansion (1.65 ± 0.76 vs. 6.12 ± 1.23) were lower in the modified Huangqi-Jianzhong decoction group (P<0.01). Thickness of regenerated mucosa (329.55 ± 32.22 μm vs. 198.22 ± 5.83 μm), the surface thickness of mucus (44.3 ± 2.8 μm vs. 24.5 ± 7.8 μm), serum 6-K-PGF1α (93.7 ± 8.9 pg/ml vs. 58.5 ± 5.8 pg/ml), EGF (2.11 ± 0.29 ng/L vs. 0.82 ± 0.20 ng/L) , NO (0.51 ± 0.03 μmol/L vs. 0.22 ± 0.05 μmol/L) was higher (P<0.01). In addition to the ulcer index, other each target improvement flavored were better in the modified Huangqi-Jianzhong decoction group than in the omeprazole group (P<0.05). Conclusion Modified Huangqi-Jianzhong decoction can improve the quality of ulcer healing by promoting gastric mucosa ulcer repair, reducimg ulcer index, mucous muscularis layer defect width and cystic glandular expansion and improving the thickness of regenerative mucosa, mucosal surface mucus thickness and cyst, EGF, NO level and thus improve the experimental gastric ulcer healing quality.

Objective To compare the differences of lifestyle factors between patients with functional constipation (FC)and constipation-predominant irritable bowel syndrome (IBS-C).Methods From February 2011 to December 2014,255 patients with chronic constipation were enrolled.Among them,there were 170 FC patients and 85 IBS-C patients.At the same period,170 healthy volunteers without symptoms of digestive diseases within one year were recruited as control.The data of demographic information and lifestyle factors were collected.First,single variant analysis was performed for statistical analysis and then the statistically significant variants were analyzed by multivariate logistic regression. Then the factors of FC and IBS-C patients were analyzed by decision tree model and the effects of factors under different categories were analyzed.Results The results of single variant analysis indicated that there was no difference in lifestyle factors between FC group and IBS-C group (all P >0.05).The results of multivariate logistic regression analysis showed that no independent protective or risk factors were found in IBS-C group compared with FC group.According to decision tree model analysis,body mass index (BMI),water intake per day and constipation family history were finally enrolled.The incidence of FC was higher in patients with BMI < 23.56 kg/m2 (except 18.74 to < 19.83 kg/m2 )(79.75 %).The incidence of FC was higher in patients with BMI from 18.74 to <19.83 kg/m2 and water intake <1 L
(66.67%).The incidence of FC was highest in patients with BMI≥23.56 kg/m2 and family history of constipation (70.00%).The total prediction accuracy of this model was 64.6% (42/65 )and area under curve (AUC)value was 0.688.Conclusions FC and IBS-C are related with many lifestyle factors.Low BMI and less water intake per day are influence factors of FC,while higher BMI and family history of constipation are influence factors of IBS-C.

Objective To explore the mechanism of radio-resistance of breast cancer stem cells by investigating the effects of ionzing radiation on the reactive oxygen species (ROS),apoptosis and cycle distribution.Methods The breast cancer MCF-7 cells were suspension cultured in serum-free medium containing a variety of growth factors. There were MCF-7 (breast cancer cells),MCF-7-S (breast cancer stem cells),MCF-7+8 Gy and MCF-7-S+8 Gy groups in the experiment. 4-24 h after 8 Gy irradiation, the ROS levels, percentages of apoptotic cells and percentages of the cells at each cycle phage were measured by FCM with 2′, 7′-dichlorodihydrofluorescin diacetate (DCFH-DA ), Annexin Ⅴ-FITC/PI and PI staining, respectively. Results The breast cancer stem cell microsphere accumulated hundreds of cells were obtained successfully at 7 d after suspension culture with serum-free medium containing a variety of growth factors;the FCM results showed that CD44+CD24- phenotype breast stem cells were up to 75.20%.With the time prolongation,the ROS levels and apoptosis in MCF-7 group and MCF-7-S group showed increasing trendency, and reached for the maximum values at 12 and 24 h;the ROS levels in MCF-7-S group were significantly lower than those in MCF-7 group at 4,8,12 and 24 h (P<0.05 or P<0.01), and the percentage of apoptotic cells in MCF-7-S group was significantly higher than that in MCF-7 group only at 8 h(P<0.05);the ROS levels (4,8,12 and 24 h)and percentage of apoptotic cells(12 h)were significantly increased in MCF-7+8 Gy group (P<0.05),and the percentages of apoptotic cells (4,8,12 and 24 h)in MCF-7-S +8 Gy group were significantly decreased (P<0.05 or P<0.01),but the ROS levels had no obvious change in MCF-7-S+8 Gy.At 12 h,as compared with MCF-7 group,the percentages of the cells at G0/G1 phase and G2/M phase in MCF-7-S group were significantly decreased (P<0.05),and the percentage of the cells at S phase was significantly increased (P<0.05 );the percentage of the cells at G2/M phase in MCF-7+8 Gy group was significantly increased (P<0.05 ), but there were no significant changes in MCF-7-S+ 8 Gy group. Conclusion Ionizing irradiation can cause the increasing of ROS level and apoptosis and G2/M phase arrest in breast cancer cells,but has no obvious effects on the breast cancer stem cells;it indicates that radio-resistance might be related to ROS level,apoptosis and G2/M phase arrest.

Objective To evaluate the short-term outcome of local intraarterial thrombolysis in patients with acute ischemic stroke of the anterior circulation. Methods 24 patients with acute ischemic stroke of the anterior cir-culation within 8 hours were treated by local intraarterial thrombolysis. Arterial recanalization was divided into total, partial and occlusive respectively according to angiography. Evaluation of clinical outcome was performed on the 30th day after thrombolysis,and was classified as good for Modified Rankin Scale (MRS) scores of 0 to Ⅲ and poor for MRS scores of Ⅳ to Ⅵ. Results Total recanalization was obtained in 54.2 % of patients, partial recanalization in 25.0%. Clinical outcome was good in 15 patients (62.5%). Cerebral hemorrhage occurred in 4 patients (16.7%). Four patients died (16.7%). Conclusion Local intraarterial thrombolysis is an effective method for treatment of a-cute iachemic stroke of the anterior circulation. It needs further practice and long-term follow-up study on safety and long-term efficacy.

To obtain the functional fusion protein of rhoptry protein 2, compound rhoptry protein2 and surface antigen 1 of Toxoplasma gondii. the ROP2 and P30 genes from genomic DNA of T.gondii RH strain were amplified by PCR, and were inserted into pMD18-T cloning vector. Then the ROP2 fragment was subcloned to pET-30a(+) plasmid digested by EcoRⅠand Hind Ⅲ to construct plasmid pET-ROP2. Furthermore,the P30 fragment was subcloned into pET-ROP2 digested by BglⅡand EcoRⅠto create plasmid pET-ROP2-P30, the resulting recombinant plasmids , transformed into E.coli BL21 (DE3), were induced with IPTG. and the proteins identified by SDS-PAGE were further purified and refolded. The biological activity was analyzed by Western blot with specific antibody. It was found that the sizes of ROP2 and ROP2-P30 were 1212 and 1896bp with corresponding molecular weight 50- kDa and 75-kDa, respectively. The recombinant protein ROP2 (50-kDa) could specifically react with rabbit-polyclonal antiserum, and complex fusion protein ROP2-P30 (75- kDa) could react with P30 monoclonal antibody.

Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to Zn2+ were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochondrial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis.
Amino Acid Sequence ; Animals ; Clonorchis sinensis/*enzymology ; Cluster Analysis ; Conserved Sequence ; DNA, Complementary/genetics ; Energy Metabolism ; Gene Expression Profiling ; Mitochondria/metabolism ; Models, Molecular ; Molecular Weight ; Phylogeny ; Protein Conformation ; Sequence Homology, Amino Acid ; Ubiquitin-Protein Ligases/chemistry/*genetics/*metabolism

Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to Zn2+ were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochondrial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis.
Amino Acid Sequence ; Animals ; Clonorchis sinensis/*enzymology ; Cluster Analysis ; Conserved Sequence ; DNA, Complementary/genetics ; Energy Metabolism ; Gene Expression Profiling ; Mitochondria/metabolism ; Models, Molecular ; Molecular Weight ; Phylogeny ; Protein Conformation ; Sequence Homology, Amino Acid ; Ubiquitin-Protein Ligases/chemistry/*genetics/*metabolism

AIK is a novel cationic peptide with potential antitumor activity. In order to construct the AIK expression vector by Gateway technology, and establish an optimal expression and purification method for recombinant AIK, a set of primers containing AttB sites were designed and used to create the AttB-TEV-FLAG-AIR fusion gene by overlapping PCR. The resulting fusion gene was cloned into the donor vector pDONR223 by attB and attP mediated recombination (BP reaction), then, transferred into the destination vector pDESTl 5 by attL and attR mediated recombination (LR reaction). All the cloning was verified by both colony PCR and DNA sequencing. The BL21 F. coli transformed by the GST-AIR expression plasmid was used to express the GST-AIK fusion protein with IPTG induction and the induction conditions were optimized. GST-AIR fusion protein was purified by glutathione magnetic beads, followed by rTEV cleavage to remove GST tag and MTS assay to test the growth inhibition activity of the recombinant AIR on human leukemia HL-60 cells. We found that a high level of soluble expression of GST-AIK protein (more than 30% out of the total bacterial proteins) was achieved upon 0.1 mmol/L ITPG induction for 4 h at 37 °C in the transformed BL21 F. coli with starting OD₆₀₀ at 1.0. Through GST affinity purification and rTEV cleavage, the purity of the resulting recombinant AIK was greater than 95%. And the MTS assays on HL-60 cells confirmed that the recombinant AIK retains an antitumor activity at a level similar to the chemically synthesized AIK. Taken together, we have established a method for expression and purification of recombinant AIK with a potent activity against tumor cells, which will be beneficial for the large-scale production and application of recombinant AIK in the future.
Antimicrobial Cationic Peptides ; biosynthesis ; Antineoplastic Agents ; metabolism ; Escherichia coli ; metabolism ; Genetic Vectors ; HL-60 Cells ; Humans ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; Sequence Analysis, DNA

Embryonic stem (ES) cells have the unique capacity to proliferate extensively and maintain the potential to differentiate into advanced derivatives of all three primary germ layers. ES cell lines can also be generated from human blastocyst embryos and are considered promising donor sources for cell transplantation therapies for diseases such as juvenile diabetes, Parkinson's disease, and heart failure. However, as for organ transplants, tissue rejection remains a significant concern for ES cell transplantation. Another concern is the use of human embryos. One possible means to avoid these issues is by reprogramming the nuclei of differentiated cells to ES cell-like, pluripotent cells. This review discusses the potential of these strategies to generate tailor-made pluripotent stem cells and the role of transcription factors in the reprogramming process.
Cell Culture Techniques ; Cell Differentiation ; physiology ; Cells, Cultured ; Cellular Reprogramming ; Humans ; Nuclear Transfer Techniques ; Pluripotent Stem Cells ; cytology

Objective To study the protective effect of ROP2 nuclei acid vaccine in mice.Methods Forty-two BALB/c mice were divided into three groups.Each mouse in experiment group was injected with 50 ?g recombinant plasmid pc-DNA3-ROP2 through musculus quadriceps fexoris.In control groups,each mouse was injected with 50 ?g blank plasmid pc-DNA3 and with 50 ?l PBS respectively.All mice were immunized for three times with an interval of three weeks.The volume was doubled for the final injection in the two plasmid groups.Blood,spleens and lymph nodes of 4 mice in each group were taken for the detection of CD4+,CD8+ T cells and cytokines 2 weeks after the final immunization.The rest mice in 3 groups were challenged with 500 tachyzoites of Toxoplasm gondii RH strain for further observation.Results The vaccine induced strong cellular and humoral immune response.The titer of antibody in serum was high after inoculation and recognized ROP2 protein antigen expressed in vitro.The lymphocyte phenotype was analyzed.CD4+ T cells proliferated sharply(69.5?3.4)%,and the ratio of CD4+/CD8+ increased considerably by(4.69?1.32)%(P