Objective To investigate the relationship between TNF-α, IFN-γand intestinal muco-sal permeability in a mouse colitis model and its inhibiting effect by balsalazide. Methods Forty-five C57BL/6J mice were divided randomly into five groups. Normal group was only fed with distilled water, DSS group and balsalazide groups at doses of 42, 141,423 mg/kg were both fed with 5% DSS. Balsalazide was given by intragastric administration. At the end of the experiment, colon tissue was collected for assessment of histological index(HI) and the MPO activity. Small intestinal mucosa was collected for assessment of the content of TNF-α and IFN-γ,transmission electron microscope(TEM), and detection of permeability by Ev-arts blue method. Results Compared with normal group, DSS group mice all manifested severe weight loss associated with hematocbezia and diarrhea, HI score, and the colon MPO activity and the content of TNF-α and IFN-γ were increased significantly. Intestinal mucosa showed a thinning of microvillous carpet, with de-curtated and broaden junctional complex and enlarged intercollutar space under TEM observations. The amount of Evans blue permeated into intestinal wall was obvious. Compared with DSS group, the HI score, the MPO activity and the content of TNF-α and IFN-γ were decreased by balsalazide. The amount of Evans blue permeated into intestinal wall was less. Ileal microvillous carpet was ameliorated dose dependently by balsalazide. Conclusion In DSS-induced colitis model, the change of the content of the TNF-α and IFN-γ, was accordance with the increase of intestinal mucosal permeability while balsalazide can significantly amelio-rate intestinal mucosal permeability by its anti-colitis effect.

AIM: To construct the mammalian cell expression vector for enhanced green fluorescent protein (EGFP) and HLA-A*0201 fusion protein and analyze its expression and subcellular localization in the transfected K562 cells. METHODS: The HLA-A*0201 cDNA was cloned by RT-PCR and the gene was inserted into pEGFP-N1 to construct a vector for the fusion protein. The expression of the fusion protein in K562 cells transfected with the vector was evaluated by flow cytometry and its subcellular localization was investigated by confocal microscopy. RESULTS: The full-length encoding region of HLA-A*0201 cDNA was cloned from two HLA-A2 positive donors and the expression vector for the HLA-A*0201-EGFP fusion protein was constructed by PCR using a primer pair to introduce a Kozak sequence before ATG and the stop codon was deleted. Five hours after K562 cells was transfected with the vector, the expression percentages of HLA-A*0201 and EGFP were 25.12?2.26 and 27.37?3.59, respectively and no significant increase was observed after 24 h. The fusion protein was predominantly located on the membrane with low level distribution within the cells. In contrast, no HLA-A*0201 but only EGFP was detected in the empty vector transfected K562 cells and the EGFP was dispersed within the cells. CONCLUSIONS: The expression vector for HLA-A*0201-EGFP fusion protein was constructed and the fusion protein expressed in K562 cells was primarily distributed on the membrane. The results suggest that the transfected K562 cells are potential antigen-presenting cells.

BACKGROUND:Central nerve damage and peripheral nerve injury are common clinical problems that have no ideal treatment. Nerve growth factor has an important role in neuronal repairing and growth. But its local injections may have shorts of inactivation and loss. OBJECTIVE:To construct human nerve growth factor beta recombinant plasmids, which are transfected into bone marrow mesenchymal stem cells from the rabbit mandible by lentiviral vectors, and to investigate the bioactivity of human nerve growth factor beta. METHODS:pDC316-hNGFβ-mCMV-EGFP plasmids were constructed via lentiviral vectors using Hind III+Not I digestion. Bone marrow mesenchymal stem cells from the rabbit mandible were isolated and cultured, and then transfected by recombinant plasmids. The expression of human nerve growth factor beta in transfected cells was detected by ELISA method. RESULTS AND CONCLUSION:pDC316-hNGFβ-mCMV-EGFP plasmids were proved to be constructed successful y by gene sequencing and enzyme identification. The transfected cells under a fluorescence microscope emitted green fluorescence, and the fluorescence intensity had no change with incubation time. The expression of human nerve growth factor beta was maintained at a level of 25μg/L at 7 days after celltransfection, and the bioacitivty was increased significnalty.

Objective To explore the clinical characteristics and medicine treatment of patients with severe ulcerative colitis (UC),the efficacy of rescue treatment in patients with glucocorticoid (GCS) resistant severe UC,and the clinical risk factors in patients with GCS-refractory severe UC.Methods From January 2001 to December 2012,clinical,laboratory,endoscopy,imaging data and medication of treatment of 106 patients with severe UC were retrospectively analyzed.Then the patients were followed up,and the clinical efficacy and under endoscopic presentation of Mayo score were evaluated.Logistic regression analysis was performed to analyze the high risk factor of GCS-refractory severe UC.Results Among 106 patients with severe UC,95 were chronic relapse type accounting for 89.6 ％.The percentage of patients with defecation times over six was 73.6％ (78/106),with severe purulent bloody stool was 51.0％ (54/106),and with moderately or severe abdominal pain was 83.0％ (88/106).The percentage of diffuse colon type was 83.0％ (88/106),endoscopic presentation of Mayo score over two was 87.7％ (93/106).Hemoglobin decreased in 65.1％ (69/106) patients,blood platelet increased in 48.1％ (51/106) patients,C-reaction protein elevated in 88.7％ (94/106) patients,and hypoalbuminemia decreased in 42.5％ (45/106) patients.Account to 89.6％ (95/106) of patients with severe UC received GCS treatment,and the percentage of induced remission was 64.2％ (61/95),effective rate was 16.8％ (16/95),and ineffective rate was 18.9％ (18/95).The percentage of GCS refractory was 35.8％(34/95).There were 23 patients with GCS resistance and 11 patients with GCS dependence.Ten patients with GCS resistant severe UC accepted medicine rescue therapy.Five cases were treated with cyclosporin A,of which two cases induced remission,one case was effective,and two cases were ineffective.Another five cases were treated with infliximab,of which three cases induced remission,and two cases were ineffective.The results of Logistic regression analysis showed that severe anemia (OR=6.750,95％CI:2.656 to 17.152,P＜0.01),elevated blood platelets (OR=4.032,95％CI:1.226 to 13.261,P=0.015) and albumin level less than 25 g/L (OR =3.022,95 ％ CI:1.236 to 7.390,P =0.022) were risk factors of GCS-refractory severe UC.Conclusions GCS resistant or dependent occurred in part of patients with severe UC.Patients with G-CS resistant severe UC receive rescue treatment of cyclosporin A or infliximab.Severe anemia,elevated blood platelets,albumin less than 25 g/L may be clinical predicting factors in patients with GCS-refractory severe UC.

Objective To explore the clinical correlation of the variation of plasma homocysteine (HCY), melatonin (MLT) and ulceative colitis (UC). Methods The clinical data of 112 UC patients was collected, and 110 normal healthy persons as control. The level of plasma HCY and MLT was detected by high pressure liquid chromatography-fluorescence detection (HPLC-FD) method. The level of plasma folate ( FA) and vitamin B12 was detected by enzyme-linked immunosorbent assay (ELISA) method. The correlation of these four indexes and UC was analyzed. Results The serum level of HCY in UC patients was significantly higher than that in normal healthy persons [(11. 27± 7.26) μmol/L vs (8. 19±4. 81) μmol/L, P = 0. 000]. The serum level of MLT in UC patients was significantly lower than that in normal healthy persons [(49. 06 + 31. 40) pg/ml vs (64. 28±41. 16) pg/ml,P=0. 008]. The serum level of FA in UC patients was significantly lower than that in normal healthy persons [(7. 64 + 1.95) nmol/L vs (9. 14 + 1.23) nmol/L, P = 0. 005]. The serum level of vitamin B12 in UC patients was significantly lower than that in normal healthy persons [(108. 64 ±32. 22) pmol/L vs (112. 64±33. 33) pmol/L, P = 0. 004]. There was no correlation between plasma HCY, MLT and UC disease activity degree, range, disease duration, erythrocyte sedimentation rate (ESR), or C reactive protein (CRP) in UC patients. There was no significant correlation between MLT and HCY in UC patients. Conclusions The serum level of HCY is higher in UC patients than that in normal control, and MLT is lower than that in normal control. However there is no significant correlation between them.

AIM: The expression of CD28, CD56 and CD57 on CD8+T cells in the peripheral blood of young (age range 20-35) and elderly(age range 60-75) healthy donors were compared to explore the change of the cellular immune function with aging.METHODS: Three-color fluorescent flow cytometry was performed to analyze the differences in percentage of CD8+CD28+, CD8+CD56+ and CD8+CD57+T cells in the peripheral blood between the young and elderly groups.RESULTS: CD8+CD28+T cells in the peripheral blood of the elderly group was significantly lower than those in the young group, with percentage of 34.07?5.28 and 49.84?7.43,respectively (P

Objective To investigate the effect and mechanism of anti-tumor necrosis factor (TNF)-α on the intestinal mucosal permeability in dextran sulfate sodium (DSS) induced colitis mice.Methods Eighteen C57BL/6J mice were evenly divided into healthy control group,model group and anti-TNF-α treated group.The mice of model group and anti-TNF-α treated group were fed with 5％DSS solution for 7 days.The mice of anti-TNF-α treated group were injected anti-TNF-α (5 mg/kg)intraperitoneally on the first and fourth day; control group and model group were substituted with equal volume saline injection.The mice were sacrificed at 7 days after modeling.The disease activity index (DAI) score was evaluated everyday.The intestinal permeability was examined by Evan′s blue (EB) method and FITC-dextran (FITC-D) method.The colon tissue was collected for observation under microscope and histological index (HI).The small intestinal tissues were examined under electron microscope.The 10％ homogenate of colon and intestinal mucosa was prepared,the activity of myeloperoxidase (MPO),the content of TNF-α and epithelial myosin light chain kinase (MLCK) concentration were determined with kits respectively.The expression of MLCK in intestinal mucosa was tested by Western blot assay.Single factor of variance between groups were analyzed.Results Compared with control group,the DAI of model group increased daily.Compared with model group,the DAI of anti-TNF-α treated group improved.In model group,mice intestinal epithelial cells junctional complex shortened and widened and the cell gap expanded.In anti-TNF-α treated group,the connection structure of mice intestinal epithelial cells was tighter.The activity of HI and MPO and the content of TNF-α of model group were higher than those of control group (P ＝ 0.008,0.006 and 0.001,respectively),all of those of anti-TNF-α treated group were lower than those of model group (P=0.004,0.008 and 0.005,respectively).The F value of three groups was 131.98,218.28 and 58.93,respectively.The contents of EB in mice intestinal wall and serum FITC-D of model group were higher than those of control group (P=0.003 and 0.010),and those of anti-TNF-α treated group were lower model group (P＝0.001 and 0.009).The F value of three groups was 69.36 and 17.96.The MLCK concentration in mice intestinal mucosa of model group [(71.10± 7.52) ng/g] was higher than that of control group [(18.56±9.92) ng/g,P＜0.01],that of anti-TNF-α treated group [(37.56±15.84) ng/g] was lower than model group (P=0.008),and the difference among these three groups was statistically significant (F＝ 17.23).The Western blot results indicated the expression of MLCK in intestinal mucosa of model group was higher than that of control group,and that of anti-TNF-α treated group was lower than model group.Conclusions Anti-TNF-α play an important role in improving colitis,and the intestinal mucosal permeability.The mechanism may be related with the regulation of MLCK expression.

Objective To investigate the association between MYO9B rs962917 and rs1545620 gene polymorphism and clinical pathological characteristics of patients with inflammatory bowel disease (IBD) and permeability of intestinal mucosa.Methods From September 2010 to May 2012,a total of 196 cases of patients with IBD were collected,100 cases were ulcerative colitis (UC) and 96 cases were Crohn's disease (CD).At the same time,99 gender and age matched healthy individuals were collected as healthy controls.The 5 mL blood of participants was obtained and DNA was extracted.The MYO9B gene rs962917 and rs1545620 polymorphism was detected by polymerase chain reaction (PCR) and ligase detection reaction (LDR).After 60 patients with UC and 58 patients with CD orally took intestinal permeability testing fluid (with lactulose and mannitol),the urine of the patients was analyzed with high pressure liquid chromatography-pulsed lectrochemical dection (HPLC-PED).The permeability of intestinal mucosa was determined according to the ratio of lactulose and mannitol.Chisquare test was used for count data.Results Compared with healthy control group,there was no significant difference in genotype and allelic gene distribution of MYO9B rs962917 and rs1545620 of IBD group,UC group and CD group (all P＞0.05).The genotype of MYO9B rs962917 and rs1545620 of patients with UC was not related with the disease activity and location of lesions (rs962917:x2 =0.481 and 3.812,rs1545620..x2 =0.398 and 4.543 ;all P＞0.05).The genotype of MYO9B rs962917 of patients with CD was not related with the disease activity,lesion type and occurrence of perianal lesions (x2 =0.384,0.476 and 3.486,all P＞0.05) and was related with location of lesions (x2=15.926,P＜0.05).The genotype of MYO9B rs1545620 of patients with CD was not related with the disease activity and lesion type (x2 =1.407 and 5.126,both P＞0.05),however was related with location of lesions and occurrence of perianal lesions (x2 =18.165 and 7.629,both P＜0.05).The permeability of intestinal mucosa of all 58 patients with CD was high.The genotype of MYO9B rs962917 and rs1545620 of patients with UC was not related with the permeability of intestinal mucosa (x2=1.508 and 1.025,both P ＞ 0.05).Conclusion MYO9B rs962917 and rs1545620 gene polymorphism is related with the location of lesions in CD and is not related with the permeability of intestinal mucosa of patients with UC.

BACKGROUND:Distraction osteogenesis is one of the most important tissue engineering technologies. However, the exact signaling pathway controling mesenchymal stem cel-osteoblast lineage (MSC-OB) migration during distraction osteogenesis has not yet been elucidated. More efforts should be paid to make a ful understanding of the mechanism on MSC-OB lineage migration, which can improve the clinical efficacy of distraction osteogenesis.
OBJECTIVE:To evaluate the effects of mechanical stretch on the ability of MSC-OB mobility and expression of mammalian target of rapamycin (mTOR) signaling pathway as wel as matrix metaloproteinases (MMPs) in MSC-OB, and to make clear the mechanism by which controls MSC-OB migration during distraction osteogenesis.
METHODS:Twelve Sprague-Dawley rats were randomized into two groups: experimental group (n=6), anin vivo rat mandibular distraction osteogenesis model was established on the right side of rats; non-stretch group (n=6), only the mandibular resection was done but with no distraction osteogenesis. Immunohistochemical staining was used to detect phosphorylated mTOR expression in new osteotylus at 15 days after operation. In addition, an in vitro cel stretch model was made in the mandibular mesenchymal stem cels from healthy Sprague-Dawley rats under resting tension force (6%, 4 hours); no distraction was done in control group. The ability of MSC-OB mobility, the expression of mTOR, Raptor, p70S6K and MMPs were evaluated using experiment methods including immunohistochemistry staining, real-time PCR and scratch assay.
RESULTS AND CONCLUSION: The expression of phosphorylated mTOR in MSC-OB was upregulated in the mandibular bone calus of the stretch group than the non-stretch group (P < 0.05). In thein vitro experiments, MSC-OB applied with mechanical stretch (6%, 4 hours) showed elevated gene expression levels of mTOR, Raptor, p70S6K, MMP-2, MMP-9 and MMP-13 compared with the control group (0%, 4 hours). Meanwhile, MSC-OB in the experiment group (6%, 4 hours) showed a greater ability of mobility, as demonstrated by a farther distance after 48 hours of observation (P < 0.05). The present study suggests that the enhancement of MSC-OB mobility correlates with increase of the gene expression of MMPs and mTOR signaling pathway. Mechanical stretch may promote MSC-OB migration through activation of mTOR/MMPs signaling pathway.

Aim To investigate whether Hcy influenced the intestinal mucosal permeability by regulating MEK-ERK-MLCK pathway. Methods SD rats were divided into 4 groups:normal group, normal+Hcy group, TN-BS/ethanol group, TNBS/ethanol+Hcy group. Experi-mental colitis model with hyperhomocystinemia was es-tablished in rats with intracolonic administration of TN-BS and subcutaneous injection of Hcy. The colonic mucosal tissue was collected for histopathological exam-ination and activity of myeloperoxidase ( MPO ) . The protein expression of MLCK, p-MLCK, MEK, ERK and p-ERK in intestinal mucosal tissues was examined by Western blot method. The mRNA expression of ML-CK was examined by RT-qPCR method. Result Com-pared with the normal group and TNBS group, the DAI and HI scores and the MPO activity were increased in TNBS/ethanol+Hcy group ( P <0. 01 ) . Western blot and RT-qPCR showed that expression of MLCK, p-ML-CK, MEK, ERK and p-ERK increased in small intes-tine in TNBS/ethanol+Hcy group. Conclusion Hcy can increase intestinal permeability in TNBS-induced colitis rats by regulating the expression of MEK-ERK-MLCK signal pathway.