BACKGROUND: The glial scar formation after spinal cord injury in mammals is the physical and chemical barriers for neural regeneration, and relieving or delaying glial scar formation can provide benefit conditions for the regeneration of injured spinal cord.OBJECTIVE: To design and screen short hairpin RNA (shRNA) interfere molecular targeting the gene coding region of glial fibrillary acidic protein (GFAP) in rat, and reconstruct the eukaryotic vector of shRNA.DESIGN: An observational animal experiment.SETTING: Department of Spine Surgery, Shenzhen Hospital affiliated to Southern Medical University.MATERIALS: Twenty-five Wistar rats of clean degree, either male or female, weighing 20-25 g, were used. DMEM/F12,lipofectamine2000, Trizol RNA separating kits); fetal bovine serum (Hyclone); BamH Ⅰ, HindⅢ, Pstl, Sail and T4 ligases;Plasmid mini preparation kit and DNA gel extraction kit.METHODS: The experiments were carried out in the Shenzhen Hospital affiliated to Southern Medical University from October 2005 to June 2006. Three pairs of shRNA template which composed of 19 bp reverse repeated motif of GFAP target sequence with 9 bp spacer were designed and synthesized, then they were inserted directionally into plasmid Psilencer 2.1 respectively to generate small interfering RNA (siRNA) eukaryotic expression vector. ShRNA molecules were transfected by liposome via ex vivo expression repressive model of GFAP of rat spinal astrocytes. The effects of expressive suppression were detected with real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, and then the optimal shRNA eukaryotic vector of repressive expression of GFAP was screened.MAIN OUTCOME MEASURES: ① Interfering sequence specific shRNA template synthesis; ② Constructing specific recombinant plasmid eukaryotic expression vector. ③ Culturing rat spinal astrocytes in vitro; ④ Effects of expressive suppression on GFAP in primary astrocytes after RNA interference detected with real-time fluorescence quantitative RT-PCR and Western blot.RESULTS: Sequence analysis showed that GFAP-shRNA recombinant plasmid eukaryotic expression vector was successfully constructed, and optimal GFAP-shRNA eukaryotic vector was screened using real-time fluorescence quantitative RT-PCR and Western blot. The GFAP expressions were reduced by 81%, 63% and 56% at the levels of mRNA and protein respectively.CONCLUSION: GFAP-shRNA eukaryotic expression vectors were successfully constructed and screened. The gene expression GFAP of primitive rat astrocyte can be suppressed significantly by the GFAP-shRNA eukaryotic expression recombinant optimized via ex vivo cellular expression suppression model, which should pave the way for the following multiple targets of RNAi genetic manipulation in the treatment of suppression of glial scar formation after spinal cord injury.

Objective To evaluate the role of autophagy on acute kidney injury after liver transplantation.Method Fifty-six healthy male Sprague-Dawley rats were randomly assigned into 4 groups:sham group,orthotopic liver transplantation (OLT) group,sirolimus pretreated (SRL) group and 3-methyladenine pretreated(3-MA) group.OLT model was established.Then the rats were sacrificed at 6 h after reperfusion.The renal function and the extent of oxidative stress relative proteins malondialdehyde (MDA) and superoxide dismutase (SOD) were observed.The levels of apoptosis relative genes caspase-3 and cyt c and the expression of autophagy relative proteins were detected.The pathological changes were microscopically examined in renal tissues.TUNEL staining was used to observe the apoptosis of tubular epithelial cells.Transmission electron microscopy was applied to observe the ultrastructure changes of tubular epithelial cells.Result As compared with sham group,OLT and 3-MA groups showed a serious renal injury including cellular vacuolization,loss of brush borders,and a significant rise in BUN,Cr and MDA,while a decrease in SOD activity.The levels of caspase-3 mRNA and cyt c rnRNA were increased significantly.Whereas compared to OLT and 3-MA groups,renal function and oxidative stress levels in SRL group ameliorated,and histopathologic damage and apoptosis alleviated after OLT.Simultaneously,the levels of caspase-3 mRNA and cyt c mRNA were decreased.The expression of beclin-1 and LC3-]Ⅱ was effectively upregulated.Conclusion Autophagy could alleviate acute kidney injury after liver transplantation through inhibiting oxidative stress and apoptosis.

Objective To study the effects of extracellular-signal regulated kinase mitogenactivated protein kinase (ERK-MAPK) signaling pathway inhibition on histone phosphorylation and the related gene expression in human colorectal cancer cells.Methods Two human colorectal cancer cell lines (SW1116 and HCT116) were cultured and treated with gradient(0,20,40/μmol/L) doses of ERK-MAPK signaling pathway inhibitor U0126.Cell viability was determined by cell counting kit 8 (CCK-8) assay.Cell cycle distribution was assessed by flow cytometry.The expression levels of histone H3 kinases including ribosomal S6 serine-threonine kinase (RSK-2) and mitogen-and stressactivated protein kinase 1 and 2 (MSK1 and MSK2),and the levels of histone H3 (Ser10) phosphorylation and c-Fos protein were detected using Western blotting.Results Treatment of these two human colorectal cancer cell lines with ERK-MAPK inhibitor resulted in a time and dose-dependent inhibition of cell proliferation significantly. Proliferation rate of HCT116 was reduced to 47% at 72 hours after 40/μmol/L U0126 treatment. Cell cycle analysis showed that the percentage of phase G0/G1 cells significantly increased (P<0. 01) and the percentage of phase S cells decreased (P<0.01) after treatment with ERK-MAPK inhibitor. The expression of MSK1 and RSK2 reduced obviously in both of human colorectal cancer cell lines treated with U0126, which resulted in a 28% and 40% reduction of levels of MSK1 and RSK2 as compared with control HCT116 cells respectively,while no detectable change in the expression of MSK2 was found. Consistent with this, the expression level of histone H3 (ser10) phosphorylation was markedly down-regulated by ERK-MAPK inhibitor, and the related protein c-Fos expression decreased accordantly. Conclusions Decreased ERK-MAPK signaling pathway may reduce histone H3 (Ser10) phosphorylation via suppression of the activity of histone H3 kinase including MSK1 and RSK2, but not MSK2, consequently decrease the expression of c-Fos protein, which results in the inhibition of colorectal cancer cells proliferation.

BACKGROUND: It is demonstrated that bone marrow stem cells (BMSCs) can generate neurosphere structures, which is similar to cloning sphere of neuron-specific enolase (NSE), in a specially induced system in vitro; therefore, BMSCs draw more and more attention as seed cells to repair central nerve injury.OBJECTIVE: To investigate the differentiation from BMSCs into neuron-like calls induced by fetal spinal cord tissue.DESIGN: Observational study.SETTING: Department of Spine Surgery, Second People's Hospital of Shenzhen.MATERIALS: SD rats (16 pregnant days old and 2 months old) were provided by Animal Center, Tongji Medical College,Huazhong University of Science and Technology. Single antibody of NSE, multi-antibody of glial fibrillary acidic protein (GFAP) and single antibody of neurofilament (NF200) were provided by Wuhan Boster Company.METHODS: The experiment was carried out in Immune Opening Laboratory and Central Laboratory, Basic Medical Department, Tongji Medical College, Huazhong University of Science and Technology in September 2006. Bones of lower extremities of rats were collected to centrifuge BMSCs. Fetal spinal cord tissue homogenate was extracted from 16 pregnant days old rats to make inducing solution and induce differentiation of BMSCs. Otherwise, embryo muscle tissue was used to make muscle tissue homogenate as the same way so as to regard as the controls.MAIN OUTCOME MEASURES: BMSCs underwent morphological observation after induction; in addition, anti-NSE, NF200 and anti-GFAP were used to label neurons and astrocytes, respectively. Ten non-overlapping sights were randomly selected from positive reactive-induced cells after immunohistochemical staining under optic microscope to calculate ratio of positive cells of NSE and NF200 counting for total numbers of cells.RESULTS: ① Morphological changes of BMSCs after induction: During early induction, optic microscope indicated that soma of partial cells rebounded; whose apophysis was long and thin; apophysis of differentiated cells grew gradually and intercrossed each other. It was similar to nerve cells and some branches were similar to dendrite branches. However,morphological changes of cells in the control group were not obvious. ② Expression of relevant antigens differentiated from BMSCs into neuron-like cells at one week after induction: Most cells in spinal cord homogenate group expressed as positive NSE and NF200; a few of cells expressed as GFAP. While, positive staining of nerve cell antibody was not observed in the control group; meanwhile, positive reaction of nerve cell antigen was not observed in the control group,too. Immunohistochemistry examination demonstrated that positive rates of NSE and NF200 expressions were (68±1.7)%and (76.2±2.9)%, respectively.CONCLUSION: Fetal spinal cord tissue homogenate can induce differentiation from BMSCs into neuron-like cells.

Objective To observe the effect of aloe on intestine motility in the old costive mice and investigate the mechanism for aloe promoting an intestinal motility. Methods The content of aloin in aloe powder was determined by high performance liquid chromatography(HPLC).The old mice aged 15 months were randomly divided into 6 groups(10 in each group):blank control,positive control,constipation model,low-dose aloe,middle-dose aloe,high-dose aloe plus model.The mice of with equivalent volume of distilled water.On the eighth day,the mice except control group were given Compound Diphenoxylate to establish constipation model. With the black Indian ink as marker,the first time of black stool discharge,the character and weight of the stool,and the ink propulsion rate by intestines in mice were observed respectively.The serum level of nitric oxide(NO)was determined by spectrophotometry. Results The content of aloin in aloe powder was 0.266%.Compared with constipation model group,aloe groups in different dose decreased the first black stool time and increased stool grains and weight in 6 hours of constipated mice.The ink propulsion rates of intestines in the aloe groups were significantly higher than that of model group as well.The NO level in high-dose aloe group decreased more significantly compared with model group(P＜0.05),and there was a negative correlation between the serum NO level and propulsion rate of intestines(r=-0.346.P＜O.05). Conclusions Aloe could promote the mobility of intestine and ameliorate the constipation of mice,which might attribute to the decrease of the serum NO level.

Objective To evaluate the diagnosis and surgical treatment of primary hyperparathyroidism (PHPT). Methods 53 patients with PHPT who were treated in our hospital from 1997 to 2009 were analyzed retrospectively. Results All patients showed hypercalcemia and elevated parathyroid hormone (PTH). Among them, 43 were diagnosed as parathyroid adenoma, 6 were parathyroid hyperplasia and 4 were parathyroid cancer.The accuracy rate of parathyroid tumor localization was above 94. 3%. All patients presented temporary hypocalcemia after surgery. Conclusions Parathyroidectomy is an effective approach for patients with PHPT. Preoperative localization is essential to the surgery.

Objective To investigate the role of nuclear factor erythroid 2-related factor 2 (Nrf2) / heme oxygenase-1 (HO-1) signaling pathway in reduction of acute kidney injury following orthotopic liver transplantation (OLT) by hydrogen-rich saline in rats.Methods Thirty-two healthy adult male SpragueDawley rats,weighing 220-250 g,were randomly assigned into 4 groups (n =8 each) using a random number table:sham operation group (S group),OLT group,hydrogen-rich saline group (HS group),and all-trans retinoic acid (ATRA) group.Laparotomy was performed,and the related blood vessels were isolated in group S.The model of orthotopic autologous liver transplantation was established in OLT,HS and ATRA groups.Normal saline and hydrogen-rich saline 6 ml/kg were injected through the inferior vena cava at 5 min before the portal vein was clamped in OLT and HS groups,respectively.In group ATRA,Nrf2 inhibitor ATRA 7 mg/kg was injected intraperitoneally once a day for 2 consecutive days,the model of orthotopic autologous liver transplantation was established at 16 h after the last injection of ATRA,and the other treatments were similar to those previously described in group HS.At 6 h of reperfusion,blood samples were collected for determination of serum blood urea nitrogen (BUN),creatinine (Cr),interleukin10 (1L-10) and tumor necrosis factor-alpha (TNF-α) concentrations.After blood sampling,the lungs were removed for determination of malondialdehyde (MDA) content,superoxide dismutase (SOD) activity,expression of HO-1,Bcl-2 and Bax mRNA (by using real-time reverse transcriptase polymerase chain reaction),and HO-1 protein expression in lung tissues (by Western blot) and for microscopic examination.The damage to the renal tubules was scored.Results Compared with group S,the serum BUN,Cr and TNF-α concentrations were significantly increased,the serum IL-10 concentrations were decreased,the MDA content and renal tubular damage score were increased,the SOD activity was decreased,and the expression of HO-1 protein and mRNA,and Bcl-2 and Bax mRNA was up-regulated in group OLT (P＜ 0.05).Compared with group OLT,the serum BUN,Cr and TNF-α concentrations were significantly decreased,the serum IL-10 concentrations were increased,the MDA content and renal tubular damage score were decreased,the SOD activity was increased,the expression of HO-1 protein and mRNA and Bcl-2 mR-NA was up-regulated,and the expression of Bax mRNA was down-regulated in group HS (P＜0.05).Compared with group HS,the serum BUN,Cr and TNF-α concentrations were significantly increased,the serum IL-10 concentrations were decreased,the MDA content and renal tubular damage score were increased,the SOD activity was decreased,the expression of HO-1 protein and mRNA and Bcl-2 mRNA was down-regulated,and the expression of Bax mRNA was up-regulated in group ATRA (P＜0.05).Conclusion The mechanism by which hydrogen-rich saline reduces acute kidney injury following OLT is probably associated with activation of Nrf2/HO-1 signaling pathway in rats.

Objective To evaluate the role of autophagy in hydrogen-induced inhibition of apoptosis in hippocampal neurons in a rat model of orthotopic liver transplantation (OLT).Methods Fifty-six pathogen-free healthy adult male Sprague-Dawley rats,aged 8-10 weeks,weighing 220-250 g,were used in the study.Thirty-two rats were selected and assigned into 4 groups (n =8 each) using a random number table:sham operation group (group S),OLT group,hydrogen-rich saline group (group HS) and chloroquine group (group CQ).The other 24 rats severed as the donors.In group S,laparotomy was performed,and the related blood vessels were isolated.The model of OLT was established in OLT,HS and CQ groups.In group OLT,normal saline 6 ml/kg was slowly injected via the inferior vena cava at 5 min before anhepatic phase.In group HS,hydrogen-rich saline 6 ml/kg was slowly injected via the inferior vena cava at 5 min before anhepatic phase.In group CQ,autophagy inhibitor chloroquine 60 mg/kg was injected intraperitoneally at 1 h before establishment of the model,and the other treatments were similar to those previously described in group HS.At 6 h of reperfusion,the rats were sacrificed and hippocampi were isolated for determination of malondialdehyde (MDA) content and superoxide dismutase (SOD) activity,for pathological examination (with light microscope),and for detection of cell apoptosis (by TUNEL staining) and expression of autophagy-and apoptosis-related proteins caspase-3,cytochrome c (Cyt c),microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ),Beclin-1 and p53 in hippocampal tissues (by Western blot analysis).Apoptosis index (AI) was calculated.Results Compared with group S,the MDA content and AI were significantly increased,the SOD activity was decreased,and the expression of caspase-3,Cyt c,LC3 Ⅱ,Beclin-1 and p53 was up-regulated in OLT,HS and CQ groups (P＜0.05).Compared with group OLT,the MDA content and AI were significantly decreased,the SOD activity was increased,the expression of caspase-3 and Cyt c was down-regulated,and the expression of LC3 Ⅱ,Beclin-1 and p53 was up-regulated in group HS (P＜0.05).Compared with group HS,the MDA content and AI were significantly increased,the SOD activity was decreased,and the expression of caspase-3 and Cyt c was up-regulated,and the expression of LC3 Ⅱ,Beclin-1 and p53 was down-regulated in group CQ (P＜0.05).Conclusion The mechanism by which hydrogen inhibits apoptosis in hippocampal neurons is related to promotion of autophagy in a rat model of OLT.

Objective To investigate the expression and the role of Tim-3 and Th-17 in ITP patients and to research their clinical application. Methods Total 42 active ITP patients and 39 healthy donors were recruited in this research. The expressions of Th17 and CD4+ CD25+ Treg were measured with flow cytometer. IL-17, IFN-γ levels as well as IL-4 plasma levels were determined by ELISA. The mRNA expression of Tim-3, IFN-γ, IL-4 and T-bet were measured using RT-PCR in all samples. Results The expression of Th17 cells in ITP patients was (2.41 ± 1.43 )%, which was significantly higher than control group ( 1.08 ± 0.59)% ( t = 5.35, P < 0.05 ). But the percentage of Treg in ITP patients was ( 1.64 ±0.74)%, which was lower than control group (3.12 ±0.52)% (t = 10.33, P <0.05). The levels of IL-17 in plasma of ITP and controls were ( 14.42 ±6.37) ng/L and ( 13.91 ±4.47) ng/L respectively (t =0.42, P > 0.05). The level of IFN-γin plasma of ITP was (55.74 ± 15.25 ) ng/L, which was higher than control group (31.33 ± 12.99) ng/L (t = 7.72, P < 0.05 ). The level of IL-4 in the plasma of ITP was (7.42 ± 1.50) ng/L, which was lower than controls ( 18.17 ± 5.19) ng/L ( t = 12.87, P ＜ 0.05 ). Both IFN-γand T-bet mRNA levels were up-regulated in active ITP patients by the factor ( 8.57 ± 3.44 ) -fold and (3.34 ± 1.32)-fold than control group (t = 13.21,6.41 ,P <0.05). The decreases observed in IL-4 and Tim-3 were (0.25 ±0.15 )-fold and (0.29 ±0.15)-fold respectively in ITP patients compared with control group (t=10.02,9.61,P＜0.05 ). Conclusion The imbalance of Th17/Treg and the decrease of Tim-3 might be important determinants in the evolution of ITP.

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