Study on protein expression and antigenicity detection of hepatitis C Virus envelope protein E2 in prokaryotic and eucaryotic expression systems.The gene that encoding.HCV E2 protein was cloned in pQE30 and pEF1/HisC.After the expression of E2 protein in E.Coli M15 and COS 7 cell,the expressed proteins were used to detect their antigenicity with ELISA and WB.The results showed that protein E2 was expressed in both prokaryotic and eucaryotic cells.Special reaction could be detected using the expressed proteins and sera from HCV infected people.The studied E2 gene could express the desired proteins in both prokaryotic and eucaryotic expression systems,and glycosylation of the E2 protein happened in COS 7 cell.

objective To investigate the effects of different sections of receptor associated protein (RAP) on the expression and distribution of TRPC6,synaptopodin and podocalyxin in passive Heymann nephritis(PHN). Methods Male Sprague-Dawley rats were injected with three kinds of antisera (anti-RAP full-length serum,anti-RAP N-terminal serum and anti-RAP C-terminal serum)to establish three kinds of PHN models.The control group was injected with normal rabbit serum.The quatitation of 24 h urinary protein,serum albumin and creatinine were taken before injection and one week after PHN model successfully induced.The histopathologic changes of renal tissues were observed by light microscopy.The expression and distribution of TRPC6,synaptopodin and podocalyxin in glomerular podocytes were observed by laser scanning confocal microscopy and analyzed by fluorescence quantitative software after indirect immunofluorescence double staining.Results The quantities of 24 h urinary protein in the three model groups were significantly higher than those of themselves before injection and control groups (P<0.01,respectively).The values of serum albumin and creatinine were not significantly different before and after injection (P>0.05).The expression of TRPC6 in podocytes was higher in the PHN model groups than that of control group.Fluorescence intensity of TRPC6 in RAP full-length group was stronger than that in RAP N-terminal or C-terminal groups.The expressions of synaptopodin and podocalyxin distributed along the glomerular basement membrane as spot,discontinuous short line and defect of some segments,and were lower in three PHN groups than those of control group.Fluorescence intensity of synaptopodin and podocalyxin among three PHN groups had no differences. Conclusions RAP full-length and N-terminal or C-terminal parts can increase the expression of podocyte TRPC6,but decrease the expressions of synaptopodin and podocalyxin,and alter their distribution,which may be associated with the proteinuria,however,their role in the PHN pathogenesis needs further study.

To observe the effects of Danshen-containing serum on SuFu and DYRK2 expression in the HSCs stimulated by leptin. SD rats (n = 60) were used to make danshen-containing serum by gastric perfusion for ten days with Danshen water decoction, normal saline and colchicine. The HSCs that were cultured in vitro would be stimulated for 24 hours by leptin (100 μg x L(-1)) except blank control group, after being intervened, the drug serum in each group would be cultured at 37 degrees C in 5% incubator. The cells would be collected after 24 hours, then the effects of danshen-containing serum on the proliferation of HSCs were detected by MTT, the expression of SuFu mRNA and DYRK2 mRNA were detected by RT-PCR, the expression of SuFu and DYRK2 proteins were tested by Western blot. Compared with blank control group, the expression of DYRK2 mRNA and DYRK2 proteins were enhanced obviously after stimulated the HSCs of rats by leptin (P < 0.01), but the expression of SuFu mRNA and SuFu proteins were decreased significantly (P < 0.01). Compared with the model group, after cyclopamine group (Hh pathway inhibitor), Danshen-containing serum and colchicine were interfered, the expression of DYRK2 mRNA and DYRK2 proteins were decreased clearly (P < 0.01), but the expression of SuFu mRNA and SuFu proteins were increased significantly (P < 0.01 or P < 0.05). Compared with model group, adding purmorphamine (Hh pathway agonist) to model group and making it activate could increase the expression of DYRK2 mRNA and DYRK2 proteins, but the expression of SuFu mRNA and SuFu proteins were decreased significantly (P < 0.01). Compared with the model group, using the Danshen-containing serum to interfere the purmorphamine group could make the expression of DYRK2 mRNA and DYRK2 proteins decrease and the expression of SuFu mRNA and SuFu proteins increase significantly (P < 0.01). Danshen-containing serum would inhibition the activation and increment of HSCs by interfering the expression of SuFu and DYRK2 which were induced by leptin.
Animals ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Hepatic Stellate Cells ; drug effects ; metabolism ; Humans ; Liver Cirrhosis ; drug therapy ; genetics ; metabolism ; Male ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Protein-Tyrosine Kinases ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Repressor Proteins ; genetics ; metabolism ; Salvia miltiorrhiza ; chemistry

Objective To evaluate biochemical characteristics and the trend of diabetic complications in patients with newly-diagnosed type 2 diabetes from 1994 to 2008. Methods We utilized the database of the diabetes complications assessment and analyzed the metabolic disorder and the diabetic complications in the patients with newly diagnosed diabetes. Results 2 085 cases were collected, including 1189 males and 896 females. The average age of onset of diabetes was 51.6±13.1 and 54.6±7.9 yrs respectively in 2008 and 1994. During 1994,no case was found in subjects aged 20-29 yrs and 5% of the patients were aged 30-39; but 2% of patients aged 20-29 and 16% aged 30-39 yrs were found in 2008. BMI was increased from 24.48±4.15 in 1994 to 26.03±3.63 in 2008. Percentage of patients with abnormal BMI ( ≥25 kg/m2 ), WHR [≥0.90 (male) or ≥0.85 (female)]increased significantly from 63.6%, 75.0%, and 71.4% in 1994 to 79.6%, 95.2%, and 93.8% in 2008,respectively. Both SBP and DBP were not significantly changed. The fasting blood and postprandial blood glucose,HbA1c decreased from 10.3 mmol/L, 15.2 mmol/L, 11.1% in 1994 to 9.0 mmol/L, 14.3 mmol/L, and 8.6% in 2008, respectively. The average TG level increased from 1.7 mmol/L in 1994 to 2. 1 mmol/L in 2008,however, TC and HDL level were not significantly changed. The prevalence of diabetic retinopathy decreased from 28.2% in 1994 to 3.9% in 2008. The prevalence of diabetic nephropathy increased from 17.7% in 1994 to 24.6% in 2008. The prevalence of diabetic cardiovascular disease increased from 14.3% in 1994 to 24. 1% in 2008. Compared with the patients without microvascular complications, the patients with microvascular complications had higher SBP, DBP, and HbA1c( 136/78 vs 130/77 mm Hg, 9.41% vs 9.11% ). The patients with macrovascular complications had older age, higher SBP, TC, and TG than those without macrovascular complications (53.4 vs 50.0 yrs; 132 vs 129 mm Hg ; 5.3 vs 5.1 mmol/L and 2.6 vs 2.1 mmol/L). Conclusions In the studied newly-diagnosed diabetic patients from 1994 to 2008, there were increasing incidences of obesity and hypertriglyceridemia. However, the prevalence of diabetic retinopathy decreased significantly, while that of nephropathy showed no significant change.Cardiovascular complications were markedly increased.

Objective:To study the construction of matrix metalloproteinase 3 (MMP3)biosensor vector,and to illuminate the activated process of MMP3 in the living cells.Methods:The ECFP-MMP3-YPet biosensor vector anchored on cellular surface was constructed and identified.The MMP3 biosensor was transfected into the 293T cells.The transfection efficiency was observed 24 h after transfection.The flurorescence resonance energy transfer (FRET )-based MMP3 biosensor was observed by inversion fluorescence microscope. Results:The MMP3 biosensor vector was successfully constructed.The length of MMP3-YPet identified by double enzyme digestion and PCR was about 780 bp.The transfection efficiency of MMP3 biosensor was about 40%,and which was evenly presented in cytoplasm of 293T cells.And the FRET ratio of MMP3 biosensor was decreased after stimulation with uPA on the 293T cells. The FRET ratio reached its minimum about 30 min later. Conclusion:The MMP3 biosensor can sensitively and reliably monitor the MMP3 activation in living cells.

Objective To evaluate the effects of transforming growth factor β1 (TGF-β1 )on migration, adhesion and proliferation of periodontal ligament stem cells (PDLSCs)and explore the mechanisms of PDLSCs-induced periodontal remodeling.Methods PDLSCs were isolated and identified from human teeth.The effect of TGF-β1 on migration of PDLSCs was evaluated using transwell migration assay.Cells attachment assay was used to test the effect of TGF-β1 on the adhesion of PDLSCs.In addition,the effect of TGF-β1 on the proliferation of PDLSCs was evaluated by MTT and cell growth rate assay.Results The results showed that TGF-β1 induced the migration of PDLSCs in a dose-dependent manner,improved the adhesion and proliferation of PDLSCs.So we propose that TGF-β1 may promote periodium remodeling by inducing PDLSCs migration,following adhesion and proliferation in these areas.Conclusion This study demonstrated for the first time that TGF-β1 increases the adhesion and migration of PDLSCs in vitro .The signal pathway is involved in the TGF-β1-induced migration of PDLSCs and the mechanical-chemical interaction during the orthodontic periodontal remodeling will be researched in our further studies.

OBJECTIVETo review the recent studies about human umbilical cord mesenchymal stem cells (hUCMSCs) and advances in the treatment of spinal cord injury. Data sources Published articles (1983 - 2007) about hUCMSCs and spinal cord injury were selected using Medline. Study selection Articles selected were relevant to development of mesenchymal stem cells (MSCs) for transplantation in spinal cord injury therapy. Of 258 originally identified articles 51 were selected that specifically addressed the stated purpose.

RESULTSRecent work has revealed that hUCMSCs share most of the characteristics with MSCs derived from bone marrow and are more appropriate to transplantation for cell based therapies.

CONCLUSIONSHuman umbilical cord could be regarded as a source of MSCs for experimental and clinical needs. In addition, as a peculiar source of stem cells, hUCMSCs may play an important role in the treatment of spinal cord injury.

Humans ; Mesenchymal Stromal Cells ; cytology ; physiology ; Models, Biological ; Spinal Cord Injuries ; pathology ; therapy ; Stem Cell Transplantation ; Umbilical Cord ; cytology

Objective To construct the eukaryotic expression vector urokinase-type plasminogen activator (uPA) biosensor which was the composition of the fusion protein enhanced cyan fluorescent protein-uPA (substrate)-yellow fluorescent protein variant (ECFP-uPA substrate-linker-YPet).Methods By the template Src-biosensor, the YPet primers were designed by Primer Premier 5.0 software,and the restriction enzyme sites,uPA substrate gene sequence and linker were added in its 5′ end. With the intermediate vector pDMTM-18T, an eukaryotic expression vector which contained a fusion protein of ECFP-uPA substrate-linker-YPet was constructed by genetic engineering.Then the uPA biosensor was transfected into 293T cells.The transfection efficiency and expression of fusion proteins were observed after 24 h.Fluorescence resonance energy transfer (FRET)was observed by the inversion fluorescence microscope and measured by the MetaFlour FRET 4.6 software. Results The uPA biosensor vector was confirmed by the fragment of PCR and double restriction enzyme digestion.The transfection efficiency was nearly 40%.The immunofluorescence detection results displayed that uPA biosensor fusion protein expressed in the 293T cells membrane and the FRET of uPA biosensor in the living 293T cells was observed after incubation with the recombinant human uPA (rhuPA).Conclusion uPA biosensor is successfully constructed and it could be used as a molecular probe to study the temporal and spatial variation of uPA in living cells.

Objective To investigate the effect of different Chinese herbs on cell proliferation and cartilage oligomeric matrix protein(COMP)expression in chondrocyte culture.Methods Chondrocytes isolated from rabbit knee cartilage were cultured for 3 generations with the density of 2?10~4/cm~2 and were verified by collagenⅡimmunohistochemical staining.Rabbit sera containing herbs were obtained after animals orally ad- ministrated herbs at the dosage equivalent to human.At 5% and 10% serum density,cells were cultured in the medium that contained liver-softening herbal compound sera.Subgroups setting at 1,3 and 5 hours after herb intervention were observed.Rabbit and bovine sera were control groups.Seven days after intervention,chon- drocytes proliferation was observed using the MTT assay kit.For the study of COMP expression,chondrocytes were isolated from human knee cartilage supematant.Superuatant COMP level was tested by enzyme-linked immunoabsorbent assays(ELISA)after directly adding compound and extract from liver-softening herbs to the culture at the final concentration of 10 mg/ml for 3 days.Results Liver-softening herbal compound group had significant effect on cell proliferation compared to control,of which,3-hour subgroup was more significant than 1-and 5-hour subgroups(P

Objective To study the prevention and treatment of complications occurring in anterior cervical spinal surgery.Methods3163cases with cervical spondylotic meylopathy,spinal injury,spinal tu-mor and spinal tuberculosis treated by anterior cervical surgery,were included in this study.Trephination,corpectomy,disectomy and debridement were used for decompression,iliac bone autograft,BAK cage,cubic shaped cages and titanium mesh cages were used for bone grafting,while anterior cervical spine locking plates were performed in some of the cases.1848cases were followed-up from6months to8years with an aver-age of 2years and3months.646cases suffered from operative complications,372cases were immediate due to anterior cervical surgery,and the incidence of morbidity was11.76%.Results26cases of transient laryn-geal nerve or superior laryngeal nerve injury recovered in4to12weeks without special treatment.Most of 16cases with cervical hemotoma were caused by bleeding of smaller blood vessels and obstruction of drainage.13cases of spinal cord or nerve root irritation or injury were treated with medicine for dehydration,12of them had good result.11cases of CSF leakage were cured with cervical spinal immobilization and moderate local compression.8cases of local infection were cured with antibiotics or combined with debride-ment and suturing.2cases of esophagus perforation were repaired and healed.8cases of grafted bone dis-placement were re-operated on the day or second day of occurring.17cases of pseudoarthritis had revision surgery.6of 35cases of adjacent segments degeneration with new symptoms of spinal cord compression were treated with anterior cervical decompression again,and had good results.Most of 342cases of iliac donor side com plications were local pain or lateral femoral cutaneous nerve injury,infection occurred in some cases.Compli cations related to instruments included10cases of BAK subsidence,1case of plate breakage,1case of screws and plate back-out and7cases of titanium mesh cage subsidence.216cases of cervical axial pain were cured3to6months later with medicine.There was1case of sudden death in this group.Conclusion Many kinds of operative complications could occur in anterior cervical surgery.A standardized procedure in diagnosis and surgery methods are the key points to decrease and prevent operative related complications.