BACKGROUND: Previous studies demonstrated that recombinant human bone morphogenetic pretein-7 (hBMP-7) can effectively promote the synthesis of extracellular matrix, repair of damaged disc matrix, restore of degenerative disc height. It is hoped that BMP-7 can be used to control and reverse the intervertebral disc degeneration. However, because of the short half-life of recombinant protein and low biological activity, it is difficult to maintain BMP-7 high concentrations on degenerative disc. Gene therapy can prevent these defects effectively.OBJECTIVE: To study the effect of hBMP-7 gene transfeotion on biological activity of primary cultured rabbit nucleus pulposus cells in vitro, to determine the feasibility of hBMP-7 gene which will on gene therapy research of intervertebral disc degeneration,and to provide basis for further study.DESIGN, TIME AND SETTING: A randomized and controlled observation was performed at the Institute of Cardiothoracic Surgery of Changhai Hospital from December 2005 to September 2006.MATERIALS: Six healthy New Zealand white rabbits of either gender, averaging 4 weeks old and weighing 500 g, were used in this study, and Ad-hBMP7 was constructed by the Cardiothoracic Surgery Institute of Changhai Hospital.METHODS: After rabbits sacrifice under aseptic condition, the nucleus pulposus was got. After digested with Pronase, type Ⅱcollagenase and type Ⅱ DNAase (4 hours, 37 "(2), the cells were seeded in the Petri dishes and were kept in the incubator. After 7 days and then twice a week, the media were refreshed. The nucleus pulposus cells were infected by adenovirus integrated with hBMP7 gene. The cells which were transfected by adenovirus vector for Lac-Z gene and which were not transfected were adopted as control.MAIN OUTCOME MEASURES: The expression of hBMP-7 was determined by RTopCR and Western blot. The effect of hBMP-7 on cell proliferation was surveyed by MTT. Furthermore, the effect of hBMP-7 gene on synthesis of proteoglycan and type Ⅱcollagen was detected by modified dimethylmethylene blue method and ELISA, respectively.RESULTS: Gene sequencing and PCR showed that hBMP-7 gene was inserted correctly and no mutation happened. The pdmary cultured rabbit nucleus pulposus cells were identical with those reported in literature. After Ad-hBMP7 transduct into the nucleus pulposus cells, high level of hBMP-7 expression was observed and lasted over 3 weeks. Also hBMP-7 gene can promote cell proliferation and synthesis of proteoglycan and type II collagen with significant difference compared with control groups (P < 0.05).CONCLUSION: hBMP-7 gene mediated by adenovirus can be the target gene for the further study on gene therapy of intervertebral disc degeneration.

Objective To analyze the characteristics and influencing factors of sleep disorders and nocturnal hypoxemia of patients with different degrees of obstructive sleep apnea-hypopnea syndrome (OSAHS). Methods Four hundred and twenty-five patients with snoring were scored by Epworth Sleepiness Scale ( ESS), and monitored by polysomnography (PSG). The possible correlations between sleep structure, hypoxia parameters, ESS and clinical features were analyzed and compared in those patients. Results Four hundred and twenty-five patients were divided into 4 groups according to the apnea-hypopnea index (AHI). There were 65 primary snoring patients (15.3%) and 360 OSAHS patients (84. 7% ) including 187 patients (44. 0% ) in severe OSAHS group. ESS was increased as aggravation of OSAHS. There were significant statistical differences in ESS among each group. Compared with primary snoring group, sleep efficiency, NREM1 + 2, oxygen desaturation index ( ODI), time with pulse oxygen saturation below 90% (T(SpO2 <90% ) ) were significantly higher in the OSAHS groups, and NREM3 +4, lowest pulse oxygen saturation level ( LSpO2 ) were lower. ESS was correlated positively with AHI (r= 0. 474,P <0. 01 ). They were both correlated positively with ODI, T (SpO2 <90% ) and NREM1 + 2( ESSr =0. 392, 0. 356,0. 194;AHI r = 0. 714, O. 682, 0. 365, all P < 0. 01 ), and correlated negatively with LSpO2, NREM3 + 4 ( ESS r = - 0. 414, - 0. 196; AHI r = - 0. 740, - 0. 385, both P < 0. 01 ). LSpO2, ODI and T (SpO2 < 90% ) were the primary influencing factors. Common clinical presentations and subjective symptoms were presented including daytime sleepiness, impaired memory, fatigue, dry mouth, oppressive wake and morning headache, etc. Percentage of individuals with daytime sleepiness in the severe OSAHS group was 73. 3% (137/187). These had serious impact on the patients' quality of life, leading to difficulty concentrating, poor memory and cognitive impairment. Conclusions Sleep disorders are found in the patients with different degrees of OSAHS. The excessive daytime sleepiness interrelated partly with the structure of sleep, and totally with hypoxia parameters. The more severity the patients have, the more nocturnal hypoxia, sleep disorders and higher ESS are found.

Objective To investigate radiological features of patients with heroin spongiform leukoencephalopathy(HSLE)of different clinical stages and discuss the evolutional characteristics of the disease.Methods Thirty two patients with HSLE underwent precontrast MRI and postcontrast MRI.The history of addiction,clinical presentations,and brain MRI were analyzed and summarized according to the patient's clinical staging.There are 6 cases in Ⅰ stage,21 cases in Ⅱ stage,5 cases in Ⅲ stage.Results All patients had history of heroin vapor inhalation.Most of the cases developed subacute cerebellar impairment in earlier period.Brain MRI revealed symmetrical lesion within bilateral cerebellum in all patients.Splenium of the corpus callosum,posterior limb of the internal capsule,deep white matter of the occipital and parietal lobes,were gradually involved with progressive deterioration of HSLE.The brain stem and deep white matter of the frontal and temporal lobes were involved in some cases.Conclusions The history of heated heroin vapor inhalation was the prerequisite for the diagnosis of HSLE.Brain MRI presented the characteristic lesion and its evolution of HSLE.Brain MRI was very important for accurate diagnosis and helpful to judge the clinical stages according to the involved brain region.

AIM: To investigate the effect of enhanced green fluorescence protein (EGFP) gene transfection on the cell cycle distribution of primary cultured human chondrocytes in order to establish a tracking method of cultured human nasoseptal chondrocytes. METHODS: pEGFP-N1 plasmid was amplified in E.coli, and purified by high purity kit. Primary cultured human chondrocytes,which were initially obtained from the nasoseptal cartilage, were cultured in vitro and transferred with pEGFP-N1 by means of electroporation with Amaxa nucleofector device. Transfering process and transient expression were evaluated by laser scanning confocal microscope (LSCM), the transfer efficiency and the cell cycle distribution were evaluated by flow cytometry. RESULTS: There was significant expression of EGFP at 24 h after transferring. The transfection efficiency of pEGFP-N1 into primary cultured human chondrocytes reached 35 37% at 48 h. It didn't affect the process of cell adherance and had no effect on the cell cycle distribution. CONCLUSION: Primary cultured human chondrocytes, which were transfected with pEGFP, are alive in vitro, and the transferring process doesn't affect the cell cycle distribution. These results suggest that pEGFP-N1 is an ideal transient expression vector for primary cultured human chondrocytes and it might be a well tracer in construction tissue engineered cartilage.

The mechanism of injury on the human glomerular endothelial cells (ciGENC) induced by preeclampsia serum was investigated. Concentration of maternal serum sFlt-1 protein was detected by ELISA. Fluorescently-labeled bovine serum albumin infiltrating through lower chamber of Transwell was measured by multifunction microplate reader. Morphologic change of ciGENC was observed under inverted phase contrast microscope. The concentration of sflt-1 in preeclampsia groups was significantly increased as compared with control group (P<0.01). Permeability in preeclampsia groups was significantly increased as compared with control group (P<0.01). By contrast with severe preeclampsia group, the permeability of ciGENC monolayer in mild preeclampsia group was decreased significantly (P<0.05). Intervention of exogenous VEGF significantly decreased permeability of ciGENC in preeclampsia groups. It was concluded that sFlt-1 increased ciGENC permeability by damaging integrity of endothelial barrier function.

This study examined the effect of over-expression of sFlt-1 by trophoblasts on the barrier function of glomerular endothelial cells and the role of VEGF in this process in order to explore the pathogenesis of glomerular disease in preeclampsia. SFlt-1 expression in the human trophoblasts (TEV-1 cells) was enhanced by transfecting sFlt-1 plasmid DNA into TEV-1 cells. The monolayer barrier function of glomerular endothelial cells (ciGEnCs) was determined by measuring the fluorescence intensity of bovine serum albumin (BSA) that crossed the monolayer of glomerular endothelial cells. The results showed that the over-expression of sFlt-1 by TEV-1 cells led to the barrier dysfunction of ciGEnCs, and the exogenous VEGF could alleviate the ciGEnCs dysfunction resulting from the over-expression of sFlt-1 to a certain extent. It was concluded that the dysregulation of sFlt-1 and VEGF in preeclamptic pregnancy may contribute to the barrier dysfunction of glomerular endothelial cells, and VEGF may play an important role in maintaining the barrier function of glomerular endothelial cells, but it may not be the sole factor.

This study investigated the effect of epigenetic modification of maspin on extravillous trophoblastic function. The mRNA expression of maspin in placentae from normotensive and preeclamptic pregnant women was detected by RT-PCR. TEV-1 cells, a human first-trimester extravillous trophoblast cell line, were cultured and treated with CoCl(2) (300 μmol/L) to induce chemical hypoxia and with 5-aza (500 nmol/L) to induce demethylation. The mRNA expression of maspin in TEV-1 cells subjected to different treatments was determined by RT-PCR, and the proliferative and migratory abilities of TEV-1 cells were assessed by cell counting kit-8 (CCK-8) and Transwell assays. Our results showed that the maspin mRNA expression level in placentae from preeclamptic women was much higher than that from normotensive women. CoCl(2) or 5-aza could up-regulate the mRNA expression of maspin and significantly suppress the proliferation and migration of TEV-1 cells. It was concluded that the epigenetic modification in promoter region of maspin contributes to incomplete trophoblast invasion, which offers a novel approach for predicting and treating placental dysfunction.

Objective: To establish a method for determining the molecular weight and molecular weight distribution of Poly(Lactide-co-Glycolide Acid) (PLGA) using Size Exclusion Chromatography-Refractive Index-Multiangle Laser Light Scattering (SEC-RI-MALLS) and Size Exclusion Chromatography-Refractive Index (SEC-RID), and to compare the results obtained from these two methods.
Methods: For SEC-RI-MALLS, tetrahydrofuran was used as the mobile phase, Shodex GPC KF-803L was employed as the chromatographic column with a flow rate of 1 mL·min^-1, column temperature at 30 ℃, and an injection volume of 100 μL. For SEC-RID, tetrahydrofuran was also used as the mobile phase, Agilent PLgel 5 μm MIXD-D was used as the chromatographic column with a flow rate of 1 mL·min^-1, column temperature at 30 ℃, differential detector temperature at 35 ℃, and an injection volume of 20 μL. The molecular weight and molecular weight distribution were calculated using Agilent’s GPC software. The newly established methods were validated methodologically, and the molecular weight and molecular weight distribution of 13 batches of samples were determined.
Results: The precision, accuracy, stability, and repeatability tests for SEC-RI-MALLS showed RSD values of 1.35%, 1.58%, 1.53%, and 1.26%, respectively. The SEC-RID method exhibited good linearity (r=0.999 9), with RSD values for precision, accuracy, stability, and repeatability tests (n=6) of 2.05%, 1.62%, 1.30%, and 2.97%, respectively. The results obtained from SEC-RI-MALLS were lower than those from SEC-RID, and the molecular weight distribution coefficient was smaller, but the results from the paired T-test performed with the value measured by SEC-RID method and the value measured by SEC-RI-MALLS method multiplied a conversion coefficient of 1.5 showed no significant difference between the two methods.
Conclusion: Both methods are stable and reliable, and can be used for the determination of PLGA molecular weight and molecular weight distribution based on the specific situations.

Objective To investigate the distribution and role of α,βand γ subunits of epithelial sodium channel(ENaC) in the cochlea and endolymphatic sac of guinea pig. Methods The expression of α-, β- and γ-ENaC subunits proteins was studied by immunohistochemistry with the specific polyclonal rabbit antibodies against the α,β and γ subunits of rat ENaC. α-ENaC mRNA was detected by in situ hybridization with digoxin labeled cDNA probe. Results All three subunits of ENaC, α-, β- and γ-, were widely distributed in the labyrinth. In the cochlea, strong labeling of α-ENaC protein was found in the spiral limbns, and to a less extent, in the spiral ligament, organ of Corti and Reissner's membrane. The immunoreactivity of β-ENaC was observed in the spiral ligament, spiral limbus, spiral ganglion, organ of Corti and Reissner's membrane with a less intensity than that of α-ENaC. γ-ENaC was presented primarily in the superior part of the spiral ligament, spiral limbus, spiral ganglion, and weakly in the organ of Corti and Reissner's membrane. In the endolymphatic sac, intensive immunoreactivities of all three subunits were seen in the epithelial cells and the subepithelial cells at similar intensity, α-ENaC mRNA was localized in the spiral limbus, the inferior part of spiral ligament, stria vascularis, and epithelial cells and subepitbelial cells of endolymphatic sac. Conclusion Different subunits of the ENaC expressed in various cell regions of the cochlea and endolymphatic sac in distinct patterns may form the functional sodium channel to regulate the endolymph, thus serve to maintain homeostasis in inner ear.

Objective To investigate the antibiotic susceptibilities and the profiles of virulence genes of clinically isolated Salmonella enterica serovars Schwarzengrund ( S. Schwarzengrund) strains for bet-ter understanding the epidemiological trend of this type of non-typhoidal Salomonella and to provide guide-lines for the prevention and treatment of S. Schwarzengrund infection. Methods Stool samples and clinical data of patients with acute diarrhea who received treatment in the Second Hospital of Tianjin Medical Univer-sity during May, 2014 to October, 2014 were collected for this study. Enrichment culture and biochemical identification were used to isolate and identify the S. Schwarzengrund strains. The isolated strains were fur-ther analyzed with serotyping analysis, drug susceptibility test, pulsed field gel electrophoresis ( PFGE) and multiple locus sequence typing ( MLST ) . The representative genes carried by Salmonella pathogenicity islands (SPI) 1-5, SPI regulators and virulence plasmids were amplified by PCR. The coding genes of CdtB-islet, which were cdtB, pltA and pltB were amplified and sequenced. Results In total, 16 (14. 8%) out of 108 non-typhoidal Salmonella strains were identified as S. Schwarzengrund strains and all of them were sus-ceptible to 11 kinds of antibiotics such as fluoroquinolone, ampicillin, ceftriaxone and trimethoprim-sulfame-thoxazole. PFGE categorized the 16 S. Schwarzengrund strains into 3 clusters including A clone ( 14 strains), B clone (1 strain) and C clone (1 strain). The strains that isolated from 8 patients who ate the same food belonged to one cluster ( A clone ) , suggesting that it was an outbreak of infection. The 16 S. Schwarzengrund strains showed identical MLST type, which was ST241. The representative genes carried by SPI1-5 ( invA, sitC, hilA, sseL, sifA, mgtC, siiE and sopB) , the regulatory gene ( phoP) and the cytole-thal distending toxin islet (CdtB-islet) coding genes (cdtB, pltA and pltB) were positive, while the genes carried by virulence plasmids (pefA, prot6E and spvB) were negative. The similarities in CdtB-islet coding genes and amino acids sequences between Salmonella typhi and S. Schwarzengrund strains in this study were more than 97% and 98%, respectively. Conclusion In this study, polyclonal S. Schwarzengrund strains of ST241 type were isolated from the patients. They were susceptible to common antibiotics, but carried the virulence genes contained in SPI1-5 and CdtB-islet coding genes and might cause an outbreak of infection. Attention should be paid to the tendency and threat of clinical S. Schwarzengrund infection and continuous surveillance and investigation should be performed.