Objective To construct the third-generation self-inactivating lentiviral vectors including BMP-2,BMP-4,BMP-6,BMP-7,BMP-9 and Wnt3a,then integrate them into the genome of mouse embryonic osteoblast cell,MC3T3-E1,and to explore the capability of osteogenic differentiation of individual bone morphogenetic control factor and the effective approaches to further improve the capability of osteogenic differentiation.Methods The plasmid vectors of gene expression including BMP-2,BMP-4,BMP-6,BMP-7,BMP-9 and Wnt3a were constructed,which were identified through enzyme cutting and further confirmed through sequencing.After packing pELNS-BMP-2,BMP-4,BMP-6,BMP-7,BMP-9 and Wnt3a,mouse embryonic osteoblast cell,MC3T3-E 1 was transfected,and the transfection efficiency was confirmed by GFP fluorescence imaging.The expression level of Runx2 mRNA and the transfection efficiency of individual bone morphogenetic control factor was detected by Real time PCR.Eight groups of MC3T3-E1 were dual-gene co-transfected,and the transfection efficiency was verified by GFP fluorescence imaging.ELISA was adopted to detect the expression level of BGP and ALP in MC3T3-E1culture supernatants;Real time PCR was adopted to detect the expression level of Runx2 mRNA;Western blot was adopted to detect the expression level of protein of BMP-2,BMP-4,BMP-6,BMP-7,BMP-9 and Wnt3a.Thus,the effectiveness of osteogenic differentiation of dual-gene co-transfection were evaluated.Results The recombination of lentiviruses,pELNS-BMP-2,pELNS-BMP-4,pELNS-BMP-6,pELNS-BMP-7,pELNS-BMP-9 and pELNS-Wnt3a were successfully constructed.MC3T3-E1 was successfully transfected.The expression levels of Runx2 mRNA were:BMP-2 ＞ BMP-4 ＞ BMP-9 ＞ BMP-7 ＞ Wnt3a ＞ BMP-6.Successful transfection of the dual-gene co-transfection of eight groups of MC3T3-E1 were verified by GFP fluorescence imaging.The expression level of Runx2 mRNA,the expression of BGP and ALP showed BMP-2 and BMP-7 co-transfection group was the most efficient in osteogenesis transfection.Western blot revealed thatthe protein expression of BMP-2,BMP-4,BMP-6,BMP-7,BMP-9 and Wnt3a increased after cotransfection of MC3T3-E1 by BMP-2 and BMP-7.Conclusion The third-generation lentiviral vector,pELNS can lead BMP-2,BMP-4,BMP-6,BMP-7,BMP-9 and Wnt3a into MC3T3-E1,mouse embryonic osteoblast cell,and stabilize its expression.Individual bone morphogenetic control factors can promote MC3T3-E1's differentiation to osteoblasts.The dual-gene co-transfection of BMP-2 and BMP-7 can effectively promote osteoblast conversion,which provides significant theoretical basis and technical support for remodeling of tissue engineering bone.

Objective To investigate clinical features of lower motor neuron lesion (LMNL) caused by the single level lower thoracic disc protrusion (LTDP),and to observe clinical outcomes of surgical treatment.Methods Between January 1997 and December 2009,17 patients with LMNL caused by single level LTDP underwent en bloc resection of the superior articular process,Cave-in 360° circumferential decompression and internal fixation in our hospital.MRI and CT scans were taken to confirm lesion levels:T10-11 in 4 patients of whom 3 had patellar clonus and ankle clonus,T11-12 in 5 patients of whom 4 had ankle clonus,and T12L1 in 8 patients who only had positive Babinski sign.The neurologic status was assessed using the Japanese Orthopaedic Association (JOA) scoring system.The muscle strength of the tibialis anterior was assessed using the Manual Muscle Test (MMT).Sagittal Cobb angle and cross-sectional area of the dural sac at the level of maximal compression in MRI were also observed.Results All patients were followed up for 22 to 76 months (average,48.6 months).The mean JOA score increased from preoperative 5.88±1.11 to 9.53±0.94 at final follow-up (t=16.143,P＜0.05).The muscle strength of the tibialis anterior recovered to more than grade 4 in all patients.Postoperative Cobb angle was unchanged compared with that before operation.MRI indicated that the cross-sectional area of the dural sac at the level of maximum compression increased from preoperative 35.8±7.3 mm2 to postoperative 132.9±6.5 mm2 (t=70.78,P＜0.05).Conclusion LMNL can be caused by LTDP.The eu bloc resection of the superior articular process,Cave-in 360° circumferential decompression and internal fixation can provide a satisfactory decompression effect and marked recovery of neurological function.

The optimization on liquid fermentation and purification of the fibrinolytic enzyme from Stenotrophomonas maltophilia(DR-929) was investigated.The results showed the best fermentation are amidulin 2.0%,soya flour 1.0%,yeast extract 0.5%,NaCl 1.0%,CaCl2 0.02%,MgSO4 0.05%,inoculum of 36 hours,fermental time 4d,initial pH 8.0 or 9.0,temperature 25℃,volume of media 30ml,volume of inoculum 5% or 6%.The purification process includes the following steps: removing cells by the centrifugation,25%~70% saturation ammonium sulfate precipitation,HIC with Phenyl FF(high sub),IEC with Q-Sepharose FF,gel filtration chromatography with Superdex 75.SDS-PAGE electrophoresis was used to examine the purification effect,and the results indicated that homogeneous strap in SDS-PAGE and has a molecular weight around 28.3kDa.The purification factor and activity recovery of the fibrinolytic enzyme are 271.5 and 24.5%,respectively.

AIM :To investigate the association of primary open-glaucoma (POAG), intraocular pressure (IOP) and systemic blood pressure in a rural population aged 50 years old or above in Shaanxi Province, China.METHODS: In the population-based, cross-sectional study, 1 775 (83.53%) residents, aged 50 years old or above, from 3 counties of Shaanxi Province, China, undertook an interview with a standard questionnaire and a detailed eye examination, including logarithm of the minimum angle of resolution visual acuity, slit-lamp biomicroscopy, applanation tonometry and dilated fundus examination. Two blood pressure reading in the sitting position were taken. Gonioscopy was performed if a narrower peripheral anterior chamber (less than one fourth of the corneal thickness) was indicated. Automated visual field testing was performed by participants assessed to have suspicion of glaucomatous disc damage or if IOP was 22mmHg or higher.RESULTS: IOP significantly correlated with systemic blood pressure, and both IOP and systolic blood pressure increased significantly with increasing age. No association between POAG and hypertension was found. The frequency of POAG increased significantly with lower diastolic perfusion pressure.CONCLUSION: Data in our study are accordance with those reported in other population-based studies, and confirm that lower diastolic perfusion pressure is a significant risk factor for primary open angle glaucoma.

AIM: To assess the prevalence and risk factors for age-related macular degeneration (AMD) in a rural population in Shaanxi Province of China.METHODS: A total of 2 835 (81.00%) people aged 40 years old or more, from Fuping county, Jingbian county and Yang county of Shaanxi Province, China, underwent a comprehensive interview and a relative eye examination. The present of AMD was classified into neovascular AMD (NV) and pure geographic atrophy (GA) by using direct ophthalmoscopy for fundus examination according to International Classification System.RESULTS: The prevalence (95% CI) of AMD was 3.00% (2.42, 3.71) in this population, of which NV accounted for 1.45% (1.05, 1.98) and 1.55% (1.14, 2.10)for GA. The prevalence of AMD increased significantly with increasing age (P ＜0.001). AMD was present in 0.47% of participants aged 40 to 49 years, rising to 11.90% of participants older than 80 years, of which the corresponding data increased from 0.28% to 4.76% for NV and from 0.19% to 7.14% for GA. No significant difference was found in the prevalence of NA and GA between genders in this population. With multiple logistic analyses, apart from advancing age, only smoking was found to have a strong association with any type of AMD.CONCLUSION: The prevalence of AMD in the rural population of Shaanxi Province of China is lower than that reported from other population-based studies in different provinces of China, less than that reported in whites, more than that reported in blacks. Except increasing age, smoking is also a significant well-known risk factor for AMD.

· AIM: To assess the prevalence and related risk factors for primary angle-closure glaucoma in a rural population for 40 years of age or older in Shaanxi Province.· METHODS: By using a stratified, cluster-based, random sampling technique, 8 500 persons of all ages were selected randomly, including 3 500 those aged 40 years or more, from the North, the South and the Middle of Shaanxi Province in Western China from July to December in 2003. All participants had an interview with a standard questionnaire and those questions related to glaucoma of previous diagnosis and treatment, family history and outbreak history. Then a detailed and relative eye examination was performed, including logarithm of the minimum angle of resolution visual acuity, external eye examination with slit-lamp biomicroscopy and fundus examination. The intraocular pressure was measured with Perkins applanation tonometry to those persons aged 50 years old or more and those with suspected increased IOP. A further examination was performed to those persons with suspicious glaucoma, including repeated tonometric examination, gonioscopy, dark room test, automated visual field testing, et al.·RESULTS: 6 815 of the eligible 8 500 persons of all ages were interviewed and examined from July to December in 2003, a response rate of 80.18%, including 2835 of the eligible 3 500 persons of 40 years old or more with a response rate of 81.00%. According to Van Herick method, the percentage of shallow peripheral anterior chamber was 13.6%, of which 10.4% was in grade 2, 3.0% in grade 1, and 0.2% in grade 0. In this rural population for 40 years of age or older 31 participants were found to have primary angle-closure glaucoma, with the prevalence of 1.09%. With multiple logistic analyses, the prevalence increased significantly with age (P=0.008), whereas no significant difference (Pgender=0.180, Peducation=0.199) was found in the rate with gender and education, although women and illiteracy seemed to be at increased risk of the disease (OR: 1.77, 95%CI,0.77-4.10; OR: 1.71, 95%CT, 0.76-3.87). Of 31 participants, 21 persons (67.74%) had not been previously diagnosed and treated. Of 10 participants previously diagnosed, 6 (60%) had been received peripheral iridectomy; nevertheless, no any other treatments were performed. 48.39% participants suffered from visual impairment in various degrees, of which the percentage of blindness in either eye accounted for 29.03%. Of those with primary angle-closure glaucoma, 22 persons (70.97%) were classified as having chronic from of the disease.· CONCLUSION: In this rural population in Shaanxi Province of China, the rate of occludable angles is 13.6%, more common than that reported previously in other Chinese populations. The prevalence of primary angle-closure glaucoma is close to that from other Asian populations, increasing with age. A majority of glaucoma was undiagnosed and untreated previously.

· AIM: To assess the prevalence and related risk factors of primary open angle glaucoma (POAG) in a rural population in Shaanxi Province.· METHODS: By using a stratified, duster-based, random sampling technique, 8 500 persons of all ages were selected randomly, from the North, the South and the Middle of Shaanxi Province from July to December in 2003. All participants had an interview with a standard questionnaire and those questions related to glaucoma of previous diagnosis and treatment, family history and outbreak history. Then a detailed and relative eye examination was performed, including logarithm of the minimum angle of resolution visual acuity, external eye examination with slit-lamp biomicroscopy and fundus examination. The intraocular pressure was measured with Perkins applanation tonometry to those persons aged 50years old or more and those with suspected increased IOP. A further examination was performed to those persons with suspicious glaucoma, including repeated tonometric examination, gonioscopy, dark room test, automated visual field testing, et al.· RESULTS: 6 815 of the eligible 8 500 persons of all ages were interviewed and examined from July to December in 2003, a response rate of 80.18%. 9 participants were found to have POAG, with the prevalence of 0.13%, age ranging from 38 to 80 years (mean year:62.0). In those aged 30 years old or more, the prevalence of the disease was 0.23%, 0.28% and 0.39% for those more than 40 and 50 years old, respectively. The rate of suspicious POAG was 0.18% (12 cases) with age ranging from 35 to 77 years (mean year: 54.7). With multiple logistic analyses, the prevalence increased significantly with age (P=0.023). Other than increasing age, myopia was also a strong risk factor for POAG. Of 9participants with POAG, only 2 cases (22.22%) had been previously diagnosed. No one with POAG was received any treatment previously. 66.67% (6 cases) participants with POAG suffered from visual impairment in various degrees secondary to POAG. The percentage of blindness in either eye was 33.33%.· CONCLUSION: The prevalence of POAG is close to that from other Chinese populations, increasing with age. A majority of glaucoma was undiagnosed and untreated previously.

In traditional clinical application, Coptidis Rhizome and Evodiae Fructus have been combined to treat various stomach heat and cold syndromes, gastritis, gastric ulcer and the like. With the application of modem instruments and the development of molecular pharmacologic theory, their chemical constituents and pharmacological effects have been sufficiently studied. In this paper, literatures from Pubmed were adopted, with particular emphasis on findings of international counterparts and studies on compatibility of main chemical components in Coptidis Rhizoma and Evodiae Fructus, in order to elaborate on the scientific comparability of Coptidis Rhizoma and Evodiae Fructus through chemical analysis, and pharmacological and biopharmaceutics studies and introduce the future development trend of the studies.
Animals ; Drug Interactions ; Drugs, Chinese Herbal ; analysis ; pharmacology ; Evodia ; chemistry ; Fruit ; chemistry ; Humans ; Ranunculaceae ; chemistry ; Rhizome ; chemistry

Objective To explore the regulation function of Icariine on the expression of bone morphogenetic protein signaling pathway Smad1,Smad5,Smad4 and to explore the mechanism of promoting MC3T3-E1 cell proliferation and differentiation.Methods MC3T3-E1 cells were treated by 0,10-9,10-8 and 10-7 mol/L Icariine respectively.After stimulated by Icariine 1 d,2 d and 3 d,MTT method and population diploid time were used to observe the cell proliferation,and the cell alkaline phosphatase (ALP) level was assayed.At 21 days later,the alizarin red staining was proceeded.At 1,2 and 3 days later,the RT-PCR was used to detect the mRNA expression level about Smad1,Smad5 and Smad4,and the Western blot was to detect the Smad1,5 and Smad4 protein.At 2 days later,the RT-PCR was used to detect the mRNA expression level about Runx2,BMP-2 and osteoprotegerin (OPG),and the Western blot was used to analyze osteocalcin (OCN) protein level.Results After simulated by Icariine,the proliferation (MTT test),the ALP activity and mineralization of osteoblasts were increased,the cell population diploid was reduced (P＜0.05).At 1,2 and 3days later,the results of RT-PCR showed that Icariine continued increasing the mRNA level of Smad1,5 and Smad4 in 10-8 mol/L.At 2 days later,Smad1,Smad5 and Smad4 mRNA expression were obviously reduced in 0 mol/L group,and At 3 days later,Smad1,Smad5 and Smad4 mRNA expression were obviously reduced in 10-9 and 10-7 mol/L groups.At 2 days later,BMP-2,Runx2 and OPG mRNA were obviously increased in 10-8 mol/L group.The results of Western blot showed that OCN,Smad1,Smad5 and Smad4 protein were obviously up-regulated in 10-8 mol/L group.Conclusion Icariine occurs the expressions of BMP-2,Runx2,Smad1,Smad5 and Smad4 by stimulating the bone morphology of MC3T3-E1 cells directly,and promotes the osteogenic differentiation in the manner of expression level synchronous rising.

Animals ; Dose-Response Relationship, Drug ; Male ; Mice ; Mice, Inbred Strains ; Nickel ; toxicity ; Oxidative Stress ; physiology ; Testis ; drug effects ; metabolism