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.

This paper discussed the roots of American ethical review system, the development of twists and turns, and puts forward the enlightenment to our country, the ethical review system mainly include:perfecting the ethical review at the top of specification design, improve specification status;improve the system of education and training, to strengthen the construction of information communication channels, establish standard operating proce-dures, improve the regulatory system;improve the system of continuous review, formulate specific implementation specification.

Objective To evaluate effects of chronic arsenic exposure and arsenic exposure time on oxidative DNA lesions in humans. Methods A cross-sectional study was conducted in 108 subjects exposed to high concentrations of arsenic in drinking water and 75 control subjects. A cohort study was conducted in 64 subjects exposed to high levels of arsenic in drinking water for 7 or 9 years. Urinary 8-oxo-7,8-dihydredeoxygnanine(8-OHdG) levels were analyzed by the enzyme-linked immunosorbent assay kit(ELISA). Urinary arsenic concentration was detected with hydride generation atomic absorption spectroscopy. Results In the cross-sectional study, the median of urinary arsenic concentration was 484.17 mg/kg Cr for the arsenic-exposed group, and 13.80 mg/kg Cr for the control group, and the difference between the two groups was statistically significant (t=32.57, P<0.01). The median of urinary 8-OHdG levels was 16.60 and 21.88 mg/kg Cr for arsenic-exposed children and adults respectively, much higher than control children(10.50 mg/kg Cr) and adults (9.11 mg/kg Cr), and the difference was statistically significant (t=5.049, 6913, all P<0.01). Urinary 8-OHdG levels were signifieandy lower for children than adults in the exposed group(t=-1.997, P<0.05). In the cohort study, the median of urinary arsenic concentration was 461.3 mg/kg Cr for the 7-year-exposed subjects and 422.90 mg/kg Cr for the 9-year-expesed subjects, and no significant difference was observed(t=-0.250, P 0.05). The median of urinary 8- OHdG levels for 9-year-exposed children and adults were 23.46 and 24.30 mg/kg Cr respectively, significantly increased compared with those of 7-year-exposed(14.29 and 18.38 mg/kg Cr), and the difference had statical signhqcanees (t= -2.949,-3.055, all P<0.01). Conclusions Chronic arsenic exposure can lead to oxidative DNA lesions in humans. The arsenic-induced DNA lesions may aggravate with the exposure time in a certain period.

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

Objective To investigate the effects and mechanisms of 1,25-Dihydroxyvitamin D3 [1,25(OH)2 D3] on hyperoxia-induced lung injury of neonatal rats.Methods Neonatal Sprague-Dawley rats were randomly divided into air group,hyperoxia group and 1,25(OH)2D3 group within 12 hours after birth,eight in each group.Rats in air group were exposed to air,while those in hyperoxia and 1,25 (OH) 2 D3 group were exposed to hyperoxia (≥85 ％ oxygen concentration).Rats in 1,25(OH)2D3 group were injected with 1,25(OH)2D3 0.5 μg/(kg · d) intraperitoneally once a dayfor seven days,meanwhile the rats in the other two groups received 0.9 ％ saline in the same way.All rats were sacrificed on day 7.Lung tissue sections were HE stained in order to assess lung histological changes and lung radical alveolar count (RAC).The levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-1β and IL-6 mRNA were measured by real time-polymerase chain reaction.Analysis of variance and LSD test were applied for statistics.Results Compared with the air group,the weight of rats in the hyperoxia group was significantly lower on day 7 [(8.48±1.34) g vs (12.51±0.47) g,t=8.05,P＜0.05],while the weight of rats in 1,25(OH)2D3 group [(10.29±1.00) g] was higher than that in the hyperoxia group (t=3.61,P＜0.05).Lung tissue structure was normal in the air group.In the hyperoxia group,inflammatory exudation was observed in pulmonary interstitial,the alveolar size was uneven,and the RAC was lower than that in the air group (5.6±0.1 vs 6.8±0.2,t=21.45,P＜0.05).The RAC in 1,25(OH)2D3 group (6.2±0.1) was significantly increased compared with that in the hyperoxia group (t=11.76,P＜0.05),but still lower than that in the air group (t=9.69,P＜0.05).The expressions of TNF-α,IL-1β and IL-6 mRNA in hyperoxia group (0.0348±0.0006,0.0269±0.0003 and 0.0368 ± 0.0006) were higher than those in the air group (0.0111±0.0007,0.0040±0.0003 and 0.0162 ±0.0007,t=56.54,111.12 and 49.26,P＜0.05,respectively).The expressions of TNF-α,IL-1β and IL-6 mRNA in the 1,25 (OH)2D3 group (0.0203±0.0009,0.0141±0.0004 and 0.0251±0.0009) were lower than those in the hyperoxia group (t=34.44,61.93 and 27.99,P＜0.05,respectively),but higher than those in the air group (t=22.10,49.19 and 21.27,P＜0.05,respectively).Conclusions 1,25(OH)2D3 could attenuate hyperoxia-induced lung injury by inhibiting the expression of inflammatory cytokines.

Objective To evaluate the role of calcium channel in the mechanism of the generation and maintenance of bursting firing of substantia nigra pars compacta (SNc) dopaminergic neurons in rats.Methods Using the patch clamp technique,we observed the firing pattern switching features after adding 10 μmol/L N-methyl-D-aspartic acid (NMDA),compared the changes of whole-calcium current and L-type calcium current with or without NMDA,and analyzed the correlation between the generation of burst firing and L-type calcium channel activation.Results After NMDA treatment,the firing pattern of SNc dopaminergic neurons changed to burst firing,which was compromised by a charastistic high plateau potential and series of action potential on it.The current density of L-type calcium current increased significantly after adding NMDA,which,from (2.86 ±0.26) pA/pF (n =28),significantly increased to (3.75 ± 0.18) pA/pF (n =34 ; t =7.52,P =0.002 8).The high plateau potential was almost abolished with the application of verapamil,a specific antagonist of L-type calcium channel.Consiusion NMDA could induce the firing pattern changed to burst firing in SNc dopaminergic neurons,while L-type calcium channel contributes to the process of generation and maintenance of burst firing.

Objective To investigate and analyze the mineral and bone disorder (MBD) in the patients with chronic kidney disease (CKD),reveal the change of related indexes of CKD-MBD.Methods A cross-sectional study was carried out in the First Affiliated Hospital of Harbin Medical University.From October 2011 to May 2014,1318 inpatients and hemodialysis outpatients were enrolled.Parameters related to MBD,including serum phosphorus (P),total calcium (t-Ca),intact parathyroid hormone (iPTH) and alkaline phosphatase (AKP) were analyzed.Last,it was analyzed with multiple regression analysis to related factors of the secondary hyperparathyroidism (SHPT) in patients with CKD.Results Serum calcium,phosphorus and iPTH had no obvious abnormalities at the early stages of CKD [GFR ＞ 60 ml· min-1· (1.73 m2)-1],and relatively stable before GFR ＞ 30 ml· min-1· (1.73m2)-1.After entering the CKD4 stage,serum phosphorus,iPTH increased sharply and serum calcium decreased obviously along with the decreased glomerular filtration rate (GFR).Serum P,t-Ca and iPTH levels were statistically significant in CKD 1 to 5D patients,respectively,serum P:(1.13±0.20) mmol/L,(1.14±0.22) mmol/L,(1.26±0.23) mmol/L,(1.48±0.34) mmol/L,(2.05±0.61) mmol/L and (2.08±0.58)mmol/L;serum t-Ca (mmol/L) (2.35±0.13) mmol/L,(2.35±0.12) mmol/L,(2.35±0.15) mmol/L,(2.26± 0.18) mmol/L,(2.07±0.29) mmol/L and (2.31±0.26) mmol/L;iPTH:57.8(45.6,91.8) ng/L,54.1(37.8,74.6) ng/L,71.6(45.8,102.2) ng/L,131.1(81.7,205.1) ng/L,277.5(173.6,395.3) ng/L and 354.9 (194.4,720.3) ng/L;The stepwise logistic regression analysis showed:hypocalcemia (OR=3.32,P ＜ 0.01) and decreased GFR (OR=5.28,P ＜ 0.01) were independent risk factors of iPTH elevation at stage CKD3～ 5.Conclusions From the beginning of the CKD3 stage,serum t-Ca,P,iPTH level began to be relatively abnormal as renal function declined.Hyperphosphatemia,SHPT has not been improved significantly in CKD5D stage patients even with hemodialysis.The regulation of hemodialysis on serum calcium showed overcorrecting phenomenon.

Objective To apply 3C3R model in problem design of PBL for pediatrics teaching.Methods The 3C3R model comprises two classes of components:core components and processing components.Core components of the model are 3C,which are content,context and connection while processing components are 3R,which are researching,reasoning and reflecting.3C3R model was used in the problem design for the PBL case of ‘ Why the mouth of Baobao became purple when he was crying?' Totally 76 eight-year program medical students and 7 tutors were enrolled as teaching object.The anonymous questionnaires from the students were collected for assessment of PBL teaching.Results The percentage of students with scores ≥4 for content in PLB problem design was 90.8％,for context was 80.3％,for connection was 64.5％,for researching was 81.6％,for reasoning was 69.7％ and for reflecting was 40.8％.The percentage of tutors with scores ≥4 for content in PLB problem design was 100％,for context was 71.4％,for connection was 28.6％,for researching was 71.4％,for reasoning was100％,for reflecting was57.1％.Both students and tutors held a positive attitude towards the component of content,context,researching and reasoning in problem design model.But the components of connection and reflection needed to be improved.Conclusion The 3C3R model is helpful for problem design of PBL in pediatric teaching.

Antineoplastic Agents ; administration & dosage ; therapeutic use ; Child ; Female ; Humans ; Injections, Intralesional ; Interferon-alpha ; administration & dosage ; therapeutic use ; Male ; Neoplasms ; drug therapy ; Recombinant Proteins ; Treatment Outcome

Objective To study the clinical features and surgical strategies of thoracic spinal stenosis caused by ossification of posterior longitudinal ligament(OPLL).Methods From January 2004 to March 2009,21 cases of thoracic spinal stenosis casued by OPLL,including 13 males and 8 females,received surgical treatments.Those cases aged from 34 to 71 years,with an average of 51.2 years old.The courses of disease were from 2 to 50 months,averaged 11 months.The lesions located in upper thoracic(T1-T4)for 4 cases,in middle thoracic(T5-T8)for 7 cases,in lower thoracic(T9-T12)for 10 cases.Nine cases were associated with ossification of ligamentum flavum(OLF),and 8 cases combined with cervical OPLL.Eleven cases received laminectomy and 10 cases received anterolateral decompression.Results The operation time was 90 to 240 min for posterior laminectomy with an average of 140 min,and 110 to 360 min for anterolateral decompression with an average of 240 min.All cases had no worse postoperative symptoms,neurological complications,subarachnoid cavity or wound infection.Japanese Orthopaedic Association(JOA)score was 8 to 15 with an average of 9.17±1.63 in 6 months after surgery.Nerve function improvement was excellent for 8 cases,good for 6 cases,fair for 5 cases and poor for 2 cases.The excellent and good rate was 66.7%.JOA score was 8-15,averaged 10.23±1.64,in 12 months after surgery.Nerve function improvement was excellent for 8 cases,good for 7 cases,fair for 4 cases,and poor for 2 cases.The excellent and good rate was 71.4%.Conclusion Thoracic spinal stenosis result from OPLL,which often combine with cervical OPLL and OLF,often show multiple manifestations.Posterior laminectomy and anterolateral decompression are suitable for those conditions.