BACKGROUND: Lentivirus-mediated RNA interference technology has been widely used in the study of gene function because of its specificity, high inhibition efficiency and persistent effects. Virus packaging, transfection, as well as small hairpin RNA (shRNA)-coding sequence could affect the inhibition efficiency. Therefore, impact factors of RNA interference for human Bax inhibitor 1 (BI-1) was studied in this experiment.OBJECTIVE: To find the valid small interference RNA (siRNA) targeting human BI-1 gene by Lentivirus.DESIGN, TIME AND SETTING: Single sample observation was completed in Department of Medical Genetics, Basic Medical College, Peking University Health Science Center from September 2007 to December 2008.MATERIALS: 293T cell and SH-SY5Y cells were preserved in this laboratory; 4 shRNAs were designed through a use of online design tool by Ambion (www.ambion.com). METHODS: Several recombinant plasmids which expressed shRNAs targeting different regions of the BI-1 gene and labeled enhanced green fluorescent protein as a fusion gene were constructed. Four types of shRNA-expressing virions were obtained by cotransfection of shRNA-expressing plasmid and package protein-expressing plasmids into 293T cells. Green fluorescent protein expression was determined using flow cytometry to search the optimal package conditions. Real-time PCR and beta actin mRNA was used as an internal control, four types of recombinant viral supernatant and control viral supernatant were added on the SH-SY5Y cells, knock-down efficacies of the endogenous BI-1 gene were determined, and the RNA interference effective sequences were optimized. MAIN OUTCOME MEASURES: Intracellular green fluorescent protein expression rate of the cells infected with different packaging virus. RESULTS: The optimal infective packaging virus could be obtained when the ratio of plasmids pLentiLox3.7, REV, VSVG and RRE was 2:1:1:1 and the supernatants were collected 48 hours after transfection. Replacement of culture media 24 hours after transfection could increase the infection efficacy of the virions. shRNA targeting-2-17nt (start coding region) of BI-1 gene was most valid interference efficiency and could decrease the gene expression to 40%. CONCLUSION: Influencing factors of Lentivirus-mediated RNA interference technology virus packages were investigated in this study, which could be a foundation for constructing stable BI-1 knocked down neuronal cell model and for studying the abnormal expression of BI-1 related to the neuron apoptosis disease.

Background Corneal neovascularization (CNV) is one of the causes of corneal blindness.Studies showed that ADP-ribosylation factor (ARF) can regulate the growth of tumor cells,and inhibiting ARF will decrease angiogenesis.However,whether ARF antagonist plays an action on CNV is unclear.Objective The aim of this study was to explore the effect and mechanism of ARF inhibitor on alkali-burn induced CNV.Methods Sixty clean male BABL/c mice aged 7-8 weeks were divided into PBS group and ARF antagonist group according to randomized number table.CNV models were induced by NaOH burn method in all the mice.ARF at the concentration of 0.5 g/L(0.5 ml) was intraperitoneally injected 3 times per week for 1 week followed the induction of CNV in the ARF antagonist group,and 0.5 ml PBS was used in the PBS group.CNV was examined 2,4,7,14 days after injection by the slit lamp microscope and the CNV related area in the cornea was calculated.Betore modeling(0 day) and 4,7,14 days after modeling,real-time PCR and Western blot were used to analyze the expressions of ARF mRNA and protein in the corneas.Forteen days after modeling,the expression of the CD31 in the CNV was detected using immnofluorescence of corneal whole mount;the expression of vascular endothelial growth factor(VEGF) in the cornea was assayed by Western blot.Cellular wound scratch test was employed to evaluate the effects of ARF antagonist on proliferation and migration of human retinal vascular endothelial cells (RECs).All animal experiments were done in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and Guideline for the Care and Use of Laboratory Animals on the the Soochow University Animal Care Committee.Results ARF mRNA and protein were expressed in the mice corneas in both the PBS group and the ARF antagonist group at various time points.The expression of ARF mRNA in mice corneas was enhanced with the lapse of the time (Ftime =65.17,P =0.00),but no significant difference was found among the groups (Fsroup =1.98,P=0.18).There was also significant difference in the expression of ARF protein in mice corneas at different time points in the ARF antagonist group (F =10.77,P =0.00).The related CNV area was 0.45±0.05 in the ARF antagonist group,and that in the PBS group was 0.72±0.11,with significant difference between them (t =-3.87,P ＜ 0.05).The green fluorescence area of C D31 expression in the cornea was smaller in the ARF antagonist group than that of the PBS group.Expression level of VEGF in the ARF antagonist group was 1.20±0.21,and that in the PBS group was 2.47±0.33,showing a significant difference (t =-5.62,P ＜ 0.05).As the increase of ARF antagonist concentration,the inhibiting rate of cell proliferation was reinforced among 10,100 and 1 000 μg/L ARF antagonist groups (F=8.47,P =0.02).Twenty-four hours after scratch test,the migrating distance of human RECs was (5.46±1.32) μm and (5.04±1.68) μm in the 100 μg/L and 1 000 μg/L ARF antagonist groups,respectively,which were shorter than (8.49± 1.18) μm of the PBS group (t=-2.94,-2.91,both at P＜0.05).Conclusions ARF inhibitor can reduce CNV by down-regulating the expression of VEGF in alkaline burn cornea and inhibiting the proliferation and migration of vascular endothelial cells.

Background Researches showed that ADP-ribosylation factor (ARF) promotes intracorneal secretion of multiple angiogenesis-related factors,such as vascular endothelial growth factor (VEGF) and nitricoxide synthase (NOS) etc., and therefore results in corneal neovascularization.However, whether ARF affects the tube formation of human retinal endothelial cells(HRECs) is unelucidated.Understanding the effect of ARF tube formation of HRECs is important for the target treatment of retinal vascular diseases.Objective The aim of this study was to explore the effect and mechanism of ARF inhibitor on tube formation of HRECs in vitro.Methods HRECs (HREC line) were cultured and passaged.The growth-well cells were harvested and divided into two groups.The cells were regularly cultured in the control group,and ARF antagonist (lml) was added in the culture medium in the ARF antagonist group.The expression levels of ARFmRNA and protein in the cells were examined by reverse transcription (RT)-PCR and Western blot.The morphology and number of HREC tube formation were detected by using three-dimensional Matrigel assay.The relative expression levels of VEGF, NOS, focal adhesion kinase (FAK) and heat shock protein 90 (HSP90) at gene level and protein level were examined by RT-PCR and Western blot in vitro.Results The relative expression levels of ARFmRNA in the cells were 0.65 ±0.14 and 0.32±0.10, and those of ARF protein were 0.85±0.15 and 0.24±0.17 in the control group and ARF antagonist group,showing significant differnces between the two groups (t =7.32, P =0.00;t =5.15, P =0.00).The number of HREC tube formation was (34.66±8.57)/field in the ARF antagonist group, which was significantly lower than (51.46±7.12)/field in the control group (t=2.99 ,P=0.04).The relative expression levels of VEGF mRNA, NOSmRNA and their proteins in the cells were significantly lower than those of the control group (t =3.02, P =0.04;t =3.68, P =0.02;t =3.33,P=0.03;t=2.89 ,P=0.04).The relative expression levels of FAKmRNA and HSP90mRNA in the ARF antagonist group were 0.65±0.18 and 0.28±0.05 ,which were significantly lower than 0.76±0.25 and 0.46±0.09 in the control group (all at P＜0.05).Conclusions ARF antagonist appears to have an inhibitory effect on the tube formation ability of HRECs propably by down-regulating the expressions of VEGF, NOS and the downstream signal transduction factors FAK and HSP90 in HRECs in vitro.

Objective To obtain Chinese hamsterovary (CHO) cell line expressing human decay accelerating activity (hDAF) stably and to observe the protective effect of hDAF on heterologous cells under the circumstance of complement activation. Methods The eukaryotic expression vector DAF pcDNA3.1 was constructed and then transfected into CHO cells by lipofection. Monoclones of cells expressing hDAF stably were screened by the method of limiting dilution. hDAF expression was detected by flow cytometry. The decay accelerating activity of hDAF was determined by assay of C3 deposition and 51Cr release. Results The expression vector DAF pcDNA3.1 was successfully constructed, and monoclones of cells expressing hDAF were obtained. CHO cells expressing hDAF could decrease C3 deposition and attenuate the killing effect of activation of the complement system. Conclusion We have obtained CHO cell clones expressing hDAF stably, which is helpful for the further studies of the relationship of the structure with the functions of hDAF.

Objective:To compare the effect of age on clinical outcome of laparoscopic sleeve gastrectomy (LSG) in the obese patients.Methods:A total of 113 patients who underwent LSG due to obesity and metabolic disorders between 2013 and 2018 at Fudan University Pudong Hospital, and completed the scheduled follow-up (1, 3, 6, and 12 months after surgery) were included for the retrospective analysis. The patients were divided into three groups based upon pre-operative age, including 15 to 30 year-old group ( n=58), 31 to 45 year-old group ( n=32), and 45 to 65 year-old group ( n=23). The body weight related parameters, glycemic and metabolic related parameters, lipid panel as well as arterial blood pressure were compared at pre-operative baseline, 1, 3, 6, and 12 months after surgery. The quantitative data were analyzed by repeated measurement ANOVA, and the P value was corrected by Bonferroni method. And the categorical variables were analyzed by chi square test. Results:The preoperative baseline data showed that with the increase of age, the preoperative body mass index gradually decreased, which were (40.1±5.9) kg/m 2, (37.1±6.6) kg/m 2 and (35.3±7.4) kg/m 2 in 16 to 30, 31 to 45 and 46 to 65 year-old groups, respectively. Otherwise, other metabolic related parameters were comparable. At 12 months after LSG, there was no significant difference in the amount of weight loss among the groups, but the percentage of total weight loss (% TWL) and the percentage of total BMI loss (%TBMIL) decreased significantly with age increasing. The %TBMIL in 16 to 30, 31 to 45 and 46 to 65 year-old groups were 32.3±7.5%, 28.4±8.4% ( P<005 compared with 16 to 30 year-old group) and 25.7±8.2% (compared with 16 to 30 year-old group P<0.001), respectively. In the patients with preoperative HbA1c>7%, HbA1c reduction in the three groups at 12 months after operation were 3.20% (compared with 46-65 year-old group P<0.001), 2.64% (compared with 46-65 year-old group P<0.05) and 1.34%, respectively. The proportions of patients with HbA1c < 6.5% were 95.8%, 88.9% and 50.0%, respectively. LDL, triglyceride and arterial blood pressure in all groups decreased and HDL increased rapidly within 3 months after operation, but there was no significant difference among the three groups. Conclusions:The improvement of blood glucose metabolism and the remission rate of type 2 diabetes mellitus (T2DM) in the patients with older age were worse than those in the younger patients, and the T2DM in the younger patients tends to obtain better clinical remission after LSG; in terms of weight loss, with the increase of age, %TWL and %TBMIL also showed a decreasing trend; however, the improvement of blood lipid and blood pressure after LSG was not affected by the factor of age. This study implies that patients who meet the indications of metabolic and bariatric surgery should be suggested to receive surgical treatment early in order to achieve better clinical outcomes.

Objective To evaluate the efficacy and safety of domestic olmesartan in treatment of mild to moderate essential hypertension in comparison with losartan. Methods Two hundred and thirty-seven patients with mild to moderate essential hypertension were enrolled in a randomized, double-blind, multi-center, paralleded and active-controlled trial, and were divided into olmesartan group (olmesartan 20 mg + losartan 50 mg placebo) and losartan group (losartan 50 mg + olmesartan 20 mg placebo) for a 8-week therapy. Four weeks after treatment, dosages of drugs were doubled in patients with seated diastolic blood pressure ≥90 mmHg (1 mmHg =0.133 kPa). All patients were followed up every two weeks, and the efficacy and adverse effects were observed. Another 32 patients with mild to moderate essential hypertension were enrolled and given olmesartan only, and ambulatory blood pressure monitoring was performed before and 8 weeks after treatment. Results Compared with those before treatment, both systolic blood pressure and diastolic blood pressure significantly decreased in olmesartan group and losartan group 8 weeks after treatment [(15.2 ±13.3) mmHg and (19.5 ±11.8) mmHg, respectively for systolic blood pressure (P <0.001); (15.9 ±7.48) mmHg and (16.2 ± 5.95) mmHg, respectively for diastolic blood pressure (P < 0.01) ], while there was no significant difference between these two groups (P > 0.05). There was no significant difference in total effective rate and incidence of adverse effect between these two groups (86.9% vs 93.7% and 7.63% vs 5.88% , P > 0.05) . Ambulatory blood pressure monitoring demonstrated that trough to peak ratios of systolic blood pressure and diastolic blood pressure were 86% and 71%, respectively. Conclusion Domestic olmesaratan provides an effective, safe and long action in the treatment of mild to moderate essential hypertension.

OBJECTIVEIn order to find out the cause of molar intrusion and how to avoid molar intrusion, we analyze the movement tendency of tooth by changing the angles of upper accentuated-curve and the height of free traction hook.

METHODSSpiral CT scanning and Mimics 10.0 software were employed in this study to construct the three dimensional finite element model of maxillary teeth, periodontal ligaments and alveolar bone as well as the inch stainless steel upper accentuated-curve archwires with different angles (5 degrees, 10 degrees, 15 degrees, 20 degrees, 25 degrees) and the free traction hook with different height (2.1, 4.0, 5.5 mm). By exerting 1.5 N backward force to analysis the displacement trend of maxillary tooth.

RESULTSUpper accentuated-curve archwires made the incisors labially tip, intruse, distally upright and mesilabially torsion; made the canines labially tip, prolong, mesial-tipped and mesi-labially torsion; made the second premolars and the first molars buccal tip, intruse, distal upright and mesi-labially torsion. With the angle of upper accentuated-curve archwires increased, all tooth movement tendency increased; while with the height of free traction hook increased, all tooth movement tendency decreased.

CONCLUSIONWhen the angle of upper accentuated-curve archwires increased, the extent of molar intrusion increased, but when the height of free traction hook increased, the degree of molar intrusion decreased. The degree of molar intrusion was minimum using 5 degrees upper accentuated-curve archwires cooperated with 5.5 mm free traction hook.

Bicuspid ; Bone Screws ; Finite Element Analysis ; Humans ; Incisor ; Maxilla ; Molar ; Periodontal Ligament ; Tooth ; Tooth Movement Techniques

Background and purpose:Perioperative anesthetic management is thought to be critical to the success of free flap breast reconstruction. The purpose of this study was to discuss intraoperative fluid, hemodynamic and temperature management in patients undergoing deep inferior epigastric perforator (DIEP) flap breast reconstruction.Methods:From Jun. 2011 to Dec. 2015, 126 patients underwent DIEP lfap breast reconstruction. Postoperative complications were reviewed. Intraoperative fluid infusion rate was analyzed. Mean arterial blood pressure (MAP) and core temperature were measured before induction (T0), after lfap elevation but before lfap transfer (T1), 15 min after flap revascularization (T2), and at the end of surgery (T3).Results:Nine patients developed flap compromised: 7 were salvaged and 2 failed. The mean intraoperative lfuid infusion rate was (5.44±1.66) (mL?kg-1)/h. MAP at T0, T1, T2 and T3 were (87.45±8.90), (74.19±8.63), (74.60±8.71) and (79.62±7.88) mmHg, respectively. Core temperature at T0, T1, T2 and T3 were (36.69±0.14), (36.36±0.18), (36.27±0.14) and (36.21±0.15)℃, respectively. Conclusion:Standard practice focusing on intraoperative lfuid management, hemodynamic adjustment and temperature control in microsurgical reconstruction of the breast should be established to further improve free lfap outcome.

10.3969/j.issn.1000-3606.2013.06.019

Objective: To explore the value of preventive nursing in the treatment of neonatal respiratory distress syndrome (NRDS) mechanical ventilation in premature infants. Methods: A total of 45 NRDS preterm infants who received treatment during the preventive care program from February 1, 2018 to November 1, 2018 were selected as group A, 45 cases of NRDS preterm infants who received treatment during the preventive care program from January 1, 2017 to January 10, 2018 were included in group B for retrospective study. Baseline data, mechanical ventilation index, complications, and family satisfaction of the children were observed. Results: The number of pulmonary surfactant applications, mechanical ventilation time, hospitalization time and hospitalization cost were (1.5±0.5) times, (92.5±13.8) h, (12.5±1.3) d, (26±4) thousand yuan in group A, and (2.6±0.4) times, (131.4±23.1) h, (16.0±2.8) d, (33±5) thousand yuan in group B, the highest oxygen saturation value was 468.9±42.1 in group A,401.2±22.3 in group B, there were significant difference between the two groups(t=7.334-11.524, P<0.05). The total incidence of complications during treatment was 15.6%(7/45) in group A and 48.9%(22/45), there was significant difference between the two groups (χ²=11.447, P<0.05). The total satisfaction rate of family members was 95.6%(43/45) in group A, which was significantly higher than that in group B (64.4%, 29/45). The difference was statistically significant (χ²=13.611, P<0.05). Conclusions Prophylactic care can be used in the mechanical ventilation of premature infants with NRDS, which can significantly reduce the incidence of complications, reduce the amount of pulmonary surfactant, shorten the mechanical ventilation time, and save the cost of treatment. It is worth promoting.