Objective To screen and determine the effective silencing targets of β2-microglobulin(B2m)gene at the cellular level in marmoset.Methods By homology comparison of the b2m gene in human and the B2m gene in marmoset, choose homology small hairpin RNA(shRNA)sequences targeting marmoset B2m gene were designed, We choose homology small hairpin RNA(shRNA)sequences targeting designed B2m gene to make homology analysis, and insert into lentivirus-based gene silencing constructs FUGW-TDT.The vectors were transfected into HEK293T cells induced by polyethylenimine(PEI).The suppression of B2m mRNA was detected by real-time PCR.Results Two gene-silencing sequences were screened that lied in 290~310 bp and 665~685 bp of the marmoset B2m mRNA, and have statistical significance in the silencing rate:(46.54±7.91)% (P < 0.05) and(83.22±4.37)%(P < 0.0001).Conclusions Two effective silencing target sequences are screened at cellular level, which can be further used in studies on gene silencing in marmoset.

Objective To evaluate the factors affecting the treatment of old persons diagnosed hypertension. Method A sample of 55566 persons from the community of Yanji area of Shanghai was recruited. Results Among the 19295 persons over sixty there are 5842 patients with hypertension, the morbidity was 30.3%. Among the 36271 persons aged fifteen to fifty-nine there are 3536 patients with hypertension, the morbidity is 9.7%. The ratio of non-treatment in the old age group is 3.1% and in younger group is 8.8%. However,the compliance to the treatment was better in the younger than in old group(73.7% vs 56.2%). The age,hypertension grade and sex were all accounted for the differences in the regularity of the hypertension treatment. Conclusion Further effort on the education of non-old hypertensive patients is essential to decrease the damage from hypertension.

Objective To explore the relationship between the level of morning blood pressure surge (MBPS) and High-sensitivity C-reactive protein( hs-CRP) in acute coronary events among old patients and to evaluate their clinical value. Methods One hundred and twenty - three patients with coronary disease were divided into two groups, each of which was old myocardial infarction(OMI, n = 55) and old myocardial infarction with acute cardiovascular syndrome (OMI + ACS, n =68). The control group includes the patients without coronary disease( n =50). The patients who have infection, malignant tumors, liver and kidney dysfunction, blood diseases, valvular heart diseases, congenital heart diseases, aortic dissection,secondary hypertension were not included. All groups were received 24 hours ambulatory blood pressure monitoring to calculate MBPS and the determination of hs-CRP in serum. Results Compared with each two groups, the levels of MBPS and hs-CRP in OMI group were significantly higher than those of the control group (0. 01 ＜ P ＜0. 05) , and had more significance in the other groups ( P ＜0.01). The levels of MBPS and hs-CRP had positive relationship in coronary events in the elderly ( r =0. 76, P ＜0. 01) . Conclusion The level of MBPS is positively correlated with serum hs-CRP which is one of key risk factors of inflammatory reactions by impairing vessels in body. The two factors both play important roles in the occurrence and the development of coronary events among old patients. They both have important clinical value in recurrent acute coronary events among old patients.

AIM: To observe the effects of Egr-1 gene knockout on the expression of inflammatory-related factors in pancreatic tissue in a mouse acute pancreatitis model.METHODS: The experimental pancreatitis was induced by high-dose of cearulein in wildtype mice and Egr-1 knockout mice.The pancreatitis indexes,such as serum amylase,pancreata edema,and myeloperoxidase(MPO) levels in pancreata and lungs were recorded.The mRNA levels of tissue factor(TF),plasminogen activator inhibitor(PAI-1),monocyte chemoattractant protein(MCP-1),Gro-1,IL-6 and ICAM-1 were measured by quantitative PCR.RESULTS: Contrary to wildtype mice,typical pancreatitis was not induced by high-dose cearulein in the Egr-1 knockout mice,not only markedly reduced edema in pancreata and lungs,but decreased MPO levels in lungs as well were found.Furthermore,the mRNA of TF,PAI,MCAP,ICAM-1 and IL-6 in pancreata were significantly decreased in Egr-1 knockout mice.CONCLUSION: The severity of pancreatitis and lung damage is ameliorated in Egr-1 knockout mice stimulated by high-dosage of cearulein,which was probably mediated by decreasing expression of inflammatory-related factors in pancreata,such as TF,PAI,MCP-1,ICAM-1 and IL-6.

BACKGROUND:Bone marrow mesenchymal stem cells (BMSCs) have been induced to differentiate into cardiomyocyte-like cells in vitro.OBJECTIVE:To explore the association between GATA-4, Nkx-2.5 and α-myosin heavy chain (α-MHC) expression and cell morphological changes and structure formation in the process of BMSCs differentiation into cardiomyocyte-like cells.METHODS:By using myocardial lysate, BMSCs were induced to differentiate into cardiomyocytes.Immunocytochemistry staining was used to detect cardiac troponin T (cTnT) and connexin43, for the identification of cardiomyocytes. In the process of directional differentiation, RT-PCR was used to detect the expression of GATA-4,Nkx2.5 and α-MHC.RESULTS AND CONCLUSION:During the directional differentiation of BMSCs, the cells were changed from long fusiform to short rod, forming protrusions that were interconnected to form mesh-like, bamboo-like or myotube-like structure. When the cells were interconnected like a bamboo, cTNT and connexin43 positive cells were visible, and then the number of positive cells increased with the presence of myotube-like structure. RT-PCR results showed that during the induced directional differentiation of BMSCs, GATA-4, Nkx2.5 and α-MHC mRNA levels increased continuously. When interconnected cells formed a mesh-like structure, GATA-4 expression reached the peak and then kept a high level. When adjacent cells were fused into a myotube-like structure, α-MHC reached the peak. Additionally, the expression of Nkx2.5presented a time-dependent increase trend. Overall, during the induced differentiation of BMSCs into cardiomyocyte-like cells, the expression of cardiomyocyte specific genes, characterized by temporality and spatiality, is related to the changes of cell morphology and special structure formation.

Objective To assess the efficacy of urethroplasty by using the mucosa of diverticulum as the distal urethral for the recurrence of urethral stricture.Methods We reviewed our experience about 6 cases from Jan.2007 to Oct.2012,including 5 hypospadias and 1 epispadias.The mean age of the patients was 28 ± 16 (12-45)month,range 12 to 45 months.The patients presenting urethral diverticulum should last for 6 months after urethroplasty,which should last for 6 months.The preoperative mean urinary flow rate was 5.1 ± 1.4 (range 3.1 to 7.3) ml/s.All the patients took the retrograde urethrography and cystoscopy to confirm the position and length of urethrostenosis.The positions of urethrostenosis were at the penis coronary in 5 cases and glans penis in 1 case.The mean length of stricture was 13.3 ±4.2(range 8.8 to 20.5) mm.The mean length of diverticulum was 37.5 ± 15.3 (range 21.8 to 55.2) mm.All patients received the urethral reconstruction by using the pedicel diverticulum mucosa.After resecting the stenosis urethra and exposed the whole diverticulum,we opened the diverticulum laterally and halved it.One was used for tabularized urethroplasty in situ,the other was flipped to the distal urethral as pedicel flap to remedy the defect of the urethral.Results Totally 6 cases received the procedure.The mean operating time was 133 ± 48 (range 84 to 192) min.Postoperatively,urethral catheter was remained 2 weeks.All the patients can urinate smoothly after removing the catheter.The postoperative mean urinary flow rate was 10o 9 ± 3.3 (range 6.3 to 15.9) ml/s.The mean follow-up time was 2.2 yrs(range 1 to 4 years).At the end point of follow-up,all patients had not experienced a recurrent diverticulum or stricture at the anastomotic site.Urethrocutaneous fistula in the coronary glan was reported in 2 patients,which were finally cured after 2nd repair.No complication was found in other 4 patients.Conclusions Urethroplasty by flipping the mucosa of diverticulum takes full advantage of urinary mucosa that already existing,which can be very useful in those patients who are lack of foreskin.This approach can also reduce the patient's pain by completing the urethroplasty in situ and avoiding staging operation.But up till now,it can only be applied to the urethral diverticulum secondary to urethrostenosis.It also demands flip flap skilled.And the long-term effects and complications still remain to be seen.

Objective To decrease the p53 gene expression at cellular and animal levels in marmoset using RNA interference technique.Methods The shRNA interference sequences were designed and inserted into the adeno-associated virus vector plasmid after bioinformatics analysis.The plasmids were transfected into African green monkey kidney cos-7 cells.The suppression of p53 mRNA was detected by real-time PCR, and the changes of p53 protein expression were detected by Western bolt.The adeno-associated virus-8 was injected through the hind leg vein.The changes of p53 protein expression in the liver tissue was detected by Western blot and immunohistochemistry.Results We screened two RNA interference effective arget sequences.The expression of p53 mRNA was suppressed ( 82.7 ±8.1 )% and ( 80.7 ± 7.5)%, respectively (P<0.05), and the expression of p53 protein was decreased (77.3 ±11.5)% and (73.7 ± 10.7)%, respectively (P<0.05).The two marmosets after virus infection showed that there were virus distributions in the liver, testes, and neck detected by in vivo fluorescence imaging.The expression of p53 in the marmoset liver was detected by western blot, immunohistochemistry analysis showing no obvious changes.Conclusions In the present study, the decrease of P53 gene expression at cellular level is achieved, however, the liver P53 protein in the marmoset liver is not significantly changes.Further optimization of the way of infection is needed in the future.

AIM:To discuss the effect of Shenmai injection on insulin resistance ( IR) in 3T3-L1 cells and its mechanisms.METHODS:3T3-L1 preadipocytes were induced by chemical reagents to differentiate into fully differentiated adipocytes.Oil red O staining was used to detect the differentiation level of the adipocytes .The insulin-resistant 3T3-L1 cell model was demonstrated using insulin , which was confirmed by glucose concentration in cell supernatant .The IR cell model was given 10 μmol/L rosiglitazone , 25 and 50 g/L Shenmai injection and normal saline for comparison .MTT assay was used to assess the cell activity of 3T3-L1 cells which was treated with drugs for 8, 16, 24 and 36 h.Glucose oxidase method was used to detect the glucose concentration in the cell supernatant at 8, 16 and 24 h.The protein levels of glucose transporter-4 (GLUT4), phosphatidylinositol 3-kinase (PI3K), AKT and p-AKT were determined by Western blot .RE-SULTS:3T3-L1 adipocytes were successfully induced as shown by the positive oil red O staining .The IR cell model was demonstrated , and glucose concentration in the cell supernatant after treatment with Shenmai injection showed that Shenmai injection reduced the IR in 3T3-L1 cell model.The protein levels of GLUT4, PI3K and p-AKT increased compared to con-trol group.CONCLUSION:Shenmai injection reduces the IR in 3T3-L1 cell model, which functions by increasing the protein levels of GLUT4, PI3K and p-AKT.

Objective:To investigate the prevalence of physical violence against children (PVAC)by parents and to explore possible related factors.Methods:In two primary schools in Liaoning Province,1164 parents of pupils in grade 1 to 6 were surveyed by a self-administered questionnaire anonymously to analyze situation about PVAC and related factors.PVAC was defined as having one or more following parent-to-child behaviors in the past three months:push,or shook a child;pinch,screw,or scratch a child;hit child's buttocks with hand;hit child's hand,foot,arm,leg or back with hand;hit child's buttocks with an object;hit child's face or head with hand;hit elsewhere (not buttocks)with an object;kicked a child with a foot or hit with a fist.Results:Of 1164 parents,53.1% reported that they had minor PVAC (51.0%)or/and severe PVAC (19.8%).Multivariate logistic regres-sion analysis indicated that following factors increased the risk of PVAC:child's male gender (OR =1.49),younger age of children (OR =1.38),poor school performance of children (OR =1.85 ),mother (OR =2.09),parents'childhood physical violence victimization experiences (OR =1.53),parents'supportive or tolerant attitudes towards corporal punishment (OR =3.15),parents'lower awareness of the harmfulness of physical violence against children (OR =2.31),and lower social economic status (OR =1.47).Conclusion:The PVAC by their parents may be com-mon in the present study.Parents'supportive or tolerant attitudes toward corporal punishment and low awareness of the harmfulness of physical violence is the main risk factors of PVAC.

OBJECTIVETo investigate the acquaintance to orthodontics and possible factors that obstruct orthodontic treatment in middle school students in zones with different economic levels in Jiangjin, Chongqing.

METHODSThe students were randomly divided into four groups: A class from a junior high school in town (group 1), a class from a senior high school in town (group 2), a class from a junior high school in the countryside (group 3), a class from a senior high school in the countryside (group 4). The information was collected by questionnaire, and the statistical analysis of results was carried out by SPSS 17.0 software.

RESULTSSex made difference in the acquaintance to orthodontics treatment. Girls worried more about teeth extraction and pain that may occur during treatment than the boys. Students in the city had more access to related consultations compared to those in the countryside. Different economic conditions made much contribution to the proportion to go for orthodontics treatment in cities and rural areas. The evaluation from families and friends may affect patients' subjective estimation to themselves.

CONCLUSIONOrthodontists should learn more about student patiens from various aspects, and make different therapies according to their sexes, economic conditions of family, education levels, etc. Only so can orthodontists get better compliance from their patients.