Abstract:Objective:To investigate the effects of glycine on hypoxic impair in cells. Methods:MDCK cells were cultured in a modular incubator chamber full of 95% N2 and 5% CO2 in the presence or absence of glycine for 24 h,36 h,48 h,72 h and 84 h. We observed the effects of glycine on cellular proliferation under hypoxic conditions by MTT method. Western blot was used to detect the phosphorylation activities of ERK1/2,p38MAPK and Akt in cells. Results:The proliferation of MDCK cells cultured under hypoxic conditions decreased significantly,while the proliferation increased after glycine treatment. Protective effect of glycine on cells was found at 24 h,36 h and 48 h of hypoxia. Activation of p38MAPK and depression of ERK1/2 and Akt in MDCK cells were detected when oxygen was deprived. Treatment with glycine antagonized the hypoxia-induced phosphorylative changes in ERK1/2,Akt and p38MAPK. Conclusion:Glycine could protect MDCK cells from early hypoxic injury. Activation of ERK1/2 and Akt but suppression of p38MAPK may contribute to the cytoprotective mechanisms of glycine on hypoxic impair in MDCK cells.

Abstract:Objective:To investigate the proliferative lesions of glomerular mesangial cell (GMC) in Thy-1 nephritis (Thy-1N) rats and the role of sublytic C5b-9 complexes in GMC proliferation. Methods:Thy-1N rat model was constructed. The expression of cyclin D2,proliferative cell nuclear antigen(PCNA) in the renal tissues of rats at mRNA and protein levels were observed at 18 h,on day 3 (3 d) and day 7 (7 d) after Thy-1 Ab injection. The deposition of C5b-9 was detected in the glomerulus. Meanwhile,the proliferation of glomerular cells in rats was assessed by BrdU incorporation,light microscopy(LM) and electron microscopy (EM). Furthermore,rat GMC were cultured and divided into different groups,and then the proliferation of GMC exposed to sublytic C5b-9 was evaluated by 3H-thymidine incorporation(3H-TdR) analysis. Results:The expression of cyclin D2 and PCNA in the renal tissues and the number of glomerular BrdU-positive cells became increasing at 18 h in Thy-1N rats,and continue increasing on 3 d and 7 d. The deposition of C5b-9 was found in the glomerulus,and some cells were intact. Moreover,the number of GMC and the secretion of ECM were increased on 3d and became obvious on 7 d under LM and EM. The proliferation of GMC was increased at 18 h after sublytic C5b-9 stimulation in vitro. The supernatant of GMC induced by sublytic C5b-9 could also promote GMC proliferation of rat. Conclusion:Glomerular lesions in rats with Thy-1N include early and secondary GMC proliferation,while sublytic C5b-9 complexes might be involved in early GMC proliferation.

Abstract:Objective:To investigate the effect of genistein on the invasion of breast cancer cells (MDA-MB-231) and its possible mechanism. Methods:MDA-MB-231 cells were co-cultured with genistein at various concentrations (25 to 100 μmol/L) for 24 h. Cell invasion ability was studied by Transwell invasion assay. Phosphorylated ERK and total ERK proteins were quantified by Western blot analysis. Distribution of cellular phosphorylated ERK was detected by fluorescence microscope. Intracellular superoxide anion production was stained by DHE staining and detected by fluorescence microscope. Results:After treated with genistein for 24 h,the invasion ability of MDA-MB-231 cells was significantly reduced in a dose-dependent manner compared to the control cells. Consistently,ERK activity and p-ERK location in the nucleus also decreased after genistein treatment. Furthermore,ERK inhibitor U0126 could inhibit cell invasion similarly as genistein. In addition,superoxide anion production was suppressed when treated with genistein,and superoxide anion scavenger TEMPOL could not only suppress ERK phosphorylation,but also inhibit cell invasion. Conclusion:Genistein can inhibit invasive ability of MDA-MB-231 cells,probably through inhibiting the superoxide anion/ERK pathway.

Abstract:Objective:To construct plasmid pcDNA3.1/HNF4α,transfect it into human umbilical cord mesenchymal stem cells (HUMSCs),and detect the expression in HUMSCs. Methods:pcDNA3.1/HNF4α vector was constructed with recombinant DNA technology. After detected by enzyme digestion and DNA sequencing,the plasmid was transfected into HUMSCs. The expression of HNF4α was detected simultaneously by using RT-PCR and Western blot. The hepatic specific protein ALB and AFP was detected by immunofluorescence after one week’s transfection. Results:The plasmid pcDNA3.1/HNF4α was constructed successfully. RT-PCR and Western blot results confirmed that HUMSCs expressed HNF4α after transfection. The green fluorescent protein could be identified by immunofluorescence after one week’s transfection. Conclusion:Recombinant eukaryote plasmid pcDNA3.1/HNF4α was successfully constructed,and the HNF4α protien can be expressed in HUMSCs.

Abstract:Objective:To investigate the effect of tissue engineered bone of the chitosan/phosphonic chitosan sponge combined with human umbilical cord mesenchymal stem cells (HUMSCs) on repairing bone defects. Methods:First,The tissue engineered bone was constructed in vitro;Second,New Zealand rabbit model with bone defect were divided into groups A,B,and C. The complex sponge was implanted into bone defects in group A;cell scaffold composites were implanted in group B;and group C was as control. The defective bone specimen was collected at 4th,8th,12th week after implantation sugery. The gross and histological changes and the imaging of bone specimen were observed. Results:After 12 weeks,new bone have completely filled the bone defects in group A. Moreover,bone stump have been connected by some cortical bone. While part of porosis can be found in group B,but no continuous sclerotin was observed. Only little porosis can be found in group C. Conclusion:The tissue engineered bone by combining the HUMSCs with chitosan/phosphonic chitosan sponge has the potential of osteogenic ability in rabbit,and can become material substitution for bone transplantation.

Abstract:Objective:To examine whether activation of calcium-sensing receptor(CaSR) is associated with apoptosis and inflammatory reaction of cardiomyocytes stimulated by ischemia/reperfusion(I/R). Methods:Primary neonatal rat ventricular cardiomyocytes were incubated in anoxia/reoxygenation method to establish a model of simulated I/R. And neonatal rat ventricular cardiomyocytes were randomly divided into control group,I/R group,GdCl3 (CaSR agonist) group and GdCl3+SB203058 (a specific inhibitor of p38MAPK) group. Cardiomyocyte apoptosis was detected by TUNEL assay. The expressions of CaSR,tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were detected by real reverse transcriptase polymerase chain reaction(RT-PCR). The expressions of Caspase-3,Bcl-2 and p38MAPK were detected by Western blot. Results:The simulated I/R enhances cardiomyocyte apoptosis and the expression of CaSR,TNF-α,IL-6 and p38MAPK. GdCl3 can further increase cardiomyocyte apoptosis and expression of above proteins,along with up-regulation of Caspase-3 and down-regulation of Bcl-2. Compared with GdCl3 group,the mRNA expression of TNF-α and IL-6 were markedly decreased with no significant changes of cardiomyocytes apoptosis and CaSR mRNA in GdCl3+SB203058 group. Conclusion:Activation of CaSR is not only associated with increased cardiomyocytes apoptosis after I/R injury via suppressing Bcl-2 and promoting Caspase-3 expression,but also induces inflammatory reaction via p38MAPK signaling pathway at I/R area.

Abstract:Objective:To detect the human-specific protein kinase-15(NYD-SP15) gene expression in normal gingival tissue and hyperplastic gingival tissues,observe the expression of NYD-SP15 protein in gingival tissues,and to explore the relationship of NYD-SP15 and gingival hyperplasia initially. Methods:Reverse transcription-polymerase chain reaction(RT-PCR) and immunohistochemical method were applicated to detect the expression of NYD-SP15 gene in gingival tissues of 27 cases of normal people and 40 cases of gingival hyperplasia patients. The normal gingival tissues were the excision of gingival tissue during crown lengthening surgery (the teeth were traumatic crown fractures),recorded as normal control group. The hyperplasia samples were the excision of gingival tissue during gingivectomy,recorded as hyperplasia group. The half of all specimens were removed and stored in nitrogen liquid immediately. The remaining half of the tissues with formalin,embedded in paraffin storage. Staining positive cells were counted under the microscope and classificated by positive degree. The data of the experiment was analyzed by Stata 9.2(StataCorporation College Station,TX) software. Results:The expression of NYD-SP15 mRNA in tissues of hyperplasia group was significantly higher than that of control group(P < 0.05). The immunohistochemistry results showed that the NYD-SP15 protein was highly expression in the cytoplasm in normal gingival tissue. But,in the hyplasia gingival tissues,the expression of NYD-SP15 protein was highly in the nucleus. Conclusion:The level of NYD-SP15 expression and location in the gingival tissues may be associated with the gingival hyperplasia. However,the relationship between them remains to be further studied.

Abstract:Objective:To construct the prokaryotic plasmid expressing the S100A16 and prepare the rabbit polyclonal antibody against S100A16. Methods:S100A16 gene was amplified by RT-PCR and cloned into the expression vector pET-28a,then the recombinant plasmid was transformed into E. coli BL21 (DE3) and induced to express recombinant protein with IPTG. The fusion protein was further purified by affinity chromatography and identified by SDS-PAGE. The rabbit was immunized with fusion protein to produce polyclonal antibody,and the sensitivity and specificity of the antibody was evaluated by enzymelinked immunosorbent assay (ELISA) and Western blot. Results:The recombinant expression plasmid has been successfully constructed,which was confirmed by the restriction enzyme digestion and DNA sequencing analysis. The recombinant protein could be expressed effectively,and the antibody was specific and effective. Conclusion:The S100A16 prokaryotic expression plasmid was constructed,and polyclonal antibody directed against this protein has been successfully prepared,which will provide a useful tool for the further research of S100A16.

Abstract:Objective:To analyse the value of cardiac troponin I(cTnI) test and echocardiography in evaluating acute radiation induced heart disease(RIHD) after a single dose radiation in a Newzealand rabbit model. Methods:Each rabbit was irradiated with a single dose. According to the radiation dose,25 rabbits were divided into group A(clinical dose group) and group B(high dose group),then each group was divided into several subgroups according to the radiation dose. The subgroup’s dose and the number of group A was 0,10,14,18 Gy and 3,3,6,7 respectively;the dose and the number of group B was 22,30,40,54 Gy and 3,1,1,1 respectively. Echocardiography and cTnI were detected before and after radiation. The echocardiographic parameters were:① flow pulse doppler Tei index;② tissue doppler imaging:mitral annulus peak velocities in systole (Sa),early disatiole (Ea),late diastole (Aa) and Ea/Aa;③ conventional parameters:left ventricular diameter,ejection fraction(EF) and fractional shortening(FS). Then the pathologic changes of heart were semiquantitatively analyzed after 4 months follow-up. Results:① cTnI began to increase 3 hours after radiation,reached the apex after 14 to 24 hours,then decreased and stayed at a low level about 1 week;moreover,the level of group B was much higher than that of group A; ② after radiation,changes in diastolic and systolic ventricular function were observed with a decrease of Sa,Ea,Aa and a increase of the Tei index;furthermore,with the radiation dose increased,the value of Sa,Ea and Aa deceased,and the Tei index increased. Moreover,there were no changes in the conventional parameters. ③ The average severity grade of RIHD in group A and group B was 1 and 1. 5 respectively. Conclusion:Single high dose radiation can lead severe acute RIHD and the severity is related to the radiation dose.

Abstract:Objective:To investigate the expression of osteoprotegerin(OPG)/receptor activator of nuclear factor-κB (RANK)/RANK ligand(RANKL) axis in stabilization of spontaneously restored sinus rhythm in permanent atrial fibrillation patients after mitral valve (MV) surgery and study its clinical significance. Methods:Clinical data,biopsies of right atrial appendage were collected from 135 atrial fibrillation patients who spontaneously restored sinus rhythm after conventional isolated MV replacement. Comparison was made between patients who had recurrence of atrial fibrillation within 7 days(AF group) vs. patients with persisted sinus rhythm for more than 7 days(SR group). Results:The result of Western blot and immunohistochemistry showed an increased expression of RANK,RANKL and RANKL/OPG ratio in AF group than SR group. The degree of interstitial fibrosis reflected by collagen volume fraction(CVF) was also more severe in AF group than SR group,which was positively correlated with the expressions of RANK and RANKL and RANKL/OPG ratio. Conclusion:These findings suggest that the OPG/RANK/RANKL axis plays important roles in stabilization of restored sinus rhythm after MV surgery by stimulating AF-related atrial remodeling in atrial fibrillation patients,and possibly represents a new target for upstream treatment of atrial fibrillation.

Abstract:Objective:To determine the effects of parenteral nutrition (PN) and enteral nutrition (EN) on biochemical and clinical outcomes in patients who underwent pancreaticoduodenectomy. Methods:Eighty-four patients who underwent pancreaticoduodenectomy were enrolled in this study. They were randomly divided into the EN group and the PN group. The program of isonitrogenous and isocaloric intake was taken in each group. The biochemical and clinical parameters were recorded and analyzed between the two groups. Results:Significant differences were found in total complications,initiation of soft diet,hospital cost and liver function on the 7th postoperative day (P < 0.05). The incidence rate of complications was higher in PN group(33. 3%) than PD group(14.3%)(χ2 = 4.200,P = 0.040);The hospital cost was higher and initiation of soft diet was later in PN group than in PD goup[(64 650 ± 11 977)Yuan vs. (51 358 ± 5 114)Yuan,t = 6.614,P = 0.003;(76.1 ± 12.3)h vs. (44.5 ± 7.1)h,t = 14.375,P = 0.017]. And liver function was much better in EN group than in PN group. No significant differences were found in mortality,anastomotic leakage,hemorrhage,delayed gastric emptying,nutritional status and postoperative hospital stay between EN group and PN group. Conclusion:EN support can improve the liver function,reduce the complications and hospital cost,and help soft diet initiation of patients undergoing pancreatoduodenectomy. EN should be recommended as a regular treatment for patients who received pancreaticoduodenectomy.

Abstract:Objective:To investigate the expression and significance of cancer stem cell markers CD133 and TROP-2 in non-small cell lung carcinoma(NSCLC). Methods:The expression of CD133 and TROP-2 in NSCLC was detected by immunohistochemistry and double-labelling immunofluorescence. The location and the colocalization of the two proteins were observed under light microscope and confocal laser scanning microscope. Results:①The expression rate of CD133 and TROP-2 were 30.0% and 41.25% in 80 NSCLC tissues respectively,which were significantly higher than that in the tumor-adjacent normal tissues or benign lesion tissues(P < 0.01);②CD133-positive expression was significantly higher in poor differentiated tumor than that in the well differentiated tumor(P = 0.024);③ Positive expression of CD133 and TROP-2 in the lymph node metastasis group was significantly higher than that in the non-lymph node metastasis group(P < 0.05). The positive expression rate of TROP-2 increased with the increasing Tumor-Nodes-Metastasis (TNM) stage(P < 0.05). ④CD133 expression was correlated with TROP-2 expression (P < 0.001). And there was collocation between the CD133 and TROP-2 proteins. Conclusion:The expression CD133 and TROP-2 was associated with the invasion and metastasis of NSCLC. Their colocalization in NSCLC was detected preliminarily. CD133 and TROP-2 may be used as two important markers in judging the cancer stem cell of NSCLC.

Abstract:Objective:To explore the relationship between platelet parameters and microangiopathy and peripheral arterial disease (PAD) in type 2 diabetes. Methods:Total 650 patients with type 2 diabetes were collected. The levels of platelet count (PLT),mean platelet volume(MPV),platelet distribution width(PDW),clinical and biochemical characteristics,microalbuminuria(MAU) and ankle brachial index(ABI) were measured. The patients were divided into MAU group and normal abuminuria group according to presence or absence of MAU. The patients were also divided into PAD group and non-PAD group according to the ABI value. The comparison of platelet parameters and biochemical indices between groups was performed. The correlation of the risk factors for MAU and PAD were analyzed. Results:The level of SBP,TC,TG,MPV and PDW in MAU group were significantly higher than those in normal abuminuria group,and the level of HDL in MAU group were lower than that in normal abuminuria group(P < 0.05). The differences in WHR,SBP and TC between PAD and non-PAD group were statistically significant(P < 0.05). However,there was no significant difference in platelet parameters between two groups(P > 0.05). Logistic multiple regression analysis shown that SBP,TC,TG and PDW were the independent risk factors of the progress of MAU. SBP,WHR and HDL were significant factors for PAD(P > 0.05). Conclusion:Monitoring the platelet parameters,especially MPV and PDW,has very important prognostic value in the early diagnosis of microangiopathy in type 2 diabetes.

Abstract:Objective:To investigate the clinical effects of several treatments for vascular cognitive impairment not dementia (VCIND). Methods:Total 90 patients with VCIND were randomly divided into the cerebellar fastigial nucleus electrical stimulation (FNS) group,oxiracetam group and nimodipine group,with 30 cases in each group. On the base of all groups receiving conventional treatments,the treatment of FNS was applied in the FNS group,oxiracetam in the oxiracetam group and nimodipine in nimodipine group. The Event-Related Potentials P300 (ERP-P300),Montreal cognitive assessment (MoCA) and transcranial dopplor ultrasonic monitor(TCD) of all patients were observed and compared before and after the treatment. Results: FNS group:After the treatment,the scores of MoCA showed improvement(P < 0.01),the latency period of P300 was significantly shorten(P < 0.01) and the amplitude was significantly increased(P < 0.01). TCD result showed the mean velocity of blood flow improved significantly(P < 0.01). Oxiracetam group:After the treatment,the scores of MoCA showed improvement (P < 0.01),the latency period of P300 was significantly shorten (P < 0.01) and the amplitude was significantly increased(P < 0.01),but the TCD result had no obvious improvement (P > 0.05). Nimodipine group:After the treatment,TCD result showed the mean velocity of blood flow improved significantly (P < 0.01),but the MoCA the latency period and the amplitude of P300 had no obvious improvement (P > 0.05). After the treatment,the score of MoCA in the FNS group and oxiracetam group were both higher than that in nimodipine group(P < 0.01),and the score of the FNS group was higher than that of the oxiracetam group (P < 0.01). After the treatment,the latency of P300 of the FNS group was shorten than that of the oxiracetam group and nimodipine group (P < 0.01),and there was no significant difference between the oxiracetam group and nimodipine group(P > 0.05);the amplitude of P300 in the oxiracetam group was higher than that in the FNS group and nimodipine group (P < 0.05),but there were no significant differences between the FNS group and nimodipine group (P > 0.05). After the treatment,TCD result showed the mean velocity of blood flow of MCA in FNS group was higher than those in oxiracetam group and nimodipine group(P < 0.01),but there were no significant differences between the oxiracetam group and nimodipine group (P > 0.05);The mean velocity of blood flow of ACA,PCA ans VA in FNS group and nimodipine group were higher than those in oxiracetam group(P < 0.05),ACA and VA of FNS group was higher than those of nimodipine group (P < 0.05),but there was no significant difference in PCA between FNS group and nimodipine group(P > 0.05). Conclusion:The FNS,oxiracetam and nimodipine are all beneficial in treatment of VCIND. Oxiracetam can effectively improve cognitive function;nimodipine can effectively improve blood supply to the brain;FNS can effectively improve cognitive function and blood supply to the brain in patients with VCIND.

Abstract:Objective:To analyze the survival rate of AIDS patients who initially received anti-retroviral therapy in 2005-2008 by the end of 2011,and to determine factors associated with survival status. Methods:Information of AIDS patients initially starting anti-retroviral therapy during 2005-2008 was collected from Chinese HIV/AIDS Integrated Control System. Kaplan-Meier was applied to make survival curve;and Cox proportional hazard regression model was used to determine associated factors of survival status. Results:Totally 451 cases were analyzed. The gender ratio of man to women was 1.75∶1. Most of participants were married or cohabitated,with 20 to 50 years old. The major transmitted route was heterosexual. Most of participants were from southern area of Jiangsu. At baseline,average CD4+ T cell counts were 107/mm3. Accumulative survival rates in 12th,24th,36th,48th and 60th after therapy were 0.88,0.85,0.84,0.78 and 0.78 respectively. 53.6% of participants died within the first six months of therapy. CD4+ T cell counts at baseline,initial therapy schedule,and therapy schedule changed were found to be associated with survival time of patients. Conclusion:Anti-retroviral therapy improved the survival rate of AIDS patients. Starting therapy earlier can get better treatment result.

Abstract:Objective:To describe the distribution and characteristics of children falling injury in surveillance areas in Jiangsu province,and to provide basis for injury prevention and control. Methods:Data of children falling injury incidence during 2006 to 2010 were collected and analyzed from provincial injury surveillance system. Results:Falling was the first leading cause(40.4%) of children injury in outpatients. Falling injury occurrence in boys was much higher than that in girls,gender ratio of boy to girl was 2.1∶1. The injury occurred in all ages and the proportion of each age was similar. The top three frequently occurred location of injury was home(32.9%),school and public place(32.0%),public living place(18.1%). Most injury events took place in leisure activities,accounted for 66.5%. Regarding the character of wound,the first one was contusions and abrasions,accounted for 55.3%. The most three common body regions injured were head(39.9%),higher limbs(28.9%) and lower limbs(24.6%). About 80.3% cases were minor injuries,and only 0.4% cases were serious wound. Most injury events were accident,accounted for 99.3%. Comparing between urban and rural areas,no significant difference was observed in gender,intention,season and time. Whereas,significant differences were found in terms of characteristics of age,education background,location,activities and time at injury,character and sites of wounds the body,severity and outcome of injury(P < 0.05). Conclusion:In Jiangsu Province,falling has become the first leading cause of children injury. We should carry out more intervention activities according to the specific injury distributions and characteristics.

Abstract:Objective:To explore a new and suitable technical scheme of establishing type 1 diabetes (T1DM) rats model by STZ injection for beginners. Methods:Total 100 male Wistar rats were divided into diabetes mellitus model (DMM) group and the control group (NC) randomly. The technical scheme for establishing animal model is different from the existing literature. The DMM group of two batches were followed by a single intraperitoneal injection 60 mg/kg(DMM1) or 70 mg/kg(DMM2) STZ solution respectively. the NC group was injected with the same amount of citrate buffer. The rats in DMM1 and DMM2 goups,which didn’t develop T1DM,were compiled into DMM3 group(55 mg/kg STZ by a intraperitoneal injection). After 72 h of STZ treatment,the rats were considerd as T1DM model with fasting plasma glucose (FPG) after 17 h fasting≥ 11.1 mmol /L and with corresponding physiological indicators. Results:The rat model can’t be constructed successfully by a single intraperitoneal injection STZ of 60 mg/kg. The FPG of rats in DMM2 group changed significantly before and after STZ injection(P < 0.05),and 75% of rats developed T1DM. The rats that didn’t develop T1DM with first injection of STZ developed T1DM after supplementary dose in DMM3 group. Conclusion:Constructing T1DM rats induced by STZ injection in batch is best for beginners to use.