Objective　To study the feature of immunoglobulin complementarity determining region 3 (CDR3) gene in neonates of different gestational age (GA), and the effect of neonatal maturity on the diversity of CDR3 nucleotide sequence. Methods　DNA were extracted from cord blood of 10 neonates of very immature (25-30 weeks), 12 immature (31-36 weeks), and 11 mature (37-41 weeks). CDR3 sequence was amplified suing nested PCR technique then cloned and sequenced. Results　1. There was a CDR3 length of 29.4±7.8, 32.4±9.2 and 40.8±10.7bp, including NDN length of 13.5±5.6, 16.1±7.8 and 22.0±8.5bp respectively in very immature, immature and mature neonates. 2. DP73 and DP75 were preferentially used in very immature and immature neonates, VH5, DP73 and DP75 were used in mature neonates. The usage rate of DP73 and DP75 reduced, whereas VH5 was raised with GA increasing. 3. For D gene segment, there was a frequent use of DN, DQ52 and DXP in very immature neonates, whereas DXP, DLR and DN in immature and mature neonates. 4. JH4 usage was preferential, followed by JH6 in all neonates, and their usage rate was decreased with GA increasing. 5. 63.3%, 68.8% and 92.0% of CDR3 sequence respectively in very immature, immature and mature neonates have an open reading frame with 303bp long encoding 101 residues. Conclusion　During the early life of neonates, VH-D-JH rearrangement of IgH gene is in an active condition, but the diversity is limited. Humoral immunity is a gradual development with increasing GA. Both heterogeneity and similarity of CDR3 sequence exists in neonates with different GA.

0.05).CONCLUSION:Nedaplatin combined with docetaxel is effective and safe for advanced NSCLC with high short-term efficacy and mild toxic side reaction in digestive tract and kidney.but the long-term efficacy of it require further study.

AIM: To observe the effects of toddalia asiatica aqueous extract (Fei Long No 1, F01) on cardiac function and hemodynamics of acute myocardial ischemia in the animal model. METHODS: High positioned double-ligation of the anterior descending left coronary artery induced acute myocardial ischemia in New Zealand rabbits. F01 268 mg/kg were ip into the acute myocardial ischemic model, Cardiac function and hemodynamic measurements were performed before and after ligation and administration of F01. t -test paired was used for statistical analysis. RESULTS: After ligation all indices was reduced significantly except that LVEDP was markedly increased and t-dp/dt max had little change. After administration of F01 changes of most indices were reversed, and returned to or were close to the baseline. 1.5 h after administration of F01 action was more markedly. But the indices of left ventricle work and consumption of oxygen ( HR, TTI and TTI?HR) were reducing continuously. CONCLUSION: F01 markedly decreases ventricle work and consumption of oxygen of acute ischemic myocardium, so that the contractility, diastolic function of myocardium and cardial output are improved. These are the mechanism of protective effect on myocardial ischemia.

AIM: To study the effect of xin-tong capsule on nitric oxide production and cGMP content of ventricular myocardium in rats with experimental myocardial ischemia. METHODS: Rat model of acute myocardial ischemia induced by pituitrin was used in this study, and the lift of ST segment in ECG was used as the index of myocardial ischemia. The NO - 2/NO - 3 and cGMP contents of ventricular myocardium in rats with myocardial ischemia were determined.RESULTS: NO - 2/NO - 3 and cGMP contents in ventricular myocardium of myocardial ischemia were (486?59) nmol/g protein and (0.38?0.08) nmol/g protein, respectively, and markedly lower than those of control [(1057?145) nmol/g protein and (0.70?0.09) nmol/g protein] ( P

AIM: To study the effect of shenmai injection (SMI), a Chinese medicine, on nitric oxide and the expression of endothelial nitric oxide synthase (eNOS) mRNA in myocardium of rats with experimental myocardial ischemia. METHODS: Rats were randomly divided into four groups: control, model (ischemia), SMI 1 and SMI 2 group. A rat model of acute myocardial ischemia was established by isoprenaline treatment and the lift of ST segment in ECG was used as the index of myocardial ischemia. The nitric oxide (NO) contents in serum and myocardium and the expression of eNOS mRNA in myocardium were measured. RESULTS: Compared with control group, ST segment of ECG was significantly elevated 20, 30, 40 min after myocardial ischemia in model group, the lift peak of ST segment occurred 20 min after myocardial ischemia, the concentration of NO in the serum and myocardium and the expression of eNOS mRNA in myocardium were significantly lowered in model group. Compared with the model group, in SMI 1 group and SMI 2 group, the concentration of NO in the serum and myocardium and the expression of eNOS mRNA in myocardium were significantly increased, the lift of ST segment were significantly reduced 20, 30, 40 min after myocardial ischemia. Compared with SMI 1 group, the concentration of NO in the serum and myocardium and the expression of eNOS mRNA in myocardium and the lift of ST segment were not statistically different in SMI 2 group. CONCLUSION: Shenmai injection can increase the expression of eNOS mRNA in myocardium and the content of NO, and protect against myocardial ischemia in rats.

Objectives To investigate the effects of sodium arsenite (NaAsO2) on the expression ot microRNA-191 (miR-191) and tissue inhibitor of metalloproteinase 3 (TIMP-3) in human normal hepatic cells (L-02 cells).Methods L-02 cells were exposed to different doses of NaAsO2 [0 (control group),5,25,50 and 75 μmol/L]for 24 h,or treated with 5 and 25 μmol/L NaAs02 for 0 (control group),12,24 and 48 h.The miR-191 inhibitor was used to suppress the expression of miR-191.qRT-PCR was performed to detect the expression level of miR-191 and TIMP-3 mRNA,and the protein level of TIMP-3 was analyzed by Western blotting.Results Dose-effect study:There were significant differences in the expressions of miR-191,TIMP-3 mRNA and protein between the 5 groups (F =85.674,20.952,123.393,all P ＜ 0.05).The expressions of miR-191 in all groups (1.702 ± 0.124,2.077 ±0.234,2.145 ± 0.105,2.003 ± 0.077) were higher than that of control group (0.990 ± 0.035,all P ＜ 0.05);the mRNA expressions of TIMP-3 in 25,50,75 μmol/L groups (0.848 ± 0.067,0.804 ± 0.081,0.813 ± 0.076) were all lower than that of control group (0.996 ± 0.007,all P ＜ 0.05),but there was no significant difference in the mRNA expression of TIMP-3 between the 5 μmol/L group and control group (0.939 ± 0.133 vs 0.996 ± 0.007,P＞ 0.05),and the protein expressions of TIMP-3 in all groups (0.846 ± 0.093,0.611 ± 0.123,0.554 ± 0.098,0.529 ± 0.067) were lower than that of control group (1.006 ± 0.003,all P ＜ 0.05).Time-effect study:there were significant differences in the expressions of miR-191,TIMP-3 mRNA and protein between the exposure groups of 5 and 25 μmol/L (For 5 μmol/L:F =86.355,16.404,22.898,all P ＜ 0.05;For 25 μmol/L:F =104.321,20.123,52.321,all P ＜ 0.05).The expressions of miR-191 in all exposure groups of 5 and 25 μmol/L (1.392 ± 0.152,1.691 ± 0.167,2.018 ± 0.130 and 1.456 ± 0.167,1.946 ± 0.178,2.259 ± 0.256) were higher than those of control groups (1.001 ± 0.014,1.008 ±0.027,all P ＜ 0.05);the mRNA expressions of TIMP-3 in 48 h exposure group of 5 μmol/L and all exposure groups of 25 μmol/L (0.824 ± 0.093 and 0.897 ± 0.033,0.815 ± 0.089,0.709 ± 0.103) were lower than those of control groups (1.004 ± 0.018,0.997 ± 0.057,all P ＜ 0.05),but there were no significant differences in the mRNA expressions of TIMP-3 between the 12,24 h exposure groups of 5 μmol/L and control group (0.952 ± 0.072,0.929 ± 0.121 vs1.004 ± 0.018,all P ＞ 0.05);the protein expressions of TIMP-3 in all exposure groups of 5 and 25 μmol/L (0.857 ±0.068,0.832 ± 0.106,0.691 ± 0.112 and 0.785 ± 0.097,0.620 ± 0.066,0.453 ± 0.075) were lower than those of control groups (1.006 ± 0.045,1.004 ± 0.078,all P ＜ 0.05).The treatment of miR-191 inhibitor:there were significant differences in the expressions of miR-191 and TIMP-3 protein between different groups (F =104.306,67.015,all P ＜ 0.05).The elevated expression level of miR-191 induced by NaAsO2 was significantly suppressed after transfected with miR-191 inhibitor (0.314 ± 0.094 vs 2.051 ± 0.371,P ＜ 0.05),which in turn up-regulated the protein expression of TIMP-3 (1.965 ± 0.277 vs 0.541 ± 0.183,P ＜ 0.05).Conclusion The expression level of miR-191 is elevated in response to NaAsO2 exposure,and miR-191 has subsequently suppressed the expression of TIMP-3,a potential target of miR-191.

Objective To investigate the effect of sodium arsenite (NaAsO2) on the expression of miRNA-21 (miR-21) mediated by transcription factor ETS-1 in human normal hepatocytes (L-02).Methods Dose-effect study:The L-02 cells were treated with different doses of NaAsO2 [0.0 (control),2.5,5.0,10.0,20.0,40.0 μmol/L] for 24 h.Time-effect study:L-02 cells were exposed to 0 (control) and 20 μmol/L NaAsO2 for 12,24,36 and 48 h (n =6).ETS-1 and miR-21 were treated with ETS-1 shRNA and miR-21 inhibitor,respectively.The cells treated with ETS-1 shRNA (100 nmol/L) were divided into 4 groups:①ETS-1 shRNA NC treatment alone (control group);②ETS-1 shRNA NC combined with NaAsO2 (20 μ,mol/L) treatment group;③ETS-1 shRNA treatment alone group;④Treatment with ETS-1 shRNA and NaAsO2 (20 μmol/L) group.The MiR-21 inhibitor (100 nmol/L) treated cells were also divided into 4 groups:① miR-21 inhibitor NC treatment (control group);② miR-21 inhibitor NC combined with NaAsO2 (20 μmol/L);③miR-21 inhibitor group;④miR-21 inhibitor combined with NaAsO2 (20 μ mol/L) treatment group.The expression of ETS-1 mRNA and miR-21 were detected by quantitative real-time PCR (qRT-PCR);the protein expression of ETS-1 was detected by Western blotting.Results Dose-effect study:The expression of ETS-1 mRNA in the groups of 0.0 (control),2.5,5.0,10.0,20.0 and 40.0 μmol/L was 1.008 ± 0.028,1.552 ± 0.029,1.697 ± 0.050,1.842 ± 0.077,2.233 ± 0.096 and 2.235 ± 0.092;miR-21 expression was 1.025 ± 0.094,1.552 ± 0.072,1.683 ± 0.066,1.915 ± 0.171,2.337 ± 0.195 and 2.592 ± 0.177;the expression of ETS-1 protein was 1.060 ± 0.045,1.267 ± 0.160,1.386 ± 0.087,1.723 ± 0.196,2.208 ± 0.122 and 2.284 ± 0.224,respectively,and the differences were statistically significant (F =47.797,8.959,65.748,all P ＜ 0.05),the NaAsO2 dose groups were significantly higher than those of the control group (all P ＜ 0.05),and there was a dose-effect relationship.Time-effect study:The expression of ETS-1 mRNA in L-02 cells was 1.253 ± 0.175,1.623 ± 0.220,1.771 ± 0.324 and 1.913 ± 0.251,respectively at 12,24,36 and 48 h;the expression of miR-21 was 1.502 ± 0.111,1.716 ± 0.113,1.979 ± 0.186 and 2.452 ± 0.304;the expression of ETS-1 protein was 1.196 ± 0.105,1.502 ± 0.076,1.651 ± 0.074 and 1.839 ± 0.139,respectively,there were significant differences between the groups (F =14.936,39.180,39.441,all P ＜ 0.05).The expression of various time points of exposure to NaAsO2 was significantly higher than those in the control group (1.044 ± 0.115,1.044 ± 0.124,1.108 ± 0.088,1.053 ± 0.061;1.092 ± 0.061,1.096 ± 0.169,1.024 ± 0.111,1.057 ± 0.146;1.020 ± 0.017,1.049 ± 0.121,1.024 ± 0.089,1.031 ± 0.124,all P＜ 0.05),and there was a time-effect relationship.ETS-1 shRNA and miR-21 inhibitor treatment:compared with ETS-1 shRNA NC combined with NaAsO2 (20 μmol/L),ETS-1 shRNA and NaAsO2 (20 μmol/L) could significantly inhibit the expression of ETS-1 (0.912 ± 0.238 vs 1.641 ± 0.225,P ＜ 0.05),and down-regulated the expression of miR-21 (1.313 ± 0.334 vs 2.363 ± 0.252,P ＜ 0.05).There was no significant difference of ETS-1 mRNA expression between miR-21 inhibitor and NaAsO2 (20.μmol/L) group (1.580 ± 0.077 vs 1.576 ± 0.065,P ＞ 0.05) compared with miR-21 inhibitor NC and NaAsO2 (20 μmol/L).Conclusions The expression of ETS-1 and miR-21 in L-02 cells is significantly higher than those in control.The high expression of ETS-1 mediates NaAsO2-induced miR-21 overexpression,which may be an important molecular mechanism of arsenic-induced expression dysregulation of human hepatic miRNAs and liver damage.

Objective To observe the effects of curcumin on hepatic oxidant stress in water arsenic-exposed rats and to study its mechanism,which can offer references for curcumin used in antioxidant therapy of arsenic poisoning.Methods Thirty-two SD rats were divided into 4 groups according to body weight by random number table,half male and half female.Including control group (lavaged 135 days with deionized water),arsenic poisoning group (lavaged 45 days with deionized water after lavaging 90 days with 10 mg/kg sodium arsenite),pure curcumin group (lavaged 135 days with 1 000 mg/kg curcumin solution) and curcumin treatment group (lavaged 45 days with 1 000 mg/kg curcumin solution after lavaging 90 days with 10 mg/kg sodium arsenite),8 rats in each group.The arsenic contents of urine (urine creatinine corrected) and liver were detected by hydride generation inductively coupled plasma optical emission spectrometer (HG-ICP-OES);the activity of Cu/Zn-superoxide dismutase (SOD1) and catalase (CAT),the contents of malondialdehyde (MDA) in serum and liver homogenate by colorimetric method;the protein expression of liver antioxidant enzyme (SOD 1 and CAT) was assayed by Western blotting.Results The arsenic contents of urine and liver in arsenic poisoning group [(5.83 ± 0.29)μg/g Cr,(15.76 ± 1.65)μg/g] and the arsenic contents of urine in curcumin treatment group [(1.07 ± 0.14)μg/g Cr] were obviously higher than those of control group [(0.40 ± 0.14)μg/g Cr,(4.56 ± 1.05)μg/g,all P ＜ 0.05];compared to arsenic poisoning group,the arsenic contents of urine and liver in curcumin treatment group [(1.07 ± 0.14)μg/g Cr,(5.42 ± 1.76)μg/g] were obviously lower (all P ＜ 0.05).The contents of serum and liver SOD1,CAT and MDA in control group respectively were (102.46 ± 5.03),(29.33 ± 8.13)U/ml,(3.11 ± 0.49)μ mol/L and (204.05 ± 18.33),(126.26 ± 13.19)U/mg prot,(1.62 ± 0.42) μmol/g prot.Compared to the control,the activity of serum and liver SOD1 and CAT in arsenic poisoning group [(60.97 ± 7.94),(13.56 ± 5.14)U/ml and (133.66 ± 11.51),(74.01 ± 13.30)U/mg prot] were lower,the contents of MDA [(7.26 ± 0.54)μmol/L and (2.61 ± 0.52)μmol/g prot] were higher (all P ＜ 0.05).Compared to arsenic poisoning group,the activity of serum and liver SOD1 and CAT in curcumin treatment group [(87.39 ± 9.38),(20.45 ± 6.49) U/ml and (178.27 ± 9.32),(93.70 ± 20.35)U/mg prot] were higher,the contents of MDA [(4.34 ± 0.79)μmol/L and (1.92 ± 0.18)μmol/g prot] were lower (all P ＜ 0.05).The protein expressions of SOD1 and CAT in control group respectively were 0.64 ± 0.32 and 0.72 ± 0.31.Compared to the control group,the protein expressions of SOD1 and CAT in pure curcumin group (1.03 ± 0.23,1.02 ± 0.20) were significantly higher (all P ＜ 0.05) and in arsenic poisoning group (0.34 ± 0.12,0.39 ± 0.11) were lower (all P ＜ 0.05);Compared with the arsenic poisoning group,the protein expressions of SOD1 and CAT in curcumin treatment group (0.58 ± 0.09,0.68 ± 0.29) were significantly higher (all P ＜ 0.05).The arsenic content of urine in rats were positively related with arsenic content of liver and the content of MDA [correlation coefficient (r) =0.952,0.732,all P ＜ 0.05],but negativity related with the activity of SOD1 and CAT in liver (r =-0.874,-0.679,all P ＜ 0.05);the activity of SOD1 and CAT and the content of MDA in serum and liver were positively related (r =0.796,0.484,0.607,all P ＜ 0.05),the activity and protein expression of SOD1 and CAT in liver were positively related (r =0.748,0.424,all P ＜ 0.05).Conclusion The curcumin may improve the activity of hepatic antioxidant enzyme in water arsenic-exposed rats and effectively decrease lipid poroxidation damage caused by arsenic via promoting the excretion of arsenic and the protein expression of hepatic antioxidant enzyme.

AIM: To investigate the changes of potassium channels in thoracic aorta of streptozotocin-induced diabetic mouse in the early stage of diabetes mellitus. METHODS: The effects of 60 mmol/L KCl, phenylephrine (PE), sodium nitroprusside (SNP) were measured and concentration-response curves to SNP were determined in the presence and in the absence of the inhibitors of potassium channels on the thoracic aortic rings of diabetic and age-matched control mice in vitro. RESULTS: STZ-diabetic mice showed a significant increase in the maximum contractile response and sensitivity of thoracic aorta to 60 mmol/L KCl and PE. The endothelium-independent relaxation response to SNP was increased by diabetes and were decreased significantly by pretreatment of the vessels with 1 mmol/L tetraethylammonium (TEA), 1 mmol/L 4-aminopyridine (4-AP) and 10 ?mol/L glibenclamide in diabetes thoracic aorta. Only 4-AP decreased relaxation response to SNP in age-matched control mice. The -logIC 50 difference of TEA in thoracic aorta rings of diabetes was significantly higher than age-matched control mice.CONCLUSION: In early stage of diabetes mellitus, the opening or expression of K Ca channels is significantly enhanced.The opening of K ATP channels is also enhanced in this stage.

AIM: To determine EGF contents in human milk, frech cow's milk and cow's milk-based infant formulas and the relationship between EGF content of human milk and neonatal maturity.METHODS: EGF contents in 57 human colostrum from mothers delivering prematurely and at term, 4 different fresh cow's milk and 8 different cow's-milk-based infant formulas with hydrolyzed and non-hydrolyzed proteins were determined by radioimmunoassay (RIA). RESULTS: Human milk from mothers of premature infants had higher EGF content compared to that from mothers of term infants[(28.2?10.3) nmol/L vs(17.3?9.6) nmol/L]. There was a negative correlation between EGF content of human milk and gestational age, birth weight of neonates. The values in fresh cow's milk [(16.6?3.8) nmol/L]were similar to that in human term milk. The contents in non-hydrolyzed protein formulas[(7.5?1.9) nmol/L]were much lower than that in human milk and fresh cow's milk. No immunoreactive EGF was detected in all hydrolyzed protein formulas. CONCLUSION: The occurrence of high EGF concentration in premature milk may represent a maternal compensatory mechanism to accelerate the growth and maturation in immature infants. Lack of EGF in formulas suggests that they may not suitable for those newborns with immature or damaged gastrointestinal tract.