AIM:To observe the change of nitric oxide(NO)and hydrogen sulfide(H_2S)in blood and lung homogenate of hypoxic pulmonary hypertension(HPH)rat model,and to discuss the meaning of inhalation sodium nitrite and these factors in the treatment of HPH. METHODS:Fifty healthy male Wistar rats were assigned randomly into 5 groups(10 rats each):normoxia control group(NC),normoxia sodium nitrite group(NNI),hypoxic control group(HC),hypoxic normal saline group(HNS)and hypoxic sodium nitrite group(HNI). The mean pulmonary arterial pressure(mPAP),weight of right ventricle,weight of left ventricle plus septum,and the ratio of the weight of right ventricle to that of left ventricle plus septum(right ventricle hypertrophy index,RVHI)were also determined. The serum level of NO and plasma level of H_2S were measured,and at the same time the levels of NO in the lung homogenate were detected. The structures in pulmonary arteries were examined using optical microscope. RESULTS:After model established,compared to that in the normoxia groups,the body weight decreased significantly in hypoxia groups(P<0.05),although no difference of body weight in five groups before producing model was observed. Compared to that in normoxia groups,the levels of mPAP and RVHI increased significantly in hypoxia groups,and compared to that in hypoxia control groups and hypoxia normal saline group,mPAP and RVHI levels decreased significantly in hypoxia sodium nitrite group(P<0.05). Compared to that in normoxia groups,the serum level of NO decreased significantly in hypoxia groups(P<0.05). NO level in lung homogenate decreased significantly in hypoxia control group and hypoxia normal saline group as compared to that in normoxia groups(P<0.05),and no obvious difference between hypoxic sodium nitrite group and normoxia groups was found. The plasma level of H_2S was decreased significantly in hypoxia groups(P<0.05)as compared to that in normoxia groups. H_2S level increased significantly in hypoxia sodium nitrite group as compared to that in hypoxia control groups and hypoxia normal saline group(P<0.05). Observation under optical microscope,the lumen structure of lung in normoxia control group was normal. No significant change in normoxia sodium nitrite group was found. The proliferation of smooth muscle cells(SMCs),the collagen fiber deposition in the vessel wall and every caliber thickening was observed in hypoxic control group. The same changes were also observed in hypoxic normal saline group. The thickened caliber was relieved significantly in hypoxic nitrite group. CONCLUSION:Pulmonary hypertension and right ventricle reconstitution can be relieved by inhalation of sodium nitrite,and can be regulated by the level of NO and H_2S in rats. Above all,inhalation of sodium nitrite may degrade HPH directly or by affecting the externalization and synthesizing of gas signaling molecule indirectly.

Objective To investigate the effect of hyperoxia on growth of type Ⅱ alveolar epithelial cells (AECⅡ).MethodsLungs of fetal rats at 19 days of pregnancy were collected,and AEC Ⅱ was isolated and cultured by differential adherence method.Cells were randomly divided into air group and hyperoxia group.In air group,cells were cultured in 5％ CO2 incubator.And cells in hyperoxia group were cultured in 5％ CO2+95％ O2 incubator.The growth,activity,cell cycle,cell apoptosis of AEC Ⅱ were observed at 2,4,6 and 8 days of culture.The interaction between different time and groups were analyzed by ANOVA of factorial design.Comparison of means was done by two-sample independent t test and one-way analysis of variance.Bonferroni correction was used during the comparisons.Results(1) Cell growth situation:in hyperoxia group,cell number was decreased from2 hto 8 h [(7.29±0.43)×105/ml,(2.68±0.37)×105/ml,(0.23±0.10)×105/ml and (0.00±0.00) × 105/ml],and lower than those in air group [(10.41 ± 0.24) × 105/ml,(27.90±1.91) × 105/ml,(27.12±0.85) ×105/ml and (26.29±1.59) × 105/ml](t=10.992,38.912,94.166and 49.696,P=0.000 respectively). (2) Cell activity:the living cells ratio in hyperoxia group at 2 d[(79.00±0.71) ％],4 d [(52.80±1.14)％] and 6 d [(31.60±1.52)％] was lower than those [(97.00±0.71)％,(97.20±0.84)％ and (95.00±0.71)％] ir air group (t=31.213,70.519 and 84.722,P=0.000 respectively).(3) Cell cycle:the cell ratios of G1 phase and S phase in hyperoxia group at day 4 [(66.82±1.20) ％ and (27.31±1.16) ％] and day 6 [(70.22±1.27) ％ and (30.31±1.40) ％] were significantly higher than that at day 2 and that in air group (P＜0.05 respectively).(4) Cell apoptosis:in hyperoxia group,the cell ratio of Annexin-V+/PI- subgroup at 4 h was the highest [(23.89 ± 0.52)％],followed by those at day 2 and 6 [(21.32 ± 0.43)％ and (1.47 ±0.61)％].While the cell ratio of Annexin-V+/PI+ was the highest at 6 h [(53.92± 1.64)％],followed by those at 4 h and 2 h [(45.03±1.01)％ and (12.17±0.60)％],which were all different with those in air group(P＜0.05 respectively).ConclusionsHyperoxia might inhibit cell activity and cell cycle of AEC Ⅱ and promote apoptosis.

Objective To study the influence of alprostadil on the serum adiponectin and leptin in patients with early diabetic nephropathy.Methods 98 patients with early diabetic nephropathy were randomly divided into two groups,the control group(n =48 cases) and the treatment group(n =50 cases).The patients of the control group were treated through the conventional treatment.However,the patients of the treatment group were treated plus alprostadil.They were treated for 21 days.The urinary albumin excretion rate (UAER),adiponectin,leptin and insulin resistance were detected before and after treatment.Results The UAER,adiponectin leptin and insulin resistance were improved after treatment,while those of the treatment group were more significantly improved than those of the control group(P ＜ 0.05 or P ＜ 0.01).Conclusion Alprostadil can improve the conditions of early diabetic nephropathy which is through improving the adiponectin and insulin resistance.

Objective To study the effects of the Notch ligands Dlk1 and recombinant human nucleu factorκB (Jagged1) on the proliferation and transdifferentiation of the type Ⅱ alveolar epithelial cells when the Notch signaling pathway activated.Methods The primary type Ⅱ alveolar epithelial cells (AEC Ⅱ) cultured with recombinant protein Dlk1 and recombinant human nucleu factor-κB (rhNF-κB) (activator of Jagged1),respectively,and then cultured with DMEM (containing 120 mL/L FBS) as controls.Proliferation and differentiation conditions of the AEC Ⅱ were observed at 48 h,72 h,96 h time point by the light microscope and electron microscopes separately.Cell number was counted with hemacytometer; the proliferation rate was measured by methyl thiazolyl tetrazolium (MTT) ; Immunofluorescence double standard method was used to detect the AEC Ⅱ specific surfactant protein C (SP-C) and AEC Ⅰ specific protein aquaporin5 (AQPS) ;the expression of SP-C,AQPS,Dlk1,Jagged1,Notch1 and Hes1 mRNA were detected by real time-PCR.Results The cell population and proliferation:compared with control group,AEC Ⅱ proliferation was promoted in the Dlk1 group [cell numbers (× 109/L) 9.05 ± 0.45 vs 7.95 ± 0.65,11.68 ± 0.43 vs 8.68 ± 0.52,11.55 ± 0.17 vs 8.73 ± 0.48,all P ＜ 0.05 ; MTT results (value A) 0.699 ± 0.050 vs 0.462 ± 0.080,0.912 ± 0.080 vs 0.535 ±0.040,0.726 ±0.050 vs 0.540 ±0.020,all P ＜0.05] and decelerated AEC Ⅱ transdifferentiation into AEC Ⅰ ; while AEC Ⅱ proliferation was inhibited in rhNF-κB group [cell numbers (× 109/L) 4.95 ± 0.33 vs 7.95 ± 0.65,4.73 ±0.71 vs 8.68 ± 0.52,4.04 ± 0.11 vs 8.73 ± 0.48,all P ＜ 0.05; MTT results (value A) 0.398 ± 0.030 vs 0.462 ± 0.080,0.402 ± 0.070 vs 0.535 ± 0.040,0.380 ± 0.110 vs 0.540 ± 0.020,all P ＜ 0.05] and accelerated AEC Ⅱ transdifferentiation into AEC Ⅰ.One-Way ANOVA showed that the difference among the 3 groups had statistical significance (cell numbers:F =486.73,P =0.02; cell proliferation:F =37.16,P =0.02).The mRNA expression:compared with control group,the expression of SP-C mRNA of Dlk1 group was significantly higher (P ＜ 0.05) while the expression of AQP5 mRNA was remarkably lower and delayed (P ＜ 0.05),the expression of Jagged1 mRNA was weak or little,Dlk1 and Notch1 mRNA were up-regulated (P ＜ 0.05),and the Hes1 mRNA was reduced (P ＜ 0.05) ; the expression of SP-C mRNA of rhNF-κB group was significantly reduced (P ＜ 0.05),while the AQP5 mRNA expressed ahead of time and increased (P ＜ 0.05),Jagged1,Hes1 and Notch1 mRNA were higher (P ＜ 0.05),and the Dlk1 mRNA was weak.One-Way ANOVA showed that the difference in the expressions of SP-C,AQP5,D1k1,Jagged1,Hes1 and Notch1 mRNA among the 3 groups had staistical significance (F =96.80,P =0.01 ; F =82.55,P =0.01 ; F =269.80,P=0.00;F =312.34,P =0.00;F =169.17,P =0.01;F =19.85,P =0.02).Conclusions There are varied effects on proliferation and differentiation of the AEC Ⅱ when the Notch signaling is activated by different ligands:Dlk1 promoted proliferation and inhibited differentiation,while Jagged1 inhibited proliferation and promoted transdifferentiation.

Background and purpose:The pro-oncogene Bmi-1 is a member of the polycomb-group family, can regulation of the proliferation and self-renewal of normal and tumor stem cells. In recent years, Bmi-1 has been found that it is overexpressed in varieties of human malignant tumors. The study aimed to observe the effects of Bmi-1-siRNA on the growth capacity of lung cancer cell line A549 in vivo and in vivo, and explore its mechanism. Methods:The most effective one as a target sequence was chosen from four Bmi-1 siRNA sequences which were designed by our lab, and one random sequence was chosen as a negative control. In short, the chemically synthesized siRNA and control sequences were connected to a retrovirus expressing vector, pSUPERretro-Neo plasmid, and then transfected into A549 cells. The stably transfected cells were cultured and passed. The level of mRNA and protein of Bmi-1 in A549 cells were assessed by RT-PCR and Western blot respectively. The proliferations of A549 cells in vivo was analyzed with MTT, trypan blue exclusion and plate colony forming methods. Flow cytometry was used for cell cycle analysis. The potency of tumorigenesis was observed in nude mouse through hypodermic inoculation of A549 cells. The expressions of cyclin D, p21/27, p-AKT and PTEN were analyzed by Western blot. Results:Compared to A549-ctr and A549-wt cells, Bmi-1 mRNA and protein levels all signiifcantly reduced in A549-Bmi-1-siRNA cells. Bmi-1-siRNA inhibited the growth, colony formation in vitro and tumorigenesis in vitro of A549 cells, and the interference cells cell cycle arrested in G1 phase. In A549-Bmi-1-siRNA cells, p-AKT and cyclinD1 expression were down-regulated while p21/p27 and PTEN were up-regulated. Conclusion:Silencing Bmi-1 gene inhibits the proliferation of A549 cells through G1 phase arrest, which involves the downregulation of cyclin D/p-AKT and upregulation of p21/p27/PTEN.

Background and purpose:The human oncogene B-cell-speciifc moloney murine leukemia virus integration site 1 (Bmi-1) is an important member of the polycomb group family, and it regulates cell proliferation and senescence via INK4a/ARF locus. This study investigated the effects of Bmi-1-siRNA on the proliferation of lung adenocarcinoma cell line SPC-A1 cells with INK4a/ARF locus and clarify the mechanism of Bmi-1-mediated effect on proliferation of lung adenocarcinoma cells. Methods:In this study, we chose the most efifcient siRNA chain the pGeneshl-2-Bmi-1 sense-1 and inserted into a pSUPER-retro-neo retroviral vector. The packaged si-Bmi-1 pSUPERret-ro-neo retroviral vector was stably transfected into lung adenocarcinoma SPC-A1 cell line. The stably transfected cells were cultured and passed. After transfection, the levels of Bmi-1 mRNA and protein expression of SPC-A1 cells were analyzed by RT-PCR and Western blot respectively. Trypan blue, MTT and plate colony forming assay were performed to observe the proliferation capibility of SPC-A1 cells and evaluate the cloning forming ability in vitro. The potency of tumorigenesis was observed in nude mouse through hypodermic inoculation of SPC-A1 cells. Cell cycle distribu-tion was analyzed by lfow cytometry (FCM) in SPC-A1 cells. The expression levels of proliferation proteins including p16INK4a, p53, Cyclin D1, PTEN, Akt and Ser473p-Akt were analyzed by Western blot. Results:The mRNA and protein expression levels of Bmi-1 were signiifcantly reduced in SPC-A1-Bmi-1-siRNA cells transfected with pSUPER-retro-neo retroviral vector. Knockdown of Bmi-1 could inhibit the growth, colony formation in vitro and tumorigenesis in vivo of SPC-A1 cells (P<0.01). The transfected SPC-A1 cells were arrested in G1 phase [(64.6±1.2)%, P<0.05]. Compared with two control groups, p16INK4a, p53 and Akt were not affected (P>0.05), while Cyclin D1 and Ser473p-Akt were downregulated (P<0.01) and PTEN was up-regulated (P<0.01) in the SPC-A1-Bmi-1-siRNA cells. SPC-A1-Bmi-1-siRNA cells were treated with various concentrations of PTEN inhibitor to determine expression levels of PTEN, Bmi-1 and Ser473p-Akt protein. Ablation of PTEN rescued Bmi-1 and Ser473p-Akt expression in SPC-A1-Bmi-1-siRNA cells. Conclusion:Knockdown of Bmi-1 gene can arrest the proliferation of SPC-A1 cells through G0/G1 phase arrest by inhibiting Cyclin D1 expression indirectly, which may be not associated with p16INK4a signaling pathway.

Objective To evaluate the correlation between diffusion tensor imaging(DTI)measurements,fiber tracking(FT)and the clinical symptoms in patients with cervical spondylotic myelopathy.Methods According to the Japanese orthopaedics association score(JOA),104 patients with cervical spondylopathy were divided into 4 groups:mild in 31 patients with 13-16 scores,moderate in 27 with 9-12 scores,severe in 25 with 5-8 scores,and serious in 21 with 0-4 scores.According to the lesion signal characters,all patients were divided into 3 groups:Group A with normal signal in both T1 WI and T2WI in 33 patients,Group B with normal signal in T1WI but high signal in T2WI in 30 patients,and Group C with low signal in T1 WI and high signal in T2WI in 41 patients.Apparent diffusion coefficient (ADC),fractional anisotropy(FA),λ1,λ2,λ3 were measured in the spinal cord at the serious pressed section,and fiber tractography was performed.The Spearman correlation analyses was used to correlate each of the DTI measurement with JOA score.Group difference was tested with one-way ANOVA method.Results High quality of DTI was acquired in all patients.The FA values in the mild,moderate,severe,and serious groups were respectively 0.69 ±0.13,0.58 ±0.03,0.46 ±0.08,and 0.37 ±0.11 and significant difference was found in different groups(F =100.59,P ＜ 0.05)and positively correlated with JOA scores (r =0.883,P ＜ 0.05).There was no statistical significance between JOA scores and ADC,λ1,λ2,λ3(r=0.232,0.217,0.113,0.127,P ＞0.05).The FA values in group A,B,and C were respectively 0.67 ±0.33,0.51 ±0.21,0.38 ±0.03,and significant difference was found among different groups(F =50.05,P ＜ 0.05).Decrease of JOA score and high signal in T2 companied with decrease of FA value.Decrease of FA values was found associated with increase of fiber bundle damage.The ADC,λ2,λ3 but not λ1 were significantly different among the JOA groups and the group A,B,and C.Conclusions The FA values are positively correlated with clinical symptoms.Decrease of FA values is found associated with increase of fiber bundle damage.DTI can show the severity and extent of damage of spinal cord in patients with cervical spondylotic myelopathy.

AIM:To observe the expression of calcium-sensing receptor(CaSR) of rat cardiomyocytes in anoxia-reoxygenation(A/R) injury and CaSR-induced changes of intracellular calcium;involvement of CaSR in apoptosis and relevant signaling transduction pathway.METHODS:The A/R injury was remodeled in vitro;CaSR,caspase 3 and caspase 9 were deteced by Western blotting.LSCM was used to observe changes of intracellular calcium during reperfusion with or without CaSR agonist.Morphological changes in different groups were observed by light microscopes.Apoptotic cells were measured by TUNEL assay.RESULTS:By LSCM,it was found that the intracellular calcium was significantly increased during reperfusion both in A/R and activator group.Severe injury was found by HE staining in the above two groups,the number of apoptotic cells was significantly increased according to TUNEL assay.The expression of CaSR,caspase 3 and caspase 9 was significantly increased in A/R group and activator group compared with control.CONCLUSION:CaSR is involved in intracellular calcium overload in A/R cardiomyocyte,which can accelerate apoptosis during A/R.

Objective: To investigate the clinical value of quantitative detection of DNA aneuploidy in the diagnosis and treatment of cervical lesions in middle-aged and senile women. Methods: A total of 1 404 middle-aged and elderly women who underwent screening for early cervical lesions were retrospectively studied.Patients were divided into the two groups: the 40-49 years old group(n=897)and the 50-78 years old group(n=507). Cervical lesions were screened by DNA ploidy analysis and the results were compared with those screened by liquid-based cytology, colposcopy and high-risk human papillomavirus(HPV). Results: The positive detection rate of HPV by DNA ploidy analysis was 54.4%(764/1 404). Of 1 404 patients, HPV16/18 infection accounted for 21.3%(299/1 404). The detection rate of heteroploid cells was 50.92%(715/1 404). There was a significant positive correlation between HPV infection type and cervical epithelial cell ploidy changes(r=870, P=0.001). The detection rate of HPV by liquid-based cytology was 45.08%, which was lower than that by DNA aneuploidy(χ²=9.594, P=0.002). The differences in the incidences of low-grade squamous intraepithelial lesion(LSIL)and high-grade squamous intraepithelial lesion(HSIL)and above categories of lesions were statistically significant(χ²=289.598, P=0.000)between patients with and without DNA aneuploidy.The statistically significant differences were found between the 40-49 years old group and 50-78 years old group(P<0.05)in the occurrence of abnormal DNA ploidy cells, HPV infection rate, the proportion of LSIL, HSIL and above categories of lesions. Conclusions Compared with the conventional cytology, DNA aneuploidy quantitative detection has higher sensitivity and better specificity, and has no significant difference from the high-risk HPV detection.It can be used as one of methods for screening cervical lesions in middle-aged and elderly women, especially those with high-risk HPV infection.

Objective To evaluate the effect of acarbose on the circulating concentration of inflammatory factors in patients with type 2 diabetes mellitus(T2DM). Methods Total 118 patients with T2DM who did not take acarbose before enrollment within 4 weeks were recruited by a randomizing formula into 2 groups ( group A and B). 57 healthy subjects were included into group C as control. After excluding those of inadequate samples, 57 patients with T2DM were enrolled into group A in which acarbose was prescribed 50 mg three times a day, 59 patients with T2DM were enrolled into group B in which acarbose was not given and other hypoglycemic approaches were similar to group A. Serum samples at the time of enrollment and at the end of 4 weeks intervention were collected and stored in refrigerator at -80℃ until analysis. Analysis of biochemical indexes was performed in central lab of the institution,inflammatory factors were determined with commercial ELISA kits. Results (1) The metabolic indexes were significantly decreased after intervention in two diabetic groups. (2) The baseline levels of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α( TNF-α), prothrombin activator inhibitor-1 (PAI-1), lipopolysaccharide (LPS), high sensitive C reactive protein ( hs-CRP), and advanced glycation index (AGI) in diabetic patients were significantly higher than in group C MCP-1 [( 463.4± 187.1 vs 267.1 ± 158.3 ) pg/ml, TNF-α( 12.07 ± 19.59 vs 4.18 ±3.03 ) pg/ml, PAI-1 ( 2.47 ± 1.87 vs 1.38 ± 2.37 )ng/ ml, LPS ( 130.6 ± 128.5 vs 29.39 ± 17.93 ) pg/ml, hsCRP(4.25 ±2.29 vs 2.11 ± 1.07 ) μg/ml, AGI (3.78 ± 2.61 vs 0. 74 ± 0. 15 ) AU, all P ＜ 0. 05]. (3) Repeated measurement ANOVA analysis showed that after four weeks of intervention, MCP-1 [F( 1,106 ) = 19. 830, P＜0.001],LPS[F(1,106)=7.815, P＜0.01], PAI-1 [F(1,106)= 7.792, P＜0.01], TNF-α[F(1,106=24. 656, P=0.001 )] ,AGI[F( 1,106)= 12. 971 ,P=0. 01] decreased significantly in group A than in group B. Although hsCRP decreased in both group A and group B, but the trend was not different [F( 1,102 )= 0. 915, P = 0. 342].Conclusion The levels of inflammatory factors were elevated in patients with type 2 diabetes mellitus, which could be mostly reduced by acarbose.