Objective:To observe the effects of pyrroloquinoline quinine (PQQ) on the mitochondrial function and cell survival of rat bone marrow mesenchymal stem cells (BMSCs) under oxidative stress, and to explore its mechanism.Methods:BMSCs of rats were cultured in vitro with Dulbecco′s minimum essential medium/F12 medium containing fetal bovine serum in the volume fraction of 10% (hereinafter referred to as normal medium). The rat BMSCs of third to fifth passages in logarithmic growth phase were selected for the following experiments. (1) The cells were divided into normal control group, normal control+ PQQ group, hydrogen peroxide (H 2O 2) alone group, and H 2O 2+ PQQ group. The cells in normal control group were cultured in normal medium for 24 hours; the cells in normal control+ PQQ group were cultured in normal medium containing 100 μmol/L PQQ for 24 hours; the cells in H 2O 2 alone group were cultured in normal medium containing 200 μmol/L H 2O 2 for 24 hours; the cells in H 2O 2+ PQQ group were pre-incubated with normal medium containing 100 μmol/L PQQ for 2 hours, and then with H 2O 2 added to the concentration of 200 μmol/L and cultured for 24 hours. The cell morphology of each group was observed under the inverted phase contrast microscope, and the cell survival rate was detected by cell count kit 8 method. (2) Five batches of cells were collected, and the cells of each batch were divided into normal control group, H 2O 2 alone group, and H 2O 2+ PQQ group. The cells in each group received the same treatment as that in the corresponding group of experiment (1). After 24 hours of culture, one batch of cells was collected for apoptosis detection by flow cytometry, and the apoptosis rate was calculated. One batch of cells was subjected to mitochondrial membrane potential assay and JC-1 fluorescent staining observation using the JC-1 mitochondrial membrane potential detection kit and the inverted phase contrast fluorescence microscope, respectively. One batch of cells was collected for mitochondrial morphology observation under the transmission electron microscope. One batch of cells was subjected to catalase (CAT) and superoxide dismutase (SOD) activity assay by CAT activity assay kit and SOD activity assay kit, respectively. One batch of cells was subjected to Western blotting for determination of protein level of Epac1, adenine monophosphate activated protein kinase (AMPK), phosphorylated AMPK, cysteinyl aspartate-specific proteinase 3 (caspase-3), and cleaved caspase-3, and the phosphorylation level of AMPK and cleaved caspase-3/caspase-3 ratio were calculated. Six replicates were measured in each group for each index except for morphological observation. Data were statistically analyzed with one-way analysis of variance and independent sample equal variance t test. Results:(1) After 24 hours of culture, compared with those in normal control group (the cell survival rate was set to 100.0%), there was an increase in cell vacuole and a decrease in cell number in H 2O 2 alone group, and the cell survival rate was significantly reduced to (74.3±2.9)% ( t=6.39, P<0.01). Compared with those in H 2O 2 alone group, the cell morphology of H 2O 2+ PQQ group was significantly improved, and the cell survival rate was significantly increased to (116.9±4.2)% ( t=6.92, P<0.01); the cell survival rate in normal control+ PQQ group was (101.2±1.1)%, close to that of control group ( t=1.06, P>0.05). (2) After 24 hours of culture, compared with (13.6±1.0)% in normal control group, the apoptosis rate of cells in H 2O 2 alone group was significantly increased to (37.1±2.0)% ( t=10.57, P<0.01). Compared with that in H 2O 2 alone group, the apoptosis rate of cells in H 2O 2+ PQQ group was significantly declined to (17.0±0.7)% ( t=9.49, P<0.01). (3) After 24 hours of culture, compared with those in normal control group, the mitochondrial membrane potential of cells in H 2O 2 alone group was depolarized, the JC-1 fluorescent dye mainly existed in the cytoplasm in the form of monomer, which emitted green fluorescence, and a significant decrease in mitochondrial membrane potential was shown ( t=4.18, P<0.01). Compared with those in H 2O 2 alone group, the mitochondrial membrane potential of cells in H 2O 2+ PQQ group was increased to normal level ( t=4.43, P<0.01), and the JC-1 fluorescent dye accumulated in mitochondria following the polarized mitochondrial membrane potential and emitted red fluorescence. (4) After 24 hours of culture, compared with that in normal control group, the mitochondrial structure of cells in H 2O 2 alone group was disordered, with disappeared mitochondrial cristae and decreased mitochondrial matrix density. Compared with that in H 2O 2 alone group, the mitochondrial structure of cells in H 2O 2+ PQQ group was regular and intact, with clearly visible mitochondrial cristae and increased mitochondrial matrix density. (5) After 24 hours of culture, compared with those in normal control group, the CAT activity of cells in H 2O 2 alone group was significantly increased ( t=4.54, P<0.05), and the SOD activity was significantly decreased ( t=3.93, P<0.05). Compared with those in H 2O 2 alone group, the CAT activity of cells in H 2O 2+ PQQ group was obviously increased ( t=8.65, P<0.01), while there was no significant change in the SOD activity ( t=0.72, P>0.05). (6) After 24 hours of culture, compared with those in normal control group, the protein expression of Epac1 of cells in H 2O 2 alone group was significantly decreased ( t=4.67, P<0.01), while the AMPK phosphorylation level and the cleaved caspase-3/caspase-3 ratio were significantly increased ( t=7.88, 3.62, P<0.01). Compared with those in H 2O 2 alone group, the protein expression of Epac1 and the AMPK phosphorylation level of cells in H 2O 2+ PQQ group were both significantly increased ( t=4.34, 16.37, P<0.01), while the cleaved caspase-3/caspase-3 ratio was significantly declined ( t=3.17, P<0.05). Conclusions:Pretreatment with PQQ can improve the mitochondrial function, reduce cell apoptosis rate, and enhance cell survival rate of rat BMSCs under oxidative stress, which may be related to the up-regulation of Epac1 protein expression, activation of AMPK signaling pathway, and down-regulation of cleaved caspase-3 protein level.

Objective To observe Hedgehog signaling pathways of liver cancer cell growth and the influence of the metastatic potential targeted inhibit Hedgehog.Methods Construction of Smo shRNA plasmid,The stable and low-expressed Smo-expressing HCC QGY-7701 cell line was screened after lipofection.The stable and low-expressed Smo-expressing HCC QGY-7701 cell line was screened,The cell cycle,apoptosis,invasion and metastasis of QGY-7701 cells were detected by Western blot,flow cytometry,CCK8 and transwell assay.Subcutaneous implantation of hepatocarcinoma cells in nude mice.Study on the growth and metastasis of hepatocarcinoma cells with low expression of Smo in.The ultrastructural changes of hepatoma cells with low expression of Smo were observed under electron microscope.Results RT-PCR and Western blot showed stable shR-Smo cell line was successfully constructed.Cell cycle test showed that compared with the control group,G0/G1 cells increased in shR-Smo,cells in S phase decreased;apoptosis,CCK8 and Transwell tests showed that Smo-gene silencing could significantly increase the apoptosis percentage of the hepatic cancer cells to (5.46％ ± 1.46％),proliferation activity decreasedand and the migration rates reduced to (7.82％ ±2.14)％;nude mice model showed that Smo-gene silencing could inhibit the growth of hepatocellular carcinoma cells in vivo,electron microscopy revealed that lysosomes increased significantly in Smo-gene silence cells.Conclusions Blocking Hh signaling pathways,liver cancer cells in vitro malignant degree of decline.Hedgehog in treating liver cancer have hidden meaning.

Objective To analyze the relationship between tumor top,tumor neck,tumor rupture point of intraoperative aneurysm rupture (IAR) and the prognosis in patients with anterior circulation aneurysms (ACA) by craniotomy clipping.Methods Clinical materials of 123 patients with ACA (135 aneurysms) by craniotomy clipping were analyzed retrospectively,including 40 patients had IAR (41 aneurysms) during operation.The general informations of IAR patients were analyzed.Prognostic factors of IAR were analyzed by single and multiple factor analysis.Results The sex,age,history of hypertension,aneurysm diameter,the use of temporary blockage and blocking time,intraoperative blood loss showed no significant influences on the prognosis of patients with IAR (P ＞0.05),while preoperative Hunt-Hess classification and intraoperative rupture were the influencing factors for the prognosis of IAR patients (P ＜ 0.01).The preoperative Hunt-Hess classification and intraoperative rupture were the independent risk factors for the prognosis of IAR patients (P ＜0.01).Conclusion In craniotomy clipping,the different aneurysm rupture points can significantly affect the prognosis of patients with ACA,and aneurysm neck rupture is a main factor for poor prognosis of patients.

Objective To analyze the relationship between tumor top,tumor neck,tumor rupture point of intraoperative aneurysm rupture (IAR) and the prognosis in patients with anterior circulation aneurysms (ACA) by craniotomy clipping.Methods Clinical materials of 123 patients with ACA (135 aneurysms) by craniotomy clipping were analyzed retrospectively,including 40 patients had IAR (41 aneurysms) during operation.The general informations of IAR patients were analyzed.Prognostic factors of IAR were analyzed by single and multiple factor analysis.Results The sex,age,history of hypertension,aneurysm diameter,the use of temporary blockage and blocking time,intraoperative blood loss showed no significant influences on the prognosis of patients with IAR (P ＞0.05),while preoperative Hunt-Hess classification and intraoperative rupture were the influencing factors for the prognosis of IAR patients (P ＜ 0.01).The preoperative Hunt-Hess classification and intraoperative rupture were the independent risk factors for the prognosis of IAR patients (P ＜0.01).Conclusion In craniotomy clipping,the different aneurysm rupture points can significantly affect the prognosis of patients with ACA,and aneurysm neck rupture is a main factor for poor prognosis of patients.

Objective: To investigate the effect of AKT1 deSUMOylation induced by Ubc9 silencing on the proliferation and metastasis of hepatocellular carcinoma (HCC) cells. Methods: The Ubc9 gene was silenced using RNA interference, and the expression levels of Ubc9, SUMO1 and AKT1 protein were detected by Western blot. Cell proliferation and cell cycle was analyzed by MTT and flow cytometry. Wound healing and transwell assays were used to detect the cell migration ability. Furthermore, the xenograft model was established, and tumor growth curves were drawn. The in situ apoptotic rates was measured using TUNEL Apoptosis Assay. The expression of proliferating cell nuclear antigen (PCNA), matrix metalloproteinase (MMP)-2 and MMP-9 were evaluated by immunohistochemical staining. Results: Knockdown of Ubc9 gene significantly decreased the protein expression levels of Ubc9, conjugated SUMO1, free SUMO1 and AKT1 in HCC cells (P<0.05 for all). In control, siR-neg and siR-Ubc9 groups, the cell proliferation indexes were 53.19%, 54.25% and 39.17%, respectively. Moreover, cell migration distance and migrating cells per low power field for all these three groups were (59.47±4.66) μm and 89.44±8.36, (56.56±5.37) μm and 93.84±8.79, as well as (34.57±6.61) μm and 41.67±5.39, respectively. In the xenograft model, the weights of subcutaneous tumors for these three groups were (3.78±0.69) g, (3.72±0.72) g and (2.09±0.61) g, respectively. The corresponding apoptotic cell rates were (7.79±2.21)%, (6.45±2.48)% and (33.59±5.44)%, respectively. The expression levels of PCNA, MMP-2 and MMP-9 protein were significantly decreased in siR-Ubc9 group (P<0.05). Conclusions Ubc9 silencing in HCC cells induces AKT1 deSUMOylation, and then inhibits the proliferation and metastasis. These results provide a new therapeutic strategy for liver cancer in the future.

Objective To determine the expression and distribution of Disabled-2(Dab2) in normal human adrenal glands, and further to study the expression of Dab2 in tissues of different adrenocortical adenomas, and to elucidate whether Dab2 can be a specific molecular marker in the pathology of primary aldosteronism. Methods Real-time PCR and immunohistochemical staining were used to detect Dab2 expression in 10 aldosterone-producing adenoma (APA) samples, 8 cortisol-producing adenoma ( CPA) samples, 8 non-functioning adenoma ( NFA) samples and 6 normal adrenal samples. Results Immunohistochemical staining showed that Dab2 was significantly highly expressed in zona glomerulosa of normal human adrenal glands. Sporadical cluster of ZG cells with moderate Dab2 staining were demonstrated in APAs. In all CPA and NFA tumors, weak dab2 staining was detected. According to the results of real-time PCR, Dab2 mRNA expression was increased significantly in APAs compared with normal adrenal glands. There was no significant difference between normal adrenal glands, CPAs, and NFAs in regard to Dab2 mRNA expression. Compared to nontumor portions, APAs also showed higher Dab2 mRNA expression in the tumor( P<0. 05). Conclusion Dab2 was predominantly localized in zona glomerulosa in normal adrenal gland. Increased Dab2 mRNA expression was detected in APAs compared with normal adrenal glands. Whereas, Dab2 protein expression was just moderate increased in APAs. Weather Dab2 can be a specific molecular marker in the pathology of primary aldosteronism has to be further studied.

Objective Previous study found TGF-βpathway might be the molecular pathway influencing the prognosis of colo-rectal cancer, while it was uncertain whether Chinese population is associated with the disease.The article was to evaluate the genetic factors associated with prognosis in colorectal cancer. Methods 52 cases patients with colorectal cancer were followed-up for 36 months in our hospitals from January 2013 to August 2014.Their DNAs were extracted and stored and gene typing were carried out in 5 candidate genes to detect the association between SNPs and the prognosis in colorectal cancer. Results The results showed that within the TGF-βsignaling pathway, after adjusting for Bonferroni multiple testing, allele A of SNP rs10749971 located in gene POU2AF1 was associated with the recurrence of patients with stage III disease under additive and recessive genetic models ( HR =1.968, P=0.004;HR=2.174, P=0.010).Allele C of SNP rs961253 in the gene BMP2 could increase the recurrence risk (HR=1.992, P=0.005) and the death risk (HR=3.161, P=0.007) of patients with stage III disease under recessive genetic models.Allele A of SNP rs4464148 in SMAD7 gene could significantly decrease the death risk of patients with stage II and III colorectal cancer under dominant genetic model (HR=0.382, P=0.017;HR=0.230, P=0.006).In addition, accumulated effects of several adverse genes showed gene high risk group could increase the risk of death for patients with stage III colorectal cancer significantly ( HR=15.512, P=0.036;95%CI:1.611-149.360). Conclusion In different genetic models, SNP locus mutation within gene POU2AF1, BMP2 and SMAD7 on TGF-βpathway was associated with the prognosis of patients with colorectal cancer.With the increase of the number of unfavorable genes, the death risk increases accordingly.

Objective:To explore the benefit effects of ?-3 polyunsaturated fatty acids on bone biomechanics in ovariectomized(OVX) rats.Methods: 60 virgin female Sprague-Dawley rats were randomized by the stratified weight method into 6 groups: ① Sham group:deionized water 2 mL/d;②OVX group:deionized water 2 mL/d;③E2 group: OVX +17?-ethinylestradiol 0.1 mg/(kg?d);④ A group: OVX+?-3 PUFAs 75 mg/(kg?d);⑤B group: OVX+?-3 PUFAs 150 mg/(kg?d);⑥ C group: OVX+?-3 PUFAs 300 mg/(kg?d).Each group had 10 rats.After feeding for 90 days,the rats were killed and femur were separated.The maximum load,maximum deformation,elastic load,elastic deformation and energy absorption were measured from three point bending test.Bone wet weights were measured.Result: The femur biomechanics markers of OVX group decreased obviously,while that of all medicated group increased obviously(P

Objective:To study the early postoperative nutrition support of the liver transplantation.Methods:The early intake and nutrition were analysed in 44 patients with liver transplantation . The patients were divided into group A (22 patients , high amount of nitrogen inake ) and group B ( 22 patients , low amount of nitrogen intake ). The changes of the nitrogen balance,body weight, serum albumin, hemoglobin, serum lipids, renal function and liver function were evaluated.Results:There were no significant difference in body weight, serum albumin, hemoglobin, liver function between the two groups. The nitrogen balance of group A was better than that of group B,while the renal function of group B was better than that of group A.Conclusion:The amount of caloric and nitrogen intake in early postoperative stage of liver transplantation should be controlled.

Objective: To study the effect of ?-3 polyunsaturated fatty(?-3 PUFA) on the serum lipid of ovariectomized rats.Methods: 60 virgin female Sprague-Dawley rats,3 months of age,were randomized by the stratified weight method into 6 groups: Sham group:deionized water 2 mL/d,OVX group:eionized water 2 mL/d,E2 group: OVX+17?-ethinylestradiol 0.1 mg/(kg?d),A group: OVX+?-3 PUFA 75 mg/(kg?d),B group: OVX+?-3 PUFA 150 mg/(kg?d) and C group: OVX+?-3 PUFA 300 mg/(kg?d).Each group had 10 rats.Experiment period was 90 days.Body weight were measured every week.Serum were collected to measure Cholesterol(TC),Triglyceride(TG),High-density lipoprotein-cholesterol(HDL-C),and Low-density lipoprotein-cholesterol(LDL-C).Results: There were significant difference between groups in the serum lipid.In the OVX group,TC and LDL-C increased.In the E2 group,TC increased compared with that in the Sham group,and HDL-C,LDL-C and HDL-C/TC decreased significantly compared with that in the OVX group.In the A,B and C groups HDL-C/TC increased significantly compared with those in the E2 group.Conclusion: The ovariectomized rats have lipid metabolism disorderd.The ?-3 PUFA and estrogen replacement therapy plays a role in the lipid metabolism.The ?-3 PUFA has the best efficiency in reducing the level of serum lipid.