Objective To observe the changes of the cells of human hepatocellular carcinoma (HepG2)using RNA for silencing the expression of COMMD7 gene,and investigate related mechanism of COMMD7 gene promoting HepG2 proliferation.Methods COMMD7 gene shRNA was designed and constructed into COMMD7-shRNA plasmid.HepG2 cells were divided into the HepG2 group,control-shRNA group (empty vectors were infected) and COMMD7-shRNA group (positive vectors were infected).Cells shapes were observed by fluorescence microscope after infecting.The expression of COMMD7 and expression and phosphosylation of extracellular regulated protein kinase1/2 (ERK1/2) and MEK1/2 protein were measured by Western blot.The cell vitality was measured by cholecystokinin octapeptide (CCK-8),and the apoptosis of cell was detected by flow cytometry.The measurement data with normal distribution were presented as (x) ± s.The comparisons among groups were evaluated with the one-way ANOVA,and pairwise comparison was analyzed by the LSD-t test.Results The cells were oval or spindle shapes and displayed green fluorescent after infected successfully.The results of Western blot showed that the relative quantitative expression of COMMD7 protein in the HepG2 group,control-shRNA group and COMMD7-shRNA group were 0.90 ±0.18,1.03 ±0.05 and 0.23 ±0.03,respectively,with a significant difference among the 3 groups (F =152.08,P ＜ 0.05),and the expression of COMMD7 protein in the COMMD7-shRNA group was significantly lower than those in the other 2 groups (t =20.74,21.16,P ＜ 0.05).The results of CCK-8 showed that the scores of the HepG2 vitality in the HepG2 group,control-shRNA group and COMMD7-shRNA group were 1.193 ±0.024,1.225 ±0.034 and 1.147 ±0.021,respectively,with a significant difference among the 3 groups (F =6.90,P ＜ 0.05),and the HepG2 vitality in the COMMD7-shRNA group was significantly lower than those in the other 2 groups (t =3.53,3.69,P ＜ 0.05).The results of flow cytometry showed that the apoptosis rate of HepG2 in the HepG2 group,control-shRNA group and COMMD7-shRNA group were 6.1％ ± 0.3％,7.8％ ± 0.5％ and 20.9％ ± 1.4％,showing a significant difference among the 3 groups (F =270.80,P ＜0.05),and the apoptosis rate of HepG2 in the COMMD7-shRNA group was significant higher than those in the other 2 groups (t =21.77,19.36,P ＜0.05).The results of Western blot showed that the relative quantitative expression of phosphorylation (p)-ERK1/2 and p-MEK1/2 in the HepG2 group,control-shRNA group and COMMD7-shRNA group were 0.932 ±0.046,0.945 ±0.017,0.553 ±0.052 and 0.452 ±0.031,0.468±0.027,0.263 ± 0.022,respectively,showing significant differences among the 3 groups (F =93.61,49.16,P ＜ 0.05),and the relative quantitative expression of p-ERK1/2 and p-MEK1/2 in the COMMD7-shRNA group were significantly lower than those in the other 2 groups (t =11.94,12.17,9.33,8.65,P ＜ 0.05).Conclusions COMMD7 gene can promote HepG2 proliferation via activating ERK/mitogen-activated protein kinase (MAPK) signaling pathway,and its mechanism may be promoting the phosphorylation of expression of ERK1/2 and MEK1/2.

Objective To explore the 3-D anatomical structure of blood vessel around the pancreas and its clinical significance.Methods Fifty objects were scanned by 64-slice CT of GE,the artery,portal vein,spleen vein,superior mesenteric vein and inferior mesenteric vein were 3-D reconstructed by Myrian system,and the scientific data were recorded.Results The artery,portal vein,spleen vein,superior mesenteric vein and inferior mesenteric vein were all reconstructed;the length of portal vein was(44.28±10.23)mm and the length of post-pancreas trunk was(32.13±7.08)mm;the angle between portal vein and spleen vein were classified into three types:the angle of type Ⅰ was<90°,angle of type Ⅱ was=90°,and angle of type Ⅲ was>90 °.30％of the inferior mesenteric vein fed into the spleen vein,56％fell into the superior mesenteric vein and 14％came into the the meeting point of the spleen vein and superior mesenteric vein.Conclusion The 3-D reconstruction of blood vessels around the pancreas can provide the anatomical basis for surgeon and reduce the risk of pancreatic surgery before operation.

Objective To determine the effect of COMMD7 inhibition on invasion and migration in liver cancer stem cells (LCSCs),and investigate the possible mechanism.Methods After LCSCs were infected by shRNA lentiviral vectors of COMMD7,adhesion assay and Transwell assay were used to detect the invasion and migration,and phalloidin staining was employed to observe the morphological changes.Western blotting was adopted to measure the expression of E-cadherin,N-cadherin and Vimentin.Results COMMD7 knockdown significantly inhibited the invasion and migration of LCSCs.The relative cell quantity of adhesion was 1.00 ± 0.12 and 2.35 ± 0.20 respectively in control cells and infected cells,suggesting there were significantly more adhesive cells in the infected group (P ＜ 0.05).The relative cell quantity per visual field of migration was 1.00 ±0.04 and 0.24±0.03,and that of invasion was 1.00 ±0.05 and 0.24 ±0.04 respectively in the control cells and infected cells,and there were significantly less invasive and migrated cells in the infected group (P ＜0.05).What's more,COMMD7 knockdown also induced some morphological changes of cells corresponding to the weakened abilities of migration and invasion.All the changes above were associated with up-regulation of E-cadherin (P ＜ 0.05) and down-regulation of N-cadherin and Vimentin (P ＜0.05),the molecules related to mesenchymal-epithelial transition (MET).Conclusion COMMD7 knockdown inhibits the invasion and migration in LCSCs,which may be through its regulation on the MET course.

Objective To investigate the mechanism of COMMD7,a hepatocellular carcinoma gene,promoting human hepatocellular carcinoma cell line (HePG2 cells)proliferation and migration.Methods The specific siRNA (small interference RNA)was designed for COMMD7 gene,siRNA transfected HepG2 cells was set as Si-HePG2 group,and the PTEN inhibitor treating group (Si +BpV-HePG2),the control group (HePG2),the empty vector group (HePG2 was infected empty vector,N-HePG2 group)were also set up.qRT-PCR was per-formed to evaluate the mRNA expression changes of COMMD7 and PTEN,and Western blot was performed to test the expression changes of COMMD7,PTEN,p-AKT,and AKT.MSP method was performed to detect methylation level.CCK8 was performed to test cell proliferation a-bility.Transwell was performed to detect the invasion of cells.Results The results of qRT-PCR showed that the expression of COMMD7 gene in Si-HePG2 group was lower (0.101 times)than the control group and the N-HePG2 group,and the expression of PTEN gene was higher (2.841 times)than the control group and the N-HePG2 group,and all the results above were of statistically singificant difference (P <0.05). The results of Western blot showed that in Si-HePG2 group,the expression of COMMD7 reduced obviously,the expression of PTEN increased and the expression of p-AKT was significantly inhibited.In Si +Bpv-HePG2 group,the expression of PTEN was significantly inhibited and the expression of p-AKT was increased.The result of MSP showed that compared with the control group and the N-HePG2 group,Si-HePG2 group was more obviously demethylated,which indecated that the expression of COMMD7 gene could induce PTEN demethylation.The results of CCK8 showed that the proliferation in Si-HePG2 group was decreased compared with the control group,N-HePG2 group and Si +BpV-HePG2 group,and the difference was singificant (P <0.05).The results of Transwell showed that the numbers of cell permeating septum in Si-HePG2 group,N-HePG2 group,control group and PTEN group were (17.4 ±2.7),(36.2 ±3.2),(41.6 ±4.5)and (47.6 ±1.8)respec-tively,which were obviously decreased with a significant difference (P <0.05).Conclusion siRNA interference decreased the expression of COMMD7.It can induced the demethylation of PTEN gene in HePG2 and increase the expression of PTEN gene to inhibit PI3K/AKT signaling pathway,thus decreasing the proliferation,migration and invasion ability of HePG2 cells.

DNA, Viral ; analysis ; False Positive Reactions ; Hepatitis B ; virology ; Hepatitis B virus ; genetics ; isolation & purification ; Humans ; Polymerase Chain Reaction ; methods ; standards ; Quality Control ; Reproducibility of Results ; Sensitivity and Specificity

OBJECTIVETo explore the silencing effects of RNA interference on the expression of 3-phosphoinositide-dependent protein kinase 1 (PDK1) gene, and the effects on malignant phenotypes of esophageal carcinoma EC9706 cells.

METHODSPDK1 siRNAs was transfected into the EC9706 cells. The expression of PDK1 mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR). At the same time, expressions of PDK1, Akt and phosphorylated Akt proteins were detected by Western blot. Methyl thiazolyl tetrazolium assay (MTT) was used to examine the cell proliferation after transfection. Flow cytometry was used to determine the percentage of apoptosis cells, and Transwell chambers were used to detect the invasion ability of the cells. Tumor formation in nude mice was used to assess the tumorigenic characteristics in vivo.

RESULTSCompared with the non-transfected group, PDK1 siRNA effectively inhibited the expression of PDK1 mRNA in EC9706 cells, with an inhibition rate of (28.5 ± 4.2)% at 24 h, (51.1 ± 5.7)% at 48 h and (60.6 ± 4.1)% at 72 h after transfection. The expressions of PDK1 and phosphorylated Akt protein were also knocked down by PDK1 siRNA (P < 0.05). PDK1 siRNA significantly inhibited the cell proliferation and invasion, promoted the cell apoptosis, and inhibited the EC9706 cells proliferation in vivo and the expression of PDK1 protein in the transplanted tumors (P < 0.05).

CONCLUSIONPDK1 may play an important role in esophageal cancer cell proliferation, invasion and apoptosis, and may serve as an effective target for cancer gene therapy.

3-Phosphoinositide-Dependent Protein Kinases ; Animals ; Apoptosis ; Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Esophageal Neoplasms ; genetics ; metabolism ; pathology ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Invasiveness ; Neoplasm Transplantation ; Phosphorylation ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Transfection ; Tumor Burden

OBJECTIVETo establish a model for one choosing controls with a suitable concentration for internal quality control (IQC) with qualitative ELISA detection, and a consecutive plotting method on Levey-Jennings control chart when reagent kit lot is changed.

METHODSFirst, a series of control serum with 0.2, 0.5, 1.0, 2.0 and 5.0ng/ml HBsAg respectively were assessed for within-run and between-run precision according to NCCLs EP5 document. Then, a linear regression equation (y=bx + a) with best correlation coefficient (r > 0.99) was established based on S/CO values of the series of control serum. Finally, one could choose controls with S/CO value calculated from the equation (y = bx + a) minus the product of the S/CO value multiplying three-fold between-run CV to be still more than 1.0 for IQC use. For consecutive plotting on Levey-Jennings control chart when ELISA kit lot was changed, the new lot kits were used to detect the same series of HBsAg control serum as above. Then, a new linear regression equation (y2 = b2x2 + a2) with best correlation coefficient was obtained. The old one (y1 =b1x1 + a1) could be obtained based on the mean values from above precision assessment. The S/CO value of a control serum detected by new lot kit could be changed to that detected by old kit lot based on the factor of y2/y1. Therefore, the plotting on primary Levey-Jennings control chart could be continued.

RESULTSThe within-run coefficient of variation CV of the ELISA method for control serum with 0.2, 0.5, 1.0, 2.0 and 5.0ng/ml HBsAg were 11.08%, 9.49%, 9.83%, 9.18% and 7.25%, respectively, and between-run CV were 13.25%, 14.03%, 15.11%, 13.29% and 9.92%. The linear regression equation with best correlation coefficient from a test at random was y = 3.509x + 0.180. The suitable concentration of control serum for IQC could be 0.5ng/ml or 1.0ng/ml. The linear regression equation from the old lot and other two new lots of the ELISA kits were y1 = 3.550(x1) + 0.226, y2 = 3.238(x2) +0.388, and y3 =3.428(x3) + 0.148, respectively. Then, the transferring factors of 0.960 (y2/y1) and 0.908 (y3/y1) were obtained.

CONCLUSIONThe results shows that the model established for IQC control serum concentration selecting and for consecutive plotting on control chart when the reagent lot is changed is effective and practical.

Enzyme-Linked Immunosorbent Assay ; methods ; standards ; Evaluation Studies as Topic ; Hepatitis B Surface Antigens ; blood ; Humans ; Quality Control ; Reagent Kits, Diagnostic ; standards ; Reproducibility of Results

Objective To investigate the effects of different culture conditions on the isolation and expansion of stem cells from second-trimester amniotic fluids.Methods Amniotic fluids were obtained from 15 pregnant women undergone amniocenteses for medical indications between 16-24 gestation weeks by transabdominal amniocenteses from September 2007 to June 2008.Amniotic fluids(10-20 ml)samples were collected and each WaS cultured under different conditions or groups.(1)Low-glucose DMEM(LD) medium supplemented with 10%of fetal bovine serum(group of 10% FBS);(2)LD medium with 20%of FBS(group of 20%FBS);(3)LD medium with 15%of FBS and 4 ng/ml of basic fibroblast growth factor (group of bFGF);(4)LD medium with 10%of FBS as well ag the culture plate coated with gelatin(group of gelatin).The effects of different conditions were evaluated by comparing the number of primary colonies,the cell morphology and the ability of expansion.The isolated stem cells were identified by flow cytometry,RT-PCR and differentiation ability to edipocyte.Resuits (1)The success rates of primary culture of the group of 10%FBS,20%FBS,bFGF and gelatin were 60%,73%,73%and 60% respectively(P>0.05).The numbers of colonies were 0.9±0.5,2.6±1.5,2.9±1.5,1.1±0.8(P<0.01 when group of 10%FBS and gelatin compared with group of 20%FBS and bFGF);among the primary colonies,fibroblast-like colonies accounted for 46%,49%,64%.44%respectively(P>0.05).(2)The second passage cells obtained from all of these four groups could difierentiate into adipocyte after induction.(3)In the group of bFGF,stem cells were isolated from 5 samples and expanded to nearly 107 cells after 5 passages(P<0.01 compared with other groups).(4)Karyotype were normal in all samples.(5)Stem cells from bFGF group showed positive expression of SSEA-4.Oct-4 and Nanog gene detected by flow cytometry and RT-PCR.Conclusion Stem cells can be isolated from second-trimester amniotic fluids;moderate serum concentration and supplementation of bFGF can improve the efficiency of isolation and expansion of amniotic fluid of stem cells.

Objective To establish and evaluate two protocols for the noninvasive visualization and assessment of coronary artery bypass graft (CABG) patency on electron beam tomography (EBT).Methods Two hundred and fourteen consecutive patients who underwent coronary artery bypass graft surgery were scanned using both EBT angiography with 3-dimensional reconstruction and EBT flow study with time-density-curve analysis.Results There were 589 CABGs evaluated in this study (10 grafts were excluded because of artifacts). Among them, 133 (98.5％) of 135 arterial grafts were patent, and 345 (77.7％) of 444 saphenous-vein grafts were patent. Within 5 years or between 5 and 10 years after operation, arterial graft patency exceeded venous graft patency (P ＜ 0.001 ). Three-dimensional EBT angiography achieved higher sensitivity, specificity and accuracy (97.7％, 94.1％ and 96.7％, respectively) than did EBT flow study (88.4％, 82.4％ and 85.2％, respectively) for evaluating occlusion or patency of CABG. The intra-graft flow of patent arterial and venous grafts were 4.9 ± 2.2 mi · min-1 · g-1 and 6.9 ± 2.8 mi · min-1 · g-1,respectively (P＜0.001).Conclusion The combination of EBT three-dimensional reconstruction and flow study can be more effective in the assessment of CABG anatomy and quantification of patent CABG blood flow.