RNA interference targeting gene of RING finger protein inhibits proliferation and migration of pancreatic cancer cell line PANC-1 / 基础医学与临床

Objective To investigate the effect of siRNA-mediated silencing of RNF2 on cell proliferation , migra-tion, cell cycle and apoptosis in human pancreatic cancer PANC-1 cells and its possible mechanism .Methods The siRNA interference was used to down-regulate RNF2 expression.Meanwhile, there were also empty transfection group whose cells were transfected with the control siRNA and mock group without any treatment .The result of transfection was evaluated by fluorescence microscope .The expression of RNF2 mRNA was detected by RT-qPCR. Western blot was applied to detect the expression of RNF 2 and p53.Cell proliferation and migration were analyzed by MTS assay and cell scratch assay , respectively .The transient transfection efficiency , apoptosis rate and cell cy-cle were measured by flow cytometry .Results Compared to the normalized human pancreatic duct epithelial cells , RNF2 expression in pancreatic cancer cells were higher ( P<0.05) .The expression of RNF2 mRNA and protein was decreased in PANC-1 cells by siRNA-RNF2 at 48 h post-transfection.Transfection with siRNA-RNF2 inhibited the proliferation and migration of PANC-1 cells (P<0.05), induced cell apoptosis (P<0.05), increased cell counts in phase G0/G1 and decreased in S and G2/M phase (P<0.05).What's more, after siRNA-RNF2 transfec-tion, the expression of p 53 protein was decreased .Conclusions siRNA-RNF2 can specifically knockdown the ex-pression of RNF2 gene and then inhibit the proliferation and migration of PANC-1 cells.These results indicate RNF2 may be a potential target of gene therapy for pancreatic cancer .

Suppression of vascular endothelial growth factor expression by adenovirus-mediated RNA interference in treatment of lung adenocarcinoma cells / 第二军医大学学报

Objective: To investigate the inhibitory effects of RNA silencing via adenovirus mediated vascular endothelial growth factor(VEGF) shRNA on proliferation of lung adenocarcinoma cells in vitro and in vivo. Methods: PCR method was used to construct a pAd Easy/VEGF adenovirus vector containing enhanced green fluorescent protein (EGFP) gene and expressing VEGF shRNA. The 293 cells were transfected with the linearized pAd Easy/VEGF using Lipofectamine2000. Then lung adenocarcinoma cells A459 were transfected with the constructed vector. The EGFP expression was detected by fluorescent microscopy and flow cytometry. VEGF mRNA expression was examined by RT-PCR and Western blotting. The cell growth was observed with MTT method and the growth curve was plotted. Meanwhile, nude mice were transplanted with A549 cells to establish tumor models and the growth of tumors were observed. Results: The recombinant pAd-Easy carrying shRNA targeting VEGF had been constructed and the aim sequence had been obtained. The transfection efficiencies in pAd-Easy/VEGF and blank vector transfected A549 cells were 100% and 99.7%, respectively. RT PCR and Western blotting showed a remarkable decrease of VEGF expression in the pAd-Easy/VEGF group compared with normal saline group. The tumor growth in pAd-Easy/VEGF group was obviously slowed down and the weight and volume of tumors were both significantly lower than those of the control group (all P<0.01). Conclusion: The shRNA targeting VEGF constructed in the present study can efficiently decrease the VEGF expression in A549 cells in vitro and suppress the growth of A549 cells in vivo.

Study on expression of mineralocorticoid receptor in human atria during atrial fibrillation / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the mRNA and protein expression of mineralocorticoid receptor (MR) in patients with atrial fibrillation.</p><p><b>METHODS</b>Twenty-five patients with rheumatic heart valve disease, 12 in sinus rhythm and 13 in chronic atrial fibrillation (>or= 6 months), underwent transthoracic echocardiography and right and left atrial lateral wall tissue samples were obtained from these patients during mitral/aortic valve replacement operation. Realtime quantitative PCR and Western blot were used to determine the mRNA and protein expression of MR in atria specimens. The distribution of MR in human atria was analyzed by specific immunohistochemical staining.</p><p><b>RESULTS</b>The left atrial diameters increased markedly in atrial fibrillation group compared with that in sinus rhythm group (P<0.01). And the results showed that the level of mRNA and protein of MR were increased significantly in atrial fibrillation group compared with those in sinus rhythm group (P<0.01 or 0.05), whereas the expression of mRNA and protein of MR were found to be no difference between left atria and right atria both in fibrillation and sinus groups (all P>0.05). The special immunohistochemical staining demonstrated that MR was abundant in the human atrial myocardium and MRs were located mainly in the cytoplasm of atrial cells, which were more evident in atrial fibrillation group than those in sinus rhythm group.</p><p><b>CONCLUSION</b>These findings suggested that MRs were upregulated in atrial fibrillation and aldosterone antagonists may be effective in treating atrial fibrillation.</p>

Effect of external stents on prevention of intimal hyperplasia in a canine vein graft model / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>External stents have been used to reduce intimal hyperplasia of vein grafts. The aim of the present study was to define the size of an external stent appropriate for a particular graft by comparing vein grafts with different sizes of external stents.</p><p><b>METHODS</b>A series of paired trials was performed to compare femoral vein grafts with different sizes of external stents, where 30 modeled canines were equally divided into three groups: 6-mm external stent vs non-stent control, 4-mm vs 6-mm external stent, and 4-mm vs 8-mm external stent. At day 3 after operation, color Doppler flow imaging (CDFI) was done to observe blood flow in the lumen. Four weeks later, CDFI was re-checked and the veins were harvested, stained and measured.</p><p><b>RESULTS</b>All grafts were patent without formation of thrombosis. External stents significantly reduced intimal thickness of the vein grafts with a 6-mm external stent compared with the vein grafts without external stents (P < 0.05). The vein grafts with the 4-mm external stent had similar intimal, medial and adventitial thicknesses compared with those with the 6-mm external stent and the 8-mm external stent.</p><p><b>CONCLUSIONS</b>External stents can reduce intimal hyperplasia of vein grafts. Stents of different diameters exert the similar effect on prevention of intimal hyperplasia.</p>

Protective effects of adenovirus-mediated gene transfer of soluble complement receptor type 1 on acute myocardium ischemia: An experimental study / 第二军医大学学报

Objective: To evaluate the protective effects of adenovirus-mediated gene transfer of soluble complement receptor type 1 (sCR1) on acute myocardium ischemia in mice. Methods: Twenty-seven SD rats were subjected to left anterior descending coronary artery (LAD) occlusion (30 min) and reperfusion. In the treatment group (n = 14), a mixture of adenovirus carrying sCR1 and LacZ was injected into the ischemic zone(100 μl, 1010 pfu) 5 min before reperfusion; in the control group, only adenovirus carrying LacZ was injected (n = 13). Echocardiography was performed 2 weeks later, followed immediately by pathologic examination. Results: Echocardiographic results of the treatment group were better than those of the control group (P<0.05) 2 weeks after the operation, especially when it comes to the size of infarction area (P<0.01). H-E staining showed more surviving myocardial cells in the treatment group than in the control group. Conclusion: Transfer of sCR1 gene by adenovirus can reduce the size of infarction area and preserve heart function in a long-term period.

Study of adenovirus-mediated vascular endothelial growth factor 165 gene transfer into bone marrow stromal cells and its expressive product promoting proliferation of endothelial cells / 中华外科杂志

<p><b>OBJECTIVE</b>To study transcription, expression, and bioactivity of expressive protein of vascular endothelial growth factor 165 (VEGF(165)) gene, after bone marrow stromal cells (BMSCs) were transferred by VEGF(165) gene mediated by adenovirus vector ex vivo.</p><p><b>METHODS</b>BMSCs were Isolated and cultured by gradient centrifugation method. The cells cultured are transferred by recombinant adenovirus vector that carry VEGF(165) gene. Transcription and expression of VEGF(165) gene in BMSCs and secretion of VEGF protein in culture medium were measured by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot and enzyme linked immunosorbent assay (ELISA) methods. The activity of VEGF protein in culture media was detected by proliferation effects on vascular endothelial cells.</p><p><b>RESULTS</b>When Ad. VEGF(165) transferred into BMSCs, there was an effective transcription and expression. The expressed product in the media of transferred cells had highly biological activity on proliferation of rat aortic vascular endothelial cells (P < 0.01).</p><p><b>CONCLUSIONS</b>Adenovirus vector can be transferred into BMSCs efficiently and safely. Adenovirus mediated VEGF(165) gene transferred into BMSCs could express VEGF protein with highly biological activity, which provided foundation on BMSCs based gene therapy for ischemic disease.</p>

A novel regulable sutureless aortic arch prosthesis:an experimental study / 第二军医大学学报

Objective:To develop a novel regulable sutureless aortic arch prosthesis and to apply it in an animal experimental study.Methods:The arch skeleton of the prosthesis was made of tandem Z-shape NiTiNOL wire;the branch skeleton was made of laser-cut NiTiNOL tube;and the whole skeleton was coated with thin ePTFE film.The blood vessel was anastomosed by di- rect ligature,needing no manual suturing.The prosthesis was applied in swine aortic arch operations under the bypass condi- tion.The practicality for surgery and the feasibility of anastomosis of the prosthesis were assessed.Results:Aortic arch opera- tions were successfully performed in 6 of the 8 experimental animals.The prostheses were easy to use,and the mean bypass time was only 10 min.The blood loss of the anastomoses was less than 100 ml within 8 h postoperatively in 5 animals;one had more blood loss due to prosthesis mismatch.Conclusion:The novel regulable sutureless aortic arch prosthesis has satisfactory practicality for surgery and reliable anastomosis,making it promising in future clinical application.

Establishment of human lung squamous carcinoma cell line CHLH-1 / 第二军医大学学报

Objective:To establish a human lung squamous carcinoma cell line and to study its biological characteristics. Methods:Lung squamous carcinoma specimens were freshly resected during operation;the tissues were incubated in vitro and the cell line was named CHLH-1.The biological characteristics of the cells were studied by light microscopy,electron microsco- py,chromosome analysis and transplantation experiment.Results:Cells from the specimens of the primary tumor,the CHLC- 1 cell line and the cells from transplanted tumor possessed the characteristics of malignant squamous epithelium under light and electron microscope.The cell growth curve,doubling time and mitotic index were also observed in vitro.Nuclear chromosome analysis revealed that the tumor was a subtriploid with a mode of 60-68 per cell.Tumor nodes were observed under the skin of nude mice by heterogenic transplantation.Conclusion:The characteristics of the established cell line suggest that it is a newly established human squamous carcinoma cell line.

Cancer stem cells:current status / 第二军医大学学报

Recently,study on cencer stem cells has been a focus of study.Cancer stem cell is a small population of cencer cells possessing the properties of stem cells:self-renewal,differentiation and proliferation.To date,the existence of cancer stem cells has been proven in acute and chronic myeloid leukemia,breast cancer,brain tumors,liver cancer and colon cancer,etc..In this article we reviews the current progress on cancer stem cells,including the defination,existing evidence,research methods, and challenges in clinical application.