Objective To construct an eukaryotic expression vector with human adipose most abundant gene transcript 1 (APM1) gene,and to investigate the transfection and expression of pCDEF-APM1 eukaryotic expression plasmid in HEK293 cells.Methods pCDEF-APM1 eukaryotic expression plasmid was constructed by DNA recombinant method.Expression vector pCDEF-APM1 was transfected into HEK293 cells with Effectene reagent.The level of human adiponectin protein in the supernatant of cell culture media was detected with double antibody sandwich ELISA.Results The sequence of DNA fragment from constructed pCDEF-APM1 plasmid was identical to that published in GenBank.There was raised human adiponectin protein level in culture supernatant of HEK293 cells tnmsfected with pCDEF-APM1.Conclusion The pCDEF-APM1,an eukaryotic expression plasmid for APM1 gene is successfully constructed.High protein expression of adiponectin can be obtained in HEK293 cells transfected with pCDEF-APM1 eukaryotic expression plasmid.

Objective To analyze the clinical efficacy of physical vibration lithecbole in treatment of urinary calculi. Methods Ana-lysed the efficacy of 80 patients who underwent physical vibration lithecbole only or combination therapy with surgery in urinary calculi in our hospital from February 2014 to July 2014. Result There were 1 to 4 times calculi discharge among the 80 patients. One month after the sur-gery, the calculi discharge rate was 33. 3% and the calculi clean rate was 22. 2% in the upper ureteral; the calculi discharge rate was 16. 7%and the calculi clean rate was 50. 0% in the distal ureteral; the calculi discharge rate was 40. 0% and the calculi clean rate was 23. 3% in the upper renal calyx;the calculi discharge rate was 27. 7% and the calculi clean rate was 38. 8% in the middle renal calyx;the calculi discharge rate was 60. 0% and the calculi clean rate was 20. 0% in the lower renal calyx. One month after the surgery of physical vi-bration lithecbole combined with Holium laser lithotripsy, the calculi discharge rate was 52. 1% and the calculi clean rate was 39. 1%. Con-clusion Physical vibration lithecbole is a noninvasive treatment for urinary calculi. It has good efficacy in calculi discharge and it can relieve the pain caused by calculi.

ObjectiveTo explore the clinical application of ultrasound-guided radiofrequency thermocoagulaion in brachial plexus block.MethodsC5-C7 brachial plexus block was performed by 6-13 MHz high-frequency ultrasound probe in 65 patients with cervical spondylotic radiculopathy. Visual analogue scale (VAS) score were compared before and after treatment.ResultsThe brachial plexus was showed clearly in 62 patients; however, 3 patients had to be confi rmed by nerve stimulation positioning. The percentage of successful rate is 100%. There was no operation related nerve injury and other complications. The VAS score of preoperation and 1st, 4th and 12nd week after treatment was 8.67±0.76, 3.58±0.62, 2.46±0.2 and 1.77±0.28, respectively. There were significantly difference between before and after treatment (t=58.71, 6.23, 107.72, allP<0.01).ConclusionThe brachial plexus block using radiofrequency thermocoagulaion combined with ultrasound guidance is a safe and radiation-free treatment and warrants to be promoted in clinical practices.

OBJECTIVE: To evaluate the prognostic implication of human papillomavirus (HPV) viral load in cervical cancer patients who underwent radical hysterectomy. METHODS: We conducted a retrospective review of patients with stage IA2 through stage IIIA cervical carcinoma who underwent radical hysterectomy at Sun Yat-sen University Cancer Center between January 2005 and December 2009. Patients who had undergone preoperative hybrid capture 2 testing to detect HPV DNA were included. A total of 346 patients positive for HPV DNA were enrolled and stratified into two groups according to the median HPV viral load. RESULTS: HPV viral load was significantly correlated with lymphovascular space invasion (p=0.026) and deep stromal invasion (p=0.024). However, other factors, such as age, stage, histologic grade, histologic type, lymph node metastasis, and tumor size, were not significantly associated with viral load. Low HPV viral load was correlated with poor disease-free survival in univariate analysis (p=0.037) and multivariate analysis (p=0.027). There was no significant difference in overall survival with regard to initial HPV viral load. CONCLUSION: Low initial HPV viral load may be a poor prognostic factor for cervical cancer patients who have undergone radical hysterectomy.
Adult ; Aged ; Aged, 80 and over ; Carcinoma, Squamous Cell/*diagnosis/surgery/virology ; Female ; Humans ; Middle Aged ; Papillomaviridae/*isolation & purification ; Papillomavirus Infections/complications/diagnosis/surgery/virology ; Prognosis ; Retrospective Studies ; Treatment Outcome ; Uterine Cervical Neoplasms/*diagnosis/surgery/virology ; *Viral Load ; Young Adult

OBJECTIVETo provide reliable seed cells with unlimited passage proliferation and stable biological characteristics for periodontal tissue engineering research through infecting a retrovirus carrying SV40Tag into SD rat dental follicle cells.

METHODSRetroviral virus vector containing SV40Tag by 293 cells was packaged and infected into SD rat dental follicle cells. Normal dental capsule cells were used as control group. The cell morphology and vitality were observed by inverted microscope, telomerase activity, osteogenic differentiation and proliferation of dental follicle cells were analyzed.

RESULTSThe SD rat dental follicle cells infected with SV40Tag could be passaged for 60 generations in vitro culture with strong growth activity. The telomerase activity was significantly enhanced compared with the control group (P = 0.033). The expression of alkaline phosphatase, osteocalcin, bone morphogenesis protein-2, Runx2, basic fibroblast growth factor, insulin-like growth factors had no statistical difference compared with control cells (P > 0.05).

CONCLUSIONThe SD rat dental follicle cells infected with SV40Tag not only have a strong growth activity and infinite passaged capacity, but also have a stable biological property as normal dental follicle cells, so it can be regarded as the excellent seed cells in periodontal tissue engineering.

Alkaline Phosphatase ; Animals ; Cell Differentiation ; Cells, Cultured ; Dental Sac ; Fibroblast Growth Factor 2 ; Osteoblasts ; Osteocalcin ; Osteogenesis ; Rats ; Rats, Sprague-Dawley ; Tissue Engineering

OBJECTIVETo investigate the frequency of numerical aberrations for chromosomes 7 and 8 in the sperms of workers exposed to benzene series.

METHODSNumerical aberrations in the sperms of workers were detected by two-color fluorescence in situ hybridization with biotin labeled chromosome 7 probe (D7Z1) and digoxingenin labeled chromosome 8 probe (D8Z1).

RESULTSThe time-weight average air concentration (TWA) of benzene in the workshop was 42.29 mg/m(3), which was significantly higher than that of the national maximum allowable concentration [6 mg/m(3)]. The concentration of urinary trans,trans-muconic acid(ttMA) in the exposed group was also higher than that of the control group. In all, 155721 sperm nuclei from 15 workers in the exposed group and 123771 sperm nuclei from 12 individuals in the control group were examined. The results showed that the frequency of diploidy sperms and the frequencies of disomic sperm for chromosomes 7 and 8 in the exposed group (0.129%, 0.170%, 0.078%) were significantly higher than those of the control group (0.055%, 0.053%, 0.033%), respectively. The frequencies of nullisomic sperm for chromosomes 7 and 8 in the exposed group (0.165%, 0.088%) were also significantly increased, compared with those of the control group (0.056%, 0.029%). A statistically significant difference in the frequency of overall numerical chromosome aberrations was seen between the exposed group (0.745%) and the control group (0.289%).

CONCLUSIONThe results suggested that higher concentration of benzene may induce higher frequencies of numerical aberrations in the sperms of workers exposed to benzene series.

Adult ; Benzene ; poisoning ; Chromosome Aberrations ; chemically induced ; Chromosomes, Human, Pair 7 ; genetics ; Chromosomes, Human, Pair 8 ; drug effects ; genetics ; Female ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Male ; Occupational Diseases ; diagnosis ; genetics ; Occupational Exposure ; analysis ; Spermatozoa ; drug effects ; metabolism

OBJECTIVETo assess the larvicidal effects of recombinant Escherichia coli expressing scorpion neurotoxin AaIT or Bacillus thuringiensis subsp israelensis (B.t.i) toxin Cyt2Ba against the second instar larvae of Culex pipiensquinquefasciatus and Aedes albopictus and compare different formulations for their larvicidal effects.

METHODSThe AaIT- or Cyt2Ba-coding sequences were cloned into pET28a(+) and the recombinant plasmids were transformed into E. coli BL21(DE3). After induction with IPTG, the recombinant proteins expressed by the recombinant E. coli were detected and identified by SDS-PAGE and Western blotting, respectively. The larvicidal activity of the bacterial suspension was tested at different concentrations against mosquitoes. The effective engineered bacteria were prepared into dry powder with different formulations, and their larvicidal activity was tested.

RESULTSAaIT and Cyt2Ba proteins were successfully expressed in E. coli. The recombinant AaIT protein showed no virulence to the mosquito larvae. The suspension of the recombinant E. coli expressing Cyt2Ba protein exhibited a stronger killing effect on Aedes albopictus larvae than on Culex pipiens quinquefasciatus larvae at 48 h (P<0.001) with LCof 3.00×10cells/mL and 1.25×10cells/mL, respectively. The dry powder of the engineered bacteria formulated with yeast extract, wheat flour or white pepper powder at the mass ratio of 1:1 showed the strongest killing effect on mosquito larvae (P=0.044), and the formulation with white pepper powder produced a stronger killing effect than formulations with yeast extract or wheat flour (P=0.002).

CONCLUSIONThe B.t.i Cyt2Ba protein expressed in E. coli BL21(DE3) shows a good larvicidal activity against mosquitoes, and appropriate formulations of the engineered bacteria can enhance its efficiency in mosquito control.

OBJECTIVETo evaluate the effects of recombinant adenovirus encoding human apM1 gene on proliferation and nitric oxide synthase (NOS) activity in human umbilical vein endothelial cells (HUVECs).

METHODSProtein expression of apM1 in cell culture supernatant of HUVECs transfected with human Ad-apM1 was detected by double antibody sandwich ELISA. The effect of human adiponectin on cell proliferation was assessed by MTT assay. The total NOS and iNOS expressions were measured by chromatometre.

RESULTSHuman adiponectin protein level and total NOS and eNOS expressions were significant increased and iNOS expression significantly reduced in culture supernatant of HUVECs infected with Ad-apM1 compared to that in control HUVECs. The recombinant adenovirus had no influence on HUVECs growth as determined by MTT assay.

CONCLUSIONSHuman Ad-apM1 can be effectively expressed in HUVECs and do not influence HUVECs growth. Increased total NOS and eNOS expressions and decreased iNOS expression in HUVECs transfected with Ad-apM1 gene suggest a potential role of Ad-apM1 gene transfer for the prevention and treatment of arteriosclerosis.

Adenoviridae ; genetics ; Adiponectin ; genetics ; Cell Proliferation ; Cells, Cultured ; Endothelial Cells ; cytology ; metabolism ; Endothelium, Vascular ; cytology ; Gene Expression ; Gene Transfer Techniques ; Humans ; Nitric Oxide Synthase ; metabolism ; Umbilical Veins ; cytology

OBJECTIVETo construct SD rat immortalized dental follicle cells (rDFC) induced by simian virus 40 large tumor antigen (SV40Tag) gene to provide a reliable cell source for periodontal tissue engineering research.

METHODSThe rDFC was isolated by tissue mass method combined with enzyme digestion method and evaluated by immunohistochemistry. Cell293 were transfected with plasmid pSSR69/pAmpho containing SV40Tag gene by mediating liposome. Normal rDFC were infected with virus-contained supernate and the successfully transfected cell lines were screened with hygromycin, and positive clones were cultured. While non-transfected cells served as negative controls, the cell morphology was observed, the proliferation characteristics was evaluated by calculating cell population. The expression of SV40Tag gene and telomerase in cells was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting respectively. The biological property of immortalized rDFC was assessed with calculating formation rate of flat cloning, soft agar colony formation test and tumor-forming test.

RESULTSMorphology of immortalized rDFC was not different from that of normal rDFC. The RT-PCR results of SV40Tag revealed amplification band at 357 bp, while no band was seen in the normal cells. The expression of telomerase in immortalized rDFC was higher than that in normal rDFC. The two groups had no significant difference in growth curves, but the immortalized rDFC exhibited stronger proliferative activity. No significant differences of formation rate in flat cloning were observed between the immortalized rDFC [34% (33/96)] and normal rDFC at passage four [22% (21/96)] (χ(2) = 3.71, P > 0.05). No cell cloning was seen in soft agar and the tumor formation was not observed in nude mice.

CONCLUSIONSThe rDFC induced by SV40Tag gene could be cultured and passaged in vitro, which retained the stable proliferation and differentiation characteristics and could be used for periodontal tissue engineering research.

Animals ; Antigens, Viral, Tumor ; genetics ; metabolism ; Cell Differentiation ; Cell Proliferation ; Cell Transformation, Viral ; Cells, Cultured ; Dental Sac ; cytology ; immunology ; metabolism ; HEK293 Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Plasmids ; Rats ; Rats, Sprague-Dawley ; Simian virus 40 ; genetics ; immunology ; Telomerase ; metabolism ; Transfection

OBJECTIVETo evaluate the ultrasonographic changes in the epididymis after long-term vasectomy.

METHODSSixty-four patients with a history of vasectomy for more than 10 years (vasectomy group) and another 60 without vasectomy (control group) were included in the study. The patients were referred to scrotal ultrasonography for epididymal indications. The shape, thickness and internal echoes of the head, body and tail of the epididymis were observed with high frequency ultrasonography (HFU), and the blood flow changes were observed with color Doppler flow imaging (CDFI) or color Doppler power imaging (CDPI).

RESULTSSignificantly higher rates were found in the vasectomy group than in the control: thickened body (64.1% vs 15.0%) and tail (78.1% vs 51.7%) of the epididymis, thickened head, body and tail (42.2% vs 8.3%) of the epididymis, and epididymal tubular ectasia (54.7% vs 8.3%). However, increased blood flow in the epididymis was seen at a significantly lower rate in the vasectomy group than in the control (15.6% vs 61.7%).

CONCLUSIONThe ultrasonographic changes in the epididymis after long-term vasectomy were mainly epididymis thickening and epididymal tubular ectasia, mostly with no or diminished blood flow in the epididymis.

Adult ; Aged ; Epididymis ; diagnostic imaging ; Humans ; Male ; Middle Aged ; Postoperative Period ; Ultrasonography ; Vasectomy