Objective To construct a prokaryotic expression plasmid for CPSIT_p7 gene from Chlamydophila psittaci ( Cps) 6BC strain and to evaluate immunogenicity of the recombinant protein His-CPSIT_p7 and detect its dynamic expression at mRNA and protein levels in HeLa cells during persistent Cps infection.Methods The fusion protein His-CPSIT_p7 was expressed in E.coli BL21 and purified by Ni-NTA affinity chromatography .BALB/c mice were immunized with the recombinant protein to prepare polyclonal antibody for evaluation of the immunogenicity of His-CPSIT_p7 by ELISA.Penicillin sodium was used to establish a model of Cps persistence infection .RT-PCR and Western blot assay were performed to de-tect the expression of CPSIT_p7 at mRNA and protein levels during Cps persistent infection .Results The fusion protein His-CPSIT_p7 was successfully expressed with the use of constructed recombinant expression plasmid pET30 a-CPSIT_p7 and purified .ELISA result showed that the specific antibody titer against CPSIT_p7 reached 1 ∶1 000 000 on the 40th days after immunization .The expression of CPSIT_p7 at mRNA and protein levels were increased in a time-dependent manner in Cps-infected HeLa cells .The peak of mRNA level was reached at the time point of 36 hours after infection , followed by a time-dependent decrease during Cps acute infection .However , the expression of CPSIT_p7 at mRNA and protein levels were not decreased until 60 hours after infection during Cps persistent infection .Conclusion His-CPSIT_p7 protein was suc-cessfully expressed in the prokaryotic expression system and purified , showing an advantage of good immuno-genicity.Highly expressed CPSIT_p7 at mRNA and protein levels were detected during Cps persistent infection.

Objective To investigate the effect of antisense oligonucleotides (ASODN) targeting livin on the inhibition of livin mRNA and protein expression and the apoptosis of human renal carcinoma cell line 786-O cells. Methods Specific phosphorothioate antisense oligodeoxynucleotides targeting livin were synthesized and then transfected into 786-O cells. The expressions of livin mRNA were detected by RT-PCR. Expression and location of livin protein were observed by confocal laser scanning microscope (CLSM). Apoptosis rate of 786-O cells was investigated by flow cytometer. The activity of Caspase-3 was detected by colorimetric assay. Results After the transfection of ASODN, the expression of livin mRNA was decreased (P

Objective:To observe the effects of moxibustion on serum levels of interleukin (IL)-6, IL-8 and tumor necrosis factor-α (TNF-α), and to explore the effects of moxibustion on inflammatory damaging factors in experimental rheumatoid arthritis (RA) model rats; the relationship between the therapeutic effect of moxibustion on RA and the change in the Toll-like receptor (TLR) signaling pathway was analyzed using Toll-like receptor 4 (TLR4) antagonists and agonists. Methods:Fifty Sprague-Dawley (SD) rats were divided into a normal group, a model group, a moxibustion group, a moxibustion plus TLR4 agonist group (agonist group) and a moxibustion plus TLR4 antagonist group (antagonist group) according to the random number table, with 10 rats in each group. Except the normal group, rats in the other four groups were subjected to model preparation with the wind, cold and wet environmental factors plus Freund's complete adjuvant (FCA). Rats in the normal and model groups were not treated; rats in the moxibustion, agonist and antagonist groups started to be treated with the moxibustion (cigarette-type moxa) at bilateral Shenshu (BL 23) and Zusanli (ST 36) from the 4th day after the successful modeling, for 20 min each time with a total of 10 d. Rats in the agonist and the antagonist groups were injected with TLR4 agonist or antagonist [0.1 mg/(kg·bw)] via the tail vein 30 min before moxibustion. The concentrations of serum IL-6, IL-8 and TNF-α in each group were determined by enzyme-linked immunosorbent assay (ELISA). Results:Compared with the normal group, in the model group, the rat's right hind paw swelling was significantly obvious (P<0.01), there was a lot of inflammatory infiltration in the synovial tissues, the surface of the synovial membrane was unsmooth, the synovial membrane was hyperplasia and thicker, and the serum IL-6, IL-8 and TNF-α concentrations increased significantly (P<0.05). Compared with the model group, the paw swelling degrees of the rats in the moxibustion, the agonist and the antagonist groups reduced significantly (allP<0.01); the swelling degree in the antagonist group was milder than that in the agonist group, but the between-group difference was not statistically significant (P>0.05); inflammatory infiltration and synovial membrane hyperplasia in the synovial tissues of the moxibustion group and the antagonist group were all relieved differently; the decrease of synovial layer number in the moxibustion group was more obvious, and there were no obvious improvements in inflammatory infiltration and synovial thickness in the agonist group; the concentrations of IL-6, IL-8 and TNF-α in the moxibustion group were decreased, and the differences in the IL-6 and TNF-α concentrations were statistically significant (allP<0.01); there was no significant between-group difference in the IL-8 concentration (P>0.05); the concentrations of serum IL-8 and TNF-α in the agonist group increased significantly (both P<0.01), while the IL-6 concentration decreased without significant difference (P>0.05); the concentrations of IL-6 and IL-8 in the antagonist group decreased but the between-group differences were statistically insignificant (bothP>0.05), and the TNF-α concentration significantly increased (P<0.05). Compared with the moxibustion group, IL-6, IL-8 and TNF-α concentrations increased in the agonist group, and the differences in the IL-8 and TNF-α concentrations were statistically significant (both P<0.01); the concentrations of IL-6, IL-8 and TNF-α increased in the antagonist group, and the differences in the IL-6 and TNF-α concentrations were statistically significant (bothP<0.01); there was no significant difference in the IL-8 concentration between the groups (P>0.05). The serum levels of IL-6, IL-8 and TNF-α in the antagonist group were lower than those in the agonist group (allP<0.05). Conclusion:Moxibustion at Shenshu (BL 23) and Zusanli (ST 36) can reduce the joint swelling degree and inflammation in synovial tissue of RA model rats, decrease the serum levels of IL-6, IL-8 and TNF-α in RA model rats; the decreases of IL-6 and TNF-α are more significant than the decrease of IL-8; TLR4 agonist and antagonist can significantly attenuate the effect of moxibustion in inhibiting releases of IL-6, IL-8 and TNF-α, so that the change in TLR signaling pathway affects the effect of moxibustion in inhibiting the releases of IL-6, IL-8 and TNF-α.

Abstract: Objective - - To establish a pre column derivatization high performance liquid chromatography method for detecting Methods dimethyl sulfate (DMS) in workplace air. DMS in workplace air was collected with mercaptopyridine impregnated （ silicone tube. The derivative of DMS and mercaptopyridine was eluted by mobile phase phase A: water, phase B: acetonitrile, ∶ the volume ratio was 40 60) , and separated with a C18 column, then detected with diode array detector and quantitated by a Results - standard curve. The linear range of DMS was 0.17 40.00 mg/L, with the correlation coefficient of 0.999 95. The detection limit and the lower limit of quantitation were 0.05 and 0.17 mg/L respectively. The minimum detection concentration and minimum quantitation concentration were 0.02 and 0.04 mg/m³, respectively (air sample volume of 4.5 L, 1.0 mL sample - - - solution). The average desorption efficiency was 98.40% 102.00%. The within run and between run relative standard deviations - - were 0.61% 3.92% and 1.71% 6.00%, respectively. The samples could be stored at room temperature for at least 14 days. Conclusion This method can be used to detect DMS in workplace air.

Objective:To compare and observe the different clearance effect of milk containing anti-Helicobacter pylori specific antibody. Methods:Four H. pylori strains were used to immune dairy cows to obtain milk containing anti-Helicobacter pylori specific antibody,of which,one was standardized strain and the other three were locally epidemic. Totally 148 people were screended,in which 72 were C-14 urea breath test positive, finally 39 meet the criteria. They were divided into two groups, the test group contained 21 subjects,were treated milk containing anti-Helicobacter pylori specific antibody;the 18 subjects in control group with common milk. The study was continued for 2 months. Results:Conducting the C-14 urea breath test,9 subjects in test group were negative,but no one was changed in control group. The effective clearance rate of the test group was 42. 86%,and there was no effective clearance in the control group,so there was significant difference in the two groups(P=0. 005,P<0. 05). Conclusion: The milk containing anti-Helicobacter pylori specific antibody is polyclonal and has higher valence,and could clear H . Pylori effectively.

Objective To assess the value of preoperative neutrophil-lymphocyte ratio ( NLR) for predict the prognosis in patients with high grade T1 bladder.Methods From January 2004 to December 2014, the data of 307 patients diagnosed as bladder cancer of Stage 1 and high grade after undergoing TURBT were analyzed, including gender, age, smoking status, tumor number and size, hydronephrosis, intravesical instillations and preoperative blood transfusion of 307 patients diagnosed as bladder cancer of stage 1 and high grade after undergoing TURBT were analyzed retrospectively.All patients were primary urothelial carcinoma.According to preoperative NLR,patients were divided into the low NLR group( NLR≤2.42,n=197) and the high NLR group(NLR >2.42,n =110).Recurrence-free survival (RFS) and progression-free survival ( PFS) were calculated according to the Kaplan-Meier model and compared by the log-rank model.Cox regression models were used for multivariate analyses of the association between NLR and bladder cancer, then the prognostic factors affecting RFS and PFS were evaluated.Result of these 307 patients, the low NLR group accounted for 64.2%(197/307), and the high NLR group accounted for 35. 8%(110/307).The mean follow-up period was 71(range, 1-123)months.The recurrence rate in the low NLR group and the high NLR group recurrence rate were 19.2%( 38/197 ) and 34.5%( 38/110 ) respectively, RFS were 73.0(range, 2-123)months and 67.5(range, 1-122)months respectively.The progression rates were 4.1%(8/197) and 10.9%(12/110) respectively.The recurrence and progression rates in the high NLR group is higher than those in the low NLR group(P<0.01 and P=0.008), and RFS was shorter( P=0.002).Multivariable Cox regression analysis showed that NLR>2.42(P=0.007,HR=1.912)and hydronephrosis (P<0.01, HR =2.485 ) are associated with higher risk of recurrence.Conclusion Elevated preoperative NLR is an independent predictor of RFS and PFS in patients with high grade T1 bladder cancer.

OBJECTIVETO establish a RP-HPLC method for the determination of calycosin-7-O-beta-D-glucopyranoside in Radix Astragali, and to analyse the calycosin-7-O-beta-D-glucopyranoside content of ten samples of Radix Astragali, collected from different regions.

METHODA Polaris C18(250 mm x 4.6 mm, 5 microns) column was used and a mixture of methanol-water (30:70) was used as the mobile phase at a flow rate of 1.0 mL.min-1. The column temperature was 25 degrees C and the UV detection wavelength was 254 nm.

RESULTThe calibration curve was in good linearity over the range of 0.0106-2.12 micrograms with the regression equation Y = 3035. 97 X - 14.85(r = 0.9999). The average recovery was 95.8% (n = 5, RSD = 1.3%).

CONCLUSIONThe method is simple, quick, sensitive and reproducible. In all of the samples, the calycosin-7-O-beta-D-glucopyranoside contents differ markedly.

Astragalus membranaceus ; chemistry ; classification ; China ; Chromatography, High Pressure Liquid ; Ecosystem ; Glucosides ; analysis ; Isoflavones ; analysis ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Quality Control ; Species Specificity

OBJECTIVETo explore the effect of electroporation mediated gene therapy on bone mineral density and strength of new-formed bone in mandibular distraction gap, so as to enhance the osteogenesis and shorten the distraction term.

METHODSNew-Zealand rabbits were employed. The distraction began after 3 days of latency period at the rate of 0. 8 mm per day for 7 days. After distraction, the rabbits were randomly divided into 5 groups to receive injection in the distraction gap with recombinant plasmid 2 microg (0.1 microg/microl) pIRES-hVEGF165-hBMP2 in group A, with recombinant plasmid pIRES-hBMP2 in group B, with recombinant plasmid pIRES-hVEGF165 in group C, with pIRES in group D, and with normal saline (NS) in group E. After injection, electroporation was performed in all the groups. After 1 week, 2 weeks, 4 weeks and 8 weeks of consolidation, all the animals underwent X-ray and quantitative computed tomography (QCT). The new-formed bone in distraction gap was selected as regions of interest (ROI) to measure the bone mineral density(BMD). Then the rabbits were sacrificed and the new-formed bone samples were harvested to detect 3-point crushing strength.

RESULTSBMD of newly formed bone in group A, B and C was markedly higher than that in group D and E (P < 0.01). After 2 weeks of consolidation, BMD in group A was much higher than that in the other groups, but there was no difference between group B and C. After 4 weeks of consolidation, BMD in group A and B was markedly higher than that in group C, D and E (P < 0.01). After 8 weeks of consolidation, BMD in group A was markedly higher than that in the other groups. While the BMD was not significantly different between group B and C, but the BMD in group B and C was higher than that in group D and E (P < 0.01). After 4 weeks of consolidation, the 3-point crushing strength of newly formed bone in group A was markedly higher than that in group B,C, D and E (P < 0.01), which was still the same after 8 weeks of consolidation. And the crushing strength in group B was higher than that in group C, D and E (P < 0. 05).

CONCLUSIONSElectroporation-mediated transfection of recombinant plasmid pIRES-hVEGF165-hBMP2 could greatly enhance osteogenesis and calcification. A combination of VEGF and BMP may promote osteogenesis and angiogenesis simultaneously, so as to magnify the effect of each growth factor, resulting a synergetic effect.

Animals ; Bone Density ; Bone Regeneration ; Electroporation ; Genetic Therapy ; Mandible ; physiology ; surgery ; Osteogenesis, Distraction ; Rabbits

OBJECTIVETo investigate the effect of gene transfection at different time on bone mineral density and strength of newly formed bone in mandibular distraction gap in rabbit, so as to explore the optimal time for gene therapy and enhance the therapeutic effect.

METHODS48 New-Zealand rabbits were employed to receive mandibular osteotomy and implantation of distraction devices bilaterly. Then the rabbits were randomly divided into 4 groups as group A, B and C and D. The animals in group A, B, and C were transfected with recombinant plasmids pIRES-hBMP2-hVEGF165 via electroporation-mediated approach at latency period, distraction period, consolidation period respectively. Group D was used as control group without gene transfection. After 3 days of latency period, the distraction devices were activated at the rate of 0.8 mm per day for 10 days. Three rabbits in each group were sacrificed at 1 wk, 2 wk, 4 wk and 8 wk of consolidation respectively. The mandibles were harvested and the left mandible received X-ray examination for bone healing, and quantitative computed tomography (QCT) dectection for the bone mineral density (BMD) of newly formed bone in the distraction gap. The biomechanical properties of the new generation bone at 4 th and 8 th week of consolidation in each group were detected by three point bending test.

RESULTSThe bone mineral density and the biomechanical strength of newly formed bone increased along the length of consolidation in each group. After 1 week of consolidation, there was no significant difference in BMD among group A (83.43 +/- 9.96), group B (92.29 +/- 11.25), group C (89.93 +/- 14.15), P > 0.05. But the BMD of group A, B and C was higher than that of group D (70. 31 +/- 3.30), P < 0.05. After 2wk, 4 wk and 8 wk of consolidation, the BMD of group B (137.54 +/- 7.20,492.93 +/- 17.57, 790.48 +/- 12.19) was significantly higher than those of group A (121.44 +/- 9.27, 396.15 +/- 15.70, 603.39 +/- 16.46), C (125.06 +/- 7.24, 464.15 +/- 15.45, 764.15 +/- 17.28), and D (98.86 +/- 8.13, 336.45 +/- 11.95, 577.89 +/- 18.43), P < 0.05. The biomechanical parameters were also higher in group B than those of group A, C and D after four and eight weeks of consolidation (P < 0.05).

CONCLUSIONIt is better to transfect gene at the beginning of distraction (distraction period) than at other stages of DO. In this way, more remarkable effect could be obtained on new bone formation. It suggests that the distraction stage is the optimal time for gene therapy.

Animals ; Bone Density ; genetics ; physiology ; Electroporation ; Genetic Therapy ; Mandible ; physiology ; surgery ; Osteogenesis, Distraction ; Osteotomy ; Rabbits ; Time Factors ; Transfection

OBJECTIVETo discuss the method, the advantages and disadvantages of endoscopic thyroidectomy.

METHODSEndoscopic thyroidectomy via areola of breasts approach was performed in 150 patients, including 41 cases of thyroid adenoma, 64 cases of nodular goiter, 40 cases of Graves' disease, and 5 cases of thyroid carcinoma.

RESULTSThe endoscopic thyroidectomy was successfully carried out in 144 cases, including tumor dissection in 32 cases, one lobe partial thyroidectomy in 54 cases, two lobe partial thyroidectomy in 19 cases, subtotal thyroidectomy in 37 cases of Graves' disease, and radical thyroidectomy in 2 cases of thyroid carcinoma. The operative time length ranged from 50 to 270 min (mean 80 min). There were no complications such as damage to recurrent laryngeal nerve or parathyroid glands. Postoperative hospital stay ranged from 3 to 7 days (mean 4 days). The post-operative following-up for 1 approximately 13 months indicated that all the patients were satisfied with the cosmetic results and the same curative effects as conventional surgery were obtained. However, the operations were converted into open surgery in 6 cases.

CONCLUSIONSEndoscopic thyroidectomy is a safe and effective method of thyroid surgery. Since all the minimal incisions are on concealed parts of the body, the obvious cosmetic effect of this method is guaranteed.

Adolescent ; Adult ; Endoscopy ; Female ; Follow-Up Studies ; Graves Disease ; surgery ; Humans ; Male ; Thyroidectomy ; methods ; Treatment Outcome