Objective:To clone and express active domain of human granzyme A ( aGzmA ) and detect its biological activity.Methods:Human aGzmA gene was amplified by PCR from the full-length human granzyme A and inserted into prokaryotic ex-pression vector pET24a(+).The constructed recombinant plasmid pET24a-aGzmA was transformed to E.coli BL21(DE3) and induced with IPTG.The expressed product was identified by SDS-PAGE and Western blot.The recombinant protein was purified by the Ni2+affinity column chromatography and the enzyme activity was assayed with BLT substrate.Results: A DNA fragment of 700 bp was amplified by PCR.The recombinant plasmid pET24a-aGzmA identified by enzyme-digesting analysis and sequencing showed that aGzmA gene was inserted into vector plasmid correctly.SDS-PAGE analysis showed that there was a specific protein with a relative molecular mass of about 26 kD.Western blot analysis indicated that the protein could react with mouse anti-His monoclonal antibody specifically.The recombinant protein with high purity could be acquired from the inclusion bodies by the Ni2+ affinity column chromatography and the purified protein had good enzyme activity.Conclusion: The recombinant human granzyme A with good biological activity was prepared successfully.

Abstract A rapid gas chromatographic ( GC ) method was established for the determination of short-chain fatty acids ( SCFAs ) in human feces. Feces samples were directly extracted by 1% HCl-75% ethanol solution, and then centrifuged at high speed for GC analysis. The chromatographic separation was performed on a DB-FFAP capillary column ( 30 m í 0 . 25 mm í 0 . 25 μm ) with a temperature program ( initial temperature at 50℃ held for 1 min, ramped to 190℃ at 10℃/min ) . The injection port temperature was 250℃ with split ratio at 50:1 . The carrier gas was high purity nitrogen with a constant linear velocity at 1. 0 mL/min. A flame ionization detector was employed to quantify SCFAs. The proposed method had been certified by systematic method validation, and used to determine feces samples. Subsequently, the health volunteer and colorectal cancer patient groups could be distinguished successfully by multivariate statistical analysis. Compared with health volunteers, the acetic acid and butyric acid of feces from colorectal cancer patients were reduced obviously, indicating that SCFAs particularly butyric acid could be considered as candidate markers for colorectal cancer diagnosis. In summary, this study provides a rapid method for the determination of SCFAs in feces form health volunteers and colorectal cancer patients. The method had a prospect for screening and diagnosing colorectal cancer rapidly.

Objective:To detect the immune response in mice inoculated by DNA vaccine expressing chimeric gene gag gp120 of HIV 1.Methods:After BALB/C mice were immunized by the eukaryotic expression plasmid pVAXGE,the numbers of T lymphocyte subgroups of spleen,the killing rate of specific CTL from the immunized mice and the titers of serum antibodies during the different time were observed.Results:The lymphocytes of the immunized group by recombinant plasmid pVAXGE proliferated,the specific CTL killing rate of spleen was higher significantly than those of the control group( P

Objective To develop an UPLC-MS/MS method for simultaneously determination of five components (adenosine,cytidine,guanosine,mannitol and adenine) in Qiangshenpaidu capsules. Methods The UPLC separation was performed on an Agilent ZOBAX SB-C18(2.1 mm×150 mm,5 μm) column.Isocratic elution was carried out with mobile phase consisting of methanol-0.1%formic acid (5∶95) at a flow rate of 0.2 mL.min-1.The mass spectrometer was operated in the positive ionization electrospray ( ESI) mode using multiple monitoring ( MRM) for analysis of five components. Results Adenosine,cytidine,guanosine,mannitol and adenine were all analyzed with good precision and accuracy. The linear ranges were 35-1 120 ng.mL-1( r=0.998 1) ,10-320 ng.mL-1( r=0.996 4) , 30-980 ng.mL-1( r=0.999 3) , 40-1 280 ng.mL-1( r=0.993 4), 25-800 ng.mL-1(r=0.996 5),respectively.The recoveries of six analytes ranged from 97.4% to 103.6% and the relative standard deviations were all below 4.7%. Conclusion A sensitive,accurate and suitable UPLC-MS/MS method has been developed,and the method could be applied for the determination of adenosine,cytidine,guanosine,mannitol,and adenine in Qiangshenpaidu Capsules.

Aim To investigate the influence of sarpog-relate hydrochloride (SH)on the pharmacokinetic pro-file of dextromethorphan (DM),the typical substrate of CYP2D1 /2,in rats when they were administered co-instantaneously.Methods A total of 1 2 SD rats were randomly divided into two groups:the control group (DM,1 0 mg·kg-1 )and the sarpogrelate group (SH, 1 0 mg·kg-1 ;DM,1 0 mg·kg-1 ),which received in-tragastric administration.Plasma samples were collected immediately before and at different time points after drug administration.A LC-MS /MS method was used to determine the concentrations of DM in rat plasma. Pharmacokinetic parameters were analyzed using Drug and Statistics (DAS 2.0).Results There were signif-icant differences in the pharmacokinetic parameters of DM,including T1 2 (2.49 h ±0.93 h vs 1 .47 h ±0.20 h,P <0.05 ),Cmax (325.7 μg·L -1 ±1 33.2 μg· L -1 vs 1 04.5μg·L -1 ±52.4 μg·L -1 ,P <0.05), AUC0 -t(785.5 μg·L -1 ·h ±451 .9 μg·L -1 ·h vs 244.8 μg·L -1 ·h ±1 68.3μg·L -1 ·h,P <0.05) and AUC0 -∞(804.7 μg·L -1 ·h ±445.6 μg·L -1 ·h vs 251 .4 μg·L -1 ·h ±1 73.4 μg·L -1 ·h,P<0.05 )between the two groups.Conclusion SH could significantly inhibit the elimination of DM,the substrate of CYP2D1 /2 in rats.

Objective To investigate the value of dynamic three-dimensional contrast-enhanced ultrasound (3D-CEUS) in evaluating therapeutic response of hepatoma treated with radiofrequency ablation (RFA).Methods Totally 48 cases of patients with hepatic carcinoma (48 lesions) admitted in Xiangya Hospital of Central South University from September 2012 to January 2014 were selected.All patients underwent radiofrequency ablation,of which 30 patients were diagnosed by pathology after surgery,18 patients by clinical diagnosis.All patients underwent two-dimensional contrast-enhanced ultrasound (2D-CEUS) and 3D-CEUS 1 month and 3 months after RFA treatment to evaluate the therapeutic response,and the results of contrast-enhanced ultrasound and enhanced computed tomography (CT) [or magnetic resonance imaging (MRI)] were compared.The final diagnostic results of pathologic biopsy or more than two imaging examinations [ultrasonography,CT,MRI,positron emission tomography (PET)],tumor markers,and more than 3 months follow-up of patients were used as the gold standard.The sensitivity,specificity and accuracy of dynamic 3D-CEUS,2D-CEUS,enhanced CT (or MRI) in the diagnosis of tumor inactivation were calculated respectively.Results After radiofrequency ablation,dynamic 3D-CEUS could provide more valuable information in 75.0％ (36/48) lesions,which contribute to assess the efficacy of radiofrequency ablation.While compared with 2D-CEUS,3D-CEUS did not change the diagnosis or clinical management in 12 (25.0％) lesions.40 of 48 lesions were found no-enhancement in entire CEUS procedure suggesting that the tumor completely inactivated,while 8 lesions showed local enhancement on the edge of lesion suggesting that part of the tumors were active.39 of 48 lesions showed no-enhancement and other 9 with irregular enhancement on enhanced CT (or MRI).The sensitivity,specificity and accuracy of CEUS and enhanced CT (or MRI) in detection of residual tumor after radiofrequency ablation were 80.0％,100％,95.8％ and 80.0％,97.4％,93.8％,respectively.Conclusions There was no statistical significance among 3D-CEUS,2D-CEUS and enhanced CT or MRI in evaluating therapeutic response of hepatoma treated with radiofrequency ablation.But 3D-CEUS can provide more valuable information,3D-CEUS has potential usefulness in the evaluation of percutaneous radiofrequency ablation of hepatic tumors.

Objective:To explore the clinical effects of autologous skin paste in repairing medium-thickness skin donor site wounds.Methods:The prospective randomized controlled research method was applied. From October 2018 to December 2019, 18 patients with flame burn or hydrothermal scald, conforming to the inclusion criteria were admitted to Jinhua Hospital Affiliated to Zhejiang University School of Medicine, including 15 males and 3 females, aged (45±6) years. The wounds were repaired with medium-thickness skin grafts from thigh, and the wound area was (121±33) cm 2 after medium-thickness skin grafting. The medium-thickness skin donor site wound in each patient was divided into 2 wounds in equal area and allocated into autologous skin paste group and conventional treatment group by flipping a coin, with 18 wounds in each group. The wounds in autologous skin paste group were repaired with skin paste prepared with remaining skin fragments after autologous medium-thickness skin grafting, and the wounds in conventional treatment group were covered with petroleum jelly gauze and fixed with sterile gauze. On 3, 7, 14, and 21 d after operation, the wound healing in 2 groups was observed, and the wound healing rate was calculated. The wound healing time in 2 groups was recorded. Occurrences of wound subcutaneous effusion and infection on 3, 7, 14, and 21 d after operation and wound ulceration in 3 months after operation were observed. In 6 months after operation, the Vancouver Scar Scale (VSS) was used to evaluate the scar formation of wounds in 2 groups. Data were statistically analyzed with analysis of variance for repeated measurement, chi-square test, and group t test. Results:The wounds in 2 groups did not heal on 3 and 7 d after operation. The wound healing rate in autologous skin paste group was (29.8±2.5)% and (95.6±4.7)% on 14 and 21 d after operation, which were significantly higher than (25.8±2.9)% and (82.6±8.9)% in conventional treatment group ( t=4.3, 5.6, P<0.01). The wound healing time in autologous skin paste group was (21.8±1.6) d, which was significantly shorter than (25.6±2.0) d in conventional treatment group ( t=6.24, P<0.01). On 3, 7, 14, and 21 d after operation, there were no complications such as subcutaneous effusion or infection in wounds of 2 groups. In 3 months after operation, ulceration occurred in wounds of 2 patients in autologous skin paste group, which was significantly less than 12 patients in conventional treatment group ( χ2=11.688, P<0.01). The ulcerated wounds healed after dressing changes. In 6 months after operation, the VSS score of wounds in autologous skin paste group was (9.1±1.1) points, which was significantly lower than (11.3±1.2) points in conventional treatment group ( t=-5.75, P<0.01). Conclusions:The remaining skin fragments after autologous medium-thickness skin grafting prepared into skin paste to repair medium-thickness skin donor site wounds can shorten wound healing time, improve wound healing quality, and reduce degree of scar hyperplasia, with a good clinical effect.

Objective To investigate the constitutive characteristics and the change trend of gynecologic malignant tumors in hospitalized patients in Guangxi Zhuang Autonomous Region over the recent 20 years.Methods Clinical data of 8009 in-patients who suffered from gynecologic malignant tumors in 23 hospitals from 1985 to 2004 in Guangxi Zhuang Autonomous Region were analyzed,with respect to the tumor types and change trend.Results(1)The leading 4 types of malignant tumors were cervical cancers, ovarian cancers,endometrial cancers,and malignant trophoblastic tumors according to the constitutive ratios of the tumors.The constitutive ratio of cervical cancer patients rose year by year,from 17.48% during the 1985-1989 period to 49.25% during the 2000-2004 period(P0.05).(2)The occurring age of cervical cancers became younger obviously,from≥60 years old dropped to

Objective To explore the correlation between contrast sensitivity and binocular visual function in indi-viduals with high myopia,aiming to understand how binocular visual function impacts contrast sensitivity in this popula-tion.Methods In this cross-sectional study,a total of 33 patients(66 eyes)with high myopia attending the Optometric Center of Tianjin Eye Hospital from March to December 2020 were included.They were measured for oculomotor parame-ters as well as binocular contrast sensitivity in photopic and scotopic conditions.According to the type of functional indica-tors to which the oculomotor parameters belong,they were divided into two groups:accommodation and vergence.The oculomotor parameters of accommodation include accommodative amplitude(AMP),monocular accommodative facility(MAF)and binocular accommodative facility(BAF);the oculomotor parameters of vergence include distance lateral pho-ria(DLP),near lateral phoria(NLP),positive fusional vergence(PFV),negative fusional vergence(NFV),and vergence facility(VF),among them,PFV includes the near and distance PFV breaking points and recovery points,and NFV includes the near and distance NFV breaking points and recovery points.Pearson bivariate correlation analysis was used to analyze the bivariate correlations between binocular contrast sensitivity and the oculomotor parameters of accommodation and ver-gence.In addition,canonical correlation analysis was used to analyze the associations between binocular contrast sensitivi-ty and accommodation and vergence indicators in patients with high myopia.Results The spherical equivalent refraction of the participants was(-9.51±2.71)D in the left eye and(-10.00±2.88)D in the right eye.The bivariate correlation analysis showed that among the oculomotor parameters of accommodation,the AMP of the left eye was negatively correla-ted with the contrast sensitivity at 18 c·d-1 under scotopic conditions(r=-0.406,P=0.019);among the oculomotor pa-rameters of vergence,the near PFV breaking point was positively correlated with the contrast sensitivity at 1.5 c·d-1(r=0.458)and3.0 c·d-1(r=0.441)under scotopic conditions(both P＜0.05),the near PFV recovery point was positively correlated with the contrast sensitivity at 3.0 c·d-1(r=0.351)and 6.0 c·d-1(r=0.396)under photopic conditions(both P＜0.05)and positively correlated with contrast sensitivity at 1.5 c·d-1(r=0.449),3.0 c·d-1(r=0.537)and 12.0 c·d-1(r=0.375)under scotopic conditions(all P＜0.05),and VF was positively correlated with contrast sensitivity at 1.5 c·d-1(r=0.358)under photopic conditions and 3.0 c·d-1(r=0.458)under scotopic conditions(both P＜0.05).Except for the above indicators,other oculomotor parameters of accommodation and vergence were not correlated with contrast sensitivity(all P＞0.05).Canonical correlation analysis showed that only the oculomotor parameters of vergence were correlated with the first pair of canonical correlation coefficients of contrast sensitivity under scotopic conditions(P=0.008)and the canonical correlation coefficient was 0.912.The oculomotor parameters of convergence U1 were mainly de-termined by NLP,near NFV recovery point,near PFV breaking point,and near PFV recovery point.Contrast sensitivity V,under scotopic conditions was mainly determined by contrast sensitivity at 1.5 c·d-1 and 6.0 c·d-1under scotopic condi-tions.The typical structural analysis showed that there was a strong positive correlation of U1 with near PFV breaking point and contrast sensitivity at 1.5 c·d-1 under scotopic conditions;there was a strong positive correlation of V1 with near PFV breaking point and contrast sensitivity at 1.5 c·d-1 under scotopic conditions.Conclusion There is an association be-tween vergence and contrast sensitivity under scotopic conditions among high myopia patients.Improving binocular visual function may enhance contrast sensitivity and overall visual quality in high myopia patients.

OBJECTIVE：To investigate t he effects of different compatibility ratio of Gardenia jasminoides to fermented soybean on the content of genistein and total flavonoids ，and to investigate the compatibility regularity of Zhizichi decoction. METHODS：The decoction method was used to prepare the mixed decoction with different compatibility ratio of G. jasminoides to fermented soybean （2∶1，1∶1，1∶2，1∶4，m/m，the same hereinafter ）. UPLC-MS/MS method was used to determine the content of genistein in Zhizichi decoction with different compatibility ratio and corresponding fermented soybean single decoction. UV method was used to determine the content of total flavonoids in Zhizichi decoction with different compatibility ratio and corresponding gardenia single decoction and fermented soybean single decoction. RESULTS ：The established method had good linearity ， precision，repeatability，stability and accuracy. Compared with single decoction ，the content of genistein in the mixed decoction with different compatibility ratio of G. jasminoides to fermented soybean （2∶1，1∶1，1∶2，1∶4）was decreased to different extents ， while the content of total flavonoids was increased to different extents. With the increase of fermented soybean ，the content of genistein in the decoction increased at first and then decreased. When the compatibility ratios of G. jasminoides to fermented soybean were 1 ∶ 1 and 1 ∶ 2，the content of genistein in the decoction was the highest （all 0.071 μg/mL）. With the increase of fermented soybean ，the content of total flavonoids in the decoction did not change regularly ；when the ratio of G. jasminoides to fermented soybean was 1 ∶ 1，the content of total flavonoids in the decoction was the highest （1.861 μg/mL）. CONCLUSIONS ： When the compatibility ratio of G. jasminoides to fermented soybean was 1 ∶ 1，the content of flavonoids in the decoction is the highest.