Objective:To prepare and purify recombinant human zona pellucida 3 protein,and to study its immunologic activity.Methods:The E.coli BL21 containing recombinant plasmid pGEX4T-1/ huZP3 was induced to express GST-fusion protein by IPTG.After a series of purification procedure,the purified protein was analyzed by SDS-PAGE.After the mice immunized with rhuZP3,the antibody responses against rhuZP3 were detected by ELISA.Results:The soluble fusion protein was expressed,and purity of rhuZP3 was 95%.Moreover, purity of rhuZP3 could be recognized by anti-human ZP3 in ELISA.Conclusion:The rhuZP3 is obtained through the preparation of prokaryotic expression system and anti-rhuZP3 antibody has immunological activity.

Objective To investigate the transactivating effect of hepatitis C virus(HCV)core protein on inducible nitric oxide synthase(iNOS)gene promoter and the molecular biological mecha- nisms of HCV pathogenesis.Methods Polymerase chain reaction(PCR)technique was employed to amplify the sequence of iNOS promoter by using HepG2 genomic DNA as template,and the product was cloned into pGEM-T vector.The iNOSp gene was cut from T-iNOSp by KpnⅠand XhoⅠ,and then was cloned into pCAT3-Basic,the constructed vector was named as pCAT3-iNOSp,pCAT3-iN- OSp was transfected into the LO_2 cell line.LO_2 cell was also cotransfected with pcDNA3.1(-)-core and pCAT3-iNOSp by FuGENE 6 transfection reagents.The LO_2 cells transfected with pCAT3-Basic was used as negative control.The activity of CAT in LO_2 cells was detected by an ELISA kit after 48 hours,which reflected the transactivating function of HCV core protein to iNOS gene promoter.Re- sults The expressive vector pcDNA3.1(-)-core and report vector pCAT3-iNOSp had been construc- ted and confirmed by restriction enzyme digestion and sequencing.The expression of CAT in LO_2 cells transfected with pCAT3-iNOSp and peDNA3,1(-)-core was 11 times as higher as that of pCAT3-bas- ic,and 6 times as higher as that of pCAT3-iNOSp.Conclusion It is suggested that HCV core protein can transactivate iNOS gene promoter.

Objective To investigate the feasibility of video-assisted thoracoscopic sympathetic trunk clipping in the treatment of craniofacial hyperhidrosis. Methods A total of 10 patients were operated on under general anesthesia with double-lumen endotracheal intubation. The patients were placed in lateral recumbent position with one-lung ventilation. A 7 mm trocar and a 4.5 mm trocar were inserted at the 2~3 intercostal space on the midaxillary line and at the 4~5 intercostal space on the posterior axillary line, respectively, to introduce surgical instruments and thoracoscopic camera. Alongside the sympathetic chain, the sympathetic nerve trunk immediately below the second costal margin was blocked with small-sized titanium clips. Then the lung was inflated and the incision sutured. Afterwards, the procedure in the contralateral hemithorax was performed using the same method. Results The operating time was 55~130 min (mean, 110 min). Symptoms of craniofacial hyperhidrosis disappeared in the 10 patients, all of who were satisfied with curative results. The postoperative hospital stay was 2~3 days. Neither Horner’s syndrome nor other serious complications were observed. Seven of the patients developed slight compensation hyperhidrosis in their chest, abdomen, back or legs. All the patients had normally returned to work and physical exercises in 7~10 days. Postoperative follow-up for 1~9 months (mean,6.3 months) in all the patients found no recurrence. Conclusions Video-assisted thoracoscopic block of sympathetic trunk below the second costal margin for craniofacial hyperhidrosis is safe and effective.

OBJECTIVETo explore the effect differences between twirling-rotating reinforcing and reducing technique of acupuncture on cardiac damage in spontaneous hypertensive rats (SHR).

METHODSSixty male 11-week-old SHR were randomly divided into four groups: a model control group (group A), a twirling-rotating reinforcing technique group (group B), a twirling-rotating reducing technique group (group C) and a needle retaining group (group D), 15 rats in each one. In addition, twelve male 11-week-old Wistar rats were used as a blank control group (group E). Acupuncture was not used in group A and group E, only with grasp, capture and binding stimulation that was also adapted in the rest groups. Rats in the group B were treated with acupuncture at "Taichong" (LR 3) by twirling-rotating reinforcing technique for 1 min and then the needles were retained for 9 min; rats in the group C were treated with acupuncture at "Taichong" (LR 3) by twirling-rotating reducing technique for 1 min and then the needles were retained for 9 min; rats in the group D were treated with acupuncture at "Taichong" (LR 3) but without any technique and then needles were retained for 10 min. Before and after acupuncture, blood pressure monitor was used to measure the rats' systolic pressure and diastolic pressure every 6 days. Twenty-eight days after the treatment, HE and Masson staining were adopted to observe the status of left ventricular hypertrophy and myocardial fibrosis. ELISA method was applied to test the content of endothelin-1 (ET-1). PCR semiquantitative method was used to analyze Type I and III collagen mRNA in the left ventricular.

RESULTS(1) Blood pressure: after the treatment, the systolic pressure and diastolic pressure were both increased in the group A and the group B (P < 0.05); while the two pressures were both lowered in the group C and the group D (P < 0.05), which was more obvious in the group C (P < 0.05). (2) According to HE and Masson staining, except for the group E, the myocardial hypertrophy and fibrosis could be found in the rest groups, in which the group C was the modest, followed by the group D, while the group A and the group B were more severe. (3) Concentration of ET-1: there were differences of concentration of ET-1 among 5 groups (P < 0.05), and the concentration value from high to low was the group A, B, C, D and E. (4) Type I collagen mRNA: the difference of level of Type I collagen mRNA between group C and D was not statistically significant (P > 0.05); compared with the group A and B, the level was lower in the group C; the level was the lowest in the group E. Type III collagen mRNA: the difference between the group A and B was not statistically significant (P > 0.05); compared with the group A, B and D, the level was lower in the group C.

CONCLUSIONThe twirling-rotating reducing could reduce the systolic pressure and diastolic pressure in SHR, effectively prohibit the production of ET-1 and expression of Type I and III collagen mRNA, and it has more obvious inhibiting effect on Type III collagen mRNA. There is biological effect difference between twirling-rotating reinforcing and reducing technique.

Acupuncture Therapy ; instrumentation ; methods ; Animals ; Collagen ; genetics ; metabolism ; Endothelin-1 ; genetics ; metabolism ; Heart Ventricles ; metabolism ; pathology ; Humans ; Hypertension ; genetics ; metabolism ; pathology ; therapy ; Male ; Rats ; Rats, Inbred SHR ; Rats, Wistar

Objective To investigate the protective effect of the extract of ginkgo biloba on myocardial tissue in aged rat.Methods The rats aged 20 months were given the extract of ginkgo biloba (EGB) and ALT-711 respectively by garage for 16 weeks.The aged controls and adult rats were infused with the same volume saline.The superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), malonyt diadehyde (MDA) in blood samples and advanced glycation end products (AGEs) in the myocardial tissue were measured .The myocardial histopathological changes under electron microscope and the mitochondrial DNA (mtDNA) deletion rate in myocardial cells were observed.Results Compared with the adult rats, the content of AGEs in myocardial cells in aged rats was significantly increased [(33.5±1.3)AU/mgHYP us.(18.1±1.2)AU/mgHYP, t= 7.18,P<0.05] and the levels of SOD and GSH-PX in blood samples were decreased [(138.4±3.8) U/mlvs.(227.7±13.8)U/ml, (1283.8±28.8) U/ml vs.(2114.1 ±135.9)U/ml, t=-19.59, -18.79;both P<0.01].The MDA level in the serum and mtDNA deletion rate in aged rats were higher than in adult rats[(6.7±0.6) mmol/ml vs.(4.1±1.0) mmol/ml, (0.18054±0.0718) % vs.( 0.0060±0.0001)%, t=7.18,6.98;both P<0.05].Compared with the aged controls, the content of AGEs in myocardial cells, the level of MDA and mtDNA deletion rate were significantly decreased in EGB and ALT-711 treatment adults (all P<0.05).The SOD and GSH-PX in blood samples were increased in EGB and ALT-711 treatment adults (all P<0.05).Conclusions Nonenzymatic glycation may play an important role in myocardial aging, which may be amplified by oxidative stress.EGB and ALT-711 may have the same anti-aging effects by inhibiting nonenzymatic glycation and oxidative stress.

Objective To investigate the clinical value of Fluorine-18-fluorodeoxyglucose (18 F-FDG) PET/CT examination to predict the prognosis of patients after colorectal cancer operation.Methods The retrospective cross-sectional study was adopted.The clinicopathological data of 80 patients with colorectal cancer who were admitted to the First Hospital of Nanjing Medical University from March 2007 to October 2015 were collected.Eighty patients received first preoperative 18 F-FDG PET/CT examination and underwent operations under decisions of patients and their families,and then adjuvant chemotherapy were performed according to the patients' condition.Observation indicators included:(1) preoperative imaging examination,(2) situations of treatment and follow-up,(3) analysis of prognostic factors.The patients were followed up by outpatient examination and telephone interview once every 3 months within postoperative 1 year,once every half a year within postoperative 2 years and then once a year up to May 2016.The follow-up included tumor recurrence or progression and survival of patients.Tumor-free survival time was from postoperative day 1 to tumor recurrence or progression and death or end of follow-up.Overall survival time was from postoperative day 1 to death or end of follow-up.Measurement data with skewed distribution were represented as M (Qn) and M (range).The optimal cutoff point of tumor-free survival of maximum standardized uptake value (SUVmax),mean standardized uptake value (SUV),metabolism of volume (MTV) and total lesion of glycolysis (TLG) were investigated using the ROC curve analysis,and calculating area under the curve (AUC).The median was used as a cutoff point if there was smaller AUC.The Kaplan-Meier method and Log-rank test were respectively used for survival analysis and univariate analysis,and COX proportional hazards model for multivariate analysis.Results (1) Results of preoperative imaging examination:results of PET/CT in patients with colorectal cancer showed there were circumscribed thickening of bowel wall,intestinal cavity strictures,fuzzy fat space around the some lesions,enlarged lymph node and 18 F-FDG uptake increased abnormally.The SUV SUV MTV and TLG of 80 patients were 11.83(4.26,35.42),7.06(2.38,20.92),20.47 cm3 (1.29 cm3,161.50 cm3) and 138.58 (14.17,857.89),respectively.ROC curve showed that the AUC of SUV SUV MTV and TLG were 0.453,0.448,0.815 and 0.749 [95％ confidence interval (CI):0.307-0.600,P ＞0.05;0.303-0.594,P ＞ 0.05;0.717-0.913,P ＜ 0.05;0.635-0.863,P ＜ 0.05],respectively.The median SUVmax (11.83) and SUV (7.06) were used as the cutoff points due to smaller AUC of SUVmax and SUV.The cutoff point of MTV was 18.79 cm3 (sensitivity =86.2％ and specificity =68.3％),and the cutoff point of TLG was 142.05 (sensitivity =75.9％ and specificity =70.7％).(2) Situations of treatment and follow-up:among 80 patients,13 underwent the radical resection of colorectal cancer and resection of liver metastasis and 67 underwent radical resection of colorectal cancer.Sixty-two patients received postoperative chemotherapy,including 45 with chemotherapy regimens of capecitabine and 17 with fluorouracil.Eighty patients were followed up for 41.8 months (range,6.5-109.1 months).During the follow-up,29 patients had tumor recurrence or progression,and 19 patients were dead.The median tumor-free survival time,1-,3-and 5-year tumor-free survival rates in 80 patients were 19.5 months (range,2.0-109.1 months),73.7％,36.3％ and 18.8％,respectively.The median overall survival time,1-,3-and 5-year overall survival rates were 31.8 months (range,3.3-109.1 months),76.3％,37.5％ and 20.0％,respectively.(3) Analysis of prognostic factors:results of univariate analysis showed that tumor location,N staging,M staging,clinical staging,postoperative chemotherapy,MTV and TLG were relative factors affecting postoperative tumor-free survival rate (HR =3.469,5.325,5.295,8.605,2.630,7.388,5.155,95％ CI:1.522-7.906,2.256-12.568,2.405-11.657,2.969-24.937,1.063-6.504,2.550-21.403,2.178-12.204,P ＜ 0.05).The tumor location,tumor differentiation,N staging,M staging,clinical staging,MTV and TLG were relative factors affecting postoperative overall survival rate (HR =2.697,2.814,3.083,2.916,4.193,5.450,4.876,95％ CI:1.011-7.197,1.121-7.062,1.166-8.149,1.140-7.454,1.386-12.678,1.581-18.786,1.727-13.766,P ＜ 0.05).In multivariate analysis,stage Ⅲ-Ⅳ of clinical staging and TLG≥ 142.05 were independent risk factors affecting postoperative tumor-free survival rate (HR =9.879,3.569,95％ CI:1.854-22.836,1.127-11.306,P＜0.05).The stage M1,stage Ⅲ-Ⅳ of clinical staging and TLG≥ 142.05 were independent risk factors affecting postoperative overall survival rate (HR =4.522,9.315,10.120,95％ CI:1.223-16.717,1.338-24.864,2.385-12.947,P＜0.05).Conclusion TLG through 18 F-FDG PET/CT examination is an independent prognostic factor affecting postoperative tumor-free survival rate and overall survival rate in patients with colorectal cancer after curative resection,and it has certainly reference value for prognosis.

Objective To investigate the effects of recombinant human erythropoietin(rhEPO)on expressions of tumon necrosis factor-alpha(TNF-α) and inter leukin-6(IL-6) in rats after focal cerebral ischemia and to explore its neuroprotective mechanism.Methods A total of 36 healthy male SD rats were randomly divided into sham-operated group (n=12),model group (n=12) and rhEPO treatment group (n=12).The suture method to make permanent middle cerebral artery occlusion model was adopted.rhEPO treatment group was injected with rhEPO 5000 U/kg intraperitoneally after 2 h of ischemia,whereas model group and sham-operated group were given identical saline at the same time.All rats were decapitated after 24 h of ischemia.6 rats were randomly selected in each group and the infarct volume of groups were measured by Triphenyl tetrazolium chloride (TTC)staining method.The expressions of TNF-α,IL-6 in other rats were detected by immunohistochemistry.Results No infarction was found in sham-operated group.Percentage of infarct volume in model group and rhEPO group were (36.672.40)％ and (27.49± 1.47)％,respectively.Compared with the model group,the volume of infarction in rhEPO group was significantly reduced.Cells stained by immunohistochemistry showed that The numbers of TNF-α-positive cells in the 3 groups were 9.001.41,27.83±2.48,17.50±1.87 and IL 6 positive cells were 8.94±2.31,20.33±3.53,14.83±1.70,respectively.Compared with sham operated group,the expressions of TNF-α and IL 6 in model group were significantly increased (q=16.1,19.6,P＜0.01).Compared with the model group,the expressions of TNF α and IL-6 in rhEPO group were significantly decreased (q=8.19,3.44,all P＜0.01).Conclusions rhEPO can decrease the infarct volume in SD rats after acute focal cerebral ischemic injure.rhEPO might exert its neuroprotective effect by reducing the expressions of TNF α and IL-6.

Objective To explore the role of Th17 cells in systemic lupus erythematosus (SLE)with cardiovascular disease (CVD).Methods 61 patients of SLE were collected from September 2011 to March 2013 in the Second Hospital of Hebei Medical University by revised SLE classification standards of ACR in 1997.These patients were divided two groups:22patients of SLE with CVD and 39 patients of SLE without CVD;the control group include 20 healthy.Th17 cells were measured by flow cytometry,IL-1 7 was detected by enzyme-linked immunosorbent assay.The correlation among them and the disease active index were analyzed.Results ①The percent of Th1 7 cells in the group of SLE with CVD,that in the group of SLE without CVD and that in control group were (2.09±0.98)%,(1.75±0.75)% and (0.89±0.44)%,respec-tively.The percent of Th1 7 cells in healthy group were lower than that in SLE with CVD and SLE without CVD group (t=4.717~5.030,P<0.001).The level of IL-17 in the group of SLE with CVD,that in the group of SLE without CVD and that in control group were 85.64±20.76 pg/ml,75.25±28.14 pg/ml and 35.06±6.58 pg/ml respectively,and the serum of IL-17 in healthy group were lower than in SLE with CVD and SLE without CVD group (t=6.275~9.954,P<0.001). There were no significant difference of Th1 7 cells and IL-1 7 between SLE with CVD and SLE without CVD groups (t=1.520,P>0.05;t=1.513,P>0.05).②The level of IL-17 were correlated positively with SLEDAI and the anti-double strand DNA (r=0.393,P=0.008;r=0.558,P<0.001),were correlated negatively with complement C3 (r=-0.423,P=0.005).The percent of Th17 cells in CD4+T cells were correlated positively with SLEDAIand the anti-double strand DNA (r=0.681,P<0.001;r=0.492,P=0.015)were correlated negatively with complement C3 (r=-0.534,P=0.027).Con-clusion The level of Th1 7 cells and IL-1 7 were high in SLE,and they were related with the disease activity.The cardiovas-cular factor had not affect the expression of Th1 7 cells and IL-1 7 in SLE.

Objective To explore the expression and the role of Toll-like receptor(TLR3 and TLR4) in rats with nephrotic syndrome induced by respiratory syncytial virus(RSV).Methods SD rats were inoculated intranasally and intraperitoneally with 6?106 plaque for-ming unit(PFU) RSV to construct RSV-induced nephropathy in rat model.Rats were anesthetized and blood was withdrawn from cardiac on day 4,14,30,60 after inoculation.The normal ones without intervention were set as control group.The renal histology was observed by light microscope and electron microscope.The urinary protein collected in 24 hours were measured.Meanwhile,the expressions of TLR3 and TLR4 were detected by indirect immunofluorescence staining and flow cytometry in peripheral blood mononuclear cells of rats.The results were analyzed by SPSS 13.0 softwore.Results After inoculation,the proteinuria increased and under the electron microscope the foot processes of glomerular epithelial cells were fused which resembled human minimal change nephrotic syndrome.Proteinuria reached the peak and the fusion of foot processes were most extensive in rats of RSV at 60 d.The expressions of TLR3 and TLR4 in each group of RSV-induced nephropathy in rat models were significantly higher than those in normal control group(Pa0.05).Conclusions TLR3 and TLR4 in peripheral blood mononuclear cells of RSV-induced nephropathy rat mo-dels had being significantly activated until 60 d after RSV inoculation.TLR signaling pathway may play an important role in nephrotic syndrome of rats induced by RSV.

Objective To observe the effects of infrasound(IS)of different frequencies and intensities on the pathological morphology of the gastric mucosa(GM)in rats.Methods One hundred and forty male Sprague- Dawley rats were randomly and evenly divided into a control group(group A),an 8 Hz 90 dB group(group B),an 8 Hz 130 dB group(group C)and a 16 Hz 130 dB group(group D).IS with these frequencies and intensities was administered daily for 2 h to all groups except group A,which received sham infrasound.The other 70 rats were ran- domly and evenly divided into a second control group(group E)and an exposure group(group F),in which the rats were continuously stimulated by IS at 8 Hz and 130 dB for 2 h a day for 14 d.The pathological morphology of the GM in each group was observed at 1 d,7 d,14 d,21 d and 28 d after IS exposure.Results①Compared with group A,GM lesion scores were significantly increased in groups B,C and D at 1 d,7 d,14 d,21 d and 28 d(P＜0.01),but not in group B at 1 d.②Compared with group B,the GM lesion scores in group C were obviously in- creased at 1 d,7 d,14 d,21 d and 28 d,while scores were also obviously improved in group C in comparison with those in group D at 14 d,21 d and 28 d(P＜0.01).③The GM lesion scores in group F decreased gradually after IS,but were still higher than those in group E at 1 d,7 d,14 d,21 d and 28 d after IS.④The ultrastructures of the chondrosome and endocytoplasmic reticulum in GM cells were deformed after 8 Hz and 16 Hz IS.Conclusion 8 Hz 90 dB,8 Hz 130 dB and 16 Hz 130 dB IS can all result in GM damage in rats.The injury severity was closely related to the frequency,intensity and duration of the IS.Rats can adapt to IS after several exposures,and the damage tends to recover automatically.