The E. coli BL21(DE3) strain bearing the plasmid(T_7lac/His6-tag) with the h?TNF?recombinant gene was grown in LB medium containing SO?g/ml kanamycin, at 37 ℃ , up to the late logarithmic phase(OD590 ~ 0.5)then induced with IPTG (final concentration lmmol/L)for 5 hours. SDS-PAGE analysis revealed that expression level of the product(His6-?TNF?was up to 45% of the total bacterial proteins and was mainly as insoluble inclusion bodies ( IBs) . After cell lysis, the IBs was separated by centrifugation, dissolved in 7mol/L urea, then purified by Ni column ( Ni~(2+) -Sepharose 6B) . And the purity of more than 95% and the recovery of about 90% were obtained. The purified product was refolded with the renaturation buffer under low temperafure(

Objective To discuss the diagnostic value of 3D-ultrasound for cord entanglement in monoam-niotic twins. Methods From November 2013 to March 2016,a total of 65 monoamniotic twinsunderwent routine obstetric ultrasonography to screen fetal abnormalities in our hospital. The cord was meticulously observedusing a systematic continuous sequence approach. Fetuses who showed cord entanglement by 2-D ultrasound or 3-D flow Doppler were all recruited. The gestational weeks,fetal malformation,ultrasonic image feature of cord entangle-ment,and clinicaloutcome were recorded and analyzed retrospectively. Results 4 monoamniotic twins showed cord entanglement. Three-dimensional color Doppler clearly delineated thefeatures of entanglement. At following-up of 8 fetuses,3 fetuses were found intrauterine fetal death and 3 fetuses with structural abnormalities except for 2 well infants.Conclusion Cord entanglement may threaten the safety of fetusesin monoamniotic pregnancies.In aid of 2-D ultrasound or 3-D flow Doppler in monoamniotic twins can help to find it in time ,monitor it carefully and provide evidence for obstetric treatment .

Objective To study the effect of chloride channel blocker(niflumic acid,NFA) on pulmonary hypertension induced by high pulmonary blood flow in rats.Methods Fifty male or female Sprague-Dawley rats were randomly divided into 5 groups:normal group,sham group,model group,drug 1 group,and drug 2 group,with 10 rats in each group.After subjected to an abdominal aorta-inferior vena cava shunt,all the rats were reared under the same condition for 11 weeks.Then,mean pulmonary artery pressure(mPAP) and right ventricular hypertrophy index(RVHI) of each rat were measured.In addition,arterial wall area/vessel area (W/V) and arterial wall thickness/vessel external diameter(T/D) of each rat were also measured.Results 1.The mPAP of model group [(25.79 ± 4.03) mmHg,1 mmHg =0.133 kPa] was significantly higher than those of normal group [(16.48 ± 1.70) mmHg],sham group [(17.03 ± 2.01) mmHg],drug 1 group [(21.78 ± 2.77) mmHg] and drug 2 group [(20.31 ± 2.15) mmHg] (F =18.983,P ＜0.01).Although the mPAP of drug 1 group was a little higher than drug 2 group,there was no significant difference (P ＞ 0.05).Compared with normal group and sham group,the mPAP of drug 1 group and drug 2 group increased(P ＜0.01,respectively).2.The W/V and T/D of model group were significantly higher than those of normal group,sham group,drug 1 group and drug 2 group (F =26.135,15.527,all P ＜ 0.001).The W/V and T/D of two drug groups showed no significant difference,but they were higher than those of normal group and sham group (P ＜ 0.01,respectively).Conclusions Chloride channel blocker NFA partly decrease mPAP of pulmonary hypertension indnced by high pulmonary blood flow in rats,and inhibit proliferation of vascular smooth muscle cells.These results suggest that NFA had part of therapeutic effect to pulmonary hypertension induced by high pulmonary blood flow.

Objective To study the pulmonary interstitial changes in pulmonary hypertension induced by high pulmonary blood flow.Methods Sixty-five male or female Sprague-Dawley rats (180-230 g) were used and randomly divided into 3 groups:normal group (n =20) ; sham group (n =20),only exposing the abdominal aorta and inferior vena cava about 10-20 minutes;model group(n =25),rats in this group were subjected to an abdominal aorta-inferior vena cava shunt to create animal models of high pulmonary.After operation,all the rats were reared under the same conditions for 11 weeks.Then,the systolic pulmonary artery pressure (sPAP) and mean pulmonary artery pressure (mPAP) of every rat were determined by means of homemade right heart catheterization.After that,the right ventricle (RV) was separated from the left ventricle (LV) and septum (S),then weighed.And right ventricular hypertrophy index (RVHI) was measured by the ratio of RV to LV + S [RV/(LV + S)].In addition,the morphological changes of pulmonary interstitial of rats were observed under optical microscope by means of hematoxylin-eosin (HE) staining.In the end,single pulmonary artery smooth muscle cell (PASMC) was isolated through acute enzyme separation.Then membrane capacitance (Cm) was recorded through in the method of patch clamp technique.Results 1.Compared with sham group and normal group,the sPAP,mPAP and RVHI of model group increased significantly(F =17.293,16.259,12.878,all P ＜ 0.01).2.In contrast to sham group and normal group,arterial wall area/vessel area(W/V) and arterial wall thickness/vessel external diameter(T/D) in model group increased significantly(F =85.717,22.795,all P ＜0.01).3.The membrane capacitance of model group was bigger than that of sham group and normal group(F =8.704,P ＜ 0.01).4.mPAP was positively correlated with W/V,T/D and Cm (r =0.669,0.662,0.663,all P ＜ 0.01).Conclusions Shunts from abdominal aorta-inferior vena cava in SD rats caused high pulmonary blood flow-induced pulmonary hypertension,and these rats appeared with pulmonary smooth muscle cells hypertrophy,pulmonary vascular wall thickening and inflammatory cells infiltration.