Aim To investigate the effects of lentivirus mediated nerve growth factor ( NGF) gene silencing on pheochromocytoma cells ( PC12 ) and the possible mechanisms .Methods The NGF shRNA expression vector was constructed .PC12 cells were randomly divi-ded into five groups (n=3 each) as follows: negative control group ( NC ) , control lentivirus group ( LV CON) , lentivirus NGF shRNA1 group ( LV shNGF1 ) , lentivirus NGF shRNA2 group(LV shNGF2), lentivir-us NGF shRNA3 group(LV shNGF3).The cells in NC group were cultured in DMEM/HG and polybrene me-dium, while others were cultured in DMEM/HG, poly-brene and corresponding lentivirus medium .After the treatment, the infection efficiency was determined by fluorescent microscope .Relative expression of NGF , extracellular signal-regulated kinase ( ERK1/2 ) and p-ERK1/2 were assessed by Western blot .The expres-sion of NGF mRNA was analyzed by quantitative re-verse transcription polymerase chain reaction ( qRT-PCR) .The differentiation degree was valued according to the length of neuritis and max diameter of cells .The cell viability was detected by CCK-8.Results The in-fection efficiency in PC12 cells reached over 90%. Compared with NC group , the relative expression of NGF mRNA and NGF protein was significantly down-regulated ( P<0.05 ) .There was no difference in the expression of ERK1/2 protein and cell viability .The expression of p-ERK1/2 protein was markedly down-regulated in LV shNGF3 group ( P<0.01 ) .The cells morphology was changed , and the length of neuritis and max diameter of cells were strained in LV shNGF 3 group than those in NC group ( P<0.01 ) .Conclusion Lentivirus-mediated NGF gene silencing inhibits the differentiation of PC12 cells through suppressing the activation of ERK1/2.

Purpose To explore the clinical value of reducing the incidence of reflux and improving patients' comfort by low pressure contrast injection during transvaginal ultrasound real time three-dimensional hysterosalpingo-contrast sonography (TVS RT-3D-HyCoSy).Materials and Methods Data ofTVS RT-3D HyCoSy using low pressure contrast injection of 498 infertile women were analyzed.The peak pressure,the incidence of reflux and adverse reactions were recorded.Compared these parameters between bilateral tubals.Results The peak pressure in patency,partially patency and fully obstructed of bilateral tubals were (22.47±5.96) kPa,(37.24±8.83) kPa and (44.64±7.73) kPa.There was significant difference among the three groups (F=70.35,P＜0.05).Among all 498 patients,the incidence of reflux was 26.31％.The incidence in patency,partially patency and fully obstructed of bilateral tubals were 18.00％,30.21％ and 43.59％.There was significant difference among the three groups (x2=15.59,P＜0.05).Different degrees of adverse reactions were 88.96％,10.64％ and 0.4％.Conclusion By using low pressure contrast injection during HyCoSy,the incidence of reflux can be reduced and examination comfort can be improved.

Objective To investigate the effect of intrathecal morphine preconditioning on the expression of nerve growth factor (NGF) in the dorsal root ganglia (DRG) in a rat model of myocardial ischemia-reperfusion (I/R).Methods Thirty healthy adult male Sprague-Dawley rats in which intrathecal catheters were successfully placed without complications,weighing 250-350 g,were randomly divided into 5 groups (n =6 each) using a random number table:sham operation group (S group),I/R group,intrathecal morphine preconditioning group (ITMP group),μ receptor antagonist CTOP + intrathecal morphine preconditioning group (CTOP + ITMP group),and CTOP control group (CTOP group).Myocardial ischemia was induced by 30 min of occlusion of the anterior descending branch of the left coronary artery followed by 120 min of reperfusion in all the groups except S group.Intrathecal morphine preconditioning was produced by 3 cycles of 5 min intrathecal injection of morphine 3 μg/kg (10 μl) at 5 min intervals within 30 min before ischemia in ITMP group.In CTOP+ITMP and CTOP groups,1 μg/μ1 CTOP 10 μl was injected intrathecally at 10 min before morphine preconditioning and 40 min before ischemia,respectively.At 120 min of reperfusion,the rats were sacrificed,and myocardial specimens were obtained for determination of myocardial infarct size,and DRGs were removed for determination of the expression of NGF by using immunohistochemistry and Western blot.Results Compared with S group,the myocardial infarct size was significantly increased,and the expression of NGF in DRGs was significantly up-regulated in I/R group (P＜0.05).Compared with I/R group,the myocardial infarct size was significantly decreased,and the expression of NGF in DRGs was significantly down-regulated in ITMP group (P＜ 0.05),and no significant change was found in the parameters mentioned above in CTOP group (P＞0.05).Compared with ITMP group,the myocardial infarct size was significantly increased,and the expression of NGF in DRGs was significantly up-regulated in CTOP+ITMP and CTOP groups (P＜0.05).Conclusion The mechanism by which intrathecal morphine preconditioning reduces myocardial I/R injury is related to activation of spinal μ receptors,inhibition of NGF expression in DRGs,and reduction of responses to noxious stimulation in the rats.

Objective To investigate the effect of intrathecal morphine preconditioning (ITMP) on the excitability of substantia gelatinosa (SG) neurons in the dorsal horn of the spinal cord in a rat model of myocardial ischemia-reperfusion (I/R).Methods Thirty-six adult male Sprague-Dawley rats,weighing 200-300 g,in which intrathecal catheters were successfully placed without complications,were randomly divided into 3 groups (n =12 each) using a random number table:sham operation group (group S),group I/R,and group ITMP.Myocardial I/R injury was produced by occlusion of the left anterior descending branch of the coronary artery for 30 min followed by 120 min reperfusion.In group ITMP,the rats received intrathecal morphine 3 μg/kg (10 μl) by three cycles of 5 min infusions interspersed with 5 min infusion-free periods starting from 30 min before ischemia,and the equal volume of normal saline was given instead of morphine in group I/R.At 10 min of reperfusion,6 rats randomly selected in each group were sacrificed,and the T2-6 segments of the spinal cords were acutely isolated to prepare spinal cord slices.The resting potential,threshold of action potential (APT),peak of action potential (APP) and action potential duration in SG neurons in the dorsal horn of spinal cord slices were determined using the whole-cell patch-clamp technique,and the number of action potentials evoked by currents of 40,60,80 and 100 pA was recorded.At 120 min of reperfusion,6 rats randomly selected in each group were sacrificed,and myocardial specimens were obtained for determination of myocardial infarct size (IS) and area at risk (AAR),and IS/AAR ratio was calculated.The expression of c-fos in the T2-5 dorsal horns of the spinal cords was detected by Western blot.Results Compared with group S,the IS/AAR ratio was significantly increased,the expression of c-fos was up-regulated,the number of action potentials in SG neurons in dorsal horns of spinal cord was increased,APT was decreased,and APP was increased in group I/R (P＜0.05).Compared with group I/R,the IS/AAR ratio was significantly decreased,the expression of c-fos was down-regulated,the number of action potentials in SG neurons in dorsal horns of spinal cord was decreased,APT was increased,and APP was decreased in group ITMP (P＜0.05).Conclusion The mechanism by which ITMP attenuates myocardial I/R injury is related to decrease in the excitability of SG neurons in the dorsal horn of the spinal cord and reduction of responses to nociceptive stimuli in rats.

Objective To investigate the parental origin for a rare case of complete hydatidiform mole and coexisting fetus and to discuss its diagnosis and differential diagnosis.Methods Tissues from the fetus,mole and placenta were collected and pathology analysis and chromosome analysis were done.The DNA from the fetus,mole and parents' peripheral blood leukocytes was amplified with five short tandem repeat (STR) markers (D4S2460,D18S488,D21S2039,DXS1205 and DYS219) at the same time to confirm the parental source of the hydatidiform.Results (1) Casereport:A 27-year-old woman,gravida 1,para 0,was found high risk for neural tube defects at 20 weeks of gestation.At 24+5 weeks of gestation,ultrasound examination demonstrated a normal fetus,a normal placenta and a huge mass with a multicystic appearance attached to the placenta with an obvious demarcation.The fetus died at 26 weeks of gestation.Serum human chorionic gonadotropin-β(β -hCG) level decreased obviously during the first two weeks after artificial induction,but elevated at the third week,and β-hCG titers fell to normal after 2 courses of chemotherapy.Fetus autopsy showed no structure abnormality.Histopathologic examination of the hydatidiform showed swelling of chorionic villi with hyperplasia of the trophoblast and formation of central cisterns suggesting of a twin pregnancy consisting of a complete hydatidiform mole and coexisting fetus.(2) Genetic analysis:The karyotype analysis of the normal placental villi was 46,XY; the cell cultures of fetal cartilage tissue and hydatidiform were failed.STR analysis showed that the fetus was diploid from biparental source;the mole was androgenetic source.And the mole had locus both from Y and X chromosome of the father,so it was heterozygous.It was suggested that this case was derived from one single oocyte fertilized with three spermatozoas.Conclusions STR analysis could be used to confirm the diagnosis of complete hydatidiform mole and coexisting fetus and to find the pathogenetic rnechanism.

Objective To investigate the eftect of radiation dose of dual-source computed tomography (CT) dual energy single-phase enhanced scan in patients with esophageal cancer.Methods The prospective study was conducted.The clinicopathological data of 56 patients with esophageal cancer who were admitted to the Lishui Hospital of Zhejiang University between January 2015 and December 2016 were collected.All the patients were divided into the experimental group (undergoing dual-source CT dual energy single-phase enhanced scan) and control group (undergoing dual-phase CT enhanced scan) bv randomised block method.TNM classification of esophageal cancer (Seventh Edition) published by American Joint Committee on Cancer (AJCC) was used as a standard TNM staging.Two observers independently read films.All the patients underwent radical resection of esophageal cancer or palliative surgery,and then received adjuvant radiochemotherapy.Follow-up using outpatient examination and telephone interview was performed to detect postoperative survival of patients up to March 2017.Observation indicators:(1) consistencies of T staging,N staging and M staging;(2) accuracies of T staging,N staging and M staging (pathological results as a gold standard);(3) radiation dose of CT scan;(4) treatment and follow-up situations.The Kappa test was used for evaluating the consistency,κ≥0.75 as a good consistency,0.40≤κ＜0.75 as a normal consistency and κ＜0.40 as a poor consistency.Comparisons of count data and ratio were done by the chi-square test.Comparisons of measurement data were analyzed by the t test.Results A total of 50 patients were enrolled in the study,including 25 in the experimental group and 25 in the control group.(1) Consistencies of T staging,N staging and M staging:all the 50 patients finished successfully CT scans.Two observers considered that consistencies of T staging,N staging and M staging in the 2 groups were normal (κ =0.452,0.618,0.729,P＜0.05).Consistencies of N staging and M staging were superior to T staging.(2) The pathological results were used as a gold standard.Accuracies of T staging,N staging and M staging in the experimental and control groups were 72％,76％ and 88％,84％ and 92％,88％,respectively,with no statistically significant difference between the 2 groups (x2 =0.10,0.37,0.50,P＞0.05).(3) Radiation dose of CT scan:volume CT dose index (CTDIvol),dose length production (DLP) and effective radiation dose (E) were (10.35±2.01) mGy,(400.63± 34.13) mGy · cm,(5.61 ± 0.47) mSv in the experimental group and (3.55 ± 0.60)mGy,(140.66± 10.89) mGy · cm,(1.98±0.17) mSv in the control group,respectively.There were statistically significant differences in CTDIvol and E between the 2 groups (t =16.23,36.30,P＜0.05).(4) Treatment and follow-up situations:of 50 patients,43 patients received treatments,including 32 undergoing radical resection (11 receiving postoperative adjuvant chemotherapy),6 undergoing palliative surgery,3 receiving single radiotherapy and 2 receiving single chemotherapy.Thirty-six of 43 patients were followed up for 3-18 months,with a median time of 6 months.During follow-up,1-year survival rate was 61.1％.Conclusion Dual-source CT dual energy single-phase enhanced scan in patients with esophageal cancer cannot reduce accuracy of TNM staging,but decreased effectively radiation dose.

Objective:To clone the human cell death-inducing DFF45-like effectors 3(CIDE3) full length cDNA for construction the eukaryotic expression vector pcDNA3.l(+)-CIDE3 and to study its bioactivity.Methods:① Total RNA was extracted from human white adipose tissues and the desired cDNA fragment was obtained by RT-PCR.After the fragment had being inserted into an eukaryotic expression vector pcDNA3.l(+),the resulted recombinant plasmid pcDNA3.l(+)-CIDE3 was transformed into DH5?.The positive clone was selected and confirmed to contain full length of CIDE3 cDNA by agarose gel and DNA sequence analysis.②After the pcDNA3.l(+)-CIDE3 plasmid was transfected into HeLa cells under lipofectamine mediation,the effect of target gene expression on growth of HeLa cells was analysed by TUNEL staining. Results:① The recombinant eukaryotic expression plasmid pcDNA3.l(+)-CIDE3 was constructed successfully and the sequence of CIDE3 was consistent with that of genebank.②After transfected pcDNA3.l(+)-CIDE3,HeLa cells presented distinguished apoptosis(about 15%),compared with that of transfected plasmid pcDNA3.l(+)(

Aim To evaluate the effects of morphine preconditioning ( MPC ) on the expression of microR-NAs ( miRNAs ) induced by hypoxia-reoxygenation (H/R) in H9c2 myocardial cells. Methods H9c2 cells were randomly divided into 3 groups ( n=4 each) as follows:control group ( CON) , hypoxia/ reoxygen-ation group ( H/R ) and morphine preconditioning group ( MPC+H/R) . The cells were cultured in nor-mal condition in CON group. The cells were subjected to 5 h hypoxia followed by 1 h reoxygenation in H/R group and MPC+H/R group. Specifically, the cells in MPC+H/R group were preconditioned with morphine with the final concentration of 1 μmol·L-1 for 10 min before H/R. After the treatment, CCK-8 was used to detect cell viability and chemical colorimetry was used to detect lactate dehydrogenase ( LDH ) activity in the culture medium. Cell apoptosis was assessed by An-nexin-V-FITC/PI flow cytometry. Relative expression of Fas protein was detected by Western blot. The ex-pression of miRNA in myocardial cells was analyzed by quantitative reverse transcription polymerase chain re-action ( qRT-PCR ) . Results Compared with CON group, the cell viability was significantly decreased, while the LDH activity, apoptotic rate and Fas protein expression were dramatically increased in group H/R (P<0. 01). However, MPC significantly increased the cell viability, whereas it decreased the LDH activity, apoptotic rate and Fas protein expression induced by H/R injury ( P < 0. 01 ) . The expressions of miR-133a-5p, miR-133b-5p, miR-664-1-5p, miR-6216 and let-7 e-5 p were markedly down-regulated by H/R as compared to CON group ( P <0. 05 ) , while MPC inhibited these miRNAs which were significantly down-regulated by H/R group ( P <0. 01 ) . Conclusion Morphine preconditioning might protect H9 c2 myocar-dial cells against H/R injury by regulating the expres-sion of miRNAs such as miR-133a-5p, miR-133b-5p, miR-664-1-5p, miR-6216 and let-7e-5p.

Objective To evaluate the role of microRNA-133b-Sp (miR-133b-Sp) in apoptosis hypoxia/reoxygenation (H/R) induced by in rat cardiomyocytes.Methods Rat myocardial cell line H9c2 was cultured in DMEM/F12 culture medium supplemented with 10％ fetal bovine serum.The cells were seeded in 96-well or 6-well plates and randomly divided into 4 groups (n=64 wells each):control group (group C);group H/R;miR-133b-5p mimic + H/R group (group M+H/R);miR-133b-Sp negative control + H/R group (group NC+H/R).The cells were exposed to 95％ N2-5％ CO2for 5 h at 37 ℃ followed by 1 h reoxygenation in DMEM/F12 culture medium supplemented with 10％ fetal bovine serum in all the groups except group C.The cells were cultured in normal culture atmosphere in group C.In M+H/R and NC+H/R groups,the cells were transfected with miR-133b-5p mimic (final concentration 30 nmol/L) and miR-133b-5p negative control (final concentration 30 nmol/L),respectively,for 24 h before H/R.Total RNA was extracted from cells to detect the expression of miR-133b-5p using quantitative real-time PCR.The cell viability (by CCK-8) and lactic dehydrogenase (LDH) activity in the culture medium were detected.Cell apoptosis was assessed by Annexin V/PI flow cytometry.Apoptotic rate was calculated.Result Compared with group C,the cell viability was significantly decreased,and the LDH activity and apoptotic rate were increased in H/R,M+H/R and NC+H/R groups,the expression of miR-133b-5p was down-regulated in H/R and NC+H/R groups,and the expression of miR-133b-Sp was up-regulated in group M+H/R.Compared with group H/R,the cell viability was significanttly increased,the LDH activity and apoptotic rate were decreased,and the expression of miR-133b-5p was up-regulated in group M+H/R,and no significant change was found in the parameters mentioned above in group NC+H/R.Conclusion H/R in rat cardiomyocytes can induce cell apoptosis possibility through down-regulating the expression of miR-133b-5p

Objective To evaluate the effect of morphine preconditioning on the expression of miR-133b-Sp and Fas in rat cardiomyocytes subjected to hypoxia/reoxygenation (H/R).Methods Cardiomyocytes were isolated from healthy adult male Sprague-Dawley rats by using Langendorff perfusion.The cells were seeded into 24-well plates or 60 mm diameter dishes and randomly divided into 3 groups (n =24 each) using a random number table:control group (group C),group H/R,and morphine preconditioning group (group MPC).The cells in group C were cultured in normal culture atmosphere.In H/R and MPC groups,the cells were exposed to 95％ N2-5％ CO2 for 90 min followed by 120 min reoxygenation.In group MPC,the cells were cultured for 10 min in serum-free DMEM liquid culture medium containing morphine 1 μmol/L,and then were cultured for 30 min in morphine-free DMEM liquid culture medium before hypoxia.At 120 min of reoxygenation,the cells in 24-well plates were selected to detect the cell viability (by MTT),lactate dehydrogenase (LDH) activity in the culture medium,and cell apoptosis (by Hoechst 33234 staining).Apoptosis rate was calculated.Total RNA and protein were extracted from the cells in 60 mm dishes to detect the expression of miR-133b-5p and Fas mRNA (by quantitative real-time PCR) and Fas protein (by Western blot).Results Compared with C group,the cell viability was significantly decreased,LDH activity and apoptosis rate were increased,the expression of miR-133b-Sp was down-regulated,and the expression of Fas mRNA and protein was up-regulated in H/R group.Compared with H/R group,the cell viability was significantly increased,LDH activity and apoptosis rate were decreased,the expression of miR-133b-5p was up-regulatcd,and the expression of Fas mRNA and protein was down-regulated in MPC group.Conclusion The mechanism by which morphine preconditioning reduces H/R injury to rat cardiomyocytesis related to up-regulation of the expression of miR-133b-Sp and down-regulation of the expression of Fas.