AIM: Constructing plasmid that expresses human plasminogen kringle 5 gene to analyze the gene expression in E.coli. METHODS: The gene of human plasminogen kringle 5 was inserted into plasmid PBV220 EcoRI site by gene manipulation techniques and was transformed to E.coli TGI. The gene expression was observed by SDS-PAGE. RESULTS: Expression vector PBVK5 was constructed, and human plasmingen kringle 5 gene product was obtained at 42℃ induction. CONCLUSLON: Expression product of human plasminogen kringle 5 gene was soluble form of proteins, and the expression amount was 9.8% in E.coli TGI total proteins.

Objective:To develop lipiodol-containing calcium alginate microspheres (LAMs) for embolization,and study the characterization for emoblization and the radiopacity. Methods:LAMs were prepared by dripping method. The preparation of LAMs was optimized by orthogonal experiment which involved effects of three factors (the volume ratio of lipiodol to the external aqueous solution,airflow rate,and the weight pushing the injector) at three levels on the responses to the size,polydisperse index and entrapment efficiency of LAMs. The morphology of LAMs was observed under microscope. The elasticity of LAMs was investigated by texture analyzer. The capability injected through catheter of LAMs was monitored by video spinning-drop tensionmeter. The radiopacity of LAMs was measured by X-ray imaging system after LAMs were injected into vas of a rat. Results:The optimal condition for preparation of LAMs was:the volume ratio of lipiodol to the external aqueous solution was 3∶ 10,airflow rate was 40 g/mL and the weight pushing the injector was 100 g. According to the optimized condition,LAMs were prepared and characterized. The mean diameter of LAMs was (493.9?42.6) ?m,the polydisperse index was 1.02 and the entrapment efficiency was (88.97?1.09)%. The LAMs were with round shape and smooth surface in view of photograph of microscope. The maximum average load was (1.09?0.18) N when LAMs were compressed to 60%. The LAMs were injected through catheter without much difficulty. The radiopacity of LAMs in rats was demonstrated to be visible under X-ray photography system. Conclusion:The radiopaque LAMs developed are suitable for the arterial embolization,with round shape,proper size,good elasticity,easy handling character and visible property under X-ray imaging. The radiopaque embolic agent is supposed to be useful for emoblization therapy.

5-Flucytosine (5-FC) could be changed to 5-fluorouracil (5-FU) by cytosine deaminase (CD), the latter is able to kill cancer cells. However, there is no efficient method to deliver the CD gene into the tumor cells, which hampers the application of the suicide gene system. In this experiment, for the first time, the NDV has been utilized as a vector to deliver the CD gene into the cancer cells, the virus can infect the cancer cells specifically, replicate and assemble, while the cytosine deaminase is expressed. Then the CD converts the prodrug 5-FC into 5-FU to achieve the purpose of inhibiting tumor. Firstly, the whole genome of E. coli JM109 was extracted, and the CD gene was obtained by cloning method. Then the CD and IRES-EGFP were ligated into the pEE12.4 expression vector to become a recombinant pEE12.4IE-CD eukaryotic expression plasmid. The human liver cancer cells were transfected with the plasmid. The cells were treated with different concentrations of 5-FC, MTT method was used to determine the killing effect of CD/5-FC system on the human liver cancer cells. The cell deaths were 18.07%, 42.98% and 62.20% respectively when the concentrations of prodrug were at 10, 20 and 30 mmol x L(-1). In 5-FC acute toxicity experiment, Kunming mice were injected with different concentrations of 5-FC at intervals of 1:0.5. The LD50 of 5-FC through iv injection was determined by improved Karber's method, the LD50 was 507 mg x kg(-1) and the 95% confidence limit was 374-695 mg x kg(-1). According to the maximum LD0 dose of the LD50, the maximum safe dose was 200 mg x kg(-1). Body weight and clinic symptoms of the experimental animals were observed. These results laid the foundation to verify the antitumor effect and safety of CD/5-FC system in animal models. The CD gene was ligated into the NDV (rClone30) carrier, then the tumor-bearing animal was established to perform the tumor inhibiting experiment. The result showed that the recombinant rClone30-CD/5-FC system has a high antitumor activity in vivo. To summarize, CD gene has been cloned and its bioactivity has been confirmed in the mammalian cells. It is the first time in this study to utilize the recombinant NDV to deliver the CD gene into the tumor cells; our result proves the rClone30-CD/5-FC system is a potential method for cancer therapy.

Objective To discuss the treatment experience of adrenocorticotropic hormone independent macronodular adrenal hyperplasia (AIMAH).Methods The clinical data of 24 cases of AIMAH from August 1972 to December 2016 were retrospectively analyzed.The cases included 10 males and 14 females with a mean age of 43 (range 26 to 59) years.16 patients presented with typical Cushing syndrome (CS) and 8 patients had weight gain, hypertension or diabetes mellitus without any sign of CS.The circadian rhythm of serum cortisol was abnormal.Low and high dose dexamethasone suppression tests failed to suppress cortisol secretion.The preoperative CT scan showed bilateral enlargement of the adrenal glands with multiple macmnodules.14 patients underwent open surgery, including 5 cases of unilateral adrenalectomy, 6 cases of adrenalectomy combined with contralateral subtotal adrenalectomy and 3 cases of bilateral adrenalectomy simuhaneously.6 patients underwent conventional laparoscopic adrenalectomy,including 2 cases of unilateral adrenalectomy, 2 cases of adrenalectomy combined with contralateral subtotal adrenalectomy and 2 cases of bilateral adrenalectomy.4 patients underwent robot-assisted laparoscopic bilateral adrenalectomy.Results The postoperative pathological examination showed that all patients had bilateral adrenal macronodular or adenomatoid hyperplasia.During the mean follow-up of 68 months (range 3 to 192 months), the clinical symptoms of CS disappeared after surgery in all cases.7 patients who received unilateral adrenalectomy had urinary free cortisol and serum cortisol within normal ranges and no further enlargement of the contralateral gland was noticed.Among the 3 patients who received bilateral adrenalectomy simultaneously, 1 case died of adrenal crisis on the seventh day post-operation.The remaining 2 cases presented with adrenal insufficiency but returned to normal after glucocorticoid replacement therapy.Nelson's syndrome was not observed in other patients.Conclusions The use of bilateral adrenalectomy to treat AIMAH may involve risk.Unilateral adrenalectomy is recommended for an alternative treatment for AIMAH.Controlateral adrenalectomy with lifelong corticosteroid replacement or contralateral subtotal adrenalectomy may be performed if the symptoms have not improved or recurred after unilateral adrenalectomy.

Objective To investigate the value of baseline levels of aldosterone(ALD)in predicting the cardiovascular events and in-stent restenosis12 months after coronary stent implantation. Methods 268 patients with coronary heart disease admitted to the department of cardiology from January 2014 to July 2016 were selected (96 cases of stable angina pectoris and 172 cases of acute coronary syndrome).The ALD level in the preprocedural serum was detected before coronary stent implantation. According to the level of ALD,the patients were divided into two groups:ALD<130 pg/mL(n=127)and ALD≥130 pg/mL(n=141).The cardiovascular events(angina pectoris,myocardial infarction,death)and in-stent restenosis were observed and recorded 12 months after the procedures. Logistic regression analysis was done to analyze the independent risk factors of ISR. Results The baseline levels of ALD in the patients with acute coronary syndrome were significantly higher than those with stable angina pectoris[(160 ± 58)pg/mL vs.(118 ± 46)pg/mL,P < 0.05]. The cardiovascular events and those in in-stent restenosis in the patients with high baseline levels of ALD were significantly higher than those with normal baseline levels of ALD 12 months too(24.1% vs 3.1%;P<0.05;16% vs 7.4%;P<0.05).Logistic regression analysis showed that serum ALD levels were the strongest predictors of ISR,with the odds ratio of 2.56 per 10 pg/mL. Conclusions Preprocedural ALD level is a predictor for 12 months outcome of in-stent restenosis for the patents undergoing coronary stent implantation. It indicates that the complications and clinical restenosis in 12 months are markedly influenced by the activation of the rennin angiotensin aldostenone system.

Objective:To explore the value of using bipolar electrogram to guide target selection in patients with frequent premature ventricular contractions in the right ventricular outflow tract (RVOT-PVC).Methods:The clinical data of 115 patients with idiopathic and frequent RVOT-PVC from October 2018 to January June 2020 in Guangzhou First People′s Hospital were retrospectively analyzed. The number of PVCs in Holter 24 h before ablation was 19 802.6±4916.7, and the load was (20.3±5.0)%. The Johnson & Johnson Carto 3.0 system was used to guide RVOT-PVC radiofrequency ablation, and the morphological characteristics of the bipolar electrogram in the cavity of the successful ablation target were observed. According to whether the starting part of the bipolar electrogram of the distal ablation catheter showed a steep negative shape recorded by the Carto 3.0 system, the patients were divided into positive group and negative group. The differences in ablation success rate, effective discharge time, total ablation time and other indicators of the two groups were compared.Results:Steep negative wave was recorded in the initial part of the effective target site of 87 patients (75.7%). The ablation success rate of the patients was 95.4%(83/87) based on the excitation mapping and unipolar morphology combined with the above initial part of the bipolar electrogram. Compared with the negative group, the PVC disappeared faster in patients of positive group [(6.9±2.3)s vs (10.2±2.9)s, P<0.05] and the total ablation time was shorter [(187.5±35.7)s vs (267.3±54.1)s, P<0.05]. Ambulatory electrocardiogram (ECG) was rechecked at 3 months. At 3 months, there was 1 case recurrence in the positive group and 1 case recurrence in the negative group, and there was no significant difference in the long-term recurrence rate between the two groups ( P=0.422). Conclusions:On the basis of traditional mapping, the bipolar electrogram combined with the steep negative shape of the initial part can be used as an alternative RVOT-PVC ablation strategy.

Objective To explore the clinical efficacy and safety of off-clamping in robotic-assisted partial nephrectomy (RAPN) for the treatment of renal tumors.Methods From January 2015 to March 2017,the data of 48 patients who underwent off-clamping RAPN were reviewed retrospectively.There were 31 males and 17 females,and the mean age was 57 years (range:23-84 years).The mean tumor size was 3.1 cm (range:1.2-6.4 cm),with the upper,middle,and lower polar tumors account for 35.4％,27.1％,and 37.5％,respectively.The clinical tumor stage was T1N0M0 in all 48 cases,according to the AJCC tumor staging system for renal cancer.Results RAPNs were performed successfully in all 48 cases,without conversion to open surgery.In those patients,the application of off-clamping in robotic-assisted partial nephrectomy was performed in 44 cases.The renal artery and vein was exposed,dissected,isolated and then clamped in 4 cases due to bleeding.The mean surgical time was 85 min (range:75-185 min).The mean estimated blood loss was 134 ml (range:60-270 ml),and no blood transfusion was needed.The wound surface was closed using interrupted suture with Hem-o-lok clips securing each needle point.The mean time for renorrhaphy was 22 min (rang:11-31 min).No intraoperative severe complications such as vascular injury,trauma of abdominal organ occurred.There were 5 complications,including 2 cases of hematuria,2 cases of delayed healing of incision,and 1 case of pneumohypoderma.The pathological diagnosis included 40 cases of renal clear cell carcinoma,3 cases of papillary renal cell carcinoma,and 5 cases of angiomyolipomas.No tumor recurrence or distant metastasis was observed during the average follow-up of 17 months (range 3-27 months).Conclusions Off-clamping RAPN is safe and feasible approach to excise certain kidney tumors.It carries the benefits of less complication,quick recovery,and less ischemia reperfusion renal injury.Off-clamping RAPN would be suitable for those patients with solitary kidneys,renal insufficiency,and bilateral tumors.

Objective:To investigate the effect of apatinib on radiosensitivity of glioma cells U87MG and its potential mechanism.Methods:U87MG cells were divided into control group, apatinib group, radiation group and combination group treated with apatinib and radiation. The effect of different concentrations of apatinib (5, 10, 20, 40, 80 μmol/L) on cell proliferation was detected by CCK8 assay. The effect of apatinib on cell migration and invasion was detected by wound-healing assay and transwell assay, respectively. The effect of apatinib on cell radiosensitivity was detected by plate cloning assay, the cell apoptosis rate was detected by flow cytometry, and the protein expressions of Bax and Bcl-2 were detected by Western blot.Results:Apatinib significantly inhibited the proliferation of U87MG cells in a manner depended on the drug treatment time and radiation. Compared with the radiation group, the cell proliferation, migration and invasion in the combination group were inhibited much significantly ( t=9.857, 18.704, 4.197, P<0.05), so that the value of D0, Dq and SF2 of the combination group was lower, resulting in a radiosensitivity enhancement ratio (SER D0 ) of 1.3. Moreover, compared with the radiation group, the apoptosis rate of the combination group was increased, the expression of Bcl-2 protein was decreased, and the expression of Bax protein was increased ( t=16.187, 8.890, 5.222, P< 0.05). Conclusions:Apatinib inhibits cell proliferation, invasion and migration, induces apoptosis and increases radiosensitivity of glioma cells.

Objective:To establish a sensitive and specific real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) method for rapid detection of Getah virus (GETV).Methods:All the gene sequences of GETV were downloaded from GenBank database. Clustal X was used for sequence alignment, and specific primers and probes were designed according to highly conserved regions; we established a standard curve using the nucleic acid of GETV as a standard, and the sensitivity, specificity and stability of this method were evaluated respectively.Results:This method could specifically detect GETV and has no cross-reactivity with multiple arboviruses; the sensitivity was 1.0×10 pfu/ml, and the intra-assay and inter-assay coefficients of variation were less than 1%. One case was GETV positive in 196 batches of mosquitoes collected from Hunan province, Hebei province, Fujian province and Chongqing city.Conclusions:We established a TaqMan probe real-time quantitative RT-PCR with high sensitivity and specificity which can be used for screening.

Objective To compare cortical inhibition(CI)function between patients with schizophrenia and depression,and to explore the correlation between CI function and clinical symptoms.Methods A total of 35 first episode schizophrenia(FES)patients,41 depression patients(21 with first episode depression,20 with recurrent depression),and 35 healthy controls(HC)were recruited.The positive and negative syndrome scale(PANSS)was used to assess symptoms in FES patients,while the 17-item Hamilton depression scale(HAMD-17)and Hamilton anxiety scale(HAMA)were used to assess symptoms in depression patients.All participants'cortical inhibition and excitation measures were examined using single or paired pulses transcranial magnetic stimulation.Analysis of covariance/generalized linear model was employed to compare cortical inhibition and excitation measures among groups including age,gender,and medication status as covariates.The correlations between cortical inhibition and excitation measures and clinical symptoms were analyzed.Results The cortical silent period(CSP)in FES group was longer than that in control group and depression group[(92.08±35.43)ms vs.(70.27±22.12)ms vs.(70.81±29.29)ms,P<0.05].Depression group was further divided into first episode depression(FED)and recurrent depression(RD)subgroups.The short-interval cortical inhibition(SICI)was weaker in FED group than in the RD group and the control group(0.76±0.44 vs.0.43±0.32 vs.0.45±0.20,P<0.05).In FED group,CSP was negatively correlated with the general symptom score of PANSS(r=-0.544,P<0.001),and SICI was negatively correlated with the negative symptom score of PANSS(r=-0.501,P=0.005).In the FED group,SICI was positively correlated with HAMD-17 score(r=0.605,P=0.028).Conclusion Both FES patients and FED patients exhibit abnormal CI.There are distinct characteristics between FES and FED.FES patients exhibit prolonged CSP,while FED patients exhibit decreased SICI.The abnormal CI in FES and FED are correlated with their clinical symptoms.