Objiective:To construct human single-chain antibody gene fragment for breast cancer.Methods:Total RNA was isolated from tumor adjacent lymphatic tissue of breast cancer patients.Then heavy and light chain(VH and VL) genes of antibody were amplified separately by RT-PCR,and assembled into ScFv genes by SOE-PCR.Results:The human single-chain antibody gene fragment for breast cancer was constructed and its size was about 750bp.Conclusion:The human single-chain antibody gene fragments were constructed successfully,which provides a way for constructing the human single-chain antibody library of anti-breast cancer.

Aim To construct human phage single-chain antibody library associated with esophageal cancer and to screen the specific scFv against Eca109 cells from the liberary. Methods Metastatic periesophageal lymph nodes of esophageal cancer patients were used as the B cells source, the total RNA of these B cells was extracted and prepared as the template of RT-PCR. First, we screened graticulely two pairs of primers of the heavy and light regions separately, then the V_H and V_L fragments were first amplified from the cDNA by the polymerase chain reaction (PCR). Second, the V_H-linker and V_L-linker were amplified from the V_H and V_L fragments. Last, the V_H-linker and V_L-linker were assembled into scFv gene fragments by SOE-PCR,and then Sfi I and Not I restriction site were inlet in it. ScFv gene was cloned into the pCANTAB-5E phagemid. Phagemids were introduced into E.coli TG1 by electrotransformation, followed by rescue of antibody-expressing phage using M13K07 helper-phage superinfection. Recombinant scFv phage library was constracted and PCR was used to identify the insert ratio of scFv antibodies library. Results of SfiI/Not I double digestion reaction positive insert clone were identified by 1.5% agarose gel electrophoresis. The phage library was panned with NHEEC and Eca109 cancer cells in suspension for four rounds. Strongly positive recombinant phage clones were used to infect E.coli HB2151. Expression of soluble scFv was induced by IPTG. Soluble scFv from periplasm were purified by affinity chromatography and identified by SDS-PAGE and Western blot. Cell ELISA , immunohistochemical staining and immunocytochemical staining were used to identify the activity of the soluble scFv. Results The result of agarose gel electrophoresis showed that total RNA of these B cells had two bands of 28 S and 18 S. The size of V_H fragment is about 450 bp,V_L fragment is about 350 bp and scFv is about 850 bp. The competence is 108 cfu??g-1 pUC18 DNA. Randomly digestive reac-tion showed that the positive insert ratio was 91.7% (22/24). After four rounds of panning, the fourth phage yield is 141 times as much as that of the first one. SDS-PAGE and Western blot showed that the MW of the soluble scFv was about 30 ku and the brand of 30 ku was stained. Immunohistochemical staining showed strong stainning of the tissue of esophageal cancer, but not the liver and gastric cancer tissue. Immunocytochemical staining showed significant staining of the esophageal cancer line Eca109. The result of cell ELISA assay revealed that soluble scFv had highly specific and could combined with Eca109 cells, but not with BGC-823 and NHEEC. Conclusion A human scFv phage display library associated with esophageal cancer has been constructed successfully and the specific scFv antibody against Eca109 has been identified from the liberary.

Objective To investigate the efficacy and safety of tirofiban for emergency interventional therapy in patients with acute ST segment elevation myocardial infarction.Methods The 96 acute ST segment elevation myocardial infarction patients having accepted emergency percutaneous coronary intervention (PCI) were divided into treatment group and control group according to the treatment method with 48 cases each.The treatment group was given conventional standard treatment combined with tirofiban treatment [tirofiban intracoronary injection 10 μ g/kg and then intravenous 0.15 μ g/(kg· min) for 24-48 h].The control group was given conventional standard treatment only.The postoperative 60 min electrocardiogram ST segment recovery,the coronary blood flow TIMI grade,the major adverse cardiac events (angina,myocardial infarction,heart failure,death) and postoperative bleeding in 4 weeks were compared between the two groups.Results The patients having postoperative 60 min electrocardiogram ST segment recovery in treatment group was 45 cases (93.8％,45/48),in control group was 35 cases (72.9％,35/48),and there was statistical difference (P ＜ 0.05).The incidence rate of infarction vascular TIMI grade ≥ 3 grade flow in treatment group was 95.8％ (46/48),in control group was 75.0％ (36/48),and there was statistical difference (P ＜ 0.05).The incidence rate of the major adverse cardiac events 4 weeks in treatment group was significantly lower than that in control group,and there was statistical difference (P ＜ 0.05).There was no statistical difference in the incidence rate of bleeding between the two groups (P ＞ 0.05).Conclusion In patients with acute ST segment elevation myocardial infarction,the emergency interventional therapy with tirofiban is efficacy and safety.

Objective To summarize our clinical experience of surgical treatment for pediatric patients with ventricular septal defect(VSD) and mitral regurgitation(MR).Methods A retrospective study was performed including consecutive 84 patients with VSD and MR receiving mitral valvuloplasty(MVP) and VSD closure from January 2006 to January 2012 in Shanghai Xinhua Hospital.All patients were associated with pulmonary hypertension(PH,32-85 mm Hg).The diameters of ventricular septal defects were between 0.7 and 1.6 cm.Echocardiography showed that trivial MR (+) in 9 cases,mild MR (++)in 18 cases,moderate MR(+++) in 33 cases,and severe MR(++++) in 24 cases.VSD closure and MVP were performed with cardiopulmonary bypass under moderate systemic hypothermia.The results of repair were evaluated by transesophageal echocardiography (TEE) during operation.Results Intra-operative TEE results: no residual shunt of VSD,none MR in 80 cases,residual trivial MR in 4 cases.Mean Cardiopulmonary bypass (CPB) time was (84.6 ± 18.5) mins.Mean Aortic clump time was(50.8 ± 11.5) mins.Mean postoperative ventilation time was (38.7 ± 30.2) hours,and mean postoperative inhosptial time was(10.5 ±4.6) days.The in-hospital mortality was 1.2％ (1 case died).78 cases were fully followed up.There was no late death.Echocardiography showed that none MR in 62 cases,trivial MR in 10 cases,mild MR in 4 cases,moderate MR in 2 patients.The overall freedom from reoperation at 5 years was (97.4 ± 1.8) ％.Conclusion Ventricular septal defect with pulmonary hypertension need early surgical repair.MR was treated at the same time of VSD closure could effectively improve the surgical outcome of pediatric patients with ventricular septal defect and mitral regurgitation.

Objective To study the correlation between morphological characteristics of coronary artery calcified plaques and luminal stenosis of local coronary artery segments with 64-slice computed tomography.Methods One hundred and eleven patients who had undergone 64-slice computed tomographic coronary angiography(CTCA)and conventional coronary angiography(CAG)were retrospectively analyzed.The calcified plaques were classified as punctate,nodular,strip-like and nubbly in long-axis view of coronary artery lumen,and were classified as crescent,semilunar,round moon and circinate in short-axis view.The morphologic characteristics of these calcified plaques on CTCA were retrospectively analyzed and compared with luminal stenosis of CAG results.Results Among 528 calcified coronary segments which were analyzed in 111 patients,there were 383(72.5%)punctate calcified plaque segments and 145(27.5%)of non-punctate plaques.There were 34(23.4%,34/145)non-punctate calcified plaques which caused severe stenosis(≥75%),including 4(11.8%)nodular,8(23.5%)stripe-like and 22(64.7%)nubbly calcified plaques on the long-axis view,and 0(0.0%)cresent,8(23.5%)semilunar,18(52.9%)round moon and 8(23.5%)circinate calcified plaques on the short-axis view.The ratios of different morphological coronary artery calcifications which caused severe stenoses were significantly different with each other(all P＜0.01).Conclusion Different figures of coronary artery calcified plaques demonstrate different degrees of stenoses of local coronary artery lumen.Severe stenoses were mostly caused by nubbly calcified plaque on long-axis view,round moon and circinate calcified plaque on short-axis view.

Objective To review the surgical methods and mid-term results of mitral valve repair in pediatric patients with moderate to severe mitral regurgitation (MR).Methods 132 children with moderate to severe MR,aged (18.9 ± 7.2)months,weighted(11.3 ±4.8) kg.The etiology for mitral regurgitation is congenital heart disease in 126 cases,infective endocarditis in 5 cases and Marfan syndrome in 1 case.Mitral valvuloplasty(MVP) was performed with cardiopulmonary bypass under moderate systemic hypothermia.The methods of MVP included annuloplasty,annuloplasty ring,cleft closure,reconstruction of posterior leaflet.The coucomitant cardiac anomalies were treated at the same time.The results of repair were evaluated by saline injection test and transesophageal echocardiography (TEE) during operation.Results Intra-operative TEE results: 131 cases had none to mild MR,and only one case had moderate MR.The patient underwent second repair immediately,subsequent TEE was mild.Mean cardiopulmonary bypass (CPB) time was (80.0 ± 31.1) minutes.Mean aortic clump time was (48.0 ± 17.9) minutes.The in-hospital mortality was 2.3％ (3 cases died).One died of heart failure on postoperative day 7,the other died of low cardiac output syndrome resulting on postoperative day 2.Another one was large ventricular septal defect(VSD) with pulmonary hypertension (PH),died of pulmonary infection.Mean postoperative ventilation time was (34.4 ± 31.9) hours,and mean postoperative inhosptial time was (9.0 ± 5.4) days.The average follow-up period was (40.5 ± 8.3) months (2 to 74 months).122 cases were fully followed up.Echocardiography showed that moderate MR was in 7 patients,and 3 patients had severe MR.4 patients underwent re-do mitral valve repair or mitral valve replacement.There was no late death.The overall survival rate at 5 years was 97.7％ and the overall freedom from reoperation at 5 years was 92.0％.Conclusion Pediatric patients with moderate to severe MR need early surgical treatment,the early and mid-term results were satisfactory.Individualized treatment protocol based on specific pathology was the keypoint of surgical therapy.

Objective:To explore the effects of sandplay combined with sensory integration therapy on cognitive function in children with attention deficit hyperactivity disorder (ADHD).Methods:60 children with ADHD diagnosed in Baoji Maternal and Child Health Hospital from June 2018 to June 2019 were randomly divided into study group and control group.The children in the control group were treated by sandplay, while the patients in the study group were treated by sandplay combined with sensory integration.Results:There was no significant difference in Parent Symptom Questionnaire (PSQ) score, Combined Raven Test (CRT) results and attention test results between the two groups before treatment ( P>0.05), and there was no significant difference in PSQ score of control group after treatment ( P>0.05); The behavioral problems (0.92±0.23), anxiety (0.51±0.26), impulse/hyperactivity (1.06±0.31) and hyperactivity index (0.88±0.14) in the study group were significantly lower than those in the control group [behavioral problems (1.12±0.21), anxiety (0.79±0.45), impulse/hyperactivity (1.42±0.34) and hyperactivity index (1.16±0.17) ( P<0.05)]. There was no significant difference in the scores of mental disorders and learning problems between the two groups [(0.42±0.20), (1.28±0.44) vs (0.52±0.28), (1.37±0.48)] ( P>0.05). The results of CRT in the study group were (6.6±0.3, 7.3±0.2, 9.1±0.1, 5.5±0.2, 2.7±0.1, 117.3±4.4), which were higher than those in the control group (6.2± 0.1, 6.7±0.1, 8.7±0.1, 5.0±0.1, 2.2±0.1, 110.0±3.8) ( P<0.05). The slip time (52.4±0.1), error number (55.9±0.2) and missed report number (60.2 ±0.1) of the study group were significantly lower than those of the control group [slip time (56.1±0.2), error number (60.3±0.1) and missed report number (70.8±0.3)] ( P<0.05). Conclusions:Combination of sandplay and sensory integration can significantly improve the cognitive and behavioral abilities of children with attention deficit hyperactivity disorder, and improve the balance function of children, which is conducive to clinical application.

Objective: To explore the correlation between antithrombotic strategy and its prognostic value in coronary artery disease (CAD) patients combining with atrial ifbrillation (AF) after percutaneous coronary intervention (PCI).
Methods: A total of 570 CAD patients with AF received PCI in our hospital from 2012-01 to 2013-12 were retrospectively analyzed by CHADS2 and HAS-BLED Score system. According to CHADS2 < 2 and CHADS2 ≥ 2 or with/without warfarin medication, the patients were divided into 2 groups:①CHADS2 < 2 (Low risk of stroke) group,n=339 including 309 patients without warfarin and 30 with warfarin medication;②CHADS2 ≥ 2 (High risk of stroke) group,n=231 including 200 patients without warfarin and 31 with warfarin medication. All patients were followed-up for 15 months to compare the different anti-platelet/ anti-coagulation strategies for the occurrence rate of MACCE, ischemic and bleeding events.
Results: Compared with Low risk of stroke group, the patients in High risk of stroke group had the worse prognosis and higher rate of MACCE occurrence,P<0.001, HR=2.677, 95% CI (1.535-4.635), more ischemic events,P=0.013, HR=2.080, 95% CI (1.167-3.709). Multi-factor Cox surving analysis indicated that compared with low risk patients without warfarin medication, the high risk patients without warfarin had the higher rate of MACCE occurrence,P=0.001, HR=2.985, 95% CI (1.532-5.816), more ischemic events,P=0.026, HR=2.068, 95% CI (1.090-3.925). Whereas, the occurrence rates of MACCE and ischemic events in high risk, low risk patients with warfarin and low risk patients without warfarin were similar, all P>0.05. The major bleeding events in patients with warfarin were a little higher than those without warfarin,P>0.05. The minor bleeding events were increased in both Low risk stroke group asP<0.001, OR=4.458, 95% CI (1.934-10.277) and High risk stroke group asP=0.002, OR=4.155, 95% CI (1.717-10.055).
Conclusion: Warfarin medication may obviously decrease the occurrence rates of MACCE and ischemic events in high risk of stroke patients, while in low risk patients, warfarin could not further decrease the occurrence of MACCE and ischemic events. Warfarin could increase the risk for minor bleeding in both low risk and high risk of stroke in CAD patients combining with AF after PCI.

Gallbladder carcinoma (GBC) is the most common malignancy of the biliary tract, radical resection is the only effective treatment for GBC at present. However, the postoperative effect is still poor. Therefore, identifying the key prognostic factors and establishing an individual and accurate survival prediction model for GBC are critical to prognosis assessment, treatment options and clinical decision support in patients with GBC. The prediction value of current commonly used TNM staging system is limited. Cox regression model is the most commonly used classical survival analysis method, but it is difficult to establish the association between prognostic variables. Nomogram and machine learning techniques including Bayesian network have been used to establish survival prediction model of GBC in recent years, which representing a certain degree of advancement, however, the model precision and clinical application still need to be further verified. The establishment of more accurate survival prediction models for GBC based on machine learning algorithm from Chinese multicenter large sample database to guide the clinical decision-making is the main research direction in the future.

Gallbladder carcinoma (GBC) is the most common malignancy of the biliary tract, radical resection is the only effective treatment for GBC at present. However, the postoperative effect is still poor. Therefore, identifying the key prognostic factors and establishing an individual and accurate survival prediction model for GBC are critical to prognosis assessment, treatment options and clinical decision support in patients with GBC. The prediction value of current commonly used TNM staging system is limited. Cox regression model is the most commonly used classical survival analysis method, but it is difficult to establish the association between prognostic variables. Nomogram and machine learning techniques including Bayesian network have been used to establish survival prediction model of GBC in recent years, which representing a certain degree of advancement, however, the model precision and clinical application still need to be further verified. The establishment of more accurate survival prediction models for GBC based on machine learning algorithm from Chinese multicenter large sample database to guide the clinical decision-making is the main research direction in the future.