Objective To investigate the screening and evaluating methods of positive recombinant clones for small fragments such as LoxP sequence. Methods Synthesized LoxP and vector complementary sequence were used as the upper and lower primer respectively, and colonies were used directly as the templates of polymerase chain reaction (PCR). The presence of 784 bp strap in electrophoresis was seen as positive. The positive recombinant clones screened by PCR were evaluated contrastively by restriction endonuclease digestion and verified by DNA sequence analysis. Results Among the six colonies randomly screened by PCR, three showed positive straps and one was verified by DNA sequence analysis. However, the electrophoresis only showed unclear and clouding straps when the three positive recombinant clones were evaluated by restriction endonuclease digestion. Conclusion Self-primer colony PCR is a high-speed, convenient, economic and effective method for screening and evaluating of positive clones recombinated by small fragments such as LoxP sequence.

AIM: To investigate DNA damage and cell stress (heat shock protein 70 expression) in human bronchi smooth muscle cells by cigarette smoke extract (CSE) in vitro. METHODS: 30 mL smog was dissolved in 1 mL culture medium as stock solution of CSE. Human bronchi smooth muscle cells were cultured 3 hours with 1∶16, 1∶10, 1∶8, 1∶6 and 1∶4 of CSE. The DNA damage and HSP70 expression were determined by single cell gel assay (comet assay) and Western blot, respectively. RESULTS: Associated with rising CSE concentration, DNA damage aggravated. Compared with the untreated group, except 1∶16 of CSE, the level of DNA damage was significantly different (P

Objective To observe the targeting effect of curcumin gelatin microsphere in rats in vivo. Methods Injections of curcumin gelatin microsphere and curcumin were injected via tail vein, respectively. HPLC was used to determine the content of curcumin in different organs. The pharmacokinetic parameters were calculated on the basis of compartment models by using DAS 2. 0 program. Targeting efficiency was used to evaluate tissue distribution of curcumin. Results Targeting efficiency of curcumin gelatin microsphere in heart, liver, spleen, lung and kidney was 0. 875, 0. 121, 1. 182, 5. 834 and 0. 896, respectively. Conclusion Curcumine gelatin microspheres can improve lung-targeting efficiency, and benefit for study on lung targeting therapeutic effect.

Objective To investigate the role of human cytomegalovirus (CMV) in the occurrence of drug eruptions.Methods Peripheral blood samples were collected from 44 patients with drug eruptions (including 13 severe cases) and 50 healthy human controls.Taqman fluorescent real-time quantitative PCR (RT-PCR) was performed to determine the positive rate and load of CMV DNA in peripheral blood mononuclear cells (PBMCs).Enzyme-linked immunosorbent assay (ELISA) was conducted to detect anti-CMV IgM antibodies in sera.Results The positive rate of CMV DNA was significantly higher in the patients than in the controls (65.91％ (29/44) vs.28.00 ％ (14/50),x2 =13.552,P ＜ 0.05),significantly different among patients with severe drug eruptions (11/13),patients with mild drug eruptions (58.06％ (18/31)) and the controls (x2 =16.153,P ＜ 0.05).In addition,patients with severe drug eruptions showed a higher positive rate of CMV DNA compared with patients with mild drug eruptions (x2 =13.817,P ＜ 0.05) and the controls (x2 =7.237,P ＜ 0.05).CMV DNA load was significantly higher in the patients than in the controls ((28 183.829 ± 19 527.654) vs.(3 019.952 ± 1 760.952) copies,t' =8.517,P ＜ 0.05).No significant difference was found in CMV DNA load between patients with severe drug eruptions ((554 813.389 ± 722 642.498) copies),patients with mild drug eruptions ((13 290.558 ± 14 082.356) copies)) and the controls (P ＞ 0.05).The positive rate of anti-CMV IgM antibodies was similar between the patients and controls (13.64％ (6/44) vs.6.00％ (3/50),P ＞ 0.05),but significantly different among patients with severe drug eruptions (4/13),patients with mild drug eruptions (6.45％,2/31) and the controls (x2 =7.832,P ＜ 0.05),and significantly higher in patients with severe drug eruptions than in the controls (x2 =6.409,P ＜ 0.05).Conclusions CMV infection exists in patients with drug eruptions,and might be a factor associated with the initiation and aggravation of drug eruptions.

In order to investigate whether polymorphism in gene for heat shock protein 70 (HSP70) has any bearing on individual susceptibility to the development of chronic obstructive pulmonary disease (COPD), the geotypes of 88 patients with COPD and 87 healthy smoking control subjects were tested by polymerase chain reaction followed by restriction fragment polymorphism analysis for HSP70 gene. In COPD group, HSP70-1 genotype A/A, A/B and B/B was 59.1%, 35.2% and 5.7%, HSP70-2 genotype A/A, A/B and B/B was 26.1%, 54.6% and 19.3%, and HSP70-hom genotype A/A, A/B and B/B was 70.4%, 27.3% and 2.3% respectively. In the control group, it was 60.9%, 27.5% and 3.5%, 20.7%, 56.3% and 23.0%, and 54.0%, 42.5% and 3.5%, respectively. The frequency of polymorphic genetypes showed no difference between the COPD group and the control group (P>0.05). It was suggested that geneic polymorphism in HSP70 is not associated with development of COPD in Han nationality of China.
China/ethnology ; Ethnic Groups ; Genetic Predisposition to Disease/*genetics ; HSP70 Heat-Shock Proteins/*genetics ; *Polymorphism, Restriction Fragment Length ; Pulmonary Disease, Chronic Obstructive/*genetics ; Smoking

Objective To evaluate the value of ghrelin on predicting prognosis in patients with chronic heart failure (CHF) after hospital discharge.Methods Totally 145 patients withCHF (age≥60 years,83 males and 62 females) were divided into 3 subgroups by New York Heart Association classification (NYHA):class Ⅱ (n=48),class Ⅲ(n=57) and class Ⅳ(n =40).According to the basic diseases,the CHF group was divided into five subgroups.All patients were followed up for about 2 years.The study included 55 healthy control subjects (30 males and 25 females).Results Plasma ghrelin level was lower in CHF cases (1.66±0.28) μg/L than in control subjects (2.27±0.26) μg/L (t 3.77,P＜0.01).The ghrelin level in NYHA Ⅱ(1.85±0.13) μg/L were higher than in NYHA Ⅲ (1.56±0.28) μg/L,the latter were higher than in NYHA Ⅳ (1.27±0.24) μg/L (P＜0.05).The plasma ghrelin level of patients after treatment (1.98±0.25) μg/L was increased compared with that of before treatment (1.66±0.28) μg/L (P＜0.05).No significant difference was found among the five basic disease groups (P＞0.05).During the follow up periods of (637±97)days,plasma ghrelin level was decreased in patients with cardiovascular event (1.26±0.38) μg/L than in patients without cardiovascular event (1.86±0.34) μg/L.The plasma ghrelin was negatively correlated with left ventricular end-diastolic diameter (LVEDD) and left ventricular ejection fraction (P＜0.05).Conclusions The plasma ghrelin in elderly patients with CHF is decreased than in healthy adults,and its level is lower in patients with severe heart failure.The plasma ghrelin is a predictor of cardiovascular event and death in elderly patients with CHF.

Objective To investigates the role of duodenum and bile duct preserving pancreatic head resection (DBPPHR) in treatment of benign or low-grade malignant diseases located in the head of pancreas.Methods The clinical data of 31 patients who underwent DBPPHR between April 2012 to May 2016 in Zhejiang Provincial People's Hospital and Zhangzhou Municipal Hospital of Fujian Province were analyzed retrospectively.Results Of the 31 patients,4 patients underwent laparoscopic DBPPHR.One patient in the open group was converted to pancreaticoduodenectomy.For the open group,the mean operation time was (165.3 ±63.6) min;the mean estimated blood loss was (258.1 ± 156.9) ml;and the mean postoperative stay was (11.7 ± 6.3) days.The postoperative complications included 1 reoperation due to postoperative bleeding,1 bile leakage and 13 patients developed grade A pancreatic fistula (48.2％).For the laparoscopic group,the mean operation time was 350.0 (280.0 ～ 450.0) min;the mean estimated blood loss was 425.0 (250.0 ～600.0) ml;and the mean postoperative stay was 14 days.Three patients developed postoperative pancreatic fistula (grade A).The pathological diagnosis were:12 patients with pancreatolithiasis,8 patients with serous cystadenoma,4 patients with branched intraductal papillary mucinous neoplasm,5 patients with neuroendocrine tumor and 2 patients with mucinous cystadenoma.The follow-up period was 1 ～ 48 month,and there was no patient with diabetes or diarrhea.Conclusions DBPPHR was safe and efficacious.It is less invasive to treat benign or low-grade malignant diseases located in the head of pancreas.

Objective To accurately judge the tumor-feeding artery is the most important basis for a successful treatment of hepatocellular carcinoma (HCC) with super-selective hepatic arterial chemoembo lization therapy. This study aims to assess the clinical value of cone-beam CT hepatic arteriography (CBCT-HA) in detecting tumor-feeding arteries during the performance of conventional transarterial chemoembo lization (TACE), and to compare the diagnostic effects between CBCT-HA and non-selective hepatic DSA. Methods Twenty-three consecutive patients with inoperable HCC were enrolled in this study. TACE was carried out in all patients. During the performance of TACE, the DSA-HA, CBCT-HA, Lipiodol-TACE and Lipiodol-CBCT were performed separately. The imaging materials, including DSA-HA and CBCT-HA, were analyzed by two experienced interventional physicians together to judge the tumor-feeding arteries. Statistic analysis was conducted by using chi square test. Results Tumor stain and lipiodol accumulation were regarded as the “gold standard” of the presence of tumor-feeding artery, based on which the tumor-feeding artery was confirmed in 75 lesions. DSA-HA demonstrated positive tumor-feeding artery in 40 lesions, among which true-positive tumor-feeding artery was seen in 32 and false-positive one in 8. CBCT-HA showed positive tumor-feeding artery in 72 lesions, which included true-positive tumor-feeding artery in 68 and false-positive one in 4. The sensitivity of CBCT-HA in judging tumor-feeding artery was 90.7% (68/75), which was much higher than that of DSA-HA (42.6%, 32/75), the difference was statistically significant(P<0.001). The positive predictive value of CBCT-HA in detecting tumor-feeding artery was also higher than that of DSA-HA (94.4% vs. 80.0%; P=0.040). Conclusion Cone-beam CT hepatic arteriography is obviously superior to DSA hepatic arteriography in identifying tumor-feeding arteries, which is very helpful in guiding super-selective TACE for HCC.

Objective To investigate the effects of acupoint therapy on inflammatory factors and its clini-cal efficacy in relieving bronchial asthma. Methods Selected patients with bronchial asthma which was in remis-sion were randomly divided into a treatment group that was treated with acupoint therapy and a control group that was given Seretide. Each group had 30 cases. The treatment period was 4 weeks. Both groups were evaluated in terms of Asthma Control Test ( ACT) scores and the serum content of interleukin-5 ( IL-5) and interleukin-10 ( IL-10) before and at one month ( short-term) , as well as three months after the end of the treatment ( long-term) . The asthma control situation ( fully controlled, partially controlled or uncontrolled) was evaluated. Results Before treatment the average ACT scores of the two groups were not significantly different. After the treatment both the short-term and long-term average ACT scores of the treatment group were significantly higher than those of the con-trol group. The total effectiveness rate of asthma control in the treatment group in the short term ( 93%) was signifi-cantly higher than that in the control group ( 70%) . After the treatment the IL-5 and IL-10 levels in the treatment group were improved to a significantly greater extent than those in the control group. Conclusion Acupoint thera-py can reduce airway inflammation, control bronchial asthma symptoms and show good clinical efficacy, probably by regulating IL-5 and IL-10 levels.

Objective To approach the effect of protective mechanical ventilation on acute lung injury after orthotopic liver transplantation, by observing changes of plasma markers of lung injury and inflammatory mediators.Methods Sixty patients scheduled for liver transplantation under general anesthesia, 42 males and 18 females, aged 21-62 years, weighing 43-80 kg, ASA physical status Ⅱ-Ⅳ, were randomly divided into 2 groups: protective mechanical ventilation group (group P) and unprotective mechanical ventilation group (group U).Pulmonary artery blood for plasma markers of lung injury and inflammatory mediators were collected at the following time points: before operation (T1), 3 hours after mechanical ventilation (T2), 2 hours (T3) and 4 hours in neohepatic stage (T4).These mediators included clara cell secretory protein (CC16), surfactant proteins (SP-D), soluble receptor for advanced glycation end-products (sRAGE), TNF-α, IL-6 and IL-8.Moreover, blood gas results were recorded at these 7 time points: T1-T4, 2 hours after operation (T5), before tracheal extubation (T6) and 2 days after operation (T7).The postoperative awakening time, tracheal extubation time, ICU stay time and the incidence of ALI were recorded.Results Compared with T1, plasma level of CC16 in the two groups increased at T2 and T3 (P<0.05 or P<0.01), however, plasma level of SP-D, sRAGE, TNF-α, IL-6 and IL-8 did not increase until T3 (P<0.01).Moreover, plasma level of sRAGE, TNF-α, IL-6 and IL-8 at T4 were higher than those at T1 (P<0.05 or P<0.01).Compared with T1, OIs in the two groups increased at T2, T5 and T6 (P<0.05 or P<0.01), while decreased at T4 in group P (P<0.01) and at T3 and T4 in group U (P<0.01).In group P, patients showed a lower plasma level of CC16 at T2 and T3 (P<0.05 or P<0.01), a higher OI at T3 (P<0.05) and an earlier tracheal extubation after operation [(8.9±3.2) h vs (9.3±2.8) h, P<0.05] compared with group U.There was no significant difference of acute lung injury incidence between the two groups after operation, which was 5(16.6%) and 7 (23.3%), respectively.Conclusion Protective mechanical ventilation may promote oxygenation index, and shorten tracheal extubation time, thus protect lung function of patients in liver transplantation to some extend.