克隆人血小板源性生长因子B基因构建腺相关病毒载体及其病毒滴度测定

目的:克隆人血小板源性生长因子B基因于腺相关病毒载体中,以获得高感染滴度的重组人血小板源性生长因子B基因腺相关病毒。方法:实验于2005-10/2006-11在青岛大学医学院附属医院中心实验室完成。①健康足月产妇胎盘组织由青岛大学医学院附属医院妇产科提供,产妇及其家属均知情同意。DH5α宿主菌由青岛大学医学院附属医院中心实验室制备保存。②Trizol试剂提取健康足月产妇胎盘组织总RNA,反转录-聚合酶链反应一步法克隆人血小板源性生长因子B基因全长开放读框。聚合酶链反应产物定向克隆于腺相关病毒载体,重组人血小板源性生长因子B基因腺相关病毒载体进行双酶切和测序鉴定。③采用磷酸钙转染法将重组人血小板源性生长因子B基因腺相关病毒载体、腺相关病毒包装载体,腺相关病毒辅助载体共转染腺相关病毒293细胞,包装得到重组腺相关病毒并回收病毒原液,显微镜下观察细胞及培养基的变化。④以携带β-半乳糖苷酶基因的腺相关病毒作为报告基因同重组人血小板源性生长因子B基因腺相关病毒载体一样共转染相关病毒293细胞,对报告基因行X-Gal染色,通过蓝染的细胞计算病毒滴度。结果:①重组人血小板源性生长因子B基因腺相关病毒载体酶切鉴定及序列分析:聚合酶链反应产物经电泳证明大小正确,重组人血小板源性生长因子B基因腺相关病毒载体酶经双酶切和测序鉴定证实将血小板源性生长因子B基因正确插入。②腺相关病毒293细胞转染过程中病毒颗粒的产生:腺相关病毒293细胞转染6h后培养基底部出现无数小黑色颗粒,为加入的转染复合物颗粒。24h后腺相关病毒293细胞部分变圆,随着病毒增殖,细胞成串浮起,出现细胞病理效应。48h后细胞变形,逐渐从壁上脱落。72h后培养基的颜色从红色变化为橙色或黄色。③病毒滴度检测:通过携带β-半乳糖苷酶基因的腺相关病毒感染细胞后X-gal染色计数,测定重组腺相关病毒滴度为1×1010 L-1。结论:成功构建表达人血小板源性生长因子B基因的重组腺相关病毒载体,为进一步研究血小板源性生长因子B基因治疗角膜病和相关细胞系与组织的增殖提供实验基础。     

Abnormal heart rate recovery on exercise in ankylosing spondylitis

Objectives

This study evaluated the heart rate recovery response in ankylosing spondylitis (AS) patients and control subjects.

Background

Delayed heart rate recovery after exercise reflects AD and independently predicts adverse cardiac outcome.

Methods

Fifty-one patients with AS and 50 age- and matched controls received electrocardiography, echocardiography, and treadmill exercise testing. The heart rate recovery (HRR) index was calculated as the reduction in heart rate from the rate at peak exercise to the rate at the 1st (HRR₁), 2nd (HRR₂), 3rd (HRR₃) and 5th (HRR₅) minute after the cessation of exercise stress testing.

Results

There were significant differences in HRR₁ and HRR₂ indices between patients and controls (24.8 ± 12.1 vs 34.9 ± 11.0; p < 0.001 and 41.2 ± 14.2 vs 54.3 ± 11.8; p < 0.001, beats/min, respectively). Similarly, HRR₃ and HRR₅ indices were lower in patients than controls (51.3 ± 15.1 vs 65.2 ± 14.0; p < 0.001 and 61.0 ± 14.2 vs 76.1 ± 14.8; p < 0.001). In addition, exercise capacity was markedly lower (8.1 ± 2.0 vs 10.5 ± 2.5 METs; p < 0.001) in AS than controls.

Conclusion

The HRR index is impaired in AS patients, implying the occurrence of autonomic dysfunction even without active joint disease or frank cardiac involvement.     

Factors influencing choice of surgical route of repair of genitourinary fistula, and the influence of route of repair on surgical outcomes: findings from a prospective cohort study

Please cite this paper as: Frajzyngier V, Ruminjo J, Asiimwe F, Barry T, Bello A, Danladi D, Ganda S, Idris S, Inoussa M, Lynch M, Mussell F, Podder D, Barone M. Factors influencing choice of surgical route of repair of genitourinary fistula, and the influence of route of repair on surgical outcomes: findings from a prospective cohort study. BJOG 2012;119:1344-1353. Objective The abdominal route of genitourinary fistula repair may be associated with longer term hospitalisation, hospital-associated infection and increased resource requirements. We examined: (1) the factors influencing the route of repair; (2) the influence of the route of repair on fistula closure 3?months following surgery; and (3) whether the influence of the route of repair on repair outcome varied by whether or not women met the published indications for abdominal repair. Design Prospective cohort study. Setting Eleven health facilities in sub-Saharan Africa and Asia. Population The 1274 women with genitourinary fistula presenting for surgical repair services. Methods Risk ratios (RRs) and 95% confidence intervals (95% CIs) were generated using log-binomial and Poisson (log-link) regression. Multivariable regression and propensity score matching were employed to adjust for confounding. Main outcome measures Abdominal route of repair and fistula closure at 3?months following fistula repair surgery. Results Published indications for abdominal route of repair (extensive scarring or tissue loss, genital infibulation, ureteric involvement, trigonal, supratrigonal, vesico-uterine or intracervical location or other abdominal pathology) predicted the abdominal route [adjusted risk ratio (ARR), 15.56; 95% CI, 2.12-114.00]. A vaginal route of repair was associated with increased risk of failed closure (ARR, 1.41; 95% CI, 1.05-1.88); stratified analyses suggested elevated risk among women meeting indications for the abdominal route. Conclusions Additional studies powered to test effect modification hypotheses are warranted to confirm whether the abdominal route of repair is beneficial for certain women.     

Prevalence and determinants of the practice of genital mutilation of girls in Khartoum, Sudan

BACKGROUND: Female genital mutilation (FGM) is widely practised in Sudan, despite many decades of attempts to prevent it. AIMS: To estimate the prevalence of FGM, identify the types performed in Khartoum and investigate whether FGM is associated with various social factors. METHODS: Girls aged 4-9 years (n=255) presenting to a paediatric emergency ward were recruited. A detailed history was obtained and full examination, including inspection of the genitalia, was performed to verify the type and extent of FGM. RESULTS: Twenty per cent of the study group had undergone FGM, 50% of guardians indicated that it would be done later, and 29% stated that the child would not undergo FGM. In 66% of those who had undergone FGM, it was WHO type III. All operations had been performed by health professionals, mainly midwives. Those who had allowed or intended to allow their daughters to undergo FGM were of significantly lower socio-economic status (p=0.0008) and had spent significantly fewer years in school (both mothers, p=0.0015, and fathers, p=0.0266) than those who had not/would not. All who had undergone FGM were Muslims. None of the 16 Christians had undergone FGM. In girls over 7 years of age, there was a higher risk of having FGM in those who attended school than in those who did not. CONCLUSION: FGM is still practised widely in Khartoum and probably in many parts of northern Sudan and the type undertaken is often the most severe. Parental education, socio-economic level and religion are important determinants of the practice, but social pressure on parents and girls seems to play an important role.     

Protein kinase C-beta inhibition and diabetic microangiopathy: effects on endothelial permeability responses in vitro

Protein kinase C (PKC)-beta and other PKC isozymes have been implicated in the loss of endothelial barrier function in diabetic microangiopathy. The effects of a PKC-beta-specific inhibitor, LY379196, on hyperpermeability responses to high-glucose, angiotensin II, alpha-thrombin and endothelin-1 were evaluated using an in vitro model of human pulmonary artery endothelial cell monolayers. LY379196 attenuated the increase in transendothelial albumin flux induced by glucose 40 mM (e.g. 411+/-160% [high-glucose] vs. 167+37% [high-glucose+LY379196], P<0.001) and angiotensin II 10 microM (e.g. 121+/-12% vs. 246+/-35%, P<0.01); endothelin-1 had no significant effect on monolayer permeability. LY379196 had no significant effect on the marked hyperpermeability response to alpha-thrombin 1 microM. Thus, two major pathways involved in vascular leakage in diabetic microangiopathy are amenable to therapeutic blockade by PKC-beta inhibition.