bFGF基因转染MSC目的基因检测

目的：探讨碱性成纤维细胞生长因子（bFGF）基因外源性转染到MSC（骨髓间充质细胞）后，bFGF基因的表达情况。方法选取SD大鼠，获取骨髓间充质细胞，分为对照组、阴性对照组（转染外源性GFP）和实验组（外源性bFGF转染MSC），经过体外培养后，应用RT-PCR和ELISE法检测各组MSC的bFGF基因阳性表达情况。结果 RT-PCR法显示实验组bFGF基因表达量（0.73±0.15）高于对照组和阴性对照组（P＜0.05）。ELISA法显示bFGF基因表达主要集中在胞浆，实验组bFGF基因表达量[上清为（5.38±0.45）ng/L，胞浆为（8.27±0.82）ng/L]高于其他两组（P＜0.05）。结论采用病毒介导基因转染技术能够将外源性bFGF转染至MSC，并能够成功实现bFGF基因表达。     

Clinical significance of changes in Torque teno virus DNA titer after chemotherapy in patients with primary lung cancer

Background

Several studies have reported that viral infections are related to lung cancer. We previously reported the involvement of Torque teno virus (TTV) in patients with lung cancer and idiopathic pulmonary fibrosis. However, the role of TTV in lung cancer growth, and its influence on changes in TTV DNA titers due to idiopathic pulmonary fibrosis (IPF) in lung cancer patients are poorly understood.

Slowing down as an early warning signal for abrupt climate change

In the Earth's history, periods of relatively stable climate have often been interrupted by sharp transitions to a contrasting state. One explanation for such events of abrupt change is that they happened when the earth system reached a critical tipping point. However, this remains hard to prove for events in the remote past, and it is even more difficult to predict if and when we might reach a tipping point for abrupt climate change in the future. Here, we analyze eight ancient abrupt climate shifts and show that they were all preceded by a characteristic slowing down of the fluctuations starting well before the actual shift. Such slowing down, measured as increased autocorrelation, can be mathematically shown to be a hallmark of tipping points. Therefore, our results imply independent empirical evidence for the idea that past abrupt shifts were associated with the passing of critical thresholds. Because the mechanism causing slowing down is fundamentally inherent to tipping points, it follows that our way to detect slowing down might be used as a universal early warning signal for upcoming catastrophic change. Because tipping points in ecosystems and other complex systems are notoriously hard to predict in other ways, this is a promising perspective.     

Measurement of long‐term iron absorption and loss during iron supplementation using a stable isotope of iron (57Fe)

We report the first measurements of long‐term iron absorption and loss during iron supplementation in African children using a stable isotope of iron (⁵⁷Fe). After uniform labelling of body iron with ⁵⁷Fe, iron absorption is proportional to the rate of decrease in the ⁵⁷Fe tracer concentration, while iron loss is proportional to the rate of decrease in the ⁵⁷Fe tracer amount. Anaemic Gambian toddlers were given 2 mg ⁵⁷Fe orally to equilibrate with total body iron over 8–11 months. After assignment to the positive control arm of the HIGH study, 22 toddlers consumed a micronutrient powder containing 12 mg iron for 12 weeks followed by 12 weeks without iron supplementation. Their daily iron absorption increased 3·8‐fold during the iron supplementation period compared to the control period [median (interquartile range, IQR): 1·00 (0·82; 1·28) mg/day vs. 0·26 (0·22; 0·35) mg/day; P = 0·001]. Unexpectedly, during the supplementation period, daily iron loss also increased by 3·4‐fold [0·75 (0·55; 0·87) mg/day vs. 0·22 (0·19; 0·29) mg/day; P = 0·005]. Consequently, most (~72%) of the absorbed iron was lost during supplementation. Long‐term studies of iron absorption and loss are a promising and accurate method for assessing and quantifying long‐term iron balance and may provide a reference method for evaluating iron intervention programs in vulnerable population groups. This study was registered as ISRCTN 0720906.     

Long-term in vivo gene expression in mouse kidney using ϕC31 integrase and electroporation

Background: Achieving long-term gene expression in kidney will be beneficial for gene therapy of renal and congenital diseases, genetic studies constructing animal disease models, and the functional analysis of disease-related genes.

Purpose: The purpose of this study was to develop an in vivo long-term gene expression system in murine kidney using ?C31 integrase.

Methods: Gene expression in cultured RENCA, TCMK-1, and HEK293 cells was assessed. The long-term in vivo gene expression system in the kidney was achieved by co-transfecting 5?µg of pORF-luc/attB as a donor plasmid and 20?µg of pCMV-luc as a helper plasmid into the right kidney of mice by electroporation. Luciferase expression levels were measured to determine longevity of the expression.

Results: Significantly high luciferase expression levels were observed in cultured RENCA, TCMK-1, and HEK293 cells over 1 month compared with controls (non-integrase system). The luciferase cDNA sequence was integrated at a pseudo attP site termed mpsL1. In vivo luciferase expression levels in the integrase group were sustained and significantly higher than those in the control group over 2 months. Furthermore, ?C31 integrase-transfected cells had less genomic DNA damage caused by integrase expression.

Discussion and conclusion: These results demonstrated that the ?C31 integrase system could produce long-term (2 months) in vivo gene expression in mouse kidney.     

聚乙烯亚胺-蛋白纳米复合体转染蛋白进入骨髓间充质干细胞的应用研究

目的:探索纳米材料聚乙烯亚胺(PEI)携载不同分子量的蛋白质转染进人骨髓间充质干细胞(h BMSCs)的作用及优化其最佳配比条件。方法:利用物理化学作用构建6组PEI-DNase I(DNA酶I)复合体,运用激光散射技术初步摸索其最佳摩尔比,用透射电镜直观复原纳米-蛋白复合体外貌;同时,原代分离培养健康人的骨髓间充质干细胞并进行体外扩增,通过构建成功的4组PEI-GFP(绿色荧光蛋白)纳米蛋白复合体对h BMSCs进行转染,共聚焦荧光显微镜下观察荧光表达情况,再通过MTT法检测该复合体对细胞增殖的毒性影响,并用β-半乳糖苷酶实验验证其转入蛋白的活性表现。结果:当PEI与蛋白质的摩尔比为4∶1时,形成的复合体转染效率最高,β-半乳糖苷酶实验细胞染色变蓝。结论:PEI能与蛋白质形成纳米复合体,并能转染多种蛋白质进入人骨髓间充质干细胞,所转染的蛋白质分子仍具有活性,为细胞重编程技术提供了新途径。     

慢性稳定性心绞痛的护理及中医辨证施护干预

慢性稳定性心绞痛是危害人类健康的常见病、多发病,临床现代护理已经较完善,中医护理遵循"整体观念""辨证施护"的原则,进行辨证施护,取得良好的效果。     

过表达NK4对人肺腺癌A549细胞增殖和凋亡的影响

目的:构建含有NK4基因的重组慢病毒载体(LV-NK4),感染人肺腺癌A549细胞后观察NK4基因的表达,并研究NK4对肺癌A549细胞增殖和凋亡的影响?方法:采用DNA重组技术,将NK4基因克隆至带增强型绿色荧光蛋白(EGFP)的慢病毒载体GV358,脂质体介导法将其与慢病毒包装系统共转染293T细胞,包装为慢病毒,感染A549细胞,观察感染效率?采用逆转录聚合酶链反应(RT-PCR)及免疫印迹法(Western blot)检测NK4基因和蛋白的表达?建立A549(空白对照组)?A549/NK4(实验组)?A549/LV(空病毒对照组)3组细胞;RT-PCR 法测定各组细胞c-met基因水平;噻唑蓝(MTT)比色法测定各组细胞第1~7天增殖情况,绘制生长曲线;流式细胞术检测各组细胞凋亡率?结果:经基因测序证实LV-NK4构建成功;感染后A549细胞中可见明显的NK4蛋白表达,Western blot显示50 000处有蛋白条带;RT-PCR结果显示实验组NK4 mRNA水平较空白对照组和空病毒对照组明显升高(P < 0.01),c-met mRNA水平较其他两组下降(P < 0.05);MTT结果显示实验组细胞从第4天起生长比正常对照组和空病毒对照组均缓慢(P < 0.05);流式细胞术结果显示实验组细胞凋亡率高于正常对照组和空病毒对照组(P < 0.05)?结论:成功构建NK4慢病毒载体且有效转染A549细胞;NK4具有抑制A549细胞增殖并促进其凋亡的作用,可能与下调c-met基因水平有关?     

A Multicenter,Randomized,Double-blind Clinical Study on Wufuxinnaoqing Soft Capsule(五福心脑清胶囊) in Treatment of Chronic Stable Angina Patients with Blood Stasis Syndrome

Objective:To confirm the efficacy and safety of Wufuxinnaoqing Soft Capsule(五福心脑清胶囊,WSC)in the treatment of chronic stable angina(blood stasis syndrome).Methods:A multicenter,randomized,double-blind,placebo-controlled trial with superiority test was designed.A total of 240 patients with chronic stable angina(blood stasis syndrome)from multiple centers were randomly and equally assigned to the treatment group and the control group.Based on standard treatment of Westem medicine,the treatment group was given WSC,while the control group was given WSC mimetic,both for 12 weeks.Observed indicators included the efficacy in angina,the efficacy in Chinese medicine syndrome,the withdrawal or reduce rate of nitroglycerin and routine safety indices.Results:After 12-week treatment,the significant effective rate and total effective rate of the treatment group were significantly better than those of the control group(23.5%vs.9.2%,64.7%vs.30.8%),respectively,with statistically significant difference(P0.01).After 12-week treatment,the decreased points and the decreased rate of angina symptom score in the treatment group were better than in the control group(5.1±4.2 points vs.2.8±3.5 points,44.9%±37.2%vs.25.4%±30.7%)respectively,with significant difference(P0.01).After 12-week treatment,the significant effective rate and total effective rate of the treatment group were better than the control group(respectively,30.3%vs.15.0%,67.2%vs.45.0%,P0.01).After 8-or 12-week treatment,the decreased points and the decreased rate of Chinese medicine syndrome score in the treatment group were better than the control group(P0.05 or P0.01).After 12-week treatment,nitroglycerin withdrawal rate and the withdrawal or reduce rate in treatment group were better than the control group(P0.01).On safety evaluation,the incidence of adverse events(7.563%vs.7.500%)and the incidence of cardiovascular events(0.840%vs.0.000%)in the treatment group were similar with the control group,and the difference was not statistically significant(P0.05).Conclusion:In treatment of chronic stable angina(blood stasis syndrome),WSC can reduce angina attacks and consumption of nitroglycerin,decrease angina severity degree,effectively relieve the blood stasis syndromes,such as chest pain,chest tightness,palpitations,dark purple tongue and other symptoms.Besides,adverse events and cardiovascular adverse events in the treatment group and the control group showed no difference.All shows that the drug is safe and effective.[This study was registered in Chinese Clinical Trial Registry(ChiCTR),with registration number:ChiCTR-TRC-14005158.]