成熟心肌细胞的分离与培养方法

0　引言　我们介绍一种在无血清培养基中分离和培养成熟心肌细胞的方法[1,2],着重强调对优化心肌细胞培养至关重要的分离过程.1　材料　分离装置可用典型的Langandorff装置,配合无菌操作要求,对心脏进行逆行灌注.基本灌流液(溶液A),是用纯水和高纯度试剂制备,其成分有(mmol·L-1):NaCl130;HEPES23;葡萄糖21;牛磺酸20;肌酸5;MgCl25;丙酮酸钠5;KCl4.5;NaH2PO41;pH7.3.过滤(0.2μm滤纸片)溶液除去微生物和水颗粒.A溶液是分离过程中使用的其他4种溶液的原料:1750μmol·L-1Ca2+溶液(300mLA溶液+750μmol·L-1Ca2+);2游离Ca2++EGTA…     

Performance evaluation of five commercial real-time PCR reagent systems using TaqMan assays for B. anthracis detection

BackgroundReal-time PCR assay sensitivity is affected by the choice and concentrations of reaction mix constituents among other factors such as primers, probes, and analytical assay platforms. Commercially available reagent mixes facilitate PCR assay set-up with fewer steps and timeliness. However, determination of analytical assay framework is important for ready-to-use real-time PCR reagent systems for rapid, quantitative and accurate detection of bioterror pathogens such as Bacillus anthracis.MethodsIn this study, performance characteristics of five commercially available quantitative PCR reagent mixes were evaluated using TaqMan-based real-time PCR. The reagent systems were tested for compatibility on the ABI 7000 assay platform and compared for their distinctive analytical characteristics using the B. anthracis rpoB and pag gene real-time PCR assays.Results and conclusionsKnowledge of distinctive assay performance characteristics of commercially available qPCR reagent mixes is critical for carefully designing analytical assay systems. The ABI, ABgene and Eppendorf reagent systems performed consistently overall for the two TaqMan assays for B. anthracis detection that were used in the current study. However, the use of Eppendorf reagent system requires shorter thermal cycling time. In addition, while the ABI and Eppendorf systems have similar assay sensitivity for both the rpoB and pag assays, the Eppendorf system achieves the same with lower C_T values.     

Microarray and microfluidic methodology for genotyping cytokine gene polymorphisms

Cytokine genetic polymorphisms are the subject of disease-association studies that require large-scale human genotyping. Polymerase chain reaction based custom microarrays and microfluidics systems were used to develop genotyping assays for following cytokine polymorphisms: tumor necrosis factor-alpha G-308A, interleukin-4 (IL-4) C-589T, interferon-gamma (CA)n repeats, IL-1RN 86-bp variable number of tandem repeats (VNTR), and CCR5 32-bp indel. For G-308A, 70.9% of DNA samples assayed were homozygous for wild type, 25.5% were heterozygous, and none were homozygous for variant allele. For C-589T, 35.5% of DNA samples were homozygous for wild type, 38% were heterozygous, and 22% were homozygous for variant. For IL-1RN VNTR, 71% of DNA samples were homozygous and the remainder were heterozygous. For CCR5, 96.4% of amplicons were homozygous for wild type, and 3.6% were heterozygous containing deletion. For IFN-gamma (CA)n repeats, 35.6% had 2,2 alleles, 42.2% had 2,3 alleles, and 11% had 3,3 alleles with alleles 1 through 5 corresponding to 11 through 15 repeats, respectively. There was good concordance between the results we obtained and current "gold-standard" methodologies for analyzing single nucleotide polymorphisms and size polymorphisms. Electronic DNA concentration with high stringency predisposes microarray technology to hybridization fidelity and accuracy, and microfluidics systems outperform conventional methodologies for size polymorphisms. Comprehensive genotyping can be achieved for clinical epidemiologic studies on cytokine gene polymorphisms using this approach.     

Effect of dietary curcumin and dibenzoylmethane on formation of 7,12- dimethylbenz[a]anthracene-induced mammary tumors and lymphomas/leukemias in Sencar mice

Female Sencar mice (6 weeks old) were administered 1 mg of 7,12- dimethylbenz[a]anthracene (DMBA) by oral gavage once a week for 5 weeks. At 20 weeks after the first dose of DMBA, 68% of mice developed mammary tumors (the average 1.08 tumors per mouse) and 45% had lymphomas/leukemias. Feeding 1% dibenzoylmethane (DBM) in AIN 76A diet, starting at 2 weeks before the first dose of DMBA and continuing until the end of the experiment, inhibited both the multiplicity and incidence of DMBA-induced mammary tumor by 97%. The incidence of lymphomas/leukemias was completely inhibited by 1% DBM diet. In contrast, feeding 2% curcumin diet had little or no effect on the incidence of mammary tumors, and the incidence of lymphomas/leukemias was reduced by 53%.      

Hepatoprotective effect of a curcumin/absinthium compound in experimental severe liver injury

OBJECTIVE: A preliminary in vitro study with hepatocyte culture showed that concentrations as low as 10 µg/mL of PN‐M001 are able to significantly mitigate CCl4 hepatocyte damage (P < 0.05) comparable to 100 µg/mL silymarin, and 100 µg/mL proved to be more protective than either silymarin 100 µg/mL or glycyrrhizin 10 µg/mL (P < 0.05). METHODS: Wistar rats were allocated into three groups: (A) 0.1 mL/100 g body weight (BW) mixture of CCl₄ in olive oil (1 : 1 v/v) subcutaneous injection twice daily for 4 weeks; (B) as A, plus oral administration of 50 mg/kg of K‐17.22 dissolved in 5% glucose; (C) as B but with PN‐M001 given 1 week after the first injection of CCl₄. Rats were killed at the end of the study and blood and liver samples were obtained. RESULTS: When compared with a control, group A showed a significant decrease of glutathione (GSH;>45%, P < 0.001) and oxidized GSH (GSSG; P < 0.01) liver content, a lower liver wet weight (P < 0.01) together with an increase of both transaminases (>15‐fold, P < 0.001) whereas groups B and C both showed only a mild increase in transaminases (<4‐fold, P < 0.05). Group A showed a significant decrease of Y‐protein fraction and of GST activity, as tested by both substrates (P < 0.01 vs control). However, both these parameters were reverted to normal by PN‐M001 (P < 0.05 vs A). CONCLUSIONS: These preliminary data suggest that PN‐M001 exerts a highly protective and prolonged effect (either preventive or therapeutic) on GSH depletion in CCl₄‐induced liver injury, which suggests its potential use in the clinical setting.     

Multipotent adult progenitor cells

We here discuss the potency and characteristics of various adult derived adherent stem cells with special focus on multipotent adult progenitor cells (MAPC) isolated first in 2002 in our lab. We describe the potency of MAPC, our current understanding in relationship with novel insights gained in epigenetic modifications that increase cellular potency, and their possible clinical applications.     

Born Too Soon: Care before and between pregnancy to prevent preterm births: from evidence to action

Providing care to adolescent girls and women before and between pregnancies improves their own health and wellbeing, as well as pregnancy and newborn outcomes, and can also reduce the rates of preterm birth. This paper has reviewed the evidence based interventions and services for preventing preterm births; reported the findings from research priority exercise; and prescribed actions for taking this call further. Certain factors in the preconception period have been shown to increase the risk for prematurity and, therefore, preconception care services for all women of reproductive age should address these risk factors through preventing adolescent pregnancy, preventing unintended pregnancies, promoting optimal birth spacing, optimizing pre-pregnancy weight and nutritional status (including a folic acid containing multivitamin supplement, and ensuring that all adolescent girls have received complete vaccination. Preconception care must also address risk factors that may be applicable to only some women. These include screening for and management of chronic diseases, especially diabetes; sexually-transmitted infections; tobacco and smoke exposure; mental health disorders, notably depression; and intimate partner violence. The approach to research in preconception care to prevent preterm births should include a cycle of development and delivery research that evaluates how best to scale up coverage of existing, evidence-based interventions, epidemiologic research that assesses the impact of implementing these interventions, and discovery science that better elucidates the complex causal pathway of preterm birth and helps to develop new screening and intervention tools. In addition to research, policy and financial investment is crucial to increasing opportunities to implement preconception care, and rates of prematurity should be included as a tracking indicator in global and national maternal child health assessments.

Declaration

This article is part of a supplement jointly funded by Save the Children's Saving Newborn Lives programme through a grant from The Bill & Melinda Gates Foundation and March of Dimes Foundation and published in collaboration with the World Health Organization (WHO). The original article was published in PDF format in the WHO Report "Born Too Soon: the global action report on preterm birth (ISBN 978 92 4 150343 30). The article has been reformatted for journal publication and has undergone peer review according to Reproductive Health's standard process for supplements and may feature some variations in content when compared to the original report. This co-publication makes the article available to the community in a full-text format.

     

Preconception care: preventing and treating infections

Introduction

Infections can impact the reproductive health of women and hence may influence pregnancy related outcomes for both the mother and the child. These infections range from sexually transmitted infections (STIs) to TORCHS infections to periodontal disease to systemic infections and may be transmitted to the fetus during pregnancy, labor, delivery or breastfeeding.

Methods

A systematic review and meta-analysis of the evidence was conducted to ascertain the possible impact of preconception care for adolescents, women and couples of reproductive age on MNCH outcomes. A comprehensive strategy was used to search electronic reference libraries, and both observational and clinical controlled trials were included. Cross-referencing and a separate search strategy for each preconception risk and intervention ensured wider study capture.

Results

Preconception behavioral interventions significantly declines re-infection or new STI rates by 35% (95% CI: 20-47%). Further, condom use has been shown to be the most effective way to prevent HIV infection (85% protection in prospective studies) through sexual intercourse. Intervention trials showed that preconception vaccination against tetanus averted a significant number of neonatal deaths (including those specifically due to tetanus) when compared to placebo in women receiving more than 1 dose of the vaccine (OR 0.28; 95% CI: 0.15-0.52); (OR 0.02; 95% CI: 0.00-0.28) respectively.

Conclusion

Preconception counseling should be offered to women of reproductive age as soon as they test HIV-positive, and conversely women of reproductive age should be screened with their partners before pregnancy. Risk assessment, screening, and treatment for specific infections should be a component of preconception care because there is convincing evidence that treatment of these infections before pregnancy prevents neonatal infections.