浅谈血液制品生产厂房工艺设计

结合具体项目,介绍了按照国家2010版GMP规范及参照欧盟GMP规范设计的某血液制品生产车间的工艺布局特点和设计创新点,该设计突破了以往设计思路,引入了国外先进的设计理念,最大限度的减少了交叉污染。     

氯丁二烯对小鼠血浆及脾脏淋巴细胞中cAMP和cGMP含量的影响

氯丁二烯经皮下注射给小鼠染毒,可引起血浆及脾脏淋巴细胞中cAMP含量升高和cGMP含量降低,二者含量的比值亦明显升高。结果提示,氯丁二烯可能通过改变血浆中cAMP和cGMP的含量而影响机体的生理功能及代谢;氯丁二烯所致淋巴细胞中的cAMP和cGMP含量的变化可能是引起细胞免疫和体液免疫毒性的机理之一。     

过表达RIP140的肿瘤相关巨噬细胞抑制肝癌细胞的侵袭和增殖

目的探讨肿瘤相关巨噬细胞（TAMs）中RIP140 的表达对肝癌细胞侵袭、增殖的影响。方法慢病毒介导小鼠腹腔巨 噬细胞（PMs）RIP140 的过表达,Western blot 和Real-time PCR（qRT-PCR）分别检测PMs中RIP140 蛋白以及核酸表达水平, 流式细胞仪分析慢病毒转染率;Western blot、细胞免疫荧光和qRT-PCR 检测肝癌条件培养基（HCM）刺激PMs 后TAMs中 RIP140 的表达变化;HCM刺激PMs以及HCM刺激过表达RIP140 的PMs,qRT-PCR检测TAMs极化指标以及NF-κB和IL-6 的表达;Transwell 实验和细胞流式凋亡实验检测肝癌细胞的侵袭和凋亡;肝癌细胞和PMs以4∶1 比例注射于BALB/c裸鼠皮 下,建立裸鼠皮下肝癌模型,成瘤癌组织HE染色和免疫组化评定肝癌组织大体生长情况和肝癌细胞增殖能力。结果慢病 毒介导PMs RIP140 的过表达,病毒转染效率高,RIP140 过表达明显;HCM刺激PMs后,TAMs中RIP140 呈低表达;HCM诱 导TAMs呈M2 型极化,并且与肿瘤生长密切相关的NF-κB-IL-6 轴处于活化状态;TAMs可促进肝癌细胞侵袭和增殖,抑制 肝癌细胞凋亡。TAMs过表达RIP140 可抑制HCM介导的TAMs M2 型极化并抑制NF-κB/IL-6 通路的激活,减少IL-6 的释 放;除此之外,TAMs过表达RIP140 可抑制肝癌细胞的侵袭和增殖,促进肝癌细胞的凋亡。结论过表达RIP140 的TAMs可 抑制肝癌细胞的侵袭和增殖。其机制可能与TAMs过表达RIP140后抑制TAMs M2型极化有关。     

Circ_0008039 supports breast cancer cell proliferation,migration, invasion,and glycolysis by regulating the miR‐140‐3p/SKA2 axis

Circular RNAs (circRNAs) have been shown to modulate gene expression and participate in the development of multiple malignancies. The purpose of this study was to investigate the role of circ_0008039 in breast cancer (BC). The expression of circ_0008039, miR‐140‐3p, and spindle and kinetochore‐associated protein 2 (SKA2) was detected by qRT‐PCR. Cell viability, colony formation, migration, and invasion were evaluated using methylthiazolyldiphenyl‐tetrazolium bromide (MTT) assay, colony formation assay, and transwell assay, respectively. Glucose consumption and lactate production were measured using commercial kits. Protein levels of hexokinase II (HK2) and SKA2 were determined by western blot. The interaction between miR‐140‐3p and circ_0008039 or SKA2 was verified by dual‐luciferase reporter assay. Finally, a mouse xenograft model was established to investigate the roles of circ_0008039 in BC in vivo. We found that circ_0008039 and SKA2 were upregulated in BC tissues and cells, while miR‐140‐3p was downregulated. Knockdown of circ_0008039 suppressed BC cell proliferation, migration, invasion, and glycolysis. Moreover, miR‐140‐3p could bind to circ_0008039 and its inhibition reversed the inhibitory effect of circ_0008039 interference on proliferation, migration, invasion, and glycolysis in BC cells. SKA2 was verified as a direct target of miR‐140‐3p and its overexpression partially inhibited the suppressive effect of miR‐140‐3p restoration in BC cells. Additionally, circ_0008039 positively regulated SKA2 expression by sponging miR‐140‐3p. Consistently, silencing circ_0008039 restrained tumor growth via increasing miR‐140‐3p and decreasing SKA2. In conclusion, circ_0008039 downregulation suppressed BC cell proliferation, migration, invasion, and glycolysis partially through regulating the miR‐140‐3p/SKA2 axis, providing an important theoretical basis for treatment of BC.

Abbreviations

ANOVA

analysis of variance

BC

breast cancer

circRNAs

circular RNAs

DMSO

dimethyl sulfoxide

ECAR

extracellular acidification rate

ECL

enhanced chemiluminescence

FBS

fetal bovine serum

HK2

hexokinase II

MEGM

mammary epithelial growth medium

miR‐140‐3p

microRNA‐140‐3p

MTT

methylthiazolyldiphenyl‐tetrazolium bromide

PBS

phosphate‐buffered saline

PRKAR1B

protein kinase A regulatory subunit R1‐beta

SD

standard ± deviation

SKA2

spindle and kinetochore‐associated protein 2

     

Signal transduction by cGMP in heart

Summary Early studies in whole heart indicated that cGMP antagonized the positive inotropic effects of catecholamines and cAMP. However, the regulation of cGMP levels by a variety of agents was not always consistent with their effects on contractility. It is now clear that at least two major cell types in whole heart, cardiac myocytes and vascular smooth muscle cells, differ markedly in their mechanisms of cGMP regulation and response to cGMP. Furthermore, experiments on isolated cardiac myocytes indicate that the mechanism of cGMP action even in this single cell type can be multifaceted. Cyclic GMP inhibits the L-type calcium channel current (I_Ca), which is the major source of Ca⁺⁺ entry into heart cells, and which plays a predominant role in the initiation and regulation of cardiac electrical and contractile activities. Patch-clamp measurements of I_Ca indicate that in isolated frog myocytes cGMP inhibits I_Ca by stimulation of cAMP phosphodiesterase (cGS-PDE), whereas in purified rat ventricular myocytes, cGMP predominantly inhibits I_Ca via a mechanism involving cGMP-dependent protein kinase (cGMP-PK). Under certain conditions, cGMP can also inhibit a cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) and thereby produce a stimulatory effect on I_Ca. Biochemical characterization of the endogenous PDEs and cGMP-PK in purified cardiac myocytes provided further evidence in support of these mechanisms of cGMP action on I_Ca.     

Development of a parent-proxy quality-of-life chronic cough-specific questionnaire: clinical impact vs psychometric evaluations

BACKGROUND: Chronic cough affects at least 7% of children, and the impact of this on families is significant. Although adult cough-specific quality-of-life (QOL) instruments have been shown to be a useful cough outcome measure, no suitable cough-specific QOL for parents of children with chronic cough exists. This article compares two methods of item reduction (clinical impact and psychometric) and reports on the statistical properties of both QOL instruments. METHOD: One hundred seventy children (97 boys and 73 girls; median age, 4 years; interquartile range, 3 to 7.25 years) and one of their parents participated. A preliminary 50-item parent cough-specific QOL (PC-QOL) questionnaire was developed from conversations with parents of children with chronic cough (ie, cough for > 3 weeks). Parents also completed generic QOL questionnaires (eg, Pediatric Quality of Life Inventory, version 4.0 [PedsQL4.0] and the 12-item Short Form Health Survey, version 2 [SF-12v2]). RESULTS: The clinical impact and psychometric method of item reduction resulted in 27-item and 26-item PC-QOL questionnaires, respectively, with approximately 50% of items overlapping. Internal consistency among the final items from both methods was excellent. Some evidence for concurrent and criterion validity of both methods was established as significant correlations were found between subscales of the PC-QOL questionnaire and the scales of the SF-12v2 and PedsQL4.0 scores. The PC-QOL questionnaire derived from both methods was sensitive to change following an intervention. CONCLUSION: Chronic cough significantly impacts on the QOL of both parents and children. Although the PC-QOL questionnaires derived from a clinical impact method and from a psychometric method contained different items, both versions were shown to be internally consistent and valid. Further testing is required to compare both final versions to objective and subjective cough measures.     

Zinc chloride (smoke bomb) inhalation lung injury: clinical presentations, high-resolution CT findings, and pulmonary function test results

STUDY OBJECTIVES: Zinc chloride smoke inhalation injury (ZCSII) is uncommon and has been rarely described in previous studies. We hypothesized that structural changes of the lung might correlate with pulmonary function. To answer this question, we correlated findings from high-resolution CT (HRCT) scan and the results of pulmonary function tests (PFTs) in patients with ZCSII. DESIGN: Retrospective cohort study. SETTING: University hospital. PATIENTS: Twenty patients who had been hospitalized with ZCSII-related conditions. MEASUREMENTS: The study included HRCT scan scores (0 to 100), static and dynamic lung volumes, and diffusing capacity of the lung for carbon monoxide (D(LCO)). RESULTS: HRCT scans and PFTs were performed initially after injury (range, 3 to 21 days) in all patients and during the follow-up period (range, 27 to 66 days) in 10 patients. The predominant CT scan findings were patchy or diffuse ground-glass opacities with or without consolidation. The majority of patients showed a significant reduction of FVC, FEV1, total lung capacity, and D(LCO), but normal FEV1/FVC ratio values. Changes of functional parameters correlated well with HRCT scan scores. Substantial improvements in CT scan abnormalities and pulmonary function were observed at follow-up. CONCLUSIONS: The majority of our patients with ZCSII presented with a predominant parenchymal injury of the lung that was consistent with a restrictive type of functional impairment and a reduction in Dlco rather than with obstructive disease. Our results suggest that HRCT scanning and pulmonary function testing may reliably predict the severity of ZCSII.     

Differential immune responses to HIV-1 envelope protein induced by liposomal adjuvant formulations containing monophosphoryl lipid A with or without QS21

Liposomes have shown promise as constituents of adjuvant formulations in vaccines to parasitic and viral diseases. A particular type of liposomal construct, referred to as Army Liposome Formulation (ALF), containing neutral and anionic saturated phospholipids, cholesterol, and monophosphoryl lipid A (MPLA), has been used as an adjuvant for many years. Here we investigated the effects of physical and chemical changes of ALF liposomes on adjuvanted immune responses to CN54 gp140, a recombinant HIV-1 envelope protein. While holding the total amounts of liposomal MPLA and the gp140 antigen constant, different liposome sizes and liposomal MPLA:phospholipid molar ratios, and the effect of adding QS21 to the liposomes were compared for inducing immune responses to the gp140. For liposomes lacking QS21, higher titers of IgG binding antibodies to gp140 were induced by small unilamellar vesicle (SUV) rather than by large multilamellar vesicle (MLV) liposomes, and the highest titers were obtained with SUV having the MPLA:phospholipid ratio of 1:5.6. ALF plus QS21 (ALFQ) liposomes induced the same maximal binding antibody titers regardless of the MPLA:phospholipid ratio. ALF MLV liposomes induced mainly IgG1 and very low IgG2a antibodies, while ALF SUV liposomes induced IgG1 ≥ IgG2a > IgG2b antibodies. Liposomes containing QS21 induced IgG1 > IgG2a > IgG2b > IgG3 antibodies. ELISPOT analysis of splenocytes from immunized mice revealed that ALF liposomes induced low levels of IFN-γ, but ALFQ induced high levels. ALF and ALFQ liposomes each induced approximately equivalent high levels of IL-4. Based on antibody subtypes and cytokine secretion, we conclude that ALF liposomes predominantly stimulate Th2, while ALFQ strongly induces both Th1 and Th2 immunity. When CN54 gp140 was adjuvanted with either ALF or ALFQ liposomes, antibodies were induced that neutralized two HIV-1 tier 1 clade C strain pseudoviruses.     

氧化型低密度脂蛋白促进单核细胞－内皮细胞体外粘附反应

体外培养猪胸主动脉内皮细胞，观察氧化型低密度脂蛋白对内皮细胞－单核细胞体外粘附反应的影响，同时观察氧化型低密庆脂蛋白对内皮细胞表面粘附分子颗粒膜蛋白－１４０及内皮细胞分泌前列环素的影响。结果发现氧化型低密度脂蛋白（０、５０、１００和２００ｍｇ／Ｌ）增加内皮细胞－单核细胞粘附率并呈剂量依赖性，其中以１００ｍｇ／Ｌ作用最强；氧化型低密度脂蛋白（１００ｍｇ／Ｌ）与内皮细胞孵育０．５ｈ，粘附率上升但无显著性，１ｈ后显著升高，３ｈ达高峰，４８ｈ回复接近正常水平。同时发现１００ｍｇ／Ｌ氧化型低密度脂蛋白使内皮细胞表面颗粒膜蛋白－１４０含量从９２．８±４７．６上升到２９３．０±１４０．７μｇ／Ｌ（Ｐ＜０．０５），内皮细胞分泌前列环素量从８４．６±１８．７降低到６．０±３．１ｎｇ／Ｌ（Ｐ＜０．０１）．结果表明氧化型低密度脂蛋白可显著促进单核细胞－内皮细胞粘附，其作用机制可能同内皮细胞表面粘附分子颗粒膜蛋白－１４０增加有关。关键词     

HN-10200 causes endothelium-independent relaxations in isolated canine arteries

Summary HN-10200, a nonselective inhibitor of phosphodiesterase, has positive inotropic and vasodilator activity. The present study was designed to determine the role of endotheliumin in causing relaxation to HN-10200 in isolated canine femoral and basilar arteries. Rings with and without endothelium were suspended for isometric tension recording in Krebs-Ringer bicarbonate solution bubbled with 94% O₂, 6% CO₂ (t=37°C; pH=7.4). HN-10200 and another nonselective phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX), caused similar concentration-dependent relaxations in femoral arteries with and without endothelium. In femoral arteries without endothelium, HN-10200 and IBMX significantly augmented relaxations to prostacyclin, but did not affect relaxations to a nitric oxide donor 3-morpholinosydnonimine (SIN-1) or endothelium-derived relaxing factor (EDRF) released by bradykinin. In basilar arteries, relaxations to HN-10200 were augmented by the removal of endothelium, whereas relaxations to IBMX were not affected. Relaxations to prostacyclin, SIN-1, and EDRF were not affected by the presence of phosphodiesterase inhibitors. The results of the present study suggest that HN-10200 causes endothelium-independent relaxations. In addition, it may augment relaxations to prostacyclin but does not affect relaxations to EDRF.