Oxygen saturation measurements in canine blood containing hemoglobin glutamer-200 (Bovine): in vitro validation of the NOVA CO-Oximeter

Abstract: This study was designed to validate in vitro oxygen saturation (SO₂) measurements with the NOVA CO-Oximeter (Nova Biomedical Corp, Waltham, Mass, USA) in canine blood containing hemoglobin (Hb) glutamer-200 bovine (Hb-200; Oxyglobin, Biopure, Cambridge, Mass, USA) as a Hb-based oxygen carrier recently introduced into clinical practice. In the first set of experiments, stored blood from 6 mixed-breed canine blood donors was used. Target PO₂ levels were reached in aliquots of blood samples by tonometry. Oxygen saturation was then measured with the test device and calculated based on known PO₂ values. In the second set of experiments, total oxygen content was directly measured by means of an oxygen-specific electrode in aliquots of fresh whole arterial, venous, and mixed (arterial-venous) blood withdrawn from the same canine blood donors. Hb-200 was added to those blood samples to yield plasma Hb concentrations of 1.62, 3.25, 6.50, and 9.75 g/dL. Based on Hb content and SO₂ measured by the NOVA CO-Oximeter in these samples, total oxygen content was also calculated for each sample and compared with measured values. A strong correlation was found between SO₂ values measured with the co-oximeter in samples after tonometry, and calculated SO₂ based on known PO₂. Directly measured total blood O₂ content varied by ≤ 5% from values computed based on co-oximeter measurements of Hb content and SO₂. These results did not change with different levels of oxygenation of the samples or different plasma Hb-200 concentrations. In conclusion, the NOVA CO-Oximeter is an accurate analyzer for measurement of SO₂ after Hb-200 administration to canine blood.     

miRNA-200a通过靶向ZEB1参与调节鸡胸肌细胞增殖分化

ZEB1在细胞增殖分化中发挥关键作用,然而关于ZEB1在鸡胸肌细胞增殖分化过程中的功能及其与miRNA互作的研究极少。为探索miRNA如何通过靶向ZEB1参与调节鸡胸肌细胞增殖分化,实验检测了ZEB1在55周龄和20周龄鸡胸肌组织中的表达,使用Target Scan及miRDB在线软件预测鸡ZEB1基因的靶向miRNA,构建ZEB1野生型、突变型双荧光报告载体,并在DF1细胞中验证了ZEB1的靶向miRNA,双荧光素酶报告实验结果说明miR-200a通过特异性结合ZEB1 3'非编码区种子序列直接靶向并抑制ZEB1基因的表达。结果表明:由于miR-200a在55周龄鸡胸肌表达下调,对ZEB1的抑制作用减弱,导致ZEB1在55周龄固始鸡胸肌组织表达较20周龄显著升高(P0.01)。本研究首次在鸡上证明miR-200a是ZEB1的靶向miRNA,且miR-200a可能通过靶向ZEB1参与调节鸡胸肌细胞的增殖分化,为深入理解miRNAs在家禽及其他鸟类中的分子调节机制提供了基础与依据。     

Effects of isovolemic resuscitation with hemoglobin-based oxygen carrier Hemoglobin glutamer-200 (bovine) on systemic and mesenteric perfusion and oxygenation in a canine model of hemorrhagic shock: a comparison with 6% hetastarch solution and shed blood

OBJECTIVE: To study Hemoglobin glutamer-200 bovine (Hb-200), 6% hetastarch (HES) and shed whole blood (WB) resuscitation in canine hemorrhagic shock. STUDY DESIGN: Prospective laboratory investigation. Animals Twelve adult dogs [29 +/- 1 kg (mean +/- SD)]. METHODS: Anesthetized dogs were instrumented for recording systemic and mesenteric hemodynamic parameters and withdrawal of arterial, mixed and mesenteric venous blood, in which hematological, oxygenation, blood gas and acid-bases variables were determined. Recordings were made before [baseline (BL)], after 1 hour of hypovolemia and immediately and 3 hours post-resuscitation with 30 mL kg(-1) of either Hb-200, HES, or WB. RESULTS: Blood withdrawal (average 34 +/- 2 mL kg(-1)) caused significant hemodynamic changes, metabolic acidosis and hyperlactatemia characteristic for hemorrhagic shock. Only WB transfusion restored all variables. Hemoglobin glutamer-200 bovine infusion returned most hemodynamic parameters including cardiac output and mesenteric arterial blood flow to BL but increased mean arterial pressure above BL (p < 0.05). However, Hb-200 failed to restore total Hb and arterial oxygen content (CaO2), leaving systemic (DO2I) and mesenteric O2 delivery (DO2Im) below BL (p < 0.05). Nevertheless, acid-base variables recovered completely after Hb-200 resuscitation, and met-hemoglobin (Met-Hb) levels increased (p < 0.05). Hetastarch resuscitation returned hemodynamic variables to or above BL but further decreased total Hb and CaO2, preventing recovery of sDO2I and mDO2I (p < 0.05). Thus, systemic and mesenteric O2 extraction stayed above BL (p < 0.05) while acid-base variables recovered to BL, although slower than in Hb-200 and WB groups (p < 0.05). CONCLUSIONS AND CLINICAL RELEVANCE: Resuscitation with Hb-200 seemed to resolve metabolic acidosis and lactatemia more rapidly than HES, but not WB; yet it is not superior to HES in improving DO2I and DO2Im. The hyperoncotic property of solutions like Hb-200 that results in rapid volume expansion with more homogenous microvascular perfusion and the ability to facilitate diffusive O2 transfer accelerating metabolic recovery may be the key mechanisms underlying their beneficial effects as resuscitants.     

牦牛VEGFA双荧光素酶载体构建及与miR-200b的靶向验证

为探究微小RNA(miR-200b)与血管内皮生长因子A(VEGFA)基因之间的靶向关系，采用miRanda和Targetscan软件预测牦牛miR-200b与VEGFA可能存在结合位点，构建了VEGFA基因3′UTR区的野生型和突变型双荧光素酶载体。利用与目的miRNA靶种子序列相结合基因的突变序列，构建该靶基因的突变型重组质粒(pGL3-promoter-mut VEGFA)，再进行重组质粒和目的miRNA的miRNA mimic的共转染实验。结果表明，预测到的miR-200b与VEGFA基因的3′UTR区存在结合位点。PCR进行扩增和测序之后，VEGFA基因3′UTR区的野生型、突变型载体构建成功。共转染VEGFA野生型载体和miR-200b mimic的双荧光素酶活性极显著低于对照组(P < 0.05)，VEGFA突变型载体和miR-200b mimic共转染的双荧光素酶活性与对照组无显著差异(P ＞ 0.05)。说明VEGFA基因的3′UTR区能与miR-200b结合并抑制双荧光素酶活性，验证了VEGFA是miR-200b的靶基因。