Mineralization of 3D Osteogenic Model Based on Gelatin-Dextran Hybrid Hydrogel Scaffold Bioengineered with Mesenchymal Stromal Cells: A Multiparametric Evaluation

Gelatin–dextran hydrogel scaffolds (G-PEG-Dx) were evaluated for their ability to activate the bone marrow human mesenchymal stromal cells (BM-hMSCs) towards mineralization. G-PEG-Dx1 and G-PEG-Dx2, with identical composition but different architecture, were seeded with BM-hMSCs in presence of fetal bovine serum or human platelet lysate (hPL) with or without osteogenic medium. G-PEG-Dx1, characterized by a lower degree of crosslinking and larger pores, was able to induce a better cell colonization than G-PEG-Dx2. At day 28, G-PEG-Dx2, with hPL and osteogenic factors, was more efficient than G-PEG-Dx1 in inducing mineralization. Scanning electron microscopy (SEM) and Raman spectroscopy showed that extracellular matrix produced by BM-hMSCs and calcium-positive mineralization were present along the backbone of the G-PEG-Dx2, even though it was colonized to a lesser degree by hMSCs than G-PEG-Dx1. These findings were confirmed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), detecting distinct lipidomic signatures that were associated with the different degree of scaffold mineralization. Our data show that the architecture and morphology of G-PEG-Dx2 is determinant and better than that of G-PEG-Dx1 in promoting a faster mineralization, suggesting a more favorable and active role for improving bone repair.     

小鼠心肌组织裂解上清液诱导小鼠骨髓瘤Sp2/0细胞凋亡

目的：研究小鼠心肌上清液诱导小鼠骨髓瘤Sp2／0细胞凋亡的作用．方法：在Sp2／0细胞培养孔中,分别加入不同稀释度（1：6,1：12,1：24）的小鼠心肌上清液和环磷酰胺,24h后镜下观察细胞形态,并于48h后流式细胞仪检测凋亡细胞的比率;同时,Sp2／0细胞和小鼠心肌细胞共培养,10h后观察Sp2／0细胞形态学变化;并用不同稀释度（1：5,1：10,1：20,1：40）小鼠心肌裂解上清液、肝脏裂解上清液、胸腺裂解上清液及环磷酰胺诱导Sp2／0,HeLa,Hep-2和Vero细胞凋亡,并比较其差异．结果：不同稀释度的心肌裂解上清液均可诱导Sp2／0细胞的凋亡,尤以高稀释度更明显;小鼠心肌细胞与Sp2／0细胞共培养10h后,少部分Sp2／0细胞出现凋亡;小鼠心肌裂解上清液、肝脏裂解上清液、胸腺裂解上清液均可引起Sp2／0,HeLa,Hep-2和Vero细胞发生凋亡,但小鼠心肌裂解上清液作用明显,且作用时间持久．结论：小鼠心肌组织裂解上清液具有明显诱导小鼠骨髓瘤Sp2／0细胞凋亡的作用．     

茵栀黄注射液中细菌内毒素的检测

目的:研究细菌内毒素检察法应用于茵栀黄注射液的可行性,为临床安全用药和基层快速检验提供依据。方法:按照中国药典2000年版二部附录ⅪE复核鲎试剂灵敏度并进行样品干扰试验和样品细菌内毒素测定。结果:样品稀释18倍可排除干扰,稀释20倍为最佳测定浓度。结论:用鲎试剂检测茵栀黄注射液中的细菌内毒素灵敏、快速,结果可靠,可代替兔法检查其热原。     

人骨髓间充质干细胞体外分离无需红细胞裂解

目的观察红细胞裂解液对人骨髓间充质干细胞（hMSCs）体外分离、培养及增殖特性的影响。方法将22份骨髓随机分为两组：（1）碎红组11份,即骨髓样本在处理过程中给予Tris-NH。C1红细胞裂解剂;（2）未碎红组11份,即骨髓样本处理过程中未加红细胞裂解剂。观察两组细胞生长情况,第1、2、3代细胞达到传代的时间和细胞扩增达到107数量的时间。结果（1）碎红组与未碎红组在P1传代时间分别为（9．3±4．9）d和（7．2-4-1．0）d、P2传代时间（14．4±4．7）d和（14．5±3．5）d、P3传代时间（18．5±5．0）d和（20．1±4．4）d,两组在传代时间上差异均无统计学意义（f值分别为1．39、0．06、0．80,P均〉0．05）;（2）碎红组与未碎红组在细胞培养达到10’数量的时间差异无统计学意义[（19．5±7．9）、（22．1±7．3）d,t=0．80,P〉0．05]。结论添加与不添加Tris-NH4C1红细胞裂解剂对hMSCs的体外分离、培养和细胞增殖速度方面分析均无影响。故hMSCs体外分离培养可省略红细胞裂解步骤。     

卡介苗热休克蛋白70对肿瘤细胞裂解物免疫原性的影响

的：观察卡介苗热休克蛋白70（BCGHSP70）对肿瘤细胞裂解物（TCL）免疫原性的影响,为TCL相关肿瘤疫苗的制备提供依据。方法：应用冻融法制备B16黑色素瘤细胞裂解物B16TCL,将BCGHSP70与B16TCL在体外混合制备BCGHSP70-B16TCL。将C57BL/6小鼠随机分为BCGHSP70-B16TCL组、PBS组、BCGHSP70组和B16TCL组,分别于第1和14天皮下免疫BCGHSP70-B16TCL、PBS、BCGHSP70和B16TCL。于末次免疫后第7天处死小鼠,MTT法观察不同免疫组小鼠脾淋巴细胞在B16TCL体外刺激后的增殖情况,计算刺激指数（SI）。结果：应用冻融法能够使B16细胞完全裂解,所制备的B16TCL中含有丰富的蛋白成分。与PBS组、BCGHSP70组和B16TCL组比较,BCGHSP70-B16TCL组免疫小鼠脾脏明显增大;BCGHSP70-B16TCL免疫组SI高于PBS免疫组（P=0.00）、 B16TCL免疫组（P=0.01）和BCGHSP70免疫组（P=0.00）。结论：BCGHSP70　能够增强B16TCL的免疫原性,提示BCGHSP70可能作为有效的免疫佐剂用于TCL相关肿瘤疫苗的研究。     

醋酸氯己定冲洗剂细菌内毒素检查法研究

目的建立醋酸氯己定冲洗剂细菌内毒素的检查方法。方法按2015年版《中国药典(四部)》通则1143内毒素检查法,建立醋酸氯己定冲洗剂细菌内毒素的检查方法,并使用2个厂家的鲎试剂进行方法验证。结果使用稀释剂Ⅰ将醋酸氯己定冲洗剂稀释2倍后,再用细菌内毒素检查用水稀释至8倍,可消除其对鲎试剂凝集反应的干扰,细菌内毒素限值为0.5EU/mL。结论该方法可用于醋酸氯己定冲洗剂细菌内毒素的检查,为可能导致鲎试剂中单价和二价离子缺失样品的细菌内毒素检查方法的建立提供参考。     

HEMOXCell,a New Oxygen Carrier Usable as an Additive for Mesenchymal Stem Cell Culture in Platelet Lysate‐Supplemented Media

Human mesenchymal stem cells (MSCs) are promising candidates for therapeutic applications such as tissue engineering. However, one of the main challenges is to improve oxygen supply to hypoxic areas to reduce oxygen gradient formation while preserving MSC differentiation potential and viability. For this purpose, a marine hemoglobin, HEMOXCell, was evaluated as an oxygen carrier for culturing human bone marrow MSCs in vitro for future three‐dimensional culture applications. Impact of HEMOXCell on cell growth and viability was assessed in human platelet lysate (hPL)‐supplemented media. Maintenance of MSC features, such as multipotency and expression of MSC specific markers, was further investigated by biochemical assays and flow cytometry analysis. Our experimental results highlight its oxygenator potential and indicate that an optimal concentration of 0.025 g/L HEMOXCell induces a 25%‐increase of the cell growth rate, preserves MSC phenotype, and maintains MSC differentiation properties; a two‐fold higher concentration induces cell detachment without altering cell viability. Our data suggest the potential interest of HEMOXCell as a natural oxygen carrier for tissue engineering applications to oxygenate hypoxic areas and to maintain cell viability, functions and “stemness.” These features will be further tested within three‐dimensional scaffolds.     

细菌溶解产物防治儿童反复呼吸道感染的临床疗效观察

目的 本研究旨在观察细菌溶解产物(泛福舒)在呼吸道感染急性期及预防再发呼吸道感染的疗效.方法 选取2014年4月至2015年4月中国医科大学附属盛京医院小儿呼吸病房因反复呼吸道感染住院的80例患儿,随机分为治疗组及对照组各40例,对照组给予常规抗感染及对症治疗,治疗组在常规抗感染及对症治疗基础上加用口服泛福舒3个月,观察2组患儿在呼吸道感染急性期各临床症状持续时间、抗生素使用及住院时间变化;观察1年内再次发生呼吸道感染次数;测定两组治疗前及治疗后6个月血清免疫球蛋白指标.结果 与对照组比较,治疗组呼吸道感染急性期发热、咳嗽等症状持续时间、肺部啰音持续时间、抗生素使用及住院时间等均明显缩短,再次发生呼吸道感染次数明显减少,差异有统计学意义(P<0.05);治疗组治疗后血清免疫球蛋白IgG、IgA较治疗前明显升高,治疗组治疗后免疫球蛋白IgG、IgA较对照组治疗后免疫球蛋白IgG、IgA明显升高,差异有统计学意义(P<0.05).结论 口服泛福舒防治呼吸道感染,不仅可以减轻呼吸道感染急性期各种临床症状持续时间,缩短抗生素使用时间及住院时间,同时可以提高患儿免疫力,减少再次呼吸道感染次数.     

Hemolymph coagulation and phenoloxidase activity in Uca tangeri induced by Escherichia coli endotoxin

Uca tangeri is a marine fiddler crab found commonly in the West African coast and is often exposed to Gram-negative pathogens upon injury. The aim of this study was to document the patterns of endotoxin-induced protein coagulation and phenoloxidase (PO) activity in hemolymph fractions of Uca tangeri. Hemolymph from live crabs was obtained by carapace puncture, pooled. and then separated into plasma, hemocyte Lysate (HL), hemocyte lysate supernatant (HLS) and hemocyte lysate debris (HLD). The effect of Escherichia coli (O1111:B4) endotoxin and calcium ion (Ca²⁺) on protein coagulation in the presence/absence of endotoxin and the endotoxin dose-dependence of coagulation and PO activity were each studied in the plasma, HL, HLS and HLD. The results showed Ca²⁺ was required to induce coagulation, and was endotoxin concentration-dependent in the plasma. PO activity was highest in the HLS but PO specific activity was highest in HLD. PO activity remained relatively constant with increased LPS concentration in the range studied 0–10 EU/ml. From the data we conclude that endotoxin-induced protein coagulation occurs in the plasma alone and might be mediated by trans-glutaminases, while PO activity is localized inside hemocytes and cell membranes in Uca tangeri.