Rapid and simple isolation of DNA from agarose gels

Summary We present a simple method for the isolation of DNA from agarose gels that is economic, fast, and independent of electrical equipment. DNA fragments of up to 6 kb can be easily extracted within 5 min using a disposable plastic syringe and filter paper. Total extraction of DNA fragments between 10 and 20 kb in size is achieved by concentrating the DNA flushed from the gel in a DNA-binding column.     

琼脂糖胶/聚苯乙烯磺酸微囊化胰B细胞株MIN6作为人工生物胰的实验研究

应用琼脂糖胶／聚苯乙烯磺酸微囊化胰B细胞林MIN6作为人工生物胰进行大鼠皮下异种移植。结果表明，在不应用任何免疫抑制剂的情况下，大鼠皮下异种移植一周后，重新取出的微囊仍保持光滑完整的球状，其内MIN6细胞仍清晰可见，囊周无明显纤维化及粘着。胰岛素释放试验结果显示，低糖浓度刺激时胰岛素的释放量为19．20±1．35μU·10microbead－1／h；高糖浓度刺激时胰岛素的释放量达到51．47±3．80μU·100microbeads-1／h，约为低糖浓度时的2．7倍，与低糖浓度时比较其差异有高度显著性（P＜0．001）。提示琼脂糖胶／聚苯乙烯磺酸微囊化胰B细胞林MIN6作为人工生物胰，既能保护移植物膜B细胞株MIN6免受排斥反应，也能维护膝B细胞株MIN6对糖刺激的有效的胰岛素分泌反应性。     

改良琼脂糖凝胶电泳法测定血清LDH同工酶的应用

目的 寻求更加准确、简便、快速、实用的乳酸脱氢酶同工酶（LDH）测定方法。方法 据LDH同工酶等电点不同,选择琼脂糖胶作支持物,对以往LDH同工酶测定方法进行改进,将五型LDH同工酶进行分离并定量测定。结果 改进后的LDH同工酶测定方法稳定,区带清晰,重复性好,结果可靠,试剂可长期保存。结论 改良琼脂糖凝胶电泳法测定血清LDH同工酶应用前景良好。     

琼脂糖火箭电泳法测定唾液溶菌酶活性

本文采用琼脂糖火箭电泳法测定唾液溶菌酶活性,将琼脂扩散方法和电泳技术结合一起,使溶菌酶在电场中泳动,在通过含有作用底物的凝胶时与溶壁微球菌发生水解作用,形成可见的溶菌透明峰,根据峰值与溶菌酶浓度的时数值成正比的关系求得样品溶菌酶含量。该方法简便、快速、稳定性好,灵敏度高,结果准确可靠。在本实验条件下最低检出量为1.25 μg/ml,在5.O-100.0μg/ml范围内呈良好的浓度-峰高的线性关系,r=O.9947,P<0.001,批内重复变异系数CV=4.15％,批间重复变异系数CV=7.42％,平均回收率为95％～114％。     

Tuning mechanical performance of poly(ethylene glycol) and agarose interpenetrating network hydrogels for cartilage tissue engineering

Hydrogels are attractive for tissue engineering applications due to their incredible versatility, but they can be limited in cartilage tissue engineering applications due to inadequate mechanical performance. In an effort to address this limitation, our team previously reported the drastic improvement in the mechanical performance of interpenetrating networks (IPNs) of poly(ethylene glycol) diacrylate (PEG-DA) and agarose relative to pure PEG-DA and agarose networks. The goal of the current study was specifically to determine the relative importance of PEG-DA concentration, agarose concentration, and PEG-DA molecular weight in controlling mechanical performance, swelling characteristics, and network parameters. IPNs consistently had compressive and shear moduli greater than the additive sum of either single network when compared to pure PEG-DA gels with a similar PEG-DA content. IPNs withstood a maximum stress of up to 4.0 MPa in unconfined compression, with increased PEG-DA molecular weight being the greatest contributing factor to improved failure properties. However, aside from failure properties, PEG-DA concentration was the most influential factor for the large majority of properties. Increasing the agarose and PEG-DA concentrations as well as the PEG-DA molecular weight of agarose/PEG-DA IPNs and pure PEG-DA gels improved moduli and maximum stresses by as much as an order of magnitude or greater compared to pure PEG-DA gels in our previous studies. Although the viability of encapsulated chondrocytes was not significantly affected by IPN formulation, glycosaminoglycan (GAG) content was significantly influenced, with a 12-fold increase over a three-week period in gels with a lower PEG-DA concentration. These results suggest that mechanical performance of IPNs may be tuned with partial but not complete independence from biological performance of encapsulated cells.     

Determination of glycosylated hemoglobin by affinity electrophoresis on agarose gel

We describe a method for electrophoretic determination of glycosylated hemoglobin (GHb) on agarose gel membrane. Clear separation of GHb from non-GHb is achieved after 30 min electrophoresis at 60 V using a sodium citrate buffer solution (34 mmol/l, pH 6.5) containing 0.6 g/l dextran sulfate. The results obtained by this method correlate well with those by agar gel electrophoresis (r = 0.98) and column chromatography (r = 0.96). However, unlike column chromatographic methods, GHb values obtained on agarose gel are not affected by fluctuations in ambient temperature (18-28 degrees C), changes in pH (6.2-6.6) and ionic strength of buffer solution (26-40 mmol/l), or variant hemoglobin S and C. Also, electrophoresis on agarose gel membrane eliminates the lengthy preparative steps required for cellulose acetate electrophoresis. We conclude that agarose gel electrophoresis is a simple method for quantitative determination of GHb.     

兔关节软骨细胞的琼脂糖凝胶培养及其力学性质的研究

目的对兔膝关节软骨细胞进行体外琼脂糖凝胶三维培养,并对其力学性能进行检测。方法将琼脂糖凝胶、细胞、血清和培养基在正确的比例下混合,形成软骨细胞凝胶块。取培养7d、14d和21d时的软骨细胞凝胶块分别对细胞外基质进行组织学观察和免疫组化染色,同时采用Instron 5544材料试验机检测其极限应力、极限应变、抗压模量和切线模量的力学性能变化。结果在琼脂糖凝胶中培养的关节软骨细胞具有典型的软骨细胞特性,能够正常合成细胞外基质,形成有一定弹性和抗压缩能力的软骨样凝胶块。而且随着培养时间增长,基质合成增加,培养21d时的极限应力(0.0226±0.006)N/mm、抗压模量(0.608±0.061)N/mm和切线模量(0.096±0.004)N/mm,比7d时的(0.0204±0.004)MPa,(0.558±0.036)N/mm,(0.029±0.002)N/mm和14d时的(0.0213±0.008)N/mm,(0.586±0.095)N/mm,(0.049±0.005)N/mm有明显增加,但极限应变却没有明显差异(P>0.05)。结论软骨细胞-琼脂糖凝胶块的力学性能与细胞外基质的合成直接相关。     

琼脂糖凝胶基片用于蛋白质与抗原分子的固定研究与表征

目的 探索一种新型的适用于生物分子偶联的固相载体，并对其用于蛋白质与抗原分子微阵列制作的实验条件和检测结果进行评价。方法 于氨基硅烷化的载玻片表面制作琼脂糖凝胶膜，经NaIO4氧化和戊二醛分子偶联后，得到3种不同的凝胶表面，分别考察其固定生物分子的偶联效率、检测敏感度和稳定性能，并用原子力显微术和X射线光电子能谱对其表面进行表征和对比研究。结果 未经氧化的琼脂糖凝胶基片固定生物分子的能力和检测敏感度低于经NaIO4氧化与后续醛化处理的基片，其中又以后者的应用效果最佳，温度因素（实验温度分别为37℃、25℃和4℃）对用此法制作的蛋白质与抗原分子微阵列的使用无显著影响。表征结果提供了琼脂糖凝胶表面固定生物分子的直接证据和内在机制。结论 经修饰处理的琼脂糖凝胶基片可有效固定不同的生物分子，用其作为固相表面制作的蛋白质与抗原分子微阵列可取得较为理想的实验效果。     

氧化型极低密度脂蛋白诱导小鼠腹腔巨噬细胞凋亡

探讨氧化型极低密度脂蛋白诱导小鼠腹腔巨噬细胞凋亡的作用。应用低温超速离心法分离健康人血浆极低密度脂蛋白，用CuCl2氧化得到氧化型极低密度脂蛋白。琼脂糖凝胶电泳观察巨噬细胞凋亡DNA断裂“梯状”图谱；流式细胞仪和二苯胺法分别测定凋亡巨噬细胞百分率和：DNA片断百分率；光镜观察凋亡巨噬细胞形态变化。结果发现：①氧化型极低密度脂蛋白能诱导巨噬细胞发生凋亡及DNA断裂；②抗氧化剂二甲基硫脲能部分抑制氧化型极低密度脂蛋白诱导的：DNA断裂；③核转录因子核因子kB抑制剂吡咯烷二硫代氨基甲酸盐可完全抑制氧化型极低密度脂蛋白诱导的DNA断裂。提示氧化型极低密度脂蛋白诱导巨噬细胞凋亡可能是动脉粥样硬化形成机制之一；氧化型极低密度脂蛋白诱导巨噬细胞凋亡的作用可能与自由基和激活核因子kB有关。     

基于琼脂糖/壳聚糖共混凝胶模型的壳聚糖生物相容性的机理研究

目的 以琼脂糖/壳聚糖共混凝胶为模型,研究壳聚糖材料生物相容性的可能机理.方法 通过共混法,制备出一系列不同壳聚糖含量的琼脂糖/壳聚糖共混凝胶.利用傅里叶变换红外光谱(FTIR)分析共混凝胶的化学基团,利用荧光素-4-异硫氰酸酯(FITC)标记法观察琼脂糖和壳聚糖之间的可共混性.通过Zeta电势测量共混凝胶的电荷,利用二喹啉甲酸(BCA)法分别测定胎牛血清(FBS)总蛋白和牛血清白蛋白(RSA)在共混凝胶上的吸附,利用酶联免疫吸附(ELISA)法测定纤黏连蛋白(FN)在共混凝胶上的吸附.细胞实验以人微血管内皮细胞系(HMEC-1)为模型,通过观测细胞的黏附、增殖和形态来评价共混凝胶的细胞相容性.结果 琼脂糖/壳聚糖共混凝胶含有壳聚糖特征性的化学基团.琼脂糖和壳聚糖之间存在着良好的可共混性,壳聚糖的氨基基团在共混凝胶中呈均匀分布.在pH酸性条件下(pH 3.0)共混凝胶带有较强的正电荷,然而在pH中性条件下(pH 7.4)所有共混凝胶的Zeta电势均降低至0 mV附近.各组共混凝胶之间对FBS总蛋白以及BSA的吸附差异无统计学意义,但是共混凝胶对FN的吸附却随着壳聚糖含量的升高而显著升高.细胞实验结果显示:随着壳聚糖含量的提高,共混凝胶的细胞相容性有明显改善,HMECs在壳聚糖含量较高的凝胶上表现出良好的黏附、铺展和增殖.结论 相对于血清中的其他蛋白,壳聚糖组分对FN存在优先吸附,从而能够促进细胞在共混凝胶表面的黏附铺展.与传统观点不同,本研究发现壳聚糖的生物相容性与其所携带的正电荷无关.