重组双特异性抗体药物电荷异质性分析iCIEF方法新载体基质研究及验证

目的:建立应用成像毛细管等电聚焦电泳(iCIEF)法测定重组双特异性抗体(bsAb)电荷异质性的新载体基质的方法。方法:首先,对2种bsAbs在传统的载体基质甲基纤维素(MC)/羟丙甲基纤维素(HPMC)与新的甘油基质中的电荷异构体分离效果进行对比;然后,对甘油基质方法进行进一步优化,以研究甘油浓度、两性电解质种类及其终浓度、不同pH范围两性电解质的组合方式等对电荷异构体分离效果的影响,获得基于甘油基质最佳的分析条件;最后,应用甘油基质的最优分离条件进行了iCIEF方法验证,包括精密度、准确度、线性范围及稳定性指示性验证等。结果:2种bsAbs在甘油基质中的电荷异构体的分离效果均优于HPMC载体基质,且方法的稳健性较好;方法验证结果显示:对于bsAb 1,酸区、主峰、碱区峰峰面积百分比的RSD均小于2.0%,精密度良好;在蛋白终浓度0.10~0.30 mg·mL^(-1)范围内,主峰平均峰面积与蛋白浓度回归曲线r大于0.99,线性良好;每个浓度点的回收率均在95%~105%范围内,准确度良好。对于bsAb 2,酸区、主峰峰面积百分比RSD均小于2.0%,碱区峰面积百分比小于20.0%,精密度良好。该方法能够指示出产品电荷异构体的含量变化情况,可用于产品的质控。结论:使用甘油作为新的载体基质代替传统的HPMC载体基质进行iCIEF分析,具有分离度较好,稳定性强,准确性高,成本低等优势,解决了结构较复杂的重组双特异性抗体类药物在传统MC/HPMC载体基质中无法实现电荷异构体良好分离的难题,尤其适用于复杂的双特异抗体类药物的产品质量评估及质量控制。

生物活性物质递送体系下丝素蛋白微胶囊的研究进展

药物、香味剂、营养成分等生物活性物质易受到外界环境影响,导致其活性迅速降低。在使用过程中,如何长久稳定其活性是当前亟须解决的问题。丝素蛋白具有出色的生物相容性、安全性及优异的可塑性,基于丝素蛋白材料对生物活性物质具有高度的亲和性和持久的稳定性。近年来,热点之一的丝素蛋白微胶囊在生物活性物质的装载、稳定和递送等方面已被深入研究。本文从递送体系角度出发,分别从丝素蛋白微胶囊的材料选择、成囊机理、制备方法和理化性能的影响因素及应用现状5个方面进行了综述,并重点比较了丝素蛋白微胶囊的制备方法及理化性能的影响因素。综合分析表明,丝素蛋白微胶囊的研究应着重围绕制备方法的开发、理化性能的提升、生物活性物质长久高效的稳定及特定递送体系下的功能化和智能化等方面入手,为应用于疾病治疗、食品保护、生物传感器等领域提供材料选择。

常压鼓风干燥法制备直投式纳豆菌剂工艺

建立采用常压鼓风干燥法制备直投式纳豆菌剂的工艺。通过优化直投式纳豆菌剂的制备条件,得到其最佳制备工艺,并测定该菌剂的发酵性能和贮藏稳定性。结果表明,直投式纳豆菌剂的最佳制备工艺为:复合干燥保护剂为11.07%脱脂乳粉、6.41%谷氨酸钠和14.76%柠檬酸钠;纳豆菌菌泥与复合保护剂的混合条件为:菌泥与复合保护剂的混合比例(g/m L)1∶7、复合保护剂的p H为6.0、菌泥与复合保护剂的平衡时间35 min;纳豆菌的载体基质为碎米粉,菌悬液与碎米粉的混合比例(m L/g)为1∶2;菌剂的干燥条件为50℃干燥9 h,在此条件下制备出的纳豆菌剂活菌率达82.66%,含菌量为7.93×10^(10)cfu/g,该菌剂发酵性能和贮藏稳定性较好,在4℃或-20℃条件下贮藏180 d后活菌数及发酵活力无明显变化。故采用常压鼓风干燥法制成的直投式纳豆菌剂符合市售纳豆菌剂的标准要求,可推广至工业生产中,简化生产过程。

KISS-1 inhibits the proliferation and invasion of gastric carcinoma cells

AIM:To investigate the function of the KISS-1 gene in gastric carcinoma cells and to explore its potential mechanism.METHODS:A KISS-1 eukaryotic expression vector was constructed and transfected into BGC-823 cells.Resistant clones were obtained through G418 selection.reverse transcription-polymerase chain reaction and western blotting were used to detect KISS-1 and matrix metalloproteinase-9(MMP-9)expression in transfected cells.The growth of transfected cells was investigated by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide(MTT)proliferation assays,and the cells'invasive potential was analyzed by basement membrane(Matrigel)invasion assays.The anti-tumor effects of KISS-1 were tested in vivo using allografts in nude mice.RESULTS:The expression level of KISS-1 mRNA andprotein in BGC-823/KISS-1 transfected cells were significantly higher than in BGC-823/pcDNA3.1 transfected cells(P<0.05)or the parental BGC-823 cell line(P< 0.05).The expression level of MMP-9 mRNA and protein in BGC-823/KISS-1 were significantly less than in BGC-823/pcDNA3.1(P<0.05)or BGC-823 cells(P< 0.05).MTT growth assays show the proliferation of BGC-823/KISS-1 cells at 48 h(0.642±0.130)and 72 h(0.530±0.164)were significantly reduced compared to BGC-823/pcDNA3.1(0.750±0.163,0.645±0.140)(P<0.05)and BGC-823 cells(0.782±0.137,0.685± 0.111)(P<0.05).Invasion assays indicate the invasive potential of BGC-823/KISS-1 cells(16.50±14.88)is significantly reduced compared to BGC-823/pcDNA3.1(20.22±14.87)(P<0.05)and BGC-823 cells after 24 h(22.12±16.12)(P<0.05).In vivo studies demonstrate the rate of pcDNA3.1-KISS-1 tumor growth is significantly slower than pcDNA3.1 and control cell tumor growth in nude mice.Furthermore,tumor volume of pcDNA3.1-KISS-1 tumors(939.38±82.08 mm3)was significantly less than pcDNA3.1(1250.46±44.36 mm3) and control tumors(1284.36±55.26 mm3)(P<0.05).Moreover,the tumor mass of pcDNA3.1-KISS-1 tumors(0.494±0.84 g)was significantly less than pcDNA3.1(0.668±0.55 g)and control tumors(0.682±0.38 g)(P <0.05).CONCLUSION:KISS-1 may inhibit the proliferation and invasion of gastric carcinoma cells in vitro and in vivo through the downregulation of MMP-9.

Optimization of the construction of recombinant plasmids PPARγ-pSUPER-EGFP for RNA interference

Objective：To construct and identify the recombinant plasmids PPARγ-pSUPER-EGFP for RNA interference. Methods： The pSUPER-EGFP vectors were used to transcribe functional short interfering RNA （siRNA）. Four pairs of 64 nt PPARγ siRNA encoding sequences were inserted into the downstream of the H1 promoter. The recombinant plasmids were confirmed by double digestion with the enzymes and sequencing. Western blotting was used to examine the silencing effect of PPARγ gene in RAW264.7 cells. Following procedures were used to optimize the experiments： the oligonucleotides were incubated 5 min at 95 C and cooled automatically in boiled water bath to anneal, and then phosphorylated oligonucleotides, pSUPER-EGFP plasmids was digested with Bgl Ⅱ and Hind Ⅲ , and the product was ligated into digested pSUPER-EGFP plasmids, and transforming the ligation products followed by screening and identifying positive clones. Results ：Four kinds of positive clones producing 285 bp fragments were selected. Sequencing further proved their correctness. Four recombinant plasmids containing corresponding PPARγ gene-specific target sequences induced the silencing of its target gene more or less. Conclusion： The optimizing method in constructing these recombinant plasmids serves other plasmid-based RNA interference research. The final plasmids PPARγ-pSUPER-EGFP established the basis for research on the function of PPARγ gene.

纳米制剂技术改善中药精油稳定性的研究进展与思考

作为从天然植物中提取的活性成分,中药精油具有广泛的药理活性,在医药、食品、化妆品以及农业等方面应用广泛。然而大多数精油主要成分均为挥发性成分,其受光、热、湿及氧气等影响稳定性很差,易挥发、氧化分解及异构化等,导致精油生物活性下降甚至产生毒副产物。如何通过制剂技术解决中药精油稳定性差的问题,以保护其活性成分,成为制约其储存及应用的一大挑战。纳米制剂技术为改善中药精油的稳定性提供了一种有效的方法,为此综述了纳米制剂技术改善中药精油稳定性的研究进展与存在问题,以期为中药精油的制剂稳定化应用提供参考。