实时荧光定量PCR法检测外源性DNA在重组Ⅲ型人源化胶原蛋白冻干纤维中的残留

目的对实时荧光定量PCR(qPCR)法检测重组Ⅲ型人源化胶原蛋白冻干纤维中的外源性DNA残留量进行探究和分析,确定该方法用于DNA质量控制的可行性。方法采用宿主细胞残留DNA样本前处理试剂盒(磁珠法)提取DNA,利用E.coli残留DNA检测试剂盒(PCR-荧光探针法)进行扩增反应,绘制标准曲线,建立重组Ⅲ型人源化胶原蛋白冻干纤维中外源性DNA残留量的qPCR检测方法。结果对8批重组Ⅲ型人源化胶原蛋白冻干纤维进行测定,外源性DNA残留量在0.03~300 pg/μl内,重复性良好,均远低于每剂10 ng,线性良好(R^(2)>0.99),回收率均在50%~150%之间。结论该方法可用于重组Ⅲ型人源化胶原蛋白冻干纤维中外源性DNA残留量的定量测定。

消毒剂对重组Ⅲ型人源化胶原蛋白促细胞黏附活性的影响

通过CCK8实验探究75%乙醇、碘伏、葡萄糖酸洗必泰、苯扎氯铵、苯扎溴铵对细胞的毒性以及对重组Ⅲ型人源化胶原蛋白(rhColⅢ)促细胞的黏附活性的影响,预测消毒剂的使用在面部填充时对胶原蛋白活性的影响,为临床应用提供指导。结果显示:各消毒剂对NIH/3T3小鼠胚胎成纤维细胞和HFB人皮肤成纤维细胞均表现出细胞毒性,比较结果为:葡萄糖酸洗必泰<苯扎氯铵、苯扎溴铵<75%乙醇、碘伏,该结果也间接提示75%乙醇、碘伏消毒效果较佳;将各消毒剂原液稀释100倍后,其对rhColⅢ促细胞黏附活性影响比较为:葡萄糖酸洗必泰、苯扎氯铵<75%乙醇<碘伏、苯扎溴铵。碘伏和苯扎溴铵工作液直接接触rhColⅢ均显著降低其促细胞黏附活性,碘伏、葡萄糖酸洗必泰、苯扎溴铵工作液直接接触rhColⅢ可导致其发生聚集。

亚洲系与非洲系寨卡病毒株对神经胶质细胞感染性及稳定性差异研究

目的明确亚洲系ZIKV与非洲系ZIKV感染性与稳定性的差异。方法选择亚洲系SZ01与非洲系MR766病毒株在神经胶质细胞中展开研究。使用CCK8法检测SZ01与MR766对星形胶质细胞U251、U87和神经胶质母细胞瘤细胞T98G感染性差异;qRT-PCR或病毒空斑法检测SZ01与MR766在游离状态和感染细胞状态时,对37℃和40℃的耐受能力;最后,通过使用病毒空斑法检测反复冻融对SZ01与MR766稳定性的影响。结果ZIKV对U251、U87的感染性高于T98G,SZ01对星形神经胶质细胞的感染性强于MR766;游离状态下,SZ01对40℃的耐受能力高于MR766;在40℃高温下,SZ01在神经胶质细胞内的增殖速度快于MR766;SZ01对于反复冻融的耐受性高于MR766。结论亚洲系ZIKV的感染性及稳定性均显著高于非洲系ZIKV,或与其持续性感染致病有关。

Skin care efficacy study of recombinant humanized collagen based on in vitro level

Studying the skin care efficacy of recombinant humanized collagen based on in vitro level.The stability of the recombinant humanized collagen was first analyzed by treating at different temperatures,then its skincare efficacy based on in vitro level was evaluated by detecting the inhibition rate of elastase,the inhibition rate of collagenase,the protein content of type I collagen in human fibroblasts,the inhibition of reactive oxygen species(ROS)with human keratinocytes,and the effects of the recombinant humanized collagen on the expression of hyaluronic acid(HA),filaggrin(FLG)and transglutaminase 1(TGM1)in keratinocytes.The results showed that recombinant humanized collagen was able to maintain stability at temperatures below 70℃.With regard to its skincare efficacy,recombinant humanized collagen could inhibit elastase and collagenase activities and promote the increase of type I collagen content in human fibroblasts.It also showed good inhibition of ROS in keratinocytes in vitro and could increase the expression of HA,FLG,and TGM1 in keratinocytes.In short,the recombinant humanized collagen exhibited a favourable skin care effect in vitro level.This study proved that it has potential firming,anti-wrinkle,moisturizing,and repairing efficacy,and is a valuable cosmetic raw material.