Background Different basal media have very different effects on cell growth, proliferation and differentiation potential of cultured human umbilical cord mesenchymal stem cell (hUC-MSC). This issue has not gotten enough research. The goal of this study is to select high safety and low cost basal media for hUC-MSC, which performs best in combination with commercial human platelet lysate product for cell proliferation and differentiation functions, especially suitable for high-passage hUC-MSC cultures. Methods Three different basal media were combined with 5% UltraGROGAdvanced to culture P0 to P8 passage hUC-MSC. Cell morphology, cell expansion ratio, proliferation, differentiation and cell surface markers were examined and analyzed in order to draw conclusions. Results When observing the same passage under different culture systems, the number of cells obtained in MSCBM and α-MEM were higher than that cultured in IMDM (0.01 < P < 0.05). The results between MSCBM and α-MEM were not significantly different (P > 0.05). Combining growth curve measurement and cell doubling time calculation, it was found that the cell proliferation functions of the three culture media were ranked as MSCBM, α-MEM and IMDM from high to low. The P8 cells cultured in different media all yield good clonal formation, and also all differentiated well. The number of cells differentiated into osteoblasts and chondrocytes cultured in different media was not significantly different (P > 0.05), while the number of adipocyte differentiation in MSCBM and α-MEM cultures were significantly higher than that of IMDM culture (0.01 < P < 0.05). The cells in all cultures uniformly express high levels of MSC surface markers CD73, CD90, CD105, and negatively express CD34, CD45. Conclusions Taking all indicators into consideration, we conclude that the culture system of MSMBM supplemented with 5% UltraGROGAdvanced is the best for culturing hUC-MSC withα-MEM follows right after. This study revealed the importance of basal media in MSC cultures, and offer guidance on their selective use.
目的 探讨抗生素(antibiotic,ATB)联合抗程序性细胞死亡蛋白1(programmed cell death protein 1,PD1)抗体免疫治疗对小鼠肝癌疗效的影响.方法 建立雄性BALB/cH22荷瘤小鼠模型,于建模后腹腔注射抗PD1抗体(250μg/只)免疫治疗,同时给予小鼠灌胃头孢曲松钠(100 mg/kg)+盐酸林可霉素(200 mg/kg),设灌胃生理盐水的正常对照组(0.2 mL/只),每组8只.通过小鼠肿瘤体积、肿瘤组织的苏木素-伊红(HE)染色和脾指数评估ATB与抗PD1抗体联合治疗的抗肿瘤效果;ELISA法检测小鼠血清中IL-2、IL-10、IFNγ细胞因子分泌水平;流式细胞术分析小鼠淋巴细胞中CD3+和CD8+T细胞比例;免疫组化(immunohistochemistry,IHC)法检测小鼠肿瘤组织中CD3和CD8表达水平.结果 ATB与抗PD1抗体联合治疗对肿瘤生长有显著抑制作用,上调了T淋巴细胞中CD3+和CD8+T细胞比例及IL-2、IL-10、IFNγ细胞因子分泌水平;通过上调肿瘤组织中CD3和CD8表达水平来调节T细胞免疫功能,以持续激活免疫系统,抑制肿瘤.结论 窄谱ATB可能通过改善免疫功能以促进抗体免疫治疗对肿瘤的抑制效果.
目的 探讨螺旋藻藻蓝蛋白对乙醇致氧化应激小鼠抗氧化能力的影响.方法 将48只雌性KM小鼠随机分为空白、模型、低剂量、高剂量组,每组12只.低、高剂量组小鼠经灌胃分别给予藻蓝蛋白0.15和0.30 g/kg体质量,1次/d,连续灌胃42 d,称量小鼠体质量;末次灌胃后禁食16 h(过夜),1次性灌胃50％乙醇,12 mL/kg体质量,模型组小鼠仅进行50％乙醇灌胃,空白组不灌胃.6 h后经摘眼球采血,分离血清,相应试剂盒检测8-表氢氧异前列腺素(8-iso-prostane)含量.无菌取各组小鼠肝组织,经研磨离心后,取上清,相应试剂盒检测蛋白质羰基、谷胱甘肽抗氧化酶(glutathione peroxidase,GSH-Px)、还原型谷胱甘肽(reduced glutathione,GSH)含量,并分析小鼠体质量与GSH的相关性.结果 与空白组比较,模型组小鼠体质量增加,但差异无统计学意义(F=1.585,P＞0.05);血清中8-isoprostane、肝组织中蛋白质羰基和GSH含量均显著升高(F分别为11.697、13.582、17.213,P均＜0.05);肝组织中GSH-Px含量下降,但差异无统计学意义(F=5.978,P＞0.05).与模型组比较,低、高剂量组小鼠体质量明显下降(F分别为4.125和18.842,P均＜0.05);血清中8-isoprostane及肝组织中蛋白质羰基含量均明显降低(F=10.695～40.512,P均＜0.01);肝组织中GSH-Px和GSH含量均显著提高(F=42.654～76.379,P均＜0.01).小鼠肝组织中GSH含量与小鼠体质量呈负相关(R2=0.013 49,P＞0.05).结论 藻蓝蛋白可降低乙醇至氧化应激小鼠的氧化损伤,提高抗氧化能力,同时可降低小鼠体质量.
血脂异常是动脉粥样硬化性心血管疾病(atherosclerotic cardiovascular disease,ASCVD)最主要的致病因素,降脂治疗是预防和管理ASCVD的主要方式之一.前蛋白转化酶枯草溶菌素9(proprotein convertase subtilisin/kexin type 9,PCSK9)可通过升高低密度脂蛋白胆固醇(low-density lipoprotein cholesterol,LDL-C)、炎性反应等促进动脉粥样硬化,而PCSK9抑制剂可靶向降低PCSK9水平,降脂效率高.联合他汀类或依折麦布治疗的基础上,还能带来更多的临床效益,随着研究的不断深入,已成为近年降脂药研究的热点.本文就PCSK9抑制剂的研发进展作一综述,以期为该类药物的临床应用提供参考.
目的 在大肠埃希菌(E.coli)中表达登革病毒(dengue virus,DENV)NS2B-NS3蛋白酶,对表达条件进行优化,并测定酶活性,为抗DENV药物先导化合物的筛选与发现奠定基础.方法 将密码子优化的NS2B-NS3基因连接到pET-28a载体中,构建重组原核表达质粒pET-28a-NS2B-NS3,转化至E.coli Rosetta(DE3)感受态细胞中,IPTG诱导NS2B-NS3蛋白酶表达.通过优化诱导时间、诱导温度和IPTG诱导浓度,确定NS2B-NS3蛋白酶在E.coli中的最佳表达条件.使用HisTrapTM亲和层析柱分离纯化NS2B-NS3蛋白酶,通过荧光共振能量转移(fluorescence resonance energy transfer,FRET)试验测定酶活性.结果 重组原核表达质粒pET-28a-NS2B-NS3经双酶切(Nhe Ⅰ/Xho Ⅰ)及测序证明构建正确.NS2B-NS3蛋白酶在E.coli中的最佳表达条件为:诱导温度20℃,诱导时间10 h,IPTG浓度0.2 mmol/L,表达量达20 mg/L.纯化的NS2B-NS3蛋白酶纯度大于90％,可与小鼠抗His-tag单克隆抗体特异性结合,且具有良好的水解活性,其比活力为16 111 U/mg、米氏常数(Km)值为16.46 μmol/L、催化常数(kcat)值为0.028/s.结论 成功制备了高纯度、高活性的DENV NS2B-NS3蛋白酶,为NS2B-NS3蛋白酶抑制剂高通量筛选模型的建立奠定了实验基础.
