酸性微环境下大蒜素增强大鼠自然杀伤细胞的功能活性

Objective To investigate the role of allicin on rat nature killer (NK) cell activity in vitro under acidic microenvironment, and its possible mechanism. Methods CD3- NKR-P1+ NK cells isolated from the rat spleen were cultured in the complete RPMI 1640 medium ( pH 5. 6, pH 6. 5, or pH 7. 2 respectively), and treated with allicin at final concentration of 30 mg/L. Enzyme linked immunosorbent assay (ELISA) was used to determine supernatant interferon (IFN)-γ levels. The percentage of NK cells proliferation and apopotosis was analyzed by flow cytometry. NK cell cytotoxicity toward YAC-1 tumor cells was detected by LDH release assay. Results Proliferation and cytotoxicity of NK cells were significantly suppressed by acidic microenvironment in vitro. Under the cultured condition of acidic pH below 7. 2, allicin seemed to promote NK cells proliferation, which reached to highest level of 33% at pH 5. 6 cultured for 16 h. Correspondingly, at pH of 5. 6, allicin induced a marked increase of IFN-γ concentration in the supernatant from (50. 07 ± 0. 38) (cultured for 4 h) to (64. 59 ± 0. 09) ng/L ( cultured for 16 h). The cytotoxicity of NK cells toward YAC-1 tumor cells was also found strongest under the condition of pH 5. 6 cultured for 16 h. Conclusion Allicin favored to enhance the cytotoxicity of NK cells under the acidic cultured condition, which might be related to the increase of IFN-γ production.     

Vps4A对肝癌细胞SMMC-7721的Wnt途径的影响

【摘要】 目的 探讨Vps4A对肝癌细胞SMMC-7721中Wnt途径的影响。方法〓构建过表达Vps4A的SMMC-7721细胞稳定株,通过RT-qPCR的方法检测19个WNT基因的转录水平的变化,进一步通过Western blot及检测相关下游基因Notch2的变化进行确定。结果〓在SMMC-7721中,经典Wnt亚型Wnt10a和Wnt10b的mRNA水平较高,而在过表达Vps4A之后,其蛋白及mRNA水平均显著下降。同时,在过表达Vps4A细胞株中,与经典Wnt通路相关的下游靶基因Notch2在转录及表达水平同样明显下调。结论〓Vps4A选择性抑制肝癌细胞SMMC-7721的经典Wnt/Notch2途径。     

Decorin/fusin双基因修饰骨髓间充质干细胞趋化动力学及抗纤维化的研究

目的 观察双基因(Decorin/fusin)修饰骨髓间充质干细胞(MSCs)体外趋化动力学及对纤维合成的影响.方法 pLVX-puro为载体,pLTR-CMV-DCN、pLTR-CMV-FIB-DCN、pLTR-CMV-FIB-Fusin为质粒,构建慢病毒嵌合体融合基因载体;HEK293细胞包装,滴度1×107cfu/ml病毒液转染MSCs,puromycin抗性筛选、纯化.检测转染效率、基因表达情况、修饰MSCs体外趋化动力学及Transwell纤维合成.结果 基因转染效率为11%,筛选纯化后达30%;外源基因可稳定表达;修饰MSCs具有微环境依赖性体外趋化、穿膜及抗纤维合成能力.结论 双基因修饰MSCs可影响其基因表达和趋化动力学并因微环境变化而降低体外纤维合成.     

Decorin/fusin双基因修饰骨髓间充质干细胞趋化动力学及抗纤维化的研究

Objective To investigate in vitro chemotaxis dynamics and anti-fibrillar synthesis of dual-genes-modified mesenchymal stem cells ( MSCs). Methods A recombinant lentiviral-mediated chimera fusion genes was constructed using pLTR-CMV-DCN, pLTR-CMV-FIB-DCN, pLTR-CMV-FIB-Fusin plasmids and a pLVX-puro vector. Viral particles titer 1×107 cfu/ml obtained through HEK 293 cells packages were transfected into MSCs followed by puromycin selection. Transfection efficiency, gene expression were analyzed;chemotaxis,Transwell in vitro assay of fibrillar synthesis were assessed. Results Transfection efficiency 11%, after selection and purification 30%. Migrational ability and anti-fibrillar synthesis were shown under various conditions. Conclusion Dual-gene-modified mesenchymal stem cells could undergo a conformational structural change under specific microenvironment, affecting its genes expression, chemotaxis dynamics,and in vitro fibrillar synthesis.     

多因子联合诱导人胚胎干细胞定向分化为肝细胞样细胞

目的 建立有效的体外诱导人胚胎干细胞(hESCs)分化为肝细胞样细胞的培养体系.方法 将H9 hESC细胞株接种到基底膜提取物包被的培养板上,序贯加入含有下列诱导因子的分化培养基诱导分化:100μg/L细胞因子活化素A( activin A)诱导3d,20 μg/L骨形成蛋白4(BMP-4)和10 μg/L碱性成纤维细胞生长因子(bFGF)诱导4d,10μg/L肝细胞生长因子(HGF)诱导5d,最后以含10 μg/L制瘤素M(OSM)及1×10-7 mol/L地塞米松(Dex)的培养液继续诱导4d.于细胞诱导分化第16天,采用逆转录-聚合酶链反应(RT-PCR)及免疫荧光检测分化细胞肝脏特异性基因和蛋白表达水平;过碘酸-雪夫反应(PAS)试验和吲哚氰绿(ICG)摄取试验检测分化细胞是否具备肝细胞功能.结果 activin A、BMP-4、bFGF、HGF、OSM和Dex联合诱导的分化细胞具有类似肝细胞的形态结构,呈多角形或卵圆形;RT-PCR结果显示分化第16天的细胞表达甲胎蛋白(AFP)、细胞角蛋白7(CK7)、细胞角蛋白19(CK19)、肝细胞核因子-4α(HNF4-α)、1-抗胰蛋白酶(AAT)等肝脏特异性基因;免疫荧光化学检测结果显示分化第16天的细胞表达AFP、ALB、CK19、CYP7A1等肝脏特异性蛋白;PAS试验及ICG试验显示分化细胞具备糖原合成和ICG摄取释放等肝细胞功能.结论 体外联合多种诱导因子可诱导hESCs定向分化为肝细胞样细胞.     

大鼠原位肝移植胆道并发症的原因分析

目的研究胆道并发症的发生原因,建立稳定的大鼠原位肝移植模型。方法“二袖套法”行大鼠原位肝脏移植180例,即肝上下腔静脉（SVC）采用连续缝合法吻合,门静脉（PV）以及肝下下腔静脉（IVC）采用袖套法吻合,胆总管采用内支架胆管端端吻合法。结果模型稳定后,供肝冷缺血时间为（50．1±12．0）min,无肝期为（16．0±3．1）min,受体手术时间为（54．4±10．6）min,术后胆道并发症的发生率为40％,主要表现为肝脓肿、肝内外胆管扩张、胆泥形成等。结论胆管内支架管的选择以及手术技巧是影响大鼠肝移植术后胆道并发症发生发展的重要因素。     

大鼠肝脏NK细胞的分离、纯化与超微结构特征

目的： 建立一种稳定而高效的大鼠全肝自然杀伤细胞（NK细胞）的分离方法,并利用该方法获得目的细胞,研究肝脏NK细胞的超微结构特征。方法：通过灌注和消化法分离大鼠肝脏非实质细胞,经大鼠淋巴细胞分离液密度梯度离心后收集单个核细胞层,磁珠分选得到肝脏NK细胞,流式细胞仪检测及免疫荧光鉴定其纯度,并通过光镜、透射电镜及扫描电镜的方法对目的细胞进行形态学特征研究。结果：分离所得NK细胞纯度为（88.64±4.36）%,存活率＞95%。肝脏NK细胞具有典型大颗粒淋巴细胞的形态学特征,胞内可见多量与其细胞毒性相关的分泌颗粒及NK细胞特有的囊泡结构。随着其被激活,细胞的形态会发生变化。结论：该方法是大鼠肝脏NK细胞分离的一种简单可行的方法。肝脏NK细胞的超微结构特征为其细胞功能发挥提供可能。