常见肝癌细胞系的转铁蛋白受体表达差异分析及主动靶向载体效率比较

转铁蛋白受体1(transferrin receptor 1,TfR1)可介导细胞内吞过程,从而摄取与之特异结合的纳米颗粒,因此成为许多主动靶向型纳米载体的靶点。研究表明,肝癌细胞存在TfR1高表达现象,可作为肿瘤治疗纳米药物递送系统的关键性靶点。体外评价是TfR1靶向纳米载体的重要研究环节,然而肝癌细胞模型种类繁多,其TfR1表达水平可能存在一定差异。选择了几种常见的肝癌细胞系,包括HepG2、Hep3B、MHCC97-H以及Huh-1,分别从mRNA水平以及蛋白水平测定了细胞系TfR1的表达情况,考察了转铁蛋白(Tf)以及转铁蛋白核酸适配体(transferrin nucleic acid aptamer, Tf-APT)对不同细胞的亲和效率。同时,制备了包载紫杉醇的TfR1靶向脂质体,并考察其对不同细胞系的细胞生长抑制作用。结果表明,4种肝癌细胞系在mRNA水平以及蛋白水平均存在TfR1的表达差异;同时,体外抗肿瘤结果显示,不同肝癌细胞系对紫杉醇-TfR1靶向脂质体的敏感性也存在显著不同。     

可跨膜SOD对肝癌及正常肝细胞增殖的作用

可跨膜融合蛋白PTD-SOD与肝癌细胞和正常肝细胞共培养,使细胞内SOD活力分别增加了52%和23%,提高胞内总抗氧化能力(T-AOC)水平达100%,同时降低ROS水平.实验结果显示,肝癌细胞增殖受到显著抑制,最高抑制率达91.91%,对正常肝细胞抑制率较小,为45.50%.     

正常细胞及肿瘤细胞内核苷酸库的分析

通过离子对反相高效液相色谱 (IP RPLC)同时检测细胞内的核糖核苷三磷酸及脱氧核糖核苷三磷酸现已成为可能 ,采用这种方法检测了 16种正常及肿瘤细胞内的ADP、CTP、dCTP、GTP、UTP、dGTP、dTTP、ATP、dATP等核苷酸的含量 ,并进行了简要的对比及方法学验证。采用 6 %三氯乙酸分离提取样品 ,并用 5mol l碳酸钾中和后进行HPLC分析。采用Waters 6 0 0E 4 86色谱系统 ,SymmetryC1 8(3 5 μm ,4 6× 15 0mm)分析柱及Nova -PakC1 8SentryGuardColumn进行色谱分析 ,柱温 2 7℃。流动相由两种缓冲液组成 ,采用梯度洗脱 ,流速 1 0ml min。缓冲液A包含 10mmol l氢氧化四丁基胺 ,10mmol lKH2 PO4及 0 2 5 %甲醇 ,并用HCl调pH至 6 9;缓冲液B包括 5 6mmol l氢氧化四丁基胺 ,5 0mmol lKH2 PO4及 30 %甲醇 ,并用NaOH调pH至 7 0。流动相开始时为 6 0 %A及 40 %B ,30min达 40 %A及 6 0 %B ,此比例一直持续到 6 0min。采用各标准品的水溶液作标准曲线 (r均大于 0 99)。各物质测定的精密度 (CV %)平均为日内 0 9%,日间 5 0 %。各物质的检测限 (pmol)分别为 1 39(ADP)、4 32(CTP)、15 5 (dCTP)、2 38(GTP)、4 4 2 (UTP)、9 4 5 (dGTP)、14 6 (dTTP)、2 4 4(ATP)、11 8(dATP)。该测定方法的平均回收率亦可达到 99 5 %     

基于分子印迹技术的细胞靶向识别研究进展

分子印迹技术(Molecular imprinting technology,MIT)是一项模拟抗体等靶向生物分子识别专一性的仿生分子识别技术,由于其预定性强、识别选择性好、成本低廉等优势而被广泛应用。细胞靶向识别是MIT近些年发展起来的一个重要研究方向,在分子生物学、医疗诊断等领域极具应用前景。本文从分子印迹材料的合成方法、识别原理及其应用等方面介绍了MIT在细胞靶向识别领域的研究进展,最后提出了MIT在细胞靶向识别方面尚存的若干问题并对其未来可能的发展方向进行了展望。     

Cloning of aminopeptidase N promoter and its activity in hematopoietic cell and different tumor cell lines

Aminopeptidase N (APN) promoter region was cloned and sequenced from peripheral blood mononuclear cells. The recombinant reporter construct containing the promoter and luciferase gene, designated pXP1-APNLuc, was introduced into myeloblastic cell line, T lymphocyte cell line and various tumor cell lines. Luciferase assay showed that APN upstream promoter is myeloid-specific for high expression in myeloblastic cell line and much lower expression in T lymphocyte cell line. The promoter activity was relatively high in lung adenoma cell line compared with other tumor cell lines including hepatoma cell line, tong cancer cell line and esophageal cancer cell line in which the promoter activity significantly diminished or was almost undetectable. The characteristics of APN promoter may provide a new strategy for specific myeloprotection while tumor patients are being treated with chemotherapy and/or radiotherapy.     

Amplification of P-glycoprotein genes in multidrug-resistant mammalian cell lines

The multidrug-resistance phenotype expressed in mammalian cell lines is complex. Cells selected with a single agent can acquire cross-resistance to a remarkably wide range of compounds which have no obvious structural or functional similarities. The basis for cross-resistance seems to be a decreased net cellular accumulation of the drug involved, and has been attributed to alterations in the plasma membrane. An over-expressed plasma membrane glycoprotein of relative molecular mass (Mr) 170,000 (P-glycoprotein) is consistently found in different multidrug-resistant human and animal cell lines, and in transplantable tumours. Consequently, it has been postulated that P-glycoprotein directly or indirectly mediates multidrug resistance. Here we report the cloning of a complementary DNA encoding P-glycoprotein. Southern blot analysis of hamster, mouse and human DNA using this cDNA as a probe showed that P-glycoprotein is conserved and is probably encoded by a gene family, and that members of this putative family are amplified in multidrug-resistant cells.     

Antibody-mediated enhancement of Flavivirus replication in macrophage-like cell lines

Interactions between animal viruses and antiviral antisera may exceptionally result in an apparent increase in viral infectivity. Halstead and coworkers demonstrated enhanced replication of dengue virus (a Flavivirus, family Togaviridae) in human or simian peripheral blood leucocytes carrying Fc receptors at subneutralising concentrations of antidengue antibody. We have used three continuous cell lines which express macrophage markers to explore the mechanism of this phenomenon. Dengue virus failed to replicate in these cells, but West Nile virus, another Flavivirus, replicated in all three, and we were able to demonstrate reproducibly 50--100-fold enhancement of virus yields in the presence of Flavivirus antisera, the effect also being directly demonstrable in P388D1 cells by increased numbers of virus-induced plaques. The phenomenon of antibody-dependent enhancement of viral replication is not unique to dengue virus, and may have far wider relevance in other viral infections.     

Cell-mediated immunity to Theileria-transformed cell lines.

In East and Central Africa the protozoan parasite Theileria parva causes a disease of cattle called East Coast fever (ECF). In Kenya alone between 60,000 and 85,000 cattle die from ECF every year. Infected animals can recover from ECF either naturally or after treatment with tetracyclines or menoctone and are subsequently able to resist challenge with the homologous strain of parasite. That this acquired resistance is due to cell-mediated rather than humoral immunity has been suspected but never decisively shown. A major difficulty in studying immunity to ECF has been the lack of inbred animals for studying Theileria-specific immunity in the absence of allogeneic histocompatibility barriers. We have avoided this problem by measuring cell-mediated immune responses in a syngeneic system in vitro. Unidirectional mixed lymphocyte cultures (MLC) were set up using bovine peripheral blood lymphocytes (PBL) as responder cells and autologous cell lines transformed in vitro by T. parva as stimulator cells. In these cultures, DNA synthesis was induced in PBL from both normal and Theileria-immune animals. However, cytotoxic lymphocytes were induced only in cultures containing responder lymphocytes from Theileria-immune cattle. The results show that Theileria-transformed cells express antigens which are recognized by effector cells and provide evidence that cell-mediated cytotoxic mechanisms function in immunity to ECF.