【作者】 秦娜琳；

【导师】 李卓娅； 尹丙姣；

【作者基本信息】 华中科技大学 ， 免疫学， 2007， 硕士

【摘要】 肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)具有多种生物学功能,在体内以跨膜型(transmembrane TNF-α, TM-TNF-α)和分泌型(secreted TNF-α, S-TNF-α)两种形式发挥作用。本室及其他实验室工作表明,两型TNF-α具有不同的生物学功能,TM-TNF-α比S-TNF-α具有更广的杀瘤谱,可杀伤S-TNF-α耐受的肿瘤细胞;TM-TNF-α主要引起靶细胞凋亡,S-TNF-α则主要引起靶细胞坏死。TM-TNF-α不但可以作为配体,与受体结合传递正向信号,而且还可作为受体向效应细胞传递反向信号。由于TM-TNF-α是S-TNF-α的前体,在一级结构上,TM-TNF-α比S-TNF-α多一个由76个氨基酸组成的信号肽(signal peptide, SP),该信号肽不仅将TM-TNF-α锚定在细胞膜表面,且其胞浆段参与传递反向信号。我们的前期研究证实肿瘤细胞可通过TM-TNF-α的反向信号抵抗凋亡,为排除TM-TNF-α正向信号的影响,本研究建立稳定表达TNF-SP的乳腺癌细胞株,并初步研究TNF-SP反向信号对肿瘤行为的影响,为探讨TM-TNF-α反向信号在肿瘤生长中的作用提供了有力的实验证据。该研究主要结果如下:一、建立稳定高效表达TNF-SP的乳腺癌细胞株采用脂质体转染的方法将插入有TNF-SP的真核表达载体pIRES2-EGFP/TNF-SP和空载体pIRES2-EGFP转入人乳腺癌细胞MCF7,经G418抗性筛选,获得克隆化细胞株,借助流式细胞仪筛选出高表达TNF-SP的细胞株,进一步用Western blotting证实阳性克隆株中大约10kD处出现特异性条带,表明有TNF-SP的表达,而对照细胞未见相同分子量的条带。提示已成功建立表达TNF-SP的乳腺癌细胞株。二、TNF-SP反向信号促进肿瘤细胞的存活1.高表达TNF-SP的MCF7细胞对S-TNF-α的细胞毒作用产生抵抗:MTT的结果显示转染TNF-SP的细胞可抵抗S-TNF-α的杀伤(杀伤率3.57%),而转染空载的MCF7和未转染的MCF7细胞则对S-TNF-α杀伤活性敏感(杀伤率分别是22.45%、22.24%),前者与后两对照者之间差异显著(P<0.01),提示转染TNF-SP可使原TNF敏感株变为耐受株。2. TNF-SP通过反向信号激活NF-κB,使MCF7细胞抵抗S-TNF-α胞毒效应: ELISA结果显示转染TNF-SP可明显活化MCF7细胞的NF-κB,S-TNF-α可进一步使其活性增强,而其他两种对照细胞NF-κB并未活化。应用NF-κB抑制剂PDTC预处理细胞后,可抑制稳转细胞的NF-κB活性,恢复MCF7细胞对S-TNF-α胞毒效应的敏感性(杀伤率增加到36.4%,P<0.01),同时也能增加两种对照细胞对S-TNF-α的敏感程度,使其杀伤率明显增强(杀伤率分别上升到50.64%和53.44%,P<0.01)。3. TNF-SP反向信号促进MCF7细胞胞浆I-κB的降解:Western blotting结果显示转染TNF-SP的MCF7细胞胞浆内I-κB的水平低于未转染和转染空载体的MCF7细胞,同时发现这种高表达TNF-SP的细胞对S-TNF-α短暂刺激更敏感,胞浆内IκB-α降解增加显著,而其他两种对照MCF7细胞胞浆的I-κB含量变化不明显。4. TNF-SP反向信号促使MCF7细胞产生NO增加:NO检测显示TNF-SP稳定转染的细胞产生NO的量较未转染和转染空载体的MCF7细胞增加约50.68% (p<0.01)。综上所述,本研究成功建立了能稳定表达TNF-SP的肿瘤细胞株(TNF-SP/MCF7细胞),并证实转染TNF-SP基因能通过组成性激活NF-κB的活性而使MCF7细胞抵抗S-TNF-α介导的细胞毒效应,提高NO的产生。上述结果提示TNF-SP可能通过其胞内的一段活化基序参与TM-TNF-α的反向信号,最终活化NF-κB,促使肿瘤细胞抵抗凋亡刺激而存活。此外,本课题建立的TNF-SP/MCF7细胞系为进一步研究TM-TNF-α的反向信号提供了一个良好的实验工具。更多还原

【Abstract】 Tumor necrosis factor-a (tumor necrosis factor-α, TNF-α) is a pleiotropic cytokine and exerts its biological activities in two forms, namely secreted TNF-α(S-TNF-α) and transmembrane TNF-α(TM-TNF-α). It has been shown in our previous studies and other laboratory’s researches that TM-TNF-αhad a broader tumoricidal spectrum than S-TNF-αand was able to kill the tumor cells which are resistant to S-TNF-α. TM-TNF-αinduced mainly apoptosis while S-TNF-αcaused chiefly necrosis.In addition,TM-TNF-αis not only as a ligand that deliveries forward signal via TNF receptor to target cell but also as a receptor that transmits reverse signal to effect cells. As TM-TNF-αis the precursor of S-TNF-αand their only one difference in the structure is that TM-TNF-αhas an unusual long signal peptide (SP) consisting of 76 amino acids. This signal peptide not only anchors TM-TNF-αon cell surface membran, but also is closely related to the transmission of reverse signal via TM-TNF-α. Our previous work had confirmed that tumor cells were able to resistant apoptosis through reverse signal mediated by TM-TNF-α. Therefore, to exclude the interference of the forward signaling of TM-TNF-α, we had constructed the human breast tumor cell line expression TNF-SP and investigated the biological functional of TNF-SP on tumor cells, which provided an useful tool to further research the effect of reverse signal mediated by TM-TNF-αon tumor growth . The main results as follows:1. Construction of human breast tumor cells with expression of high levers of TNF-SPThe recombinant pIRES2-EGFP/TNF-SP, which contained human TNF-SP cDNA, and the vector pIRES2-EGFP were transduced into a human breast tumor cell line MCF-7 with Lipofectamine 2000 respectively. Cloned cell lines were obtained after G418 resistance selection. The positive cell clone, which expressed high level TNF-SP protein were selected by FACS analysis. It was found by western blotting assay that around 10kD, an apparent band was seen in the positive cell clone. No evident same band was observed in otherwise cell groups. These findings indicate that a eukaryotic cell line highly expressing TNF-SP has successfully been constructed.2. Effect of reverse signal via TNF-SP on tumor growth(1) MCF7 cells expressed high level TNF-SP can resiste the cytotoxicity of S-TNF-αThe results of MTT showed that MCF7 cells transfected with TNF-SP were resistant against the cytotoxicity of S-TNF-α(motality 3.57%, P<0.01), while transfected with pIRES2-EGFP MCF7 cells and no-transfected MCF7 cells were still sensitive to cytotoxic effect of S-TNF-α(mortality 22.45%,22.24%), suggesting that transfected with TNF-SP can make the TNF-sensitive cells become the TNF-resistant cells.(2) TNF-SP can active NF-κB by reverse signal, resulting in MCF7 cells resisting to the cytotoxicity of S-TNF-αThe results from ELISA revealed that the NF-κB activity level of TNF-SP/MCF7 cells was higher than that of the other two cells, and it significantly increased after stimulated by S-TNF-α. In addition, pre-treated with NF-κB inhibitor PDTC could inhibit the NF-κB activity of TNF-SP/MCF7cells and restore efficiently the sensibility of to S-TNF-α( the death rate increased to 36.7%, P<0.01), and it also could increase the sensitivity of the control cells to the cytotoxic effect of S-TNF-α.(3) TNF-SP can promot degradation of IκB-αby reverse signal The results of Western blotting showed that the intracellular IκB-αlevel of MCF7 transfected with TNF-SP cells was lower than that of the two kinds of control cells (control vector transfected MCF7 cells and no-transfected MCF7 cells), and the TNF-SP/MCF7 cells were more sensitive to stimulation of S-TNF-αand its level of intracellular IκB-αdecreased more significantly than the two kinds of control cells.(4) TNF-SP can increase the production of NO by reverse signal It was found that transfection with TNF-SP was capable of enhancing NO production in MCF7 cells by 50.68% (p<0.01) than the control cells.In summary, this study had successfully constructed a eukaryotic cell line highly expressing TNF-SP (TNF-SP /MCF7 cells), and demonstrated that transfected with TNF-SP gene can make MCF7 cells resiste to the cytotoxicity of TNF-αby activating NF-κB and increasing NO production, suggested that TNF-SP may be involved in the reverse signaling of TM-TNF-αvia its active modif to induce NF-κB activity increase which promoting tumor cells survival. So the TNF-SP/MCF7 cell line can provide an useful tool to further research the reverse signaling via TM-TNF-α.更多还原