【作者】 孟刚；

【导师】 马传庚；

【作者基本信息】 安徽医科大学 ， 药理学， 2001， 博士

【摘要】 细胞凋亡是细胞在有关基因调控下发生的遵循自身程序主动结束自己生命的过程。肿瘤的发生发展是细胞丧失自发凋亡反应能力的最终结果，因此最有效的治疗可能是诱导肿瘤细胞凋亡。 传统的大多数抗癌药物特异性的抗肿瘤毒性是由于它们对活跃增生的肿瘤细胞的破坏作用，研究表明，许多化学结构不同、作用靶点各异的抗肿瘤药物均可诱导肿瘤细胞的凋亡。因此药物的有效性不仅反映了它们对细胞损伤的作用，也体现在细胞对这种损伤的察觉及反应能力。而有关CDDP、Taxol诱导乳腺癌细胞株MCF-7/ADR及宫颈癌细胞株Hela凋亡的报道不多。 近年来的研究表明，细胞凋亡受一些凋亡相关基因蛋白的调控，如：bc1-2、bax、caspase-3、HSP70、NF-κB等，但这些蛋白是抑制或诱导凋亡观点不一。 金属离子如Ca²⁺，Mg²⁺，or Zn²⁺参与细胞的多种功能，如信号传导、酶活性的调节等。金属离子与凋亡的关系，尤其是Ca²⁺与凋亡的关系已见诸多报道。但Ca²⁺作为第二信使传递凋亡信号还是激活Ca²⁺/Mg²⁺依赖性核酸内切酶参与细胞凋亡过程，存在诸多矛盾的观点。 鉴于上述状况，本研究选用CDDP、Taxol、a-TNF及高热诱导乳腺癌细胞株MCF-7/ADR及宫颈癌细胞株Hela凋亡，运用HE、TUNEL、透射电镜、流式细胞仪、S-P免疫组化及荧光指示剂Fura-2等方法分别检测细胞凋亡、凋亡相关蛋白bc1-2、bax、PCNA、caspase-3、HSP70、NF-κB的表达和活性以及细胞内Ca²⁺的浓度，以探讨CDDP、Taxol以及与热、α-TNF联合应用诱导MCF-7/ADR及Hela凋亡特点和与凋亡相关蛋白表达的关系以及细胞凋亡过程中Ca²⁺的变化。 安傲医科大学博士学位论文 1．Taxo、CDDP诱导鹏F－7／ADR及湘 la细胞凋亡及与其它因素的联合 应用 用紫杉醇、顺铂及与a－TNF和高热联合应用诱导乳腺癌细胞系MCF－7／ADR及 宫颈癌细胞系Hela细胞凋亡，采用HE、TUNEL、电镜及流式细胞仪分别检测凋亡 及观察凋亡细胞的形态变化特点。结果发现①紫杉醇可诱导mF－7／ADR及Hela 细胞凋亡，而顺铂仅诱导Hela细胞凋亡，对乳腺癌细胞系MCF－7／ADR只导致细胞 坏死，不诱导其凋亡；两种药物对Hela细胞的诱导凋亡作用在一定范围内均具有 时间及剂量依赖性：②a－TNF诱导Hela及MCF－7／ADR细胞凋亡亦具有时间及剂量 依赖性，但两种细胞对其的敏感性不同；与CODP联合应用时具有协同作用，但与 Taxol联合应用无协同作用：③高热也可诱导Hela细胞凋亡，与顺铂联合具有协 同作用，但 40’C一42’C的高热反而抵消低剂量顺铂（0．ling·L‘－0．5 mg·L－‘）的诱导 凋亡作用；④紫杉醇可使MCF－7／ADR及Hela细胞出现较多的病理性核分裂像以及 细胞核的多微核化，但顺铂、a－TNF及高热（20。in）无此作用。 上述结果提示：紫杉醇和 a－TNF可诱导皿F－7／ADR及 Hela细胞凋亡：顺铂和 高热可诱导Hela细胞凋亡：紫杉醇与a－TNF联合应用时无协同作用：40’C－42℃的 高热反而抵消低剂量顺铂的诱导凋亡作用，其出现的原因有待于进一步研究。 2．He及瞩CF－7／ADR细胞凋亡的分子机制白探讨 为进一步探讨抗肿瘤药物紫杉醇及顺铂诱导肿瘤细胞凋亡及与a1NF和高热 联合应用的作用机制。采用免疫组化S干法及ELISA、酶化学法等分别检测凋亡相 关蛋白 PCNA、bclZ、bax、HSP70及 NF－xB、。aspase－3的表达和活性的动态变化。 结果发现：①Taxol诱导mF－7／ADR细胞凋亡与 hcf－2降低、bax增加及 hcf－2／bax 比值下降有关：而与Caspase－3、P53无关；a－TNF可诱导Hela细胞NF－。B表达增 强及明显的核移位，这可能与Taxol联合应用对Hela细胞凋亡无协同作用的原因 有关。欧DDP诱导Hela细胞凋亡与bclZ降低及移位及Caspase－3活化和过表达 4 安徽医科大学博士学位论文 有关；与对照组及44‘C组比较，40‘C～42’C高热可诱导Hela细胞HSP70、NF－B表 达增强，这可能与其抵消低剂量顺铂＊．l屿·L－’－0．5 mg·L‘)的诱导凋亡作用 的机制有关。③免疫组化染色表明，多微核化细胞bclZ、PCNA、阴spase亿均阳 性，其出现原因可能是细胞对外界损伤性因子的适应性反应，是否与耐药有关以 及其出现的真正意义有待于进一步研究。 上述结果提示：①不同药物及药物对不同的细胞，诱导细胞凋亡的机制有所 不同；②PCNA、HSP70?更多还原

【Abstract】 Apoptosis is a process of cell death that actively carries out the suicide process under the control of a series of gene regulators.The occurance and development of tumor are the result of the ability of loss in apoptosis of tumor cells.Thus, the effectiveness of anticancer drugs may not simply reflect their ability to cause cellular damage, but also the cell’s ability to detect and respond to this damage. However, until recently, the tumor-specific toxicity of most anticancer agents was attributed to their debilitating effects on actively proliferating cells. Most recently, it has become apparent that most anticancer agents induce apoptosis. Current evidences suggest that apoptosis is controlled by genes such as bcl-2, bax, Pcna, HSP7O, NF-KB and caspase-3 et al . Pro- and anti-apoptosis of these proteins were controversial. Metal ions such as Ca2+ Cu2+,Mg+,Zn2~ take part in the different functions such as signal transduction, apoptosis, regulation of enzymes activities etc. Especially the relationship between apoptosis and Ca2~ had been focused on. Increased in intracellular Ca2~ concentration have been shown to trigger apoptosis and numerous apoptosis inducers increase [Ca2+]i. 6 Together taken, in this study, we examined the expression levels of apoptosis-related proteins bcl-2, bax, PCNA, HSP7O, NF-kB and to determine the relationship between their changes and apoptosis. In addition, we investigated the changes of Ca2+ during the effects of CDDP, Taxol and a-TNF on Hela cell. 1. Effects of taxol, CDDP, Heat and a-TNF on apoptosis in MCF-7/ADR and Hela cells. To investigate the effects of taxol and CDDP as well as in combination with heat and a-TNF on apoptosis in MCF-7/ADR and Hela cells. The apoptotic cells were detected by TU7NEL, HE, eletronic micrpscopy and flow cytometty method .The results followed as: HeLa cells exposed to CDDP and Taxol undergo cell death, presenting morphological and biochemical characteristics typical of apoptosis and the apoptotic cells increased with time and concentration. CDDP may give rise to the death of MCF-7/ADR, but did not cause the apoptosis of MCF-7/ADR. Treatment of MCF-7/ADR cell with taxol resulted in characteristics typical of apoptosis. a-TNF induced apoptosis of MCF-7/ADR and Hela cells with time and concentration increase in apoptotic cell index. Hela cell was more susceptible to a-TNF than MCF-7/ADR. The synergistic effect of CDDP in combination with a-TNF and heat were observed. CDDP (1.5mg·L-1 in combination with heat(400C or 440C) had synergistic effect on HeIa, but (0.1-0.5mg·L-1 in combination with heat(40-420C) had antagonistic effect on Hela. Cells with multiple micronuclei were observed in Hela and MCF-7/ADR cells treated with Taxol, but not with CDDP, a-TNF and heat (20mm). Taken together, All of Taxol, CDDP, a-TN7F and heat may induced apoptosis in Hela cell. Taxol in combination with a-TNF and CDDP in combination with heat had no synergistic effect on MCF-7/ADR or Hela cell. Low concentration of CDDP in combination with moderate hyperthermia had antagonistic effect on Hela. The 7 mechanism are to be fuether studied. 2. The relationship between apoptosis induced by Taxol and CDDP as well as in combination wi更多还原