摘要
单功能烷化剂N-甲基-N-硝基-N-亚硝基胍(MNNG)是一种在环境中广泛存在的化学诱变剂和致癌剂，它能和DNA及蛋白质等生物大分子形成加合物(adduct)，其引起的与突变有关的主要DNA损伤类型是O~6-甲基鸟嘌呤，这种损伤与肿瘤尤其是胃癌的发生密切相关。但是DNA损伤并不是引起突变的必要条件，一个典型的例子就是发生在B淋巴细胞的免疫球蛋白(Ig)可变区基因上的“体细胞超突变(somatic hyermutation)”，是由表面抗原受体而不是DNA损伤驱动的主动突变。环境诱变剂引起的突变也可发生在非DNA损伤部位，我们称之为非定标性突变(nontargeted mutagenesis)。这种突变除了可能由DNA损伤途径触发外，也有可能通过非核起源的外遗传(epigenetic)途径，激活细胞信号转导通路，引起一系列基因表达的改变而最终导致突变的形成。同样地，环境诱变剂诱发的细胞应答反应也不只是单纯地由DNA损伤所触发的。自环境毒物接触细胞开始，细胞就发生了一系列广泛和复杂的应答反应以应对此不良环境。其中包括了即刻发生的并不依赖于核损伤信号的信号转导通路的激活和较迟发生的基因表达的改变。
我们实验室曾用一特殊的突变检测系统，直接证明DNA损伤剂可在哺乳动物细胞诱发非定标性突变：首先用低浓度(0.2μM)的短寿烷化剂MNNG(半寿期为1.1hr)处理细胞2.5h后，继续培养24h，将重组有用作突变检测的靶基因supFtRNA基因的穿梭质粒pZ189转入细胞复制，发现在未受致癌物直接攻击的穿梭质粒中有较自发突变率高5倍以上的靶基因突变。这种突变并非在接受MNNG攻击以
浙江大学博士学位论文 金静华
后立刻出现，而是具有时相依存性，在致癌物攻击后其发生率逐渐升高，在 12h
达最高峰，以后逐渐下降。且突变谱明显不同于由MNNG直接攻击引起的定标
性突变，有其突变好发部位的序列特异性。
进一步地，我们还证明了低浓度MNNG的作用下有广泛的细胞反应，尤其
是在信号转导通路的激活和基因表达的改变的研究中取得了一些突破性的进展。
例如细胞表面受体如表皮生长因子受体、肿瘤坏死因子受体发生聚簇，细胞信号
转导通路CAMP｝KAcgyB和JNK／SAPK被激活。更值得注意的是这些有关信
号转导通路的激活并不依赖于细胞核中的DNA损伤，因为在除细胞核的细胞中
这些细胞通路的激活仍可被诱发。我们还利用mRNA差异显示技术分离到了近
30个在 MNNG处理后表达改变的表达序列标签（expressed sequence ag，EsT）。
其中9号片段在MNNG处理后表达增高，其表达改变不受蛋白质合成抑制剂的
影响。利用反义核酸技术构建含反向插入9号片段的真核细胞表达重组体并转染
细胞，以获得反义RNA阻断vero细胞中相应基因的表达，发现MN’NG诱发的
非定标性突变频率显著增高，提示被阻断的相关基因的表达产物可能参与抑制非
定标性突变的发生。
由此可见，信号转导通路的激活和基因表达的改变可能是环境诱变剂通过非
DNA损伤途径诱发哺乳动物细胞非定标突变发生的关键所在。所以，从整体上
研究烷化剂作用后细胞基因表达谱改变，对于了解化学致癌物诱发的哺乳动物细
胞应激反应的全貌和揭示非定标性突变的发生机制具有非常重要的意义。虽然，
利用mRNA差异显示技术和基因芯片技术可在转录水平高通量的筛选基因表达
的差异，但是这些技术都不能提供转录后的尤其是蛋白质在翻译后修饰的信息，
而蛋白质通常是细胞内的功能执行者。蛋白质组学的技术——一种能同时研究成千
上万种蛋白质的技术一更适合于研究细胞对烷化剂的应答反应。因此，本研究中
我们利用蛋白质组学的技术研究了低浓度MN’NG诱发的哺乳动物细胞蛋白质的
表达差异；并且，利用双向凝胶电泳结合相应的ZD分析软件比较了反义阻断9
号片段相关基因的vero巾M－amp”习－细胞和转染空载体的对照细胞vero巾M－amp”
在MN’NG处理后细胞蛋白质组的表达差异，为阐明9号片段相关基因的作用机
制提供线索。
双向凝胶电泳方法的建立和低浓度MNNG诱发的人羊膜F’L细胞蛋白质的
2
浙江大学博士学位论文 金静华
差异表达的初步分析：FL细胞用0二sgu的MNNG处理又5小时后继续培养12
小时，对照组用溶剂二甲亚圃处理，然后提取细胞总蛋白，用双向凝胶电泳分离，
电泳后的凝胶用银染进行染色，并用专门的计算机软件对电泳结果进行数据采集
和图象分析，建立蛋白质组差异表达谱。结果：MNNG处理后的FL细胞中检测
到IO个新出现的蛋白点，同时有5个蛋白点在MN’N G处理后消失：有30个点
在表达量上有显著变化，其中16个点在MNNG处理后表达升高，另14个点则
表达量降低。结论：在低浓度烷化剂攻击的FL细胞中有一系列蛋白质表达水平
的改变，提示这些发生改变的蛋白质可能参与了哺乳类细胞非定标性突变的发
生。
低浓度MNNG诱发的人羊膜FL细胞差异表达蛋白的鉴定：为了能用质谱
的方法鉴定MN－NG诱发的FL细胞的差异表达蛋白，我们采用了上样量更大，
分离距?
Monofunctional alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is a widely spread environmental mutagen and carcinogen that targets DNA and proteins to generate adducts. Among the adducts, O6-alkyl guanine is the predominant mutagenic lesion because of its mispairing properties, which can eventually lead to chromosomal aberrations, point mutations, and cell death. This lesion also appears to be involved in tumor initiation, particularly in gastric carcinogenesis. However, DNA damage has been ruled out as the prerequisite for alkylating agents-induced mutagenesis. For example, somatic hypermutation, which occurred around the variable region in the immunoglobin gene of B cells, is driven by antigen activation not by DNA damage and is considered a kind of active mutation. Environmental mutagens can also induce mutations at undamaged sites and result in the so-called nontargeted mutation. This kind of mutation may be not only induced by DNA damage but also through the epigenetic pathway other than the nuclear origin including the activation of various signal transduction pathways and altered expressions of many genes. Similarly, the cellular response to the environmental mutagens is not always initiated by DNA damage. Exposure to genotoxic agents would trigger a series of comprehensive and complex responses in cells to counteract the abnormal conditions, which include the rapid activation of signal transduction pathways independent of the nuclear damage signal and late alteration of gene expression.
In our laboratory, a unique mutation detection system using a shuttle vector
plasmid has been established to demonstrate that a low concentration of MNNG (0.2 M) can induce nontargeted mutation in mammalian cells: the mammalian cells were exposed to 0.2M MNNG for 2.5h, then a shuttle plasmid pZ189 carrying supF tRNA gene was transfected into cells after 24h culture. We found a 5-fold higher mutation frequency of the plasmid replicated in pretreated cells than the spontaneous mutation frequency of the plasmid replicated in control cells. This kind of mutation did not occur immediately after MNNG exposure. Time-course analysis showed that the frequency of MNNG induced nontargeted mutation increased gradually, reached the peak at 12 h after MNNG treatment, and then declined. The specific nontargeted mutation spectrum is different from that of targeted mutation, whereas the mutation occurs at damaged DNA site.
Furthermore, we have demonstrated that low concentration MNNG exposure induced comprehensive cellular responses. For example, we have found the clustering of EGFR (epidermic growth factor receptor) and TNFR (tumor necrosis factor receptor) and the activation of cAMP-PKA-CREB and JNK/SAPK pathways after MNNG treatment. It is even more interesting that the activation of these pathways seems to be independent of DNA damage, because these events can still occur in enucleated cells. In addition, more than 30 differential expressed sequence tags (EST) have been isolated by mRNA differential display. Among them, fragment 9 showed enhanced expression after MNNG exposure and protein synthesis inhibitor couldn't inhibit the alteration. An expression vector with fragment 9 in antisense orientation was constructed to block the expression of the relevant gene (fragment 9 related gene, FNR gene) in vero cells. Interestingly, we found that the nontargeted mutation frequency induced by MNNG was increased significantly, implicating that the product of the blocked gene may be involved in the inhibition of nontargeted mutation. Therefore, it is important to study the profiles of gene expression, which will help understand the global cellular stress responses to chemical carcinogens, and further elucidate the mechanisms of nontargeted mutagenesis.
Currently there are many techniques available to screen the gene expression at the
transcriptional levels, such as mKNA differential display and cDNA microarray. Although these methods can provide high-throughput information about the differential gene expressi
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