【作者】 成细华；

【导师】 田道法；

【作者基本信息】 湖南中医药大学 ， 中西医结合临床， 2007， 博士

【摘要】 目的在建立二亚硝基哌嗪诱导气虚体质状态大鼠鼻咽黏膜上皮细胞癌变模型基础上,应用益气解毒方干预模型大鼠,研究“气虚染毒”病机与鼻咽上皮细胞癌变启动和发展过程中细胞凋亡活性异常改变的相关性及其信号传导机理,确定不同凋亡信号传导通路中的关键基因；探讨益气解毒方诱导鼻咽上皮癌变细胞凋亡信号传导的主要通路及其主要环节相关责任基因的表达调控和干预靶点,验证对应性治疗法则“益气解毒”对“气虚染毒”病机所诱发鼻咽上皮细胞癌变病程的阻逆效应。方法研究工作分两步进行。1.二亚硝基哌嗪诱发力竭游泳大鼠鼻咽上皮细胞癌变(“气虚染毒”)的细胞凋亡信号传导通路研究健康雄性SD大鼠56只,随机分为模型组Ⅰ24只,模型组Ⅱ24只和空白对照组8只。模型组Ⅰ腋部皮下注射二亚硝基哌嗪(DNP)和氟波酯(TPA),模型组Ⅱ于进行力竭游泳的同时同组Ⅰ注射DNP和TPA,空白组同法注射用生理盐水稀释10倍的二甲基亚砜(DMSO),共注射28次。实验周期最长达到400天。定期分批处死大鼠,取鼻咽组织进行鼻咽癌变诱发过程中各阶段的病理组织学检查,以TUNEL法检测各阶段组织标本细胞凋亡活性,应用免疫组织化学SABC法检测各组织标本PCNA指数、细胞凋亡信号传到通路线粒体途径(Bcl-2、Bax、Cyt-C和Caspase-3)及NF-κBp65信号传导途径(NF-κBp65和IκB)的主要相关蛋白活性表达,并应用RT-PCR技术检测Bcl-2和Bax mRNA的表达,以验证相关指标免疫组化检测结果。2.益气解毒方治疗效应对大鼠鼻咽上皮细胞诱发性癌变过程中细胞凋亡信号传导通路的干预作用研究制备中药复方益气解毒方浓缩煎液。取健康雄性SD大鼠72只,随机分为3组,即模型组24只,给生理盐水灌胃作为阴性对照；实验组24只,以中药益气解毒方浓缩煎液灌胃作为药效的实验观察,处理持续时间90天；阳性对照组24只,以维甲酸灌胃作为阳性对照,处理持续时间同实验组。所有动物均按照第一部分模型组Ⅱ所用诱癌方法诱导鼻咽上皮细胞癌变进程,各组动物分别同时予以相应干预措施处理,同前分期分批处死动物,采集标本进行干预结果观察。检测指标与方法同上。结果1.二亚硝基哌嗪诱发力竭游泳大鼠鼻咽上皮细胞癌变(“气虚染毒”)的细胞凋亡信号传导通路特征随DNP诱导时间的延长,大鼠鼻咽上皮细胞病理变化日趋明显,最终发生恶变。在同一实验时段,模型组Ⅱ鼻咽上皮细胞病变发生率明显高于模型组Ⅰ,只是在同一病理阶段,所检测的基因表达活性在两组间的差异无统计学意义。在正常鼻咽上皮细胞和单纯性增生病理阶段,二者之间的细胞凋亡活性和PCNA蛋白表达活性差异均无统计学意义(P>0.05)。随着鼻咽上皮细胞癌变过程的进展,异型增生组织和癌变组织中的细胞凋亡活性降低；虽然该二阶段的细胞凋亡活性差异无统计学意义(P>0.05),但分别与之前的两个阶段比较,其差异都具有统计学意义(P<0.05)。异型增生组织和癌变组织中的PCNA蛋白表达活性增强,而且其间的差异具有统计学意义(P<0.05)；分别与之前的两个病理阶段比较,指标值差异也分别具有统计学意义(P<0.05)。在该一病理进程中,鼻咽上皮细胞的细胞凋亡活性和PCNA表达活性呈现明显的负相关关系(r=-0.794,P<0.05)。正常鼻咽上皮细胞和单纯性增生病理阶段,二者之间的Bcl-2、Cyt-C和Caspase-3蛋白表达活性差异都无统计学意义(P>0.05)。随着鼻咽上皮细胞病理变化的进展,异型增生组织和癌变组织中的Bcl-2蛋白表达活性增强,分别与之前的两个病理阶段比较,其差异均具有统计学意义(P<0.05),但异型增生和癌变阶段之间的检测指标差异却无统计学意义(P>0.05)。异型增生组织和癌变组织中的Cytochrome-C与Caspase-3蛋白表达活性减弱,分别与此前的两个病理阶段比较,其差异均具有统计学意义(P<0.05),但异型增生和癌变阶段之间的检测指标差异却无统计学意义(P>0.05)。随着鼻咽上皮细胞诱发性癌变进程的发展,Bax蛋白的活性表达程度呈现逐步降低趋势,但各病理阶段间的比较结果显示,其差异均无统计学意义(P>0.05)。进一步的相关分析提示,在此病理进程中,Bax蛋白活性与Bcl-2.Cytochrome-C和Caspase-3表达活性亦无明显的相关性关系(r分别为-0.157,0.196,0.334；P分别为0.593,0.501,0.243),但Bcl-2与Cytochrome-C和Caspase-3的表达活性之间则呈现明显的负相关(r分别为-0.668,-0.871；P<0.05),Cytochrome-C与Caspase-3的表达活性又呈明显的正相关(r=0.698,P<0.05)。同时,在同一病理阶段,Bax mRNA与Bcl-2 mRNA的转录活性与各自蛋白的表达活性也是相平行的。在正常鼻咽上皮细胞和单纯性增生病理阶段,二者之间的NF-κBp65和IκBα蛋白表达活性差异均无统计学意义(P>0.05),但NF-K B p65蛋白在异型增生组织和癌变组织中的表达活性都增强,而且该二病理阶段之间的指标值差异还具有统计学意义(P<0.05)；分别与此前的两个病理阶段比较,指标值差异也具有显著性意义(P<0.05)。在异型增生组织和癌变组织中,IκBα蛋白的表达活性都减弱,只是二者之间的检测值差异无统计学意义(P>0.05)；但分别与此前的两个病理阶段比较,其差异值却具有显著性意义(P<0.05)。相关分析显示,在大鼠鼻咽癌变病理进程中,NF-KBp65表达活性与1 K Bα表达活性呈现显著的负相关关系(r=-0.541,P<0.05)。2.益气解毒方对大鼠鼻咽上皮细胞诱发性癌变过程中细胞凋亡信号传导通路的干预作用灌服益气解毒方浓缩煎液的实验组大鼠中,鼻咽上皮组织的癌变率为0%(0／5),明显低于生理盐水对照组的86.3%(5／6)。虽然维甲酸组阳性对照组大鼠的发癌率也为0%(0／5),但该组所有动物均于实验周期的第250～310d间逐渐死亡,显示该药具有明显的毒副作用。在诱发性鼻咽上皮细胞癌变进程中的单纯性增生阶段,3组大鼠的鼻咽上皮细胞凋亡活性和PCNA蛋白表达活性组间差异均无统计学意义(P>0.05)；在异型增生阶段,益气解毒方组鼻咽上皮细胞凋亡活性明显高于模型组(P<0.05), PCNA蛋白表达活性则显著低于模型组(P<0.05)。在诱发性鼻咽癌变进程的单纯性增生阶段,分别比较3组大鼠鼻咽上皮细胞的Bcl-2、Bax、Cytochrome-C和Caspase-3蛋白表达活性,其组间差异均无统计学意义(P>0.05)。而在异型增生阶段,益气解毒方组鼻咽上皮细胞的Bcl-2蛋白表达活性显著低于模型组(P<0.05), Cytochrome-C知Caspase-3蛋白表达活性又分别显著高于模型组(P<0.05)；Bax蛋白表达活性虽然高于模型组,但比较分析结果表明,组间差异却无统计学意义(P>0.05)。在同一病理阶段,各组鼻咽上皮细胞的Bax mRNA和Bcl-2 mRNA转录活性与各自蛋白的表达活性呈现平行趋势。在诱发性鼻咽癌变进程的单纯性增生阶段,3组大鼠鼻咽上皮细胞的NF-κB p65和IκBα蛋白表达活性均未出现显著性变化(P>0.05),但在异型增生阶段,益气解毒方组鼻咽上皮细胞的NF-κBp65蛋白表达活性显著低于模型组(P<0.05),而IκBα蛋白表达活性显著高于模型组(P<0.05)。结论1.气虚体质状态可能是环境致癌因素诱发性鼻咽癌发病过程的遗传易感性体质因素,接受诱癌物质作用后,便可启动鼻咽癌变病理进程,即“气虚染毒”病机假说是能够成立的。2.大鼠鼻咽上皮细胞的诱发性癌变的病理进展过程,与靶器官的细胞凋亡活性异常降低、细胞增殖活性过度升高有关,病理机制涉及细胞凋亡信号传导通路的线粒体途径和NF-κB途径信号传导异常。3.中药益气解毒方能够有效阻逆由“气虚染毒”病机诱发的大鼠鼻咽上皮细胞癌变进程。该一阻逆效应的发挥,可能与其有效抑制病变组织过度升高的细胞增殖活性并强力诱导其细胞凋亡活性有关。其诱导细胞凋亡活性效应,可能是通过干预细胞凋亡信号传导通路的线粒体途径和NF-κB途径而实现的。4.研究结果提示,二亚硝基哌嗪诱发力竭游泳大鼠鼻咽上皮细胞癌变技术,能够较好地模拟“气虚染毒”这一中医病机,由此制备的鼻咽上皮细胞癌变动物模型,可以比较有效地应用于验证鼻咽上皮细胞癌变的“气虚染毒”中医病机假说。而在此基础上设计的中药益气解毒方干预试验,不仅可以进一步反证“气虚染毒”病机假说的合理性,还可以由此而建立该一病理变化的“益气解毒”治疗法则。更多还原

【Abstract】 Objectives:There were two aims having been focused on in this study. Firstly, the key responsible genes located in various pathways of apoptotic signaling and the signaling mechanisms associated with them were to be determined among rats with nasopharyngeal cancerous lesion induced by an integrated carcinogenesis of Qi deficiency toxin with contamination pathogenesis resulting in abnormally changed apoptotic activities in the process of initiation and development of such a kind of pathogenesis of in terms of TCM. Then, to be explored were the main pathways of apoptotic signaling and the related responsible genes and intervenable targets in the associated main links with apoptotic activities in nasopharyngeal epithelia undergoing carcinogenesis during the modeling course, induced by the therapeutic effects of herbal medicine preparation Qi-Boosting Toxin-Resolving Formulae (QBTRF).Methods:This study was carried out in two steps as following.1. Exploration on the characteristics of apoptotic signaling pathways in the developing process of carcinogenesis in nasopharyngeal epithelia induced by N,N’-dinitrosopiperazine used among rats undergone exhausted swimming ("contaminating toxin due to Qi deficiency" pathogenesis)Included in this part of study were 56 SD rats, randomly divided into 3 groups, i.e. modeling group I (MG1,24 rats), modeling group II (MG2,24 rats) and healthy controlling group (HCQ 8 rats). The rats in MG1 were sub-cutaneously injected in axillary fossa with N,N’-dinitrosopiperazine (DNP) and 12-O-tetradecanoylphorbol (TPA) simply, those in MG2 were also injected with DNP and TPA in the same way but in combination with the animals undergone exhausted swimming to mimic the carcinogenesis-inducing effect of Qi deficiency toxin with contamination pathogenesis on nasopharyngeal epithelia, and ones in HCG were also sub-cutaneously injected in the same area with the same volume of dimethyl sulfoxide (DMSO) diluted with normal saline, twice a week and 28 times in total for all the animals. All the animals were put into death for taking tissue samples from the nasopharynx in a regular numbers of rats and regular intervals of period during the whole experimental course lasted for 400 days. Then, the tissue samples were cut into two pieces respectively, with one part fixed in 4%neutral paraformaldehyde for pathohistological evaluation, TUNEL (TdT-mediated x-dUTP nick end labeling) method on apoptotic index and SABC immunohistochemical study on PCNA index and the expressive activities of genes in the apoptotic signaling pathways, as Bcl-2, Bax, Cyt-C and Caspase-3 in the signaling route of mitochondrion and NF-KBp65 and IκB in the signaling route of NF-κB p65, and the other part kept in nitrogen for RT-PCR assaying on BCL-2 and Bax mRNA.2. Investigation on the intervening effects of Qi-Boosting Toxin-Resolving Formulae on the apoptotic signaling pathways in nasopharyngeal epithelia undergone the developing process of carcinogenesis induced by N,N’-dinitrosopiperazine used among rats undergone exhausted swimming ("contaminating toxin due to Qi deficiency")Included in this part of study were 72 SD rats, randomly divided into 3 groups, i.e. modeling group (MQ 24 rats) with normal saline fed, experimental group (EQ 24 rats) with concentrated decoction of Qi-Boosting Toxin-Resolving Formulae (DQBTRF) fed and positive controlling group (PCQ 24 rats) with vitamin A acid fed, all treated for 90 days. All these animals were induced a carcinogenesis process in nasophayngeal epithelia with the procedures as the same as that in part A and intervened with the associated methods for a given period of time. Then, the animals were put into death in the same way as in part A to collect tissue sample from the nasopharynx and determine the changes of indicators in nasophayngeal epithelia, also in the same as above.Results:The results obtained from the study were as following, stated in two parts as that in methods.1. The characteristics of apoptotic signaling pathways in the developing process of carcinogenesis in nasopharyngeal epithelia induced by N,N’-dinitrosopiperazine used among rats undergone exhausted swimming ("contaminating toxin due to Qi deficiency" pathogenesis)More typical and severe pathohistological changes could be observed in nasopharyngeal epithelia with the progressing of carcinogenesis-inducing program with the use of DNP and TEA among the animals undergone exhausted swimming, the effects mimicking contaminating toxin due to Qi deficiency pathogenesis on the target organ, with nasopharyngeal carcinoma (NPC) developed in the nasopharynx at last. The occurring rate of induced epithelia lesion was much higher among the animals of MG2 than that in MG1 during the same experimental stage, while there were no significant differences in the expressive activities of apoptosis-associated genes determined here between these two groups in a same pathological stage.At the lesion developing stages of normal nasopharyngeal epithelia and simple hyperplasia, there were no significant differences in apoptotic index and PCNA index between MG1 and MG2 (P>0.05). With the development of induced carcinogenesis in nasopharyngeal epithelia, apoptotic index was significantly decreased in the tissue samples of nasopharynx with atypical hyperplasia and cancerous transformation. Although there was no significant difference in the apoptotic index found between the stage of atypical hyperplasia and that of cancerous transformation, significant differences could be determined when compared this indicator of these two stages with that of normal nasopharyngeal epithelia stage and simple hyperplasia stage respectively (P<0.05).The expressive activities of PCNA were both significantly increased in atypical hyperplasia and cancerous transformation nasopharyngeal epithelia with significant difference between the two stages of pathology (P<0.05). Furthermore, there were also significant differences between different stages of pathology, when compared with that of normal nasopharyngeal epithelia or simple hyperplasia nasopharyngeal epithelia respectively (P<0.05). Moreover, could found be a significant negative correlation present between apoptotic index and PCNA expressive activity in this progress of lesion developing (r=-0.794, P<0.05).There were no significant differences found in the expressive activities of Bcl-2, Cyt-C and Caspase-3 at the pathological stages of normal nasopharyngeal epithelia and simple hyperplasia (P>0.05). However, an increased expressive activity could be determined in Bcl-2 either in atypical hyperplasia or in cancerous transformation tissue samples with the development of induced lesion in nasopharyngeal epithelia, though with no significant differences found in these two stages of pathology themselves (P>0.05). In contrast, there were significant differences present when compared them with that of normal nasopharyngeal epithelia and simple hyperplasia (P< 0.05). The expressive activities of Cytochrome-C and Caspase-3 were decreased in nasopharyngeal epithelia with the pathological changes of atypical hyperplasia or cancerous transformation. Also, no significant differences could be found in these two stages of pathology themselves (P>0.05), while significant differences were found when compared them with that of normal nasopharyngeal epithelia and simple hyperplasia (P<0.05). The expressing activity of Bax showed a gradually increased tendency with the progressing of induced cancerous lesion in the pharynx, while there were no significant differences found among any stages of pathology when comparison was made (P>0.05). Meanwhile, various associations could be found among these determined indicators from correlating analysis carried out among them, with very significant negative correlation of Bcl-2 with Cytochrome-C and Caspase-3 (r=-0.668 and-0.871, P<0.05) respectively and significant positive correlation of Cytochrome-C with Caspase-3 (r= 0.698, P<0.05) while without significant correlation of Bax with Bcl-2, Cytochrome-C and Caspase-3 (r=-0.157,0.196,0.334; P= 0.593,0.501,0.243) respectively. Furthermore, there was also a parallel tendency of expressing activity in either Bax mRNA or Bcl-2 mRNA with Bax or Bcl-2 itself respectively in any stage of pathology. As seen in the mitochondrion route of apoptotic signaling pathway, similar changes could be observed in the NF-κB route of signaling pathway. In the lesion developing stages of normal nasopharyngeal epithelia and simple hyperplasia, there were no significant differences between them in the expressive activities of NF-κB p65 and IκBα(P >0.05), while there was increased expressing activities of NF-κB p65 in the tissue samples of atypical hyperplasia and cancerous transformation nasopharyngeal epithelia, with very significant differences these two stages of pathology (P<0.05). Moreover, very significant differences could also be determined in the expressing activities of this protein, when compared these two stages with either normal epithelia stage or simple hyperplasia one (P<0.05). On the other hand, the expressing activity of IκBαwas decreased in either atypical hyperplasia or cancerous transformation of the nasopharyngeal epithelia, even though without significant difference present between these two stages of pathology (P > 0.05). When compared these data with either that of normal nasopharyngeal epithelia stage or that of simple hyperplasia stage, very significant differences were found among them (P<0.05 respectively). Once more, a negative correlation could be determined between the activity of NF-κB p65 and that of IκBαin the cancerously transformed nasopharyngeal epithelia (r=-0.541, P<0.05).2. The intervening effects of Qi-Boosting Toxin-Resolving Formulae on the apoptotic signaling pathways in nasopharyngeal epithelia undergone the developing process of carcinogenesis induced by N,N’-dinitrosopiperazine used among rats undergone exhausted swimming ("contaminating toxin due to Qi deficiency" pathogenesis) The rate of cancerously transformed nasopharyngeal epithelia was 0% (0/5) in the rats of EG receiving concentrated DQBTRF feeding, significantly lower than that of MG (86.3%,5/6) receiving normal saline feeding (P<0.05). Though such a rate was also the same (0%,0/5) as that of EG, all the animals in this group were gradually dead during the experimental period from 250 to 310 d, suggesting a very significantly toxic effect of vitamin A acid on these experimental animals.In the simple hyperplasia stage of induced cancerous transforming lesion in the nasopharyngeal epithelia, no significant differences were found in the apoptotic index and PCNA activity among the 3 groups of rats (P>0.05). Till the pathological stage of atypical hyperplasia, apoptotic index was significantly elevated (P<0.05) and PCNA activity was significantly declined (P<0.05) among the rats of EG when compared with that of MGAlso in the simple hyperplasia stage of induced cancerous transforming lesion in the nasopharyngeal epithelia, no significant differences were determined in the expressing activities of Bcl-2, Bax, Cytochrome-C and Caspase-3 (P>0.05), when compared among them respectively. At the pathological stage of atypical hyperplasia, the expressing activity of Bcl-2 was significantly lower (P<0.05) and the activities of Cytochrome-C and Caspase-3 were significantly higher in the rats of EG than that of MG (P<0.05). Though the expressing activity of Bax was higher in the rats of EG than that of MG, no significant difference was determined between them (P>0.05). As the same as above in part one, there was also a parallel tendency of expressing activity in either Bax mRNA or Bcl-2 mRNA with Bax or Bcl-2 itself respectively in any stage of pathology. No significant changes in the expressing activities of NF-κB p65 and IκB a could be determined among these 3 groups of rats (P>0.05) in the simple hyperplasia stage of induced cancerous transforming lesion in the nasopharyngeal epithelia, while significantly lower expressing activity of NF-κB p65 (P<0.05) and higher expressing activity of IκB a (P<0.05) were assayed in the rats of EG than that of MG.Conclusions:Four aspects can be concluded based on this study as followings.1. Qi-deficient status of physique can be the genetically determined susceptible factor of physique to cancerous transforming development in the nasopharynx induced by environmental carcinogenic factors. Once one with such a physique status is contacted with this kind of carcinogenesis-inducing reagent for a certain period of time, the carcinogenesis-developing process may be induced in this way in nasopharyngeal epithelia. Then, the hypothesis of "contaminating toxin due to Qi deficiency" pathogenesis can be established reasonably to explain the carcinogenesis responsible for nasopharyngeal carcinoma developing in the nasopharynx.2. The developing process of induced cancerous transforming in nasopharyngeal epithelia of rats should be closely associated with the greatly decreased activity of apoptosis and significantly increased activity of cell proliferating in the target organ, with its pathological mechanisms possibly involved in the abnormally changed apoptotic signaling pathways of mitochondrion route and NF-κB route.3. Such a cancerous transformation developing process, induced by "contaminating toxin due to Qi deficiency" pathogenesis, can be effectively blocked by the use of herbal medicine preparation of Qi-Boosting Toxin-Resolving Formulae. This blocking effect on the induced lesion of cancerous transformation in nasopharyngeal epithelia may be underlay by the mechanisms of it to effectively inhibit the significantly elevated proliferating potentiality and actively induce apoptotic activity among them. Furthermore, the inducing effect of the formulae on apoptotic activity of nasopharyngeal epithelia with the induced lesion may be brought about by via intervening the routes of mitochondrion and NF-κB in the signaling pathway of apoptosis.4. Based on the results of this study, it has been suggested that the hypothesis of "contaminating toxin due to Qi deficiency" pathogenesis in terms of TCM responsible for the cancerous transformation of nasopharyngeal epithelia can be more effectively verified by the mimicking procedures of cancinogenesis in nasopharyngeal epithelia induced by N,N’-dinitrosopiperazine used among rats undergone exhausted swimming to prepare animal model with this kind of nasopharyngeal lesion. Furthermore, the experiment based on the intervening therapy with herbal medicine Qi-Boosting Toxin-Resolving Formulae has provided much powerful evidences for establishing a therapeutic principle to such a lesion by means of boosting Qi to resolve toxin, other than to confirm the reasonability of such a hypothesis to explain the pathogenesis of the lesion through the very effective therapeutic effects on it.更多还原