气虚染毒病机诱发大鼠鼻咽上皮癌变的凋亡信号传导通路及其干预的研究
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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。
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 MG
Also 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.
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。
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 MG
Also 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.
引文
[1]秦岭,李龄.鼻咽癌遗传易感性的分子水平研究进展.医学文选；2002,21(2)：83-84.
[2]田道法主编.中西医结合耳鼻咽喉科学.北京,第一版。中国中医出版社,2002：197.
[3]田道法,周小军,唐发清.气虚证模型大鼠鼻咽组织cDNA阵列B区基因表达谱特征及其对治疗的反应.中医药学报,2003,31(5)：60-61.
[4]唐发清,龚志军,周辉,等.益气解毒颗粒对鼻咽癌裸鼠移植瘤基因表达的影响[J].实用预防医学,2004,11(4)：637-640.
[5]Boussen H, Bouaouina N, Mokni-Baizig N, Gamoudi A, Chouchane L, Benna F, Ladgham A. Nasopharyngeal carcinoma. Recent data. Pathol Biol (Paris).2005; 53(1):45-51.
[6]湖南医学院肿瘤研究室.亚硝胺类化舍诱发大鼠鼻咽癌的实验研究[J].科学通报,1978,756-759.
[7]刘宇,张幸平,邓红彬等。bcl—2、bax和p53在鼻咽鳞癌中的表达及其与瘤细胞凋亡的关系[J].现代肿瘤医学,2006,14(7)：778-779。
[8]樊晋华,侯明新,王方等.健脾增免中药对小鼠免疫机能及力竭性游泳运动能力的影响[J].中国运动医学杂志,2002,21(4)：371-374。
[9]何迎春。田道法,田甜等.外源突变型p53和潜伏膜蛋白1基因在转基因鼠不同器官组织中的表达[J]。中国临床康复,2005,9(42)：99-101
[10]周小军,田道法,陈学东。鼻咽癌高危人群体质调查研究.中国中医基础医学杂志,2003;9(8)：51-54.
[11]Tang FQ, Tian DF, et al. The inhibitory effect of Chinese herbal complex compound on telomerase activity of NPC cell. Int J Mord Cancer Therapy,2001,42:307-309
[12]田道法,唐发清,周小军。气虚证模型大鼠cDNA阵列A区基因表达谱特征的初步研究。湖南中医学院学报,2001,21：1-3
[13]Hengartner'M. The biochemistry of apoptosis [J]. Nature,2000,407 (6805):770-776
[14]李萍,张增锋,韦敏怡。鼻咽癌细胞凋亡指数与p73表达及患者临床预后的关系[J].基础医学与临床,2004,24(5)：518-521
[15]周光耀,刘亚峰,罗锋等.鼻咽癌细胞增殖与凋亡的研究[J].华西医学,2005,20(2)：212-213
[16]李杰恩,唐安洲,黄光武等。鼻咽癌细胞凋亡及凋亡相关基因的表达[J].中华肿瘤杂志.2000,22(2)：158-160
[17]金欧,冯德云,程瑞雪.鼻咽癌细胞凋亡与细胞增殖的原位观察[J].湖南医科大学学报,1999,24(5)：421-424.
[18]刘卫平Johann L, Martin LH,等.鼻T细胞性淋巴瘤细胞凋亡和增生的比较研究[J].中华病理杂志,1996,25(6)：343—346
[19]Drigo B, Rainer F, Patricia A, et, al. Cyclin/PCNA is thr auxiliary protein of DNA polymeter-b[J]. Nature,1987:326:515
[20]王均,陈洪雷,朱润庆,等,大鼠肺鳞癌癌变过程中凋亡和增殖相关基因表达的研究.癌症,2003,22(5)：471—476.
[21]金欧,冯德云,程瑞雪.鼻咽癌细胞凋亡与细胞增殖的原位观察[J].湖南医科大学学报,1999,24(5)：421-424.
[22]Li P, Nijhawan D, Budihardjo I, Srinivasula SM, AbroadM, Alnemri KS, et al. Cytochrome C and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade [J]. Ceel,1997,91 (4): 479-489.
[23]Green DR, Reed JC. Mitochondria and apoptosis[J]. Science,1998, 281(5381):1309-1312.
[24]李萍,韦敏怡.细胞凋亡基因与鼻咽癌。中国医学文摘·肿瘤学,2003,17(1)：77-79
[25]周小军,等。广西医学2002；24(5)；647-649。
[26]陈少华,邱前辉,邵建永,等。EBV潜伏膜蛋白-1表达与鼻咽癌增殖和凋亡关系的研究。耳鼻咽喉-头颈外科,2003,10(3)：168-172
[27]Metcalfe AD, Hunter HR, Bloor DJ, et al. Expression of 11 members of the Bcl-2 family of apoptosis regulatory molecules during human preimplantation embryo development and fragmentation. Mol Reprod Dev,
2004,68(1):35-50.
[28]Shi Y, Chen J, Weng C, et al. Identification of the protein-protein contact site and interaction mode of human VDAC1 with Bcl-2 family proteins. Biochem Biophys Res Commun,2003,305(4):989-996.
[29]Grander D, Shoshan MC, Panaretakis T, et al. Interferon-alpha-induced apoptosis in V266 cells is associated with activation of the proapoptotic Bcl-2 family members Bak and Bax. Oncogene,2003,22(29): 4543-4556.
[30]YinXM, WangK, Gross A, ZhaoY, et al. Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis [J]. Nature,1999,400(6747): 886-891.
[31]Yu Y, Dong W, Li X, et al. Significance of C-Myc and Bcl-2 protein expression in nasopharyngeal carcinoma[J]. Arch Otolaryngol Head Neck Surg,2003,129 (12):1322-1326.
[32]Sarac S, Akyol MU, Kanbur B, et al. Bcl-2 and LMP1 expression in nasopharyngeal carcinomas[J]. Am J Otolaryngol,2001,22(6):377-382.
[33]刘宇,张幸平,邓红彬,等.Bcl-2、bax和p53在鼻咽鳞癌中的表达及其与瘤细胞凋亡的关系[J].现代肿瘤医学,2006,(14)：278-279
[34]马薇韦,敏怡.Bcl-2和Bax蛋白在鼻咽癌中的表达及意义[J].广西医科大学学报,2003,20(1)：39-41
[35]王茂鑫.鼻咽癌组织中凋亡相关基因蛋白表达及其意义[J].福州总医院学报,2004,11(4)：262-264
[36]张敏燕,张正昆,韩仲明.凋亡调控基因Bcl-2、Bax与肿瘤[J].国外医学耳鼻咽喉科学分册,2004,28(1)：40-43。
[37]田立宁,李慧军,夏虹等.Bcl-2基因在鼻咽癌中的表达[J].哈尔滨医科大学学报.2005,39(3)：231-232.
[38]Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death[J]. Cell,1993,74 (4):609-619.
[39]Depraetere V, Golstein P. Dismantling in cell death:molecular mechanisms and relationship to caspase activation[J]. Scand J Immunol,1998.47:523-531.
[40]方希敏,陈铭珍,陈日玲等.细胞色素C对HL-60细胞凋亡作用及其与相关bcl-2、bax基因的关系[J].血液学杂志,2005,13(4)：570-574
[41]Zhivotovsky B, Orrenius S, Brustrugun OT. et al. Injected cytochrome C induces apoptosis. Nature,1998:391(6666):449—450
[42]Yang J, Liu X, Bhalla K, Kim CaN, Ibrado AM, Cai J,. Prevention of apoptosis by Bcl-2:release of cytochrome C from mitochondria blocked [J]. Science,1997,275(5303):1129-1132.
[43]Yu HG, Yu LL, Yang Y, etal. Increased expression of RelA/nuclear factor-kappa B protein correlates with colorectal tumorigenesis[J]. Oncology,2003; 65 (1)37-45
[44]Nair A. Venkatraman M,Maliekal TT,et al.NF-kappaB is constitutively activated in high-grade squamous intraepithelial lesions and squamous cell carcinomas of the human uterine cervix[J]. Oncogene,2003; 22(1):50-58
[45]Tamatani T,Azuma M,Aota K, et al. Enhanced IkappaB kinase activity is responsible for the angmented activity of NF-kappaB in human head and neck carcinoma cells [J]. Cancer Lett,2001,171 (2):165-172
[46]朱江,胡国华,孙余才.免疫印迹法检测IKK,1KB和NF-KB在喉癌中的表达[J].第四军医大学报,25(23)：2165-2166
[47]钟霞.NF-κB的活化与肿瘤的研究进展[J].医学理论与实践,2001；14(11)：1102-1105
[48]李智,林素暇,梁惠珍.鼻咽癌组织中MIF、c-Fos、NF-KB和MMP9的表达及意义肿瘤[J].2004,24(3)：290-293
[49]潘雷,顾焕华,粱,等.鼻咽癌中EBV LMP1激活NF-κB的试验研究[J].中国耳鼻喉咽颅底外科杂志,1999,5(2)：95-99。
[50]唐发清,李建玲,荆照政等.鼻咽癌变过程中基因表达的cDNA阵列研究[J].湖南医科大学学报,2002,27(5)：397-401.
[1]田道法,唐发清,陈协云,等.益气解毒颗粒对鼻咽癌高危人群EB病毒感染活性抑制作用的临床观察[J].湖南中医学院学报,2000,20(3)：47-47.
[2]杜可,田道法.益气解毒颗粒对HNE3细胞及其裸鼠移植瘤的抑制作用[J].湖中医学院学报,2005,25(3)：7-9.
[3]唐发请,谌兵来,田道法。益气解毒片对鼻咽癌细胞基因表达的影响[J].湖南中医学院学报,1998,2(16)：14-15.
[4]唐发清,田道法,龚志军,等.益气解毒颗粒干预鼻咽癌细胞HNE1蛋白质表达的研究[J]。中国中西医结合耳鼻咽喉科杂志,2004,12(3)：120-123
[5]唐发清,田道法,邓富良,等.中药益气解毒颗粒对鼻咽癌裸鼠移植瘤蛋白质表达的影响中南大学学报[J].2004,29(5)：577-582：
[6]王大海,田道法.益气解毒颗粒配合放射治疗鼻咽癌的临床研究[J].湖南中医学院学报,2006,26(1)：36-37.
[7]刘铭球,朱润庆,高文,等.中药黄芪注射液及扶正流浸膏对大鼠诱发性肺癌抑制作用的实验研究.肿瘤防治研究,1992,19(3)：133
[8]张占好,张春艳,黄芪多糖对小鼠体内六种细胞系瘤株抑瘤作用的实验研究。中医药学报,1996,[4]：55
[9]全宏勋.黄芪水煎液对环磷酰胺抗肿瘤活性与毒性的影响.中药材,1995,18(7)：356
[10]汪德清,沈文梅,田亚平,等.黄芪总黄酮对大鼠的肺癌术中故疗抗辐射损伤作用的研究.中华放射医学与防护杂志
[11]陈敏。新疆党参和潞党参对知DSOD,MDA的影响
[12]施荣山.党参的药理研究进展现代预防医学 1997,24(3),335—337,1996,16(6)：399
[13]吴波 梁谋 茯苓多糖抗肿瘤作用与机理的实验研究 中国药理学报.1994,10(4).-300-304
[14]柴宝玲。林志彬.曹似兰.羧甲基茯苓多糖对巨噬细胞吞噬功能的影响.北京医学院学报.1983.15[1]：
[15]单保恩,张金艳,杜肖娜,等.白花蛇舌草的免疫学调节活性和抗肿瘤活性
[J].中国中西医结合杂志,2001,21(5)：370.[16]李瑞,赵浩如,林以宁.白花蛇舌草水溶性提取物的抗肿瘤作用和对化疗损伤的保护作用的研究[J]. journal of Chinesep phrmaceutical sciences(中国药学：英文版),2002,11(2):58.9-11 [17] Chen KT, Hao DM, Liu ZX, et al.Effects of berberine or in combination with argon ion lascer treatment on 91 rat glioma cell line [J]Chin Med J (End) 1994,107 (11):808-812 [18] Cheng JG,Wu LT,Chu CB,etal.Effects of berberine on arylamine N-acetyltransferase activity in human bladder tumor cells[J] Food Chem Toxicol,1999,37 (4):319-326[19]黄小洁等.沈阳药学院学报1991；8(3)：204.[20]郭峰,刘小平,徐珏.天花粉对小鼠艾氏腹水瘤的功效和对红细胞免疫功能的影响.中西医结合杂志,1989,9(7)：418：4[21]徐瑛,郭峰,叶天星.天花粉治疗小鼠艾氏腹水瘤对红细胞增强吞噬作用的研究.肿瘤,1990,10(5)：224[22]朱晏伟。益气健脾法治疗恶性肿瘤机理研究的进展LjI.云南中医中药杂志,
1995,16(3)：55—58[23]王岳五,史玉荣,张海波,等.甘草残渣中多糖GPS抗肿瘤作用的研究[J2.南开大学学报,2000,33(4)：46—48。[24]唐发清,田道法。黄连及复方对鼻咽癌细胞凋亡的诱导作用[J].中国耳鼻咽喉-颅底外科杂志.1996,2(s)：6-8[25]唐发清,田道法,谌兵来,等.益气解毒颗粒对鼻咽细胞端粒酶和端粒酶RNA的抑制作用的研究[J]湖南中医学院学报,2000.20(1)：10-12。[26]汤爱国,田道法,易运连,等.中药益气解毒颗粒下调端粒酶活性抑制鼻咽癌细胞的生长。中国现代医学杂志,2005,15(1)：53-55[27]李杰恩,唐安洲,黄光武等.鼻咽癌细胞凋亡及凋亡相关基因的表达[J].中华肿瘤杂志。2000,22(2)：158-160[28]刘宇,张幸平,邓红彬,等.Bcl-2、bax和p53在鼻咽鳞癌中的表达及其与瘤细胞凋亡的关系[J].现代肿瘤医学,2006,(14)：278-279[29]唐发清,李建玲,荆照政等.鼻咽癌变过程中基因表达的cDNA阵列研究[J]. 湖南医科大学学报,2002,27(5)：397-401.
[30]王光平,唐发清,周金平,等.复方黄连对NF-κB活化相关基因在鼻咽癌移植瘤组织表达活性的影响[J].湖南中医学院学报,2004,24(6)：14-17.
[31]Nair A. Venkatraman M, Maliekal TT, et al.NF-kappaB is constitutively activated in high-grade squamous intraepithelial lesions and squamous cell carcinomas of the human uterine cervix [J].Oncogene,2003;22(1):50-58
1 黄带发,孙开来.肿瘤细胞信号传导的研究进展[J].国外医学遗传学分册,2004,27(4)：203-207
2 Ghosh S, May MJ, KoPP EB. NF-kaPPa B and Rel proteins:evolutionary conserved mediators of immune response [J]. Annu Rev Immunol,1998,16:225-260
3 陈华江,王杰军,何金,等.血管内皮细胞生长因子及其受体在结直肠癌中的表达[J].临床肿瘤学杂志,1999,4(4)：1
4 丁新民,刘凡光,顾瑛,等。线粒体在血卟啉单甲醚-PDT诱导Hela细胞调亡中的作用[J].中国激光医学杂志,2004,13(1)：18-22
5 周本杰,陈蔚文。连黛片对大鼠溃疡性胃癌相关癌基因蛋白表达的影响[J].中药新药与临床药理,2001,012(004)：249-251
6 杨江苏,秦旭平,张娜,等.两种真菌多糖对HL-60细胞酪氨峻蛋白磷酸化作用的影响[J].中国药学杂志,2000,35(5)：303
7 池岛乔,安巍巍,王敏伟.去甲斑蝥素通过caspase, MAPK和线粒体途径诱导HeLa细胞凋亡[J].中国病理生理杂志,2003,019(011)：1444
8 杨晶凡,刘丽波,于英君。树舌多糖GF对HepA瘤细胞Ras蛋白表达的影响[J]。中医药学刊,2003,021(011)：1831
9 Hu G,Han C,Chen J. Inhibition of oncogene expression by green tea and (-)epigallo catechingallate in mice [J]. Nutrb Cancer,1995,34(2):203.
10 沈世林,赵健雄,朱玉真,等.复方中药制剂对肿瘤细胞NF-κB表达及凋亡影响的实验研究[J].四川中医,2004,022(007)：12-13
11 王继伟,戴书静.金雀黄素抑制人肝癌细胞Bel7402中NF-κB表达的实验研究[J].北京中医药大学学报,2001,024(005)：6-19,44
12戴书静,王继峰.去甲斑蝥素增强Be17402中IκBα表达和抑制细胞增殖的研究[J].解剖学报,2001,032(002)：155-158
13赵健雄,曲勇,陈学忠,等.扶正抑瘤颗粒对食管癌、胃癌组织细胞周期及核转录因子的影响[J].中西医结合杂志,2003,023(012)：908-910
14 罗非君,胡智。茶多酚诱导鼻咽癌细胞caspase-3活化[J].癌 症,2000,019(012)：1082-1086
15 王玉,于英君,徐广有,等.树舌多糖对小鼠HepA P16 P27 Rb基因表达的影响[J].世界华人消化杂志,2004,012(006)：1353-1356
16 翁昔阳,于立坚.土贝母皂苷诱导人鼻咽癌细胞株CNE-2Z细胞凋亡的研究[J].中国药理学通报,2003,019(002)：181
17 袁淑兰,王艳萍.参丹酮Ⅱ A诱导人鼻咽癌细胞凋亡及其作用机制的体外实验研究[J].华西医科大学学报,2002,033(001)：84-86
18 闵存云,李庆明,吴伟康.中药复方胃康宁对人胃癌细胞VEGF及其受体表达的作用[J].世界华人消化杂志,2004,012(003)：533-536
19 孔庆志,黄冬生,黄涛,等.3种中药注射剂对小鼠移植性S180肉瘤血管形成抑制作用[J].中国医院药学杂志,2003,023(011)：646-648
20Yoshiyuki Kimura, Hiromichi Okuda. Resveratrol Isolated from Polygonum cuspidatum Root Prevents Tumors Growth andMetastasis to Lung and Tumor-induced Neovascularization in Lewis Lung Carcinoma-Bearing Mice[J]. JNutr,2001,131 (6):1844
21 唐旭东,周克元,侯敢,等.白藜芦醇诱导鼻咽癌细胞CNE-2Z凋亡的线粒体机制[J].中国药理学与毒理学杂志,2004,018(003)：171-177
22 方志俊,曹佳,敖琳,庄为笕,等.基因芯片分析显示昆明山海棠有机萃取液经NF-κB及线粒体信号传导途径诱导HL-60细胞凋亡[J].中草药,2003,034(004)：334-338
23 李宝元,梁云燕。中药白龙片与HMBA对人胃癌不同周期细胞癌基因与抑癌基因表达调控的共性研究[J].中国中西医结合杂志,2001,021(004)：286-290
[2]田道法主编.中西医结合耳鼻咽喉科学.北京,第一版。中国中医出版社,2002：197.
[3]田道法,周小军,唐发清.气虚证模型大鼠鼻咽组织cDNA阵列B区基因表达谱特征及其对治疗的反应.中医药学报,2003,31(5)：60-61.
[4]唐发清,龚志军,周辉,等.益气解毒颗粒对鼻咽癌裸鼠移植瘤基因表达的影响[J].实用预防医学,2004,11(4)：637-640.
[5]Boussen H, Bouaouina N, Mokni-Baizig N, Gamoudi A, Chouchane L, Benna F, Ladgham A. Nasopharyngeal carcinoma. Recent data. Pathol Biol (Paris).2005; 53(1):45-51.
[6]湖南医学院肿瘤研究室.亚硝胺类化舍诱发大鼠鼻咽癌的实验研究[J].科学通报,1978,756-759.
[7]刘宇,张幸平,邓红彬等。bcl—2、bax和p53在鼻咽鳞癌中的表达及其与瘤细胞凋亡的关系[J].现代肿瘤医学,2006,14(7)：778-779。
[8]樊晋华,侯明新,王方等.健脾增免中药对小鼠免疫机能及力竭性游泳运动能力的影响[J].中国运动医学杂志,2002,21(4)：371-374。
[9]何迎春。田道法,田甜等.外源突变型p53和潜伏膜蛋白1基因在转基因鼠不同器官组织中的表达[J]。中国临床康复,2005,9(42)：99-101
[10]周小军,田道法,陈学东。鼻咽癌高危人群体质调查研究.中国中医基础医学杂志,2003;9(8)：51-54.
[11]Tang FQ, Tian DF, et al. The inhibitory effect of Chinese herbal complex compound on telomerase activity of NPC cell. Int J Mord Cancer Therapy,2001,42:307-309
[12]田道法,唐发清,周小军。气虚证模型大鼠cDNA阵列A区基因表达谱特征的初步研究。湖南中医学院学报,2001,21：1-3
[13]Hengartner'M. The biochemistry of apoptosis [J]. Nature,2000,407 (6805):770-776
[14]李萍,张增锋,韦敏怡。鼻咽癌细胞凋亡指数与p73表达及患者临床预后的关系[J].基础医学与临床,2004,24(5)：518-521
[15]周光耀,刘亚峰,罗锋等.鼻咽癌细胞增殖与凋亡的研究[J].华西医学,2005,20(2)：212-213
[16]李杰恩,唐安洲,黄光武等。鼻咽癌细胞凋亡及凋亡相关基因的表达[J].中华肿瘤杂志.2000,22(2)：158-160
[17]金欧,冯德云,程瑞雪.鼻咽癌细胞凋亡与细胞增殖的原位观察[J].湖南医科大学学报,1999,24(5)：421-424.
[18]刘卫平Johann L, Martin LH,等.鼻T细胞性淋巴瘤细胞凋亡和增生的比较研究[J].中华病理杂志,1996,25(6)：343—346
[19]Drigo B, Rainer F, Patricia A, et, al. Cyclin/PCNA is thr auxiliary protein of DNA polymeter-b[J]. Nature,1987:326:515
[20]王均,陈洪雷,朱润庆,等,大鼠肺鳞癌癌变过程中凋亡和增殖相关基因表达的研究.癌症,2003,22(5)：471—476.
[21]金欧,冯德云,程瑞雪.鼻咽癌细胞凋亡与细胞增殖的原位观察[J].湖南医科大学学报,1999,24(5)：421-424.
[22]Li P, Nijhawan D, Budihardjo I, Srinivasula SM, AbroadM, Alnemri KS, et al. Cytochrome C and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade [J]. Ceel,1997,91 (4): 479-489.
[23]Green DR, Reed JC. Mitochondria and apoptosis[J]. Science,1998, 281(5381):1309-1312.
[24]李萍,韦敏怡.细胞凋亡基因与鼻咽癌。中国医学文摘·肿瘤学,2003,17(1)：77-79
[25]周小军,等。广西医学2002；24(5)；647-649。
[26]陈少华,邱前辉,邵建永,等。EBV潜伏膜蛋白-1表达与鼻咽癌增殖和凋亡关系的研究。耳鼻咽喉-头颈外科,2003,10(3)：168-172
[27]Metcalfe AD, Hunter HR, Bloor DJ, et al. Expression of 11 members of the Bcl-2 family of apoptosis regulatory molecules during human preimplantation embryo development and fragmentation. Mol Reprod Dev,
2004,68(1):35-50.
[28]Shi Y, Chen J, Weng C, et al. Identification of the protein-protein contact site and interaction mode of human VDAC1 with Bcl-2 family proteins. Biochem Biophys Res Commun,2003,305(4):989-996.
[29]Grander D, Shoshan MC, Panaretakis T, et al. Interferon-alpha-induced apoptosis in V266 cells is associated with activation of the proapoptotic Bcl-2 family members Bak and Bax. Oncogene,2003,22(29): 4543-4556.
[30]YinXM, WangK, Gross A, ZhaoY, et al. Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis [J]. Nature,1999,400(6747): 886-891.
[31]Yu Y, Dong W, Li X, et al. Significance of C-Myc and Bcl-2 protein expression in nasopharyngeal carcinoma[J]. Arch Otolaryngol Head Neck Surg,2003,129 (12):1322-1326.
[32]Sarac S, Akyol MU, Kanbur B, et al. Bcl-2 and LMP1 expression in nasopharyngeal carcinomas[J]. Am J Otolaryngol,2001,22(6):377-382.
[33]刘宇,张幸平,邓红彬,等.Bcl-2、bax和p53在鼻咽鳞癌中的表达及其与瘤细胞凋亡的关系[J].现代肿瘤医学,2006,(14)：278-279
[34]马薇韦,敏怡.Bcl-2和Bax蛋白在鼻咽癌中的表达及意义[J].广西医科大学学报,2003,20(1)：39-41
[35]王茂鑫.鼻咽癌组织中凋亡相关基因蛋白表达及其意义[J].福州总医院学报,2004,11(4)：262-264
[36]张敏燕,张正昆,韩仲明.凋亡调控基因Bcl-2、Bax与肿瘤[J].国外医学耳鼻咽喉科学分册,2004,28(1)：40-43。
[37]田立宁,李慧军,夏虹等.Bcl-2基因在鼻咽癌中的表达[J].哈尔滨医科大学学报.2005,39(3)：231-232.
[38]Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death[J]. Cell,1993,74 (4):609-619.
[39]Depraetere V, Golstein P. Dismantling in cell death:molecular mechanisms and relationship to caspase activation[J]. Scand J Immunol,1998.47:523-531.
[40]方希敏,陈铭珍,陈日玲等.细胞色素C对HL-60细胞凋亡作用及其与相关bcl-2、bax基因的关系[J].血液学杂志,2005,13(4)：570-574
[41]Zhivotovsky B, Orrenius S, Brustrugun OT. et al. Injected cytochrome C induces apoptosis. Nature,1998:391(6666):449—450
[42]Yang J, Liu X, Bhalla K, Kim CaN, Ibrado AM, Cai J,. Prevention of apoptosis by Bcl-2:release of cytochrome C from mitochondria blocked [J]. Science,1997,275(5303):1129-1132.
[43]Yu HG, Yu LL, Yang Y, etal. Increased expression of RelA/nuclear factor-kappa B protein correlates with colorectal tumorigenesis[J]. Oncology,2003; 65 (1)37-45
[44]Nair A. Venkatraman M,Maliekal TT,et al.NF-kappaB is constitutively activated in high-grade squamous intraepithelial lesions and squamous cell carcinomas of the human uterine cervix[J]. Oncogene,2003; 22(1):50-58
[45]Tamatani T,Azuma M,Aota K, et al. Enhanced IkappaB kinase activity is responsible for the angmented activity of NF-kappaB in human head and neck carcinoma cells [J]. Cancer Lett,2001,171 (2):165-172
[46]朱江,胡国华,孙余才.免疫印迹法检测IKK,1KB和NF-KB在喉癌中的表达[J].第四军医大学报,25(23)：2165-2166
[47]钟霞.NF-κB的活化与肿瘤的研究进展[J].医学理论与实践,2001；14(11)：1102-1105
[48]李智,林素暇,梁惠珍.鼻咽癌组织中MIF、c-Fos、NF-KB和MMP9的表达及意义肿瘤[J].2004,24(3)：290-293
[49]潘雷,顾焕华,粱,等.鼻咽癌中EBV LMP1激活NF-κB的试验研究[J].中国耳鼻喉咽颅底外科杂志,1999,5(2)：95-99。
[50]唐发清,李建玲,荆照政等.鼻咽癌变过程中基因表达的cDNA阵列研究[J].湖南医科大学学报,2002,27(5)：397-401.
[1]田道法,唐发清,陈协云,等.益气解毒颗粒对鼻咽癌高危人群EB病毒感染活性抑制作用的临床观察[J].湖南中医学院学报,2000,20(3)：47-47.
[2]杜可,田道法.益气解毒颗粒对HNE3细胞及其裸鼠移植瘤的抑制作用[J].湖中医学院学报,2005,25(3)：7-9.
[3]唐发请,谌兵来,田道法。益气解毒片对鼻咽癌细胞基因表达的影响[J].湖南中医学院学报,1998,2(16)：14-15.
[4]唐发清,田道法,龚志军,等.益气解毒颗粒干预鼻咽癌细胞HNE1蛋白质表达的研究[J]。中国中西医结合耳鼻咽喉科杂志,2004,12(3)：120-123
[5]唐发清,田道法,邓富良,等.中药益气解毒颗粒对鼻咽癌裸鼠移植瘤蛋白质表达的影响中南大学学报[J].2004,29(5)：577-582：
[6]王大海,田道法.益气解毒颗粒配合放射治疗鼻咽癌的临床研究[J].湖南中医学院学报,2006,26(1)：36-37.
[7]刘铭球,朱润庆,高文,等.中药黄芪注射液及扶正流浸膏对大鼠诱发性肺癌抑制作用的实验研究.肿瘤防治研究,1992,19(3)：133
[8]张占好,张春艳,黄芪多糖对小鼠体内六种细胞系瘤株抑瘤作用的实验研究。中医药学报,1996,[4]：55
[9]全宏勋.黄芪水煎液对环磷酰胺抗肿瘤活性与毒性的影响.中药材,1995,18(7)：356
[10]汪德清,沈文梅,田亚平,等.黄芪总黄酮对大鼠的肺癌术中故疗抗辐射损伤作用的研究.中华放射医学与防护杂志
[11]陈敏。新疆党参和潞党参对知DSOD,MDA的影响
[12]施荣山.党参的药理研究进展现代预防医学 1997,24(3),335—337,1996,16(6)：399
[13]吴波 梁谋 茯苓多糖抗肿瘤作用与机理的实验研究 中国药理学报.1994,10(4).-300-304
[14]柴宝玲。林志彬.曹似兰.羧甲基茯苓多糖对巨噬细胞吞噬功能的影响.北京医学院学报.1983.15[1]：
[15]单保恩,张金艳,杜肖娜,等.白花蛇舌草的免疫学调节活性和抗肿瘤活性
[J].中国中西医结合杂志,2001,21(5)：370.[16]李瑞,赵浩如,林以宁.白花蛇舌草水溶性提取物的抗肿瘤作用和对化疗损伤的保护作用的研究[J]. journal of Chinesep phrmaceutical sciences(中国药学：英文版),2002,11(2):58.9-11 [17] Chen KT, Hao DM, Liu ZX, et al.Effects of berberine or in combination with argon ion lascer treatment on 91 rat glioma cell line [J]Chin Med J (End) 1994,107 (11):808-812 [18] Cheng JG,Wu LT,Chu CB,etal.Effects of berberine on arylamine N-acetyltransferase activity in human bladder tumor cells[J] Food Chem Toxicol,1999,37 (4):319-326[19]黄小洁等.沈阳药学院学报1991；8(3)：204.[20]郭峰,刘小平,徐珏.天花粉对小鼠艾氏腹水瘤的功效和对红细胞免疫功能的影响.中西医结合杂志,1989,9(7)：418：4[21]徐瑛,郭峰,叶天星.天花粉治疗小鼠艾氏腹水瘤对红细胞增强吞噬作用的研究.肿瘤,1990,10(5)：224[22]朱晏伟。益气健脾法治疗恶性肿瘤机理研究的进展LjI.云南中医中药杂志,
1995,16(3)：55—58[23]王岳五,史玉荣,张海波,等.甘草残渣中多糖GPS抗肿瘤作用的研究[J2.南开大学学报,2000,33(4)：46—48。[24]唐发清,田道法。黄连及复方对鼻咽癌细胞凋亡的诱导作用[J].中国耳鼻咽喉-颅底外科杂志.1996,2(s)：6-8[25]唐发清,田道法,谌兵来,等.益气解毒颗粒对鼻咽细胞端粒酶和端粒酶RNA的抑制作用的研究[J]湖南中医学院学报,2000.20(1)：10-12。[26]汤爱国,田道法,易运连,等.中药益气解毒颗粒下调端粒酶活性抑制鼻咽癌细胞的生长。中国现代医学杂志,2005,15(1)：53-55[27]李杰恩,唐安洲,黄光武等.鼻咽癌细胞凋亡及凋亡相关基因的表达[J].中华肿瘤杂志。2000,22(2)：158-160[28]刘宇,张幸平,邓红彬,等.Bcl-2、bax和p53在鼻咽鳞癌中的表达及其与瘤细胞凋亡的关系[J].现代肿瘤医学,2006,(14)：278-279[29]唐发清,李建玲,荆照政等.鼻咽癌变过程中基因表达的cDNA阵列研究[J]. 湖南医科大学学报,2002,27(5)：397-401.
[30]王光平,唐发清,周金平,等.复方黄连对NF-κB活化相关基因在鼻咽癌移植瘤组织表达活性的影响[J].湖南中医学院学报,2004,24(6)：14-17.
[31]Nair A. Venkatraman M, Maliekal TT, et al.NF-kappaB is constitutively activated in high-grade squamous intraepithelial lesions and squamous cell carcinomas of the human uterine cervix [J].Oncogene,2003;22(1):50-58
1 黄带发,孙开来.肿瘤细胞信号传导的研究进展[J].国外医学遗传学分册,2004,27(4)：203-207
2 Ghosh S, May MJ, KoPP EB. NF-kaPPa B and Rel proteins:evolutionary conserved mediators of immune response [J]. Annu Rev Immunol,1998,16:225-260
3 陈华江,王杰军,何金,等.血管内皮细胞生长因子及其受体在结直肠癌中的表达[J].临床肿瘤学杂志,1999,4(4)：1
4 丁新民,刘凡光,顾瑛,等。线粒体在血卟啉单甲醚-PDT诱导Hela细胞调亡中的作用[J].中国激光医学杂志,2004,13(1)：18-22
5 周本杰,陈蔚文。连黛片对大鼠溃疡性胃癌相关癌基因蛋白表达的影响[J].中药新药与临床药理,2001,012(004)：249-251
6 杨江苏,秦旭平,张娜,等.两种真菌多糖对HL-60细胞酪氨峻蛋白磷酸化作用的影响[J].中国药学杂志,2000,35(5)：303
7 池岛乔,安巍巍,王敏伟.去甲斑蝥素通过caspase, MAPK和线粒体途径诱导HeLa细胞凋亡[J].中国病理生理杂志,2003,019(011)：1444
8 杨晶凡,刘丽波,于英君。树舌多糖GF对HepA瘤细胞Ras蛋白表达的影响[J]。中医药学刊,2003,021(011)：1831
9 Hu G,Han C,Chen J. Inhibition of oncogene expression by green tea and (-)epigallo catechingallate in mice [J]. Nutrb Cancer,1995,34(2):203.
10 沈世林,赵健雄,朱玉真,等.复方中药制剂对肿瘤细胞NF-κB表达及凋亡影响的实验研究[J].四川中医,2004,022(007)：12-13
11 王继伟,戴书静.金雀黄素抑制人肝癌细胞Bel7402中NF-κB表达的实验研究[J].北京中医药大学学报,2001,024(005)：6-19,44
12戴书静,王继峰.去甲斑蝥素增强Be17402中IκBα表达和抑制细胞增殖的研究[J].解剖学报,2001,032(002)：155-158
13赵健雄,曲勇,陈学忠,等.扶正抑瘤颗粒对食管癌、胃癌组织细胞周期及核转录因子的影响[J].中西医结合杂志,2003,023(012)：908-910
14 罗非君,胡智。茶多酚诱导鼻咽癌细胞caspase-3活化[J].癌 症,2000,019(012)：1082-1086
15 王玉,于英君,徐广有,等.树舌多糖对小鼠HepA P16 P27 Rb基因表达的影响[J].世界华人消化杂志,2004,012(006)：1353-1356
16 翁昔阳,于立坚.土贝母皂苷诱导人鼻咽癌细胞株CNE-2Z细胞凋亡的研究[J].中国药理学通报,2003,019(002)：181
17 袁淑兰,王艳萍.参丹酮Ⅱ A诱导人鼻咽癌细胞凋亡及其作用机制的体外实验研究[J].华西医科大学学报,2002,033(001)：84-86
18 闵存云,李庆明,吴伟康.中药复方胃康宁对人胃癌细胞VEGF及其受体表达的作用[J].世界华人消化杂志,2004,012(003)：533-536
19 孔庆志,黄冬生,黄涛,等.3种中药注射剂对小鼠移植性S180肉瘤血管形成抑制作用[J].中国医院药学杂志,2003,023(011)：646-648
20Yoshiyuki Kimura, Hiromichi Okuda. Resveratrol Isolated from Polygonum cuspidatum Root Prevents Tumors Growth andMetastasis to Lung and Tumor-induced Neovascularization in Lewis Lung Carcinoma-Bearing Mice[J]. JNutr,2001,131 (6):1844
21 唐旭东,周克元,侯敢,等.白藜芦醇诱导鼻咽癌细胞CNE-2Z凋亡的线粒体机制[J].中国药理学与毒理学杂志,2004,018(003)：171-177
22 方志俊,曹佳,敖琳,庄为笕,等.基因芯片分析显示昆明山海棠有机萃取液经NF-κB及线粒体信号传导途径诱导HL-60细胞凋亡[J].中草药,2003,034(004)：334-338
23 李宝元,梁云燕。中药白龙片与HMBA对人胃癌不同周期细胞癌基因与抑癌基因表达调控的共性研究[J].中国中西医结合杂志,2001,021(004)：286-290