【作者】 周昕欣；

【导师】 王彩霞；

【作者基本信息】 辽宁中医药大学 ， 中医基础理论， 2013， 博士

【摘要】 目的：恶性黑色素瘤（Malignant melanoma, MM）是由黑色素细胞恶性转化形成，具有高侵袭和高转移特性的恶性肿瘤。MM因其持续增长的发病率和死亡率日益受到人们的重视，其发病率年平均增长速度为4%～6%，在近二十年中成倍增长。到目前为止，对MM的治疗主要包括手术、生物治疗、放疗、化疗和肿瘤疫苗，然而，只要MM发生转移，几乎没有任何治疗方法可以治愈。唯一由美国食品和药品管理局批准的治疗转移性黑色素瘤的药物达卡巴嗦，其完全缓解率尚未超过5%。这可能是由多药耐药（multidrug resistance，MDR）引起，引起多药耐药最主要的机制是肿瘤细胞凋亡通路障碍。因此，如何解决化疗药物的多药耐药性，进而提高黑色素瘤肿瘤细胞凋亡是现代医学丞待解决的问题。中医学认为，中药在抗肿瘤作用中占有重要地位，尤其在抗耐药性和减毒增效方面更具有显著优势。随着相关研究的深入，业内学者认同“肺主皮毛”理论不能简单的理解为肺主管体表皮肤、粘膜、汗腺、发须等生理功能上的皮毛，而是对抗病邪时与“肌表”相似，是呼吸道和皮肤免疫功能相互影响相互协调的免疫反馈机制，大量临床观察表明肺与皮毛在发病机制和免疫机制上可能有相似之处，有人观察到湿疹性皮炎患者发病前后均患有肺部疾患；支气管哮喘患者64%患有皮肤疾病，临床同一药物既可治疗肺系病变，也可治疗皮肤疾患。有学者总结治疗肺癌最常用的中药有26味，本研究中有7味中药构成组方，其中白术、茯苓、甘草、浙贝母和莪术5味中药属于此26味中药之中，且肺癌治疗多采用以扶正为主的补气方剂，以四君子汤为基础加减使用频次最多，已有学者证明四君子汤加减对肺癌治疗疗效确切。培土生金也称补脾益肺，是治疗肺脾两虚证的一种治法。按五行相生规律肺金与脾土为子母关系，按虚则补其母的治则，运用培土生金之法，健脾胃可以益肺气，待脾气充实，健运复职，土旺则金自生，肺虚之候自去。脾肺同属太阴，主行于人身胸腹，两经密切相连，经气相通，气血相贯。现代医学研究表明，人类肿瘤特异性免疫治疗的重要物质基础是肿瘤抗原的存在，肺癌和黑色素瘤表达共同的黑色素瘤抗原（melanoma antigen, MAGE）MAGE-1和MAGE-3，提示这两种疾病可能有相似的抗肿瘤免疫应答机制。综上，从中医理论和现代医学两个层面支撑，精选四君子汤作为基础方剂，酌加治疗肺癌有效的药物，可能对治疗皮肤肿瘤有效。且现代药理学证明，贝母苷甲抑制人高转移巨细胞肺癌细胞的粘附、侵袭和迁移能力，同样土贝母苷甲对小鼠B16黑色素瘤和Lewis肺癌转移均具有抑制作用。莪术挥发油对多种癌细胞既有直接破坏作用，又能通过增强免疫系统功能发挥抗癌作用，莪术醇通过非依赖Caspase途径来介导肺癌A549细胞的凋亡，同样发现莪术水提波能够抑制黑色素瘤高转移细胞株B16的转移，可能与其能够抑制B16与细胞外基质的粘附以及降低B16的侵袭性和运动能力有关。半枝莲有增加机体免疫功能和抗肿瘤作用，对人肺巨细胞癌细胞株PG细胞端粒酶活性的影响明显下降，其提取物抗肺癌A549细胞生长。因而，本研究在四君子汤基础上，酌选浙贝母、莪术和半枝莲组成加味四君子汤治疗MM。Bcl-2和Caspase是公认的凋亡相关基因，Bcl-2家族是凋亡抑制基因，对凋亡有抑制作用，研究表明，抑制Bcl-2蛋白的表达可以促进MM细胞凋亡。凋亡相关蛋白Caspases被激活后能促进凋亡，其中Caspase3和Caspase9的激活尤为重要，引起细胞凋亡。基质金属蛋白酶（metalloprotease, MMP）是一个能够水解某些蛋白成分的蛋白家族，因其需要Ca²⁺、Zn²⁺等金属离子作为辅助因子而得名，其家族成员具有相似的结构，其中MMP2、MMP9研究的较为深入，MMPs几乎能降解细胞外基质（extracellular matrix，ECM）中的各种蛋白成分，破坏肿瘤细胞迁移侵袭的组织学屏障，在肿瘤侵袭转移中起关键性作用，从而在肿瘤迁移浸润转移中的作用日益受到重视，MMP被认为肿瘤侵袭转移过程中主要的蛋白水解酶。转录因子核因子NF-κB （nuclear factor-kappa B, NF-κB）是由五种不同蛋白（p50, p52,p65/RelA, RelB, c-Rel）组成的二聚体。通常NF-κB二聚体与抑制蛋白IκB相结合，以无活性的形式存在于细胞浆中，当多种刺激激活IκB激酶诱导IκB蛋白水解后，NF-κB的DNA结合结构域和核定位序列暴露出来，使NF-κB转位到细胞核，诱导靶基因转录。有大量文献已明确NF-κB （p65）是Bcl-2、MMP2和MMP9正性转录调控因子，而Bcl-2的低表达可以通过诱导线粒体途径和受体途径使Caspase3和Caspase9表达升高。本研究首先建立体外B16MM模型，利用血清药理学方法给药，利用MTT法确认加味四君子汤的最佳作用时间和最佳作用剂量；其次研究加味四君子汤对B16MM细胞凋亡的影响，以及调节凋亡相关基因Bcl-2、Caspase3和Caspase9表达；再次研究加味四君子汤对B16MM细胞迁移侵袭的影响，以及相关蛋白MMP2和MMP9的表达；最后研究可能参与调节上述蛋白表达的信号分子NF-κB的活性和蛋白表达，进一步明确加味四君子汤抗恶性黑色素瘤的机制。材料与方法：1.体外MM模型的制备。2.血清药理学实验方法给药。3.细胞增殖抑制试验即MTT法检测加味四君子汤含药血清对B16MM细胞增殖的影响。4.应用流式细胞仪Annexin V-FITC/PI法检测加味四君子汤含药血清对B16MM细胞凋亡的影响。5.应用细胞划痕实验和Transwell实验检测加味四君子汤含药血清对B16MM细胞迁移和侵袭的影响。6.应用半定量RT-PCR法检测迁移和侵袭相关基因MMP2和MMP9的mRNA表达；检测抑制凋亡基因Bcl-2的mRNA表达，检测促进凋亡基因Caspase3和Caspase9的mRNA表达。7.应用Western blot法检测迁移和侵袭相关基因MMP2和MMP9的蛋白表达；检测抑制凋亡基因Bcl-2的蛋白表达，检测促进凋亡基因Caspase3和Caspase9的蛋白表达；检测转录因子核因子NF-κB （p65）的蛋白表达。8.不同剂量的加味四君子汤含药血清作用于B16细胞24h后，应用Trans AMTMNF-κB （p65）活性检测试剂盒检测其活性。结果：1.成功建立了体外MM模型。2.成功制备加味四君子汤含药血清。3.加味四君子汤含药血清作用后，抑制B16MM细胞增殖，作用12，24，48h，与同时间段空白对照组和血清对照组相比，以剂量依赖方式抑制B16恶性黑色素细胞增殖，中剂量加味四君子汤含药血清组24，48h抑制率为50.22%，53.81%，且48h与24h相比没有显著性变化。4.加味四君子汤含药血清作用后，不同剂量的加味四君子汤含药血清作用于B16细胞24h后，应用Annexin V-FITC/PI双标记法检测细胞凋亡，结果表明加味四君子汤含药血清以剂量依赖方式促进B16MM细胞凋亡，各组凋亡率分别为：空白对照组（2.97±0.62）%，血清对照组（3.12±0.71）%，低剂量加味四君子汤含药血清组（7.10±0.95）%，中剂量中药组（19.70±0.98）%，高剂量中药组（25.21±1.03）%，顺铂组（21.31±1.00）%，除空白对照组与血清对照比较外，各组间比较均差异有统计学意义（P<0.01）。5.加味四君子汤含药血清作用后，细胞划痕试验观察随着加味四君子汤含药血清剂量升高，B16细胞的迁移能力逐渐降低。但顺铂组迁移能力低于中剂量中药组，高于高剂量中药组。加味四君子汤含药血清以剂量依赖方式抑制B16细胞侵袭能力，各组穿过8μm含BD基质胶的Transwell小室的细胞个数分别是78.5±3.3个，80.3±3.5个，70.6±3.0个，44.6±1.8个，38.8±1.2个，41.5±1.5个,除空白对照组与血清对照比较外，各组间比较均差异有统计学意义（P<0.01）。6.不同剂量的加味四君子汤含药血清作用于B16细胞24h后，应用半定量RT-PCR法方法检测Bcl-2、Caspase3、Caspase9、MMP2、MMP9mRNA的表达。结果表明，加味四君子汤含药血清以剂量依赖方式抑制Bcl-2mRNA表达，增加Caspase3和Caspase9mRNA表达，加味四君子汤含药血清作用后，B16MM细胞侵袭相关基因MMP2和MMP9的mRNA表达降低，且以剂量依赖方式抑制MMP2和MMP9mRNA表达；除空白对照组与血清对照比较外，各组间比较均差异有统计学意义（P<0.01）。7.应用Western Blot方法检测，加味四君子汤含药血清以剂量依赖方式抑制Bcl-2蛋白表达，增加Caspase3和Caspase9蛋白表达，除空白对照组与血清对照比较外，各组间比较均差异有统计学意义（P<0.01），加味四君子汤含药血清作用后，B16MM细胞侵袭相关基因MMP2和MMP9的蛋白表达降低，除空白对照组与血清对照比较外，各组间比较均差异有统计学意义（P<0.01）。8.加味四君子汤含药血清以剂量依赖方式抑制NF-κB（p65）蛋白表达，除空白对照组与血清对照比较外，各组间比较均差异有统计学意义（P<0.01）；加味四君子汤含药血清作用后，NF-κB （p65）活性降低，除空白对照组与血清对照比较外，各组间比较均差异有统计学意义（P<0.01）。结论：1.加味四君子汤对治疗肺癌有效的药物对B16MM细胞有作用。2.加味四君子汤含药血清是通过下调凋亡抑制基因Bcl-2的表达，上调凋亡促进基因Caspase3和Caspase9表达来实现促进B16MM细胞凋亡，抑制B16MM细胞增殖。3.加味四君子汤含药血清是通过下调MMP2和MMP9表达来实现抑制B16MM细胞迁移和侵袭。4. NF-κB（p65）是介导加味四君子汤含药血清抑制B16恶性黑色素瘤的细胞迁移侵袭和促进凋亡的信号分子。更多还原

【Abstract】 Malignant melanoma （Malignant melanoma, MM） is malignant transformation frommelanocytes, which is characteristics of high invasion and metastasis. The average annualincidence growth rate is4%to6%, the incidence is double in nearly two decades. So far, the MMtreatment methods are including surgery, biological therapy, radiation therapy, chemotherapy andtumor vaccine, however, as long as the MM is metastasis, any treatment almost can not cure thisdisease. The Dacarbazine drug was only approved by the U.S. Food and Drug Administration（FDA）, and the complete remission rate has not been more than5%. This phenomenon may becaused by multi-drug resistant （multidrug resistance, MDR）, which main mechanism is apoptosispathway handicap. Therefore, how to solve the chemotherapy drug MDR, and to increasemelanoma tumor cell apoptosis is urgent problem to be solved for the modern medicine.In medicine, traditional Chinese medicine in the anti-tumor effect plays an important role,especially in the anti-drug resistance and attenuated efficiency aspects more significant advantage.With related research, industry scholars agree "lung skin and hair" theory can not be simplyinterpreted as the competent body surface lung skin, mucous membranes, sweat glands, hair andwhiskers and other physiological functions of fur, but when the fight Evils and "muscle table"similar to the respiratory tract and skin immune function coordinated interaction of the immunefeedback mechanism, a large number of clinical observations suggest that lung and fur inpathogenesis and immune mechanisms that may have similarities, eczema and dermatitis wasobserved in all patients before and after onset there lung disease;64%of patients suffering frombronchial asthma, skin diseases, clinical same drug either treating lung lesions, but also treat skindisorders. Some scholars have summarized the most commonly used traditional Chinesemedicine treatment of lung cancer,26flavor, this study has seven herbs constitute a group ofparties, which Atractylodes, Poria, Licorice, Fritillaria and Curcuma five herbs are among this26herbs and treatment of lung cancer to use more prescription righting based qi to Sijunzitang basedsubtraction maximum frequency of use, some academics have already proved Sijunzitangsubtraction curative treatment for lung cancer. Earth into gold, also known as Spleen Yifei, is thetreatment of lung and spleen deficiency of both a treatment method. Press the five elementsregularity lungs and spleen as picture-relations, according to his mother, then fill the rule is, useearth into gold of the law, can benefit lung spleen and stomach, to be tempered enrichment, health movement reinstatement, earth wang gold autogenous, lung deficiency of waiting sincelast. Insufficiency belong lunar, the main line of the chest and abdomen in the personal, the twoare closely linked through, communicated through the air, blood intersection. Modern medicalresearch shows that human tumor-specific immunotherapy is an important material basis for thepresence of tumor antigens, lung cancer and melanoma antigen expression in common melanoma（melanoma antigen, MAGE） MAGE-1and MAGE-3, suggesting that these two diseases mayhave similar anti-tumor immune response mechanism. In summary, the theory of Chinesemedicine and modern medicine from two levels of support, as a basis for selection of Sijunzitangprescriptions Zhuojia effective drug treatment of lung cancer may be an effective for thetreatment of skin cancer. Modern pharmacology proof, Fritillaria glycosides A inhibition ofhuman highly metastatic giant cell lung cancer cell adhesion, invasion and migration, the sameTubeimoside on mouse B16melanoma and Lewis lung metastasis were inhibited. Volatile oil onboth direct damaging effects of a variety of cancer cells, but also by enhancing the immunesystem play a role in cancer, curcumol Caspase-independent pathway through mediated apoptosisof A549cells, was also found to inhibit Curcuma water Tibo highly metastatic melanoma cellline B16of the transfer, may be related to inhibition of B16cell adhesion to extracellular matrixand reduce the invasiveness of B16and exercise capacity. Banzhilian an increase in immunefunction and anti-tumor effect on human lung giant cell carcinoma cell line PG cells telomeraseactivity decreased significantly, the extract anti-lung cancer A549cell growth. Thus, the presentstudy Sijunzitang based on the discretionary selection Fritillaria, Curcuma and flavoredcomposition banzhilian Sijunzitang treatment MM.Chinese medicine believes that the traditional Chinese medicine play an key in antitumor,especially in anti-drug resistance, attenuation and increase efficiency. According to “LungRelating to Skin Theory”and “strengthening earth to generate metal”, Sijunzi Decoction as abasis prescriptions plus lung cancer and\or MM effective drug was selected to treat MM.Bcl-2and Caspase were recognized as apoptosis-related genes, Bcl-2family is an apoptosisinhibiting genes, the studies have shown that inhibition of Bcl-2gene expression can promoteapoptosis of MM cells. Caspases are apoptosis-related gene for promoting apoptosis, especiallyCaspase3and Caspase9is important to induce apoptosis.Matrix metalloproteinase （metalloprotease, MMP） is a certain family of proteins which canhydrolyze some kinds of protein component. MMPs were named as their fuction need to acofactor, as well as Ca²⁺, Zn²⁺and other metalions.This family members have similar structures,and MMP2and MMP9were in depth research. MMPs almost degradate all protein components in the ECM, and destruct the histological barrier for tumor cell migration and invasion, and playsa critical role in tumor invasion and metastasis. Thus, it is an increase important role in tumorinvasion and metastasis. MMPS is considered to be the major proteolytic enzymes in theabove-mentioned process.Methods:1. Malignant melanoma model was built in vitro.2. Plus SiJunZi Decoction was administrated by serum pharmacological method.3. Cell proliferation inhibiting test （MTT） were used to analyse the Plus SiJunZi Decoctioncontaining serum influence on B16malignant melanoma cell proliferation.4. Flow cytometry Annexin V-FITC/PI assay were used to analyse the Plus SiJunZi Decoctioncontaining serum influence on B16malignant melanoma cell proliferation.5. Cell scratch test and transwell assay were used to analyse the Plus SiJunZi Decoctioncontaining serum influence on B16malignant melanoma cell migration and invasion.6. Migration and invasion-related gene MMP2and MMP9mRNA expression were observed andanalysed by Semiquantitative RT-PCR. Inhibiting apoptotic gene Bcl-2and promoting apoptoticgenes Caspase3and Caspase9mRNA expression were also observed and analysed bySemiquantitative RT-PCR.7. Migration and invasion-related gene MMP2and MMP9protein expression were observed andanalysed by Western Blot. Inhibiting apoptotic gene Bcl-2and promoting apoptotic genesCaspase3and Caspase9protein expression were also observed and analysed by Western Blot.The expression of NF-κB（p65） in B16malignant melanoma cell were detected and analysed byWestern Blot.8. Trans AMTM NF-κB（p65） Kit was used to measure the activity of NF-kB.Results：1. Malignant melanoma model was built successfully in vitro.2. The Plus SiJunZi Decoction containing serum was prepared successfully.3. A time-dependent increase of inhibiting B16malignant melanoma cell proliferation by PlusSiJunZiDecoction containing serum was observed compared to blank control group, serumcontrol group in the same time period, and middle dose traditional Chinese medicine groupinhibition rate was50.22in12h and53.81in24h respectively（P<0.01），and reached the halfof inhibition rate, and half of inhibition rate in48hours period and24hours period werecompared to no significant change.4. Plus SiJunZi Decoction containing serum promotes significantly B16malignant melanoma cell apoptosis. Flow cytometry Annexin V-FITC/PI assay were used to analyse the plus Sijunzidecoction containing serum influence on B16malignant melanoma cell apoptosis. Atime-dependent increase of inhibiting B16malignant melanoma cell apoptosis by PlusSiJunZiDecoction containing serum was observed. apoptotic rates were in each group: controlgroup （2.97±0.62）%, serum control group （3.12±0.71）%, low-dosePlus SiJunZi Decoctioncontaining serum group （7.10±0.95）%, middle dose group （19.70±0.98）%, the high dosegroup （25.21±1.03）%, cisplatin group （21.31±1.00）%. In addition to the control groupcompared with the serum control, the comparison between the groups were statisticallysignificant （P <0.01）. The rate of Cisplatin group apoptosis is high than middle dose traditionalChinese medicine group, and lower than high dose traditional Chinese medicine group.5. A time-dependent decrease of B16malignant melanoma cell migration and invasion wasobserved used cell scratch test and transwell assay by Plus SiJunZi Decoction containing serum.Each group through8μm BD Matrigel Transwell chamber cell numbers were78.5±3.3,80.3±3.5,70.6±3.0,44.6±1.8,38.8±1.2,41.5±1.5. In addition to the control group compared with the serumcontrol, the comparison between the groups were statistically significant （P <0.01）.6. RT-PCR method was used to detect Bcl-2, Caspase3, Caspase9, MMP2, MMP9mRNAexpression. The results show that, A time-dependent decrease in MMP2and MMP9mRNAexpression by Plus SiJunZi Decoction containing serum were observed, and decreases inhibitingapoptotic gene Bcl-2mRNA expression, and increases promoting apoptotic genes Caspase3andCaspase9mRNA expression.In addition to the control group compared with the serum control,the comparison between the groups were statistically significant （P <0.01）.7. Western Blot method was used to detect Bcl-2, Caspase3, Caspase9, MMP2, MMP9proteinexpression.A dose-dependent manner inhibiting Bcl-2protein expression, increasing Caspase3and Caspase9protein expression by Plus SiJunZi Decoction containing serum were observed, inaddition to the control group compared with the serum control, the comparison between thegroups were statistically significant（P<0.01）. A dose-dependent manner inhibiting invasionrelated genes MMP2and MMP9protein expression were observed and analysed. In addition tothe control group compared with the serum control, the comparison between the groups werestatistically significant （P <0.01）.8. A time-dependent decrease in NF-κB（p65） protein expression in B16malignant melanoma cellwere detected. In addition to the control group compared with the serum control, the comparisonbetween the groups were statistically significant （P <0.01）.The activity of NF-κB（p65） was adecrease in B16malignant melanoma cell. In addition to the control group compared with theserum control, the comparison between the groups were statistically significant（P <0.01）. Conclusions：1. Plus Sijunzi Decoction as an efficient therapeutic pulmonary cancer prescriptions was also aneffective decoction on B16malignant melanoma.2. Plus SiJunZi Decoction containing serum promotes significantly B16malignant melanomacell apoptosis, which can be mediated by decreasing Bcl-2expression, and increasing Caspase3and Caspase9expression. Plus SiJunZi Decoction containing serum inhibits B16malignantmelanoma cells proliferation.3. Plus SiJunZi Decoction containing serum inhibits significantly B16malignant melanoma cellmigration and invasion, which can be mediated by decreasing MMP2and MMP9expression.4. NF-κB（p65） signal molecule can play a key role in above-mentioned process.更多还原