乳岩宁与三苯氧胺联合抗乳腺癌作用及其机制的研究

【作者】 关玉娟；

【导师】 殷东风；

【作者基本信息】 辽宁中医药大学 ， 中医内科学， 2011， 博士

【摘要】 目的:乳腺癌是女性最好发的恶性肿瘤之一,我国近年来的发病率有非常明显的上升趋势。目前乳腺癌病人因为化疗、放疗、内分泌治疗、免疫治疗及手术治疗的不断发展,其死亡率开始有所下降,但仍有相当一部分病人会复发和转移。而多数晚期乳腺癌患者对二次化疗、放疗及手术已经不能忍受。因内分泌治疗乳腺癌,有着使用方便,副作用小,缓解时间长等优势,已得到绝大多数医生及病人的认可。辽宁中医药大学附属医院肿瘤科在治疗中晚期乳腺癌患者服用三苯氧胺(TAMoxifen,TAM)同时服用乳岩宁方取得了很好的疗效。乳岩宁方是在《伤寒论》的柴胡加龙骨牡蛎汤基础上加味组成,其主要功效是调畅气机、疏肝健脾、扶正抗癌、软坚散结。肿瘤的发生发展与气机出入升降失常有密切关系,可通过调理肝、肾、脾三方面来治疗恶性肿瘤所引起的各种临床症状,进而达到提高生存质量,延长生命的目的。中医认为,肝脉挟乳,气血郁滞,肝失疏泄,结于乳中,日久则乳络不畅而发“乳岩”,故古往今来治疗乳腺疾病多从肝论治。柴胡加龙骨牡蛎汤是《伤寒论》中和解少阳,理气调肝的主要用方。吾导师殷东风教授多年来总结认为,乳腺癌患者体内癌毒四处走窜,虽变证百出,但气机不畅乃贯穿始终。故乳岩宁方以柴胡加龙骨牡蛎汤为主方,以达到枢利气机、调和肝气的目的。方中柴胡为君药,与臣药黄芩相配,而使气机通达,枢机调和;生姜与半夏相配,为小半夏汤,具有辛开散结,和胃止呕,豁痰降逆,宣畅气机之功;而人参、大枣、甘草,一者取其扶正以祛邪,二者取其“见肝之病,则知肝当传之与脾,故先实其脾气”之意。加入牡蛎、龙骨以镇定安神,辅以浙贝母、山慈菇、莪术以化瘀解毒,软坚散结。纵观全方,有升有降,共奏调畅气机、疏肝健脾之功效。乳岩宁对TAM抑瘤作用及其抗肿瘤机制尚需进一步研究。因此本文选用人雌激素依赖性乳腺癌移植瘤模型,通过观察乳岩宁与TAM联合对MCF-7荷瘤裸鼠乳腺癌的抑瘤作用、对MCF-7荷瘤裸鼠乳腺癌的细胞凋亡,免疫组化方法检测裸鼠移植瘤组织MMP-2、MMP-9蛋白表达,Western blotting法检测移植瘤p38MAPK、c-fos蛋白表达水平的影响,探讨乳岩宁对乳腺癌侵袭转移的抑制作用及其可能的机制,可能与p38MAPK信号转导通路相关。材料与方法:第一部分:MCF-7荷瘤裸鼠移植模型的建立将培养好的MCF-7细胞经消化胰酶消化后,用PBS液清洗2次,再用1%台盼蓝染色,于细胞记数板上进行细胞记数,并调节活细胞浓度为1×10~7/ml,在无菌条件下施行裸鼠右侧胸壁第二乳垫,脂肪层下接种0.2ml/只,共接种3只,术后荷瘤裸鼠继续饲养于SPF环境中,待肿瘤长至0.8cm3时,行裸鼠间原位移植。在无菌条件下取出乳腺癌组织,剪切成1mm~3左右的小块,分别移植于24只裸鼠右侧胸壁第二乳垫脂肪层下,4d后可见肿瘤生长,成瘤率100%。将24只MCF-7荷瘤裸鼠随机分为4组,每组6只。①模型组(荷瘤裸鼠)②TAM组③乳岩宁组④TAM+乳岩宁组(简称联合组)。模型组:0.9%氯化钠注射液,0.2ml/只;TAM组:为3.6mg/kg;0.2ml/只;乳岩宁组:乳岩宁1.149g/kg,0.2ml/只;联合组:TAM 3.6mg/kg+乳岩宁1.149g/kg,0.2ml/只。每日灌胃1次,连续给药28d。检测指标:①肿瘤生长曲线图:从第1次注射开始每两到三天,观察一次裸鼠的全身情况和肿瘤生长状况,无菌条件下测量肿瘤的直径,连续观察记录,并以时间为横坐标,以肿瘤的体积为纵坐标,肿瘤体积计算公式为:V=1/2×ab~2,a为肿瘤长径,b为肿瘤短径,并据此绘制移植瘤生长曲线。②抑瘤率:注射药物4周后,脱颈椎处死裸鼠,将处死小鼠用动物天平称体重,再分离瘤体用电子天平称瘤重,计算抑瘤率,抑瘤率按照公式(模型组瘤重一实验组瘤重)/模型组瘤重×100%计算。③透射电镜观察细胞凋亡:以超薄切片术快速冷冻肿瘤组织,采用戊二醛和四氧化锇的双固定法将其固定,脱水、浸透、包埋、切片、透射电镜(TEM)进行超微结构水平上的观测。④肿瘤细胞凋亡率:采用TUNNEL法检测,细胞核内出现棕色颗粒者为阳性细胞,高倍视野下计数100个细胞中的阳性细胞数,每张切片随机计数5个视野,计算细胞凋亡指数(Apoptosis Inedx ,AI),( AI =阳性细胞数/计数的细胞个数×100%)。统计学方法采用SPSS13.0软件,单因素方差分析,统计数据以均数±标准差表示,以P<0.05为显著性差异的标准。第二部分:免疫组化法(S-P法)测定裸鼠移植瘤组织MMP-2、MMP-9的表达。用已知的阳性切片做阳性对照,用PBS溶液代替一抗做阴性对照。MMP-2、MMP-9的结果判定标准:MMP-2、MMP-9的阳性细胞为细胞膜或细胞浆中出现浅黄色至棕褐色的颗粒。其判断方法为:在镜下,根据棕色反应的阳性强度及面积判断其结果。对每张切片阳性细胞的阳性强度按无着色、淡黄色、棕黄色和棕褐色分别打0,1,2,3分,着色阳性面积按无着色、着色0～30%,30%～60%,60%～100%分别打0,1,2,3分,然后根据两项打分之和判断其结果:0分为阴性(-),≥3分为阳性(+),其中5分者为强阳性(++) (注:每张切片选择有代表性的区域,在100倍视野下进行计数,共计5个视野,取其平均值以避免随意性)。统计分析:采用SPSS13.0软件进行统计,计数资料采用均数±标准差表示,以P<0.05为显著性差异的标准。第三部分用免疫印迹(Western blotting)方法检测裸鼠移植瘤肿瘤组织p38MAPK、c-fos蛋白表达。统计分析:用扫描仪将底片上的条带的条带扫描后输入计算机,用Band Scan V5.0条带分析软件进行统计分析,各组条带的表达强度以各样本与其内参的灰度比值(相对灰度值)的平均数表示。结果:1.乳岩宁抑制裸鼠移植瘤的生长,使肿瘤体积缩小,各组瘤重分别为模型组2.25g±0.51g,TAM组1.51g±0.32g,乳岩宁组1.71g±0.39g,联合组1.33g±0.18g,各用药组与模型组比较有显著性统计学差异(P<0.05)。抑瘤率分别为TAM组32.89%,乳岩宁组24.00%,联合组40.89%,各用药组与模型组比较有显著性统计学差异(P<0.05);联合组与TAM组及乳岩宁组相比,裸鼠的肿瘤体积均有显著性差异(P <0.05);但TAM组及乳岩宁组之间无统计学差异(P>0.05)。2.细胞凋亡率模型组为8.66%±3.11%,TAM组为29.14%±8.18%,乳岩宁组为22.11%±7.02%,联合组为36.60%±6.71%,各用药组与模型组比较,细胞凋亡率提高,存在显著性统计学差异(P<0.05);联合组与TAM组及乳岩宁组相比,均有显著性差异(P <0.05);但TAM组及乳岩宁组之间无统计学差异(P>0.05)。3. MMP-2、MMP-9在模型组中的表达水平最高,分别为42.35%±5.28%、52.33%±4.76%在TAM组分别为28.39%±4.21%、27.56%±3.12%,乳岩宁组分别为31.52%±3.18%,32.14%±3.98%而在联合组的表达分别为24.36%±3.23%、22.29%±2.67%。各用药组与模型组相比较,MMP-2、MMP-9的表达水平均下降,有显著性统计学差异(P<0.05),而TAM组及乳岩宁组对于MMP-2、MMP-9的表达无统计学差异。本实验证实了TAM及乳岩宁均可以下调MMP-2、MMP-9的表达,两组药物的作用无显著性差异(P>0.05),两药联合应用能起到协同效应。4.组磷酸化p38MAPK蛋白在各组的表达分别为模型组1.13±0.23,TAM组0.70±0.13,乳岩宁组0.76±0.11,联合组0.58±0.18,各用药组与模型组比较,p38MAPK蛋白表达下降,存在显著性统计学差异(P<0.05);联合组与TAM组及乳岩宁组相比,也存在显著性差异(P <0.05) , TAM组及乳岩宁组之间无统计学差异(P>0.05)。5.c-fos在各组的表达分别为模型组0.52±0.21,TAM组0.32±0.11,乳岩宁组0.35±0.16,联合组0.19±0.09,各用药组与模型组比较,p38MAPK蛋白表达下降,存在显著性统计学差异(P<0.05);联合组与TAM组及乳岩宁组相比,也存在显著性差异(P <0.05),TAM组及乳岩宁组之间无统计学差异(P>0.05)。结论:1.乳岩宁能在体内增强TAM的抗裸鼠移植瘤作用,使肿瘤体积缩小。2.乳岩宁具有诱导乳腺癌细胞凋亡的作用,与TAM合用则诱导凋亡作用更明显,可以增强TAM的细胞凋亡诱导作用,发挥抗肿瘤作用。3.TAM及乳岩宁均能下调MMP-2、MMP-9的表达,两药联合能起到协同效应。4.乳岩宁可能通过抑制p38MAPK信号通路的活性而降低c-fos蛋白的含量,从而抑制MMP-2、MMP-9蛋白的表达,产生对乳腺癌的侵袭转移的抑制作用。更多还原

【Abstract】 Object:Breast cancer is one of the malignant tumors which occur most frequently in women. The incidence rate of breast cancer in our country presented obvious increasing trend recently. At present, it starts to decrease because of the development of chemotherapy, radiotherapy, immunotherapy and surgery for treating breast cancer. But quite a few of patients would be relapse or metastasis. However, most of terminal breast cancer patients could not suffer the second chemotherapy, radiotherapy or surgery. Endocrine therapy has been confirmed by most of doctors and patients because of its advantages, such as convenience to use, gentle side effect and long alleviation time, etc. Terminal breast cancer patients were treated with taking TAM (TAMoxifen) and Ru-yan-ning decoction in oncology department of Affiliated Hospital to Liaoning University of Traditional Chinese Medicine, and the therapy obtained good efficacy. Ru-yan-ning decoction was prescribed on the basis ofChai-hui-jia-long-gu-mu-li decoction,and its main effects are adjusting the motion of Qi, flowing the liver and nourishing the spleen, strengthening vital Qi and anticancer, resolving hard lump. The occurrence and development of cancer has close relationship with the motion of Qi, including ascending, descending, entering and leaving of Qi. We could treated various symptoms induced by malignant tumor by recuperating liver, kidney and spleen, in order to enhance the quality of life and prolong the length of life. In the area of traditional Chinese medicine, it is believed that liver pulse passing breast, and stagnant of Qi and Blood induced constraining Liver Qi to result in stagnant Qi and Blood coagulating in breast, in course of time, it induce obstructing of breast collateral to develop breast cancer. Therefore, treating breast diseases of all ages mostly focused on liver. Chai-hui-jia-long-gu-mu-li decoction in Discussion of Cold-Induced Disorders is the main decoction to harmonize Shao-yang, regulate the flow of vital Qi and adjust Liver. It could smooth the motion of Qi, freely open the ascending, descending, entering and leaving of Qi, including all things from soup to nuts. My advisor, professor Yin Dongfeng, summarized his experience to assume that cancer poison flee everywhere in the body of breast cancer patients, the mechanism is always the stagnant of the motion of Qi although syndromes occurred are variational. Therefore, Ru-Yan-Ning decoction is prescribed on the basis of Xiao-Chai-Hu decoction to smooth the motion of Qi and harmonize Liver Qi. In this description, bupleurum (Chai-Hu), as the monarch drug, matches with ministerial drug Scutellaria (Huang-Qin) to smooth the motion of Qi and harmonize the helm; Ginger (Sheng-Jiang) matches with pinellia (Ban-Xia) compose Xiao-Ban-Xia decoction which could dissipate the stagnant with acrid flavor, regulate stomach and arrest vomiting, reduce spitting and descend the rebellious Qi, and smooth the motion of Qi; Ginseng (Ren-Shen), Chinese Date (Da-Zao) and glycyrrhiza (Gan-Cao) match not only present the meaning of strengthening the vital Qi to eliminate evil Qi, but also mean that seeing liver disease is the knowledge of the liver disease could transmit to spleen, so nourishing spleen Qi is necessary. Oyster (Mu-Li) and keel (Long-Gu) are added to relieve uneasiness of mind and body. Thunberg Fritillary Bulb (Zhe-Bei-Mu), Sagittaria sagittifolia (Shan-Ci-Gu) and Zedoary (E-Zhu) assist to eliminate blood stasis, detoxify the body and resolve the lump. Making a general survey of this decoction, it has the function of ascending and descending to smooth the motion of Qi, sooth liver and strengthen spleen.The effect of Ru-Yan-Ning decoction on TAM for antitumor and its mechanism need further study. Therefore, our investigation chose human estrogen dependence breast cancer transplanting model to observe Ru-Yan-Ning decoction united TAM to effect on MCF-7 tumor bearing nude mice for antitumor and the apoptosis of it effecting on MCF-7 tumor bearing nude mice, determine the expression of MMP-2 and MMP-9 in transplanting tissues of nude mice by immunohistochemistry, detect the expression of p38MAPK and c-fos in transplanting tissues by Western blotting. Then we could discuss the inhibitory effect of Ru-Yan-Ning decoction on breast cancer metastasis and its mechanism may be related to the signal transduction passage of p38MAPK.Materials and methods:The first part: The establishment of MCF-7 tumor bearing nude mice model: after trypsinization, cultured MCF-7 cells were rinsed twice with PBS, and dyed by 1% trypan blue,and counted on the cell counting chamber. Then adjust the cell concentration to 1×10~7/ml,and filled the second breast pad into the right chest wall and filled one 0.2ml pad under the adipose layer, altogether filled 3 pads. After surgery, the tumor bearing nude mice continued to be fed in SPF environment. When tumor raised to 0.8 cm~3,the mice were treated with orthotopic transplantation. The breast cancer tissues were taken out in aseptic condition, then cut into chips about 1 mm~3 size, and transplanted under the adipose layer of second pad of right chest wall of 24 nude mice. 4 days later, tumor growth was observed, tumorigenesis rate reached to 100%. 24 MCF-7 tumor bearing nude mice were randomly divided into 4 groups (6 mice in every group):①Model group (tumor bearing nude mice),②TAM group,③Ru-Yan-Ning group,④TAM+ Ru-Yan-Ning group (i.e. union group). Model group:the mice in this group were treated with 0.9% sodium chloride inoculation fluid (0.2ml per nude mouse); TAM group: 3.6mg/kg (0.2ml per nude mouse); Ru-Yan-Ning group:the mice in the group were treated with Ru-Yan-Ning 1.149g/kg (0.2ml per nude mouse); Union group:TAM 3.6mg/kg+Ru-Yan-Ning 1.149g/kg(0.2ml per nude mouse). The mice were perfused once every day for 28d continuously. Determination indices including:①Tumor growth curve: From the 1st injection, the general situation and the growth condition were observed every two or three days. The diameters of tumor were detected under the aseptic condition and record continuously. Take the time as the abscissa,and the tumor volume as the y-coordinate. The tumor volume formula is: V=1/2×ab~2,“a”is the tumor major axis,and“b”is the tumor minor axis, and draws up the transplant tumor growth curve according to the above.②Tumor inhibition rate:4 weeks after injection medicine,the nude mice were killed by breaking the cervical vertebra off,then to measure the body weight of the killed mice with the animal balance, and measure the weight of separated tumor with the electronic balance, and then the calculate the tumor inhibition rate. The tumor inhibition rate formula: (the weight of tumor in model group- the weight of the tumor in experimental groups)/ the weight of tumor in model group×100%.③Cell apoptosis was observed by the transmission electron microscope:tumor tissues were quick frozen by ultramicrotomy, fixed by double fixation methods with the glutaric dialdehyde and the perosmic anhydride osmium,then underwent dehydration, percolation, embedding and slice,and then observed on the ultrastructure level with TEM (transmission electron microscope).④Rate of tumor cell apoptosis: It was determined by TUNNEL method. The brown granules appeared in the cell nucleus presents were positive cells. Count the number of positive cells in 100 cells under the high power field and chose 5 fields randomly in each slice to calculate AI (Apoptosis Inedx) (AI = positive cells number/cell number counted×100%). The results were analyzed by SPSS13.0 software, and take the single factor variance analysis as analysis method. The statistical data were indicated by the mean value±standard deviation, take P<0.05 as the significance difference standard. The second part:The expression of MMP-2 and MMP-9 in transplanting tumor tissues of nude mice was determined by immunohistochemistry (S-P method). Take forgone positive slice as positive control and take PBS replacing the first antibody as negative control. MMP-2 and MMP-9 results determination standard: MMP-2 and MMP-9 positive cells present the buff to sepia granules in the cell membrane or the cytoplasma. The judgment method is according to the positive intensity and area of the brown granules under the microscope. Graded every slice according to the positive intensity of positive cells, graded non-coloration, faint yellow,brownish yellow and sepia as 0, 1, 2 and 3 point respectively. Graded positive area non-coloration, coloration 0~30%,30%~60%,60%~100% as 0,1,2 and 3 point respectively. Then the results were judged according to sums of the two grades: 0 point as negative (-),≥3 point as positive (+), 5 point as strong positive (++) (note: the representative regions chosen in each slice were counted under 100 time fields of vision,total 5 fields of vision, take its mean value to avoid randomness). Statistical analysis: Used SPSS13.0 software to carry on the statistics, the counting materials were indicated by the mean value±standard deviation and take P<0.05 as the significance difference standard. The third part: The expression of p38MAPK and c-fos proteins in transplanting tumor tissues in nude mice was determined by Western blotting. Statistical analysis: After scanning band on film, input it into the computer,and analyzed it with Band Scan the V5.0. The expression intensity of bands in each group was indicated by the mean of relative gray value (gray value of band in each sample/ the internal reference gray value.Results:1. The Ru-Yan-Ning inhibited the transplant tumor in nude mice to reduce the volume of tumor. The weights of tumors in each group were 2.25g±0.51g in model group, 1.51g±0.32g in TAM group, 1.71g±0.39g in the Ru-Yan-Ning group,and 1.33g±0.18g in the union group. There existed significant statistic differences between experimental groups and model group (P<0.05). Tumor inhibition rate were 32.89% in the TAM group,24.00% in the Ru-Yan-Ning group,40.89% in the union group. There existed significant statistic differences between experimental groups and model group (P<0.05);There existed significant statistic differences between the union group and TAM group ,Ru-Yan-Ning group on the volume of tumor in nude mice (P<0.05); But there had no significant statistic difference between TAM group and Ru-Yan-Ning group(P>0.05).2. Rates of cell apoptosis were 8.66%±3.11% in model group, 29.14%±8.18% in TAM the group, 22.11%±7.02% in the Ru-Yan-Ning group, and 36.60%±6.71% in the union group. Compared to model group, rates of cell apoptosis in experimental groups increased significantly (P<0.05); There existed significant statistic differences between TAM group and Ru-Yan-Ning group (P<0.05);But there had no significant statistic difference between TAM group and Ru-Yan-Ning group(P>0.05).3. MMP-2 and MMP-9 expression were highest in model group, which were 42.35 %±3.28%, 52.33%±4.76% respectively, and 28.39%±4.21%, 27.56 %±3.12% in TAM group, 31.52±3.18%、32.14%±3.98 % in Ru-Yan-Ning group, but 24.36%±3.23%, 22.29%±2.67% in union group. Compared to model group, MMP-2 and MMP-9 expression in each experimental group decreased significantly (P<0.05),but there had no significant statistic difference between TAM group and Ru-Yan-Ning group. Our investigation confirmed that TAM and Ru-Yan-Ning could down regulate the expression of MMP-2 and MMP-9,there had no significant statistic difference between the two groups on the effectiveness (P>0.05). Joint application of two medicines could play the cooperative effect.4.Phosphorylation p38MAPK protein expression in each group were 1.13±0.23 in model group, 0.70±0.13 in TAM group, 0.76±0.11 in Ru-Yan-Ning group,and 0.58±0.18 in the union group, respectively. Compared to model group,p38MAPK protein expression decreased significantly in each experimental group (P<0.05);Compared to TAM and Ru-Yan-Ning group, it existed significant differentiation in the union group (P <0.05),but there had no significant statistic difference between TAM group and Ru-Yan-Ning group(P>0.05).Conclusion:1. Ru-Yan-Ning could strengthen the inhibition effect of TAM on transplanting tumor in nude mice in vivo to reduce the tumor volume.2. Ru-Yan-Ning has the function to induce cell apoptosis of breast cancer. Jointly application with TAM could enhance inducing apoptosis effect, could strengthen TAM to induce cell apoptosis to play the role of anti-tumor.3. TAM and Ru-Yan-Ning could down regulate the expression of MMP-2 and MMP-9. Joint application of two medicines could play the cooperative effect.4. Ru-Yan-Ning could inhibit the activity of p38MAPK signal passage to reduce the content of c-fos protein, to inhibit the expression of MMP-2 and MMP-9 protein,thus to inhibit invasion and metastasis on breast cancer.更多还原