中药复方联合他莫昔芬对MCF-7乳腺癌的抑瘤作用及其机理的实验研究
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摘要
研究目的：观察中药复方乳岩宁及益气消积方联合他莫昔芬（TAM）对MCF-7荷瘤裸鼠一般状态及体内雌激素的影响，了解中药复方乳岩宁及益气消积方联合TAM是否有协同增效的作用；观察中药复方联合TAM对MCF-7乳腺癌细胞凋亡的影响及其诱导细胞凋亡的作用机制；观察中药复方联合TAM对MCF-7乳腺癌细胞周期分布、细胞周期相关调控蛋白CyclinD1及凋亡相关基因Bcl-2、Bax蛋白表达的影响，了解中药复方联合TAM抑制MCF-7乳腺癌细胞增殖的作用机理；通过观察中药复方联合TAM对MCF-7乳腺癌细胞增殖的MAPK信号转导通路的ERK1/2、ERα蛋白及对HER-2基因mRNA表达的影响，了解中药复方联合TAM抑制MCF-7乳腺癌细胞增殖的信号传导途径。
研究方法：
1.MCF-7荷瘤裸鼠移植模型的建立:
将进入对数生长期的MCF-7细胞用胰酶/EDTA消化后，PBS液清洗2次，加入细胞培养液，直接吹打均匀，0.4%台盼蓝染色，记录活细胞数（＞95%），细胞计数板上作细胞计数，调节活细胞浓度为1×107/ml，裸鼠右侧胸壁碘伏消毒，于无菌条件下施行BALB/c-nu/nu裸鼠右侧胸壁第二乳垫下接种细胞液0.2ml/只，裸鼠继续饲养于层流罩中。接种后10-15天，在接种部位乳垫处出现肿瘤结节，质地较硬，而且逐渐增大，越长越快，认为原代肿瘤造模成功。当原代肿瘤长至0.8cm3大小时，无菌手术取下肿瘤，放入DMEM培养液中，剪切成1mm3左右的小块，利用骨髓穿刺针分别移植至余40只裸小鼠乳垫下，不需要缝合伤口，移植后第4天后即可见肿瘤生长，成瘤率100%。经HE染色病理诊断为浸润性导管癌。
2.实验分组：
将40只造模成功裸鼠随机分为模型组、他莫昔芬(TAM)组、乳结合组（乳岩宁+TAM）和益结合组（益气消积方+TAM）。模型组给蒸馏水0.2ml.只-1，TAM组为3.6mg.kg-1体重(相当于临床人用量的9倍)，中药乳岩宁为33.3g.kg-1体重(相当于临床人用量的12倍)，中药益气消积方为35.3g.kg-1体重(相当于临床人用量的12倍)，中药+TAM组为中药乳岩宁或者益气消积方+TAM3.6mg·kg-1体重，每日灌胃1次，连续给药21d。
3.检测指标：
(1)隔日测量裸鼠体重，绘画体重变化曲线，观察饮食、活动、色泽、精神状态及死亡情况；分别于用药前、实验第10天、21天用小鼠自主活动测试仪测小鼠5分钟内自主活动次数；处死裸鼠前摘眼球取血约0.5ml-1ml，将血置于试管中，室温静置1-2小时后，2500转/分，离心10min，分离上清，采用酶联免疫吸附法（ELISA）检测小鼠血清中雌二醇（E2）和孕酮（PROG简称P）水平；处死裸鼠摘取子宫称重并计算子宫脏器系数。子宫脏器指数=子宫重量(mg)/体重(g)。
(2)用药21天后脱颈处死裸鼠，完整剥离肿瘤称重，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；肿瘤组织经4%多聚甲醛固定，梯度酒精脱水，二甲苯透明，浸蜡与包埋，用石蜡切片机切片，组织切片厚度为5μm，苏木素、伊红染色后光镜观察病理形态学变化；采用TUNNEL法检测肿瘤细胞凋亡率，肿瘤组织，常规石蜡包埋、切片，其余方法步骤按试剂盒说明书进行。细胞核内出现绿色荧光者为阳性细胞，荧光显微镜下观察，每张染色后的切片随机选择5个400×典型的高倍视野，每个视野分别观察100个细胞，共500个细胞，计算出每100个细胞内的阳性细胞平均数作为凋亡指数（Apoptosis Index，AI）。AI=阳性细胞数/计数的细胞个数×100%；进一步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。
(3)用药21天后完整剥离出瘤体，称重后置于冰盒上，并将标本切开取材，置于EP管中，-80℃冰箱中冻存用于蛋白测定。乳腺癌组织块剪碎后用0.25%胰酶消化30min－1h，过200－400目筛网过滤细胞，获得单细胞悬液，75％冷乙醇（－20℃预冷）固定细胞12h以上，加入PI（终浓度50ug／ml）和无DNA酶污染的RNA酶（终浓度50ug／ml）1ml染色30min－1h后上流式细胞仪检测细胞周期；采用免疫组织化学法对凋亡调控基因Bcl-2、Bax的表达进行定性分析；应用Western blot蛋白免疫印迹法检测凋亡调控基因Bcl-2、Bax及细胞周期相关调控蛋白CyclinD1的表达，并对结果进行统计分析。
(4)RT-PCR的取材是将组织置于1mlTrizol中，标本作好标记后，立即置于-80℃冰箱中冻存备用。Western blot法检测乳腺癌组织中ERK1/2、ERɑ蛋白的表达，RT-PCR法检测乳腺癌组织中HER-2基因mRNA的表达。
研究结果：
(1)体重：TAM组裸鼠体重下降最明显（3.18±0.061），乳结合组次之（2.5±0.042），益结合组（1.3±0.019），组间比较有统计学差异（p﹤0.01），各用药组裸鼠的体重均低于模型组（p﹤0.05）；自主活动次数：实验第10、21天结合组裸鼠活动次数，两个结合组明显优于TAM组（p<0.01）；子宫脏器指数：乳结合组最高，TAM次之，益结合组次之，均明显高于模型组(p <0.01)，而三个用药组之间对比无明显差异（p>0.05）；雌激素水平结果：TAM可以降低血清中E2和P的水平,与模型组相比差异显著(p<0.01)，乳结合组的E2水平明显低于TAM组(p<0.01)，益结合组的E2水平与TAM组对比无明显差异（p>0.05），而两个结合组对P水平的影响显著，明显低于模型组(p<0.01)。
(2)瘤重：TAM组、乳结合组、益结合组的的肿瘤重量均低于模型组，与模型组比较有非常显著差异（p﹤0.01），乳结合组明显低于TAM组（p﹤0.01），但是TAM组与益结合组、乳结合组与益结合组对比无明显差异（p＞0.05），TAM组、乳结合组、益结合组的抑瘤率分别为32.9%、44.9%、37.1%；TUNEL结果： TAM组、乳结合组、益结合组与模型组相比，其细胞凋亡指数(AI)比较有统计学差异(p <0.01)；其中乳结合用药组具有最高的AI，益结合组次之，二者比较无统计学差异（p＞0.05），但是与单独用药TAM组相比差异均有统计学意义(p<0.01)；HE染色提示病理为浸润性导管癌，模型组可见境界清楚的癌巢，细胞密集排列，核大深染，病理性核分裂象多见，异型性明显，而各用药组肿瘤细胞略增大，瘤组织结构破坏，部分细胞固缩成为孤立的细胞，可见大量坏死、凋亡细胞。坏死的肿瘤细胞周围有大量的淋巴细胞和白细胞浸润，显示出肿瘤细胞的生长受到抑制；透射电镜下观察发现模型组乳腺癌细胞体积大，核大，核浆比例失调，胞质内细胞器丰富，各用药组肿瘤细胞以凋亡变化为主，并可见晚期典型凋亡表现--大量凋亡小体，其中TAM组电镜下可见少量坏死细胞。
(3)流式细胞仪检测细胞周期结果显示：各用药组G0/G1期细胞的比例增多，S期比例明显减少，与模型组比较差异显著（P<0.01），但是乳结合组与益结合组分别对比G0/G1期、S期的细胞比例均无统计学差异（P﹥0.05）；免疫组化法对凋亡调控基因Bcl-2、Bax的检测：Bcl-2及Bax蛋白表达阳性主要定位于细胞浆，有的在胞膜，Bcl-2染色显示，模型组细胞浆浓染呈黄色、棕黄色甚至深棕褐色，而各用药组细胞浆着色明显减弱，呈淡黄色，有的细胞甚至呈阴性。Bax染色显示，模型组细胞浆染色呈淡黄色，而各用药组细胞浆着色则明显增强，尤以核周、胞膜明显，黄染加重。Western blot半定量提示：模型组中Bcl-2蛋白存在高表达，Bax低表达，Bcl-2/Bax的比值显著升高，与三个治疗组相比有显著性差异（p﹤0.01）；TAM组、乳结合组和益结合组，都可不同程度的降低Bcl-2蛋白的表达，并使Bax蛋白表达增加，Bcl-2/Bax的比值降低，其中乳结合组的作用更为显著，益结合组次之，TAM组再次之，两个结合组与TAM组相比较有显著性差异（p﹤0.01），而两个结合组相比较无统计学差异（p﹥0.05）。说明模型组中存在乳腺癌细胞生长失去控制，凋亡过程受阻现象。各用药组均可下调Bcl-2蛋白的表达，上调Bax蛋白的表达，使Bcl-2/Bax的比值降低，从而诱导MCF-7乳腺癌细胞凋亡，而且以二者联合应用诱导凋亡的作用最为显著。Western blot法检测Bcl-2、Bax及细胞周期相关调控蛋白CyclinD1的表达结果：Western blot法检测凋亡调控基因Bcl-2、Bax的结果与免疫组化检测结果基本一致，TAM组、乳结合组和益结合组，都可不同程度的降低Bcl-2蛋白的表达，并使Bax蛋白表达增加，降低Bcl-2/Bax的比值，其中乳结合组的作用最为显著，益结合组次之，两个结合组与TAM组相比较有显著性差异（p﹤0.01），而两个结合组相比较无统计学差异（p﹥0.05）。模型组中CyclinD1蛋白存在高表达，各用药组中CyclinD1蛋白表达水平明显下降，明显低于模型组（p﹤0.01）；其中以乳结合组的作用最为显著，益结合组次之，两个结合组与TAM组相比较有显著性差异（p﹤0.01），说明各用药组乳腺癌细胞细胞周期阻滞于G0/G1期与下调CyclinD1蛋白表达有关。
(4)Western blot检测ERK1/2、ERɑ蛋白表达的结果： TAM组、乳结合组和益结合组，都可不同程度的降低ERK1/2、ERɑ蛋白的表达，明显低于模型组（p﹤0.01）；其中乳结合组的作用最为显著，益结合组次之，两个结合组与TAM组相比较有显著性差异（p﹤0.01），而两个结合组相比较无统计学差异（p﹥0.05）。RT-PCR法检测乳腺癌组织中HER-2基因mRNA表达的结果：TAM组、乳结合组和益结合组，都可不同程度的降低HER-2基因mRNA的表达，明显低于模型组（p﹤0.01）；其中乳结合组的作用最为显著，益结合组次之，TAM组再次之，两个结合组与TAM组相比较有显著性差异（p﹤0.01），而两个结合组相比较无统计学差异（p﹥0.05）。
结论:
1.中药复方能改善荷瘤裸鼠一般状态，减轻TAM带来的裸鼠体重下降，改善裸鼠活动能力。
2.中药复方与TAM合用可明显降低体内雌激素E2、P水平。
3.中药复方联合TAM具有抑制乳腺癌生长的作用，可能与阻滞细胞周期进程及诱导细胞凋亡有关。
4.中药复方联合TAM可能是通过下调通过癌组织中HER-2基因mRNA的表达来抑制MAPK信号通路的活性，下调ERK1/2、ERɑ蛋白的表达来抑制乳腺癌细胞增殖的。
5.中药复方与TAM合用有协同增效的作用，但是中药乳岩宁方与益气消积方对比疗效无明显差异。
Objective:
To observe the effects of Ruyan Ning and Yiqi Xiaoji combined with tamoxifen(TAM) to the general state and the level of estrogen in tumor-bearing nude mice,to understand whether compound traditional chinese medicine combined withtamoxifen (TAM) had positive effect or not. To observe the influence of CompoundChinese medicine combined with tamoxifen (TAM) to induce apoptosis of MCF-7breast cancer cells in tumor-bearing nude mice and discuss the possible mechanismInhibition and pathological. To observe Compound Chinese medicine combined withTAM to affect cell cycle distribution of MCF-7breast cancer cell and the relatedregulatory protein CyclinD1,the influence to the expression of apoptosis relatedgenes Bcl-2and Bax protein, understanding the active mechanism of CompoundChinese medicine combined with TAM to inhibite proliferation of MCF-7breastcancer cell. According to observe the the influence of Compound Chinesemedicine combined with TAM to the expression of ERK1/2, ERɑ protein and HER-2gene mRNA,discussed the signal pathway of Compound Chinese medicine combinedwith TAM to inhabite the proliferation of MCF-7breast cancer cell.
Methods:
1.The establishment of MCF-7tumor-bearing nude mice model:
MCF-7cells in logarithmic growth phase were digested by trypsin/EDTA, thenwashed2times with PBS, cell culture medium was added, blow and beat it directlyto the uniform, stained by0.4%trypan blue, take the record of the number ofliving cells (>95%), counting cells on the cell counting board, adjust thedensity of living cell1×107/ml, BALB/C Nude mice were injected MCF-7on thesecond breast pad of the right chest wall subcutaneously after disinfected theright chest wall of nude mice with iodophor, vaccinated cytosol0.2ml each nudemice, then went on raising in a laminar flow hood. Tumor nodules could been seen at the inoculation site of breast pads10-15days after vaccination, thecharacter of tumor nodules was hard and gradually increasing. The longer thetime,the faster the tumor nodules, that the primary model was recognized to besuccessful. When the primary tumor grew to0.8cm3, to removed the tumor by sterilesurgery, placed in DMEM medium, cut into pieces of about1mm3, then transferredto breast pads of the remaining40nude mice taking use of bone marrow needle,4days later you could see growth of transplanted tumor, successful rate of tumorformation was100%.The diagnosis of pathology was the infiltrating ductalcarcinoma identified by HE staining.
2.Divided groups of experiment: Forty mice were randomly divided intocontrol group, tamoxifen (TAM) group, Ru combined group (the Ruyanning Recipe+TAM) and Yi combined group (YiQi Xiaoji Recipe+TAM), after establishing themodel of MCF-7cancer nude mice successfully. The Control group was givendistilled water0.2ml each one,TAM group was given TAM3.6mg.kg-1body weight(This doseage was equivalent to nine times as the clinical human’s), traditionalChinese medicine Ruyan Ning Recipe was given according to33.3g.kg-1body weight(equivalent to12times as the amount of clinical human’s dosage), Yiqi XiaojiRecipe was given35.3g.kg-1body weight (equivalent to12times as the clinicalhuman’s dosage), Traditional Chinese medicine+TAM conbining group was giventraditional Chinese medicine of Ruyan Ning Recipe or Yiqi Xiaoji Recipe andTAM3.6mg kg-1body weight, gavaging once a day for consecutive21days.
3. Detection index:
(1)The conditions of drink and food, weight of nude mice, mental state anddeath were observed and recorded every other day,drew the curve of changingweight of the mice. Take the record of autonomic activities of mice within fiveminutes before experiment, on the first,twenty-first day separately using ZZ6autonomic locomotor tester. Blood was taken approximately0.5-1ml by the removalof eyeball before mice were sacrificed,the blood was placed in a tube, then putit aside for1-2hours at room temperature, centrifuged them2500r/min for10minutes, separated supernatant serum to get ready for ELISA test. The levels of E2and progesterone (PROG referred to as P) were detected by enzyme-linkedimmunosorbent assay (ELISA) in the serum of mouse.when the mice were sacrificed,removed the uterus and weighed, calculated the index of organ. Uterine organindex=uterine weight(mg)/body weight(g).
(2)Allthe mice were sacrificed through pull off neck after given drug for21days, tumor were taken completely and weighed in each group respectively.Then the inhibition rate of tumor were calculated,The formula of tumorinhibition rate was accordance with the tumor weight of control group-tumorweight in experimental group/tumor weight of the control group×100%; Tumortissue was fixed by4%formaldehyde, graded dehydration by ethanol, xylene madeit transparent, dipped and embeded by paraffin,slicing machine for slicing atthickness of5μm, observed the pathological changes by light microscopy afterHE staining;The apoptosis rate of tumor cells was detected by TUNNEL method,tissue was embedded with paraffin, sectioned in routine, and the rest of thesteps are carried out according to instructions about Apoptosis Assay Kit,Greenfluorescence that was observed within the nucleus were positive cells byfluorescence microscopy, each stained slice was selected randomly five400×high power typical fields, each field were observed in100cells,500cells inall, calculated the average of positive cells per100,the result was regardedas the apoptotic index (AI). AI=the number of positive cells/the number of cellsthat counted×100%; ultramicrotomy and fast-frozen were taken to deal with tumortissue, using the double glutaral dehyde and osmium tetroxide to fix, dehydrated,soaked, embedded, sliced, then observed by transmission electron microscopy(TEM) on the ultrastructural level to determine whether apoptosis.
(3) All the mice were sacrificed after given drug for21days, tumor weretaken completely and weighed in each group respectively, placed on the ice box,and cut the specimen into small pieces,placed it in EP tube in-80℃refriger-ator for protein detection. Flow cytometry cell cycle: breast cancer tissue wascut into pieces and digested with0.25%trypsin for30min-1h, filtered by200-400mesh to obtain single suspension cell, fixed by75%cold ethanol for more than 12h, added PI (final concentration50ug/ml) and RNA enzyme of no DNA enzyme(finalconcentration50ug/ml)1ml,staining30min-1h before Flow cytometry detection;the expression of apoptotic regulated gene Bcl-2and Bax were detected in theway of immunohistochemistry (IHC)in qualitative analysis; then detected byWestern blot in quantitative cell analysis,detecting CyclinD1proteinsexpression using Western blot method, and the results for statistical analysis.
(4)The specimen of RT-PCR was to placed into1mlTrizol, tagged,then placedit in-80℃refrigerator immediately. To detected the expression of ERK1/2,ERɑ protein of MCF-7breast cancer cell in the way of Western blot, RT-PCR methodwas taken to detect the expression of HER-2mRNA.
Results:
(1)Weight of mice: The decreased weight of nude mice in TAM group was themost obvious(3.18±0.061),Ru combined group followed by(2.5±0.042), Yicombined group（1.3±0.019）,the difference was significant among groups(p<0.01),The weight of mice in the three treatment groups was lower than the controlgroup（p﹤0.05）;The number of autonomic activity: Two combined groups were betterthan TAM group significantly on the tenth, twenty-first day of experiment(p<0.01);The index of the uterine: The index of the uterine in Ru combined groupwas highest,TAM group was second, the Yi combined group followed, they weresignificantly higher than the control group(p<0.01);The results of estrogen’s levels: TAM group can reduce the level of E2and P,comparing with the controlgroup, there were significant differences(p<0.01),the level E2in Ru combinedgroup was significantly lower than TAM group(p<0.01),but there was meaninglessbetween Yi combined group's and TAM group about the level of E2(p>0.05), whilethe progesterone’s level in two combined groups were significantly lower thancontrol group(p<0.01).
(2)The tumor weight: The weight of tumor in TAM group, combined group andYi combined group were lower than the control group, comparing with the controlgroup,there was a significant difference(p<0.01),Ru combined group was significantly lower than the TAM group (p<0.01), but the TAM group compared withYi combined group, Ru combined group compared with Yi combined group there wasno significant difference (p>0.05).The rates of tumor inhibition in TAM groupwas32.9%, Ru combined group was44.9%,Yi combined group was37.1%; The resultsof TUNEL: Apoptosis index (AI) in control group was higher than other TAM groups(p<0.01), AI in Ru combined group was the highest, the Yi combination groupwas second, there was no significant difference statistically between the twogroups. But comparing to TAM group,the differences were significant (p<0.01);Pathological diagnosis was invasive ductal carcinoma by HE staining, in thecontrol group, realm of cancer nests could be seen clearly, tumor cells grewstrongly, packed densely, lots of mitosis and heteromorphism could be seenvisibly in cells,while in the treated groups, the volume of tumor cells increasedslightly, the structure of tumor tissue was damaged, some cells shrinked toisolated cells, a large number of necrotic and apoptotic cells was seen. Tumorcells of necrosis were surrounded by a lot of lymphocytes and leukocyte(WBC),All show that the growth of tumor cells was inhibited in the treated groups;Theresults of pathomorphology: The pathomorphological change on the ultrastructural level observed by transmission electron microscopy (TEM) found thatMCF-7cells in control group have strong ability for growth and metabolism,big cell volume and nucleus, the ratio of nucleus and cytoplasm imbalance, richin organelles, while in each treatment group could find apoptotic changes, andsee a typical apoptotic manifestations in late stage-a large number of apoptoticbodies, in the TAM group under the electron microscope can be seen a small amountof necrotic cells.
(3)Results of cell cycle detected by flow cytometry: In each treatmentgroup,the proportion of cells in G0/G1phase increased, S phase was sharplyreduced compared with the control group (P<0.01), inhibited the transformationfrom G0/G1phase to S phase in a definite range, but there were not statisticallydifferent between Ru combined group and Yi combined groups about cell ratio inthe G0/G1phase and S phase (P>0.05); Bcl-2,Bax detection by immunohistochemical method: The positive expression of Bcl-2and Bax was mainly localized in thecytoplasm, and some in the membrane.Bcl-2staining in the control group，thecytoplasm stained yellow, brown or dark brown, while in each treated groupcytoplasm coloration was significantly weakened, light yellow and some cellseven negative. Bax staining showed cytoplasm stained light yellow in the controlgroup, while in each treated group cytoplasm coloration was significantlyenhanced, especially in the part of peri nuclear and membrane significantly,yellow dying heavier. The semi-quantitative of Bcl-2and Bax by Western blot:The expression of Bcl-2protein was high, Bax with low expression, the ratioof Bcl-2/Bax was significantly increased in the control group compared withthe three treatment groups (p<0.01); The treated group could down-regulate theexpression of Bcl-2protein and up-regulate the expression of Bax protein,Bcl-2/Bax ratio was decreased to induce apoptosis of MCF-7breast cancer cellstogether. The expression results of Bcl-2and Bax detected by Western blot werebasically the same as results detected by immunohistochemistry.The expressionof cell cycle regulatory CyclinD1detected by Western blot: CyclinD1proteinin the control group were highly expressed, CyclinD1protein while in the treatedgroup expression levels were significantly decreased comparing with the controlgroup (p<0.01); in Ru combined group the expression of CyclinD1protein wasleast,the Yi combined group second, the results indicating that in eachtreatment group cell cycle blocked at G0/G1phase was related to down-regulateexpression of CyclinD1protein.
(4)The results of expression of ERK1/2and ERɑ protein: The expression ofERK1/2, ERɑ protein in treated groups was significantly lower than in the controlgroup (p<0.01); the two combined groups could reduce the expression of ERK1/2and ERɑ compared with TAM group (p<0.01), but no significant difference the twocombination groups (p>0.05). HER-2gene mRNA expression detected by RT-PCR: Theexpression of HER-2gene mRNA in control group was higher than other groupsobviously (p<0.01); Ru combined group reduced the expression of HER-2gene mRNAmost of three groups. HER-2gene mRNA expression in TAM group was less than the two combined groups (p<0.01), while no significant difference between the twocombination groups (p>0.05).
Conclusions:
1. Compound Chinese medicine can improve the general state of tumor-bearingnude mice,reducing loss of the weight brought by TAM, improve the ability ofnude mice to move.
2. Compound Chinese medicine combined with TAM can significantly reduce thelevels of E2, progesterone.
3. Compound Chinese medicine has a function inhibite growth of tumor,thepossible mechanism was closely related to inhibite cell cycle and induceapoptosis.combined groups is the most obvious, illustrate that traditionalChinese medicine can collaborate TAM to tumor inhibitation.
4. Compound Chinese medicine combined with TAM can inhibite breast cancer cellproliferation,it may be related to inhibite the activity of the MAPK signalingpathway to down-regulate ERK1/2, ERɑ protein, and it maybe by inhibiting theexpression of HER-2gene mRNA in breast cancer tissue to inhibite the activityof the MAPK signaling pathway.
5.Compound Chinese medicine can enhance efficacy of TAM,but compares theefficacy,there is no significant difference between the Ruyan ning Recipe andYi Qi Xiaoji Recipe.
研究方法：
1.MCF-7荷瘤裸鼠移植模型的建立:
将进入对数生长期的MCF-7细胞用胰酶/EDTA消化后，PBS液清洗2次，加入细胞培养液，直接吹打均匀，0.4%台盼蓝染色，记录活细胞数（＞95%），细胞计数板上作细胞计数，调节活细胞浓度为1×107/ml，裸鼠右侧胸壁碘伏消毒，于无菌条件下施行BALB/c-nu/nu裸鼠右侧胸壁第二乳垫下接种细胞液0.2ml/只，裸鼠继续饲养于层流罩中。接种后10-15天，在接种部位乳垫处出现肿瘤结节，质地较硬，而且逐渐增大，越长越快，认为原代肿瘤造模成功。当原代肿瘤长至0.8cm3大小时，无菌手术取下肿瘤，放入DMEM培养液中，剪切成1mm3左右的小块，利用骨髓穿刺针分别移植至余40只裸小鼠乳垫下，不需要缝合伤口，移植后第4天后即可见肿瘤生长，成瘤率100%。经HE染色病理诊断为浸润性导管癌。
2.实验分组：
将40只造模成功裸鼠随机分为模型组、他莫昔芬(TAM)组、乳结合组（乳岩宁+TAM）和益结合组（益气消积方+TAM）。模型组给蒸馏水0.2ml.只-1，TAM组为3.6mg.kg-1体重(相当于临床人用量的9倍)，中药乳岩宁为33.3g.kg-1体重(相当于临床人用量的12倍)，中药益气消积方为35.3g.kg-1体重(相当于临床人用量的12倍)，中药+TAM组为中药乳岩宁或者益气消积方+TAM3.6mg·kg-1体重，每日灌胃1次，连续给药21d。
3.检测指标：
(1)隔日测量裸鼠体重，绘画体重变化曲线，观察饮食、活动、色泽、精神状态及死亡情况；分别于用药前、实验第10天、21天用小鼠自主活动测试仪测小鼠5分钟内自主活动次数；处死裸鼠前摘眼球取血约0.5ml-1ml，将血置于试管中，室温静置1-2小时后，2500转/分，离心10min，分离上清，采用酶联免疫吸附法（ELISA）检测小鼠血清中雌二醇（E2）和孕酮（PROG简称P）水平；处死裸鼠摘取子宫称重并计算子宫脏器系数。子宫脏器指数=子宫重量(mg)/体重(g)。
(2)用药21天后脱颈处死裸鼠，完整剥离肿瘤称重，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；肿瘤组织经4%多聚甲醛固定，梯度酒精脱水，二甲苯透明，浸蜡与包埋，用石蜡切片机切片，组织切片厚度为5μm，苏木素、伊红染色后光镜观察病理形态学变化；采用TUNNEL法检测肿瘤细胞凋亡率，肿瘤组织，常规石蜡包埋、切片，其余方法步骤按试剂盒说明书进行。细胞核内出现绿色荧光者为阳性细胞，荧光显微镜下观察，每张染色后的切片随机选择5个400×典型的高倍视野，每个视野分别观察100个细胞，共500个细胞，计算出每100个细胞内的阳性细胞平均数作为凋亡指数（Apoptosis Index，AI）。AI=阳性细胞数/计数的细胞个数×100%；进一步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。
(3)用药21天后完整剥离出瘤体，称重后置于冰盒上，并将标本切开取材，置于EP管中，-80℃冰箱中冻存用于蛋白测定。乳腺癌组织块剪碎后用0.25%胰酶消化30min－1h，过200－400目筛网过滤细胞，获得单细胞悬液，75％冷乙醇（－20℃预冷）固定细胞12h以上，加入PI（终浓度50ug／ml）和无DNA酶污染的RNA酶（终浓度50ug／ml）1ml染色30min－1h后上流式细胞仪检测细胞周期；采用免疫组织化学法对凋亡调控基因Bcl-2、Bax的表达进行定性分析；应用Western blot蛋白免疫印迹法检测凋亡调控基因Bcl-2、Bax及细胞周期相关调控蛋白CyclinD1的表达，并对结果进行统计分析。
(4)RT-PCR的取材是将组织置于1mlTrizol中，标本作好标记后，立即置于-80℃冰箱中冻存备用。Western blot法检测乳腺癌组织中ERK1/2、ERɑ蛋白的表达，RT-PCR法检测乳腺癌组织中HER-2基因mRNA的表达。
研究结果：
(1)体重：TAM组裸鼠体重下降最明显（3.18±0.061），乳结合组次之（2.5±0.042），益结合组（1.3±0.019），组间比较有统计学差异（p﹤0.01），各用药组裸鼠的体重均低于模型组（p﹤0.05）；自主活动次数：实验第10、21天结合组裸鼠活动次数，两个结合组明显优于TAM组（p<0.01）；子宫脏器指数：乳结合组最高，TAM次之，益结合组次之，均明显高于模型组(p <0.01)，而三个用药组之间对比无明显差异（p>0.05）；雌激素水平结果：TAM可以降低血清中E2和P的水平,与模型组相比差异显著(p<0.01)，乳结合组的E2水平明显低于TAM组(p<0.01)，益结合组的E2水平与TAM组对比无明显差异（p>0.05），而两个结合组对P水平的影响显著，明显低于模型组(p<0.01)。
(2)瘤重：TAM组、乳结合组、益结合组的的肿瘤重量均低于模型组，与模型组比较有非常显著差异（p﹤0.01），乳结合组明显低于TAM组（p﹤0.01），但是TAM组与益结合组、乳结合组与益结合组对比无明显差异（p＞0.05），TAM组、乳结合组、益结合组的抑瘤率分别为32.9%、44.9%、37.1%；TUNEL结果： TAM组、乳结合组、益结合组与模型组相比，其细胞凋亡指数(AI)比较有统计学差异(p <0.01)；其中乳结合用药组具有最高的AI，益结合组次之，二者比较无统计学差异（p＞0.05），但是与单独用药TAM组相比差异均有统计学意义(p<0.01)；HE染色提示病理为浸润性导管癌，模型组可见境界清楚的癌巢，细胞密集排列，核大深染，病理性核分裂象多见，异型性明显，而各用药组肿瘤细胞略增大，瘤组织结构破坏，部分细胞固缩成为孤立的细胞，可见大量坏死、凋亡细胞。坏死的肿瘤细胞周围有大量的淋巴细胞和白细胞浸润，显示出肿瘤细胞的生长受到抑制；透射电镜下观察发现模型组乳腺癌细胞体积大，核大，核浆比例失调，胞质内细胞器丰富，各用药组肿瘤细胞以凋亡变化为主，并可见晚期典型凋亡表现--大量凋亡小体，其中TAM组电镜下可见少量坏死细胞。
(3)流式细胞仪检测细胞周期结果显示：各用药组G0/G1期细胞的比例增多，S期比例明显减少，与模型组比较差异显著（P<0.01），但是乳结合组与益结合组分别对比G0/G1期、S期的细胞比例均无统计学差异（P﹥0.05）；免疫组化法对凋亡调控基因Bcl-2、Bax的检测：Bcl-2及Bax蛋白表达阳性主要定位于细胞浆，有的在胞膜，Bcl-2染色显示，模型组细胞浆浓染呈黄色、棕黄色甚至深棕褐色，而各用药组细胞浆着色明显减弱，呈淡黄色，有的细胞甚至呈阴性。Bax染色显示，模型组细胞浆染色呈淡黄色，而各用药组细胞浆着色则明显增强，尤以核周、胞膜明显，黄染加重。Western blot半定量提示：模型组中Bcl-2蛋白存在高表达，Bax低表达，Bcl-2/Bax的比值显著升高，与三个治疗组相比有显著性差异（p﹤0.01）；TAM组、乳结合组和益结合组，都可不同程度的降低Bcl-2蛋白的表达，并使Bax蛋白表达增加，Bcl-2/Bax的比值降低，其中乳结合组的作用更为显著，益结合组次之，TAM组再次之，两个结合组与TAM组相比较有显著性差异（p﹤0.01），而两个结合组相比较无统计学差异（p﹥0.05）。说明模型组中存在乳腺癌细胞生长失去控制，凋亡过程受阻现象。各用药组均可下调Bcl-2蛋白的表达，上调Bax蛋白的表达，使Bcl-2/Bax的比值降低，从而诱导MCF-7乳腺癌细胞凋亡，而且以二者联合应用诱导凋亡的作用最为显著。Western blot法检测Bcl-2、Bax及细胞周期相关调控蛋白CyclinD1的表达结果：Western blot法检测凋亡调控基因Bcl-2、Bax的结果与免疫组化检测结果基本一致，TAM组、乳结合组和益结合组，都可不同程度的降低Bcl-2蛋白的表达，并使Bax蛋白表达增加，降低Bcl-2/Bax的比值，其中乳结合组的作用最为显著，益结合组次之，两个结合组与TAM组相比较有显著性差异（p﹤0.01），而两个结合组相比较无统计学差异（p﹥0.05）。模型组中CyclinD1蛋白存在高表达，各用药组中CyclinD1蛋白表达水平明显下降，明显低于模型组（p﹤0.01）；其中以乳结合组的作用最为显著，益结合组次之，两个结合组与TAM组相比较有显著性差异（p﹤0.01），说明各用药组乳腺癌细胞细胞周期阻滞于G0/G1期与下调CyclinD1蛋白表达有关。
(4)Western blot检测ERK1/2、ERɑ蛋白表达的结果： TAM组、乳结合组和益结合组，都可不同程度的降低ERK1/2、ERɑ蛋白的表达，明显低于模型组（p﹤0.01）；其中乳结合组的作用最为显著，益结合组次之，两个结合组与TAM组相比较有显著性差异（p﹤0.01），而两个结合组相比较无统计学差异（p﹥0.05）。RT-PCR法检测乳腺癌组织中HER-2基因mRNA表达的结果：TAM组、乳结合组和益结合组，都可不同程度的降低HER-2基因mRNA的表达，明显低于模型组（p﹤0.01）；其中乳结合组的作用最为显著，益结合组次之，TAM组再次之，两个结合组与TAM组相比较有显著性差异（p﹤0.01），而两个结合组相比较无统计学差异（p﹥0.05）。
结论:
1.中药复方能改善荷瘤裸鼠一般状态，减轻TAM带来的裸鼠体重下降，改善裸鼠活动能力。
2.中药复方与TAM合用可明显降低体内雌激素E2、P水平。
3.中药复方联合TAM具有抑制乳腺癌生长的作用，可能与阻滞细胞周期进程及诱导细胞凋亡有关。
4.中药复方联合TAM可能是通过下调通过癌组织中HER-2基因mRNA的表达来抑制MAPK信号通路的活性，下调ERK1/2、ERɑ蛋白的表达来抑制乳腺癌细胞增殖的。
5.中药复方与TAM合用有协同增效的作用，但是中药乳岩宁方与益气消积方对比疗效无明显差异。
Objective:
To observe the effects of Ruyan Ning and Yiqi Xiaoji combined with tamoxifen(TAM) to the general state and the level of estrogen in tumor-bearing nude mice,to understand whether compound traditional chinese medicine combined withtamoxifen (TAM) had positive effect or not. To observe the influence of CompoundChinese medicine combined with tamoxifen (TAM) to induce apoptosis of MCF-7breast cancer cells in tumor-bearing nude mice and discuss the possible mechanismInhibition and pathological. To observe Compound Chinese medicine combined withTAM to affect cell cycle distribution of MCF-7breast cancer cell and the relatedregulatory protein CyclinD1,the influence to the expression of apoptosis relatedgenes Bcl-2and Bax protein, understanding the active mechanism of CompoundChinese medicine combined with TAM to inhibite proliferation of MCF-7breastcancer cell. According to observe the the influence of Compound Chinesemedicine combined with TAM to the expression of ERK1/2, ERɑ protein and HER-2gene mRNA,discussed the signal pathway of Compound Chinese medicine combinedwith TAM to inhabite the proliferation of MCF-7breast cancer cell.
Methods:
1.The establishment of MCF-7tumor-bearing nude mice model:
MCF-7cells in logarithmic growth phase were digested by trypsin/EDTA, thenwashed2times with PBS, cell culture medium was added, blow and beat it directlyto the uniform, stained by0.4%trypan blue, take the record of the number ofliving cells (>95%), counting cells on the cell counting board, adjust thedensity of living cell1×107/ml, BALB/C Nude mice were injected MCF-7on thesecond breast pad of the right chest wall subcutaneously after disinfected theright chest wall of nude mice with iodophor, vaccinated cytosol0.2ml each nudemice, then went on raising in a laminar flow hood. Tumor nodules could been seen at the inoculation site of breast pads10-15days after vaccination, thecharacter of tumor nodules was hard and gradually increasing. The longer thetime,the faster the tumor nodules, that the primary model was recognized to besuccessful. When the primary tumor grew to0.8cm3, to removed the tumor by sterilesurgery, placed in DMEM medium, cut into pieces of about1mm3, then transferredto breast pads of the remaining40nude mice taking use of bone marrow needle,4days later you could see growth of transplanted tumor, successful rate of tumorformation was100%.The diagnosis of pathology was the infiltrating ductalcarcinoma identified by HE staining.
2.Divided groups of experiment: Forty mice were randomly divided intocontrol group, tamoxifen (TAM) group, Ru combined group (the Ruyanning Recipe+TAM) and Yi combined group (YiQi Xiaoji Recipe+TAM), after establishing themodel of MCF-7cancer nude mice successfully. The Control group was givendistilled water0.2ml each one,TAM group was given TAM3.6mg.kg-1body weight(This doseage was equivalent to nine times as the clinical human’s), traditionalChinese medicine Ruyan Ning Recipe was given according to33.3g.kg-1body weight(equivalent to12times as the amount of clinical human’s dosage), Yiqi XiaojiRecipe was given35.3g.kg-1body weight (equivalent to12times as the clinicalhuman’s dosage), Traditional Chinese medicine+TAM conbining group was giventraditional Chinese medicine of Ruyan Ning Recipe or Yiqi Xiaoji Recipe andTAM3.6mg kg-1body weight, gavaging once a day for consecutive21days.
3. Detection index:
(1)The conditions of drink and food, weight of nude mice, mental state anddeath were observed and recorded every other day,drew the curve of changingweight of the mice. Take the record of autonomic activities of mice within fiveminutes before experiment, on the first,twenty-first day separately using ZZ6autonomic locomotor tester. Blood was taken approximately0.5-1ml by the removalof eyeball before mice were sacrificed,the blood was placed in a tube, then putit aside for1-2hours at room temperature, centrifuged them2500r/min for10minutes, separated supernatant serum to get ready for ELISA test. The levels of E2and progesterone (PROG referred to as P) were detected by enzyme-linkedimmunosorbent assay (ELISA) in the serum of mouse.when the mice were sacrificed,removed the uterus and weighed, calculated the index of organ. Uterine organindex=uterine weight(mg)/body weight(g).
(2)Allthe mice were sacrificed through pull off neck after given drug for21days, tumor were taken completely and weighed in each group respectively.Then the inhibition rate of tumor were calculated,The formula of tumorinhibition rate was accordance with the tumor weight of control group-tumorweight in experimental group/tumor weight of the control group×100%; Tumortissue was fixed by4%formaldehyde, graded dehydration by ethanol, xylene madeit transparent, dipped and embeded by paraffin,slicing machine for slicing atthickness of5μm, observed the pathological changes by light microscopy afterHE staining;The apoptosis rate of tumor cells was detected by TUNNEL method,tissue was embedded with paraffin, sectioned in routine, and the rest of thesteps are carried out according to instructions about Apoptosis Assay Kit,Greenfluorescence that was observed within the nucleus were positive cells byfluorescence microscopy, each stained slice was selected randomly five400×high power typical fields, each field were observed in100cells,500cells inall, calculated the average of positive cells per100,the result was regardedas the apoptotic index (AI). AI=the number of positive cells/the number of cellsthat counted×100%; ultramicrotomy and fast-frozen were taken to deal with tumortissue, using the double glutaral dehyde and osmium tetroxide to fix, dehydrated,soaked, embedded, sliced, then observed by transmission electron microscopy(TEM) on the ultrastructural level to determine whether apoptosis.
(3) All the mice were sacrificed after given drug for21days, tumor weretaken completely and weighed in each group respectively, placed on the ice box,and cut the specimen into small pieces,placed it in EP tube in-80℃refriger-ator for protein detection. Flow cytometry cell cycle: breast cancer tissue wascut into pieces and digested with0.25%trypsin for30min-1h, filtered by200-400mesh to obtain single suspension cell, fixed by75%cold ethanol for more than 12h, added PI (final concentration50ug/ml) and RNA enzyme of no DNA enzyme(finalconcentration50ug/ml)1ml,staining30min-1h before Flow cytometry detection;the expression of apoptotic regulated gene Bcl-2and Bax were detected in theway of immunohistochemistry (IHC)in qualitative analysis; then detected byWestern blot in quantitative cell analysis,detecting CyclinD1proteinsexpression using Western blot method, and the results for statistical analysis.
(4)The specimen of RT-PCR was to placed into1mlTrizol, tagged,then placedit in-80℃refrigerator immediately. To detected the expression of ERK1/2,ERɑ protein of MCF-7breast cancer cell in the way of Western blot, RT-PCR methodwas taken to detect the expression of HER-2mRNA.
Results:
(1)Weight of mice: The decreased weight of nude mice in TAM group was themost obvious(3.18±0.061),Ru combined group followed by(2.5±0.042), Yicombined group（1.3±0.019）,the difference was significant among groups(p<0.01),The weight of mice in the three treatment groups was lower than the controlgroup（p﹤0.05）;The number of autonomic activity: Two combined groups were betterthan TAM group significantly on the tenth, twenty-first day of experiment(p<0.01);The index of the uterine: The index of the uterine in Ru combined groupwas highest,TAM group was second, the Yi combined group followed, they weresignificantly higher than the control group(p<0.01);The results of estrogen’s levels: TAM group can reduce the level of E2and P,comparing with the controlgroup, there were significant differences(p<0.01),the level E2in Ru combinedgroup was significantly lower than TAM group(p<0.01),but there was meaninglessbetween Yi combined group's and TAM group about the level of E2(p>0.05), whilethe progesterone’s level in two combined groups were significantly lower thancontrol group(p<0.01).
(2)The tumor weight: The weight of tumor in TAM group, combined group andYi combined group were lower than the control group, comparing with the controlgroup,there was a significant difference(p<0.01),Ru combined group was significantly lower than the TAM group (p<0.01), but the TAM group compared withYi combined group, Ru combined group compared with Yi combined group there wasno significant difference (p>0.05).The rates of tumor inhibition in TAM groupwas32.9%, Ru combined group was44.9%,Yi combined group was37.1%; The resultsof TUNEL: Apoptosis index (AI) in control group was higher than other TAM groups(p<0.01), AI in Ru combined group was the highest, the Yi combination groupwas second, there was no significant difference statistically between the twogroups. But comparing to TAM group,the differences were significant (p<0.01);Pathological diagnosis was invasive ductal carcinoma by HE staining, in thecontrol group, realm of cancer nests could be seen clearly, tumor cells grewstrongly, packed densely, lots of mitosis and heteromorphism could be seenvisibly in cells,while in the treated groups, the volume of tumor cells increasedslightly, the structure of tumor tissue was damaged, some cells shrinked toisolated cells, a large number of necrotic and apoptotic cells was seen. Tumorcells of necrosis were surrounded by a lot of lymphocytes and leukocyte(WBC),All show that the growth of tumor cells was inhibited in the treated groups;Theresults of pathomorphology: The pathomorphological change on the ultrastructural level observed by transmission electron microscopy (TEM) found thatMCF-7cells in control group have strong ability for growth and metabolism,big cell volume and nucleus, the ratio of nucleus and cytoplasm imbalance, richin organelles, while in each treatment group could find apoptotic changes, andsee a typical apoptotic manifestations in late stage-a large number of apoptoticbodies, in the TAM group under the electron microscope can be seen a small amountof necrotic cells.
(3)Results of cell cycle detected by flow cytometry: In each treatmentgroup,the proportion of cells in G0/G1phase increased, S phase was sharplyreduced compared with the control group (P<0.01), inhibited the transformationfrom G0/G1phase to S phase in a definite range, but there were not statisticallydifferent between Ru combined group and Yi combined groups about cell ratio inthe G0/G1phase and S phase (P>0.05); Bcl-2,Bax detection by immunohistochemical method: The positive expression of Bcl-2and Bax was mainly localized in thecytoplasm, and some in the membrane.Bcl-2staining in the control group，thecytoplasm stained yellow, brown or dark brown, while in each treated groupcytoplasm coloration was significantly weakened, light yellow and some cellseven negative. Bax staining showed cytoplasm stained light yellow in the controlgroup, while in each treated group cytoplasm coloration was significantlyenhanced, especially in the part of peri nuclear and membrane significantly,yellow dying heavier. The semi-quantitative of Bcl-2and Bax by Western blot:The expression of Bcl-2protein was high, Bax with low expression, the ratioof Bcl-2/Bax was significantly increased in the control group compared withthe three treatment groups (p<0.01); The treated group could down-regulate theexpression of Bcl-2protein and up-regulate the expression of Bax protein,Bcl-2/Bax ratio was decreased to induce apoptosis of MCF-7breast cancer cellstogether. The expression results of Bcl-2and Bax detected by Western blot werebasically the same as results detected by immunohistochemistry.The expressionof cell cycle regulatory CyclinD1detected by Western blot: CyclinD1proteinin the control group were highly expressed, CyclinD1protein while in the treatedgroup expression levels were significantly decreased comparing with the controlgroup (p<0.01); in Ru combined group the expression of CyclinD1protein wasleast,the Yi combined group second, the results indicating that in eachtreatment group cell cycle blocked at G0/G1phase was related to down-regulateexpression of CyclinD1protein.
(4)The results of expression of ERK1/2and ERɑ protein: The expression ofERK1/2, ERɑ protein in treated groups was significantly lower than in the controlgroup (p<0.01); the two combined groups could reduce the expression of ERK1/2and ERɑ compared with TAM group (p<0.01), but no significant difference the twocombination groups (p>0.05). HER-2gene mRNA expression detected by RT-PCR: Theexpression of HER-2gene mRNA in control group was higher than other groupsobviously (p<0.01); Ru combined group reduced the expression of HER-2gene mRNAmost of three groups. HER-2gene mRNA expression in TAM group was less than the two combined groups (p<0.01), while no significant difference between the twocombination groups (p>0.05).
Conclusions:
1. Compound Chinese medicine can improve the general state of tumor-bearingnude mice,reducing loss of the weight brought by TAM, improve the ability ofnude mice to move.
2. Compound Chinese medicine combined with TAM can significantly reduce thelevels of E2, progesterone.
3. Compound Chinese medicine has a function inhibite growth of tumor,thepossible mechanism was closely related to inhibite cell cycle and induceapoptosis.combined groups is the most obvious, illustrate that traditionalChinese medicine can collaborate TAM to tumor inhibitation.
4. Compound Chinese medicine combined with TAM can inhibite breast cancer cellproliferation,it may be related to inhibite the activity of the MAPK signalingpathway to down-regulate ERK1/2, ERɑ protein, and it maybe by inhibiting theexpression of HER-2gene mRNA in breast cancer tissue to inhibite the activityof the MAPK signaling pathway.
5.Compound Chinese medicine can enhance efficacy of TAM,but compares theefficacy,there is no significant difference between the Ruyan ning Recipe andYi Qi Xiaoji Recipe.
引文
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