氯离子通道CLC-3和线粒体在三苯氧胺诱导MCF-7乳癌细胞凋亡中的作用及分子机制的研究/

2019-05-11 07:23:36

MCF 乳腺癌 TAM antisense CLC



中文摘要
论文题目:氯离子通道CLC-3和线粒体在三苯氧胺诱导MFC-7乳腺癌细胞凋亡中的作用及分子机制的研究
专 业:胸外科
博 士 生:赵 维
指导教师:刘国津教授
乳腺癌是影响女性健康最常见的恶性肿瘤之一,无论在发达国家还是发展中国家,这种疾病的发病率都在上升。在美国,自从1960年以来,乳腺癌的发病率每年以1~2%的比例稳定上升,在日本,35~45岁的女性中,从1960年到1985年乳腺癌的发病率增加了一倍。中国是发展中国家乳腺癌的发病率却与发达国家相似,呈不断上升趋势,有资料表明,近二十年来乳腺癌的发病率增长了37.6%平均每年上升2.3%,到2000年我国乳腺癌的发病率以达28.8/10万,居女性肿瘤的第一位。总的来说,乳腺癌的发病和流行随着年龄的增加而增加。众说周知乳腺癌的发病率在30到40岁之间增加迅速,并且持续增加,在40、50~60岁以后增加的速度逐渐下降。预计,在下一个十年中,全世界将会有至少5百万女性乳腺癌。
在过去的20年间人们对各种肿瘤疾病与离子通道的关系进行了一定的研究并且得到了一定的认识,许多不同的离子通道都与乳腺癌有关,其中研究最多的细胞之一是MCF-7细胞株。
 CLC-3是氯离子通道的一种,CLC-3 通道主要表达在神经外胚层组织中,在乳腺癌组织中鲜有报道。近几年的研究发现CLC-3 可能是容量调节性Cl - 通道的分子基础,它在细胞的容量调节、增殖、分化等功能中都有重要作用。目前也有报道证明氯离子通道亦存在与线粒体外膜上,在线粒体对细胞凋亡调节方面有重要的作用。
本试验的研究目的是通过对氯离子通道CLC-3及线粒体在三苯氧胺诱导MFC-7乳腺癌细胞凋亡中的作用及分子机制的研究,发现三苯氧胺通过氯离子通道和线粒体诱导凋亡的新机制,对反义CLC-3核苷酸链在治疗乳腺癌的临床运用上提出新的理论依据。
我们将实验分为如下步骤;
一、氯离子通道CLC-3在乳腺癌中的表达
在27例行乳腺癌改良根治术的乳腺癌患者中,分别将乳腺癌癌灶和周围正常组织用抗ClC-3抗体进行免疫组织化学染色,ClC-3蛋白主要表达在瘤细胞的胞浆和/或胞膜上。由于没有组间差异,应用x2检验进行组间比较,其中乳腺癌患者21例(77.77%)阳性,6例(22.33%)阴性;乳腺正常组织中3例(11.1%%)阳性,24例(88.9%)阴性。x2为24.3,P小于0.05,说明良恶性之间有明显差异。证明临床乳腺癌的标本中有氯离子通道CLC-3的高表达。为进一步研究提供临床应用基础。
二、CLC-3反义核苷酸的设计与鉴定。
通过对氯离子通道反义链的设计与鉴定,获得以后试验步骤的工具。
三、TAM及靶向CLC-3反义核苷酸对人乳腺癌细胞系MCF-7(ER +)增殖的影响
将MCF-7细胞分成如下6组,对照组(control group);实验组1:CLC-3 antisense;实验组2:TAM 10μM;实验组3:TAM 20μM;实验组4:CLC-3 antisense+TAM 10μM;实验组5:CLC-3 antisense+TAM 20μM。单纯给予TAM时 ,于给药后24小时收集细胞。MTT比色法我们发现随着TAM浓度从0逐渐增加到25μM, MCF-7细胞较对照组的增殖抑制率逐渐增加,呈剂量依赖性。0-10(P<0.05),10-20μM(P<0.01),但是在浓度增加到30μM时细胞的增殖抑制率有下降的趋势,这可能与三苯氧胺可以部分激活雌激素受体α作用有关。
研究中发现在单纯用反义链阻断CLC-3时MCF-7细胞的增殖没有明显受到抑制。CLC-3 antisense+TAM 10μM与对照组相比有差异(p<0.05);CLC-3 antisense+TAM 20μM较对照组相比有明显差异(p<0.01)。CLC-3 antisense+TAM 10μM与TAM 10μM对比两组之间有差异(p<0.05);CLC-3 antisense+TAM20μM与TAM 20μM对比两组之间有明显差异(p<0.01)。
为了进一步证明细胞的凋亡情况,我们用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL染色)及DNAladder方法对各组细胞进行检测,发现TAM组 10μM的凋亡率为38%,TAM组 20μM为66%,TAM组20μM加CLC-3antisense为85%。从DNAladder图中可以清晰的看到TAM 10uM、TAM 20uM 、TAM 20uM+CLC-3Antisense组细胞凋亡后由于DNA裂解而出现的DNA梯状带,而对照组则没有。且通过上诉各项数值可以看出随着TAM计量的加大,细胞凋亡的程度加大。CLC-3 antisense链可以加大TAM对凋亡的诱导。
四、对TAM及CLC-3antisense对人乳腺癌细胞系MCF-7(ER+)增殖抑制的作用机制进行探讨。
通过用ER 抗体对MCF-7细胞进行免疫组织化学染色,我们发现由于MCF-7细胞株加入TAM20uM,雌激素受体ER 的表达较正常MCF-7细胞明显减低。而给予CLC-3antisense后,MCF-7细胞雌激素受体ER 的表达未见明显变化。说明TAM可以抑制MCF-7细胞中雌激素受体ER 的表达,而CLC-3antisense对细胞浆中的雌激素受体的表达未有明显影响。
对各组处理后的MCF-7细胞用RT-PCR法进行CLC-3、BCL-2和BAXmRNA检测。
结果从电泳的图中可以看出TAM和/或CLC-3antisense可以使BCL-2、CLC-3表达降低,而使BAX表达增高,并且TAM和CLC-3有协同作用。
使用DCFH-DA(2',7'-二氯荧光黄双乙酸盐)测定活细胞内氧化水平的变化,结果通过照片可以看出TAM20uM和氯离子通道CLC-3antisense分别可以使细胞内的氧化活性物质增加,并且二者有协同作用。使用分离线粒体的方法对线粒体蛋白进行检测发现CLC-3antisense 和/或TAM20uM可以使线粒体内的谷胱苷肽减低并且二者有协同作用。用western blot对细胞浆内的细胞色素C进行测定发现TAM20uM和CLC-3antisense+TAM20uM细胞色素C增多,但二者之间差别不大。
根据以上结果我们可以得到如下结论:
1、发现氯离子通道CLC-3在乳腺癌组织中表达,较正常组织有明显差异P<0.05,说明氯离子通道CLC-3在乳腺癌组织的发生,发展过程中有一定的病理生理意义。
2、单纯用反义链阻断CLC-3时MCF-7细胞的增殖没有明显受到抑制,可能与我们运用CLC-3反义链的剂量不够有关,或者是由于氯离子通道不是生命所依赖的必须通道。TAM和CLC-3 antisense联合有增强对乳腺癌细胞株MCF-7凋亡的诱导作用,且随TAM的浓度增加两者之间的协同作用增大。为临床乳腺癌的内分泌治疗及生物治疗提供了参考。
3、线粒体是细胞凋亡过程的枢纽,TAM可以拮抗线粒体上的雌激素受体,如果是线粒体的雌激素受体直接参与了雌激素对线粒体DNA表达的诱导,那么通过这些线粒体上雌激素受体介导的信号途径可能为我们提供一个调控、预防及治疗肿瘤的新靶点。
4、TAM可能通过线粒体上雌激素受体 和CLC-3通道两条途径来诱导细胞的凋亡,但主要是通过雌激素受体 的途径。适当加强氯离子通道CLC-3的拮抗功能,不仅可以使其诱导细胞凋亡的能力提高,同时也可以加强其他常规化疗药物的作用。提示在肿瘤生长过程中,是由多途径、多机制共同参与的结果,作为激素依赖性的乳腺癌也不例外。


关键词;氯离子通道CLC-3 线粒体 三苯氧胺 凋亡 氯离子通道CLC-3反义核甘酸链



ABSTRACT
Subject: Molecular Mechanism and the Effects of Mitochondrion and Chloride Channel CLC-3 on Apoptosis Induced by Tamoxifen in the MFC-7 Breast Cancer Cell Line
Majors: Surgery
Auther: Zhao Wei
Director: Professor Liu Guo Jin
The breast cancer is a common malignant tumor which threaten to the health of women.Whether in developed country or in developing country,the morbility of breast cancer increases. In America ,ever since 1960,the morbility of breast cancer rised stably at the rate of 1-2% per year.In Japan,from1960 to 1985,the morbility of breast cancer doubled.But,China,as a developing country, her breast cancer`s morbility is similar to the developed country,and has a rising tendency.Somestudies have showed that in recent 20 years ,China`s breast cancer morbility had increased 37.6% at a rate of 2.3% per year.In our country the morbility had been 2.88/million at the year 2000 , and had taken the first place of the female malignant tumor.Generally,the morbility and the epidemic goes up with the increase of age.With the best knowledge, breast cancer morbility increases rapidly between 30 and 40 years old,and increases persistently,but between 40,50 and 60 years old,the speed of increase fall-off.In the next decade,there will be 5 millon breast cancer patients all over the world.
Now breast cancer has been identified as a kind of systemic disease which has the local symptoms.Up to date, the therapy of breast cancer is to take an operation together with the chemotherapy, radiotherapy,biotherapy and traditional Chinese medicine.With the development of molecular biology and immunology, endocrine therapy and biotherapy gradually become the important part of the combined therapy.
The relationship between the ion channel and the every kind of tumors has been studied in the last two decades.There are some different kinds of ion channel relate with the breast cancer, MCF-7 cells have been studied more carefully.
  CLC-3 is a kind of the chloride channels,CLC-3 express mostly in the neuroepidermal layer,and in the breast cancer the report is seldom.Recently the studies have found that CLC-3 may be the molecular foundation of volume regulatory chloride channel,it plays the important role in the cellular volume regulatory ,proliferation and the differentiation.Up to date there are some reports to identified that the chloride channels locate at the outer mitochondrial membrane and it plays a crucial role in the cellular apoptosis process regulated by the mitochondria.
The objective of our study is to determine the molecular mechanisms and the effects of mitochondria and chloride channel CLC-3 on apoptosis induced by tamoxifen in the MCF-7 cell line, to find the new theory to guide the clinical practice of CLC-3antisense in the treatment of breast cancer.
We divide the experiment into four parts
The part 1; Expression of the CLC-3 in the breast cancer
We carry out the immunohistochemical staining in the breast cancer tissue and the same perifocal benign tissue of the 27 patients who had been taken operation. We found that the ClC-3 protein mainly expressed in the cytoplasm and on the cellular membrane.The study showed that the 21(77.77%) breast cancer tissue in 27 patients were positive and the 6(22.33%) were negative;the 3 (11.1%%) perifocal benign tissue in 27were positive,24(88.9%)were negative. x2 was 24.3, P<0.05.The result implieed that there was a obvious difference between the expression of CLC-3 in the breast cancer and the benign perifocal tissue P<0.05.This implied that the CLC-3 had some pathological and physiological significance in the process of breast cancer`s genesis and growth.This became the clinical foundation to make a further study.
The part 2 Design and the identification of the CLC-3 antisense strand
To obtain the tools which were applied in the following steps studies ,we designed and identified the CLC-3 antisense strand with the method of RT-PCR.
The part 3; Effects of the CLC-3 antisense strand and TAM on the proliferation of MCF-7 cell lines
To add the different concentration TAM in each group MCF-7 cells then to incubate the cells for 24 huors ,to conllect the cells and then with MTT chromatometry,we found that TAM could enhance the apoptosis induction in the MCF-7 cells vs the control,and this effect can be improved with the concentration increasing(from 0 to 25 of the).shows the dose dependent 0-10μM(P<0.05),10-20μM(P<0.01).
Then we divided the MCF-7 cell into 6 gruops, control group;group 1:CLC-3 antisense;group 2:TAM 10μM;group 3:TAM 20μM;group 4:CLC-3 antisense+TAM 10μM; group 5:CLC-3 antisense+TAM 20μM.To transfect CLC-3 antisense strand into the cells for 4 huors and then to incubated the cells for 24 huors ,to conllect the cells and then with MTT chromatometry.We found that the proliferation inhibition of MCF-7cells had not been affected clearly only by CLC-3 antisense strand.There were a difference of proliferation inhibition in the MCF-7cells between CLC-3 antisense+TAM 10μand the control(p<0.05);CLC-3 antisense+TAM 20μM vs the control had a big difference(p<0.01)。CLC-3 antisense+TAM 10μM vs TAM 10μM,had a difference(p<0.05);CLC-3 antisense+TAM20μM vs TAM 20μM, had a big difference(p<0.01)。
Inorder to study the apoptosis ,we detected the each group cells with the TUNEL and the DNA ladder methods .The TUNEL result showed that the apoptosis rate of TAM 10μM was 38%,TAM 20μM was 66%,TAM 20μM+CLC-3antisense was 85%.From DNA ladder electropherogram, we could see the ladders in the TAM 10uM、TAM 20uM and TAM 20uM+CLC-3Antisense strand because of the DNA fragments,but the control had no.From the date,we could find that TAM could enhance the apoptosis induction in the MCF-7 cells and this effect could be improved with the concentration increasing. CLC-3 antisense strand could increase the effects of TAM either.
Part four.Study of the molecular mechanisms and the effects of mitochondria and chloride channel CLC-3 on apoptosis induced by tamoxifen in the MCF-7 cell line.
By the method of cellular immunohistochemical staining with the estrogen receptor (ER ) antibody ,we found that in MCF-7 cells of TAM20uM,the expression of ER vs the control reduced obviously,but the CLC-3antisense had no clearly change .This could determind that TAM could inhibit the expression of the ER in MCF-7 cells,but CLC-3 antisense strand had no this effect.With the RT-PCR method,we detected the mRNA of CLC-3、BCL-2 and Bax,we found that TAM and/or CLC-3antisense made the low expression of CLC-3、BCL-2 but the high expression of Bax,and the both had the joint action.With the DCFH-DA fluorescent staining,we could detect the ROS in the cells,and we found the TAM20u and TAM20u+ CLC-3 antisense strand could increase the ROS in the cells.The both had the joint action.With the method of isolation of mitochondria we found that TAM20u and TAM20u+ CLC-3antisense strand gave rise to the decreased of GSH in the mitochondria ,and the both had the joint action..By the method of western blot we found that TAM20u and TAM20u+ CLC-3 antisense strand brought about cytochrome C increased in the cytoplasm, but there was no clear difference between the two groups.
From the above date and discussion we can drew the following conclusions:
1、There are a obvious difference between the expression of CLC-3 in the breast cancer and the benign tissue P<0.05.This implies that the CLC-3 has some pathological and physiological significance in the process of breast cancer`s genesis and growth
2、The proliferation of MCF-7 cells treated with the CLC-3 antisense isnot inhibited significantly.This may be relate with the concentration which we applied in the study or the chloride channel CLC-3 is not necessary in the cell. CLC-3 antisense srand can enhances the apoptosis induction of the TAM in the MCF-7 cells, and this effect can be improved with the TAM concentration increasing.This can be refered by the clinical Endocrine therapy and the biotherapy
3、Mitochondria plays a key role in the process of cellular apoptosis, TAM can inhibit the estrogen receptor of the mitochondria.If the mitochondrial estrogen receptor directly take part in the expression of the mitochondrial DNA induced by the estrogen, there will be a target point to regulate, prevent and treat with the tumor in the signal pathway of mitochondrial estrogen receptor
4、TAM maybe induce the apoptosis through the estrogen receptor of the mitochondria and the chloride channel CLC-3 pathway,but mainly through the estrogen receptor pathway. Proper enhancement of the chloride channel`s antagonism can improve the inducement capacity of the apoptosis. This implies that there are some many pathways and mechanisms involved together in the process of tumor growth, the breast cancer as the hormonal dependent tumor does not exclude.

Keywords: Chloride channel CLC-3; Mitochondria ; Tamxifen; Apoptosis; Chloride channel CLC-3 antisense strand;