【作者】 王蔚；

【导师】 茆灿泉；

【作者基本信息】 西南交通大学 ， 生物工程， 2019， 硕士

【摘要】 乳腺癌作为最常见的恶性肿瘤之一,严重影响女性的身心健康。手术、放疗、化疗、内分泌疗法和分子靶向广泛用于乳腺癌的治疗。中药在治疗肿瘤方面具有独特的优势,其具有毒副作用相对较小、作用靶点多样、不易产生耐药性等多种特点,在治疗肿瘤方面愈来愈受到重视。因此,研发一种适于体外给药方式、低毒副作用、抗乳腺癌效果好,用药量少的中药制剂具有重要意义与价值。本研究首先通过资料收集、文献查阅,初步整理出白花蛇舌草、苦参、防风等七十余种在临床上可有效治疗乳腺癌的中药材,再通过测定各药材对人乳腺癌细胞MDAMB-231的杀伤作用,筛选出了覆盆子、瓜蒌、紫苏、白花蛇舌草、苦参、防风等多种强抗乳腺癌作用的中药材,结合中药配伍,确定了一种抗乳腺癌的中药复方并命名为MW1712。其次通过CCK-8细胞活力测定,发现该中药复方不仅对人乳腺癌细胞MDA-MB-231有极强的抑制杀伤作用,对人黑色素瘤细胞B16、人肺癌细胞A549、人肝癌细胞HepG2均有较强的抑制作用,抑制率分别为:70.197%、69.823%、69.072%、64.653%。随后以人乳腺癌细胞MDA-MB-231为研究对象,就MW1712中药复方,进行了平板克隆、软琼脂克隆形成和细胞划痕实验,发现该复方能显著抑制MDA-MB-231细胞的增殖及迁移,这也为qRT-PCR实验结果所证实:MW1712处理过的人乳腺癌细胞MDAMB-231中的foxm1、mmp9、Vimentin以及vegf的表达分别下调了37.85%、97.27%、99.21%、87.41%。AO/EB荧光双染结果显示该中药复方显著促进人乳腺癌细胞MDAMB-231凋亡,qRT-PCR实验同样证实了这一点,被中药复方处理过的人乳腺癌细胞MDA-MB-231中的促凋亡相关基因p53表达显著上调(p<0.01)。qRT-PCR进一步发现,耐药基因mdr1显著下调(p<0.01),该中药复方还可能降低靶细胞的耐药性。最后通过DNA条形码技术,对复方中的多种药材进行了DNA水平的分子鉴定。对覆盆子、瓜蒌、紫苏三种关键药材进行基因组DNA提取,然后通过PCR扩增ITS2、psbAtrnH序列,并构建进化树,再将以上药材psbA-trnH序列在中药材DNA条形码鉴定系统进行鉴定,确定与之最为匹配的亲缘物种分别为茅莓、双边栝楼、紫苏。与ITS2结果结合,最终将待鉴定药材确定为:插田泡、双边栝楼、紫苏。综上所述,本工作研发了一种抗乳腺癌中药复方MW1712,初步确定了其抗MDAMB-231乳腺癌细胞的作用机制,并对其主要药材覆盆子、瓜蒌、紫苏进行了DNA条形码分子鉴定。本研究为抗乳腺癌中药复方的研发提供了重要的前期工作与基础。更多还原

【Abstract】 As one of the most common malignant tumors,breast cancer can seriously affect women’s physical and mental health.Surgery,radiotherapy,chemotherapy,endocrine therapy and molecular targeting are widely used in the treatment of breast cancer.Traditional Chinese medicine has unique advantages while treating tumors,such as relatively less side effects,diverse target targets and lower susceptibility to drug resistance,due to these reasons,it has been paid more and more attention in the treatment of tumors.Therefore,it is of great significance and value to develop a kind of traditional Chinese medicine preparation not only suitable for external administration,but also with low toxic and side effects,good anti-breast cancer efficacy and low dosage.By collecting data and consulting the literature,a preliminary screening of 77 Chinese herbal medicines which can effectively kill breast cancer cells clinically was carried out,and then the inhibitory effects for human breast cancer cells of MDA-MB-231 are determined by measuring each medicinal material.As a high-throughput screening,the medicinal materials such as Rubus idaeus L.,melon of Trichosanthes uniflora Hao.,Perilla frutescens(L.)Britt,Hedyotis diffusa Willd.,Sophora flavescens Ait.,and Saposhnikovia divaricata(Trucz.)Schischk.were selected.A Chinese herbal formula nominated MW1712 was formed according to the compatibility of Chinese traditional medicines.Secondly,by the cell viability assay,we found that this Chinese herbal formula MW1712 can not only strongly inhibit the vialbilitiesof human breast cancer cells MDA-MB-231,but also human melanoma cells B16,human lung cancer cells A549,human hepatoma cells HepG2,and the inhibitory rates were 70.197%,69.823%,69.072%,64.653% respectively.Subsequently,the human breast cancer cell MDA-MB-231 was used as the host to implement plate cloning,soft agar colony formation and cell scratch test.It was found that the formula can significantly inhibit the proliferation and the migration of MDA-MB-231 cells,which was also confirmed by the qRT-PCR results: the expression of foxm1,mmp9,Vimentin and vegf genes in MDA-MB-231 cells treated with MW1712 were down-regulated by 37.85%,97.27%,99.21%,87.41,respectively.Then the AO/EB fluorescence double staining observation showed that the MW1712 can significantly promote the apoptosis of MDA-MB-231,which was also confirmed by qRT-PCR results: the expression of proapoptosis-related gene p53 in MDA-MB-231 cells treated with MW1712 was significantly up-regulated.Furthermore,qRTPCR also found that the mdr1 drug resistance gene was significantly down-regulated,revealing that MW1712 may also reduce the drug resistance of target cells.Finally,we used DNA barcode technology to molecularly identify the various herbs in MW1712,laying foundation for the quality control of the formula.Firstly,the genomic DNA was extracted from the three key medicinal materials of MW1712.Then,the ITS2,psbA-trnH sequences were amplified by PCR,and subsequently the phylogenetic tree was constructed.In addition,the psbA-trnH sequence of the medicinal materials was identified in the DNA barcode identification system of Chinese medicinal materials,the results of which showed that the closest matching species were Rubus parvifolius Linn.,Trichosanthes uniflora Hao.,Perilla frutescens(L.)Britt.,respectively.Combined with the results of ITS2,the samole was identified as Rubus coreanus Miq.,Trichosanthes uniflora Hao,and Perilla frutescens(L.)Britt..In summary,MW1712,an anti-breast cancer traditional Chinese medicine formula,was developed in this study,and the mechanism of its anti-MDA-MB-231 cells was preliminarily determined.DNA barcode molecular identification of its main medicinal materials,Rubus idaeus L.,melon of Trichosanthes uniflora Hao.and Perilla frutescens(L.)Britt.,was carried out.This study provides important preliminary work and foundation for the research and development of anti-breast cancer raditional Chinese medicine formula.更多还原