长白山林蛙蛙卵Onconase基因的克隆表达及其抗人乳腺癌作用研究
发布时间：2018-08-06 13:50
【摘要】：乳腺癌是女性最常见的恶性肿瘤。乳腺癌的发生与细胞增殖和凋亡的失衡有关,是细胞经过致瘤因素的刺激,在基因水平上失去对其正常调控,使细胞凋亡受到抑制而造成的异常增生,肿瘤细胞能够和正常细胞争夺营养并造成正常组织与器官受损,而且其能够远处转移,最终造成患者死亡。随着科学技术的不断发展,在癌症的治疗手段中,抗癌药物的作用日渐重要,因此,需要寻找新的治疗乳腺癌的药物。Onconase是在北美豹蛙卵中被首次发现,其对多种肿瘤细胞,包括宫颈癌、乳腺癌、结肠癌、胰腺癌和前列腺癌细胞都具有细胞毒性,它可以通过调控细胞周期以及诱导细胞程序性死亡而发挥其抗增殖和细胞毒性作用。其抗癌机制还需要进一步研究。在本研究中,我们应用PCR技术,从长白山林蛙亚种基因组中克隆得到一个Rdchonc基因,并利用原核系统表达得到重组蛋白。经序列分析,得到的重组蛋白属于RNase A超家族,它具有RNase A家族的保守序列。通过核酸酶的活性实验测定,证明该蛋白具有较高的活性。为了研究得到的重组蛋白Rdchonc是否具有抗肿瘤活性,我们以人乳腺癌细胞MCF-7和MDA-MB-231为研究对象,通过MTT实验以及细胞侵袭实验,实验结果证明Rdchonc具有抗肿瘤细胞活性,可以抑制MCF-7和MDA-MB-231细胞的增殖及侵袭作用。为了进一步探讨其抗肿瘤的分子生物学机制,通过细胞周期实验,发现Rdchonc可以使细胞停滞在G0/G1期而发挥其抗癌作用。此外,Western blot结果表明Rdchonc通过下调Bcl-2、上调Bad蛋白的表达而促进人乳腺癌细胞MCF-7和MDA-MB-231的凋亡。由于MAPK/ERK信号通路在细胞的致癌性转化中起着重要的作用。它可以通过细胞表面生长因子受体而激活,从而调节细胞的增殖、分化和存活,同时,ERK在人乳腺癌中过度激活,MEK1/2-ERK1/2信号通路可能作为潜在的治疗途径。目前,已开始寻找针对ERK途径中各个环节的抑制物,用以切断信号转导的途径,从而达到治疗疾病的目的。因此,我们进一步探讨Rdchonc是否对MAPK/ERK信号通路起着调节作用,研究表明:Rdchonc通过下调P-MEK1/2和P-ERK1/2的表达而抑制人乳腺癌细胞MCF-7和MDA-MB-231的增殖。总之,本研究为林蛙的综合利用和精深开发,为长白山林蛙Onconase的进一步的开发及临床应用奠定了基础。
[Abstract]:Breast cancer is the most common malignant tumor in women. The occurrence of breast cancer is related to the imbalance of cell proliferation and apoptosis. Tumor cells can compete for nutrition with normal cells and cause damage to normal tissues and organs, and they can metastasize far away, resulting in the death of patients. With the development of science and technology, the role of anticancer drugs is becoming more and more important in the treatment of cancer. Therefore, the need to find a new drug for breast cancer. Onconase was first found in the eggs of the North American leopard frog. Including cervical cancer, breast cancer, colon cancer, pancreatic cancer and prostate cancer cells have cytotoxicity, which can play its anti-proliferation and cytotoxic effects by regulating cell cycle and inducing programmed cell death. Its anti-cancer mechanism needs further study. In this study, we cloned a Rdchonc gene from the subspecies genome of Rana Changbai Mountain by using PCR technique, and expressed the recombinant protein by prokaryotic system. The recombinant protein belongs to RNase A superfamily and has the conserved sequence of RNase A family. The results of nuclease activity test showed that the protein had high activity. In order to study whether the recombinant protein Rdchonc has antitumor activity or not, we studied human breast cancer cell line MCF-7 and MDA-MB-231. The results of MTT assay and cell invasion test showed that Rdchonc had anti-tumor activity. It can inhibit the proliferation and invasion of MCF-7 and MDA-MB-231 cells. In order to further study the molecular biological mechanism of its antitumor effect, it was found that Rdchonc could make the cells stagnate in G0/G1 phase and play its anticancer role by cell cycle experiment. In addition, the results of Western blot showed that Rdchonc promoted the apoptosis of MCF-7 and MDA-MB-231 by down-regulating Bcl-2 and up-regulating the expression of Bad protein. MAPK/ERK signaling pathway plays an important role in carcinogenic transformation of cells. It can be activated by cell surface growth factor receptor, thereby regulating cell proliferation, differentiation and survival. At the same time, ERK may act as a potential therapeutic pathway in human breast cancer by over-activating the MEK1 / 2-ERK1 / 2 signaling pathway. At present, we have begun to search for inhibitors of each link of ERK pathway to cut off the signal transduction pathway, so as to achieve the purpose of disease treatment. Therefore, we further investigate whether Rdchonc plays a role in regulating the MAPK/ERK signaling pathway. It has been shown that: Rdchonc inhibits the proliferation of MCF-7 and MDA-MB-231 by down-regulating the expression of P-MEK1/2 and P-ERK1/2 in human breast cancer cells. All in all, this study laid a foundation for the comprehensive utilization and deep development of Rana forest frog, and for the further development and clinical application of Onconase in Changbai Mountain Forest Frog.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R96
[Abstract]:Breast cancer is the most common malignant tumor in women. The occurrence of breast cancer is related to the imbalance of cell proliferation and apoptosis. Tumor cells can compete for nutrition with normal cells and cause damage to normal tissues and organs, and they can metastasize far away, resulting in the death of patients. With the development of science and technology, the role of anticancer drugs is becoming more and more important in the treatment of cancer. Therefore, the need to find a new drug for breast cancer. Onconase was first found in the eggs of the North American leopard frog. Including cervical cancer, breast cancer, colon cancer, pancreatic cancer and prostate cancer cells have cytotoxicity, which can play its anti-proliferation and cytotoxic effects by regulating cell cycle and inducing programmed cell death. Its anti-cancer mechanism needs further study. In this study, we cloned a Rdchonc gene from the subspecies genome of Rana Changbai Mountain by using PCR technique, and expressed the recombinant protein by prokaryotic system. The recombinant protein belongs to RNase A superfamily and has the conserved sequence of RNase A family. The results of nuclease activity test showed that the protein had high activity. In order to study whether the recombinant protein Rdchonc has antitumor activity or not, we studied human breast cancer cell line MCF-7 and MDA-MB-231. The results of MTT assay and cell invasion test showed that Rdchonc had anti-tumor activity. It can inhibit the proliferation and invasion of MCF-7 and MDA-MB-231 cells. In order to further study the molecular biological mechanism of its antitumor effect, it was found that Rdchonc could make the cells stagnate in G0/G1 phase and play its anticancer role by cell cycle experiment. In addition, the results of Western blot showed that Rdchonc promoted the apoptosis of MCF-7 and MDA-MB-231 by down-regulating Bcl-2 and up-regulating the expression of Bad protein. MAPK/ERK signaling pathway plays an important role in carcinogenic transformation of cells. It can be activated by cell surface growth factor receptor, thereby regulating cell proliferation, differentiation and survival. At the same time, ERK may act as a potential therapeutic pathway in human breast cancer by over-activating the MEK1 / 2-ERK1 / 2 signaling pathway. At present, we have begun to search for inhibitors of each link of ERK pathway to cut off the signal transduction pathway, so as to achieve the purpose of disease treatment. Therefore, we further investigate whether Rdchonc plays a role in regulating the MAPK/ERK signaling pathway. It has been shown that: Rdchonc inhibits the proliferation of MCF-7 and MDA-MB-231 by down-regulating the expression of P-MEK1/2 and P-ERK1/2 in human breast cancer cells. All in all, this study laid a foundation for the comprehensive utilization and deep development of Rana forest frog, and for the further development and clinical application of Onconase in Changbai Mountain Forest Frog.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R96
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