長(zhǎng)白山林蛙蛙卵Onconase基因的克隆表達(dá)及其抗人乳腺癌作用研究
發(fā)布時(shí)間：2018-08-06 13:50
【摘要】：乳腺癌是女性最常見(jiàn)的惡性腫瘤。乳腺癌的發(fā)生與細(xì)胞增殖和凋亡的失衡有關(guān),是細(xì)胞經(jīng)過(guò)致瘤因素的刺激,在基因水平上失去對(duì)其正常調(diào)控,使細(xì)胞凋亡受到抑制而造成的異常增生,腫瘤細(xì)胞能夠和正常細(xì)胞爭(zhēng)奪營(yíng)養(yǎng)并造成正常組織與器官受損,而且其能夠遠(yuǎn)處轉(zhuǎn)移,最終造成患者死亡。隨著科學(xué)技術(shù)的不斷發(fā)展,在癌癥的治療手段中,抗癌藥物的作用日漸重要,因此,需要尋找新的治療乳腺癌的藥物。Onconase是在北美豹蛙卵中被首次發(fā)現(xiàn),其對(duì)多種腫瘤細(xì)胞,包括宮頸癌、乳腺癌、結(jié)腸癌、胰腺癌和前列腺癌細(xì)胞都具有細(xì)胞毒性,它可以通過(guò)調(diào)控細(xì)胞周期以及誘導(dǎo)細(xì)胞程序性死亡而發(fā)揮其抗增殖和細(xì)胞毒性作用。其抗癌機(jī)制還需要進(jìn)一步研究。在本研究中,我們應(yīng)用PCR技術(shù),從長(zhǎng)白山林蛙亞種基因組中克隆得到一個(gè)Rdchonc基因,并利用原核系統(tǒng)表達(dá)得到重組蛋白。經(jīng)序列分析,得到的重組蛋白屬于RNase A超家族,它具有RNase A家族的保守序列。通過(guò)核酸酶的活性實(shí)驗(yàn)測(cè)定,證明該蛋白具有較高的活性。為了研究得到的重組蛋白R(shí)dchonc是否具有抗腫瘤活性,我們以人乳腺癌細(xì)胞MCF-7和MDA-MB-231為研究對(duì)象,通過(guò)MTT實(shí)驗(yàn)以及細(xì)胞侵襲實(shí)驗(yàn),實(shí)驗(yàn)結(jié)果證明Rdchonc具有抗腫瘤細(xì)胞活性,可以抑制MCF-7和MDA-MB-231細(xì)胞的增殖及侵襲作用。為了進(jìn)一步探討其抗腫瘤的分子生物學(xué)機(jī)制,通過(guò)細(xì)胞周期實(shí)驗(yàn),發(fā)現(xiàn)Rdchonc可以使細(xì)胞停滯在G0/G1期而發(fā)揮其抗癌作用。此外,Western blot結(jié)果表明Rdchonc通過(guò)下調(diào)Bcl-2、上調(diào)Bad蛋白的表達(dá)而促進(jìn)人乳腺癌細(xì)胞MCF-7和MDA-MB-231的凋亡。由于MAPK/ERK信號(hào)通路在細(xì)胞的致癌性轉(zhuǎn)化中起著重要的作用。它可以通過(guò)細(xì)胞表面生長(zhǎng)因子受體而激活,從而調(diào)節(jié)細(xì)胞的增殖、分化和存活,同時(shí),ERK在人乳腺癌中過(guò)度激活,MEK1/2-ERK1/2信號(hào)通路可能作為潛在的治療途徑。目前,已開(kāi)始尋找針對(duì)ERK途徑中各個(gè)環(huán)節(jié)的抑制物,用以切斷信號(hào)轉(zhuǎn)導(dǎo)的途徑,從而達(dá)到治療疾病的目的。因此,我們進(jìn)一步探討Rdchonc是否對(duì)MAPK/ERK信號(hào)通路起著調(diào)節(jié)作用,研究表明:Rdchonc通過(guò)下調(diào)P-MEK1/2和P-ERK1/2的表達(dá)而抑制人乳腺癌細(xì)胞MCF-7和MDA-MB-231的增殖。總之,本研究為林蛙的綜合利用和精深開(kāi)發(fā),為長(zhǎng)白山林蛙Onconase的進(jìn)一步的開(kāi)發(fā)及臨床應(yīng)用奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R96
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