天花粉蛋白抑制结肠癌细胞生长及分子机制研究
发布时间：2019-05-07 20:16
【摘要】：结肠癌(colorectal cancer,CRC)是人类疾病中常见的消化系统恶性肿瘤,发病率位居人类恶性肿瘤第三位。临床上多表现为消瘦、贫血、便血、腹痛等,其易发生远处转移,约50%患者发现时已发生肝转移,因此患者在病程后期常因梗阻性黄疸、肝衰竭等原因死亡。目前结肠癌治疗上虽然以外科手术为主,但是药物抗肿瘤治疗亦是重要治疗手段,有助于患者病情的控制,提高患者生命质量。人类恶性肿瘤的发生不仅与细胞的异常增生有关,还与细胞的凋亡受阻,致使肿瘤细胞无限制恶性增生有密切关系。恶性肿瘤的转移过程中包括血管生成、细胞粘附、侵袭、转移及增殖等等。因此,细胞凋亡在控制肿瘤细胞增殖、肿瘤发生及肿瘤生长过程中起到至关重要的作用。现代药理学研究发现,一些中国传统中药具有很好的抑制肿瘤生长作用。天花粉为葫芦科植物瓜萎或双边瓜萎的干燥根,现代药理学研究发现其具有终止妊娠、抗肿瘤、抗炎抑菌、抗病毒等多种药理活性。天花粉蛋白(Trichosanthin,TCS)是一种从中药瓜萎块根中分离提纯的有效成分,属于Ⅰ型核糖体失活蛋白,具有RNAN-糖苷酶活性,能够水解真核细胞核糖体28SrRNA特定位点的腺苷酸,从而抑制细胞蛋白质的合成。既往实验发现,TCS具有潜在的抗肿瘤作用。本文研究天花粉蛋白抑制结肠癌细胞生长作用,并在细胞内进行天花粉蛋白及其突变体Y(酪氨酸)70A(丙氨酸)、R(精氨酸)163Q(谷氨酰胺)TCS生物活性功能研究。结果显示:(1)TCS对结肠癌HCT116细胞的生长有明显的抑制作用,HCT116细胞出现细胞皱缩、变形,并且细胞凋亡呈现明显的浓度和时间依赖性;(2)TCS可将结肠癌HCT116细胞阻滞于S期;(3)TCS可诱导HCT116细胞发生凋亡;(4)Western blotting检测结果显示caspase-3酶原,caspase-9酶原,PARP表达降低,呈现浓度依赖性。TCS处理可活化Akt、pAkt上升;(5)在细胞内,野生种TCS能抑制荧光素酶活性,突变体Y70A则不能抑制荧光素酶活性,而突变种R163Q对荧光素酶活性抑制作用强于野生种TCS。本研究共分为两部分内容。第一部分TCS抑制结肠癌HCT116细胞生长作用的研究研究目的:研究天花粉蛋白(Trichosanthin,TCS)对人结肠癌细胞生长抑制的影响。研究方法:人结肠癌HCT116细胞,分设TCS实验组(浓度分别为7.0μM、14μM、28μM)和阴性对照组。实验中(1)采用倒置显微镜观察TCS对HCT116细胞形态及增殖的影响;(2)采用流式细胞仪检测TCS对HCT116细胞周期以及TCS对HCT116细胞凋亡影响;(3)应用 Western blotting 检测结肠癌 HCT116 细胞 caspase-3 酶原,caspase-9 酶原;PARP;pAkt;Akt变化。研究结果:(1)TCS处理明显抑制结肠癌HCT116细胞的生长,HCT116细胞出现细胞皱缩、变形,体积变小,并且细胞凋亡呈现明显的浓度和时间依赖性;(2)TCS可将结肠癌HCT116细胞阻滞于S期;(3)TCS可诱导HCT116细胞发生凋亡;(4)Western blotting法检测结果显示caspase-3酶原,caspase-9酶原,PARP表达降低,呈浓度依赖性。TCS处理激活Akt,磷酸化Akt升高。研究结论:TCS可抑制结肠癌HCT116细胞生长,呈药物浓度和作用时间依赖性。TCS可将HCT116细胞阻滞于S期,并诱导HCT116细胞凋亡。caspase-9、caspase-3的激活为介导TCS诱导HCT116细胞发生凋亡的通路,结果提示与PI3K/Akt通路调节有关系。第二部分天花粉蛋白及其突变体Y70A、R163Q TCS生物活性研究研究目的:在细胞内进行天花粉蛋白野生种(TCS)及天花粉蛋白突变体Y70A、R163Q TCS生物活性比较;研究方法:以pAAV-CMV-TCS为载体,通过定点突变、质粒转化、扩增、提取、测序、转染等方法构建突变种Y70A、R163Q TCS。在细胞内通过荧光素酶活性测定,进行野生种TCS与突变种TCS活性比较。研究结果:荧光素酶报告基因检测,单独或与野生种TCS、突变种Y70A、R163Q共转染293T细胞。在细胞内,野生种TCS能抑制荧光素酶活性,突变种Y70A则不能抑制荧光素酶活性,而突变种R163Q对荧光素酶活性抑制作用强于野生种TCS。研究结论:通过野生种TCS,突变种Y70A和R163Q在细胞内对荧光素酶活性影响的比较,结果显示Tyr70、Arg163为天花粉蛋白活性结构中重要氨基酸。
[Abstract]:Colon cancer (CRC) is a common malignant tumor of the digestive system in human diseases, and the incidence is the third of the human malignant tumor. There are more clinical manifestations of emaciation, anemia, hematochezia, and abdominal pain. It is easy for distant metastasis. In some 50% of the patients, the liver metastasis has occurred. Therefore, the patients often died due to obstructive jaundice and liver failure at the later stage of the course of the disease. At present, while the treatment of colon cancer is the main surgical procedure, the anti-tumor therapy of the drug is also an important means of treatment, which is helpful to the control of the patient's condition and to improve the quality of life of the patient. The occurrence of human malignant tumor is related not only to the abnormal proliferation of the cells, but also to the apoptosis of the cells, so that the tumor cells are not restricted to the malignant proliferation. The metastasis of malignant tumor includes angiogenesis, cell adhesion, invasion, metastasis and proliferation. Therefore, apoptosis plays an important role in the control of tumor cell proliferation, tumorigenesis and tumor growth. Modern pharmacology studies have found that some Chinese traditional Chinese medicines have a good effect on tumor growth. Trichosanthis is a dry root of the plant of the Cucurbitaceae, and the modern pharmacological research has found that it has many pharmacological activities such as termination of pregnancy, anti-tumor, anti-inflammatory and antibacterial, and antiviral. Trichosanthis (TCS) is an effective component isolated and purified from the root tuber of the Chinese traditional medicine, and belongs to the type I ribosome inactivating protein, and has the activity of RNAN-glycosidase and can hydrolyze the adenoid acid of the site of the ribosome 28SrRNA of the eukaryotic cell, thereby inhibiting the synthesis of the cell protein. The previous experiments have shown that TCS has a potential anti-tumor effect. This paper studies the effect of trichosanthin on the growth of colon cancer cells, and studies the biological activity of trichosanthin and its mutant Y (tyrosine) 70A (alanine), R (arginine)163 Q (glutamate) TCS in the cells. The results showed that: (1) TCS can inhibit the growth of colon cancer HCT116 cells, and the cells of the HCT116 cells are shriveled and deformed, and the apoptosis of the cells is obvious; (2) TCS can block the colon cancer HCT116 cells in the S phase; (3) TCS can induce the apoptosis of the HCT116 cells; (4) The results of Western blotting showed that the expression of caspase-3, caspase-9 and PARP decreased and the concentration-dependent was present. TCS treatment can activate Akt and pAkt to rise; (5) in the cell, the wild type TCS can inhibit the luciferase activity, and the mutant Y70A can not inhibit the luciferase activity, and the mutant R163Q has stronger effect on luciferase activity than the wild type TCS. The study is divided into two parts. The purpose of this study is to study the effect of trichosanthin (TCS) on the growth inhibition of human colon cancer cells. Methods: Human colon cancer HCT116 cells were divided into TCS group (7.0. mu.M,14. mu.M,28. mu.M) and negative control group. In the experiment (1), the effect of TCS on the morphology and proliferation of the HCT116 cells was observed by an inverted microscope. (2) The cell cycle of the HCT116 and the effect of TCS on the apoptosis of the HCT116 cells were detected by flow cytometry. (3) The caspase-3 zymogen, the caspase-9 zymogen, the PARP, and the pAkt of the colon cancer HCT116 were detected by Western blotting. Akt changes. The results of the study: (1) TCS treatment significantly inhibited the growth of colon cancer HCT116 cells, and the cells of the HCT116 cells were collapsed, deformed, and the volume decreased, and the apoptosis of the cells showed a significant concentration and time-dependent manner; (2) TCS can block the colon cancer HCT116 cells in the S phase; (3) TCS can induce the apoptosis of the HCT116 cells; (4) The results of Western blotting showed that the expression of caspase-3, caspase-9 and PARP decreased and the concentration-dependent. The TCS treatment activates Akt and the phosphorylation Akt increases. Conclusion: TCS can inhibit the growth of colon cancer HCT116 cells and show the dependence of drug concentration and time on time. The TCS can block the HCT116 cells in the S phase and induce the apoptosis of the HCT116 cells. The activation of caspase-9 and caspase-3 was a pathway to mediate the apoptosis of the TCS-induced HCT116 cells. The results suggest that there is a relationship with the regulation of PI3K/ Akt pathway. The biological activity of the second part of trichosanthin and its mutant Y70A and R163Q TCS was studied. The results of the study on the biological activity of the wild type (TCS) and the trichosanthin mutant Y70A and R163Q TCS were carried out in the cells. The methods of the study were as follows: the pAAV-CMV-TCS was used as the carrier, and the plasmid was transformed with the site-directed mutagenesis and the plasmid. The mutant Y70A and R163Q TCS were constructed by amplification, extraction, sequencing and transfection. The activity of the wild type TCS and the mutant TCS was compared with the activity of the luciferase in the cell. Results: The luciferase reporter gene was detected, and the 293T cells were co-transfected with the wild type TCS, the mutant Y70A and the R163Q. In the cell, the wild type TCS can inhibit the luciferase activity, and the mutant Y70A can not inhibit the luciferase activity, and the mutant R163Q has stronger effect on the luciferase activity than the wild type TCS. The results show that Tyr70 and Arg163 are important amino acids in the active structure of the trichosanthin protein by comparison of the effect of wild type TCS, mutant Y70A and R163Q on the activity of luciferase.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.35
，
本文编号：2471350
[Abstract]:Colon cancer (CRC) is a common malignant tumor of the digestive system in human diseases, and the incidence is the third of the human malignant tumor. There are more clinical manifestations of emaciation, anemia, hematochezia, and abdominal pain. It is easy for distant metastasis. In some 50% of the patients, the liver metastasis has occurred. Therefore, the patients often died due to obstructive jaundice and liver failure at the later stage of the course of the disease. At present, while the treatment of colon cancer is the main surgical procedure, the anti-tumor therapy of the drug is also an important means of treatment, which is helpful to the control of the patient's condition and to improve the quality of life of the patient. The occurrence of human malignant tumor is related not only to the abnormal proliferation of the cells, but also to the apoptosis of the cells, so that the tumor cells are not restricted to the malignant proliferation. The metastasis of malignant tumor includes angiogenesis, cell adhesion, invasion, metastasis and proliferation. Therefore, apoptosis plays an important role in the control of tumor cell proliferation, tumorigenesis and tumor growth. Modern pharmacology studies have found that some Chinese traditional Chinese medicines have a good effect on tumor growth. Trichosanthis is a dry root of the plant of the Cucurbitaceae, and the modern pharmacological research has found that it has many pharmacological activities such as termination of pregnancy, anti-tumor, anti-inflammatory and antibacterial, and antiviral. Trichosanthis (TCS) is an effective component isolated and purified from the root tuber of the Chinese traditional medicine, and belongs to the type I ribosome inactivating protein, and has the activity of RNAN-glycosidase and can hydrolyze the adenoid acid of the site of the ribosome 28SrRNA of the eukaryotic cell, thereby inhibiting the synthesis of the cell protein. The previous experiments have shown that TCS has a potential anti-tumor effect. This paper studies the effect of trichosanthin on the growth of colon cancer cells, and studies the biological activity of trichosanthin and its mutant Y (tyrosine) 70A (alanine), R (arginine)163 Q (glutamate) TCS in the cells. The results showed that: (1) TCS can inhibit the growth of colon cancer HCT116 cells, and the cells of the HCT116 cells are shriveled and deformed, and the apoptosis of the cells is obvious; (2) TCS can block the colon cancer HCT116 cells in the S phase; (3) TCS can induce the apoptosis of the HCT116 cells; (4) The results of Western blotting showed that the expression of caspase-3, caspase-9 and PARP decreased and the concentration-dependent was present. TCS treatment can activate Akt and pAkt to rise; (5) in the cell, the wild type TCS can inhibit the luciferase activity, and the mutant Y70A can not inhibit the luciferase activity, and the mutant R163Q has stronger effect on luciferase activity than the wild type TCS. The study is divided into two parts. The purpose of this study is to study the effect of trichosanthin (TCS) on the growth inhibition of human colon cancer cells. Methods: Human colon cancer HCT116 cells were divided into TCS group (7.0. mu.M,14. mu.M,28. mu.M) and negative control group. In the experiment (1), the effect of TCS on the morphology and proliferation of the HCT116 cells was observed by an inverted microscope. (2) The cell cycle of the HCT116 and the effect of TCS on the apoptosis of the HCT116 cells were detected by flow cytometry. (3) The caspase-3 zymogen, the caspase-9 zymogen, the PARP, and the pAkt of the colon cancer HCT116 were detected by Western blotting. Akt changes. The results of the study: (1) TCS treatment significantly inhibited the growth of colon cancer HCT116 cells, and the cells of the HCT116 cells were collapsed, deformed, and the volume decreased, and the apoptosis of the cells showed a significant concentration and time-dependent manner; (2) TCS can block the colon cancer HCT116 cells in the S phase; (3) TCS can induce the apoptosis of the HCT116 cells; (4) The results of Western blotting showed that the expression of caspase-3, caspase-9 and PARP decreased and the concentration-dependent. The TCS treatment activates Akt and the phosphorylation Akt increases. Conclusion: TCS can inhibit the growth of colon cancer HCT116 cells and show the dependence of drug concentration and time on time. The TCS can block the HCT116 cells in the S phase and induce the apoptosis of the HCT116 cells. The activation of caspase-9 and caspase-3 was a pathway to mediate the apoptosis of the TCS-induced HCT116 cells. The results suggest that there is a relationship with the regulation of PI3K/ Akt pathway. The biological activity of the second part of trichosanthin and its mutant Y70A and R163Q TCS was studied. The results of the study on the biological activity of the wild type (TCS) and the trichosanthin mutant Y70A and R163Q TCS were carried out in the cells. The methods of the study were as follows: the pAAV-CMV-TCS was used as the carrier, and the plasmid was transformed with the site-directed mutagenesis and the plasmid. The mutant Y70A and R163Q TCS were constructed by amplification, extraction, sequencing and transfection. The activity of the wild type TCS and the mutant TCS was compared with the activity of the luciferase in the cell. Results: The luciferase reporter gene was detected, and the 293T cells were co-transfected with the wild type TCS, the mutant Y70A and the R163Q. In the cell, the wild type TCS can inhibit the luciferase activity, and the mutant Y70A can not inhibit the luciferase activity, and the mutant R163Q has stronger effect on the luciferase activity than the wild type TCS. The results show that Tyr70 and Arg163 are important amino acids in the active structure of the trichosanthin protein by comparison of the effect of wild type TCS, mutant Y70A and R163Q on the activity of luciferase.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.35
，
本文编号：2471350
本文链接：https://www.wllwen.com/yixuelunwen/zlx/2471350.html
