天花粉蛋白抑制結(jié)腸癌細胞生長及分子機制研究
發(fā)布時間：2019-05-07 20:16
【摘要】：結(jié)腸癌(colorectal cancer,CRC)是人類疾病中常見的消化系統(tǒng)惡性腫瘤,發(fā)病率位居人類惡性腫瘤第三位。臨床上多表現(xiàn)為消瘦、貧血、便血、腹痛等,其易發(fā)生遠處轉(zhuǎn)移,約50%患者發(fā)現(xiàn)時已發(fā)生肝轉(zhuǎn)移,因此患者在病程后期常因梗阻性黃疸、肝衰竭等原因死亡。目前結(jié)腸癌治療上雖然以外科手術(shù)為主,但是藥物抗腫瘤治療亦是重要治療手段,有助于患者病情的控制,提高患者生命質(zhì)量。人類惡性腫瘤的發(fā)生不僅與細胞的異常增生有關(guān),還與細胞的凋亡受阻,致使腫瘤細胞無限制惡性增生有密切關(guān)系。惡性腫瘤的轉(zhuǎn)移過程中包括血管生成、細胞粘附、侵襲、轉(zhuǎn)移及增殖等等。因此,細胞凋亡在控制腫瘤細胞增殖、腫瘤發(fā)生及腫瘤生長過程中起到至關(guān)重要的作用�，F(xiàn)代藥理學研究發(fā)現(xiàn),一些中國傳統(tǒng)中藥具有很好的抑制腫瘤生長作用。天花粉為葫蘆科植物瓜萎或雙邊瓜萎的干燥根,現(xiàn)代藥理學研究發(fā)現(xiàn)其具有終止妊娠、抗腫瘤、抗炎抑菌、抗病毒等多種藥理活性。天花粉蛋白(Trichosanthin,TCS)是一種從中藥瓜萎塊根中分離提純的有效成分,屬于Ⅰ型核糖體失活蛋白,具有RNAN-糖苷酶活性,能夠水解真核細胞核糖體28SrRNA特定位點的腺苷酸,從而抑制細胞蛋白質(zhì)的合成。既往實驗發(fā)現(xiàn),TCS具有潛在的抗腫瘤作用。本文研究天花粉蛋白抑制結(jié)腸癌細胞生長作用,并在細胞內(nèi)進行天花粉蛋白及其突變體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。結(jié)果顯示:(1)TCS對結(jié)腸癌HCT116細胞的生長有明顯的抑制作用,HCT116細胞出現(xiàn)細胞皺縮、變形,并且細胞凋亡呈現(xiàn)明顯的濃度和時間依賴性;(2)TCS可將結(jié)腸癌HCT116細胞阻滯于S期;(3)TCS可誘導HCT116細胞發(fā)生凋亡;(4)Western blotting檢測結(jié)果顯示caspase-3酶原,caspase-9酶原,PARP表達降低,呈現(xiàn)濃度依賴性。TCS處理可活化Akt、pAkt上升;(5)在細胞內(nèi),野生種TCS能抑制熒光素酶活性,突變體Y70A則不能抑制熒光素酶活性,而突變種R163Q對熒光素酶活性抑制作用強于野生種TCS。本研究共分為兩部分內(nèi)容。第一部分TCS抑制結(jié)腸癌HCT116細胞生長作用的研究研究目的:研究天花粉蛋白(Trichosanthin,TCS)對人結(jié)腸癌細胞生長抑制的影響。研究方法:人結(jié)腸癌HCT116細胞,分設TCS實驗組(濃度分別為7.0μM、14μM、28μM)和陰性對照組。實驗中(1)采用倒置顯微鏡觀察TCS對HCT116細胞形態(tài)及增殖的影響;(2)采用流式細胞儀檢測TCS對HCT116細胞周期以及TCS對HCT116細胞凋亡影響;(3)應用 Western blotting 檢測結(jié)腸癌 HCT116 細胞 caspase-3 酶原,caspase-9 酶原;PARP;pAkt;Akt變化。研究結(jié)果:(1)TCS處理明顯抑制結(jié)腸癌HCT116細胞的生長,HCT116細胞出現(xiàn)細胞皺縮、變形,體積變小,并且細胞凋亡呈現(xiàn)明顯的濃度和時間依賴性;(2)TCS可將結(jié)腸癌HCT116細胞阻滯于S期;(3)TCS可誘導HCT116細胞發(fā)生凋亡;(4)Western blotting法檢測結(jié)果顯示caspase-3酶原,caspase-9酶原,PARP表達降低,呈濃度依賴性。TCS處理激活Akt,磷酸化Akt升高。研究結(jié)論:TCS可抑制結(jié)腸癌HCT116細胞生長,呈藥物濃度和作用時間依賴性。TCS可將HCT116細胞阻滯于S期,并誘導HCT116細胞凋亡。caspase-9、caspase-3的激活為介導TCS誘導HCT116細胞發(fā)生凋亡的通路,結(jié)果提示與PI3K/Akt通路調(diào)節(jié)有關(guān)系。第二部分天花粉蛋白及其突變體Y70A、R163Q TCS生物活性研究研究目的:在細胞內(nèi)進行天花粉蛋白野生種(TCS)及天花粉蛋白突變體Y70A、R163Q TCS生物活性比較;研究方法:以pAAV-CMV-TCS為載體,通過定點突變、質(zhì)粒轉(zhuǎn)化、擴增、提取、測序、轉(zhuǎn)染等方法構(gòu)建突變種Y70A、R163Q TCS。在細胞內(nèi)通過熒光素酶活性測定,進行野生種TCS與突變種TCS活性比較。研究結(jié)果:熒光素酶報告基因檢測,單獨或與野生種TCS、突變種Y70A、R163Q共轉(zhuǎn)染293T細胞。在細胞內(nèi),野生種TCS能抑制熒光素酶活性,突變種Y70A則不能抑制熒光素酶活性,而突變種R163Q對熒光素酶活性抑制作用強于野生種TCS。研究結(jié)論:通過野生種TCS,突變種Y70A和R163Q在細胞內(nèi)對熒光素酶活性影響的比較,結(jié)果顯示Tyr70、Arg163為天花粉蛋白活性結(jié)構(gòu)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.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.35
，
本文編號：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.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.35
，
本文編號：2471350
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