本文選題：環(huán)境因素 + 遺傳因素��； 參考：《昆明理工大學》2017年博士論文

【摘要】：癌癥是人類復雜疾病中最難攻克的一種,目前我國的癌癥發(fā)病率持續(xù)上升,惡性腫瘤已經(jīng)成為致死率最高的疾病。癌癥的發(fā)生和發(fā)展是遺傳因素和環(huán)境因素共同作用的結(jié)果,環(huán)境因素包括多種化學致癌物、物理輻射、微生物類如病毒感染等,遺傳因素則包括原癌基因的激活及抑癌基因的突變或表達被抑制導致其功能丟失或降低。在攻克癌癥的道路上,各國科學家致力于抑癌基因的研究和探索,通過充分了解其作用機制進而嘗試對其進行多方面的調(diào)控,從而完成對腫瘤的抑制。在眾多抑癌基因中,p53作為最重要的抑癌基因之一,在腫瘤抑制中發(fā)揮重要作用。p53通過周期阻滯完成對DNA損傷的修復,在修復無法完成時誘導細胞凋亡和衰老,進而抑制腫瘤的發(fā)生和發(fā)展。在多種惡性腫瘤中p53水平很低,其表達水平受到多種因素的調(diào)控,其中轉(zhuǎn)錄水平的調(diào)控是基因表達的關(guān)鍵環(huán)節(jié)�；蜣D(zhuǎn)錄水平的升高離不開上游轉(zhuǎn)錄因子對其啟動子的轉(zhuǎn)錄激活,因此我們利用p53基因啟動子特有的DNA損傷感應能力,克隆了p53基因核心啟動子在內(nèi)的起始密碼子上游約750bp的序列,并構(gòu)建了以綠色熒光蛋白(GFP)作為報告基因的遺傳毒性篩選系統(tǒng)。該系統(tǒng)能對環(huán)境中的多種化合物進行高通量篩選,快速鑒別出具有遺傳毒性的化合物。除了遺傳毒性篩選之外,有研究證明在p53基因未突變的腫瘤細胞中再激活p53可以有效抑制腫瘤的發(fā)展甚至誘導腫瘤消退,因此我們預期可以通過該篩選系統(tǒng)對多種天然提取化合物進行篩選,以期篩選到以p53基因啟動子作為靶點的低毒性抗腫瘤藥物。除了 p53這一重要的抑癌基因外,研究發(fā)現(xiàn)在多種惡性腫瘤中存在抑癌基因Ink4a/ARF的變異,包括基因的純合性缺失、雜合性缺失、突變以及啟動子甲基化等。p16和p19蛋白功能分別屬于Rb和p53信號途徑,在監(jiān)控細胞周期,抑制腫瘤發(fā)生等過程中起到了重要作用。有研究證明p16Ink4a基因的表達異常和惡性腫瘤的發(fā)生和發(fā)展有關(guān),例如在黑色素瘤中經(jīng)常發(fā)現(xiàn)p16Ink4a基因啟動子甲基化導致的基因沉默。而ARF基因被證明能被多種原癌基因激活信號所激活,通過p53介導的腫瘤抑制對抗癌癥的發(fā)生和發(fā)展。因此我們同樣構(gòu)建了以GFP作為報告基因,分別以p16Ink4a和p19ARF基因的啟動子區(qū)域作為靶點的抗腫瘤藥物篩選系統(tǒng),以期能夠篩選到能夠有效激活I(lǐng)nk4a/ARF)基因啟動子的低毒性抗腫瘤天然化合物。在本研究中,我們選擇的第四代啟動子報告載體pGL4.82是在前幾代的基礎(chǔ)上進行了改良,減少了過多的轉(zhuǎn)錄因子結(jié)合位點并提高了其在細胞內(nèi)表達的穩(wěn)定性。為了提高檢測效率,我們將載體上的熒光素酶報告基因替換為GFP,這樣我們可以通過熒光顯微鏡對報告基因進行快速觀察,構(gòu)建得到的載體命名為pGL4-GFP。隨后我們將上面三種重要的抑癌基因啟動子構(gòu)建入載體,并穩(wěn)定轉(zhuǎn)染到遺傳背景清晰的細胞系里,通過抗性篩選得到基于三種抑癌基因啟動子的穩(wěn)定篩選細胞系 p53-GFP,p16-GFP 和 p19-GFP。篩選系統(tǒng)建立后,我們首先對p53-GFP細胞系功能進行驗證:對p53-GFP篩選系統(tǒng)我們選擇了阿霉素、環(huán)磷酰胺、順鉑、苯并芘等DNA毒性劑進行測試。在阿霉素處理6h后就可以在熒光顯微鏡下觀察到綠色熒光蛋白GFP的表達,且隨著藥物處理時間的增加,熒光強度逐步升高,在24h時達到高峰,而陰性對照則沒有觀察GFP表達。其他幾種DNA毒性劑也觀察到了相同的結(jié)果,蛋白免疫印跡確證了 GFP蛋白的表達。這一實驗證明了該系統(tǒng)可以有效響應DNA損傷,因此我們可以將其應用于環(huán)境遺傳毒性化合物的監(jiān)測。同時,臨床上使用的大量癌癥化療藥物都屬于DNA損傷劑,這些藥物對于活躍增殖的正常細胞,如骨髓造血細胞、小腸上皮細胞等具有較大的殺傷,從而普遍存在藥物毒副作用較強的問題。我們構(gòu)建的這一高通量篩選系統(tǒng)是基于p53啟動子的轉(zhuǎn)錄激活,因此我們預期能夠篩選到能有效激活抑癌基因表達同時細胞毒性較弱的天然化合物。接下來,我們應用該系統(tǒng)篩選了多種天然化合物包括昆明植物研究所提供的升麻類提取物及黃楊類生物堿。結(jié)果表明,多種升麻類提取物均不能有效激活p53啟動子,對野生型MEF細胞的毒性也較小。而黃楊生物堿化合物中有部分化合物如 KBA01、KBA02、KBA03、KBA07、KBR11 等能有效激活 GFP 表達,為了尋找到細胞毒性較低,又能在細胞內(nèi)再激活p53的天然化合物,我們進一步將這些篩選出的化合物對野生型MEF細胞進行了處理,均有效升高了 p53水平。隨后我們對這些化合物處理后的細胞形態(tài)進行拍攝并通過流式細胞儀對細胞凋亡水平和周期分布進行檢測,發(fā)現(xiàn)其中多數(shù)化合物均對細胞增殖產(chǎn)生了抑制,且凋亡細胞明顯增多,例如KBA03誘導了細胞G1期的阻滯,KBR18處理后凋亡細胞明顯增多。大多數(shù)篩選出的黃楊生物堿均帶有不同程度的基因毒性,但我們發(fā)現(xiàn)其中的KBA07能有效激活p53基因啟動子,且不具有明顯的遺傳毒性,其處理后的野生型MEF細胞在周期和凋亡水平與對照均沒有顯著差異。我們認為該化合物可能是通過p53基因上游的某些轉(zhuǎn)錄因子的激活或相互作用激活了p5 基因啟動子,但本身并不具有強遺傳毒性,對KBA07需要進一步研究其作用機制。在實驗室之前的普洱茶抗腫瘤機制的研究中,我們發(fā)現(xiàn)普洱茶能顯著提高p16的mRNA水平,用普洱茶對p16-GFP細胞進行處理后,能觀察到報告基因GFP的表達升高,證明普洱茶是通過作用于p16Ink4p基因啟動子激活了 p16的表達,但并不存在劑量效應。在多種惡性腫瘤中均發(fā)現(xiàn)p16的失活,因此能激活p16啟動子的化合物就具備一定的抗腫瘤潛能。我們用該系統(tǒng)篩出了如升麻提取物KY01、KY40等能有效激活p16的化合物,對它們的作用機制還需要進一步研究。在驗證p19-GFP篩選系統(tǒng)時,我們對該篩選系統(tǒng)轉(zhuǎn)染了 pBabe-RasG12V外源表達載體,過表達的RasG12V能有效激活p1p基因啟動子,在轉(zhuǎn)染后24h就觀察到明顯的GFP表達。之前有研究證明p19基因啟動子對原癌基因激活信號敏感且存在特異性,這一特質(zhì)也是該基因啟動子的寶貴特點,區(qū)別于p53會被多種損傷信號激活從而有可能造成過度應激。通過對多種化合物進行篩選也發(fā)現(xiàn)鮮有能激活p19啟動子的化合物,但我們欣喜的發(fā)現(xiàn)KBA07能有效激活p19啟動子,從而我們覺得KBA07具有較高的研究價值。為了進一步研究KBA07的作用機制,我們用KBA07處理了野生型MEF細胞和多種遺傳背景清晰的腫瘤細胞系,發(fā)現(xiàn)p53和p1p基因的轉(zhuǎn)錄水平明顯升高,但升高的p53并沒有激活其下游分子p21,證明其并不具備強遺傳毒性,其對p53的激活主要是通過升高其轉(zhuǎn)錄水平。在p53為野生型的Hela細胞進行KBA07處理后,觀察到p53升高,對細胞增殖產(chǎn)生了抑制。當KBA07與阿霉素和順鉑聯(lián)合使用時對細胞增殖的抑制更為明顯,提示我們KBA07可能可以作為一種化療的輔助藥物,通過提高細胞內(nèi)的p53蛋白本底水平,并在DNA毒性劑作用下,快速激發(fā)細胞阻滯、凋亡和衰老進而完成腫瘤細胞的抑制和清除。為了進一步研究KBA07對p53基因啟動子的作用機制,我們通過數(shù)據(jù)庫預測和查詢找到了 10個位于p53啟動子的轉(zhuǎn)錄因子結(jié)合位點,通過人工合成這些轉(zhuǎn)錄因子結(jié)合序列并轉(zhuǎn)染到p53-GFP細胞中,從而和啟動子序列產(chǎn)生競爭抑制。研究發(fā)現(xiàn)在轉(zhuǎn)入了NF-κB與HIF-1這兩種轉(zhuǎn)錄因子結(jié)合序列的p53-GFP細胞中,KBA07對報告基因的轉(zhuǎn)錄激活受到了明顯抑制,證明KBA07的作用機制可能與這兩個轉(zhuǎn)錄因子有關(guān)。通過蛋白免疫印跡證明KBA07能有效激活HIF-1,進一步的研究還在進行中。綜上所述,我們構(gòu)建了分別基于三種重要的抑癌基因p53、Ink4a、ARF啟動子的以綠色熒光蛋白GFP作為報告基因的穩(wěn)定篩選細胞系p53-GFP,p16-GFP和p19-GFP,并對這三個篩選系統(tǒng)進行了驗證實驗,證明了它們能夠快速、特異性的對作用于這三種抑癌基因的啟動子的天然化合物小分子產(chǎn)生響應。該系統(tǒng)能對環(huán)境中的遺傳毒性物質(zhì)進行快速篩選并能對多種化合物或小分子進行篩選,快速找到靶向性明確的具有抗腫瘤潛力的化合物,對于抗腫瘤藥物的開發(fā)具有重要意義。同時我們篩選出了一些有抗腫瘤潛力的化合物例如黃楊生物堿KBA07,并將其進一步處理多種遺傳背景清晰的腫瘤細胞,對其抗腫瘤活性進行研究,證明了其抗腫瘤潛力。同時通過針對p53基因啟動子的轉(zhuǎn)錄因子結(jié)合序列競爭實驗,對KBA07的分子機制進行了初探。
[Abstract]:Cancer is the most difficult to attack in human complex diseases. At present, the incidence of cancer in our country is rising, and the malignant tumor has become the most fatal disease. The occurrence and development of cancer are the result of the combination of genetic and environmental factors, and the environmental factors include a variety of carcinogens, physical radiation, and the sense of microorganism like virus. The genetic factors include the activation of the proto oncogene, the mutation of the tumor suppressor gene or the inhibition of the expression of the gene, which leads to the loss or reduction of its function. In the road to attack cancer, the scientists of all countries are committed to the research and exploration of the tumor suppressor genes. In many tumor suppressor genes, p53 is one of the most important tumor suppressor genes and plays an important role in tumor inhibition..p53 can repair DNA damage through periodic block, induce cell apoptosis and senescence when the repair is unable to complete, and then inhibit the occurrence and development of tumor. In many malignant tumors, the level of p53 is very low. The level of the transcriptional level is the key link in gene expression. The elevation of the transcriptional level can not be separated from the transcriptional activation of the upstream transcription factor to its promoter. Therefore, we use the specific DNA damage induction ability of the p53 gene promoter to clone the initial density of the core promoter of the p53 gene. A sequence of about 750bp upstream, and a genotoxicity screening system with green fluorescent protein (GFP) as a reporter gene is constructed. This system can screen a variety of compounds in the environment by high throughput screening, and quickly identify the compounds with genetic toxicity. Besides the genetic toxicity screening, there is a study to prove that the p53 gene is not mutated. The reactivation of p53 in the tumor cells can effectively inhibit the development of tumor and even induce tumor regression. Therefore, we expect to screen various natural extracts through this screening system, in order to screen the low toxic antitumor drugs that target the p53 gene promoter as a target. In addition to the important tumor suppressor gene of p53, we have found that Mutations in the tumor suppressor gene Ink4a/ARF exist in a variety of malignant tumors, including homozygous deletion of genes, deletion of heterozygosity, mutation and promoter methylation of.P16 and P19 proteins, which belong to Rb and p53 signaling pathways, which play an important role in monitoring cell cycle and inhibiting the occurrence of tumor. The abnormal expression is associated with the occurrence and development of malignant tumors, such as the gene silencing caused by p16Ink4a gene promoter methylation in melanoma, and the ARF gene has been proved to be activated by a variety of proto oncogene activation signals, and p53 mediated tumor inhibition against the occurrence and development of cancer. The anti tumor drug screening system with GFP as the reporter gene, using the promoter region of the p16Ink4a and p19ARF genes as the target, is expected to be able to screen the low toxic antitumor natural compounds that can effectively activate the Ink4a/ARF gene promoter. In this study, our selected fourth generation promoter report carrier pGL4.82 is in the front of this study. In order to improve the detection efficiency, we replace the luciferase reporter gene on the carrier to GFP so that we can quickly observe the reporter gene through a fluorescence microscope and construct the obtained vector. The three important tumor suppressor promoter were built into the carrier and transfected into the cell lines with clear genetic background, and the stable screening cell line p53-GFP based on the promoter of the tumor suppressor gene was obtained by resistance screening. After the establishment of the p16-GFP and p19-GFP. screening system, we first introduced p53-GFP to p53-GFP The function of cell line was verified: the p53-GFP screening system was tested with adriamycin, cyclophosphamide, cisplatin, benzopyrene and other DNA toxic agents. After adriamycin treatment 6h, the expression of green fluorescent protein GFP could be observed under the fluorescence microscope, and the fluorescence intensity increased gradually with the increase of the time of drug treatment, and reached the time of 24h. At the peak, the negative control did not observe the expression of GFP. Several other DNA toxicities also observed the same results. The protein immunoblotting confirmed the expression of GFP protein. This experiment proved that the system could respond effectively to DNA damage, so we can apply it to the monitoring of environmental genotoxic compounds. The large number of cancer chemotherapy drugs used are DNA damage agents. These drugs have a great killing effect on the active and proliferating normal cells, such as bone marrow hematopoietic cells, small intestinal epithelial cells, and so on. Therefore, there is a widespread problem of drug toxicity. The high flux screening system constructed by us is based on the transcription activation of the p53 promoter. We expect to be able to screen the natural compounds that can effectively activate the expression of the tumor suppressor gene with weak cytotoxicity. Next, we used this system to screen a variety of natural compounds, including the cohosh extracts and boxwood alkaloids provided by the Kunming Plant Research Institute. The active p53 promoter is less toxic to the wild type MEF cells. Some compounds in the alkaloid of Euonymus japonicus, such as KBA01, KBA02, KBA03, KBA07, KBR11, can effectively activate the GFP expression. In order to find the low cytotoxicity and can reactivate the natural compounds of p53 in the cells, we further apply these screened compounds to the compounds. Wild type MEF cells were treated and p53 levels were increased effectively. Subsequently, we detected the cell morphology after these compounds and detected the cell apoptosis and the cycle distribution by flow cytometry. Most of them inhibited the proliferation of cells, and the apoptotic cells were significantly increased, such as KB. A03 induced the cell G1 phase block, and the apoptotic cells increased significantly after KBR18 treatment. Most of the selected poplar alkaloids had different degree of gene toxicity, but we found that the KBA07 could effectively activate the p53 gene promoter and did not have obvious genetic toxicity, and the wild type MEF cells after treatment were in the cycle and apoptosis level. There is no significant difference from the control. We think that the compound may activate the P5 gene promoter through the activation or interaction of some transcription factors upstream of the p53 gene, but it does not have strong genotoxicity itself. It is necessary to further study the mechanism of action for KBA07. In the study of the antitumor mechanism of Pu'er tea before the laboratory, We found that Pu'er tea can significantly increase the mRNA level of p16. After treating p16-GFP cells with Pu'er tea, the expression of the reporter gene GFP can be observed. It proves that Pu'er tea is activated by p16Ink4p gene promoter to activate the expression of p16, but does not exist in the dose effect. The deactivation of p16 in many malignant tumors is found, therefore, the inactivation of p16 is found in many malignant tumors. The compounds that can activate the p16 promoter have a certain antitumor potential. We use this system to screen compounds such as cohosh extract KY01, KY40 and so on, which can effectively activate p16. The mechanism of their action needs further study. When the p19-GFP screening system is verified, we transfect the pBabe-RasG12V exogenous expression to the screening system. The overexpressed RasG12V can effectively activate the p1p gene promoter and observe the obvious GFP expression in 24h after transfection. Previous studies have shown that the P19 gene promoter is sensitive and specific to the activation signal of the proto oncogene. This trait is also a valuable feature of the gene promoter, which is different from that p53 will be activated by a variety of damage signals. It may cause excessive stress. By screening a variety of compounds, there are few compounds that can activate the P19 promoter, but we are pleased to find that KBA07 can effectively activate the P19 promoter, so we think that KBA07 has high research value. In order to further study the mechanism of KBA07, we used KBA07 to treat the wild type MEF fine. Cell and a variety of genetic background clear tumor cell lines found that the transcriptional level of p53 and p1p genes increased significantly, but the elevated p53 did not activate its downstream molecule p21, which proved that it did not possess strong genotoxicity. The activation of p53 was mainly by increasing its transcriptional level. After p53 for the wild type Hela cells, the observation was observed. P53 increases and inhibits cell proliferation. When KBA07 is combined with adriamycin and cisplatin, the inhibition of cell proliferation is more obvious, suggesting that KBA07 may act as a adjuvant for chemotherapy by increasing the level of p53 protein background in cells and rapidly stimulating cell block, apoptosis and failure under the action of DNA toxicities. In order to further study the inhibition and removal of tumor cells, in order to further study the mechanism of KBA07's action on the p53 gene promoter, we found 10 transcription factor binding sites located in the p53 promoter by database prediction and query, and transfected into p53-GFP cells by synthesizing these transcription factors and transfected into p53-GFP cells. The subsequence produced competitive inhibition. The study found that in the p53-GFP cells transferred to the two transcription factor binding sequences of NF- kappa B and HIF-1, KBA07 inhibited the transcriptional activation of the reporter gene. It was proved that the mechanism of KBA07 may be related to the two transcription factors. By protein immunoblotting, it is proved that KBA07 can activate HIF-1 effectively. One step of the study is still in progress. To sum up, we have constructed a stable screening cell line p53-GFP, p16-GFP and p19-GFP based on three important tumor suppressor genes, p53, Ink4a and ARF promoters, using green fluorescent protein GFP as the reporter gene, and tested these three screening systems, proving that they can be fast and specific. Sexual response to small molecules of natural compounds that act on the promoter of these three tumor suppressor genes. The system can quickly screen the genotoxic substances in the environment and screen a variety of compounds or small molecules to quickly find targeted compounds with specific antitumor potential and develop antitumor drugs. It is of great significance. At the same time, we have screened some antitumor potential compounds, such as boxwood alkaloid KBA07, and further treated a variety of tumor cells with clear genetic background, and studied its anti-tumor activity and proved its anti-tumor potential. At the same time, the transcription factor binding sequence of the p53 gene promoter was competing with the needle. In this experiment, the molecular mechanism of KBA07 is discussed.

【學位授予單位】：昆明理工大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：Q523

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