本文選題：子宮頸癌 + 紫杉醇耐藥性��； 參考：《山東大學(xué)》2014年碩士論文

【摘要】：子宮頸癌是一種常見的婦科腫瘤,其發(fā)病率、死亡率在女性腫瘤中居首位。目前,手術(shù)治療是早期子宮頸癌的主要治療方法,而化療和放療是中晚期子宮頸癌重要的治療措施。作為一線用藥,基于紫杉醇(paclitaxel, PTX)的聯(lián)合化療是中晚期子宮頸癌患者的主要療法,普遍應(yīng)用于臨床。然而,多數(shù)子宮頸癌細(xì)胞會逐漸對PTX產(chǎn)生耐藥性,PTX耐藥性的發(fā)展已經(jīng)成為臨床上很棘手的問題。因此找到提升子宮頸癌對紫杉醇敏感性的方法很重要。 子宮頸癌的發(fā)生和發(fā)展及對化療藥物的耐受與其細(xì)胞內(nèi)活性氧(reactive oxygen species, ROS)水平和抗氧化系統(tǒng)的狀態(tài)有密切的關(guān)系,且紫杉醇的抗癌機(jī)制和效果亦與細(xì)胞氧化還原因素有密切的關(guān)系。 紫杉醇耐藥基因1(taxol resistance gene1, Txrl)是新近發(fā)現(xiàn)的與紫杉醇耐藥性有關(guān)的基因。它編碼一個18-kDa的富含脯氨酸和絲氨酸的核蛋白,其表達(dá)水平的升高被認(rèn)為可以引起PTX耐藥性。 N-乙酰半胱氨酸(N-acetylcysteine, NAC)為硫醇類抗氧化劑,它能清除細(xì)胞內(nèi)自由基,降低ROS水平,增加細(xì)胞內(nèi)的GSH水平,也可增強(qiáng)多種藥物的抗腫瘤療效。 [實(shí)驗(yàn)?zāi)康腯 建立耐紫杉醇子宮頸癌細(xì)胞株HeLa/PTX,觀察和檢測其細(xì)胞形態(tài)、細(xì)胞生長曲線、耐藥指數(shù)、氧化還原狀態(tài)及Txrl的表達(dá),并探討NAC對子宮頸癌紫杉醇耐藥性的影響。 [實(shí)驗(yàn)方法] 1.用用濃度不斷增加的PTX處理并培養(yǎng)子宮頸癌HeLa細(xì)胞,建立耐PTX子宮頸癌細(xì)胞株HeLa/PTX; 2.用倒置顯微鏡觀察HeLa和HeLa/PTX兩種細(xì)胞的形態(tài),分別繪制細(xì)胞生長曲線,并進(jìn)行細(xì)胞耐藥指數(shù)檢測； 3.用流式細(xì)胞儀檢測兩種細(xì)胞ROS水平；測定細(xì)胞還原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)水平及超氧化物歧化酶(SOD)、過氧化氫酶(CAT)和谷胱甘肽過氧化物酶(GPx)的活性水平；用RT-PCR檢測紫杉醇耐藥基因1(Txrl) mRNA的表達(dá)； 4.用NAC處理HeLa/PTX細(xì)胞,MTT檢測NAC對PTX細(xì)胞作用的影響,并檢測ROS水平及Txrl mRNA的表達(dá)的變化,來觀察NAC對其耐受PTX的影響。 [實(shí)驗(yàn)結(jié)果] 1.用PTX處理HeLa細(xì)胞10個月后,獲得在500μg/L PTX中生長狀態(tài)良好的HeLa/PTX耐藥細(xì)胞株。 2. HeLa/PTX細(xì)胞較HeLa細(xì)胞體積偏大,胞漿內(nèi)顆粒較多,增殖速度減慢,分裂高峰后移,群體倍增時(shí)間是親代細(xì)胞的1.32倍,對PTX具有顯著耐藥性。 3.與HeLa細(xì)胞相比,HeLa/PTX細(xì)胞有高水平的ROS水平,且Txrl mRNA表達(dá)明顯增高,但GSH水平及SOD、CAT和GPx的活性卻在不同程度上有所降低,而GSSG水平和GSH/GSSG比值則沒有明顯變化。 4.NAC處理后,HeLa/PTX細(xì)胞ROS水平和Txr1mRNA水平都下降,NAC可增強(qiáng)PTX對HeLa/PTX細(xì)胞的細(xì)胞毒作用,在一定程度上恢復(fù)對PTX敏感性。 [結(jié)論] 成功建立耐紫杉醇子宮頸癌細(xì)胞株HeLa/PTX的氧化還原狀態(tài)失衡,Txrl基因表達(dá)增加。NAC能降低HeLa/PTX細(xì)胞的ROS水平,降低其Txrl表達(dá),在一定程度上逆轉(zhuǎn)HeLa/PTX細(xì)胞對PTX的耐藥性。 阿爾茨海默癥(Alzheimer's disease, AD)是一種神經(jīng)系統(tǒng)退行性疾病,具有起病隱匿和進(jìn)行性發(fā)展的特點(diǎn),其發(fā)病機(jī)制及發(fā)病進(jìn)展尚未被完全了解,目前沿?zé)o有效的治療措施能阻止或逆轉(zhuǎn)病變。研究顯示,編碼淀粉樣前體蛋白(amyloid precursor protein, APP)、早老素(presenilins)1和2三種蛋白的基因突變后,可引起β-淀粉樣肽(P-amyloid peptide, Aβ)表達(dá)增加。Aβ的沉積和聚集是AD主要病理學(xué)特征之一,也是AD發(fā)病的關(guān)鍵因素之一。 神經(jīng)內(nèi)肽酶(neprilysin, NEP)是一種Ⅱ型鋅依賴的膜結(jié)合型肽鏈內(nèi)切酶,近年來被認(rèn)為是腦內(nèi)催化Aβ降解的關(guān)鍵酶,也是維持腦內(nèi)Aβ合成與降解平衡的重要因素。由于提高NEP的酶活性或者增加其表達(dá),有助于Aβ的降解,因此NEP是治療的潛在的新靶點(diǎn)。 人參皂苷Rg3(ginsenoside Rg3),是從人參中提取的特有活性物質(zhì)之一,能提升記憶和認(rèn)知等能力,具有神經(jīng)保護(hù)作用。研究顯示,在AD的細(xì)胞模型和動物模型中,Rg3可以明顯減少Aβ的沉積,因此在AD治療中很有應(yīng)用前景。 [研究目的] 本研究選用人參皂苷Rg3,處理NEP啟動子缺失體-熒光素酶報(bào)告基因質(zhì)粒轉(zhuǎn)染的人神經(jīng)母細(xì)胞瘤SH-SY5Y細(xì)胞,探查NEP上游調(diào)控區(qū)域與Rg3影響NEP啟動子活性有關(guān)的位點(diǎn)及相應(yīng)轉(zhuǎn)錄因子表達(dá)情況,初步探討Rg3促進(jìn)NEP基因表達(dá)的分子機(jī)制。 [研究方法] 1.用pGL3-nep2.4重組質(zhì)粒瞬時(shí)轉(zhuǎn)染人神經(jīng)瘤細(xì)胞母細(xì)胞SH-SY5Y,并用Rg3 處理轉(zhuǎn)染細(xì)胞,檢測其雙熒光素酶活性。 2.用Rg3處理前期實(shí)驗(yàn)篩選出的Region Ⅰ、Ⅱ、Ⅲ、Ⅳ的缺失前后突變體質(zhì)粒轉(zhuǎn)染的SH-SY5Y細(xì)胞,檢測其雙熒光素酶活性。 3.用轉(zhuǎn)錄因子分析軟件TFSearch與TFBIND分析Rg3發(fā)揮調(diào)控作用的序列,檢索相關(guān)轉(zhuǎn)錄因子的識別序列。 4.用Rg3處理SH-SY5Y細(xì)胞,采用RT-PCR, Western blot法檢測相關(guān)轉(zhuǎn)錄因子的表達(dá)。 [實(shí)驗(yàn)結(jié)果] 1.熒光素酶活性檢測顯示,轉(zhuǎn)染pGL3-nep2.4質(zhì)粒細(xì)胞熒光素酶活性介于轉(zhuǎn)染pGL3-basic和pGL3-control的之間,是轉(zhuǎn)染pGL3-basic的13.1倍。Rg3處理后,轉(zhuǎn)染pGL3-nep2.4細(xì)胞熒光素酶活性是未處理的1.98倍。 2.Rg3處理后,Region Ⅰ、Ⅲ和Ⅳ缺失前后缺失體質(zhì)粒轉(zhuǎn)染細(xì)胞熒光素酶活性均與未處理的無明顯差異,而Region Ⅱ未缺失質(zhì)粒轉(zhuǎn)染細(xì)胞的熒光素酶活性是未處理的3.20倍,Region Ⅱ缺失質(zhì)粒轉(zhuǎn)染細(xì)胞的熒光素酶活性與未處理的無明顯差異。 3.轉(zhuǎn)錄因子分析軟件檢索提示,Region Ⅱ序列可能結(jié)合的轉(zhuǎn)錄因子有HSF1、SRY、C/EBPβ、CP2、GATA3等。 4. RT-PCR結(jié)果顯示,Rg3可以增加C/EBPβ mRNA水平,降低HSF1mRNA水平。Western Blot分析結(jié)果顯示,Rg3可明顯增加C/EBPβ蛋白的水平。 [結(jié)論] 1.重組質(zhì)粒pGL3-nep2.4在SH-SY5Y細(xì)胞中表現(xiàn)出很強(qiáng)的啟動子活性,且Rg3能明顯增加其啟動子活性； 2.NEP基因2.4kb啟動調(diào)控片段有4個調(diào)控區(qū)域(Region Ⅰ、Ⅱ、Ⅲ和Ⅳ),而Rg3通過Region Ⅱ發(fā)揮調(diào)控作用。 3.Rg3可明顯增加轉(zhuǎn)錄因子C/EBPβ的表達(dá),說明Rg3可能通過轉(zhuǎn)錄因子C/EBPβ來影響NEP基因的表達(dá)。
[Abstract]:Cervical cancer is a common gynecologic tumor. Its incidence and mortality are the first in female tumors. Currently, surgical treatment is the main treatment for early cervical cancer. Chemotherapy and radiotherapy are important treatment measures for middle and late cervical cancer. As a first-line drug, combined chemotherapy based on paclitaxel (PTX) is in the middle and late stage. The main treatment of cervical cancer is widely used in clinical practice. However, most cervical cancer cells will gradually develop resistance to PTX. The development of PTX resistance has become a difficult problem in clinical. Therefore, it is important to find a way to improve the sensitivity of cervical cancer to taxol.
The occurrence and development of cervical cancer and the tolerance to chemotherapy drugs are closely related to the level of intracellular reactive oxygen species (reactive oxygen species, ROS) and the state of antioxidant system, and the anticancer mechanism and effect of paclitaxel are also closely related to the redox factors of cell.
The taxol resistance gene 1 (taxol resistance gene1, Txrl) is a newly discovered gene associated with taxol resistance. It encodes a 18-kDa rich in proline and serine nucleoprotein, which is believed to cause PTX resistance.
N- acetyl cysteine (N-acetylcysteine, NAC) is a thiol antioxidant. It can remove free radicals in cells, reduce ROS level, increase the level of GSH in cells, and enhance the antitumor effect of various drugs.
[the purpose of the experiment]
The cell morphology, cell growth curve, drug resistance index, redox state and Txrl expression of taxol resistant cervical cancer cell line HeLa/PTX were observed and detected, and the effect of NAC on taxol resistance of cervical cancer was investigated.
[experimental method]
1. the PTX cervical cancer HeLa cells were treated and cultured with increasing concentration of PTX, and PTX resistant cervical cancer cell line HeLa/PTX was established.
2. the morphology of HeLa and HeLa/PTX two kinds of cells were observed by inverted microscope, and cell growth curves were drawn respectively, and the cell resistance index was detected.
3. the level of two cells ROS was detected by flow cytometry; the levels of glutathione (GSH) and oxidized glutathione (GSSG), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were measured, and the expression of taxol resistance gene 1 (Txrl) mRNA was detected by RT-PCR.
4. HeLa/PTX cells were treated with NAC, and MTT was used to detect the effect of NAC on PTX cells, and the changes of ROS level and the expression of Txrl mRNA were detected to observe the effect of NAC on its tolerance to PTX.
[experimental results]
1. after 10 months of HeLa treatment with PTX, the HeLa/PTX resistant cell line, which grew well in 500 g/L PTX, was obtained.
The volume of 2. HeLa/PTX cells was larger than that of HeLa cells, with more granules in the cytoplasm, slower proliferation rate, and after the peak shift. The time of population multiplication was 1.32 times more than that of the parent cells, and the resistance to PTX was significant.
3. compared with HeLa cells, HeLa/PTX cells had a high level of ROS level, and the expression of Txrl mRNA increased significantly, but the GSH level and the activity of SOD, CAT and GPx decreased in varying degrees, while the GSSG level and GSH/GSSG ratio did not change significantly.
After 4.NAC treatment, both the level of ROS and the level of Txr1mRNA decreased in HeLa/PTX cells. NAC enhanced the cytotoxic effect of PTX on HeLa/PTX cells and, to a certain extent, restored the sensitivity of PTX to PTX.
[Conclusion]
The redox state of the cell line of taxol resistant cervical cancer cell line HeLa/PTX was successfully established, and the expression of.NAC could reduce the ROS level of HeLa/PTX cells and reduce the Txrl expression in HeLa/PTX cells, and to a certain extent reversed the resistance of HeLa/PTX cells to PTX.
Blzheimer (Alzheimer's disease, AD) is a kind of neurodegenerative disease with the characteristics of the occult and progressive development of the disease. Its pathogenesis and progress have not been fully understood. At present, there is no effective treatment to prevent or reverse the disease. The study shows that the amyloid precursor prot (amyloid precursor prot) is encoded. Ein, APP), the gene mutation of presenilins 1 and 2 proteins can cause the expression of beta amyloid peptide (P-amyloid peptide, A beta) to increase the deposition and accumulation of.A beta, which is one of the main pathological characteristics of AD, and also one of the key factors for the pathogenesis of AD.
Neprilysin (NEP) is a kind of type II zinc dependent membrane bound endopeptidase. In recent years, it has been considered as the key enzyme to catalyze the degradation of A beta in the brain. It is also an important factor to maintain the balance of A beta synthesis and degradation in the brain. It is potential for NEP to improve the degradation of A beta because of increasing the enzyme activity of NEP or increasing its appearance. New targets.
Ginsenoside Rg3 (ginsenoside Rg3), one of the endemic active substances extracted from ginseng, can enhance memory and cognition, and has a neuroprotective effect. The research shows that Rg3 can obviously reduce the deposition of A beta in the cell and animal models of AD, so it is very promising in the treatment of AD.
[research purposes]
In this study, ginsenoside Rg3 was used to treat human neuroblastoma SH-SY5Y cells transfected by NEP promoter deletion luciferase reporter gene plasmid, and to explore the molecular mechanism of Rg3 to promote the expression of NEP gene by exploring the loci of the upstream regulation area of NEP and the expression of the corresponding transcription factors related to the activity of NEP promoter by Rg3.
[research methods]
1. transiently transfected human neuroma cell mother cell SH-SY5Y with pGL3-nep2.4 recombinant plasmid, and using Rg3
The transfected cells were treated and their double luciferase activity was detected.
2. the SH-SY5Y cells transfected with mutant plasmid were detected by Rg3 before and after the deletion of Region I, II, III and IV, and their dual luciferase activity was detected.
3. using transcription factor analysis software TFSearch and TFBIND to analyze the sequence of Rg3 regulatory action and retrieve the recognition sequence of related transcription factors.
4. SH-SY5Y cells were treated with Rg3, and the transcription factors were detected by RT-PCR and Western blot.
[experimental results]
1. luciferase activity assay showed that the luciferase activity of transfected pGL3-nep2.4 plasmids was between pGL3-basic and pGL3-control, and the 13.1 times.Rg3 treatment of pGL3-basic transfected, the luciferase activity of transfected pGL3-nep2.4 cells was 1.98 times that of untreated.
After 2.Rg3 treatment, the luciferase activity of Region I, III and IV deleted plasmid transfected cells were not significantly different from that of untreated cells, but the luciferase activity of Region II plasmid transfected cells was 3.20 times that of untreated cells, and the luciferase activity of Region II deleted plasmids transfected cells was not significantly different from that of untreated cells.
3. transcription factor analysis software indicates that the transcription factors associated with Region II sequence are HSF1, SRY, C/EBP beta, CP2, GATA3 and so on.
4. RT-PCR results showed that Rg3 could increase the level of C/EBP beta mRNA and reduce the HSF1mRNA level..Western Blot analysis showed that Rg3 could significantly increase the level of C/EBP beta protein.
[Conclusion]
1. recombinant plasmid pGL3-nep2.4 showed strong promoter activity in SH-SY5Y cells, and Rg3 significantly increased its promoter activity.
2.NEP gene 2.4kb has 4 regulatory regions (Region I, II, III and IV), and Rg3 plays a regulatory role through Region II.
3.Rg3 can significantly increase the expression of transcription factor C/EBP beta, indicating that Rg3 may affect the expression of NEP gene through transcription factor C/EBP beta.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.33

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