子宫颈癌细胞紫杉醇耐药性的研究与RG3影响NEP基因启动子活性的研究

发布时间：2018-05-23 16:17

本文选题：子宫颈癌 + 紫杉醇耐药性　；参考：《山东大学》2014年硕士论文

【摘要】：子宫颈癌是一种常见的妇科肿瘤,其发病率、死亡率在女性肿瘤中居首位。目前,手术治疗是早期子宫颈癌的主要治疗方法,而化疗和放疗是中晚期子宫颈癌重要的治疗措施。作为一线用药,基于紫杉醇(paclitaxel, PTX)的联合化疗是中晚期子宫颈癌患者的主要疗法,普遍应用于临床。然而,多数子宫颈癌细胞会逐渐对PTX产生耐药性,PTX耐药性的发展已经成为临床上很棘手的问题。因此找到提升子宫颈癌对紫杉醇敏感性的方法很重要。子宫颈癌的发生和发展及对化疗药物的耐受与其细胞内活性氧(reactive oxygen species, ROS)水平和抗氧化系统的状态有密切的关系,且紫杉醇的抗癌机制和效果亦与细胞氧化还原因素有密切的关系。紫杉醇耐药基因1(taxol resistance gene1, Txrl)是新近发现的与紫杉醇耐药性有关的基因。它编码一个18-kDa的富含脯氨酸和丝氨酸的核蛋白,其表达水平的升高被认为可以引起PTX耐药性。 N-乙酰半胱氨酸(N-acetylcysteine, NAC)为硫醇类抗氧化剂,它能清除细胞内自由基,降低ROS水平,增加细胞内的GSH水平,也可增强多种药物的抗肿瘤疗效。 [实验目的] 建立耐紫杉醇子宫颈癌细胞株HeLa/PTX,观察和检测其细胞形态、细胞生长曲线、耐药指数、氧化还原状态及Txrl的表达,并探讨NAC对子宫颈癌紫杉醇耐药性的影响。 [实验方法] 1.用用浓度不断增加的PTX处理并培养子宫颈癌HeLa细胞,建立耐PTX子宫颈癌细胞株HeLa/PTX; 2.用倒置显微镜观察HeLa和HeLa/PTX两种细胞的形态,分别绘制细胞生长曲线,并进行细胞耐药指数检测； 3.用流式细胞仪检测两种细胞ROS水平；测定细胞还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)水平及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GPx)的活性水平；用RT-PCR检测紫杉醇耐药基因1(Txrl) mRNA的表达； 4.用NAC处理HeLa/PTX细胞,MTT检测NAC对PTX细胞作用的影响,并检测ROS水平及Txrl mRNA的表达的变化,来观察NAC对其耐受PTX的影响。 [实验结果] 1.用PTX处理HeLa细胞10个月后,获得在500μg/L PTX中生长状态良好的HeLa/PTX耐药细胞株。 2. HeLa/PTX细胞较HeLa细胞体积偏大,胞浆内颗粒较多,增殖速度减慢,分裂高峰后移,群体倍增时间是亲代细胞的1.32倍,对PTX具有显著耐药性。 3.与HeLa细胞相比,HeLa/PTX细胞有高水平的ROS水平,且Txrl mRNA表达明显增高,但GSH水平及SOD、CAT和GPx的活性却在不同程度上有所降低,而GSSG水平和GSH/GSSG比值则没有明显变化。 4.NAC处理后,HeLa/PTX细胞ROS水平和Txr1mRNA水平都下降,NAC可增强PTX对HeLa/PTX细胞的细胞毒作用,在一定程度上恢复对PTX敏感性。 [结论] 成功建立耐紫杉醇子宫颈癌细胞株HeLa/PTX的氧化还原状态失衡,Txrl基因表达增加。NAC能降低HeLa/PTX细胞的ROS水平,降低其Txrl表达,在一定程度上逆转HeLa/PTX细胞对PTX的耐药性。阿尔茨海默症(Alzheimer's disease, AD)是一种神经系统退行性疾病,具有起病隐匿和进行性发展的特点,其发病机制及发病进展尚未被完全了解,目前沿无有效的治疗措施能阻止或逆转病变。研究显示,编码淀粉样前体蛋白(amyloid precursor protein, APP)、早老素(presenilins)1和2三种蛋白的基因突变后,可引起β-淀粉样肽(P-amyloid peptide, Aβ)表达增加。Aβ的沉积和聚集是AD主要病理学特征之一,也是AD发病的关键因素之一。神经内肽酶(neprilysin, NEP)是一种Ⅱ型锌依赖的膜结合型肽链内切酶,近年来被认为是脑内催化Aβ降解的关键酶,也是维持脑内Aβ合成与降解平衡的重要因素。由于提高NEP的酶活性或者增加其表达,有助于Aβ的降解,因此NEP是治疗的潜在的新靶点。人参皂苷Rg3(ginsenoside Rg3),是从人参中提取的特有活性物质之一,能提升记忆和认知等能力,具有神经保护作用。研究显示,在AD的细胞模型和动物模型中,Rg3可以明显减少Aβ的沉积,因此在AD治疗中很有应用前景。 [研究目的] 本研究选用人参皂苷Rg3,处理NEP启动子缺失体-荧光素酶报告基因质粒转染的人神经母细胞瘤SH-SY5Y细胞,探查NEP上游调控区域与Rg3影响NEP启动子活性有关的位点及相应转录因子表达情况,初步探讨Rg3促进NEP基因表达的分子机制。 [研究方法] 1.用pGL3-nep2.4重组质粒瞬时转染人神经瘤细胞母细胞SH-SY5Y,并用Rg3 处理转染细胞,检测其双荧光素酶活性。 2.用Rg3处理前期实验筛选出的Region Ⅰ、Ⅱ、Ⅲ、Ⅳ的缺失前后突变体质粒转染的SH-SY5Y细胞,检测其双荧光素酶活性。 3.用转录因子分析软件TFSearch与TFBIND分析Rg3发挥调控作用的序列,检索相关转录因子的识别序列。 4.用Rg3处理SH-SY5Y细胞,采用RT-PCR, Western blot法检测相关转录因子的表达。 [实验结果] 1.荧光素酶活性检测显示,转染pGL3-nep2.4质粒细胞荧光素酶活性介于转染pGL3-basic和pGL3-control的之间,是转染pGL3-basic的13.1倍。Rg3处理后,转染pGL3-nep2.4细胞荧光素酶活性是未处理的1.98倍。 2.Rg3处理后,Region Ⅰ、Ⅲ和Ⅳ缺失前后缺失体质粒转染细胞荧光素酶活性均与未处理的无明显差异,而Region Ⅱ未缺失质粒转染细胞的荧光素酶活性是未处理的3.20倍,Region Ⅱ缺失质粒转染细胞的荧光素酶活性与未处理的无明显差异。 3.转录因子分析软件检索提示,Region Ⅱ序列可能结合的转录因子有HSF1、SRY、C/EBPβ、CP2、GATA3等。 4. RT-PCR结果显示,Rg3可以增加C/EBPβ mRNA水平,降低HSF1mRNA水平。Western Blot分析结果显示,Rg3可明显增加C/EBPβ蛋白的水平。 [结论] 1.重组质粒pGL3-nep2.4在SH-SY5Y细胞中表现出很强的启动子活性,且Rg3能明显增加其启动子活性； 2.NEP基因2.4kb启动调控片段有4个调控区域(Region Ⅰ、Ⅱ、Ⅲ和Ⅳ),而Rg3通过Region Ⅱ发挥调控作用。 3.Rg3可明显增加转录因子C/EBPβ的表达,说明Rg3可能通过转录因子C/EBPβ来影响NEP基因的表达。
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

【共引文献】