CXCL1基因在卵巢恶性肿瘤生物学作用的研究
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摘要
第一章卵巢肿瘤血清诊断标志物筛选及临床验证研究近况(文献综述)
卵巢上皮癌是妇科恶性肿瘤中发病率占第三位,死亡率却居第一的恶性疾病。由于卵巢解剖上隐匿于盆腔深处,在发病早期缺乏特异性症状和体征,很难在早期发现和诊断,约60%-70%的卵巢癌患者确诊时已属于晚期。近30年来,虽然经过全世界妇科肿瘤专家的不懈努力,不断改进手术方式,发现更新更好的化疗药物,增加新的化疗方案,但是5年生存率一直徘徊在20%-30%左右。所以,寻找新的早期筛查,发现卵巢癌的方法和新的治疗手段是目前卵巢癌研究的重点。本研究采用实时荧光定量PCR的方法检测了CXCL1在卵巢癌和卵巢良性病变组织及正常组织中的表达情况,并进一步研究揭示了其表达和卵巢癌临床资料之间的关系。接着构建了CXCL1基因慢病毒表达载体,并通过一系列体外实验,对CXCL1在卵巢癌生长增殖过程中的作用及其机制做了研究。
第二章CXCL1基因在卵巢组织中的表达检测及临床意义初探
目的：探讨CXCL1 (Chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha)基因mRNA在卵巢肿瘤患者组织中的表达与其临床病理的相关性。方法：采用Trizol一步法提取卵巢肿瘤患者卵巢组织总RNA和实时荧光定量PCR技术对80例卵巢恶性肿瘤,40例卵巢良性病变及20例正常卵巢组织中CXCL1基因表达进行定量分析,并分析其与临床病理的相关性。结果：CXCL1mRNA在卵巢癌中表达量比在卵巢良性病变中明显升高(P值<0.01)；卵巢恶性肿瘤晚期(Ⅲ-Ⅳ)患者组织CXCL1表达量显著高于早期(Ⅰ～Ⅱ)患者,差异有统计学意义(P=0.005)。但Kaplan-Meier生存曲线结果显示,肿瘤组织中CXCL1基因表达与其预后无关,Cox比例风险模型分析未显示CXCL1表达量是独立判断卵巢恶性肿瘤患者预后的因素。结论：卵巢癌患者肿瘤组织CXCL1mRNA水平显著高于良性及正常对照组,可能与恶性肿瘤的发生发展有关。
第三章CXCL1基因表达载体的构建及鉴定
目的：采用第二代慢病毒表达载体系统,构建了CXCL1基因的慢病毒表达载体,摸索出较传统转染方法更为高效、可靠的转基因方法,为今后进一步的基因靶向治疗研究打下良好的基础。方法：首先扩增CXCL1全长序列,将其与慢病毒载体pWPI连接后测序,通过BLAST检索,鉴定慢病毒表达载体构建成功。将重组慢病毒质粒转染293T细胞,48小时后荧光显微镜鉴定,慢病毒转染并包装成功。结果：重组慢病毒质粒CXCL1-pWPI测序结果经BLAST,与CXCL1基因的同源性达99%。结论：成功的构建了CXCL1基因的重组慢病毒表达系统。为今后的CXCL1基因功能研究奠定了基础。
第四章CXCL1基因介导对卵巢上皮癌生物学功能影响体外实验研究
目的：了解CXCL1对卵巢癌细胞功能的影响,分析CXCL1在卵巢癌生长以及浸润转移过程中所起的作用。方法：成功提取含有CXCL1基因的慢病毒颗粒后,将其感染无CXCL1基因表达的卵巢癌细胞株A2780,经荧光显微镜和RT-PCR鉴定确认获得稳定转染的A2780细胞株。然后进行细胞生长特性、细胞周期变化、黏附能力的实验。结果：通过慢病毒转染的方法,获得能够稳定表达CXCL1基因的卵巢癌细胞株。MTT法检测表明,CXCL1组和对照组的生长曲线有显著差异,CXCL1组的细胞生长速度明显加快。CXCL1组的克隆形成率大于对照组和空白组,差异有统计学意义。CXCL1对A2780细胞的细胞周期和侵袭能力有影响。结论：细胞功能实验结果表明,CXCL1能够增强卵巢癌A2780细胞的生长和增殖能力。对A2780细胞的迁移能力无影响。
Chapter 1 The study of the biological effects of CXCL1 gene in ovarian cancer
Ovarian cancer is the gynecologic malignancy in the incidence of third place in the mortality rate ranks,but No.1 in the maliganat disease.As the ovary in pelvic anatomy on the hidden depths of the early onset of the lack of specific symptoms and signs, difficulty in early detection and diagnosis, about 60%-70% of ovarian cancer when patients are diagnosed late. The past 30 years, gynecologic oncology experts around the world despite the tireless efforts to continuously improve the surgical procedure and found that newer and better chemotherapeutic drugs, the addition of new chemotherapy regimens, but the 5-year survival rate has hovered around 20%-30%. Therefore, the search for new early screening methods for ovarian cancer was found and a new ovarian cancer treatment is the focus of the study. In this study, real time quantitative PCR method to detect ovarian cancer and ovarian CXCL1 in benign lesions and normal tissues, and to further reveal its expression and clinical data of ovarian cancer between. Then constructed lentiviral CXCL1 gene expression vector, and through a series of in vitro experiments, CXCL1 in the process of growth and proliferation of ovarian cancer and its mechanism were investigated.
Chapter 2 The study on the relationship between the expression of CXCUmRNA and the Clinical-pathology factor in ovarian tumor
Objective:To investigate the relationship between the expression of CXCL1 (Chemokine (CXC motif) ligand 1 (melanoma growth stimulating activity, alpha) gene mRNA in ovarian tumor and the clinical-pathology factor.
Methods:Trizol one-step method was used to extract the total RNA in ovarian tissue, and real-time fluorescence quantitative PCR technique was used to detect the expression copy number of CXCL1 gene in 80 cases of ovarian cancer,40 cases of ovarian benign lesions and 20 normal control. As well as the relationship between the expression of CXCL1 gene mRNA in ovarian tumor and the clinical-pathology factor was analyzed.
Results:The expression copy number of CXCL1 mRNA in ovarian malignancy was higher than that in benign lesions as well as normal control. There was a difference significantly (P<0.01); In advanced ovarian cancer (Ⅲ-Ⅳstage), The expression of CXCL1 mRNA was also significantly higher than that in the patients with early stage (Ⅰ～Ⅱstage)(P=0.005). The results of Kaplan-Meier survival curve showed that there was no relationship between the expression of CXCL1 mRNA and the prognosis in ovarian cancer, and the analysis of Cox proportional hazard model also showed CXCL1 expression was not independent prognostic factors. For patients with ovarian cancer.
Conclusions:The expression copy number of CXCL1 mRNA in ovarian malignant was higher significantly than that in benign lesions as well as normal control. It was suggesting that the over-expression of CXCL1mRNA should be related with occurrence and development of ovarian malignant
Chapter 3 The construction and identification of CXCL1 gene expression vector
Objective:Using the second-generation lentiviral vector system to constructe lentiviral CXCL1 gene expression vector, and exploring the methods of gene transfer more efficient and reliable than traditional methods, laying a good basis for gene targeted therapy in the further.
Methods:Amplification CXCL1 full sequence of its connection with the lentiviral vector pWPI sequenced by BLAST search, identification of lentiviral expression vectors. The recombinant lentiviral vector transfected 293T cells, identified by fluorescence microscopy after 48 hours, lentivirus infection and packaging success.
Results:The recombinant lentiviral vector CXCL1-pWPI sequencing by BLAST, and the CXCL1 gene homology 99%.
Conclusion:Successfully constructed recombinant lentivirus CXCL1 expression system. Laying the foundation for CXCL1 gene function in future research.
Chapter 4 Experimental study of CXCL1-mediated gene on the biological function of epithelial ovarian cancer in vitro
Objective:To understand the function of CXCL1 impact on ovarian cancer,analysis CXCL1 in ovarian cancer growth and metastasis the role of the process.
Methods:The successful extraction of CXCL1 gene containing lentiviral particles, will be transfected into non-CXCL1 gene expression in ovarian cancer cell line A2780, by fluorescence microscopy and RT-PCR to confirm identification of stable transfected A2780 cell lines. And then cell growth, cell cycle experiment.
Results:Lentiviral infection, we obtain stable expression of CXCL1 gene ovarian cancer cell line. MTT assay showed that, CXCL1 and control groups of growth curves were significantly different, CXCL1 group significantly faster cell growth. CXCL1 group of colony formation rate than the control group and the control group, the difference was statistically significant.
Conclusion:The results show that cell function, CXCL1 can enhance the A2780 ovarian cancer cell growth and proliferation.
卵巢上皮癌是妇科恶性肿瘤中发病率占第三位,死亡率却居第一的恶性疾病。由于卵巢解剖上隐匿于盆腔深处,在发病早期缺乏特异性症状和体征,很难在早期发现和诊断,约60%-70%的卵巢癌患者确诊时已属于晚期。近30年来,虽然经过全世界妇科肿瘤专家的不懈努力,不断改进手术方式,发现更新更好的化疗药物,增加新的化疗方案,但是5年生存率一直徘徊在20%-30%左右。所以,寻找新的早期筛查,发现卵巢癌的方法和新的治疗手段是目前卵巢癌研究的重点。本研究采用实时荧光定量PCR的方法检测了CXCL1在卵巢癌和卵巢良性病变组织及正常组织中的表达情况,并进一步研究揭示了其表达和卵巢癌临床资料之间的关系。接着构建了CXCL1基因慢病毒表达载体,并通过一系列体外实验,对CXCL1在卵巢癌生长增殖过程中的作用及其机制做了研究。
第二章CXCL1基因在卵巢组织中的表达检测及临床意义初探
目的：探讨CXCL1 (Chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha)基因mRNA在卵巢肿瘤患者组织中的表达与其临床病理的相关性。方法：采用Trizol一步法提取卵巢肿瘤患者卵巢组织总RNA和实时荧光定量PCR技术对80例卵巢恶性肿瘤,40例卵巢良性病变及20例正常卵巢组织中CXCL1基因表达进行定量分析,并分析其与临床病理的相关性。结果：CXCL1mRNA在卵巢癌中表达量比在卵巢良性病变中明显升高(P值<0.01)；卵巢恶性肿瘤晚期(Ⅲ-Ⅳ)患者组织CXCL1表达量显著高于早期(Ⅰ～Ⅱ)患者,差异有统计学意义(P=0.005)。但Kaplan-Meier生存曲线结果显示,肿瘤组织中CXCL1基因表达与其预后无关,Cox比例风险模型分析未显示CXCL1表达量是独立判断卵巢恶性肿瘤患者预后的因素。结论：卵巢癌患者肿瘤组织CXCL1mRNA水平显著高于良性及正常对照组,可能与恶性肿瘤的发生发展有关。
第三章CXCL1基因表达载体的构建及鉴定
目的：采用第二代慢病毒表达载体系统,构建了CXCL1基因的慢病毒表达载体,摸索出较传统转染方法更为高效、可靠的转基因方法,为今后进一步的基因靶向治疗研究打下良好的基础。方法：首先扩增CXCL1全长序列,将其与慢病毒载体pWPI连接后测序,通过BLAST检索,鉴定慢病毒表达载体构建成功。将重组慢病毒质粒转染293T细胞,48小时后荧光显微镜鉴定,慢病毒转染并包装成功。结果：重组慢病毒质粒CXCL1-pWPI测序结果经BLAST,与CXCL1基因的同源性达99%。结论：成功的构建了CXCL1基因的重组慢病毒表达系统。为今后的CXCL1基因功能研究奠定了基础。
第四章CXCL1基因介导对卵巢上皮癌生物学功能影响体外实验研究
目的：了解CXCL1对卵巢癌细胞功能的影响,分析CXCL1在卵巢癌生长以及浸润转移过程中所起的作用。方法：成功提取含有CXCL1基因的慢病毒颗粒后,将其感染无CXCL1基因表达的卵巢癌细胞株A2780,经荧光显微镜和RT-PCR鉴定确认获得稳定转染的A2780细胞株。然后进行细胞生长特性、细胞周期变化、黏附能力的实验。结果：通过慢病毒转染的方法,获得能够稳定表达CXCL1基因的卵巢癌细胞株。MTT法检测表明,CXCL1组和对照组的生长曲线有显著差异,CXCL1组的细胞生长速度明显加快。CXCL1组的克隆形成率大于对照组和空白组,差异有统计学意义。CXCL1对A2780细胞的细胞周期和侵袭能力有影响。结论：细胞功能实验结果表明,CXCL1能够增强卵巢癌A2780细胞的生长和增殖能力。对A2780细胞的迁移能力无影响。
Chapter 1 The study of the biological effects of CXCL1 gene in ovarian cancer
Ovarian cancer is the gynecologic malignancy in the incidence of third place in the mortality rate ranks,but No.1 in the maliganat disease.As the ovary in pelvic anatomy on the hidden depths of the early onset of the lack of specific symptoms and signs, difficulty in early detection and diagnosis, about 60%-70% of ovarian cancer when patients are diagnosed late. The past 30 years, gynecologic oncology experts around the world despite the tireless efforts to continuously improve the surgical procedure and found that newer and better chemotherapeutic drugs, the addition of new chemotherapy regimens, but the 5-year survival rate has hovered around 20%-30%. Therefore, the search for new early screening methods for ovarian cancer was found and a new ovarian cancer treatment is the focus of the study. In this study, real time quantitative PCR method to detect ovarian cancer and ovarian CXCL1 in benign lesions and normal tissues, and to further reveal its expression and clinical data of ovarian cancer between. Then constructed lentiviral CXCL1 gene expression vector, and through a series of in vitro experiments, CXCL1 in the process of growth and proliferation of ovarian cancer and its mechanism were investigated.
Chapter 2 The study on the relationship between the expression of CXCUmRNA and the Clinical-pathology factor in ovarian tumor
Objective:To investigate the relationship between the expression of CXCL1 (Chemokine (CXC motif) ligand 1 (melanoma growth stimulating activity, alpha) gene mRNA in ovarian tumor and the clinical-pathology factor.
Methods:Trizol one-step method was used to extract the total RNA in ovarian tissue, and real-time fluorescence quantitative PCR technique was used to detect the expression copy number of CXCL1 gene in 80 cases of ovarian cancer,40 cases of ovarian benign lesions and 20 normal control. As well as the relationship between the expression of CXCL1 gene mRNA in ovarian tumor and the clinical-pathology factor was analyzed.
Results:The expression copy number of CXCL1 mRNA in ovarian malignancy was higher than that in benign lesions as well as normal control. There was a difference significantly (P<0.01); In advanced ovarian cancer (Ⅲ-Ⅳstage), The expression of CXCL1 mRNA was also significantly higher than that in the patients with early stage (Ⅰ～Ⅱstage)(P=0.005). The results of Kaplan-Meier survival curve showed that there was no relationship between the expression of CXCL1 mRNA and the prognosis in ovarian cancer, and the analysis of Cox proportional hazard model also showed CXCL1 expression was not independent prognostic factors. For patients with ovarian cancer.
Conclusions:The expression copy number of CXCL1 mRNA in ovarian malignant was higher significantly than that in benign lesions as well as normal control. It was suggesting that the over-expression of CXCL1mRNA should be related with occurrence and development of ovarian malignant
Chapter 3 The construction and identification of CXCL1 gene expression vector
Objective:Using the second-generation lentiviral vector system to constructe lentiviral CXCL1 gene expression vector, and exploring the methods of gene transfer more efficient and reliable than traditional methods, laying a good basis for gene targeted therapy in the further.
Methods:Amplification CXCL1 full sequence of its connection with the lentiviral vector pWPI sequenced by BLAST search, identification of lentiviral expression vectors. The recombinant lentiviral vector transfected 293T cells, identified by fluorescence microscopy after 48 hours, lentivirus infection and packaging success.
Results:The recombinant lentiviral vector CXCL1-pWPI sequencing by BLAST, and the CXCL1 gene homology 99%.
Conclusion:Successfully constructed recombinant lentivirus CXCL1 expression system. Laying the foundation for CXCL1 gene function in future research.
Chapter 4 Experimental study of CXCL1-mediated gene on the biological function of epithelial ovarian cancer in vitro
Objective:To understand the function of CXCL1 impact on ovarian cancer,analysis CXCL1 in ovarian cancer growth and metastasis the role of the process.
Methods:The successful extraction of CXCL1 gene containing lentiviral particles, will be transfected into non-CXCL1 gene expression in ovarian cancer cell line A2780, by fluorescence microscopy and RT-PCR to confirm identification of stable transfected A2780 cell lines. And then cell growth, cell cycle experiment.
Results:Lentiviral infection, we obtain stable expression of CXCL1 gene ovarian cancer cell line. MTT assay showed that, CXCL1 and control groups of growth curves were significantly different, CXCL1 group significantly faster cell growth. CXCL1 group of colony formation rate than the control group and the control group, the difference was statistically significant.
Conclusion:The results show that cell function, CXCL1 can enhance the A2780 ovarian cancer cell growth and proliferation.
引文
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