CK2抑制剂CX4945逆转胃癌耐药细胞放射抗性的分子基础及临床研究
发布时间：2018-08-04 08:56
【摘要】：第一部分CK2抑制剂CX-4945逆转胃癌耐药细胞放射抗性的作用机制在我国,胃癌的发病与死亡处于恶性肿瘤第三位。据统计,2012年全国胃癌发病数约40万,死亡数约32万,胃癌病人五年生存率不足20%。手术、化疗、放疗是治疗胃癌的主要手段。早期胃癌可通过手术方式切除局部肿瘤病灶,晚期胃癌由于发生广泛浸润和转移,失去手术机会,则以放化疗联合治疗方案为主。肿瘤细胞对各种放化疗措施的耐受性往往导致放化疗失败。顺铂是胃癌化疗一线用药,主要通过诱导DNA交联—双链断裂损伤杀伤肿瘤细胞。肿瘤细胞可以通过发展多种耐药机制抵抗顺铂诱导的细胞凋亡,其中DNA损伤修复能力增强是最主要的机制。而放疗主要靶向DNA,通过诱导DNA双链断裂损伤抑制细胞增殖,引发细胞凋亡。对顺铂耐受的肿瘤细胞同时也会对放疗产生交叉耐受性。因此,研发针对放化疗交叉耐受关键分子的干预措施,对于有效逆转放化疗交叉耐受和提高胃癌治疗效果,具有十分重要的科学意义,也是目前临床肿瘤治疗急需解决的重大科学问题。酪蛋白激酶2(Casein Kinase 2,CK2),是一个多效的丝氨酸/苏氨酸蛋白激酶。CK2在细胞生理活动的调节中处于中心地位,广泛参与细胞增殖、分化、死亡等过程,CK2表达及功能的异常往往导致肿瘤发生发展。CX-4945是近年来研发的特异性针对CK2的抑制剂,体内外研究发现CX-4945通过抑制CK2活性发挥抗肿瘤作用,可提高化疗效果,而CX-4945能否提高胃癌放疗效果,逆转胃癌细胞放疗抗性则未见报道。本实验室前期用浓度梯度法诱导建立了胃癌顺铂耐药细胞模型,发现DNA修复蛋白XRCC1表达异常升高介导顺铂所致DNA损伤修复过程是导致胃癌细胞产生顺铂耐受性的重要原因。进一步研究发现,顺铂耐药细胞中CK2表达升高是导致XRCC1高表达的重要机制,采用CX-4945能有效杀伤耐药细胞,提高其对顺铂敏感性。XRCC1是参与修复放射诱导的DNA损伤的重要分子,提示胃癌顺铂耐药细胞可能对放疗具有交叉耐受,而通过抑制CK2可逆转顺铂耐药细胞的放疗耐受。目的:研究胃癌顺铂耐药细胞放射抗性产生及逆转的分子基础。方法:在已经成功构建的胃癌顺铂耐药细胞株中采用细胞分子生物学技术比较耐药细胞与敏感细胞对辐射损伤修复能力的差异、XRCC1表达差异,探讨CK2、XRCC1等分子表达与胃癌顺铂耐药细胞放射抗性关系及其分子机制。结果:1.XRCC1介导胃癌顺铂耐药细胞放射抗性。在用浓度梯度法构建的两株耐药细胞BGC823/DDP、SGC7901/DDP及其敏感性亲本株中采用高能X-射线处理,结果显示DNA损伤程度显著低于相同剂量处理的敏感细胞。而胃癌顺铂耐药细胞中P-XRCC1水平显著高于敏感细胞。在敏感细胞中转染GFP-XRCC1表达质粒上调XRCC1表达可以显著抑制高能X-射线引起的DNA损伤;在耐药细胞中转染XRCC1 siRNA抑制XRCC1表达可以显著增加高能X-射线引起的DNA损伤。2.CK2抑制剂CX-4945通过抑制XRCC1增加X-射线诱导的DNA损伤。耐药细胞对CX-4945保持敏感性,CX-4945可抑制P-XRCC1水平,从而提高X-射线诱导的DNA损伤程度。3.CX-4945逆转顺铂耐药细胞对放射的抗性。克隆形成实验表明,顺铂耐药细胞对X-射线引发的细胞死亡具有显著的抵抗能力。而采用CX-4945可以提高X-射线诱导的顺铂耐药细胞死亡率。4.CX-4945逆转胃癌顺铂耐药细胞对于紫外UVB辐射的抗性。胃癌顺铂耐药细胞对于紫外UVB辐射引起的DNA损伤修复能力也显著增强,抵抗辐射诱导的细胞凋亡;当CK2抑制剂联合紫外UVB辐射后可以抑制XRCC1磷酸化使细胞不能修复紫外UVB诱导的DNA损伤,促进其损伤加重及凋亡。结论:本研究首次发现胃癌顺铂耐药细胞对放疗及紫外UVB辐射具有交叉耐受,阐明了 XRCC1在胃癌顺铂耐药细胞内修复放疗及紫外UVB引起DNA损伤的作用,首次提出通过抑制胃癌耐药细胞中CK2的表达,可以逆转其放射抗性。第二部分胃贲门癌组织CK2α的表达与放疗疗效的关系目的:采用免疫组化(immunohistochemistry,IHC)检测胃贲门癌单纯放疗患者胃镜活检组织中CK2和XRCC1的表达,探讨二者相关性以及和胃贲门癌放射治疗效果及与病人临床指标及预后生存的关系。明确以CK2为靶点,判断胃贲门癌患者放疗敏感度,作为个体化放疗的依据。方法:1.本研究所用资料来自中国人民解放军第八二医院肿瘤中心2010年1月至2013年12月收治的有手术禁忌或本人拒绝等原因未经手术治疗的首诊、无放疗禁忌症,年龄50岁以上、KPS评分大于70分并且病例资料完备的胃责门癌病人。46例患者都具备胃镜下活检病理诊断支持。患者中有36例男性,10例女性,年龄平均71.8±10.23岁,中位数71.2岁。分期选择我国目前普遍使用的非手术患者临床病理分期,Ⅰ期0例,Ⅱ期22例,Ⅲ期18例,Ⅳ期6例(其中肝转移4例,左锁骨上淋巴结转移2例)。2.应用免疫组织化学方法(SP法),检测46例胃贲门癌组织蜡块中CK2和XRCC1的表达情况,分析二者相关性以及和胃贲门癌放射治疗效果及与病人临床一般资料的关系。3.采用PASW Statistics 18统计软件包进行数据录入和分析,计数数据采用X2检验。采用X2检验分析CK2及XRCC1表达与临床资料参数间关系;采用单因素分析Kaplan-Meier法进行生存分析研究胃贲门癌患者预后影响因素。P0.05差异有统计学意义。结果:1.贲门癌组织中CK2的表达水平与临床病理指标的关系贲门癌组织中CK2高表达定位于细胞浆,阳性表达率为45.7%(21/46)。卡方检验结果,CK2阳性表达和患者胃癌家族史、淋巴结转移、临床分期有关,存在统计学差异(P0.05)。CK2在有胃癌家族史人群中阳性表达率为75.0%(9/12);有淋巴结转移组阳性率为56.3%(18/32)、无淋巴结转移组阳性率为21.4%(3/14);病例中无Ⅰ期患者,CK2阳性表达率Ⅱ期27.3%(6/22)、Ⅲ期66.7%(12/18)、ⅣV期50.0%(3/6)。CK2阳性表达和病人性别、年龄、KPS评分、慢性胃病史、主要症状表现、血红蛋白浓度、腺癌分化级别、远处转移没有关系,无统计学差异(P0.05)。2.责门癌组织中XRCC1的表达水平与临床病理指标的关系责门癌组织中XRCC1高表达定位在细胞核,阳性表达率为65.2%(30/46)。卡方检验结果,XRCC1阳性表达和患者淋巴结转移、临床分期有关,存在统计学差异(P0,05)。XRCC1在有淋巴结转移组阳性率为78.1%(25/32)、无淋巴结转移组阳性率为35.7%(5/14);病例中无Ⅰ期患者,XRCC1阳性表达率Ⅱ 期 45.5%(10/22)、Ⅲ 期 88.9%(16/18)、Ⅳ 期 66.7%(4/6)。XRCC1 阳性表达和病人性别、年龄、KPS评分、慢性胃病史、胃癌家族史、主要症状表现、血红蛋白浓度、腺癌分化级别、远处转移没有关系,无统计学差异(P0.05)。3.贲门癌组织中CK2与XRCC1表达水平存在相关性CK2在46例胃癌患者中的表达21例,XRCC1在46例胃癌患者中的表达30例,卡方检验显示,两者表达有相关性(x2=4.267,P0.05)。4.CK2和XRCC1的表达与胃癌放疗疗效的及预后的关系46例胃贲门癌患者中,CK2表达阳性者21例,放射治疗后复查影像学指标评价疗效5例CR,2例PR,有效缓解率RR(CR+PR)为33.3%(7/21);CK2表达阴性者25例,复查疗效评价12例CR,8例PR,有效缓解率RR(CR+PR)为80.0%(20/25),有统计学差异(P0.05),说明CK2表达与放疗后近期疗效相关,CK2表达较低者放疗有效率较高(x2=10.252,P0.05)。XRCC1表达阳性者30例,放射治疗后复查影像学指标评价疗效9例CR,4例PR,有效缓解率RR(CR+PR)为43.3%(13/30);XRCC1表达阴性者16例,复查疗效评价8例CR,6例PR,有效缓解率RR(CR+PR)为87.5%(14/16),有统计学差异(P0.05),说明XRCC1表达与放疗后近期疗效也相关,XRCC1表达较低者放疗有效率较高(x2=8.396,P0.05)。CK2高表达组和低表达组1、2年生存率分别为 33.3%(7/21)、14.3%(3/21)和 68.0%(17/25)、48.0%(12/25);XRCC1高表达组和低表达组1、2年生存率分别为40.0%(12/30)、23.3%(7/30)和 75.0%(12/16)、50.0%(8/16)。Kaplan-Meier 曲线生存分析结果,CK2 与XRCC1的表达水平和预后生存相关,二者表达阳性者生存期较短,表达阴性者生存期较长(P0.05)。而且,CK2与XRCC1两个分子联合起来判断胃癌预后表现出比单个分子更大的预测效能。结论:1.CK2和XRCC1在胃贲门癌组织中表达均较高,并且有相关性;CK2与XRCC1的表达和淋巴结转移、临床病理分期相关,提示期别越晚,二者阳性越高。可能与胃癌家族史有关。与患者性别、年龄、KPS评分、慢性胃病史、临床主要症状(上腹痛、吞咽困难、消瘦)、血红蛋白浓度、分化级别、远处转移没有关系。2.CK2及XRCC1高表达患者放疗有效率显著低于低表达患者,CK2和XRCC1高表达者1年、2年生存率显著低于低表达者。本研究通过CK2蛋白在体内表达程度的差异和临床放疗及预后的关系,说明CK2作为胃癌患者个体化放疗分子标志物的潜在价值以及CK2作为胃癌放化疗交叉耐受逆转治疗的新靶点,为CK2抑制剂CX-4945应用于胃癌临床放疗增敏提供了理论依据。
[Abstract]:In the first part, the mechanism of CK2 inhibitor CX-4945 reverses the radiation resistance of gastric cancer resistant cells in China. The incidence and death of gastric cancer are in third malignant tumors. According to the statistics, the number of gastric cancer in China is about 400 thousand in 2012, the number of deaths is about 320 thousand. The five year survival rate of gastric cancer patients is less than 20%. operation, chemotherapy and radiotherapy are the main means for the treatment of gastric cancer. Early gastric cancer can excision local tumor by surgical procedure. Advanced gastric cancer has extensive infiltration and metastasis and loss of operation opportunity. The combination of radiotherapy and chemotherapy is the main method. The tolerance of tumor cells to various chemoradiotherapy often leads to the failure of radiotherapy and chemotherapy. Cisplatin is the first line of chemotherapy for gastric cancer, mainly by inducing DNA delivery. The tumor cells can kill tumor cells. The tumor cells can resist cisplatin induced apoptosis by developing a variety of resistance mechanisms. The enhancement of DNA damage repair ability is the most important mechanism. Radiotherapy is mainly targeted to DNA, which induces cell proliferation by inducing DNA double strand breakage and induces apoptosis. The tumor cells also have cross tolerance to radiotherapy. Therefore, it is of great scientific significance for the effective reversal of the cross tolerance of chemoradiotherapy and the improvement of the therapeutic effect of gastric cancer. It is also a major scientific problem to be solved urgently in clinical cancer treatment. Casein stimulated by the intervention measures of key molecules of cross tolerance of radiotherapy and chemotherapy. Enzyme 2 (Casein Kinase 2, CK2), a multipotent serine / threonine protein kinase.CK2, is in a central position in the regulation of cell physiological activity, and is widely involved in cell proliferation, differentiation, and death. The abnormal expression of CK2 and the abnormal function of CK2 often lead to the development of.CX-4945 as a specific inhibitor for CK2. Internal and external studies have found that CX-4945 can improve the effect of chemotherapy by inhibiting the activity of CK2, and can improve the effect of chemotherapy, but whether CX-4945 can improve the radiotherapy effect of gastric cancer and reverse the radiation resistance of gastric cancer cells has not been reported. In the early stage of the laboratory, the cisplatin resistance fine cell model of gastric cancer was induced by concentration gradient method, and the abnormal expression of DNA repair protein XRCC1 was found to be abnormal. The increase of DNA damage repair induced by cisplatin is an important cause of the tolerance to cisplatin induced by gastric cancer cells. Further studies have found that the increase of CK2 expression in cisplatin resistant cells is an important mechanism for the high expression of XRCC1. The use of CX-4945 to effectively kill drug resistant cells and to improve the sensitivity of.XRCC1 to cisplatin is involved in the repair of radiation induced by cisplatin. The important molecules of DNA damage suggest that cisplatin resistant cells may have cross tolerance to radiotherapy, and by inhibiting CK2 can reverse the radiotherapy tolerance of cisplatin resistant cells. Objective: To study the molecular basis of radiation resistance production and reversal of cisplatin resistant cells in gastric cancer. Methods: in the successful construction of cisplatin resistant cell lines of cisplatin, a successful cell line of cisplatin resistance in gastric cancer Cell molecular biology technique was used to compare the difference of radiation damage repair ability between drug resistant and sensitive cells, the difference of XRCC1 expression and the relationship between CK2, XRCC1 and other molecular mechanisms of cisplatin resistant cells in gastric cancer and its molecular mechanism. Results: 1.XRCC1 mediated radiation resistance of cisplatin resistant cells in gastric cancer. Two strains of resistant cells BGC823/DDP, SGC7901/DDP and their sensitive parent strains were treated with high energy X- ray. The results showed that the degree of DNA damage was significantly lower than that of the same dose treated sensitive cells. The P-XRCC1 level of cisplatin resistant cells in gastric cancer was significantly higher than that of the sensitive cells. The GFP-XRCC1 expression plasmid was transfected in the sensitive cells to up regulate the XRCC1 table. The DNA damage caused by high energy X- ray can be significantly inhibited, and the conversion of XRCC1 siRNA to XRCC1 expression in the drug resistant cells can significantly increase the DNA damage of DNA damage caused by high energy X- ray, CX-4945 by inhibiting XRCC1 to increase X- ray induced DNA damage. The increase of X- ray induced DNA damage.3.CX-4945 reverses the radiation resistance of cisplatin resistant cells. Cloning and formation experiments showed that cisplatin resistant cells had significant resistance to X- ray induced cell death. The use of CX-4945 could improve the X- ray induced cisplatin resistance cell mortality.4.CX-4945 to reverse cisplatin in gastric cancer. Resistance cells are resistant to ultraviolet UVB radiation. The ability to repair DNA damage caused by UVB radiation from cisplatin is also significantly enhanced to resist radiation induced apoptosis. When CK2 inhibitors combined with UV UVB radiation, XRCC1 phosphorylation can inhibit the cell failure to repair DNA damage induced by ultraviolet UVB and promote its damage. Conclusion: the cross tolerance of cisplatin resistant cells to radiotherapy and ultraviolet UVB radiation was first found in this study, and the effect of XRCC1 on the repair of cisplatin resistant cells in gastric cancer and DNA damage caused by ultraviolet UVB was first proposed. The first proposed to reverse the expression of CK2 in gastric cancer resistant cells could reverse its radiation resistance. The relationship between the expression of CK2 alpha and the effect of radiotherapy in the two part of gastric cardia carcinoma: immunohistochemistry (IHC) was used to detect the expression of CK2 and XRCC1 in the gastric biopsies of gastric cardia cancer patients. The correlation between the gastric cardia cancer and gastric cardia cancer patients was examined and the effects of radiotherapy on the gastric cardia cancer and the clinical indicators and survival of the patients were discussed. CK2 as a target to determine the sensitivity of radiotherapy for gastric cardia cancer patients as a basis for individualized radiotherapy. Methods: 1. the data from the No.82 Hospital of PLA tumor center from January 2010 to December 2013 were treated with surgical taboos or refusal of the first diagnosis of untreated surgery, and no contraindications of radiotherapy. Patients with age over 50 years old, KPS score greater than 70 points and.46 patients with complete case data have a pathological diagnosis support for endoscopic biopsy. There are 36 males and 10 females with an average age of 71.8 + 10.23 years, with a median of 71.2 years. 0 cases, 22 cases in stage II, 18 cases in stage III, 6 cases in stage IV (4 cases of hepatic metastases and 2 cases of left supraclavicular lymph node metastasis).2. application immuno histochemical method (SP) to detect the expression of CK2 and XRCC1 in 46 cases of gastric cardia cancer tissue, analyze the correlation between two and the effect of radiotherapy for gastric cardia cancer and the general clinical data of the patients. .3. using PASW Statistics 18 statistical software package for data entry and analysis, counting data using X2 test. X2 test was used to analyze the relationship between the expression of CK2 and XRCC1 and the parameters of clinical data, and the survival analysis of gastric cardia cancer patients by the single factor analysis Kaplan-Meier method was statistically significant. Results: the expression level of CK2 in 1. cardiac carcinoma tissues was related to the clinicopathological index. The high expression of CK2 was located in the cytoplasm and the positive expression rate was 45.7% (21/46). The result of chi square test was that the positive expression of CK2 was related to the family history of gastric cancer, lymph node metastasis and clinical stage, and there was a statistical difference (P0.05).CK2 in the family history of gastric cancer The positive rate in the population was 75% (9/12), the positive rate in the lymph node metastasis group was 56.3% (18/32), the positive rate in the non lymph node metastasis group was 21.4% (3/14). There were no stage I patients, CK2 positive expression stage II 27.3% (6/22), stage III 66.7% (12/18), 50% (3/6).CK2 positive expression in stage IV V and patients' sex, age, KPS score, and chronic stomach disease history. Symptoms, hemoglobin concentration, grade of differentiation of adenocarcinoma, distant metastasis, no statistical difference (P0.05) the relationship between the expression level of XRCC1 and the clinicopathological index in.2. imputation carcinoma tissue, the high expression of XRCC1 in the cell carcinoma tissue is located in the nucleus, the positive expression rate is 65.2% (30/46). The result of chi square test, the positive expression of XRCC1 and the patient Lymph node metastasis, clinical staging, statistical difference (P0,05).XRCC1 in lymph node metastasis group positive rate was 78.1% (25/32), no lymph node metastasis group positive rate was 35.7% (5/14); no stage I patients, XRCC1 positive expression stage II 45.5% (10/22), stage III 88.9% (16/18), stage IV 66.7% (4/6).XRCC1 positive expression and patient sex No, age, KPS score, history of chronic stomach disease, family history of gastric cancer, main symptoms, hemoglobin concentration, grade of adenocarcinoma differentiation, distant metastasis, no statistical difference (P0.05), there is a correlation between CK2 and XRCC1 expression in.3. cardiac cancer tissue, CK2 in 46 patients with gastric cancer, and XRCC1 in 46 cases of gastric cancer 30 The relationship between the expression of x2=4.267, P0.05,.4.CK2 and XRCC1 and the relationship between the expression of.4.CK2 and XRCC1 and the prognosis and prognosis of gastric cancer were 46 cases of gastric cardia cancer, 21 cases of CK2 expression positive, 5 cases of CR, 2 PR, 33.3% (7/21) effective remission rate RR (CR+PR), CK2 expression 2. 5 cases, 12 cases of CR and 8 cases of PR, effective remission rate RR (CR+PR) was 80% (20/25), and there were statistical differences (P0.05), indicating that the expression of CK2 was related to the short-term effect after radiotherapy, 30 cases with higher efficiency of radiotherapy (x2=10.252, P0.05).XRCC1 expression in the lower CK2 expression. 9 cases of CR and 4 cases were evaluated after radiological treatment. There were 4 cases. The remission rate of RR (CR+PR) was 43.3% (13/30) and 16 cases with negative XRCC1 expression, 8 cases of CR and 6 cases of PR were rechecked. The effective remission rate RR (CR+PR) was 87.5% (14/16), and there was a statistical difference (P0.05), indicating that XRCC1 expression was also related to the short-term effect after radiotherapy, and the high expression and low expression of radiotherapy were higher in the lower XRCC1 table. The survival rate of group 1,2 was 33.3% (7/21), 14.3% (3/21) and 68% (17/25), 48% (12/25), and the survival rate of 1,2 year in XRCC1 high expression group and low expression group was 40% (12/30), 23.3% (7/30) and 75% (12/16), 50% (8/16).Kaplan-Meier curve survival analysis. The survival rate was related to the expression level and the prognosis of the prognosis, and the two expressed positive. The survival period was shorter and the expression negative person had a longer life period (P0.05). Moreover, CK2 and XRCC1 were combined to judge the prognosis of gastric cancer to be more predictive than single molecule. Conclusion: the expression of 1.CK2 and XRCC1 in gastric cardia cancer tissues were higher and correlated, and the expression of CK2 and XRCC1, lymph node metastasis, and clinicopathological stages Close, the more late the period, the more positive of the two. It may be related to the family history of gastric cancer. Sex, age, KPS score, history of chronic gastropathy, clinical major symptoms (upper abdominal pain, dysphagia, emaciation), hemoglobin concentration, differentiation grade, and distant metastasis are not related to.2.CK2 and XRCC1 high expression, the effective rate of radiotherapy is significantly lower than that of low expression patients. The 2 year survival rate of the high expression of CK2 and XRCC1 was significantly lower than that of low expression in 1 years. This study demonstrated the potential value of CK2 as a molecular marker of individualized radiotherapy for gastric cancer patients and the new target of CK2 as a cross tolerance reversal therapy for gastric cancer and chemotherapy through the relationship between the difference in the expression of CK2 protein in the body and the relationship between the clinical radiotherapy and prognosis. 2 inhibitor CX-4945 provides a theoretical basis for clinical radiotherapy sensitization of gastric cancer.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2
本文编号：2163295
[Abstract]:In the first part, the mechanism of CK2 inhibitor CX-4945 reverses the radiation resistance of gastric cancer resistant cells in China. The incidence and death of gastric cancer are in third malignant tumors. According to the statistics, the number of gastric cancer in China is about 400 thousand in 2012, the number of deaths is about 320 thousand. The five year survival rate of gastric cancer patients is less than 20%. operation, chemotherapy and radiotherapy are the main means for the treatment of gastric cancer. Early gastric cancer can excision local tumor by surgical procedure. Advanced gastric cancer has extensive infiltration and metastasis and loss of operation opportunity. The combination of radiotherapy and chemotherapy is the main method. The tolerance of tumor cells to various chemoradiotherapy often leads to the failure of radiotherapy and chemotherapy. Cisplatin is the first line of chemotherapy for gastric cancer, mainly by inducing DNA delivery. The tumor cells can kill tumor cells. The tumor cells can resist cisplatin induced apoptosis by developing a variety of resistance mechanisms. The enhancement of DNA damage repair ability is the most important mechanism. Radiotherapy is mainly targeted to DNA, which induces cell proliferation by inducing DNA double strand breakage and induces apoptosis. The tumor cells also have cross tolerance to radiotherapy. Therefore, it is of great scientific significance for the effective reversal of the cross tolerance of chemoradiotherapy and the improvement of the therapeutic effect of gastric cancer. It is also a major scientific problem to be solved urgently in clinical cancer treatment. Casein stimulated by the intervention measures of key molecules of cross tolerance of radiotherapy and chemotherapy. Enzyme 2 (Casein Kinase 2, CK2), a multipotent serine / threonine protein kinase.CK2, is in a central position in the regulation of cell physiological activity, and is widely involved in cell proliferation, differentiation, and death. The abnormal expression of CK2 and the abnormal function of CK2 often lead to the development of.CX-4945 as a specific inhibitor for CK2. Internal and external studies have found that CX-4945 can improve the effect of chemotherapy by inhibiting the activity of CK2, and can improve the effect of chemotherapy, but whether CX-4945 can improve the radiotherapy effect of gastric cancer and reverse the radiation resistance of gastric cancer cells has not been reported. In the early stage of the laboratory, the cisplatin resistance fine cell model of gastric cancer was induced by concentration gradient method, and the abnormal expression of DNA repair protein XRCC1 was found to be abnormal. The increase of DNA damage repair induced by cisplatin is an important cause of the tolerance to cisplatin induced by gastric cancer cells. Further studies have found that the increase of CK2 expression in cisplatin resistant cells is an important mechanism for the high expression of XRCC1. The use of CX-4945 to effectively kill drug resistant cells and to improve the sensitivity of.XRCC1 to cisplatin is involved in the repair of radiation induced by cisplatin. The important molecules of DNA damage suggest that cisplatin resistant cells may have cross tolerance to radiotherapy, and by inhibiting CK2 can reverse the radiotherapy tolerance of cisplatin resistant cells. Objective: To study the molecular basis of radiation resistance production and reversal of cisplatin resistant cells in gastric cancer. Methods: in the successful construction of cisplatin resistant cell lines of cisplatin, a successful cell line of cisplatin resistance in gastric cancer Cell molecular biology technique was used to compare the difference of radiation damage repair ability between drug resistant and sensitive cells, the difference of XRCC1 expression and the relationship between CK2, XRCC1 and other molecular mechanisms of cisplatin resistant cells in gastric cancer and its molecular mechanism. Results: 1.XRCC1 mediated radiation resistance of cisplatin resistant cells in gastric cancer. Two strains of resistant cells BGC823/DDP, SGC7901/DDP and their sensitive parent strains were treated with high energy X- ray. The results showed that the degree of DNA damage was significantly lower than that of the same dose treated sensitive cells. The P-XRCC1 level of cisplatin resistant cells in gastric cancer was significantly higher than that of the sensitive cells. The GFP-XRCC1 expression plasmid was transfected in the sensitive cells to up regulate the XRCC1 table. The DNA damage caused by high energy X- ray can be significantly inhibited, and the conversion of XRCC1 siRNA to XRCC1 expression in the drug resistant cells can significantly increase the DNA damage of DNA damage caused by high energy X- ray, CX-4945 by inhibiting XRCC1 to increase X- ray induced DNA damage. The increase of X- ray induced DNA damage.3.CX-4945 reverses the radiation resistance of cisplatin resistant cells. Cloning and formation experiments showed that cisplatin resistant cells had significant resistance to X- ray induced cell death. The use of CX-4945 could improve the X- ray induced cisplatin resistance cell mortality.4.CX-4945 to reverse cisplatin in gastric cancer. Resistance cells are resistant to ultraviolet UVB radiation. The ability to repair DNA damage caused by UVB radiation from cisplatin is also significantly enhanced to resist radiation induced apoptosis. When CK2 inhibitors combined with UV UVB radiation, XRCC1 phosphorylation can inhibit the cell failure to repair DNA damage induced by ultraviolet UVB and promote its damage. Conclusion: the cross tolerance of cisplatin resistant cells to radiotherapy and ultraviolet UVB radiation was first found in this study, and the effect of XRCC1 on the repair of cisplatin resistant cells in gastric cancer and DNA damage caused by ultraviolet UVB was first proposed. The first proposed to reverse the expression of CK2 in gastric cancer resistant cells could reverse its radiation resistance. The relationship between the expression of CK2 alpha and the effect of radiotherapy in the two part of gastric cardia carcinoma: immunohistochemistry (IHC) was used to detect the expression of CK2 and XRCC1 in the gastric biopsies of gastric cardia cancer patients. The correlation between the gastric cardia cancer and gastric cardia cancer patients was examined and the effects of radiotherapy on the gastric cardia cancer and the clinical indicators and survival of the patients were discussed. CK2 as a target to determine the sensitivity of radiotherapy for gastric cardia cancer patients as a basis for individualized radiotherapy. Methods: 1. the data from the No.82 Hospital of PLA tumor center from January 2010 to December 2013 were treated with surgical taboos or refusal of the first diagnosis of untreated surgery, and no contraindications of radiotherapy. Patients with age over 50 years old, KPS score greater than 70 points and.46 patients with complete case data have a pathological diagnosis support for endoscopic biopsy. There are 36 males and 10 females with an average age of 71.8 + 10.23 years, with a median of 71.2 years. 0 cases, 22 cases in stage II, 18 cases in stage III, 6 cases in stage IV (4 cases of hepatic metastases and 2 cases of left supraclavicular lymph node metastasis).2. application immuno histochemical method (SP) to detect the expression of CK2 and XRCC1 in 46 cases of gastric cardia cancer tissue, analyze the correlation between two and the effect of radiotherapy for gastric cardia cancer and the general clinical data of the patients. .3. using PASW Statistics 18 statistical software package for data entry and analysis, counting data using X2 test. X2 test was used to analyze the relationship between the expression of CK2 and XRCC1 and the parameters of clinical data, and the survival analysis of gastric cardia cancer patients by the single factor analysis Kaplan-Meier method was statistically significant. Results: the expression level of CK2 in 1. cardiac carcinoma tissues was related to the clinicopathological index. The high expression of CK2 was located in the cytoplasm and the positive expression rate was 45.7% (21/46). The result of chi square test was that the positive expression of CK2 was related to the family history of gastric cancer, lymph node metastasis and clinical stage, and there was a statistical difference (P0.05).CK2 in the family history of gastric cancer The positive rate in the population was 75% (9/12), the positive rate in the lymph node metastasis group was 56.3% (18/32), the positive rate in the non lymph node metastasis group was 21.4% (3/14). There were no stage I patients, CK2 positive expression stage II 27.3% (6/22), stage III 66.7% (12/18), 50% (3/6).CK2 positive expression in stage IV V and patients' sex, age, KPS score, and chronic stomach disease history. Symptoms, hemoglobin concentration, grade of differentiation of adenocarcinoma, distant metastasis, no statistical difference (P0.05) the relationship between the expression level of XRCC1 and the clinicopathological index in.2. imputation carcinoma tissue, the high expression of XRCC1 in the cell carcinoma tissue is located in the nucleus, the positive expression rate is 65.2% (30/46). The result of chi square test, the positive expression of XRCC1 and the patient Lymph node metastasis, clinical staging, statistical difference (P0,05).XRCC1 in lymph node metastasis group positive rate was 78.1% (25/32), no lymph node metastasis group positive rate was 35.7% (5/14); no stage I patients, XRCC1 positive expression stage II 45.5% (10/22), stage III 88.9% (16/18), stage IV 66.7% (4/6).XRCC1 positive expression and patient sex No, age, KPS score, history of chronic stomach disease, family history of gastric cancer, main symptoms, hemoglobin concentration, grade of adenocarcinoma differentiation, distant metastasis, no statistical difference (P0.05), there is a correlation between CK2 and XRCC1 expression in.3. cardiac cancer tissue, CK2 in 46 patients with gastric cancer, and XRCC1 in 46 cases of gastric cancer 30 The relationship between the expression of x2=4.267, P0.05,.4.CK2 and XRCC1 and the relationship between the expression of.4.CK2 and XRCC1 and the prognosis and prognosis of gastric cancer were 46 cases of gastric cardia cancer, 21 cases of CK2 expression positive, 5 cases of CR, 2 PR, 33.3% (7/21) effective remission rate RR (CR+PR), CK2 expression 2. 5 cases, 12 cases of CR and 8 cases of PR, effective remission rate RR (CR+PR) was 80% (20/25), and there were statistical differences (P0.05), indicating that the expression of CK2 was related to the short-term effect after radiotherapy, 30 cases with higher efficiency of radiotherapy (x2=10.252, P0.05).XRCC1 expression in the lower CK2 expression. 9 cases of CR and 4 cases were evaluated after radiological treatment. There were 4 cases. The remission rate of RR (CR+PR) was 43.3% (13/30) and 16 cases with negative XRCC1 expression, 8 cases of CR and 6 cases of PR were rechecked. The effective remission rate RR (CR+PR) was 87.5% (14/16), and there was a statistical difference (P0.05), indicating that XRCC1 expression was also related to the short-term effect after radiotherapy, and the high expression and low expression of radiotherapy were higher in the lower XRCC1 table. The survival rate of group 1,2 was 33.3% (7/21), 14.3% (3/21) and 68% (17/25), 48% (12/25), and the survival rate of 1,2 year in XRCC1 high expression group and low expression group was 40% (12/30), 23.3% (7/30) and 75% (12/16), 50% (8/16).Kaplan-Meier curve survival analysis. The survival rate was related to the expression level and the prognosis of the prognosis, and the two expressed positive. The survival period was shorter and the expression negative person had a longer life period (P0.05). Moreover, CK2 and XRCC1 were combined to judge the prognosis of gastric cancer to be more predictive than single molecule. Conclusion: the expression of 1.CK2 and XRCC1 in gastric cardia cancer tissues were higher and correlated, and the expression of CK2 and XRCC1, lymph node metastasis, and clinicopathological stages Close, the more late the period, the more positive of the two. It may be related to the family history of gastric cancer. Sex, age, KPS score, history of chronic gastropathy, clinical major symptoms (upper abdominal pain, dysphagia, emaciation), hemoglobin concentration, differentiation grade, and distant metastasis are not related to.2.CK2 and XRCC1 high expression, the effective rate of radiotherapy is significantly lower than that of low expression patients. The 2 year survival rate of the high expression of CK2 and XRCC1 was significantly lower than that of low expression in 1 years. This study demonstrated the potential value of CK2 as a molecular marker of individualized radiotherapy for gastric cancer patients and the new target of CK2 as a cross tolerance reversal therapy for gastric cancer and chemotherapy through the relationship between the difference in the expression of CK2 protein in the body and the relationship between the clinical radiotherapy and prognosis. 2 inhibitor CX-4945 provides a theoretical basis for clinical radiotherapy sensitization of gastric cancer.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2
【相似文献】
相关博士学位论文 前1条
1 耿炜;CK2抑制剂CX4945逆转胃癌耐药细胞放射抗性的分子基础及临床研究[D];南京医科大学;2016年
，本文编号：2163295
本文链接：https://www.wllwen.com/yixuelunwen/zlx/2163295.html
