BRCA1蛋白表达在骨肉瘤细胞MG-63放化疗疗效评估中的作用及其机制研究
发布时间：2019-05-28 16:25
【摘要】：背景:骨肉瘤是最常见的原发性恶性骨肿瘤,其起源于骨间叶细胞,好发于儿童及青少年,第二个发病高峰出现在60-80岁之间。发病有性别差异,男性多于女性。发病位置多见于长骨,例如肱骨和胫骨近端、股骨远端。大约20%患者确诊时已经为晚期转移患者,肺转移居多,远处转移是骨肉瘤患者治疗失败和死亡的主要原因。目前治疗以新辅助化疗结合手术为主,虽然局部病变患者5年生存期从1970年的20%上升至如今的60%以上,但是对于晚期转移的骨肉瘤患者来说仍是一个不小的挑战,5年生存期降至30%以下,近30年针对骨肉瘤的治疗手段未有实质性进展,急需更加有效的治疗方案。综上,考虑骨肉瘤患者发病年龄轻、恶性程度极高、治疗方法单一和毒副作用大,并且总体效果不佳的情况下,寻找更加新颖、安全有效、低毒副作用的新兴治疗方案已成为国内外学者瞩目的研究之一。目前与骨肉瘤发生发展相关的关键基因包括TP53、Rb1、FAS、ATRX、PTEN、COX-2、HER2等。尽管已经有这些研究进展,但是仍缺乏一个能准确预测骨肉瘤进展、转移和对化疗反应性的标志性分子。BRCA1是乳腺癌易感基因,位于染色体17q21,并且编码1863个氨基酸蛋白,BRCA1有24个外显子,最大的是第11外显子,在第11外显子中富含乳腺癌患者最重要也是最常见的突变基因。BRCA1是抑癌基因,能通过多条细胞转导通路抑制细胞增殖、细胞生长,阻滞细胞于G2/M期,并诱导细胞凋亡。BRCA1的发挥抑癌作用可能与多条信号通路有关,如BRCA1-MAPK通路、BRCA1-p53通路、BRCA1-Gdd45通路有关,其中BRCA1-p53通路涉及细胞凋亡,BRCA1可以调节p53基因表达,而且p53直接调控凋亡蛋白Bcl-2及其家族蛋白Bax的表达,因此BRCA1可以通过调节p53的表达从而影响凋亡蛋白Bcl-2及其家族Bax的表达。BRCA1还涉及DAN损伤、修复过程,并且与同源重组修复(HRR)密切相关,因此BRCA1作为抑癌基因参与了乳腺癌、肺癌等许多恶性肿瘤的发生发展过程。但是,到目前为止国内外对于BRCA1与骨肉瘤的相关性研究却较为缺乏,因此本研究将重点关注BRCA1与骨肉瘤的相关性研究方面,尤其侧重在BRCA1与骨肉瘤细胞MG-63放化疗的疗效评估方面进行研究。目的:研究BRCA1在人骨肉瘤组织和骨肉瘤细胞中的表达情况及其临床意义。观察骨肉瘤细胞MG-63在放化疗的作用下,BRCA1表达水平对肿瘤细胞增殖、侵袭、细胞周期和凋亡的影响,从而探讨BRCA1蛋白表达在骨肉瘤细胞MG-63放化疗疗效评估中的潜在价值。方法:利用免疫组化的方法检测BRCA1在45例人骨肉瘤组织、22例软骨肉瘤以及骨肉瘤细胞MG-63中的表达,探讨BRCA1的表达情况与临床病理因素的联系,以及在骨肉瘤细胞MG-63中的表达水平。利用RT-PCR、Western Blot方法检测骨肉瘤细胞MG-63在放疗(分别为0Gy、0.5Gy、1.0Gy、1.5Gy、2.0Gy)、化疗(分别为0μg/ml、5μg/ml、10μg/ml、20μg/ml、40μg/ml)及放化疗联合(分别为0Gy+0μg/ml、2Gy、5μg/ml、2Gy+5μg/ml)作用下的BRCA1、p53、Bax和Bcl-2的表达水平。利用CCK-8细胞增殖实验、Transwell细胞侵袭实验、Annexin V-Fitc和PI双染细胞凋亡实验对骨肉瘤细胞MG-63在放、化疗及放化疗联合作用下肿瘤细胞的增殖能力、侵袭能力和肿瘤细胞凋亡等情况进行检测。结果:1.临床病理证实BRCA1在人骨肉瘤组织高表达(29/45),软骨肉瘤(3/22),在骨肉瘤癌旁组织中无BRCA1表达,在骨肉瘤细胞MG-63表达。骨肉瘤组织的BRCA1阳性表达率显著高于软骨肉瘤组织,二者相比差异有统计学意义(P0.05)。BRCA1表达情况与骨肉瘤临床病理因素中的肿瘤坏死存在显著关联,二者相比差异有统计学意义(P0.05),BRCA1表达情况与年龄、性别、WHO分型、病变位置、遗传因素、肺转移、Codman三角、病理性骨折和病理边界相比无统计学意义(P0.05)。2.在骨肉瘤细胞MG-63中,放疗可以抑制骨肉瘤细胞MG-63的增殖,且随剂量的增加,抑制作用越明显;促进骨肉瘤细胞MG-63凋亡;引起骨肉瘤细胞G2期阻滞,且随着剂量的增加阻滞加剧;抑制骨肉瘤细胞MG-63的侵袭,且随剂量的增加,细胞数减少越明显;抑制骨肉瘤细胞MG-63中BRCA1、p53基因和蛋白的表达;抑制Bax蛋白的表达,促进Bcl-2蛋白的表达。3.在骨肉瘤细胞MG-63中,顺铂可以抑制骨肉瘤细胞MG-63的增殖,且随剂量的增加,抑制作用增强;促进骨肉瘤细胞MG-63凋亡,且随剂量的增加,凋亡细胞数也增加;引起MG-63细胞G2期阻滞,且随着剂量的增加阻滞加剧;抑制骨肉瘤细胞MG-63的侵袭,且随剂量的增加,细胞数减少越明显;抑制BRCA1、p53基因和蛋白的表达;抑制Bax蛋白表达,促进Bcl-2蛋白的表达。4.在骨肉瘤细胞MG-63中,放化疗联合作用抑制骨肉瘤细胞MG-63的增殖更显著;其促进细胞凋亡效果介于放疗和化疗之间;引起骨肉瘤细胞MG-63G2期阻滞的效果介于放疗和化疗之间;抑制骨肉瘤细胞MG-63的侵袭,抑制细胞侵袭的作用优于单纯放疗组和化疗组;放化疗联合作用对骨肉瘤细胞MG-63中BRCA1蛋白表达和p53基因表达的抑制效果优于单纯放疗组和化疗组;对Bax蛋白表达的抑制作用和对Bcl-2蛋白表达的促进作用优于单纯放疗组和化疗组。结论:1、BRCA1在骨肉瘤组织中高表达,在骨肉瘤癌旁组织中不表达,且骨肉瘤组织的BRCA1阳性表达率显著高于软骨肉瘤组织。提示其表达可能与骨肉瘤的发生、发展密切相关,检测BRCA1的表达将对骨肉瘤的早期诊断、判断恶性程度及预后具有指导意义。2、放疗、化疗及放化疗联合均可以抑制骨肉瘤细胞MG-63的增殖、侵袭,引起细胞周期G2期阻滞,并促进细胞凋亡;同时抑制BRCA1和p53的表达,抑制Bax表达而促进Bcl-2的表达。提示BRCA1可能通过BRCA1-p53通路发挥作用。3、放化疗联合对骨肉瘤细胞MG-63的增殖、侵袭的抑制作用、对BRCA1和p53表达的抑制作用、对Bax表达的抑制和Bcl-2表达的促进作用较单纯放疗组和单纯化疗组更加显著。提示放化疗联合治疗是骨肉瘤治疗的较好手段,并认为BRCA1蛋白的表达可以作为人骨肉瘤细胞MG-63放化疗疗效的评价指标之一。
[Abstract]:Background: Osteosarcoma is the most common primary malignant bone tumor, which is derived from inter-bone leaf cells, which is good for children and adolescents. The second peak is between 60 and 80 years of age. There is a gender difference in the incidence of the disease, and more males than females. The location of the disease is usually found in the long bone, such as the proximal and distal femur of the humerus and the tibia. Approximately 20% of the patients were diagnosed with advanced metastases, with most lung metastases, and distant metastasis was the main cause of the failure and death of the patients with osteosarcoma. The current treatment is mainly combined with neoadjuvant chemotherapy, although the survival time of the patients with local lesions increased from 20% in 1970 to more than 60% today, it is still a challenge for patients with advanced metastatic osteosarcoma, and the 5-year survival rate is reduced below 30%. There is no substantial progress in the treatment of osteosarcoma in recent 30 years, and it is in urgent need of a more effective treatment plan. In order to find a more novel, safe and effective and low-toxic side-effect new treatment scheme, it has become one of the most important researches at home and abroad in the light of the low incidence of the patients with osteosarcoma, the extremely high degree of malignancy, the single treatment method and the large toxic and side effect, and the overall effect is not good. The key genes related to the development of osteosarcoma include TP53, Rb1, FAS, ATRX, PTEN, COX-2, HER2 and so on. Despite these advances, there is a lack of a signature molecule that can accurately predict the progression, metastasis and response to chemotherapy. BRCA1 is a breast cancer susceptibility gene, located on chromosome 17q21 and encodes 1863 amino acid proteins, and BRCA1 has 24 exons, the largest is exon 11, and the most important and most common mutation gene in exon 11 is breast cancer. BRCA1 is a tumor suppressor gene, which can inhibit cell proliferation and cell growth through a plurality of cell transduction pathways, block cells in G2/ M phase, and induce cell apoptosis. The role of BRCA1 may be associated with multiple signal pathways, such as the BRCA1-MAPK pathway, the BRCA1-p53 pathway, the BRCA1-Gdd45 pathway, where the BRCA1-p53 pathway involves cell apoptosis, the BRCA1 can modulate the expression of the p53 gene, and p53 directly regulates the expression of the apoptosis protein Bcl-2 and its family protein Bax, Therefore, BRCA1 can influence the expression of Bcl-2 and its family Bax by regulating the expression of p53. BRCA1 also involved DAN injury and repair, and is closely related to homologous recombination repair (HRR), so BRCA1 is involved in the development of many malignant tumors such as breast cancer and lung cancer. However, so far, the study on the relationship between BRCA1 and osteosarcoma has been lacking, so this study will focus on the study of the relationship between BRCA1 and osteosarcoma, especially in the evaluation of the curative effect of the chemotherapy and chemotherapy of the MG-63 in the BRCA1 and the osteosarcoma cell. Objective: To study the expression of BRCA1 in human osteosarcoma and osteosarcoma and its clinical significance. To investigate the effect of the expression of BRCA1 on the proliferation, invasion, cell cycle and apoptosis of the tumor cells, the potential value of the expression of BRCA1 protein in the treatment of osteosarcoma cell MG-63 chemoradiotherapy was discussed. Methods: The expression of BRCA1 in 45 human osteosarcoma tissues,22 chondrosarcoma and MG-63 of osteosarcoma cells was detected by immunohistochemistry. The relationship between the expression of BRCA1 and clinicopathological factors and the level of expression of BRCA1 in osteosarcoma cell MG-63 were discussed. BRCA1 was detected by RT-PCR and Western Blot method in the treatment of osteosarcoma cell MG-63 in radiotherapy (0 Gy, 0.5 Gy, 1.0 Gy, 1.5 Gy, 2.0 Gy, respectively), chemotherapy (0. mu.g/ ml,5. mu.g/ ml,10. mu.g/ ml,20. mu.g/ ml,40. mu.g/ ml, respectively) and chemoradiotherapy (0 Gy + 0. mu.g/ ml,2 Gy,5. mu.g/ ml,2 Gy + 5. mu.g/ ml, respectively). The expression level of p53, Bax and Bcl-2. The proliferation, invasion and apoptosis of the tumor cells were detected by using CCK-8 cell proliferation experiment, Transwell cell invasion experiment, Annexin V-Fitc and PI double-staining cell apoptosis experiment. Results:1. The expression of BRCA1 in human osteosarcoma tissues (29/45) and chondrosarcoma (3/22) was confirmed by clinical pathology. The positive expression rate of BRCA1 in osteosarcoma was significantly higher than that of chondrosarcoma (P0.05). The expression of BRCA1 was significantly associated with the tumor necrosis in the clinical and pathological factors of osteosarcoma (P0.05), and the expression of BRCA1 and age. There was no significant difference in sex, WHO type, position of lesion, genetic factor, lung metastasis, Codman's triangle, pathological fracture and pathological boundary (P0.05). In the osteosarcoma cell MG-63, the proliferation of the osteosarcoma cell MG-63 can be inhibited, and the more obvious the inhibition effect with the increase of the dose, promote the apoptosis of the osteosarcoma cell MG-63, cause the G2 phase block of the osteosarcoma cell, and increase the block of the osteosarcoma cell MG-63, and inhibit the invasion of the osteosarcoma cell MG-63, And the expression of BRCA1, p53 gene and protein in the osteosarcoma cell MG-63 is inhibited, the expression of the Bax protein is inhibited, and the expression of the Bcl-2 protein is promoted. in that osteosarcoma cell MG-63, cisplatin can inhibit the proliferation of the osteosarcoma cell MG-63 and increase the inhibition effect with the increase of the dose, promote the apoptosis of the osteosarcoma cell MG-63, increase the number of the apoptotic cells with the increase of the dose, cause the G2 phase block of the MG-63 cell, And the expression of the Bax protein is inhibited, and the expression of the Bcl-2 protein is promoted. In the osteosarcoma cell MG-63, the combination of radiotherapy and chemotherapy can inhibit the proliferation of the osteosarcoma cell MG-63, and the effect of promoting the cell apoptosis is between the radiotherapy and the chemotherapy; the effect of the MG-63G2 phase block in the osteosarcoma cell is between the radiotherapy and the chemotherapy; and the invasion of the osteosarcoma cell MG-63 is inhibited, The effect of chemotherapy and chemotherapy on the expression of BRCA1 and the expression of p53 gene in osteosarcoma cell MG-63 was superior to that of simple radiotherapy group and chemotherapy group. The inhibition of the expression of Bax protein and the effect on the expression of Bcl-2 protein were superior to that of simple radiotherapy group and chemotherapy group. Conclusion:1. The expression of BRCA1 in the tissue of osteosarcoma is not expressed, and the expression rate of BRCA1 in osteosarcoma is significantly higher than that of chondrosarcoma. It is suggested that the expression of BRCA1 may be closely related to the occurrence and development of osteosarcoma. The detection of BRCA1 expression will have a guiding significance for the early diagnosis of osteosarcoma, the determination of the degree of malignancy and the prognosis. The expression of Bcl-2 was promoted by inhibiting the expression of BRCA1 and p53, and inhibiting the expression of Bax. It was suggested that BRCA1 might play a role in the BRCA1-p53 pathway. The effect of chemotherapy and chemotherapy on the proliferation and invasion of osteosarcoma cells MG-63, the inhibition of the expression of BRCA1 and p53, the inhibition of the expression of Bax and the expression of Bcl-2 were more significant than that of the simple radiotherapy group and the simple chemotherapy group. It is suggested that the combination of chemotherapy and chemotherapy is a good method for the treatment of osteosarcoma, and it is considered that the expression of BRCA1 protein can be used as one of the evaluation indexes for the treatment of human osteosarcoma cell MG-63.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R738.1
[Abstract]:Background: Osteosarcoma is the most common primary malignant bone tumor, which is derived from inter-bone leaf cells, which is good for children and adolescents. The second peak is between 60 and 80 years of age. There is a gender difference in the incidence of the disease, and more males than females. The location of the disease is usually found in the long bone, such as the proximal and distal femur of the humerus and the tibia. Approximately 20% of the patients were diagnosed with advanced metastases, with most lung metastases, and distant metastasis was the main cause of the failure and death of the patients with osteosarcoma. The current treatment is mainly combined with neoadjuvant chemotherapy, although the survival time of the patients with local lesions increased from 20% in 1970 to more than 60% today, it is still a challenge for patients with advanced metastatic osteosarcoma, and the 5-year survival rate is reduced below 30%. There is no substantial progress in the treatment of osteosarcoma in recent 30 years, and it is in urgent need of a more effective treatment plan. In order to find a more novel, safe and effective and low-toxic side-effect new treatment scheme, it has become one of the most important researches at home and abroad in the light of the low incidence of the patients with osteosarcoma, the extremely high degree of malignancy, the single treatment method and the large toxic and side effect, and the overall effect is not good. The key genes related to the development of osteosarcoma include TP53, Rb1, FAS, ATRX, PTEN, COX-2, HER2 and so on. Despite these advances, there is a lack of a signature molecule that can accurately predict the progression, metastasis and response to chemotherapy. BRCA1 is a breast cancer susceptibility gene, located on chromosome 17q21 and encodes 1863 amino acid proteins, and BRCA1 has 24 exons, the largest is exon 11, and the most important and most common mutation gene in exon 11 is breast cancer. BRCA1 is a tumor suppressor gene, which can inhibit cell proliferation and cell growth through a plurality of cell transduction pathways, block cells in G2/ M phase, and induce cell apoptosis. The role of BRCA1 may be associated with multiple signal pathways, such as the BRCA1-MAPK pathway, the BRCA1-p53 pathway, the BRCA1-Gdd45 pathway, where the BRCA1-p53 pathway involves cell apoptosis, the BRCA1 can modulate the expression of the p53 gene, and p53 directly regulates the expression of the apoptosis protein Bcl-2 and its family protein Bax, Therefore, BRCA1 can influence the expression of Bcl-2 and its family Bax by regulating the expression of p53. BRCA1 also involved DAN injury and repair, and is closely related to homologous recombination repair (HRR), so BRCA1 is involved in the development of many malignant tumors such as breast cancer and lung cancer. However, so far, the study on the relationship between BRCA1 and osteosarcoma has been lacking, so this study will focus on the study of the relationship between BRCA1 and osteosarcoma, especially in the evaluation of the curative effect of the chemotherapy and chemotherapy of the MG-63 in the BRCA1 and the osteosarcoma cell. Objective: To study the expression of BRCA1 in human osteosarcoma and osteosarcoma and its clinical significance. To investigate the effect of the expression of BRCA1 on the proliferation, invasion, cell cycle and apoptosis of the tumor cells, the potential value of the expression of BRCA1 protein in the treatment of osteosarcoma cell MG-63 chemoradiotherapy was discussed. Methods: The expression of BRCA1 in 45 human osteosarcoma tissues,22 chondrosarcoma and MG-63 of osteosarcoma cells was detected by immunohistochemistry. The relationship between the expression of BRCA1 and clinicopathological factors and the level of expression of BRCA1 in osteosarcoma cell MG-63 were discussed. BRCA1 was detected by RT-PCR and Western Blot method in the treatment of osteosarcoma cell MG-63 in radiotherapy (0 Gy, 0.5 Gy, 1.0 Gy, 1.5 Gy, 2.0 Gy, respectively), chemotherapy (0. mu.g/ ml,5. mu.g/ ml,10. mu.g/ ml,20. mu.g/ ml,40. mu.g/ ml, respectively) and chemoradiotherapy (0 Gy + 0. mu.g/ ml,2 Gy,5. mu.g/ ml,2 Gy + 5. mu.g/ ml, respectively). The expression level of p53, Bax and Bcl-2. The proliferation, invasion and apoptosis of the tumor cells were detected by using CCK-8 cell proliferation experiment, Transwell cell invasion experiment, Annexin V-Fitc and PI double-staining cell apoptosis experiment. Results:1. The expression of BRCA1 in human osteosarcoma tissues (29/45) and chondrosarcoma (3/22) was confirmed by clinical pathology. The positive expression rate of BRCA1 in osteosarcoma was significantly higher than that of chondrosarcoma (P0.05). The expression of BRCA1 was significantly associated with the tumor necrosis in the clinical and pathological factors of osteosarcoma (P0.05), and the expression of BRCA1 and age. There was no significant difference in sex, WHO type, position of lesion, genetic factor, lung metastasis, Codman's triangle, pathological fracture and pathological boundary (P0.05). In the osteosarcoma cell MG-63, the proliferation of the osteosarcoma cell MG-63 can be inhibited, and the more obvious the inhibition effect with the increase of the dose, promote the apoptosis of the osteosarcoma cell MG-63, cause the G2 phase block of the osteosarcoma cell, and increase the block of the osteosarcoma cell MG-63, and inhibit the invasion of the osteosarcoma cell MG-63, And the expression of BRCA1, p53 gene and protein in the osteosarcoma cell MG-63 is inhibited, the expression of the Bax protein is inhibited, and the expression of the Bcl-2 protein is promoted. in that osteosarcoma cell MG-63, cisplatin can inhibit the proliferation of the osteosarcoma cell MG-63 and increase the inhibition effect with the increase of the dose, promote the apoptosis of the osteosarcoma cell MG-63, increase the number of the apoptotic cells with the increase of the dose, cause the G2 phase block of the MG-63 cell, And the expression of the Bax protein is inhibited, and the expression of the Bcl-2 protein is promoted. In the osteosarcoma cell MG-63, the combination of radiotherapy and chemotherapy can inhibit the proliferation of the osteosarcoma cell MG-63, and the effect of promoting the cell apoptosis is between the radiotherapy and the chemotherapy; the effect of the MG-63G2 phase block in the osteosarcoma cell is between the radiotherapy and the chemotherapy; and the invasion of the osteosarcoma cell MG-63 is inhibited, The effect of chemotherapy and chemotherapy on the expression of BRCA1 and the expression of p53 gene in osteosarcoma cell MG-63 was superior to that of simple radiotherapy group and chemotherapy group. The inhibition of the expression of Bax protein and the effect on the expression of Bcl-2 protein were superior to that of simple radiotherapy group and chemotherapy group. Conclusion:1. The expression of BRCA1 in the tissue of osteosarcoma is not expressed, and the expression rate of BRCA1 in osteosarcoma is significantly higher than that of chondrosarcoma. It is suggested that the expression of BRCA1 may be closely related to the occurrence and development of osteosarcoma. The detection of BRCA1 expression will have a guiding significance for the early diagnosis of osteosarcoma, the determination of the degree of malignancy and the prognosis. The expression of Bcl-2 was promoted by inhibiting the expression of BRCA1 and p53, and inhibiting the expression of Bax. It was suggested that BRCA1 might play a role in the BRCA1-p53 pathway. The effect of chemotherapy and chemotherapy on the proliferation and invasion of osteosarcoma cells MG-63, the inhibition of the expression of BRCA1 and p53, the inhibition of the expression of Bax and the expression of Bcl-2 were more significant than that of the simple radiotherapy group and the simple chemotherapy group. It is suggested that the combination of chemotherapy and chemotherapy is a good method for the treatment of osteosarcoma, and it is considered that the expression of BRCA1 protein can be used as one of the evaluation indexes for the treatment of human osteosarcoma cell MG-63.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R738.1
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