【摘要】：背景:骨肉瘤是最常見(jiàn)的原發(fā)性惡性骨腫瘤,其起源于骨間葉細(xì)胞,好發(fā)于兒童及青少年,第二個(gè)發(fā)病高峰出現(xiàn)在60-80歲之間。發(fā)病有性別差異,男性多于女性。發(fā)病位置多見(jiàn)于長(zhǎng)骨,例如肱骨和脛骨近端、股骨遠(yuǎn)端。大約20%患者確診時(shí)已經(jīng)為晚期轉(zhuǎn)移患者,肺轉(zhuǎn)移居多,遠(yuǎn)處轉(zhuǎn)移是骨肉瘤患者治療失敗和死亡的主要原因。目前治療以新輔助化療結(jié)合手術(shù)為主,雖然局部病變患者5年生存期從1970年的20%上升至如今的60%以上,但是對(duì)于晚期轉(zhuǎn)移的骨肉瘤患者來(lái)說(shuō)仍是一個(gè)不小的挑戰(zhàn),5年生存期降至30%以下,近30年針對(duì)骨肉瘤的治療手段未有實(shí)質(zhì)性進(jìn)展,急需更加有效的治療方案。綜上,考慮骨肉瘤患者發(fā)病年齡輕、惡性程度極高、治療方法單一和毒副作用大,并且總體效果不佳的情況下,尋找更加新穎、安全有效、低毒副作用的新興治療方案已成為國(guó)內(nèi)外學(xué)者矚目的研究之一。目前與骨肉瘤發(fā)生發(fā)展相關(guān)的關(guān)鍵基因包括TP53、Rb1、FAS、ATRX、PTEN、COX-2、HER2等。盡管已經(jīng)有這些研究進(jìn)展,但是仍缺乏一個(gè)能準(zhǔn)確預(yù)測(cè)骨肉瘤進(jìn)展、轉(zhuǎn)移和對(duì)化療反應(yīng)性的標(biāo)志性分子。BRCA1是乳腺癌易感基因,位于染色體17q21,并且編碼1863個(gè)氨基酸蛋白,BRCA1有24個(gè)外顯子,最大的是第11外顯子,在第11外顯子中富含乳腺癌患者最重要也是最常見(jiàn)的突變基因。BRCA1是抑癌基因,能通過(guò)多條細(xì)胞轉(zhuǎn)導(dǎo)通路抑制細(xì)胞增殖、細(xì)胞生長(zhǎng),阻滯細(xì)胞于G2/M期,并誘導(dǎo)細(xì)胞凋亡。BRCA1的發(fā)揮抑癌作用可能與多條信號(hào)通路有關(guān),如BRCA1-MAPK通路、BRCA1-p53通路、BRCA1-Gdd45通路有關(guān),其中BRCA1-p53通路涉及細(xì)胞凋亡,BRCA1可以調(diào)節(jié)p53基因表達(dá),而且p53直接調(diào)控凋亡蛋白Bcl-2及其家族蛋白Bax的表達(dá),因此BRCA1可以通過(guò)調(diào)節(jié)p53的表達(dá)從而影響凋亡蛋白Bcl-2及其家族Bax的表達(dá)。BRCA1還涉及DAN損傷、修復(fù)過(guò)程,并且與同源重組修復(fù)(HRR)密切相關(guān),因此BRCA1作為抑癌基因參與了乳腺癌、肺癌等許多惡性腫瘤的發(fā)生發(fā)展過(guò)程。但是,到目前為止國(guó)內(nèi)外對(duì)于BRCA1與骨肉瘤的相關(guān)性研究卻較為缺乏,因此本研究將重點(diǎn)關(guān)注BRCA1與骨肉瘤的相關(guān)性研究方面,尤其側(cè)重在BRCA1與骨肉瘤細(xì)胞MG-63放化療的療效評(píng)估方面進(jìn)行研究。目的:研究BRCA1在人骨肉瘤組織和骨肉瘤細(xì)胞中的表達(dá)情況及其臨床意義。觀察骨肉瘤細(xì)胞MG-63在放化療的作用下,BRCA1表達(dá)水平對(duì)腫瘤細(xì)胞增殖、侵襲、細(xì)胞周期和凋亡的影響,從而探討B(tài)RCA1蛋白表達(dá)在骨肉瘤細(xì)胞MG-63放化療療效評(píng)估中的潛在價(jià)值。方法:利用免疫組化的方法檢測(cè)BRCA1在45例人骨肉瘤組織、22例軟骨肉瘤以及骨肉瘤細(xì)胞MG-63中的表達(dá),探討B(tài)RCA1的表達(dá)情況與臨床病理因素的聯(lián)系,以及在骨肉瘤細(xì)胞MG-63中的表達(dá)水平。利用RT-PCR、Western Blot方法檢測(cè)骨肉瘤細(xì)胞MG-63在放療(分別為0Gy、0.5Gy、1.0Gy、1.5Gy、2.0Gy)、化療(分別為0μg/ml、5μg/ml、10μg/ml、20μg/ml、40μg/ml)及放化療聯(lián)合(分別為0Gy+0μg/ml、2Gy、5μg/ml、2Gy+5μg/ml)作用下的BRCA1、p53、Bax和Bcl-2的表達(dá)水平。利用CCK-8細(xì)胞增殖實(shí)驗(yàn)、Transwell細(xì)胞侵襲實(shí)驗(yàn)、Annexin V-Fitc和PI雙染細(xì)胞凋亡實(shí)驗(yàn)對(duì)骨肉瘤細(xì)胞MG-63在放、化療及放化療聯(lián)合作用下腫瘤細(xì)胞的增殖能力、侵襲能力和腫瘤細(xì)胞凋亡等情況進(jìn)行檢測(cè)。結(jié)果:1.臨床病理證實(shí)BRCA1在人骨肉瘤組織高表達(dá)(29/45),軟骨肉瘤(3/22),在骨肉瘤癌旁組織中無(wú)BRCA1表達(dá),在骨肉瘤細(xì)胞MG-63表達(dá)。骨肉瘤組織的BRCA1陽(yáng)性表達(dá)率顯著高于軟骨肉瘤組織,二者相比差異有統(tǒng)計(jì)學(xué)意義(P0.05)。BRCA1表達(dá)情況與骨肉瘤臨床病理因素中的腫瘤壞死存在顯著關(guān)聯(lián),二者相比差異有統(tǒng)計(jì)學(xué)意義(P0.05),BRCA1表達(dá)情況與年齡、性別、WHO分型、病變位置、遺傳因素、肺轉(zhuǎn)移、Codman三角、病理性骨折和病理邊界相比無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。2.在骨肉瘤細(xì)胞MG-63中,放療可以抑制骨肉瘤細(xì)胞MG-63的增殖,且隨劑量的增加,抑制作用越明顯;促進(jìn)骨肉瘤細(xì)胞MG-63凋亡;引起骨肉瘤細(xì)胞G2期阻滯,且隨著劑量的增加阻滯加劇;抑制骨肉瘤細(xì)胞MG-63的侵襲,且隨劑量的增加,細(xì)胞數(shù)減少越明顯;抑制骨肉瘤細(xì)胞MG-63中BRCA1、p53基因和蛋白的表達(dá);抑制Bax蛋白的表達(dá),促進(jìn)Bcl-2蛋白的表達(dá)。3.在骨肉瘤細(xì)胞MG-63中,順鉑可以抑制骨肉瘤細(xì)胞MG-63的增殖,且隨劑量的增加,抑制作用增強(qiáng);促進(jìn)骨肉瘤細(xì)胞MG-63凋亡,且隨劑量的增加,凋亡細(xì)胞數(shù)也增加;引起MG-63細(xì)胞G2期阻滯,且隨著劑量的增加阻滯加劇;抑制骨肉瘤細(xì)胞MG-63的侵襲,且隨劑量的增加,細(xì)胞數(shù)減少越明顯;抑制BRCA1、p53基因和蛋白的表達(dá);抑制Bax蛋白表達(dá),促進(jìn)Bcl-2蛋白的表達(dá)。4.在骨肉瘤細(xì)胞MG-63中,放化療聯(lián)合作用抑制骨肉瘤細(xì)胞MG-63的增殖更顯著;其促進(jìn)細(xì)胞凋亡效果介于放療和化療之間;引起骨肉瘤細(xì)胞MG-63G2期阻滯的效果介于放療和化療之間;抑制骨肉瘤細(xì)胞MG-63的侵襲,抑制細(xì)胞侵襲的作用優(yōu)于單純放療組和化療組;放化療聯(lián)合作用對(duì)骨肉瘤細(xì)胞MG-63中BRCA1蛋白表達(dá)和p53基因表達(dá)的抑制效果優(yōu)于單純放療組和化療組;對(duì)Bax蛋白表達(dá)的抑制作用和對(duì)Bcl-2蛋白表達(dá)的促進(jìn)作用優(yōu)于單純放療組和化療組。結(jié)論:1、BRCA1在骨肉瘤組織中高表達(dá),在骨肉瘤癌旁組織中不表達(dá),且骨肉瘤組織的BRCA1陽(yáng)性表達(dá)率顯著高于軟骨肉瘤組織。提示其表達(dá)可能與骨肉瘤的發(fā)生、發(fā)展密切相關(guān),檢測(cè)BRCA1的表達(dá)將對(duì)骨肉瘤的早期診斷、判斷惡性程度及預(yù)后具有指導(dǎo)意義。2、放療、化療及放化療聯(lián)合均可以抑制骨肉瘤細(xì)胞MG-63的增殖、侵襲,引起細(xì)胞周期G2期阻滯,并促進(jìn)細(xì)胞凋亡;同時(shí)抑制BRCA1和p53的表達(dá),抑制Bax表達(dá)而促進(jìn)Bcl-2的表達(dá)。提示BRCA1可能通過(guò)BRCA1-p53通路發(fā)揮作用。3、放化療聯(lián)合對(duì)骨肉瘤細(xì)胞MG-63的增殖、侵襲的抑制作用、對(duì)BRCA1和p53表達(dá)的抑制作用、對(duì)Bax表達(dá)的抑制和Bcl-2表達(dá)的促進(jìn)作用較單純放療組和單純化療組更加顯著。提示放化療聯(lián)合治療是骨肉瘤治療的較好手段,并認(rèn)為BRCA1蛋白的表達(dá)可以作為人骨肉瘤細(xì)胞MG-63放化療療效的評(píng)價(jià)指標(biāo)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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R738.1

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