卵巢癌PDX模型的建立及鉴定

发布时间：2018-09-09 12:11

【摘要】：研究背景:卵巢癌(OC)是死亡率最高的妇科恶性肿瘤,其中最常见的是上皮性卵巢癌(EOC),约占卵巢癌的70%,在女性恶性肿瘤中死亡率居第五位[1]。由于临床症状不明显,患者在出现严重腹痛或消化道症状时才就诊,初诊患者中有2/3已属晚期,其中超过70%已有盆腔转移或淋巴结转移。目前NCCN指南的晚期卵巢癌标准治疗方案为“肿瘤细胞减灭术联合铂类药物化疗”,卵巢癌的主要综合治疗化疗方案是紫杉醇联合铂类化疗[2]。虽然外科手术技巧在不断进步,铂类为基础的联合化疗也取得了新的进展,但卵巢癌患者的5年生存率却并没有明显提高。主要原因是卵巢肿瘤患者在化疗过程中出现耐药现象,严重影响化疗效果。现在常用的卵巢癌研究模型是细胞模型和细胞株移植模型,由于细胞模型的缺陷,如体外培养时间过长导致难以避免的遗传改变及生物学行为变化,难以真实模拟卵巢癌的异质性和宿主微环境。因此我们迫切需要一个合适的临床前模型来研究卵巢癌发生、发展、转移的机制,研究化疗治疗及化疗耐药的机制,研究卵巢癌新的治疗方案。本研究将建立人源化的卵巢癌鼠模型(Patient-derived xenograft model,PDX模型)并鉴定模型与原肿瘤组织的相似性,为卵巢癌的精准治疗提供合适的模型。目的:1.建立人源化的卵巢癌鼠模型,即卵巢癌PDX模型;2.鉴定卵巢癌PDX模型与原肿瘤组织的相似性,包括组织病理学,分子特点的比较。方法:本研究已经通过了伦理委员会的批准1.收集了42例西京医院妇产科术中经冰冻证实且术后病理结果明确为卵巢恶性肿瘤的新鲜标本移植到SCID鼠皮下建立P1代鼠模型;2.P1代成瘤后剥除移植瘤组织传代到裸鼠皮下建立P2代鼠模型,成瘤后继续传代,分别命名为P3,P4,P5代;3.收集的临床标本记录临床资料,包括患者的年龄、临床分期、组织学亚型、手术时间、有无腹水及复发时间,成瘤后统计分析成瘤率与临床资料的相关性;4.成瘤入库的缓冻组织复苏检测组织的活性,成瘤能力;5.原组织与移植瘤组织均留部分组织固定,做HE染色及CA125免疫组化染色;6.原组织与移植瘤组织提DNA,P53外显子5-9基因测序分析。结果:1.卵巢癌PDX模型建模成功率35.7%(15/42),P2,P3代成瘤率100%,移植瘤均生长良好;统计分析发现病人的年龄与成瘤率无关(P=0.137),与组织学亚型无明显关系(P=0.461),与病人手术时间无关(P=0.694),与病人手术病理分期无关(P=0.222),但是与腹水有一定的相关性(P=0.047),与复发时间有相关性(P=0.001);2.复苏检测移植成功率100%,移植瘤均生长良好;3.经HE染色原组织与移植瘤组织病理学形态一致;4.CA125免疫组化染色原组织与移植瘤组织阳性率一致;5.P53外显子5-9基因测序结果表明原组织与移植瘤组织DNA序列一致。结论:1.本实验建模成功率35.7%(15/42),移植瘤均生长良好,且移植瘤的成功率与恶性腹水的存在有关,成瘤率或许可以作为卵巢癌患者预后的一项预测因素;2.经鉴定移植瘤与原组织的组织病理学和分子特点相似,初步判定本研究的PDX模型可以为卵巢癌的精准治疗提供临床前研究平台。
[Abstract]:Background: Ovarian cancer (OC) is the most lethal gynecologic malignancy, the most common of which is epithelial ovarian cancer (EOC), accounting for 70% of ovarian cancer, and the fifth leading cause of mortality in female malignancies. At present, the standard treatment regimen for advanced ovarian cancer under NCCN guidelines is "cytoreductive surgery combined with platinum-based chemotherapy". The main combined chemotherapy regimen for ovarian cancer is paclitaxel plus platinum-based chemotherapy [2]. New progress has been made in combination chemotherapy, but the 5-year survival rate of patients with ovarian cancer has not been significantly improved. The main reason is that patients with ovarian cancer develop drug resistance during chemotherapy, which seriously affects the effectiveness of chemotherapy. It is difficult to simulate the heterogeneity and host microenvironment of ovarian cancer because of the unavoidable genetic and biological behavior changes caused by long incubation time in vitro. Therefore, we urgently need a suitable preclinical model to study the mechanism of ovarian cancer occurrence, development and metastasis, the mechanism of chemotherapy and chemotherapy resistance, and the ovarian cancer. This study will establish a humanized mouse model of ovarian cancer (PDX model) and identify the similarity between the model and the original tumor tissue, so as to provide a suitable model for accurate treatment of ovarian cancer. Methods: This study has been approved by the Ethics Committee. 42 fresh specimens of ovarian malignancies confirmed by frozen surgery in Xijing Hospital were transplanted into SCID mice to establish the P1 generation mouse model. The P2 generation mouse model was established by peeling off the transplanted tumor tissue and passing it to the nude mice subcutaneously, and then passing it on, named P3, P4 and P5, respectively. 3. The clinical data were collected and recorded, including age, clinical stage, histological subtype, operation time, ascites and recurrence time. Result: 1. PDX model of ovarian cancer was successfully established by 35.7% (P 53 exon 5-9 gene sequencing). 15/42, P 2, P 3 generation tumorigenesis rate was 100%, the transplanted tumors grew well; statistical analysis showed that the patient's age was not related to the tumorigenesis rate (P = 0.137), had no significant relationship with histological subtypes (P = 0.461), had no correlation with the operation time (P = 0.694), had no correlation with the operation pathological stage (P = 0.222), but had a certain correlation with ascites (P = 0.047), and had no correlation with the recurrence time (P = 0.047). Resuscitation test showed that the success rate of transplantation was 100%, and the transplanted tumors grew well. 3. HE staining was consistent with the histopathological morphology of the transplanted tumors. 4. The positive rate of CA125 immunohistochemical staining was consistent with that of the transplanted tumors. 5. P53 exon 5-9 gene sequencing showed that the DNA sequence of the original tissues was consistent with that of the transplanted tumors. Conclusion: 1. The success rate of this model was 35.7% (15/42), and the transplanted tumors grew well, and the success rate of transplanted tumors was related to the presence of malignant ascites. Tumor formation rate may be a predictor of the prognosis of ovarian cancer patients. 2. The transplanted tumors were identified to be similar to the histopathological and molecular characteristics of the original tissues, and the PDX model of this study was preliminarily determined. It can provide a preclinical research platform for precise treatment of ovarian cancer.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.31;R-332

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