技术领域technical field
本发明属微生物动物细胞系技术领域，涉及卵巢透明细胞癌细胞 系，具体涉及一种中国人卵巢透明细胞癌细胞系FDOV1及其建立方 法。The invention belongs to the technical field of microbial animal cell lines, relates to ovarian clear cell cancer cell lines, in particular to a Chinese ovarian clear cell cancer cell line FDOV1 and its establishment method.
背景技术Background technique
现有技术公开了卵巢癌是恶性程度最高的妇科恶性肿瘤。卵巢透 明细胞癌(Ovarian Clear Cell Carcinoma，OCCC)，是第二大常见组 织学亚型，尤见于亚洲女性，目前已发表的研究结果多来自日本。国 内有研究发表了一系列文章，其中较全面分析了我国患者的特点，提 示卵巢透明细胞癌有特殊的临床病理特点和生物学行为，如，恶性程 度高、预后差，传统化疗不敏感，复发后缺少有效的治疗手段等等， 因此，进一步探索卵巢透明细胞癌的生物学行为和分子机制在临床研 究与实践中显得尤为重要。The prior art discloses that ovarian cancer is the most malignant gynecological malignancy. Ovarian Clear Cell Carcinoma (OCCC) is the second most common histological subtype, especially in Asian women, and most of the published research results are from Japan. A series of articles have been published in domestic research, which comprehensively analyzed the characteristics of Chinese patients, suggesting that ovarian clear cell carcinoma has special clinicopathological characteristics and biological behaviors, such as high degree of malignancy, poor prognosis, insensitivity to traditional chemotherapy, and recurrence Therefore, it is particularly important to further explore the biological behavior and molecular mechanism of ovarian clear cell carcinoma in clinical research and practice.
业内知晓，人肿瘤细胞系的建立和鉴定，对肿瘤生物学行为及分 子机制的研究尤为重要。据知，目前美国标准生物品收藏中心 (ATCC)仅存有2株人卵巢透明细胞癌细胞系(ES-2、TOV-21G)， 而我国国家实验细胞资源共享平台仅存有1株(ES-2)。2016年发表 的文献公开了迄今为止建立和鉴定的人卵巢透明细胞癌细胞系：1987 年至2016年间共有14株，仅有1株来自中国香港，而其他细胞株均为 日本学者建立。It is known in the industry that the establishment and identification of human tumor cell lines is particularly important for the study of tumor biological behavior and molecular mechanism. As far as is known, there are only two human ovarian clear cell carcinoma cell lines (ES-2, TOV-21G) in the American Standard Biological Collection (ATCC), and only one strain (ES-2, TOV-21G) in the National Experimental Cell Resource Sharing Platform in my country. -2). The literature published in 2016 disclosed the human ovarian clear cell carcinoma cell lines established and identified so far: from 1987 to 2016, there were 14 strains, only 1 came from Hong Kong, China, while the other cell lines were established by Japanese scholars.
基于现有技术的现状，本申请的发明人拟建立和鉴定我国患者来 源的卵巢透明细胞癌细胞系，具体涉及一种中国人卵巢透明细胞癌细 胞系FDOV1及其建立方法。Based on the current state of the prior art, the inventors of the present application intend to establish and identify ovarian clear cell carcinoma cell lines derived from Chinese patients, and specifically relate to a Chinese ovarian clear cell carcinoma cell line FDOV1 and its establishment method.
于本发明相关的现有技术以及参考文献有，The prior art and references related to the present invention are,
1.Chen W,Zheng R,Baade PD,Zhang S,Zeng H,Bray F,et al.Cancerstatistics in China,2015.CA Cancer J Clin.2016；66:115-32.1. Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, et al. Cancer statistics in China, 2015. CA Cancer J Clin. 2016; 66:115-32.
2.Sung PL,Chang YH,Chao KC,Chuang CM.Global distribution pattern ofhistological subtypes of epithelial ovarian cancer:a database analysis andsystematic review.Gynecol Oncol.2014；133:147-54.2. Sung PL, Chang YH, Chao KC, Chuang CM. Global distribution pattern of histological subtypes of epithelial ovarian cancer: a database analysis and systematic review. Gynecol Oncol. 2014; 133:147-54.
3.Okamoto A,Glasspool RM,Mabuchi S,Matsumura N,Nomura H, Itamochi H,et al.Gynecologic Cancer InterGroup(GCIG)consensus review for clear cellcarcinoma of the ovary.Int J Gynecol Cancer. 2014；24:S20-5.3. Okamoto A, Glasspool RM, Mabuchi S, Matsumura N, Nomura H, Itamochi H, et al. Gynecologic Cancer InterGroup (GCIG) consensus review for clear cell carcinoma of the ovary. Int J Gynecol Cancer. 2014; 24:S20-5 .
4.Ye S,Yang J,You Y,Cao D,Bai H,Lang J,et al.Comparative study ofovarian clear cell carcinoma with and without endometriosis in People'sRepublic of China.Fertil Steril.2014；102:1656-62.4. Ye S, Yang J, You Y, Cao D, Bai H, Lang J, et al. Comparative study of ovarian clear cell carcinoma with and without endometriosis in People's Republic of China. Fertil Steril. 2014; 102:1656-62 .
5.Ye S,You Y,Yang J,Cao D,Bai H,Huang H,et al.Comparison of pure andmixed-type clear cell carcinoma of the ovary:a clinicopathological analysisof 341chinese patients.Int J Gynecol Cancer. 2014；24:1590-6.5. Ye S, You Y, Yang J, Cao D, Bai H, Huang H, et al. Comparison of pure and mixed-type clear cell carcinoma of the ovary: a clinicopathological analysis of 341 Chinese patients. Int J Gynecol Cancer. 2014; 24 :1590-6.
6.Ye S,Yang J,Cao D,Bai H,Huang H,Wu M,et al.Characteristic andprognostic implication of venous thromboembolism in ovarian clear cellcarcinoma:a 12-year retrospective study.PLoS One.2015；10:e0121818. 7.Ye S,Yang J,You Y,Cao D,Huang H,Wu M,et al.Comparison of Clinical Characteristicand Prognosis between Ovarian Clear Cell Carcinoma and Serous Carcinoma:A 10-Year Cohort Study of Chinese Patients.PLoS One.2015；10:e0133498.6. Ye S, Yang J, Cao D, Bai H, Huang H, Wu M, et al. Characteristic and prognostic implications of venous thromboembolism in ovarian clear cell carcinoma: a 12-year retrospective study. PLoS One. 2015; 10: e0121818. 7. Ye S, Yang J, You Y, Cao D, Huang H, Wu M, et al. Comparison of Clinical Characteristic and Prognosis between Ovarian Clear Cell Carcinoma and Serous Carcinoma: A 10-Year Cohort Study of Chinese Patients. PLoS One. 2015;10:e0133498.
8.Yamada T,Hattori K,Satomi H,Okazaki T,Mori H,Hirose Y.Establishment and characterization of a cell line(HCH-1)originating from ahuman clear cell carcinoma of the ovary.J Ovarian Res. 2016；9:32.。8. Yamada T, Hattori K, Satomi H, Okazaki T, Mori H, Hirose Y. Establishment and characterization of a cell line (HCH-1) originating from a human clear cell carcinoma of the ovary. J Ovarian Res. 2016; 9: 32.
发明内容Contents of the invention
本发明的目的是克服现有技术存在的缺陷，提供一种卵巢透明细 胞癌细胞系，具体涉及一种中国人卵巢透明细胞癌细胞系FDOV1及其 建立方法。The purpose of the present invention is to overcome the defects in the prior art and provide a clear cell ovarian cancer cell line, in particular to a Chinese ovarian clear cell cancer cell line FDOV1 and its establishment method.
本发明建立了一种来源于中国患者的卵巢透明细胞癌细胞系，该 细胞系可稳定传代，成瘤性好，能适用于建立动物模型。该细胞系可 用于卵巢透明细胞癌的发生发展及转移机制，耐药原理及新药筛选等 的研究。The present invention establishes an ovarian clear cell carcinoma cell line derived from a Chinese patient. The cell line can be passed down stably, has good tumorigenicity, and is suitable for establishing an animal model. This cell line can be used in the research of the occurrence, development and metastasis mechanism of ovarian clear cell carcinoma, the mechanism of drug resistance and the screening of new drugs, etc.
本发明提供的一种中国人卵巢透明细胞癌细胞系，命名为人卵巢透明 细胞癌细胞株FDOV1，已于2017年3月16日由中国微生物菌种保藏管理 委员会普通微生物中心(CGMCC)保藏，保藏单位地址：北京市朝阳区北 辰西路1号院3号，保藏编号为CGMCCNO.13812。A Chinese ovarian clear cell carcinoma cell line provided by the present invention, named as human ovarian clear cell carcinoma cell line FDOV1, has been preserved by the General Microorganism Center (CGMCC) of China Committee for the Collection of Microorganisms on March 16, 2017. Unit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCCNO.13812.
本发明的中国人卵巢透明细胞癌细胞系通过下述方法制得，The Chinese ovarian clear cell carcinoma cell line of the present invention is prepared by the following method,
(一)取材(1) Materials
获得离体新鲜的卵巢肿瘤切除标本，病灶组织浸入无菌PBS溶 液清洗；Obtain fresh ovarian tumor resection specimens in vitro, and immerse the lesion tissue in sterile PBS solution for cleaning;
(二)原代培养(2) Primary culture
选取较硬的肿瘤组织，将其切成米粒大小的组织数块，均匀的接 种于10cm大皿中，静置培养，每4天半量换液1次，约1周后细胞 从组织块长出；Select hard tumor tissue, cut it into several pieces of rice grain-sized tissue, inoculate evenly in a 10cm large dish, and culture it statically, change the medium once every 4 days, and the cells grow out of the tissue piece after about 1 week;
(三)细胞纯化(3) Cell purification
接种约2周后传第1代，用0.25％胰蛋白酶消化，将细胞悬液接 种于培养皿中，静置约15～20分钟后于倒置相差显微镜下观察，见 部分细胞贴壁，吸取细胞悬液入另一培养瓶中继续培养；按上述方法， 重复2次传代，使肿瘤细胞与成纤维细胞完全分开；Passage the first generation about 2 weeks after inoculation, digest with 0.25% trypsin, inoculate the cell suspension in a culture dish, and observe it under an inverted phase contrast microscope after standing for about 15 to 20 minutes. Put the suspension into another culture flask to continue culturing; according to the above method, repeat the passage twice to completely separate the tumor cells from the fibroblasts;
(四)传代培养(4) Subculture
当细胞布满瓶底时，吸除培养液，PBS缓冲液漂洗，0.25％胰蛋 白酶消化，按1:2的比例传代，目前已传至50余代；When the cells are all over the bottom of the bottle, remove the culture medium, rinse with PBS buffer, digest with 0.25% trypsin, and pass passage at a ratio of 1:2, which has been passed to more than 50 generations;
经生物学特性鉴定获得中国人卵巢透明细胞癌细胞系，命名为 FDOV1，保藏号为NO.13812。The Chinese ovarian clear cell carcinoma cell line was obtained through the identification of biological characteristics, named FDOV1, and the preservation number was NO.13812.
本发明中：体外常规培养条件为含10％FBS、1％双抗的 Medium199细胞培养基，培养温度为37℃，气体环境为5％CO2/95％ 空气，湿度为饱和湿度。In the present invention: the conventional in vitro culture condition is Medium199 cell culture medium containing 10% FBS and 1% double antibody, the culture temperature is 37°C, the gas environment is 5% CO2 /95% air, and the humidity is saturated humidity.
本发明的细胞系FDOV1具有以下生物学特性：The cell line FDOV1 of the present invention has the following biological characteristics:
1.单层贴壁生长，失去接触抑制，形态不一，呈圆形椭圆形或纺 锤形扁平状，或不规则形；1. Single-layer adherent growth, loss of contact inhibition, different shapes, round, oval, spindle-shaped, flat, or irregular;
2.体外培养生长良好，每4天传代一次，能够连续稳定传代，目 前已传至50余代；2. The in vitro culture grows well and can be passaged every 4 days, which can be continuously and stably passed down to more than 50 generations;
3.染色体核型分析结果提示该细胞为近二倍体细胞系，存在复杂 的易位,缺失等染色体畸变；3. The results of chromosome karyotype analysis suggest that the cell is a nearly diploid cell line with complex translocations, deletions and other chromosomal aberrations;
4.流式细胞周期检测提示：G1期细胞42.28％；G2期细胞36.10％； S期细胞21.62％。4. The detection of flow cytometry revealed: 42.28% of cells in G1 phase; 36.10% of cells in G2 phase; 21.62% of cells in S phase.
5.肿瘤标志物检测：CA125:33.7U/ml；CA199:0.78U/ml； CA153:<1.00U/ml；CA724:2.96U/ml；AFP:<0.61ng/ml；CEA:0.21ng/ml； HE4:<15nmol/L。5. Tumor marker detection: CA125:33.7U/ml; CA199:0.78U/ml; CA153:<1.00U/ml; CA724:2.96U/ml; AFP:<0.61ng/ml; CEA:0.21ng/ml ; HE4:<15nmol/L.
6.二代测序结果：FDOV1及肿瘤组织同时检测到ARID1A fs突 变及PIK3CA H1047R突变。6. Next-generation sequencing results: ARID1A fs mutation and PIK3CA H1047R mutation were both detected in FDOV1 and tumor tissue.
7.FDOV1免疫细胞化学染色与临床肿瘤组织免疫化学染色结果 一致，证实FDOV1来源于肿瘤组织。7. FDOV1 immunocytochemical staining was consistent with clinical tumor tissue immunochemical staining, confirming that FDOV1 originated from tumor tissue.
8.将第18代FDOV1细胞按1*107数量级接种于NOD/SKID裸 鼠，20天后移植瘤形成，病理切片提示移植瘤组织学特性与原发瘤 相似。8. The 18th generation of FDOV1 cells was inoculated into NOD/SKID nude mice on the order of 1*107 , and transplanted tumors formed after 20 days. Pathological sections showed that the histological characteristics of the transplanted tumors were similar to those of the primary tumors.
本发明提供了一种中国人卵巢透明细胞癌细胞系FDOV1，该细 胞系形态稳定，可以稳定多次传代，可用于卵巢透明细胞癌生物学行 为的研究；具有较强的致瘤性，可用于建立卵巢透明细胞癌动物模型， 所制得的动物模型适用于卵巢透明细胞癌发生发展的分子机制研究， 可进一步探究其对传统化疗药物的耐药机理并筛选有效的靶向药物， 该细胞系是人卵巢透明细胞癌基础研究及临床前期应用的理想细胞 系。The present invention provides a Chinese ovarian clear cell carcinoma cell line FDOV1, the cell line is stable in shape, can be stably passed down for multiple times, and can be used for the research on the biological behavior of ovarian clear cell carcinoma; it has strong tumorigenicity and can be used for The animal model of ovarian clear cell carcinoma is established. The obtained animal model is suitable for the study of the molecular mechanism of the occurrence and development of ovarian clear cell carcinoma. It can further explore its resistance mechanism to traditional chemotherapy drugs and screen effective targeted drugs. The cell line It is an ideal cell line for basic research and preclinical application of human ovarian clear cell carcinoma.
附图说明Description of drawings
图1.FDOV1细胞形态学观察(倒置相差显微镜100×)。Figure 1. Morphological observation of FDOV1 cells (inverted phase-contrast microscope 100×).
图2.FDOV1细胞生长曲线。Figure 2. Growth curve of FDOV1 cells.
图3.FDOV1染色体核型分析。Figure 3. FDOV1 chromosome karyotype analysis.
图4.流式细胞仪分析FDOV1细胞周期。Figure 4. Flow cytometry analysis of FDOV1 cell cycle.
图5.二代测序结果。A：FDOV1及肿瘤组织单核苷酸变异(SNV) 结果分析；B：FDOV1及肿瘤组织基因拷贝数变异(CNV)结果分 析。Figure 5. Next-generation sequencing results. A: Analysis of FDOV1 and tumor tissue single nucleotide variation (SNV) results; B: Analysis of FDOV1 and tumor tissue gene copy number variation (CNV) results.
图6.FDOV1细胞在NOD/SCID小鼠体内成瘤MRI成像。Figure 6. MRI imaging of tumor formation of FDOV1 cells in NOD/SCID mice.
图7.FDOV1细胞、临床肿瘤组织及移植瘤病理切片免疫化学 分析。Figure 7. Immunochemical analysis of FDOV1 cells, clinical tumor tissues and pathological sections of transplanted tumors.
具体实施方式Detailed ways
实施例1制备中国人卵巢透明细胞癌细胞系FDOV1Example 1 Preparation of Chinese ovarian clear cell carcinoma cell line FDOV1
(1)取材(1) Take materials
2016年5月获得离体的新鲜的卵巢透明细胞癌手术切除标本(标 本来源自复旦大学附属肿瘤医院卵巢透明细胞癌患者，女，67岁， 病理诊断：左附件卵巢透明细胞癌)，取病灶组织1cm3浸入无菌PBS 溶液清洗，并去除结缔组织和坏死组织；In May 2016, a fresh surgical resection specimen of ovarian clear cell carcinoma was obtained in vitro (the specimen was obtained from a patient with ovarian clear cell carcinoma in Fudan University Cancer Hospital, female, 67 years old, pathological diagnosis: left adnexal ovarian clear cell carcinoma), and the lesion was taken Tissue 1cm3 was immersed in sterile PBS solution to clean, and remove connective tissue and necrotic tissue;
(2)原代培养(2) Primary culture
将肿瘤组织剪成约1mm3大小的小块，将组织块均匀接种于10cm 大皿中，静置培养，每4天半量换液1次，约1周后观察到细胞从组 织块长出；Cut the tumor tissue into small pieces with a size of about 1 mm3 , inoculate the tissue pieces evenly in a 10cm large dish, culture them statically, change the medium every 4 days in half, and observe cells grow out of the tissue pieces after about 1 week;
(3)细胞纯化(3) Cell purification
接种约2周后传第1代，用0.25％胰蛋白酶消化，离心后用无血 清培养基重悬接种于一个培养皿中，静置15～20min，于倒置相差显 微镜下观察，见部分细胞贴壁，小心吸取细胞悬液入另一培养瓶中继 续培养。按上述方法传代处理2次，可使肿瘤细胞与成纤维细胞完全 分开；About 2 weeks after inoculation, pass the first generation, digest with 0.25% trypsin, resuspend and inoculate in a culture dish with serum-free medium after centrifugation, let stand for 15-20min, observe under an inverted phase-contrast microscope, see some cell stickers Carefully pipette the cell suspension into another culture flask to continue culturing. Passage treatment twice according to the above method can completely separate tumor cells from fibroblasts;
(4)传代培养(4) Subculture
当细胞布满瓶底时，吸除原瓶中的旧培养液，用PBS缓冲液漂洗1～2 遍，用0.25％胰蛋白酶消化，按1:2的比例传代，目前已传至50余代；制 得中国人卵巢透明细胞癌细胞系，命名为人卵巢透明细胞癌细胞株FDOV1， 已于2017年3月16日由中国微生物菌种保藏管理委员会普通微生物中心 (CGMCC)保藏，保藏单位地址：北京市朝阳区北辰西路1号院3号，保 藏编号为CGMCC NO.13812。When the cells are all over the bottom of the bottle, suck off the old culture medium in the original bottle, rinse with PBS buffer 1-2 times, digest with 0.25% trypsin, and pass passage at a ratio of 1:2. It has been passed to more than 50 generations ; The Chinese ovarian clear cell carcinoma cell line was obtained, named as the human ovarian clear cell carcinoma cell line FDOV1, which was preserved by the General Microorganism Center (CGMCC) of the China Committee for the Collection of Microorganisms on March 16, 2017. The address of the preservation unit is: No. 3, No. 1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.13812.
本实验体外常规培养条件为含10％FBS、1％双抗的Medium199 细胞培养基，培养温度为37℃，气体环境为5％CO2/95％空气，湿度 为饱和湿度；The conventional culture conditions in this experiment were Medium199 cell culture medium containing 10% FBS and 1% double antibody, the culture temperature was 37°C, the gas environment was 5% CO2 /95% air, and the humidity was saturated humidity;
(5)生物学特性的鉴定(5) Identification of biological characteristics
细胞形态：倒置相差显微镜下观察，FDOV1细胞单层贴壁生长， 失去接触抑制，形态不一，呈圆形椭圆形或纺锤形扁平状，或不规则 形(如图1所示)；Cell morphology: Observed under an inverted phase-contrast microscope, FDOV1 cells grow adherently in a single layer, lose contact inhibition, and have different shapes, such as round, oval, spindle-shaped, flat, or irregular (as shown in Figure 1);
细胞生长曲线：取对数生长期的细胞，调整细胞密度为每孔 2*103/100ul，接种于96孔板，设6个复孔；接种后连续10天在同一 时间加入100ul CCK-8试剂与培养基1:9混合液反应2小时，用酶标 仪测定在450nm处的吸光度(OD)，以时间为横轴，OD均值为纵 轴绘制细胞生长曲线(如图2所示)；Cell growth curve: Take cells in the logarithmic growth phase, adjust the cell density to 2*103 /100ul per well, inoculate in a 96-well plate, set up 6 duplicate wells; add 100ul CCK-8 at the same time for 10 consecutive days after inoculation The reagent was reacted with the 1:9 mixture of the medium for 2 hours, and the absorbance (OD) at 450nm was measured with a microplate reader, and the time was taken as the horizontal axis, and the OD mean value was the vertical axis to draw the cell growth curve (as shown in Figure 2);
染色体核型分析：取对数生长期细胞，用0.25μg/ml秋水仙素处 理6小时，37℃过夜，采集分裂中期细胞，固定液(甲醇:冰醋酸＝3:1) 固定，标本经胰蛋白酶消化处理后，用Giemsa染液染色，于显微镜下 观察计数，随机计数45个细胞，其中2n＝45/46有35个(78％)，2n ＝47条有3个(7％)，4n＝86-90有6个(13％)，其它数目1个(2％)， 染色体存在复杂的易位,缺失等染色体畸变，结果符合恶性肿瘤的遗 传学特征(如图3所示)；Chromosomal karyotype analysis: cells in logarithmic growth phase were taken, treated with 0.25 μg/ml colchicine for 6 hours, overnight at 37°C, metaphase cells were collected, fixed in fixative solution (methanol:glacial acetic acid = 3:1), and the specimen was passed through pancreas After protease digestion, stain with Giemsa staining solution, observe and count under a microscope, randomly count 45 cells, wherein 2n=45/46 have 35 (78%), 2n=47 have 3 (7%), 4n = 6 (13%) in 86-90, and 1 (2%) in the other number, complex translocations, deletions and other chromosomal aberrations in the chromosomes, the results conform to the genetic characteristics of malignant tumors (as shown in Figure 3);
流式细胞周期检测：收集1*106细胞于15ml离心管中，预冷的PBS 重悬两次后，离心弃上清。细胞沉淀用3ml－20℃75％乙醇重悬混匀， 置－20℃过夜，离心，弃上清，加入500ulPI染色液，在暗处4℃孵育20min，流式细胞仪分析标本，用488mm激发光，600nm波长滤器检 测PI荧光；结果表明：G1期细胞42.28％；G2期细胞36.10％；S期细胞 21.62％(如图4所示)；Flow cytometry detection: Collect 1*106 cells in a 15ml centrifuge tube, resuspend twice in pre-cooled PBS, and discard the supernatant by centrifugation. Resuspend the cell pellet with 3ml-20°C 75% ethanol and mix well, put it at -20°C overnight, centrifuge, discard the supernatant, add 500ulPI staining solution, incubate at 4°C in the dark for 20min, analyze the specimen by flow cytometry, and excite with 488mm Light, 600nm wavelength filter detects PI fluorescence; The result shows: G1 phase cell 42.28%; G2 phase cell 36.10%; S phase cell 21.62% (as shown in Figure 4);
肿瘤标志物检测准备2*10^6个细胞(约2个25T培养瓶，每瓶约7ml 培养)，培养48h后，收集上清，换液，48h后，再次收集上清，送检. CA125:33.7U/ml；CA199:0.78U/ml；CA153:<1.00U/ml； CA724:2.96U/ml；AFP:<0.61ng/ml；CEA:0.21ng/ml；HE4:<15nmol/L；Tumor marker detection Prepare 2*10^6 cells (about 2 25T culture flasks, about 7ml culture in each bottle), after 48 hours of culture, collect the supernatant, change the medium, after 48 hours, collect the supernatant again, and send it for inspection. CA125 :33.7U/ml; CA199:0.78U/ml; CA153:<1.00U/ml; CA724:2.96U/ml; AFP:<0.61ng/ml; CEA:0.21ng/ml; HE4:<15nmol/L;
二代测序基因谱分析：按Qiagen DNA提取试剂盒内实验步骤对 病人肿瘤组织、血及FDOV1DNA进行提取，经酶标仪检测DNA浓度， 凝胶电泳检查DNA完整程度，利用Covaris将gDNA随机打断，用 sample purification beads进行纯化，产物的末端经过修补成平末端，接着用磁珠进行片段筛选，然后在3’末端加A，连接PCR反应接头， 进行PCR扩增，然后与使用Sure Select capture library在杂交缓冲液中 进行杂交，使用Dynal磁珠捕获杂交的目的片段并分离纯化，PCR扩 增捕获的DNA片段并纯化PCR产物，然后进行捕获文库的质检，包括 琼脂糖凝胶电泳质检、Qubit浓度测定和2100Bioanalyzer片段长度测 定，将待测DNA文库浓度在cBot上完成cluster generation，然后将 Flowcell转移到测序系统上，按照Illumina的标准流程进行二代测序及 CNV、SNV数据分析(样本有效深度>250X,target区域捕获率约70％)， 结果显示，FDOV1及肿瘤组织同时检测到ARID1A fs突变及PIK3CA H1047R突变(如图5所示)；Next-generation sequencing gene profile analysis: extract the patient's tumor tissue, blood and FDOV1 DNA according to the experimental procedures in the Qiagen DNA extraction kit, detect the DNA concentration with a microplate reader, check the integrity of the DNA by gel electrophoresis, and use Covaris to randomly interrupt the gDNA , purified with sample purification beads, the end of the product was repaired to blunt end, then fragment screening was performed with magnetic beads, then A was added at the 3' end, the PCR reaction adapter was connected, and PCR amplification was performed, and then used with the Sure Select capture library in Hybridization is carried out in the hybridization buffer, using Dynal magnetic beads to capture the hybridized target fragments and separate and purify them, PCR amplifies the captured DNA fragments and purifies the PCR products, and then performs quality inspection of the capture library, including agarose gel electrophoresis quality inspection, Qubit Concentration determination and 2100Bioanalyzer fragment length determination, the concentration of the DNA library to be tested is completed on the cBot to complete the cluster generation, and then the Flowcell is transferred to the sequencing system, and the next-generation sequencing and CNV and SNV data analysis are performed according to Illumina's standard procedures (the sample effective depth> 250X, the capture rate of the target area is about 70%), the results showed that both ARID1A fs mutation and PIK3CA H1047R mutation were detected in FDOV1 and tumor tissue (as shown in Figure 5);
FDOV1细胞及肿瘤组织免疫化学鉴定：将手术标本以及单克隆 FDOV1细胞用福尔马林固定，石蜡包埋切片，进行HE染色(如图6 所示)，选取HNF1β、PAX8对单克隆FDOV1细胞进行免疫细胞化 学染色，与肿瘤组织免疫组织化学染色比对(如图7所示)，结果表 明，免疫细胞化学染色与免疫组织化学染色结果一致，证实其保留了 原始肿瘤细胞的分化表型；Immunochemical identification of FDOV1 cells and tumor tissues: the surgical specimens and monoclonal FDOV1 cells were fixed in formalin, embedded in paraffin, and stained with HE (as shown in Figure 6). HNF1β and PAX8 were selected for monoclonal FDOV1 cells. Immunocytochemical staining was compared with tumor tissue immunohistochemical staining (as shown in Figure 7). The results showed that the immunocytochemical staining was consistent with the immunohistochemical staining results, confirming that it retained the differentiated phenotype of the original tumor cells;
细胞成瘤性：体外大规模扩增细胞，将第18代FDOV1细胞以1*107数量级皮下接种NOD/SCID小鼠3只，20天后可观察到3只裸鼠全部 移植瘤长出，继续生长20天后，小动物MRI成像显示移植瘤呈结节状， 外有包膜(如图6所示)；Cell tumorigenicity: large-scale expansion of cells in vitro, the 18th generation FDOV1 cells were subcutaneously inoculated into 3 NOD/SCID mice on the order of 1*107 , and all transplanted tumors in the 3 nude mice could be observed 20 days later. Continue After growing for 20 days, MRI imaging of small animals showed that the transplanted tumor was nodular and had a capsule (as shown in Figure 6);
肿瘤的病理学鉴定：继续培养一周后，常规处理2只小鼠(剩余1 只小鼠在继续培养过程中死于肺炎)，肿瘤组织经福尔马林固定，石 蜡包埋切片，进行HE染色及免疫组织化学染色，结果表明，移植瘤 免疫组织化学染色跟原临床标本免疫组织化学染色结果一致，形成对 应关系(如图7所示)。Pathological identification of the tumor: After continuing to culture for one week, 2 mice were routinely treated (the remaining 1 mouse died of pneumonia during the continuing culture), the tumor tissue was fixed in formalin, embedded in paraffin and sectioned for HE staining And immunohistochemical staining, the results showed that the immunohistochemical staining of transplanted tumors was consistent with the immunohistochemical staining results of the original clinical specimens, forming a corresponding relationship (as shown in Figure 7).
本发明提供了一种中国人卵巢透明细胞癌细胞系FDOV1，该细胞 系形态稳定，可以稳定多次传代，用于卵巢透明细胞癌生物学行为的 研究；具有较强的致瘤性，可用于建立卵巢透明细胞癌动物模型，制 得的动物模型适用于卵巢透明细胞癌发生发展的分子机制研究以及 药物筛选。The invention provides a Chinese ovarian clear cell carcinoma cell line FDOV1, which is stable in shape and can be stably passed down for multiple times, and is used for the research on the biological behavior of ovarian clear cell carcinoma; it has strong tumorigenicity and can be used for The animal model of ovarian clear cell carcinoma is established, and the obtained animal model is suitable for molecular mechanism research and drug screening of the occurrence and development of ovarian clear cell carcinoma.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710554757.9ACN108504637A (en) | 2017-07-10 | 2017-07-10 | Chinese ovarian clear cell carcinoma cell line |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710554757.9ACN108504637A (en) | 2017-07-10 | 2017-07-10 | Chinese ovarian clear cell carcinoma cell line |
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| CN108504637Atrue CN108504637A (en) | 2018-09-07 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710554757.9APendingCN108504637A (en) | 2017-07-10 | 2017-07-10 | Chinese ovarian clear cell carcinoma cell line |
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| CN (1) | CN108504637A (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486767A (en)* | 2018-11-20 | 2019-03-19 | 上海市奉贤区中心医院 | A kind of people's epithelial ovarian cancer cell SHOV4 and its application |
| CN109666747A (en)* | 2019-02-20 | 2019-04-23 | 天津脉络医学检验有限公司 | A kind of primer combination of probe object and its application |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5710038A (en)* | 1994-11-25 | 1998-01-20 | Universite De Montreal | Primary cultures of normal and tumoral human ovarian epithelium |
| CN1490403A (en)* | 2003-08-25 | 2004-04-21 | 复旦大学附属肿瘤医院 | Human inflammatory breast cancer cell line and its establishment method |
| CN103026227A (en)* | 2010-04-22 | 2013-04-03 | 不列颠哥伦比亚省癌症分社 | Novel biomarkers and targets for ovarian carcinoma |
| US20130210900A1 (en)* | 2010-09-03 | 2013-08-15 | The Johns Hopkins University | ARID1A and PPP2R1A Mutations in Cancer |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5710038A (en)* | 1994-11-25 | 1998-01-20 | Universite De Montreal | Primary cultures of normal and tumoral human ovarian epithelium |
| CN1490403A (en)* | 2003-08-25 | 2004-04-21 | 复旦大学附属肿瘤医院 | Human inflammatory breast cancer cell line and its establishment method |
| CN103026227A (en)* | 2010-04-22 | 2013-04-03 | 不列颠哥伦比亚省癌症分社 | Novel biomarkers and targets for ovarian carcinoma |
| US20130210900A1 (en)* | 2010-09-03 | 2013-08-15 | The Johns Hopkins University | ARID1A and PPP2R1A Mutations in Cancer |
| Title |
|---|
| RONALD L. CHANDLER ET AL.,: "Coexistent ARID1A-PIK3CA mutations promote ovarian clear-cell tumorigenesis through pro-tumorigenic inflammatory cytokine signaling", 《NAT COMMUN》* |
| WEI JIANG ET AL.,: "Establishment and molecular characterization of a human ovarian clear cell carcinoma cell line (FDOV1)", 《JOURNAL OF OVARIAN RESEARCH》* |
| YAMADA ET AL.,: "Establishment and characterization of a cell line (HCH-1) originating from a human clear cell carcinoma of the ovary", 《JOURNAL OF OVARIAN RESEARCH》* |
| 吴慕云等: "应用下一代测序技术检测肿瘤单核苷酸多态性研究进展", 《广东药学院学报》* |
| 贾连群等: "《现代基础医学理论与技术进展》", 31 August 2015, 中国医药科技出版社* |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486767A (en)* | 2018-11-20 | 2019-03-19 | 上海市奉贤区中心医院 | A kind of people's epithelial ovarian cancer cell SHOV4 and its application |
| CN109666747A (en)* | 2019-02-20 | 2019-04-23 | 天津脉络医学检验有限公司 | A kind of primer combination of probe object and its application |
| Publication | Publication Date | Title |
|---|---|---|
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