CN1336431A - Establishment of immortal human ovary carcinoma cell strain - Google Patents

Abstract

Translated fromChinese

本发明公开了一种人卵巢癌永生化细胞株的建立方法和依照该方法建立的人卵巢肉瘤样癌永生化细胞株,属于微生物动物细胞株领域。人卵巢癌永生化细胞株的建立方法是将永生化基因——SV40 T抗原基因导入人卵巢癌原代细胞,经筛选,抗性克隆扩大培养,得到永生化细胞株。依照该方法建立的人卵巢肉瘤样癌细胞株命名为BUPH:OVSC－2,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,保藏号为NO.0606,其生物学特征在于:侵袭力强,细胞呈现为梭形肉瘤细胞形态,为上皮来源,异型性明显,保留其原始细胞的生物学特性。本发明的建系方法和建立的BUPH:OVSC－2细胞株,可用于研究人卵巢癌的病因学、转移机制、耐药机理、新药筛选等。

The invention discloses a method for establishing an immortalized cell line of human ovarian cancer and an immortalized cell line of human ovarian sarcoma-like carcinoma established according to the method, belonging to the field of microbial animal cell lines. The establishment method of immortalized human ovarian cancer cell line is to introduce the immortalized gene——SV40 T antigen gene into the primary human ovarian cancer cell, and after screening, the resistant clone is enlarged and cultivated to obtain the immortalized cell line. The human ovarian sarcoma-like cancer cell line established according to this method was named BUPH:OVSC-2, and was preserved by the General Microorganism Center (CGMCC) of the China Committee for the Collection of Microbial Cultures (CGMCC). The preservation number is No. 0606. Its biological characteristics are: The cells showed the shape of spindle sarcoma cells, which were of epithelial origin, with obvious atypia, and retained the biological characteristics of the original cells. The line establishment method and the established BUPH:OVSC-2 cell line of the present invention can be used to study the etiology, transfer mechanism, drug resistance mechanism, new drug screening and the like of human ovarian cancer.

Description

Translated fromChinese

人卵巢癌永生化细胞株的建立Establishment of immortalized human ovarian cancer cell line

技术领域：Technical field:

本发明属于微生物动物细胞株领域，具体涉及一种人卵巢癌永生化细胞株的建立方法和依该方法建立的人卵巢肉瘤样癌永生化细胞株。The invention belongs to the field of microbial animal cell lines, and in particular relates to a method for establishing an immortalized human ovarian cancer cell line and an immortalized human ovarian sarcoma-like carcinoma cell line established according to the method.

背景技术：Background technique:

在女性生殖系统恶性肿瘤中，卵巢癌的发病率位居第二，但死亡率却居于首位。虽然手术、化疗和放疗技术不断改进，但近十年来卵巢癌的五年生存率仍徘徊在30％左右。主要原因在于卵巢癌病理类型复杂、恶性度高、个体差异大，其分子及细胞水平上的发生、发展机制不明。由于从患者体内获得的新鲜癌细胞寿命过短，无法解决临床上指导用药及生物治疗等研究的全过程。为加深对卵巢癌的理解，需要有大量的来源于不同类型及个体的卵巢癌细胞株作为体外研究的模型。目前建立卵巢癌细胞株的方法有经原代培养自发成系或经裸鼠体内种植传代成系，前者成系率非常低，在大量的原代培养标本中只有个别细胞成为细胞株；后者费事费力，且所建系的细胞存在人细胞表面抗原减弱或消失的缺点。因此，目前建立卵巢癌细胞株的方法远远不能满足体外研究的需求，以至限制了在细胞及分子生物学水平上对卵巢癌发病机理、耐药机制、转移相关因素等的理解，迫切需要建立一套可重复建立卵巢癌细胞株的方法。Among the malignant tumors of the female reproductive system, the incidence of ovarian cancer ranks second, but the mortality rate ranks first. Despite continuous improvements in surgery, chemotherapy, and radiation, the five-year survival rate for ovarian cancer has hovered around 30 percent for the past decade. The main reason is that the pathological types of ovarian cancer are complicated, the degree of malignancy is high, the individual differences are large, and the mechanism of its occurrence and development at the molecular and cellular levels is unknown. Since the lifespan of fresh cancer cells obtained from patients is too short, it is impossible to solve the whole process of clinically guiding drug and biotherapy research. In order to deepen the understanding of ovarian cancer, a large number of ovarian cancer cell lines from different types and individuals are needed as models for in vitro research. At present, the methods for establishing ovarian cancer cell lines include spontaneous line formation through primary culture or planting and passage in nude mice. The line formation rate of the former is very low, and only a few cells become cell lines in a large number of primary culture specimens; the latter It takes a lot of time and effort, and the cells of the established line have the disadvantage of weakening or disappearing of human cell surface antigens. Therefore, the current methods for establishing ovarian cancer cell lines are far from meeting the needs of in vitro research, which limits the understanding of ovarian cancer pathogenesis, drug resistance mechanisms, and metastasis-related factors at the level of cell and molecular biology. It is urgent to establish A set of methods for the reproducible establishment of ovarian cancer cell lines.

细胞永生化(Immortalization)也称不死性，是细胞获得可持续生长与增殖能力的特性，永生化细胞具有无限生长和增殖的能力，长期传代不死。将SV40T抗原基因导入原代细胞内建立相应的细胞株是最常用的细胞永生化的方法，参考文献见Jha KK，BangaS，Palejwala V，et al.SV40-mediated immortalization.Exp Cell Res，1998，245：1-7。利用SV40 T抗原基因已先后使支气管、肝内胆管、宫颈等上皮细胞永生化。它可提高细胞永生化的效率，其永生化作用除使细胞生命周期延长外，还能够保留其原始细胞的分化表型，在细胞水平上可反映其原始细胞的生物学特性，具有成系率高、可重复的特点，参考文献见Hopfer UJW，Jacobberger DC，Gruenert RL，et al.Immortalizationof epithelial cells.Am J Physiol，1996，270(Cell Physiol.39)：C1-C11。但是，目前国内外尚无利用SV40T建立人卵巢癌永生化细胞株的报道。Cell immortalization (Immortalization), also known as immortality, is the characteristic of cells to obtain sustainable growth and proliferation capabilities. Immortalized cells have the ability to grow and proliferate indefinitely, and are immortal for long-term passage. Introducing the SV40T antigen gene into primary cells to establish corresponding cell lines is the most commonly used method for cell immortalization. For reference, see Jha KK, BangaS, Palejwala V, et al.SV40-mediated immortalization. Exp Cell Res, 1998, 245 : 1-7. Epithelial cells such as bronchi, intrahepatic bile duct, and cervix have been immortalized successively by using the SV40 T antigen gene. It can improve the efficiency of cell immortalization. In addition to prolonging the life cycle of cells, its immortalization can also retain the differentiation phenotype of its original cells. It can reflect the biological characteristics of its original cells at the cellular level and has a lineage rate. High, reproducible characteristics, references see Hopfer UJW, Jacobberger DC, Gruenert RL, et al. Immortalization of epithelial cells. Am J Physiol, 1996, 270 (Cell Physiol.39): C1-C11. However, there is no report on the establishment of immortalized human ovarian cancer cell lines using SV40T at home and abroad.

关于由癌和肉瘤组成的恶性肿瘤的诊断名称多达119种，目前的统一名称是肉瘤样癌。医学界对这种恶性肿瘤缺乏认识，在临床诊治过程中容易引起混乱和争论。肉瘤样癌属上皮细胞源性，本质是一种分化差的癌。卵巢肉瘤样癌十分少见，易引起误诊，临床上病情进展很快，生长迅速，侵袭力强，5年生存率很低，多数在诊断后半年内死亡。肿瘤学家和病理医师对卵巢肉瘤样癌的临床诊治缺乏认识，对其生物学特性更是知之甚少。因此，极其需要建立相应病理类型的细胞株供体外深入研究，但目前国内外尚无一株建系的报道。There are as many as 119 diagnostic names for malignant tumors composed of carcinoma and sarcoma, and the current unified name is sarcomatoid carcinoma. The lack of awareness of this malignant tumor in the medical community is likely to cause confusion and controversy in the process of clinical diagnosis and treatment. Sarcomatoid carcinoma is of epithelial cell origin and is essentially a poorly differentiated carcinoma. Sarcomatoid carcinoma of the ovary is very rare, and it is easy to cause misdiagnosis. Clinically, the disease progresses rapidly, grows rapidly, and has strong invasiveness. The 5-year survival rate is very low, and most of them die within half a year after diagnosis. Oncologists and pathologists are poorly aware of the clinical diagnosis and management of ovarian sarcomatoid carcinoma, let alone its biological characteristics. Therefore, it is extremely necessary to establish cell lines of corresponding pathological types for in-depth research in vitro, but there is no report on the establishment of a line at home and abroad.

发明内容：Invention content:

本发明的目的是提供一种建立人卵巢癌永生化细胞株的方法，其特征在于将永生化基因——SV40 T抗原基因导入人卵巢癌原代细胞，经筛选，抗性克隆扩大培养，及SV40 T基因在转染细胞中表达的鉴定，得到永生化细胞株。The object of the present invention is to provide a method for establishing human ovarian cancer immortalized cell lines, which is characterized in that the immortalization gene——SV40 T antigen gene is introduced into human ovarian cancer primary cells, after screening, the resistant clones are expanded and cultivated, and Identification of SV40 T gene expression in transfected cells, resulting in immortalized cell lines.

本发明的另一目的还在于利用上述永生化的方法提供一种人卵巢肉瘤样癌永生化细胞株，其生物学特征在于：侵袭力强，细胞呈现为梭形肉瘤细胞形态，为上皮来源，异型性明显，并保留其原始细胞的生物学特性。Another object of the present invention is to provide an immortalized human ovarian sarcoma-like carcinoma cell line by using the above-mentioned immortalization method, which has the following biological characteristics: strong invasiveness, the cells are in the form of spindle-shaped sarcoma cells, and are of epithelial origin. The atypia is obvious and the biological characteristics of the original cells are retained.

本发明的人卵巢肉瘤样癌永生化细胞株，命名为BUPH：OVSC-2，该细胞株已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，保藏号为NO.0606。The immortalized human ovarian sarcoma-like carcinoma cell line of the present invention is named BUPH: OVSC-2, and the cell line has been preserved by the General Microorganism Center (CGMCC) of the China Committee for the Collection of Microorganisms, and the preservation number is NO.0606.

一、本发明的人卵巢癌永生化细胞株的建立方法：One, the establishment method of human ovarian cancer immortalized cell line of the present invention:

1.SV40 T抗原基因真核表达载体的构建1. Construction of SV40 T antigen gene eukaryotic expression vector

pSV₃neo含SV40 T抗原(含大T和小T抗原)基因片段，pcD2为高效表达真核载体质粒。利用基因重组技术将SV40 T抗原基因片段克隆入pcD2载体中，构建成pcD2-SV40。重组质粒经酶切鉴定。pSV₃ neo contains SV40 T antigen (including large T and small T antigen) gene fragments, and pcD2 is a high-efficiency expression eukaryotic vector plasmid. The SV40 T antigen gene fragment was cloned into the pcD2 vector by gene recombination technology to construct pcD2-SV40. Recombinant plasmids were identified by enzyme digestion.

2.原代培养：2. Primary culture:

手术室无菌取材之卵巢癌新鲜标本，按原代培养常规处理组织。用终浓度200U/ml胶原酶I、100U/ml透明质酸酶消化组织。消化产物经45％和20％Percoll溶液进行密度梯度离心，来分离不同的细胞组分，收集所分离的细胞，接种至塑料培养瓶中。培养基为MCDB105∶M199 1∶1添加15％胎牛血清、0.2U/ml胰岛素、2mmol/ml L-谷氨酰胺培养，37℃、5％CO₂培养箱内培养，675U/ml胶原酶I消化细胞进行传代。The fresh specimens of ovarian cancer were collected aseptically in the operating room, and the tissues were processed according to the routine of primary culture. The tissue was digested with a final concentration of 200 U/ml collagenase I and 100 U/ml hyaluronidase. The digested product was subjected to density gradient centrifugation with 45% and 20% Percoll solutions to separate different cell components, and the separated cells were collected and inoculated into plastic culture flasks. The culture medium is MCDB105:M199 1:1 supplemented with 15% fetal bovine serum, 0.2U/ml insulin, 2mmol/ml L-glutamine, cultured in 37°C, 5%_CO2 incubator, 675U/ml collagenase I Digest cells for passaging.

3.永生化细胞株的建立：3. Establishment of immortalized cell lines:

挑选生长状态良好、80％汇合的早期传代细胞以脂质体Lipofectin2000为载体转染质粒pcD2-SV40。转染后细胞经G418筛选，抗性克隆扩大培养，SV40 T抗原基因在转染细胞中表达的鉴定，建成永生化细胞株。转染后，细胞在稳定生长及传代一定时期后，换成含15％小牛血清的培养液培养及0.25％胰蛋白酶：0.02％ETDA 1∶1消化传代。成系后细胞按细胞培养的常规方法进行冻存与复苏。The early passage cells with good growth state and 80% confluence were selected and the plasmid pcD2-SV40 was transfected with liposome Lipofectin2000 as the carrier. After transfection, the cells were screened by G418, the resistant clones were expanded and cultivated, the expression of SV40 T antigen gene in the transfected cells was identified, and an immortalized cell line was established. After transfection, the cells were grown stably and passaged for a certain period of time, then replaced with culture medium containing 15% calf serum and digested with 0.25% trypsin: 0.02% ETDA 1:1 for passage. After forming a line, the cells were cryopreserved and recovered according to the conventional method of cell culture.

二、人卵巢肉瘤样癌永生化细胞株(BUPH：0VSC-2)2. Human ovarian sarcoma-like carcinoma immortalized cell line (BUPH: 0VSC-2)

细胞来源于北京大学人民医院妇科住院手术患者，取材于人卵巢肉瘤样癌腹壁转移结节，术后HE染色、免疫组化及电镜证实腹壁转移结节性质与卵巢原发灶相同，为卵巢肉瘤样癌。The cells were derived from patients undergoing inpatient surgery in the Department of Gynecology, Peking University People's Hospital. The cells were obtained from metastatic nodules on the abdominal wall of human ovarian sarcoma-like carcinoma. Postoperative HE staining, immunohistochemistry and electron microscopy confirmed that the metastatic nodules on the abdominal wall were the same as the primary tumor of the ovary. They were ovarian sarcoma. like cancer.

按照本发明的建立细胞株的方法建立的人卵巢肉瘤样癌永生化细胞株BUPH：OVSC-2，培养基为MCDB105∶M199 1∶1添加15％胎牛血清、0.2U/ml胰岛素、2mmol/ml L-谷氨酰胺培养，37℃、5％CO₂培养箱内培养，0.25％胰蛋白酶：0.02％ETDA 1∶1消化传代。按细胞培养的常规方法进行冻存与复苏，复苏后BUPH：OVSC-2细胞接种率约为80％。目前已传至80余代，能够长期生长及稳定传代。The human ovarian sarcoid carcinoma immortalized cell line BUPH:OVSC-2 established according to the method for establishing cell lines of the present invention, the medium is MCDB105:M199 1:1 adding 15% fetal bovine serum, 0.2U/ml insulin, 2mmol/ Cultured in ml L-glutamine, cultivated in a 37°C, 5% CO₂ incubator, digested and passaged with 0.25% trypsin: 0.02% ETDA 1:1. Cryopreservation and recovery were carried out according to the conventional method of cell culture. After recovery, the inoculation rate of BUPH:OVSC-2 cells was about 80%. At present, it has been passed down to more than 80 generations, capable of long-term growth and stable passage.

经实验观察和验证，体外培养的BUPH：OVSC-2具有如下特征：According to experimental observation and verification, BUPH:OVSC-2 cultured in vitro has the following characteristics:

形态学观察：光镜下观察培养瓶内活细胞呈梭形和多角形，分裂期细胞变成圆形，传代后细胞单层贴壁散在生长，密度达0.5×10⁵/cm²时，见堆积现象，失去接触抑制(见附图1)。HE染色可见细胞大小不等，形态不规则，细胞核与细胞质比例增大，核内染色质增加，核仁增大，可见多个核仁。透射电镜观察，细胞间多为一般连接(见附图2)，可见桥粒(见附图3)、紧密连接、绒毛、胞膜下中间丝和张力丝，胞浆内存在分泌颗粒，证实细胞为上皮起源；细胞核形状不规则，核浆比例大，核内异染色质丰富，核仁大且边集，可见两个以上核仁；胞浆内细胞器少。Morphological observation: under the light microscope, the living cells in the culture flask are spindle-shaped and polygonal, and the cells in the division stage become round.^After passage, the cells grow in a single layer adherent and^scattered . Accumulation phenomenon, loss of contact inhibition (see Figure 1). HE staining showed that the cells were of different sizes and irregular shapes, the ratio of nucleus to cytoplasm was increased, the chromatin in the nucleus was increased, the nucleolus was enlarged, and multiple nucleoli could be seen. Observation by transmission electron microscope showed that the cells were mostly general junctions (see attached figure 2), desmosomes (see attached figure 3), tight junctions, villi, submembrane intermediate filaments and tension filaments were visible, and there were secretory granules in the cytoplasm, confirming that the cells It is of epithelial origin; the shape of the nucleus is irregular, the ratio of nucleus to cytoplasm is large, the nucleus is rich in heterochromatin, the nucleolus is large and bordered, and more than two nucleoli can be seen; there are few organelles in the cytoplasm.

细胞倍增时间：约42小时。Cell doubling time: about 42 hours.

克隆形成率：11.8％。Colony formation rate: 11.8%.

软琼脂培养：将第30代细胞进行双层软琼脂培养，于接种后12天见有细胞集落形成，说明该细胞具有集落形成能力。Soft agar culture: The 30th passage cells were cultured in double-layer soft agar, and cell colonies were observed 12 days after inoculation, indicating that the cells had the ability to form colonies.

染色体分析：观察第40代细胞染色体，随机计数50个分裂像，染色体在43-46条之间，众数为46条。随机挑选15个分散好的G显带标本，进行核型分析，全部缺少1条X染色体，多一条20号染色体，且每个核型都能见到3条标记染色体，其中两条为1q与12q相互易位，另一条为14p⁺。其染色体结构存在显著异常，而非正常二倍体核型，呈假二倍体(见附图4)。Chromosome analysis: observe the chromosomes of the 40th generation cells, count 50 mitotic images randomly, the number of chromosomes is between 43-46, and the mode is 46. Randomly selected 15 scattered G-banded specimens for karyotype analysis, all of which lacked one X chromosome and had one extra chromosome 20, and each karyotype could see three marker chromosomes, two of which were 1q and 12q reciprocal translocation, the other is 14p⁺ . There was significant abnormality in the chromosome structure, instead of the normal diploid karyotype, it was pseudodiploid (see Figure 4).

凝集实验：细胞与刀豆球蛋白A有较强的凝集反应，并随着刀豆球蛋白浓度的增加，凝集反应逐渐增强，其具有凝集力。Agglutination test: cells have a strong agglutination reaction with concanavalin A, and with the increase of concanavalin A concentration, the agglutination reaction is gradually enhanced, which has agglutinative force.

友原体检测：细胞经肉汤培养法培养，支原体检测阴性。Mycoplasma detection: The cells were cultured by the broth culture method, and the mycoplasma test was negative.

异种动物接种：将35代1×10⁷细胞接种裸鼠皮下，7天见皮下小结节，30天肿瘤长至约1.5cm×1.5cm大小，说明其具有致瘤性。60天后切取肿瘤组织，HE染色显示种植肿瘤与原肿瘤组织类型及分化程度一致，细胞呈现梭形成束排列的肉瘤样形态，未见癌巢及腺上皮(见附图5、6)，说明该细胞株仍保留其原有的生物学特性。Xenogeneic animal inoculation: 35 passages of 1×10⁷ cells were inoculated subcutaneously in nude mice, small subcutaneous nodules were seen in 7 days, and the tumor grew to a size of about 1.5cm×1.5cm in 30 days, indicating that it was tumorigenic. The tumor tissue was excised 60 days later. HE staining showed that the implanted tumor had the same tissue type and degree of differentiation as the original tumor. Cell lines still retain their original biological characteristics.

免疫组化：单克隆细胞蜡块的免疫组化结果角蛋白和波形蛋白染色阳性，胞浆内呈现棕色不均匀颗粒，说明其为上皮起源。AB-PAS染色阳性，胞浆呈现蓝色颗粒，表明该细胞具有分泌粘液的功能。以上结果与原组织免疫组化结果一致，证实其保留了原始细胞的分化表型。Immunohistochemistry: The immunohistochemistry results of the monoclonal cell wax block stained positively for keratin and vimentin, and brown uneven particles appeared in the cytoplasm, indicating that it was of epithelial origin. AB-PAS staining was positive, and the cytoplasm showed blue granules, indicating that the cells had the function of secreting mucus. The above results were consistent with the immunohistochemical results of the original tissue, confirming that it retained the differentiation phenotype of the original cells.

上清液标记物检测：ELISA法检测细胞分泌CEA、AFP、CA15-3、CA125、CA19-9抗原情况，细胞培养上清中可检测到CEA和CA19-9抗原。Detection of supernatant markers: ELISA method was used to detect the secretion of CEA, AFP, CA15-3, CA125, and CA19-9 antigens from cells, and CEA and CA19-9 antigens could be detected in the cell culture supernatant.

本发明的利用SV40 T抗原基因进行原代细胞的永生化是一种可行的、可重复的建立人卵巢癌细胞株的方法，这种方法可使不同组织类型、不同分期、不同分化程度的人卵巢癌细胞获得永生，建立相应的人卵巢癌永生化细胞株，成系效率较高，能极大地丰富人卵巢癌体外细胞库。Immortalization of primary cells using the SV40 T antigen gene of the present invention is a feasible and reproducible method for establishing human ovarian cancer cell lines. This method can make human cells of different tissue types, stages, and degrees of differentiation Ovarian cancer cells are immortalized, and the corresponding immortalized cell lines of human ovarian cancer are established. The lineage efficiency is high, which can greatly enrich the human ovarian cancer cell bank in vitro.

本发明建立的细胞株BUPH：OVSC-2，恶性度高，在细胞水平上能够反映其原始细胞的生物学特性，为研究人卵巢肉瘤样癌的病因学、转移机制、耐药机理、新药筛选等提供理想的实验对象。The cell line BUPH:OVSC-2 established by the present invention has a high degree of malignancy and can reflect the biological characteristics of its original cells at the cellular level. provide ideal experimental subjects.

附图说明：Description of drawings:

图1为倒置显微镜下BUPH：OVSC-2细胞形态(×200)照片。可见细胞呈梭形和多角形，可重叠生长，失去接触抑制。Fig. 1 is a photo of BUPH:OVSC-2 cell morphology (×200) under an inverted microscope. It can be seen that the cells are spindle-shaped and polygonal, can overlap and grow, and lose contact inhibition.

图2为BUPH：OVSC-2细胞裸鼠种植瘤透射电镜观察照片。可见三个上皮细胞间的一般连接。Fig. 2 is a transmission electron microscope observation photo of BUPH: OVSC-2 cell implanted tumor in nude mice. A general junction between the three epithelial cells is seen.

图3为BUPH：OVSC-2细胞裸鼠种植瘤透射电镜观察照片，箭头所示为桥粒结构。Figure 3 is a transmission electron microscope image of BUPH: OVSC-2 cell implanted tumor in nude mice, and the arrow shows the desmosome structure.

图4为BUPH：OVSC-2细胞染色体核型分析照片。可见缺少1条X染色体，多一条20号染色体，能见到3条标记染色体，其中两条为1q与12q相互易位，另一条为14p⁺，呈假二倍体。Figure 4 is a photo of karyotype analysis of BUPH:OVSC-2 cells. It can be seen that one X chromosome is missing, one more chromosome 20, and three marker chromosomes can be seen, two of which are 1q and 12q reciprocal translocation, and the other is 14p⁺ , showing pseudodiploidy.

图5为取材部位肿瘤组织HE染色照片。可见细胞呈现梭形成束排列的肉瘤样形态，异型性明显，未见癌巢及腺上皮。Figure 5 is a HE staining photo of the tumor tissue at the sampling site. It can be seen that the cells showed a sarcomatoid morphology arranged in spindle fascicles, with obvious atypia, and no cancer nests and glandular epithelium were seen.

图6为BUPH：OVSC-2细胞裸鼠种植瘤HE染色照片。可见与取材部位肿瘤组织形态、分化一致。Fig. 6 is HE staining photos of BUPH:OVSC-2 cell tumors implanted in nude mice. It can be seen that the morphology and differentiation of the tumor tissue at the sampling site are consistent.

实施例Example

一、SV40基因真核表达载体的构建One, the construction of SV40 gene eukaryotic expression vector

pSV₃neo含SV40 T抗原(含大T和小T抗原)基因片段，BamHI单酶切得到3kb的SV40 T基因片段，电泳回收。pcD2为高效表达真核载体质粒，经多克隆位点处的BamHI单酶切，电泳回收线性载体，再经小牛肠碱性磷酸酶处理，使其不会自连。将其与回收的SV40 T基因片段在T4 DNA连接酶的作用下进行连接，得到重组质粒pcD2-SV40。重组质粒经酶切鉴定。经SmaI和HpaI双酶切，筛选出pcD2-SV40正向克隆。pSV₃ neo contains SV40 T antigen (including large T and small T antigen) gene fragments, which can be digested with BamHI to obtain a 3kb SV40 T gene fragment, which is recovered by electrophoresis. pcD2 is a high-efficiency expression eukaryotic vector plasmid. It is single-digested with BamHI at the multiple cloning site, and the linear vector is recovered by electrophoresis, and then treated with calf intestinal alkaline phosphatase to prevent self-ligation. Ligate it with the recovered SV40 T gene fragment under the action of T4 DNA ligase to obtain the recombinant plasmid pcD2-SV40. Recombinant plasmids were identified by enzyme digestion. The positive clone of pcD2-SV40 was screened out by SmaI and HpaI double digestion.

二、人卵巢肉瘤样癌细胞的原代培养2. Primary culture of human ovarian sarcoma-like carcinoma cells

细胞来源于北京大学人民医院妇科住院手术患者，取材于卵巢肉瘤样癌腹壁转移结节，术后HE染色、免疫组化及电镜证实腹壁转移结节性质与卵巢原发灶相同，为卵巢肉瘤样癌。于手术室无菌取材之新鲜标本，立即送回实验室。将人卵巢肉瘤样癌组织于PBS中清洗，剪去坏死组织、脂肪组织及包膜，切成1cm³小块，置入青霉素小瓶中。将组织块剪切成1mm³小块，加入5-6ml PBS吹打，令其自然沉降，吸出上清。于青霉素小瓶中加入终浓度200U/ml胶原酶I、100U/ml透明质酸酶及完全培养基，37℃以150转/分钟振摇1小时。离心收集消化产物，用PBS洗2次。重悬细胞，将其通过400目的滤网，以保证为单细胞悬液。于10ml塑料离心管中依次缓慢加入45％Percoll溶液、20％Percoll溶液以及单细胞悬液各3ml，3000g离心30分钟。将分出的上层细胞吸出，用PBS洗2次，接种入50ml塑料培养瓶中培养。以培养基MCDB105∶M199 1∶1添加15％胎牛血清、0.2U/ml胰岛素、2mmol/ml L-谷氨酰胺培养。5日后，见细胞呈梭型或多角型，无旋涡走向，继续培养。于接种11日后以675U/ml胶原酶I消化传代。当传至第10代，挑选生长状态良好、80％汇合的细胞用于转染。The cells were obtained from patients undergoing inpatient surgery in the Department of Gynecology of Peking University People's Hospital. The cells were obtained from the metastatic nodules on the abdominal wall of ovarian sarcoma-like carcinoma. Postoperative HE staining, immunohistochemistry and electron microscopy confirmed that the metastatic nodules on the abdominal wall were the same as the primary ovarian tumor and were ovarian sarcoma-like. cancer. Fresh specimens collected aseptically in the operating room were sent back to the laboratory immediately. The human ovarian sarcoid carcinoma tissue was washed in PBS, the necrotic tissue, adipose tissue and capsule were cut off, cut into small pieces of 1 cm³ , and placed in penicillin vials. Cut the tissue block into 1mm³ pieces, add 5-6ml PBS and pipette, let it settle naturally, and suck out the supernatant. Add a final concentration of 200 U/ml collagenase I, 100 U/ml hyaluronidase and complete medium into a penicillin vial, shake at 150 rpm for 1 hour at 37°C. The digested product was collected by centrifugation and washed twice with PBS. Resuspend the cells and pass them through a 400-mesh filter to ensure a single-cell suspension. Slowly add 3ml each of 45% Percoll solution, 20% Percoll solution and single cell suspension to a 10ml plastic centrifuge tube successively, and centrifuge at 3000g for 30 minutes. The separated upper layer cells were aspirated, washed twice with PBS, and inoculated into 50ml plastic culture flasks for culture. Culture medium MCDB105:M199 1:1 supplemented with 15% fetal bovine serum, 0.2 U/ml insulin, 2 mmol/ml L-glutamine. After 5 days, the cells were found to be spindle-shaped or polygonal, without vortex orientation, and continued to be cultured. 11 days after inoculation, they were digested and passaged with 675U/ml collagenase I. When passing to the 10th passage, cells with good growth status and 80% confluence were selected for transfection.

三、人卵巢肉瘤样癌细胞的转染及筛选3. Transfection and screening of human ovarian sarcoma-like cancer cells

1.转染第一天，取上述80％左右汇合的第10代人卵巢癌细胞，换成无青、链霉素的完全培养基，继续培养24小时。1. On the first day of transfection, take the above 80% confluent human ovarian cancer cells of the 10th generation, replace them with complete medium without penicillin and streptomycin, and continue to culture for 24 hours.

2.第二天，在无菌玻璃管中制备下列质粒与脂质体混合液，用于转染6孔培养板内(50ml塑料培养瓶内)培养的细胞。溶液A：将2μg(4μg)质粒DNA溶于250μl(500μl)无血清培养基中。溶液B：将6μl(12μl)Lipofectin2000试剂溶于250μl(500μl)无血清培养基中，室温5～30分钟。合并溶液A和溶液B，轻轻混合，置室温20～50分钟。弃去细胞培养液。加2ml(4ml)无青、链霉素完全培养基至Lipofectin2000-DNA混合液中，混匀后加至细胞上，37℃、5％CO₂培养箱培养24小时。第三天，弃去转染液，继续培养。2. The next day, prepare the following plasmid and liposome mixture in a sterile glass tube for transfection of cells cultured in a 6-well culture plate (in a 50 ml plastic culture bottle). Solution A: Dissolve 2 μg (4 μg) of plasmid DNA in 250 μl (500 μl) of serum-free medium. Solution B: Dissolve 6 μl (12 μl) of Lipofectin2000 reagent in 250 μl (500 μl) of serum-free medium, and keep at room temperature for 5-30 minutes. Combine solution A and solution B, mix gently, and let stand at room temperature for 20-50 minutes. Discard the cell culture medium. Add 2ml (4ml) complete culture medium without penicillin and streptomycin to the Lipofectin2000-DNA mixture, mix well and add to the cells, and incubate for 24 hours in a 37°C, 5%_CO2 incubator. On the third day, the transfection solution was discarded and culture continued.

3.转染细胞的筛选3. Screening of Transfected Cells

(1)转染后48～72小时，待细胞生长接近汇合时按1∶4比例传代。(1) 48 to 72 hours after transfection, when the cells grow close to confluence, they are passaged at a ratio of 1:4.

(2)继续培养，至细胞密度达50％～70％汇合。(2) Continue culturing until the cell density reaches 50%-70% confluence.

(3)弃去培养液，更换成浓度为100μg/ml的G418培养液，同时用未加转染(3) Discard the culture medium, and replace it with G418 culture medium with a concentration of 100 μg/ml, and at the same time, use no transfection medium

   液的细胞作对照。The cells in the solution were used as a control.

(4)此后每3～4天更换一次含100μg/ml的G418培养液，2～3周后，可见抗(4) Thereafter, the G418 culture medium containing 100 μg/ml was replaced every 3 to 4 days. After 2 to 3 weeks, the anti-

   性克隆形成而对照细胞全部死亡，待克隆逐渐增大，将其逐一移至24孔Sexual clones were formed while all control cells died. After the clones gradually increased, they were moved to 24 wells one by one.

   板继续培养。The plates continued to be cultured.

4.克隆细胞株的转移与扩增4. Transfer and expansion of cloned cell lines

(1)将已形成的克隆于显微镜下，用记号笔标记出克隆的位置。(1) Place the formed clone under a microscope, and mark the position of the clone with a marker pen.

(2)在超净台内，倾去培养基，用PBS洗一次。(2) In the ultra-clean bench, discard the culture medium and wash once with PBS.

(3)以长柄镊子，夹取一块0.2cm×0.3cm的无菌滤纸片，醮取消化液，覆于(3) With long-handled tweezers, pick up a 0.2cm × 0.3cm piece of sterile filter paper, dip it to remove the digestion solution, and cover it with

   细胞克隆之上，约10秒左右。On the cell clone, about 10 seconds.

(4)将滤纸取出(取出时在原处涂擦一下)，立即置于含培养基的24孔板中，(4) Take out the filter paper (smear it on the original place when taking it out), and immediately place it in a 24-well plate containing medium,

   继续培养。Continue to cultivate.

(5)待细胞长满后，转移至6孔板继续扩增培养。(5) After the cells are confluent, transfer to a 6-well plate to continue expansion and culture.

四、SV40 T基因在转染的人卵巢癌永生化细胞株中的表达4. Expression of SV40 T gene in transfected human ovarian cancer immortalized cell lines

1. SV40T基因片段的PCR1. PCR of SV40T gene fragment

根据SV40大T抗原cDNA基因序列内部设计一对特异引物，扩增片段应为372bp大小。pcD2-SV40作阳性对照，未转染的原代细胞作阴性对照，以人卵巢癌永生化细胞株的基因组DNA为模板扩增。经30个循环，PCR产物进行1％琼脂糖凝胶电泳分析。可见两样品分别在370bp处有一特异扩增条带，与阳性对照条带相同，而未转染的原代细胞基因组DNA中无扩增条带。说明SV40 T基因已成功导入细胞内。Design a pair of specific primers based on the SV40 large T antigen cDNA gene sequence, and the amplified fragment should be 372bp in size. pcD2-SV40 was used as a positive control, untransfected primary cells were used as a negative control, and the genomic DNA of an immortalized human ovarian cancer cell line was used as a template for amplification. After 30 cycles, the PCR products were analyzed by 1% agarose gel electrophoresis. It can be seen that there is a specific amplification band at 370bp in the two samples, which is the same as the positive control band, but there is no amplification band in the genomic DNA of the untransfected primary cells. It shows that the SV40 T gene has been successfully introduced into the cells.

2.SV40T基因片段的RT-PCR2. RT-PCR of SV40T gene fragment

利用随机引物进行转染细胞和未转染的原代细胞mRNA的反转录，以pcD2-SV40作阳性对照，未转染的原代细胞作阴性对照。用SV40大T抗原cDNA基因序列内部设计的一对特异引物(同PCR引物)，对这些反转录产物进行扩增，产物为372bp大小。同时另管以内参引物GAPDH进行扩增。PCR产物进行1％琼脂糖凝胶电泳分析。结果经转染的永生化细胞及阴性对照均可见300bp左右的内参引物的扩增条带，证实反转录成功；永生化细胞可见370bp的SV40 T基因特异引物的扩增条带，与阳性对照条带相同，而未转染的原代细胞未见条带。说明转染的永生化细胞株存在SV40 T基因在转录水平的表达。Random primers were used to reverse transcribe the mRNA of transfected cells and untransfected primary cells, pcD2-SV40 was used as a positive control, and untransfected primary cells were used as a negative control. These reverse transcription products were amplified with a pair of specific primers (same as PCR primers) designed inside the SV40 large T antigen cDNA gene sequence, and the product was 372bp in size. At the same time, another tube was amplified with the internal reference primer GAPDH. PCR products were analyzed by 1% agarose gel electrophoresis. Results The transfected immortalized cells and the negative control could see the amplified band of the internal reference primer of about 300bp, which confirmed the success of reverse transcription; The bands were the same, but no bands were seen in untransfected primary cells. It shows that the transfected immortalized cell line has the expression of SV40 T gene at the transcriptional level.

3.SV40 T基因片段PCR产物的Sothem Blot3. Sothem Blot of SV40 T gene fragment PCR product

切取载体pcD2-SV40中的SV40 T基因片段，采用随机引物法标记上地高辛(DIG)作为探针。将永生化细胞基因组DNA中SV40 T特异引物的扩增产物及质粒pcD2-SV40的扩增产物(阳性对照)于1％琼脂糖凝胶中电泳，经转膜、预杂交、杂交、洗膜、显色，结果两样品均出现棕黑色的显色带，与阳性对照相同。说明所扩增出的产物能够与SV40基因杂交，两细胞内表达的基因为SV40 T基因。The SV40 T gene fragment in the vector pcD2-SV40 was excised, and the random primer method was used to label digoxigenin (DIG) as a probe. The amplified products of the SV40 T-specific primers in the genomic DNA of the immortalized cells and the amplified products of the plasmid pcD2-SV40 (positive control) were electrophoresed in 1% agarose gel, and transferred to the membrane, pre-hybridized, hybridized, washed, As a result, brown-black color bands appeared in both samples, which was the same as the positive control. It shows that the amplified product can hybridize with the SV40 gene, and the gene expressed in the two cells is the SV40 T gene.

Claims (2)

1. the foundation of immortal human ovary carcinoma cell strain is characterized in that step is as follows:

(1) utilize gene recombination technology with pSV₃SV40 T antigen among the neo (containing big T and little T antigen) gene fragment clone goes into to efficiently express among the gram nuclear vector plasmid pcD2, is built into pcD2-SV40, and recombinant plasmid is cut evaluation through enzyme;

(2) with final concentration 200U/ml collagenase I and 100U/ml hyaluronic acid enzymic digestion ovarian cancer tissue sample, digestion product through 45% and 20%Percoll solution carry out density gradient centrifugation, with substratum MCDB105: M1991: 1 adds 15% foetal calf serum, 0.2U/ml Regular Insulin, 2mmol/ml L-glutaminate cultivates, and 675U/ml collagenase I peptic cell goes down to posterity;

(3) select that growth conditions is good, the 80% early stage passage cell that converges is carrier transfection plasmid pcD2-SV40 with liposome Lipofectin2000, cell screens through G418 after the transfection, the resistance clone enlarged culturing, reach the evaluation that the SV40T gene is expressed in transfectional cell, obtaining thus can be in external growth steady in a long-term and the ovarian cancer cell strain of going down to posterity.

2. people's ovarian sarcoma sample cancer immortalized cell line of setting up according to the establishment method of immortal human ovary carcinoma cell strain according to claim 1, called after BUPH:OVSC-2, by China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation, preserving number is NO.0606, can be external long term growth and stable going down to posterity, have following feature: 1. cellular form is based on fusiformis and polygon; 2. cell doubling time is about 42 hours; 3. karyomit(e) is pseudodiploid, and chromosome structure all lacks 1 X chromosome, many No. 20 karyomit(e), and each caryogram can both see 3 marker chromosomess, is 1q, 12q and 14P⁺4. cell has muciparous function; 5. cell expressing Keratin sulfate and vimentin; 6. nude mice plantation tumor growth is fast, keeps former tumour form; 7. with in the SV40T antigen gene transfered cell and become immortalized cell line; The biological characteristics that 8. on cell levels, can reflect its initiating cell.

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