CN111662874A - Chinese esophageal squamous carcinoma cell line and application thereof - Google Patents
Chinese esophageal squamous carcinoma cell line and application thereof Download PDFInfo
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- CN111662874A CN111662874A CN202010558292.6A CN202010558292A CN111662874A CN 111662874 A CN111662874 A CN 111662874A CN 202010558292 A CN202010558292 A CN 202010558292A CN 111662874 A CN111662874 A CN 111662874A
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Abstract
The invention provides a Chinese esophageal squamous carcinoma cell line and application thereof, which is named as the Chinese esophageal squamous carcinoma cell line EC-113, and the preservation number is as follows: CCTCC NO: C2019257. The cell line of the invention has similar protein expression with the primary tumor tissue, and simultaneously has chromosome abnormal karyotype and tumor characteristics of in vitro tumor forming capability. The cell line of the invention can be continuously subcultured in vitro while keeping the characteristics of the esophageal squamous carcinoma tumor cells unchanged, and is suitable for being used as a cell material for researching and developing esophageal squamous carcinoma-related immunotherapy, diagnosis and medicaments.
Description
Technical Field
The invention relates to the technical field of tumor biology, in particular to a Chinese esophageal squamous carcinoma cell line and application thereof.
Background
Esophageal cancer is a common tumor of the digestive tract, about 30 thousands of people die of esophageal cancer every year worldwide, and the morbidity and mortality of the esophageal cancer vary greatly from country to country. China is one of the high-incidence areas of esophageal cancer in the world, and the average death rate of people is about 15 ten thousand every year. Esophageal cancer typically has progressive dysphagia, which is characterized by difficulty swallowing dry food, followed by semifluid food, and finally, water and saliva.
The incidence of esophageal cancer is at the 5 th position of the tumor of the whole body in China, and the mortality rate is at the 4 th position. Two major subtypes of esophageal cancer: squamous cell carcinoma and adenocarcinoma, of which squamous cell carcinoma accounts for about 90%. The esophageal cancer treatment is mainly surgical treatment, radiotherapy, chemotherapy and the like, in recent years, comprehensive treatment mainly based on surgery has reached a bottleneck, and chemotherapy for esophageal cancer is slow in development, and no clear conclusion or standard scheme is available.
In order to deeply understand the pathogenesis of esophageal squamous carcinoma and research on medicaments and other treatment methods, a proper research model must be established. At present, esophageal cancer cell strains commonly used at home and abroad are mainly derived from Japan, including KYSE series and TE series, and the genetic background of the esophageal cancer cell strains is greatly different from Chinese ethnic group in Asian regions. Few cell strains established in a few laboratories in China are few, and part of widely used cell strains are polluted by other cells, so that the research on the esophageal cancer cannot be deeply carried out.
In order to more effectively research pathogenesis and treatment method of esophageal squamous cell carcinoma of Chinese, a Chinese esophageal squamous cell carcinoma cell line is newly established by adopting a tumor cell primary culture method, a new model is provided for the research of esophageal squamous cell carcinoma of Chinese, and a better platform is provided for the mechanism research of esophageal squamous cell carcinoma.
Disclosure of Invention
The first purpose of the invention is to provide a Chinese esophageal squamous carcinoma cell line which has similar protein expression with primary tumor tissues, has chromosome abnormal karyotype, has in vitro tumorigenicity capability and keeps the characteristics of esophageal squamous carcinoma tumor cells unchanged by in vitro continuous subculture.
The second purpose of the invention is to provide the application of the Chinese esophageal squamous carcinoma cell line EC-113 in the preparation of esophageal squamous carcinoma diagnostic reagents.
The third purpose of the invention is to provide the application of the Chinese esophageal squamous carcinoma cell line EC-113 in preparing the medicines for treating esophageal squamous carcinoma.
The fourth purpose of the invention is to provide the application of the Chinese esophageal squamous carcinoma cell line EC-113 in the preparation of an esophageal squamous carcinoma animal model.
The fifth purpose of the invention is to provide the application of the Chinese esophageal squamous carcinoma cell line EC-113 as a drug target in the preparation of drugs for inhibiting esophageal squamous carcinoma.
The sixth purpose of the invention is to provide the progeny cells of the Chinese esophageal squamous carcinoma cell line EC-113.
In order to achieve the first object, the invention provides a Chinese esophageal squamous carcinoma cell line, which is named as the Chinese esophageal squamous carcinoma cell line EC-113 and has the preservation number as follows: CCTCC NO: C2019257.
In order to realize the second purpose, the invention provides the application of the Chinese esophageal squamous carcinoma cell line EC-113 in preparing an esophageal squamous carcinoma diagnostic reagent.
In order to realize the third purpose, the invention provides application of the Chinese esophageal squamous carcinoma cell line EC-113 in preparing a medicament for treating esophageal squamous carcinoma.
In order to realize the fourth purpose, the invention provides application of the Chinese esophageal squamous carcinoma cell line EC-113 in preparing an esophageal squamous carcinoma animal model.
In order to realize the fifth purpose, the invention provides application of the Chinese esophageal squamous carcinoma cell line EC-113 as a drug target in preparing drugs for inhibiting esophageal squamous carcinoma. The Chinese human esophageal squamous carcinoma cell line EC-113 can form tumor under the skin of a nude mouse, and an antibody capable of inhibiting and killing EC-113 cells can be prepared and screened, and the antibody is used as a main component of an anti-esophageal squamous carcinoma medicament.
In order to achieve the sixth object, the present invention provides progeny cells of the Chinese esophageal squamous carcinoma cell line EC-113. The progeny cells retain substantially or all of the characteristics of the parent cell.
The cell line to be protected by the invention is named as a Chinese esophageal squamous cell line EC-113, is preserved in the China center for type culture Collection (preservation address: Lo Jia mountain road 16 Wuhan university China center for type culture Collection, Wuhan mountain, Hubei province), has the preservation date of 2019, 12 and 17 days, and has the preservation number: CCTCC NO: C2019257.
The human esophageal squamous carcinoma cell line EC-113 of the invention has similar protein expression with the tissue of the esophageal squamous carcinoma of origin, and is positive to TTF1, CEA, vimentin and pan-keratin. The chromosome structure and the number of the Chinese esophageal squamous carcinoma cell line EC-113 are abnormal and are heteroploid or polyploid, 41 metaphase cell division phases are analyzed under a mirror, the cell line is a nearly 3-fold karyotype, and the chromosome karyotype has complex chromosome aberrations such as deletion, translocation and the like.
The invention has the advantages that the cell line of the invention has similar protein expression with the primary tumor tissue, and simultaneously has chromosome abnormal karyotype and tumor characteristics of in vitro tumor forming capability. The cell line of the invention can be continuously subcultured in vitro while keeping the characteristics of the esophageal squamous carcinoma tumor cells unchanged, and is suitable for being used as a cell material for researching and developing esophageal squamous carcinoma-related immunotherapy, diagnosis and medicaments.
Drawings
FIG. 1 shows the cell morphology (100X) of a Chinese esophageal squamous carcinoma cell line EC-113 under an inverted microscope.
FIG. 2 is a cell growth and proliferation curve of a human esophageal squamous carcinoma cell line EC-113 in the invention.
FIG. 3 is a chromosome karyotype analysis chart of the human esophageal squamous carcinoma cell line EC-113 of the present invention.
FIG. 4 is a graph of subcutaneous tumor formation in nude mice of a national esophageal squamous carcinoma cell line EC-113 of the present invention, wherein a is a tumor growth curve, and b is a photograph of subcutaneous tumor formation in nude mice.
Detailed Description
Hereinafter, the technique of the present invention will be described in detail with reference to specific embodiments. It should be understood that the following detailed description is only for the purpose of assisting those skilled in the art in understanding the present invention, and is not intended to limit the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
Example 1.
First, primary tumor cell separation treatment
The isolation culture of tumor primary cells is carried out on fresh diseased Chinese tumor tissue blocks obtained from operating tables of hospitals under a sterile environment.
1. Tumor tissue blocks were first placed in 50ml centrifuge tubes and washed 5 times with 10-fold double antibody in PBS by shaking until there was no blood. The tissue mass is placed in a petri dish and excess tissue, such as fat or necrotic tissue, is excised.
2. The tissue was transferred to another dish and fine cut to approximately 1mm with a cross scalpel3Tissue mass of size. A70 um filter sieve was taken and placed in a 50ml centrifuge tube. The tissue is forced through the mesh into the medium by gentle pressure with the core of the syringe, and the medium is aspirated and blown through the screen to flush the cells.
3. 5ml of trypsin/collagenase II was added to the dish of the remaining tissue mass and digested in an incubator at 37 ℃ for 2 hours, while being blown up once every 15 minutes. After digestion was complete, the 70um filter was filtered 1 time, and the filtered cell sap was washed once by centrifugation at 1200rpm for 5 minutes with PBS added to 5 ml.
4. And transferring the cells obtained by centrifugation to a culture dish for culture, and simultaneously putting the tissue block into other culture dishes for standing culture. Absolutely standing for 12-24 hours, slowly taking out the culture dish, supplementing the culture solution, continuously culturing, and standing for 2-3 days. After 3 days the flasks were carefully removed and the floating pieces were removed and the culture was continued by changing the medium.
Second, in vitro culture of primary tumor cells
1. The interval is 24-48 hours.
2. Observing the growth condition of the cells under a light mirror, and replacing the proper culture solution at proper time. If the adherent cells grow, the culture fluid amount can be properly increased to meet the requirement of the growth of the cells.
3. Of the culture of the inventionThe culture conditions were: at 37 deg.C, 5% CO2Culturing in environment with LONZA BEBMBasal Medium bronchial epithelial cell basic culture Medium.
Third, monoclonal culture of primary tumor cells
1. Cells were trypsinized to prepare single cell suspensions, cells were counted, and cells were diluted to 1X10 cells/mL. The method comprises the following specific steps:
a) the digested cells were diluted to 1 × 105one/mL.
b) Take 1 × 105200uL of cell suspension/mL, added to 20mL, became 1 × 103one/mL.
c) 1 × 103200uL of cell suspension was added to 20mL to give 1 × 10 cells/mL.
2. 100uL of 1X 10/mL cell suspension was added to a 96-well microtiter plate, and one colony was formed per well.
3. After a few hours of inoculation, culture wells with only one cell were found by microscopy. The wells are marked and followed, and a decrease in PH, indicative of cell growth, is confirmed by microscopic observation. After the cells overgrow, pancreatin digestion is connected into a 6-well plate for subculture.
Obtaining a cell line through culture, wherein the cell line is named as a Chinese esophageal squamous carcinoma cell line EC-113 and is preserved in the China center for type culture Collection with the preservation number: CCTCC NO: C2019257.
Example 2 detection and identification of Chinese esophageal squamous carcinoma cell line EC-113
1. Cytomorphological observation of primary tumor cell strain
Most of tumor cells under a light mirror are fusiform and oval, a few of the tumor cells are polygonal, the two poles of the cells are sharp, the cell nucleus is obvious, a few of the cells are multinuclear, the nucleolus is clearly visible, and the ratio of the nucleoplasm is large. The cells in the logarithmic growth phase are spread well, EC-113 is randomly stretched to be mostly elliptical epithelioid cells, and the cells are polygonal and have more regular shapes. As shown in fig. 1.
2. Immunofluorescence detection of cell marker proteins
1)1ml of complete medium after cell digestion by pancreatin was suspended and blown into single cellsSuspending the solution, counting the cells, and inserting 5 × 10 into 24-well plate according to the requirement4And (4) cells. Before dropping cell suspension, a small amount of culture medium is added into each hole to make the slide and the culture dish adhere together to reduce cell entry so as to prevent the slide from floating.
2) Slides that were seeded with cells were washed twice with fresh PBS. The PBS was blotted dry and fixed with 4% paraformaldehyde for 40min, and after fixation with methanol, the PBS was washed twice, 5 min each time, and excess PBS was blotted dry. 0.2% Triton X-100, dissolved in PBS, left at room temperature for 15 minutes, and perforated. The punch was blotted dry, PBS was added and washed twice. Dropping goat serum and sealing for 1 h.
3) Primary antibody was dissolved in goat serum, dropped onto a cell-mounting slide, and incubated overnight in a refrigerator at 4 ℃. The next day, PBS was washed three times and the fluorescent secondary antibody was incubated at room temperature for 1h, taking care to keep out of the light. The PBS was washed three times and mounted with 90% glycerol.
The immunofluorescence staining result of the Chinese esophageal squamous carcinoma cell line EC-113 shows that the Chinese esophageal squamous carcinoma cell line EC-113 and the derived esophageal squamous carcinoma tissues have similar protein expressions and are positive in TTF1, CEA, vimentin and pan-keratin.
3. Proliferation curve detection of primary tumor cell strain
1) Digesting the cells with pancreatin to prepare single cell suspension, counting the concentration of the cell suspension by using a counting plate, and preparing the cell suspension into the cell concentration 1 × 10 required by the experiment3one/mL.
2) Cell suspensions (100 ml/well) were seeded in 96-well plates. The plates were pre-incubated in an incubator (5% CO at 37 ℃)2Under the conditions of (a).
3) To each well was added 10ml of CCK-8 solution (care was taken not to generate bubbles in the wells which would affect the OD reading).
4) The plates were incubated in an incubator for 1-4 hours.
5) Absorbance at 450nm was measured with a microplate reader.
The growth curve of the Chinese esophageal squamous carcinoma cell line EC-113 of the invention is shown in FIG. 2, and the logarithmic growth phase is 22.7 hours.
Example 3.
The karyotype analysis detects the karyotype characteristics, chromosome number and the like.
1) The cells in logarithmic growth phase were added with colchicine to a final concentration of 0.1ug/ml and cultured for 3 h.
2) The cells were digested and transferred to a 10ml tip centrifuge tube, centrifuged at 1000rpm for 10min, the supernatant was removed, 0.075mol/L KC110ml was added, incubated at 37 ℃ for 30min, and then fresh fixative (glacial acetic acid: methanol 3:1)1ml, mixed well, 1000rpm, centrifuged for 10min, and the supernatant discarded.
3) Slowly adding 10ml of fresh fixing solution, gently blowing uniformly, fixing at room temperature for 20min, centrifuging, removing supernatant, and fixing again; centrifuge at 1000rpm for 10min and discard the supernatant.
4) Adding 250ul of fresh fixing solution, blowing gently and uniformly, dripping a drop of suspension at a height of 15cm from the glass slide, fully drying air, dyeing by fresh Giemsa, washing by tap water, airing, enabling xylene to be transparent, sealing by neutral resin, and observing under a mirror.
The chromosome structure and the number of the Chinese esophageal squamous carcinoma cell line are abnormal and are heteroploid or polyploid, 41 metaphase cell division phases are analyzed under a microscope, the cell line is a nearly 3-fold karyotype, 3n is 59-64[ 88% ], the chromosome karyotype has complicated chromosome aberrations such as deletion, translocation and the like, (possibly including structural change of sex chromosomes, and the whole chromosome sequencing in an analysis map refers to a human chromosome G standard pattern map). As shown in fig. 3.
Example 4.
The cell suspension is inoculated into the heterogeneous animal body, and the tumor forming capability is observed.
1) Injecting Chinese esophageal squamous carcinoma cell line EC-113 cell suspension into the skin of the root of the sterilized hindlimb of 3 nude mice with the ages of 4 weeks, wherein the numbers are 1, 2 and 3, and each cell suspension is 1x107Individual cells, gently pressed after injection, without outflow.
2) On day 12, 3 nude mice all showed white protrusions in the cell injection region, and on day 40, tumors were seen to grow in leaf form, with a tumor length of 12mm-15mm, and the nude mice were sacrificed after anesthesia. As shown in fig. 4a and 4 b.
The Chinese human esophageal squamous carcinoma cell line can form tumor under the skin of a nude mouse, and can be used for preparing and screening an antibody capable of inhibiting and killing EC-113 cells, wherein the antibody is used as a main component of an anti-esophageal squamous carcinoma medicament.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (6)
1. A Chinese esophageal squamous cancer cell line is characterized in that the cancer cell line is named as a Chinese esophageal squamous cancer cell line EC-113, and the preservation number of the cancer cell line EC-113 is as follows: CCTCC NO: C2019257.
2. The use of a Chinese esophageal squamous carcinoma cell line of claim 1 in the preparation of an esophageal squamous carcinoma diagnostic reagent.
3. The use of a Chinese esophageal squamous carcinoma cell line of claim 1 in the preparation of a medicament for the treatment of esophageal squamous carcinoma.
4. The use of a Chinese esophageal squamous carcinoma cell line of claim 1 in the preparation of an esophageal squamous carcinoma animal model.
5. The application of the Chinese esophageal squamous cancer cell line as a drug target in preparing drugs for inhibiting esophageal squamous cancer according to claim 1.
6. A progeny cell of a Chinese esophageal squamous carcinoma cell line according to claim 1.
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| CN112210538A (en) * | 2020-10-23 | 2021-01-12 | 中国医学科学院肿瘤医院 | Human esophageal squamous carcinoma cell line NCCE1, and establishment method and application thereof |
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| CN105969734A (en) * | 2016-07-22 | 2016-09-28 | 浙江省肿瘤医院 | Esophageal cancer cell line and application thereof |
| CN106011068A (en) * | 2016-07-22 | 2016-10-12 | 浙江省肿瘤医院 | Esophageal cancer cell line and application thereof |
| CN106011069A (en) * | 2016-07-22 | 2016-10-12 | 浙江省肿瘤医院 | Esophageal cancer cell line and application thereof |
| CN106047815A (en) * | 2016-07-22 | 2016-10-26 | 浙江省肿瘤医院 | Esophageal cancer cell line and application thereof |
| CN107541496A (en) * | 2016-06-27 | 2018-01-05 | 安徽省立医院 | A kind of human esophageal carcinoma cell line and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107541496A (en) * | 2016-06-27 | 2018-01-05 | 安徽省立医院 | A kind of human esophageal carcinoma cell line and its application |
| CN105969734A (en) * | 2016-07-22 | 2016-09-28 | 浙江省肿瘤医院 | Esophageal cancer cell line and application thereof |
| CN106011068A (en) * | 2016-07-22 | 2016-10-12 | 浙江省肿瘤医院 | Esophageal cancer cell line and application thereof |
| CN106011069A (en) * | 2016-07-22 | 2016-10-12 | 浙江省肿瘤医院 | Esophageal cancer cell line and application thereof |
| CN106047815A (en) * | 2016-07-22 | 2016-10-26 | 浙江省肿瘤医院 | Esophageal cancer cell line and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112210538A (en) * | 2020-10-23 | 2021-01-12 | 中国医学科学院肿瘤医院 | Human esophageal squamous carcinoma cell line NCCE1, and establishment method and application thereof |
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