CN108977410B - Pancreatic cancer in-situ cancer cell line of Chinese people - Google Patents

Pancreatic cancer in-situ cancer cell line of Chinese people Download PDF

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CN108977410B
CN108977410B CN201810869616.0A CN201810869616A CN108977410B CN 108977410 B CN108977410 B CN 108977410B CN 201810869616 A CN201810869616 A CN 201810869616A CN 108977410 B CN108977410 B CN 108977410B
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郭世伟
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Abstract

The invention relates to a pancreatic cancer in-situ cancer cell line of Chinese people, which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of C201623. The invention also provides application of the cell line and a cell line establishment method. The advantages are as follows: the cell line of the invention is directly established in the pancreatic cancer in-situ cancer of Chinese, thus enriching the cell bank of the pancreatic cancer of human. Has the tumorigenicity, and the inoculation of nude mice has strong tumorigenicity, and tissue sections indicate that the histology characteristics of transplanted tumors are similar to those of primary tumors. Can provide materials for further researching the occurrence mechanism of pancreatic cancer and also can provide a platform for screening medicines for treating pancreatic cancer. The pancreatic cancer in-situ cancer cell line of the Chinese people has stable character and can be stably passaged for multiple times.

Description

Pancreatic cancer in-situ cancer cell line of Chinese people
Technical Field
The invention relates to the technical field of cell lines, in particular to a pancreatic cancer in-situ cancer cell line of Chinese people.
Background
Pancreatic cancer has a very high mortality rate and is the main cause of death in malignant tumors. In developed countries, the disease with the highest tumor-related mortality rate; the incidence rate in China also tends to rise year by year. In recent years, with the development of economy, improvement of living conditions and aging of population, the incidence of pancreatic cancer has been continuously increasing, and has become a major disease affecting people's health.
The clinical symptoms of pancreatic cancer often appear later and early diagnosis is difficult, peripheral organs are easy to invade and transfer due to the anatomical and biological characteristics of pancreas, most patients are in advanced stage of the disease and transfer to organs such as liver, lung, spine, kidney or adrenal gland and the like when being diagnosed, only exploration or palliative surgery can be performed, but the long-term effect is not ideal, and most patients die from liver transfer and local recurrence; the number of patients who can perform surgical excision and radical cure is only 5 to 30 percent; the postoperative recurrence and metastasis are early, the incidence rate is high, the treatment effect of single radiotherapy or chemotherapy is not ideal, and the prognosis is extremely poor. Surgical statistics in China show that the survival rate of 5 years is only about 5%.
The pathogenesis of pancreatic cancer is the same as that of most malignant tumors, and is the result of the combined action of environmental factors and genetic factors, so far, the exact cause of which is not clear. The biological mechanisms associated with the occurrence, development, metastasis and resistance of pancreatic cancer are currently not well understood. At present, experimental materials which are close to clinical tumor biological characteristics and are used for pancreatic cancer occurrence mechanism and development of anti-pancreatic cancer drugs are lacking.
Since pancreatic cancer is mostly found later, the surgical excision rate is extremely low, and the source of cultured cells is difficult; in addition, pancreatic cancer tissues often have a large amount of fibrous connective tissue and pancreatin, and thus, it is difficult to construct human pancreatic cancer in vitro. Currently, human pancreatic cancer cell lines are mostly established from metastatic cancer, nude mouse engrafted tumor or ascites tumor cells.
Chinese patent zl201010178581.X discloses a human pancreatic cancer cell line. Chinese patent ZL200610118934.0 discloses a pancreatic cancer high liver metastasis cell line. Chinese patent ZL201010225741.1 discloses a human pancreatic cancer cell line resistant to gemcitabine. Chinese patent ZL201110077923.3 discloses a human pancreatic adenosquamous carcinoma cell line. Chinese patent ZL201110383666.6 discloses a pancreatic cancer cell line with high lymphatic channel metastasis activity. However, the in-situ cancer cell line of pancreatic cancer of Chinese people is not reported at present.
Disclosure of Invention
The invention aims at overcoming the defects in the prior art and providing a pancreatic cancer in-situ cancer cell line for Chinese people.
It is a further object of the present invention to provide the use of a cancer cell line in situ in pancreatic cancer in chinese.
Another object of the invention is to provide a method for establishing a cancer cell line in situ of pancreatic cancer in Chinese.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: said method
In order to achieve the second purpose, the invention adopts the following technical scheme:
the application of the in-situ cancer cell line of pancreatic cancer in Chinese is in screening medicines for treating pancreatic cancer.
The application of the pancreatic cancer in-situ cancer cell line in the preparation of a reagent for generating pancreatic cancer in a non-human mammal.
The mammal is a rodent.
The rodent is a mouse.
In order to achieve the third object, the present invention adopts the following technical scheme: the establishing method comprises the following steps:
(1) Obtaining a fresh clinical pancreatic cancer in-situ cancer surgical excision specimen, cutting into small blocks, and inoculating a mammal;
(2) Tumor-bearing animals are sacrificed, tumor tissues are taken out, and primary culture and subculture of cancer cells are performed.
The surgical excision specimens were rinsed with sterile HBSS buffer containing 500U/ml penicillin G, 500. Mu.g/ml streptomycin sulfate and 1.50. Mu.g/ml amphotericin B and inoculated.
The primary culture method comprises the following steps: cutting tumor tissue into small pieces, placing into culture flask, incubator at 37deg.C, 5% CO 2 Is cultured under the condition of (2); the next day, the flask was turned over slowly and laid flat, and the culture medium was added to the flask.
The subculture comprises the following steps: sucking and discarding old culture solution, adding trypsin solution into the bottle, slightly moistening and washing the adherent cell layer, sucking and discarding, adding trypsin solution, incubating in an incubator at 37 ℃, adding DMEM/F12 culture solution after the cells fall off, and carefully blowing to separate from the bottle wall to form cell suspension; a small amount of rounded cells without falling off on the wall of the flask, gently hanging and wiping the surface of the flask with a sterile cell scraper, collecting all cells, centrifuging, counting, inoculating to a new flask respectively, and gradually changing the culture solution into RPMI 1640 culture solution after the cells are passaged to the 5 th generation.
The invention has the advantages that:
1. the cell line of the invention is directly established in the pancreatic cancer in-situ cancer of Chinese, thus enriching the cell bank of the pancreatic cancer of human.
2. The pancreatic cancer in-situ cancer cell line of the Chinese has the advantages of tumorigenicity, nude mice inoculated, strong tumorigenicity, tissue section prompting the histological characteristics of transplanted tumor to be similar to that of primary tumor. Can provide materials for further researching the occurrence mechanism of pancreatic cancer and also can provide a platform for screening medicines for treating pancreatic cancer.
3. The pancreatic cancer in-situ cancer cell line of the Chinese people has stable character and can be stably passaged for multiple times.
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FIG. 1: morphological observation of HPPS-1 cells.
Fig. 2: chromosome analysis of HPPS-1 cells. A: HPPS-1 cells, B: nude mouse chromosome.
Fig. 3: HPPS-1 cell immunohistochemical staining. A: cytokeratin, B: CA19-9, C: carcinoembryonic antigen.
Detailed Description
The following provides a detailed description of embodiments of the present invention with reference to examples.
Biological material preservation information
The pancreatic cancer in-situ cancer cell line of the invention is preserved in China center for type culture collection (address: china, wuhan, university of Wuhan, post code: 430072) at 5-11 days of 2016, and preservation number: cctccc NO: C201623, culture name: the cancer cell line HPPS-1 in situ of pancreatic cancer of Chinese people.
EXAMPLE 1 establishment of HPPS-1 cell line
Nude mice: 5 females, 16.3+ -1.2 g in weight, were kept in SPF environment for 5 weeks of age.
Fresh clinical pancreatic carcinoma in situ resections (female, 63 years old, pancreatic carcinoma in situ) were obtained from Shanghai, changhai Hospital and immediately immersed in pre-chilled sterile HBSS buffer (containing 500U/ml penicillin G, 500. Mu.g/ml streptomycin sulfate and 1.50. Mu.g/ml amphotericin B). In a biosafety cabinet, the specimen is washed by fresh sterile HBSS buffer solution, cut into small blocks of 20-50 mg, and inoculated under the back skin of the armpit of a nude mouse by puncture. After the animals are inoculated, the health state is good and the behavior is normal. After 40 days of inoculation, the subcutaneous tumor nodules were found by touching, and the nodules began to grow significantly after 50 days of inoculation, and the tumor volume exceeded 300mm by 70 days after inoculation 3 。
Primary culture: after inoculating human pancreatic cancer by subcutaneous puncture for 70-90 days, tumor-bearing mice are sacrificed by anesthesia with excessive carbon dioxide gas, sterilized dissected, and tumor tissues are taken out for primary culture, and the method comprises the following steps: by HBSS bufferingRinsing tumor tissue block 3 times, removing connective tissue and necrotic tissue, cutting tumor tissue into pieces of about 1mm with sterile surgical blade 3 A small block; feeding the sheared tissue blocks into a culture flask by using an inoculating needle, uniformly placing the tissue blocks at intervals of 0.5cm, and covering the flask cap; gently turn over the flask to make the bottom up, incubate 5% CO at 37deg.C 2 Culturing under the condition; the next day, the flask was turned over slowly and laid flat, 10ml of DMEM/F12 broth (containing 5% fetal calf serum, 10. Mu.g/ml recombinant human insulin, 6.7ng/ml sodium selenite, 5.5. Mu.g/ml transferrin, 2. Mu.g/ml ethanolamine, 100U/ml penicillin G, 100. Mu.g/ml streptomycin sulfate and 0.25. Mu.g/ml amphotericin B) was added to the flask, and the flask was subjected to stationary culture; primary culture was changed every 3 days to remove floating tissue mass and residual blood cells.
Subculture: after the bottom of the culture flask is fully filled with cells growing from the tissue mass, the cells are passaged, and the specific steps are as follows: sucking and discarding old culture solution, adding 1ml of fresh 0.05% trypsin solution into a bottle, slightly washing an adherent cell layer, sucking and discarding, adding 1ml of fresh 0.05% trypsin solution, incubating in a incubator at 37 ℃, observing that cytoplasm is retracted and cell gap is increased, adding 3ml of fresh DMEM/F12 culture solution after cell shedding, and carefully blowing to separate from the bottle wall to form cell suspension; a small amount of cells which are rounded on the wall of the flask but not shed off are gently scraped on the surface of the flask by a sterile cell scraper, all cells are collected, centrifuged, counted and inoculated into a new flask respectively. After passage of the cells to passage 5, the culture solution was gradually changed to RPMI 1640 culture solution (containing 10% fetal calf serum, 10. Mu.g/ml recombinant human insulin, 100U/ml penicillin G, 100. Mu.g/ml streptomycin sulfate), and the cells grew well and had a relatively uniform morphology. Passaging to more than 50 generations.
EXAMPLE 2 biological Properties of HPPS-1 cell line
The invention adopts RPMI 1640 culture solution containing fetal calf serum and insulin for culture, so that the strain can grow in vitro for a long time and can be stably passaged. When the cells are transferred to more than 20 generations, the cell characteristics are gradually stabilized, and related biological, genetic and tissue source identification is carried out until the 50 th generation has the same stable characteristics. Through experimental observation and verification, cells grown in vitro are in an epithelial shape, are grown on the wall of the cell under an inverted microscope, are uniformly dispersed and have unequal sizes, are in a paving stone shape, have a typical epithelial shape, lose contact growth inhibition and are in malignant growth. Genetic studies confirm that the cells are heteroploid, have serious chromosome number and structure aberration, and accord with the genetic characteristics of malignant tumors.
(1) Morphological observation
The culture flask of the cultured HPPS-1 cells is placed under an inverted microscope, the HPPS-1 cells lose contact inhibition and grow maliciously, the culture flask has the characteristic of overlapping growth, the adherent growth part is flat, the irregular paving stone is the main part, and the culture flask accords with the characteristics of epithelial-like cells.
(2) Identification of chromosomes
After incubating the cultured HPPS-1 cells at 4℃for 12 hours, colchicine was added to a final concentration of 0.4. Mu.g/ml, and the incubation was continued for 10 hours in a 37℃incubator. Metaphase cells were collected, fixed with fixative, then cell suspensions were dropped onto pre-chilled slides, stained with Giemas stain, and the chromosome number counted under a microscope. After serial passage of HPPS-1 cells, the chromosome still maintains the characteristics of the chromosome of the humanized tumor cells, and the chromosome is represented by polyploidy, the mode of the chromosome is concentrated between 76 and 84, and most central and sub-central centromere chromosomes exist. HPPS-1 cells are heteroploids, have serious chromosome number and structure aberration, and accord with the genetic characteristics of malignant tumors.
(3) Tissue origin identification
HPPS-1 cells were inoculated on coverslips and cultured, after expansion of the cells, fixed with 4% formaldehyde, and immunohistochemical staining was performed. The results show that cytokeratin and CA19-9 are strong positive, carcinoembryonic antigen is weak positive, and the cell is pancreatic cancer cell combined with pathological diagnosis.
(4) Cell dynamics
HPPS-1 cells were seeded at 2000/well in 96-well plates, cultured, fixed at 24 hours, 36 hours, 48 hours, 72 hours and 96 hours, and PI stained, and the number of cells per well was measured with a high content cell sorter. The results of the cell kinetic studies showed that the population doubling time of HPPS-1 cells was 49.39 hours.
EXAMPLE 3 application of HPPS-1 cell line to animal model establishment
Experimental animals: nude mice, males, 5-7 weeks old, 18-20 g. Raising in the environment without special pathogen, maintaining the temperature at 25-27 deg.c, and raising in each raising box in 4-6 nude mice.
The experimental method comprises the following steps: 0.1ml (1X 10) of cells was taken 7 cell/ml) was inoculated into the subcutaneous sites inside the left paw pads of the above nude mice, 8 nude mice per group were bred in the environment without special pathogen, and after 8-10 weeks, treated conventionally.
Experimental results: the experimental nude mouse claw mat was transplanted subcutaneously with a tumor rate of 100%.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and additions may be made to those skilled in the art without departing from the method of the present invention, which modifications and additions are also to be considered as within the scope of the present invention.

Claims (4)

1. The in-situ cancer cell line of the pancreatic cancer of the Chinese people is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of C201623.
2. Use of the chinese pancreatic cancer in situ cancer cell line of claim 1 in the preparation of an agent for producing pancreatic cancer in a non-human mammal.
3. The use according to claim 2, wherein the mammal is a rodent.
4. The use of claim 3, wherein the rodent is a mouse.
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