Human pancreatic cancer cell line and its application

Abstract

The present invention relates to the field of biomedicine, and discloses a human pancreatic cancer cell line and its application. The pancreatic cancer cell line is a human pancreatic cancer cell line Pahz-1R1 with a biological preservation number of CCTCC NO: C2022354. The tumor formation rate of the cell line is as high as 100%, and it can be used to establish an animal pancreatic cancer model to study biomedical issues such as the mechanism of pancreatic cell carcinogenesis, tumor metastasis, and the preparation of drugs for treating pancreatic cancer; at the same time, the antibodies produced by the cell line can be used to culture 3D organs or organoids, and have broad application value and application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a human pancreatic cancer cell line and an application thereof. Background Art

[0002] The surface of the pancreas is covered with a layer of connective tissue capsule, which extends into the gland and divides the substance into many lobules. The pancreatic substance is composed of the exocrine part and the endocrine part. The exocrine part secretes a variety of digestive enzymes, which constitute the main component of pancreatic juice, and the endocrine part is called the pancreatic islet, which secretes insulin.

[0003] Human pancreatic islets mainly consist of four types of cells: A, B, D, and PP. Among them, A cells secrete glucagon, which promotes the decomposition of glycogen and increases blood sugar; B cells secrete insulin, which promotes the synthesis of glycogen and lowers blood sugar; D cells secrete growth hormone; PP cells secrete pancreatic polypeptide, which inhibits gastrointestinal motility, the secretion of pancreatic juice, and the contraction of the gallbladder.

[0004] Based on the physiological function of the pancreas and animal cell culture technology, researchers have made great efforts to establish insulin-secreting β-cell lines that maintain normal insulin secretion regulation, but only a few of these attempts have been successful. In order to overcome the limitations of primary β-cells, many researchers have used X-ray or virus-induced insulinomas, in vitro transformation, and cell-islet cells derived from transgenic mice or even non-transgenic mice to produce immortalized β-cell lines. The most widely used insulin-secreting cell lines are RIN, HIT, MIN, INS-1, and TC cells. These cells produce insulin and small amounts of glucagon and somatostatin. Only some of these cell lines have a high sensitivity to glucose, but their concentration-dependence curves are clearly shifted toward higher sensitivity. Despite the problems associated with β-cell culture, these cell lines provide some valuable information about physiological processes.

[0005] In 2010, a new "normal" beta cell line was established to explore the application of pancreatic beta cell lines in diabetes research. Beta cell replacement therapy through pancreatic islet transplantation is a promising possibility for the optimal treatment of type 1 diabetes. However, this approach is severely limited by the shortage of donor organs (Skelin M, Rupnik M, Cencic A. Pancreatic beta cell lines and their applications in diabetes mellitus research. ALTEX. 2010; 27(2): 105-13.).

[0006] Pancreatic stem / progenitor cells have become a useful target for β-cell replacement therapy in diabetic patients because these cells are present in large quantities in the pancreas and donor organs of these patients. Based on this, a mouse pancreatic stem cell line that can be used to analyze pancreatic stem cell differentiation was established (Noguchi H, Oishi K, Ueda M, Yukawa H, Hayashi S, Kobayashi N, Levy MF, Matusmoto S. Establishment of mouse pancreatic stem cell line. Cell Transplant. 2009; 18(5): 563-71.).

[0007] In addition, it has been reported that since the pancreatic cell β lines established in the past often lose the mature β cell characteristics in long-term culture, a long-term stable β cell line has recently been established as a valuable tool for studying the regulatory mechanism of insulin secretion (Miyazaki S, Tashiro F, Tsuchiya T, et al. Establishment of a long-term stable β-cell line and its application to analyze the effect of Gcg expression on insulin secretion. Sci Rep. 2021 Jan 12; 11(1): 477.).

[0008] Pancreatic cancer has an insidious onset, lacks early and accurate diagnosis methods, and has a very poor prognosis for patients. Its morbidity and mortality rates are comparable, so it is called the "king of cancer." Over the past few decades, despite the continuous decline in the incidence of pancreatic cancer, the mortality rate of pancreatic cancer patients remains high. Often, when diagnosed, the disease has already progressed locally or has distant metastases, and the opportunity for radical surgical treatment has been lost. In order to improve the prognosis of pancreatic cancer patients, we need to conduct more in-depth research on the related mechanisms of the occurrence and development of pancreatic cancer.

[0009] With the rapid development of social economy and the increasing degree of population aging, the number of pancreatic cancer patients has continued to increase, becoming one of the burdens of my country's social economy. According to the latest statistics from the National Cancer Center, among the incidence rates of malignant tumors in Chinese cities, pancreatic cancer ranks 8th in the incidence rate of male malignant tumors and 6th in the mortality rate of malignant tumors in large cities. In addition, the age of onset of pancreatic cancer is also showing a trend of getting younger.

[0010] Abnormal cell proliferation is a common feature of all malignant tumors, including pancreatic cancer. Looking at the existing published literature, existing pancreatic cancer cell lines include but are not limited to: AsPC-1 (Chen WH, Horoszewicz JS, Leong SS, et al. Human pancreatic adenocarcinoma: in vitro and in vivo morphology of a new tumor line established from ascites. In Vitro. 1982; 18: 24–34.), BxPC-3 (Tan MH, Nowak NJ, Loor R, et al. Characterization of a new primary human pancreatic tumor line. Cancer Invest. 1986; 4: 15–23.), Capan-1 (Kyriazis AP, Kyriazis AA, Scarpelli DG, et al. Human pancreatic adenocarcinoma line Capan-1 in tissue culture and the nude mouse: morphologic, biologic, and biochemical characteristics. Am J Pathol. 1982; 106:250–260.), Capan-2 (Kyriazis AA, KyriazisAP, Sternberg CN, et al. Morphological, biological, biochemical, and karyotypiccharacteristics of human pancreatic ductal adenocarcinoma Capan-2 in tissue culture and the nude mouse. Cancer Res. 1986; 46:5810–5815), CFPAC-1 (SchoumacherRA, Ram J, Iannuzzi MC, et al. A cystic fibrosis pancreatic adenocarcinoma cellline. ProcNatlAcadSci US A. 1990; 87:4012–4016.), HPAC (Gower WRRR, Jr, GodellasCV, Fabri PJ.HPAC,a new human glucocorticoid-sensitive pancreatic ductaladenocarcinoma cell line.In Vitro Cell Dev Biol.1994;30A:151–161.),HPAF-II(Metzgar RS,Gaillard MT,Levine SJ,et al.Antigens of human pancreaticadenocarcinoma cells defined by murine monoclonal antibodies.Cancer Res.1982;42:601–608.),Hs766T(Smith HS.In vitro properties of epithelial cell linesestablished from human carcinomas and nonmalignant tissue.J Natl CancerInst.1979;62:225–230.),MIA PaCa-2(unis AA,Arimura GK,Russin DJ.Humanpancreatic carcinoma(MIA PaCa-2)in continuous culture:sensitivity toasparaginase.Int J Cancer.1977;19:218–235.),PANC-1(Lieber M,Mazzetta J,Nelson-Rees W,et al.Establishment of a continuous tumor-cell line(panc-1)froma human carcinoma of the exocrine pancreas.Int J Cancer.1975;15:741–747.),SU.86.86(Drucker BJ,Marincola FM,Siao DY,et al.A new human pancreaticcarcinoma cell line developed for adoptive immunotherapy studies withlymphokine-activated killer cells in nude mice.In Vitro Cell Dev Biol.1988;24:1179–1187.).

[0011] Currently, the most commonly used pancreatic cancer cell lines have limited sources, and there is a lack of cell lines that are closer to domestic sources.

[0012] Based on this, the necessity of developing new pancreatic cancer cell lines for cancer research is highlighted. Summary of the invention

[0013] The purpose of the present invention is to provide a new human pancreatic cancer cell line for in vitro research of pancreatic cancer.

[0014] In order to achieve the above-mentioned object, the first aspect of the present invention provides a human pancreatic cancer cell line, the human pancreatic cancer cell line Pahz-1R1 having a biological deposit number of CCTCC NO: C2022354.

[0015] The second aspect of the present invention provides a use of the human pancreatic cancer cell line described in the first aspect in 3D organ or organoid culture.

[0016] The third aspect of the present invention provides a use of the human pancreatic cancer cell line described in the first aspect in establishing an animal pancreatic cancer model.

[0017] The fourth aspect of the present invention provides a use of the human pancreatic cancer cell line described in the first aspect in the preparation of a drug for treating pancreatic cancer.

[0018] The present invention obtains a human pancreatic cancer cell line Pahz-1R1 with a tumor formation rate of up to 100% by enzymatically digesting the tumor tissue obtained by pathological section screening of lesions of patients with pancreatic ductal adenocarcinoma and then forming a tumor in mice. The cell line can be used to establish an animal pancreatic cancer model to study biomedical issues such as the mechanism of pancreatic cell carcinogenesis, tumor metastasis, and the preparation of drugs for treating pancreatic cancer. At the same time, the antibodies produced by the cell line can be used to culture 3D organs or organoids, and have broad application value and application prospects.

[0019] Other features and advantages of the present invention will be described in detail in the following detailed description.

[0020] Biological Deposit

[0021] The human pancreatic cancer cell line Pahz-1R1 of the present invention was deposited in the China Center for Type Culture Collection (abbreviated as CCTCC, addressed at Wuhan University, China) on December 21, 2022, with the deposit number CCTCC NO: C2022354. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the result of HE staining;

[0023] Figure 2 This is a diagram of the growth status of tumor cell I-2;

[0024] Figure 3 This is a status diagram of mice at day 51 after tumor cells I-2 were used to form tumors;

[0025] Figure 4 This is a status diagram of mice at day 56 after tumor cells I-2 were used to form tumors;

[0026] Figure 5 This is a picture of the state of mice on the 19th day after tumor formation with Pahz-1R1 cells;

[0027] Figure 6 Growth curve of mouse tumors. DETAILED DESCRIPTION

[0028] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0029] As mentioned above, the first aspect of the present invention provides a human pancreatic cancer cell line, the human pancreatic cancer cell line Pahz-1R1 with biological deposit number CCTCCNO: C2022354.

[0030] According to a preferred embodiment, the cell line is prepared by a method comprising the following steps:

[0031] (1) Pathological sections were performed on the lesions of patients with pancreatic ductal adenocarcinoma to screen and obtain tumor tissue I;

[0032] (2) digesting the tumor tissue I with trypsin I to obtain tumor cells I-1; and primary culturing the tumor cells I-1 I to obtain single tumor cells I-2;

[0033] (3) transplanting the tumor cells I-2 into BALB / c-nu mice subcutaneously to form tumor I, thereby obtaining tumor tissue II;

[0034] (4) Digesting the tumor tissue II with collagenase type D and deoxyribonuclease type I to obtain tumor cells II-1; and primary culturing the tumor cells II-1 and subculturing them for more than 20 times.

[0035] Preferably, the conditions of the primary culture I and the primary culture II each independently include: culturing at 37° C. in a 5% CO 2 atmosphere.

[0036] Preferably, the transplantation amount of the tumor cells I-2 is 4.5×10 6 -5.5×10 6 Piece / piece.

[0037] Preferably, the conditions of digestion I include: temperature of 36-38° C. and time of 15-20 min.

[0038] Preferably, the conditions of digestion II include: temperature of 36-38°C and time of 40-50 min.

[0039] As mentioned above, the second aspect of the present invention provides a use of the human pancreatic cancer cell line described in the first aspect in 3D organ or organoid culture.

[0040] As mentioned above, the third aspect of the present invention provides a use of the human pancreatic cancer cell line described in the first aspect in establishing an animal pancreatic cancer model.

[0041] As mentioned above, the fourth aspect of the present invention provides a use of the human pancreatic cancer cell line described in the first aspect in the preparation of a drug for treating pancreatic cancer.

[0042] The present invention will be described in detail below by way of examples. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions, such as conditions described in literature or books, or methods recommended by kit manufacturers. Various commonly used chemical reagents used in the examples are all commercially available products.

[0043] In the present invention, unless otherwise specified, the percentages used are all expressed by volume fraction, and the solvent is water. For example, 95% ethanol means a solution of 95% ethanol by volume and 5% water by volume; 70% ethanol, 4% paraformaldehyde, etc. have similar explanations, except that the volume fractions of ethanol, paraformaldehyde, and water are different.

[0044] Xylene I and xylene II both represent xylene with the same composition, and the two xylenes are independent and numbered I and II; anhydrous ethanol I and anhydrous ethanol II both represent anhydrous ethanol and are numbered I and II.

[0045] 5% CO2 represents a gas environment of 5% by volume CO2 and 95% by volume air.

[0046] Room temperature refers to a temperature of 25±5°C.

[0047] Experimental Materials

[0048] Nine 6- to 8-week-old immunodeficient BALB / c-nu mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.;

[0049] DMEM cell culture medium, fetal bovine serum, and DPBS were purchased from Gibco;

[0050] Penicillin-streptomycin antibody and trypsin were purchased from Themofisher Company;

[0051] Hematoxylin dye and eosin dye were purchased from Aladdin Reagent Company;

[0052] Tissue embedding medium was purchased from Sakura;

[0053] Collagenase type D and deoxyribonuclease type I were purchased from Roche;

[0054] Screening of tumor tissue I

[0055] The tumor tissues separated from the pancreatic mass tissues of male patients were cut into slices of 2±1 mm thickness and fixed in 4% paraformaldehyde for 2 days;

[0056] The tissue was rinsed with PBS (0.01 mol / L, pH 7.4) for 1 h and then dehydrated as follows:

[0057] Soak in 70% ethanol for 30 min, 80% ethanol for 30 min, 90% ethanol for 30 min, 95% ethanol for 30 min, anhydrous ethanol I for 30 min, anhydrous ethanol II for 30 min, xylene I for 30 min, and xylene II for 30 min;

[0058] After the tissue was immersed in tissue embedding medium at 60°C overnight, the slices were taken out and placed at room temperature to dry;

[0059] After the sections were stained with 0.1% hematoxylin dye for 10 minutes, they were rinsed in a decolorizing jar with running distilled water for 5 minutes (avoid running water directly washing the tissue slides); then immersed in 0.5% eosin dye up and down 12 times, 1 minute each time; then washed in running distilled water until the eosin dye no longer faded (avoid excessive washing); then immersed in 50% ethanol up and down 10 times, 1 minute each time; then immersed in 70% ethanol up and down 10 times, 1 minute each time; then balanced in 95% ethanol for 30 seconds and then balanced in anhydrous ethanol for 1 minute; then immersed in xylene 10 times, 1 minute each time, and dried naturally in a fume hood;

[0060] Clean the slides, seal them with neutral resin, fix the coverslips with nail polish, and finally observe under a microscope and take photos for analysis.

[0061] Figure 1 This is the HE staining result. Figure 1 It can be seen that the patient meets the criteria for poorly differentiated cancer, with sarcomatoid carcinoma as the main component and a small part of moderately to poorly differentiated ductal adenocarcinoma. The cancer tissue invades the peripheral fibrofatty tissue of the pancreas, and cancer metastasis can be seen.

[0062] Preparation of tumor tissue II

[0063] The tumor tissue I of the patient was immersed in DMEM medium containing 5% fetal bovine serum for standby use, taken out from the clean bench, and washed with PBS to remove excess blood stains and fat;

[0064] Use sterilized scissors and forceps to cut the tissue into 1 mm 3 Place the tissue in a culture dish, add DMEM medium containing 10% fetal bovine serum, and cover the tissue block; place it in an incubator at 37°C and 5% CO2 and invert it for overnight culture, so that the tissue block is adsorbed on the bottom of the culture dish; restore the inverted culture dish and continue to culture until cells are observed crawling out from around the tissue block;

[0065] After observing the cell clones growing in the culture dish, wash the cells once with PBS, drop 0.25% trypsin on the cell clones, place them in a 37°C, 5% CO2 incubator for digestion I, take them out after 15 minutes, transfer the digested cell clones (tumor cells I-1) to a 12-well plate and continue to place them in a 37°C, 5% CO2 incubator for primary culture I; after the cell clones are fully grown in the 12-well plate, transfer them to a 6-well plate and continue to place them in a 37°C, 5% CO2 incubator for primary culture I; after the cell clones are fully grown in the 6-well plate, transfer them to a 10 cm culture dish; obtain a single tumor cell I-2 (i.e., tumor cell Pahz-1);

[0066] Prepare 5 immunodeficient female nude mice BALB / c-nu, 5 × 10 6 Tumor cells I-2 were implanted subcutaneously to form tumor I, and tumor tissue II was obtained after 50 days.

[0067] Figure 2 This is a diagram of the growth status of tumor cell I-2. Figure 2 It can be seen that most of the tumor cells I-2 are fibrous and have a good growth state, which is consistent with the state of poorly differentiated adenocarcinoma cells.

[0068] Figure 3 This is a picture of the state of mice on the 51st day after tumor cells I-2 were used to form tumors. Figure 3 It can be seen that the tumor volume value is 108mm 3 ,

[0069] Figure 4 This is a picture of the state of mice on the 56th day after tumor cells I-2 were used to form tumors. Figure 4 It can be seen that the tumor volume is 224mm 3 .

[0070] In this tumor formation, the tumor formation rate was 40% (2 / 5), and the average tumor volume was 166mm 3 It can be seen that in this tumor model, the tumor formation rate is low and the growth is slow.

[0071] Preparation of human pancreatic cancer cell lines

[0072] Soak the tumor tissue II in DMEM culture medium for later use. Transfer the tumor tissue II to a culture dish on a clean bench and cut it into 1 mm thick pieces using sterilized scissors and tweezers. 3 Transfer the tissue pieces to a sterile 50 mL centrifuge tube, add collagenase D (1.0 mg / mL) and deoxyribonuclease I (50 mg / mL) at a volume ratio of 2:5, cover the tissue pieces, and perform digestion II in a 37°C water bath for 45 min, shaking the centrifuge tube every 5 min;

[0073] The substance in the centrifuge tube was filtered using a 40 μm cell filter, the cell suspension obtained by filtration was carefully blown away, centrifuged at 1200 rpm for 5 min, and the supernatant was removed; then washed once with 2 mL of DMEM medium, centrifuged at 1200 rpm for 5 min, and the supernatant was removed to obtain tumor cells II-1;

[0074] Then, 1 mL of PDAC primary culture medium (culture medium formula: DMEM F-12 (45 mL, purchased from Gibco), fetal bovine serum (5 mL) inactivated at 56° C. for 30 min, P / S (100×, 500 μL), L-Glutamine (100×, 500 μL, purchased from Gibco), EGF (100 μg / mL, 50 μL, purchased from Gibco), insulin Human Recombinant (100×, 10 μL, purchased from Themofisher)) was used to resuspend the cells, and the cells were carefully blown away, and all the cells were transferred to a culture dish containing 9 mL of PDAC primary culture medium, shaken at an infinity angle, and primary culture II was carried out in a 37° C., 5% CO2 incubator and subcultured for more than 20 times; Pahz-1R1 cells (i.e., human pancreatic cancer cell line) were obtained.

[0075] Example 1: Tumor formation analysis

[0076] Prepare 4 immunodeficient female nude mice BALB / c-nu, 5 × 106 Pahz-1R1 cells were subcutaneously cultured for tumor formation and analyzed. The tumor formation rate was 100% (4 / 4).

[0077] Figure 5 This is a picture of the state of mice on the 19th day after tumor formation with Pahz-1R1 cells. Figure 5 It can be seen that the tumor volume is about 516mm 3 .

[0078] Figure 6 This is a graph showing the growth of mouse tumors. Figure 6 It can be seen that Pahz-1R1 cells formed tumors 10 days after implantation, and the average tumor volume was 516mm on the 19th day. 3 , indicating that it grows rapidly and can be quickly modeled for pancreatic cancer treatment research.

[0079] Example 2

[0080] Pahz-1 cells and Pahz-1R1 cells were subjected to STR detection. The specific detection method is as follows:

[0081] The genomic DNA of the above cells was extracted using Tsingke's animal genome extraction kit, amplified using 20 site-specific fluorescent primers, and 20 known STR loci and the sex gene Amelogenin were continuously detected and analyzed on an ABI 3730xl Genetic Analyzer (Beijing Qingke Biotechnology Co., Ltd.), and compared with the cell line STR data included in the ATCC, DSMZ, JCRB and RIKEN databases. The analysis results are shown in Tables 1 and 2, respectively.

[0082] Table 1: Typing results of Pahz-1 cells

[0083]

[0084]

[0085] Table 2: Typing results of Pahz-1R1 cells

[0086]

[0087] From the results in Tables 1 and 2, it can be seen that the comparison of the STR information of Pahz-1 cells and Pahz-1R1 cells with the STR information of the cell bank NRS-1 cells showed that both Pahz-1 cells and Pahz-1R1 cells are a new cell line.

[0088] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A human pancreatic cancer cell line, characterized in that: The human pancreatic cancer cell line Pahz-1R1 has a biological deposit number of CCTCC NO: C2022354.

2. Use of the human pancreatic cancer cell line according to claim 1 in 3D organ or organoid culture.

3. Use of the human pancreatic cancer cell line according to claim 1 in establishing an animal pancreatic cancer model.

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