Papillary mucous tumor model mouse in pancreatic duct and preparation method and application of tumor cells of papillary mucous tumor model mouse

Abstract

The invention discloses a papillary mucous tumor model mouse in a pancreatic duct and a preparation method and application of tumor cells of the papillary mucous tumor model mouse. The invention provides a method for constructing a KPPS mouse, which comprises the following steps: hybridizing an S positive mouse and a K hybrid P pure mouse as parents to obtain the KPPS mouse. The invention also provides a method for preparing a papillary mucous tumor model mouse in a pancreatic duct. The method comprises the following steps: (1) constructing a KPPS mouse according to the method; and (2) inducing the KPPS mouse by using an inducer to obtain the papillary mucous tumor model mouse in the pancreatic duct. The obtained papillary mucous tumor model mouse in the pancreatic duct completely shows pathological progress, and can be used for mechanism research, drug screening and treatment strategy development of the IPMN progress process.

Description

Technical Field

[0001] The present invention relates to a pancreatic intraductal papillary mucinous tumor model mouse and a preparation method and application of tumor cells thereof. Background Art

[0002] Intraductal papillary mucinous neoplasm (IPMN) is one of the most common pancreatic cystic tumors and a precursor lesion of pancreatic cancer. The pathological characteristics of IPMN are lesions with branching papillary protrusions and lined with tumor epithelial cells. These tumor epithelial cells extend into the dilated pancreatic ducts and express abundant intracellular and extracellular mucins, which may fill the ductal lumen. Different gene mutations can be detected in the development of IPMN lesions, including driver gene mutations such as KRAS and GNAS, and malignant progression mutations such as TP53 and CDKN2A. With the deepening understanding of the basic gene mutations of tumorigenesis, researchers have developed various genetically engineered mouse models (GEMMs), which reproduce genetic events in the in vivo environment and allow the formation of new tumors in a microenvironment with good natural immunity. KPPC ( Kras LSL-G12D / + ;Trp53 fl / fl ;Pdx1-CreER ) mouse model is widely used in various pancreatic cancer studies, but due to the ubiquitous (non-ductal) expression of Cre, specific characterization of IPMN-associated invasive carcinoma (IPMN-IC) is lacking.

[0003] Given that the Sox9 gene is specifically expressed in pancreatic ductal cells of adult mice, Sox9-CreER tool mice can provide a genetic manipulation platform for establishing an IPMN animal model of ductal cell origin, helping to explore its carcinogenesis mechanism. Studies have shown that inducing Kras mutations in ductal cells alone is not sufficient to produce IPMN. von Figura et al. specifically expressed Sox9 in pancreatic ductal cells. Kras G12D Mutation and knockout Brg1The chromatin regulator Brg1 suppresses formation of intraductal papillarymucinous neoplasm and pancreatic ductal adenocarcinoma. Nat. Cell Biol. 16, 255–267. https: / / doi.org / 10.1038 / ncb2916.] Janel L. Kopp et al. express Brg1 specifically in ductal cells. Kras G12D Mutation-specific knockout Pten Gene expression in this model also induces IPMN-like lesions, which can eventually progress to IPMN-related invasive cancer. However, this model has a long tumor progression period, and tumors are not observed until the mice are 6-14 months old [Kopp, JL,Dubois, CL, Schaeffer, DF, Samani, A., Taghizadeh, F., Cowan, RW, Rhim,AD, Stiles, BL, Valasek, M., and Sander, M. (2018). Loss of Pten andActivation of Kras Synergistically Induce Formation of Intraductal Papillary Mucinous Neoplasia From Pancreatic Ductal Cells in Mice. Gastroenterology154, 1509-1523.e5. https: / / doi.org / 10.1053 / j.gastro.2017.12.007.]. Summary of the Invention

[0004] In response to the technical defects of the existing IPMN genetically engineered mouse models, such as long tumor progression and / or low malignant transformation rate and / or inability to fully present pathological progression, the present invention provides a pancreatic intraductal papillary mucinous neoplasia model mouse and a method for preparing and using its tumor cells.

[0005] The present invention provides a method for constructing a genetically engineered mouse for preparing a pancreatic intraductal papillary mucinous neoplasia model mouse (named as Method I), characterized in that: The genetically engineered mice used to prepare the pancreatic intraductal papillary mucinous neoplasia model mice were named KPPS mice; The method comprises the following steps: hybridizing S-positive mice and K-hybrid P-pure mice as parents to obtain KPPS mice; The S-positive mouse is a transgenic mouse carrying Sox9-CreER; the Sox9-CreER is a DNA molecule having, from upstream to downstream, a promoter and a Cre recombinase gene transcribed by the promoter; the promoter is a pancreatic duct cell-specific promoter; The K hybrid P pure mouse has a pair of homologous chromosomes No. 11 both carrying loxP-Trp53-loxP, and a chromosome No. 6 carrying Promoter-LSL-Kras G12D ; The loxP-Trp53-loxP is obtained by modifying the Trp53 gene located on chromosome 11, wherein the modification refers to inserting two loxP sites in the same direction into the Trp53 gene, and the two loxP sites are inserted at different positions in the Trp53 gene; the loxP site recombination of the loxP-Trp53-loxP causes the Trp53 gene to be knocked out; and the loxP site recombination is mediated by the Cre recombinase; The Promoter-LSL-Kras G12D The Promoter-LSL-Kras is obtained by modifying the region from the promoter of the Kras gene on chromosome 6 to the end of the Kras gene. G12D From upstream to downstream, there are the promoter of Kras gene, LSL sequence and Kras G12D Mutant gene; LSL sequence is a DNA fragment having two loxP sites in the same direction and a termination box located between the two loxP sites; Kras G12D The expression of the mutant gene is blocked by the LSL sequence; the Promoter-LSL-Kras G12D The loxP site recombination results in the expression of Kras blocked by the LSL sequence. G12DThe mutant gene is restored to expression; the loxP site recombination is mediated by the Cre recombinase; The KPPS mouse is an S-positive K hybrid pure mouse; the S-positive K hybrid pure mouse means: carrying the Sox9-CreER, and both a pair of homologous chromosomes No. 11 carry the loxP-Trp53-loxP, and a chromosome No. 6 carries the Promoter-LSL-Kras G12D .

[0006] As an example, the method I includes the following steps: ① Crossbreed S-positive mice and K-hybrid P-positive mice as parents to obtain offspring mice, and select S-positive K-hybrid P-hybrid mice from the offspring mice; ② The S-positive K-hybrid P-hybrid male mice obtained in step ① were mated with the S-positive K-hybrid P-hybrid female mice obtained in step ① to obtain offspring mice, and S-positive K-hybrid P-pure mice were screened from the offspring mice.

[0007] The meaning of the Syang K hybrid P hybrid is: carrying the Sox9-CreER, and one chromosome 11 carries the loxP-Trp53-loxP, and one chromosome 6 carries the Promoter-LSL-Kras G12D .

[0008] As an example, the method I further includes the following step ③: the S-positive K-hybrid P-pure male mouse obtained in step ② is mated with the S-positive K-hybrid P-pure female mouse obtained in step ② to obtain offspring mice, and S-positive K-hybrid P-pure mice are screened from the offspring mice.

[0009] As an example, the method I further includes the following step ③: the Syang K hybrid P hybrid mice obtained in step ① are mated with K hybrid P pure mice (such as commercially available mice) to obtain offspring mice, and Syang K hybrid P pure mice are screened from the offspring mice.

[0010] As an example, the method I further includes the following step ③: mating the Syang K hybrid P hybrid mice obtained in step ① with Syin K hybrid P pure mice to obtain offspring mice, and screening Syang K hybrid P pure mice from the offspring mice.

[0011] The meaning of S-yin K-hybrid P-pure is: a pair of homologous chromosomes No. 11 both carry the loxP-Trp53-loxP, and one chromosome No. 6 carries the Promoter-LSL-Kras G12D , and does not carry the Sox9-CreER.

[0012] As an example, the S-yin K-hybrid P-pure mice can be screened from offspring mice obtained by hybridizing S-yang mice (such as commercially available mice) and K-hybrid P-pure mice (such as commercially available mice) as parents.

[0013] As an example, the S-yin K-hybrid P pure mice can be screened from the offspring mice obtained by mating the S-yang K-hybrid P-hybrid male mice obtained in step ① and the S-yang K-hybrid P-hybrid female mice obtained in step ①.

[0014] In some specific embodiments of the present invention, the method for identifying K hybrid is as follows: using mouse total DNA as a template, PCR amplification is performed using a primer set (consisting of the primer shown in SEQ ID NO: 1, the primer shown in SEQ ID NO: 2, and the primer shown in SEQ ID NO: 3). If two amplification products of 100 bp and 250 bp are obtained, it is determined to be K hybrid.

[0015] In some specific embodiments of the present invention, the method for identifying K negative is as follows: using total DNA of mice as a template, PCR amplification is performed using a primer set (consisting of a primer shown in SEQ ID NO: 1, a primer shown in SEQ ID NO: 2, and a primer shown in SEQ ID NO: 3). If a 250 bp amplification product is obtained and no 100 bp amplification product is obtained, it is judged to be K negative.

[0016] In some specific embodiments of the present invention, the method for identifying P-purity is as follows: using mouse total DNA as a template, PCR amplification is performed using a primer pair (consisting of the primer shown in SEQ ID NO: 4 and the primer shown in SEQ ID NO: 5). If a 300 bp-400 bp amplification product is obtained and a 270 bp amplification product is not obtained, it is judged to be P-pure.

[0017] In some specific embodiments of the present invention, the method for identifying P heterozygosity is as follows: using mouse total DNA as a template, PCR amplification is performed using a primer pair (consisting of the primer shown in SEQ ID NO: 4 and the primer shown in SEQ ID NO: 5). If a 300 bp-400 bp amplification product and a 270 bp amplification product are obtained, it is determined to be P heterozygosity.

[0018] In some specific embodiments of the present invention, the method for identifying S positive is as follows: using the total DNA of the mouse as a template, PCR amplification is performed using primer pair A (consisting of the primer shown in SEQ ID NO: 6 and the primer shown in SEQ ID NO: 7) and primer pair B (consisting of the primer shown in SEQ ID NO: 8 and the primer shown in SEQ ID NO: 9), respectively. If a 290bp-310bp amplification product is obtained using primer pair A and a 521bp amplification product is obtained using primer pair B, it is judged to be S positive.

[0019] In some specific embodiments of the present invention, the method for identifying S Yin is as follows: using the total DNA of the mouse as a template, PCR amplification is performed using primer pair A (consisting of the primer shown in SEQ ID NO: 6 and the primer shown in SEQ ID NO: 7) and primer pair B (consisting of the primer shown in SEQ ID NO: 8 and the primer shown in SEQ ID NO: 9), respectively. If no 290bp-310bp amplification product is obtained using primer pair A and a 521bp amplification product is obtained using primer pair B, it is judged to be S Yin.

[0020] As an example, the Cre recombinase is an inducible Cre recombinase.

[0021] As an example, the inducible Cre recombinase is tamoxifen-inducible Cre recombinase.

[0022] As an example, the tamoxifen-inducible Cre recombinase is CreER or CreERT2.

[0023] As an example, the pancreatic duct cell-specific promoter is the Sox9 gene promoter.

[0024] The Kras G12D Mutant gene expression Kras G12D Compared with the protein Kras (wild-type protein), Kras G12D The only difference in the amino acid sequence of the mutant protein is that the 12th amino acid residue mutates from G to D, and the other amino acid residues are the same.

[0025] As an example, the S-positive mouse is a Sox9-cre / ERT2 mouse (Sox9CreER T2 ).

[0026] As an example, the S-positive mouse is a mouse with the Jackson Laboratory No. 018829.

[0027] As an example, the K hybrid P pure mouse is a KP mouse (LSL-K-ras G12D; p53 LoxP ).

[0028] As an example, the K hybrid P pure mouse is the mouse with the Jackson Laboratory No. 032435.

[0029] The present invention also provides a method for preparing a pancreatic intraductal papillary mucinous tumor model mouse (named as method II), comprising the following steps: (1) Construct KPPS mice according to Method I; (2) Inducing the KPPS mice with an inducer to obtain pancreatic intraductal papillary mucinous tumor model mice.

[0030] In the step (2), the induction time is 6 days.

[0031] As an example, the inducer is tamoxifen.

[0032] As an example, the induction method is: administering the drug continuously for 6 days, injecting tamoxifen solution once a day (the dosage of tamoxifen is 100 mg / kg·body weight).

[0033] As an example, the induction method is: administering the drug continuously for 6 days, with a tamoxifen solution injected intraperitoneally once a day (the dosage of tamoxifen is 100 mg / kg·body weight).

[0034] Tamoxifen solution: Tamoxifen was dissolved in corn oil to a concentration of 10 mg / mL.

[0035] The course of the pancreatic intraductal papillary mucinous tumor model mice is up to 24 weeks.

[0036] The day on which the last administration is completed is designated as day 0. In step (2), pancreatic intraductal papillary mucinous neoplasm model mice at the IPMN stage are obtained after day 28 (including day 28). In some embodiments of the present invention, in step (2), the day after day 28 refers to day 28 (including day 28) to day 168 (including day 168). In some embodiments of the present invention, in step (2), pancreatic intraductal papillary mucinous neoplasm model mice at the LG-IPMN stage are obtained from day 28 to day 112. In some embodiments of the present invention, in step (2), pancreatic intraductal papillary mucinous neoplasm model mice at the HG-IPMN stage are obtained from day 112 to day 140. In some embodiments of the present invention, in step (2), pancreatic intraductal papillary mucinous neoplasm model mice at the IPMN-IC stage are obtained from day 84 to day 168.

[0037] In the step (2), starting from the 4th week after the completion of the induction, a pancreatic intraductal papillary mucinous neoplasm model mouse at the IPMN stage is obtained. In some specific embodiments of the present invention, in the step (2), starting from the 4th week after the completion of the induction, the 4th week refers to the 4th week to the 24th week. In some specific embodiments of the present invention, in the step (2), starting from the 4th week after the completion of the induction, a pancreatic intraductal papillary mucinous neoplasm model mouse at the LG-IPMN stage is obtained from the 4th week to the 16th week. In some specific embodiments of the present invention, in the step (2), starting from the 16th week to the 20th week after the completion of the induction, a pancreatic intraductal papillary mucinous neoplasm model mouse at the HG-IPMN stage is obtained. In some specific embodiments of the present invention, in the step (2), starting from the 12th week to the 24th week after the completion of the induction, a pancreatic intraductal papillary mucinous neoplasm model mouse at the IPMN-IC stage is obtained.

[0038] The present invention also protects the application of the KPPS mice prepared by the above method; the application is to prepare pancreatic intraductal papillary mucinous tumor model mice.

[0039] The present invention also protects the use of the pancreatic intraductal papillary mucinous tumor model mouse prepared by the above method; the use is as follows (a) or (b) or (c): (a) Presents the pathological progression during IPMN progression; (b) Conduct mechanistic studies on the progression of IPMN; (c) Drug screening during IPMN progression.

[0040] The IPMN progression process refers to the progress of the IPMN from the LG-IPMN stage to the HG-IPMN stage to the IPMN-IC stage.

[0041] The present invention also provides a tumor cell (named KPPS cell or KPPS cell line), which is a tumor cell from a pancreatic intraductal papillary mucinous tumor model mouse prepared by any of the above methods II.

[0042] In a specific embodiment of the present invention, the pancreatic intraductal papillary mucinous neoplasm model mouse is a mouse at the IPMN-IC stage.

[0043] The tumor cells are isolated from pancreatic tumor tissue of the pancreatic intraductal papillary mucinous tumor model mouse.

[0044] The present invention also provides a tissue or organ, characterized in that: the tissue or organ is derived from a tumor of a pancreatic intraductal papillary mucinous tumor model mouse prepared by any of the above methods.

[0045] In China, the tissues or organs do not include embryos.

[0046] The present invention also provides tissues or organs, characterized in that: the tissues or organs are derived from tumors of tumor-bearing mice; the tumor-bearing mice are mice in which the tumor cells are inoculated and tumors are generated.

[0047] The starting mouse may be a mouse that does not contain the tumor cells.

[0048] As an example, the starting mice are C57BL / 6J mice.

[0049] Specifically, the method for preparing tumor-bearing mice is as follows: KPPS cells were injected into C57BL / 6J mice at a dose of 5×10 6 Cells / cells.

[0050] Specifically, the method for preparing tumor-bearing mice is as follows: KPPS cells were subcutaneously injected into the unilateral scapular region of 8-week-old C57BL / 6J mice, with each mouse being injected with 5×10 6 cells.

[0051] The present invention is helpful for the study of the mechanism of IPMN progression, drug screening and treatment strategy development. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 These are exemplary slices of LG-IPMN, HG-IPMN, and IPMN-IC.

[0053] Figure 2 is the survival curve.

[0054] Figure 3 Exemplary photographs of immunofluorescence staining. DETAILED DESCRIPTION

[0055] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0056] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified. Unless otherwise specified, the quantitative tests in the following examples were performed in triplicate, and the results were averaged. All animal experiments complied with the relevant regulations of the Animal Ethics Committee of Beijing Vidatong Biotechnology Co., Ltd. (Ethics Approval Number: VST-SY-20210108). All primers were single-stranded DNA molecules. Tamoxifen: CAS Registry Number: 10540-29-1.

[0057] Tamoxifen solution: Tamoxifen was dissolved in corn oil to a concentration of 10 mg / mL.

[0058] IPMN: Intraductal papillary mucinous neoplasm of the pancreas. IPMN is a precancerous lesion characterized by ductal dilatation, mucinous secretion, and papillary projections. IPMNs have dendritic papillary projections composed of neoplastic epithelial cells that extend into the dilated pancreatic ducts and display abundant intracellular and extracellular mucin, which may fill the ductal lumen.

[0059] IPMN-IC: IPMN-associated invasive carcinoma. IPMN-IC is an invasive carcinoma that has progressed from an IPMN to invade below the basement membrane. Characteristics of IPMN-IC include the presence of small, structurally abnormal glands within the ductal walls, with distorted glandular architecture and an elevated nuclear-to-cytoplasmic ratio.

[0060] LG-IPMN: Low-grade IPMN. LG-IPMN is an early, benign stage of IPMN. LG-IPMN is characterized by branching papillary structures within the pancreatic ducts, lined with mildly atypical epithelial cells and with intracellular and extracellular mucus accumulation.

[0061] HG-IPMN: High-grade IPMN. Compared to LG-IPMN, HG-IPMN is more malignant and has a high risk of developing invasive cancer. Characteristics of HG-IPMN include more numerous and complex papillary structures, significant cellular atypia, increased mitotic figures, increased nuclear-cytoplasmic ratio, loss of nuclear polarity, and the absence of signs of invasive cancer.

[0062] GEMMs: Genetically Engineered Mouse Models. GEMMs mimic the genetic characteristics of human diseases through gene editing technology.

[0063] KP mice (LSL-K-ras G12D; p53 LoxP = ): Jackson Laboratory, No. 032435; KP mice are K hybrid P pure mice. Sox9-cre / ERT2 mice (Sox9CreER T2 ): Jackson Laboratory, No. 018829; Sox9-cre / ERT2 mice are S-positive mice. KPPC mice, also known as KPC mice (Kras LSL-G12D ;p53 LoxP ;Pdx1-CreER): Jackson Laboratory, No. 032429; KPPC mice are genetically engineered mice with triple mutations of KrasLSL-G12D;p53LoxP;Pdx1-CreER, and are used to develop a pancreatic ductal adenocarcinoma model induced by tamoxifen.

[0064] Example 1: Preparation of KPPS mice 1. Preparation and Screening of S-positive, K-hybrid, and P-hybrid Mice KP mice were co-bred with Sox9-cre / ERT2 mice (four cages total; each cage contained one female and one male mouse, one KP mouse and one Sox9-cre / ERT2 mouse). Produced offspring mice were approximately one-quarter S-positive, K-positive, and P-positive. The offspring mice were screened for S-positive, K-positive, and P-positive mice.

[0065] In the above steps, the female mice gave birth once every 4-6 weeks, and gave birth multiple times, obtaining a total of 82 offspring mice, from which 20 S-positive, K-hybrid, and P-hybrid mice were screened.

[0066] 2. Preparation and Screening of S-positive K-hybrid P-pure Mice The S-positive male mice obtained in step 1 were mated with the S-positive female mice obtained in step 2 (six cages total; one female and one male mouse per cage) to produce offspring mice. Approximately 3 / 32 of the offspring mice were S-positive pure. Screen the offspring mice for S-positive pure mice. Approximately 1 / 32 of the offspring mice were S-negative pure mice, and these S-negative pure mice can be used in step 4. Approximately 3 / 32 of the offspring mice were S-positive pure mice, and these S-positive pure mice can be used in step 6. Approximately 3 / 16 of the offspring mice were S-positive pure mice, and these S-positive pure mice can be used in step 5.

[0067] In the above steps, the female mice gave birth once every 4-6 weeks, and gave birth multiple times, obtaining a total of 116 offspring mice, from which 11 S-positive K-hybrid P-pure mice were screened.

[0068] 3. Further preparation and screening of S-positive K-hybrid P-pure mice The S-positive K-hybrid male mice obtained in step 2 were caged and mated with the S-positive K-hybrid female mice obtained in step 3 (five cages total; one female and one male mouse per cage) to obtain offspring mice. Approximately 3 / 8 of the offspring mice were S-positive K-hybrid. Screen the offspring mice for S-positive K-hybrid mice. Approximately 1 / 8 of the offspring mice were S-negative K-hybrid mice, and these S-negative K-hybrid mice can be used in step 4. Approximately 3 / 8 of the offspring mice were S-positive K-hybrid mice, and these S-positive K-hybrid mice can be used in step 6.

[0069] In the above steps, female mice give birth once every 4-6 weeks, and produce an average of 23-25 ​​offspring mice per month (8-10 of which are S-positive K-hybrid P-pure mice).

[0070] 4. Preparation of K-hybrid P-pure mice A S-yin K-hybrid P-pure male mouse is mated with a S-yin K-hybrid P-pure female mouse to produce offspring mice. Approximately half of the offspring mice are S-yin K-hybrid P-pure mice. The S-yin K-hybrid P-pure mice used as parents can be KP mice, S-yin K-hybrid P-pure mice from the offspring obtained in step 2, or S-yin K-hybrid P-pure mice from the offspring obtained in step 3.

[0071] S-negative K-hybrid P-pure mice can be used instead of KP mice for step 1.

[0072] S-negative K-hybrid P-pure mice can be used as parents for step five.

[0073] 5. Preparation of Mice The S-positive K-hybrid mice are mated with S-negative K-hybrid mice to obtain offspring mice. The S-positive K-hybrid mice serving as parents can be S-positive K-hybrid mice among the offspring mice obtained in step 1, or S-positive K-hybrid mice among the offspring mice obtained in step 2. The S-negative K-hybrid mice serving as parents can be KP mice, S-negative K-hybrid mice among the offspring mice obtained in step 2, S-negative K-hybrid mice among the offspring mice obtained in step 3, or S-negative K-hybrid mice among the offspring mice obtained in step 4.

[0074] Among the offspring mice, about 1 / 8 were SPEK hybrid P pure mice.

[0075] Approximately 1 / 8 of the offspring mice are S-negative, K-hybrid, and P-pure. S-negative, K-hybrid, and P-pure mice can be used in step 1 instead of KP mice. S-negative, K-hybrid, and P-pure mice can be used in step 4.

[0076] About 1 / 8 of the offspring mice are S-positive, K-negative, and P-pure. These S-positive, K-negative, and P-pure mice can be used in step 6.

[0077] Among the offspring mice, about 1 / 8 were S-positive, K-positive, and P-positive mice.

[0078] VI. Preparation of Mice S-positive hybrid mice are mated with S-positive hybrid mice to obtain offspring mice. The S-positive hybrid mice used as parents can be S-positive hybrid mice among the offspring mice obtained in step 2, S-positive hybrid mice among the offspring mice obtained in step 3, or S-positive hybrid mice among the offspring mice obtained in step 5. The S-positive hybrid mice used as parents can be S-positive hybrid mice among the offspring mice obtained in step 2, S-positive hybrid mice among the offspring mice obtained in step 3, or S-positive hybrid mice among the offspring mice obtained in step 5.

[0079] Among the offspring mice, about 3 / 8 are SPEK hybrid mice. They can replace the SPEK hybrid mice in the previous step as parents.

[0080] Among the offspring mice, about 1 / 8 are S-negative K-hybrid P-hybrid mice. These can replace the S-negative K-hybrid P-hybrid mice in the previous step as parents.

[0081] Among the offspring mice, about 3 / 8 are S positive K negative P pure mice. They can replace the S positive K negative P pure mice in the previous step as parents.

[0082] Among the offspring mice, about 1 / 8 are S-positive, K-positive, and P-positive mice. These can replace the S-positive, K-positive, and P-positive mice in the previous steps as parents.

[0083] VII. Methods for Identifying Mice in Steps 1 to 6 Tail tissues were collected from mice 10 days after birth, total DNA was extracted, and then PCR identification was performed.

[0084] 1. Identification of K-hybrid or K-negative Primer sequences used for Kras PCR identification: Mutant upstream primer (SEQ ID NO: 1): 5′-GCAGGTCGAGGGACCTAATA-3′; Wild-type upstream primer (SEQ ID NO: 2): 5′-TGTCTTTCCCCAGCACAGT-3′; Universal downstream primer (SEQ ID NO: 3): 5'-CTGCATAGTACGCTATACCCTGT-3'.

[0085] The target fragment size of the mutant target sequence is 100 bp, and the target fragment size of the wild-type target sequence is 250 bp.

[0086] Perform PCR amplification using total DNA as a template with the three primers described above, followed by agarose gel electrophoresis. If two amplification products of 100 bp and 250 bp are obtained, the gene is considered K heterozygous (K heterozygous mutant). If a 250 bp amplification product is obtained but no 100 bp amplification product is obtained, the gene is considered K negative (K wild-type).

[0087] 2. Identification of P-pure and P-impure Primer sequences used for Trp53 PCR identification: Upstream primer (SEQ ID NO: 4): 5′-GGTTAAACCCAGCTTGACCA-3′; Downstream primer (SEQ ID NO: 5): 5′-GGAGGCAGAGACAGTTGGAG-3′.

[0088] The target fragment size of the mutant target sequence is 300bp-400bp, and the target size of the wild-type target sequence is 270bp.

[0089] Perform PCR amplification using total DNA as a template using the two primers described above, followed by agarose gel electrophoresis. If a 300-400 bp amplification product is obtained and no 270 bp amplification product is obtained, the gene is considered P-homozygous (P-homozygous mutant). If a 300-400 bp amplification product and a 270 bp amplification product are obtained, the gene is considered P-heterozygous (P-heterozygous mutant).

[0090] 3. Identification of S positive and S negative Primer sequences used for Sox9-cre / ERT2 PCR identification: Transgene upstream primer (SEQ ID NO: 6): 5′-CGGTTTCGTTCTCTGTTTTCC-3′; Transgene downstream primer (SEQ ID NO: 7): 5′-AGGCAAATTTTGGTGTACGG-3′.

[0091] Internal positive control upstream primer (SEQ ID NO: 8): 5′-AGTGGCCTCTTCCAGAAATG-3′; Internal positive control downstream primer (SEQ ID NO: 9): 5'-TGCGACTGTGTCTGATTTCC-3'.

[0092] The target fragment size of the transgenic target sequence is 290 bp-310 bp, and the target fragment size of the internal positive control is 521 bp.

[0093] Using total DNA as a template, PCR amplification was performed using the transgenic primer pair (composed of the aforementioned transgenic upstream primer and transgenic downstream primer) and the positive control primer pair (composed of the aforementioned internal positive control upstream primer and internal positive control downstream primer), followed by agarose gel electrophoresis. If PCR amplification using the transgenic primer pair yields a 290-310 bp amplification product, and PCR amplification using the positive control primer pair yields a 521 bp amplification product, the test was considered S-positive. If PCR amplification using the transgenic primer pair fails to yield a 290-310 bp amplification product, and PCR amplification using the positive control primer pair yields a 521 bp amplification product, the test was considered S-negative.

[0094] The SPE K-hybrid P-pure mice obtained in this example are KPPS mice.

[0095] Example 2: Preparation of Pancreatic Intraductal Papillary Mucinous Tumor Model Mice Test mice: 8-week-old KPPS mice (prepared in step 3 of Example 1).

[0096] 1. Tamoxifen induction Test mice were administered tamoxifen solution (100 mg / kg body weight) intraperitoneally once daily for 6 consecutive days. The day of the 6th administration was designated as Day 0, and the days thereafter were counted continuously.

[0097] 2. Pancreatic Histopathological Evaluation Mice were sampled on days 28, 56, 84, 112, 140, and 168, and pancreatic tissue was obtained after dissection to prepare sections, which were then subjected to H&E staining. After H&E staining, two professional pathologists analyzed the tissue sections. Characteristics of LG-IPMN: branching papillary structures in the pancreatic ducts, covered with mildly atypical epithelial cells, accompanied by intracellular / extracellular mucus accumulation. Characteristics of HG-IPMN: more and more complex papillary structures, significant cellular atypia, increased mitotic figures, increased nuclear-to-cytoplasmic ratio, disappearance of nuclear polarity, and no signs of invasive cancer. Characteristics of IPMN-IC: small glands with abnormal structures can be seen in the duct wall, with distorted glandular structure and increased nuclear-to-cytoplasmic ratio.

[0098] The results are shown in Table 1. LG-IPMNs were observed in pancreatic tissue sections of KPPS mice after 4 weeks of tamoxifen treatment, and IPMN-ICs were observed in KPPS mice after 12 weeks of tamoxifen treatment. HG-IPMNs and IPMN-ICs were also observed in KPPS mice. Exemplary sections of LG-IPMNs, HG-IPMNs, and IPMN-ICs are shown in Table 1. Figure 1 .

[0099] Table 1 Number and proportion of mice in different disease stages at different sampling times

[0100] Example 3: Survival analysis Test mice: 46 8-week-old KPPS mice (prepared in step 3 of Example 1) or 25 KPPC mice (purchased).

[0101] Test mice were administered tamoxifen solution (100 mg / kg body weight) intraperitoneally once daily for 6 consecutive days. The day of the 6th administration was designated as Day 0, and the days thereafter were counted continuously.

[0102] Starting from day 1, every 7 days was considered as a week, and the survival rate of mice was counted every week to generate a survival curve.

[0103] See the results Figure 2 The median survival of KPPS mice was 20 weeks after the completion of tamoxifen infusion (n=46). The median survival of KPPC mice was 13 weeks after the completion of tamoxifen infusion (n=25). There was a significant difference in median survival between the two mouse models (P<0.0001), with KPPS mice having a significantly longer median survival than KPPC mice.

[0104] Example 4: Obtaining KPPS cell lines and using them for mouse modeling 1. KPPS Cell Construction 1. The pancreas of KPPS mice at the IPMN-IC stage (prepared as in Example 2) was aseptically harvested to isolate the tumor. The tumor was rinsed in PBS buffer to remove excess blood, and non-tumor tissue, such as blood vessels, from the tumor was sheared to remove. The tumor was then transferred to a new 6 cm diameter culture dish and minced with sterile scissors until it resembled minced meat.

[0105] 2. After completing step 1, remove the culture dish, add 3 mL of 0.25% trypsin solution, and digest at 37°C for 15-30 minutes (the following morphology is observed under the microscope: cells are free from cell clumps, and the edges of the cell clumps become transparent).

[0106] 3. After completing step 2, take the culture dish, add 6 mL of DMEM complete medium to terminate the digestion, and then filter with a cell sieve with a pore size of 70 μm to collect the filtrate.

[0107] 4. Take the filtrate obtained in step 3, centrifuge at 800g for 10 min, discard the supernatant, and resuspend the cell pellet in 5 mL of DMEM complete medium to obtain a cell suspension.

[0108] 5. Inoculate a 6-well plate with the cell suspension obtained in step 4 and culture until confluence reaches 90% (replace the culture medium every 2-3 days). The cells in this step are referred to as P0 cells.

[0109] 6. After completing step 5, discard the supernatant and add 0.25% trypsin solution for digestion for 2-3 minutes. Then, subculture at a 1:3 ratio. After the 10th passage, the cells are considered to be successfully established if they have uniform morphology and a relatively stable growth rate.

[0110] Named KPPS cells (also known as KPPS cell line).

[0111] 2. Cell Immunofluorescence Staining 1. Place a standard clean glass slide in a 6-well plate and inoculate 2×10 5 KPPS cells were cultured overnight.

[0112] 2. After completing step 1, discard the supernatant and wash twice with PBS buffer.

[0113] 3. After completing step 2, add 1 mL of fixative (4% paraformaldehyde solution) to each well and fix at room temperature for 20 minutes.

[0114] 4. After completing step 3, discard the fixative and wash three times with PBS buffer (5 minutes each time, gently shaking).

[0115] 5. After completing step 4, add 0.6 mL of PBS buffer containing 0.5% Triton X-100 to each well to permeabilize the cells. Incubate at room temperature for 20 minutes, then discard the liquid and wash three times with PBS buffer.

[0116] 6. After completing step 5, add 0.6 mL of blocking solution (i.e., goat serum) to each well, incubate for 30 minutes, and then discard the blocking solution.

[0117] 7. After completing step 6, add 0.5 mL of primary antibody working solution to each well and incubate at 4°C with shaking overnight.

[0118] Primary antibody (recombinant anti-pan cytokeratin antibody): Abcam, product catalog number ab7753.

[0119] 8. After completing step 7, discard the primary antibody working solution and wash three times with PBS buffer.

[0120] 9. After completing step 8, add 0.5 mL of secondary antibody working solution to each well and incubate at room temperature in the dark for 60 minutes with gentle shaking.

[0121] Secondary antibody (goat anti-mouse IgG H&L, Alexa Fluor® 488): Abcam, catalog number ab150113.

[0122] 10. After completing step 9, discard the secondary antibody working solution and wash three times with PBS buffer.

[0123] 11. After completing step 10, add 0.2 mL of DAPI solution to each well and incubate in the dark for 5 minutes.

[0124] 12. After completing step 11, discard the DAPI solution and wash the slides four times with PBS buffer (5 minutes each time). Then, use filter paper to absorb excess liquid on the slides. Add anti-fading mounting solution and gently place the coverslip over the cells, ensuring that there are no bubbles and that the cells are in contact with the mounting solution. Finally, use a confocal microscope to image.

[0125] See the exemplary imaging photos Figure 3 Immunofluorescence staining confirmed that KPPS cells had an epithelial phenotype (PanCK+) and exhibited significant mitotic activity.

[0126] 3. Cell Proliferation Assay 1. Collect KPPS cells in the logarithmic growth phase, digest them with 0.25% trypsin solution, and then centrifuge at 1000 rpm for 1 minute to collect the cell pellet. Then, wash the cell pellet with PBS buffer and resuspend the cell pellet in DMEM complete medium to obtain a cell suspension. Count the cells using trypan blue staining.

[0127] 2. Inoculate 1 mL of cell suspension (containing 3000 cells) into each well of a 64-well plate, then add 0.2 mL of DMEM complete medium into each well and culture for 1 day.

[0128] 3. After completing step 2, continue culturing until the cell density reaches above 90%. During the culturing process, add 0.05 mL of fixative (10% trichloroacetic acid solution) every two days.

[0129] 4. After completing step 3, continue culturing for one day, discard the liquid in the wells, wash the cells twice with PBS, and then air dry for 30 minutes.

[0130] 5. After completing step 4, add 0.05 mL of SRB (Suphoramide B) dye solution to each well, incubate for 30 minutes, then wash twice with 1% acetic acid and air dry.

[0131] 6. After completing step 5, add 0.05 mL of Tris base solution (containing 10 mM 2-amino-2-hydroxymethylpropane-1,3-diol, pH = 10.5) to each well to resuspend the cells, and then measure the absorbance at a wavelength of 564 nm using a microplate reader.

[0132] The results of the cell proliferation experiment showed that KPPS cells grew vigorously, with a doubling time of approximately 31 hours.

[0133] IV. Establishment of Subcutaneous Tumor Mouse Model KPPS cells were subcutaneously injected into the unilateral scapular region of 8-week-old C57BL / 6J mice (12 mice, half male and half female), with 5×10 6 Continuous monitoring after injection revealed that mice could grow subcutaneous tumors, and on day 14 after injection, the maximum diameter of most tumors exceeded 10 mm.

[0134] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A method for constructing a genetically engineered mouse model for pancreatic intraductal papillary mucinous neoplasia, characterized in that: The genetically engineered mice used to prepare the pancreatic intraductal papillary mucinous neoplasia model mice were named KPPS mice; The method comprises the following steps: hybridizing S-positive mice and K-hybrid P-pure mice as parents to obtain KPPS mice; The S-positive mouse is a transgenic mouse carrying Sox9-CreER; the Sox9-CreER is a DNA molecule having, from upstream to downstream, a promoter and a Cre recombinase gene transcribed by the promoter; the promoter is a pancreatic duct cell-specific promoter; The K hybrid P pure mouse has a pair of homologous chromosomes No. 11 both carrying loxP-Trp53-loxP, and a chromosome No. 6 carrying Promoter-LSL-Kras G12D ; The loxP-Trp53-loxP is obtained by modifying the Trp53 gene located on chromosome 11, wherein the modification refers to inserting two loxP sites in the same direction into the Trp53 gene, and the two loxP sites are inserted at different positions in the Trp53 gene; The loxP site recombination of loxP-Trp53-loxP results in the knockout of the Trp53 gene; the loxP site recombination is mediated by the Cre recombinase; The Promoter-LSL-Kras G12D The Promoter-LSL-Kras is obtained by modifying the region from the promoter of the Kras gene on chromosome 6 to the end of the Kras gene. G12D From upstream to downstream, there are the promoter of Kras gene, LSL sequence and Kras G12D Mutant gene; LSL sequence is a DNA fragment having two loxP sites in the same direction and a termination box located between the two loxP sites; Kras G12D The expression of the mutant gene is blocked by the LSL sequence; the Promoter-LSL-Kras G12D The loxP site recombination results in the expression of Kras blocked by the LSL sequence. G12D The mutant gene is restored to expression; the loxP site recombination is mediated by the Cre recombinase; The KPPS mouse is an S-positive K hybrid pure mouse; the S-positive K hybrid pure mouse means: carrying the Sox9-CreER, and both a pair of homologous chromosomes No. 11 carry the loxP-Trp53-loxP, and a chromosome No. 6 carries the Promoter-LSL-Kras G12D .

2. A method for preparing a pancreatic intraductal papillary mucinous neoplasia model mouse comprises the following steps: (1) Constructing KPPS mice according to the method of claim 1; (2) Inducing the KPPS mice with an inducer to obtain pancreatic intraductal papillary mucinous tumor model mice.

3. The method according to claim 2, wherein: In the step (2), pancreatic intraductal papillary mucinous tumor model mice at the IPMN stage are obtained starting from the fourth week after the completion of the induction plan.

4. Use of the KPPS mice prepared by the method of claim 1; the use is to prepare pancreatic intraductal papillary mucinous tumor model mice.

5. Use of the pancreatic intraductal papillary mucinous neoplasia model mouse prepared by the method of claim 2 or 3; the use is as follows (a), (b), or (c): (a) Presents the pathological progression during IPMN progression; (b) Conduct mechanistic studies on the progression of IPMN; (c) Drug screening during IPMN progression.

6. A tumor cell derived from a pancreatic intraductal papillary mucinous neoplasm model mouse prepared by the method of claim 2 or 3.

7. The tumor cell according to claim 6, wherein: The tumor cells are isolated from pancreatic tumor tissue of the pancreatic intraductal papillary mucinous tumor model mouse.

8. Tissue or organ, characterized in that: The tissue or organ comes from the tumor of the pancreatic intraductal papillary mucinous tumor model mouse prepared by the method of claim 2 or 3.

9. Tissue or organ, characterized in that: The tissue or organ is from a tumor of a tumor-bearing mouse; the tumor-bearing mouse is a mouse in which the tumor cells according to claim 6 or 7 are inoculated and a tumor is developed.

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