A method for constructing an animal model of pituitary tumor based on primary pituitary tumor cells

Abstract

This invention discloses a method for constructing an animal model of pituitary tumors based on primary pituitary tumor cells. First, tumor tissue from a patient with a pituitary adenoma is obtained in vitro. This tumor tissue is then cultured in its primary form. Finally, the cultured cells are injected in situ into the pituitary fossa of the experimental animal. The model constructed using this method exhibits imaging and pathological manifestations consistent with the intracranial space-occupying effect and hormonal level disorders associated with pituitary adenomas. It demonstrates excellent clinical pathological and symptom simulation, making it suitable for pituitary adenoma pathological research and drug screening, with broad application prospects.

Description

Technical fields:

[0001] This invention belongs to the field of biology and relates to a method for constructing an animal model, specifically a method for constructing a pituitary tumor animal model based on primary pituitary tumor cells. Background technology:

[0002] A pituitary adenoma is a benign tumor that occurs in the brain, originating from the pituitary gland. The prevalence of pituitary adenoma is relatively high in adults, estimated at approximately 1%. Although most pituitary adenoma grows slowly, they can cause problems such as visual disturbances, headaches, fatigue, and abnormal hormone levels. Treatment typically includes medication, radiation therapy, and surgery. However, sometimes pituitary adenoma may recur despite treatment, and the treatment outcome is not ideal. Basic research on pituitary adenoma has progressed slowly due to the lack of effective animal models.

[0003] Mouse models of pituitary adenomas offer significant advantages in medical research on pituitary adenomas. They are among the most valuable tools for gaining a deeper understanding of the pathogenesis and treatment of pituitary adenomas, enabling advancements in understanding disease mechanisms, evaluating therapies, and developing personalized medicine. Mice have a shorter lifespan and a faster reproductive cycle; compared to larger animal models, mouse models are less costly to maintain and conduct experiments, making large-scale studies and replicated experiments possible.

[0004] Animal models serve as a bridge between clinical and basic research, and establishing suitable animal models that closely align with clinical needs for basic research is crucial. Currently used pituitary adenoma models primarily involve manipulating the genes of experimental animals to induce pituitary adenoma development. However, standardized model evaluation methods are lacking, and due to the diverse pathological features and endocrine characteristics of pituitary adenomas, research that closely aligns with clinical needs and personalized treatment plans is not feasible. The limited number of animal models of pituitary adenomas reported in domestic and international literature hinders medical research on pituitary adenomas and the screening of clinical candidate drugs. Summary of the Invention:

[0005] To address the aforementioned technical problems in the prior art, this invention provides a method for constructing a pituitary tumor animal model based on primary pituitary tumor cells. This method aims to solve the technical problem that the lack of standardized model evaluation methods for pituitary tumor animal models in the prior art is detrimental to medical research on pituitary adenomas and the screening of clinical candidate drugs.

[0006] This invention provides a method for constructing a pituitary tumor animal model based on primary pituitary tumor cells, characterized by the following steps:

[0007] 1) Obtain tumor tissue from isolated pituitary adenoma patients;

[0008] 2) Culture the above-mentioned tumor tissue in its primary form;

[0009] 3) Inject the cells cultured in step 2) into the pituitary fossa of the experimental animal in situ.

[0010] Furthermore, the construction method is as follows:

[0011] i. Preserve isolated pituitary adenoma tumor tissue in sterile, serum-free culture medium to ensure tumor stability.

[0012] The tumor tissue was completely immersed and kept sterile;

[0013] ii. After completing i), perform suspension culture of human primary pituitary tumor cells;

[0014] iii. Observe cell growth. During the logarithmic growth phase, use a stereotaxic instrument to inject the cells cultured in step ii into the pituitary fossa of the experimental animal in situ. After the procedure, the experimental animal is kept in an SPF environment for continued feeding.

[0015] Furthermore, the tumor tissue was minced, digested, centrifuged, and then cultured in suspension using DMEM complete medium.

[0016] Furthermore, the experimental animal was a SCID mouse.

[0017] Furthermore, the in situ injection coordinates in the mouse pituitary fossa were fixed at 3.30 mm posterior to the anterior fontanelle and at a depth of 6.30 mm.

[0018] Furthermore, the number of cells injected in situ was 5 × 10⁶. 5 .

[0019] Furthermore, the average feeding period for experimental animals after in situ injection was 30 days.

[0020] The present invention also provides an animal model of pituitary tumor established using primary pituitary tumor cells obtained by any of the above-described construction methods.

[0021] The present invention also provides the application of the above-mentioned animal model in the pathological study of pituitary tumors or in the screening of candidate drugs for the treatment of pituitary tumors.

[0022] The present invention also provides a method for screening candidate drugs for the treatment of pituitary tumors, the method comprising: administering the above-mentioned animal model by gavage daily for at least 30 days, evaluating the efficacy, and screening candidate drugs based on tumor pathological characteristics and size changes.

[0023] The construction method provided by this invention is based on the pituitary adenoma tissue removed from a clinical pituitary adenoma patient. Primary culture of tumor cells is performed, and the cells are injected into the pituitary fossa within one hour using a stereotaxic instrument and fixed coordinates. After feeding, a mouse tumor-bearing model with pathological results and endocrine characteristics completely consistent with the source tumor cells is obtained.

[0024] This invention verifies the correct tumor location using mouse pituitary fossa MRI and small animal imaging results. After tumor tissue resection, comparison with tumor tissue of the same origin shows identical pathological changes and alterations in serum hormone levels. The pathological manifestations and serum hormone level changes in this model are consistent with those of tumor tissue originating from pituitary adenomas, demonstrating excellent clinical symptom simulation. It can be used for further medical research on pituitary adenomas and for screening candidate therapeutic drugs, showing broad application prospects. It will play an important role in the study of the pathogenesis of pituitary adenomas and in personalized treatment.

[0025] Compared with existing technologies, the technical effects of this invention are positive and significant. This invention provides a method and application for establishing an animal model of pituitary tumors based on primary pituitary tumor cells. The imaging and pathological manifestations of this model are consistent with the intracranial space-occupying effect and hormonal level disorders of pituitary adenomas, exhibiting excellent clinical pathological and symptom simulation. It can be used for pituitary adenoma pathological research and is also a good model for drug screening, with broad application prospects. The model constructed by this invention is consistent with clinical pathology and is a pituitary adenoma animal model with good reproducibility, high stability, and a comprehensive evaluation system, laying the foundation for further research on pituitary adenomas. Attached image description:

[0026] Figure 1 A schematic diagram showing the anatomical location and stereotactic injection site of the mouse pituitary fossa.

[0027] Figure 2 The results of pituitary MRI images in mice 30 days after in situ injection of cells.

[0028] Figure 3 To compare biochemical markers between animal model tumor tissue and source patient tissue after successful modeling.

[0029] Figure 4 To assess serum hormone levels in tumor-bearing mice.

[0030] Figure 5 This is a schematic diagram for model making. Detailed implementation method:

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] The experimental materials and instruments used in the following examples are:

[0033] 1. Laboratory animals

[0034] Forty male SPF-grade SCID mice, aged 4–6 weeks and weighing 20–25 g, were purchased from Spiford (Suzhou) Biotechnology Co., Ltd. (Experimental Animal License No.: SCXK(Su)2022-0006, Experimental Animal Qualification Certificate No.: A202306270493). The experimental animals were housed in the standard animal room of Jiangnan University at a room temperature of 23±2℃, relative humidity of 55%±5%, and a light-dark cycle of 12h (6:00–18:00). The environment was quiet, and the animals had free access to food and water. This experimental study was approved by the Ethics Review Committee of Jiangnan University (JN.No20230830m1000115

[332] ).

[0035] 2. Main Reagents and Instruments

[0036] Stereotype system (Baoding Dichuang Electronic Technology Co., Ltd., product model: L0107-1B), miniature handheld cranial drill (Shenzhen Ruiwode Life Technology Co., Ltd., specification model: 78001), laboratory micro-injection pump (Baoding Dichuang Electronic Technology Co., Ltd., product model: TJ-2A), CO2 incubator (Thermo Fisher Scientific XII (China) Co., Ltd., model: 371GP), biosafety cabinet (Shanghai Lishen Scientific Instruments Co., Ltd., model: HFsafe-1500LC(A2)), laser confocal microscope (Carl Zeiss, model: LSM880), fully automatic cryostat (Leica, model: CM3050S), fully digital magnetic resonance system (Philips, model: Ingenia) 3.0T), Ki67 (ABclonal, part number: A20018), CAM5.2 (Biotechnologies, part number: MAB-0687), Synapsin (Proteintech, part number: 20258-1-AP), ACTH (Biotechnologies, part number: RAB-0010), hGH (Biotechnologies, part number: RAB-0084), PRL (Biotechnologies, part number: RAB-0109), CK14 (Proteintech, part number: 10143-1-AP).

[0037] Example 1: Construction of an animal model of pituitary tumor based on primary pituitary tumor cells

[0038] Tumor tissue from clinical cases of pituitary adenomas was collected and placed in serum-free DMEM culture medium under aseptic conditions. The tissue was immediately transported to the laboratory, where it was washed 2-3 times with sterile PBS solution in a laminar flow hood to remove connective tissue, necrotic tissue, and blood. The tissue was then minced to 1mm size using surgical scissors. 3 Tissue blocks were then digested with 5 times their volume of trypsin at 37°C for 6 minutes. After pipetting and digestion again, this process was repeated 3 times. After stopping digestion, the cells were filtered through a 100-mesh cell filter, centrifuged at 1200 rpm for 3 minutes, and the supernatant was discarded. The cells were resuspended and dispersed in DMEM complete medium, counted under a microscope, and the cell concentration was adjusted to 1.0 × 10⁶ cells / cm². 6 / mL.

[0039] Cells were seeded into 24-well plates pre-coated with poly-L-lysine, 1 mL per well, and the culture medium was changed every two days.

[0040] Human pituitary tumors are cultured in suspension, and cell growth is observed daily under a microscope. Cells are passaged when they reach approximately 70% confluence. Once the cells reach the logarithmic growth phase, the next step of constructing an animal model can be performed.

[0041] like Figure 1 , 5 As shown, male 6-week-old SCID mice were selected, and primary human pituitary tumor cells in the logarithmic growth phase were injected into the pituitary fossa of anesthetized SCID mice using a stereotaxic instrument. The cell count was 5 × 10⁶ cells / mL. 5 The injection site was located 3.3 mm posterior to the anterior fontanelle, with a depth of 6.3 mm.

[0042] Example 2 Evaluation of a human primary pituitary tumor model

[0043] 1. After injecting human primary pituitary tumor cells into SCID mice using a stereotaxic instrument, the mice were fed for 30 days before the animal model was evaluated.

[0044] 2. Evaluation Methods

[0045] 2.1 General Condition Evaluation of SCID Mice: During the experiment, the general morphological and behavioral characteristics of each group of SCID mice were observed and recorded, mainly including feeding status, drinking status, weight changes, fur luster, and activity level. Compared with normal mice, the survival status of the mice was judged by whether there was an increase or decrease in weight and whether the fur was shiny. There were no scoring criteria or scores assigned.

[0046] 2.2 SCID mice underwent enhanced pituitary MRI to observe tumor formation.

[0047] Before scanning, Gd-DTPA (100 μl / 20 g) was injected intraperitoneally, and T1-enhanced scanning was performed 3 minutes later to observe the formation of tumors in the pituitary fossa.

[0048] 2.3 Histopathological evaluation of pituitary tumor tissue in SCID mice

[0049] Pituitary tumor tissue from fixed SCID mice was paraffin-embedded, sectioned at 3 μm, and stained with hematoxylin and eosin (HE). HE staining was observed under a microscope and compared with HE staining of primary cell-derived tumor tissue. Morphological changes in the tumor tissue were evaluated by observing the pathological sections. Simultaneously, frozen sections (16 μm) were stained with immunofluorescence for ACTH, CAM5.2, CK14, Ki67, and Synapsin, PRL, and GH indicators. These sections were then compared with primary cell-derived tumor tissue to evaluate the pathological condition of the tumor tissue.

[0050] 2.4 Evaluation of plasma ACTH (pg / ml), PRL (ng / ml), and GH (ug / ml) levels in SCID mice

[0051] Three ml of peripheral blood from SCID mice was drawn into an EDTA-K2 vacuum blood collection tube and centrifuged at 4000 r·min⁻¹ for 10 min (centrifugation radius 16 cm) at 4°C. The plasma sample was separated and stored at -20°C for later analysis. The plasma ACTH (pg / ml), PRL (ng / ml), and GH (ug / ml) levels of SCID mice were measured according to the ELISA kit instructions.

[0052] 3. Results

[0053] 3.1 General condition and behavioral scores of SCID mice

[0054] As shown in Table 1, compared with normal mice, the model group mice showed changes in body weight, water intake, and fur luster due to the different pathological characteristics of tumor tissue derived from primary cells.

[0055] Table 1 shows the survival status of mice after orthotopic cell injection.

[0056]

[0057] 3.2 SCID mice undergoing enhanced pituitary MRI

[0058] like Figure 2 As shown, after the model group mice were fed, they underwent enhanced MRI imaging of the pituitary gland. Tumors were visible in the pituitary fossa of the mice that were successfully modeled.

[0059] 3.3 Histopathological characteristics of mouse pituitary tumors

[0060] like Figure 3 As shown, after tumor formation in the model group mice, tumor tissue was removed and subjected to immunofluorescence staining and HE staining for ACTH, CAM5.2, CK14, Ki67, Synapsin, PRL, and GH indicators. The results were compared with those of primary cell-derived tumor tissue, indicating that the model mice retained the pathological characteristics of primary tumor cells.

[0061] 3.4 Plasma ACTH (pg / ml), PRL (ng / ml), and GH (ug / ml) levels in SCID mice

[0062] like Figure 4 As shown, after tumor formation in the model group mice, plasma was extracted, and the levels of ACTH (pg / ml), PRL (ng / ml), and GH (ug / ml) were detected and compared with plasma samples from patients with primary cell sources. This indicated that the model mice retained the endocrine characteristics of primary tumor cells.

[0063] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A method for constructing a pituitary tumor animal model based on pituitary tumor primary cells, characterized in that, The construction method is: i) The tumor tissue of the patient with pituitary adenoma is preserved in a sterile serum-free medium to ensure that the tumor tissue is completely soaked and kept sterile; ii) After the end of i), the tumor tissue is cut, digested, centrifuged, and suspended in DMEM complete medium for culture; iii) observing the cell growth, in the logarithmic growth phase, using a stereotaxic apparatus to inject the cells cultured in step ii) into the pituitary fossa of a test animal, which is a SCID mouse, the coordinates of the pituitary fossa of the mouse for injection are 3.30 mm behind the bregma, the depth is 6.30 mm, and the number of cells injected in situ is 5 x 10 5 The average duration of feeding after the test animal is injected in situ is 30 days, and after the end, the test animal is continuously fed in an SPF environment.

2. The use of the pituitary tumor animal model obtained by the method of claim 1 in the pathological study of pituitary tumor or the screening of candidate drugs for treating pituitary tumor.

3. A method for screening a candidate drug for pituitary tumor treatment, characterized by, The method comprises: daily gavage administration of the pituitary tumor animal model obtained by the method of claim 1 for at least 30 days, then performing efficacy evaluation, and screening candidate drugs according to the pathological characteristics and size changes of the tumor.

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