Construction method and application of peritoneal mesothelium-mesenchymal transition cell and animal model

Abstract

The invention discloses a construction method and application of a peritoneal mesothelium-mesenchymal transition cell and animal model, and relates to the technical field of cell biology, the key points of the technical scheme are as follows: the peritoneal mesothelium-mesenchymal transition cell and animal model is established by adopting a metabolite 4-HNE of a lipid peroxidation reaction; the 4-HNE can better simulate the process of tumor peritoneal metastasis when being used for establishing peritoneal mesothelium-mesenchymal transition cells and animal models, and the 4-HNE has the advantages of being stable in property, low in cost and capable of being stored for a long time. The peritoneal mesothelium-mesenchymal transition cell and the animal model show typical mesenchymal transition characteristics, and can well simulate the real process of peritoneal mesothelium-mesenchymal transition. The invention provides an ideal experimental platform for researching a basic mechanism of abdominal tumor metastasis and exploring clinical new drug target screening.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and more specifically, to a method for constructing peritoneal mesothelial-mesenchymal transition cells and an animal model and their application. Background Art

[0002] The peritoneum is a thin membrane that covers the abdominal cavity, pelvic cavity, and the surface of the internal organs, and is composed of a single layer of mesothelial cells. This serous membrane has important physiological functions, such as reducing friction between organs, absorbing body fluids, providing defense mechanisms, and repairing after injury. Under stimulation factors such as inflammation and trauma, peritoneal mesothelial cells promote the repair of peritoneal injuries through a process called mesothelial-to-mesenchymal transition (MMT). However, when this process is abnormally regulated, it may cause excessive repair, leading to pathological phenomena such as peritoneal fibrosis and adhesions. These abnormal changes are also closely related to the occurrence of a series of peritoneal-related diseases.

[0003] Mesothelial-mesenchymal transition is not only associated with diseases such as fibrosis and adhesion, but is also considered a key step in the metastasis of tumor cells on the peritoneal surface. In particular, mesothelial-mesenchymal transition plays a core role in the metastasis of tumors such as colorectal cancer, pancreatic cancer, gastric cancer, and ovarian cancer into the peritoneum. During the process of tumor peritoneal metastasis, mesothelial cells secrete various proteins that regulate extracellular matrix remodeling, such as metal matrix proteinases and lysyl oxidase, into the extracellular space through mesothelial-mesenchymal transition. These molecules can degrade and remodel the extracellular matrix of the peritoneum, providing favorable conditions for the invasion and metastasis of tumor cells.

[0004] In addition, mesothelial-mesenchymal transition can also help tumor cells adhere and colonize on the peritoneal surface by secreting extracellular matrix components such as collagen and fibronectin, further promoting their metastasis. At the same time, after mesothelial-mesenchymal transition, peritoneal mesothelial cells will also secrete vascular endothelial growth factor and basic fibroblast growth factor, etc., to promote the proliferation and growth of tumor cells in the peritoneum. Therefore, the establishment of in vitro and in vivo peritoneal mesothelial-mesenchymal transition models is of great significance for studying the molecular mechanism of tumor peritoneal metastasis and developing targeted therapeutic drugs.

[0005] At present, common peritoneal mesothelial-mesenchymal transition cell models are mainly induced by cytokines such as transforming growth factor β1 (TGF-β1) or interleukin-6 (IL-6). However, these methods face multiple challenges in practical applications. First, exogenous cytokines are expensive and easily inactivated in animals, which limits their application in animal models. In addition, these cytokines are not specifically related to tumor peritoneal metastasis, and therefore cannot fully simulate the physiological mechanism of mesothelial-mesenchymal transition during tumor peritoneal metastasis.

[0006] 4-Hydroxynonenoic acid (4-HNE) is a metabolite of lipid peroxidation, which plays an important role in many pathological and physiological processes, such as inflammation and apoptosis. 4-HNE has been shown to be associated with the occurrence and development of a variety of diseases, including ischemic heart disease and pulmonary fibrosis. Studies have found that 4-HNE is highly expressed in peritoneal tumors such as colorectal cancer, and due to its stable chemical properties, 4-HNE can diffuse to distant tissues and organs through the blood and other pathways, forming a pre-metastatic microenvironment that is conducive to tumor implantation and metastasis before tumor cells reach secondary tissues and organs. However, as an endogenous metabolite, the specific role of 4-HNE in peritoneal mesothelial-mesenchymal transition and tumor peritoneal metastasis has not been fully studied.

[0007] The present invention proposes a new method for inducing peritoneal mesothelial-mesenchymal transformation using 4-HNE, and successfully establishes an in vitro peritoneal mesothelial-mesenchymal transformation cell model and an in vivo animal model through this method. This provides a new experimental tool and theoretical basis for studying the molecular mechanism of tumor peritoneal metastasis and developing targeted treatments for tumor metastasis. Compared with traditional cytokine induction methods, the use of 4-HNE is not only less expensive, but also has a broader prospect for application in animal models due to its strong stability. Summary of the invention

[0008] The purpose of the present invention is to provide a method for constructing a peritoneal mesothelial-mesenchymal transition cell and an animal model and its application, so as to solve the problems in the prior art of the peritoneal mesothelial-mesenchymal transition model being unstable and unable to effectively simulate the pre-metastatic and metastatic microenvironment of tumor peritoneum.

[0009] The above technical objective of the present invention is to construct a peritoneal mesothelial-mesenchymal transition cell model achieved by the following technical scheme, comprising the following steps:

[0010] S1: Use complete medium containing 4-HNE at a dose range of 0.5-15 μM to culture peritoneal mesothelial cells for 10-30 days;

[0011] S2: performing cell phenotype analysis on the peritoneal mesothelial cells obtained in step S1;

[0012] S3: Study the protein functions and characteristics of the peritoneal mesothelial cells;

[0013] S4: Based on the results of step S2 and step S3, summarize and establish a peritoneal mesothelial-mesenchymal transition cell model.

[0014] The present invention is further configured as follows: the dosage of 4-HNE used in step S1 is specifically 2 μM, and the cell culture time is specifically 21 days.

[0015] The present invention is further configured as follows: the cell phenotype analysis described in step S2 includes cell morphology observation: using an inverted microscope to continuously observe and record the morphological changes of peritoneal mesothelial cells; cell immunophenotype detection: using immunofluorescence staining to detect the expression changes of epithelial and mesenchymal cell markers; transcription factor expression detection: using protein immunoblotting western blot to detect the expression of transcription factors that regulate mesothelial-mesenchymal transition.

[0016] The present invention is further configured that the protein function and property research in step S3 comprises the following steps:

[0017] Protein stability and modification analysis: Liquid chromatography-tandem mass spectrometry was used to analyze 4-HNE-modified proteins, and the binding sites of 4-HNE to cysteine ​​residues of SDHA protein were screened and determined;

[0018] Analysis of SDH activity and protein expression: The protein expression of the four subunits SDHA, SDHB, SDHC, and SDHD was analyzed by western blot, and the effect of 4-HNE on SDH enzyme activity and the accumulation of succinic acid were analyzed;

[0019] Species homology analysis: Compare the protein sequence differences of the helical domain of SDHA proteins from different animals to confirm the conservation of 4-HNE modification sites among different species.

[0020] By adopting the above technical scheme, the present invention uses a complete medium containing 4-HNE in a dosage range of 0.5-15 μM to culture peritoneal mesothelial cells, which can induce mesothelial-mesenchymal transformation of cells without causing excessive cytotoxicity, thereby better simulating the transformation process under pathological and physiological conditions, and compared with the experimental scheme of high-dose toxic substances, the present invention adopts a low-dose concentration, which helps to maintain the vitality and function of the cells;

[0021] The present invention adopts a variety of analysis methods in step S2 to comprehensively evaluate the cell phenotype: by continuously observing the changes in cell morphology through an inverted microscope, the transition from epithelial to mesenchymal cell morphology is intuitively recorded; by the expression changes of epithelial cell markers such as E-cadherin and mesenchymal cell markers such as α-SMA, it is verified that the cells have undergone epithelial to mesenchymal transformation; transcription factor detection: by detecting the expression levels of transcription factors (such as SNAIL and TWIST) closely related to mesenchymal transformation through western blot, the occurrence of transformation is further proved at the molecular level. This multi-angle and multi-indicator evaluation method makes the verification of the transformation process more comprehensive and accurate.

[0022] In step S3, the present invention is not limited to observing phenotypic changes, but also deeply explores the function and mechanism of action of proteins by the following means: using liquid chromatography-tandem mass spectrometry technology, in-depth analysis of the sites and stability of 4-HNE modification of specific proteins (SDHA), which provides a strong basis for studying the molecular mechanism induced by 4-HNE; through the expression changes and enzyme activity evaluation of SDHA and its related proteins (SDHB, SDHC, SDHD), the potential effects of 4-HNE on mitochondrial metabolism are revealed, further indicating that the disorder of cellular metabolism may play a key role in mesenchymal transformation. Compared with conventional protein expression detection, the in-depth study of modification sites and enzyme activity of the present invention provides a more detailed basis for understanding the biochemical mechanism of peritoneal mesothelial-mesenchymal transformation.

[0023] The present invention also confirmed the conservation of 4-HNE modification sites in different species by comparing the protein sequences of the helical domains of SDHA proteins in different species. This analysis result shows that the modification of SDHA by 4-HNE has broad biological applicability, which means that this model is not only suitable for human cell research, but may also provide a reference for peritoneal mesothelial-mesenchymal transition research in other species. This cross-species applicability enhances the universality and promotion value of the model.

[0024] The present invention provides a reliable and innovative method for constructing a peritoneal mesothelial-mesenchymal transition cell model through experimental design. Its advantage lies not only in the successful model construction, but more importantly, through in-depth discussions on key protein modifications, transcription factor expression and cell metabolism, it broadens the understanding of the mesenchymal transition mechanism and provides an important experimental platform for future drug screening and disease treatment.

[0025] Another object of the present invention is to provide a method for constructing a peritoneal mesothelial-mesenchymal transition animal model, comprising the following steps:

[0026] SS2 selected healthy adult mice as experimental animals, and injected intraperitoneally with phosphate buffer containing 4-HNE at a dose of 50-100 μg / kg, once every 4 days, and the feeding period of mice was 15-30 days;

[0027] SS2: the experimental animals were sacrificed and peritoneal tissues were obtained, and routine pathological tests and immunophenotypic tests were performed on the peritoneal tissues;

[0028] SS3: Based on the results of step SS2, summarize and establish an animal model of peritoneal mesothelial-mesenchymal transition.

[0029] The present invention is further configured as follows: the dosage of 4-HNE in the above step SS1 is specifically 80 μg / kg, and the feeding cycle of mice is 21 days.

[0030] The present invention is further configured as follows: the specific methods of conventional pathological detection and immunophenotypic detection in the above step SS2 are respectively:

[0031] Routine pathological examination: HE staining and VG collagen staining were performed on the peritoneal tissue to observe and record the changes in the peritoneal tissue;

[0032] Immunophenotypic detection: Immunohistochemical staining was used to detect the expression of epithelial markers and mesenchymal markers of peritoneal surface mesothelial cells, and the immunophenotypic changes of peritoneal mesothelial cells in the control group and 4-HNE-treated animals were recorded.

[0033] By adopting the above technical scheme, the present invention uses adult mice as experimental animals to study the mesenchymal transformation of peritoneal mesothelial cells in an in vivo environment. Compared with in vitro cell models, in vivo models can better simulate real pathophysiological conditions and reflect more comprehensive biological reactions, such as the interaction between cells and matrix, immune system and other microenvironmental factors, so that research results closer to the human body can be obtained, providing more powerful support for the mechanism research and development of treatment strategies for related diseases; in step SS2, the present invention adopts a variety of detection methods to verify the construction effect of the animal model: HE staining is used to observe the changes in cell morphology and tissue structure, and VG collagen staining is used to detect the changes in collagen fibers of the peritoneal matrix. These methods can provide intuitive evidence of tissue structure changes and show the mesenchymal transformation process induced by 4-HNE; immunohistochemical staining is used to detect the expression changes of epithelial markers and mesenchymal markers, and the occurrence of mesenchymal transformation is further verified at the molecular level. This multi-angle evaluation method makes the verification of the model more comprehensive and powerful.

[0034] The construction of the animal model makes the present invention not only suitable for in vitro cell research, but also for more complex biological and pharmacological studies, such as drug screening, toxicity assessment, and interaction studies with the immune system. Compared with cell models, animal models can provide a more comprehensive research platform for studying the various mechanisms and potential therapeutic targets of peritoneal mesothelial cell transformation. The present invention provides a cutting-edge experimental platform for future clinical research and drug development through the successful construction of an animal model of peritoneal mesothelial-mesenchymal transformation. The model can be used to evaluate the effectiveness and safety of new anti-fibrotic drugs or treatment methods, as well as to further explore biomarkers and mechanisms related to peritoneal mesothelial-mesenchymal transformation, providing an important basis for the diagnosis, prevention and treatment of peritoneal fibrosis-related diseases.

[0035] Another object of the present invention is to provide an application of a cell model constructed by the method for constructing a peritoneal mesothelial-mesenchymal transition cell model in peritoneal metastasis of tumors.

[0036] By adopting the above technical scheme, the peritoneal mesothelial-mesenchymal transition cell model constructed by the present invention successfully simulates the process of peritoneal mesothelial cells transforming to a mesenchymal phenotype, which plays an important role in tumor cell invasion and spread. Through this model, the dynamic changes of peritoneal mesothelial cells in tumor metastasis and their interaction with tumor cells can be studied more accurately, thereby revealing the mechanism of peritoneal metastasis; the mesenchymal transformation of peritoneal mesothelial cells is similar to the epithelial-mesenchymal transformation (EMT) occurring in tumor cells, and EMT is a key process for tumor cells to acquire migration and invasion capabilities. The model constructed by the present invention not only simulates the mesenchymal transformation process of peritoneal cells, but can also be used to study the interaction between peritoneal mesothelial cells and metastatic tumor cells, especially the promotion of mesenchymal-mesenchymal transformation to tumor invasion and metastasis in the microenvironment. This provides a new idea for studying how to inhibit mesenchymal-mesenchymal transformation to prevent tumor peritoneal metastasis.

[0037] The cell model of the present invention is not only suitable for the study of a single type of tumor, but can also be widely used in the study of various abdominal metastasis tumors by introducing tumor cells from different sources. The flexibility of this model makes it have potential application value in a wide range of tumor research, and helps to promote the in-depth exploration of the mechanism of abdominal metastasis of various tumors. The cell model provided by the present invention can be used to discover biomarkers related to peritoneal mesothelial-mesenchymal transformation and abdominal metastasis of tumors. By conducting in-depth genetic and proteomic analysis of the cells in the model, specific molecular markers can be screened out, which not only help to understand the molecular mechanism of the metastasis process, but also may become clinically used biomarkers for detecting abdominal metastasis, which has important diagnostic and prognostic value.

[0038] Another object of the present invention is to provide an application of the animal model constructed by the method for constructing the peritoneal mesothelial-mesenchymal transition animal model in the peritoneal metastasis of tumors.

[0039] By adopting the above technical scheme, the present invention can more comprehensively reveal the specific role of peritoneal mesothelial-mesenchymal transition in tumor metastasis through the study of the in vivo environment. Through the pathological and immunophenotypic analysis of peritoneal tissue, the interaction between tumor cells and peritoneal mesothelial cells and their promotion of tumor metastasis can be deeply studied. Through this animal model, the interaction between tumors and host microenvironment can be further explored, thereby providing support for the development of microenvironment-based treatment methods. By using different types of mouse models, the peritoneal metastasis process of different tumor types can be simulated, and the impact of individual differences on the metastasis process can be studied. This provides an experimental basis for the individualized treatment of tumor patients and helps to optimize treatment plans.

[0040] The animal model of the present invention can be widely used in the study of abdominal metastasis of various tumors such as ovarian cancer, pancreatic cancer, gastric cancer, colorectal cancer, etc., and helps to deeply explore the mechanism of abdominal metastasis of different tumor types. This wide application helps to promote the development of anti-metastasis treatment strategies for different tumor types. The present invention uses an innovative method of intraperitoneal injection of 4-HNE to induce mesenchymal transformation, which can more directly study the process of constructing a microenvironment before peritoneal metastasis induced by the metabolites of lipid peroxidation reactions secreted by tumor cells in an in vivo environment, which provides an unprecedented experimental approach for the study of abdominal metastasis.

[0041] In summary, the present invention has the following beneficial effects:

[0042] 1. The present invention uses 4-HNE, a metabolite of lipid peroxidation, for the first time to establish a cell and animal model of peritoneal mesothelial-mesenchymal transition. Compared with traditional cytokines such as transforming growth factor β1 and interleukin-6, 4-HNE has the advantages of stable properties, low cost, and easy storage, which solves the problem of easy inactivation and high cost of factor-type inducers;

[0043] 2. Since exogenous cytokines are easily inactivated in animals, it is difficult to construct an animal model. The present invention successfully uses 4-HNE to construct an animal model of peritoneal mesothelial-mesenchymal transformation, which makes up for the shortcomings of the prior art and makes the model operable and effective. In addition, 4-HNE is an endogenous metabolite secreted by tumor cells. Using it to construct an animal model of peritoneal mesothelial-mesenchymal transformation can better simulate the process of tumor metastasis in vivo. The peritoneal mesothelial-mesenchymal transformation cells and animal models of the present invention show typical mesenchymal transformation characteristics and can well simulate the real process of peritoneal mesothelial-mesenchymal transformation. This provides an ideal experimental platform for studying the basic mechanism of peritoneal tumor metastasis and exploring the screening of clinical new drug targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a diagram of the dynamic observation results of cell morphology in Example 1 of the present invention;

[0045] Figure 2 This is an example diagram of the immunofluorescence staining results in Example 1 of the present invention;

[0046] Figure 3 is a graph showing the results of transcription factor expression detection in Example 1 of the present invention;

[0047] Figure 4 is the effect of 4-HNE on the expression of EMT-related transcription factors and HIF-1α protein in Example 1 of the present invention;

[0048] Figure 5 is the effect of the small molecule inhibitor in Example 1 of the present invention on HIF-1α and EMT-related transcription factors;

[0049] Figure 6 This is a diagram showing the results of liquid chromatography-tandem mass spectrometry analysis of the binding sites of 4-HNE and SDHA protein in Example 1 of the present invention;

[0050] Figure 7 The effect of 4-HNE on SDH protein expression, enzyme activity and succinate accumulation in Example 1 of the present invention;

[0051] Among them, Figure 7 A shows the effect of 4-HNE on SDH protein expression; Figure 7 B is the effect of 4-HNE on enzyme activity; Figure 7 C is the effect of 4-HNE on succinate accumulation;

[0052] Figure 8 This is a schematic diagram of the conservation analysis of the 4-HNE modification sites in the helical domains of SDHA proteins of different species in Example 1 of the present invention;

[0053] Fig. 9 2. HE staining and VG collagen staining results of peritoneal tissue in Example 2 of the present invention;

[0054] Fig.10 This is a schematic diagram of the implantation of HCT116 cells in the abdominal cavity of BALB / c nude mice in Example 3 of the present invention;

[0055] Fig.11 This is a schematic diagram of the implantation of MC38 cells in the peritoneal cavity of mice in Example 4 of the present invention. DETAILED DESCRIPTION

[0056] The present invention is further described in detail below in conjunction with the accompanying drawings.

[0057] Example 1: A method for constructing a peritoneal mesothelial-mesenchymal transition cell model

[0058] Materials and methods: Human peritoneal mesothelial cell line HMrSV5 cells were purchased from Genio Biotechnology Co., Ltd. (Guangzhou, China). They were cultured in DMEM complete medium containing 10% fetal bovine serum and placed in an incubator at 37°C. 4-HNE was purchased from GLPBIO, with the product number GC42410 and the molecular formula C9H16O2. DMEM (Dulbecco's Modified Eagle Medium) medium and fetal bovine serum (FBS) were purchased from Thermo Company, USA. Antibodies used for immunofluorescence staining and western blot detection were purchased from Abcam and Affinity Company, USA. Liquid chromatography-tandem mass spectrometry detection was performed by Shanghai Jiyun Biotechnology Co., Ltd.

[0059] The method for constructing the peritoneal mesothelial-mesenchymal transition cell model specifically includes the following steps:

[0060] S1: Experimental group: HMrSV5 peritoneal mesothelial cells were cultured in complete medium containing 4-HNE with a concentration gradient of 0.5-15 μM to induce mesothelial-mesenchymal transition in the cells;

[0061] Control group: Peritoneal mesothelial cells were cultured in complete medium containing DMSO as a solvent control group to exclude the influence of the solvent on the cells.

[0062] S2: performing cell phenotype analysis on the peritoneal mesothelial cells obtained in step S1;

[0063] Dynamic observation of cell morphology: Through continuous microscopic observation, the morphological changes of cells during the experiment are dynamically recorded, such as the attached Figure 1 As shown;

[0064] Immunofluorescence staining: Immunofluorescence staining was used to detect the expression of epithelial cell marker E-cadherin and mesenchymal cell marker α-SMA in the control group and experimental group, respectively. Figure 2 As shown;

[0065] Transcription factor expression detection: Western blot was used to detect the expression of five transcription factors that regulate mesothelial-mesenchymal transition, including Twist1, Snail, Slug, ZEB1, and ZEB2. Figure 3 shown.

[0066] Cell phenotype analysis results:

[0067] In the control group, HMrSV5 cells showed typical epithelial cell morphology. The cells were polygonal with uneven edges, and the cells maintained tight connections. The cells formed a paving stone-like appearance. The typical epithelial phenotype showed its high cell polarity and dense arrangement. Under the action of 4-HNE, HMrSV5 cells gradually lost the typical characteristics of epithelial cells. The tight junctions between cells were destroyed, and the polarity of cells was weakened, which was manifested as the cells gradually becoming wider and flatter. Over time, the cell morphology changed further, developing into a polygonal and multi-branched structure, showing the morphological characteristics of mesothelial to mesenchymal transformation.

[0068] In the control group HMrSV5 cells, the epithelial cell-specific marker E-cadherin was strongly expressed, showing the epithelial phenotype of the cells, and the mesenchymal cell marker α-SMA was almost not expressed, indicating that the mesothelial-mesenchymal transition did not occur. Under the action of 4-HNE, the HMrSV5 cells in the experimental group gradually lost the expression of E-cadherin, indicating a decrease in epithelial characteristics. At the same time, the expression of α-SMA gradually increased, indicating that the cells had transformed into a mesenchymal phenotype, as shown in the attached Figure 2 As shown, 4-HNE induced mesothelial-mesenchymal transition.

[0069] The results of transcription factor expression detection are shown in the attached Figure 3 As shown, the expression results showed that after 21 days of 4-HNE treatment, the expression levels of these EMT-related transcription factors in 4-HNE-treated cells were significantly increased compared with those in the control group, indicating that molecular events of epithelial to mesenchymal transition occurred in the cells.

[0070] S3: Study the protein functions and characteristics of the peritoneal mesothelial cells;

[0071] As attached Figure 4 As shown in Figure 2, 4-HNE promoted the expression of EMT-related transcription factors Twist1, Snail1, and Snail2 in a dose-dependent manner, and also significantly increased the protein expression level of hypoxia-inducible factor HIF-1α. In order to further verify the role of HIF-1α in 4-HNE regulating mesothelial-mesenchymal transition, small molecule inhibitors (such as PX478, LW6, etc.) were used to block the expression of HIF-1α in HMrSV5 cells. The experimental results showed that small molecule inhibitors could partially block the induction of 4-HNE on the mRNA expression of Twist1, Snail1, and Slug. Figure 5 This suggests that HIF-1α plays an important regulatory role in 4-HNE-mediated mesothelial-mesenchymal transition.

[0072] 4-HNE is an active electrophilic molecule that can quickly react with nucleophilic substances in cells and interfere with the normal function of cells. In order to study the mechanism of action of 4-HNE on the stability of HIF-1α protein, liquid chromatography-tandem mass spectrometry analysis was used to screen out that 4-HNE can bind to the cysteine ​​residue at site 536 of the SDHA protein responsible for the tricarboxylic acid cycle in the inner membrane of mitochondria. Figure 6 shown.

[0073] SDHA is a catalytic subunit of the succinate dehydrogenase SDH complex, which catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid cycle. The SDH complex consists of four subunits: SDHA, SDHB, SDHC, and SDHD, of which SDHA and SDHB have catalytic activity, while SDHC and SDHD are responsible for anchoring SDHA and SDHB on the inner membrane of mitochondria.

[0074] The experimental results showed that although 4-HNE had no significant effect on the protein expression of SDHA, SDHB, SDHC and SDHD, Figure 7 A, but it inhibited the enzyme activity of SDH in a dose-dependent manner. Figure 7 B, leading to the accumulation of succinic acid, such as Figure 7 C. The accumulation of succinate inhibits the degradation of HIF-1α by prolyl hydroxylase, thereby stabilizing the expression of HIF-1α and further activating its downstream transcriptional signaling pathway.

[0075] In summary, 4-HNE inhibits the enzymatic activity of SDH by binding to SDHA, leading to the accumulation of succinate, which in turn stabilizes the expression of HIF-1α. HIF-1α then promotes the expression of EMT-related transcription factors, thereby regulating the mesothelial-mesenchymal transition process of peritoneal mesothelial cells. This molecular mechanism provides a new perspective for further understanding the mesothelial-mesenchymal transition in tumor peritoneal metastasis.

[0076] The present invention compares the protein sequences of the helical domain of SDHA protein of various animals such as humans, mice, and rats, and studies show that the 4-HNE modification sites of the helical domain, such as the attached Figure 8 As shown in the figure, it is highly conserved among different species. The conservation of this modification site indicates that 4-HNE can bind to SDHA proteins of different species, thereby inhibiting their activity and triggering subsequent biological effects.

[0077] Since the 4-HNE modification sites in the helical domain of SDHA are conserved in multiple species, it indicates that the modification effect of 4-HNE on SDHA is not limited by species. This means that the mechanism of regulating peritoneal mesothelial-mesenchymal transition by 4-HNE is cross-species feasible. Specifically, 4-HNE can not only play a role in human cell models cultured in vitro, but can also be used to establish peritoneal mesothelial-mesenchymal transition models in experimental animals including mice and rats. This provides a wide range of possibilities for further studying the role of 4-HNE in mesothelial-mesenchymal transition and tumor metastasis in animal models.

[0078] S4: Based on the results of step S2 and step S3, summarize and establish a peritoneal mesothelial-mesenchymal transition cell model.

[0079] Example 2: A method for constructing an animal model of peritoneal mesothelial-mesenchymal transition

[0080] Materials and methods: Healthy adult C57BL / 6 mice were used as experimental animals. The animal husbandry and operation procedures were strictly carried out in accordance with the animal ethics standards of the First Affiliated Hospital of Naval Medical University and approved by the Ethics Committee.

[0081] The method for constructing a peritoneal mesothelial-mesenchymal transition animal model specifically includes the following steps:

[0082] SS1: Experimental group: Healthy adult C57BL / 6 mice were selected as experimental animals and injected intraperitoneally with phosphate buffer containing 4-HNE at a dose of 80 μg / kg. The injection frequency was once every 4 days, and the mice were kept for 21 days.

[0083] Control group: healthy adult C57BL / 6 mice were selected as experimental animals and injected intraperitoneally with PBS phosphate buffer; injection frequency: once every 4 days; the dosage and feeding cycle were the same as those of the experimental group.

[0084] SS2: the experimental animals were sacrificed and peritoneal tissues were obtained, and routine pathological tests and immunophenotypic tests were performed on the peritoneal tissues;

[0085] At the end of the experiment, the experimental animals were killed and their peritoneal tissues were subjected to routine pathological examinations, including HE staining and VG collagen staining. The results are shown in the attached Fig. 9 As shown:

[0086] Control group: The peritoneal structure was normal, showing a single layer of flat mesothelial cells, without obvious tissue thickening and cell infiltration;

[0087] Experimental group: The peritoneum was significantly thickened, and the mesothelial cells changed from the original single-layer flat morphology to spindle-shaped and multi-layered distribution. The mesothelial cells infiltrated deep into the peritoneum, indicating that the tissue structure had changed significantly. VG collagen staining showed obvious collagen deposition in the submesothelial tissue, indicating fibrosis.

[0088] The above pathological changes indicate that 4-HNE treatment can significantly induce morphological changes in peritoneal mesothelial cells and promote tissue fibrosis.

[0089] Immunophenotyping: Immunohistochemical staining was used to further verify the mesothelial-mesenchymal transition induced by 4-HNE. The results are as follows:

[0090] Control group: Mesothelial cells on the peritoneal surface showed positive expression of the typical epithelial marker Calretinin, indicating that these cells maintained an epithelial phenotype, and negative expression of the mesenchymal marker α-SMA, indicating that no mesenchymal transition occurred;

[0091] Experimental group: Calretinin expression in peritoneal mesothelial cells was negative, indicating that the cells had lost their epithelial phenotype, and α-SMA expression was positive, indicating that these cells had transformed into a mesenchymal phenotype, which was consistent with the characteristics of mesothelial-mesenchymal transition.

[0092] The experimental results showed that low-dose 4-HNE successfully induced mesothelial-mesenchymal transition of peritoneal mesothelial cells, which was manifested in peritoneal thickening, cell morphology changes and the occurrence of fibrous collagen deposition. In addition, immunohistochemical results further confirmed that the cells in the 4-HNE-treated group transformed from an epithelial phenotype to a mesenchymal phenotype. Given that the SDHA protein modification sites of 4-HNE in different species are highly conserved, it is shown that 4-HNE can be used not only in mouse models, but also in other animal models, which provides an experimental basis for the establishment of peritoneal mesothelial-mesenchymal transition models in various animals.

[0093] SS3: Based on the results of the above histopathological evaluation, summarize and establish an animal model of peritoneal mesothelial-mesenchymal transition.

[0094] Example 3: Application of a peritoneal mesothelial-mesenchymal transition cell model in peritoneal tumor metastasis

[0095] Materials and methods: Human colorectal cancer cell line HCT116 cells were purchased from Biopharmaceuticals Co., Ltd. (Shanghai, China). Human peritoneal mesothelial cell line HMrSV5 cells were purchased from Biopharmaceuticals Co., Ltd. (Shanghai, China). BALB / c nude mice were used as experimental animals. The animal husbandry and operation procedures were strictly carried out in accordance with the animal ethics standards of the First Affiliated Hospital of Naval Medical University and approved by the Ethics Committee.

[0096] The specific method for validating the peritoneal mesothelial-mesenchymal transition cell model in tumor peritoneal metastasis includes the following steps:

[0097] Model establishment: According to Example 1, a peritoneal mesothelial-mesenchymal transition cell model was first established. The specific steps were as follows: mesothelial cells with a mesenchymal phenotype were obtained by induction, and normal mesothelial cells and mesothelial cells with a mesenchymal phenotype were used for subsequent experiments respectively.

[0098] Cell inoculation: Different cells were mixed with HCT116 cells and inoculated into the peritoneal cavity of BALB / c nude mice. The experimental group was inoculated with a mixture of mesothelial cells with mesenchymal phenotype and HCT116 cells; the control group was inoculated with a mixture of normal mesothelial cells and HCT116 cells.

[0099] Feeding and observation: Feed the animals for 3 weeks after inoculation to observe the peritoneal implantation. After 3 weeks, the experimental animals were killed to detect the peritoneal implantation of HCT116 cells.

[0100] Peritoneal implantation detection: At the end of the experiment, the experimental animals were sacrificed, and the implantation of HCT116 cells in the peritoneal cavity of BALB / c nude mice was examined by laparotomy, and the number and distribution of implanted tumors were recorded.

[0101] The experimental results are as attached Fig.10 As shown, there was a significant difference in the peritoneal implantation between the experimental group and the control group. In the experimental group, after HCT116 cells were mixed with mesothelial cells with a mesenchymal phenotype, the implantation ability of HCT116 cells in the peritoneal cavity of nude mice was significantly enhanced, and the number of implanted tumors formed was significantly higher than that in the control group. In contrast, the implantation ability of HCT116 cells mixed with normal mesothelial cells was lower, and the number of implanted tumors was smaller.

[0102] The experimental results showed that mesothelial cells with mesenchymal phenotype can significantly promote the implantation of HCT116 cells in the peritoneal cavity of BALB / c nude mice, indicating that mesothelial-mesenchymal transition may enhance the colonization and spread of tumor cells in the peritoneal environment. This discovery is of great significance for studying the mechanism of peritoneal tumor metastasis, and also verifies the potential role of mesenchymal mesothelial cells with mesenchymal phenotype in promoting peritoneal implantation.

[0103] Example 4: Application of a peritoneal mesothelial-mesenchymal transition animal model in peritoneal tumor metastasis

[0104] Materials and methods: C57BL / 6 mouse-derived colorectal cancer cell line MC38 was purchased from CyBiotech Co., Ltd. (Shanghai, China). C57BL / 6 mice were used as experimental animals. The animal husbandry and operation procedures were strictly carried out in accordance with the animal ethics standards of the First Affiliated Hospital of Naval Medical University and approved by the Ethics Committee.

[0105] The specific method for validating the peritoneal mesothelial-mesenchymal transition animal model in terms of tumor peritoneal metastasis includes the following steps:

[0106] Model establishment: A peritoneal mesothelial-mesenchymal transition mouse model was established according to Example 2, and the specific steps were as follows: a peritoneal mesothelial-mesenchymal transition mouse model was established to simulate the mesenchymal transition of peritoneal mesothelial cells; mice were treated with a solvent control to establish mice without peritoneal mesothelial-mesenchymal transition as a control;

[0107] MC38 cell inoculation: MC38 cells were inoculated into the peritoneal cavity of two groups of mice, respectively. The experimental group was inoculated with MC38 cells into the peritoneal cavity of mice with peritoneal mesothelial-mesenchymal transformation; the control group was inoculated with MC38 cells into the peritoneal cavity of control mice without peritoneal mesothelial-mesenchymal transformation.

[0108] Feeding and observation: The animals were fed for 3 weeks after inoculation. After 3 weeks, the experimental animals were killed and the peritoneal implantation of MC38 cells was detected.

[0109] Peritoneal implantation detection: At the end of the experiment, the experimental animals were sacrificed, and the implantation of MC38 cells in the mouse peritoneal cavity was examined by laparotomy, and the number and distribution of implanted tumors were recorded.

[0110] The results of the abdominal implant test are as follows Fig.11 As shown, there were significant differences in the peritoneal implantation between the experimental group and the control group: the peritoneal implantation rate of MC38 cells in the experimental group in peritoneal mesothelial-mesenchymal transition mice was significantly increased, and the number of implanted tumors formed was significantly higher than that in the control group; in contrast, the implantation rate of MC38 cells in the control group in the solvent control mice was lower, and the number of implanted tumors was significantly reduced.

[0111] The experimental results showed that peritoneal mesothelial-mesenchymal transition significantly improved the ability of MC38 cells to implant in the mouse peritoneal cavity. Compared with the control group, the number of peritoneal tumors in the experimental group was significantly increased. This indicates that peritoneal mesothelial-mesenchymal transition plays a promoting role in the process of peritoneal tumor implantation, which may provide a new perspective and model for studying the regulatory mechanism of the microenvironment before tumor metastasis.

[0112] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A method for constructing a peritoneal mesothelial-mesenchymal transition cell model, characterized by: The following steps are involved: S1: Use complete medium containing 4-HNE at a dose range of 0.5-15 μM to culture peritoneal mesothelial cells for 10-30 days; S2: performing cell phenotype analysis on the peritoneal mesothelial cells obtained in step S1; S3: Study the protein functions and characteristics of the peritoneal mesothelial cells; S4: Based on the results of step S2 and step S3, summarize and establish a peritoneal mesothelial-mesenchymal transition cell model.

2. The method for constructing a peritoneal mesothelial-mesenchymal transition cell model according to claim 1, characterized in that: The specific dosage of 4-HNE used in step S1 is 2 μM, and the specific cell culture time is 21 days.

3. The method for constructing a peritoneal mesothelial-mesenchymal transition cell model according to claim 1, characterized in that: The cell phenotype analysis described in step S2 includes cell morphology observation: using an inverted microscope to continuously observe and record the morphological changes of peritoneal mesothelial cells; cell immunophenotype detection: using immunofluorescence staining to detect the expression changes of epithelial and mesenchymal cell markers; transcription factor expression detection: using protein immunoblot western blot to detect the expression of transcription factors that regulate mesothelial-mesenchymal transition.

4. The method for constructing a peritoneal mesothelial-mesenchymal transition cell model according to claim 1, characterized in that: The protein function and property study described in step S3 includes the following steps: Protein stability and modification analysis: Liquid chromatography-tandem mass spectrometry was used to analyze 4-HNE-modified proteins, and the binding sites of 4-HNE to cysteine ​​residues of SDHA protein were screened and determined; Analysis of SDH activity and protein expression: The protein expression of the four subunits SDHA, SDHB, SDHC, and SDHD was analyzed by western blot, and the effect of 4-HNE on SDH enzyme activity and the accumulation of succinic acid were analyzed; Species homology analysis: Compare the protein sequence differences of the helical domain of SDHA proteins from different animals to confirm the conservation of 4-HNE modification sites among different species.

5. A method for constructing an animal model of peritoneal mesothelial-mesenchymal transition, characterized by: The following steps are involved: SS1: Healthy adult mice were selected as experimental animals and injected intraperitoneally with phosphate buffer containing 50-100 μg / kg of 4-HNE once every 4 days. The culture period of mice was 10-30 days. SS2: the experimental animals were sacrificed and peritoneal tissues were obtained, and routine pathological tests and immunophenotypic tests were performed on the peritoneal tissues; SS3: Based on the results of step SS2, summarize and establish an animal model of peritoneal mesothelial-mesenchymal transition.

6. The method for constructing a peritoneal mesothelial-mesenchymal transition animal model according to claim 5, characterized in that: The specific dosage of 4-HNE in step SS1 is 80 μg / kg, and the feeding period of mice is 21 days.

7. The method for constructing a peritoneal mesothelial-mesenchymal transition animal model according to claim 5, characterized in that: The specific methods of routine pathological testing and immunophenotyping testing in step SS2 are: Routine pathological examination: HE staining and VG collagen staining were performed on the peritoneal tissue to observe and record the changes in the peritoneal tissue; Immunophenotypic detection: Immunohistochemical staining was used to detect the expression of epithelial markers and mesenchymal markers of peritoneal surface mesothelial cells, and the immunophenotypic changes of peritoneal mesothelial cells in the control group and 4-HNE-treated animals were recorded.

8. Use of the peritoneal mesothelial-mesenchymal transition cell model as claimed in claim 1 in peritoneal metastasis of tumors.

9. Use of the peritoneal mesothelial-mesenchymal transition animal model as claimed in claim 5 in peritoneal metastasis of tumors.

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