Liver tissue regeneration regulation and control method based on epigenetic state regulation and control

Abstract

The invention discloses a liver tissue regeneration regulation and control method based on epigenetic state regulation and control, and relates to the technical field of biology. Comprising the following steps: sampling the liver tissue at a plurality of preset time points after the liver tissue is damaged or partially resected, selecting a target gene related to liver tissue regeneration regulation, and detecting an epigenetic state of a promoter region of the target gene; comparing and analyzing the change of the epigenetic state of the target gene promoter region on the basis of detection results obtained at different time points, and determining epigenetic modification characteristics in the liver tissue regeneration process; according to an analysis result, intervening the expression level of a regulatory factor related to DNA methylation or DNA hydroxymethylation so as to regulate the epigenetic state of the target gene promoter region; after intervention is completed, the transcription expression condition of a target gene and the activity change of a cell signal channel related to liver tissue regeneration are detected, and the liver tissue regeneration process can be effectively regulated and controlled through the method.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for regulating liver tissue regeneration based on epigenetic state regulation. Background Technology

[0002] The liver is one of the most important metabolic and detoxification organs in the human body, and also one of the few solid organs with significant regenerative capacity. After liver tissue injury or partial resection, the remaining liver tissue can gradually restore liver volume and physiological function through hepatocyte proliferation and functional reconstruction. Therefore, the regulatory mechanisms and intervention methods for liver tissue regeneration have always been an important research direction in the fields of biomedicine and regenerative medicine.

[0003] Current research on liver tissue regeneration largely focuses on signaling pathway regulation, cytokine activity, and changes in the expression of molecules related to cell proliferation. For example, some studies have shown that the Hippo signaling pathway and its downstream effector molecules participate in regulating hepatocyte proliferation and tissue homeostasis during liver tissue regeneration. However, most of these studies emphasize detection and analysis at a single time point or single molecule level, making it difficult to reflect the dynamic regulatory characteristics during liver tissue regeneration.

[0004] In recent years, epigenetic regulation, as an important regulatory mechanism connecting the genome and phenotype, has gradually attracted attention in liver tissue regeneration research. Among these, epigenetic modifications such as DNA methylation and DNA hydroxymethylation can participate in regulating cell proliferation, differentiation, and tissue remodeling by affecting the transcriptional activity of gene promoter regions. Some studies have reported changes in the methylation levels of related gene promoter regions during liver tissue regeneration; however, current techniques still lack a systematic regulatory method that combines multi-timepoint detection, change characteristic analysis, and targeted intervention.

[0005] Meanwhile, existing technologies have the following shortcomings in practical applications: First, they lack a comprehensive analytical method for the changes in epigenetic state during liver tissue regeneration, making it difficult to fully reflect the regulatory characteristics of different regeneration stages; second, the connection between epigenetic regulation and subsequent functional verification is insufficient, failing to form a complete regulation and evaluation process; and third, existing methods are mostly fragmented operations in terms of technical processes, lacking a reproducible and implementable overall technical solution. Therefore, a liver tissue regeneration regulation method based on epigenetic state regulation is proposed. Summary of the Invention

[0006] The main objective of this invention is to provide a liver tissue regeneration regulation method based on epigenetic state regulation, which can effectively solve the problems in the background art.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A method for regulating liver tissue regeneration based on epigenetic state includes the following steps:

[0009] S1. After liver tissue is damaged or partially removed, liver tissue of the target object is sampled at multiple preset time points to obtain liver tissue samples containing hepatocytes, and at least one target gene related to liver tissue regeneration regulation is selected. The promoter region of the target gene is detected to obtain the epigenetic status of the promoter region of the target gene as the detection result.

[0010] S2. Based on the detection results obtained at multiple preset time points as described in step S1, compare and analyze the changes in the epigenetic state of the target gene promoter region, determine the epigenetic modification characteristics of the target gene promoter region during liver tissue regeneration, and use the analysis results as the basis for subsequent intervention.

[0011] S3. Based on the analysis results obtained in step S2, intervene in the expression levels of regulatory factors related to DNA methylation or DNA hydroxymethylation to regulate the epigenetic state of the promoter region of the target gene.

[0012] S4. After completing the intervention described in step S3, detect the transcriptional expression of the target gene and the changes in the activity of cell signaling pathways related to liver tissue regeneration, and obtain the results of the intervention's impact on molecular events related to liver tissue regeneration.

[0013] S5. Based on the results obtained in step S4, and combined with liver tissue regeneration evaluation indicators, assess the liver tissue regeneration status and characterize the effect of epigenetic state regulation on the liver tissue regeneration process.

[0014] Furthermore, the epigenetic state of the target gene promoter region includes at least the DNA methylation level or DNA hydroxymethylation level of CpG sites within the promoter region, and the detection of the epigenetic state is performed by genome bisulfite conversion treatment combined with sequencing analysis, high-throughput sequencing of methylated CpG islands, methylation-specific polymerase chain reaction, or a combination thereof.

[0015] Furthermore, the target gene is a gene involved in regulating hepatocyte proliferation, cell cycle progression, or organ volume changes during liver tissue regeneration.

[0016] Furthermore, the target gene is the LATS1 gene, and the promoter region of the LATS1 gene contains at least one CpG island region.

[0017] Furthermore, the regulatory factors related to DNA methylation are enzymes involved in the DNA methylation modification process, and the regulatory factors related to DNA hydroxymethylation are enzymes involved in the DNA demethylation or hydroxymethylation modification process.

[0018] Furthermore, the enzyme factor involved in the DNA methylation modification process includes DNMT3b, and the enzyme factor involved in the DNA demethylation or hydroxymethylation modification process includes TET1.

[0019] Furthermore, intervention on the expression level of the aforementioned regulatory factors can be achieved through viral vector-mediated gene delivery, RNA interference, antisense nucleic acid delivery, or a combination thereof.

[0020] Furthermore, the multiple preset time points correspond to different stages in the liver tissue regeneration process, and are used to obtain epigenetic state change information at each stage.

[0021] Furthermore, the evaluation indicators for liver tissue regeneration include at least two or more of the following indicators: changes in the ratio of liver weight to body weight, liver tissue structural remodeling, expression levels of hepatocyte proliferation-related markers, and changes in apoptosis-related indicators.

[0022] Furthermore, the activity of the cell signaling pathways related to liver tissue regeneration is characterized by changes in the phosphorylation state of downstream effector proteins or changes in the distribution of these downstream effector proteins in the cell nucleus and cytoplasm.

[0023] The present invention has the following beneficial effects:

[0024] Compared with existing technologies, this approach detects and compares the epigenetic status of the promoter region of target genes related to liver tissue regeneration at multiple preset time points after liver tissue injury or partial resection. This allows for the systematic acquisition of the changes in epigenetic modifications during liver tissue regeneration, thereby avoiding the one-sidedness caused by relying solely on detection at a single time point and improving the accuracy of characterizing the regulatory state of liver tissue regeneration.

[0025] Based on the analysis of the epigenetic state changes in the promoter region of the target gene, this scheme intervenes in the regulatory factors related to DNA methylation or DNA hydroxymethylation, combining epigenetic regulation with gene expression changes and downstream signaling pathway activity detection, forming a complete technical process from epigenetic state regulation to molecular event verification, which is conducive to improving the feasibility and reproducibility of the regulation scheme.

[0026] This scheme introduces liver tissue regeneration evaluation indicators to comprehensively assess the state of liver tissue regeneration, achieving multi-dimensional characterization of the regulatory effect of epigenetic status. It helps to reflect the regulatory effect of the liver tissue regeneration process from multiple levels such as tissue structure, cell proliferation and functional changes. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the present invention. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0029] Example 1

[0030] like Figure 1 As shown, a liver tissue regeneration regulation method based on epigenetic state regulation is proposed, which is applicable to the regulation of the regeneration process after liver tissue injury or partial resection.

[0031] I. Establishment and sampling of a liver tissue regeneration model;

[0032] Healthy adult mice were selected as experimental subjects, and a liver tissue regeneration model was established through partial hepatectomy. The partial hepatectomy involved removing approximately 70% of the liver tissue to simulate liver tissue damage or loss. After surgery, the mice were placed under standard feeding conditions for recovery.

[0033] Samples were taken from the experimental subjects at multiple predetermined time points following partial liver resection. These predetermined time points included at least several time points selected before surgery and postoperatively at 3h, 6h, 12h, 24h, 36h, 48h, 72h, 5d, and 7d. The corresponding experimental subjects were sacrificed at each time point, and their liver tissue samples were collected for subsequent testing and analysis.

[0034] II. Detection of the epigenetic status of the target gene promoter region;

[0035] Genomic DNA was extracted from liver tissue samples obtained at various time points, and at least one target gene related to the regulation of liver tissue regeneration was selected as the detection target. In this embodiment, the target gene is the LATS1 gene.

[0036] Epigenetic status analysis was performed on the promoter region of the LATS1 gene, including the DNA methylation and hydroxymethylation levels at CpG sites in the promoter region. Specifically, the methylation status of CpG sites in the LATS1 gene promoter region was detected using a combination of genomic bisulfite transformation and sequencing analysis; the results were then validated using high-throughput sequencing of methylated CpG islands or methylation-specific polymerase chain reaction.

[0037] The epigenetic state of the promoter region of the target gene at each preset time point is obtained through the above detection steps as the detection result.

[0038] III. Analysis of the characteristics of changes in epigenetic status;

[0039] Based on the detection results obtained at different preset time points, the changes in the epigenetic state of the target gene promoter region are compared and analyzed to determine the epigenetic modification characteristics of the LATS1 gene promoter region during liver tissue regeneration.

[0040] The analysis includes a comparison of the trends in DNA methylation and hydroxymethylation levels at different time points, thereby obtaining information on the dynamic changes of the target gene promoter region during liver tissue regeneration, and using the analysis results as the basis for subsequent regulatory interventions.

[0041] IV. Intervention by epigenetic regulatory factors;

[0042] Based on the above analysis results, the expression levels of regulatory factors related to DNA methylation or DNA hydroxymethylation were intervened to regulate the epigenetic state of the promoter region of the target gene.

[0043] In this embodiment, the regulatory factors related to DNA methylation include DNMT3b, and the regulatory factors related to DNA hydroxymethylation include TET1. By constructing viral vectors targeting DNMT3b or TET1 and introducing these viral vectors into experimental subjects via tail vein injection, intervention on the expression levels of the corresponding regulatory factors can be achieved. The viral vector can be an adeno-associated virus vector (such as AAV8) to improve its expression efficiency in liver tissue.

[0044] V. Detection of gene expression and signaling pathway activity;

[0045] After completing the above-mentioned intervention on the expression of regulatory factors, liver tissue samples were obtained again at preset time points. The transcriptional expression of the target gene LATS1 was detected by real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot.

[0046] Simultaneously, changes in the activity of cell signaling pathways related to liver tissue regeneration were detected. In this embodiment, the activity of related signaling pathways was characterized by detecting the phosphorylation status of the downstream effector protein YAP and its distribution in the cell nucleus and cytoplasm.

[0047] VI. Evaluation of liver tissue regeneration status;

[0048] Based on the above test results, and combined with liver tissue regeneration evaluation indicators, the liver tissue regeneration status is assessed. These liver tissue regeneration evaluation indicators include at least two of the following: changes in the liver weight-to-body weight ratio, liver tissue structural remodeling, and the expression levels of hepatocyte proliferation-related markers.

[0049] The remodeling of liver tissue structure was observed by hematoxylin-eosin (HE) staining; hepatocyte proliferation-related markers included Ki67, BrdU, PCNA or p-H3S10, and their expression levels were detected by immunohistochemistry or immunofluorescence staining.

[0050] The effects of epigenetic state regulation on liver tissue regeneration were characterized through the above evaluation steps.

[0051] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for regulating liver tissue regeneration based on epigenetic state regulation, characterized in that, Includes the following steps: S1. After liver tissue is damaged or partially removed, liver tissue of the target object is sampled at multiple preset time points to obtain liver tissue samples containing hepatocytes, and at least one target gene related to liver tissue regeneration regulation is selected. The promoter region of the target gene is detected to obtain the epigenetic status of the promoter region of the target gene as the detection result. S2. Based on the detection results obtained at multiple preset time points as described in step S1, compare and analyze the changes in the epigenetic state of the target gene promoter region, determine the epigenetic modification characteristics of the target gene promoter region during liver tissue regeneration, and use the analysis results as the basis for subsequent intervention. S3. Based on the analysis results obtained in step S2, intervene in the expression levels of regulatory factors related to DNA methylation or DNA hydroxymethylation to regulate the epigenetic state of the promoter region of the target gene. S4. After completing the intervention described in step S3, detect the transcriptional expression of the target gene and the changes in the activity of cell signaling pathways related to liver tissue regeneration, and obtain the results of the intervention's impact on molecular events related to liver tissue regeneration. S5. Based on the results obtained in step S4, and combined with liver tissue regeneration evaluation indicators, assess the liver tissue regeneration status and characterize the effect of epigenetic state regulation on the liver tissue regeneration process.

2. The method for regulating liver tissue regeneration based on epigenetic state regulation according to claim 1, characterized in that, The epigenetic state of the target gene promoter region includes at least the DNA methylation level or DNA hydroxymethylation level of CpG sites within the promoter region, and the detection of the epigenetic state is performed by genome bisulfite conversion treatment combined with sequencing analysis, high-throughput sequencing of methylated CpG islands, methylation-specific polymerase chain reaction, or a combination thereof.

3. The method for regulating liver tissue regeneration based on epigenetic state regulation according to claim 1, characterized in that, The target gene is a gene that participates in regulating hepatocyte proliferation, cell cycle progression, or organ volume changes during liver tissue regeneration.

4. The method for regulating liver tissue regeneration based on epigenetic state regulation according to claim 1, characterized in that, The target gene is the LATS1 gene, and the promoter region of the LATS1 gene contains at least one CpG island region.

5. The method for regulating liver tissue regeneration based on epigenetic state regulation according to claim 1, characterized in that, The regulatory factors related to DNA methylation are enzymes involved in the DNA methylation modification process, and the regulatory factors related to DNA hydroxymethylation are enzymes involved in the DNA demethylation or hydroxymethylation modification process.

6. The method for regulating liver tissue regeneration based on epigenetic state regulation according to claim 5, characterized in that, The enzymes involved in the DNA methylation modification process include DNMT3b, and the enzymes involved in the DNA demethylation or hydroxymethylation modification process include TET1.

7. The method for regulating liver tissue regeneration based on epigenetic state regulation according to claim 1, characterized in that, Intervention on the expression level of the aforementioned regulatory factors is achieved through viral vector-mediated gene delivery, RNA interference, antisense nucleic acid delivery, or a combination thereof.

8. The method for regulating liver tissue regeneration based on epigenetic state regulation according to claim 1, characterized in that, The multiple preset time points correspond to different stages in the liver tissue regeneration process and are used to obtain epigenetic state change information at each stage.

9. The method for regulating liver tissue regeneration based on epigenetic state regulation according to claim 1, characterized in that, The evaluation indicators for liver tissue regeneration include at least two or more of the following indicators: changes in the ratio of liver weight to body weight, liver tissue structural remodeling, expression levels of hepatocyte proliferation-related markers, and changes in apoptosis-related indicators.

10. The method for regulating liver tissue regeneration based on epigenetic state regulation according to claim 4, characterized in that, The activity of the cell signaling pathways associated with liver tissue regeneration is characterized by changes in the phosphorylation state of downstream effector proteins or changes in the distribution of downstream effector proteins in the cell nucleus and cytoplasm.

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