A method for extracting interstitial fluid from heart tissue

Abstract

This invention discloses a method for extracting interstitial fluid from cardiac tissue, belonging to the field of biomedical technology. Based on the unique characteristics of the heart, a organ closely related to blood circulation, and its differences in tissue physical properties compared to other organs, this invention designs and develops this extraction method, which specifically includes: rinsing isolated cardiac tissue with pre-cooled physiological saline; low-speed centrifugation to remove residual rinsing fluid from the heart; and centrifugation again to obtain interstitial fluid from the cardiac tissue. Comparison of the obtained interstitial fluid with cardiac cell fluid and serum confirms the accuracy and specificity of the extraction scheme of this invention. The centrifugation process of this invention does not damage cells, preserving its unique differences from serum and cardiac cell fluid. The extracted interstitial fluid has a high protein concentration, which can better reflect changes in the cardiac microenvironment, facilitating subsequent related medical and basic research.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for extracting interstitial fluid from cardiac tissue. Background Technology

[0002] The heart participates in blood circulation through contraction and relaxation, and its metabolism and energy production are fundamental to its normal function. Cardiac extracellular fluid (EF) is a crucial biological fluid present between cardiomyocytes, providing a medium between cardiomyocytes and their microenvironment. The relationship between the two helps to reveal the dynamic changes in cardiac metabolism. The composition of cardiac extracellular fluid dynamically reflects the metabolic state, inflammation level, and microcirculatory function of the heart. Precise analysis of EF can provide crucial information for research on the mechanisms of cardiac diseases, biomarker discovery, and pharmacokinetics.

[0003] Currently, there are three main limitations in the extraction of cardiac interstitial fluid (EF): (1) Component degradation: Since the extraction of cardiac interstitial fluid uses an ex vivo heart, it will lead to a certain degree of degradation, and the operation needs to be improved to minimize degradation; (2) Poor specificity of interstitial fluid components and easy sample contamination: The heart is an important organ involved in blood circulation and contains rich blood components; therefore, blood components may be an important interfering factor in the extraction of cardiac interstitial fluid; (3) Structural damage: In the extraction of cardiac interstitial fluid, operations such as heart dissection and centrifugation may damage the cell structure, causing the leakage of intracellular components, resulting in the interstitial fluid components being contaminated by intracellular substances, making it impossible to accurately obtain the changes in the cardiac microenvironment under the studied state.

[0004] The patent "CN 220867382 U A Device for Extracting Interstitial Fluid by Centrifugation" mentions that preliminary explorations have been made into methods for extracting interstitial fluid from tumor tissues, and low-speed centrifugation has been proven to be an effective method. However, due to the significant structural differences between tumor tissue (as a diseased tissue) and normal heart tissue, methods applied to tumor tissue cannot solve the problems of low interstitial fluid extraction concentration, high sample contamination rate, poor component specificity, and significant operational damage in current methods for extracting interstitial fluid from heart tissues. Therefore, developing a high-concentration, high-fidelity, high-specificity, and low-damage method for extracting interstitial fluid from heart tissues is of great significance for improving the accuracy and reliability of cardiac microenvironment research and for the study of cardiovascular disease mechanisms and precision diagnosis and treatment. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide a method for extracting interstitial fluid from cardiac tissue.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides a method for extracting interstitial fluid from cardiac tissue, comprising the following steps: (1) Take a fresh heart and irrigate it with pre-cooled physiological saline until the irrigating solution has no obvious blood color; (2) The heart was cut open and centrifuged at low speed to remove residual lavage fluid. (3) Centrifuge the heart again to obtain cardiac interstitial fluid.

[0007] First, the mouse heart was rapidly excised and thoroughly perfused with pre-cooled lavage fluid to remove as much blood as possible, minimizing interference from blood components (such as high-abundance proteins) during subsequent extraction. Then, the heart was cut open and briefly centrifuged at low speed to remove residual lavage fluid, improving the concentration, purity, and accuracy of the interstitial fluid. Finally, the heart was appropriately minced and centrifuged for 20 minutes to maximize the extraction volume and accuracy of the interstitial fluid, providing a foundation for further clinical and basic research. Furthermore, multi-stage centrifugation during the extraction process better prevents the rupture of cardiomyocytes.

[0008] Furthermore, in step (1), the temperature of the pre-cooled saline solution is 4°C.

[0009] Furthermore, in step (2), the perfused heart is cut open and placed on a cell filter with a pore size of 40µm for low-speed centrifugation.

[0010] Furthermore, in step (2), the temperature of the low-speed centrifugation is 2-4℃, the speed is 600-700rpm, and the centrifugation time is 3-5min.

[0011] Furthermore, in step (3), the heart is further cut into smaller pieces and placed on a cell filter with a pore size of 40µm for centrifugation.

[0012] Furthermore, in step (3), the centrifugation temperature is 2-4℃, the centrifugation force is 1600rpm, and the centrifugation time is 20-30min.

[0013] Furthermore, in step (3), after centrifugation, the collected liquid is transferred to a SpinX filter tube and centrifuged for 1 minute to finally obtain the required cardiac interstitial fluid.

[0014] Furthermore, the fresh heart is a rapidly isolated mouse heart.

[0015] The beneficial effects of this invention are: This invention, based on the unique characteristics of the heart, a organ closely related to blood circulation, and its differences in tissue physical properties compared to other organs, designs and develops a fluid extraction method. Specifically, the method includes: rinsing isolated heart tissue with pre-cooled physiological saline; low-speed centrifugation to remove residual rinsing fluid from the heart; and centrifugation to obtain cardiac interstitial fluid. Comparison of the obtained cardiac interstitial fluid with cardiac cell fluid and serum confirms the accuracy and specificity of the extraction scheme of this invention. The centrifugation process of this invention does not damage cells, preserving its unique differences from serum and cardiac cell fluid. The extracted cardiac interstitial fluid has a high protein concentration, which can better reflect changes in the cardiac microenvironment, facilitating subsequent related medical and basic research. Attached Figure Description

[0016] Figure 1 This refers to the volume of interstitial fluid in the heart tissue obtained under centrifugation conditions.

[0017] Figure 2 This represents the ratio of HSA concentration in cardiac interstitial fluid to its concentration in serum under centrifugation conditions.

[0018] Figure 3 Comparison of protein components in cardiac tissue lysate, serum, and cardiac interstitial fluid.

[0019] Figure 4 This represents the expression levels of marker proteins for intracellular components in cardiac tissue lysate and cardiac interstitial fluid.

[0020] Figure 5 GO functional enrichment analysis for differentially expressed proteins.

[0021] Figure 6 This study analyzed the expression of marker proteins that are key intracellular components. Detailed Implementation

[0022] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0023] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0024] Cardiac interstitial fluid (EF), as a component of the microenvironment in which cardiomyocytes directly infiltrate, carries important biological information such as cellular metabolites, signaling molecules (e.g., cytokines, hormones), exosomes, and drug metabolites. Compared to blood or tissue homogenates, which are commonly used as traditional research targets, EF more accurately reflects the pathophysiological dynamics of the cardiac microenvironment, such as elucidating the mechanisms of cardiovascular diseases, identifying biomarkers, and optimizing drug development. Therefore, actively developing extraction strategies for cardiac interstitial fluid is of great significance for the diagnosis and treatment of heart-related diseases.

[0025] Unless otherwise specified, all experimental materials used in the embodiments of this invention are conventional experimental materials in the art and can be purchased through commercial channels. Cy5-HSA (Cy5-labeled human serum albumin) is a complex formed by the covalent bonding of the near-infrared fluorescent dye Cy5 and human serum albumin (HSA), possessing both biological carrier and fluorescent tracer functions, and is widely used in the field of biomedical research. It was purchased from Xi'an Qiyue Biotechnology Co., Ltd.

[0026] Example 1: Extraction of interstitial fluid from cardiac tissue The specific steps are as follows: (1) Bilateral renal ligation: The kidneys of mice were ligated and Cy5-HSA (2μg) was injected into the tail vein; after 20 minutes, when the systemic circulation was sufficient, the serum of the mice was collected. (2) Heart extraction: The mouse heart was quickly removed and rinsed rapidly and thoroughly with pre-cooled physiological saline to flush out as much blood as possible from the heart.

[0027] (3) Obtaining cardiac interstitial fluid: After irrigation, the heart was cut open and placed on a 40µm pore size cell filter for centrifugation at 4℃, 50g (653rpm) for 3 minutes; the heart was then minced and placed on a 20µm pore size filter membrane for centrifugation. The samples were divided into four groups of equal mass and processed separately at centrifugation speeds of 1400rpm, 1600rpm, 1800rpm, and 2000rpm for 20 minutes each. The collected interstitial fluid was transferred to a SpinX filter tube and centrifuged for 1 minute to obtain the desired cardiac interstitial fluid. Test Example 1: Ratio of interstitial fluid volume to HSA concentration The volume of interstitial fluid collected from cardiac tissue and the concentration of HSA in cardiac tissue and serum were monitored at different rotation speeds. Optimal extraction conditions were selected by comparing the collected interstitial fluid volume and the ratio of HSA concentration in the interstitial fluid to that in serum. The extracted interstitial fluid volume is shown in the figure. Figure 1 The corresponding HSA concentration ratio is shown in the figure. Figure 2 .

[0028] according to Figure 1 As a result, the volume of EF obtained gradually increased with the increase of centrifugation speed; according to Figure 2 The results showed that the concentration ratios of HSA obtained at 1600 rpm and 1800 rpm were basically the same; to better ensure the quality of the obtained EF, 1600 rpm was ultimately selected as the optimal centrifugation extraction condition. Subsequent extraction of interstitial fluid was performed under this condition.

[0029] Test Example 2: Differences in interstitial fluid protein composition Proteins extracted from cardiac interstitial fluid, serum, and cardiac tissue obtained at 1600 rpm were subjected to Western blotting analysis. The differences in protein composition among the three samples were compared to verify the reliability of the cardiac interstitial fluid extraction protocol.

[0030] First, proteins were extracted from heart tissue and their concentrations were measured in cardiac interstitial fluid and serum. Then, based on the measured protein concentrations, 10 µg of total protein was used for Western blotting. Finally, SDS-PAGE gels were stained with silver to determine the distribution of proteins (expression of high / low abundance proteins).

[0031] The SDS-PAGE silver staining process includes the following steps: (1) Fixation: After electrophoresis, place the gel into approximately 100 ml of fixative and shake on a shaker at room temperature for 20 minutes at a speed of 60-70 rpm. Fix for 50 minutes to fully reduce the background. Preparation of fixative: Add 50 mL of ethanol, 10 mL of acetic acid, and 40 mL of Milli-Q grade pure water or double-distilled water in sequence, mix well to obtain 100 mL of fixative.

[0032] (2) Washing with 30% ethanol: Discard the fixative, add 100 mL of 30% ethanol, and shake on a shaker at room temperature for 10 minutes at a shaking speed of 60-70 rpm. Preparation of 30% ethanol: Add 30 mL of ethanol to 70 mL of Milli-Q grade pure water or double-distilled water, mix well and you will have 100 mL of 30% ethanol.

[0033] (3) Washing with water: Discard 30% ethanol, add 200 ml of Milli-Q grade pure water or double distilled water, and shake on a shaker at room temperature for 10 minutes at a shaking speed of 60-70 rpm.

[0034] (4) Sensitization: Discard the water, add 100 mL of silver staining sensitizing solution (1X), and shake on a shaker at room temperature for 2 minutes at a shaking speed of 60-70 rpm. Preparation of silver staining sensitizing solution (1X): Add 1 mL of silver staining sensitizing solution (100X) to 99 mL of Lmilli-Q grade pure water or double-distilled water, mix well, and the silver staining sensitizing solution (1X) is ready. Silver staining sensitizing solution (1X) should be used within 2 hours after preparation.

[0035] (5) Washing with water (twice): Discard the original solution, add 200 mL of Milli-Q grade pure water or double-distilled water, and shake on a shaker at room temperature for 1 minute at a shaking speed of 60-70 rpm. Discard the water, add another 200 mL of Milli-Q grade pure water or double-distilled water, and shake on a shaker at room temperature for 1 minute at a shaking speed of 60-70 rpm.

[0036] (6) Silver staining: Discard the water, add 100 mL of silver solution (1X), and shake on a shaker at room temperature for 10 minutes at a shaking speed of 60-70 rpm. Preparation of silver solution (1X): Add 1 mL of silver solution (100X) to 99 mL of Milli-Q grade pure water or double-distilled water, mix well, and the silver solution (1X) is ready. The silver solution (1X) should be used within 2 hours after preparation.

[0037] (7) Washing with water: Discard the original solution, add 100 mL of Milli-Q grade pure water or double-distilled water, and shake on a shaker at room temperature for 1 minute at a shaking speed of 60-70 rpm.

[0038] (8) Color Development: Discard the water, add 100 mL of silver staining developing solution, and shake on a shaker at room temperature for 10 minutes until the desired protein band appears. The shaking speed is 60-70 rpm. Preparation of Silver Staining Developing Solution: Add 20 mL of basic silver staining developing solution (5X) to 80 mL of Milli-Q grade pure water or double-distilled water, then add 0.05 mL of silver staining accelerating solution (2000X), and mix well to obtain the silver staining developing solution. The silver staining developing solution should be used within 20 minutes after preparation.

[0039] (9) Termination: Discard the silver staining developer, add 100 mL of silver staining termination solution (1X), and shake on a shaker at room temperature for 10 minutes at a speed of 60-70 rpm. Preparation of silver staining termination solution (1X): Add 5 mL of silver staining termination solution (20X) to 95 mL of Milli-Q grade pure water or double-distilled water, mix well, and the silver staining termination solution (1X) is ready for use on the same day it is prepared.

[0040] (10) Washing with water: Discard the silver staining stop solution, add 100 mL of Milli-Q grade pure water or double distilled water, and shake on a shaker at room temperature for 2-5 minutes at a shaking speed of 60-70 rpm.

[0041] (11) Taking photos Figure 3 The results showed that high-abundance proteins were abundant in serum; cardiac tissue lysate showed uniform expression of proteins of various abundances due to the enrichment of a large number of intracellular structural and metabolic proteins; while interstitial fluid lacked the large number of circulating high-abundance proteins found in serum, and its proteins mainly originated from local tissue secretion and micro-exudation. Therefore, it was dominated by low-abundance, tissue-specific proteins, and interstitial fluid was more suitable for detecting low-abundance signaling molecules related to intercellular communication.

[0042] Test Example 3: Detection of Interstitial Fluid Component Contamination To assess whether the extraction of interstitial fluid during cell-tissue extraction would damage cells and thus lead to contamination of the interstitial fluid components, the following experiment was conducted: Protein immunoblotting was performed on the extracted cardiac tissue lysate and interstitial fluid to observe the expression levels of labeled proteins in important organelles such as mitochondria, nucleus, endoplasmic reticulum, and lysosomes.

[0043] Figure 4 The results showed that there was significant expression of organelle marker proteins in the lysate of cardiac tissue, while no related proteins were expressed in the extracted interstitial fluid of cardiac tissue, which proves that there was no cell damage during the extraction of interstitial fluid of cardiac tissue.

[0044] Test Example 4: Interstitial Fluid Proteomics Detection To verify the specific effects of this extraction method on the components of cardiac interstitial fluid and the extent of operational damage, proteomics analysis was performed on the three samples, and the following analyses were conducted: Principal component analysis of the three; (1) GO functional enrichment analysis of differentially expressed proteins; (2) Expression analysis of major intracellular marker proteins; The results show: (1) The protein components in the interstitial fluid of the heart are significantly different from those in serum and heart tissue. Figure 5 This illustrates the unique characteristics of the components extracted from cardiac interstitial fluid. (2) Marker proteins of major intracellular components are significantly expressed in the interstitial fluid of the heart tissue, but are expressed at low levels or not at all in the heart tissue and serum. Figure 6 This indicates that the cells were almost undamaged during the extraction of interstitial fluid from the heart tissue.

[0045] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for extracting interstitial fluid from cardiac tissue, characterized in that, Includes the following steps: (1) Take a fresh heart and irrigate it with pre-cooled physiological saline until the irrigating solution has no obvious blood color; (2) The heart was cut open and centrifuged at low speed to remove residual lavage fluid. (3) Centrifuge the heart again to obtain cardiac interstitial fluid.

2. The method for extracting interstitial fluid from cardiac tissue according to claim 1, characterized in that, In step (1), the temperature of the pre-cooled saline solution is 4°C.

3. The method for extracting interstitial fluid from cardiac tissue according to claim 1, characterized in that, In step (2), the heart after irrigation is cut open and placed on a cell filter with a pore size of 40µm for low-speed centrifugation.

4. The method for extracting interstitial fluid from cardiac tissue according to claim 3, characterized in that, In step (2), the temperature for low-speed centrifugation is 2-4℃, the rotation speed is 600-700rpm, and the centrifugation time is 3-5min.

5. The method for extracting interstitial fluid from cardiac tissue according to claim 1, characterized in that, In step (3), the heart is further cut into smaller pieces and placed on a cell filter with a pore size of 40µm for centrifugation.

6. The method for extracting interstitial fluid from cardiac tissue according to claim 5, characterized in that, In step (3), the centrifugation temperature is 2-4℃, the centrifugation force is 1600rpm, and the centrifugation time is 20-30min.

7. The method for extracting cardiac interstitial fluid according to claim 5, characterized in that, In step (3), after centrifugation, the collected liquid is transferred to a SpinX filter tube and centrifuged for 1 minute to finally obtain the required cardiac interstitial fluid.

8. The method for extracting interstitial fluid from cardiac tissue according to claim 1, characterized in that, The fresh heart mentioned is a rapidly isolated mouse heart.

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