Method for evaluating treatment effect of alveolar echinococcosis

Abstract

The invention relates to the technical field of medical detection, in particular to a method for evaluating the treatment effect of alveolar echinococcosis, which comprises the following steps: acquiring an echinococcosis multilocularis sample after radiation treatment; determining the mortality rate of the protoscolex through a trypan blue dyeing method; observing the superstructure change of the hair growth layer cells through a transmission electron microscope; detecting mitochondrial DNA copy number change through qPCR (quantitative polymerase chain reaction); the cell apoptosis degree is evaluated through caspase-3 activity detection; the treatment effect is evaluated according to the mortality rate of the protoscolex, the ultrastructure change, the mitochondrial DNA copy number change and the cell apoptosis degree.

Description

Technical Field

[0001] This invention relates to the field of medical testing technology, specifically a method, system, and storage medium for evaluating the treatment effect of alveolar echinococcosis. Background Technology

[0002] Alveolar echinococcosis (AE) is a serious zoonotic parasitic disease caused by infection with the larvae of *Echinococcus multilocularis*. The disease primarily affects the liver, exhibiting infiltrative growth and clinically resembling malignant tumors. Without timely treatment, the mortality rate can exceed 90%. Currently, surgical resection combined with drug therapy is the main treatment method; however, for patients in advanced stages or those who are not candidates for surgery, the treatment effect is limited.

[0003] In recent years, radiotherapy has shown potential application value in the treatment of alveolar echinococcosis as an emerging treatment method. Carbon ion radiation, in particular, due to its Bragg peak effect and high relative biological effect (RBE), can more precisely target lesions while reducing damage to surrounding normal tissues. However, there is still a lack of unified and objective standards for evaluating the efficacy of radiotherapy for alveolar echinococcosis.

[0004] Traditional methods for evaluating treatment efficacy primarily rely on imaging examinations to observe changes in lesion size. However, this method suffers from drawbacks such as low sensitivity and significant lag. While histopathological examination can directly observe morphological changes in parasites, it is an invasive procedure, limiting its clinical application. Furthermore, existing evaluation methods often focus on only a single indicator, failing to comprehensively reflect treatment effectiveness.

[0005] At the molecular level, studies have shown that radiation can cause mitochondrial DNA damage and apoptosis in parasites, but the correlation between these changes and treatment efficacy remains unclear. Therefore, there is an urgent need to establish a comprehensive evaluation method based on multiple parameters to provide objective and accurate criteria for judging the efficacy of radiotherapy for alveolar echinococcosis. Summary of the Invention

[0006] This invention addresses the problems existing in the prior art by proposing a novel method for evaluating the treatment efficacy of alveolar echinococcosis. This method, by combining protocercariae mortality detection and ultrastructural observation, can more comprehensively and accurately assess treatment effectiveness, providing important evidence for the formulation and optimization of clinical treatment plans.

[0007] This invention provides a method for evaluating the treatment efficacy of alveolar echinococcosis, comprising: Obtain samples of multilocular Echinococcus larvae after radiation treatment; The mortality rate of protocercariae was determined by trypan blue staining. Observation of ultrastructural changes in germinal layer cells using transmission electron microscopy; Changes in mitochondrial DNA copy number were detected by qPCR; The degree of cell apoptosis was assessed by caspase-3 activity assay; Treatment efficacy was evaluated based on protocercariae mortality rate, ultrastructural changes, mitochondrial DNA copy number changes, and the degree of apoptosis.

[0008] Furthermore, radiation treatment includes carbon ion radiation or X-ray radiation.

[0009] Furthermore, the dose of carbon ion radiation is 20-60 Gy, and the linear energy transfer value is 50-200 keV / μm.

[0010] Furthermore, the trypan blue staining method includes: Mechanically break up multilocular Echinococcus samples to release protocercariae; Stain with 0.4% trypan blue solution for 5-10 minutes; Under a microscope, live protocercariae that refused to be stained and dead protocercariae that were stained were counted.

[0011] Further transmission electron microscopy observations included: Detect at least one ultrastructural change among mitochondrial cristae breakage, nuclear chromatin condensation, and cell membrane integrity disruption in germinal layer cells.

[0012] Furthermore, the treatment efficacy was evaluated based on protocercariae mortality rate, ultrastructural changes, mitochondrial DNA copy number changes, and the degree of apoptosis. The evaluation criteria formula is as follows: T=α×(Mor)+β×Mit+γ×(1-mtDNA)+δ×Cas; in: T represents the treatment effectiveness score; Mor is the mortality rate of protocercariae measured by trypan blue staining, measured in % (%). Mit is the mitochondrial damage score observed by transmission electron microscopy. mtDNA: The relative copy number of mitochondrial DNA detected by qPCR, compared with the control group; Cas: fold increase in activity, compared to the control group; Weight coefficient; α=0.4, β=0.3, γ=0.2, δ=0.1.

[0013] Furthermore, the mitochondrial damage scoring criteria observed by Mit transmission electron microscopy: 0 points: No significant damage; 1 point: ≤30% of mitochondria show cristae breakage; 2 points: 30-70% of mitochondria show cristae breakage; 3 points: ≥70% of mitochondria show cristae breakage.

[0014] Furthermore, when T≥0.85, the treatment is considered significantly effective; when 0.65≤T<0.85, the treatment is considered effective; when 0.45≤T<0.65, the treatment is considered partially effective; and when T<0.45, the treatment is considered ineffective.

[0015] In summary, the present invention has the following beneficial effects: The method for evaluating the treatment efficacy of alveolar echinococcosis provided by this invention has several significant advantages: By combining a dual evaluation mechanism—quantitative determination of protocercarial mortality using trypan blue staining and observation of ultrastructural changes using transmission electron microscopy—the efficacy assessment becomes more objective and comprehensive. This method enables early efficacy prediction, significantly earlier than the time window of traditional imaging assessments. In clinical applications, it features small sample requirements, short testing cycles, and low cost. It can effectively guide clinical treatment decisions and provide a reliable basis for optimizing treatment plans. It has strong technical compatibility and is applicable to the evaluation of various types of radiotherapy. Simultaneously, it has significant scientific research value, providing a research platform for elucidating treatment mechanisms and developing novel treatment methods. Compared to existing technologies, this method significantly improves the accuracy of efficacy assessment, greatly shortens the evaluation cycle, and provides strong support for timely adjustments to clinical treatment plans. Detailed Implementation

[0016] To further illustrate the technical means and effects adopted by the present invention to achieve the intended purpose, the specific implementation method, features and effects of a method for evaluating the treatment effect of alveolar echinococcosis proposed according to the present invention are described in detail below.

[0017] This specific embodiment provides a method for evaluating the treatment effect of alveolar echinococcosis, including: S1. Obtain samples of multilocular Echinococcus larvae after radiation treatment; S2. The mortality rate of protocercariae was determined by trypan blue staining. S3. Observe the ultrastructural changes of germinal layer cells using transmission electron microscopy; S4. Detect changes in mitochondrial DNA copy number by qPCR; S5. Assess the degree of cell apoptosis by detecting caspase-3 activity; S6. Evaluate the treatment effect based on the mortality rate of protocercariae, ultrastructural changes, changes in mitochondrial DNA copy number, and degree of apoptosis.

[0018] Understandably, this specific implementation method, based on the principles of radiobiology and parasitic pathology, establishes a multi-dimensional method for evaluating the treatment efficacy of alveolar echinococcosis. This method uses trypan blue staining to detect protocercariae mortality, reflecting the degree of cell membrane integrity damage; transmission electron microscopy to observe ultrastructural changes such as mitochondrial cristae breakage; and combines qPCR detection of mitochondrial DNA copy number changes and caspase-3 activity assays to comprehensively evaluate the radiation therapy effect at the cellular, subcellular, and molecular levels. It integrates macroscopic mortality and microstructural damage indicators to improve evaluation accuracy; it enables efficacy prediction through early changes in mitochondrial DNA and apoptosis markers; it has high detection sensitivity, requires a small sample size, and has strong clinical applicability; and it provides objective evidence for adjusting treatment regimens. This method not only accurately determines treatment efficacy but also reveals the molecular mechanism of radiation effects, providing important technical support for the precision treatment of alveolar echinococcosis.

[0019] In some preferred embodiments, the radiation treatment includes carbon ion radiation or X-ray radiation.

[0020] In some preferred embodiments, the carbon ion radiation dose is 20-60 Gy, and the linear energy transfer value is 50-200 keV / μm.

[0021] In some preferred embodiments, the trypan blue staining method includes: Mechanically break up multilocular Echinococcus samples to release protocercariae; Stain with 0.4% trypan blue solution for 5-10 minutes; Under a microscope, live protocercariae that refused to be stained and dead protocercariae that were stained were counted.

[0022] Understandably, the principle of trypan blue staining is based on the selective permeability of the cell membrane of living cells. The intact cell membrane of a living protocercariae can repel trypan blue dye and remain colorless and transparent, while the cell membrane of a dead protocercariae is damaged, allowing the dye to penetrate and appear blue.

[0023] This specific implementation method preferably uses a 0.4% concentration staining solution for 5-10 minutes, achieving a staining specificity of 98.2±1.3% (live cell rejection rate) and avoiding false positives due to excessive staining time (live cell staining rate increases to 12.7% after 15 minutes). Mechanical disruption using optimized parameters of 3000 rpm / 30s achieves a protocercariae release rate of 95.3±2.1%, significantly higher than the enzymatic digestion method (87.5±3.6%), while avoiding the influence of enzyme activity. The standardized counting procedure (double-blind hemocytometer counting) ensures a repeatability CV value of <5%, improving accuracy by 3 times compared to traditional visual methods. This method can complete the detection within 15 minutes, providing immediate and reliable cell activity data for efficacy evaluation.

[0024] In some preferred embodiments, transmission electron microscopy observation includes: Detect at least one ultrastructural change among mitochondrial cristae breakage, nuclear chromatin condensation, and cell membrane integrity disruption in germinal layer cells.

[0025] In some preferred embodiments, the therapeutic effect is evaluated based on protocercariae mortality, ultrastructural changes, mitochondrial DNA copy number changes, and the degree of apoptosis. The evaluation criteria formula is as follows: T=α×(Mor)+β×Mit+γ×(1-mtDNA)+δ×Cas; in: T represents the treatment effectiveness score; Mor is the mortality rate of protocercariae measured by trypan blue staining, measured in % (%). Mit is the mitochondrial damage score observed by transmission electron microscopy. mtDNA: The relative copy number of mitochondrial DNA detected by qPCR, compared with the control group; Cas: fold increase in activity, compared to the control group; Weight coefficient; α=0.4, β=0.3, γ=0.2, δ=0.1.

[0026] Understandably, the above formula integrates key indicators from four dimensions: cell death rate (Mor), mitochondrial damage score (Mit), mitochondrial DNA damage (1-mtDNA), and apoptosis (Cas). Through scientifically set weighting coefficients (0.4 / 0.3 / 0.2 / 0.1), it achieves multi-parameter quantitative evaluation, overcoming the limitations of single indicators and establishing a three-tiered evaluation system of "cell-subcellular-molecular." The weight allocation has been experimentally verified, highlighting both primary and secondary indicators while retaining their sensitivity. Normalization standardizes the scores, and the T-score grading (0.45 / 0.65 / 0.85) is clearly defined. The formula structure reflects radiobiological mechanisms, such as (1-mtDNA) reflecting radiation-specific damage. It has strong clinical guidance, capable of identifying special cases such as non-specific damage or apoptosis inhibition. It also has good compatibility, with detection methods and weights adjustable as technology advances.

[0027] In some preferred embodiments, the mitochondrial damage scoring criteria observed by Mit transmission electron microscopy are as follows: 0 points: No significant damage; 1 point: ≤30% of mitochondria show cristae breakage; 2 points: 30-70% of mitochondria show cristae breakage; 3 points: ≥70% of mitochondria show cristae breakage.

[0028] Understandably, the scientific basis for setting the mitochondrial damage scoring criteria in this specific implementation method is as follows: According to radiobiological studies, the degree of mitochondrial cristae breakage is significantly positively correlated with radiation dose (r=0.89, p<0.01). This scoring system divides the degree of damage into four levels: 0 points (no significant damage) corresponds to radiation not causing observable substructural changes; 1 point (≤30% cristae breakage) characterizes the early reversible damage stage; 2 points (30-70% breakage) indicates that irreversible damage has been entered; and 3 points (≥70% breakage) marks the collapse of the cellular energy metabolism system. This grading standard has been validated by animal models, showing that: a 1-point threshold corresponds to a 20Gy radiation dose, 2 points to 40Gy, and 3 points to 60Gy (p<0.05). The differences in the magnitude of mitochondrial membrane potential decrease between each level are significant (ΔΨm decreased by 15±3%, 42±5%, and 78±7%, respectively), which can objectively reflect the progressive damage process induced by radiation. This semi-quantitative scoring method is both feasible and scientific, with a Kappa value of 0.82 for inter-observer consistency in electron microscopy.

[0029] In some preferred embodiments, when T≥0.85, the treatment is considered to be significantly effective; when 0.65≤T<0.85, the treatment is considered to be effective; when 0.45≤T<0.65, the treatment is considered to be partially effective; and when T<0.45, the treatment is considered to be ineffective.

[0030] The present invention will be further described below with reference to specific embodiments.

[0031] Example 1: Evaluation of the efficacy of carbon ion radiation therapy for alveolar echinococcosis Biological materials: sheep liver tissue samples infected with Echinococcus multilocularis (treated with 40 Gy carbon ion radiation) and healthy sheep liver tissue (negative control).

[0032] The main reagents used in this example are: 0.4% trypan blue solution (prepared with PBS); TRIzol reagent (for RNA extraction); Caspase-3 activity assay kit (containing Ac-DEVD-pNA substrate); and transmission electron microscopy fixative (2.5% glutaraldehyde + 1% osmium tetroxide).

[0033] The instruments and equipment used in this embodiment are: a carbon ion radiotherapy system (LET=100keV / μm); a transmission electron microscope (JEM-1400Flash); a real-time quantitative PCR instrument (QuantStudio 5); and an enzyme-linked immunosorbent assay (ELISA) reader (detection wavelength 405nm). The method for evaluating the treatment effect of alveolar echinococcosis in this embodiment includes the following steps: S1. Obtain samples of multilocular Echinococcus larvae after radiation treatment: The patient will receive carbon ion radiation at a dose of 40 Gy (single irradiation, LET = 100 keV / μm); 48 hours after irradiation, liver lesion tissue will be surgically obtained, and approximately 1 cm will be removed under aseptic conditions. 3 sample.

[0034] S2. Determining the mortality rate of protocercariae using the trypan blue staining method: Tissue blocks were rinsed in PBS containing penicillin-streptomycin, vesicles were gently disrupted using a tissue homogenizer, filtered through a 200-mesh sieve, and protocercariae were collected by centrifugation at 1000 rpm for 5 minutes. 50 μl of the protocercariae suspension was mixed with an equal volume of 0.4% trypan blue solution and incubated at room temperature for 8 minutes. The protocercariae were counted using a hemocytometer. Live protocercariae (anti-stained) and dead protocercariae (blue-stained) were counted separately. The mortality rate (%) was calculated as: (Number of dead protocercariae / Total number of protocercariae) × 100.

[0035] S3. Observe the ultrastructural changes of germinal layer cells using transmission electron microscopy: Take about 1mm 3 Tissue blocks were fixed with 2.5% glutaraldehyde at 4°C for 24 hours, followed by fixation with 1% osmium tetroxide for 2 hours, dehydrated with graded ethanol, embedded in EPON 812 resin, cut into 70 nm sections using an ultramicrotome, and stained with uranium acetate-lead citrate double staining. Observation was performed under an 80 kV accelerating voltage, focusing on the mitochondrial cristae structure and nuclear chromatin distribution of germinal layer cells. Ten fields of view were randomly selected from each sample for photographic recording.

[0036] S4. Detect changes in mitochondrial DNA copy number by qPCR; Extract total DNA from tissues; qPCR primers: COX1-F: 5'-GTTCGTTGGTGGAGTGTTT-3'; COX1-R: 5'-CACCAGCAATACCATAAGG-3'; β-actin is used as an internal parameter, and the relative copy number is 2 - ΔΔCt.

[0037] S5. Assess the degree of apoptosis using caspase-3 activity detection: Prepare tissue homogenate supernatant (4℃, 10000g, 10min); add Ac-DEVD-pNA substrate (final concentration 200μM); react at 37℃ for 2h; measure absorbance at 405nm; activity factor = experimental group OD value / control group OD value.

[0038] S6. Evaluate the treatment effect based on the mortality rate of protocercariae, ultrastructural changes, changes in mitochondrial DNA copy number, and degree of apoptosis.

[0039] The evaluation criteria formula used is as follows: T=α×(Mor)+β×Mit+γ×(1-mtDNA)+δ×Cas; in: T represents the treatment effectiveness score; Mor is the mortality rate of protocercariae measured by trypan blue staining, measured in % (%). Mit is the mitochondrial damage score observed by transmission electron microscopy. mtDNA: The relative copy number of mitochondrial DNA detected by qPCR, compared with the control group; Cas: fold increase in activity, compared to the control group; Weight coefficient; α=0.4, β=0.3, γ=0.2, δ=0.1.

[0040] Furthermore, the mitochondrial damage scoring criteria observed by Mit transmission electron microscopy: 0 points: No significant damage; 1 point: ≤30% of mitochondria show cristae breakage; 2 points: 30-70% of mitochondria show cristae breakage; 3 points: ≥70% of mitochondria show cristae breakage.

[0041] The data results are shown in the table below:

[0042] T=0.4×(68 / 100)+0.3×2+0.2×(1-0.58)+0.1×1.9=0.272+0.6+0.084+0.19=1.146.

[0043] According to the T-criterion: T=1.146>0.85, therefore it is judged as "significantly effective".

[0044] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been shown above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for evaluating the therapeutic effect of alveolar echinococcosis, characterized in that, include: Obtain samples of multilocular Echinococcus larvae after radiation treatment; The mortality rate of protocercariae was determined by trypan blue staining. Observation of ultrastructural changes in germinal layer cells using transmission electron microscopy; Changes in mitochondrial DNA copy number were detected by qPCR; The degree of cell apoptosis was assessed by caspase-3 activity assay; Treatment efficacy was evaluated based on protocercariae mortality rate, ultrastructural changes, mitochondrial DNA copy number changes, and the degree of apoptosis.

2. The method for evaluating the therapeutic effect of alveolar echinococcosis according to claim 1, characterized in that, The radiation treatment includes carbon ion radiation or X-ray radiation.

3. The method for evaluating the therapeutic effect of alveolar echinococcosis according to claim 2, characterized in that, The dose of the carbon ion radiation is 20-60 Gy, and the linear energy transfer value is 50-200 keV / μm.

4. The method for evaluating the therapeutic effect of alveolar echinococcosis according to claim 1, characterized in that, The trypan blue staining method includes: Mechanically break up multilocular Echinococcus samples to release protocercariae; Stain with 0.4% trypan blue solution for 5-10 minutes; Under a microscope, live protocercariae that refused to be stained and dead protocercariae that were stained were counted.

5. The method for evaluating the therapeutic effect of alveolar echinococcosis according to claim 1, characterized in that, The transmission electron microscopy observations included: Detect at least one ultrastructural change among mitochondrial cristae breakage, nuclear chromatin condensation, and cell membrane integrity disruption in germinal layer cells.

6. The method for evaluating the therapeutic effect of alveolar echinococcosis according to claim 1, characterized in that, The evaluation criteria for assessing treatment efficacy based on protocercariae mortality, ultrastructural changes, mitochondrial DNA copy number changes, and the degree of apoptosis are as follows: T=α×(Mor)+β×Mit+γ×(1-mtDNA)+δ×Cas; in: T represents the treatment effectiveness score; Mor is the mortality rate of protocercariae measured by trypan blue staining, measured in % (%). Mit is the mitochondrial damage score observed by transmission electron microscopy. mtDNA: The relative copy number of mitochondrial DNA detected by qPCR, compared with the control group; Cas: fold increase in activity, compared to the control group; Weight coefficient; α=0.4, β=0.3, γ=0.2, δ=0.

1.

7. The method for evaluating the therapeutic effect of alveolar echinococcosis according to claim 6, characterized in that, Mitochondrial damage scoring criteria observed by transmission electron microscopy: 0 points: No significant damage; 1 point: ≤30% of mitochondria show cristae breakage; 2 points: 30-70% of mitochondria show cristae breakage; 3 points: ≥70% of mitochondria show cristae breakage.

8. The method for evaluating the therapeutic effect of alveolar echinococcosis according to claim 6, characterized in that, When T ≥ 0.85, the treatment is considered significantly effective; when 0.65 ≤ T < 0.85, the treatment is considered effective; when 0.45 ≤ T < 0.65, the treatment is considered partially effective. When T < 0.45, the treatment is considered ineffective.

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