Photodynamic therapy curative effect evaluation method based on salivary gland tumor organ model

Abstract

The invention relates to the technical field of biological medicines, in particular to a photodynamic therapy curative effect evaluation method based on a salivary gland tumor organoid model, which comprises the following steps: S1, after a mypofine drug solution of an organoid culture medium is taken by cells, irradiating the organoid model by using different laser groups, and determining the treatment effect of the salivary gland tumor organoid model; observing the morphological change of the organ-like model through an optical microscope, and photographing and recording; s2, after a certain period of time, adding a Cell Titer-Glo3D Reagent of which the volume is the same as that of the organoid culture medium containing the mycipofine medicine into each hole, and fully mixing to perform cell lysis; and S3, continuously placing the pore plate at room temperature for re-incubation, then transferring the solution of each pore into a lightproof pore white board according to groups, recording a chemiluminescence value by using a microplate reader, and evaluating the curative effect of photodynamic therapy. The photodynamic therapy is applied to salivary gland tumor organs, so that the advantages of small trauma, high targeting property, repeated treatment and the like are realized, and the surgical risk is reduced; the effectiveness of the photodynamic therapy on tumors is evaluated, and clinical medication reference is provided.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for evaluating the efficacy of photodynamic therapy based on a salivary gland tumor organoid model. Background Art

[0002] Photodynamic therapy (PDT) is a treatment method that uses the interaction of light of a specific wavelength and photosensitizers to induce biological tissue damage. It mainly includes three steps: photosensitizer input, light source irradiation, and cell damage and death. According to the different mechanisms of action of photosensitizers, photodynamic therapy is divided into type I and type II. Studies have shown that photodynamic therapy can be used to treat skin tumors such as superficial basal cell carcinoma and squamous cell carcinoma in situ. In addition, it may play a role in the treatment of other types of tumors, but the specific efficacy and safety still need further research and clinical trials to verify. Studies have shown that photodynamic therapy has the unique advantage of selectively killing diseased cells or tissues, and is used in many fields. It has been proven to have good effects in preserving organ function, skin lesions, and tumor treatment (laryngeal cancer). However, so far, there have been no reports on its application to salivary gland tumors.

[0003] Salivary gland tumors are a type of head and neck tumor with characteristic lesions, especially malignant salivary gland tumors. In addition to conventional surgical treatment and radiotherapy and chemotherapy, there is a lack of targeted drugs. For patients with recurrence, metastasis and advanced stages, there is a lack of treatment options and the prognosis is often poor. Summary of the invention

[0004] The purpose of the present invention is to solve the shortcomings of the prior art and provide a method for evaluating the efficacy of photodynamic therapy based on a salivary gland tumor organoid model, comprising the following steps: S1: After the hemoporfin drug solution in the organoid culture medium is taken up by cells, the organoid model is irradiated with different laser groups, and the morphological changes of the organoid model are observed by optical microscopy and photographed and recorded; S2: After a certain period of time, add the same volume of CellTiter-Glo®3D Reagent as the organoid culture medium containing the hemoporfin drug to each well of the well plate, mix thoroughly to lyse the cells, wherein the CellTiter-Glo®3D Reagent is added after 24 hours; S3: The well plate is placed at room temperature for further incubation, and then the solutions in each well of the well plate are transferred to a light-proof white plate according to the groups, and the chemiluminescence values ​​are recorded with an ELISA instrument, and the efficacy of photodynamic therapy is evaluated according to the chemiluminescence values.

[0005] Preferably, the organoid model is a malignant pleomorphic adenoma PDO model.

[0006] Preferably, the hemoporfin drug solution has different concentration gradients.

[0007] Preferably, the organoid model containing a helminthine drug solution with different concentration gradients is added, comprising: S11: Grouping the well plates, constructing and inoculating a malignant pleomorphic adenoma PDO model in the well plates, discarding the original culture medium when the cells grow to the logarithmic growth phase, and washing with a buffer solution PBS; S12: Add different concentration gradients of helminthic drug solutions diluted in advance with organoid culture medium to each group, and continue to culture the organoid models added with the different concentration gradients of helminthic drug solutions in an incubator.

[0008] Preferably, after the organoid model is cultured in the incubator for 3 hours, the hemoporfin drug solution is taken up by the cells.

[0009] Preferably, in step S11, the well plate is a 96-well plate, and each well of the 96-well plate has cells.

[0010] Preferably, in step S12, the hemoporfin drug solution with different concentration gradients is set to 200 , 150 , 100 , 80 , 50 , 40 , 20 , 0 .

[0011] Preferably, in step S1, the organoid model is irradiated with 532 nm and 410 nm lasers for 5 min at a laser intensity of 100 .

[0012] Preferably, in step S2, the CellTiter-Glo® 3D Reagent is thawed at 4°C overnight in advance, placed in a 22°C water bath for 20 min before use, equilibrated to room temperature for 30 min, and gently shaken upside down.

[0013] Preferably, in step S3, the chemiluminescence value significantly decreases with the increase of the administration concentration of the hemoporfin HMME drug, indicating that the intracellular ATP content is significantly reduced after the administration of hemoporfin HMME-mediated photodynamic therapy, indicating a significant weakening of cell viability, and the therapeutic effect is better.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The photodynamic therapy of the present invention is applied to salivary gland tumor organoids, which can achieve small trauma, strong targeting, repeatable treatment, and small side effects, thereby reducing surgical risks; The present invention evaluates the effectiveness of photodynamic therapy for tumors by operating under different photodynamic intensities and different drug concentrations, thereby providing a reference for clinical medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the following detailed description of the preferred embodiment.The drawings are only for the purpose of illustrating the preferred embodiments and are not to be construed as limiting the invention.

[0016] Figure 1 This is a cell diagram showing the efficacy of photodynamic therapy mediated by different concentrations of HMME under different laser irradiation of the present invention; Figure 2 This is a graph showing the results of detecting the efficacy of HMME-mediated photodynamic therapy using CellTiter-Glo® 3D Reagent after irradiation under different lasers in the present invention; Figure 3 This is a graph showing the results of detecting the efficacy of HMME-mediated photodynamic therapy using CellTiter-Glo® 3D Reagent after 532 nm green laser irradiation. DETAILED DESCRIPTION

[0017] In this article, the range expressed by "a value to another value" is a summary expression method to avoid listing all the values ​​in the range one by one in the specification. Therefore, the description of a specific numerical range covers any value in the numerical range and a smaller numerical range defined by any value in the numerical range, just as if the arbitrary value and the smaller numerical range were explicitly written in the specification.

[0018] In this application, the use of "or" means "and / or" unless otherwise stated. In the case of multiple dependent claims, use of "or" refers to more than one of the preceding independent or dependent claims only in the alternative.

[0019] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0020] The technical terms involved in the embodiments of the present invention are defined as follows: Hemoporfin (HMME), also known as Fumida, is a new type of monomeric porphyrin drug, and its chemical name is hematoporphyrin monomethyl ether.

[0021] Type II photodynamic therapy is also widely used in dermatology, oncology and other fields. It is particularly suitable for treating diseased tissues that absorb photosensitizers well and have high oxygen content. Generally speaking, type II photodynamic therapy may have faster treatment speed and better treatment effect in some cases because the singlet oxygen produced has stronger oxidizing properties. The three elements for its occurrence are photosensitizer, light source and oxygen. Therefore, this scheme explores the treatment based on type II photodynamic PDT. This embodiment chooses to use a 410nm blue light source and a 532nm green light source to mediate photodynamic therapy of salivary gland tumor organoid models. This treatment method has achieved remarkable results in the treatment of skin diseases such as port wine stains, but so far, there has been no report on its application to salivary gland tumors.

[0022] This embodiment provides a method for evaluating the efficacy of photodynamic therapy based on a salivary gland tumor organoid model.

[0023] Grouping of well plates The 96-well plate is divided into two groups according to different photodynamic intensities and different drug concentrations. In other embodiments, well plates with different numbers of wells may be used, and the well plates may be divided into more groups as needed.

[0024] (2) Cultivation (2.1) Cultivation in original culture medium The malignant pleomorphic adenoma PDO model was constructed and seeded in a 96-well plate, where each well of the 96-well plate has When the cells grow to the logarithmic growth phase, discard the original culture medium and wash with buffer PBS; The PDO model of malignant pleomorphic adenoma was constructed and inoculated (2.1.1) Sample collection Malignant pleomorphic adenoma samples were collected under sterile conditions and processed as quickly as possible to maintain cell viability.

[0025] (2.1.2) Organizational processing The malignant pleomorphic adenoma tissue was washed under sterile conditions to remove blood and necrotic tissue; Mechanical and enzymatic digestion methods are used to break down malignant pleomorphic adenoma tissue into single cells or small cell clumps. Commonly used enzymes include collagenase, trypsin, etc.

[0026] (2.1.3) Organoid models Inoculate the digested cells into a suitable three-dimensional culture system, such as Matrigel; Use specific organoid culture media that contains multiple growth factors, cytokines, and nutrients to promote tumor cell growth and organoid formation; Place the culture system under appropriate culture conditions, such as 37°C, 5% CO 2 The growth of organoids was observed regularly in the environment.

[0027] (2.1.4) Identification and verification The constructed PDO was identified by morphological observation, immunohistochemical staining, gene expression analysis and other methods to confirm its similarity with primary tumor tissue.

[0028] The expression of tumor-related markers in PDO, such as cytokeratin and S-100 protein, was detected to verify that the results were consistent with the primary tumor.

[0029] (2.1.5) Animal selection Select appropriate immunodeficient animals, such as nude mice, NOD / SCID mice, etc., as hosts for the inoculation model.

[0030] (2.1.6) Vaccination method PDO can be inoculated into animals by subcutaneous injection, orthotopic transplantation, etc.; For subcutaneous injection, PDO was mixed with Matrigel and injected into the subcutaneous tissue of the animal; Orthotopic transplantation involves transplanting PDO into an animal organ that corresponds to the site where the human tumor occurs.

[0031] (2.1.7) Observation and monitoring The animals' health status, tumor growth, and metastasis were regularly observed after inoculation; The development of the tumor is monitored using methods such as measuring tumor size and imaging tests (such as ultrasound, CT, etc.).

[0032] (2.1.8) Experimental analysis Tumor tissues and animal samples were collected at appropriate time points for pathological analysis, molecular biology analysis, etc. to evaluate the effectiveness and reliability of the PDO model.

[0033] (2.2) Placing the organoid model administered with hemoporfin in an incubator Different concentration gradients of Hemoporfin drug solutions diluted in advance with organoid culture medium were added to the organoid models of each of the two groups, and the organoid models were cultured in a 37°C incubator for 3 h. 100 μg of Hemoporfin solution was added to each well. Hemoporfin drug solutions with different concentration gradients are obtained by diluting the organoid culture medium.

[0034] (2.2.1) Set up different concentration gradients of Hemoporfin drug solution at 200 , 150 , 100 , 80 , 50 , 40 , 20 , 0 Or set up different concentration gradients of Hemoporfin drug solution to 200 , 150 , 100 , 80 , 50 , 40 , 20 , 10 , 5 , 0 In other embodiments, other concentration gradients of hemoporfin drug solutions can be set according to the tolerance of different tumor cells.

[0035] (3) Laser irradiated organoid model After 3 hours, the hemoporfin drug solution in the organoid model was taken up by cells. The first group of organoid models was irradiated with a 532nm green laser for 5 minutes, and the second group of organoid models was irradiated with a 410nm blue laser for 5 minutes. The laser intensity was 100 During this period, the morphological changes of the organoid model were observed with an optical microscope and photographed and recorded. Specifically, in this embodiment, after administration, a large amount of hemoporfin was enriched in tumor cells and vascular endothelial cells. At this time, after a certain wavelength of light was applied, the photodynamic reaction was activated, and a large amount of reactive oxygen species (ROS) was generated. These oxygen toxic substances can not only oxidatively damage tumor cells, directly kill them or promote them to release tumor-associated antigens, induce tumor immunogenic death, activate anti-tumor immune response, but also damage tumor blood vessels, block them, deprive tumors of nutritional support, and further kill tumor cells.

[0036] See also Figure 1As shown in the figure, since the penetration ability of 410nm laser is weaker than that of 532nm green laser, the photodynamic therapy mediated by 410nm laser has a weakened ability to kill cells deep inside the organoid model. According to the representative microscopic images of each group, the salivary gland tumor organoids in the 410nm blue laser irradiation group still have a relatively complete 3D spherical structure even under a higher concentration of HMME-mediated photodynamic therapy; while the salivary gland tumor organoids in the 532nm green laser irradiation group become chaotic and disordered with the increase of HMME concentration, and the cell arrangement loses its original rules and hierarchy. This phenomenon suggests that the photodynamic therapy under 532nm laser irradiation has a better killing effect.

[0037] (4) Record chemiluminescence value (4.1) Thaw the CellTiter-Glo® 3D Reagent at 4°C overnight. Place in a 22°C water bath for 20 min before use. Equilibrate to room temperature for 30 min and shake gently by inversion.

[0038] (4.2) After 24 hours of culture, add the same volume of CellTiter-Glo® 3D Reagent as the organoid culture medium containing hemoporfin to each well of the well plate and mix thoroughly to lyse the cells.

[0039] (4.3) Incubate the plate at room temperature for another 25 min to stabilize the luminescent signal. Then transfer the solutions in each well of the plate to a light-proof white plate according to the grouping, and record the chemiluminescent RLU value using an ELISA reader.

[0040] (5) Evaluation of the efficacy of photodynamic therapy based on chemiluminescence RLU value According to the significance test, the results of different groups under 532nm green laser irradiation group and 410nm blue laser were analyzed. If the P value is less than 0.05, it means that there is a statistical difference between the 532nm green laser irradiation group and the 410nm blue laser. If the P value is less than 0.001, it means that the data difference is greater and the significance is greater.

[0041] See also Figure 2 As shown, the chemiluminescence RLU value of the 532nm green laser irradiation group was significantly lower than that of the 410nm blue laser irradiation group with the increase of HMME administration concentration. The statistical analysis showed that the P value was less than 0.001, which was significantly different. This result shows that the intracellular ATP content was significantly reduced after HMME-mediated photodynamic therapy, indicating a significant weakening of cell viability, proving that photodynamic therapy under 532nm laser irradiation has a good killing effect on the organoid model of malignant pleomorphic adenoma (high-grade adenocarcinoma of glandular epithelium).

[0042] See also Figure 3As shown in the figure, the efficacy of photodynamic therapy mediated by different concentrations of HMME was evaluated in the organoid model of malignant pleomorphic adenoma (high-grade adenocarcinoma of glandular epithelium) under 532nm green excitation using CellTiter-Glo® 3D Reagent. That is, under 532nm green laser irradiation, the chemiluminescence RLU value decreased significantly with the increase of HMME administration concentration, indicating that the intracellular ATP content was significantly reduced after HMME-mediated photodynamic therapy, indicating a significant weakening of cell viability. The concentration of hemoporfin was 200 The anti-tumor effect of photodynamic therapy mediated by

[0043] The above disclosure is only the preferred embodiment of the present invention. The preferred embodiment does not describe all the details in detail, nor does it limit the invention to the specific implementation method described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can make good use of the present invention. The present invention is only limited by the claims and their full scope and equivalents. Under the guidance of the present invention and the above embodiments, it is easy for those skilled in the art to foresee that each raw material or its equivalent substitute, each processing method or its equivalent substitute listed or exemplified in the present invention can realize the present invention, and the upper and lower limit values ​​and interval values ​​of the parameters of each raw material and processing method can realize the present invention, and the embodiments are not listed one by one here.

Claims

1. A method for evaluating the efficacy of photodynamic therapy based on a salivary gland tumor organoid model, characterized in that: The following steps are involved: S1: After the hemoporfin drug solution in the organoid culture medium is taken up by cells, the organoid model is irradiated with different laser groups, and the morphological changes of the organoid model are observed by optical microscopy and photographed and recorded; S2: After a certain period of time, add the same volume of CellTiter-Glo®3D Reagent as the organoid culture medium containing the hemoporfin drug to each well of the well plate, mix thoroughly and lyse the cells; S3: The well plate is placed at room temperature for further incubation, and then the solutions in each well of the well plate are transferred to a light-proof white plate according to the groups, and the chemiluminescence values ​​are recorded with an ELISA instrument, and the efficacy of photodynamic therapy is evaluated according to the chemiluminescence values.

2. The method for evaluating the efficacy of photodynamic therapy based on a salivary gland tumor organoid model according to claim 1, characterized in that: The organoid model is a malignant pleomorphic adenoma PDO model.

3. The method for evaluating the efficacy of photodynamic therapy based on a salivary gland tumor organoid model according to claim 2, characterized in that: The hemoporfin drug solution has different concentration gradients.

4. The method for evaluating the efficacy of photodynamic therapy based on a salivary gland tumor organoid model according to claim 3, characterized in that: The organoid culture medium containing a hemoporfin drug solution with different concentration gradients is added, comprising: S11: Grouping the well plates, constructing and inoculating a malignant pleomorphic adenoma PDO model in the well plates, discarding the original culture medium when the cells grow to the logarithmic growth phase, and washing with a buffer solution PBS; S12: Add different concentration gradients of helminthic drug solutions diluted in advance with organoid culture medium to each group, and continue to culture the organoid models added with the different concentration gradients of helminthic drug solutions in an incubator.

5. The method for evaluating the efficacy of photodynamic therapy based on a salivary gland tumor organoid model according to claim 4, characterized in that: After the organoid model was cultured in the incubator for 3 hours, the hemoporfin drug solution was taken up by the cells.

6. The method for evaluating the efficacy of photodynamic therapy based on a salivary gland tumor organoid model according to claim 4, characterized in that: In step S11, the well plate is a 96-well plate, and each well of the 96-well plate has cells.

7. The method for evaluating the efficacy of photodynamic therapy based on a salivary gland tumor organoid model according to claim 4, characterized in that: In step S12, a 200 μg / ml hemoporfin drug solution with different concentration gradients is set. , 150 , 100 , 80 , 50 , 40 , 20 , 0 .

8. The method for evaluating the efficacy of photodynamic therapy based on a salivary gland tumor organoid model according to claim 1, characterized in that: In step S1, the organoid model was irradiated with 532 nm and 410 nm lasers for 5 min at a laser intensity of 100 .

9. The method for evaluating the efficacy of photodynamic therapy based on a salivary gland tumor organoid model according to claim 1, characterized in that: In step S2, thaw the CellTiter-Glo® 3D Reagent at 4°C overnight, place in a 22°C water bath for 20 min before use, equilibrate to room temperature for 30 min and gently shake by inversion.

10. The method for evaluating the efficacy of photodynamic therapy based on a salivary gland tumor organoid model according to claim 7, characterized in that: In step S3, the chemiluminescence value significantly decreases with the increase of the administration concentration of the hemoporfin HMME drug, indicating that the intracellular ATP content is significantly reduced after the administration of hemoporfin HMME-mediated photodynamic therapy, indicating that the cell viability is significantly weakened, and the therapeutic effect is better.

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