An in vitro rapid detection method for predicting the radiosensitivity of tumor patients for non-diagnostic purposes

Abstract

The present invention provides a rapid in vitro detection method for predicting the radiosensitivity of tumor patients for non-diagnostic purposes. The method includes: preprocessing cancer tissue samples and adjacent tissue samples; transferring tissue blocks into a well plate and grouping them; irradiating each group of tissue blocks at a second preset time according to the radiation time and a preset dose rate; detecting the absorbance values of each group and calculating the inhibition rate of the cancer tissue and the activity value of the adjacent tissue. Furthermore, the radiosensitivity corresponding to each radiation dose is predicted. The rapid in vitro detection method for predicting the radiosensitivity of tumor patients for non-diagnostic purposes provided by the present invention can rapidly and accurately predict the radiosensitivity of tissues, which is beneficial to providing a strong basis for routine clinical implementation.

Description

Technical Field

[0001] The present invention relates to the technical field of detecting the radiosensitivity of tumor cells, and particularly relates to a rapid in vitro detection method for predicting the radiosensitivity of tumor patients for non-diagnostic purposes. Background Art

[0002] Tumor radiotherapy is a local treatment method that uses radiation to treat tumors. The radiation includes α, β, γ rays generated by radioactive isotopes and x-rays, electron beams, proton beams, and other particle beams generated by various x-ray therapy machines or accelerators. The success of radiotherapy mainly depends on the total administered dose, because there are significant individual differences in the sensitivity of tissues to ionizing radiation damage. Worldwide every year, patients receiving radiotherapy often show adverse reactions due to high sensitivity.

[0003] That is to say, the main factor determining the radiotherapy effect is the radiosensitivity of the tissue. Different tissues, organs, and different types of cancer tissues have obvious differences in their responses after receiving radiation. Generally, it is considered that the radiosensitivity is related to the proliferation cycle and pathological grade of tumor cells. It is shown that proliferating active cells are more sensitive than non-proliferating cells, and the higher the degree of cell differentiation, the lower the radiosensitivity, and vice versa. The oxygen content is a very important environmental factor affecting the radiosensitivity of cancer tissues. For example, early-stage tumors are small in size, have good blood circulation, sufficient oxygen content, and high radiosensitivity; while late-stage tumors, due to their large size, poor blood circulation in the tumor, and even central necrosis, have low radiosensitivity. The radiation dose is also an important variable determining the radiotherapy sensitivity. The higher the radiation dose, the correspondingly higher the radiotherapy sensitivity.

[0004] However, high-dose radiation exposure will also cause more serious damage to normal tissues. Therefore, theoretically, the radiation dose cannot be increased indefinitely. In addition, the physical fitness and physiological conditions of patients will also affect the radiotherapy sensitivity. The sensitivities of patients' normal tissues to treatment vary greatly, and the side effects of radiotherapy are usually relatively serious. If the radiosensitivity of patients to radiation can be predicted before radiotherapy, then a radiation dose with higher radiotherapy efficacy and lower damage to normal tissues can be obtained. Therefore, there is an urgent need for a solution that can quickly test the results of radiotherapy sensitivity. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to propose a rapid in vitro detection method for predicting the radiosensitivity of tumor patients for non-diagnostic purposes, so as to quickly and accurately predict the radiosensitivity of tissues, which is beneficial to providing a strong basis for clinical routine implementation.

[0006] According to a rapid in vitro detection method for predicting the radiosensitivity of tumor patients for non-diagnostic purposes proposed by the present invention, before a tumor patient undergoes radiotherapy, the method includes:

[0007] Obtain the tumor patient number, obtain the cancer tissue sample and the adjacent tissue sample stored in the tissue preservation solution respectively according to the tumor patient number, and perform sample pretreatment on the cancer tissue sample and the adjacent tissue sample to obtain a number of cancer tissue blocks and adjacent tissue blocks of a first preset size;

[0008] Transfer the cancer tissue blocks and the adjacent tissue blocks into a number of well plates, with at most one tissue block inoculated in each well of each well plate. Divide the tissue blocks in the well plates into several groups according to the number of the cancer tissue blocks or the adjacent tissue blocks, and calculate and label the radiation doses corresponding to each group of tissue blocks respectively according to the preset radiation dose peak and the number of divided groups. The grouping results include a blank group, a control group and an experimental group;

[0009] Calculate the radiation time corresponding to each group of tissue blocks respectively according to the radiation dose corresponding to each group of tissue blocks and the preset dose rate. Place the well plates in the irradiation area of the radiation instrument, and control the radiation instrument to irradiate each group of tissue blocks a first preset number of times every second preset time according to the radiation time and the preset dose rate;

[0010] Detect the absorbance values of each well in the blank group, the control group and the experimental group after irradiation respectively, and calculate the inhibition rate of the cancer tissue and the activity value of the adjacent tissue at each radiation dose according to the absorbance values of each well in the blank group, the control group and the experimental group, so as to predict the sensitivity corresponding to each tumor patient at each radiation dose according to the inhibition rate of the cancer tissue and the activity value of the adjacent tissue.

[0011] Further, the step of performing sample pretreatment on the cancer tissue sample and the adjacent tissue sample to obtain a number of cancer tissue blocks and adjacent tissue blocks of a first preset size includes:

[0012] Rinse the cancer tissue sample and the adjacent tissue sample 2-4 times respectively with a tissue culture medium, with each time lasting 0.5-2 min;

[0013] Remove the necrotic or poorly shaped areas in the rinsed cancer tissue sample and adjacent tissue sample, and after soaking in iodophor disinfectant for 10-60 s, divide both the cancer tissue sample and the adjacent tissue into micro tissue blocks of a first preset size.

[0014] Further, the step of transferring the cancer tissue blocks and the adjacent tissue blocks into a number of well plates, with at most one tissue block inoculated in each well of each well plate, dividing the tissue blocks in the well plates into several groups according to the number of the cancer tissue blocks or the adjacent tissue blocks, and calculating and labeling the radiation doses corresponding to each group of tissue blocks respectively according to the preset radiation dose peak and the number of divided groups includes:

[0015] The number of microtissue blocks separated from one of the tissue samples and the specifications of the well plate are used to calculate the number of well rows or well columns for each tissue sample, so as to divide the tissue blocks into several categories according to the number of well rows or well columns. Each category includes one group of blank group, control group and experimental group, where:

[0016] The wells divided for the control group and the experimental group in the same category are inoculated with the same type of tissue blocks, the wells divided for each blank group are not inoculated with tissue blocks, the blank group and the experimental group in the same category are marked with equal radiation doses, and the radiation dose corresponding to each control group is zero.

[0017] Further, the step of calculating the radiation time corresponding to each group of tissue blocks according to the radiation dose and the preset dose rate corresponding to each group of tissue blocks, placing the well plate in the irradiation area of the radiation instrument, and controlling the radiation instrument to irradiate each group of tissue blocks for the first preset number of times every second preset time includes:

[0018] After grouping, the blank group or the experimental group is irradiated with rays for the first time using the calculated radiation time and the preset dose rate. After the irradiation is completed, 50 - 200 μL of tissue culture medium is added to each well, and it is placed in a cell culture incubator for culture. After the second preset time, the tissue culture medium is aspirated, and the second ray irradiation is performed until the first preset number of ray irradiations is completed.

[0019] Further, the step of respectively detecting the absorbance values of each well in the blank group, the control group and the experimental group after irradiation, and calculating the inhibition rate of the cancer tissue and the activity value of the adjacent cancer tissue at each radiation dose according to the absorbance values of each well in the blank group, the control group and the experimental group includes:

[0020] After the irradiation is completed, the samples are taken out from the cell incubator, the tissue culture medium is aspirated, and they are washed 2 - 4 times with PBS, then CCK8 reaction solution is added and they are put back into the cell culture incubator for incubation for the fourth preset time. After incubation, the absorbance values of each well are detected in sequence;

[0021] The average absorbance value of each group is calculated according to the absorbance value of each well, and the inhibition rate of the cancer tissue and the activity value of the adjacent cancer tissue are calculated according to the average absorbance value of each group.

[0022] Further, the step of calculating the average absorbance value of each group according to the absorbance value of each well, and calculating the inhibition rate of the cancer tissue and the activity value of the adjacent cancer tissue according to the average absorbance value of each group includes:

[0023] The activity value of the adjacent cancer tissue is calculated according to the following formula:

[0024] γ i0= (A si0 - A bi0 ) / (A ci0 - A bi0 )

[0025] where γ i0 represents the activity value corresponding to the adjacent cancer tissue in the i-th category grouping of the adjacent cancer tissue samples, A si0 represents the average absorbance value corresponding to the experimental group in the i-th category grouping of the adjacent cancer tissue samples, A bi0 represents the average absorbance value corresponding to the blank group in the i-th category grouping of the adjacent cancer tissue samples, A ci0 represents the average absorbance value corresponding to the control group in the i-th category grouping of the adjacent cancer tissue samples;

[0026] The inhibition rate of the cancer tissue is calculated according to the following formula:

[0027] γ i1 = (A ci1 - A si1 ) / (A ci1 - A bi1 )

[0028] where γ i1 represents the inhibition rate corresponding to the cancer tissue in the i-th category grouping of the cancer tissue samples, A si1 represents the average absorbance value corresponding to the experimental group in the i-th category grouping of the cancer tissue samples, A bi1 represents the average absorbance value corresponding to the blank group in the i-th category grouping of the cancer tissue samples, A ci1 represents the average absorbance value corresponding to the control group in the i-th category grouping of the cancer tissue samples.

[0029] Furthermore, the step of predicting the sensitivity corresponding to each radiation dose of the tumor patient according to the inhibition rate of the cancer tissue and the activity value of the adjacent cancer tissue further includes:

[0030] Judging whether the inhibition rate of the cancer tissue in the same category is greater than or equal to the first preset inhibition rate threshold, and whether the activity value of the adjacent cancer tissue is greater than or equal to the first preset activity value;

[0031] If the inhibition rate of the cancer tissue in the same category is greater than or equal to the first preset inhibition rate threshold and the activity value of the adjacent cancer cells is greater than or equal to the first preset activity value, it is determined that the sensitivity of the radiation dose to the tissue block under this category grouping is qualified.

[0032] Furthermore, after the step of if the inhibition rate of the cancer tissue in the same category is greater than or equal to the first preset inhibition rate threshold and the activity value of the adjacent cancer cells is greater than or equal to the first preset activity value, it is determined that the sensitivity of the radiation dose to the tissue block under this category grouping is qualified, the following steps are further included:

[0033] Perform a first function fitting on the inhibition rate data of the obtained cancer tissue and its corresponding radiation dose to obtain a first function. Perform a second function fitting on the activity value data of the obtained adjacent cancer tissue and the expected corresponding radiation dose to obtain a second function;

[0034] Traverse the first function and the second function simultaneously according to the same radiation dose to obtain all the test data with qualified sensitivity.

[0035] Furthermore, the components in the tissue preservation solution include tissue culture medium, fetal bovine serum, and antibiotics.

[0036] Furthermore, in the tissue preservation solution:

[0037] The proportion of the tissue culture medium is 85%-94%, the proportion of the fetal bovine serum is 5%-15%, and the proportion of the antibiotics is 0.5%-2%.

[0038] Compared with the prior art, the in vitro rapid detection method for predicting the radiosensitivity of tumor patients proposed by the present invention for non-diagnostic purposes is a brand-new laboratory detection method, which can quickly, comprehensively and specifically predict the sensitivity of any patient to radiotherapy before radiotherapy, thereby providing an experimental basis for the individualized radiotherapy of tumor patients.

[0039] The additional aspects and advantages of the present invention will be partially given in the following description, partially become obvious from the following description, or be understood through the embodiments of the present invention. Description of the Drawings

[0040] Figure 1 It is a flowchart of the in vitro rapid detection method for predicting the radiosensitivity of tumor patients proposed by the first embodiment of the present invention for non-diagnostic purposes;

[0041] Figure 2 It is a flowchart of the in vitro rapid detection method for predicting the radiosensitivity of tumor patients proposed by the second embodiment of the present invention for non-diagnostic purposes;

[0042] Figure 3 It is a schematic diagram of the test data proposed by the second embodiment of the present invention.

[0043] The following specific embodiments will further illustrate the present invention in conjunction with the above-mentioned drawings. Specific Embodiments

[0044] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. The drawings illustrate several embodiments of the present invention. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present invention.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0046] See also Figure 1 , which shows a non-diagnostic in vitro rapid detection method for predicting the radiosensitivity of tumor patients in a first embodiment of the present invention, the method includes steps S01 to S04, wherein:

[0047] Step S01: Obtaining a tumor patient number, obtaining cancer tissue samples and adjacent tissue samples pre-stored in tissue preservation fluid according to the tumor patient number, and performing sample pre-processing on the cancer tissue samples and adjacent tissue samples to obtain a plurality of cancer tissue blocks and adjacent tissue blocks of a first preset size;

[0048] It should be noted that each tumor patient number corresponds to a cancer tissue sample and a paracancerous tissue sample. The cancer tissue sample and paracancerous tissue sample are obtained based on the tumor patient. The specific acquisition method can be direct excision and collection, or in vitro cell culture, etc. The cancer tissue sample or paracancerous tissue sample preserved in tissue preservation fluid has a corresponding number. This is mainly to take into account the obvious differences in the sensitivity of different individuals to radiotherapy. In this way, the radiosensitivity of each individual patient can be predicted in a targeted manner.

[0049] In this embodiment, the main costs of the tissue preservation solution include tissue culture medium, fetal bovine serum and antibiotics. The tissue culture medium generally adopts RPMI-1640 culture medium, and the antibiotics can be penicillin or streptomycin.

[0050] Specifically, in the tissue preservation solution: the proportion of the tissue culture medium is 85%-94%. Exemplarily, the proportion of the tissue culture medium can be 85%, 89% or 95%, etc.; the proportion of the fetal bovine serum is 5%-15%. Exemplarily, the proportion of the fetal bovine serum can be 5%, 10%, 15%, etc.; the proportion of the antibiotic is 0.5%-2%. The proportion of the antibiotic can be 0.5%, 1%, 1.5%, 2%, etc. It can be understood that the total proportion of the tissue culture medium, the fetal bovine serum and the antibiotic is 100%.

[0051] When specifically preserving tissue samples, put the tissue samples into the prepared tissue preservation solution, control the temperature at 4-8°C, and then mark the types and numbers of the tissue samples, so as to achieve the purpose of long-term preservation or transfer of the tissue samples. Specifically, the preservation time generally does not exceed one week.

[0052] The first preset size is set to unify the sizes of all tissue blocks to be detected, and at the same time to be applicable to the specifications of the well plates for inoculating tissue blocks in the follow-up. It can be understood that in this embodiment, 96-well plates are used, and the first preset size is set to 2*2*2 mm. The experimenter can also flexibly preprocess the tissue block samples into other sizes, and at the same time can select other specifications such as 24-well plates for subsequent inoculation of tissue blocks.

[0053] Step S02: Transfer the cancer tissue blocks and the para-cancer tissue blocks into several well plates. Each well of each well plate is inoculated with at most one tissue block. Divide the tissue blocks in the well plates into several groups according to the number of the cancer tissue blocks or the para-cancer tissue blocks, and calculate and mark the radiation doses corresponding to each group of tissue blocks respectively according to the preset peak radiation dose and the divided groups. The grouping results include a blank group, a control group and an experimental group;

[0054] In this step, in order to comprehensively predict the radiation sensitivity of tissue blocks to cells, it is necessary to comprehensively detect and evaluate the cancer tissue blocks and the para-cancer tissue blocks. When transferring the obtained tissue blocks to a 96-well plate, the blank group corresponds to the wells in the well plate that are not inoculated with tissue blocks, and the control group and the experimental group correspond to the wells in the well plate that are inoculated with one tissue block respectively. After grouping, calculate the radiation dose corresponding to each group according to the number of grouped cancer tissue blocks and the preset peak radiation dose. Specifically, the initial radiation dose is defaulted to zero, and the difference in radiation dose between any two adjacent groups is equal, so as to calculate the radiation dose corresponding to each group according to the preset peak radiation dose and the number of groups. By way of example and not limitation, please refer to Table 1 below for the grouping situation of cancer tissue and para-cancer tissue obtained in this step and the radiation dose corresponding to each group:

[0055] Table 1

[0056]

[0057] The preset peak radiation dose is obtained by the experimenter based on clinical data. This embodiment is for sensitivity detection of breast cancer, and the preset peak radiation dose is within 10 Gy.

[0058] Step S03: Calculate the radiation time corresponding to each group of tissue blocks according to the radiation dose and the preset dose rate corresponding to each group of tissue blocks, place the well plate in the irradiation area of the radiation instrument, and control the radiation instrument to irradiate each group of tissue blocks for a first preset number of times every second preset time according to the radiation time and the preset dose rate;

[0059] It should be noted that, in order to improve the accuracy of the evaluation of radiation sensitivity, when irradiating each group, it is not just irradiated once. The second preset time is set to continuously irradiate the tissue blocks multiple times to accurately simulate the actual radiotherapy scenario. At the same time, in order to take into account the detection cycle, the first preset number of times generally does not exceed 5 times, and the second preset time, that is, the interval time, is generally 24 h - 48 h.

[0060] Furthermore, the radiation dose is equal to the product of the preset dose rate and the radiation time. When the radiation dose and the preset dose rate are known, the radiation time of the radiation instrument can be calculated. It should be noted that the preset radiation dose rate in this embodiment is 200 cGy / min, and the preset radiation dose rate is determined by the radiation instrument, tumor type, etc. In other embodiments of the present invention, it can also be other values.

[0061] Step S04: Detect the absorbance value of each well in the blank group, the control group, and the experimental group after the irradiation is completed, and calculate the inhibition rate of the cancer tissue and the activity value of the adjacent cancer tissue at each radiation dose according to the absorbance values of each well in the blank group, the control group, and the experimental group, so as to predict the sensitivity corresponding to each radiation dose of the tumor patient according to the inhibition rate of the cancer tissue and the activity value of the adjacent cancer tissue.

[0062] It should be noted that after the ray irradiation of the blank group, the control group, and the experimental group is completed, the samples are taken out of the cell incubator, the tissue culture medium is aspirated, washed 3 times with PBS, then CCK8 reaction solution is added and put back into the cell incubator for incubation. After 4 hours, the tissue blocks are taken out with ophthalmic forceps, and then the absorbance value of each well is detected by a spectrophotometer or an enzyme-linked immunosorbent assay (ELISA) reader, and then the average absorbance value of each group under the condition of the set irradiation dose is calculated, and then the inhibition rate of the cancer tissue and the activity value of the adjacent cancer tissue are calculated, so as to evaluate the effectiveness of different radiation doses on the cancer tissue and the side effects on normal tissues (adjacent cancer tissues), and then it is beneficial to find a better radiation dose.

[0063] In summary, the in vitro rapid detection method for predicting the radiosensitivity of tumor patients for non-diagnostic purposes proposed according to the above embodiments is a brand-new laboratory detection method, which can quickly, comprehensively and specifically predict the sensitivity of any patient to radiotherapy before radiotherapy, thereby providing an experimental basis for the individualized radiotherapy of tumor patients.

[0064] Please refer to Figure 2 , which shows the flowchart of the in vitro rapid detection method for predicting the radiosensitivity of tumor patients for non-diagnostic purposes in the second embodiment of the present invention. The method includes steps S101 to S10, where:

[0065] Step S101: Rinse the cancer tissue sample and the adjacent tissue sample with tissue culture medium 2-4 times, each time lasting 0.5-2 min;

[0066] It should be noted that the distance between the cancer tissue sample and the adjacent tissue sample is generally more than 3 cm, that is, the cancer tissue sample corresponds to the cancerous part, and the adjacent tissue corresponds to the non-cancerous part. The tissue culture medium is RPMI-1640 medium.

[0067] Step S102: Remove the necrotic or poorly shaped areas in the rinsed cancer tissue sample and adjacent tissue sample, soak them in iodophor disinfectant for 10-60 s, and then divide the cancer tissue sample and the adjacent tissue into microtissue blocks of a first preset size;

[0068] Step S103: Calculate the number of well rows or well columns of each tissue sample according to the number of microtissue blocks separated from one of the tissue samples and the specifications of the well plate, and divide the tissue blocks into several categories according to the number of well rows or well columns. Each category includes a blank group, a control group, and an experimental group;

[0069] It should be pointed out that the wells divided into the control group and the experimental group in the same category are inoculated with the same type of tissue blocks, the wells divided into each blank group are not inoculated with tissue blocks, the blank group and the experimental group in the same category are marked with equal radiation doses, and the radiation dose corresponding to each control group is zero. When grouping, considering the limited number of tissue blocks obtained after pretreatment, generally one row or one column, or multiple rows and multiple columns in the well plate are divided into one group. If the number of wells divided into each group is less than one well plate, then when irradiating each group, it is necessary to use a lead plate to completely block the wells corresponding to other groups.

[0070] Step S104: After grouping, perform the first ray irradiation on the blank group or the experimental group using the calculated irradiation time and the preset dose rate. After the irradiation is completed, add 50 - 200 μL of tissue culture medium to each well, and place it in a cell culture incubator for cultivation. After the second preset time, aspirate the tissue culture medium, and perform the second ray irradiation until the first preset number of ray irradiations is completed;

[0071] By way of example rather than limitation, the specific process of ray irradiation is as follows: Place the grouped 96-well plate in the irradiation area of the radiation instrument, and irradiate each group according to the corresponding data in Table 1. If the radiation dose is zero, no irradiation is performed. After irradiation, add 100 μL of tissue culture medium to each well, and place it in a cell culture incubator for cultivation. After 24 h, aspirate the tissue culture medium, perform the second ray irradiation. After 48 h, aspirate the culture medium again and perform the third ray irradiation until the first preset number is completed. Since ionizing radiation directly acts on water to generate free radicals to kill tissues, if the culture medium is not removed during irradiation, the presence of a large amount of water will affect the accuracy of the results. Based on this, it is necessary to aspirate the tissue culture medium in the tissue block during each irradiation, and add tissue culture medium after irradiation.

[0072] Step S105: After the irradiation is completed, take out the samples from the cell incubator, aspirate the tissue culture medium, wash with PBS 2 - 4 times, then add CCK8 reaction solution and place it back in the cell culture incubator for incubation for the fourth preset time. After incubation, measure the absorbance value of each well in sequence;

[0073] Step S106: Calculate the average absorbance value of each group based on the absorbance value of each well, and calculate the inhibition rate of the cancer tissue and the activity value of the adjacent cancer tissue based on the average absorbance value of each group;

[0074] Specifically, after obtaining the average absorbance value corresponding to each group, calculate the activity value of the adjacent cancer tissue according to the following formula:

[0075] γ i0 =(A si0 -A bi0 ) / (A ci0 -A bi0 )

[0076] where γ i0 represents the activity value corresponding to the adjacent cancer tissue in the i-th category grouping of the adjacent cancer tissue sample, A si0 represents the average absorbance value corresponding to the experimental group in the i-th category grouping of the adjacent cancer tissue sample, A bi0 represents the average absorbance value corresponding to the blank group in the i-th category grouping of the adjacent cancer tissue sample, A ci0 represents the average absorbance value corresponding to the control group in the i-th category grouping of the adjacent cancer tissue sample;

[0077] The inhibition rate of cancer tissue is calculated according to the following formula:

[0078] γ i1 =(A ci1 -A si1 ) / (A ci1 -A bi1 )

[0079] where γ i1 represents the inhibition rate corresponding to the cancer tissue in the i-th category grouping of the cancer tissue sample, A si1 represents the average absorbance value corresponding to the experimental group in the i-th category grouping of the cancer tissue sample, A bi1 represents the average absorbance value corresponding to the blank group in the i-th category grouping of the cancer tissue sample, A ci1 represents the average absorbance value corresponding to the control group in the i-th category grouping of the cancer tissue sample.

[0080] By setting up the blank group, the control group and the experimental group, and then using the above specific formula to calculate the inhibition rate and the activity value, the detection error can be eliminated. At the same time, according to the ratio of absorbance, the inhibition rate and the activity value of the tissue before and after irradiation are obtained, ensuring efficient detection and making the detection results more reliable.

[0081] Step S107: Determine whether the inhibition rate of the cancer tissue in the same category is greater than or equal to the first preset inhibition rate threshold, and whether the activity value of the adjacent cancer tissue is greater than or equal to the first preset activity value;

[0082] Step S108: If the inhibition rate of the cancer tissue in the same category is greater than or equal to the first preset inhibition rate threshold and the activity value of the adjacent cancer cells is greater than or equal to the first preset activity value, then determine that the sensitivity of the radiation dose to the tissue block under this category grouping is qualified;

[0083] It should be noted that each radiation dose corresponds to an inhibition rate and an activity value. The radiation doses of the cancer tissue and the adjacent cancer tissue in the same category are equal. Then, it is determined whether the inhibition rate calculated by using the equal radiation dose under each category is at a relatively high level and whether the activity value is at a relatively low level, so as to analyze whether there is a suitable radiation dose.

[0084] By way of example and not limitation, the range of the first preset inhibition rate threshold is 15%-30%, and the first preset activity value is 70%-85%. Exemplarily, if the inhibition rate is greater than or equal to 30%, it is determined that the radiation to the cancer tissue is effective; if the activity value of the normal tissue is greater than or equal to 70%, it is determined that there is no obvious toxicity. It can be understood that in other embodiments of the present invention, the first preset inhibition rate threshold and the first preset activity value can also be set to other values to achieve the effect of determining whether the tissue block is sensitive to different radiation doses.

[0085] Step S109: Perform a first function fitting on the obtained inhibition rate data of the cancer tissue and the corresponding radiation dose to obtain a first function, and perform a second function fitting on the obtained activity value data of the tissue adjacent to the cancer and the expected corresponding radiation dose to obtain a second function;

[0086] Please refer to Figure 3 , which shows a schematic diagram of the detection data. According to Figure 3 it can be seen that as the radiation dose continuously increases, the normal tissue adjacent to the cancer in the patient becomes more and more sensitive to radiotherapy, and the activity value becomes lower and lower, that is, the side effects become greater and greater. There is no radiation dose that can satisfy both the inhibition rate and the activity value at the same time. The above results suggest that the radiotherapy benefit of this patient may not reach the expectation and may cause serious side effects, thereby providing a reliable experimental basis for clinical radiotherapy.

[0087] Step S110: Traverse the first function and the second function simultaneously according to the same radiation dose to obtain all the detection data with qualified sensitivity.

[0088] It should be noted that after it is determined that there is a radiation dose with qualified sensitivity, in order to obtain a better radiation dose, at this time, function fittings will be performed on the inhibition rate - radiation dose and the activity value - radiation dose respectively, and then the two functions obtained by fitting will be traversed according to the same radiation dose, so as to screen out all the radiation doses with qualified sensitivity and the corresponding inhibition rates and activity values, so as to comprehensively predict the sensitivity situation under the qualified radiation dose.

[0089] In the description of this specification, the descriptions with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0090] The above - described embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but should not be construed as limiting the scope of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. A rapid in vitro detection method for predicting the radiosensitivity of tumor patients for non-diagnostic purposes, characterized in that, Before radiotherapy for cancer patients, the method includes: Obtaining the cancer patient number, obtaining the cancer tissue sample and the adjacent tissue sample pre-stored in the tissue preservation solution respectively according to the cancer patient number, and performing sample pretreatment on the cancer tissue sample and the adjacent tissue sample to obtain a number of cancer tissue blocks and adjacent tissue blocks of a first preset size; Transferring the cancer tissue blocks and the adjacent tissue blocks into a number of well plates, with at most one tissue block inoculated in each well of each well plate, dividing the tissue blocks in the well plates into several groups according to the number of the cancer tissue blocks or the adjacent tissue blocks, and calculating and labeling the radiation doses corresponding to each group of tissue blocks respectively according to the preset peak radiation dose and the number of divided groups. The grouping results include a blank group, a control group and an experimental group; Calculating the radiation time corresponding to each group of tissue blocks respectively according to the radiation dose corresponding to each group of tissue blocks and the preset dose rate, placing the well plates in the irradiation area of the radiation instrument, and controlling the radiation instrument to irradiate each group of tissue blocks a first preset number of times every second preset time according to the radiation time and the preset dose rate; Detecting the absorbance values of each well in the blank group, the control group and the experimental group after irradiation respectively, and calculating the inhibition rate of the cancer tissue and the activity value of the adjacent tissue at each radiation dose according to the absorbance values of each well in the blank group, the control group and the experimental group, so as to predict the sensitivity corresponding to each radiation dose of the cancer patient according to the inhibition rate of the cancer tissue and the activity value of the adjacent tissue.

2. The in vitro rapid detection method for predicting the radiosensitivity of tumor patients for non-diagnostic purposes according to claim 1, characterized in that, The step of performing sample pretreatment on the cancer tissue sample and the adjacent tissue sample to obtain a number of cancer tissue blocks and adjacent tissue blocks of a first preset size includes: Rinsing the cancer tissue sample and the adjacent tissue sample 2-4 times respectively with a tissue culture medium, each time lasting 0.5-2 min; Removing the necrotic or poorly shaped areas in the rinsed cancer tissue sample and adjacent tissue sample, soaking them in iodophor disinfectant for 10-60 s, and then dividing the cancer tissue sample and the adjacent tissue into micro tissue blocks of a first preset size.

3. The in vitro rapid detection method for predicting the radiosensitivity of tumor patients for non-diagnostic purposes according to claim 2, characterized in that, The step of transferring the cancer tissue blocks and the adjacent tissue blocks into a number of well plates, with at most one tissue block inoculated in each well of each well plate, dividing the tissue blocks in the well plates into several groups according to the number of the cancer tissue blocks or the adjacent tissue blocks, and calculating and labeling the radiation doses corresponding to each group of tissue blocks respectively according to the preset peak radiation dose and the number of divided groups includes: Calculating the number of well rows or well columns of each tissue sample according to the number of micro tissue blocks separated from one of the tissue samples and the specifications of the well plates, so as to divide the tissue blocks into several categories according to the number of well rows or well columns. Each category includes one group of the blank group, the control group and the experimental group respectively, where: The wells divided in the control group and the experimental group in the same category are inoculated with the same type of tissue blocks, the wells divided in each blank group are not inoculated with tissue blocks, the blank group and the experimental group in the same category are labeled with equal radiation doses, and the radiation dose corresponding to each control group is zero.

4. The in vitro rapid detection method for predicting the radiosensitivity of tumor patients for non-diagnostic purposes according to claim 3, characterized in that, The step of calculating the irradiation time corresponding to each group of tissue blocks according to the radiation dose and the preset dose rate corresponding to each group of tissue blocks respectively, placing the well plate in the irradiation area of the radiation instrument, and controlling the radiation instrument to irradiate each group of tissue blocks for a first preset number of times every second preset time includes: After grouping, perform the first ray irradiation on the blank group or the experimental group using the calculated irradiation time and the preset dose rate. After the irradiation is completed, add 50 - 200 μL of tissue culture medium into each well, and place it in a cell incubator for culture. After the second preset time, aspirate the tissue culture medium, and perform the second ray irradiation until the first preset number of ray irradiations is completed.

5. The in vitro rapid detection method for predicting the radiosensitivity of tumor patients for non-diagnostic purposes according to claim 4, wherein The step of respectively detecting the absorbance values of each well in the blank group, the control group, and the experimental group after the irradiation is completed, and calculating the inhibition rate of the cancer tissue and the activity value of the adjacent cancer tissue at each radiation dose according to the absorbance values of each well in the blank group, the control group, and the experimental group includes: After the irradiation is completed, take out the samples from the cell incubator, aspirate the tissue culture medium, and wash them 2 - 4 times with PBS. Then add the CCK8 reaction solution and put it back into the cell incubator for incubation for the fourth preset time. After incubation, detect the absorbance value of each well in turn; Calculate the average absorbance value of each group according to the absorbance value of each well, and calculate the inhibition rate of the cancer tissue and the activity value of the adjacent cancer tissue according to the average absorbance value of each group.

6. The in vitro rapid detection method for predicting the radiosensitivity of tumor patients for non-diagnostic purposes according to claim 5, characterized in that, The step of calculating the average absorbance value of each group according to the absorbance value of each well, and calculating the inhibition rate of the cancer tissue and the activity value of the adjacent cancer tissue according to the average absorbance value of each group includes: Calculate the activity value of the adjacent cancer tissue according to the following formula: γ i0 = (A si0 - A bi0 ) / (A ci0 - A bi0 ) Among them, γ i0 represents the activity value corresponding to the adjacent tissue in the i-th group of adjacent tissue samples, A si0 represents the average absorbance value corresponding to the experimental group in the i-th group of adjacent tissue samples, A bi0 represents the average absorbance value corresponding to the blank group in the i-th group of adjacent tissue samples, A ci0 represents the average absorbance value corresponding to the control group in the i-th group of adjacent tissue samples; Calculate the inhibition rate of the cancer tissue according to the following formula: γ i1 = (A ci1 - A si1 ) / (A ci1 - A bi1 ) Among them, γ i1 represents the inhibition rate corresponding to the cancer tissue in the i-th group of cancer tissue samples, A si1 represents the average absorbance value corresponding to the experimental group in the i-th group of cancer tissue samples, A bi1 represents the average absorbance value corresponding to the blank group in the i-th group of cancer tissue samples, A ci1 represents the average absorbance value corresponding to the control group in the i-th group of cancer tissue samples.

7. The in vitro rapid detection method for predicting the radiosensitivity of tumor patients for non-diagnostic purposes according to claim 6, characterized in that, The step of predicting the sensitivity corresponding to each radiation dose of the tumor patient according to the inhibition rate of the cancer tissue and the activity value of the adjacent cancer tissue further includes: Judge whether the inhibition rate of the cancer tissue in the same category is greater than or equal to the first preset inhibition rate threshold, and whether the activity value of the adjacent cancer tissue is greater than or equal to the first preset activity value; If the inhibition rate of the cancer tissue in the same category is greater than or equal to the first preset inhibition rate threshold and the activity value of the adjacent cancer cells is greater than or equal to the first preset activity value, then judge that the radiation dose of this category of grouping is qualified for the sensitivity of the tissue block.

8. The in vitro rapid detection method for predicting the radiosensitivity of tumor patients for non-diagnostic purposes according to claim 7, characterized in that, After the step of if the inhibition rate of the cancer tissue in the same category is greater than or equal to the first preset inhibition rate threshold and the activity value of the adjacent cancer cells is greater than or equal to the first preset activity value, then judge that the radiation dose of this category of grouping is qualified for the sensitivity of the tissue block, it further includes: Perform a first function fitting on the obtained inhibition rate data of the cancer tissue and its corresponding radiation dose to obtain a first function, and perform a second function fitting on the obtained activity value data of the adjacent cancer tissue and the expected corresponding radiation dose to obtain a second function; Traverse the first function and the second function simultaneously according to the same radiation dose to obtain all the detection data with qualified sensitivity.

9. The in vitro rapid detection method for predicting the radiosensitivity of tumor patients for non-diagnostic purposes according to claim 1, characterized in that, The components in the tissue preservation solution include tissue culture medium, fetal bovine serum, and antibiotics.

10. The in vitro rapid detection method for predicting the radiosensitivity of tumor patients for non-diagnostic purposes according to claim 9, characterized in that, In the tissue preservation solution: The proportion of the tissue culture medium is 85%-94%, the proportion of the fetal bovine serum is 5%-15%, and the proportion of the antibiotic is 0.5%-2%.

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