CN113416629B - Tumor chemotherapeutic drug sensitivity detection kit and detection method - Google Patents
Tumor chemotherapeutic drug sensitivity detection kit and detection method Download PDFInfo
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- CN113416629B CN113416629B CN202110621228.2A CN202110621228A CN113416629B CN 113416629 B CN113416629 B CN 113416629B CN 202110621228 A CN202110621228 A CN 202110621228A CN 113416629 B CN113416629 B CN 113416629B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M37/00—Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/44—Means for regulation, monitoring, measurement or control, e.g. flow regulation of volume or liquid level
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
Abstract
The invention discloses a tumor chemotherapeutic drug sensitivity detection kit and a detection method, and particularly relates to the technical field of tumor chemotherapeutic drug sensitivity detection, comprising the following steps: firstly, picking up tumor tissues and partial normal cell tissues around tumors in a sterile environment, then treating the picked tumor tissues and cell tissues, and cutting the tumor tissues and cell tissues into tissue blocks with the size of 1 cubic millimeter by scissors in the sterile environment; step two, preparing a culture medium: extracting serum of tumor patients as a culture medium, inactivating the serum before use, and preserving at-20deg.C; step three, selecting and preparing other common medicaments; and step four, preparing a culture solution. According to the invention, the serum of a patient is used as a culture medium, and tumor cells and complete cell tissues around the tumor cells are cultured together, so that the physiological characteristics of the patient can be simulated to the greatest extent, and then the material-dissolving medicine is added into the culture medium, so that the accuracy of a measurement result can be improved.
Description
Technical Field
The embodiment of the invention relates to the technical field of tumor chemotherapeutic drug sensitivity detection, in particular to a tumor chemotherapeutic drug sensitivity detection kit and a detection method.
Background
A tumor (tumor) is a genetic disease, but is not inherited. It refers to a new organism formed by monoclonal abnormal proliferation caused by the fact that the cell is changed under the action of cancerogenic factors and the normal regulation and control of the growth of the cell are lost. This neoplasm often forms local masses and is therefore named. Tumors are classified into benign tumors and malignant tumors, benign tumors generally recover health after treatment by surgery, but malignant tumors are not easily cured because of their recalcitrance. Malignant tumors are cancers which are commonly known by people, and after suffering from the cancers, a plurality of people consider to be judged as dead criminals, and the heart state is extremely negative. In recent years, a new method for treating malignant tumor is developed, namely, air negative ion natural therapy. A large number of clinical experiments prove that the air negative ion physiotherapy cancer effect is remarkable, and the method is a new method besides radiotherapy, chemotherapy and operation treatment. In the common treatment of tumors, tumor chemotherapy resistance is one of the main reasons for chemotherapy failure, and how to formulate an individual chemotherapy scheme to improve the chemotherapy curative effect is a difficult problem to be solved in clinic. The tumor in-vitro drug sensitivity detection technology can detect the sensitivity of tumor cells of a patient to drugs before chemotherapy of the patient, accurately foresee the in-vivo treatment effect, thereby screening out relatively effective chemotherapeutic drugs for the patient and providing scientific basis for a clinician to determine a chemotherapeutic scheme and develop a certain radiotherapy and vector personalized treatment. The method has the characteristics of good stability and repeatability, high sensitivity, high ratable rate, high throughput screening, computer scanning, software analysis and the like, so that the in-vitro detection result and in-vivo treatment reaction of the technology have high consistency, the total prediction accuracy is more than 85%, and the positive and negative prediction values, sensitivity and specificity are higher than those of other technologies. A large number of clinical researches show that the in vitro drug sensitivity detection of the tumor has a certain correlation with the clinical curative effect, and the in vitro drug sensitivity detection can better guide the clinical medication and improve the clinical curative effect. The current tumor drug sensitivity detection methods are as follows: soft agar clone formation assay (HTCA), tetrazolium blue colorimetric Method (MTT), differential staining cytotoxicity detection method (disec), and adenosine triphosphate bioluminescence method.
In the prior art, when tumor chemotherapeutic drug sensitivity detection is carried out, a human body is simulated to prepare a culture medium, so that tumor cells can be successfully cultured, but because of large individual differences of patients, physiological conditions of each patient are different, the response of each patient to the chemotherapeutic drug is also different, and certain errors exist in the detection results for different patients.
Disclosure of Invention
Therefore, the embodiment of the invention provides a tumor chemotherapeutic drug sensitivity detection kit and a detection method, which can simulate physiological characteristics of a patient to the greatest extent by using serum of the patient as a culture medium and culturing tumor cells and complete cell tissues around the tumor cells together, and then adding a chemical drug into the culture medium, so that the accuracy of a detection result can be improved, and the problem that in the prior art, the detection result has errors due to individual variability of different patients is solved.
In order to achieve the above object, the embodiment of the present invention provides the following technical solutions: the utility model provides a tumour chemotherapy drug sensitivity detection kit and detection method, includes the kit, the inside two reagent cups that are equipped with of kit, two reagent cup bottom threaded connection has first base, first base bottom is fixed and is equipped with the standpipe, standpipe outer end is fixed and is equipped with first solenoid valve and flowmeter, the flowmeter is established in first solenoid valve bottom, standpipe outer end fixedly connected with a plurality of first connecting pipes, first connecting pipe other end is fixed and is equipped with the second base, second base and first base top are all fixed and are equipped with the outlet pipe, a plurality of through-holes have been seted up at outlet pipe outer end top, second base top threaded connection has a batching section of thick bamboo, the outlet pipe top is pegged graft inside the batching section of thick bamboo, batching section of thick bamboo and inside bottom of thick bamboo are pegged graft and are equipped with the rubber stopper, rubber stopper bottom is fixed and is equipped with a plurality of springs, spring bottom and the inside bottom fixed connection of batching section of thick bamboo, the inside is equipped with the rubber piston, the rubber piston is established at the rubber stopper top, batching section of thick bamboo top is fixed and is equipped with the second connecting pipe, second connecting pipe top is fixed and is equipped with second solenoid valve, second pipe top is equipped with the inside the mechanism of pipe, the pipeline is equipped with the inside the joint, the second is equipped with the pipeline.
Further, the air supply mechanism comprises two fans, the two fans are respectively and fixedly arranged on two sides of the kit, a third connecting pipe is fixedly arranged at the top of the fan, one end of the third connecting pipe extends into the kit, a runner is arranged in the side wall of the kit, one end of the pipeline joint is communicated with the runner, and one end of the third connecting pipe extends into the runner and is communicated with the runner.
Further, the sterilization mechanism comprises a box cover, the box cover is arranged at the top of the kit, the rear end of the box cover is movably connected with the rear side of the kit through a damping rotating shaft, a plurality of ultraviolet sterilization lamps are fixedly arranged at the top end inside the box cover, a plurality of controllers are fixedly arranged in the middle of the top of the box cover, a control panel is arranged at the top of each controller, and each controller is electrically connected with the box cover, the first electromagnetic valve, the flowmeter and the second electromagnetic valve.
The invention also comprises a detection method using the kit for detecting the sensitivity of the tumor chemotherapeutic medicine, and the specific steps are as follows:
firstly, picking up tumor tissues and partial normal cell tissues around tumors in a sterile environment, then treating the picked tumor tissues and cell tissues, and cutting the tumor tissues and cell tissues into tissue blocks with the size of cubic millimeters by scissors in the sterile environment;
preparing a culture medium by using the kit: extracting serum of a tumor patient to serve as a culture medium, inactivating the serum before use, preserving the serum under the condition of minus ℃ and adding the extracted serum into a reagent cup at the left side in the reagent box, then respectively placing reagents required for treating the serum into a plurality of dosing cylinders around the reagent cup, closing a controller, starting an ultraviolet sterilizing lamp through the controller, sterilizing the inside of the reagent box by using the ultraviolet sterilizing lamp, starting a blower at the left side through the controller, sequentially opening a second electromagnetic valve at the outer ends of a plurality of second connecting pipes at the left side, enabling gas generated in the working process of the blower to enter the inside of the second connecting pipes through a third connecting pipe, a runner and a pipeline joint, and enter the inside of the dosing cylinders through the second connecting pipe, then pushing a rubber piston in the inside of the dosing cylinders to move downwards along the inner wall of the dosing cylinders, so as to push the reagents in the dosing cylinders to move into the inside of the second base through holes on a water outlet pipe, and move into the inside of the reagent cup through the second base, and starting the ultraviolet sterilizing lamp through the first connecting pipe and the first base, and then starting a blower at the controller, and sequentially opening a second electromagnetic valve at the outer ends of the left side of the second connecting pipe, and opening a second electromagnetic valve at the first electromagnetic valve, and a second electromagnetic valve, and a reagent can be quantitatively added into the dosing cylinder when the reagents and the reagent can be quantitatively added into the dosing cylinder through the reagent cup through the first electromagnetic valve and the electromagnetic valve;
selecting and preparing other common medicaments by using a kit, wherein the other common medicaments comprise balanced salt solution, digestive juice, pH adjusting solution, antibiotic solution and glutamine supplementing solution, wherein Hank's balanced solution is selected as the balanced salt solution, EDTA and pancreatin mixed solution is selected as the digestive juice, HEPES solution is selected as the pH adjusting solution, penicillin and streptomycin mixed solution is selected as the antibiotic solution, the glutamine supplementing solution is prepared, and the selected reagents are sequentially added into a plurality of batching cylinders on the right side;
step four, preparing a culture solution: pouring serum inside a reagent cup processed on the left side inside a reagent box into the reagent cup on the right side, and in the process of taking out, only rotating the reagent cup to enable the reagent cup to be separated from a first base, then automatically plugging the bottom end of the rubber plug into the bottom end inside the reagent cup under the action of the elasticity of a spring so as to prevent water leakage, then starting a blower on the right side, sequentially opening a second electromagnetic valve at the outer ends of a plurality of second connecting pipes on the right side, enabling gas generated in the working process of the blower to enter the second connecting pipe through a third connecting pipe, a runner and a pipeline joint, and enter the inside of a batching cylinder through the second connecting pipe, then pushing a rubber piston inside the batching cylinder to move downwards along the inner wall of the batching cylinder, and then enabling a reagent inside the batching cylinder to move into the inside of the second base through a through hole on a water outlet pipe, and enabling the first connecting pipe, and the first electromagnetic valve to be in an opened state, and then calculating the flow of the reagent flowing through the first electromagnetic valve, so that the reagent can be quantitatively added into the reagent cup, and when the blower is automatically controlled, a proper amount of reagent is automatically added into the batching medium, and a proper amount of reagent is added into the reagent cup after a proper amount of the reagent is filled with a proper amount of medium, and a proper amount of reagent is prepared into a medium, and a proper amount of a reagent medium is prepared by adding a proper amount of a reagent solution, and a proper amount of a reagent buffer solution and a reagent tank, and a proper amount of a reagent tank is prepared and a proper amount and a reagent buffer tank and a reagent;
step five, adding a proper amount of antibiotic solution into Hank's balance liquid, then respectively rinsing the cut tumor tissue blocks and cell tissue blocks by utilizing mixed liquid of Hank's balance liquid and the antibiotic solution, rinsing for a plurality of times to wash off blood on the tissue surfaces, and then respectively transferring the rinsed tumor tissue blocks and cell tissue block mixtures into two groups of sterilized culture bottles;
step six, standing two groups of culture bottles in an incubator for 1-2h, adding culture solution into the culture bottles, turning over the culture bottles for 180 degrees, standing the turned-over culture bottles for 1-2h, and turning over the culture bottles to enable primary cells to start growing and adhere to the wall;
step seven, measuring the number and shape change of tumor cells in a tissue block by adopting a label-free dynamic cell analysis technology, recording the measurement result, adding a mixed solution of a proper amount of material medicine and a culture solution into one group of culture bottles, adding only the culture solution into the other group of culture bottles without adding the material medicine as a control group, and culturing in an incubator for 36 hours;
and step eight, taking out the two groups of culture flasks, then measuring the number and shape change of tumor cells in the tissue block by adopting a label-free dynamic cell analysis technology again, recording the measurement result, comparing the measurement result with the last measurement result, analyzing the number and shape change of the tumor cells in the tissue block between the two measurements, and further judging the sensitivity of the chemical material medicine.
Further, in the second step, the inactivation treatment of the serum requires that the serum be allowed to stand for 30 minutes under sterile, ambient conditions at 56 ℃.
Further, in the third step, the temperature is required to be increased by 30 ℃ in the preparation process of the glutamine replenishment liquid, and after glutamine is completely dissolved, the glutamine is filtered and sterilized, and is packaged into small bottles, and the small bottles are stored at the temperature of 4 ℃, so that the storage time cannot exceed 7 days.
Further, in the label-free dynamic cell analysis technology in the seventh and eighth steps, microelectronic cell sensor chips are integrated on the microporous membrane of the bottom of a cell detection plate or a cell infiltration migration plate, the cell sensor chips are suitable for cell attachment and growth, so as to construct a cell impedance detection sensing system for real-time, dynamic and quantitative tracking of changes of cell morphology, proliferation and differentiation and the like, when cells growing on the surface of the microelectrodes in an attached mode cause changes of interface impedance of the attached electrode, the changes are related to the real-time functional state of the cells, biological information related to physiological functions of the cells can be obtained through the real-time dynamic electrode impedance detection, including cell growth, stretching, morphology change, infiltration, migration, death, attachment and the like, and a real-time label-free cell function analysis system is used in the specific use process, and is an analysis instrument for the fields of biology, preventive medicine and public health.
Further, in step five, the number of flasks in each set is 3 and the plurality of tissue pieces are spaced apart, then a culture medium is added to the flasks and allowed to infiltrate the tissue pieces.
Further, in the second step, the medicine selected when the serum is inactivated is formaldehyde, and the ratio of the serum to the formaldehyde is that the ratio of the serum to the formaldehyde is 1:4000, and washing with physiological saline for 3-5 times after inactivation.
The embodiment of the invention has the following advantages:
1. according to the invention, serum is extracted from the body of a patient to be used as a culture medium and a culture solution is prepared, and part of normal cell tissues around a tumor are extracted while tumor tissues are extracted, so that the normal cell tissues and the tumor tissues can exist in the culture medium simultaneously, the physiological condition of the patient can be simulated to the greatest extent, the detection result of the chemotherapeutic medicine has pertinence, the influence of individual difference of the patient on the detection result is eliminated, and compared with the prior art, the influence of the individual difference of the patient on the detection result is effectively eliminated, so that the more accurate chemotherapeutic medicine sensitivity detection result can be obtained;
2. according to the invention, the tumor cells are cultured in the form of tissue blocks, so that the damage to the cells in the cell dispersion process is reduced, more active tumor cells can be stored, and the cell activity is high, so that the waste of a large amount of detection samples can be avoided, and the extraction times of the samples can be effectively reduced.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are exemplary only and that other implementations can be obtained from the extensions of the drawings provided without inventive effort.
The structures, proportions, sizes, etc. shown in the present specification are shown only for the purposes of illustration and description, and are not intended to limit the scope of the invention, which is defined by the claims, so that any structural modifications, changes in proportions, or adjustments of sizes, which do not affect the efficacy or the achievement of the present invention, should fall within the ambit of the technical disclosure.
FIG. 1 is a schematic view of the overall structure provided by the present invention;
FIG. 2 is a schematic view of the structure of the ultraviolet germicidal lamp and the box cover provided by the invention;
FIG. 3 is a top view of the present invention;
FIG. 4 is a schematic diagram of the structure of two reagent cups and a reagent kit provided by the invention;
FIG. 5 is a schematic diagram of the flow channel and the kit according to the present invention;
FIG. 6 is a schematic diagram of the standpipe, the first solenoid valve, and the flow meter according to the present invention;
FIG. 7 is a schematic view of the structure of the reagent cup and the plurality of dispensing cartridges provided by the present invention;
fig. 8 is an enlarged view of a in fig. 7 provided by the present invention.
In the figure: 1 a reagent box, 2 a reagent cup, 3 a first base, 4 a vertical pipe, 5 a first connecting pipe, 6 a second base, 7 a water outlet pipe, 8 a rubber plug, 9 a spring, 10 a batching barrel, 11 a second connecting pipe, 12 a pipeline joint, 13 a runner, 14 a fan, 15 a third connecting pipe, 16 a first electromagnetic valve, 17 a flowmeter, 18 a second electromagnetic valve, 19 a box cover, 20 an ultraviolet sterilization lamp and 21 a controller.
Detailed Description
Other advantages and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, which, by way of illustration, is to be read in connection with certain specific embodiments, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The embodiment of the invention provides the following technical scheme: the utility model provides a tumour chemotherapy drug sensitivity detection kit and detection method, includes kit 1, kit 1 inside is equipped with two reagent cups 2, two reagent cups 2 bottom threaded connection has first base 3, first base 3 bottom is fixed and is equipped with standpipe 4, standpipe 4 outer end is fixed and is equipped with first solenoid valve 16 and flowmeter 17, flowmeter 17 sets up in first solenoid valve 16 bottom, standpipe 4 outer end fixedly connected with a plurality of first connecting pipes 5, first connecting pipe 5 other end is fixed and is equipped with second base 6, second base 6 and first base 3 top all are fixed and are equipped with outlet pipe 7, a plurality of through-holes have been seted up at outlet pipe 7 outer end top, second base 6 top threaded connection has batching section of thick bamboo 10, outlet pipe 7 top is pegged graft in batching section of thick bamboo 10 inside, batching section of thick bamboo 10 and the inside bottom of thick bamboo 2 of thick bamboo of reagent is pegged graft and is equipped with rubber stopper 8, rubber stopper 8 bottom is fixed and is equipped with a plurality of springs 9, spring 9 bottom and batching section of thick bamboo 10 and reagent cup 2 inside is fixedly connected with a plurality of first connecting pipes 5, the first connecting pipe 5 other end is fixedly equipped with second base 6, second base 6 and is equipped with outlet pipe 7 outside is equipped with a plurality of through-holes at the top of the outlet pipe 7, a plurality of top is offered at the outlet pipe 7 outside the top, a plurality of through-holes, batching section of thick bamboo 6 top threaded connection has a plurality of proportioning tube 10 inside, rubber stopper 8 inside is pegged graft and is equipped with a rubber stopper 8 inside the top end contact with 7, contact.
Further, the air supply mechanism comprises two fans 14, the two fans 14 are respectively and fixedly arranged on two sides of the kit 1, a third connecting pipe 15 is fixedly arranged at the top of each fan 14, one end of each third connecting pipe 15 extends into the kit 1, a runner 13 is arranged in the side wall of the kit 1, one end of each pipeline joint 12 is communicated with the runner 13, and one end of each third connecting pipe 15 extends into the runner 13 and is communicated with the runner 13.
Further, the sterilization mechanism comprises a box cover 19, the box cover 19 is arranged at the top of the kit 1, the rear end of the box cover 19 is movably connected with the rear side of the kit 1 through a damping rotating shaft, a plurality of ultraviolet sterilization lamps 20 are fixedly arranged at the top end inside the box cover 19, a plurality of controllers 21 are fixedly arranged in the middle of the top of the box cover 19, control panels are arranged at the tops of the controllers 21, and the controllers 21 are electrically connected with the box cover 19, the first electromagnetic valve 16, the flowmeter 17 and the second electromagnetic valve 18.
The invention also comprises a detection method using the kit for detecting the sensitivity of the tumor chemotherapeutic medicine, and the specific steps are as follows:
firstly, picking up tumor tissues and partial normal cell tissues around tumors in a sterile environment, then treating the picked tumor tissues and cell tissues, and cutting the tumor tissues and cell tissues into tissue blocks with the size of 1 cubic millimeter by scissors in the sterile environment;
step two, preparing a culture medium by using the kit 1: extracting serum of a tumor patient to serve as a culture medium, inactivating the serum before use, preserving the serum at-20 ℃, adding the extracted serum into a reagent cup 2 on the left side in the kit 1, then respectively placing reagents required for treating the serum into a plurality of dosing cylinders 10 around the reagent cup 2, closing a controller 21, starting an ultraviolet sterilizing lamp 20 through the controller 21, sterilizing the inside of the kit 1 by using the ultraviolet sterilizing lamp 20, starting a blower 14 on the left side through the controller 21, sequentially opening a second electromagnetic valve 18 at the outer ends of a plurality of second connecting pipes 11 on the left side, so that gas generated in the working process of the blower 14 can enter the second connecting pipes 11 through a third connecting pipe 15, a flow passage 13 and a pipeline joint 12, and will enter the inside of the batching cylinder 10 through the second connecting pipe 11, then the gas will push the rubber piston in the batching cylinder 10 to move downwards along the inner wall of the batching cylinder 10, so that the reagent in the batching cylinder 10 can be pushed to move into the second base 6 through the through hole on the water outlet pipe 7, and will move into the reagent cup 2 through the second base 6, the first connecting pipe 5, the vertical pipe 4 and the first base 3, at this moment, the first electromagnetic valve 16 is in an opened state, the flow meter 17 will calculate the flow of the reagent flowing through, so that the reagent can be quantitatively added into the reagent cup 2, so that the serum in the reagent cup 2 can be inactivated by the reagent, when the reagent in the batching cylinder 10 is added to a certain amount, the controller 21 will automatically close the second electromagnetic valve 18 on the second connecting pipe 11 connected with the batching cylinder 10 containing the reagent, to prevent excessive addition of reagents; the serum needs to be kept stand for 30 minutes under the condition of sterility and environmental temperature of 56 ℃; the medicine selected when the serum is inactivated is formaldehyde, and the ratio of the serum to the formaldehyde is 1:4000, and washing 3-5 times by using normal saline after inactivation;
selecting and preparing other common medicaments by using the kit 1, including balanced salt solution, digestive juice, PH regulating solution, antibiotic solution and glutamine supplementing solution, wherein Hank's balanced solution is selected as the balanced salt solution, EDTA and pancreatin mixed solution is selected as the digestive juice, HEPES solution is selected as the PH regulating solution, penicillin and streptomycin mixed solution is selected as the antibiotic solution, glutamine supplementing solution is prepared, and the selected reagents are sequentially added into a plurality of batching cylinders 10 on the right side;
heating to 30deg.C, dissolving glutamine completely, filtering, sterilizing, packaging into small bottles, and storing at 4deg.C for 7 days;
step four, preparing a culture solution: the serum inside the reagent cup 2 processed on the left side inside the reagent box 1 is poured into the reagent cup 2 on the right side, in the process of taking out, the reagent cup 2 is only required to be rotated, so that the reagent cup 2 is separated from the first base 3, then the rubber plug 8 at the bottom end inside the reagent cup 2 can be automatically inserted into the bottom end inside the reagent cup 2 under the action of the elastic force of the spring 9 to prevent water leakage, then the right blower 14 is started, the second electromagnetic valves 18 at the outer ends of the plurality of second connecting pipes 11 on the right side are sequentially opened, so that gas generated in the working process of the blower 14 can enter the second connecting pipes 11 through the third connecting pipes 15, the flow channels 13 and the pipeline joints 12, and can enter the inside of the batching cylinder 10 through the second connecting pipes 11, then the gas can push the rubber piston inside the batching cylinder 10 to move downwards along the inner wall of the batching cylinder 10, so that the reagent in the dosing cylinder 10 can be pushed to move into the second base 6 through the through hole on the water outlet pipe 7, and can move into the reagent cup 2 through the second base 6, the first connecting pipe 5, the vertical pipe 4 and the first base 3, at the moment, the first electromagnetic valve 16 is in an opened state, the flow meter 17 calculates the flow of the reagent flowing through, and can quantitatively add the reagent into the reagent cup 2, when one of the reagent is added, the controller 21 automatically closes the second electromagnetic valve 18 on the second connecting pipe 11 connected with the dosing cylinder 10 containing the reagent to prevent the excessive added reagent, and then the steps are repeated to add other reagents into the reagent cup 2 until a proper amount of Hank's balance liquid, a proper amount of HEPES solution, a proper amount of mixed liquid of penicillin and streptomycin and a proper amount of glutamine supplement liquid are added into the serum culture medium, to obtain a culture solution;
step five, adding a proper amount of antibiotic solution into Hank's balance liquid, then respectively rinsing the cut tumor tissue blocks and cell tissue blocks by utilizing mixed liquid of Hank's balance liquid and the antibiotic solution, rinsing for a plurality of times to wash off blood on the tissue surfaces, and then respectively transferring the rinsed tumor tissue blocks and cell tissue block mixtures into two groups of sterilized culture bottles; the number of culture flasks in each set was 3 and the plurality of tissue pieces were spaced apart, then culture broth was added to the flasks and allowed to infiltrate the tissue pieces;
step six, standing two groups of culture bottles in an incubator for 1-2h, adding culture solution into the culture bottles, turning over the culture bottles for 180 degrees, standing the turned-over culture bottles for 1-2h, and turning over the culture bottles to enable primary cells to start growing and adhere to the wall;
step seven, measuring the number and shape change of tumor cells in a tissue block by adopting a label-free dynamic cell analysis technology, recording the measurement result, adding a mixed solution of a proper amount of material medicine and a culture solution into one group of culture bottles, adding only the culture solution into the other group of culture bottles without adding the material medicine as a control group, and culturing in an incubator for 36 hours;
and step eight, taking out the two groups of culture flasks, then measuring the number and shape change of tumor cells in the tissue block by adopting a label-free dynamic cell analysis technology again, recording the measurement result, comparing the measurement result with the last measurement result, analyzing the number and shape change of the tumor cells in the tissue block between the two measurements, and further judging the sensitivity of the chemical material medicine.
The technology of label-free dynamic cell analysis in the seventh and eighth steps is to integrate microelectronic cell sensor chip to the bottom of cell detection plate or cell infiltration migration plate with surface suitable for cell attachment and growth, to construct cell impedance detection sensing system for real-time, dynamic and quantitative tracking of cell morphology and proliferation differentiation, when the cell growing on the microelectrode surface causes the change of interface impedance of the adherence electrode, the change is correlated with the real-time function state of the cell, biological information related to physiological function of the cell can be obtained through real-time dynamic electrode impedance detection, including cell growth, extension, morphology change, infiltration and migration, death and adherence, etc., and in the specific use process, a real-time label-free cell function analysis system is used, which is an analysis instrument for biology, preventive medicine and public health fields.
While the invention has been described in detail in the foregoing general description and specific examples, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (9)
1. The kit for detecting the sensitivity of the tumor chemotherapeutic drug comprises a kit (1) and is characterized in that: the reagent kit is characterized in that two reagent cups (2) are arranged inside the reagent kit (1), two first bases (3) are fixedly arranged at the bottoms of the reagent cups (2) in a threaded manner, a vertical pipe (4) is fixedly arranged at the bottoms of the first bases (3), a first electromagnetic valve (16) and a flowmeter (17) are fixedly arranged at the outer ends of the vertical pipe (4), a plurality of first connecting pipes (5) are fixedly connected to the outer ends of the vertical pipe (4), a second base (6) is fixedly arranged at the other ends of the first connecting pipes (5), a plurality of through holes are fixedly arranged at the tops of the second bases (6) and the first bases (3), a dispensing cylinder (10) is fixedly arranged at the tops of the outer ends of the outlet pipes (7), the top ends of the outlet pipes (7) are inserted into the interior of the dispensing cylinder (10), rubber plugs (8) are inserted into the inner bottom ends of the dispensing cylinder (2), the bottom ends of the outlet pipe (8) are fixedly connected with a plurality of first connecting pipes (5), a plurality of water outlet pipes (6) are fixedly arranged at the tops of the second bases (3), a plurality of water outlet pipes (7) are fixedly arranged at the tops of the bottoms of the second bases (3), a plurality of dispensing cylinders (10) are fixedly connected with the bottom ends of the dispensing cylinder (10), the utility model discloses a reagent kit, including rubber piston, batching section of thick bamboo (10), rubber piston is established at rubber buffer (8) top, batching section of thick bamboo (10) top is fixed to be equipped with second connecting pipe (11), second connecting pipe (11) outer end is fixed to be equipped with second solenoid valve (18), second connecting pipe (11) one end threaded connection has pipe joint (12), inside reagent kit (1) is pegged graft to pipe joint (12) one end, inside air supply mechanism that is equipped with of reagent kit (1), reagent kit (1) top is equipped with sterilizing mechanism.
2. The kit for detecting sensitivity of a chemotherapeutic agent for tumor according to claim 1, wherein: the air supply mechanism comprises two fans (14), the two fans (14) are respectively and fixedly arranged on two sides of the kit (1), a third connecting pipe (15) is fixedly arranged at the top of the fans (14), one end of the third connecting pipe (15) extends into the kit (1), a runner (13) is arranged in the side wall of the kit (1), one end of the pipeline joint (12) is communicated with the runner (13), and one end of the third connecting pipe (15) extends into the runner (13) and is communicated with the runner (13).
3. The kit for detecting sensitivity of a chemotherapeutic agent for tumor according to claim 1, wherein: the sterilizing mechanism comprises a box cover (19), the box cover (19) is arranged at the top of the kit (1), the rear end of the box cover (19) is movably connected with the rear side of the kit (1) through a damping rotating shaft, a plurality of ultraviolet sterilizing lamps (20) are fixedly arranged at the top end of the inside of the box cover (19), a plurality of controllers (21) are fixedly arranged in the middle of the top of the box cover (19), control panels are arranged at the tops of the controllers (21), and the controllers (21) are electrically connected with the box cover (19), the first electromagnetic valve (16), the flowmeter (17) and the second electromagnetic valve (18).
4. A detection method using the kit for detecting sensitivity of a tumor chemotherapeutic according to any one of claims 1 to 3, comprising the following steps:
firstly, picking up tumor tissues and partial normal cell tissues around tumors in a sterile environment, then treating the picked tumor tissues and cell tissues, and cutting the tumor tissues and cell tissues into tissue blocks with the size of 1 cubic millimeter by scissors in the sterile environment;
preparing a culture medium by using the kit (1): extracting serum of a tumor patient to serve as a culture medium, inactivating the serum before use, preserving the serum at minus 20 ℃, adding the extracted serum into a reagent cup (2) on the left side inside a reagent box (1), then placing reagents required for treating the serum into a plurality of dosing cylinders (10) around the reagent cup (2) respectively, closing a controller (21), starting an ultraviolet sterilizing lamp (20) through the controller (21), sterilizing the inside of the reagent box (1) by using the ultraviolet sterilizing lamp (20), starting a fan (14) positioned on the left side through the controller (21), sequentially opening a second electromagnetic valve (18) at the outer end of a plurality of second connecting pipes (11) on the left side, enabling gas generated in the working process of the fan (14) to enter the inside of the second connecting pipes (11) through a third connecting pipe (15), a runner (13) and a pipeline joint (12), and enter the inside of the dosing cylinders (10) through the second connecting pipes (11), and then pushing the inside of the dosing cylinders (10) to move from the inner walls of the dosing cylinders (10) to the inside of the dosing cylinders (6) through the second connecting pipes (6), and moving from the inner walls of the dosing cylinders (6) to the base (6) through the second connecting pipes (6), and moving the base (6) to the base (6) through the base (6) of the base plate) and the base plate) of the base plate The vertical pipe (4) and the first base (3) move into the reagent cup (2), at the moment, the first electromagnetic valve (16) is in an opened state, the flow meter (17) can calculate the flow of the reagent flowing through, so that the reagent can be quantitatively added into the reagent cup (2), the serum in the reagent cup (2) can be inactivated by the reagent, and when the reagent in the batching barrel (10) is added to a certain amount, the controller (21) can automatically close the second electromagnetic valve (18) on the second connecting pipe (11) connected with the batching barrel (10) containing the reagent, so as to prevent excessive added reagent;
selecting and preparing other common medicaments by using a kit (1), wherein the other common medicaments comprise balanced salt solution, digestive juice, PH regulating solution, antibiotic solution and glutamine supplementing solution, the Hank's balanced solution is selected as the balanced salt solution, EDTA and pancreatin mixed solution is selected as the digestive juice, HEPES solution is selected as the PH regulating solution, penicillin and streptomycin mixed solution is selected as the antibiotic solution, the glutamine supplementing solution is prepared, and the selected reagents are sequentially added into a plurality of dosing cylinders (10) on the right side;
step four, preparing a culture solution: the serum inside the reagent cup (2) processed on the left side inside the reagent box (1) is poured into the reagent cup (2) on the right side, only the reagent cup (2) needs to be rotated in the process of taking out, so that the reagent cup (2) is separated from the first base (3), then the rubber plug (8) at the bottom end inside the reagent cup (2) can be automatically spliced at the bottom end inside the reagent cup (2) under the elastic force of the spring (9) to prevent water leakage, then the right-side fan (14) is started, the second electromagnetic valve (18) at the outer ends of the plurality of second connecting pipes (11) on the right side is sequentially opened, gas generated in the working process of the fan (14) can enter the second connecting pipes (11) through the third connecting pipes (15), the flow channels (13) and the pipeline connectors (12), and can enter the inside of the dosing cylinder (10) through the second connecting pipes (11), then the rubber piston inside the dosing cylinder (10) can be pushed to move downwards along the inner wall of the dosing cylinder (10), the gas can be pushed to move on the dosing cylinder (10) through the first connecting pipes (7) to the first base (6) and the first connecting pipes (6) through the first connecting pipes (6) and the first connecting pipes (6), at this time, the first electromagnetic valve (16) is in an opened state, the flow meter (17) calculates the flow rate of the flowing reagent, so that the reagent can be quantitatively added into the reagent cup (2), when one reagent is added, the controller (21) automatically closes the second electromagnetic valve (18) on the second connecting pipe (11) connected with the batching barrel (10) containing the reagent, so as to prevent excessive added reagent, and then the steps are repeated to add other reagents into the reagent cup (2) until a proper amount of Hank's balance liquid, a proper amount of HEPES solution, a proper amount of mixed liquid of penicillin and streptomycin and a proper amount of glutamine supplement liquid are added into the serum culture medium, so that a culture liquid is prepared;
step five, adding a proper amount of antibiotic solution into Hank's balance liquid, then respectively rinsing the cut tumor tissue blocks and cell tissue blocks by utilizing mixed liquid of Hank's balance liquid and the antibiotic solution, rinsing for a plurality of times to wash off blood on the tissue surfaces, and then respectively transferring the rinsed tumor tissue blocks and cell tissue block mixtures into two groups of sterilized culture bottles;
step six, standing two groups of culture bottles in an incubator for 1-2h, adding culture solution into the culture bottles, turning over the culture bottles for 180 degrees, standing the turned-over culture bottles for 1-2h, and turning over the culture bottles to enable primary cells to start growing and adhere to the wall;
step seven, measuring the number and shape change of tumor cells in a tissue block by adopting a label-free dynamic cell analysis technology, recording the measurement result, adding a mixed solution of a proper amount of material medicine and a culture solution into one group of culture bottles, adding only the culture solution into the other group of culture bottles without adding the material medicine as a control group, and culturing in an incubator for 36 hours;
and step eight, taking out the two groups of culture flasks, then measuring the number and shape change of tumor cells in the tissue block by adopting a label-free dynamic cell analysis technology again, recording the measurement result, comparing the measurement result with the last measurement result, analyzing the number and shape change of the tumor cells in the tissue block between the two measurements, and further judging the sensitivity of the chemical material medicine.
5. The method for detecting sensitivity of a chemotherapeutic agent for tumor according to claim 4, wherein: in the second step, the serum is inactivated by standing the serum for 30 minutes under the conditions of sterility and ambient temperature of 56 ℃.
6. The method for detecting sensitivity of a chemotherapeutic agent for tumor according to claim 4, wherein: in the third step, the preparation process of the glutamine replenishment liquid needs to be heated to 30 ℃, and after glutamine is completely dissolved, the glutamine is filtered and sterilized, and is packaged into small bottles, and the small bottles are stored at the temperature of 4 ℃, so that the storage time cannot exceed 7 days.
7. The method for detecting sensitivity of a chemotherapeutic agent for tumor according to claim 4, wherein: the technology of label-free dynamic cell analysis in the seventh and eighth steps is to integrate microelectronic cell sensor chip to the microporous membrane of the bottom of cell detection plate or cell infiltration migration plate with the surface suitable for cell attachment and growth, to construct a cell impedance detection sensing system for real-time, dynamic and quantitative tracking of cell morphology and proliferation differentiation change, when the cell growing on the microelectrode surface causes the change of the interface impedance of the adherence electrode, the change is related to the real-time functional state of the cell, biological information related to the physiological function of the cell can be obtained through the real-time dynamic electrode impedance detection, including cell growth, extension, morphology change, infiltration and migration, death and adherence, and the real-time label-free cell function analysis system can be used in the specific use process.
8. The method for detecting sensitivity of a chemotherapeutic agent for tumor according to claim 4, wherein: in step five, the number of flasks in each set is 3 and the plurality of tissue pieces are spaced apart, and then culture medium is added to the flasks and allowed to infiltrate the tissue pieces.
9. The method for detecting sensitivity of a chemotherapeutic agent for tumor according to claim 4, wherein: in the second step, the medicine selected in the process of inactivating the serum is formaldehyde, and the ratio of the serum to the formaldehyde is 1:4000, and washing with physiological saline for 3-5 times after inactivation.
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| CN101235350A (en) * | 2007-03-06 | 2008-08-06 | 上海市环境科学研究院 | Soil DNA sample preparation kit and preparation method thereof |
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