CN114885900A - Construction and evaluation method of in vivo chemotherapy drug side effect research model - Google Patents
Construction and evaluation method of in vivo chemotherapy drug side effect research model Download PDFInfo
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Abstract
The invention discloses a construction method and an evaluation method of a model for researching side effects of in vivo chemotherapeutic drugs, wherein the construction method comprises the following steps: preparing bromphenol blue staining food, adding medicaments, collecting fruit fly groups, putting the fruit fly groups into a fruit fly tube, and administering the medicaments to obtain a fruit fly animal model; the evaluation method comprises the following steps: and detecting the survival index, climbing index, abdominal score after dyeing and antioxidant index of the model group and the normal control group. The method uses the dyed food containing the chemotherapeutic drug to feed the drosophila to construct an animal model for researching the side effect of the in vivo chemotherapeutic drug, uses normal food as a control to count the survival rate of the drosophila, and carries out the determination of the movement ability, the determination of the anti-oxidative stress ability and the determination of the food intake when the drosophila begins to die after the food containing cytarabine/irinotecan hydrochloride is fed.
Description
Technical Field
The invention belongs to the technical field of animal model evaluation, and particularly relates to a construction and evaluation method of an in vivo chemotherapy drug side effect research model.
Background
According to the statistics of the world health organization, the number of cancer death cases in the world is 996 ten thousand in 2020, which accounts for about 50% of new cases, and the incidence and mortality of cancer in the world are on the rise. Patients with different stages of disease progression will have different treatment regimens, and chemotherapy will often dominate. However, chemotherapy drugs have general killing property on cells, and when cancer cells are killed, the chemotherapy drugs also cause certain damage to normal cells, which causes cardiotoxicity, lung injury, nephrotoxicity, hepatotoxicity, gastrointestinal toxicity, neurotoxicity and the like, affects the chemotherapy process of tumor patients, and even causes life risks for serious patients.
The side effects of chemotherapy drugs are mostly studied in vivo or in vitro cell experiments, and in the in vivo experiments, the mammal is long in modeling time, high in cost, ethical to animals and the like, so that the research is limited. In vitro experiments only perform toxicity evaluation at the level of single cells in vitro, and tested animals are much more complex relative to single cells, and the metabolism, immunity and other systems are mutually linked, so that even the test of single toxicant is also subjected to unpredictable interference, the metabolic kinetics verification at the level of the whole organism is not available, and the whole toxicity after the action of each system in the in vivo environment cannot be accurately presumed. Therefore, a more suitable animal model for the side effect of the chemotherapeutic medicament is lacked, so that the research and screening of the side effect mechanism of the chemotherapeutic medicament for relieving the side effect thereof are in the bottleneck.
Drosophila melanogaster (Drosophila melanogaster) is an ideal model organism for researching human disease generating mechanism, and with the continuous abundance of Drosophila pathological models and transgenic Drosophila strains, Drosophila melanogaster models are widely applied to the research of reproductive, immune, neural, cardiovascular and other systems and disease mechanisms such as neurodegenerative diseases, metabolic syndrome, sleep and the like. The drosophila melanogaster has a digestive system and a metabolic system similar to those of mammals, is applied to drug toxicity evaluation, and has potential application value in the research of side effects of chemotherapeutic drugs.
Disclosure of Invention
The invention aims to provide a construction method of a model for researching the side effect of an in vivo chemotherapeutic medicament;
the second purpose of the invention is to provide an evaluation method of a side effect research model of the in vivo chemotherapy medicament;
the animal model is constructed and evaluated to establish a high-throughput screening platform for reducing the spectrum of the medicine for reducing the body injury caused by the chemotherapeutic medicine.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for constructing a model for researching side effects of in vivo chemotherapeutic drugs comprises the following steps:
s1: adding bromophenol blue dye into fruit fly food, uniformly mixing to obtain dyed food, equally dividing the dyed food, then placing the dyed food into fruit fly tubes, respectively placing the prepared cytarabine and irinotecan hydrochloride into the fruit fly tubes, and marking to obtain cytarabine fruit fly tubes and irinotecan hydrochloride fruit fly tubes;
s2: using CO for fruit fly 2 After anesthesia, collecting 3-day-old male and female drosophila melanogaster, randomly grouping, wherein each tube is provided with 20 flies, the male and female drosophila melanogaster are separated, each group is repeated for 3 times and respectively placed in the cytarabine drosophila arabica tube and the irinotecan hydrochloride drosophila tube obtained from S1;
s3: the stained food is renewed every 2-3 days, and the drosophila animal model can be obtained after administration for 9-10 days.
To further implement the present invention, the concentration of cytarabine in S1 is 1mM-10mM and the concentration of irinotecan hydrochloride is 1mM-5 mM.
To further implement the present invention, the concentration of the bromophenol blue dye in S1 is 0.01% to 0.5%.
The evaluation method of the in vivo chemotherapy drug side effect research model comprises the steps of establishing a normal control group, taking the drosophila animal model obtained from S3 as a model group, and detecting the following indexes of the model group and the normal control group:
(1) survival index;
(2) a climbing index;
(3) scoring the abdominal area after staining;
(4) anti-oxidation index;
and (3) carrying out difference analysis on the index data and the scores of the model group and the normal control group by using a statistical method, establishing a qualified value for the indexes, and if the index data and the scores of the model group are significantly different from those of the normal control group and the model group reaches the qualified value, determining that the model is successfully constructed.
In order to further realize the invention, the method for detecting the survival index comprises the following steps: and (3) continuously administering the drosophila animal model obtained in the step (S3), observing the drosophila state for 3-4 times every day, recording the death condition of the drosophila until all drosophila in a model group with cytarabine administration concentration being more than or equal to 5mM or irinotecan hydrochloride administration concentration being more than or equal to 2.5mM die, and calculating the survival rate of the model group:
compared with the survival rate of a normal control group, the survival rate of the cytarabine administration model group is lower than 70% at the 22 th day, and the survival rate of the irinotecan hydrochloride administration model group is lower than 80% at the 22 th day.
In order to further realize the invention, the detection method of the climbing index comprises the following steps: transferring the fruit fly animal model obtained in the step S3 and the normal control group to a climbing pipe with the length of 15cm, recording the number of fruit flies which climb 8cm in the climbing pipe for 8S, and calculating the climbing index of the model group:
compared with the survival rate of a normal control group, the survival rate of the model group has significant difference, and the climbing index of the model group is lower than 65 percent, namely the model group is qualified.
In order to further realize the invention, the detection method of the abdominal score after dyeing comprises the following steps: transferring the fruit fly animal model obtained in S3 and normal control group into fruit fly tube containing 1% agar only, starving for 16-21h, transferring into fruit fly tube containing 2% bromophenol blue dye agar, feeding for 3-8h, and collecting fruit fly CO 2 After anaesthesia in CO 2 Abdomen scoring of each group of drosophila on air-permeable plate:
(1) 1 point (drosophila) with light blue dye or stained food less than one third of the abdominal volume;
(2) the blue dye food accounts for 2 minutes of half of the abdomen of the drosophila melanogaster;
(3) the content of the blue dye is 3 minutes more than half of the abdomen;
and the abdominal score of the research model group is qualified when the abdominal score is lower than 1.75 after the dyeing.
In order to further realize the invention, the method for detecting the antioxidant stress index comprises the following steps: transferring the fruit fly animal model obtained in the step S3 and the normal control group into an empty fruit fly tube for hungry for 1h, transferring into a fruit fly tube paved with filter paper, dropping 300 mu L of hydrogen peroxide solution, transferring the hungry fruit fly into the tube after the hydrogen peroxide solution completely permeates the filter paper, carrying out 3-4 times of fruit fly state observation every day, changing agar every 2-3 times until all the fruit flies of the research model group and the normal control group die, and calculating the survival rate:
in the research model group, the survival rate of the male fruit flies at 42h is lower than 30%, and the survival rate of the female fruit flies at 42h is lower than 70%, and the fruit flies are qualified.
The utility model provides a fruit fly pipe that is used for chemotherapy medicine side effect research model to construct in vivo, fruit fly pipe includes body (1) and lid (2), and the bottom of body (1) sets up dyeing food layer (3), and the top surface of dyeing food layer (3) sets up tray (4), sets up in tray (4) and doses layer (5).
Compared with the prior art, the invention has the beneficial effects that:
the invention uses the drosophila food containing the chemotherapeutic drug cytarabine or irinotecan hydrochloride to feed drosophila melanogaster of 3 days old so as to construct an animal (drosophila) model for researching the side effect of the chemotherapeutic drug in vivo, uses normal food as a control, counts the survival rate of the drosophila melanogaster, and carries out the exercise capacity measurement, the anti-oxidative stress capacity measurement and the food intake measurement when the drosophila melanogaster begins to die after the food containing cytarabine/irinotecan hydrochloride is fed, and the result shows that cytarabine/irinotecan hydrochloride causes the lethal increase of the drosophila melanogaster, the exercise capacity, the anti-stress capacity and the food intake reduction, and is used for evaluating whether the model is successfully constructed or not. Compared with other traditional model organisms, the drosophila melanogaster experimental system has the advantages that within an experimental period of 25 days, drosophila melanogaster propagation speed is high, feeding cost is low, the experimental period is short, and experimental effects are remarkable, and the drosophila melanogaster experimental system can be used for establishing and evaluating an animal injury (drosophila melanogaster) model with side effects caused by chemotherapy drugs in drosophila melanogaster bodies, so that a high-throughput screening platform for reducing the spectrum of the drugs for body injury caused by the chemotherapy drugs is established.
The traditional drug administration mode is improved, the traditional drug administration mode is that the drugs are dissolved in basic food and poured into the bottom of the fruit fly tube, fruit flies cannot independently select to eat but only take food in a forced mode, the fruit flies can meet daily consumption only by needing less food, the area of the fruit fly tube is large, reagent waste and the like exist to a certain extent, and the experiment cost is high. In contrast, the novel fruit fly tube is designed for administration, non-metabolizable bromophenol blue dye is added into food, whether blue areas exist in the abdomen of the fruit flies can be observed in real time to ensure that the fruit flies take in the food containing the medicament, meanwhile, because the periphery of the tray is 1% of agar, the fruit flies can independently choose to drink or eat water, the independent diet of the fruit flies is greatly increased, and the independent diet selectivity similar to that of mammals is simulated as much as possible.
The invention also supplements the model organisms for researching the side effect of the chemotherapy drugs, makes up the defect of lack of integrity of in vitro cell experiments, enriches the types of animals in vivo experiment modes, and greatly reduces the research economy and time cost. The research on the side effect pathogenesis of the chemotherapeutic drug and the high-throughput screening of the anti-chemotherapeutic drug are very favorable.
Drawings
FIG. 1 is a graph showing the survival curves of Drosophila raised by cytarabine with different concentrations in the present invention, expressing the effect of cytarabine with different concentrations on the life span of Drosophila, wherein the graph A is the survival curve of male flies raised by cytarabine with different concentrations (n = 100-. P <0.0001 indicates that the difference is statistically significant;
FIG. 2 is a graph showing the survival curves of male flies raised with different concentrations of irinotecan hydrochloride, expressing the effect of irinotecan hydrochloride at different concentrations on the longevity of fruit flies, graph A shows the survival curves of male flies raised with irinotecan hydrochloride at different concentrations (n = 74-120), graph B shows the survival curves of female flies raised with irinotecan hydrochloride at different concentrations (n = 73-120), and graph P <0.01, wherein P <0.0001 shows that the difference is statistically significant;
FIG. 3 is a climbing index of feeding Drosophila with cytarabine of different concentrations, expressing the effect of cytarabine of different concentrations on the movement ability of Drosophila, wherein A is a climbing index (n = 8) of 8cm climbed by 8s of male flies fed with cytarabine of different concentrations for 10 days, and B is a climbing index (n = 8) of 8cm climbed by 8s of female flies fed with cytarabine of different concentrations for 10 days, p <0.01, p <0.0001 indicates that the difference is statistically significant;
FIG. 4 is the abdominal score and absorbance of Drosophila fed with cytarabine at different concentrations, and the influence of expression of cytarabine at different concentrations on the feed intake of Drosophila, wherein A is the abdominal score of male flies (n = 79) fed with cytarabine at different concentrations for 10 days, and B is the abdominal score of female flies (n = 90) fed with cytarabine at different concentrations for 10 days;
FIG. 5 is a male fly survival curve of different concentrations of cytarabine under oxidative stress conditions for 10 days, expressing the effect of different concentrations of cytarabine on the antioxidant stress capacity of drosophila, A is a male fly survival curve (n = 160) of different concentrations of cytarabine under oxidative stress conditions for 10 days, B is a female fly survival curve (n = 140) of different concentrations of cytarabine under oxidative stress conditions for 10 days, p is less than 0.001, and p is less than 0.0001, which shows that the difference has statistical significance;
FIG. 6 is a schematic structural view of a Drosophila tube according to the present invention;
the reference numerals have the following meanings: 1. a pipe body; 2. a cover body; 3. an agar layer; 4. a tray; 5. an administration layer.
Detailed Description
The invention is further described with reference to the following figures and detailed description.
A method for constructing a model for researching side effects of in vivo chemotherapeutic drugs comprises the following steps:
s1: adding bromophenol blue dye into fruit fly food, uniformly mixing to obtain dyed food, equally dividing the dyed food, then placing the dyed food into fruit fly tubes, respectively placing the prepared cytarabine and irinotecan hydrochloride into the fruit fly tubes, and marking to obtain cytarabine fruit fly tubes and irinotecan hydrochloride fruit fly tubes;
s2: using CO for fruit fly 2 After anesthesia, collecting 3-day-old male and female drosophila melanogaster, randomly grouping, wherein each tube is provided with 20 flies, the male and female drosophila melanogaster are separated, each group is repeated for 3 times and respectively placed in the cytarabine drosophila arabica tube and the irinotecan hydrochloride drosophila tube obtained from S1;
s3: the stained food is renewed every 2-3 days, and the drosophila animal model can be obtained after administration for 9-10 days.
The concentration of cytarabine in S1 is 1mM-10mM, the concentration of irinotecan hydrochloride is 1mM-5mM, and the concentration of bromophenol blue dye is 0.01% -0.5%.
Example 1, the method for constructing a model for studying the side effects of an in vivo chemotherapeutic agent comprises the following steps:
s1: adding bromophenol blue dye into drosophila melanogaster food, uniformly mixing to obtain dyed food, equally dividing the dyed food, filling the dyed food into drosophila melanogaster tubes, preparing cytarabine-containing and irinotecan hydrochloride with concentrations of 1mM, 5mM and 10mM respectively, and placing and marking the prepared cytarabine and the irinotecan hydrochloride into the drosophila melanogaster tubes respectively to obtain cytarabine drosophila melanogaster tubes of a low administration group, a middle administration group and a high administration group and irinotecan hydrochloride drosophila tubes of the low administration group, the middle administration group and the high administration group;
s2: using CO to fruit fly 2 After anesthesia, collecting 3-day-old male and female drosophila melanogaster, randomly grouping, wherein each tube is provided with 20 flies, the male and female drosophila melanogaster are separated, each group is repeated for 3 times and respectively placed in the cytarabine drosophila arabica tube and the irinotecan hydrochloride drosophila tube obtained from S1;
s3: the stained food is renewed every 2-3 days, and the drosophila animal model can be obtained after administration for 9-10 days.
Example 2, the chemotherapy drugs have general lethality to organisms, the viability is one of important indexes for reflecting whether the drugs have toxicity, and the toxicity of the chemotherapy drugs to the organisms can be intuitively explained by detecting the survival index of the drosophila flies fed with the chemotherapy drugs. The survival index detection method comprises the following steps:
and (3) continuing to administer the drosophila animal model obtained in the step S3, observing the drosophila state 3-4 times a day, recording the death condition of the drosophila until all the drosophila die in a model group with cytarabine administration concentration being more than or equal to 5mM or irinotecan hydrochloride administration concentration being more than or equal to 2.5mM, and calculating the survival rate of the model group:
compared with the survival rate of a normal control group, the survival rate of the cytarabine administration model group is lower than 70% at the 22 th day, and the survival rate of the irinotecan hydrochloride administration model group is lower than 80% at the 22 th day.
As can be seen from fig. 1-2, cytarabine and irinotecan hydrochloride were able to significantly shorten the life span of drosophila, and as the concentration of chemotherapeutic agent increased, the life span of drosophila gradually shortened. Research shows that the chemotherapeutic medicine has lethal effect. Wherein the survival rate of the male flies is 40.09 percent and the survival rate of the female flies is 65.50 percent when 1mM cytarabine is fed for 22 days, the survival rate of the male flies is 0 when 5mM cytarabine and cytarabine are fed for 22 days, the survival rate of the female flies is 1.65 percent, and the survival rates of the male flies and the female flies are 0 when 10mM cytarabine and cytarabine are fed for 22 days. The survival rate of male flies is 10.42 percent and 78.28 percent when 1mM irinotecan hydrochloride is fed for 22 days, the survival rate of male flies is 0 when 2.5mM irinotecan hydrochloride is fed for 22 days, the survival rate of female flies is 32.61 percent, and the survival rates of male flies and female flies are both 0 when 5mM irinotecan hydrochloride is fed for 22 days.
From the above, the survival rates of the cytarabine low-dose group, the cytarabine medium-dose group and the cytarabine high-dose group and the survival rates of the irinotecan hydrochloride low-dose group, the cytarabine medium-dose group and the irinotecan hydrochloride high-dose group have significant differences compared with the survival rate of a normal control group, and the survival indexes are qualified in detection, namely the model is successfully constructed.
Example 3 climbing is the embodiment of the fruit fly's motor ability, the nervous system of fruit fly can regulate its motor ability, when chemotherapy drug causes the nerve injury of organism, then can reduce the motor ability of fruit fly. The strength of the fruit fly movement ability can be reflected by detecting the fruit fly climbing index. The detection method of the climbing index comprises the following steps:
transferring the fruit fly animal model obtained in the step S3 and the normal control group to a climbing pipe with the length of 15cm, recording the number of fruit flies which climb 8cm in the climbing pipe for 8S, and calculating the climbing index of the model group:
compared with the survival rate of a normal control group, the survival rate of the model group has significant difference, and the climbing index of the model group is lower than 65 percent, namely the model group is qualified.
As can be seen from FIG. 3, cytarabine can reduce the motility of fruit flies, and the motility and climbing ability of the fruit flies is gradually reduced along with the increase of the concentration. The research shows that cytarabine can cause nerve injury, so that the motor ability is reduced. Wherein the climbing index of the male flies in the normal control group is 91.23%, the climbing indexes of the male flies in the cytarabine low administration group, the cytarabine middle administration group and the cytarabine high administration group are 64.12%, 29.17% and 16.38% respectively, and the climbing indexes of the male flies in the normal control group are 70.28%, 31.98% and 17.95% respectively; the climbing index of the female flies in the normal control group is 83.74 percent, the climbing indexes of the female flies in the cytarabine low-dose group, the cytarabine middle-dose group and the cytarabine high-dose group are 72.62 percent (without statistical significance), 40.13 percent and 18.88 percent respectively, and the climbing indexes of the female flies in the normal control group are 86.73 percent, 47.92 percent and 22.55 percent respectively
From the above, the climbing indexes of the cytarabine low-dose group, the cytarabine medium-dose group and the cytarabine high-dose group have significant difference compared with the climbing index of the normal control group, and the climbing index detection is qualified, namely the model construction is successful.
Example 4, food intake is one of the indexes of normal physiology of the body, when the chemotherapy drug causes damage to the body, the food intake of the drosophila is influenced, and the food intake can be reflected by the detection of abdominal scores and absorbance of the drosophila. The detection method of the abdominal score after staining comprises the following steps:
transferring the fruit fly animal model obtained in S3 and normal control group into fruit fly tube containing 1% agar only, starving for 16-21h, transferring into fruit fly tube containing 2% bromophenol blue dye agar, feeding for 3-8h, and collecting fruit fly CO 2 After anaesthesia in CO 2 Abdomen scoring of each group of drosophila on air-permeable plate:
(1) 1 point for (drosophila) with light blue dye or colored food occupying less than one third of the abdominal volume;
(2) the blue dye food accounts for 2 minutes of the belly of the fruit fly;
(3) the content of the blue dye is 3 minutes more than half of the abdomen;
and the abdominal score of the research model group is qualified when the abdominal score is lower than 1.75 after the dyeing.
As can be seen from FIG. 4, cytarabine was able to decrease feed intake by Drosophila, which decreased gradually with increasing concentration. The research shows that the cytarabine can cause metabolic disorder and reduce the food intake. The abdominal score of the male flies in the normal control group was 2.07 points, and the abdominal scores of the male flies in the cytarabine low-dose group, the cytarabine medium-dose group and the cytarabine high-dose group were 1.88 points (no statistical significance), 1.76 points (no statistical significance) and 1.39 points, which were 90.82%, 85.02% and 67.15% of the normal control group, respectively; the abdominal scores of the female flies in the normal control group were 2.03 points, and the abdominal scores of the female flies in the cytarabine-low administration group, the cytarabine-medium administration group and the cytarabine-high administration group were 1.54, 1.64 and 1.52 points, which were 75.86%, 80.79% and 78.88% of the normal control group, respectively.
As can be seen from the above, the stained abdominal scores of the cytarabine low-dose group, the cytarabine medium-dose group and the cytarabine high-dose group have significant differences compared with the stained abdominal score of the normal control group, and the stained abdominal scores are all qualified, i.e., the model construction is successful.
Example 5 in a normal state, the body has an extremely strong antioxidant stress ability, and when the body is damaged, the antioxidant stress ability of the body is lowered, thereby damaging the body. The damage condition of the medicament to the organism can be reflected by detecting the antioxidant index of the drosophila melanogaster. The detection method of the antioxidant stress index comprises the following steps: transferring the fruit fly animal model obtained in the step S3 and the normal control group into an empty fruit fly tube for hungry for 1h, transferring into a fruit fly tube paved with filter paper, dropping 300 mu L of hydrogen peroxide solution, transferring the hungry fruit fly into the tube after the hydrogen peroxide solution completely permeates the filter paper, carrying out 3-4 times of fruit fly state observation every day, changing agar every 2-3 times until all the fruit flies of the research model group and the normal control group die, and calculating the survival rate:
in the research model group, the survival rate of the male fruit flies at 42h is lower than 30%, and the survival rate of the female fruit flies at 42h is lower than 70%.
As can be seen in FIG. 5, cytarabine can reduce the antioxidant stress capacity of Drosophila, indicating that cytarabine can cause oxidative damage to the body. The survival rate of the normal control group male flies is 42.17% when the normal control group male flies are stimulated by hydrogen peroxide for 42 hours, and the survival rates of the cytarabine low-dose group male flies, the cytarabine medium-dose group male flies and the cytarabine high-dose group male flies are 41.31% (without statistical significance), 38.96% (without statistical significance) and 27.96% respectively; the survival rate of the female flies in the normal control group was 87.62%, and the survival rates of the female flies in the cytarabine-low administration group, the female flies in the medium administration group and the female flies in the high administration group were 88.76% (not statistically significant), 65.16% and 63.99%, respectively.
From the above, the survival rates of the cytarabine low-dose group, the cytarabine medium-dose group and the cytarabine high-dose group are remarkably different from the survival rate of the normal control group, and the detection of the antioxidant stress indexes is qualified, namely the model construction is successful.
As shown in fig. 6, a drosophila tube for the in vivo chemotherapy drug side effect research model construction according to claim 1, the drosophila tube described in S2 comprises a tube body 1 and a cover body 2, wherein the bottom of the tube body 1 is provided with a dyed food layer 3, the top of the dyed food layer 3 is provided with a tray 4, and an administration layer 5 is arranged in the tray 4.
Claims (9)
1. A construction method of a model for researching side effects of in vivo chemotherapeutic drugs is characterized by comprising the following steps:
s1: adding bromophenol blue dye into fruit fly food, uniformly mixing to obtain dyed food, equally dividing the dyed food, then placing the dyed food into fruit fly tubes, respectively placing the prepared cytarabine and irinotecan hydrochloride into the fruit fly tubes, and marking to obtain cytarabine fruit fly tubes and irinotecan hydrochloride fruit fly tubes;
s2: using CO for fruit fly 2 After anaesthesia, 3 days old male and female fruit flies were collected and randomly grouped, 20 flies per tube, male and female separated, each group was repeated 3 timesRespectively placing the obtained product in the cytarabine drosophila tube obtained in the step S1 and an irinotecan hydrochloride drosophila tube;
s3: the stained food is renewed every 2-3 days, and the drosophila animal model can be obtained after administration for 9-10 days.
2. The method for constructing a model for studying side effects of an in vivo chemotherapeutic agent according to claim 1, wherein: the concentration of the cytarabine in S1 is 1mM-10mM, and the concentration of the irinotecan hydrochloride is 1mM-5 mM.
3. The method for constructing a model for studying side effects of an in vivo chemotherapeutic agent according to claim 1, wherein: the concentration of the bromophenol blue dye in S1 is 0.01% -0.5%.
4. The method for evaluating a model for studying adverse effects of an in vivo chemotherapeutic agent according to any of claims 1-3, wherein a normal control group is established, the drosophila animal model obtained in S3 is used as the model group, and the following criteria are examined for the model group and the normal control group:
(1) survival index;
(2) a climbing index;
(3) abdominal scoring after staining;
(4) anti-oxidation index;
and (3) carrying out difference analysis on the index data and the scores of the model group and the normal control group by using a statistical method, establishing a qualified value for the indexes, and if the index data and the scores of the model group are significantly different from those of the normal control group and the model group reaches the qualified value, determining that the model is successfully constructed.
5. The method for evaluating the model for studying the side effect of the in vivo chemotherapeutic agent according to claim 4, wherein: the survival index detection method comprises the following steps: and (3) continuing to administer the drosophila animal model obtained in the step S3, observing the drosophila state 3-4 times a day, recording the death condition of the drosophila until all the drosophila die in a model group with cytarabine administration concentration being more than or equal to 5mM or irinotecan hydrochloride administration concentration being more than or equal to 2.5mM, and calculating the survival rate of the model group:
compared with the survival rate of a normal control group, the survival rate of the cytarabine administration model group is lower than 70% at the 22 th day, namely the cytarabine administration model group is qualified, and the survival rate of the irinotecan hydrochloride administration model group is lower than 80% at the 22 th day, namely the cytarabine hydrochloride administration model group is qualified.
6. The method for evaluating the model for the study of side effects of an in vivo chemotherapeutic agent according to claim 5, wherein: the detection method of the climbing index comprises the following steps: transferring the fruit fly animal model obtained in the step S3 and the normal control group to a climbing pipe with the length of 15cm, recording the number of fruit flies which climb 8cm in the climbing pipe for 8S, and calculating the climbing index of the model group:
compared with the survival rate of a normal control group, the survival rate of the model group has significant difference, and the climbing index of the model group is lower than 65 percent, namely the model group is qualified.
7. The method for evaluating the model for studying the side effect of the in vivo chemotherapeutic agent according to claim 6, wherein: the detection method of the abdominal score after staining comprises the following steps: transferring the fruit fly animal model obtained in S3 and normal control group into fruit fly tube containing 1% agar only, starving for 16-21h, transferring into fruit fly tube containing 2% bromophenol blue dye agar, feeding for 3-8h, and collecting fruit fly CO 2 After anaesthesia in CO 2 Abdomen scoring of each group of drosophila on air-permeable plate:
(1) 1 point for (drosophila) with light blue dye or colored food occupying less than one third of the abdominal volume;
(2) the blue dye food accounts for 2 minutes of half of the abdomen of the drosophila melanogaster;
(3) the content of the blue dye is 3 minutes more than half of the abdomen;
and the abdominal score of the research model group is qualified when the abdominal score is lower than 1.75 after the dyeing.
8. The method for evaluating the model for studying the side effect of an in vivo chemotherapeutic agent according to claim 7, wherein: the detection method of the antioxidant stress index comprises the following steps: transferring the fruit fly animal model obtained in the step S3 and the normal control group into an empty fruit fly tube for hungry for 1h, transferring into a fruit fly tube paved with filter paper, dropping 300 mu L of hydrogen peroxide solution, transferring the hungry fruit fly into the tube after the hydrogen peroxide solution completely permeates the filter paper, carrying out 3-4 times of fruit fly state observation every day, changing agar every 2-3 times until all the fruit flies of the research model group and the normal control group die, and calculating the survival rate:
in the research model group, the survival rate of the male fruit flies at 42h is lower than 30%, and the survival rate of the female fruit flies at 42h is lower than 70%, and the fruit flies are qualified.
9. A drosophila tube for use in the in vivo chemotherapeutic drug side effect study model construction of claim 1, wherein: the fruit fly tube comprises a tube body (1) and a cover body (2), wherein a dyed food layer (3) is arranged at the bottom of the tube body (1), a tray (4) is arranged on the top surface of the dyed food layer (3), and an administration layer (5) is arranged in the tray (4).
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| US6291739B1 (en) * | 2000-03-24 | 2001-09-18 | Council Of Scientific And Industrial Research | Method for screening of potential anti-epileptic drugs using a Drosophila melanogaster model |
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