Language
动物实验
z-bo
2020-07-16
951
When you are doing animal experiments, there are often problems with unstable models, confusing results, or too large individual differences in data. Based on some experiments I have done, I try to summarize some experiences, which is more suitable for comrades who are new to animal experiments. For the masters of animal experiments, please also advise a lot, and hope to talk to the post and talk about your own experience
Most of the comrades-in-arms need to do animal experiments when they do graduation projects, and some are based on animal experiments. Many of the students who are mainly graduate students have not been exposed to animal experiments, and the foundation is relatively weak. For this part of the students, you can first find previous posts, look at other people’s discussions, read more literature, and clarify ideas, but we will also It is found that there are many details in the literature, and the experience of others may not be suitable for you. So only by doing more pre-experiments and thinking can you complete a animal experiment better. I will talk about my views from the following aspects:
1. Animal sex and weight selection:
The most commonly used are large mice, rabbits, dogs and monkeys are often used in some evaluation institutions. Big mice have canceled the ordinary level, only clean level and higher. For the sex chosen by the animal, it is better to choose the male and the female half, because female mice are affected by their own hormones and may get different results. Kunming mice are not very recognized abroad, so it is best not to use Kunming mice if you want to post a foreign language. Because Kunming mice are hybrids, the immunity is relatively strong, so it is better to avoid choosing Kunming mice when making similar models of immunocompromise. It is better to use bablc. In the model of drug modeling, sometimes the dose of Kunming mice is also used. Higher than other rats. For hot plate experiments, male mice should be selected, female mice should be selected for ovulation, etc. It is basically wrong to look at the literature; rats often use SD and Wistar, and the difference between the two is not very big.
The weight of the mice is preferably 18-22g. The weight of the mice is too large, and the results are often not very good. The difference is relatively large, especially for acute toxicity tests. The weight should not be too large, and the range of weight fluctuations should not be too large. This guiding principle has written. For some experiments on nude mice, if the experiment period is relatively long, it is best to choose 3 or 4w mice, because nude mice are easy to die due to aging in the later period. This has had a bitter lesson. Rats use 180-220g as a routine choice. Regardless of rats and mice, I personally recommend not to give too much food and grow too fat. Although generally a free diet required, one reason is that it is wasteful (many of them are molars) Lost), one is to increase the dosage (the volume of the gavage needs to be adjusted according to the dosage, as will be described below), and the other, based on experience, some test data are not very good. The above is more conventional. Of course, some special experiments choose the species and weight of the mouse because of the experiment. For example, the experiment of hair growth. C57 and C3H are more selected at home and abroad, and the hair has a growth cycle. Yes, you have to check the animal hair growth cycle. For example, when C3H is at 7-8 weeks, the hair is in the resting period. It is more suitable for drug research. For example, if you study sexual function, the mouse is only sexually mature at 8w. Therefore, we must choose a rat of about 8w. Of course, the specific problems should be analyzed in detail. Here, it is only said that the choice of rat species and weight is the first step to success.
2. Administration dose, administration volume, administration method, administration time:
The dosage of positive drugs is generally based on human conversion, or direct reference to the literature. There is a lot of discussion about the dosage forum, but the FDA has a guideline that has changed the conversion factor of humans and mice to 12.3. Moreover, some books in China are based on the weight of 70kg, and some are based on 60kg. Personally, 60kg is more reasonable. The dose conversion of young rats is different from that of adult rats, and it cannot be converted in the same way. If it is a brand-new drug, it should be a toxicology. According to the results of acute toxicity, three gradients are designed for the experiment. Generally, the gradient is designed with a gradient of 2 or 3 times. Some students did not do toxicology, only made cells. The dose of cells could not be calculated on animals. If you really don’t have enough energy and drugs to do toxicology, you can find the dosage of compounds with similar structure as For reference, the dose of western medicine and the dose of traditional Chinese medicine are not on the same order of magnitude. If you design an animal to be administered at a too large dose, you will be questioned about the problem of medicine and research significance. For example, some new compounds are calculated on the human body. , I have to eat a few kilograms a day. Obviously, such research is meaningless, even if the animal has an effect, it will be questioned. Some traditional Chinese medicine extracts are designed to have a too small dose, which is smaller than the dose of positive drugs (chemical drugs), so it is obviously difficult to make a result. Some doses are reasonable, but compared with the same type of positive drugs, the dose is too large, then others will question the advantages of your compound (generally, the greater the dose, the greater the toxic side effects), if the drug effect It can't be significantly better than the positive drug, then your compound's disadvantage is obvious. By the way, let’s take a positive drug. The positive drug should be comparable to the drug you are studying. For example, if you are a Chinese medicine extract, then you must design a control drug for Chinese medicine on the market, and you should choose a similar structure and a similar way of action. To increase comparability.
After the dosage is determined, the administration volume should be calculated. The administration volume should not be too large or too small. For example, 0.05 mL of tail vein injection is a bit too small. If you accidentally expose some or remain some, the dose is very inaccurate, for example Rats are gavaged with 10mL per day, which is too large and easy to flow out after gavage. If the gavage is divided twice in the afternoon and again, it is not ethical, so we still have to calculate the best administration volume and increase the drug concentration.
About the administration method, it is better to be close to the clinical administration method. If there is really no way to give tail vein injection, you can choose intraperitoneal injection instead. In addition, the suspension can not be injected intraperitoneally. When the stomach is lavaged, the suspension should be shaken every few times, but it is best to make a solution, such as adding CMC-Na, DMSO, ultrasound, heating, etc. If the oil solution can be dissolved, the control group should choose an oil solution for gastric gavage. If some of the test drugs are dissolved in oil and normal saline, then the control group can choose to use only the oil solution for long-term intragastric administration. The solution will affect the biochemical indicators, so the control group must be set. Sometimes the animal serum given to the oil solution is white, indicating that the impact is still very large. Regardless of intragastric administration, intraperitoneal administration, and intravenous administration, practicing techniques is the most basic. Poor intragastric irrigation can easily cause animal death or esophageal injury. It is difficult to administer intragastric administration in the future. Well, if you hit the liver or bladder, the effect of the drug will not work properly. Many of these techniques have also been discussed by comrades in arms, so it is no longer cumbersome. In addition, the normal control group must also be administered. For example, some control groups are given normal saline. In order to save trouble, they are not given normal saline every time. As a result, when the material is found, the control group is very important and you cannot judge the weight of the model group. The decline is caused by gavage or drugs, which leads to many unexplainable problems, so it is still necessary to standardize operations and unify operations.
Dosing time: It is best to fix it every day. The evaluation of hormone drugs should be combined with the hormone release rule of animals. The blood sugar and blood pressure drugs should be selected at the appropriate time. There is an important one-time administration in the time of administration, which is the last administration. The following focuses on this.
3. Random grouping, last dose, parallel trial
Let’s talk about random grouping first. Most trials are randomized by weight, but some have also been grouped with biochemical indicators, such as blood sugar and blood pressure. Then the indicators must be measured again and then grouped. White mice can be smeared with ***, black mice can reduce ears , Ear tags and other methods, it is recommended to use ear tags, it is not easy to make mistakes (but ear tags are not available for ear tests, such as xylene-induced ear swelling test). If it is modeled first, it should be grouped according to the model, such as diabetes experiments. If it is surgical modeling, there are no specific indicators, you can make a score and group by score. The last administration is very important for the experiments of lowering blood pressure and blood sugar, but it is not very important for long-term administration, adjuvant therapy, traditional Chinese medicine. Taking blood pressure as an example, the best time to lower blood pressure for this medicine is How much, if it is 5h, and your blood pressure is measured 1h after the administration, it is obviously unreasonable. Even if it is a long-term administration, the last administration is also very important. Here again involves the problem of parallel trials. This I think is The key to the success or failure of many experiments must be listed at a time point, so that the experimenter does not know the group, use the serial number operation to avoid the impact of psychological effects, the person who picks the material should also be fixed, the heart is taken only the heart, the lung Just take the lungs and so on. The parallel experiment will greatly improve the quality of the experimental data. It is particularly important in experiments like blood pressure lowering, uric acid lowering, and blood sugar lowering. If you have time to talk about it in detail, I hope that experienced comrades can put forward their opinions.
The above is a relatively basic process of doing animal experiments. To a certain extent, it can improve the stability of the model and data. There are materials, measurement indicators, and data processing later. I have time to share and discuss with you. Most of the articles in this article are my personal views. If there are any improper issues, I would also like to ask professionals to point out and discuss together to make progress together.