阅读说明：本技术 一种利用蝗虫构建泌尿系结石动物模型的方法 (Method for constructing urinary calculus animal model by using locust ) 是由 刘耀明 于 2020-05-07 设计创作，主要内容包括：一种利用蝗虫构建泌尿系结石动物模型的方法,属于生物医学领域；它包括：(1)第一次向蝗虫腹腔内注入4-8μL且浓度为5.0-10.5 mM的氯化铜溶液,在光照与黑暗时间比为13:11小时,光照时温度24-28±2℃,黑暗时温度20-26±2℃,湿度为60-85%的条件下,用新鲜麦苗饲养蝗虫；(2)第二次向蝗虫腹腔内注入4-8μL的氯化铜溶液,在光照与黑暗时间比为14:10小时,光照时温度24-28±2℃,黑暗时温度20-26±2℃,湿度为60-85%的条件下,用新鲜麦苗饲养蝗虫；(3)注入氯化铜溶液的蝗虫经24-48小时生理代谢后其马氏管内逐渐产生不同形态的草酸钙晶体。本发明具有材料成本低、稳定性好、过程可控、周期短优点。(A method for constructing a urinary calculus animal model by using locust belongs to the field of biomedicine; it includes: (1) injecting 4-8 μ L of 5.0-10.5 mM copper chloride solution into locusts abdominal cavity for the first time, and feeding locusts with fresh wheat seedlings under the conditions that the ratio of light to dark is 13:11 hours, the temperature in light is 24-28 + -2 ℃, the temperature in dark is 20-26 + -2 ℃, and the humidity is 60-85%; (2) injecting 4-8 μ L of copper chloride solution into abdominal cavity of locust for the second time, and feeding locust with fresh wheat seedling under the conditions of light illumination and dark time ratio of 14:10 hr, light illumination temperature of 24-28 + -2 deg.C, dark temperature of 20-26 + -2 deg.C, and humidity of 60-85%; (3) the locust injected with the copper chloride solution is physiologically metabolized for 24 to 48 hours to gradually generate calcium oxalate crystals with different forms in the Malpighian tube. The invention has the advantages of low material cost, good stability, controllable process and short period.)

1. A method for constructing a urinary calculus animal model by using locust is characterized by comprising the following steps:

(1) injecting 4-8 μ L of 5.0-10.5 mM copper chloride solution into locusts abdominal cavity for the first time, and feeding locusts with fresh wheat seedlings under the conditions that the ratio of light to dark is 13:11 hours, the temperature in light is 24-28 + -2 ℃, the temperature in dark is 20-26 + -2 ℃, and the humidity is 60-85%;

(2) injecting 4-8 μ L of copper chloride solution into abdominal cavity of locust for the second time, and feeding locust with fresh wheat seedling under the conditions of light illumination and dark time ratio of 13:11 hr, light illumination temperature of 24-28 + -2 deg.C, dark temperature of 20-26 + -2 deg.C, and humidity of 60-85%;

(3) the locust injected with the copper chloride solution is physiologically metabolized for 24 to 48 hours to gradually generate calcium oxalate crystals with different forms in the Malpighian tube.

2. The method as claimed in claim 1, wherein the locust is adult locusts of Oryza glutinosa captured from rice field, and the captured adult locusts is cultured in laboratory for 2-3 days.

3. The method for constructing the animal model of urinary calculus by using grasshopper as claimed in claim 1, wherein the formulation of the insect normal saline is: NaCl: 0.138M, KCl: 0.0027M, Na₂HPO₄：0.0073 M，KH₂PO₄：0.00147 M，pH=7.4。

4. The method as claimed in claim 1, wherein the cupric chloride solution is prepared from normal saline solution of insects, and is sterilized by steam sterilization at 120 ℃ for 20 min.

5. The method as claimed in claim 1, wherein the step of injecting the cupric chloride solution into abdominal cavity of the locust is to inject 4-8 μ L of the cupric chloride solution into abdominal cavity of the locust from the internode membrane of the third and fourth abdominal segments of the locust.

6. The method for constructing the animal model of urinary calculus by using locust as claimed in claim 1, wherein the locust injected with the first time of cupric chloride solution or normal saline is separately placed in a ventilated gauze cage and kept in a climatic incubator for 24 hours.

7. The method for constructing the animal model of urinary calculus by using locust as claimed in claim 1, wherein the locust injected with the second injection of cupric chloride solution or normal saline is separately placed in a ventilated gauze cage and raised in a climatic incubator for 1-3 days.

8. The method as claimed in claim 1, wherein the locust is dissected under a dissecting lens to obtain the Mahalanobis duct, and different forms of crystals in the Mahalanobis duct can be observed by a polarizing microscope.

9. The method as claimed in claim 8, wherein the different crystal forms emit blue fluorescence under the irradiation of ultraviolet light.

Technical Field

The invention belongs to the field of biomedical research, and particularly relates to a method for constructing an animal metabolic urinary calculus model by using Chinese rice locust.

Background

Metabolic urinary calculus (kidney calculus or urinary calculus) is a common disease of animal urinary systems including human beings, and researches show that the calculus incidence rate of people in different regions is between 3% and 12%. Due to the particularity of human as a treatment object and scientific research materials, research on related urinary calculi needs to rely on other animals as substitution and test models, proper animals are selected, and the animals are induced or stressed to generate symptoms related to metabolic calculi and corresponding pathological changes through a normative method and a normative program to effectively develop corresponding scientific research tests.

Animals such as mice, rats, rabbits, dogs, pigs and the like are selected to establish animal metabolic calculus models and carry out related researches in modeling experiments according to different symptoms and characteristics, and ethylene glycol (ethylene glycol + ammonium chloride), oxalamide, terephthalic acid, melamine and the like are applied to the existing reports, so that the animals are induced to generate and have main symptoms and physical sign indexes of related diseases through continuous quantitative administration, and corresponding animal models are obtained. The limitations of these models have somewhat hindered and limited the effective medical treatment, preventive measures, medical transformation applications. In order to comprehensively recognize metabolic urinary calculi, diversified animal models need to be objectively expanded. In recent years, the study found that fruit flies (A), (B), (CDrosophila melanogaster) Representative insects are becoming transformation models for animal diseases. The insect model can provide convenient operation and high-efficiency tests for physiological processes, pathogenesis, prevention strategies and drug screening of disease formation.

The locusts of China are distributed in rice growing areas all over the country, and mainly take stems and leaves of rice as food. A tissue called Ma tube is arranged in the Chinese rice locust body, is a hollow tube with a blind end, is soaked in haemolymph in an abdominal cavity, and the blind end can creep. From the aspects of animal development and insect physiology, the genetic composition, anatomical structure and physiological function of the kidney of an insect Marek's tube and a mammal (human) are very similar, and the insect Marek's tube can absorb metabolites from blood lymph to enter the tube to form protourine, so that calcium oxalate crystals generated in the Marek's tube are equivalent to kidney stones or urinary stones of the mammal.

In the early period of intensive research, the copper chloride is used to form urate, namely calcium oxalate, in the Ma-tube of the Chinese rice locust. Based on the physiological phenomenon, a metabolic insect urinary calculus animal model can be established. The formation process of calcium oxalate crystals can be adjusted by artificially controlling physiological conditions, and a scientific and convenient experimental model can be provided for people to understand the formation and pathogenesis of urinary calculus and screen and test related medicaments.

Disclosure of Invention

Aiming at the defects of the existing animal disease model, the invention provides the animal disease model which is easy to obtain and simple to feed; the method for constructing the urinary calculus animal model by using the locust is low in cost, short in modeling period, good in test repeatability and simple to operate.

In order to achieve the purpose, the method is specifically realized by the following technical method:

placing locust in ventilated gauze cage, placing in climate incubator, and adaptively feeding with fresh wheat seedling for 2-3 days;

feeding environmental conditions, climate incubator relevant settings: the illumination and dark time is 14:10 hours, the temperature is 24-28 +/-2 ℃ in the illumination, the temperature is 20-26 +/-2 ℃ in the dark and the humidity is 60-85 percent;

injecting a copper chloride solution with the concentration of 5.0-10.5 mM prepared by using normal saline of insects into the abdomen of the locust; sterilizing the copper chloride solution by steam sterilization at 120 ℃ for 20 min;

the formula of the insect normal saline comprises: NaCl: 0.138M, KCl: 0.0027M, Na₂HPO₄：0.0073 M，KH₂PO₄：0.00147 M，pH=7.4；

Randomly selecting locust which is subjected to adaptive feeding, and slowly injecting 4-8 mu L of copper chloride solution into the locust from the abdomen by using an injector;

placing locust injected with copper chloride into a gauze cage, and placing in a climatic incubator for feeding for 1 day; the breeding environment in the incubator is as follows: the illumination and dark time is 13:11 hours, the temperature is 24-28 +/-2 ℃ in the illumination, the temperature is 20-26 +/-2 ℃ in the dark and the humidity is 60-85 percent;

randomly selecting the locusts which are fed and injected with copper chloride, injecting 4-8 mu L of copper chloride solution again, and feeding for 2-3 days;

dissecting locusts, placing under a dissecting mirror to obtain Mariothis, and observing with a polarizing microscope to show crystals in the Mariothis;

the crystals stay in the grasshopper Marek's tube for 24-48 hours and are finally transported into the hindgut;

the chemical composition of the crystal obtained by separating in the Mariothis tube or the hindgut feces is determined to be calcium oxalate through an X-ray fluorescence spectrometer (XRF) test;

the locust is adult Chinese rice locust captured in rice field, and can be used in both male and female.

The invention has the advantages that the main material, namely the Chinese rice locust, is a field insect, is easy to obtain and simple to feed; the used medicine is copper chloride, the cost is low, the modeling period is short (20-48 hours), the test repeatability is good, and the operation is simple. The physiological aspects of the Chinese rice locust and the fruit fly are very similar, and compared with a fruit fly model, the fruit fly model has the advantages that the size is larger than that of the fruit fly, the number of the Malpighian is more than that of the Malpighian of the fruit fly, and the calcium oxalate crystal is convenient to observe, dissect and separate. As a transformation model of human metabolic urinary calculus, the pathogenesis can be artificially controlled to obtain calcium oxalate crystals at different stages.

The method and the model constructed by the invention are economical, convenient, simple to operate, short in period and high in efficiency, have remarkable superiority compared with other animal models used at present, and can be used as supplement or substitute of the existing model.

Drawings

FIG. 1 is a photomicrograph of crystals formed in Ma tube 48 hours after the adult cells of Piperda huashanensis were injected with a copper chloride solution.

Detailed Description