CN102524178A - Screening method of febrile convulsion model rat and application thereof - Google Patents
Screening method of febrile convulsion model rat and application thereof Download PDFInfo
- Publication number
- CN102524178A CN102524178A CN2011100251701A CN201110025170A CN102524178A CN 102524178 A CN102524178 A CN 102524178A CN 2011100251701 A CN2011100251701 A CN 2011100251701A CN 201110025170 A CN201110025170 A CN 201110025170A CN 102524178 A CN102524178 A CN 102524178A
- Authority
- CN
- China
- Prior art keywords
- screening
- rat
- temperature
- febrile convulsion
- time
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to the technical field of establishment of an animal model used for screening drug and researching disease mechanism, in particular to a screening method of a febrile convulsion model rat, which can be used for obtaining the model rat by screening in a way of combining a typical water bath heating induction model with artificial directional selection and mating breeding. The method is unique and can be used for successfully obtaining the good febrile convulsion sensitive model rat and a tolerance model rat.
Description
Technical field
The present invention relates to be used for the technical field of foundation of the animal model of drug screening and disease mechanisms research; Being specifically related to a kind of screening technique of febrile convulsion model mouse, is that a kind of mode of breeding with artificial orientation's selection and mating through the febrile convulsion model is screened the method that obtains model mouse.
Background technology
Febrile convulsion is the unusual urgent symptom of modal central nervous system function in children's's period, particularly in the infant sees that its total incidence is about 2%～4% more, and the America and Europe is 2%～5%, and Japan is 6%～9%, certain areas even up to 14%.Febrile convulsion can cause neural sequelae; Retrospective study shows; 40% has the patient of complexity febrile convulsion morbidity history can produce hippocampal sclerosis and temporal epilepsy, and the result of epidemiology survey simultaneously shows that the relative risk of infant generation epilepsy is 10 times of normal children.Therefore, the febrile convulsion prevention and treatment of diseases is just seemed very important.But still lack a kind of special model mouse that is used for febrile convulsion Mechanism Study and medicine evaluation and screening at present.
The foundation of the disease model of febrile convulsion is of long duration, and mainly is divided into following several types: the direct heating induction model of body surface, heating using microwave guidance model, lipopolysaccharide-induced fever model, thermal cycle air-flow heating induction model and hot bath heating induction model.Wherein, The body surface snead process can cause the body surface of experimental rats to scald and lethality high (Holtzman, D., Obana; K.; And Olson, J.Hyperthermia-induced seizures in therat pup:A model for febrile convulsions.Science.1981,213 (4511): 1034-1036.).Lipopolysaccharide-induced convulsions method success rate is low; Be merely 50%; And can't get rid of of the influence of this allogenic material, therefore can not become a kind of suitable animal model (Heida JG, Boisse L model construction; Lipopolysaccharide-induced febrile convulsions in the rat:short-term sequelae.Epilepsia.2004,45 (11): 1317-1329.).
Summary of the invention
To the deficiency that exists in the prior art, one object of the present invention has been to provide a kind of screening technique that obtains responsive model mouse of febrile convulsion and febrile convulsion tolerance model mouse.The onset temp of the responsive model mouse of the febrile convulsion that obtains through the method is low, the incidence of disease is high and near clinical manifestation; The onset temp of the febrile convulsion tolerance model mouse that obtains is high, and the incidence of disease is low.These two kinds of model mouses are not only applicable to the structure of febrile convulsion model, and help the Study on Pathogenesis of febrile convulsion.
Another object of the present invention be to provide the responsive model mouse of a kind of aforesaid febrile convulsion the drug evaluation of control febrile convulsion disease, in the screening application and be used for Study on Pathogenesis.
A further object of the present invention is to provide a kind of aforesaid febrile convulsion tolerance model mouse for the febrile convulsion Study on Pathogenesis.
In order to realize first purpose, the technical measures that the present invention takes are following:
The screening technique of responsive model mouse of a kind of febrile convulsion and tolerance model mouse, step is following:
1) first screening is carried out febrile convulsion water-bath experiment with 100 (male and female half and half) 21 age in days SPF level rats, and temperature is set in 43.0 ℃-45.0 ℃.
1.1) screening of responsive group rat: 5 grades of standards of grading according to the outbreak grade of febrile convulsion are screened, and be sensitivity more than 3 grades, and grade is high more more responsive; Under the identical situation of outbreak grade more relatively latent period length, latent period is short more more responsive; Relatively faint from fear again under the outbreak grade situation all identical with the latent period length of duration, the time is grown more responsive more.Select 4 of the most responsive rats of febrile convulsion with this standard finishing screen, wherein male and female half and half, forms responsive group rat;
The screening of tolerance group rat: the outbreak class 5 level standards of grading according to febrile convulsion are screened, and 0 grade or 1 grade is tolerance, the low more tolerance more of grade; Under the identical situation of outbreak grade more relatively latent period length, grow latent period and more tolerate; Relatively faint from fear again under the outbreak grade situation all identical with the latent period length of duration, the time lacks and more tolerates.Select febrile convulsion with this standard finishing screen and tolerate 4 of rats most, wherein male and female half and half, forms tolerance group rat.
1.2) the sensitivity group rat that will filter out and tolerance group rat organize respectively in mating breed and obtain second generation rat, the second generation rat that is obtained all is used for next step experiment.
2) treat that responsive group of the second generation and tolerance group rat grow to 21 ages in days, carry out febrile convulsion water-bath experiment.
2.1) bath temperature of the responsive group of febrile convulsion rat is set in 43.0 ℃-45.0 ℃.According to the same screening criteria of above-mentioned responsive group rat, therefrom select 4 of the most responsive rats of febrile convulsion (male and female half and half), mating is bred and is obtained third generation rat in organizing, and the third generation rat that is obtained all is used for next step experiment.
2.2) bath temperature of febrile convulsion tolerance group rat is set in 44.0 ℃-46.0 ℃.According to the same screening criteria of above-mentioned tolerance group rat, therefrom select febrile convulsion and tolerate 4 of rats (male and female half and half) most, mating is bred and is obtained third generation rat in organizing, and the third generation rat that is obtained all is used for next step experiment.
When 3) treating that responsive group of the third generation and tolerance group rat grow to 21 ages in days, continue to change bath temperature and carry out screening experiment.
3.1) bath temperature of the responsive group of febrile convulsion rat is set in 40.0 ℃-42.0 ℃.According to the same screening criteria of above-mentioned responsive group rat, filter out responsive more rat.
3.2) bath temperature of febrile convulsion tolerance group rat is set in 45.0 ℃-46.0 ℃.According to the same screening criteria of above-mentioned tolerance group rat, filter out the rat of tolerance more.
(the convulsions grading: 0 grade for not fainting from fear; Twitch rapidly for the rat face for 1 grade; Nod or whipping for rat for 2 grades; 3 grades are lifted clonic spasm for rat one side forelimb; 4 grades are lifted spasm for the tetanic or bilateral forelimb of rat body; 5 grades are the tetanic clonic spasm of rat body or fall.)
(faint from fear latent period: put into water to the time that just begins to faint from fear from rat.)
(faint from fear the duration: rat is by the time that just begins to faint from fear and stop to convulsions.)
The present invention utilizes classical water-bath heating induction model, screens the responsive rat that obtains with the mode that the artificial orientation selects and mating is bred, and its responsive to temperature degree and the incidence of disease increase with the increase of algebraically, and trend can continue to keep; The tolerance rat that the inventive method screening obtains, the low and incidence of disease of responsive to temperature degree reduces with the increase of algebraically, and trend can continue to keep.
In order to realize second purpose of the present invention, the responsive model mouse that checking is filtered out in research febrile convulsion pathogenesis effect and in the advantage aspect the drug screening, we have selected for use clinical anticonvulsant drug phenobarbital commonly used to carry out pharmacological experiment.
Experimental technique is following:
(1) treats that the responsive rat of third generation febrile convulsion grows to 21 ages in days, is divided into 2 groups at random and is used for pharmacological evaluation (phenobarbital group and febrile convulsion group) that other gets normal SPF level 21 age in days rats as the normal control group.Give continuous 8 days handled, write down latent period, duration and the outbreak grade of its outbreak.
1. phenobarbital group: every day lumbar injection phenobarbital 25mg/kg, 43 ℃ of water-bath 4min;
2. febrile convulsion group: every day intraperitoneal injection of saline 50ml/kg, 43 ℃ of water-bath 4min;
3. normal control group: every day intraperitoneal injection of saline 50ml/kg, 33 ℃ of water-bath 4min;
Carry out the Morris water maze laboratory when (2) treating rat 30 ages in days, be mainly used in the ability of learning and memory of checking rat.Experiment comprises two parts: orientation navigation experiment and space orientation experiment.Through relatively, all significantly be superior to the febrile convulsion group in febrile convulsion grade and phenobarbital group on latent period, the while, the phenobarbital group obviously was superior to the febrile convulsion group aspect ability of learning and memory.
(3) we utilize experimental techniques such as the experiment of TUNEL method apoptosis, Nissl's staining experiment and electron microscope experiment to prove at last: the extent of damage of febrile convulsion group is the highest, and the phenobarbital group extent of damage alleviates with respect to the febrile convulsion group to some extent.
In order to realize the 3rd purpose of the present invention, further study the pathogenesis of febrile convulsion, we utilize chip gene expression profile to analyze the mRNA differential expression situation of responsive mouse of febrile convulsion and tolerance mouse.
Experimental technique is following:
(1) animal screening: from the SPF level rat of 100 of the above-mentioned first generation (male and female half and half), 21 ages in days, adopt above-mentioned method, finally from third generation rat, select the most responsive mouse of febrile convulsion and tolerate each 3 of mouse most by the generation screening.
(2) animal is drawn materials: extract the brain tissue hippocampus of the rat that filters out, put it in the frozen pipe of no RNA enzyme, place liquid nitrogen to preserve.
(3) genetic chip analysis: 6 preservation sample appearance are delivered to Shanghai Bai Hao Bioisystech Co., Ltd carry out the genetic chip analysis.
Compared with prior art, advantage of the present invention and beneficial effect are following:
Screening technique of the present invention mainly is to utilize the most classical febrile convulsion hot bath heating induction model, and combines the method that the artificial orientation selects and animal mating is bred to obtain responsive model mouse of febrile convulsion and tolerance model mouse, and this model induction time is short; Observe directly; And model induces success rate high, and from the first generation to the third generation, the temperature sensitivity difference of responsive to temperature group and tolerance group is with algebraically and enlarges; And continue to cultivate, this trend can be kept.The screening that the combination of these two kinds of methods is applied to the febrile convulsion model mouse does not at home and abroad appear in the newspapers as yet.Can keep the genetic predisposition better through this screening technique, and more and more littler to the influence of growing.
The screening technique that the artificial orientation that the inventive method relates to selects and mating is bred can screen and obtain the temperature susceplibility height, the responsive rat of the febrile convulsion that the incidence of disease is higher, and the clinical manifestation of approaching human febrile convulsion; And temperature susceplibility is low, the febrile convulsion tolerance rat that the incidence of disease is lower.Can find out no matter be all significantly to be superior to the febrile convulsion group from the experimental applications aspect in the convulsions grade or in phenobarbital group aspect the learning and memory; The responsive mouse that utilizes screening to obtain simultaneously carries out the analysis of gene expression profile with the tolerance mouse, obtains febrile convulsion correlated expression differential gene.Thereby proved that febrile convulsion model mouse that the present invention obtains more helps studying the evaluation and the screening of febrile convulsion pathogenesis and control medicine.
Description of drawings
Fig. 1: responsive group rat febrile convulsion outbreak mean temperature.
1-3 is respectively 44.3 ± 0.79 ℃, 43.5 ± 0.53 ℃ and 41.9 ± 1.1 ℃ for the outbreak mean temperature.
In 1 generation and 2 generations, compare, P＜0.05; In 2 generations and 3 generations, compare, P＜0.01; In 3 generations and 1 generation, compare, P＜0.001.
Fig. 2: under 43.0 ℃ of water bath condition (other conditions are with embodiment 1 and 2), the responsive group rat febrile convulsion incidence of disease (being morbidity more than 2 grades).
1-3 is respectively 25%, 35.2% and 72.7% for the incidence of disease.
In 3 generations and 1,2 generations, compared P＜0.05.
Fig. 3: febrile convulsion tolerance group rat convulsions mean temperature.
1-3 is respectively 43.9 ± 0.72 ℃, 44.7 ± 0.61 ℃ and 45.4 ± 0.50 ℃ for the outbreak mean temperature.
In 2 generations and 1 generation, 3 generations, compared P＜0.05; In 1 generation, compared comparison with 3 generations, P＜0.001.
Fig. 4: under 45.0 ℃ of water bath condition (other conditions are with embodiment 1 and 2), the tolerance group rat febrile convulsion incidence of disease (being morbidity more than 2 grades).
1-3 is respectively 62.5%, 29.5% and 20.8% for the incidence of disease.In 1 generation and 2,3 generations, compared, P＜0.05.
Fig. 5: hippocampal tissue Electronic Speculum Ultrastructural observation experimental result sketch map.
A is that normal control group, B are that phenobarbital group, C are the febrile convulsion group among the figure.
Embodiment
Further set forth the present invention below in conjunction with embodiment, should be appreciated that these embodiment only are used to explain the present invention, and do not limit protection scope of the present invention.
Embodiment 1:
The screening technique of the responsive model mouse of a kind of febrile convulsion, step is following:
(1) selects 100 (male and female half and half) 21 age in days SPF level normal rats for use in the first screening experiment.Rat is put into a transparent glass infuser (diameter 6cm, high 20cm), and a plurality of apertures and exterior are arranged at this glass infuser bottom.Tube is erected in the constant water bath box,, when barrel is stood, only can exposes head with rat and be as the criterion through putting plastic spacer to the glass infuser heelpiece to regulate the degree of depth of water in the tube.For the first time screening temperature is 43.0 ℃, and the programmed screening temperature is 44.0 ℃, and screening temperature for the third time is 45.0 ℃.Each water-bath is taken out rat and is observed after 4 minutes.5 grades of standards of grading according to the outbreak grade of febrile convulsion are screened, and are responsive more than 3 grades, and grade is high more more responsive; Under the identical situation of outbreak grade more relatively latent period length, latent period is short more more responsive; Relatively faint from fear again under the outbreak grade situation all identical length of duration with latent period; Time is long more more responsive; Finishing screen is selected 4 of the most responsive rats of febrile convulsion (male and female half and half), continues then to cultivate to body weight to reach more than the 250g, and reaches 90 ages in days at least; Carry out the random mating breeding in the group, the second generation rat that is obtained all is used for next step experiment.
(2) treat that second generation rat grows to 21 ages in days, experimentize with identical method, only change bath temperature, the screening temperature reduces gradually.For the first time screening temperature is 45.0 ℃, and the programmed screening temperature is 44.0 ℃, and screening temperature for the third time is 43.0 ℃.According to above-mentioned same standard, finishing screen is selected 4 rats the most responsive (male and female each 2), continue then to cultivate to body weight more than 250g, and reach 90 ages in days at least, carry out the random mating breeding in the group, the third generation rat that is obtained all is used for next step experiment.
(3) treat that third generation rat grows to 21 ages in days, still with identical method screening, the screening temperature further reduces.For the first time screening temperature is 42.0 ℃, and the programmed screening temperature is 41.0 ℃, and screening temperature for the third time is 40.0 ℃.According to above-mentioned same standard, finishing screen is selected 4 rats the most responsive (each 2 of male and female).
Results of statistical analysis shows (seeing Fig. 1 and 2), and the convulsive attack mean temperature of the responsive rat that the mode of breeding through artificial orientation's selection and mating obtains is pursued generation and reduced, and the incidence of disease pursues for rising, and significant difference is all arranged.Show that through this screening technique successfully obtained the responsive model mouse of a kind of febrile convulsion, its onset temp is low, the incidence of disease is high and near clinical manifestation, more help studying the pathogenesis and the drug screening of febrile convulsion.
Embodiment 2:
A kind of screening technique of febrile convulsion tolerance model mouse, step is following:
(1) selects 100 (male and female half and half) 21 age in days SPF level normal rats for use in the first screening experiment.Rat is put into a transparent glass infuser (diameter 6cm, high 20cm), and a plurality of apertures and exterior are arranged at this glass infuser bottom.Tube is erected in the constant water bath box,, when barrel is stood, only can exposes head with rat and be as the criterion through putting plastic spacer to the glass infuser heelpiece to regulate the degree of depth of water in the tube.For the first time screening temperature is 43.0 ℃, and the programmed screening temperature is 44.0 ℃, and screening temperature for the third time is 45.0 ℃.Each water-bath is taken out rat and is observed after 4 minutes, screen according to the outbreak class 5 level standards of grading of febrile convulsion, and 0 grade or 1 grade is tolerance, the low more tolerance more of grade; Under the identical situation of outbreak grade more relatively latent period length, grow latent period and more tolerate; Relatively faint from fear again under the outbreak grade situation all identical length of duration with latent period; Time lacks and more tolerates; Finishing screen is selected 4 of the rats (male and female half and half) that febrile convulsion tolerates most, continues then to cultivate to body weight to reach more than the 250g, and reaches 90 ages in days at least; Carry out the random mating breeding in the group, the second generation rat that is obtained all is used for next step experiment.
(2) treat that second generation rat grows to 21 ages in days, experimentize that bath temperature only raises with identical method.For the first time screening temperature is 44.0 ℃, and the programmed screening temperature is 45.0 ℃, and screening temperature for the third time is 46.0 ℃.Finally select according to above-mentioned same tolerance mouse screening criteria and to tolerate 4 of rats (male and female half and half) most; Continue then to cultivate to body weight and reach more than the 250g; And reach 90 ages in days at least, carry out the random mating breeding in the group, the third generation rat that is obtained all is used for next step experiment.
(3) treat that third generation rat grows to 21 ages in days, still with identical method screening.For the first time screening temperature is 45.0 ℃, and the programmed screening temperature is 45.5 ℃, and the temperature of screening is 46.0 ℃ for the third time.Finally select according to above-mentioned same tolerance mouse screening criteria and to tolerate 4 of rats (male and female half and half) most.
Results of statistical analysis shows (seeing Fig. 3 and 4), and the convulsive attack mean temperature of the tolerance model mouse that the mode of breeding through artificial orientation's selection and mating obtains is pursued generation and risen, and the incidence of disease pursues for decline, and significant difference is all arranged.Show through screening successfully to have obtained a kind of febrile convulsion tolerance model mouse, more help studying the pathogenesis and the drug screening of febrile convulsion.
Embodiment 3: the gene chip expression spectral analysis of febrile convulsion sensitivity and tolerance model mouse
The information of utilizing chip gene expression profile to provide is carried out high-throughout information analysis, aspects such as the gene function that acquisition febrile convulsion relevant difference expressing gene is participated in, signal path, metabolic pathway, thus the febrile convulsion pathogenesis is carried out more deep understanding.The cluster thermal map is that sample and gene are carried out respectively being drawn in thermal map after the hierarchical clustering.The expression of same gene in different samples can be observed through thermal map, the grouping situation of sample can also be verified according to expression conditions.
Experimental procedure is following:
1. animal screening: from the SPF level rat of 100 of the above-mentioned first generation (male and female half and half), 21 ages in days, adopt above-mentioned method, finally from third generation rat, select the most responsive mouse of febrile convulsion and tolerate each 3 of mouse most by the generation screening.
2. animal is drawn materials: according to the standard of drawing materials of genetic chip company, extract the rat cerebral tissue hippocampus, put it in the frozen pipe of no RNA enzyme, place liquid nitrogen to preserve.
3. genetic chip analysis: 6 preservation sample appearance are delivered to genetic chip company (Shanghai Bai Hao Bioisystech Co., Ltd) carry out gene expression spectrum analysis.
Gained genetic chip cluster thermal map is the result show: the responsive model mouse of febrile convulsion is remarkable with the gene expression difference of tolerance model mouse.P in all genes＜0.05 have 1582, wherein the gene of P＜0.01 has 312, satisfying P value and difference multiple (FC) has 89 greater than 2 gene.In these differential genes, some gene is verified to be the febrile convulsion related gene, like GPR98.We can be through the functional dependency of gene simultaneously, and through other genes to be selected are further screened, analyze and research, this provides strong support and help for exploring new febrile convulsion pathogenesis.
Embodiment 4: utilize the responsive model mouse of febrile convulsion to carry out pharmacological experiment
Random packet when third generation febrile convulsion sensitivity rat 21 ages in days of embodiment 1 gained; 12 of phenobarbital groups; 11 of febrile convulsion groups, alternative are used without 6 of 21 age in days SPF level rats of any processing as normal control group (the subsequent implementation example is also undertaken by this grouping).When rat 21 ages in days, begin to carry out continuous processing below 8 days, and write down latent period, duration and the outbreak grade of its outbreak.
1. phenobarbital group: every day lumbar injection phenobarbital 25mg/kg, 43.0 ℃ of water-bath 4min;
2. febrile convulsion group: every day intraperitoneal injection of saline 50ml/kg, 43.0 ℃ of water-bath 4min;
3. normal control group: every day intraperitoneal injection of saline 50ml/kg, 33.0 ℃ of water-bath 4min;
Results of statistical analysis shows that the febrile convulsion group is compared with the phenobarbital group, and latent period is short, and the outbreak grade is high, and all has significant difference.Show that the responsive model mouse of febrile convulsion that the inventive method filters out has and human similar pharmacological characteristic, is more suitable for the structure of febrile convulsion model.Because normal group convulsive attack grade is 0, so does not relate to its latent period and duration in the subordinate list 1.
Table 1
# and febrile convulsion group relatively have significant difference, P＜0.05
Embodiment 5: the learning and memory function of the responsive model mouse of febrile convulsion and the experiment of spatial cognition Function detection
When in embodiment 4, carrying out third generation febrile convulsion sensitivity rat 30 ages in days of packet transaction, detect phenobarbital group and febrile convulsion group and normal group learning and memory function and spatial cognition function through Morris water maze method.The experimental water groove is zinc-plated circular groove, diameter 150cm, and high 50cm, the dark 25cm of pool inner water, water temperature 22-23 ℃, add prepared Chinese ink in the water, make the pond opaque.The pond is divided into four quadrants, and the platform at certain quadrant mid point placement one high 23.5cm, diameter 8cm apart from pool side 25cm place is hidden in underwater 1.5cm.
Experiment is divided into two parts:
(1) orientation navigation experiment: mainly be to detect the animal learning memory function.
Experiment lets rat free swimming 2min in tank noon before that day, to be familiar with environment.Experiment was carried out 4 days altogether; Every day at the upper and lower noon is respectively carried out 4 tests (every rat is at random from the formula entry 1 time of facing the wall and meditating of the mid point of each quadrant); Escape flood after the rat entry as possible; When finding then to stay on it behind the platform, IMAQ and analytical system (EthoVision 3.0) automatically record preserve rat movement locus, find latent period (rat from entry to the time of climbing up platform), swimming time, swimming rate, the swimming distance of platform and seek the information such as search strategy of platform.Do not find platform as if 120 seconds yet, by the operator rat is drawn and had a rest for 4 seconds on the platform and will be designated as 120 seconds latent period.
(2) space exploration experiment: mainly be to detect animal spatial cognition ability.
After accomplishing 4 orientation navigation afternoon on the 4th platform is removed, carried out the space exploration test.Selected and the relative quadrant mid point in platform zone is a place of entry, and rat is with the mode entry of facing the wall and meditating, and the record rat is searched for the number of times that platform passes former platform zone in 120 seconds.
More than the statistical result of experiment sees the following form 2:
Table 2
| Divide into groups | Latent period (s) | Wear the platform number of times |
| The febrile convulsion group | 38.28±20.83＊＊ | 7.20±2.48＊ |
| The phenobarbital group | 32.55±19.29＊ | 8.80±1.64 |
| The normal control group | 14.14±5.87 | 10.67±2.49 |
* with the normal control group significant difference is arranged relatively, P＜0.05
* and normal control group relatively have significant difference, P＜0.01
Above-mentioned experimental result shows, compares with the phenobarbital group with the normal control group, and febrile convulsion all causes the significance damage to learning and memory function and spatial cognition ability.The memory function of phenobarbital group and cognitive ability obviously are superior to the febrile convulsion group.
Embodiment 6TUNEL method detects organizes the Apoptosis experiment
The fracture of chromosomal DNA is a progressive process stage by stage in the Apoptosis, and chromosomal DNA at first is degraded to the big fragment of 50-300kb under the effect of endogenic hydrolase nucleic acid.About then 30% chromosomal DNA is at Ca
2+And Mg
2+Under the endonuclease effect that relies on, between nucleosome unit, cut off at random, formed 180-200bp nucleosome DNA polymer.The 3 '-OH that occurs a series of DNA that breach can produce on dna double chain fracture or the chain is terminal, under the effect of deoxyribonucleotide terminal transferase (TdT), with deoxyribonucleotide with
FluorescenceThe derivative mark that element, peroxidase, alkaline phosphatase enzyme or vitamin h form just can carry out the detection of apoptotic cell to the 3 ' end of DNA, and these class methods are commonly referred to as the breach end-labelling (TUNEL) of deoxyribonucleotide terminal transferase mediation.Owing to almost do not have the fracture of DNA normally or just in proliferating cells, thereby do not have 3 '-OH to form, seldom can be colored.Apoptotic cell is early stage to be distributed in the cell nucleus position it is thus clear that dye dark brown yellow ring-type hollow-core construction, and the nuclei dyeing chromaticness is concentrated into and is semilune or ring-type under the nuclear membrane, and late period, visible karyopycnosis diminished.
Experimental procedure is following:
Apoptosis detection kit content:
End deoxyribonucleic acid transferase (10 *)
The reactant liquor (10 *) that contains the nucleic acid mixed liquor
Enzyme labeling fluorescence antibody (instant)
(1) solution preparation: negative control: reagent 2 100ul;
The TUNEL reactant mixture: reagent 1 50ul and reagent 24 50ul mix.
(2) get the conventional dewaxing of 4 paraffin sections water pretreatment down for every group.
(3) with Proteinase K (20ug/ml is dissolved in Tris/HCl pH7.4-8.0) incubated at room 15-30min.PBS washes twice, each 5min.
(4) dry water around the sample, the TUNEL reaction mixture of Dropwise 5 0ul adds behind the cover glass in wet box and hatches 60min for 37 ℃.PBS flushing 3 times, each 5min.
(5) information transforms and analyzes: dry the moisture around the sample, in wet box, hatch 30min after adding cover glass.PBS flushing 3 times adds the 750ulDAB substrate solution, incubated at room 10min, PBS flushing 3 times, each 5min.
(6) haematoxylin redyeing: embathe acetate solution (acidifying solution) several seconds of 5%; The haematine dye liquor is handled 3-5min, and it is orange red that karyon is, and running water washes to color and becomes blue.
(7) the transparent mounting that dewaters: through 70%-80%-90%-95% alcohol at different levels, every grade of 1-5min dehydration.Go in the xylol 3min to transparent.Drip a droplet neutral gum and with the careful mounting of cover glass.
(8) microscopically is observed and taken a picture: microscope (* 200) is observed each cerebral cortex zone positive cell number of counting.5 different loci in cerebral cortex district are chosen in every section, and each site is counted from shallow to deep 6 visual field inner cell sums and positive cell number respectively, and pale brown look dyeing occurring with kytoplasm is the positive apoptosis cells sign.Calculate the apoptotic cell percentage by following formula: apoptotic cell percentage=positive cell number/(positive cell number+negative cells number) * 100, get the apoptotic cell percentage of its average as this sample.
(9) experimental result: the visible more Tunel stained positive cell of febrile convulsion group, the apoptosis neuron is distributed in the cell nucleus position it is thus clear that dye dark brown yellow ring-type hollow-core construction; The phenobarbital group also has Tunel stained positive cell to a certain degree, but comparing positive cell quantity with the febrile convulsion group has minimizing to a certain degree, and the cell regularity is better; Normal control group nerve cell is in good condition, and regular shape is normal, does not almost have apoptotic cell.Embodiment 7 Nissl's stainings detect the neuron state experiment
Tigroid body is an index of reflection neuron state: under the normal physiological situation, tigroid body is big and quantity is many, and the function of reflection nerve cell synthetic protein is stronger, and when neuronal damage, the quantity of tigroid body can reduce even disappear.
Experimental procedure is following:
(1) gets the conventional dewaxing of 4 paraffin sections water pretreatment down for every group.
(2) paraffin section is put into buffering methylene blue staining liquid and dye 10min (no more than 12min).
(3) in 0.2mol/L acetate buffer (PH=4.6), break up 1-3 second, and examine under a microscope, carry out next step test when tigroid body is clear.
(4) handle section 3-5min with 4% ammonium molybdate aqueous solution.
(5) transparent, the neutral gum mounting of conventional dehydration behind the distilled water wash-out.
(6) light microscope (* 200) is observed the variation of hippocampus CA1 district tigroid body.
(7) experimental result shows: the normal control group tigroid body in febrile convulsion group rat hippocampus CA1 district obviously reduces, and kytoplasm is light to be dyed, and the phenomenon of the sparse disorder of hippocampus layering is arranged.The normal control group hippocampal neuron tigroid body of phenobarbital group slightly reduces, and kytoplasm has light dying slightly, the sparse disorder of hippocampus layering.Above result shows: the hippocampal neuron of febrile convulsion group rat is badly damaged, phenobarbital group hippocampus of rats slightly damaged.
Embodiment 8: the experiment of hippocampal tissue Electronic Speculum Ultrastructural observation
Experimental procedure is following:
(1) perfusion is fixing: during rat 36 ages in days, each group is fixing by 2 rat perfusions of random digit method picked at random.
(2) separate hippocampal tissue: open cranium and get brain, remove the hippocampus cerebral cortex, and with hippocampus and separate tissue on every side.Get the brain tissue several piece in hippocampus CA1 district with syringe needle (the about 1mm of diameter) point, be immersed in 4 ℃ of 2.5% glutaraldehyde fixer.
(3) epoxy resin embedding: tissue fixing before 2.5% glutaraldehyde is taken out 0.1mol/L phosphate buffer rinsing three times, osmic acid (O
3O
4, 1%) and back fixing, 0.1mol/L phosphate buffer rinsing three times, gradient alcohol dehydration (50%, 70%, 80%, 95%, 100%) 2 times, each 15min.
(4) ultra-thin section sample preparation: dehydration is 2 times in the pure acetone, each 15min, and EPON812: acetone (1: 1) soaks into 30min, and pure embedding liquid soaks into 1h, 37 ℃ of pure liquid-solidization of embedding, 60 ℃ of pure liquid-solidization of embedding 48h behind the 24h.
(5) preparation before the ultra-thin section:
A. repair piece: embedded block is clipped on the special clamper, is placed under the disecting microscope,, expose tissue with the sharp blade embedding medium on surface of pruning earlier, then around tissue with the angle that becomes 45 degree with the horizontal plane embedding medium of pruning, accomplish cone-shaped.
B. semithin section is located: utilize microtome to cut the section of thickness for 1-10um.Section is transferred to drip in advance with tweezers to be had on the clean glass slide of distilled water, heats, and section is flattened.Drying is after Toluidine blue staining, and observation by light microscope is positioned hippocampus CA1 district.After the semithin section location, embedded block further is trimmed to trapezoidal or rectangle, the length on every limit is 0.2-0.3mm.
C. make cutter: the system cutter mechanism is made glass cutter, makes a tank around the edge of a knife so that ultra-thin section is swum on the water surface, install tank after, with melting wax sealing interface, prevent to leak.
(6) ultra-thin section: embedded block is installed, glass cutter is installed, regulate the distance of cutter and piece of tissue; Regulate tank liquid level and light position; Regulate heating current and section speed, LKB-V type microtome section (1-2um), it is online that carrying of supporting film reaped in section.
(7) dyeing: uranium, plumbous dyeing (uranium acetate, lead citrate).
(8) H-600 type transmission electron microscope observing.
(9) neuron electron microscopic observation result in hippocampus CA1 district shows: (mitochondria ovalize, size are normally in the hippocampus CA1 district neuron of Fig. 5-A), ridge is regular for the normal control group; (a small amount of mitochondrial swelling in the hippocampus CA1 district neuron of Fig. 5-B), ridge is smudgy or disappearance for the phenobarbital group; (Fig. 5-C) neuron a large amount of mitochondrial swellings in hippocampus CA1 district are obvious, mitochondrial cristae is smudgy or lack the functional disorder that causes hippocampal cell for the febrile convulsion group.Experimental result shows that the phenobarbital group can alleviate the mitochondrial degree of injury of hippocampus CA1 district neuron.
Claims (3)
1. the screening technique of the responsive model mouse of a febrile convulsion, its step is following:
A, 21 age in days rats with 100 male and female half and half when screening for the first time carry out febrile convulsion water-bath experiment, and temperature is set in 43.0 ℃-45.0 ℃, and screening temperature for the first time is 43.0 ℃; The programmed screening temperature is 44.0 ℃, and screening temperature for the third time is 45.0 ℃, and each water-bath is taken out rat and observed screening after 4 minutes; Finally obtain 4 of the most responsive rats of febrile convulsion, wherein male and female half and half, forms responsive group rat; Then sensitivity being organized rat continues to cultivate to body weight and reaches more than the 250g; And reach 90 ages in days at least, carry out the random mating breeding in the group, the second generation rat that is obtained all is used for next step experiment;
B, treat that second generation rat grows to 21 ages in days, experimentize, only change bath temperature with identical method; For the first time screening temperature is 45.0 ℃, and the programmed screening temperature is 44.0 ℃, and screening temperature for the third time is 43.0 ℃; Finishing screen is selected 4 rats the most responsive, wherein male and female each 2, continue then to cultivate to body weight more than 250g; And reach 90 ages in days at least, carry out the random mating breeding in the group, the third generation rat that is obtained all is used for next step experiment;
C, treat that third generation rat grows to 21 ages in days, still with identical method screening, the screening temperature further reduces; For the first time screening temperature is 42.0 ℃, and the programmed screening temperature is 41.0 ℃, and screening temperature for the third time is 40.0 ℃; Finishing screen is selected 4 rats the most responsive, and wherein male and female are each 2.
2. the screening technique of febrile convulsion tolerance model mouse, its step is following:
A, 21 age in days rats with 100 male and female half and half when screening for the first time carry out febrile convulsion water-bath experiment, and temperature is set in 43.0 ℃-45.0 ℃, and screening temperature for the first time is 43.0 ℃; The programmed screening temperature is 44.0 ℃, and screening temperature for the third time is 45.0 ℃, and each water-bath is taken out rat and observed screening after 4 minutes; Finally obtain febrile convulsion and tolerate 4 of rats most, wherein male and female half and half, forms tolerance group rat; Then rat being organized in tolerance continues to cultivate to body weight and reaches more than the 250g; And reach 90 ages in days at least, carry out the random mating breeding in the group, the second generation rat that is obtained all is used for next step experiment;
B, treat that second generation rat grows to 21 ages in days, experimentize that bath temperature only raises with identical method; For the first time screening temperature is 44.0 ℃, and the programmed screening temperature is 45.0 ℃, and screening temperature for the third time is 46.0 ℃; Finishing screen is selected 4 and is tolerated rat most, wherein male and female each 2, continue then to cultivate to body weight more than 250g; And reach 90 ages in days at least, carry out the random mating breeding in the group, the third generation rat that is obtained all is used for next step experiment;
C, treat that third generation rat grows to 21 ages in days, still with identical method screening, the screening temperature further raises; For the first time screening temperature is 45.0 ℃, and the programmed screening temperature is 45.5 ℃, and screening temperature for the third time is 46.0 ℃; Finishing screen is selected 4 rats that tolerate most, and wherein male and female are each 2.
3. the application of the model mouse that method according to claim 1 and 2 filters out in screening febrile convulsion medicine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110025170 CN102524178B (en) | 2011-01-24 | 2011-01-24 | Screening method of febrile convulsion model rat and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110025170 CN102524178B (en) | 2011-01-24 | 2011-01-24 | Screening method of febrile convulsion model rat and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102524178A true CN102524178A (en) | 2012-07-04 |
| CN102524178B CN102524178B (en) | 2013-08-14 |
Family
ID=46333231
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110025170 Expired - Fee Related CN102524178B (en) | 2011-01-24 | 2011-01-24 | Screening method of febrile convulsion model rat and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102524178B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102919199A (en) * | 2012-11-21 | 2013-02-13 | 复旦大学附属中山医院 | Preparation method for rat mild hypothermia animal model |
| CN109528341A (en) * | 2018-11-15 | 2019-03-29 | 南方医科大学第三附属医院（广东省骨科研究院） | A kind of molding apparatus, modeling method and the application of mouse intervertebral disc degeneration model |
-
2011
- 2011-01-24 CN CN 201110025170 patent/CN102524178B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| 常杏芝 等: "幼年大鼠反复热性惊厥对惊厥易感性的远期影响", 《中华儿科杂志》 * |
| 李娟: "妥泰、丙戊酸钠、苯巴比妥预防热性惊厥及惊厥性脑损伤的实验研究", 《中国优秀硕士学位论文全文数据库电子期刊》 * |
| 袁晓冉 等: "惊厥相关动物模型的研究进展", 《卒中与神经疾病》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102919199A (en) * | 2012-11-21 | 2013-02-13 | 复旦大学附属中山医院 | Preparation method for rat mild hypothermia animal model |
| CN109528341A (en) * | 2018-11-15 | 2019-03-29 | 南方医科大学第三附属医院（广东省骨科研究院） | A kind of molding apparatus, modeling method and the application of mouse intervertebral disc degeneration model |
| CN109528341B (en) * | 2018-11-15 | 2021-06-04 | 南方医科大学第三附属医院（广东省骨科研究院） | Molding equipment, molding method and application of mouse intervertebral disc degeneration model |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102524178B (en) | 2013-08-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Milkman | Genetic and developmental studies on Botryllus schlosseri | |
| CN205030365U (en) | Soil animal separator | |
| Gamble et al. | The bionomics of Convoluta roscoffensis, with special reference to its green cells | |
| CN105961251B (en) | A kind of construction method of lower oxygen concentration resistance megalobrama amblycephala | |
| CN107099595A (en) | Fish natural propagation monitoring method based on environment DNA technology | |
| CN102524178B (en) | Screening method of febrile convulsion model rat and application thereof | |
| O'Hara | Habitat selection behavior in three species of anuran larvae: environmental cues, ontogeny, and adaptive significance | |
| Kim et al. | An array microhabitat system for high throughput studies of microalgal growth under controlled nutrient gradients | |
| CN204086271U (en) | A kind ofly study the proving installation of ultraviolet spectrum on the ethological impact of planktonic organism | |
| CN109574237A (en) | A kind of devices, systems, and methods for long term monitoring stain disease comprehensive ecological toxicity up to standard | |
| Nishikata et al. | Monoclonal antibodies against components of the myoplasm of eggs of the ascidian Ciona intestinalis partially block the development of muscle-specific acetylcholinesterase | |
| CN106221887B (en) | A kind of method of the total rouge of high efficiency extraction ocean ballstone frustule | |
| AU2021105143A4 (en) | A method and a device for quickly detecting the influence on the growth of duckweed | |
| CN102600161B (en) | Application of sitagliptin phosphate in preparation of medicament for preventing and treating febrile convulsion | |
| CN209456163U (en) | A kind of device and system for long term monitoring stain disease comprehensive ecological toxicity up to standard | |
| CN108034691A (en) | A kind of method of the abundance of microorganism in accurate counting invertebrate hemolymph | |
| Lavery | Investigating the effects of highly preferred environmental enrichment on the behaviour and welfare of laboratory zebrafish (Danio rerio) | |
| CN110724724A (en) | Method for measuring and calculating semi-effective concentration of environmental pollutant ecotoxicity effect | |
| CN105388263A (en) | Method for evaluating shallow lake fishery environment by using microbial food web efficiency | |
| Liu et al. | Swimming Characteristics of Chlamydomonas reinhardtii | |
| Lamberts | Alizarin deposition by corals | |
| Kairesalo et al. | Effect of light and water flow on the spatial distribution of littoral Bosmina longispina Leydig (Cladocera) | |
| CN210008408U (en) | Experimental device for simulating and researching stress tolerance and tolerance range of aquatic animals | |
| Borges et al. | Short-term successional dynamics of a macroalgal community in a stream from northwestern São Paulo State, Brazil | |
| ROBERTSON et al. | An electron microscopic and behavioral study of tactile learning and memory in Octopus vulgaris |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130814 Termination date: 20160124 |
|
| EXPY | Termination of patent right or utility model |