CN104524545A - Model making method for cold-coagulation blood-stasis type primary dysmenorrheal animal model - Google Patents
Model making method for cold-coagulation blood-stasis type primary dysmenorrheal animal model Download PDFInfo
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Abstract
The invention discloses a model making method for a cold-coagulation blood-stasis type primary dysmenorrheal animal model. The model making method comprises the following steps: producing mice cold-coagulation blood-stasis by combining ice bath with epinephrine injection; then, preparing a primary dysmenorrheal mice model by combining estrogen with oxytocin, thereby obtaining the cold-coagulation blood-stasis type primary dysmenorrheal animal model. According to the theory of the traditional Chinese medicines and the pathogenesis of dysmenorrheal diseases, the mice cold-coagulation blood-stasis is produced by combining ice bath with epinephrine injection through large-scale experimental selection. The model making method for preparing mice cold-coagulation blood-stasis by combining ice bath with epinephrine injection is simple to operate, high in model making success rate, easy to popularize and apply, important in application value, and can be used for solving the defect that the cold-coagulation blood-stasis type primary dysmenorrheal animal model is absent at present, so that a novel method is provided for normative, standard and scientific traditional Chinese medicine evaluation.
Description
Technical field
The present invention relates to a kind of modeling method of animal model, be specifically related to a kind of modeling method and the application of this model in the cold-resistant blood stasis type primary dysmenorrhea medicine of screening thereof of cold blood stasis type primary dysmenorrhea animal model.
Background technology
Dysmenorrhea in love will of falling ill hindered, daily life accidentally or six climate exopathogens the different pathogeny such as to cause harm, and relevant with physiological environment special before and after ferritic and menstrual period, menstrual period.Its pathogeny is mainly subject to the impact of paathogenic factor during this, causes punching to appoint stasis blocking or cold accumulating in meridian, and make QI-blood circulation not smooth, the circulation of uterus menses is hindered, so that " stagnation of QI and blood may bring about pain "; Or punching is appointed, uterus loses in moistening foster, not flourish and pain.Its sick position is appointed in punching, uterus, changes in QI and blood, shows as pain card.It is so with menstrual cycle outbreak, is rush with menstrual period to appoint QI and blood to change relevant.Between non-menstrual period, punching appoints QI and blood gentle, and paathogenic factor not yet can cause that punching is appointed, uterus stagnation of QI-blood or deficiency, therefore pain does not occur; And before and after menstrual period or menstrual period, because the sea of blood rushes down excessive by being completely full of, QI and blood change is hurried, paathogenic factor is taken the opportunity and does, and just dysmenorrhea can occur.Dysmenorrhea is divided into two large classes by modern medicine: primary dysmenorrhea and secondary dysmenorrhea, and primary dysmenorrhea refers to that the general menophania of the obvious pathological changes of non-reproductive system starts to occur.
On the clinical bed of primary dysmenorrhea, common syndrome excess syndrome is more than deficiency syndrome, and the most common with cold blood stasis card in excess syndrome.
In recent years there are some researches show, immunocyte and the immunoreation of dysmenorrhea patient change, and mononuclear cell in blood, Content of B-ep is apparently higher than matched group.Other peptide classes and autonomic nervous system, Endothelin, norepinephrine increase uterus muscle and uterine vascular also can be caused to shrink and cause dysmenorrhea.
In recent years, metabolism group is applied more and more extensive in modern study of TCM.Its core concept is that using modern analytical technique to measure organism (before and after physio-pathological condition, pharmaceutical intervention etc.) under different conditions quantitatively participates in the small molecule metabolites matter of metabolic regulation and the changes of metabolome such as material Transfer, energy metabolism and Information Conduction, and this response is associated with biological event in body by Land use models identification, the target organ that locating events occurs, thus determine biomarker, characterize or disclose organism functional status overall under special time, environment.In the pathological change process occurred after organism irriate, its biological metabolism network must be affected and produce corresponding change, the final change of biological function state by metabolic manifestations out, objectifying function is played in the evaluation of metabolism group to syndrome model, sieve and ancient etc. sets up stagnation of liver-QI with deficiency of the spleen rat model with chronic constriction method, adopt NMR technical Analysis to show, normal group, model group metabolite have notable difference, relate to energy, fat and protein metabolism abnormal.
At present, the report of the animal model not yet having the disease of cold blood stasis type primary dysmenorrhea to combine.The medicine evaluating, screen cold-resistant blood stasis type primary dysmenorrhea is clinically subject to certain restrictions.
Summary of the invention
Goal of the invention: technical problem to be solved by this invention overcomes the deficiencies in the prior art, from the angle that disease combines, adopts ice bath and Injection of Adrenaline to manufacture rat cold blood stasis, combines prepare rat primary dysmenorrhea with estrogen and oxytocin.Finally, by measuring the related biochemical indicator such as beta-endorphin, Endothelin, progesterone, and the change of combining endogenous small molecule metabolite in rat plasma, urine before and after metabolism group method observation modeling preferably obtains cold blood stasis type primary dysmenorrhea animal model.
Technical scheme: in order to realize above object, the technical scheme that the present invention takes is:
A modeling method for cold blood stasis type primary dysmenorrhea animal model, it comprises the following steps:
(1) select female rats to be placed in frozen water ice bath continuously 5 ~ 7 days, then take out injection adrenalin hydrochloride, an adrenalin hydrochloride after 3 ~ 4 hours, then is injected in interval, with obtained cold blood stasis type rat;
(2) then combine with estrogen and oxytocin and prepare primary dysmenorrhea rat model, to the rat of above step (1) continuous 5 ~ 7 days subcutaneous injection estradiol benzoates, with synchronization uterus, and uterus is improved the sensitivity of oxytocin, finally give rats by intraperitoneal injection oxytocin, obtain cold blood stasis type primary dysmenorrhea rat animal model.
0.16ml//time, four hours, interval
Preferably, the modeling method of above-described cold blood stasis type primary dysmenorrhea animal model, it comprises the following steps:
(1) select female rats to be placed in frozen water ice bath continuously 7 days, then take out injection adrenalin hydrochloride, 0.16ml/ only/time, an adrenalin hydrochloride after 4 hours, then is injected in interval, 0.16ml/ only/time, with obtained cold blood stasis type rat;
(2) then combine with estrogen and oxytocin and prepare primary dysmenorrhea rat model, to the rat of above step (1) continuous 7 days subcutaneous injection estradiol benzoates, 1mL/kg/ days, with synchronization uterus, and uterus is improved the sensitivity of oxytocin, within 8th day, give rats by intraperitoneal injection oxytocin, 1mL/kg, obtains cold blood stasis type primary dysmenorrhea rat animal model.
Preferably, the modeling method of above-described cold blood stasis type primary dysmenorrhea animal model, described female rats is SD female rats.
The present invention prepare cold blood stasis type primary dysmenorrhea animal model screening anti-dysmenorrhea medicine in application.
The present invention prepare cold blood stasis type primary dysmenorrhea animal model screening cold-resistant blood stasis type primary dysmenorrhea medicine in application.
Beneficial effect: compared to the prior art the modeling method of cold blood stasis type primary dysmenorrhea animal model provided by the invention has the following advantages:
The present invention, according to the pathogenesis of Chinese medical theory and gynaecopathia, is screened by great many of experiments, manufactures rat cold blood stasis with ice bath joint injection epinephrine; Combine with estrogen and oxytocin and prepare primary dysmenorrhea rat model.Experimental result shows, model group rats plasma biochemical index detects has significant difference with matched group, and metabonomic analysis result shows, rat model urine is separated on PLS-DA figure completely with blood plasma metabolic profile, depart from normal condition, therefore cold blood stasis type primary dysmenorrhea animal model modeling success, this model can well be used for the medicine screening cold-resistant blood stasis type primary dysmenorrhea, has important in being worth.
Detailed description of the invention
According to following embodiment, the present invention may be better understood.But those skilled in the art will readily understand, concrete material proportion, process conditions and result thereof described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.
Embodiment 1
1, the modeling method of cold blood stasis type primary dysmenorrhea animal model, other comprises the following steps:
(1) select SD female rats to be placed in frozen water ice bath continuously 5 days, then take out injection adrenalin hydrochloride, 0.16ml/ only/time, an adrenalin hydrochloride after 3 hours, then is injected in interval, 0.16ml/ only/time, with obtained cold blood stasis type rat;
(2) then combine with estrogen and oxytocin and prepare primary dysmenorrhea rat model, to the rat of above step (1) continuous 5 days subcutaneous injection estradiol benzoates, 1mL/kg/ days, with synchronization uterus, and uterus is improved the sensitivity of oxytocin, within 6th day, give rats by intraperitoneal injection oxytocin, 1mL/kg, obtains cold blood stasis type primary dysmenorrhea rat animal model.
Embodiment 2
1, the modeling method of cold blood stasis type primary dysmenorrhea animal model, other comprises the following steps:
(1) select SD female rats to be placed in frozen water ice bath continuously 7 days, then take out injection adrenalin hydrochloride, 0.16ml/ only/time, an adrenalin hydrochloride after 4 hours, then is injected in interval, 0.16ml/ only/time, with obtained cold blood stasis type rat;
(2) then combine with estrogen and oxytocin and prepare primary dysmenorrhea rat model, to the rat of above step (1) continuous 7 days subcutaneous injection estradiol benzoates, 1mL/kg/ days, with synchronization uterus, and uterus is improved the sensitivity of oxytocin, within 8th day, give rats by intraperitoneal injection oxytocin, 1mL/kg, obtains cold blood stasis type primary dysmenorrhea rat animal model.
Embodiment 3
1, experiment grouping: select the cold blood stasis type primary dysmenorrhea rat 16 obtained by the modeling of above embodiment 2 method, about body weight 220g, be divided into model group 8 and administration group (SHAOFU ZHUYN DECOCTION) 8, select the rat of 8 non-modelings as Normal group, about body weight 220g simultaneously.
2, the mensuration of biochemical indicator
Prostaglandin (prostaglandins, PGs) is closely related with primary dysmenorrhea, PGF
2 αcan stimulate uterine contraction, uterus tension force is raised, there are some researches show, dysmenorrhea is more serious, PGF in blood
2 αcontent is higher; Oxytocin (oxytocin) oxytocin can act on myometril cell and cause uterine contraction, stimulates endo cell release prostaglandin simultaneously; Neuro-endocrinology hormone β-endorphin is a kind of neuro-endocrinology hormone relevant with pain; Progesterone (progesterone), estradiol (estradiol) and Endothelin (endothelin) are also all relevant to dysmenorrhea.
Respectively to the rat model that the above embodiment of the present invention 2 modeling obtains, administration group (SHAOFU ZHUYN DECOCTION) and Normal group carry out biochemical indicator detection, and concrete test experience result is as shown in table 1 below:
The associated biochemical factor in table 1 cold blood stasis primary dysmenorrhea rat model body and the intervention effect (n=8) of SHAOFU ZHUYN DECOCTION
Shown by the experimental result of table 1, the biochemical indicator relevant to cold blood stasis type primary dysmenorrhea, significant change (P<0.05) is all had before and after modeling, modeling of the present invention success is described, and shown by table result, after adopting classical side's SHAOFU ZHUYN DECOCTION to intervene, index of correlation obviously has reduction, shows that cold blood stasis type primary dysmenorrhea model that modeling of the present invention obtains may be used for screening the medicine of cold-resistant blood stasis type primary dysmenorrhea.
3, metabolism group measurement result
Metabolism group method is adopted to observe the change of endogenous small molecule metabolite in rat plasma, urine before and after modeling.Chromatographic condition: Acquity UHPLC BEH-C18 chromatographic column (2.1mm × 50mm, 1.7 μm); Mobile phase: A (0.1% aqueous formic acid), B (acetonitrile); Gradient elution.Flow: 0.4mL/min; Sample size 2 μ L, column temperature 35 DEG C.Mass Spectrometry Conditions: electric spray ion source (ESI source), scan mode: ESI
+, pattern, ion source temperature: 120 DEG C, desolventizing temperature degree: 350 DEG C, capillary voltage: 1500V, taper hole voltage: 45V, nitrogen be used for taper hole gas, argon be used for collision gas.Taper hole throughput: 50L/h, desolventizing flow: 600L/h.
(1) modeling is to the effect tendency of rat urine metabolite spectrogram: testing result shows, model group is on PLS-DA analysis chart the 4th, 7,8 days with modeling before all occur obviously to depart from, 8th day with modeling before (the 0th day) to depart from trend maximum, depart from normal condition, show cold blood stasis type primary dysmenorrhea model modeling success.
(2) cold blood stasis type primary dysmenorrhea rat model urine and blood plasma metabolic profiling analysis: modeling the 8th day model group is separated on PLS-DA analysis chart completely with blank group, depart from normal condition, show cold blood stasis type primary dysmenorrhea model modeling success.
The present invention for instructing, manufactures rat cold blood stasis with ice bath joint injection epinephrine with " sick-card " binding isotherm of the traditional Chinese medical science; Combine with estrogen and oxytocin and prepare primary dysmenorrhea rat model.Result shows, model group rats plasma biochemical index detects has significant difference with matched group, and metabonomic analysis result shows, rat model urine is separated on PLS-DA figure completely with blood plasma metabolic profile, depart from normal condition, show cold blood stasis type primary dysmenorrhea animal model modeling of the present invention success.
Claims (5)
1. a modeling method for cold blood stasis type primary dysmenorrhea animal model, is characterized in that, it comprises the following steps:
(1) select female rats to be placed in frozen water ice bath continuously 5 ~ 7 days, then take out injection adrenalin hydrochloride, an adrenalin hydrochloride after 3 ~ 4 hours, then is injected in interval, with obtained cold blood stasis type rat;
(2) then combine with estrogen and oxytocin and prepare primary dysmenorrhea rat model, to the rat of above step (1) continuous 5 ~ 7 days subcutaneous injection estradiol benzoates, with synchronization uterus, and uterus is improved the sensitivity of oxytocin, finally give rats by intraperitoneal injection oxytocin, obtain cold blood stasis type primary dysmenorrhea rat animal model.
2. the modeling method of cold blood stasis type primary dysmenorrhea animal model according to claim 1, it is characterized in that, it comprises the following steps:
(1) select female rats to be placed in frozen water ice bath continuously 7 days, then take out injection adrenalin hydrochloride, 0.16ml/ only/time, an adrenalin hydrochloride after 4 hours, then is injected in interval, 0.16ml/ only/time, with obtained cold blood stasis type rat;
(2) then combine with estrogen and oxytocin and prepare primary dysmenorrhea rat model, to the rat of above step (1) continuous 7 days subcutaneous injection estradiol benzoates, 1mL/kg/ days, with synchronization uterus, and uterus is improved the sensitivity of oxytocin, within 8th day, give rats by intraperitoneal injection oxytocin, 1mL/kg, obtains cold blood stasis type primary dysmenorrhea rat animal model.
3. the modeling method of cold blood stasis type primary dysmenorrhea animal model according to claim 1 and 2, is characterized in that, described female rats is SD female rats.
4. claim 1 or 2 prepare cold blood stasis type primary dysmenorrhea animal model screening anti-dysmenorrhea medicine in application.
5. claim 1 or 2 prepare cold blood stasis type primary dysmenorrhea animal model screening cold-resistant blood stasis type primary dysmenorrhea medicine in application.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106963772A (en) * | 2017-05-31 | 2017-07-21 | 成都中医药大学 | A kind of method for building up of qi stagnation and blood stasis type hysteromyoma model |
| CN109662802A (en) * | 2018-12-26 | 2019-04-23 | 南京大学 | The method and application of motor coordination performance during a kind of detection pain measurement |
| CN110794074A (en) * | 2019-11-18 | 2020-02-14 | 广西医科大学 | Angelica sinensis Sini decoction cold-resistant blood coagulation stasis syndrome differential metabolite metabolic pathway and research method |
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| 卢阿娜等: "原发性痛经寒凝血瘀证药效评价模型的建立", 《中国药理学通报》 * |
| 朱敏等: "四物汤及其组方药对与药味对小鼠原发性痛经模型的影响", 《中国实验方剂学杂志》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106963772A (en) * | 2017-05-31 | 2017-07-21 | 成都中医药大学 | A kind of method for building up of qi stagnation and blood stasis type hysteromyoma model |
| CN109662802A (en) * | 2018-12-26 | 2019-04-23 | 南京大学 | The method and application of motor coordination performance during a kind of detection pain measurement |
| CN110794074A (en) * | 2019-11-18 | 2020-02-14 | 广西医科大学 | Angelica sinensis Sini decoction cold-resistant blood coagulation stasis syndrome differential metabolite metabolic pathway and research method |
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