CN106310238A - Application of Ad-sAxl in establishment of preeclampsia rat model and establishment method of preeclampsia rat model - Google Patents
Application of Ad-sAxl in establishment of preeclampsia rat model and establishment method of preeclampsia rat model Download PDFInfo
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Abstract
The invention provides application of Ad-sAxl in the establishment of a preeclampsia rat model and an establishment method of the preeclampsia rat model. The preeclampsia rat model is established by injecting Ad-sAxl into a pregnant rat through in a caudal vein manner and has the preeclampsia characteristic symptoms and symptomatic changes including elevation of blood pressure, injuries of important organs such as proteinuria, heart, liver and kidney, and the characteristic symptoms and symptomatic changes are accordant with preeclampsia symptom, signs and pathological processes of human and can reflect the systematicness and severity of diseases; and the operational controllability and the stability are good. The application can be applied to the research of occurrence and development mechanisms and control measures of preeclampsia.
Description
Technical field
The invention belongs to biomedical sector, be specifically related to Ad-sAxl answering in the foundation of preeclampsia in rat model
With and the method for building up of preeclampsia in rat model.
Background technology
Preeclampsia is to occur that after pregnant 20 weeks blood pressure raises and the albuminuria peculiar disease of the trimester of pregnancy as principal character,
Can involve the multiple organ of whole body, serious threat mother's tire is healthy.Attack of Preeclampsia mechanism is still not clear, but research thinks it at present
Occur closely related with trophocyte invasion and attack obstacle and spiral artery double teeming obstacle in early days, and body vascular endothelial injury widely
It is that this disease develops further and presents multiple degree and differs the key link of clinical manifestation.Wherein Angiogensis and anti-blood
It is unbalance not only in the invasion and attack of local influence trophocyte and spiral artery double teeming that pipe generates, moreover it is possible to causes body Ink vessel transfusing widely
Skin lesion is hindered, and is the key link of preeclampsia generation development.
Owing to the medical ethics of human experimentation restricts, animal model becomes research preeclampsia and development occurs and explores anti-
Control the important tool of measure, but can not react the animal model of this disease severity of preeclampsia at present very well.
Sawtooth class animal placenta generation type is close with the mankind with trophocyte invasion and attack degree, and parent has the table that uterus thin vessels is reinvented
Existing, after modeling success, the key phenotypic of preeclampsia can partly or entirely occur.The preeclampsia in rat model previously used is
By the sFlt-1 of rat tail vein injection adenovirus construction, but this model does not typically have the infringement of multiple organ internal organs, no
Can well react the order of severity of this disease.
Axl is receptor tyrosine kinase subfamily member, has Angiogensis, maintains blood after being combined with its receptor Gas6
Pipe permeability and the effect of integrity.SAxl is the soluble component of Axl, research confirm sAxl can with Gas6 competitive binding,
Thus block the combination of Axl Yu Gas6, and the signal path of suppression Gas6-Axl mediation, ultimately result in angiogenesis exception, tremulous pulse
Double teeming obstacle, vascular endothelial cell damage and vascular permeability sexually revise.Our early-stage Study shows that sAxl suffers from preeclampsia
The notable order of severity raised and can reflect this disease in person's blood plasma, this is that the preeclampsia animal model setting up sAxl is established
Scientific basic.
Summary of the invention
Present invention seek to address that prior art passes through the eclamposia that the sFlt-1 of rat tail vein injection adenovirus construction sets up
Early stage rat model does not haves the infringement of multiple organ internal organs, it is impossible to the problem well reacting the order of severity of this disease, carries
For a kind of rat model by tail vein injection Ad-sAxl to pregnant dams construct in vitro preeclampsia and the rat of preeclampsia
The concrete method for building up of model, the rat model of this preeclampsia has the important organs such as blood pressure rising, albuminuria and the heart, liver, kidney
The characteristic symptom of the preeclampsias such as infringement and sign change, and are consistent with symptom and sign and the pathological process of mankind's preeclampsia,
And systematicness and the order of severity of disease can be reacted, operation controllability is good, good stability.Can be used for studying the generation of preeclampsia
Development mechanism and prophylactico-therapeutic measures.
For solving above-mentioned technical problem, the present invention is achieved through the following technical solutions:
A kind of Ad-sAxl application in the foundation of preeclampsia in rat model.Ad-sAxl is applied to eclamposia by the present invention first
In the foundation of early stage rat model.
The method for building up of a kind of preeclampsia in rat model, comprises the steps:
(1) selecting cleaning grade SD rat, male and female mate ratio 1:3, when simultaneously observing cloudy bolt and sperm determines gestation and is set to
Gestation 0 day；
(2) pregnant rats the 8th day through tail vein injection Ad-sAxl；
(3) monitor its blood pressure, urine protein, biochemical indicator, observe main organs pathological change and determine model construction
The most successful.
Preferably, in step (1), described SD rat body weight is 250-280g, week old 8 weeks.
Preferably, in step (2), the dosage of described Ad-sAxl is 2 × 109 pfu。
Described method also includes determining rat body weight baseline value by pregnant 0 day body weight of detection, by detection pregnant before and pregnant 1,
3,5,7 days blood pressures determine blood pressure baseline value, are determined the baseline of urine protein by pregnant front and pregnant 4 days the twenty-four-hour urine albumen of detection
Value.
In the present invention, after determining that preeclampsia in rat model foundation the most successfully method includes detection injection Ad-sAxl
The non-invasive blood pressure of rat, invasive blood pressure, twenty-four-hour urine albumen；Pregnant 19 days pregnant Mus body weight, peripheral blood routine blood test, biochemical indicators；Pregnant
19 days row Cesarean esction take out that filial mice count and survey crown-rump length, body weight, Placenta Hominis are weighed and to pass through pathological study rat main
The pathological change of internal organs.
Monitoring rat non-invasive blood pressure at pregnant 9,11,13,15,17 days with non-invasive blood pressure detector, monitoring 24 in pregnant 12,16 days is little
Time urine protein；Within pregnant 19 days, survey pregnant Mus invasive blood pressure.
Described main organs includes heart, liver, kidney, Placenta Hominis.
The method have the advantages that
1, the present invention is by tail vein injection Ad-sAxl to pregnant rat body, builds the rat model of preeclampsia, the son of foundation
Epilepsy early stage rat model has the characteristic disease of the preeclampsias such as important organ infringement such as blood pressure rising, albuminuria and the heart, liver, kidney
Shape and sign change, and are consistent with symptom and sign and the pathological process of mankind's preeclampsia, and can react the systematicness of disease with tight
Weight degree, operation controllability is good, good stability.Can be used for studying generation development mechanism and the prophylactico-therapeutic measures of preeclampsia.
The male and female of the present invention mate ratio and control to be 1:3, can at utmost ensure Mating success, thus ensure that model is built
Carrying out in order of vertical experiment, it is to avoid experimentation for a long time can not the copulation problem that successfully causes lengthening experimental period.
Rat Pregnancy is divided into First Trimester (pregnant 0-7 days), mid trimester of pregnancy (pregnant 8-14 days) and (pregnant 15-21 late trimester of pregnancy
My god), and the morbidity of mankind's preeclampsia be gestation 20 weeks and after (early stage of being equivalent to mid trimester of pregnancy) present invention through excessive
Amount practical studies, selects pregnant rats the 8th day through tail vein injection Ad-sAxl, is equivalent to the mid trimester of pregnancy (8-14) of rat, separately
The Ad-sAxl maintaining constant density in blood is can guarantee that through tail vein injection Ad-sAxl, therefore, quiet through tail pregnant rats the 8th day
Arteries and veins injection Ad-sAxl can the morbidity of at utmost simulating human preeclampsia, it is ensured that the reliability of model.
2, a large amount of practical studies prove, the present invention selects body weight to be 250-280g, the week old SD rat of 8 weeks, this body weight
With the rat of week old, there is the strongest reproductive performance, fully meet the needs of experiment.
3, Ad-sAxl dosage too small there will be experiment pregnant Mus can not occur all of blood pressure raise, twenty-four-hour urine albumen increase
Adding and body important organ pathological change, the reliability of the rat model obtained is set up in impact；Again because Ad-sAxl cost is held high
Expensive, if dosage is excessive, then can increase considerably the cost setting up model, be unfavorable for that the later stage promotes the use of, the present invention is through excessive
Amount practical studies, finds that the dosage at Ad-sAxl is 2 × 109In the case of pfu, can make experiment pregnant Mus that blood pressure liter occurs
High, twenty-four-hour urine albumen increases and the pathological change of body important organ, meets the pathological change of preeclampsia；Ad-sAxl's
Cost has been got back effective control.
4, the present invention monitored rat blood pressure, monitoring in pregnant 12,16 days at pregnant 9,11,13,15,17 days with non-invasive blood pressure detector
Twenty-four-hour urine albumen；Within pregnant 19 days, survey pregnant Mus invasive blood pressure, the ambulatory blood-pressure change situation of the pregnant Mus of dry Prognostic Assays can be objectively responded
And the dynamic situation of change of twenty-four-hour urine albumen is so that experimental result is true, credible, more convincing.
Accompanying drawing explanation
Fig. 1 is that SD rat shrinks pressure measured value variation diagram；
Fig. 2 is SD rat diastolic pressure measured value variation diagram；
Fig. 3 is SD Rat 24 h urine protein measured value variation diagram；
Fig. 4 is Ad-sAxl group SD rat kidney HE dyeing pathological change figure；
Fig. 5 is Ad-Fc group SD rat kidney HE dyeing pathological change figure；
Fig. 6 is normal saline group SD rat kidney HE dyeing pathological change figure；
Fig. 7 is Ad-sAxl group SD rat kidney CD31 immunofluorescence dyeing pathological change figure；
Fig. 8 is Ad-Fc group SD rat kidney CD31 immunofluorescence dyeing pathological change figure；
Fig. 9 is normal saline group SD rat kidney CD31 immunofluorescence dyeing pathological change figure.
Detailed description of the invention
The present invention is described in detail below in conjunction with specific embodiment.
Main agents and material: cleaning grade SD rat (Da Shuo bio tech ltd, Chengdu), recombinant adenoviral vector
(Ad-sAxl) (Sigma Co., USA) is measured by the synthesis of Shanghai Ji Kai genome company, urine protein.
Key instrument: BP-98A type intelligence non-invasive blood pressure-Mus instrument (Ruan Long bio tech ltd, Beijing), BL-420S
Biological functional system (BL series of biologic Functional Experiment system, Chengdu TME Technology Co., Ltd.), rat metabolism cage (Sichuan
University's West China Hospital animal experimental center), fully automatic blood cytoanalyze BC-3000(Shenzhen Mai Rui biologic medical company),
Automatic clinical chemistry analyzer BS-200(Shenzhen Mai Rui biologic medical company), scanning electron microscope (Japan Olympus company).
The foundation of preeclampsia in rat model and qualification:
(1) choose the week old SD rat of 8 weeks, body weight 250-280g, mate ratio 1:3 in male and female, determine after gestation by female Mus with
Machine is divided into 3 groups, often 6 female Mus of group, pregnant 0 day row measured body weight, pregnant front and pregnant 1,3,5,7 heavenly prison or jail's measuring blood pressures, Yun Qianjiyun 4 heavenly prison or jail
Survey twenty-four-hour urine albumen；
(2) within the 8th day, 2 × 10 are injected respectively through tail vein in pregnant rats9The Ad-sAxl(experimental group of pfu), Ad-Fc(carrier pair
According to group) and normal saline (blank group)；
(3) rat blood pressure, pregnant 12,16 days monitoring twenty-four-hour urines were monitored at pregnant 9,11,13,15,17 days with non-invasive blood pressure detector
Albumen；Within pregnant 19 days, survey pregnant Mus invasive blood pressure, weigh, and take pregnant Mus peripheral blood promoting the circulation of blood routine, Biochemical Indexes；Within pregnant 19 days, row cuts open
Palace is produced and is taken out that filial mice count and survey crown-rump length, body weight, Placenta Hominis are weighed, and passes through pathological study body main organs simultaneously
Pathological change.
Interpretation of result:
1, blood pressure determination value changes
Blood pressure determination value as shown in Figure 1 and Figure 2 is calculated,*: P < 0.05, Ad-sAxl group compares with Ad-Fc group；#: P <
0.05, Ad-sAxl group compares with normal saline group.
2, rat urine protein determination value change
As shown in Figure 3 and calculate,*: P < 0.05, Ad-sAxl group compares with Ad-Fc group；#: P < 0.05, Ad-sAxl group is with raw
Reason saline group compares.
3, rat kidney pathological change
Did rat kidney HE dyeing and rat kidney CD31 immunofluorescence dyeing at pregnant 19 days respectively, observe under 40 power microscopes.
As Figure 4-Figure 6, mesangial region cell number seen from Ad-sAxl group rat kidney increases, the slight swelling of glomerule, Ad-Fc group and
Saline rats kidney shows no obvious abnormalities.As Figure 7-9, Ad-sAxl group glomerule blood vessel there are no the most glimmering
Light is expressed, and in Ad-Fc group and normal saline group, there is CD31 luciferase expression at place's glomerule blood vessel shown in arrow.
4, Rat Placenta interpretation of result
The Placenta Hominis counting of row Cesarean esction taking-up in pregnant 19 days is weighed, Ad-sAxl experimental group, Ad-Fc vehicle Control group and normal saline
Rat Placenta number no significant difference (P ＞ 0.05) between group, and the placental weight of Ad-sAxl experimental group rat (0.32 ±
0.06g) being substantially less than Ad-Fc negative control group (0.52 ± 0.08g) and normal saline group (0.54 ± 0.07 g), difference has system
Meaning (the equal ＜ of P 0.05) learned by meter.
5, the pregnant 19 days row Cesarean esction that each group of detection obtains are taken out filial mice and count and survey crown-rump length, body weight, filial mice weight
And add up in table 1 below；
Table 1 filial mice number, weight and crown-rump length compare (n=6,± SD)
It is computed,*: P < 0.05, Ad-sAxl group compares with Ad-Fc group；
#: P < 0.05, Ad-sAxl group compares with normal saline group.
6, pregnant 19 days biochemical desired values, routine blood tests compare
The biochemical indicator value, the routine blood test that detected pregnant 19 days are added up in table 2 below；
Table 2 biochemical indicator value, routine blood test compare (n=6,± SD)
Calculating is known:
*: P < 0.05, Ad-sAxl group compares with Ad-Fc group；
#: P < 0.05, Ad-sAxl group compares with normal saline group.
ALT: glutamate pyruvate transaminase；AST: glutamic oxaloacetic transaminase, GOT；LDH: lactic acid dehydrogenase；Cr: creatinine；UA: blood urea nitrogen；HGB: blood
Hemoglobin concentration；PLT: platelet count.
Claims (8)
1. the Ad-sAxl application in the foundation of preeclampsia in rat model.
2. the method for building up of a preeclampsia in rat model as claimed in claim 1, it is characterised in that: include following step
Rapid:
(1) selecting cleaning grade SD rat, male and female mate ratio 1:3, when simultaneously observing cloudy bolt and sperm determines gestation and is set to
Gestation 0 day；
(2) pregnant rats the 8th day through tail vein injection Ad-sAxl；
(3) by monitoring whether its blood pressure, urine protein, biochemical indicator and observation main organs pathological change determine model construction
Success.
The method for building up of a kind of preeclampsia in rat model the most according to claim 2, it is characterised in that: in step (1)
In, described SD rat body weight 250-280g, week old 8 weeks.
The method for building up of a kind of preeclampsia in rat model the most according to claim 2, it is characterised in that: in step (2)
In, the dosage of described Ad-sAxl is 2 × 109 pfu。
The method for building up of a kind of preeclampsia in rat model the most according to claim 2, it is characterised in that: described method is also
Rat body weight baseline value is determined, by detection is pregnant front and pregnant 1,3,5,7 days blood pressures determine blood pressure including by pregnant 0 day body weight of detection
Baseline value, determines the baseline value of urine protein by pregnant front and pregnant 4 days the twenty-four-hour urine albumen of detection.
The method for building up of a kind of preeclampsia in rat model the most according to claim 2, it is characterised in that: before determining eclamposia
Phase Establishment of Rat Model whether successfully method includes the non-invasive blood pressure of rat after detection injection Ad-sAxl, invasive blood pressure, 24 little
Time urine protein；Pregnant 19 days pregnant Mus body weight, peripheral blood routine blood test, biochemical indicators；Pregnant 19 days row Cesarean esction take out filial mice and count and survey
Crown-rump length, body weight, Placenta Hominis are weighed and by the pathological change of pathological study rat main organs.
The method for building up of a kind of preeclampsia in rat model the most according to claim 6, it is characterised in that: pregnant 9,11,
13, within 15,17 days, rat blood pressure, pregnant 12,16 days monitoring twenty-four-hour urine albumen are monitored with non-invasive blood pressure detector；Within pregnant 19 days, survey pregnant Mus
Invasive blood pressure.
The method for building up of a kind of preeclampsia in rat model the most according to claim 2, it is characterised in that: described the dirtiest
Device includes heart, liver, kidney, Placenta Hominis.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112136763A (en) * | 2020-09-25 | 2020-12-29 | 四川大学华西医院 | Manba gene knockout renal fibrosis animal model and application thereof |
| CN114766427A (en) * | 2022-04-24 | 2022-07-22 | 四川大学华西第二医院 | BCAM target-based severe preeclampsia animal model and construction method and application thereof |
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| CN103993015A (en) * | 2014-05-23 | 2014-08-20 | 山西医科大学 | Peripheral white blood cell miRNA markers associated with onset of human preeclampsia and application of miRNA markers |
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| CN103993015A (en) * | 2014-05-23 | 2014-08-20 | 山西医科大学 | Peripheral white blood cell miRNA markers associated with onset of human preeclampsia and application of miRNA markers |
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| Title |
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| SOICHIRO UEHARA ET AL.: "Plasma levels of growth arrest specific protein(Gas6) and the soluble form of its tyrosine kinase receptor Axl(sAxl) in patients with hepatocellular carcinoma.", 《JOURNAL OF CANCER THERAPY》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112136763A (en) * | 2020-09-25 | 2020-12-29 | 四川大学华西医院 | Manba gene knockout renal fibrosis animal model and application thereof |
| CN114766427A (en) * | 2022-04-24 | 2022-07-22 | 四川大学华西第二医院 | BCAM target-based severe preeclampsia animal model and construction method and application thereof |
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