CN105853468A - Building method of porcine circovirus II type induced mice in-vivo immune cell oxidation stress model - Google Patents
Building method of porcine circovirus II type induced mice in-vivo immune cell oxidation stress model Download PDFInfo
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- CN105853468A CN105853468A CN201610254793.9A CN201610254793A CN105853468A CN 105853468 A CN105853468 A CN 105853468A CN 201610254793 A CN201610254793 A CN 201610254793A CN 105853468 A CN105853468 A CN 105853468A
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Abstract
The invention discloses a building method of a porcine circovirus II type induced mice in-vivo immune cell oxidation stress model. The method is characterized by infecting Kunming strain mice PCV2 with combination of an oral administration manner, a nasal dripping manner and an intraperitoneal injection manner to build the mice in-vivo immune cell oxidation stress model which is used for detecting relevant indexes of cell oxidation stress. The model has the characteristics of convenience in building, low cost and high practicability; an ideal in-vitro model is provided for studying the action mechanism of the PCV2 infected in-vivo immune cell in the process of resisting virus infection and immune diseases; furthermore, some new ideas are possibly provided for treatment on the animal diseases caused by PCV2 infection.
Description
Technical field
The invention belongs to immunocyte oxidative stress modelling technique field, particularly relate to a kind of porcine circovirus 2 type inducing mouse
The construction method of vivo immunization cellular oxidation stress model.
Background technology
Postweaning multisystemic wasting syndrome (PMWS) causes heavy losses to whole world pig industry, although PMWS clinically
Mostly it is to be infected by multiple cause of disease mixed infection and porcine circovirus 2 type (Porcine circovirus type 2, PCV2)
Other pathogen infections of rear secondary cause, but PCV2 is still main pathogen, its main infection suckling pig and growing and fattening pigs.PCV2
After virus individually infects, disease pig often shows as Subclinical, and when PCV2 and other cause of disease mixed infections, or because infecting
When PCV2 causes immunocompromised to cause secondary infection, the clinical symptoms that sick pig shows is multiformity and does not have typicality.Multiple
Virus infects and is responsible for animal body immunosuppressant, and PCV2 is a member therein, and its infection can make other cause of diseases more susceptible,
Or cause the best even immuning failure of vaccine effect, reduce the protective rate of vaccine immunity.Therefore, PCV2 infection is caused
The research of relevant disease the most significant, but, about immunocyte in PCV2 infectosome at antiviral
Infect and also lack understanding with the mechanism of action in immunity pathogenic course, it would be highly desirable to study further.
Summary of the invention
The technical problem to be solved in the present invention be to provide a kind of build conveniently, 2 porcine circovirus that cost is relatively low, practical
The construction method of type inducing mouse vivo immunization cellular oxidation stress model, for immunocyte in research PCV2 infectosome anti-
Virus infects provides comparatively ideal external model with the mechanism of action in immunity pathogenic course.
For solve above-mentioned technical problem, the present invention by the following technical solutions:
The construction method of porcine circovirus 2 type inducing mouse vivo immunization cellular oxidation stress model, uses 2 porcine circovirus
Type is by oral, collunarium, three kinds of approach co-infection mices of lumbar injection, and porcine circovirus 2 type virus titer is 104
TCID50/0.1mL。
Infective dose is PCV2 stock solution 1mL, and infection time is the 1st, 2,3 days.
The construction method of above-mentioned porcine circovirus 2 type inducing mouse vivo immunization cellular oxidation stress model, comprises the following steps:
(1) selection of PCV2 experimental infection mouse protocol
Choose SPF level body weight 20 ± 2g kunming mouse 108, be randomly divided into 6 groups, often group 18, male and female half and half,
Raising temperature is constant at 22 ± 2 DEG C；It fully adapts to environment temporarily to support 5 angels, with reduce stress, and allow its free choice feeding and
Drinking-water, mice starts front 12h fasting in experiment, and 6h prohibits water in advance；
Wherein, A1 group, B1 group, blank 1 group: the 1st, 2,3d infect PCV2,10-1PCV2, normal saline,
1mL//d；A2 group, B2 group, blank 2 groups: the 1st, 3,5,7d infect PCV2,10-1PCV2, normal saline,
1mL//d；
7d, 14d, 21d often organize to cut open respectively and kill mice 6 after virus inoculation and after inoculation, and PCR detects mice spleen
PCV2 antigen in dirty, lungs, determines that PCV2 infects scheme；
(2) PCV2 infects the foundation of kunming mouse oxidative stress model
72 kunming mouses are randomly divided into 6 groups, male and female half and half, often group 12, the scheme determined according to step (1)
Infecting PCV2, normal saline compares group；
Wherein, A group, B group, C group: infect PCV2,1mL//d, process mice at 7d, 14d, 21d respectively；
D group, E group, F group: give normal saline, process mice at 7d, 14d, 21d respectively by 1mL//d；
Separating spleen cell, for carrying out the mensuration of oxidative stress index of correlation.
Oxidative stress index of correlation include spleen cell reactive oxygen species, splenic T-GSH, GSH, GSSG content and XOD, MPO,
INOS activity.
Mice and pig have nearer sibship, and it is short to have experimental period, easily raise the advantages such as easy operation, and have a large amount of
Research shows that PCV2 can success infecting mouse.Inventor utilizes it to design and establishes a kind of porcine circovirus 2 type and induces little
The construction method of Mus vivo immunization cellular oxidation stress model, this method is combined by oral, collunarium, three kinds of approach of lumbar injection
Infect kunming mouse PCV2, establish immunocyte oxidative stress model in Mice Body, stress be correlated with finger for cellular oxidation
Target measures.System of the present invention uses porcine circovirus 2 type (PCV2) to set up immunocyte oxygen in Mice Body as inducement first
Change stress model, and use three kinds of approach co-infection kunming mouse PCV2 to guarantee that mice can more effectively infect first
PCV2, is also to use the index such as ROS level, T-GSH, GSH, GSSG content to refer to as the relevant evaluation of oxidative stress first
Mark.This model has the features such as structure is convenient, cost is relatively low, practical, and it is thin that it infects vivo immunization for research PCV2
Born of the same parents' mechanism of action in viral infection resisting and immunity pathogenic course provides comparatively ideal external model, and then it would be possible to is
PCV2 infects the treatment of the Animal diseases caused provides some new approaches.Condition of culture and method according to the present invention, it is possible to side
Help and carry out immunocyte oxidation in the personnel of immunocyte oxidative stress research set up Mice Body more smoothly in Mice Body for the first time
Stress model model.The application present invention, inventor further defines the optimal infection scheme of PCV2 infecting mouse.
Accompanying drawing explanation
Fig. 1 is the electrophoresis result figure detecting PCV2 virus after virus inoculation 7d.
Fig. 2 is the electrophoresis result figure detecting PCV2 virus after virus inoculation 14d.
Fig. 3 is the electrophoresis result figure detecting PCV2 virus after virus inoculation 21d.
Fig. 4 is the PCV2 variation diagram to mouse spleen lymphocyte ROS level.
Fig. 5 is the variation diagram that PCV2 infects to mouse spleen GSH level.
Fig. 6 is the variation diagram that PCV2 infects to mouse spleen GSSG level.
Fig. 7 is the variation diagram that PCV2 infects to mouse spleen T-GSH level.
Fig. 8 is the variation diagram that PCV2 infects to mouse spleen XOD activity.
Fig. 9 is the variation diagram that PCV2 infects to mouse spleen MPO activity.
Figure 10 is the variation diagram that PCV2 infects to mouse spleen iNOS activity.
In Fig. 5 to Figure 10: each block diagram that on time shaft, same time point is corresponding is from left to right respectively blank group,
PCV2 infected group.
Detailed description of the invention
One, experimental technique
(1) selection of PCV2 experimental infection mouse protocol
Choose SPF level body weight 20 ± 2g kunming mouse 108, be randomly divided into 6 groups, often group 18, male and female half and half,
Raising temperature is constant at 22 ± 2 DEG C.It fully adapts to environment temporarily to support 5 angels, with reduce stress, and allow its free choice feeding and
Drinking-water, mice starts front 12h fasting in experiment, and 6h prohibits water in advance.
A1 group, B1 group, blank 1 group: the 1st, 2,3d inoculation PCV2,10-1PCV2, normal saline, 1mL/ is only
/d。
A2 group, B2 group, blank 2 groups: the 1st, 3,5,7d inoculation PCV2,10-1PCV2, normal saline, 1mL/
Only/d.
7d, 14d, 21d often organize to cut open respectively and kill mice 6 after virus inoculation and after inoculation, and PCR detects mice spleen
PCV2 antigen in dirty, lungs, determines that mice PCV2 infects scheme (virus inoculation time and virus concentration).
(2) PCV2 infects the foundation of kunming mouse oxidative stress model
72 kunming mouses are randomly divided into 6 groups, male and female half and half, often group 12, the sense determined according to step (1)
Dye regime PCV2, normal saline compares group.
A group, B group, C group: PCV2,1mL//d of inoculation, process mice at 7d, 14d, 21d respectively.
D group, E group, F group: give normal saline, process mice at 7d, 14d, 21d respectively by 1mL//d.
(3) DCFH-DA fluorescent probe detection cell ROS level
The aseptic mouse spleen that takes, separating mouse lymphocyte, adjustment cell is 1*105, it being laid on 20 orifice plates, every hole adds 500
10 μMs of DCFH-DA fluorescent probes of μ L, 37 DEG C, 5%CO2Hatch 60min, every 15min to shake up gently once.Hatch
After abandon supernatant, PBS washes 3 times, adds 2000 μ L PBS, is blown down by cell and blow even, in fluorescence spectrophotometry
Meter measures fluorescent value (excitation wavelength 488nm launches wavelength 525nm).
(4) mouse spleen T-GSH, GSH, GSSG content and the detection of XOD, MPO, iNOS activity
Mouse spleen is by weight (g): the ratio of volume (m1)=1: 9 adds the pre-cold saline of 9 times of volumes, in homogenate tube,
It is homogenized under the conditions of ice-water bath.4 DEG C, 5000rpm, centrifugal 10min, take supernatant subpackage, carry out labelling, be used for
Detection T-GSH, GSH, GSSG content and XOD, MPO, iNOS activity.During detection, in strict accordance with T-GSH, GSH, GSSG,
XOD, MPO, iNOS measure test kit explanation and operate.
(5) data analysis
Experimental data uses SSP21.0 statistical software to carry out one factor analysis of variance (One-Way ANOVA), and result is with averagely
Value ± standard deviation represents.Shoulder mark * represents and there is significant difference (P ＜ 0.05) compared with blank group, shoulder mark * * represent with
Cell controls group is compared and be there is pole significant difference (P ＜ 0.01).
Two, experimental result
The infection scheme of<a>PCV-2 Experimental Infected mice model determines (Fig. 1, Fig. 2, Fig. 3)
Experimental group inoculation in 1,2,3 days and the mice of 1,3,5,7 days inoculation PCV2 virus and Normal group mice are the most not
Showing obvious clinical symptoms, and the mental status is normal, diet situation is normal.Cut open its heart, liver, spleen, lung, kidney when killing
Etc. being showed no obvious pathological change.
As Fig. 1 shows, after inoculation PCV27d, the PCV2 in PCR augmentation detection mice lungs, spleen tissue is sick
Poison nucleic acid, A1, A2 group of inoculation PCV2 stock solution has strong specific amplification band at 1154bp；And inoculate 10-1PCV2
The specific fragment that B1, B2 group of virus detects is more weak；Blank group is not detected by specific fragment.As Fig. 2,3 display,
At inoculation PCV214d, 21d, A1, A2 group of inoculation PCV2 stock solution goes out to have strong specific amplification band at 1154bp, and
Specific band detected by A1 group is relatively strong, A2 group is weak compared with A1 group；And inoculate 10-1B1, B2 group of PCV2 detects
Specific fragment fainter；Blank group is not detected by specific fragment.
Result shows, mice uses lumbar injection and collunarium combine with oral three kinds of approach and repeatedly inoculation PCV2 can become
Merit infecting mouse, sets up PCV2 infecting mouse model, and 1,2,3 days virus inoculation infectious effects are better than 1,3,5,7
It virus inoculation, PCV2 stock solution infectious effect is better than 10-1PCV2 virus.According to experimental result, select subsequent experimental infection side
Case is: the 1st, 2,3 day every day is through three kinds of approach co-infections 100PCV2 virus, 1mL//d.
<b>pCV2 infects the impact (Fig. 4) producing mouse spleen lymphocyte ROS
Fig. 4 shows, after PCV2 infecting mouse 7d, 14d, 21d, mouse spleen lymphocyte ROS level the most slightly drops
Low.Compared with blank group, PCV2 infecting mouse 7d, 14d, 21d all raise intracellular ROS level, right with blank
Compare according to group and there is pole significant difference (P ＜ 0.01).Result shows, in the PCV2 infecting mouse significantly raised Mice Body of energy, spleen drenches
Bar cell produces ROS level, and splenocyte is in oxidative stress state.
<c>PCV2 infects the impact (Fig. 5, Fig. 6, Fig. 7) on mouse spleen redox state
Fig. 5 shows, compared with normal mouse in blank group, after PCV-2 infecting mouse 7d, 14d, 21d, no
Mouse spleen GSH level, 7d, 14d mouse spleen GSH level and blank group ratio after wherein infecting is reduced with degree
Relatively there is significant difference (P ＜ 0.05), after infection, 21d mouse spleen GSH level and blank group comparing difference are without system
Meaning (P ＞ 0.05) learned by meter.
Fig. 6 shows, compared with normal mouse in blank group, after PCV-2 infecting mouse 7d, 14d, 21d, no
Increasing mouse spleen GSSG level with degree, after wherein infecting, 7d mouse spleen GSSG level is apparently higher than blank
Group, difference has statistical significance (P ＜ 0.01), and after infection, 14d, 21d mouse spleen GSSG level is right higher than blank
According to group, but no significant difference (P ＞ 0.05).
Fig. 7 shows, compared with normal mouse in blank group, after PCV-2 infecting mouse 7d, 14d, 21d, no
Reducing mouse spleen T-GSH level with degree, after wherein infecting, 7d mouse spleen T-GSH level is substantially less than blank
Group, and difference has statistical significance (P ＜ 0.05), 14d mouse spleen T-GSH level and blank group ratio after infection
Relatively there is pole significant difference (P ＜ 0.01), after infection, 21d mouse spleen T-GSH level is less than blank group, but difference
Not statistically significant (P ＞ 0.05).Test result indicate that, the PCV-2 infecting mouse significantly raised mouse spleen GSSG level of energy,
Reduce mouse spleen GSH level, make mouse spleen T-GSH aggregate level reduce, thus change the mice spleen that PCV-2 infects
Dirty redox state.
<d>PCV2 infects the impact (Fig. 8, Fig. 9, Figure 10) on mouse spleen XOD, MPO, iNOS activity
Fig. 8 shows, compared with normal mouse in blank group, after PCV2 infecting mouse 7d, 14d, 21d, and different journeys
Degree increases mouse spleen XOD activity, and after wherein infecting, 7d spleen XOD activity compares with blank group and there is significance
Difference (P ＜ 0.05), 14d, 21d mouse spleen XOD activity and blank group comparing difference not statistically significant after infection
(P ＞ 0.05).
Fig. 9 shows, compared with normal mouse in blank group, after PCV2 infecting mouse 7d, 14d, 21d, and different journeys
Degree increases mouse spleen MPO activity, and after wherein infecting, 7d mouse spleen MPO activity compares existence and shows with blank group
Writing sex differernce (P ＜ 0.05), after infection, 14d, 21d mouse spleen MPO activity and blank group comparing difference are without statistics
Meaning (P ＞ 0.05).
Figure 10 shows, compared with normal mouse in blank group, after PCV2 infecting mouse 7d, 14d, 21d, and different journeys
Degree increases mouse spleen iNOS activity, 7d mouse spleen iNOS activity and blank group comparing difference after wherein infecting
Significantly (P ＜ 0.05), after infection, 14d, 21d mouse spleen iNOS activity is anticipated without statistics with blank group comparing difference
Justice (P ＞ 0.05).Test result indicate that, PCV2 infecting mouse energy significantly raised mouse spleen XOD, MPO, iNOS activity,
Thus be catalyzed and change the mouse spleen redox state that PCV2 infects.
Three, research conclusion
This experiment uses lumbar injection, collunarium, the associating of three kinds of approach of per os and repeatedly mode of infection, after infection 7d,
When 14d, 21d, being respectively adopted PCR method specific amplification PCV2 viral nucleic acid, result shows, either PCV2 stock solution
Infect kunming mouse, its lungs, spleen tissue DNA through PCR expand after, electrophoresis result all detects specific band.
Additionally, the specific band relatively 10 that PCV2 virus stock solution used infected group detects-1PCV2 infected group is strong, the 1st, 2,3d continuous
Infected group mice measure the specific band of PCV-2 viral nucleic acid be better than 1,3,5,7d infected group.Therefore the sense of subsequent experimental
Dye scheme is defined as using PCV-2 stock solution, uses lumbar injection, collunarium, three kinds of approach associatings of per os and 1,2,3 days even
The mode of continuous many subinfections carries out PCV2 inoculation.And significantly raised mouse spleen XOD during PCV2 infecting mouse 7d, MPO,
INOS activity, and after infecting, when 14d, 21d, XOD, MPO, iNOS activity gradually trends towards normal mouse enzyme activity level,
Show that PCV2 significantly changes mice XOD, MPO, iNOS enzymatic activity after infecting 7d, thus be catalyzed the various peroxide of generation,
Mouse spleen lymphocyte is caused oxidative stress.
Originally test result indicate that, PCV2 success infecting mouse, virus inoculation (virus infects) mode is oral, collunarium, abdomen
Three kinds of approach co-infections of chamber injection；PCV2 infects scheme: the infection virus time is the 1st, 2,3 days, infects virus concentration
It is 10 for titre4TCID50The PCV2 stock solution of/0.1mL, final infection time is (i.e. to infect PCV2 the 7th first in 7 days
It processes mice).By raising infecting mouse splenocyte ROS level, raising cell XOD, MPO, iNOS activity,
Reduce GSH and T-GSH level, to change infecting mouse spleen redox state, be successfully established immunocyte oxidative stress
Animal model.
Claims (4)
1. the construction method of a porcine circovirus 2 type inducing mouse vivo immunization cellular oxidation stress model, it is characterised in that:
Use porcine circovirus 2 type by oral, collunarium, three kinds of approach co-infection mices of lumbar injection, 2 porcine circovirus
Type virus titer is 104TCID50/0.1mL。
The structure side of porcine circovirus 2 type inducing mouse vivo immunization cellular oxidation stress model the most according to claim 1
Method, it is characterised in that: described infective dose is PCV2 stock solution 1mL, and infection time is the 1st, 2,3 days.
The structure side of porcine circovirus 2 type inducing mouse vivo immunization cellular oxidation stress model the most according to claim 1
Method, it is characterised in that: comprise the following steps:
(1) selection of PCV2 experimental infection mouse protocol
Choose SPF level body weight 20 ± 2g kunming mouse 108, be randomly divided into 6 groups, often group 18, male and female half and half,
Raising temperature is constant at 22 ± 2 DEG C；It fully adapts to environment temporarily to support 5 angels, with reduce stress, and allow its free choice feeding and
Drinking-water, mice starts front 12h fasting in experiment, and 6h prohibits water in advance；
Wherein, A1 group, B1 group, blank 1 group: the 1st, 2,3d infect PCV2,10-1PCV2, normal saline,
1mL//d；A2 group, B2 group, blank 2 groups: the 1st, 3,5,7d infect PCV2,10-1PCV2, normal saline,
1mL//d；
7d, 14d, 21d often organize to cut open respectively and kill mice 6 after virus inoculation and after inoculation, and PCR detects mice spleen
PCV2 antigen in dirty, lungs, determines that PCV2 infects scheme；
(2) PCV2 infects the foundation of kunming mouse oxidative stress model
72 kunming mouses are randomly divided into 6 groups, male and female half and half, often group 12, the scheme determined according to step (1)
Infecting PCV2, normal saline compares group；
Wherein, A group, B group, C group: infect PCV2,1mL//d, process mice at 7d, 14d, 21d respectively；
D group, E group, F group: give normal saline, process mice at 7d, 14d, 21d respectively by 1mL//d；
Separating spleen cell, for carrying out the mensuration of oxidative stress index of correlation.
The structure side of porcine circovirus 2 type inducing mouse vivo immunization cellular oxidation stress model the most according to claim 4
Method, it is characterised in that: described oxidative stress index of correlation include spleen cell reactive oxygen species, splenic T-GSH, GSH,
GSSG content and XOD, MPO, iNOS activity.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106479972A (en) * | 2016-10-17 | 2017-03-08 | 广西大学 | PRRSV induces the method for building up of pig spleen cell oxidative stress model |
| CN106489828A (en) * | 2016-10-17 | 2017-03-15 | 广西大学 | The method for building up of oxidative stress model in the infection induced Mice Body of PRRSV |
| CN107875177A (en) * | 2017-09-29 | 2018-04-06 | 广西大学 | The construction method of pig blue-ear disease poison induction piglet vivo immunization cellular oxidation stress model |
-
2016
- 2016-04-22 CN CN201610254793.9A patent/CN105853468A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| 尹丹等: "PCV2诱导小鼠体内免疫细胞氧化胁迫模型的建立", 《中国畜牧兽医学会兽医药理毒理学分会第十一届会员代表大会暨第十三次学术讨论会与中国毒理学会兽医毒理专业委员会第五次学术研讨会论文集》 * |
| 尹丹等: "田七总皂苷对PCV2体外诱导3D4/2细胞氧化应激的调节作用", 《中国畜牧兽医学会兽医药理毒理学分会第十一届会员代表大会暨第十三次学术讨论会与中国毒理学会兽医毒理专业委员会第五次学术研讨会论文集》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106479972A (en) * | 2016-10-17 | 2017-03-08 | 广西大学 | PRRSV induces the method for building up of pig spleen cell oxidative stress model |
| CN106489828A (en) * | 2016-10-17 | 2017-03-15 | 广西大学 | The method for building up of oxidative stress model in the infection induced Mice Body of PRRSV |
| CN107875177A (en) * | 2017-09-29 | 2018-04-06 | 广西大学 | The construction method of pig blue-ear disease poison induction piglet vivo immunization cellular oxidation stress model |
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