CN109943529A - A kind of application method of taurine to IUGR tire mouse neuron proliferation - Google Patents
A kind of application method of taurine to IUGR tire mouse neuron proliferation Download PDFInfo
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- CN109943529A CN109943529A CN201910215482.5A CN201910215482A CN109943529A CN 109943529 A CN109943529 A CN 109943529A CN 201910215482 A CN201910215482 A CN 201910215482A CN 109943529 A CN109943529 A CN 109943529A
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Abstract
The invention belongs to bio-medical technology field, a kind of taurine is disclosed to the application method of IUGR tire mouse neuron proliferation.The present invention, which provides taurine, can be successfully established IUGR newborn rat and tire mouse model to the application method of IUGR tire mouse neuron proliferation；Pregnant mouse supplementation of taurine can obviously reduce the generation of IUGR, reduces the death rate, increases brain again；IUGR tire mouse intracerebral NSC quantity is reduced, and IUGR tire mouse NSC quantity increases after pregnant mouse supplementation of taurine；IUGR tire mouse NSC in-vitro multiplication ability reduces, and taurine can promote IUGR tire mouse NSC in-vitro multiplication；Taurine may be promoted IUGR tire mouse NSC proliferation by up-regulation cAMP-PKA-CREB signal path activity, improve brain growth；Meanwhile neural stem cell can be made to stablize proliferation by being separately cultured neural stem cell method.
Description
Technical field
The invention belongs to bio-medical technology fields more particularly to a kind of taurine to answer IUGR tire mouse neuron proliferation
Use method.
Background technique
Fetal Growth Restriction (Intrauterine growth restriction, IUGR) refers to that various factors causes fetus
It is restricted in growth in utero, best potential cannot be played, birth weight is significantly lower than normal.Mother, fetus and placenta
Etc. a variety of pathological factors can lead to the generation of IUGR.IUGR can lead to multi-organ function involvement, not to brain growth
Good influence is especially prominent, seriously affects nervous function at a specified future date.Taurine plays a significant role during brain growth, but body is certainly
Body synthesis is less, and fetus obtains enough taurines by placental transport.The experimental study of this seminar early period is shown: pregnant mouse is mended
IUGR mouse brain growth can be improved by filling taurine, such as is improved brain's operation system, increased nerve cell number, these effects may be
It is realized by adjusting cAMP-PKA-CREB signal path activity.Neural stem cell (Neural Stem is adjusted in taurine
Cells, NSC) proliferation, survival, adhesive capacity, but taurine is not yet clear to the IUGR tire mouse NSC influence being proliferated and mechanism
Chu.
In conclusion problem of the existing technology is:
Taurine is not yet clear to the IUGR tire mouse NSC influence being proliferated and mechanism；
The neural stem cell method that is separately cultured used in the prior art can not be so that neural stem cell stablizes proliferation.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of taurines answers IUGR tire mouse neuron proliferation
Use method.
The invention is realized in this way a kind of application method of taurine to IUGR tire mouse neuron proliferation, including it is following
Step:
Step 1: experimental animal and grouping: taking SD rat 60, female 30, male 30, using promoting sexual gland hormone,
Female rats 1:1 is mated every night, and vaginal fluid is taken to carry out microscopy after 8~10h, and determination is become pregnant；Female mice be randomly divided into control group,
IUGR group, taurine group, every group 10；
Step 2 establishes IUGR model, establishes IUGR model using the method for whole low protein diet；
Step 3, the pregnant mouse of IUGR group add taurine 400mg/kg/d from pregnant 10 days, weight and brain are weighed after spontaneous labor
Weight；
Step 4 takes the rat embryo Cerebral cortex separation and Extraction neuronal stem cell of pregnant 15d, trains to neural stem cell
It supports；
Step 5, carries out immunohistochemistry staining method in vitro, and cell streaming method carries out NSC judgement to each group brain tissue
And quantity determines；Inverted microscope observes cytomorphology variation, and CCK-8 method adds the proliferative capacity for surveying cell；Utilize RT-PCR
Method detection each group brain tissue in the cAMP-PKA-CREB signal path key factor expression.
Further, IUGR model is established using the method for whole low protein diet, IUGR group whole process gives Low protein diet
It feeds, Low protein diet is made of according to energy proportion albumen 7%, carbohydrate 73%, fat 20%.
Further, control group, IUGR group, taurine group use ad lib during the feeding process, free water, room temperature,
12h light and shade alternate illumination.
Further, in step 4, separation, culture of neural stem cells neural method are as follows:
(1) the pregnant mouse of pregnant 15d is pinned head dislocation to put to death, is sterilized with the ethyl alcohol bathing that volume fraction is 75%；
(2) aseptically, tissue, mechanical piping and druming and copper mesh are shredded in rat embryo Cerebral cortex to filter, 4 DEG C,
1200r/min is centrifuged 8min, separates neural stem cell；
(3) using nerve stem cell culture medium in 37 DEG C, 5%CO2Under the conditions of to separation neural stem cell cultivate, make
Neural stem cell stablizes proliferation；
It passes on 1 time within (4) 3~5 days, digests poly- cell ball using Accutase, utilize nestin after the poly- ball of cell
(Nestin) neural stem cell is identified；
(5) its adherent differentiation is induced with 12% fetal calf serum, using immunohistochemical staining method, to NSCs and its differentiation
Nervous system elementary cell afterwards is identified.
Further, culture medium, culture medium include DMDM/F12 (1:1), 1.2%B27,18ng/ml EGF, 15ng/ml
bFGF、6ug/ml Heparin。
Further, immunohistochemical staining method uses neuronspecific enolase (NSE), glial fibrillary acidic respectively
Albumen (GFAP), MBP ELISA (MBP) dyeing.
Further, in step 5, in-vitro separation control group and IUGR group tire mouse NSC, Immunofluorescence decoration method
Detect the expression of cell FABP-7 and Nestin；RT-PCR method detects taurine and signal pathway inhibitor H89 handles NSC
The expression of each group cAMP-PKA-CREB signal path key factor afterwards.
Advantages of the present invention and good effect are as follows:
Specify the influence relationship that taurine is proliferated IUGR tire mouse NSC；
The present invention provide taurine to the application method of IUGR tire mouse neuron proliferation can be successfully established IUGR newborn rat with
And tire mouse model；
Pregnant mouse supplementation of taurine can obviously reduce the generation of IUGR, reduces the death rate, increases brain again；IUGR group tire mouse brain
The quantity of interior NSC significantly reduces, and IUGR group NSC quantity obviously increases (P < 0.05) after supplementation of taurine；
The expression of the IUGR tire mouse intracerebral cAMP-PKA-CREB signal path key factor has significance difference compared with the control group
Different, the expression of taurine group key factor is substantially change compared with IUGR group, and difference is statistically significant (P < 0.05)；
IUGR tire mouse NSC in-vitro multiplication ability and cell quantity are substantially reduced, and taurine is intervened in vitro can make IUGR tire mouse
NSC proliferative capacity obviously increases, and related (P < 0.05) with concentration；
The expression of the cAMP-PKA-CREB signal path key factor is suppressed under IUGR tire mouse NSC condition of in vitro culture,
Partial factors increase in compensatory, and the expression of correlation factor occurs bright after having notable difference, taurine to intervene compared with the control group
Aobvious variation, difference have statistically significant (P < 0.05)；
IUGR tire mouse intracerebral NSC quantity is reduced, and IUGR tire mouse NSC quantity increases after pregnant mouse supplementation of taurine；
IUGR tire mouse NSC in-vitro multiplication ability reduces, and taurine can promote IUGR tire mouse NSC in-vitro multiplication；
Taurine may be promoted IUGR tire mouse NSC proliferation by up-regulation cAMP-PKA-CREB signal path activity, be improved
Brain growth；
Neural stem cell can be made to stablize proliferation by being separately cultured neural stem cell method.
Detailed description of the invention
Fig. 1 is that the present invention implements the taurine provided to the application method flow chart of IUGR tire mouse neuron proliferation.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Application principle of the invention is further described with reference to the accompanying drawing.
As shown in Figure 1, the present invention provides a kind of taurine to the application method of IUGR tire mouse neuron proliferation, including following
Step:
S101: experimental animal and grouping: SD rat 60 are taken, female 30 is 30 male, using promoting sexual gland hormone, often
Late female rats 1:1 is mated, and vaginal fluid is taken to carry out microscopy after 8~10h, and determination is become pregnant；Female mice be randomly divided into control group,
IUGR group, taurine group, every group 10；
S102 establishes IUGR model, establishes IUGR model using the method for whole low protein diet；
The pregnant mouse of S103, IUGR group added taurine 400mg/kg/d from pregnant 10 days, and weight and brain weight are weighed after spontaneous labor；
S104 takes the rat embryo Cerebral cortex separation and Extraction neuronal stem cell of pregnant 15d, trains to neural stem cell
It supports；
S105, carries out immunohistochemistry staining method in vitro, cell streaming method to each group brain tissue carry out NSC judgement and
Quantity determines；Inverted microscope observes cytomorphology variation, and CCK-8 method adds the proliferative capacity for surveying cell；Utilize RT-PCR's
Method detects the expression of the cAMP-PKA-CREB signal path key factor in each group brain tissue.
In step S102, the method provided in an embodiment of the present invention using whole low protein diet establishes IUGR model,
IUGR group whole process give Low protein diet nursing, Low protein diet according to energy proportion by albumen 7%, carbohydrate 73%,
Fat 20% forms.
In step S102, control group provided in an embodiment of the present invention, IUGR group, taurine group use during the feeding process from
By feeding, free water, room temperature, 12h light and shade alternate illumination.
In step S104, it is provided in an embodiment of the present invention separation, culture of neural stem cells neural method it is as follows:
(1) the pregnant mouse of pregnant 15d is pinned head dislocation to put to death, is sterilized with the ethyl alcohol bathing that volume fraction is 75%；
(2) aseptically, tissue, mechanical piping and druming and copper mesh are shredded in rat embryo Cerebral cortex to filter, 4 DEG C,
1200r/min is centrifuged 8min, separates neural stem cell；
(3) using nerve stem cell culture medium in 37 DEG C, 5%CO2Under the conditions of to separation neural stem cell cultivate, make
Neural stem cell stablizes proliferation；
It passes on 1 time within (4) 3~5 days, digests poly- cell ball using Accutase, utilize nestin after the poly- ball of cell
(Nestin) neural stem cell is identified；
(5) its adherent differentiation is induced with 12% fetal calf serum, using immunohistochemical staining method, to NSCs and its differentiation
Nervous system elementary cell afterwards is identified.
In step S104, culture medium provided in an embodiment of the present invention, culture medium include DMDM/F12 (1:1), 1.2%B27,
18ng/ml EGF、15ng/ml bFGF、6ug/ml Heparin。
In step S104, immunohistochemical staining method provided in an embodiment of the present invention uses neuron specific enolase respectively
Change enzyme (NSE), glial fibrillary acid protein (GFAP), MBP ELISA (MBP) dyeing.
In step S105, in-vitro separation control group and IUGR group tire mouse NSC provided in an embodiment of the present invention, immunofluorescence is thin
The expression of the detection of born of the same parents' chemical dyeing method cell FABP-7 and Nestin；RT-PCR method detects taurine and signal path inhibits
Agent H89 handles the expression of each group cAMP-PKA-CREB signal path key factor after NSC.
Application principle of the invention is further described combined with specific embodiments below；
A kind of application method of taurine to IUGR tire mouse neuron proliferation, comprising the following steps:
Step 1: experimental animal and grouping: taking SD rat 60, female 30, male 30, using promoting sexual gland hormone,
Female rats 1:1 is mated every night, and vaginal fluid is taken to carry out microscopy after 8~10h, and determination is become pregnant；Female mice be randomly divided into control group,
IUGR group, taurine group, every group 10；
Step 2 establishes IUGR model, establishes IUGR model using the method for whole low protein diet；
Step 3, the pregnant mouse of IUGR group add taurine 400mg/kg/d from pregnant 10 days, weight and brain are weighed after spontaneous labor
Weight；
Step 4 takes the rat embryo Cerebral cortex separation and Extraction neuronal stem cell of pregnant 15d, trains to neural stem cell
It supports；
Step 5, carries out immunohistochemistry staining method in vitro, and cell streaming method carries out NSC judgement to each group brain tissue
And quantity determines；Inverted microscope observes cytomorphology variation, and CCK-8 method adds the proliferative capacity for surveying cell；Utilize RT-PCR
Method detection each group brain tissue in the cAMP-PKA-CREB signal path key factor expression；
Step 6, data statistics and analysis.
The present invention provide taurine to the application method of IUGR tire mouse neuron proliferation can be successfully established IUGR newborn rat with
And tire mouse model；Pregnant mouse supplementation of taurine can obviously reduce the generation of IUGR, reduces the death rate, increases brain again；IUGR group tire
The quantity of mouse intracerebral NSC significantly reduces, and IUGR group NSC quantity obviously increases (P < 0.05) after supplementation of taurine；IUGR tire mouse brain
There were significant differences compared with the control group for the expression of the interior cAMP-PKA-CREB signal path key factor, taurine group key factor
Expression substantially change compared with IUGR group, difference is statistically significant (P < 0.05).IUGR tire mouse NSC in-vitro multiplication ability and thin
Born of the same parents' quantity is substantially reduced, and taurine is intervened in vitro can be such that IUGR tire mouse NSC proliferative capacity obviously increases, and related (the P < with concentration
0.05)；The expression of the cAMP-PKA-CREB signal path key factor is suppressed under IUGR tire mouse NSC condition of in vitro culture, portion
Molecular group increases in compensatory, and the expression of correlation factor occurs obvious after having notable difference, taurine to intervene compared with the control group
Variation, difference have statistically significant (P < 0.05).IUGR tire mouse intracerebral NSC quantity is reduced, IUGR after pregnant mouse supplementation of taurine
Tire mouse NSC quantity increases；IUGR tire mouse NSC in-vitro multiplication ability reduces, and taurine can promote IUGR tire mouse NSC in-vitro multiplication；
Taurine may be promoted IUGR tire mouse NSC proliferation by up-regulation cAMP-PKA-CREB signal path activity, improve brain growth；
Meanwhile neural stem cell can be made to stablize proliferation by being separately cultured neural stem cell method.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (7)
1. a kind of taurine is to the application method of IUGR tire mouse neuron proliferation, which is characterized in that the taurine is to IUGR tire
The application method of mouse neuron proliferation the following steps are included:
Step 1: experimental animal and grouping: SD rat 60 are taken, female 30 is 30 male, using promoting sexual gland hormone, every night
Female rats 1:1 is mated, and vaginal fluid is taken to carry out microscopy after 8~10h, and determination is become pregnant；Female mice is randomly divided into control group, IUGR
Group, taurine group, every group 10；
Step 2 establishes IUGR model, establishes IUGR model using the method for whole low protein diet；
Step 3, the pregnant mouse of IUGR group add taurine 400mg/kg/d from pregnant 10 days, weight and brain weight are weighed after spontaneous labor；
Step 4 takes the rat embryo Cerebral cortex separation and Extraction neuronal stem cell of pregnant 15d, cultivates neural stem cell；
Step 5, carries out immunohistochemistry staining method in vitro, and cell streaming method carries out NSC judgement and number to each group brain tissue
Amount determines；Inverted microscope observes cytomorphology variation, and CCK-8 method adds the proliferative capacity for surveying cell；Utilize the side of RT-PCR
Method detects the expression of the cAMP-PKA-CREB signal path key factor in each group brain tissue.
2. taurine as described in claim 1 is to the application method of IUGR tire mouse neuron proliferation, which is characterized in that described to adopt
IUGR model is established with the method for whole low protein diet, IUGR group whole process gives Low protein diet nursing, and Low protein diet is pressed
It is made of according to energy proportion albumen 7%, carbohydrate 73%, fat 20%.
3. taurine as described in claim 1 is to the application method of IUGR tire mouse neuron proliferation, which is characterized in that described right
Ad lib, free water, room temperature, 12h light and shade alternate illumination are used during the feeding process according to group, IUGR group, taurine group.
4. taurine as described in claim 1 is to the application method of IUGR tire mouse neuron proliferation, which is characterized in that the step
In rapid four, separation, culture of neural stem cells neural method are as follows:
(1) the pregnant mouse of pregnant 15d is pinned head dislocation to put to death, is sterilized with the ethyl alcohol bathing that volume fraction is 75%；
(2) aseptically, tissue, mechanical piping and druming and copper mesh are shredded in rat embryo Cerebral cortex to filter, 4 DEG C, 1200r/
Min is centrifuged 8min, separates neural stem cell；
(3) using nerve stem cell culture medium in 37 DEG C, 5%CO2Under the conditions of to separation neural stem cell cultivate, make nerve
Stem cell stablizes proliferation；
It passes on 1 time within (4) 3~5 days, digests poly- cell ball using Accutase, reflected after the poly- ball of cell using nestin (Nestin)
Determine neural stem cell；
(5) its adherent differentiation is induced with 12% fetal calf serum, using immunohistochemical staining method, after NSCs and its differentiation
Nervous system elementary cell is identified.
5. taurine as described in claim 1 is to the application method of IUGR tire mouse neuron proliferation, which is characterized in that the training
Base is supported, culture medium includes DMDM/F12 (1:1), 1.2%B27,18ng/ml EGF, 15ng/ml bFGF, 6ug/ml
Heparin。
6. taurine as described in claim 1 is to the application method of IUGR tire mouse neuron proliferation, which is characterized in that described to exempt from
Epidemic disease histochemical staining method uses neuronspecific enolase (NSE), glial fibrillary acid protein (GFAP), myelin alkali respectively
Property albumen (MBP) dye.
7. taurine as described in claim 1 is to the application method of IUGR tire mouse neuron proliferation, which is characterized in that the step
In rapid five, in-vitro separation control group and IUGR group tire mouse NSC, Immunofluorescence decoration method detect cell FABP-7 and
The expression of Nestin；RT-PCR method detects each group cAMP-PKA- after taurine and signal pathway inhibitor H89 processing NSC
The expression of the CREB signal path key factor.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018221521A1 (en) * | 2017-05-30 | 2018-12-06 | 帝人ファーマ株式会社 | Anti-igf-i receptor antibody |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018221521A1 (en) * | 2017-05-30 | 2018-12-06 | 帝人ファーマ株式会社 | Anti-igf-i receptor antibody |
Non-Patent Citations (3)
| Title |
|---|
| 刘新民等: "《中华医学百科大辞海内科学 第3卷》", 31 October 2008, 军事医学科学出版社 * |
| 王燕: "牛磺酸调节cAMP-PKA- CREB信号通路活性促进生长受限胎鼠神经干细胞增殖的实验研究", 《万方数据知识服务平台》 * |
| 魏萍: "《临床医技新编》", 31 March 2016, 云南科技出版社 * |
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Application publication date: 20190628 |