CN109777766A - A kind of external evoked decidualization method of the primary stroma cell of Mouse Uterus - Google Patents
A kind of external evoked decidualization method of the primary stroma cell of Mouse Uterus Download PDFInfo
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- CN109777766A CN109777766A CN201910140332.2A CN201910140332A CN109777766A CN 109777766 A CN109777766 A CN 109777766A CN 201910140332 A CN201910140332 A CN 201910140332A CN 109777766 A CN109777766 A CN 109777766A
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Abstract
The present invention discloses a kind of external evoked decidualization method of the primary stroma cell of Mouse Uterus, including following operating procedure: (1) taking the tissue block of the uterine tissue of the 4th day mouse of normal pregnancy；(2) by after tissue block row digestion process, the single cell suspension of the primary stroma cell of Mouse Uterus is obtained；(3) single cell suspension culture to cell density is reached after 75-85% converges, changes liquid, after removing heteroproteose cell, induced medium is added thereto, the primary stroma cell in inducing mouse uterus occurs decidualization.The external evoked decidualization method of the primary stroma cell of a kind of Mouse Uterus provided by the invention, it is easy to operate, it may make the primary stroma cell of the Mouse Uterus isolated survival rate with higher, start that decidualization success rate is higher simultaneously, lays a good foundation for researcher to the research of decidualization mechanism.
Description
Technical field
Cell inductive technology of the present invention field, and in particular to a kind of primary stroma cell of Mouse Uterus is external evoked decidualization
Method.
Background technique
With the development of industrialization with the huge change of human life style, in recent years whole world infertility patient
Ratio rises year by year.Currently, although pregnant technology is manually helped to obtain biggish development, even if the high quality embryo the case where
Under, still there is the deficiency that the implantation rate of embryo transfer is low and subsequent Pregnancy Success rate is low, this is mainly due to uterus
Caused by the dysplasia of inner membrance.The success or not of embryo implantation, on the one hand depending on early embryonic development formation has
On the other hand the blastaea of bed ability receives state, i.e. " window phase " of embryo implantation depending on the entrance of endometrium concertedness.
In mouse, after blastaea and uterine epithelium generation Adhesion, under the influence of steroid hormone, the uterus matrix around blastaea is thin
Born of the same parents start widely to be proliferated and break up (i.e. endometrium is decidualization).Decidualization effect is mainly in functional placentiform
At before, nutrition is provided for the development of blastocyst, and immunity protective effect is played to the embryo of early development.Currently, there is climax
The decidualization mechanism of Endometrium and adjusting etc. are still unclear.Therefore, the process and relevant mechanism of Endometrium decidualization are studied
It will be with important society and theory significance.In the prior art, researcher passes through the primary stroma cell in uterus frequently with mouse
As experimental subjects, to study the mechanism of Endometrium decidualization, but there is the primary stroma cells of Mouse Uterus to induce
Before decidualization, the higher deficiency of the death rate, and there is the decidualization difficulty in starting of the primary stroma cell in uterus, success rate are low
Defect.
Summary of the invention
The object of the present invention is to provide a kind of external evoked decidualization methods of the primary stroma cell of Mouse Uterus, to solve
The death rate of the primary stroma cell of Mouse Uterus existing in the prior art is high, decidualization difficulty in starting, low success rate of defect.
The present invention is achieved by the following technical solutions.
A kind of external evoked decidualization method of the primary stroma cell of Mouse Uterus, including following operating procedure:
(1) uterine tissue for taking the 4th day mouse of normal pregnancy removes epithelial cell after trypsin solution digestion process,
Obtain tissue block；
(2) it after using mass fraction to carry out digestion process again for 0.2% clostridiopetidase A II tissue block, is added thereto
The centrifugation auxiliary agent of tissue block quality 10-15%, then carries out centrifugal treating for mixture, and after discarding supernatant liquid, quality point is added
Number adjusts cell concentration for 10% PBS solution, obtains the single cell suspension of the primary stroma cell of Mouse Uterus, wherein centrifugation helps
Agent is made of the component of following parts by weight: 17-22 parts of casein sodium, 25-30 parts of Sodium Hyaluronate, 13-16 parts of α-ketoglutaric acid；
(3) single cell suspension culture to the cell density that step (2) obtains is reached after 75-85% converges, changes liquid, removes
After heteroproteose cell, induced medium is added thereto, the primary stroma cell generation in inducing mouse uterus is decidualization, wherein Fiber differentiation
Base is to contain 17 beta estradiol of 10-15nM, 1-3 μM of progesterone, 8-12 μM of isoprenaline hydrochloride and 2% mass fraction charcoal
Handle the DMEM-F12 culture medium of fetal calf serum.
Specifically, in above-mentioned steps (1), trypsin solution is that pancreatin and EDTA are dissolved in no calcium and magnesium balanced salt solution, is passed through
It filters out bacterium to be made, the whole mass fraction that wherein the whole mass fraction of pancreatin is 0.25%, EDTA is 0.02%.
Specifically, in above-mentioned steps (1) neutralization procedure (2), the operation of digestion process are as follows: be placed on 4 DEG C of refrigerators and 37 DEG C are thin
After respectively digesting 40min in born of the same parents' incubator, mass fraction is used to terminate digestion reaction reaction for 10% PBS solution.
Specifically, in above-mentioned steps (2), when centrifugal treating, the revolving speed of centrifuge is 3000rpm, and centrifugation time is
15min。
From the above technical scheme, it can be seen that the beneficial effects of the present invention are:
The external evoked decidualization method of the primary stroma cell of a kind of Mouse Uterus provided by the invention, it is easy to operate, it can
So that the primary stroma cell of the Mouse Uterus isolated survival rate with higher, while it is higher to start decidualization success rate,
It lays a good foundation for researcher to the research of decidualization mechanism.Wherein, tissue block is after clostridiopetidase A II processing, through clostridiopetidase A
The histocyte that II is isolated is mixed with the primary stroma cell of Mouse Uterus, it is difficult to and it separates, needs to carry out centrifugal treating,
The primary stroma cell of Mouse Uterus can just be obtained, during centrifugal treating, the primary stroma cell of Mouse Uterus is easy to happen extremely
It dies, and then the primary stroma cell survival rate of the Mouse Uterus caused is lower, centrifugation auxiliary agent provided by the invention can be effective
Activity of the primary stroma cell of Mouse Uterus in centrifugal process is promoted, centrifugal action is reduced and is injured caused by cell, in turn
The survival rate of the primary stroma cell of Mouse Uterus is effectively guaranteed；2% mass fraction charcoal treatment fetal calf serum can be effective
The primary stroma cell of Mouse Uterus is stimulated to complete decidualization, wherein the addition of isoprenaline hydrochloride can effectively promote small
The decidualization starting of the primary stroma cell in mouse uterus.
Specific embodiment
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.Reality used in the examples
The condition of applying can be for further adjustments according to the condition of producer, and unaccounted implementation condition is usually conventional laboratory conditions.
Embodiment 1
A kind of external evoked decidualization method of the primary stroma cell of Mouse Uterus, including following operating procedure:
(1) after taking the 4th day mouse of normal pregnancy to put to death, 2min is sterilized in 75% alcohol, is opened under aseptic condition
Normal saline flushing side uterus is used before collecting uterus, it is determined whether have the presence of embryo, it is small that one is cut at cervix in abdominal cavity
Mouthful, two sides uterine cavity is longitudinally splitted with scissors and collects uterus, and the mesentery and fat of uterus side are all removed and put as far as possible
It is filling in the 15ml centrifuge tube of PBS buffer solution, acutely concussion obtains the uterus group of mouse to remove blood and its hetero-organization
It knits, after trypsin solution is added thereto, is placed on after respectively digesting 40min in 4 DEG C of refrigerators and 37 DEG C of cell incubators, using quality point
Number terminates digestion reaction reaction for 10% PBS solution, removes epithelial cell, obtains tissue block, wherein trypsin solution is pancreatin
It is dissolved in no calcium and magnesium balanced salt solution with EDTA, is made through filtration sterilization, the whole mass fraction of pancreatin is the end of 0.25%, EDTA
Mass fraction is 0.02%；
(2) the clostridiopetidase A II that mass fraction is 0.2% into tissue block, is placed in 4 DEG C of refrigerators and 37 DEG C of cell incubators
After each digestion 40min, uses mass fraction to terminate digestion reaction reaction for 10% PBS solution, tissue block matter is added thereto
The centrifugation auxiliary agent of 10-15% is measured, mixture is then subjected to centrifugal treating, the revolving speed of centrifuge is 3000rpm, and centrifugation time is
15min after discarding supernatant liquid, is added the PBS solution that mass fraction is 10% and adjusts cell concentration, obtain the primary base of Mouse Uterus
The single cell suspension of cell plastid, wherein centrifugation auxiliary agent is made of the component of following parts by weight: 17 parts of casein sodium, Sodium Hyaluronate
25 parts, 13 parts of α-ketoglutaric acid；
(3) by single cell suspension culture to the cell density that step (2) obtains reach 75% converge after, change liquid, go to clean
After cell, induced medium is added thereto, the primary stroma cell generation in inducing mouse uterus is decidualization, wherein induced medium
To contain 17 beta estradiol of 10nM, 1 μM of progesterone, 8 μM of isoprenaline hydrochlorides and 2% mass fraction charcoal treatment tire ox blood
Clear DMEM-F12 culture medium.
Embodiment 2
A kind of external evoked decidualization method of the primary stroma cell of Mouse Uterus, including following operating procedure:
(1) after taking the 4th day mouse of normal pregnancy to put to death, 2min is sterilized in 75% alcohol, is opened under aseptic condition
Normal saline flushing side uterus is used before collecting uterus, it is determined whether have the presence of embryo in abdominal cavity.It is small that one is cut at cervix
Mouthful, two sides uterine cavity is longitudinally splitted with scissors and collects uterus, and the mesentery and fat of uterus side are all removed and put as far as possible
It is filling in the 15ml centrifuge tube of PBS buffer solution, acutely concussion obtains the uterus group of mouse to remove blood and its hetero-organization
It knits, after trypsin solution is added thereto, is placed on after respectively digesting 40min in 4 DEG C of refrigerators and 37 DEG C of cell incubators, using quality point
Number terminates digestion reaction reaction for 10% PBS solution, removes epithelial cell, obtains tissue block, wherein trypsin solution is pancreatin
It is dissolved in no calcium and magnesium balanced salt solution with EDTA, is made through filtration sterilization, the whole mass fraction of pancreatin is the end of 0.25%, EDTA
Mass fraction is 0.02%；
(2) the clostridiopetidase A II that mass fraction is 0.2% into tissue block, is placed in 4 DEG C of refrigerators and 37 DEG C of cell incubators
After each digestion 40min, uses mass fraction to terminate digestion reaction reaction for 10% PBS solution, tissue block matter is added thereto
Then mixture is carried out centrifugal treating by the centrifugation auxiliary agent of amount 15%, the revolving speed of centrifuge is 3000rpm, and centrifugation time is
15min after discarding supernatant liquid, is added the PBS solution that mass fraction is 10% and adjusts cell concentration, obtain the primary base of Mouse Uterus
The single cell suspension of cell plastid, wherein centrifugation auxiliary agent is made of the component of following parts by weight: 22 parts of casein sodium, Sodium Hyaluronate
30 parts, 16 parts of α-ketoglutaric acid；
(3) by single cell suspension culture to the cell density that step (2) obtains reach 85% converge after, change liquid, go to clean
After cell, induced medium is added thereto, the primary stroma cell generation in inducing mouse uterus is decidualization, wherein induced medium
To contain 17 beta estradiol of 15nM, 3 μM of progesterone, 12 μM of isoprenaline hydrochlorides and 2% mass fraction charcoal treatment tire ox blood
Clear DMEM-F12 culture medium.
Comparative example 1
Step does not add centrifugation auxiliary agent in (2), remaining operating procedure is identical with embodiment 1.
Comparative example 2
In step (3), isoprenaline hydrochloride is not added in induced medium, remaining operating procedure and embodiment 2 are complete
It is exactly the same.
In embodiment 1 and comparative example 1, the single cell suspension of the primary stroma cell of Mouse Uterus made from step (2) is used
0.2% Trypan blue exclusion test tests its survival rate, and test result is as shown in table 1:
The single cell suspension mesostroma cell survival rate of the primary stroma cell of 1 Mouse Uterus of table
| Project | Stroma cell survival rate, % |
| Embodiment 1 | 94 |
| Comparative example 1 | 83 |
As shown in Table 1, centrifugation auxiliary agent can effectively reduce the death rate of centrifugal process mesostroma cell.
Pass through decidualization related mark molecule Cyclin D3 whether the decidualization starting of the primary stroma cell of Mouse Uterus
Expression measure, the expression of Cyclin D3 is measured by Western blot and quantitative PCR analysis, Cyclin D3
Expression increase with the time, then the primary stroma cell of Mouse Uterus is decidualization successfully starts up, the primary matrix of Mouse Uterus
The success rate of the decidualization starting of cell is as shown in table 2:
The success rate of the decidualization starting of the primary stroma cell of 2 Mouse Uterus of table
| Project | Success rate, % |
| Embodiment 1 | 88 |
| Embodiment 2 | 91 |
| Comparative example 2 | 69 |
As shown in Table 2, isoprenaline hydrochloride can be obviously improved the decidualization starting of the primary stroma cell of Mouse Uterus
Success rate.
Certainly, the above description is not a limitation of the present invention, and the present invention is also not limited to the example above, the art
Those of ordinary skill, within the essential scope of the present invention, variation, change, addition or the replacement made all should belong to the present invention
Protection scope.
Claims (4)
1. a kind of external evoked decidualization method of the primary stroma cell of Mouse Uterus, which is characterized in that walked including following operation
It is rapid:
(1) uterine tissue for taking the 4th day mouse of normal pregnancy is removed epithelial cell, is obtained after trypsin solution digestion process
Tissue block；
(2) after using mass fraction to carry out digestion process again for 0.2% clostridiopetidase A II tissue block, tissue is added thereto
The centrifugation auxiliary agent of block quality 10-15%, then carries out centrifugal treating for mixture, and after discarding supernatant liquid, mass fraction, which is added, is
10% PBS solution adjusts cell concentration, obtains the single cell suspension of the primary stroma cell of Mouse Uterus, wherein centrifugation auxiliary agent by
The component of following parts by weight is made: 17-22 parts of casein sodium, 25-30 parts of Sodium Hyaluronate, 13-16 parts of α-ketoglutaric acid；
(3) single cell suspension culture to the cell density that step (2) obtains is reached after 75-85% converges, changes liquid, goes removal of impurities thin
After born of the same parents, induced medium is added thereto, the primary stroma cell generation in inducing mouse uterus is decidualization, and wherein induced medium is
Contain 17 beta estradiol of 10-15nM, 1-3 μM of progesterone, 8-12 μM of isoprenaline hydrochloride and 2% mass fraction charcoal treatment
The DMEM-F12 culture medium of fetal calf serum.
2. a kind of external evoked decidualization method of the primary stroma cell of Mouse Uterus as described in claim 1, feature exist
In, in above-mentioned steps (1), trypsin solution is that pancreatin and EDTA are dissolved in no calcium and magnesium balanced salt solution, it is made through filtration sterilization,
The whole mass fraction that the whole mass fraction of middle pancreatin is 0.25%, EDTA is 0.02%.
3. the external evoked decidualization method of the primary stroma cell of a kind of Mouse Uterus according to claim 1, feature
It is, in above-mentioned steps (1) neutralization procedure (2), the operation of digestion process are as follows: be placed in 4 DEG C of refrigerators and 37 DEG C of cell incubators
After each digestion 40min, mass fraction is used to terminate digestion reaction reaction for 10% PBS solution.
4. the external evoked decidualization method of the primary stroma cell of a kind of Mouse Uterus according to claim 1, feature
It is, in above-mentioned steps (2), when centrifugal treating, the revolving speed of centrifuge is 3000rpm, centrifugation time 15min.
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| CN112760278A (en) * | 2019-10-21 | 2021-05-07 | 东北林业大学 | Practical and efficient culture method of mouse uterine stromal cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104694469A (en) * | 2015-03-26 | 2015-06-10 | 江苏希瑞干细胞技术有限公司 | Preparation method for mesenchymal stem cells from placental decidua basalis |
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| CN104694469A (en) * | 2015-03-26 | 2015-06-10 | 江苏希瑞干细胞技术有限公司 | Preparation method for mesenchymal stem cells from placental decidua basalis |
Non-Patent Citations (2)
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| 于永生等: "小鼠子宫内膜诱导蜕膜化的研究", 《安徽农业科学》 * |
| 张鹏等: "小鼠子宫内膜基质细胞分离培养、鉴定及体外蜕膜化的诱导", 《中国组织工程研究》 * |
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| CN112760278A (en) * | 2019-10-21 | 2021-05-07 | 东北林业大学 | Practical and efficient culture method of mouse uterine stromal cells |
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