CN102268404B - Method for separating pig cumulus stem cells - Google Patents
Method for separating pig cumulus stem cells Download PDFInfo
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- CN102268404B CN102268404B CN201110204169A CN201110204169A CN102268404B CN 102268404 B CN102268404 B CN 102268404B CN 201110204169 A CN201110204169 A CN 201110204169A CN 201110204169 A CN201110204169 A CN 201110204169A CN 102268404 B CN102268404 B CN 102268404B
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Abstract
The invention discloses a method for separating pig cumulus stem cells, which comprises the following steps of: extracting a cumulus-oocyte complex from a young sow, performing in-vitro maturation culture on oocyte cells, digesting and separating cumulus cells, collecting digested cumulus cells for culture, performing digestive passage inoculation onto a feeder layer when a cell colony appears and a larger cell colony is formed, and culturing in a cell factor-containing culture solution, wherein the separately cultured cumulus stem cells have bird nest cell growthform, round shapes and larger nuclei, express unique markers Oct4, Nanog and SSEA1, and are subjected to in-vitro differentiation to form nerve cells, muscle cells and liver cells derived from three germ layers; and results show that the cumulus stem cells are separated from pig cumulus cells.
Description
[technical field]
The present invention relates to the separation method of stem cell animal, relate in particular to the separation method of a boar ovarian cumulus stem cell.
[background technology]
Stem cell (Stem Cell) is a kind of fully the differentiation as yet and sophisticated cell, under the induced environment condition, has to form the potential function that various histoorgans are formed cells, is referred to as by medical circle in " omnipotent cell ".The purposes of stem cell is very extensive, relates to a plurality of fields of medical science.The extensive clinical application of stem cell and derived tissues organ thereof; With producing a kind of brand-new medical skill; Just reproduce the normal even young histoorgan of human body; Thereby make the mankind can use oneself or other people stem cell or by the new histoorgan that stem cell derived, replace self pathology or old and feeble histoorgan.The research of stem cell will provide more wide application prospect for clinical medicine.Fast development along with various biotechnologys such as genetically engineered, embryo engineering, cell engineerings; According to certain purpose; Become possibility in external artificial separation, culturing stem cells; Utilize the various cells of stem cell constructing, tissue, the organ source as transplant organ, this will become the main direction that stem cell is used.Current; The research of stem cell and regenerative medicine has become the most noticeable field in the life science; Become better and approaching perfection day by day and technological fast development that it is theoretical will produce epoch-making achievement in fields such as disease treatment and biological medicines, are great revolutions to traditional medical means and medical idea.
Stem cell has the original undifferentiated cell of multidirectional differentiation potential and the of self-replication capacity as cells of origin, is the forefathers' cell that forms each histoorgan of mammals.Stem cell has general character usually on form, demonstrate circle or oval configuration, and the volume of stem cell is less, and nucleus is taller and bigger relatively.Stem cell can be divided into embryonic stem cell and adult stem cell by its source.Embryonic stem cell (Embryonic Stem cell, ES cell) is from the inner cell mass of early stage zygote blastaea, and the ES cell is a kind of height undifferentiated cell.It has the totipotency of growth, can be differentiated to form all composition cells of animal body tissue and organ, even comprise sexual cell sperm and ovum.Therefore, studying and utilize the ES cell is the focus of current bioengineering field.But because the ES cell derives from the inner cell mass of early stage blastaea, when the embryo in uterus behind the implantation inner cell mass of blastaea with living individual of bud into.Therefore, there is the contradiction of can'tting eat one's cake and have it in the birth of the separation of ES cell and a living individual.In addition, the cell that is differentiated to form of ES cell induction since the immunological rejection phenomenon can not be applied directly in patient's body.Adult stem cell derives from the many tissues or the organ of animal body as a kind of cell type in the stem cell family.Adult stem cell is used for reparation or replacement damaged cells, tissue or organ through inducing the differentiation back to form body cell, tissue or organ in short supply; Because self adult stem cell is in full accord with the animal body genotype, the immunological rejection phenomenon can be effectively avoided in stem cell transplantation.Adult stem cell is being brought into play crucial regulating effect as the cell deposit in the growth and development process of animal body, keep the running balance of tissue or organ.At present, hemopoietic stem cell has been successfully applied to clinical medicine as adult stem cell.Hemopoietic stem cell is unique seed cell source of various hemocytes in the body, and it mainly is present in marrow, peripheral blood, the Cord blood.The transplanting of hemopoietic stem cell is treatment disease in the blood system, ancestor genetic diseases and a most effectual way multiple and the transitivity malignancy disease.Hemopoietic stem cell is present in hemopoietic system, and for the serious disease of leukaemic because of hemopoietic system, the hemopoietic stem cell of self is damaged, and isolates and cultures the adult stem cell that comes from its hetero-organization or organ and has important use value and realistic meaning.
People successfully isolate adult stem cell from tissues such as skin, fat, nerve, blood at present, and wherein hemopoietic stem cell successfully is applied to human clinical.But up to the present also do not see the report of from the ovary of animal body, isolating stem cell.
[summary of the invention]
The technical problem that the present invention will solve provides the separation method of a boar ovarian cumulus stem cell.
In order to solve the problems of the technologies described above, the technical scheme that the present invention adopts is that the separation method of a boar ovarian cumulus stem cell is characterized in that, may further comprise the steps:
(1) collection of pig cumulus cell
From sow pig ovary surface extraction diameter 36mm ovarian follicle; Select cumulus cell and wrap up densification and the uniform ovarian cumulus ovocyte of kytoplasm mixture more than 2 layers; With TCM-199 oocyte maturation liquid washing 3 times, every 70-80 ovarian cumulus ovocyte mixture moved in 24 well culture plates cultivate then, every hole 400 μ lTCM-199 oocyte maturation liquid; Cover Yellow Protopet 2A above the ripe liquid, at 39 ℃, 5%CO
2, 100% humidity incubator in cultivate 42 ± 2h; Ovarian cumulus ovocyte mixture after ripe the cultivation is sloughed ovocyte cumulus cell on every side with the enzymic digestion of 1mg/ml mucinase, collects the separation and Culture that cumulus cell is used for pig ovarian cumulus stem cell;
(2) separation of pig ovarian cumulus stem cell and cultivation
Get the pig cumulus cell that collects; Behind PBS liquid centrifuge washing, add the DMEM nutrient solution of high sugar and under 37 ℃ of conditions, cultivate, cell colony that round cell forms appears in the middle of the fibrous cumulus cell to be cultivated and after; Being replaced by the high sugared DMEM nutrient solution that contains 4 μ g/ml bFGF and 10U/ml LIF continues to cultivate; Trysinization round cell colony, the cell of inoculation digestion are on mouse fetal inoblast feeder layer, and the high sugared DMEM nutrient solution that adds 4 μ g/ml bFGF and 10U/ml LIF continues to cultivate; Cell amount every day half is changed liquid, and 2-3d goes down to posterity once.
The invention has the beneficial effects as follows:
Introduce a kind of successful separation and Culture and go out the method for pig cumulus cell stem cell; Pig is because comparatively similar with human body at aspects such as individual size, physiological characteristic, histoorgan sizes; Often become the desirable animal model that is used to study human disease treatment, histoorgan transplanting; Introduction through to pig cumulus cell stem cell isolation cultivation method will lay the foundation for cumulus cell stem cell separation and Culture and the application that derives from mankind itself's body, also can lay the foundation for utilizing pig ovarian cumulus stem cell to carry out work such as breed of variety that genetic manipulation uses pig and improvement as seed cell simultaneously.
[description of drawings]
Below in conjunction with accompanying drawing and embodiment the present invention is done further detailed explanation.
Fig. 1 is the cellular form of embodiment of the invention pig cumulus cell.
Fig. 2 is the cell colony form of the pig ovarian cumulus stem cell of embodiment of the invention separation and Culture.
[embodiment]
1 test materials and method
1.1 test materials
Laboratory animal is a Du Changda three way cross sow pig;
1.2 TP
1.2.1 the separation of pig cumulus cell
From sow pig ovary surface extraction diameter 3-6mm ovarian follicle; Select cumulus cell and wrap up densification and the uniform ovarian cumulus ovocyte of kytoplasm mixture more than 2 layers; With TCM-199 oocyte maturation liquid washing 3 times, every 70-80 ovarian cumulus ovocyte mixture moved in 24 well culture plates cultivate then, every hole 400 μ lTCM-199 oocyte maturation liquid; Cover Yellow Protopet 2A above the ripe liquid, at 39 ℃, 5%CO
2, 100% humidity incubator in cultivate 42 ± 2h.Ovarian cumulus ovocyte mixture after ripe the cultivation is sloughed ovocyte cumulus cell on every side with the enzymic digestion of 1mg/ml mucinase, collects the separation and Culture that cumulus cell is used for pig ovarian cumulus stem cell.
1.2.2 separation and the cultivation of pig ovarian cumulus stem cell
Get the pig cumulus cell of collecting; Behind PBS liquid centrifuge washing, add under 37 ℃ of conditions of DMEM nutrient solution of high sugar and cultivate, cell colony that round cell forms appears in the middle of the fibrous cumulus cell to be cultivated and after; Being replaced by the high sugared DMEM nutrient solution that contains 4 μ g/ml bFGF and 10U/ml LIF continues to cultivate; Trysinization round cell colony, the cell of inoculation digestion are on mouse fetal inoblast feeder layer, and the high sugared DMEM nutrient solution that adds 4 μ g/ml bFGF and 10U/ml LIF continues to cultivate; Cell amount every day half is changed liquid, and 2-3d goes down to posterity once.
1.2.3 the detection of expression of pig ovarian cumulus stem cell versatility mark
Pig ovarian cumulus stem cell is fixed through 4% Paraformaldehyde 96, and 1% bovine serum albumin (BSA) room temperature sealing 30 minutes is added an anti-anti-Oct4 of rabbit of being, goat anti Nanog, mouse anti SSEA1 by 1: 200 back 4 ℃ of incubated overnight of dilution.PBS washes 3 times, and two of CY3 red fluorescence mark anti-is hatched 1h by 1: 200 dilution back room temperature lucifuge, and PBS washes 3 times, carries out the nucleus observations under the fluoroscope that dyes behind the 1min with the DAPI of 1 μ g/ml.
1.2.4 the detection of pig ovarian cumulus stem cell vitro differentiation ability
Dispase digestion ovarian cumulus stem cell; The cell colony piping and druming of digestion is formed less cell colony; After the DMEM medium centrifugal washing of high sugar, the inoculating cell colony adds high sugar in Micro-Organism Culture Dish DMEM nutrient solution carries out suspension culture, after the 7d suspension culture cell mass of formation is inoculated in the petridish that gelatin encapsulates and makes the cell mass adherent growth; Behind the cell mass adherent growth 7d; 4% Paraformaldehyde 96 is fixed, and it is that goat anti nerve fiber, goat anti α-Ji Dongdanbai, goat anti ALPHA-FP were by 1: 200 back 4 ℃ of incubated overnight of dilution that 1% bovine serum albumin (BSA) room temperature sealing 30 minutes, interpolation one resist.PBS washes 3 times, and two of CY3 red fluorescence mark anti-is hatched 1h by 1: 200 dilution back room temperature lucifuge, and PBS washes 3 times, carries out the nucleus observations under the fluoroscope that dyes behind the 1min with the DAPI of 1 μ g/ml.
2 test-results
2.1 the colony form of pig ovarian cumulus stem cell
Collection digests the cumulus cell that comes off from pig ovarian cumulus ovocyte mixture, in the DMEM nutrient solution of high sugar, cultivate 1d after, pig cumulus cell adherent growth; Overwhelming majority pig cumulus cell presents fibrous growthhabit (Fig. 1); The cell speed of growth is slow, and after continuing to cultivate 4d, visible few cell presents the round cell form; The round cell of division growth increases the formation cell colony gradually; Cell colony upwards protuberance forms the nest like form, and the nucleus of round cell is bigger in cell colony, the more fibrous cumulus cell fast growth of growing.Be inoculated into behind the Dispase peptic cell colony on the mouse fetal inoblast feeder layer, the dispersive cell colony presents degradation phenomena in the DMEM nutrient solution of high sugar, and cell is not bred and degenerated gradually and reduce.Cell colony is grown rapidly in containing the high sugared DMEM nutrient solution of 4 μ g/ml bFGF and 10U/ml LIF, and cell state is better, and the outstanding feeder layer surface of cell colony is apparent in view; Form typical nest like form (Fig. 2); The every 2-3d of cell colony goes down to posterity once, at every turn by carrying out had digestive transfer culture at 1: 4, still shows stable growth performance through had digestive transfer culture cell colony repeatedly; Consistent cell colony form, frozen growth performance with the recovery pair cell does not obviously influence.
2.2 the stem cell labeling of pig ovarian cumulus stem cell is expressed
The pig ovarian cumulus stem cell colonies of separation and Culture growth performance in the DMEM nutrient solution of the high sugar that contains 4 μ g/ml bFGF and 10U/ml LIF is stable; Fixing and the immunocytochemical stain check and analysis demonstration afterwards through 4% Paraformaldehyde 96; Pig ovarian cumulus stem cell is expressed stem cell specific marker Oct4, Nanog and SSEA1; Wherein Oct4 and Nanog are nuclear expression in pig ovarian cumulus stem cell, and detected result and stem cell specific marker are consistent with the result of nuclear specificity dyestuff DAPI.SSEA1 is expressed in the surface of cell membrane of pig ovarian cumulus stem cell; In pig ovarian cumulus stem cell colonies, present the cell differential expression; Promptly part cell SSEA1 membranin is expressed as strong positive in cell colony; Part cell SSEA1 membranin is expressed as the weak positive; This is the stem cell colonies of unique a kind of unique properties in the present stem cell colonies of reporting, the SSEA1 membranin is expressed and presented the homogeneous consistence in the stem cell colonies of other type, and the result demonstrates the uniqueness of pig ovarian cumulus stem cell genetic expression.
2.3 the external tridermic differentiation of pig ovarian cumulus stem cell
Pig ovarian cumulus stem cell colonies is stable at the DMEM nutrient solution growth performance of the high sugar that contains 4 μ g/ml bFGF and 10U/ml LIF; The embryoid body of making is through 7d suspension culture and 7d adherent culture; 4% Paraformaldehyde 96 shows after fixing and reaching the immunocytochemical stain check and analysis; Pig ovarian cumulus stem cell external can be spontaneous in body three germinal layer cells break up, in pig ovarian cumulus stem cell embryoid body, the part cell is differentiated to form the ectoderm neurocyte; It is positive to show as neurocyte specific antibody nerve fiber detection of expression; The part cell is differentiated to form mesoderm muscle cell, and it is positive to show as muscle cell specific antibody α-Ji Dongdanbai detection of expression, and the muscle cell of inducing differentiation to be born forms annular sphincter muscle form.Part cell expression of differentiation entoderm liver cell specific marker ALPHA-FP then in embryoid body, analyzing and testing confirm that pig ovarian cumulus stem cell has the ability to three germinal layers differentiation of body, demonstrates the distinctive characteristic of stem cell.
Claims (1)
1. the separation method of a boar ovarian cumulus stem cell is characterized in that, may further comprise the steps:
(1) collection of pig cumulus cell
From sow pig ovary surface extraction diameter 3-6mm ovarian follicle; Select cumulus cell and wrap up densification and the uniform ovarian cumulus ovocyte of kytoplasm mixture more than 2 layers; With TCM-199 oocyte maturation liquid washing 3 times, every 70-80 ovarian cumulus ovocyte mixture moved in 24 well culture plates cultivate then, every hole 400 μ lTCM-199 oocyte maturation liquid; Cover Yellow Protopet 2A above the ripe liquid, at 39 ℃, 5%CO
2, 100% humidity incubator in cultivate 42 ± 2h; Ovarian cumulus ovocyte mixture after ripe the cultivation is sloughed ovocyte cumulus cell on every side with the enzymic digestion of 1mg/ml mucinase, collects the separation and Culture that cumulus cell is used for pig ovarian cumulus stem cell;
(2) separation of pig ovarian cumulus stem cell and cultivation
Get the pig cumulus cell that collects; Behind PBS liquid centrifuge washing, add the DMEM nutrient solution of high sugar and under 37 ℃ of conditions, cultivate, cell colony that round cell forms appears in the middle of the fibrous cumulus cell to be cultivated and after; Being replaced by the high sugared DMEM nutrient solution that contains 4 μ g/ml bFGF and 10U/ml LIF continues to cultivate; Trysinization round cell colony, the cell of inoculation digestion are on mouse fetal inoblast feeder layer, and the high sugared DMEM nutrient solution that adds 4 μ g/ml bFGF and 10U/ml LIF continues to cultivate; Cell amount every day half is changed liquid, and 2-3d goes down to posterity once.
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| CN103215221A (en) * | 2013-03-06 | 2013-07-24 | 安徽农业大学 | Anti-apoptosis treatment method for in vitro culture of a goat cumulus cell |
| CN104120106B (en) * | 2014-07-01 | 2016-10-26 | 华中农业大学 | Utilize pig to dedifferente adipose cell and induce the method being differentiated to form Skeletal Muscle Cell |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101709290A (en) * | 2009-12-03 | 2010-05-19 | 安徽农业大学 | Simple, economic and efficient method for in-vitro maturity of porcine oocytes |
| EP2336297A2 (en) * | 1999-10-28 | 2011-06-22 | University of Massachusetts | Gynogenetic or androgenetic production of pluripotent cells and cell lines, and use thereof to produce differentiated cells and tissues |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2336297A2 (en) * | 1999-10-28 | 2011-06-22 | University of Massachusetts | Gynogenetic or androgenetic production of pluripotent cells and cell lines, and use thereof to produce differentiated cells and tissues |
| CN101709290A (en) * | 2009-12-03 | 2010-05-19 | 安徽农业大学 | Simple, economic and efficient method for in-vitro maturity of porcine oocytes |
Non-Patent Citations (2)
| Title |
|---|
| KAMEYAMA Y et al.In-vitro maturation of follicular oocytes obtained pig ovaries at a slaughterhouse.《Japanese journal of fertility and sterility》.1989,第34卷(第4期),5-10. * |
| 黄雅琼等.猪卵丘细胞和胎儿成纤维细胞的分离培养及传代.《中国兽医科学》.2007,第37卷(第3期),255-259. * |
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