CN109481466A

CN109481466A - Use the method and cell preparation of placenta mesenchyma stem cell treatment premature ovarian failure

Info

Publication number: CN109481466A
Application number: CN201811568010.XA
Authority: CN
Inventors: 许晓椿; 李容; 肖海蓉; 刘冰
Original assignee: BOYA STEM CELL TECHNOLOGY Co Ltd
Current assignee: BOYA STEM CELL TECHNOLOGY Co Ltd
Priority date: 2018-12-21
Filing date: 2018-12-21
Publication date: 2019-03-19
Anticipated expiration: 2038-12-21
Also published as: CN109481466B

Abstract

The present invention relates to the methods and cell preparation that use placenta mesenchyma stem cell treatment premature ovarian failure.Specifically, one aspect of the present invention is related to a kind of cell preparation, it is by making mescenchymal stem cell such as placenta mesenchyma stem cell be suspended in the cell suspension being configured in 0.9% sodium chloride solution, the cell preparation is as including made from method: mescenchymal stem cell obtained by cell passage is transferred in centrifuge tube, centrifugation, supernatant is abandoned, 0.9% sodium chloride solution is added and is resuspended, cell preparation is made.The mescenchymal stem cell is prepared by a method comprising the following steps to obtain: the processing of placental lobules, mixes enzymic digestion and termination, collects primary cell, and primary cell freezes, cell recovery, cell passage, and cell detection freezes and database association.Excellent biological effect is presented in terms for the treatment of premature ovarian failure in cell preparation produced by the present invention.

Description

Use the method and cell preparation of placenta mesenchyma stem cell treatment premature ovarian failure

Technical field

The invention belongs to biotechnology and biomedicine field, it is related to using stem-cell therapy premature ovarian failure (premature Ovarian failure, POF) method and cell preparation used.Specifically, the present invention relates to separate from placenta Stem cell, in particular to the separating mesenchymal stem cell from placenta, and then ovary is treated using such placenta mesenchyma stem cell Early ageing particularly relates to a kind of digestive enzyme compositions using unique formula of the present invention to divide from placenta tissue It sows discord mesenchymal stem cells and is trained mescenchymal stem cell, and then treat premature ovarian failure using such placenta mesenchyma stem cell. The efficiency from placenta separating mesenchymal stem cell can be effectively improved using the method for the present invention, and then can effectively improve and make With the effect of such placenta mesenchyma stem cell treatment premature ovarian failure.

Background technique

Premature ovarian failure (premature ovarian failure, POF) refer to caused by ovarian function failure 40 years old it The phenomenon that preceding amenorrhoea.Be it is a kind of by amenorrhoea, infertile, estrogen deficiency, ovarian follicle reduce and promoting sexual gland hormone increase characterized by disease Disease, and such as with a series of low estrogen symptoms: hectic fever hidrosis, flush, hyposexuality seriously affect the body and mind of women Health.In addition, the women of POF, the risk for suffering from osteoporosis, cardiovascular disease and dementia increases.POF accounts for 1- in women 3%, disease incidence is in rising trend in recent years.

The POF cause of disease is complicated, not yet illustrates completely, but with autoimmune response, infection, inherent cause, chemotherapy, radiotherapy, hand The therapeutic effects such as art and endocrine dysfunction are related.Currently, the most common treatment method of POF is hormone replacement therapy (hormone replacement therapy, HRT).Although the therapy there is certain alleviation to make the clinical symptoms of POF With however, HRT cannot fundamentally repair impaired ovary, recovery ovarian function.In addition, studies have shown that long-term HRT treatment The risk for increasing heart disease and stroke, may will increase the risk of breast cancer and autoimmune disease.Therefore, it is necessary to new to control Strategy is treated to restore the ovarian function of POF patient.

Treatment based on stem cell especially mescenchymal stem cell brings new hope for POF patient, may improve Its ovarian function restores fecundity.

The mescenchymal stem cell of mescenchymal stem cell (mesenchymal stem cell, MSC) such as mankind be earliest from It is separated in marrow, it is dry thin from mesoblastic a kind of tissue with multi-lineage potential and self-renewal capacity Born of the same parents, in vivo under external specified conditions have to osteoblast, cartilage cell, fat cell, endothelial cell, nerve cell, Ability (the Caplan AI.Mesenchymal stem cells.J of a variety of adult cell differentiation such as myocyte, liver cell Orthop Res.1991,9:641-650.Pittenger MF,Mackay AM,Beck SC,et al.Multilineage potential of adult human mesenchymal stem cells.Science.1999；284:143-147).Most It is new research shows that mescenchymal stem cell has immunological regulation and Hematopoiesis Support affect, and be easy to foreign gene and import expression. Therefore the mescenchymal stem cell not still seed cell in tissue-engineered bone, cartilage and cardiac muscle building, it is important in gene therapy Carrier cell, and since mescenchymal stem cell promotes hematopoietic reconstitution and the anti-host response function of inhibition of transplant, in hematopoiesis It is with a wide range of applications in stem cell transplantation and organ transplant.Mescenchymal stem cell has the characteristic of growth-arrested in vitro, Using this characteristic, people have succeeded to be divided from the Various Tissues such as liver, kidney, pancreas, muscle, cartilage, skin, peripheral blood From turning out mescenchymal stem cell.

The mescenchymal stem cell reported at present is mainly derived from marrow, is obtained using density-gradient centrifugation method.Although point It is easy from method, but the operation that donor takes marrow to need to undergo a comparison painful, and had very during materials and after materials High infection chance；Since the content of MSC in human bone marrow is extremely rare, every 10⁵~10⁶Only about 1 in a mononuclearcell It is a, and with the increase at age, quantity, proliferation and the differentiation capability of mescenchymal stem cell are remarkably decreased in marrow, make it It is restricted in research and application especially clinical application.Placenta originating from embryonic development period extraembryonic mesoderm be by Matter, blood vessel and trophocyte composition, contain a large amount of mesenchyma ingredient.It is newest research shows that rich in dry thin in placenta Born of the same parents, be separately cultured out from placenta these multipotential stem cells will be opened up for experimental study and clinical application one it is brand-new and abundant Source.

Existing method of the stem cell to establish placenta stem-cell library that separate from placenta still has shortcomings, such as pure Degree is insufficient, and/or quantity is not high, and then shows that these methods are not able to satisfy the expectation of people still.Such as CN101270349A Entitled " placenta mesenchyma stem cell disclosed in (Chinese Patent Application No. 200810061267.6, publication date September in 2008 24 days) The invention of separation and amplification in vitro cultural method "；CN101693884A (Chinese Patent Application No. 200910117522.9, it is open Day on April 14th, 2010) disclosed in entitled " a method of the separating and extracting stem cells from placenta, umbilical cord or adipose tissue " Invention；It is entitled disclosed in CN102146359A (Chinese Patent Application No. 201110005964.1, publication date August in 2011 10 days) The invention of " method of primary mesenchymal stem cells and serum-free amplification is extracted from placenta ".In addition, Chinese Patent Application No. 201210044648X discloses a kind of method of separating mesenchymal stem cell from placenta.Purity of these methods in extract And/or it is remained to be further improved in terms of the rate of recovery.In addition, the Invention Announce of present inventor team One kind is described in CN107299082A (Chinese Patent Application No. 201710653583.1, publication date on October 27th, 2018) to obtain The method for taking placenta mesenchyma stem cell, which has shown that, is presented some excellent performances.

This field remains a need for the new method that stem cell is separated from placenta, especially efficiently separates from placenta The method of mescenchymal stem cell.In addition, this field still need it is new for the separating mesenchymal stem cell methods from placenta Used in digestive enzyme compositions, to improve the method efficiency of the separating mesenchymal stem cell from placenta.In addition, this field is still Need new method so to treat premature ovarian failure, especially expect to have new more effective way using mescenchymal stem cell come Treat premature ovarian failure.

Summary of the invention

The purpose of the present invention includes following one or more aspects: on the one hand, it is dry thin to solve existing acquisition placenta mesenchyma The deficiency of born of the same parents' method, provide it is a kind of it is practical, simple, efficiently fill from separating mesenchymal stem cell in placenta tissue and between being trained The method of matter stem cell and the method for optionally establishing placenta stem-cell library；On the other hand, it is separated from placenta tissue to be above-mentioned The mescenchymal stem cell and method for being trained mescenchymal stem cell provides a kind of digestive enzyme compositions；Yet another aspect, to use Mescenchymal stem cell treats premature ovarian failure and provides a kind of cell preparation.The inventors discovered that using special operating method and The digestive enzyme compositions of special prescription, cell purity obtained is high and/or cell recoveries are high, and by preparing a kind of spy Fixed cell preparation can be more efficiently used for the treatment of premature ovarian failure.It is accomplished the present invention is based on such discovery.

Therefore, first aspect present invention provides a kind of cell preparation that for example can be used for treating premature ovarian failure, is logical Crossing makes mescenchymal stem cell (such as placenta mesenchyma stem cell) be suspended in the cell suspension being configured in 0.9% sodium chloride solution Liquid.

Cell preparation according to a first aspect of the present invention, wherein cell concentration is 1~10 × 10⁶A cell/ml, such as 1 ~5 × 10⁶A cell/ml, such as 1~3 × 10⁶A cell/ml.

Cell preparation according to a first aspect of the present invention, also add in 0.9% sodium chloride solution magnesium gluconate and Phosphatide.In one embodiment, the amount for adding magnesium gluconate is to make magnesium ion concentration 5mmol/L.In an embodiment party In case, the concentration for adding phosphatide is 0.2mg/ml.In one embodiment, the phosphatide is injection stage soybean-source.? Through surprisingly it has been found that cell preparation treatment ovum of the present invention can be significantly improved by passing through while adding a small amount of magnesium salts and phosphatide The biology effect of nest early ageing.The present inventor only adds magnesium in addition, it has been found that in the cell preparation of the embodiment of the present invention 4 Salt or only add phosphatide or whens above-mentioned magnesium salts is replaced with other salt such as calcium salt, zinc salt, mantoquita etc., result is far not And the effect of the PD-MSC group a of magnesium salts and phosphatide is added simultaneously, and in the result of these situations and the embodiment of the present invention 4 The result of PD-MSC group b is close.

Cell preparation according to a first aspect of the present invention is as including made from method: between obtained by cell passage Mesenchymal stem cells are transferred in centrifuge tube, and supernatant is abandoned in centrifugation, and 0.9% sodium chloride solution is added and is resuspended, cell preparation is made.

Cell preparation according to a first aspect of the present invention, wherein the mescenchymal stem cell is by including the following steps What method was prepared:

(1) processing of placental lobules: placenta is placed in ceramic whiteware disk, is rinsed with tissue-wash solution to remove placenta hemostasis, Clip 20g placental lobules is organized in steel bowl, is cleaned twice using tissue-wash solution, and after impregnating 5min, it is preferable to weigh 15g It is organized in 100mm glass dish；Add 10ml tissue-wash solution, shreds leaflet to 0.2cm³Left and right size adds 100ml tissue wash 300 mesh filter screens filtering after liquid stirs evenly, then this is operated to be cleaned twice with tissue-wash solution to remove haemocyte repeatedly；

[wherein, the tissue-wash solution is comprising 1% 0.9% dual anti-physiological saline]

(2) enzymic digestion and termination are mixed: by the leaflet tissue after cleaning be added 37 DEG C of preheatings 15~30ml (such as 20~ 25ml, such as 23ml) mix well in mixed enzyme digestive juice after, 37 DEG C of shaking table, 100rpm oscillation digestion 30min, digestion terminate Afterwards, the FBS of 2ml is added into tissue fluid to terminate digestion；

[wherein, include in the mixed enzyme digestive juice: Hank ' the s balanced salt solution of 15~30 volumes, 0.2~0.6 The Liberase MNP-S enzyme of volume, 0.2~2 volume DNA I type enzyme (such as Hank ' the s balance salt of 20~25 volumes is molten Liquid, the Liberase MNP-S enzyme of 0.3~0.5 volume, 0.5~1 volume DNA I type enzyme, such as the Hank ' s of 22 volumes is flat Weigh salting liquid, the Liberase MNP-S enzyme of 0.4 volume, 0.7 volume DNA I type enzyme)；The Liberase MNP-S enzyme example The Liberase MNP-S enzyme of Roche Holding Ag in this way, such as purchased from western precious biology, article No.: 5578582001]

(3) it collects primary cell: adding 50ml tissue-wash solution in rapid gained tissue fluid one step up, mix, 300 mesh mistakes Cell liquid is collected in filter；Wash postdigestive tissue twice repeatedly, filtrate twice is incorporated into centrifuge tube, 1500rpm from Heart 8min (acceleration 9, deceleration 7)；Remove supernatant, appropriate tissue-wash solution is added to be resuspended and be supplemented to 200ml, 1500rpm from Heart 8min (acceleration 9, deceleration 7)；Supernatant is removed, cell precipitation adds DMEM-F12 to be resuspended to 30ml, 100um strainer filtering Afterwards, then with the DMEM-F12 flushing filtering net of 10ml, 40ml cell suspension is obtained, as primary cell；

[cell suspension of this primary cell can carry out cell count with sysmex blood analyser]

(4) primary cell freezes: making cell suspension 1800rpm, is centrifuged 10min (acceleration 9, deceleration 7), leaves and takes cell Precipitating and lower liquid 5ml, are slowly added into frozen stock solution 10ml, shake well while adding after resuspension；Gained cell suspension is dispensed to 9 2ml In cryopreservation tube, every pipe 1.5ml is set in the program temperature reduction box of pre-cooling, is cooled down using programmed cooling instrument device program, then cell is transferred to It is frozen in liquid nitrogen storage tank；

[wherein, the formula of the frozen stock solution are as follows: 65% DMEM-F12,15% human serum albumins (HSA), 20% DMSO such as WAK brand DMSO]

(5) cell recovery: cell is moved to 15ml centrifuge tube, adds 8ml complete by the cell for taking 2 pipes to freeze, 37 DEG C of quick-thawings Full culture medium drop recovery；Then after 1200rpm is centrifuged 5min (acceleration 9, deceleration 7), supernatant is removed, 5ml is added to cultivate completely Base weight is outstanding；Every solencyte is inoculated in 1 T75 culture bottle, supplement complete medium to 30ml, set CO2 incubator (37 DEG C, 5% CO2, saturated humidity) in culture；It was carried out once changing liquid entirely with complete medium every 3-4 days, according to clone's shape after recovery 12 days It is counted at situation, until cell density is not less than 3000 cell/cm²When can carry out next passage；

[wherein, the complete medium is the DMEM-F12 culture medium comprising 10%FBS]

(6) cell passes on: taking P0 to be cleaned for cell with PBS, 2ml pancreatin is added to digest 2-5min, until cell is largely de- It falls, adds 5ml complete medium to terminate digestion, be transferred to cell in centrifuge tube, 1400rpm is centrifuged 5min, and (acceleration 9 slows down 7) degree, abandons supernatant, count after adding 5ml complete medium to be resuspended and be seeded to culture bottle, and cell density is 8000~12000 thin Born of the same parents/cm², the middle culture of CO2 incubator (37 DEG C, 5%CO2, saturated humidity), which is set, to cell density up to 90% or more (is generally incubated 5 It or so), it completes to pass on from the cell in P0 generation to P1 generation；Aforesaid operations are repeated in carry out P1 generation respectively to P2 generation, P2 generation Cell to P3 generation, P3 generation to P4 generation, P4 generation to P5 generation passes on, and obtains each generation mescenchymal stem cell.

Cell preparation according to a first aspect of the present invention, during preparing mescenchymal stem cell, further includes:

(7) for placenta mesenchyma stem cell obtained by step (6), detect following items at least one of: it is cell activity, thin Born of the same parents' pollution, hereditary disease, HLA-ABC/DR distribution type.

(8) each after passage obtained by step (6) is frozen in liquid nitrogen for placenta mesenchyma stem cell.

(9) database of the placenta stem-cell comprising information above is established, and keeps the database and freezing for step (8) thin Born of the same parents are associated.

Cell preparation according to a first aspect of the present invention, wherein gained is respectively big for the cell purity of placenta mesenchyma stem cell In 90%.In one embodiment, for the placenta mesenchyma stem cell after 3 more than generation pass on, cell purity is greater than 95%.

Cell preparation according to a first aspect of the present invention, wherein the Hank's balanced salt solution forms are as follows: 8.0g/L's The Na2HPO4 of MgCl26H2O, 0.06g/L of MgSO47H2O, 0.1g/L of KCl, 0.1g/L of NaCl, 0.4g/L The phenol of the glucose of KH2PO4,1.0g/L of 2H2O, 0.06g/L, NaHCO3,0.2g/L of CaCl2,0.35g/L of 0.14g/L Red, hydrochloric acid or sodium hydroxide adjust pH to 7.4.Cell preparation according to a first aspect of the present invention, wherein the mixed enzyme digests In liquid other than comprising Hank's balanced salt solution, Liberase MNP-S enzyme, DNA I type enzyme, it is also added with 0.2~0.3g/L Zinc chloride.It has been had now surprisingly been found that, using the mixed enzyme digestive juice for adding this concentration range zinc chloride, institute The CD73 expression for obtaining primary cell is greater than 60%, CD45 and does not express, and the mescenchymal stem cell content in gained primary cell Up to 60%-70%, high concentration of stem cells is shown；And when being not added with this zinc chloride in mixed enzyme digestive juice, primary cell In mescenchymal stem cell content less than 38%, usually in 31~38% ranges.

Cell preparation according to a first aspect of the present invention, wherein the cytoactive detection is to utilize trypan blue staining meter Number freezes the number of front and back living cells.

Cell preparation according to a first aspect of the present invention, wherein cell contamination detection utilizes a small amount of cell culture, inspection Survey cell whether the pollution by fungi and bacterium.In one embodiment, the cell contamination detection is to utilize aetology Method, whether detection cell is by being selected from following one or more infection: Hepatitis B virus, hepatitis, AIDS virus, big and small Cellular virus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA、TRUST。

Cell preparation according to a first aspect of the present invention, wherein hereditary disease detection is the side using molecular genetics Method, detection freeze-stored cell whether there is hereditary disease.

Cell preparation according to a first aspect of the present invention, wherein the HLA-ABC/DR distribution type is detection cell HLA-ABC/ DR phenotype.

Cell preparation according to a first aspect of the present invention, wherein the placenta mesenchyma stem cell is through program temperature-fall period It freezes in liquid nitrogen.

Cell preparation according to a first aspect of the present invention, wherein including in the database and all phases of cell for being saved The data of pass, including but not limited to: the biological characteristics testing result of cell, multi-lineage potential qualification result, cellular elements Genetic diagnosis result, fetus and its particulars of parent.

Further, second aspect of the present invention is provided from separating mesenchymal stem cell in placenta tissue and is filled between being trained The method of matter stem cell, method includes the following steps:

[wherein, the complete medium is the DMEM-F12 culture medium comprising 10%FBS]

The method of any embodiment according to a second aspect of the present invention, wherein further include:

The method of any embodiment according to a second aspect of the present invention, wherein gained is respectively for the thin of placenta mesenchyma stem cell Born of the same parents' purity is greater than 90%.In one embodiment, the placenta mesenchyma stem cell is after 3 more than generation pass on, cell purity Greater than 95%.

The method of any embodiment according to a second aspect of the present invention, wherein the Hank's balanced salt solution forms are as follows: MgCl26H2O, 0.06g/L's of MgSO47H2O, 0.1g/L of KCl, 0.1g/L of NaCl, 0.4g/L of 8.0g/L The glucose of KH2PO4,1.0g/L of Na2HPO42H2O, 0.06g/L, CaCl2,0.35g/L of 0.14g/L NaHCO3, The phenol red of 0.2g/L, hydrochloric acid or sodium hydroxide adjust pH to 7.4.The method of any embodiment according to a second aspect of the present invention, Wherein in the mixed enzyme digestive juice other than comprising Hank's balanced salt solution, Liberase MNP-S enzyme, DNA I type enzyme, Also it is added with 0.2~0.3g/L zinc chloride.It has been had now surprisingly been found that, using the mixing for adding this concentration range zinc chloride In the case where enzymic digestion liquid, the CD73 expression of gained primary cell is greater than 60%, CD45 and does not express, and in gained primary cell Mescenchymal stem cell content reach 60%-70%, show high concentration of stem cells；And works as in mixed enzyme digestive juice and be not added with this When zinc chloride, the mescenchymal stem cell content in primary cell is less than 38%, usually in 31~38% ranges.

The method of any embodiment according to a second aspect of the present invention, wherein the cytoactive detection is to utilize trypan blue Decoration method counts the number for freezing front and back living cells.

The method of any embodiment according to a second aspect of the present invention, wherein cell contamination detection utilizes a small amount of cell Culture, detection cell whether the pollution by fungi and bacterium.In one embodiment, the cell contamination detection is to utilize Aetology method, whether detection cell is by selected from following one or more infection: Hepatitis B virus, hepatitis, AIDS Poison, cytomegalovirus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV- IgM and EBV-IgA, TRUST.

The method of any embodiment according to a second aspect of the present invention, wherein hereditary disease detection is to utilize molecular genetic Method, detection freeze-stored cell whether there is hereditary disease.

The method of any embodiment according to a second aspect of the present invention, wherein the HLA-ABC/DR distribution type is detection cell HLA-ABC/DR phenotype.

The method of any embodiment according to a second aspect of the present invention, wherein the placenta mesenchyma stem cell is through program Temperature-fall period freezes in liquid nitrogen.

The method of any embodiment according to a second aspect of the present invention, wherein include in the database with saved it is thin All relevant data of born of the same parents, including but not limited to: the biological characteristics testing result of cell, multi-lineage potential qualification result, Cellular elements genetic diagnosis result, fetus and its particulars of parent.

In addition, providing a kind of placenta mesenchyma stem cell in second aspect of the present invention method.Therefore third of the present invention Aspect provides a kind of placenta mesenchyma stem cell.

The cell of placenta mesenchyma stem cell according to a third aspect of the present invention, the placenta mesenchyma stem cell in each generation is pure Degree is greater than 90%.In one embodiment, after 3 more than generation pass on, cell purity is greater than the placenta mesenchyma stem cell 95%.

Placenta mesenchyma stem cell according to a third aspect of the present invention is to be prepared by a method comprising the following steps It arrives:

[wherein, the complete medium is the DMEM-F12 culture medium comprising 10%FBS]

Placenta mesenchyma stem cell according to a third aspect of the present invention, in the preparation step, further includes:

Placenta mesenchyma stem cell according to a third aspect of the present invention, in the preparation step, gained is respectively filled between placenta The cell purity of matter stem cell is greater than 90%.In one embodiment, the placenta mesenchyma stem cell more than generation is passed on through 3 Afterwards, cell purity is greater than 95%.

Placenta mesenchyma stem cell according to a third aspect of the present invention, in the preparation step, the Hank's balances salt Solution composition are as follows: the MgCl26H2O of MgSO47H2O, 0.1g/L of KCl, 0.1g/L of NaCl, 0.4g/L of 8.0g/L, CaCl2,0.35g/L of the glucose of KH2PO4,1.0g/L of Na2HPO42H2O, 0.06g/L of 0.06g/L, 0.14g/L The phenol red of NaHCO3,0.2g/L, hydrochloric acid or sodium hydroxide adjust pH to 7.4.It is filled between placenta according to a third aspect of the present invention Matter stem cell, in the preparation step, in addition to including Hank's balanced salt solution, Liberase in the mixed enzyme digestive juice Outside MNP-S enzyme, DNA I type enzyme, it is also added with 0.2~0.3g/L zinc chloride.It has been had now surprisingly been found that, add this using In the case where the mixed enzyme digestive juice of concentration range zinc chloride, the CD73 expression of gained primary cell is greater than 60%, CD45 not table It reaches, and the mescenchymal stem cell content in gained primary cell reaches 60%-70%, shows high concentration of stem cells；And work as When being not added with this zinc chloride in mixed enzyme digestive juice, the mescenchymal stem cell content in primary cell is less than 38%, usually 31 In~38% range.

Placenta mesenchyma stem cell according to a third aspect of the present invention, in the preparation step, the cytoactive detection It is that the number for freezing front and back living cells is counted using trypan blue staining.

Placenta mesenchyma stem cell according to a third aspect of the present invention, in the preparation step, the cell contamination detection Using a small amount of cell culture, detect cell whether the pollution by fungi and bacterium.In one embodiment, the cell is dirty Dye detection is using aetology method, and whether detection cell is by selected from following one or more infection: Hepatitis B virus, Hepatitis, AIDS virus, cytomegalovirus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, TRUST.

Placenta mesenchyma stem cell according to a third aspect of the present invention, in the preparation step, the hereditary disease detection is Using the method for molecular genetics, detecting freeze-stored cell whether there is hereditary disease.

Placenta mesenchyma stem cell according to a third aspect of the present invention, in the preparation step, the HLA-ABC/DR matches Type is detection cell HLA-ABC/DR phenotype.

Placenta mesenchyma stem cell according to a third aspect of the present invention, in the preparation step, the placenta mesenchyma is dry Cell is frozen in liquid nitrogen through program temperature-fall period.

Placenta mesenchyma stem cell according to a third aspect of the present invention in the preparation step, includes in the database To all relevant data of the cell saved, including but not limited to: the biological characteristics testing result of cell, Multidirectional Differentiation are latent It can qualification result, cellular elements genetic diagnosis result, fetus and its particulars of parent.

Further, fourth aspect present invention provides one kind in the separating mesenchymal stem cell from placenta tissue and is trained Mixed enzyme digestive juice used in the method for mescenchymal stem cell, in the mixed enzyme digestive juice comprising Hank's balanced salt solution, Liberase MNP-S enzyme, DNA I type enzyme.

The mixed enzyme digestive juice of any embodiment according to a fourth aspect of the present invention, including: 15~30 volumes Hank ' s balanced salt solution, the Liberase MNP-S enzyme of 0.2~0.6 volume, 0.2~2 volume DNA I type enzyme (such as 20 Hank ' the s balanced salt solution of~25 volumes, the Liberase MNP-S enzyme of 0.3~0.5 volume, 0.5~1 volume DNA I type Enzyme, such as the DNA I type of Hank ' the s balanced salt solution of 22 volumes, the Liberase MNP-S enzyme of 0.4 volume, 0.7 volume Enzyme).

The mixed enzyme digestive juice of any embodiment according to a fourth aspect of the present invention, wherein the Hank's balanced salt solution Composition are as follows: the MgCl26H2O of MgSO47H2O, 0.1g/L of KCl, 0.1g/L of NaCl, 0.4g/L of 8.0g/L, CaCl2,0.35g/L of the glucose of KH2PO4,1.0g/L of Na2HPO42H2O, 0.06g/L of 0.06g/L, 0.14g/L The phenol red of NaHCO3,0.2g/L, hydrochloric acid or sodium hydroxide adjust pH to 7.4.Any embodiment party according to a fourth aspect of the present invention The mixed enzyme digestive juice of case, wherein other than comprising Hank's balanced salt solution, Liberase MNP-S enzyme, DNA I type enzyme, also Zinc chloride added with specified amount as described herein.It has been had now surprisingly been found that, using this concentration range zinc chloride of addition Mixed enzyme digestive juice in the case where excellent technical effect as described herein can be presented.

The mixed enzyme digestive juice of any embodiment according to a fourth aspect of the present invention, wherein described separate from placenta tissue Mescenchymal stem cell and the method for being trained mescenchymal stem cell comprising following steps:

[wherein, the complete medium is the DMEM-F12 culture medium comprising 10%FBS]

The mixed enzyme digestive juice of any embodiment according to a fourth aspect of the present invention, wherein described separate from placenta tissue Mescenchymal stem cell is simultaneously trained in the method for mescenchymal stem cell further include:

The mixed enzyme digestive juice of any embodiment according to a fourth aspect of the present invention, wherein described separate from placenta tissue Mescenchymal stem cell and to be trained cytoactive detection described in the method for mescenchymal stem cell be using trypan blue staining meter Number freezes the number of front and back living cells.

The mixed enzyme digestive juice of any embodiment according to a fourth aspect of the present invention, wherein described separate from placenta tissue Mescenchymal stem cell is simultaneously trained the detection of cell contamination described in the method for mescenchymal stem cell using a small amount of cell culture, detection Cell whether the pollution by fungi and bacterium.In one embodiment, the cell contamination detection is to utilize aetology side Method, whether detection cell is by selected from following one or more infection: Hepatitis B virus, hepatitis, AIDS virus, giant cell Virus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV- IgA、TRUST。

The mixed enzyme digestive juice of any embodiment according to a fourth aspect of the present invention, wherein described separate from placenta tissue Mescenchymal stem cell and to be trained the detection of hereditary disease described in the method for mescenchymal stem cell be the method using molecular genetics, Detecting freeze-stored cell whether there is hereditary disease.

The mixed enzyme digestive juice of any embodiment according to a fourth aspect of the present invention, wherein described separate from placenta tissue Mescenchymal stem cell and be trained HLA-ABC/DR distribution type described in the method for mescenchymal stem cell be detection cell HLA-ABC/ DR phenotype.

The mixed enzyme digestive juice of any embodiment according to a fourth aspect of the present invention, wherein described separate from placenta tissue Mescenchymal stem cell and to be trained placenta mesenchyma stem cell described in the method for mescenchymal stem cell be through program temperature-fall period It freezes in liquid nitrogen.

The mixed enzyme digestive juice of any embodiment according to a fourth aspect of the present invention, wherein described separate from placenta tissue Mescenchymal stem cell is simultaneously trained in database described in the method for mescenchymal stem cell all phases of cell for including and being saved The data of pass, including but not limited to: the biological characteristics testing result of cell, multi-lineage potential qualification result, cellular elements Genetic diagnosis result, fetus and its particulars of parent.

Further, fifth aspect present invention provides cell preparation (such as cell preparation described in first aspect present invention) Preparing the purposes in the drug for treating and/or preventing premature ovarian failure.

In the above-mentioned various operating procedures of the present invention, although its description specific steps are in certain details or language is retouched State with the preparation example of following detailed description part described in step different from, however, those skilled in the art The detailed disclosure of full text can summarize approach described above step completely according to the present invention.

Any embodiment in either present invention face can be combined with other embodiments, as long as they are not It will appear contradiction.In addition, any technical characteristic can be adapted for other realities in any embodiment of either side of the present invention The technical characteristic in scheme is applied, as long as they are not in contradiction.The invention will be further described below.

All documents recited in the present invention, their full content are incorporated herein by reference, and if these are literary When offering expressed meaning and the inconsistent present invention, it is subject to statement of the invention.In addition, the various terms that use of the present invention and Phrase has that well known to a person skilled in the art general senses, nonetheless, the present invention remain desirable at this to these terms and Phrase is described in more detail and explains, the term and phrase referred to is if any inconsistent with common art-recognized meanings, with institute's table of the present invention Subject to the meaning stated.

In the present invention, term " placenta mesenchyma stem cell " refers to the mescenchymal stem cell from placenta.Therefore exist In the present invention, more particularly in context of the invention, term " placenta mesenchyma stem cell " can with " placenta stem-cell ", " stem cell ", " mescenchymal stem cell " are used interchangeably, unless otherwise clearly indicating.

In the present invention, term " PBS buffer solution " or " PBS " refer to phosphate buffer.Those skilled in the art are ripe Know the general formula and preparation method and their general aspects such as pH value or pH of the PBS used under situation of the present invention Range, and these PBS buffer solution are usually the pre-mixing liquor (or prewired powder) that can be obtained through commercial channels, such as this The PBS of invention field is usually the commercialization buffer of pH7.4 (± 0.1), such as the PBS buffer solution of HyClone brand；Ability It include 137mM sodium chloride, 2.7nM potassium chloride and 10mM phosphate radical in PBS buffer solution composition when the application of domain classics, in this hair In bright if not otherwise specified, PBS used using when composition be the composition.

In the present invention, term " placenta " refers to newborn fetal placenta, particularly relates to the placenta within 4 hours postpartum.

Mescenchymal stem cell is a kind of adult stem cell with self-replacation and multi-lineage potential, have it is easily separated, Culture, amplification, immunogenicity is low, does not express the advantage of II type major histocompatibility complex (MHC), can be used with allosome, With powerful migration, immunoregulation capability, tissue damage reparative regeneration is promoted by paracrine mode, is regenerative medicine ideal Seed cell.

Premature ovarian failure (POF) is that occur amenorrhoea, estrogen deficiency, Gonadotropin Level liter before 40 years old with women A kind of disease that height is characterized.Disease incidence is 1%~3% in general population, accounts for 4%~8% in secondary amenorrhea women, 20 years old pervious disease incidence are 0.01%.Incidence of the premature ovarian failure in the women of child-bearing age, which has, in recent years increases year by year and to youth Change the trend of development, and its mechanism is still not clear, main inducing has inherent cause, role of autoimmune factors, medical operating wound, ring Border infection etc..There is no a kind of effective treatment method that can eradicate POF, mescenchymal stem cell (MSCs) is to belong to mesoblastic one Class multipotential stem cell, relatively light with powerful proliferative capacity and multi-lineage potential, immunoloregulation function, allograft rejection, The advantages such as distribution type requirement is not stringent, is easily isolated and cultivates.There are mainly three types of the MSCs of Typical sources for treating ovary at present Early ageing, e.g. umbilical cord mesenchymal stem cells (UCMSCs), mesenchymal stem cell (BMSCs), fat stem cell (ADSCs).In recent years, domestic and international application mescenchymal stem cell obtains preliminary research achievement in premature ovarian failure treatment.

(Sun Lin, Human plactnta mescenchymal stem cell treat the Study on Molecular Mechanism of mouse ovarian early ageing, practical woman to Sun Lin paper Section's endocrine magazine (electronic edition), 19 phases in 2016) probe into the molecule that Human plactnta mescenchymal stem cell treats mouse ovarian early ageing Mechanism Study, method are to take fully a moon placenta for caesarean birth fetus, isolate and purify to obtain placenta mesenchyma stem cell, by 20 C57 premature ovarian failure mouse is randomly divided into 2 groups, and control group injecting normal saline, experimental group injects placenta mesenchyma stem cell suspension, Continuous injection 10d, detects Zcchc11, Angpt1, the variation of Cxcr4 kind ovary related gene.The results show that flow cytometry CD29, CD44, CD90 positive expression are detected, CD45, HLA-DR are negative expression.The detection of fluorescence quantitative polymerase chain reaction method The results show that compared with the control group, the expression of the Zcchc11 and Angpt1 gene of observation group rises, under Cxcr4 gene expression Drop, difference are statistically significant (P < 0.05).These results indicate that the adjustable ovarian growth phase of Human plactnta mescenchymal stem cell The expression of correlation gene plays an important role to treatment premature ovarian failure.

Fu Xiafei paper (Fu Xiafei, etc. umbilical cord mesenchymal stem cells ovary local transplantation is intracorporal in premature ovarian failure rat Distribution, Clinical Medical Engineering, 09 phase in 2013) umbilical cord mesenchymal stem cells (UCMSCs) ovary local transplantation is observed in ovary morning The intracorporal distribution of the rat that declines, method are to establish model transplantations label UCMSCs, observe each internal organs green cells distribution feelings Condition.The results show that finding transplanted cells in ovary, the visible more transplanted cells of lung, spleen, the heart, liver, kidney are visible to be dispersed in distribution Transplanted cells.These results indicate that UCMSCs can survive in ovary tissue, it is partially trapped in the tissue such as lung, spleen.

(Zhu Shaofang, the human umbilical cord mesenchymal stem cells etc., PKH26 label are early in the chemotherapeutic ovary of rat for Zhu Shaofang paper Ovary migration in failure model, modern gynemetrics's progress, 02 phase in 2011) human umbilical cord mesenchymal stem cells are marked with PKH26, It inquires into ovary of the human umbilical cord mesenchymal stem cells in the chemotherapeutic premature ovarian failure model of rat and migrates tracer, method is to extract And human umbilical cord mesenchymal stem cells are cultivated, cell marking is carried out by PKH26 label program, transmission electron microscope observing mark group and is not marked The ultra microstructure of note group cell measures cell Proliferation, period, apoptosis situation.People's navel of fluorescence microscopy microscopic observation PKH26 label With the ovary situation after mesenchymal stem cell transplantation.The results show that the form of cell is into fiber without significant difference after label Cell sample, cell Proliferation, which has no, to be significantly affected, and cell growth state is good, and the positive cell of PKH26 label is primarily present ovary In blood vessel.These results indicate that PKH labelling technique can be used for the morning of ovary caused by human umbilical cord mesenchymal stem cells transplantation treatment chemotherapy The research go back to the nest, break up, being proliferated in declining.

Li Yongli paper (grind by Li Yongli, the experiment for repairing rat ovary early ageing etc., Human plactnta mesenchymal stem cell transplantation Study carefully, the newest medical information digest in the world, 90 phases in 2016) Human plactnta mescenchymal stem cell is transplanted to inquire into tail vein injection (hpMSCs) to the repair of premature ovarian failure rat model ovarian function caused by chemotherapy, method is identified people's tire Disk mescenchymal stem cell is transplanted through tail vein injection gives premature ovarian failure rat model, and experiment is divided into hpMSCs group and stem cell culture Liquid transplantation group (control group) every group 10, is injected in equal volume by tail vein respectively, rat ovary structural damage is observed after injection Situation compares Serum Sex Hormones (E2, AMH, INHB) concentration.The results show that rat ovary hormonal readiness is compared with the control group, HpMSCs transplanting front and back blood serum E2 and INHB level have statistical difference；The horizontal no difference of science of statistics of serum AMH (P=0.051)； Rat ovary tectology the result shows that, it is serious with the damage of model group rats ovary cortex, after tail vein injection hpMSCs Bcl-2 expression significantly reduces.These results indicate that the transplanting of hAMSCs rat tail vein improves hormonal readiness, gonad cell is reduced Apoptosis, to repair ovarian function.

Model avenges paper (Fan Xue, etc. shadow of the Bone Marrow Mesenchymal Stem Cells Transplantation to premature ovarian failure model mice ovarian function Ring, Aged in China magazine, 21 phases in 2016) influence of the mesenchymal stem cell to premature ovarian failure model mice has been inquired into, Its method is to take ICR Marrow Mesenchymal Stem Cells for cell transplantation.Healthy ICR mouse 60 be only randomly divided into control group, Model group, treatment group.Control group mice establishes chemotherapy without any intervention, model group and treatment group's mouse peritoneal injection cis-platinum Property damage mouse model of premature ovarian failure.Disposable in-situ injection mesenchymal stem cell is controlled after treatment group's modeling It treats.Rat ordinary circumstance, oestrous cycle are observed, and measures E2, FSH content and ovary tissue morphologic change in serum.As a result It has been shown that, model group Mouse Oestrous Cycle disorder, the Mouse Oestrous Cycle after stem-cell therapy return to normal.With compare Group compares, and model group E2 decline, FSH level increases (P < 0.05).E2 rises after bone mesenchymal stem cells treatment, FSH water Flat decline, almost recovery normal level.Model group folliculus is largely destroyed, folliculus quantity be considerably less than control group (P < 0.01).Folliculus and corpus luteum number obviously increase (including the corpus luteum newly formed), with model after bone mesenchymal stem cells treatment Group comparing difference is significant (P < 0.01).These results indicate that cis-platinum can cause follicular cell apoptosis, cause ovarian function too early Decline, mesenchymal stem cell can reduce damage of the cis-platinum to ovary, promote ovarian follicle by adjusting each hormone-content in serum Development, improves and enhances ovarian function.

Wang Yan paper (Wang Yan, etc., repairing research of the Bone Marrow Mesenchymal Stem Cells Transplantation to destroying in overy caused by VCD, the modern times Biomedicine progress, 10 phases in 2011) Marrow Mesenchymal Stem Cells (MSCs) transplanting has been inquired into deoxidation vinyl cyclohexyl The feasibility of the treatment of premature ovarian failure caused by alkene (VCD), method are continuously to be injected intraperitoneally using VCD (160mg/kg/d) to lure Lead mouse ovarian early ageing.Every side injection of ovary has transfected the MSCs of green fluorescence DNA murine derived from bone marrow, 14,28 after transplanting It and 45 days, take each group blood preparation and ovary tissue, while observing the variation of Mouse Oestrous Cycle；Enzyme-linked immunization detects blood Clear FSH, LH are horizontal, distribution of the microscopically observation MSC in ovary.The results show that the equal visible green of each group is glimmering after MSCs transplanting Light, and it is distributed mainly on stroma of ovary area, ovary thecacells area also shows green cells.The MSCs group oestrous cycle compared with Experimental comparison group shortens, and FSH is low compared with experimental comparison group with LH level, and difference has conspicuousness.These results indicate that being filled between marrow Matter stem cell can improve the Function of Ovary of premature ovarian failure mouse, and be present in ovary tissue for a long time.It is filled between marrow Matter stem cell is likely to become the new method of premature ovarian failure treatment.

Yingying Zhang's paper (Yingying Zhang, etc. Bone Marrow Mesenchymal Stem Cells Transplantation is in premature ovarian failure mouse ovarian reconstruction Using Agricultural University Of Anhui's journal, 01 phase in 2017) it has inquired into mesenchymal stem cell (BMSCs) and is implanted in premature ovarian failure Application value in mouse ovarian reconstruction.Cyclophosphamide is injected by disposable celiac and busulfan establishes mouse ovarian morning Decline (POF) model, and is randomly divided into modeling group, BMSCs transplantation group and blank control group.Transplantation group is in modeling 14d through tail vein Inject green fluorescent protein (GFP) trangenic mice BMSCs suspension (5 × 10⁷A/mL), every 4d is repeated 1 times, and totally 5 times；Modeling group Same amount of normal saline is injected with blank control group.The ordinary circumstance of mouse after observation modeling and after BMSCs transplanting, serum FSH and E2 is horizontal, ovarian histology changes and GFP fluorescence signal is in entovarial distribution.As a result, it has been found that modeling group mouse appetite is bright Aobvious decline, activity are reduced, and weight significantly mitigates (P < 0.05) compared with blank control group, and FSH level significantly increases (P < 0.05), E2 level significantly reduces (P < 0.05), and Ovarian Volume reduces, and interstitial fibrosis is serious, primordial follicle, growing follicle and mature egg Bubble number substantially reduces (P < 0.05).After BMSCs transplanting, transplantation group mouse weight increases, and appetite enhancing, activity increases；After 28d FSH concentration is substantially less than modeling group (P < 0.05), and E2 concentration, primordial follicle, Growing follicle and graaffian follicle number are significantly high In modeling group (P < 0.05), but there are significant differences still between blank control group for indices；It is visible green under fluorescence microscope Color fluorescence signal, which is dispersed in, to be distributed in around ovarian follicle.The prompt of this result, BMSCs can be damaged in mouse and be positioned survival in ovary tissue, There is repair to ovary tissue structure and endocrine function.

(Chen Yingxia, etc. the fat mesenchymal stem cell of, GDF-9 transfection, to treat chemotherapeutic premature ovarian failure big for Chen Yingxia paper Mouse, modern gynemetrics progress, 11 phases in 2017) research up-regulation growth differentiation factor-9 (GDF-9) expression rat fat between fill The effect of matter stem cell (ASC) infusion of therapeutic chemotherapeutic premature ovarian failure (POF) rat, method are to digest adherent method from rat ASC is cultivated in fat, liposome transfection pEGFP-N3-GDF-9 plasmid enters ASC, and verifies GDF-9 expression.It is injected intraperitoneally suitable Platinum establishes P of Rats OF model, feeds back ASC and ASC-GDF-9 cell respectively by tail vein after 1 week, observes rat body weight, ovary Estradiol (E in weight, serum₂), follicular stimulating hormone (FSH) and metakentrin (LH) value, and analyze folliculogenesis situation.Knot Fruit shows that the growth of ASC cell is vigorous GDF-9 albumen, transfection effect can be smoothly expressed after transfection to skeletonization and Adipocyte Differentiation Rate is (43 ± 9.68) %.Intraperitoneal Cisplatin injection can reduce rat body weight and reduce with Ovarian Volume, and Follicles are reduced, E₂Drop Low, FSH and LH are significantly raised.After infusion ASC-GDF-9 cell 4 times, Follicles number can be dramatically increased and stroma cell is close Degree reduces LH and FSH hormonal readiness.These results indicate that the ASC of transfection GDF-9 can preferably improve after tail vein is transfused Ovarian function restores the development of Follicles, improves hormone disturbance state.

Chen Jingjing paper (Chen Jingjing, etc. Bone Marrow Mesenchymal Stem Cells Transplantation is in premature ovarian failure mouse ovarian and reproductive function Effect in reconstruction, Medical University Of Anhui's journal, 11 phases in 2017) it has inquired into mesenchymal stem cell (BMSCs) and has been implanted in Premature ovarian failure mouse ovarian and reproductive function rebuild in effect, method is, disposable celiac injection cyclophosphamide and white disappears Peace establishes premature ovarian failure mouse model, and experimental animal is divided into modeling group, transplantation group and blank control group.14d after modeling, will be green The BMSCs of color fluorescin (GFP) transgenic mice is through taking same amount of normal saline to pass through in tail vein injection to transplantation group Mice Body In tail vein injection to modeling group and blank control group mouse body.14d and BMSCs transplants 2 months detection each group serum after modeling Follicular stimulating hormone (FSH) and estradiol (E2) are horizontal, and BMSCs transplants positioning and ovary of 2 months observation BMSCs in ovary tissue Apoptosis of granulosa cell situation obtains ovum number, rate of fertilization, cleavage rates, Blastocyst formation rate after recording each group Ovarian hyperstimulation, and with hero Property mouse mate after gestation and fertility filial generation quantity.The results show that 2 months after BMSCs transplanting, it is visible in transplantation group stroma of ovary The BMSCs of a large amount of green cells and label, but do not express fsh receptor；FSH level and apoptosis of granulosa cell index are significant Lower than modeling group (P < 0.05), E2 is horizontal and obtains ovum number and rate of fertilization, cleavage rates, Blastocyst formation rate are all remarkably higher than modeling group (P < 0.05), but indices are still significantly lower than blank control group (P < 0.05)；Modeling group is 2 small in transplantation group without mouse pregnancy Mouse gestation, gives birth to appearance normal newborn mouse 4,2 respectively.These results indicate that BMSCs can be positioned at stroma of ovary, significantly reduce Granular apoptosis of ovary index improves Function of Ovary, improves mouse fecundity.

Wang Lulu paper (Wang Lulu, etc. the chemotherapeutic premature ovarian failure of heat-shock pretreatment mesenchymal stem cell transplantation Effect, Guangdong medicine, 13 phases in 2018) inquired into heat-shock pretreatment mescenchymal stem cell (MSCs) cyclophosphamide is damaged it is big The repair of mouse ovary, method are to separate, cultivate rat marrow MSCs, with 42 DEG C of pretreatment 1h before transplanting.Experiment is divided into 4 groups: normal group, model group, MSCs group, heat shock group, every group of 25 rats.Normal group is not handled, remaining 3 groups establish it is chemotherapeutic Premature ovarian failure animal model, the interior injection 1 × 10 of MSCs group rats with bilateral ovary⁶A MSCs, in heat shock group rats with bilateral ovary Injection 1 × 10⁶The MSCs of a heat-shock pretreatment.Vaginal smear monitors the oestrous cycle of rat, is detected with chemoluminescence method female Glycol (E2), radioimmunology detect follicular stimulating hormone (FSH).Every group the 1st after modeling, blood sampling in 15,30,45,60 days and each Dead 5 rats in component other places, observation ovary weight, ovarian structure and Follicles number.The results show that heat shock group rat ratio MSCs group faster restores the oestrous cycle；Heat shock group, the ovary weight of MSCs group, ovarian follicle sum and Follicles number and model group (P < 0.05) statistically significant compared to difference, heat shock group compared between MSCs group also difference it is statistically significant (P < 0.05)；The the 30th, 45,60 day after transplanting, compared with model group, difference has statistics for MSCs group, heat shock group E2 concentration, FSH concentration It learns meaning (P < 0.05), comparing difference is also statistically significant (P < 0.05) between heat shock group and MSCs group.These result tables Bright, heat-shock pretreatment MSCs has stronger repair to chemotherapeutic premature ovarian failure.

Zhang Lingli paper (Zhang Lingli, etc. the reality of human umbilical cord mesenchymal stem cells treatment POF rat feasibility and its mechanism Test research, Chongqing Medical, 31 phases in 2018) inquire into human umbilical cord mesenchymal stem cells (hUCMSCs) treatment premature ovarian failure (POF) greatly Mouse feasibility and mechanism, method be, by 62 healthy SD rats be divided into control group (A group), model group (B group) and HUCMSCs transplantation group (C group), B, C group rats by intraperitoneal injection cyclophosphamide establish chemotherapeutic physical property P of Rats OF animal model, A group Rat injects isometric aqua sterilisa, and after modeling successfully, C group is treated by tail vein injection hUCMSCs.Compare each group rat Estradiol (E2), follicular stimulating hormone (FSH), lutropin (LH), inhibin B (INHB), Anti-Mullerian hormone in serum (AMH), the aspartic acid proteolytic enzyme 3 (caspase-3) containing cysteine is horizontal and people's ALU gene is in rat ovary Interior distribution situation, and compare ovary tissue form, follicle number and corpus luteum number after each group rat HE dyeing.The results show that having modeled At rear 1d, B group INHB, AMH level is lower than A group；17d after stem cell transplantation, C group INHB, AMH level increase, and Ovarian Volume increases Greatly, growing follicle increases, atretic follicle is reduced, statistically significant (P < 0.05) with B group comparing difference.Stroma of ovary, particle And people's ALU gene is observed in oocyte, caspase-3 signal representation amount [(4.40 ± 1.46) × 10^-3] it is significantly lower than B group (P<0.05).These results indicate that hUCMSCs is by repairing granular cell and direct combination into two kinds of granular cell oocyte Approach acts on ovary, reaches treatment POF type rat effect.

Du Jing paper (Du Jing, etc., human umbilical cord mesenchymal stem cells to premature ovarian failure rat ovary ultra microstructure and function Influence, modern gynemetrics's progress, 02 phase in 2018) human umbilical cord mesenchymal stem cells (hUCMSCs) is inquired into premature ovarian failure model The influence of rat ovary ultra microstructure and reserve function, method are that 62 healthy SD rats are randomly divided into negative control group (18), model control group (22) and hUCMSCs transplantation group (22).Negative control group rat is without any processing, mould Type control group and hUCMSCs transplantation group rats by intraperitoneal injection cyclophosphamide (CTX) establish premature ovarian failure animal model.It models successfully Afterwards, hUCMSCs transplantation group is treated by tail vein injection hUCMSCs.The ordinary circumstance of rat is observed, rat ovary is observed The change of granular cell ultra microstructure measures estradiol (E in serum₂), follicular stimulating hormone (FSH), lutropin (LH), suppression It makes element B (INHB) and Anti-Mullerian hormone (AMH) is horizontal, and analyze the variation of Ovary reserve.The results show that being shown under Electronic Speculum Show, the karyolysis of model control group granular cell, nuclear membrane disappearance, mitochondrial vacuolation, mitochondria are gradually decreased with endoplasmic reticulum quantity； HUCMSCs transplantation group granular cell nuclear membrane is gradually repaired, and kernel occurs, and endoplasmic reticulum exists on a small quantity with mitochondria.After modeling 1d, compared with negative control group, model control group and hUCMSCs transplantation group E2 content are reduced, and FSH content increases, and difference has Statistical significance (P < 0.05)；1d after hUCMSCs transplanting, model control group and hUCMSCs transplantation group INHB, AMH content compared with Negative control group is low, and difference is statistically significant (P < 0.05)；17d after hUCMSCs transplanting, the INHB water of hUCMSCs transplantation group Flat to be higher than model control group, difference is statistically significant (P < 0.05).These results indicate that hUCMSCs transplanting is to POF rat Ovary ultra microstructure and reserve function have certain repair.

Mutually beautiful paper (Xiang Li, etc. Human plactnta mesenchymal stem cell transplantation is by reducing superoxide dismutase 1 and decoupling The expression for joining albumen -2 improves ovarian function, Chinese reproduction and contraception magazine, 02 phase in 2018) to inquire into Human plactnta mesenchyma dry thin Born of the same parents' (hPMSC) transplantation treatment to caused by cyclophosphamide premature ovarian failure (POF) model SD rat superoxide dismutase 1 (SOD1) and The influence of Uncoupling protein-2 (UCP-2) expression.Its method is that 60 female sd inbred rats are randomly divided into 4 groups (n=15): blank Control group, POF model group treat control group, hPMSC transplantation group.50mg/kg, the continuous 14d of maintenance dose 8mg/kg are measured with head POF model is established to rats by intraperitoneal injection cyclophosphamide solution, observation Rats During The Estrous Cycle variation is measured using ELISA method Estradiol (E2), follicle-stimulating hormone (FSH), anti-gyneduct hormone (AMH), active chalcogen (ROS), 8- hydroxyl are de- in serum Oxygen guanosine (8-OHdG) is horizontal；HE colouring method detects ovary tissue morphology and ovarian follicle counts, and VG decoration method observes ovary group Textured fiber situation；SOD1 and UCP-2 protein expression is measured using immunohistochemistry and Western blotting method.As a result It has been shown that, ELISA as the result is shown: model group serum FSH horizontal [(10.60 ± 1.0) IU/L] is apparently higher than blank control group [(5.83 ± 0.92) IU/L] (P < 0.01), and E2, AMH level [(35.52 ± 10.27) ng/L, (2090.6 ± 397.5) ng/ L] it then is significantly lower than blank control group [(65.62 ± 3.76) ng/L, (3636.39 ± 204.46) ng/L], difference has statistics Meaning (P < 0.01).ROS and 8-OHdG increases (P < 0.05) in hPMSC transplantation group serum compared with blank control group.Ovarian follicle meter Digital display shows that hMSC transplantation group atretic follicle number (5.36 ± 1.11) is substantially less than and treats control group (8.01 ± 2.22) and POF model Group (11.21 ± 1.69) (P < 0.05).Compared with treating control group, in hPMSC transplantation group ROS and 8-OHdG be remarkably decreased (P < 0.05).VG dyeing display, with treatment control group ratio, hPMSC transplantation group fibrosis area ratio significantly reduces (P < 0.05). Western blotting is the results show that compared with treating control group, SOD1 and UCP-2 expressing quantity in hPMSC transplantation group It is remarkably decreased (P<0.05), and hPMSC transplantation group and control group then no significant difference (P>0.05)；Immunohistochemistry SOD1 and UCP-2 protein expression compared to treatment control group is to reduce in model group as the result is shown, and hPMSC transplantation group is compared In blank control group then no significant difference.It is answered these results indicate that hPMSC transplanting improves the oxidation of POF model SD rat Swash, reduce SOD1 and UCP-2 expression, plays the role of protecting ovary mitochondrial function.

The present inventor was once separately cultured mescenchymal stem cell using perfusion method from placenta, and it is very high to obtain purity Mescenchymal stem cell.But still suffer from a large amount of stem cells after perfusion and be trapped in placenta tissue, cannot effectively by It separates.It is understood that mescenchymal stem cell cannot be obtained to greatest extent using perfusion method.

The method of the invention discloses a kind of from placenta a large amount of separating mesenchymal stem cells, and save with this method Placenta mesenchyma stem cell simultaneously establishes placenta stem-cell library.The present inventor was separately cultured mesenchyma in summary in the past and did carefully It is successful from placenta in conjunction with stationary culture using Various Tissues digestive ferment mixture slaking placental lobules tissue block on the basis of born of the same parents In isolated a large amount of mescenchymal stem cells.Mescenchymal stem cell that the method for the present invention obtains purity is high, quantity are more, have and bone The identical biological characteristics of bone marrow-drived mesenchymal stem, can be thin to osteoblast, cartilage cell, fat cell, endothelial cell, nerve The differentiation such as born of the same parents.Due in placenta stem cell compared with adult stem cell naivety, rich content, before clinically having a wide range of applications Scape, we freeze mescenchymal stem cell with conventional cell freezing method as bleeding of the umbilicus, establish placenta stem-cell Library lays the foundation for the further investigation and clinical treatment of later stem cell.

Due to candidate stem cell rich in bleeding of the umbilicus, people establish unbilical blood bank, and umbilical hemopoietic stem cell, this is important Living resources store, provide a kind for the treatment of means for a variety of diseases in the blood system and disease of immune system.Same placenta As a kind of more importantly stem cell resource, we are freezed mescenchymal stem cell with conventional cell freezing method It is saved for a long time in -196 degrees Celsius of profound hypothermia liquid nitrogen, establishes placenta stem-cell library, save kind for stem-cell therapy in the future Son.

Especially, it should be noted that by the method for the invention, mescenchymal stem cell very high purity can be obtained in P0 generation Primary cell, the CD73 expression of these primary cells is greater than 60%, CD45 and does not express, and mesenchyma is dry in these primary cells Cell content reaches 60%-70%.

Having the technical effect that for method of the invention is obvious.For example, the present invention chooses mature placental samples, clip It is digested with a kind of mixing enzyme system, is purified, be can be obtained after obtaining cell by placental lobules specific position tissue 15g The purer mescenchymal stem cell of a group (CD73 expression is greater than 60%, CD45 and does not express), per gram of tissue obtains cell number up to 2.5 ×10⁷, and yield is stablized, and sample specificity is greatly reduced.By culture of recovering after this batch of primary mesenchymal stem cell cryopreserving, make Cultivated with classical complete medium formula, 4 days or so can microscopy to more spindle-type attached cell, cell melts within 10 days Conjunction rate reaches 70-80%, can pass to P1 generation.Continuous passage to P5 generation after, carry out streaming phenotypic evaluation, the cell cycle detection and The experiments such as induction differentiation, the results showed that P1-P5 is mescenchymal stem cell, positive expression (CD73, CD90, CD105) for cell Greater than 98%, feminine gender expression (CD34, CD45, CD19, CD11b, HLA-DR) is less than 2%；P5 is less than for g2 phase cell 1%, proliferative capacity is strong, does not enter division stage；Under the stimulation of specific induced medium, oriented osteoblast, lipoblast, at The ability of Chondrocyte Differentiation.

Operation of the present invention is simple, convenient and practical, can obtain a large amount of mescenchymal stem cell, and differentiation performance is good, have at The ability of the cell differentiations such as osteocyte, fat cell, cartilage cell, endothelial cell, nerve cell.Compared with existing method: MSC mainly uses modus operandi to extract donor bone marrow or perfusion method separation placenta, adhere-wall culture acquisition at present.The method gets cell number Amount is few, and donor in taking marrow and takes the possibility for having infection after marrow.Present invention success separation from placenta obtains a large amount of pure Higher mescenchymal stem cell is spent, and establishes placenta stem-cell library with this method to lay in the dry thin of this great application prospect Born of the same parents.The method is simple and easy to do, and since placenta is as bleeding of the umbilicus, Cell Component is inmatureer, from a wealth of sources, is conveniently easy to get, therefore this The method of invention will have extensive prospect in the clinical application of stem cell.

Detailed description of the invention

Fig. 1: the streaming phenotypic evaluation result figure of present invention gained primary cell.

Fig. 2: micrograph of the sample in P0 succeeding generations.

Fig. 3: micrograph of the sample in P5 succeeding generations.

Fig. 4: sample is in P5 for streaming phenotypic evaluation result.

Fig. 5: sample P5 for cell DNA content-cell number relational graph.

Fig. 6: P5 for cell induction break up test, show its have to skeletonization, at rouge, chondroblast break up energy Power.

Specific embodiment

The present invention can be further described by the following examples, however, the scope of the present invention and unlimited In following embodiments.One of skill in the art, can be with it is understood that under the premise of without departing substantially from the spirit and scope of the present invention Various change and modification are carried out to the present invention.The present invention carries out the material and test method arrived used in test general And/or specific description.Although to realize the present invention many materials and operating method used in purpose be it is known in the art that But the present invention still makees description as detailed as possible herein.

The full cell processing of embodiment 1, placenta:

1, mix synthase digestive juice preformulation: pipette respectively 22ml calcic magnesium ion HBSS (Hank's balance salt it is molten Liquid), 0.4ml Roche Liberase MNP-S enzyme (such as purchased from western precious biology, the DNA I of 0.7ml article No.: 5578582001) Type enzyme is in 50ml centrifuge tube, then adds zinc chloride (addition concentration is 0.2g/L, 0.25g/L or 0.3g/L), is mixed, 37 DEG C Preheat 20min or more.The Hank's balanced salt solution composition are as follows: KCl, 0.1g/L's of NaCl, 0.4g/L of 8.0g/L KH2PO4,1.0g/L of Na2HPO42H2O, 0.06g/L of MgCl26H2O, 0.06g/L of MgSO47H2O, 0.1g/L Glucose, 0.14g/L the phenol red of NaHCO3,0.2g/L of CaCl2,0.35g/L, hydrochloric acid or sodium hydroxide adjust pH to 7.4。

2, the preparation of placental lobules: from placenta is taken out in collection bag in ceramic whiteware disk, after tissue-wash solution rinses, tire is removed Disk hemostasis, a small amount of placental lobules tissue (20g or so) of clip is in steel bowl.Use tissue-wash solution (0.9% physiological saline+bis- Anti- (dual anti-is mycillin, content 1%)) twice of cleaning, and after impregnating 5min, it weighs 15g ± 1g and is preferably organized in 100mm In glass dish.

3, the removal of haemocyte: adding 10ml tissue-wash solution, shreds leaflet to 0.2cm³Left and right size, adds 100ml to organize 300 mesh filter screens filtering after cleaning solution stirs evenly, then cleaned with tissue-wash solution and twice (leaflet tissue is moved on in steel bowl add every time After 100ml tissue-wash solution stirs evenly, the filtering of 300 mesh).

4, enzymic digestion and termination are mixed: the leaflet tissue after cleaning being added in warmed-up 23ml mixed enzyme digestive juice and is filled Divide after mixing, 37 degree of 100rpm oscillation digestion 30min of shaking table.After digestion, tissue fluid+2ml FBS is terminated.

5, the collection of primary cell:

The dilution of 50ml tissue-wash solution is added to mix tissue fluid, the filtering of 300 mesh is collected cell liquid, washed twice postdigestive Tissue (uses 50ml tissue-wash solution) every time, and filtrate is incorporated into 1 250ml centrifuge tube, and 1500rpm is centrifuged 8min and (accelerates Degree 9, deceleration 7)；

Supernatant is removed, appropriate tissue-wash solution is added to be resuspended and is supplemented to 200ml, 1500rpm is centrifuged 8min, and (acceleration 9, subtracts Speed 7)；

Remove supernatant, cell precipitation adds DMEM-F12 to be resuspended to 30ml, after 100um strainer filtering, then with the DMEM- of 10ml F12 flushing filtering net obtains 40ml cell suspension, as primary cell；1ml suspension is taken, is carried out for sysmex blood analyser thin Born of the same parents count, and after measured, the primary cell purity is higher, and mescenchymal stem cell content is about 60%-70%.

6, primary cell freezes:

Now match frozen stock solution, the formula of frozen stock solution are as follows: 65% DMEM-F12,15% human serum albumins (HSA), 20% DMSO such as WAK brand DMSO, matching while using；

Make cell suspension 1800rpm, be centrifuged 10min (acceleration 9, deceleration 7), leaves and takes cell precipitation and lower liquid 5ml is (another Supernatant 10ml keeps sample), it is slowly added into the frozen stock solution prepared after resuspension, shakes well while adding；

Cell suspension is dispensed into 9 2ml cryopreservation tubes, every pipe 1.5ml (program temperature reduction box pre-cooling).Remaining cell suspension + the supernatant that keeps sample is used for Sterility testing；

Cooled down using programmed cooling instrument device program, cell is transferred in liquid nitrogen storage tank, freezes to gained primary cell.

The full cell treatment process of 1 placenta through the foregoing embodiment, chooses mature placental samples, and clip placental lobules is special Tissue 15g is set in positioning, is digested it with the mixed enzyme digestive juice system, is purified after obtaining cell, can be obtained one The purer primary mescenchymal stem cell of group (CD73 expression is greater than 60%, CD45 and does not express), the mesenchyma in these primary cells Stem cell content reaches 60%-70%, and the primary cell number that every gram of placental lobules tissue obtains is up to (2.4~2.8) × 10⁷It is a, And yield is stablized, and sample specificity is greatly reduced.However, when being not added with this zinc chloride in the mixed enzyme digestive juice, it is former For the mescenchymal stem cell content in cell less than 38%, usually in 31~38% ranges, and every gram of placental lobules tissue The primary cell number of acquisition is less than 5 × 10⁵It is a；In addition, the present inventor is carrying out primary cell preparation referring to other prior arts The primary cell number that every gram of placental lobules tissue of Shi Faxian obtains is less than 2 × 10⁶It is a, it is 1/10 or less the method for the present invention.This Invent it is above-mentioned the processing of full cell is carried out to placenta can efficiently obtain primary mescenchymal stem cell, to continue secondary culture thereafter It lays a good foundation at the mescenchymal stem cell with high medical value.

For example, in the present embodiment, cell yield is highly stable after placenta tissue processing, and the typical data of some tests is shown in Table 1.

Table 1: the cell yield of primary cell is obtained from placenta tissue

Handle the date	Sample registered number	Tissue mass (g)	Cell number (× 10⁸)	Cell yield (10⁸/g)
					2016.7.22	9004116082279	15	3.8	0.25
2016.7.25	9004116082301	15.9	3.9	0.25
					2016.7.26	9004116082311	14.9	3.8	0.26
2016.7.27	9004116082319	14.9	3.8	0.26
					2016.8.25	9004116082539	15	3.75	0.25

In addition, after the processing of above-mentioned placenta gained primary cell streaming phenotypic evaluation as the result is shown CD73 expression up to 60% with On, CD45 is not expressed, and shows that primary cell is the purer mescenchymal stem cell of a group, is free of haemocyte.Streaming phenotypic evaluation As a result as shown in Figure 1.

Embodiment 2, primary cell recovery and secondary culture

1, cell recovery:

Cell is moved to 15ml centrifuge tube by the cell for taking 2 pipes to freeze, 37 DEG C of quick-thawings, adds 8ml complete medium drop Recovery；If not otherwise indicated, complete medium used herein is the DMEM-F12 culture medium comprising 10%FBS；

Then after 1200rpm is centrifuged 5min (acceleration 9, deceleration 7), supernatant is removed, 5ml complete medium is added to be resuspended；Often Solencyte is inoculated in 1 T75 culture bottle, supplement complete medium to 30ml, and setting CO2 incubator, (37 DEG C, 5%CO2, be saturated Humidity) in culture；Carried out once changing liquid entirely with complete medium every 3-4 days, after recovery 12 days according to Clone formation situation into Row counts, until cell density is not less than 3000 cell/cm²When can carry out next passage；

2, cell passes on: the P0 after taking recovery is cleaned for cell with PBS, and 2ml pancreatin is added to digest 2-5min, until cell is big Partial exfoliation adds 5ml complete medium to terminate digestion, is transferred to cell in centrifuge tube, and 1400rpm is centrifuged 5min (acceleration 9, deceleration 7), supernatant is abandoned, is counted after adding 5ml complete medium to be resuspended and is seeded to culture bottle, cell density is 8000~12000 A cell/cm², set culture in CO2 incubator (37 DEG C, 5%CO2, saturated humidity) and (usually trained to cell density up to 90% or more Support 5 days or so), it completes to pass on from the cell in P0 generation to P1 generation；

The passage for being repeated in above-mentioned P0 generation to P1 generation operates, to carry out P1 generation to P2 generation, P2 generation to P3 generation, P3 generation respectively Cell to P4 generation, P4 generation to P5 generation passes on, and obtains each generation mescenchymal stem cell.

Primary inoculum density is about 5 × 10 in the present embodiment⁵cells/cm², 4 clear water surfaces of inoculation, which are inspected in open country, many patches Parietal cell is in spindle-type.P1 generation can be reached within 10 days after inoculation.High cell growth speed, amount is more, and form is in shuttle shape, full.From In P0 generation, the cell count exemplary results of some tests were as shown in table 2 below into the succeeding generations in P1 generation.Wherein PS162279 sample Originally the micrograph in succeeding generations is as shown in Figure 2.

Cell counts of the table 2:P0 generation into the succeeding generations in P1 generation

Sample registered number	P0-P1	P0 is counted
			PS162311	D10	8*10⁵(ADAM)
PS162319	D10	1.4*10⁶(ADAM)
			PS162279	D9	1.5*10⁶(ADAM)

In addition, being generally incubated 4~5 days in P1-P5 inoculation succeeding generations and harvesting and be passaged to the next generation.It is exemplary , micrograph of the PS162279 sample in P5 succeeding generations is as shown in Figure 3.

In this test, the streaming phenotypic evaluation in P1-P5 generation is carried out, the results show that CD73, CD90, CD105 positive table Up to > 98%, while CD34, CD45, CD19, HLA-DR are identified, the results are shown in Table 3, these results prove in placenta point It is mescenchymal stem cell from the cell turned out, and with high purity.

Streaming phenotypic evaluation result of the table 3:P1-P5 for cell

Illustratively, PS162279 sample P5 is as shown in Figure 4 for streaming phenotypic evaluation result.

In addition, for some samples P5 for their growth cycle of raji cell assay Raji, G2 phase cell < 1%, S as the result is shown Phase cell > 10%, it was demonstrated that these ability of cell proliferation are strong, do not enter division stage, and concrete outcome is shown in Table 4.

Table 4:P5 is for cell growth cycle measurement result

Sample registered number	The GO/G1 phase	The S phase	The G2/M phase
				PS162311-P5	84.60%	14.80%	0.64%
PS162319-P5	82.30%	16.80%	0.93%
				PS162279-P5	87.00%	11.90%	0.80%

In addition, be directed to PS162311-P5 cell, draw its DNA content-cell number relational graph, typically as the result is shown in Fig. 5.

The Identification of Biological Characteristics of embodiment 3, placenta MSC

Method progress placenta mesenchyma referring to authorized patent CN102676451A its [0062] to [0089] is dry The Identification of Biological Characteristics of cell, the results show that the MSC isolated using the method for the present invention, has to osteoblast, fat The ability of cell, Chondrocyte Differentiation, it was demonstrated that the MSC that the method for the present invention obtains has stem cell properties.

For example, illustrative, induction differentiation test is carried out for cell to P5, as the result is shown these cells have to skeletonization, The ability broken up at rouge, chondroblast.Typically Fig. 6 is seen at rouge differentiation, Osteoblast Differentiation and at the micrograph of cartilage differentiation.

The validity of embodiment 4, mescenchymal stem cell treatment POF

(1) 6 week old female C57BL/6 mouse, 50mg/kg/ days cyclophosphamide (CTX) of intraperitoneal injection, continuous 15d, daily Same time injection, establishes POF mouse model.It is commented by indexs such as follicle number, hormonal readiness, oestrous cycle and fertility tests Estimate the reserve function of ovary.

(2) 6 week old female C57BL/6 mouse, are randomly divided into: control group (n=20), POF model group (n=20), placenta Mescenchymal stem cell treatment group a (PD-MSC group a, n=20), placenta mesenchyma stem cell treatment group b (PD-MSC group b, n= 20).Treatment group mouse, the 1st day and 14 days after POF modeling, 2 placenta mesenchyma stem cell preparations are given in injection, often Mouse 2 × 10⁵A placenta mesenchyma stem cell tail vein injection.POF model group injects isometric physiological saline.In this experiment In, use placenta mesenchyma stem cell ejection preparation to prepare in this way: by the step 2 cell passage gained of embodiment 2 Mescenchymal stem cell (this example uses PS162279 sample P5 generation) is transferred in centrifuge tube, and 1500rpm is centrifuged 5min, abandons supernatant, The resuspension of 0.9% sodium chloride solution is added, it is 1~3 × 10 that cell concentration, which is made,⁶The cell system of a placenta mesenchyma stem cell/ml Agent；Wherein, additionally adding magnesium gluconate and phosphatide in 0.9% sodium chloride solution (makes magnesium ion concentration reach 5mmol/ L, phospholipid concentration 0.2mg/ml, phosphatide are injection stage soybean-sources, add magnesium gluconate and phosphatide group echo is PD-MSC Group a) or not adds magnesium gluconate and phosphatide (is not added magnesium gluconate and phosphatide group echo is PD-MSC group b).

(3) hormonal readiness

14 days and 28 days after second of placenta mesenchyma stem cell is transplanted, every group takes 10 mouse respectively, and eye socket is taken a blood sample, Serum is separated, saves -20 DEG C.Enzyme-linked immunosorbent assay (ELISA) analyzes estradiol (E2), follicle-stimulating hormone (FSH), anti-Miao Le The level of family name Guan Jisu (AMH) (specific method is carried out with reference to mutually beautiful paper method).As the result is shown: compared with POF model group, tire E2 level is each time point increases, FSH level declines in disk mescenchymal stem cell group mice serum, significant difference (p < 0.05), concrete outcome see the table below.

Compared with the POF model group of same detection day, p < 0.01 * p < 0.05, * *.

(4) ovarian follicle of Mouse Ovary Tissues counts

28 days after second of transplanting of placenta mesenchyma stem cell, every group takes 10 mouse respectively, puts to death, and takes ovum on the left of mouse Nest is organized in 4% paraformaldehyde and fixes, and the tissue after fixation is carried out serial dehydration of alcohol, dimethylbenzene is transparent, paraffin embedding, even Continuous slice, slice thickness 5um carry out HE dyeing, microscopically observation.

As the result is shown: compared to control group, POF model group mouse primary follicle, secondary follicle and graaffian follicle quantity are bright Aobvious to reduce, atretic follicle quantity obviously increases；28 days after placenta mesenchyma stem cell treatment, Follicles number has in various degree Recovery, granular cell growth increase, apoptosis reduce, ovarian epithelial cell form stable, primary follicle, secondary follicle and maturation Ovarian follicle quantity obviously increases, and atretic follicle quantity significantly reduces.28 days Follicles count after placenta mesenchyma stem cell transplanting, There were significant differences compared with POF group, and P < 0.01, concrete outcome see the table below.

	Control group	POF model group	PD-MSC group a	PD-MSC group b
					Primary follicle	26.64±3.26	7.12±1.94	21.17±4.15**	10.64±1.93
Secondary follicle	24.87±4.34	7.41±2.82	22.42±3.87**	13.26±2.24*
					Graaffian follicle	23.92±2.62	6.86±2.11	20.15±4.03**	9.83±1.74
Atretic follicle	2.64±1.04	6.03±1.36	2.87±0.92**	5.26±1.16

Compared with POF model group, p < 0.01 * p < 0.05, * *.

(5) mouse fecundity is observed:

The 28th day after placenta mesenchyma stem cell transplanting, male and female mouse is mated into raising in 2:1 ratio, counts mouse Fertility-rate, and the litter size of mouse is compared, observation placenta mesenchyma stem cell transplants the reparation to mouse ovarian function Effect, there were significant differences compared with POF group for placenta mesenchyma stem cell group, P < 0.01.Concrete outcome see the table below.

	Control group	POF model group	PD-MSC group a	PD-MSC group b
					Litter size	14.3±1.8	4.6±1.3	9.7±1.5**	6.4±1.9

Compared with POF model group, p < 0.01 * p < 0.05, * *.

According to the above results it is found that placenta mesenchyma stem cell transplantation treatment, which can obviously improve POF mouse, is damaged ovary Reserve function, follicle number increase, and estrogen and progestogen increases, and some animals fertility is restored, answers for placenta mesenchyma stem cell Clinical treatment for POF provides experimental basis.

The foundation of embodiment 5, placenta stem-cell library

1, the detection of cell activity

The number for freezing front and back living cells is counted using trypan blue staining.

2, the detection of cell contamination

Using a small amount of cell culture, detect cell whether the pollution by fungi and bacterium.Utilize aetology method, detection Cell whether by Hepatitis B virus, hepatitis, AIDS, cytomegalovirus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, TRUST infection.

3, the detection of hereditary disease

Using the method for molecular genetics, detecting freeze-stored cell whether there is hereditary disease.

4, HLA-ABC/DR distribution type

Cell HLA-ABC/DR phenotype is detected, and is placed on record.

5, the investigation of cell origin

Fetus and its particulars of parent are recorded, and are placed on record.

6, the foundation of placenta stem-cell database

After saving normal placenta stem-cell, the database of placenta stem-cell is established, including the first six data, and Foundation is associated with freeze-stored cell.

Claims

1. a kind of cell preparation is by making mescenchymal stem cell such as placenta mesenchyma stem cell be suspended in 0.9% chlorination The cell suspension being configured in sodium solution.

2. cell preparation according to claim 1, wherein cell concentration is 1~10 × 10⁶A cell/ml, such as 1~5 × 10⁶ A cell/ml, such as 1~3 × 10⁶A cell/ml；For example, also adding magnesium gluconate in 0.9% sodium chloride solution； For example, the amount of addition magnesium gluconate is to make magnesium ion concentration 5mmol/L.

3. cell preparation according to claim 1 is as including made from method: mesenchyma obtained by cell passage is dry thin Dysuria with lower abdominal colic moves in centrifuge tube, and supernatant is abandoned in centrifugation, and 0.9% sodium chloride solution is added and is resuspended, cell preparation is made.

4. cell preparation according to claim 1, wherein the mescenchymal stem cell is the method system by including the following steps For what is obtained:

(1) processing of placental lobules: placenta is placed in ceramic whiteware disk, is rinsed with tissue-wash solution to remove placenta hemostasis, clip 20g placental lobules is organized in steel bowl, is cleaned twice using tissue-wash solution, and after impregnating 5min, is weighed 15g and preferably organized In 100mm glass dish；Add 10ml tissue-wash solution, shreds leaflet to 0.2cm³Left and right size adds 100ml tissue-wash solution to stir 300 mesh filter screens filtering after even, then this is operated to be cleaned twice with tissue-wash solution to remove haemocyte repeatedly；

[wherein, include in the mixed enzyme digestive juice: Hank ' the s balanced salt solution of 15~30 volumes, 0.2~0.6 volume Liberase MNP-S enzyme, 0.2~2 volume DNA I type enzyme (such as 20~25 volumes Hank ' s balanced salt solution, 0.3 The DNA I type enzyme of the Liberase MNP-S enzyme of~0.5 volume, 0.5~1 volume, such as Hank ' the s balance salt of 22 volumes are molten Liquid, the Liberase MNP-S enzyme of 0.4 volume, 0.7 volume DNA I type enzyme)；The Liberase MNP-S enzyme is, for example, sieve The Liberase MNP-S enzyme of family name company, such as purchased from western precious biology, article No.: 5578582001]

(3) it collects primary cell: adding 50ml tissue-wash solution in rapid gained tissue fluid one step up, mix, the filtering of 300 mesh, Collect cell liquid；Wash postdigestive tissue twice repeatedly, filtrate twice is incorporated into centrifuge tube, 1500rpm centrifugation 8min (acceleration 9, deceleration 7)；Supernatant is removed, appropriate tissue-wash solution is added to be resuspended and is supplemented to 200ml, 1500rpm centrifugation 8min (acceleration 9, deceleration 7)；Remove supernatant, cell precipitation adds DMEM-F12 to be resuspended to 30ml, after 100um strainer filtering, The DMEM-F12 flushing filtering net for using 10ml again, obtains 40ml cell suspension, as primary cell；

(4) primary cell freezes: making cell suspension 1800rpm, is centrifuged 10min (acceleration 9, deceleration 7), leaves and takes cell precipitation And lower liquid 5ml, it is slowly added into frozen stock solution 10ml after resuspension, shakes well while adding；Gained cell suspension is dispensed to 9 2ml and is frozen Guan Zhong, every pipe 1.5ml, sets in the program temperature reduction box of pre-cooling, is cooled down using programmed cooling instrument device program, then cell is transferred to liquid nitrogen It is frozen in storage tank；

[wherein, the formula of the frozen stock solution are as follows: 65% DMEM-F12,15% human serum albumins (HSA), 20% The DMSO of DMSO such as WAK brand]

(5) cell recovery: cell is moved to 15ml centrifuge tube, 8ml is added to train completely by the cell for taking 2 pipes to freeze, 37 DEG C of quick-thawings Support basic point drop recovery；Then after 1200rpm is centrifuged 5min (acceleration 9, deceleration 7), supernatant is removed, 5ml complete medium weight is added It is outstanding；Every solencyte is inoculated in 1 T75 culture bottle, supplement complete medium to 30ml, set CO2 incubator (37 DEG C, 5%CO2, Saturated humidity) in culture；It was carried out once changing liquid entirely with complete medium every 3-4 days, according to Clone formation feelings after recovery 12 days Condition is counted, until cell density is not less than 3000 cell/cm²When can carry out next passage；

[wherein, the complete medium is the DMEM-F12 culture medium comprising 10%FBS]

(6) cell passes on: taking P0 to be cleaned for cell with PBS, 2ml pancreatin is added to digest 2-5min, until cell largely falls off, add 5ml complete medium terminates digestion, is transferred to cell in centrifuge tube, and 1400rpm is centrifuged 5min (acceleration 9, deceleration 7), Supernatant is abandoned, is counted after adding 5ml complete medium to be resuspended and is seeded to culture bottle, cell density is 8000~12000 cell/cm², Culture is set in CO2 incubator (37 DEG C, 5%CO2, saturated humidity) to cell density up to 90% or more (being generally incubated 5 days or so), It completes to pass on from the cell in P0 generation to P1 generation；Aforesaid operations are repeated in carry out P1 generation to P2 generation, P2 generation to P3 generation, P3 respectively The cell in generation to P4 generation, P4 generation to P5 generation passes on, and obtains each generation mescenchymal stem cell.

5. cell preparation according to claim 1, during preparing mescenchymal stem cell, further includes:

(7) for placenta mesenchyma stem cell obtained by step (6), detect following items at least one: cell activity, cell are dirty Dye, hereditary disease, HLA-ABC/DR distribution type.

6. cell preparation according to claim 1, during preparing mescenchymal stem cell, further includes:

(8) each after passage obtained by step (6) is frozen in liquid nitrogen for placenta mesenchyma stem cell；And/or

(9) establish the database of the placenta stem-cell comprising information above, and make the freeze-stored cell of the database and step (8) into Row association.

7. cell preparation according to claim 1, wherein gained is respectively greater than 90% for the cell purity of placenta mesenchyma stem cell； For example, the placenta mesenchyma stem cell, after 3 more than generation pass on, cell purity is greater than 95%.

8. cell preparation according to claim 1, wherein the Hank's balanced salt solution forms are as follows: the NaCl of 8.0g/L, The Na2HPO42H2O of MgCl26H2O, 0.06g/L of MgSO47H2O, 0.1g/L of KCl, 0.1g/L of 0.4g/L, Phenol red, the salt of the glucose of KH2PO4,1.0g/L of 0.06g/L, NaHCO3,0.2g/L of CaCl2,0.35g/L of 0.14g/L Acid or sodium hydroxide adjust pH to 7.4.

9. cell preparation according to claim 1, which is characterized in that

In the mixed enzyme digestive juice other than comprising Hank's balanced salt solution, Liberase MNP-S enzyme, DNA I type enzyme, Also it is added with 0.2~0.3g/L zinc chloride；

The cytoactive detection is that the number for freezing front and back living cells is counted using trypan blue staining；

Cell contamination detection utilizes a small amount of cell culture, detection cell whether the pollution by fungi and bacterium；

The cell contamination detection is using aetology method, and whether detection cell is by selected from following one or more senses Dye: Hepatitis B virus, hepatitis, AIDS virus, cytomegalovirus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, TRUST；

The hereditary disease detection is the method using molecular genetics, and detection freeze-stored cell whether there is hereditary disease；

The HLA-ABC/DR distribution type is detection cell HLA-ABC/DR phenotype；

The placenta mesenchyma stem cell is frozen in liquid nitrogen through program temperature-fall period；

Include in the database to all relevant data of the cell saved, including but not limited to: the biological characteristics of cell Property testing result, multi-lineage potential qualification result, cellular elements genetic diagnosis result, fetus and its detailed money of parent Material.

10. any one of claim 1~9 cell preparation is in preparing the drug for treating and/or preventing premature ovarian failure Purposes.