CN109646458A

CN109646458A - Use the method for placenta mesenchyma stem cell preparation for treating hardening illness

Info

Publication number: CN109646458A
Application number: CN201811568533.4A
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: 2019-04-19
Anticipated expiration: 2038-12-21
Also published as: CN109646458B

Abstract

The present invention relates to the methods for using placenta mesenchyma stem cell preparation for treating hardening illness.Specifically, one aspect of the present invention be related to cell preparation preparation for treat and/or the drug of prevention system hardening illness in purposes；The cell preparation 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, and cell concentration is 1~10 × 10 in the cell preparation⁶A cell/ml.The invention further relates to for treating and/or the cell preparation of prevention system hardening illness, the method for preparing the cell preparation prepares the method for mescenchymal stem cell involved in the cell preparation, the mescenchymal stem cell, the mixed enzyme digestive juice arrived used in these methods.Excellent biological effect is presented in terms for the treatment of hardening illness in cell preparation produced by the present invention.

Description

Use the method for placenta mesenchyma stem cell preparation for treating hardening illness

Technical field

The invention belongs to biotechnology and biomedicine field, it is related to hardening using placenta mesenchyma stem cell preparation for treating Placenta mesenchyma stem cell preparation used in the method and this method of disease extremely.Specifically, the present invention relates to from placenta The method of middle separation stem cell, in particular to the method for separating mesenchymal stem cell, particularly relates to one kind from placenta It to the separating mesenchymal stem cell from placenta tissue and is trained using the digestive enzyme compositions of unique formula of the present invention The method of mescenchymal stem cell.The efficiency from placenta separating mesenchymal stem cell can be effectively improved using the method for the present invention. Further, the present invention relates to using above-mentioned placenta mesenchyma stem cell to be prepared into cell preparation, and then the cell preparation is used Treatment system hardening illness.

Background technique

Systemic sclerosis (systemic sclerosis, SSc), also known as chorionitis (scleroderma), are a kind of originals Because unknown, clinically characterized by limitation or diffusivity pachyderma and fibrosis, heart, lung and gastral can also be influenced Autoimmune disease.This disease is in global distribution, and illness rate is 50-300/100 ten thousand, every annual morbidity 2.3-22.8/100 Ten thousand, at 30-50 years old onset peak age, women is common, male to female ratio 1:3~14.

According to Raynaud's phenomenon, fibrosis of skin, (anti-Scl-70 is anti-for specific internal organ involvement and Specific ANA Body and ACA), SSc can be diagnosed as.According to skin involvement situation, it is divided into diffusivity sclerosis of the skin disease (dcSSc) and limitation skin Skin sclerosis (lcSSc).Diffusivity fibrosis of skin can involve far-end of limb and proximal end, face and neck, chest and abdomen, This type disease progression is fast, and mostly with viscera, prognosis is poor, 10 years survival rates 50% or so.Limitation cutaneous lesions limitation In the distal end of elbow (knee), can there are face and neck involvement, make slow progress.2/3 or more SSc patient has pulmonary involvement, most common It is pulmonary interstitial fibrosis, is the main cause of death of this disease.

Systemic sclerosis there is no specific medicament at present.Mainly symptomatic treatment improves symptom, and glucocorticoid can reduce Acute skin oedema, but fibrosis of skin cannot be prevented.Immunosuppressor can be used by merging internal organs involvement, and there are commonly cyclosporins A, cyclophosphamide, methotrexate (MTX) etc..Traditional antifibrosis therapy has Beracilline.Muscle, arthralgia can use non-steroidal anti-inflammatory Medicine.Type is diffused since lung, kidney, Cardiac Involvemant are easy to cause death, and prognosis is poor.

It is that SSc patient brings new therapeutic choice with the treatment on stem cell basis.In recent years, some research applications are made self Hemocytoblast (HSC) transplantation treatment seriously diffuses type SSc, compared with CYC treatment, extends life span, reduces skin involvement, take Obtained certain curative effect.But there are somewhat expensives by self HSCT, and the disadvantage of high relapse rate, allosome HSC transplanting is due to rejection etc. The rate that causes death is higher.

The study found that SSc Bone Marrow of Patients mescenchymal stem cell (MSCs) is in growth, hematopoiesis support, cytokine secretion etc. Aspect existing defects, thus it is speculated that the exception of MSCs may play important function in the morbidity of SSc.MSC shows good SSc and suffers from Person's hand and facial treatments effect can improve sclerosis of the skin, the movement of finger and mouth, and reduce hand and facial pain. In newborn's placenta tissue, contain the mescenchymal stem cell (MSCs) with multi-lineage potential for having low immunogenicity.Placenta In MSCs, have immunoregulation effect, it is inhibited to T cell, NK cell, inhibit immune response, mediating tolerance and Permanent immunity escape aspect is caused to play a crucial role.Especially placenta MSC cell immunogenicity is low, and transplanting will not between allogeneic Cause immunological rejection.Antigen is not depended on, directly inhibits the T cell proliferation of activation in a dose-dependent manner.These features make Placenta MSC is obtained to be possibly realized applied to autoimmune disease.

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 It so needs new method and carrys out treatment system hardening illness, especially expect have new more effective way dry thin using mesenchyma Born of the same parents carry out treatment system hardening illness.

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 treatment system hardening illness and a kind of cell preparation is provided.The inventors discovered that using special operating method And the digestive enzyme compositions of especially prescription, cell purity obtained is high and/or cell recoveries are high, and by preparing one The specific cell preparation of kind can be more efficiently used for the treatment of systemic sclerosis.It has been able to the present invention is based on such discovery At.

For this purpose, first aspect present invention provides cell preparation in preparation for treatment and/or prevention system hardening illness Drug in purposes.

Purposes according to a first aspect of the present invention, wherein the cell preparation is by making mescenchymal stem cell (such as tire Disk mescenchymal stem cell) it is suspended in the cell suspension being configured in 0.9% sodium chloride solution.

Purposes according to a first aspect of the present invention, wherein cell concentration is 1~10 × 10 in the cell preparation⁶It is a thin Born of the same parents/ml, such as 1~5 × 10⁶A cell/ml, such as 1~3 × 10⁶A cell/ml.

Purposes according to a first aspect of the present invention, wherein also being added in 0.9% sodium chloride solution described in the cell preparation Citrate of magnesia and phosphatide.In one embodiment, the amount for adding citrate of magnesia is to make magnesium ion concentration 2.5mmol/L.? In one embodiment, the concentration for adding phosphatide is 0.2mg/ml.In one embodiment, the phosphatide is injection stage soybean Source.It has been unexpectedly discovered that cell 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 preparation for treating systemic sclerosis.The present inventor is in addition, it has been found that when the cell system in the embodiment of the present invention 4 In agent, only adds magnesium salts or only add phosphatide or above-mentioned magnesium salts is replaced with into other salt such as calcium salt, zinc salt, mantoquita etc. When, effect of the result far away from the PD-MSC group a for adding magnesium salts and phosphatide simultaneously, and the result of these situations and the present invention The result of PD-MSC group b in embodiment 4 is close.

Purposes according to a first aspect of the present invention, wherein the cell preparation is as including made from method: by cell Passage gained mescenchymal stem cell is transferred in centrifuge tube, is centrifuged, is abandoned supernatant, and 0.9% sodium chloride solution is added and is resuspended, and is made thin Born of the same parents' preparation.

Purposes according to a first aspect of the present invention, wherein mescenchymal stem cell described in the cell preparation is by including such as What the method for lower step 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.

Purposes according to a first aspect of the present invention, wherein the cell preparation is during preparing mescenchymal stem cell, Further include:

(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.

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

Purposes according to a first aspect of the present invention, 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.Cell preparation according to a second aspect of the present invention, wherein being removed in the mixed enzyme digestive juice Outside comprising Hank's balanced salt solution, Liberase MNP-S enzyme, DNA I type enzyme, it is also added with 0.2~0.3g/L chlorination Zinc.It has been had now surprisingly been found that, using the mixed enzyme digestive juice for adding this concentration range zinc chloride, gained is former CD73 expression for cell is not expressed greater than 60%, CD45, and the mescenchymal stem cell content in gained primary cell reaches 60%-70% shows high concentration of stem cells；And when being not added with this zinc chloride in mixed enzyme digestive juice, in primary cell Mescenchymal stem cell content less than 38%, usually in 31~38% ranges.

Purposes according to a first aspect of the present invention, wherein the cytoactive detection is to count to freeze using trypan blue staining Deposit the number of front and back living cells.

Purposes according to a first aspect of the present invention, wherein cell contamination detection utilizes a small amount of cell culture, detection is thin Born of the same parents whether the pollution by fungi and bacterium.In one embodiment, the cell contamination detection is to utilize aetology method, Cell is detected whether by selected from following one or more infection: Hepatitis B virus, hepatitis, AIDS virus, giant cell disease Poison, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV- IgA、TRUST。

Purposes according to a first aspect of the present invention, wherein hereditary disease detection is the method using molecular genetics, inspection Surveying freeze-stored cell whether there is hereditary disease.

Purposes according to a first aspect of the present invention, wherein the HLA-ABC/DR distribution type is detection cell HLA-ABC/DR table Type.

Purposes according to a first aspect of the present invention, wherein the placenta mesenchyma stem cell is frozen through program temperature-fall period In liquid nitrogen.

Purposes according to a first aspect of the present invention, wherein including all relevant to the cell saved in the database Data, including but not limited to: the biological characteristics testing result of cell, multi-lineage potential qualification result, cellular elements heredity Learn diagnostic 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.

Further, third aspect present invention provides a kind of cell system that for example can be used for treatment system hardening illness Agent is configured to by being suspended in mescenchymal stem cell (such as placenta mesenchyma stem cell) in 0.9% sodium chloride solution Cell suspension.

Cell preparation according to a third 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 third aspect of the present invention also adds citrate of magnesia and phosphorus in 0.9% sodium chloride solution Rouge.In one embodiment, the amount for adding citrate of magnesia is to make magnesium ion concentration 2.5mmol/L.In an embodiment In, the concentration for adding phosphatide is 0.2mg/ml.In one embodiment, the phosphatide is injection stage soybean-source.? Surprisingly it has been found that cell preparation treatment system 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 property hardening illness.The present inventor only adds in addition, it has been found that in the cell preparation of the embodiment of the present invention 4 Magnesium salts 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 remote Not as good as the effect for the PD-MSC group a for adding magnesium salts and phosphatide 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 third 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 third aspect of the present invention, wherein the mescenchymal stem cell is by including the following steps What method was prepared:

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

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

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

Cell preparation according to a third 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 third 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.

In addition, providing a kind of placenta mesenchyma stem cell in second aspect of the present invention method.Therefore the present invention the 4th Aspect provides a kind of placenta mesenchyma stem cell.

The cell of placenta mesenchyma stem cell according to a fourth 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 fourth 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 fourth aspect of the present invention, in the preparation step, further includes:

Placenta mesenchyma stem cell according to a fourth 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 fourth 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 fourth 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 fourth 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 fourth 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 fourth 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 fourth 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 fourth 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 fourth 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, fifth 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 fifth 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 DNAI 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 DNAI type enzyme of Hank ' the s balanced salt solution of 22 volumes, the Liberase MNP-S enzyme of 0.4 volume, 0.7 volume).

The mixed enzyme digestive juice of any embodiment according to a fifth 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 fifth 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 fifth 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, include in the mixed enzyme digestive juice: Hank ' the s balanced salt solution of 15~30 volumes, 0.2~0.6 The LiberaseMNP-S enzyme of volume, 0.2~2 volume DNA I type enzyme (such as 20~25 volumes Hank ' s balanced salt solution, The DNA I type enzyme of the LiberaseMNP-S enzyme of 0.3~0.5 volume, 0.5~1 volume, such as the Hank ' s of 22 volumes balance salt Solution, the Liberase MNP-S enzyme of 0.4 volume, 0.7 volume DNA I type enzyme)；The Liberase MNP-S enzyme is, for example, The Liberase MNP-S enzyme of Roche Holding Ag, such as purchased from western precious biology, article No.: 5578582001]

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

The mixed enzyme digestive juice of any embodiment according to a fifth 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 fifth 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 fifth 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 fifth 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 fifth 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 fifth 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 fifth 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.

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.

Systemic sclerosis be it is a kind of characterized by limitation or diffusivity pachyderma and fibrosis it is systemic itself Immunological disease.Lesion characteristic is that skin fiber hyperplasia and blood vessel onion-skin sample change, and eventually leads to sclerosis of the skin, blood vessel ischemic.This Disease is clinically characterized by limitation or diffusivity pachyderma and fibrosis, and in addition to skin involvement, it can also influence internal organ (organs such as the heart, lung and alimentary canal), as a kind of autoimmunity disease, often with antinuclear antibodies, anti-centromere antibody, anti-Scl-70 Equal autoantibodies.This disease women is common, and disease incidence is about 4 times of male, and children are relatively rare.

The most common initial stage performance of systemic sclerosis is Raynaud's phenomenon and acra, facial swelling, and have finger skin by Cumulative thickness.Some cases onset symptoms are Raynaud's phenomenon, and Raynaud's phenomenon can be prior to other symptoms (the finger swelling, pass of chorionitis Section is scorching, internal organ involvement) 1~2 year or occur simultaneously with other symptoms.Functional disturbances of gastrointestinal tract (stomach burn feeling and dysphagia) or Respiratory symptoms etc. are also the First presentation of this disease once in a while.Can there are irregular fever, appetite stimulator and weight before patient's onset Decline etc..Systemic sclerosis is usually in clinical manifestation in skin, bone and joint, digestive system, lung, heart, kidney.Almost All case sclerosis of the skin are all since hand.Finger, the back of the hand are shinny, tight, and finger fold disappears, and fine hair is sparse, then facial It is involved with neck.Patient's upper breast and shoulder have tight feeling.Throat may occur in which transverse thick striped, and when facing upward, patient can feel neck Portion's skin is tight, the few this phenomenons of other diseases.Skin of face involvement can behave as typical chorionitis face, show Are as follows: mask face；There is radioactivity striped week in mouth, and lip is thinning, and nose comes to a point, limitation of mouth opening.Afflicted skin can have pigmentation Or depigmentation.Cutaneous lesions can be confined to finger (toe) and face or centrality extension, involve upper arm, shoulder, shirtfront, back, abdomen And leg.What is had can involve whole skin within some months, and some is gradually in progress within a few years, some are in intermittent progress.It is clinical Upper cutaneous lesions can be divided into oedema phase, hardening period and atrophy phase, oedema phase skin in it is non-can concavity swelling, touching has tough and tensile sense； Hardening period skin is in waxy gloss, is tightly attached to subcutaneous tissue, is not easy to have pinched；Atrophy phase superficial corium is thinning to become fragile, epidermis relaxation. It is close to due to pachyderma and with its hypozygal, causes the contracture of joint and function limitation.Due to stndon sheath fibrosis, work as affected joints When actively or passively moving, especially at wrist, ankle and knee, leather sample abrasive feel can be aware of.Joint can occur in some patientss Inflammation, some of cases can have aggressive arthropathy.Since long-term chronic refers to (toe) ischemic, it can refer to that (toe) end bone is molten Solution.X-ray film shows that joint space is narrow and articular surface osteosclerosis.Alimentary canal involvement is the most common visceral lesion of chorionitis.Disappear Any position for changing road can be involved, and wherein oesophagus involvement is most common, and anus and rectum take second place, and small intestine and colon are less.Hard Involvement of lungs is generally existing in skin disease, and shortness of breath, the attenuating of activity dosis tolerata and dry cough, interstitial lung are fine when the most common symptom is movement Dimensionization and pulmonary arterial vascular lesion can exist simultaneously, but often one of which is occupied an leading position.In terms of heart, clinical manifestation is Shortness of breath, uncomfortable in chest, palpitaition, oedema.Clinical examination can have ventricular gallop, nodal tachycardia and congestive heart failure, occasionally may be used News and pericardiac friction sound.Echocardiogram shows that about half case has pericardium plump or hydrops, but clinical myocardinal inflammation and pericardium are filled out It is rare to fill in.The renal lesions of chorionitis are the most significant with interlobar arteries, arteria arcuata and parteriole, wherein most importantly small Interlobar arteries.Endangium has fibroblast proliferation, mucoid change, acidic mucopolysaccharide deposition and oedema；Vascular smooth muscle is thin Hyaline degeneration occurs for born of the same parents；Externa and surrounding interstitial have fibrosis；Glomerular basement membrane irregular thickening.Chorionitis kidney Lesion clinical manifestation is different, and some patientss have many years skin and other internal organ to be involved and the clinical picture without renal damage；Some Occur renal crises in the course of disease, i.e., severe hypertension and radical property renal failure occur suddenly.If handled not in time, often in several weeks Inside die of heart failure and uremia.Although renal crises initial stage can be asymptomatic, most of patient feels tired exacerbation, gas occurs The symptoms such as rush, severe headache, blurred vision, twitch, obnubilation.Laboratory check discovery creatinine increase, albuminuria and (or) Microscopic hematuria can have capilary hemolytic anemia and decrease of platelet.

Wu Shucai document (Wu Shucai, etc., influence and mechanism of the Human plactnta mescenchymal stem cell to mouse pulmonary fibrosis, mountain Eastern medicine, 12 phases in 2016) influence of the Human plactnta mescenchymal stem cell to mouse pulmonary fibrosis is observed, and inquire into its effect machine System.Its method is, using the separation of tissue block adherent method and vitro culture of human placenta mesenchyma stem cell.30 C57BL/6 are small Mouse is divided into observation group, control group at random, gives intratracheal Injecting Bleomycin after Retaining 8.5mg/kg and establishes pulmonary fibrosis model.It makes After mould success, (cell number is 1.0 × 10^6 to the Human plactnta mescenchymal stem cell 0.3mL of observation group's tail vein infusion in vitro culture It is a), control group injects same amount of normal saline, 1 time/d, continuous to inject 3 days；Mouse is put to death, lung tissue is taken, detects lung tissue hydroxyl dried meat Histidine content detects lung tissue vascular endothelial growth factor (VEGF), Endothelin receptor A (ET-1) using Western blotting method With angiopoietin 2 (Ang-2) albumen.As a result, observation group's lung tissue hydroxyproline content is (5.76 ± 0.13) μ g/ ML, control group are (8.13 ± 0.87) μ g/mL, and two groups are compared P < 0.01.The relative expression quantity of observation group lung tissue VEGF is 52.7 ± 4.7, it is 59.6 ± 2.8 that ET-1, which is 68.1 ± 5.4, Ang-2, and (P is < 0.05) are reduced compared with control group (100).It is tied By being that Human plactnta mescenchymal stem cell can inhibit mouse lung tissue fibrosis and be formed；Reduce lung tissue VEGF, ET-1 and Ang-2 Expression may be its mechanism of action.

Remaining intelligent document (Yu Hui, etc. embryonic stem cell treats the mouse pulmonary fibrosis of bleomycin induction, Fudan Journal (doctor Learn version), 03 phase in 2008) therapeutic effect of research intravenous injection embryonic stem cell (ESC) to pulmonary fibrosis mice.Its method It is that intratracheal instillation bleomycin 8.5mg/kg makes the pulmonary fibrosis model of C57/BL6 female mice.Treatment group (n=20) It is injected intravenously S8 mouse ESC, control group (n=10) injecting normal saline.Treatment group is divided into single therapy (n=10) again and repeats It treats (n=10), both 1h is injected intravenously ESC after modeling, and repetitive treatment group 3d after modeling is injected intravenously ESC again. The life span for recording mouse, measures the hydroxyproline content of mouse lung tissue, and lung pathology observes inflammatory conditions.Utilize order With 3 groups of mouse survival times of inspection statistics, the difference of 3 groups of mouse lung hydroxyproline contents of variance analysis.As a result, receiving dry After cell therapy, the life span (d) of pulmonary fibrosis model mouse extends, and repetitive treatment group is more obvious, and (control group, single are controlled Treatment group, repetitive treatment group are respectively 7.8 ± 2.8,8.4 ± 3.8,13.5 ± 5.6, P < 0.01)；Lung hydroxyproline content (μ g/ ML) reduce (control group, single therapy group, repetitive treatment group be respectively 8.59 ± 1.14,8.23 ± 1.09,5.51 ± 0.39, P < 0.01)；Lungs pathologic finding shows that lung tissue degree of inflammation reduces, and structure, which is destroyed, to be mitigated.Its conclusion is that intravenous injection embryo is dry Cell can mitigate the mouse lung inflammation and pulmonary fibrosis of bleomycin induction, extend the life span of pulmonary fibrosis mice.

The farsighted document of Deng Jiong (Deng Jiong is farsighted, etc., comparison of two kinds of mescenchymal stem cells to pulmonary fibrosis therapeutic effect, Guangdong doctor Learn, the S1 phase in 2018) compare mesenchymal stem cell (BM-MSCs) and umbilical cord blood mesenchymal stem cells (UCB-MSCs) to lung fibre The curative effect of dimensionization rat.Its method is that 48 male SD rats are randomly divided into 4 groups: A group in intratracheally injection 0.1mL physiology salt Water；B, C, D group make pulmonary fibrosis model in the bleomycin of intratracheally injection 0.1mL 5mg/kg.1d after modeling, A, B group warp Tail vein injection saline 1.0mL, C group is through tail vein injection BM-MSCs 1 × 10^6, and D group is through tail vein injection UCB- MSCs 1 × 10^6.28d and 42d put to death the half of each group rat after modeling, leave and take respectively lung tissue carry out pathological examination, And measure lung tissue transforming growth factor-beta 1 (TGF-β 1), hydroxyproline (HYP), MMP-2 (MMP-2), base Matter metalloproteinase inhibitor-1 (TIMP-1) expression.As a result, the visible alveolar structure serious diseases of B group, greatly Measure Collagen fiber deposition, B, C, D, A group pulmonary fibrosis scoring successively reduce [when 28d for (3.00 ± 0.00) divide, (2.17 ± 0.75) divide, (1.60 ± 0.89) point, (0.00 ± 0.00) point, be (3.00 ± 0.00) point when 42d, (2.40 ± 0.55) point, (1.75 ± 0.96) divide, (0.00 ± 0.00) point], lung tissue TGF-β 1, HYP, MMP-2/TIMP-1 level successively decline.Lung group It knits TGF-β 1, HYP, MMP-2/TIMP-1 and there is positive correlation with the scoring of pulmonary alveolitis degree, the scoring of pulmonary fibrosis degree respectively.It is tied By being that BM-MSCs and UCBMSCs can delay Pulmonary Fibrosis in Rats to be in progress, wherein curative effect is better by UCB-MSCs, therapy mechanism It may be unbalance related with 1 level of downward TGF-β, improvement MMP/TIMP.

Pan Yong document (Pan Yong, etc., Bone Marrow Mesenchymal Stem Cells Transplantation treatment silicosis mouse pneumonia and pulmonary fibrosis, China Tissue Engineering Study, 06 phase in 2013) observe therapeutic effect of the Bone Marrow Mesenchymal Stem Cells Transplantation to silicosis.Its method is, 36 C57BL/6 mouse touch ball at random and are divided into 3 groups.The intratracheal saline injection of control group mice；Silicotic model group and Bone Marrow Mesenchymal Stem Cells Transplantation group mouse intratracheally injects silica suspension and establishes Silicotic model；Medulla mesenchyma is dry thin Born of the same parents' transplantation group 6h tail vein after modeling is transfused mesenchymal stem cell.Its result and conclusion be, lung tissue hydroxyproline Content Silicotic model group and Bone Marrow Mesenchymal Stem Cells Transplantation group are higher than control group, and difference has significant (P < 0.01)； Bone Marrow Mesenchymal Stem Cells Transplantation group content is significantly lower than Silicotic model group, and difference has significant (P < 0.01).Silicosis mould The paragonimus cyst of type group and Bone Marrow Mesenchymal Stem Cells Transplantation group mouse is above control group (P < 0.01), and wherein medulla mesenchyma is dry Cell transplantation group is lower than Silicotic model group, and difference has significant (P < 0.01).The expression Silicotic model of interleukin-1 beta Group and Bone Marrow Mesenchymal Stem Cells Transplantation group are higher than control group (P < 0.01)；Bone Marrow Mesenchymal Stem Cells Transplantation group is lower than silicosis mould Type group, difference have significant (P < 0.01).Expression of the transforminggrowthfactor-β1 in lung tissue is similarly Silicotic model Group (P < 0.01) and Bone Marrow Mesenchymal Stem Cells Transplantation group (P < 0.05) are higher than control group；Bone Marrow Mesenchymal Stem Cells Transplantation group is low In Silicotic model group, difference has significant (P < 0.01).These results illustrate that Bone Marrow Mesenchymal Stem Cells Transplantation can subtract Light lung inflammation reaction and degree of fibrosis.

Zheng Kai document (Zheng Kai, etc., Bone Marrow Mesenchymal Stem Cells Transplantation treatment rat acute induced lung injury, Chinese group Knit engineering research, 50 phases in 2014) therapeutic effect of the mesenchymal stem cell to radiation pulmonary injury of rats is observed, and just Step inquires into its mechanism of action.Its method is that in-vitro separation, culture simultaneously identify male SD rat mesenchymal stem cell.Using Linear accelerator irradiates 60 female sd inbred rats chests, establishes radiation pulmonary injury of rats model, is randomly divided into medulla mesenchyma Stem-cell therapy group and saline control group.Bone mesenchymal stem cells treatment group is transfused medulla mesenchyma through rat tail vein 2 × 10^9/L of stem cell, saline control group inject same amount of normal saline, carry out phase at 1,2,4,6 weeks after irradiation Close Indexs measure.Its result and conclusion are, after irradiation at the 1st, 2,4 week, bone mesenchymal stem cells treatment group rat paragonimus cyst Significantly lower than saline control group (P < 0.05).Bone mesenchymal stem cells treatment group lung tissue of rats inflammatory seep is seen under mirror Less, alveolar, alveolar wall construction are substantially complete, and degree of fibrosis etc. is obviously lighter than saline control group.Bone at irradiation 2 weeks Mesenchymal stem cells for treatment group serum TNF-α, hydroxyprolin levels be substantially less than saline control group (P < 0.05).Bone mesenchymal stem cells treatment group lung tissue superoxide dismutase activity is significantly higher than physiology at irradiation 2,4,6 weeks Saline control group (P < 0.05), bone mesenchymal stem cells treatment group mda content is substantially less than physiology salt when irradiating 4,6 weeks Water control group (P < 0.05).The pulmonary surfactant B expression of bone mesenchymal stem cells treatment group is significantly higher than at irradiation 6 weeks Saline control group.PCR method detection lung, liver, pancreas, kidney and other organs tissue have different degrees of Sry gene table It reaches, it is especially significant with lung.The result shows that mesenchymal stem cell can move to the lung tissue of radioactive damage, promote radioactivity The reparation of injury of lungs tissue, therapy mechanism may be related with inhibition inflammatory reaction, anti-oxidant, mitigation lung fibrosis etc..

He Lifeng document (He Lifeng, etc., influence of the aging to Lung stem cells in mouse pulmonary fibrosis model, China's comparison Medical journal, 07 phase in 2013) research aging to Lung stem cells bleomycin cause pulmonary fibrosis mice model in repair ability Influence.Its method is to handle youth using bleomycin, aged mice establishes pulmonary fibrosis model, passes through flow cytometry The methods of compare lung tissue stem cell in ratio young, in aged mice pulmonary fibrosis model and proliferative conditions, study aging Influence to the injury repair ability of lung tissue stem cell.As a result, in the model that bleomycin causes pulmonary fibrosis, it is old Mouse versus young mouse lung tissue stem cells hyperplasia is substantially reduced, lung epithelial stem cell and interstitial lung stem cell ratio it is significant under Drop.Its conclusion is that lung tissue stem cell repair ability after pulmonary fibrosis damage obviously weakens in aging course.

Zhao Feng document (Zhao Feng, etc. bone marrow-derived mesenchymal stem cells on injured lung of rats transforming growth factor β and monocyte The influence of chemotactic protein 1, Chinese Tissue Engineering Study and clinical rehabilitation, 29 phases in 2008) observe mesenchymal stem cell Influence to induced lung injury in rats transforming growth factor β and MCP 1.Design, time and place: random controls, Cytology experiment in vitro is completed in 2005-05/2006-02 in liberation army The Fourth Military Medical University.Material: cleaning grade SD female is big Mouse 20, it is randomly divided into Normal group, cell controls group, injury of lungs group, cell transplantation group, 5/group.Male SD rat 5 Acquisition for mesenchymal stem cell.Its method is that injury of lungs group, cell transplantation group rat transtracheal injection 5mg/kg are rich Model of lung injury is established in 0.2~0.3mL of bleomycin induction.12h after modeling, cell transplantation group, cell controls group rat are quiet through tail Arteries and veins injects mesenchymal stem cell suspension 0.5mL, about 5 × 10^6 cell；Injury of lungs group, rats in normal control group are infused with method Enter serum-free DMEM-F120.5mL.MAIN OUTCOME MEASURES: hematoxylin eosin staining observes lung morphology variation.Acid hydrolyzation Measure lung tissue hydroxyproline content.ELISA method detection serum and BAL fluid transforming growth factor beta, monokaryon The expression of cell chemotaxis albumen 1.As a result, the alveolar space of Normal group and cell controls group is uniform after cell transplantation 2 weeks Completely；The alveolar structure of injury of lungs group destroys, and alveolar septum thickens, interstitial proliferation；Cell transplantation group injury of lungs degree obviously subtracts Gently.Compared with Normal group, injury of lungs group, cell transplantation group lung tissue hydroxyproline content significantly increase (P < 0.01 Or 0.05), cell transplantation group elevation amplitude is significantly lower than injury of lungs group (P < 0.01).Each group rat blood serum and bronchoalveolar lavage Washing lotion transforming growth factor beta, the expression of MCP 1 and lung tissue hydroxyproline content are substantially similar. Its conclusion is, mesenchymal stem cell can reduce induced lung injury in rats lung tissue hydroxyproline content, mitigate Rat Lung Injury and Degree of fibrosis, may be related with reduction transforming growth factor β, the expression of MCP 1.

(Zuo Yi, human cord blood stem cell is to lung fibrosis in rats TNF-α, NO function analysis, before Chinese medical for left virtuous document Edge, 07 phase in 2013) human cord blood stem cell is probed into lung fibrosis in rats TNF-α, the role and influence of NO.Its method is, Cleaning and healthy rat 60 are chosen, are randomly divided into treatment group and control group each 30, two groups of rats are first with bleomycin through gas The method of pipe injection manufactures pulmonary fibrosis model, and treatment group rat injects stem cell, control rats after model is successfully established It is not injected into stem cell, hereafter puts to death rat in different time, two groups of alveolar degree of injury is observed and TNF-α, NO is horizontal Change situation.As a result, control rats alveolar damage degree is more serious than the damage for the treatment of group alveolar, in pulmonary fibrosis journey Degree aspect 0-1/two groups of number of cases rat is identical, 1.1-2/, 2.1-3/treatment group's rat number of cases is relatively to impinging upon Rat is few, and furthermore low compared with control rats in different time treatment group rat TNF-α, NO level, P < 0.05, difference has statistics Meaning.Its conclusion is, after in the stem cell injection lung fibrosis in rats body in human cord blood, with the development TNF-α of time, The level of NO decreases, to a certain extent protective effect, and the expression of TNF-α, NO is suppressed, to induced lung fiber Change has inhibiting effect.

Chen Juan document (Chen Juan, etc. medulla mesenchyma sample stem cell and bone marrow mononuclear cells influence bleomycin induced lung The comparison of fibrosis, Chinese Tissue Engineering Study and clinical rehabilitation, 36 phases in 2010) compare medulla mesenchyma sample stem cell and bone Therapeutic effect of the marrow mononuclearcell to alveolar inflammation and early stage fibrosis caused by bleomycin A5.Its method is to collect The medulla mesenchyma sample stem cell of Wistar male juvenile mouse, bone marrow mononuclear cells simultaneously carry out DAPI label respectively.21 Wistar female rats intratracheally inject bleomycin A5 production model of lung injury, are divided into medulla mesenchyma sample stem cell at random Treatment group, bone marrow mononuclear cells treatment group and model group.The 7th day execution rat, takes pathologic sections observation after modeling Inflammation and fibrosis situation；DAPI is detected under fluorescence microscope marks cell；ELISA method detection lung homogenate hydroxyproline and The content of tumor necrosis factor.Its result and conclusion are, 1. in medulla mesenchyma sample stem-cell therapy group, bone marrow mononuclear cells The foreign cell of DAPI label is seen in treatment group's lung tissue frozen section.2. model group pulmonary alveolitis most serious is filled between marrow Matter sample stem-cell therapy group pulmonary alveolitis is most light, and each group compares, and difference has significant (P < 0.05).3. model group lung tissue is even Hydroxyproline and tumor necrosis factor α content are most in slurry, and medulla mesenchyma sample stem-cell therapy is minimum, and each group comparing difference has Significant (P < 0.05).Prompt medulla mesenchyma sample stem cell and bone marrow mononuclear cells can be colonized in damage lung group It knits, and can effectively prevent the alveolar inflammation and early stage fibrosis of bleomycin A5 induction, the former acts on more significant.

Wang Hongyang document (Wang Hongyang, etc. human cord blood stem cell inhibition Pulmonary Fibrosis in Rats and pulmonary macrophage TGF-β 1 Expression, cell and molecular immunology magazine, 01 phase in 2013) research Umbilical cord blood mesenchymal stem cells are to caused by bleomycin The influence of Pulmonary Fibrosis in Rats and TGF-β 1.Its method be take cleaning grade health male SD rat 60 be only randomly divided into it is rich come it is mould Plain group (P group), stem-cell therapy group (M group), dexamethasone in treatment group (D group) and negative control group (N group), take 2nd generation people's navel The culture of blood mescenchymal stem cell is to the 4th generation；Transtracheal Injecting Bleomycin after Retaining manufactures pulmonary fibrosis model respectively for P group, M group and D group, Stem cell after M group modeling immediately through bromo- 2- deoxyuridine (BrdU) label of caudal vein injection 5-, after D group modeling Start within 2nd day continuous 7d intraperitoneal injection dexamethasone, N group transtracheal injects same amount of normal saline, puts to death at the 7th, 14,28 day each Group rat, row HE, Masson dyeing, immunohistochemical method observe the expression of TGF-β 1 and label cell situation.As a result, M The stem cell of the 7th, 14, the 28 day equal witness marking of lung tissue of group；It is observed after HE and Masson dyeing, compared with N group, when P group 7d Pulmonary alveolitis is most obvious, and 28d pulmonary fibrosis degree is most heavy, and M, D group are light compared with P group and the visible M group of pathological section is compared with D group pulmonary alveolitis and fibre Dimensionization degree is slightly light；The highest in P group 7d of TGF-β 1 in lung tissue, M, D group are considerably less than P group, and the reduction of M group becomes apparent from, between group Difference is statistically significant.Its conclusion is that Umbilical cord blood mesenchymal stem cells can be colonized in lung tissue, in pulmonary fibrosis at an early stage By inhibiting the expression of TGF-β 1, pulmonary alveolitis and pulmonary fibrosis may be mitigated.

Chen Kan document (Chen Kan, Bone Marrow Mesenchymal Stem Cells Transplantation combine the research of pirfenidone treatment pulmonary interstitial fibrosis, The practical medicine of China, 01 phase in 2016) it inquires into pirfenidone (PFD) joint mesenchymal stem cell (MSCs) and is implanted into win The curative effect of mycin (BLM) inducing lung fibrosis mouse.Its method is 36 healthy cleaning grade weight 200g male SD rats, with Machine is divided into 6 groups, and A group is negative control group, B group positive controls, the simple steroid group of C group, the simple MSCs transplantation group of D group, E The simple PFD treatment group of group, F group PFD joint MSCs transplantation group, each 6.SD rats in vitro culture MSCs, injection BLM prepare lung fibre Dimensionization model, observation lung morphology variation, measures the protein expression of transforming growth factor (TGF-β 1).As a result, making After mould 7d, there is the reduction of inflammatory cell infiltration ratio B group in C, D, E, F group rat, and pulmonary fibrosis degree is substantially reduced, and F group is compared with C, D, E The pathological change of group is lighter, and pulmonary fibrosis is unobvious.The decline of B group is obvious in paragonimus cyst, statistically significant with A group comparing difference (P<0.05).B, C, D, E, F group pulmonary fibrosis scoring is higher than A group, and difference is statistically significant (P < 0.05).It is converted in lung tissue The positive cell number of growth factor collagen expression significantly reduces, and F group is reduced compared with B, C, D, E group quantity and remitted its fury is brighter Aobvious, for A group compared with B, C, D, E, F group, difference is statistically significant (P<0.05), C, D, E group difference it is not statistically significant (P> 0.05).Its conclusion is that Bone Marrow Mesenchymal Stem Cells Transplantation joint pirfenidone has certain inhibiting effect, energy to pulmonary fibrosis The expression for enough reducing lung tissue transforming growth factor, there is reparation and palingenesis, and treatment pulmonary fibrosis effect is obvious.

Once saved all document (once saved all, etc., Bone Marrow Mesenchymal Stem Cells Transplantation treats the experimental study of Pulmonary Fibrosis in Rats, Gannan Medical College's journal, 01 phase in 2016) Bone Marrow Mesenchymal Stem Cells Transplantation is inquired into the induced lung fiber of bleomycin induced Change influence and its mechanism of model pulmonary alveolitis and pulmonary fibrosis.Its method is filled between in-vitro separation, culture SD rat marrow Matter stem cell reaches forth generation for testing；60 SD rats are randomly divided into negative control group (N group), positive controls (P Group), dexamethasone in treatment group (D group) and mesenchymal stem cell transplantation group (M group), every group of each 15 rat.P group, D group and Transtracheal injection bleomycin prepares pulmonary fibrosis model to M group respectively.7 after modeling, lung tissue is left and taken within 14,21 days, HE dyeing is seen Examine the degree of pulmonary alveolitis and pulmonary fibrosis, the table of method measurement lung tissue transforminggrowthfactor-β1 (TGF-β 1) of immunohistochemistry It reaches.As a result, taking lung tissue row HE to dye after bleomycin modeling, pathology meets lung fiber；After treatment 7,14 and 21 It, compared with P group, there is pneumonia in D, M group rat and pulmonary fibrosis degree is substantially reduced, while conversion is expressed in lung tissue of rats The positive cell number of grouth factor beta 1 (TGF-β 1) significantly reduces (P < 0.05)；And D, M group in the 7th, 14,21 day pathological examination and In the comparison of transforming growth factor expression, group difference is without conspicuousness.Its conclusion is that Bone Marrow Mesenchymal Stem Cells Transplantation is to rich Lay The pulmonary alveolitis and pulmonary fibrosis degree of mycin induced rat pulmonary fibrosis model have certain inhibiting effect, and mechanism of action may It is related with the expression that it reduces lung tissue transforming growth factor (TGF-β 1).

Liu Chen document (Liu Chen, etc., Umbilical cord blood mesenchymal stem cells prevent pulmonary fibrosis induced by bleomycin in rats effect, in State's occupational medicine, 03 phase in 2013) Umbilical cord blood mesenchymal stem cells (HUCBMSC) is inquired into induced lung fiber caused by bleomycin The Primary preventive intervention effect and mechanism of change.Its method is that 2nd generation HUCBMSC is taken to cultivate to the 4th generation；Take specific pathogen free 5 week old male SD rat 120 of body grade is only randomly divided into bleomycin group, stem cell intervention group, dexamethasone intervention group and feminine gender Control group, every group 30.First 3 groups respectively transtracheal Injecting Bleomycin after Retaining establish pulmonary fibrosis model, stem cell intervention group modeling By the HUCBMSC of bromo- 2- deoxyuridine (Brdu) label of caudal vein injection 5-, after dexamethasone intervention group modeling Start within 1st day continuous intraperitoneal injection dexamethasone 7d, negative control group transtracheal injects isometric physiological sodium chloride solution, the 7,10 animals in 14, the 28 dead each groups in natural gift other places, lung tissue row hematoxylin-eosin, horse pine trichrome stain, alkali hydrolysis method are surveyed Determine the level of lung hydroxyproline.As a result, the visible Brdu label of the 7th, 14,28 day lung tissue of stem cell intervention group is dry thin Born of the same parents.The bleomycin group lung hydroxyproline level at 3 time points is in the trend that gradually rises, the 28th day up to highest level (P < 0.01).The alveolar inflammation and pulmonary fibrosis degree of 3 time point stem cell intervention groups and dexamethasone intervention group are relatively rich respectively Bleomycin group is light, and difference is statistically significant (P < 0.05).Its conclusion is that HUCBMSC can be colonized in impaired lung tissue In, and may effectively mitigate pulmonary alveolitis and pulmonary fibrosis in pulmonary fibrosis at an early stage.

Wang Xian document (Wang Xian, etc., the mechanism of action of mesenchymal stem cell inhibition Pulmonary Fibrosis in Rats, Central China science and technology College journal (medicine), 03 phase in 2014) mesenchymal stem cell (MSCs) is observed to Pulmonary Fibrosis in Rats inhibition work Mechanism.Its method is in-vitro separation, culture, the bone marrow MSCs for purifying 4 week old SD rats.SD experimental rat is randomly divided into 4 Group (every group 12): Normal group (intratracheal saline injection), model group (intratracheally injecting bleomycin), MSCs are controlled Treatment early stage group (giving tail vein injection MSCs after modeling immediately), (14d gives tail vein injection to MSCs treatment advanced stage group after modeling MSCs), intratracheally injection bleomycin dosage is 5mg/kg, and Normal group injects isometric physiological saline, tail vein injection MSCs dosage is 1.0 × 10^6/mL DMEM culture solution 1mL.Lung tissue, lung tissue disease were taken after unified execution rat in the 28th day Manage slice row hematoxylin-eosin (HE) dyeing and Masson dyeing observation lung inflammation and fibrosis situation, Western blot Method detects lung tissue transforming growth factor-beta 1 (TGF-β 1), MMP-2 (MMP-2), matrix metalloproteinase group Knit inhibitor -1 (TIMP-1) expressing quantity.As a result, being 1. successfully separated culture MSCs and identifying.2. model group pulmonary alveolitis It is obviously aggravated with pulmonary fibrosis degree compared with Normal group, MSCs treatment early stage group pulmonary alveolitis and pulmonary fibrosis degree are compared with model group Significant to mitigate, MSCs treats advanced stage group pulmonary alveolitis and pulmonary fibrosis degree and model group comparing difference is not statistically significant.3. mould Type group TGF-β 1, TIMP-1 protein expression are dramatically increased compared with Normal group, MSCs treatment group TGF-β 1, TIMP-1 protein expression It is substantially reduced compared with model group, MMP-2 protein expression no significant difference between each group rat.Its conclusion is bone marrow MSCs It can inhibit the pulmonary fibrosis of bleomycin induced, and give MSCs in injury of lungs early stage and intervene that curative effect is better, mechanism can It can be the expression for reducing by 1 albumen of TGF-β, adjust the balance between MMP-2 and TIMP-1.

Cui's good jade document (Cui's good jade, etc., the influence that mesenchymal stem cell forms pulmonary fibrosis induced by bleomycin in rats, Chinese tuberculosis and breathing magazine, 09 phase in 2007) by observing mesenchymal stem cell (MSC) to bleomycin cause induced lung The new method for the treatment of pulmonary fibrosis is inquired into the influence of fibrosis animal model.Its method is that in-vitro separation cultivates male 6 week old The marrow MSC of SD rat.48 female sd inbred rats are randomly divided into 6 groups, the 1st~5 group of transtracheal injection 5.0mg/kg is rich next mould Plain 0.3ml is injected the 1st day and the 7th day for the 1st and the 3rd group respectively at bleomycin and is injected male rat MSC liquid through tail vein 0.2ml (cell number is 2.5 × 10^6)；It injects the 1st day and the 7th day respectively at bleomycin for 2nd and the 4th group and is infused through tail vein Enter the phosphate buffer 0.2ml of equivalent；5th group is used as model positive control, does not give other processing after injecting bleomycin；The 6 groups are used as model negative control, and transtracheal injects same amount of normal saline 0.3ml and replaces bleomycin, do not give other processing.In The 28th day unified execution rat is tested, lung tissue row pathological examination, hydroxyproline content measurement are left and taken；Extract lung tissue DNA detects the sex determining gene (sry gene) of male mouse, by polymerase chain reaction (PCR)-agarose electrophoresis with judgement Whether the MSC that external source is given exists in female rats lung tissue.As a result, causing injury of lungs the 1st day and the 7th in bleomycin It is given the lungs pathological change after MSC therapeutic intervention and mitigates compared with control group, and the scoring of pulmonary fibrosis degree is respectively 1.0 ± 0.2 point, 2.5 ± 0.5 points and 1.6 ± 0.5 points, 2.3 ± 0.8 points；Lung tissue hydroxyproline content is respectively (83 ± 17) μ g/ Mg, (96 ± 20) μ g/mg and (123 ± 32) μ g/mg, (127 ± 34) μ g/mg, at bleomycin cause injury of lungs the 1st day compared with the 7th It is given MSC its effect and becomes apparent from.PCR testing result is shown, gives within the 1st day the rat of MSC group in bleomycin cause injury of lungs Lung tissue can detecte sry gene.Its conclusion be the MSC that external source is given can mitigate pulmonary fibrosis be formed in injury of lungs morning Phase gives MSC intervention, and curative effect is better.

Liu Chen document (Liu Chen, etc., influence of the human cord blood stem cell to lung fibrosis in rats TNF-α, NO, Chinese industrial Medical journal, 06 phase in 2012) probe into influence of the Umbilical cord blood mesenchymal stem cells to Pulmonary Fibrosis in Rats and TNF-α, NO.Its side Method is that cleaning grade health male SD rat 35 is taken only to be randomly divided into 15 bleomycin group 15 (P group), stem-cell therapy group (M Group) and negative control group 5 (N group), take the culture of second generation Umbilical cord blood mesenchymal stem cells to forth generation；P group, M group pass through respectively Tracheae Injecting Bleomycin after Retaining manufactures pulmonary fibrosis model, and N group injects the physiological saline of equivalent, quiet through rat-tail immediately after M group modeling Arteries and veins injects the stem cell of bromo- 2- deoxyuridine (Brdu) label of 5-.N group rat is all put to death on the 7th day after modeling, P Group and M group rat the 7th, 14,28 natural gift other places after modeling are dead.Lung tissue row HE, Masson is taken to dye, ELISA method detection lung Steep the expression of TNF-α in irrigating solution, NO.As a result, the stem cell of the 7th, 14, the 28 day equal witness marking of lung tissue of M group； It is observed after HE and Masson dyeing, compared with N group, pulmonary alveolitis is most obvious when P group 7d, and 28d pulmonary fibrosis degree is most heavy, and M group is compared with P Group pulmonary alveolitis and degree of fibrosis mitigate；Compared with N group, the TNF-α of P group rat, NO are horizontal significantly raised, in 7d Shi Dagao Peak, M group TNF-α, NO level are considerably less than P group in each period, and group difference is statistically significant.Its conclusion is people's navel Blood mescenchymal stem cell can be colonized in lung tissue, can effectively mitigate pulmonary alveolitis and pulmonary fibrosis in pulmonary fibrosis at an early stage；Suppression The expression of TNF-α, NO processed may be its mechanism of action.

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, placenta mesenchyma stem cell treatment SSc

2/3 or more SSc patient has pulmonary involvement, is that this disease is main dead former the most common are pulmonary interstitial fibrosis Cause.Therefore, the animal model using the pulmonary interstitial fibrosis mouse model that classical bleomycin induces as preclinical study is to examine Examine the validity of placenta mesenchyma stem cell treatment SSc.

With reference to Su Minhong document (Su Minhong, etc..The long-term steady of bleomycin inducing mouse pulmonary fibrosis model is injected intraperitoneally It is qualitative.Chinese Tissue Engineering Study, 2017, the 4th phase of volume 21,512-519 was tested).Female C57BL/6 is small within 6-8 weeks Mouse is dissolved in 200 μ L physiological saline with bleomycin 35mg/kg, intraperitoneal injection, and 2 times/week, continuous injection 8 times.8th abdominal cavity note After penetrating, pulmonary alveolitis, pulmonary fibrosis scoring started to gradually rise in 2 weeks, reached peak in 6-8 weeks, continued to 10 weeks still without obvious drop It is low, thus complete bleomycin pulmonary fibrosis modeling.

In test, by 6-8 weeks female C57BL/6 mouse, it is divided into 4 groups at random, is respectively as follows: the 1st group-control group (n= 20): do not give the intact animal of bleomycin, simulation modelling, through intraperitoneal injection of saline, 2 times a week, totally 8 times；2nd group- Pulmonary fibrosis model group (n=20): bleomycin pulmonary fibrosis models animal；3rd group-placenta MSC treatment group a (n=20): rich Bleomycin pulmonary fibrosis model successfully after i.e. since for the first time inject bleomycin after 10 weeks, tail vein injection Human plactnta MSC system Agent a, 2.5 × 10^5 MSC cell every time, 1 times a week, totally 2 times；4th group-placenta MSC treatment group b (n=20): bleomycin Pulmonary fibrosis model successfully after i.e. since for the first time inject bleomycin after 10 weeks, tail vein injection Human plactnta MSC preparation b, often Secondary 2.5 × 10^5 MSC cell, 1 times a week, totally 2 times.1st group and the 2nd group vein note while giving MSC for other two groups Penetrate isometric physiological saline.

In this experiment, placenta mesenchyma stem cell ejection preparation is used to prepare in this way: by the step of embodiment 2 Rapid 2 cell passage gained mescenchymal stem cell (this example uses PS162279 sample P4 generation) is transferred in centrifuge tube, 1500rpm from Heart 5min abandons supernatant, and 0.9% sodium chloride solution is added and is resuspended, and it is that 1~3 × 10^6 placenta mesenchyma is dry that cell concentration, which is made, Cell/ml cell preparation；Wherein, citrate of magnesia and phosphatide are additionally added in 0.9% sodium chloride solution (keeps magnesium ion dense Degree reaches 2.5mmol/L, phospholipid concentration 0.2mg/ml, and phosphatide is injection stage soybean-source, adds citrate of magnesia and phosphatide group It is labeled as PD-MSC group a, i.e., 3rd group above-mentioned) or citrate of magnesia is not added and phosphatide (is not added citrate of magnesia and phosphatide group echo is PD-MSC group b, i.e., 4th group above-mentioned).

7 days, the 14 days and 28 days execution each group mouse after second of tail vein injection placenta MSC is injected, every group 6, into Row detection.

(1) lung pathology changes

7 days after transplanting, 14 days, 28 days, put to death each group mouse (every group 6), by mouse lung tissue 10% neutrality Fixed in formalin acetaldehyde dehydration, paraffin embedding, histotomy is with a thickness of 5um, hematoxylin eosin staining for 24 hours, under microscope Observe alveolar structure.As the result is shown: the lung tissue of the 1st group of animal is in normal condition；2nd group the 7th day, 14 days and 28 days, it is small Mouse lung tissue has apparent cell infiltration, fibroblast focus, normal alveolar architecture deformation；3rd group of tail vein injection MSC 7 days afterwards, 14 days and 28 days, inflammatory cell infiltration is obviously few compared with the 2nd group, the basic maintains normal condition of alveolar structure；4th group of tail is quiet Arteries and veins is injected after MSC 7 days, 14 days and 28 days, than the 2nd group few but no significant difference of inflammatory cell infiltration, and obviously ratio more than the 3rd group, Alveolar structure has been shown in deformation.

(2) ELISA detects IL-1, IL-6, TNF-α and TGF-β protein level in lung tissue

Mouse lung tissue is freezed, through cracking containing protease inhibitors lysis buffer, supernatant is collected in centrifugation.ELISA It detects in mouse lung tissue lysate, the content of IL-1, IL-6, TNF-α and TGF-β albumen (cell in Unit Weight lung tissue The weight of the factor).Elisa plate is detected at 450nm, and the concentration readings of above-mentioned albumen are calculated according to standard curve.Knot Fruit shows: compared with healthy mice, MSC injection after 14 days, bleomycin cause cell factor in lung tissue (IL-1, IL-6, TNF-α, TGF-β) expression dramatically increases, these cell factors can transmitting inflammation and fibrosis (p < 0.01), and give MSC's 3rd group considerably lower (p < 0.01 or p < 0.05) relative to the 2nd group of these factor expression amounts.Concrete outcome see the table below:

Group	1st group	2nd group	3rd group	4th group
					IL-1(pg/ug)	93.2±9.3	217.2±17.2**	137.4±14.1##	185.2±16.3#
IL-6(pg/ug)	827.8±58.4	2186±141.3**	1262.6±91.3##	1764.7±137.2#
					TNF-α(pg/ug)	687.3±46.1	1732.7±143.2**	987.3±76.4##	1647.6±127.4
TGF-β(pg/mg)	7.13±1.02	23.57±2.14**	13.57±1.24##	17.83±2.02#

In table, compared with the 1st group, the 2nd group of p < 0.01st * p < 0.05, * *；Compared with the 2nd group, the 3rd group and the 4th group of #p < 0.05, ##p < 0.01.

(3) mouse lung tissue collagen content is measured

Hydroxyproline (hydroxyproline, HYP) is a kind of nonessential amino acid, be collagen tissue main component it One, and be distinctive amino acid in collagen.Pass through the measurement to lung tissue hydroxyproline (Hydroxyproline) content, assessment The variation of mouse lung tissue collagen content.Freezing mouse lung tissue is taken, is hydrolyzed in HCl, at 558nm, detects each group sample Absorbance and calculate hydroxyproline content.As the result is shown: the pulmonary fibrosis of bleomycin induced had 7 days, 14 days, 28 days Significant collagen deposition, hydroxyproline (HYP) content obviously increase.Giving the 3rd group of placenta MSC has the significant collagen that weakens to sink Long-pending effect (p < 0.01).Concrete outcome see the table below:

Measure day	1st group	2nd group	3rd group	4th group
					7 days	1.89±0.43	2.42±0.63*	1.92±0.51##	2.29±0.81
14 days	1.96±0.38	2.68±0.84*	1.97±0.63##	2.46±0.76#
					28 days	1.84±0.42	3.37±0.71*	2.21±0.72##	2.84±0.69#

(4) matrix metal proteinase activity is assessed

The activity of freezing lung tissue matrix metalloproteinase is quantitatively evaluated by density measurement and gelatinase activity.Albumen Matter content (15mg/ sample) is loaded into containing on 7.5% acrylamide and the gel of 1mg/ml gelatin.And assess MMP-2, The variation of MMP-9 and MMP-13.As the result is shown: using densitometry quantitative calibration, the various enzymatic activitys of control group mice are set as 1, calculate the various enzyme sea property values of each group.Concrete outcome see the table below:

Enzymatic activity	1st group	2nd group	3rd group	4th group
					MMP-2	1	1.07±0.13	1.71±0.21##	1.24±0.15
MMP-9	1	1.11±0.19	2.13±0.15##	1.63±0.18#
					MMP-13	1	1.28±0.08	1.87±0.11##	1.51±0.11#

In table, compared with the 2nd group, the 3rd group and the 4th group of #p < 0.05, ##p < 0.01.

Above results demonstrate that cell preparation of the present invention is used for the validity of SSc, and result also shows PD-MSC group a's Effect is substantially better than PD-MSC group b result.

The foundation of embodiment 5, placenta stem-cell library

1, the number for freezing front and back living cells the detection of cell activity: is counted using trypan blue staining.

2, the detection of cell contamination: utilize a small amount of cell culture, detection cell whether the pollution by fungi and bacterium.Benefit With aetology method, detect 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: detection cell HLA-ABC/DR phenotype, and place on record.

5, the investigation of cell origin: the particulars of record fetus and its parent, and place on record.

6, the foundation of placenta stem-cell database: after saving normal placenta stem-cell, the number of placenta stem-cell is established According to library, including the first six data, and establishes and to be associated with freeze-stored cell.

Claims

1. a kind of cell preparation preparation for treat and/or the drug of prevention system hardening illness in purposes；The cell Preparation is configured to by being suspended in mescenchymal stem cell (such as placenta mesenchyma stem cell) in 0.9% sodium chloride solution Cell suspension.

2. purposes according to claim 1, wherein cell concentration is 1~10 × 10 in the cell preparation⁶A cell/ml, such as 1~5 × 10⁶A cell/ml, such as 1~3 × 10⁶A cell/ml.

3. purposes according to claim 1, wherein also adding citrate of magnesia in 0.9% sodium chloride solution described in the cell preparation And phosphatide；For example, the amount of addition citrate of magnesia is to make magnesium ion concentration 2.5mmol/L；For example, the concentration of addition phosphatide is 0.2mg/ml；For example, the phosphatide is injection stage soybean-source；For example, the cell preparation is by being made including method : cell is passed on into gained mescenchymal stem cell and is transferred in centrifuge tube, is centrifuged, abandons supernatant, 0.9% sodium chloride solution weight is added It is outstanding, cell preparation is made.

4. purposes according to claim 1, wherein mescenchymal stem cell described in the cell preparation is by including the following steps Method be 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, 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. purposes according to claim 1, wherein the cell preparation is 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；

(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.

6. purposes according to claim 1, it is characterised in that:

Gained is respectively greater than 90% for the cell purity of placenta mesenchyma stem cell；For example, the placenta mesenchyma stem cell is through 3 generations After the above passage, cell purity is greater than 95%；

The Hank's balanced salt solution composition are as follows: the MgSO47H2O 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 of MgCl26H2O, 0.06g/L of 0.1g/L, The phenol red of NaHCO3,0.2g/L of CaCl2,0.35g/L of 0.14g/L, hydrochloric acid or sodium hydroxide adjust pH to 7.4；

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.

7. a kind of cell preparation is by making mescenchymal stem cell (such as placenta mesenchyma stem cell) be suspended in 0.9% chlorine Change the cell suspension being configured in sodium solution.

8. cell preparation according to claim 7, wherein cell concentration is 1~10 × 10⁶A cell/ml, such as 1~5 × 10⁶ A cell/ml, such as 1~3 × 10⁶A cell/ml；Such as citrate of magnesia and phosphorus are also added in 0.9% sodium chloride solution Rouge；For example, the amount of addition citrate of magnesia is to make magnesium ion concentration 2.5mmol/L；For example, the concentration of addition phosphatide is 0.2mg/ ml；For example, the phosphatide is injection stage soybean-source；For example, it is as including made from method: will be obtained by cell passage Mescenchymal stem cell is 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.

9. cell preparation according to claim 7, wherein the mescenchymal stem cell is the method system by including the following steps For what is obtained:

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

10. cell preparation according to claim 7, it is characterised in that:

During preparing mescenchymal stem cell, further includes: (7) for placenta mesenchyma stem cell obtained by step (6), detection At least one of following items: cell activity, cell contamination, hereditary disease, HLA-ABC/DR distribution type；(8) it will be passed obtained by step (6) Each being frozen in liquid nitrogen for placenta mesenchyma stem cell after generation；And/or (9) establish the placenta stem-cell comprising information above Database, and be associated the database and the freeze-stored cell of step (8)；

Gained is respectively greater than 90% for the cell purity of placenta mesenchyma stem cell；

For the placenta mesenchyma stem cell after 3 more than generation pass on, cell purity is greater than 95%；

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