CN109646458B

CN109646458B - Method for treating scleropathy using placental mesenchymal stem cell preparation

Info

Publication number: CN109646458B
Application number: CN201811568533.4A
Authority: CN
Inventors: 许晓椿; 李容; 肖海蓉; 刘冰
Original assignee: BOYALIFE Inc
Current assignee: BOYALIFE Inc
Priority date: 2018-12-21
Filing date: 2018-12-21
Publication date: 2021-02-23
Anticipated expiration: 2038-12-21
Also published as: CN109646458A

Abstract

The present invention relates to methods of treating scleroses using preparations of placental mesenchymal stem cells. In particular, one aspect of the invention relates to the use of a cell preparation in the manufacture of a medicament for the treatment and/or prevention of systemic sclerosis; the cell preparation is a cell suspension prepared by suspending mesenchymal stem cells such as placental mesenchymal stem cells in a 0.9% sodium chloride solution, and the cell concentration in the cell preparation is 1-10 × 10⁶Individual cells/ml. The invention also relates to a cell preparation for treating and/or preventing systemic sclerosis, a method for preparing the cell preparation, a method for preparing mesenchymal stem cells involved in the cell preparation, the mesenchymal stem cells, and a mixed enzyme digestive liquid used in the methods. The cell preparation prepared by the invention has excellent biological effect in treating scleroderma.

Description

Method for treating scleropathy using placental mesenchymal stem cell preparation

Technical Field

The invention belongs to the field of biotechnology and biomedicine, and relates to a method for treating scleroderma by using a placenta mesenchymal stem cell preparation, and the placenta mesenchymal stem cell preparation used in the method. In particular, the present invention relates to a method for isolating stem cells from a placenta, particularly to a method for isolating mesenchymal stem cells from a placenta, and more particularly to a method for isolating mesenchymal stem cells from a placenta tissue and culturing the mesenchymal stem cells using a digestive enzyme composition having a unique formulation according to the present invention. The method can effectively improve the efficiency of separating the mesenchymal stem cells from the placenta. Furthermore, the invention relates to a preparation of the cell by using the placenta mesenchymal stem cell, and further relates to a treatment of systemic sclerosis by using the cell preparation.

Background

Systemic sclerosis (SSc), also known as scleroderma (scleroderma), is a clinically characterized by localized or diffuse skin thickening and fibrosis, of unknown cause and can also affect autoimmune diseases of the heart, lungs and digestive tract. The disease is distributed globally, the prevalence rate is 50-300/100 ten thousand, the annual incidence rate is 2.3-22.8/100 ten thousand, the peak age of the disease is 30-50 years, the female is common, the proportion of male to female is 1: 3 to 14.

SSc can be diagnosed based on Raynaud's phenomenon, skin fibrosis, specific visceral involvement, and specific antinuclear antibodies (anti-Scl-70 antibody and ACA). According to the skin affected, there are diffuse cutaneous sclerosis (dcSSc) and localized cutaneous sclerosis (lcSSc). Diffuse skin fibrosis can affect the far end and the near end of limbs, the face and the neck, the chest and the abdomen, the disease condition is fast in progress, visceral lesions are often accompanied, the prognosis is poor, and the survival rate in 10 years is about 50%. Localized skin lesions are localized to the distal end of the elbow (knee), and may be affected on the face and neck, progressing slowly. More than 2/3 of SSc patients have lung involvement, most commonly pulmonary interstitial fibrosis, which is the main cause of death.

At present, no specific medicine exists for treating systemic sclerosis. Primarily symptomatic treatment improves symptoms, glucocorticoids relieve acute skin edema but do not prevent skin fibrosis. Immunosuppressive agents can be used for the treatment of organ involvement, and cyclosporin A, cyclophosphamide, methotrexate, etc. are commonly used. The traditional anti-fibrotic treatment is D-penicillamine. The pain of muscles and joints can be treated by non-steroidal anti-inflammatory drugs. Diffuse type is easy to cause death due to the involvement of lung, kidney and heart, and has poor prognosis.

Stem cell-based therapies have brought new treatment options for patients with SSc. In recent years, some studies have used autologous Hematopoietic Stem Cell (HSC) transplantation to treat severe disseminated SSc, which has prolonged survival time, reduced skin involvement, and achieved certain therapeutic effects compared to CYC treatment. However, autologous HSCT has the disadvantages of high cost and high recurrence rate, and allogeneic HSC transplantation has high mortality rate due to rejection and other reasons.

Research shows that bone marrow Mesenchymal Stem Cells (MSCs) of SSc patients have defects in growth, hematopoietic support, cytokine secretion and the like, and the abnormality of the MSCs is presumed to play an important role in the onset of SSc. MSCs exhibit good hand and face treatment in SSc patients, can improve skin sclerosis, finger and mouth movement, and reduce hand and face pain. Mesenchymal Stem Cells (MSCs) with low immunogenicity and multipotentiality are contained in placenta tissue of a newborn. Placental MSCs have an immunomodulatory effect, inhibitory effect on T cells and NK cells, and play a key role in suppressing immune responses, mediating tolerance, and causing long-term immune escape. Particularly, the placenta MSC has low immunogenicity, and the allogeneic transplantation does not cause immunological rejection. Antigen-independent, direct inhibition of activated T cell proliferation in a dose-dependent manner. These features make it possible to apply placental MSCs to autoimmune diseases.

Mesenchymal Stem Cells (MSCs), such as human mesenchymal stem cells, were first isolated from bone marrow and a class of tissue stem cells derived from the mesoderm, which have multipotent differentiation potential and self-renewal capacity, have the ability to differentiate into various adult cells, such as osteoblasts, chondrocytes, adipocytes, endothelial cells, nerve cells, muscle cells, hepatocytes and the like, under specific conditions in vivo and in vitro (Cap AI. mesenchymal stem cells. J Orthop Res.1991,9:641-650.Pittenger MF, Mackay AM, Beck, et al. multilineage patent sensory of epithelial man mesenchymal cells. science.1999; 284: 143-147). Recent research shows that the mesenchymal stem cells have the functions of immunoregulation and hematopoietic support, and are easy to introduce and express exogenous genes. Therefore, the mesenchymal stem cells are not only seed cells in the construction of tissue engineering bone, cartilage and cardiac muscle and important carrier cells in gene therapy, but also have wide application prospect in hematopoietic stem cell transplantation and organ transplantation because the mesenchymal stem cells promote hematopoietic reconstruction and inhibit graft-versus-host reaction. Mesenchymal stem cells have the characteristic of adherent growth in vitro, and by utilizing the characteristic, the mesenchymal stem cells are successfully separated and cultured from various tissues such as liver, kidney, pancreas, muscle, cartilage, skin, peripheral blood and the like.

At present, the reported mesenchymal stem cells are mainly derived from bone marrow and are obtained by adopting a density gradient centrifugation method. Although the separation method is simple, the donor needs to undergo a painful operation for taking marrow, and has a high infection chance in and after the material is taken; because the content of MSC in human bone marrow is very rare, every 10⁵～10⁶Only about 1 of the mononuclear cells are used, and the number, proliferation and differentiation capacity of the mesenchymal stem cells in the bone marrow are remarkably reduced along with the increase of the age, so that the mesenchymal stem cells are subjected to the reductionHave limitations in research and applications, particularly clinical applications. The placenta, which originates from the extraembryonic mesoderm during the embryonic development, is composed of mesenchyme, blood vessels and trophoblasts, and contains a large amount of mesenchyme components. Recent research shows that the placenta contains abundant stem cells, and the separation and culture of the pluripotent stem cells from the placenta opens up a brand-new and abundant source for experimental research and clinical application.

The existing methods for establishing a placental stem cell bank by isolating stem cells from placenta have many disadvantages, such as insufficient purity and/or low quantity, and thus show that these methods are not satisfactory. For example, CN101270349A (chinese patent application No. 200810061267.6, published 2008/9/24) discloses an invention entitled "placental mesenchymal stem cell isolation and in vitro expansion culture method"; CN101693884A (chinese patent application No. 200910117522.9, published 2010, 4 months and 14 days) entitled "a method for separating and extracting stem cells from placenta, umbilical cord or adipose tissue"; CN102146359A (chinese patent application No. 201110005964.1, published 2011/8/10) discloses an invention entitled "method for extracting original mesenchymal stem cells from placenta and serum-free expansion". In addition, chinese patent application No. 201210044648X discloses a method of isolating mesenchymal stem cells from placenta. These processes are to be further improved in terms of purity and/or recovery of the extract. In addition, an invention publication CN107299082A (chinese patent application No. 201710653583.1, published 2018, 10 months and 27 days) by the present inventor team describes a method of obtaining placental mesenchymal stem cells that has been shown to exhibit some excellent properties.

There remains a need in the art for new methods for isolating stem cells from placenta, and in particular for methods that efficiently isolate mesenchymal stem cells from placenta. In addition, there remains a need in the art for new digestive enzyme compositions for use in methods of isolating mesenchymal stem cells from placenta in an effort to improve the efficiency of the methods of isolating mesenchymal stem cells from placenta. In addition, there remains a need in the art for new methods for treating systemic sclerosis, and in particular, new and more effective methods for treating systemic sclerosis using mesenchymal stem cells are expected.

Disclosure of Invention

Objects of the invention include one or more of the following: on one hand, the method solves the defects of the existing method for obtaining the placenta mesenchymal stem cells, provides a practical, simple and efficient method for separating the mesenchymal stem cells from the placenta tissues and culturing the mesenchymal stem cells into the mesenchymal stem cells and optionally establishing a placenta stem cell bank; in another aspect, a digestive enzyme composition is provided for the above method of isolating mesenchymal stem cells from placental tissue and culturing into mesenchymal stem cells; in yet another aspect, a cell preparation is provided for treating systemic sclerosis using mesenchymal stem cells. The present inventors have found that by using a specific procedure and a specific formulation of the digestive enzyme composition, a high cell purity and/or a high cell recovery rate is obtained and that a specific cell preparation can be more effectively used for the treatment of systemic sclerosis by formulating it. The present invention has been completed based on such findings.

To this end, the invention provides in a first aspect the use of a cell preparation for the manufacture of a medicament for the treatment and/or prevention of systemic sclerosis.

The use according to the first aspect of the invention, wherein the cell preparation is a cell suspension formulated by suspending mesenchymal stem cells (e.g. placental mesenchymal stem cells) in a 0.9% sodium chloride solution.

The use according to the first aspect of the present invention, wherein the concentration of cells in the cell preparation is 1 to 10X 10⁶One cell/ml, e.g., 1-5X 10⁶Individual cells/ml, e.g. 1-3X 10⁶Individual cells/ml.

Use according to the first aspect of the invention, wherein said cell preparation further comprises magnesium citrate and phospholipids added to said 0.9% sodium chloride solution. In one embodiment, magnesium citrate is added in an amount to provide a magnesium ion concentration of 2.5 mmol/L. In one embodiment, the phospholipid is added at a concentration of 0.2 mg/ml. In one embodiment, the phospholipid is of injection grade soy origin. It has surprisingly been found that the biological effect of the cell preparation of the invention in the treatment of systemic sclerosis can be significantly improved by the simultaneous addition of small amounts of magnesium salts and phospholipids. The present inventors have also found that when only a magnesium salt, or only a phospholipid, or the above-mentioned magnesium salt is replaced with another salt such as a calcium salt, a zinc salt, a copper salt, or the like, is added to the cell preparation of example 4 of the present invention, the results are far inferior to those of the PD-MSC group a in which a magnesium salt and a phospholipid are added simultaneously, and the results in these cases are similar to those of the PD-MSC group b of example 4 of the present invention.

The use according to the first aspect of the invention, wherein the cell preparation is prepared by a method comprising: and transferring the mesenchymal stem cells obtained by cell passage to a centrifuge tube, centrifuging, discarding supernatant, adding 0.9% sodium chloride solution for resuspension, and thus obtaining the cell preparation.

The use according to the first aspect of the invention, wherein the cell preparation the mesenchymal stem cells are prepared by a method comprising the steps of:

(1) treatment of placental leaflets: placing placenta in a white porcelain dish, washing with tissue cleaning solution to remove blood stasis of placenta, shearing 20g of placenta lobule tissue in a steel cup, cleaning twice with the tissue cleaning solution, soaking for 5min, and weighing 15g of better tissue in a 100mm glass dish; adding 10ml tissue cleaning solution, and cutting leaflets to 0.2cm³About the size, 100ml of tissue cleaning fluid is added, the mixture is stirred evenly and filtered by a 300-mesh filter screen, and the operation is repeated to clean the tissue cleaning fluid twice so as to remove blood cells;

[ wherein the tissue cleansing solution is 0.9% physiological saline containing 1% double antibody ]

(2) Mixed enzyme digestion and termination: adding the cleaned leaflet tissue into 15-30 ml (such as 20-25 ml, such as 23ml) of mixed enzyme digestive juice preheated at 37 ℃, fully mixing uniformly, then digesting for 30min by shaking at the temperature of 37 ℃ and 100rpm by using a shaking table, and after digestion is finished, adding 2ml of FBS into the tissue juice to stop digestion;

[ wherein the mixed enzyme digestive juice contains: 15-30 volumes of Hank ' S balanced salt solution, 0.2-0.6 volumes of Liberase MNP-S enzyme, 0.2-2 volumes of DNA type I enzyme (e.g., 20-25 volumes of Hank ' S balanced salt solution, 0.3-0.5 volumes of Liberase MNP-S enzyme, 0.5-1 volumes of DNA type I enzyme, e.g., 22 volumes of Hank ' S balanced salt solution, 0.4 volumes of Liberase MNP-S enzyme, 0.7 volumes of DNA type I enzyme); the Liberase MNP-S enzyme is for example the Liberase MNP-S enzyme from Roche, for example from Siberian organisms, the cat # of which: 5578582001]

(3) Collecting primary cells: adding 50ml of tissue cleaning fluid into the tissue fluid obtained in the last step, uniformly mixing, filtering by a 300-mesh sieve, and collecting cell fluid; washing the digested tissue twice repeatedly, combining the filtrates of the two times into a centrifuge tube, and centrifuging at 1500rpm for 8min (acceleration 9 and deceleration 7); removing supernatant, adding appropriate amount of tissue washing solution, resuspending and supplementing to 200ml, centrifuging at 1500rpm for 8min (acceleration 9, deceleration 7); removing supernatant, adding DMEM-F12 into the cell sediment, resuspending the cell sediment to 30ml, filtering with a 100um filter screen, and then washing the filter screen with 10ml DMEM-F12 to obtain 40ml cell suspension as primary cells;

[ cell suspension of this primary cell can be subjected to cell counting using a sysmex hematology analyzer ]

(4) Freezing and storing primary cells: centrifuging the cell suspension at 1800rpm for 10min (acceleration 9 and deceleration 7), collecting cell precipitate and supernatant 5ml, re-suspending, slowly adding frozen stock solution 10ml, and shaking; subpackaging the obtained cell suspension into 9 freezing tubes of 2ml, each tube of 1.5ml, placing in a precooled program cooling box, performing program cooling by using a program cooling instrument, and transferring the cells into a liquid nitrogen storage tank for freezing;

[ wherein, the formula of the freezing solution is as follows: 65% DMEM-F12, 15% Human Serum Albumin (HSA), 20% DMSO, e.g. WAK brand DMSO ]

(5) Cell recovery: taking 2 tubes of frozen cells, quickly thawing at 37 ℃, transferring the cells to a 15ml centrifuge tube, and adding 8ml of complete culture base for resuscitating; centrifuging at 1200rpm for 5min (acceleration 9, deceleration 7), removing supernatant, and adding 5ml complete culture medium for resuspension; inoculating each tube of cells into 1T 75 culture bottle, supplementing complete culture medium to 30ml, and culturing in CO2 incubator (37 deg.C, 5% CO2, saturated humidity); performing total liquid change every 3-4 days with complete culture medium, recovering for 12 days, and counting according to clone formation condition until cell density is not less than 3000 cells/cm²The following passages can be performed;

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

(6) Cell passage: washing P0 generation cells with PBS, adding 2ml pancreatin for 2-5min until most of the cells fall off, adding 5ml complete culture medium to stop digestion, transferring the cells into a centrifuge tube, centrifuging at 1400rpm for 5min (acceleration 9, deceleration 7), discarding supernatant, adding 5ml complete culture medium for resuspension, counting, and inoculating to a culture bottle, wherein the cell density is 8000-12000 cells/cm²Culturing in a CO2 incubator (37 ℃, 5% CO2 and saturated humidity) until the cell density reaches over 90% (usually culturing for about 5 days), and completing cell passage from P0 generation to P1 generation; repeating the operations in sequence to perform cell passage from P1 generation to P2 generation, P2 generation to P3 generation, P3 generation to P4 generation and P4 generation to P5 generation respectively to obtain the mesenchymal stem cells of each generation.

The use according to the first aspect of the present invention, wherein the cell preparation further comprises, in the process of preparing mesenchymal stem cells:

(7) aiming at the placenta mesenchymal stem cells obtained in the step (6), detecting at least one item of the following items: cell viability, cell contamination, genetic disease, HLA-ABC/DR match.

(8) and (4) freezing and storing the passage-generated placenta mesenchymal stem cells obtained in the step (6) in liquid nitrogen.

(9) establishing a database of placental stem cells comprising the above information, and correlating the database with the cryopreserved cells of step (8).

The use according to the first aspect of the present invention, wherein the cell purity of the obtained placental mesenchymal stem cells of each generation is greater than 90%. In one embodiment, the placental mesenchymal stem cells have a cell purity of greater than 95% after more than 3 passages.

The use according to the first aspect of the invention wherein the Hank's balanced salt solution consists of: 8.0g/L NaCl, 0.4g/L KCl, 0.1g/L MgSO4 & 7H2O, 0.1g/L MgCl2 & 6H2O, 0.06g/L Na2HPO4 & 2H2O, 0.06g/L KH2PO4, 1.0g/L glucose, 0.14g/L CaCl2, 0.35 g/L3, 0.2g/L phenol red, hydrochloric acid or sodium hydroxide to adjust the pH to 7.4. The cell preparation according to the second aspect of the present invention, wherein the mixed enzyme digest further comprises 0.2-0.3 g/L zinc chloride in addition to Hank' S balanced salt solution, Liberase MNP-S enzyme, DNA type I enzyme. It has been surprisingly found that with the use of mixed enzyme digests with added zinc chloride in this concentration range, the resulting primary cells have greater than 60% CD73 expression, no CD45 expression, and mesenchymal stem cell content in the resulting primary cells of 60% -70%, showing extremely high stem cell concentrations; when the zinc chloride is not added into the mixed enzyme digestive juice, the content of the mesenchymal stem cells in the primary cells is less than 38 percent, and is usually within the range of 31-38 percent.

The use according to the first aspect of the invention, wherein the cell viability assay is counting of the number of viable cells before and after cryopreservation using trypan blue staining.

The use according to the first aspect of the present invention, wherein the cell contamination detection detects whether cells are contaminated with fungi and bacteria using a small amount of cell culture. In one embodiment, the cell contamination assay utilizes an etiological method to detect whether a cell is infected with one or more of the following: hepatitis B two-half, hepatitis C, AIDS virus, cytomegalovirus, EB virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, and TRUST.

The use according to the first aspect of the present invention, wherein the genetic disease detection is a method of molecular genetics for detecting the presence of a genetic disease in cryopreserved cells.

Use according to the first aspect of the invention, wherein said HLA-ABC/DR matching is for testing a cellular HLA-ABC/DR phenotype.

The use according to the first aspect of the present invention, wherein said placental mesenchymal stem cells are frozen in liquid nitrogen via a temperature-programmed process.

Use according to the first aspect of the invention wherein the database includes data relating to all of the cells stored, including but not limited to: the biological characteristic test result of the cell, the identification result of the multi-directional differentiation potential, the molecular genetic diagnosis result of the cell, and the detailed information of the fetus and the parents thereof.

Further, the present invention provides in a second aspect a method for isolating mesenchymal stem cells from placental tissue and culturing the mesenchymal stem cells into mesenchymal stem cells, the method comprising the steps of:

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

(6) Cell passage: washing P0 generation cells with PBS, adding 2ml pancreatin for 2-5min until most of cells fall off, adding 5ml complete culture medium to terminate digestion, transferring cells into a centrifuge tube, centrifuging at 1400rpm for 5min (acceleration 9, deceleration 7), discarding supernatant, adding 5ml complete culture medium for resuspension, counting, inoculatingTo a culture bottle, the cell density is 8000-12000 cells/cm²Culturing in a CO2 incubator (37 ℃, 5% CO2 and saturated humidity) until the cell density reaches over 90% (usually culturing for about 5 days), and completing cell passage from P0 generation to P1 generation; repeating the operations in sequence to perform cell passage from P1 generation to P2 generation, P2 generation to P3 generation, P3 generation to P4 generation and P4 generation to P5 generation respectively to obtain the mesenchymal stem cells of each generation.

The method according to any embodiment of the second aspect of the present invention, further comprising:

The method according to any embodiment of the second aspect of the present invention, wherein the cell purity of the obtained generations of placental mesenchymal stem cells is greater than 90%. In one embodiment, the placental mesenchymal stem cells have a cell purity of greater than 95% after more than 3 passages.

The method according to any embodiment of the second aspect of the present invention wherein said Hank's balanced salt solution consists of: 8.0g/L NaCl, 0.4g/L KCl, 0.1g/L MgSO4 & 7H2O, 0.1g/L MgCl2 & 6H2O, 0.06g/L Na2HPO4 & 2H2O, 0.06g/L KH2PO4, 1.0g/L glucose, 0.14g/L CaCl2, 0.35 g/L3, 0.2g/L phenol red, hydrochloric acid or sodium hydroxide to adjust the pH to 7.4. The method according to any embodiment of the second aspect of the present invention, wherein the mixed enzyme digestive juice contains Hank' S balanced salt solution, Liberase MNP-S enzyme, DNA I type enzyme, and 0.2-0.3 g/L zinc chloride. It has been surprisingly found that with the use of mixed enzyme digests with added zinc chloride in this concentration range, the resulting primary cells have greater than 60% CD73 expression, no CD45 expression, and mesenchymal stem cell content in the resulting primary cells of 60% -70%, showing extremely high stem cell concentrations; when the zinc chloride is not added into the mixed enzyme digestive juice, the content of the mesenchymal stem cells in the primary cells is less than 38 percent, and is usually within the range of 31-38 percent.

The method according to any embodiment of the second aspect of the present invention, wherein said cell viability assay is counting the number of viable cells before and after cryopreservation using trypan blue staining.

The method according to any embodiment of the second aspect of the present invention, wherein said cell contamination detection detects whether the cells are contaminated with fungi and bacteria using a small amount of cell culture. In one embodiment, the cell contamination assay utilizes an etiological method to detect whether a cell is infected with one or more of the following: hepatitis B two-half, hepatitis C, AIDS virus, cytomegalovirus, EB virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, and TRUST.

The method according to any embodiment of the second aspect of the present invention, wherein said detection of genetic disease is a method of detecting the presence of genetic disease in cryopreserved cells using molecular genetics.

A method according to any one of the embodiments of the second aspect of the invention, wherein said HLA-ABC/DR matching is the detection of the HLA-ABC/DR phenotype of the cell.

The method according to any one of the embodiments of the second aspect of the present invention, wherein said placental mesenchymal stem cells are frozen in liquid nitrogen via a temperature-programmed process.

A method according to any embodiment of the second aspect of the invention, wherein the database includes data relating to all of the cells stored, including but not limited to: the biological characteristic test result of the cell, the identification result of the multi-directional differentiation potential, the molecular genetic diagnosis result of the cell, and the detailed information of the fetus and the parents thereof.

Further, a third aspect of the present invention provides a cell preparation, for example, useful for the treatment of systemic sclerosis, which is a cell suspension formulated by suspending mesenchymal stem cells (e.g., placental mesenchymal stem cells) in a 0.9% sodium chloride solution.

The cell preparation according to the third aspect of the present invention, wherein the cell concentration is 1 to 10X 10⁶One cell/ml, e.g., 1-5X 10⁶Individual cells/ml, e.g. 1-3X 10⁶Individual cells/ml.

According to the cell preparation of the third aspect of the present invention, magnesium citrate and phospholipids are further added to the 0.9% sodium chloride solution. In one embodiment, magnesium citrate is added in an amount to provide a magnesium ion concentration of 2.5 mmol/L. In one embodiment, the phospholipid is added at a concentration of 0.2 mg/ml. In one embodiment, the phospholipid is of injection grade soy origin. It has surprisingly been found that the biological effect of the cell preparation of the invention in the treatment of systemic sclerosis can be significantly improved by the simultaneous addition of small amounts of magnesium salts and phospholipids. The present inventors have also found that when only a magnesium salt, or only a phospholipid, or the above-mentioned magnesium salt is replaced with another salt such as a calcium salt, a zinc salt, a copper salt, or the like, is added to the cell preparation of example 4 of the present invention, the results are far inferior to those of the PD-MSC group a in which a magnesium salt and a phospholipid are added simultaneously, and the results in these cases are similar to those of the PD-MSC group b of example 4 of the present invention.

A cell preparation according to a third aspect of the invention, which is prepared by a method comprising: and transferring the mesenchymal stem cells obtained by cell passage to a centrifuge tube, centrifuging, discarding supernatant, adding 0.9% sodium chloride solution for resuspension, and thus obtaining the cell preparation.

The cell preparation according to the third aspect of the present invention, wherein the mesenchymal stem cells are prepared by a method comprising the steps of:

(1) treatment of placental leaflets: placing placenta in a white porcelain dish, washing with tissue cleaning solution to remove blood stasis of placenta, shearing 20g of placenta lobule tissue in a steel cup, cleaning twice with the tissue cleaning solution, soaking for 5min, and weighing 15g of better tissue in a 100mm glass dish; adding 10ml tissue cleaning solution, and cutting leaflets to 0.2cm³About the size, 100ml of tissue is added for cleaningAfter the solution is stirred evenly, filtering the solution by a 300-mesh filter screen, and then repeating the operation to wash the solution twice by using tissue washing solution so as to remove blood cells;

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

The cell preparation according to the third aspect of the present invention, in the preparation of mesenchymal stem cells, further comprises:

The cell preparation according to the third aspect of the present invention, wherein the cell purity of each generation of the placental mesenchymal stem cells obtained is greater than 90%. In one embodiment, the placental mesenchymal stem cells have a cell purity of greater than 95% after more than 3 passages.

The cell preparation according to the third aspect of the present invention, wherein said Hank's balanced salt solution consists of: 8.0g/L NaCl, 0.4g/L KCl, 0.1g/L MgSO4 & 7H2O, 0.1g/L MgCl2 & 6H2O, 0.06g/L Na2HPO4 & 2H2O, 0.06g/L KH2PO4, 1.0g/L glucose, 0.14g/L CaCl2, 0.35 g/L3, 0.2g/L phenol red, hydrochloric acid or sodium hydroxide to adjust the pH to 7.4. According to the cell preparation of the third aspect of the invention, the mixed enzyme digestive juice contains Hank' S balanced salt solution, Liberase MNP-S enzyme, DNA I type enzyme and 0.2-0.3 g/L zinc chloride. It has been surprisingly found that with the use of mixed enzyme digests with added zinc chloride in this concentration range, the resulting primary cells have greater than 60% CD73 expression, no CD45 expression, and mesenchymal stem cell content in the resulting primary cells of 60% -70%, showing extremely high stem cell concentrations; when the zinc chloride is not added into the mixed enzyme digestive juice, the content of the mesenchymal stem cells in the primary cells is less than 38 percent, and is usually within the range of 31-38 percent.

The cell preparation according to the third aspect of the present invention, wherein said cell activity detection is counting the number of viable cells before and after cryopreservation by trypan blue staining.

The cell preparation according to the third aspect of the present invention, wherein said cell contamination detection detects whether or not the cells are contaminated with fungi and bacteria by using a small amount of cell culture. In one embodiment, the cell contamination assay utilizes an etiological method to detect whether a cell is infected with one or more of the following: hepatitis B two-half, hepatitis C, AIDS virus, cytomegalovirus, EB virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, and TRUST.

The cell preparation according to the third aspect of the present invention, wherein said detection of the genetic disease is a method of detecting the presence or absence of the genetic disease in the cryopreserved cells by using molecular genetics.

A cell preparation according to a third aspect of the invention, wherein said HLA-ABC/DR match is the detection of the HLA-ABC/DR phenotype of the cells.

The cell preparation according to the third aspect of the present invention, wherein said placental mesenchymal stem cells are frozen in liquid nitrogen by a temperature-programmed process.

A cell preparation according to the third aspect of the invention, wherein the database includes data relating to all of the cells stored, including but not limited to: the biological characteristic test result of the cell, the identification result of the multi-directional differentiation potential, the molecular genetic diagnosis result of the cell, and the detailed information of the fetus and the parents thereof.

Furthermore, in the method of the second aspect of the present invention, there is provided a placental mesenchymal stem cell. Thus in a fourth aspect the invention provides a placental mesenchymal stem cell.

The placental mesenchymal stem cells according to the fourth aspect of the invention, wherein the cell purity of each generation of placental mesenchymal stem cells is greater than 90%. In one embodiment, the placental mesenchymal stem cells have a cell purity of greater than 95% after more than 3 passages.

The placental mesenchymal stem cells according to the fourth aspect of the present invention are prepared by a method comprising the steps of:

(1) treatment of placental leaflets: placing placenta in a white porcelain dish, washing with tissue cleaning solution to remove blood stasis of placenta, shearing 20g of placenta lobule tissue in a steel cup, cleaning twice with the tissue cleaning solution, soaking for 5min, and weighing 15g of better tissue in a 100mm glass dish; adding 10ml tissue cleaning solution, and cutting leaflets to 0.2cm³Left and right size, add 100ml the tissue cleansing solution is stirred evenly and filtered by a 300-mesh filter screen, and the operation is repeated to wash twice by the tissue cleansing solution so as to remove blood cells;

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

The placental mesenchymal stem cells according to the fourth aspect of the present invention, in the preparing step, further comprise:

The placental mesenchymal stem cells according to the fourth aspect of the present invention, in the preparing step, the cell purity of each generation of placental mesenchymal stem cells is greater than 90%. In one embodiment, the placental mesenchymal stem cells have a cell purity of greater than 95% after more than 3 passages.

The placental mesenchymal stem cells according to the fourth aspect of the present invention, in the preparing step, the Hank's balanced salt solution consists of: 8.0g/L NaCl, 0.4g/L KCl, 0.1g/L MgSO4 & 7H2O, 0.1g/L MgCl2 & 6H2O, 0.06g/L Na2HPO4 & 2H2O, 0.06g/L KH2PO4, 1.0g/L glucose, 0.14g/L CaCl2, 0.35 g/L3, 0.2g/L phenol red, hydrochloric acid or sodium hydroxide to adjust the pH to 7.4. According to the placental mesenchymal stem cell of the fourth aspect of the present invention, in the preparation step, the mixed enzyme digestive fluid further comprises 0.2-0.3 g/L zinc chloride in addition to Hank' S balanced salt solution, Liberase MNP-S enzyme, and DNA type I enzyme. It has been surprisingly found that with the use of mixed enzyme digests with added zinc chloride in this concentration range, the resulting primary cells have greater than 60% CD73 expression, no CD45 expression, and mesenchymal stem cell content in the resulting primary cells of 60% -70%, showing extremely high stem cell concentrations; when the zinc chloride is not added into the mixed enzyme digestive juice, the content of the mesenchymal stem cells in the primary cells is less than 38 percent, and is usually within the range of 31-38 percent.

The placental mesenchymal stem cells according to the fourth aspect of the present invention, in the preparing step, the cell viability assay is to count the number of viable cells before and after cryopreservation using trypan blue staining method.

The placental mesenchymal stem cells according to the fourth aspect of the present invention, wherein in the preparing step, the cell contamination detection detects whether the cells are contaminated with fungi and bacteria using a small amount of cell culture. In one embodiment, the cell contamination assay utilizes an etiological method to detect whether a cell is infected with one or more of the following: hepatitis B two-half, hepatitis C, AIDS virus, cytomegalovirus, EB virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, and TRUST.

In the placental mesenchymal stem cells according to the fourth aspect of the present invention, in the preparing step, the genetic disease detection is to detect whether the genetic disease is present in the cryopreserved cells by using a molecular genetic method.

The placental mesenchymal stem cells according to the fourth aspect of the invention, wherein in the preparing step, said HLA-ABC/DR matching is a test cell HLA-ABC/DR phenotype.

The placental mesenchymal stem cells according to the fourth aspect of the present invention, in the preparing step, are frozen in liquid nitrogen through a programmed cooling process.

In the preparation step of the placental mesenchymal stem cells according to the fourth aspect of the present invention, the database includes all relevant data of the preserved cells, including but not limited to: the biological characteristic test result of the cell, the identification result of the multi-directional differentiation potential, the molecular genetic diagnosis result of the cell, and the detailed information of the fetus and the parents thereof.

Further, the fifth aspect of the present invention provides a mixed enzyme digestion solution for use in a method of separating mesenchymal stem cells from placenta tissue and culturing the mesenchymal stem cells, the mixed enzyme digestion solution comprising Hank' S balanced salt solution, Liberase MNP-S enzyme, DNA type I enzyme.

The mixed enzyme digestive juice according to any embodiment of the fifth aspect of the present invention comprises: 15-30 volumes of Hank ' S balanced salt solution, 0.2-0.6 volumes of Liberase MNP-S enzyme, 0.2-2 volumes of DNAI type enzyme (e.g., 20-25 volumes of Hank ' S balanced salt solution, 0.3-0.5 volumes of Liberase MNP-S enzyme, 0.5-1 volumes of DNA type I enzyme, e.g., 22 volumes of Hank ' S balanced salt solution, 0.4 volumes of Liberase MNP-S enzyme, 0.7 volumes of DNAI type enzyme).

The mixed enzyme digest according to any one of the embodiments of the fifth aspect of the present invention, wherein the Hank's balanced salt solution consists of: 8.0g/L NaCl, 0.4g/L KCl, 0.1g/L MgSO4 & 7H2O, 0.1g/L MgCl2 & 6H2O, 0.06g/L Na2HPO4 & 2H2O, 0.06g/L KH2PO4, 1.0g/L glucose, 0.14g/L CaCl2, 0.35 g/L3, 0.2g/L phenol red, hydrochloric acid or sodium hydroxide to adjust the pH to 7.4. The mixed enzyme digest according to any one of the embodiments of the fifth aspect of the present invention, wherein zinc chloride is added in a prescribed amount as described herein in addition to Hank' S balanced salt solution, Liberase MNP-S enzyme, DNA type I enzyme. It has been surprisingly found that superior technical effects as described in the present invention can be exhibited in the case of using a mixed enzyme digest to which zinc chloride is added in this concentration range.

The mixed enzyme digestive fluid according to any one of the embodiments of the fifth aspect of the present invention, wherein the method for separating mesenchymal stem cells from placenta tissue and culturing into mesenchymal stem cells, comprises the steps of:

[ wherein the mixed enzyme digestive juice contains: 15-30 volumes of Hank ' S balanced salt solution, 0.2-0.6 volumes of LiberasemNP-S enzyme, 0.2-2 volumes of DNA type I enzyme (e.g., 20-25 volumes of Hank ' S balanced salt solution, 0.3-0.5 volumes of LiberasemNP-S enzyme, 0.5-1 volumes of DNA type I enzyme, e.g., 22 volumes of Hank ' S balanced salt solution, 0.4 volumes of Liberase MNP-S enzyme, 0.7 volumes of DNA type I enzyme); the Liberase MNP-S enzyme is for example the Liberase MNP-S enzyme from Roche, for example from Siberian organisms, the cat # of which: 5578582001]

(5) Cell recovery: taking 2 tubes of frozen cells, quickly thawing at 37 ℃, transferring the cells to a 15ml centrifuge tube, and adding 8ml of complete culture base for resuscitating; centrifuging at 1200rpm for 5min (acceleration 9, deceleration 7), removing supernatant, and adding 5ml complete culture medium for resuspension; inoculating each tube of cells into 1T 75 culture bottle, supplementing complete culture medium to 30ml, and culturing in CO2 incubator (37 deg.C, 5% CO2, saturated humidity); performing total liquid change every 3-4 days with complete culture medium, and recoveringCounting after 12 days according to the clone formation until the cell density is not less than 3000 cells/cm²The following passages can be performed;

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

The mixed enzyme digestive juice according to any embodiment of the fifth aspect of the present invention, wherein the method for separating mesenchymal stem cells from placenta tissue and culturing into mesenchymal stem cells further comprises:

The mixed enzyme digestion solution according to any embodiment of the fifth aspect of the present invention, wherein the cell activity detection in the method of isolating mesenchymal stem cells from placental tissue and culturing into mesenchymal stem cells is counting the number of viable cells before and after cryopreservation using trypan blue staining method.

The mixed enzyme digestion solution according to any embodiment of the fifth aspect of the invention, wherein the cell contamination detection in the method for separating mesenchymal stem cells from placental tissue and culturing into mesenchymal stem cells detects whether the cells are contaminated with fungi and bacteria using a small amount of cell culture. In one embodiment, the cell contamination assay utilizes an etiological method to detect whether a cell is infected with one or more of the following: hepatitis B two-half, hepatitis C, AIDS virus, cytomegalovirus, EB virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, and TRUST.

The mixed enzyme digestion solution according to any embodiment of the fifth aspect of the invention, wherein the genetic disease detection in the method for separating mesenchymal stem cells from placenta tissue and culturing into mesenchymal stem cells is to detect whether the cryopreserved cells have genetic diseases or not by using a molecular genetic method.

The mixed enzyme digest of any embodiment of the fifth aspect of the present invention, wherein said HLA-ABC/DR typing in said method of isolating mesenchymal stem cells from placental tissue and culturing into mesenchymal stem cells is detecting the HLA-ABC/DR phenotype of the cells.

The mixed enzyme digestion solution according to any embodiment of the fifth aspect of the present invention, wherein the method for separating mesenchymal stem cells from placental tissue and culturing into mesenchymal stem cells comprises freezing the mesenchymal stem cells in liquid nitrogen through a temperature-programmed process.

The mixed enzyme digestive fluid according to any embodiment of the fifth aspect of the present invention, wherein the method for separating mesenchymal stem cells from placental tissue and culturing into mesenchymal stem cells comprises in the database data related to all of the preserved cells, including but not limited to: the biological characteristic test result of the cell, the identification result of the multi-directional differentiation potential, the molecular genetic diagnosis result of the cell, and the detailed information of the fetus and the parents thereof.

Of the various process steps described above, although specific steps are described in some detail or in language specific to the process steps described in the examples of the following detailed description, those skilled in the art will be able to fully appreciate the above-described process steps from the detailed disclosure of the invention as a whole.

Any embodiment of any aspect of the invention may be combined with other embodiments, as long as they do not contradict. Furthermore, in any embodiment of any aspect of the invention, any feature may be applicable to that feature in other embodiments, so long as they do not contradict. The invention is further described below.

All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.

In the present invention, the term "placental mesenchymal stem cells" refers to mesenchymal stem cells derived from the placenta. Thus in the present invention, and in particular in the context relating to the present invention, the term "placental mesenchymal stem cells" may be used interchangeably with "placental stem cells", "mesenchymal stem cells", unless otherwise specifically indicated.

In the present invention, the term "PBS buffer" or "PBS" refers to a phosphate buffer. The general formulation and formulation of the PBS used in the context of the present invention, as well as their general properties such as pH value or pH range, are well known to those skilled in the art and are typically commercially available pre-formulations (or powders), e.g. the PBS used in the field of the present invention is typically a commercial buffer at pH7.4(± 0.1), e.g. HyClone brand PBS buffer; in the present invention, the composition of PBS buffer solution in the classical application of the art includes 137mM sodium chloride, 2.7nM potassium chloride and 10mM phosphate, and PBS used in the present invention has the same composition as that in the present invention, unless otherwise specified.

In the present invention, the term "placenta" refers to the placenta of a newborn, and in particular to the placenta within 4 hours of birth.

The mesenchymal stem cells are adult stem cells with self-replication and multidirectional differentiation potential, have the advantages of easy separation, culture and amplification, low immunogenicity and no expression of Major Histocompatibility Complex (MHC) of type II, can be used by variant, have strong migration and immunoregulation capabilities, promote tissue damage repair and regeneration by a paracrine mode, and are ideal seed cells for regenerative medicine.

Systemic sclerosis is a systemic autoimmune disease characterized by localized or diffuse skin thickening and fibrosis. The pathological changes are characterized by skin fiber hyperplasia and skin-like changes of blood vessel onions, which finally cause skin sclerosis and blood vessel ischemia. The disease is clinically characterized by local or diffuse skin thickening and fibrosis, besides skin involvement, it can also affect internal organs (heart, lung, digestive tract and other organs), and is used as an autoimmune disease, often accompanied by autoantibodies such as antinuclear antibodies, anti-centromere antibodies, anti-Scl-70 and the like. Women with the disease are common, the incidence rate is about 4 times that of men, and children are relatively rare.

The most common initial manifestations of systemic sclerosis are Raynaud's phenomenon and swollen extremities, face, with gradual thickening of the skin of the fingers. The first symptom of some cases is the Raynaud phenomenon, and the Raynaud phenomenon can precede other symptoms (finger swelling, arthritis and viscera involvement) of scleroderma for 1-2 years or occur simultaneously with the other symptoms. Gastrointestinal dysfunction (stomach burning and dysphagia) or respiratory symptoms and the like are also the first manifestations of the disease. The patients may have irregular fever, anorexia, weight loss, etc. before onset. Systemic sclerosis is usually clinically manifested in skin, bones and joints, digestive system, lungs, heart, kidneys. In almost all cases, skin sclerosis begins in the hand. The fingers and the hand backs are shiny and tight, the finger wrinkles disappear, the sweat hairs are sparse, and then the face and the neck are affected. The patient has a tight feeling on the chest and shoulders. The transverse thick stripes can appear in the front of the neck, and when the patient lifts the head, the patient feels the skin of the neck tight, which is rarely the phenomenon in other diseases. Facial skin involvement may be manifested as a typical scleroderma face appearance, manifested as: a mask face; the radioactive stripes appear around the mouth, the mouth and the lip become thin, the nose becomes sharp, and the mouth opening is limited. Affected skin may be hyperpigmented or depigmented. Skin lesions can be localized to the fingers (toes) and face, or spread centripetally, affecting the upper arm, shoulder, forebreast, back, abdomen and legs. Some may involve the whole body of the skin within months, some gradually progress over years, and some intermittently. Clinically, the skin lesions can be divided into edema stage, hardening stage and atrophy stage, wherein the skin in the edema stage is in non-concave swelling and has tough feeling when being touched; the skin in the hardening period is in wax-like luster, is tightly attached to subcutaneous tissues and is not easy to pinch; the superficial dermis becomes thin and brittle in the atrophy period, and the epidermis is loose. The skin thickens and clings to the lower joint, so that joint contracture and function are limited. Due to the fibrosis of the tendon sheaths, a leather-like friction sensation is perceived when the affected joint is actively or passively moved, particularly at the wrist, ankle and knee. Some patients may develop joint inflammation, some of which may have aggressive joint changes. Osteolysis at the toe end can occur due to chronic ischemia of the toe. The X-ray film showed narrowing of the joint space and hardening of the articular bone. Digestive tract involvement is the most common visceral lesion of scleroderma. Any part of the digestive tract may be affected, with esophageal involvement being most common, anal and rectal secondary, and the small intestine and colon being less common. In scleroderma, lung involvement is prevalent, with the most common symptoms being shortness of breath during exercise, decreased active tolerance and dry cough, and pulmonary interstitial fibrosis and pulmonary arterial vasculopathy, although one of them is often predominant. In the aspect of heart, the clinical manifestations are short breath, chest distress, palpitation and edema. Clinical examinations may include ventricular gallop, sinus tachycardia, and congestive heart failure, with occasional audible pericardial fricatives. Echocardiography shows that there is hypertrophy or fluid accumulation in the pericardium in about half of the cases, but clinical myocarditis and pericardial tamponade are rare. Scleroderma is most marked by interlobular, segmental, and arterioles, with the interlobular artery being the most prominent. The blood vessel intima has fibroblast hyperplasia, mucus change, acid mucopolysaccharide deposition and edema; transparent degeneration of vascular smooth muscle cells; fibrosis of the adventitia and surrounding interstitium; the glomerular basement membrane is irregularly thickened. Scleroderma has different renal lesions clinical manifestations, and some patients have the clinical phenomenon that the skin and other internal organs are affected for many years without renal damage; some have renal crisis during the course of the disease, i.e. sudden onset of severe hypertension and acute renal failure. If not treated in time, it often dies in several weeks from heart failure and uremia. Although kidney crisis may be asymptomatic in the early stage, most patients suffer from fatigue and aggravation, and have symptoms of shortness of breath, severe headache, blurred vision, convulsion, obnubilation and the like. Laboratory examinations show that creatinine increase, proteinuria and/or microscopic hematuria can cause microangiohemolytic anemia and thrombocytopenia.

The wushu talent literature (wushu talent, et al, influence and mechanism of human placental mesenchymal stem cells on pulmonary fibrosis in mice, yanto medicine, 2016 (12 th year) observed the influence of human placental mesenchymal stem cells on pulmonary fibrosis in mice, and studied the mechanism of action. The method is to adopt a tissue block adherence method to separate and culture human placenta mesenchymal stem cells in vitro. 30C 57BL/6 mice are divided into an observation group and a control group randomly, and 8.5mg/kg of bleomycin is injected into the trachea to establish a pulmonary fibrosis model. After the model is successfully made, observing that 0.3mL (the cell number is 1.0 multiplied by 10^ 6) of human placental mesenchymal stem cells cultured in vitro are infused into tail veins of a group, injecting equivalent physiological saline into a control group for 1 time/d, and continuously injecting for 3 days; the mice are sacrificed, lung tissues are taken, the hydroxyproline content of the lung tissues is detected, and the Vascular Endothelial Growth Factor (VEGF), endothelin 1(ET-1) and angiopoietin 2(Ang-2) proteins of the lung tissues are detected by a Western blotting method. As a result, the hydroxyproline content in the lung tissue of the observation group was (5.76. + -. 0.13). mu.g/mL, that of the control group was (8.13. + -. 0.87). mu.g/mL, and that of the two groups was less than 0.01. The relative expression of VEGF in lung tissues of the observed group is 52.7 +/-4.7, ET-1 is 68.1 +/-5.4, and Ang-2 is 59.6 +/-2.8, which are all reduced compared with the control group (100) (P is less than 0.05). The conclusion is that the human placenta mesenchymal stem cells can inhibit the fibrosis formation of mouse lung tissues; it is possible that the reduction of the expression of VEGF, ET-1 and Ang-2 in lung tissues is the mechanism of action.

The therapeutic effect of intravenous injection of Embryonic Stem Cells (ESC) on pulmonary fibrosis mice was studied in the coma literature (coma, et al, embryonic stem cells for bleomycin-induced pulmonary fibrosis in mice, repoda (medical edition), 03 phase 2008). The method is characterized in that 8.5mg/kg of bleomycin is instilled into the trachea to prepare a pulmonary fibrosis model of C57/BL6 female mice. S8 mouse ESC was injected intravenously in the treatment group (n ═ 20), and normal saline was injected in the control group (n ═ 10). The treatment groups were again divided into single treatment (n ═ 10) and repeat treatment (n ═ 10), both with ESC injected intravenously 1h after molding, and repeat treatment groups with ESC injected intravenously again 3d after molding. Recording the survival time of the mice, measuring the hydroxyproline content of lung tissues of the mice, and observing the inflammatory state of lung pathology. The survival time of 3 groups of mice is counted by using a rank sum test, and the difference of the lung hydroxyproline content of the 3 groups of mice is analyzed by variance. As a result, after receiving the stem cell treatment, the survival time (d) of the pulmonary fibrosis model mice is prolonged, and the repeated treatment groups are more obvious (the control group, the single treatment group and the repeated treatment group are respectively 7.8 +/-2.8, 8.4 +/-3.8 and 13.5 +/-5.6, and P is less than 0.01); the content of pulmonary hydroxyproline (mu g/mL) is reduced (8.59 +/-1.14, 8.23 +/-1.09 and 5.51 +/-0.39 in a control group, a single treatment group and a repeated treatment group respectively, and P is less than 0.01); pathological examination of lung shows that the degree of inflammation of lung tissue is reduced and the structural damage is reduced. The conclusion is that the intravenous injection of the embryonic stem cells can relieve the pulmonary inflammation and pulmonary fibrosis of mice induced by bleomycin and prolong the survival time of mice with pulmonary fibrosis.

Jiong ru document (jiong ri, etc., comparison of two mesenchymal stem cells on the therapeutic effect of pulmonary fibrosis, guangdong medicine, S1 stage 2018) compares the curative effect of bone marrow mesenchymal stem cells (BM-MSCs) and umbilical blood mesenchymal stem cells (UCB-MSCs) on pulmonary fibrosis rats. The method was that 48 male SD rats were randomly divided into 4 groups: group A, injecting 0.1mL of physiological saline into trachea; B. c, D group pulmonary fibrosis model was prepared by injecting 0.1mL of bleomycin at 5mg/kg into trachea. 1D and A, B groups after modeling are injected with 1.0mL of physiological saline through tail vein, C groups are injected with BM-MSCs 1 x 10^6 through tail vein, and D groups are injected with UCB-MSCs 1 x 10^6 through tail vein. Half of each group of rats were sacrificed at 28d and 42d after modeling, lung tissues were left for pathological examination, and lung tissue transforming growth factor-beta 1 (TGF-beta 1), Hydroxyproline (HYP), matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase tissue inhibitor-1 (TIMP-1) expression levels were determined, respectively. As a result, severe disturbance of alveolar structure and deposition of a large amount of collagen fibers were observed in group B, and the score for pulmonary fibrosis was decreased in the order of B, C, D, A groups [ score (3.00. + -. 0.00), (2.17. + -. 0.75), (1.60. + -. 0.89), (0.00. + -. 0.00) at 28d, score (3.00. + -. 0.00), (2.40. + -. 0.55), (1.75. + -. 0.96), (0.00. + -. 0.00) at 42d ], and levels of TGF-. beta.1, HYP and MMP-2/TIMP-1 in lung tissues were decreased in the order of B group. The lung tissues TGF-beta 1, HYP and MMP-2/TIMP-1 are positively correlated with the alveolitis degree score and the pulmonary fibrosis degree score respectively. The conclusion is that BM-MSCs and UCBMSCs can delay the pulmonary fibrosis of rats, wherein UCB-MSCs have better curative effect, and the treatment mechanism of the UCB-MSCs is probably related to the down-regulation of TGF-beta 1 level and the improvement of MMP/TIMP imbalance.

The Pangyo literature (Pangyo, et al, research on lung pneumonia and lung fibrosis of silicosis mice treated by bone marrow mesenchymal stem cell transplantation, Chinese tissue engineering research, 2013, stage 06) observes the treatment effect of the bone marrow mesenchymal stem cell transplantation on silicosis. In this method, 36C 57BL/6 mice were divided into 3 groups by the random glowing method. Injecting normal saline into the trachea of the control group mouse; injecting a silicon dioxide suspension into the trachea of the mice in the silicosis model group and the bone marrow mesenchymal stem cell transplantation group to establish a silicosis model; and the bone marrow mesenchymal stem cell transplantation group is used for injecting the bone marrow mesenchymal stem cells into the tail vein 6h after the molding. The results and conclusions show that the content of hydroxyproline in lung tissues in a silicosis model group and a bone marrow mesenchymal stem cell transplantation group are higher than those in a control group, and the differences have significant meanings (P < 0.01); the content of the bone marrow mesenchymal stem cell transplantation group is obviously lower than that of the silicosis model group, and the difference has significant significance (P < 0.01). The lung coefficients of mice in the silicosis model group and the bone marrow mesenchymal stem cell transplantation group are higher than those of the control group (P <0.01), wherein the bone marrow mesenchymal stem cell transplantation group is lower than that of the silicosis model group, and the difference has significance (P < 0.01). The expression silicosis model group and the bone marrow mesenchymal stem cell transplantation group of the interleukin 1 beta are higher than the control group (P < 0.01); the bone marrow mesenchymal stem cell transplantation group is lower than the silicosis model group, and the differences have significance (P < 0.01). The expression of transforming growth factor beta 1 in lung tissue is also that silicosis model group (P <0.01) and bone marrow mesenchymal stem cell transplantation group (P <0.05) are higher than that of control group; the difference of the bone marrow mesenchymal stem cell transplantation group is lower than that of the silicosis model group, and the difference has significance (P < 0.01). These results indicate that bone marrow mesenchymal stem cell transplantation can reduce the inflammatory response and the degree of fibrosis in the lung.

Zhengkaiwen (Zhengkai, et al, transplantation of mesenchymal stem cells in marrow for treating acute radiation lung injury in rats, Chinese tissue engineering research, 50 th 2014) observes the treatment effect of the mesenchymal stem cells in marrow on the radiation lung injury in rats, and preliminarily discusses the action mechanism. The method comprises the steps of in vitro separation, culture and identification of the bone marrow mesenchymal stem cells of the male SD rat. A linear accelerator is used for irradiating the chest of 60 female SD rats, a radioactive lung injury model of the rats is established, and the radioactive lung injury model is randomly divided into a bone marrow mesenchymal stem cell treatment group and a normal saline control group. The bone marrow mesenchymal stem cell treatment group is infused with 2 x 10^9/L bone marrow mesenchymal stem cells through the tail vein of a rat, and the normal saline control group is injected with the same amount of normal saline and is respectively subjected to relevant index detection at 1, 2, 4 and 6 weeks after irradiation. The results and conclusions are that the lung coefficient of the rats in the mesenchymal stem cell treatment group is obviously lower than that of the normal saline control group (P <0.05) at 1, 2 and 4 weeks after irradiation. Under the microscope, the rat lung tissue of the bone marrow mesenchymal stem cell treatment group has less inflammatory exudation, the alveolar and alveolar wall structures are basically complete, and the fibrosis degree and the like are obviously lighter than those of a normal saline control group. The serum transforming growth factor beta 1 and hydroxyproline levels of the bone marrow mesenchymal stem cell treatment group at 2 weeks of irradiation are obviously lower than those of a normal saline control group (P < 0.05). The lung tissue superoxide dismutase activity of the bone marrow mesenchymal stem cell treatment group is obviously higher than that of the normal saline control group (P <0.05) at the irradiation time of 2, 4 and 6 weeks, and the malondialdehyde content of the bone marrow mesenchymal stem cell treatment group is obviously lower than that of the normal saline control group (P <0.05) at the irradiation time of 4 and 6 weeks. The expression of lung surfactant B in the bone marrow mesenchymal stem cell treatment group at 6 weeks of irradiation is significantly higher than that in the normal saline control group. The PCR method detects that the lung, liver, pancreas, kidney and other organs and tissues have Sry gene expression in different degrees, particularly the lung is obvious. The result shows that the bone marrow mesenchymal stem cells can migrate to the lung tissue with radioactive injury to promote the repair of the lung tissue with the radioactive injury, and the treatment mechanism of the bone marrow mesenchymal stem cells can be related to the inhibition of inflammatory reaction, the antioxidation, the reduction of the fibrosis of the lung tissue and the like.

The huriping literature (huriping, et al, effect of aging on lung stem cells in mouse pulmonary fibrosis model, china journal of comparative medicine, stage 07 in 2013) studies the effect of aging on the ability of lung stem cells to repair in bleomycin-induced pulmonary fibrosis mouse model. The method comprises the steps of treating young and old mice with bleomycin to establish a pulmonary fibrosis model, comparing the proportion and the proliferation condition of lung tissue stem cells in the pulmonary fibrosis model of the young and old mice by methods such as flow cytometry and the like, and researching the influence of aging on the damage repair capacity of the lung tissue stem cells. As a result, in the bleomycin-induced pulmonary fibrosis model, the lung tissue stem cell proliferation of the aged mice is obviously reduced compared with that of the young mice, and the ratio of the lung epithelial stem cells to the lung interstitial stem cells is obviously reduced. It was concluded that lung tissue stem cells have significantly diminished repair capacity following pulmonary fibrosis injury during aging.

The effect of the mesenchymal stem cells on transforming growth factor beta and monocyte chemotactic protein 1 of the lung-injured rat is observed in Zhao-kung literature (Zhao-kun, et al, the effect of the mesenchymal stem cells on transforming growth factor beta and monocyte chemotactic protein 1 of the lung-injured rat, China tissue engineering research and clinical rehabilitation, 2008, 29). Design, time and place: random controls, cytological in vitro experiments, were performed at 2005-05/2006-02 at the fourth university of military medical of the liberty military. Materials: 20 clean female SD rats were randomly divided into a normal control group, a cell control group, a lung injury group, and a cell transplantation group, 5 rats/group. Male SD rat 5 was used only for collection of bone marrow mesenchymal stem cells. The method comprises the step of injecting 0.2-0.3 mL of 5mg/kg bleomycin into rats of a lung injury group and a cell transplantation group through a trachea to induce and establish a lung injury model. Injecting 0.5mL of bone marrow mesenchymal stem cell suspension and about 5 multiplied by 10^6 cells into rats of the cell transplantation group and the cell control group through tail veins 12 hours after molding; rats in the lung injury group and the normal control group are injected with serum-free DMEM-F120.5mL by the same method. The main observation indexes are as follows: the morphological change of the lung tissue is observed by hematoxylin-eosin staining. And (3) measuring the hydroxyproline content of the lung tissue by an acidolysis method. ELISA method detects the expression of transforming growth factor beta and monocyte chemotactic protein 1 in serum and bronchoalveolar lavage fluid. As a result, after 2 weeks of cell transplantation, alveolar spaces of the normal control group and the cell control group were uniformly intact; the pulmonary alveolar structure of the lung injury group is damaged, the alveolar septum is thickened, and the interstitium is proliferated; the lung injury degree of the cell transplantation group is obviously reduced. Compared with a normal control group, the lung tissue hydroxyproline content of the lung injury group and the cell transplantation group is obviously increased (P <0.01 or 0.05), and the increase amplitude of the cell transplantation group is obviously lower than that of the lung injury group (P < 0.01). The expression levels of transforming growth factor beta and monocyte chemotactic protein 1 in the serum and bronchoalveolar lavage fluid of each group of rats are basically similar to the hydroxyproline content of lung tissues. The conclusion is that the bone marrow mesenchymal stem cells can reduce the content of hydroxyproline in lung tissue of rats with lung injury, reduce the lung injury and the fibrosis degree of the rats, and possibly relate to the reduction of the expression of transforming growth factor beta and monocyte chemotactic protein 1.

The left exemplary document (left exemplary, analysis of the effect of human umbilical cord blood stem cells on TNF-alpha and NO of rats with pulmonary fibrosis, China medical frontier, 2013, stage 07) researches the effect and influence of human umbilical cord blood stem cells on TNF-alpha and NO of rats with pulmonary fibrosis. The method comprises the steps of selecting 60 clean and healthy rats, randomly dividing the rats into 30 treatment groups and 30 control groups, manufacturing a pulmonary fibrosis model by a bleomycin tracheal injection method for the two groups of rats, injecting stem cells into the treatment group of rats after the model is successfully established, not injecting the stem cells into the control group of rats, killing the rats at different time, and observing the alveolar injury degree and TNF-alpha and NO level change conditions of the two groups of rats. As a result, the pulmonary alveolar injury degree of rats in the control group is more serious than that of rats in the treatment group, the pulmonary fibrosis degree is the same in the two groups in the range of 0-1, the pulmonary alveolar injury degree of rats in the treatment group is less than that of rats in the control group in the ranges of 1.1-2 and 2.1-3, TNF-alpha and NO levels of rats in the treatment group are lower than those of rats in the control group at different time, P is less than 0.05, and the difference has statistical significance. The conclusion is that after the stem cells in human umbilical cord blood are injected into the body of a rat with pulmonary fibrosis, the levels of TNF-alpha and NO are reduced along with the development of time, and the stem cells have protective effect to a certain extent, the expression of TNF-alpha and NO is inhibited, and the stem cells have inhibitory effect on the pulmonary fibrosis of the rat.

The aged Juan literature (aged Juan, et al, comparison of mesenchymal-like stem cells and bone marrow mononuclear cells affecting bleomycin-induced pulmonary fibrosis, Chinese tissue engineering research and clinical rehabilitation, stage 36 2010) compares the therapeutic effects of mesenchymal-like stem cells and bone marrow mononuclear cells on bleomycin A5-induced alveolitis and early fibrosis in rats. The method comprises the steps of collecting bone marrow mesenchymal stem cells and bone marrow mononuclear cells of Wistar male young mice and respectively carrying out DAPI marking. A pulmonary injury model is prepared by injecting bleomycin A5 into the trachea of 21 Wistar female rats and is divided into a bone marrow mesenchymal stem cell treatment group, a bone marrow mononuclear cell treatment group and a model group at random. The rats are sacrificed on the 7 th day after the model building, and pathological sections of lung tissues are taken to observe the inflammation and fibrosis conditions; detecting the DAPI marked cells under a fluorescence microscope; ELISA method is used for detecting the content of hydroxyproline and tumor necrosis factor in lung tissue homogenate. The results and conclusions are that DAPI marked exogenous cells are seen in lung tissue frozen sections of a bone marrow mesenchymal stem cell treatment group and a bone marrow mononuclear cell treatment group. ② the model group has the most serious alveolitis, the marrow mesenchymal stem cell treatment group has the least alveolitis, and the difference has significant meaning (P <0.05) compared with each group. ③ the lung tissue homogenate of the model group has the most contents of hydroxyproline and tumor necrosis factor alpha, the treatment of the mesenchymal stem cells is the least, and the comparative difference of each group has significance (P < 0.05). The bone marrow mesenchymal stem cells and bone marrow mononuclear cells are prompted to be planted in damaged lung tissues and can effectively prevent bleomycin A5-induced rat alveolitis and early fibrosis, and the effect of the bone marrow mesenchymal stem cells and the bone marrow mononuclear cells is more obvious.

The Wang Hongyang literature (Wang Hongyang, etc., the expression of rat pulmonary fibrosis and lung macrophage TGF-beta 1 inhibited by human umbilical cord blood stem cells, journal of cellular and molecular immunology, 2013, stage 01) researches the influence of human umbilical cord blood mesenchymal stem cells on rat pulmonary fibrosis and TGF-beta 1 caused by bleomycin. The method comprises the steps of randomly dividing 60 clean-grade healthy male SD rats into a bleomycin group (P group), a stem cell treatment group (M group), a dexamethasone treatment group (D group) and a negative control group (N group), and culturing 2 nd generation human umbilical cord blood mesenchymal stem cells to 4 th generation; injecting bleomycin into P group, M group and D group via trachea to prepare pulmonary fibrosis model, injecting 5-bromo-2-deoxyuridine (BrdU) labeled stem cells into rat tail vein immediately after M group modeling, continuously injecting dexamethasone into abdominal cavity for 7D at 2 days after D group modeling, injecting equal amount of physiological saline into N group via trachea, killing rats at 7, 14 and 28 days, performing HE and Masson staining, and observing TGF-beta 1 expression and labeled cell condition by immunohistochemistry method. As a result, labeled stem cells were observed in lung tissue on days 7, 14, and 28 in group M; observed after HE and Masson staining, compared with the group N, the alveolitis is most obvious in the group P at 7D, the pulmonary fibrosis degree of the group 28D is the heaviest, the group M, D is lighter than the group P, and pathological sections show that the alveolitis and fibrosis degree of the group M is slightly lighter than those of the group D; TGF-. beta.1 was highest in lung tissue in P group 7d, M, D groups were significantly less than in P group, M group was more significantly reduced, and differences between groups were statistically significant. The conclusion is that the human umbilical cord blood mesenchymal stem cells can be planted in lung tissues, and alveolitis and pulmonary fibrosis can be reduced by inhibiting the expression of TGF-beta 1 in the early stage of pulmonary fibrosis.

The therapeutic efficacy of Pirfenidone (PFD) in combination with bone marrow Mesenchymal Stem Cells (MSCs) transplanted into Bleomycin (BLM) induced pulmonary fibrosis mice was explored in the canon literature (canon, study of treatment of pulmonary interstitial fibrosis by bone marrow mesenchymal stem cell transplantation in combination with pirfenidone, chinese pragma drug, 2016 (01)). The method comprises the steps that 36 male SD rats with the healthy and clean weight of 200g are randomly divided into 6 groups, wherein the group A is a negative control group, a group B positive control group, a group C simple hormone treatment group, a group D simple MSCs transplantation group, a group E simple PFD treatment group and a group F PFD combined MSCs transplantation group, and 6 rats are respectively selected. SD rat cultures MSCs in vitro, injects BLM to prepare pulmonary fibrosis model, observes lung tissue morphological change, determines transforming growth factor (TGF-beta 1) protein expression. As a result, after 7d of model building, the inflammatory cell infiltration of C, D, E, F rats was reduced compared to that of group B, the degree of pulmonary fibrosis was significantly reduced, and the pathological changes of group F were mild and the pulmonary fibrosis was not significant compared to group C, D, E. The decrease in lung coefficients was significant in group B, and the difference was statistically significant compared to group A (P < 0.05). B. C, D, E, F group showed higher pulmonary fibrosis score than group A, with statistical significance of the difference (P < 0.05). The number of positive cells expressing transforming growth factor collagen in lung tissue is obviously reduced, the number and intensity of F group are more obviously reduced than B, C, D, E group, the difference between A group and B, C, D, E, F group is statistically significant (P <0.05), and the difference between C, D, E group is not statistically significant (P > 0.05). The conclusion is that the bone marrow mesenchymal stem cell transplantation and the pirfenidone have certain inhibiting effect on the pulmonary fibrosis, can reduce the expression of lung tissue transformation growth factors, have repairing and regenerating effects, and have obvious effect on treating the pulmonary fibrosis.

The research on the influence and possible mechanism of the transplantation of the mesenchymal stem cells on bleomycin-induced pulmonary fibrosis model alveolitis and pulmonary fibrosis in rats is discussed in the Zhongyao province (Zhongyao, et al, experimental study on transplantation of the mesenchymal stem cells in bone marrow for treating pulmonary fibrosis in rats, proceedings of Gannan medical college, 2016 (year 01)). The method comprises the steps of separating and culturing the bone marrow mesenchymal stem cells of the SD rat in vitro, and transferring the cells to the fourth generation for experiment; 60 SD rats were randomly divided into a negative control group (N group), a positive control group (P group), a dexamethasone-treated group (D group), and a mesenchymal stem cell transplantation-treated group (M group), each of which was 15 rats. And injecting bleomycin into the P group, the D group and the M group through the trachea respectively to prepare a pulmonary fibrosis model. Lung tissues are kept 7, 14 and 21 days after the model building, HE staining is carried out to observe the degree of alveolitis and pulmonary fibrosis, and the expression of lung tissue transforming growth factor beta 1 (TGF-beta 1) is measured by an immunohistochemical method. As a result, after the bleomycin is molded, lung tissues are taken for HE staining, and the pathology accords with lung fibers; at 7, 14 and 21 days after treatment, D, M rats exhibited significantly reduced levels of pneumonia and pulmonary fibrosis compared to the P group, while the number of positive cells expressing transforming growth factor beta 1 (TGF-. beta.1) in rat lung tissue was significantly reduced (P < 0.05); and D, M group had no significant difference between groups in comparison of pathological results and transforming growth factor expression at 7, 14 and 21 days. The conclusion is that the bone marrow mesenchymal stem cell transplantation has a certain inhibiting effect on bleomycin induced pulmonary fibrosis and pulmonary fibrosis degree of a rat pulmonary fibrosis model, and the action mechanism is probably related to the reduction of the expression of a lung tissue transformation growth factor (TGF-beta 1).

In the Liuchen literature (Liuchen, et al, the effect of human umbilical cord blood mesenchymal stem cells on preventing bleomycin-induced pulmonary fibrosis in rats, China occupational medicine, 03 in 2013), the effect and possible mechanism of preventive intervention of Human Umbilical Cord Blood Mesenchymal Stem Cells (HUCBMSC) on bleomycin-induced pulmonary fibrosis in rats are discussed. The method comprises culturing 2 nd generation HUCBMSC to 4 th generation; 120 male SD rats of 5 weeks of age without specific pathogen are randomly divided into a bleomycin group, a stem cell stem prediction group, a dexamethasone stem prediction group and a negative control group, and each group contains 30 male SD rats. The first 3 groups are respectively injected with bleomycin through a trachea to establish a pulmonary fibrosis model, after stem cell intervention and model building, 5-bromo-2-deoxyuridine (Brdu) -labeled HUCBMSC is injected through a rat tail vein, continuous intraperitoneal injection of dexamethasone 7d is started on the 1 st day after the dexamethasone intervention and model building, a negative control group is injected with an isometric physiological sodium chloride solution through the trachea, 10 animals in each group are killed on the 7 th, 14 th and 28 th days, lung tissues are stained with hematoxylin-eosin and masson, and the level of lung hydroxyproline is determined by an alkaline hydrolysis method. As a result, Brdu-labeled stem cells were observed in lung tissue on days 7, 14, and 28 in the stem cell stem pretreatment group. The pulmonary hydroxyproline levels of the bleomycin group at 3 time points tended to increase gradually, reaching the highest level at day 28 (P < 0.01). The pulmonary alveoli inflammation and pulmonary fibrosis degree of the stem cell stem prediction group and the dexamethasone stem prediction group at 3 time points are respectively lighter than those of the bleomycin group, and the difference is statistically significant (P is less than 0.05). It was concluded that HUCBMSC could colonize damaged lung tissue and be effective in reducing alveolitis and pulmonary fibrosis early in pulmonary fibrosis.

The mechanism of inhibiting the pulmonary fibrosis of the rats by the Mesenchymal Stem Cells (MSCs) is observed in the wangxian literature (wangxian, et al, the mechanism of inhibiting the pulmonary fibrosis of the rats by the mesenchymal stem cells, academy of science and technology (medical edition) in huazhong, 2014 03). The method comprises the steps of in vitro separation, culture and purification of bone marrow MSCs of SD rats of 4 weeks old. SD experimental rats were randomly divided into 4 groups (12 per group): normal control group (normal saline is injected into trachea), model group (bleomycin is injected into trachea), MSCs treatment early group (tail vein injection MSCs is given immediately after model building), MSCs treatment late group (tail vein injection MSCs is given 14 days after model building), bleomycin dosage is 5mg/kg, normal control group is injected with isovolume normal saline, tail vein injection MSCs dosage is 1.0 x 10^6/mL DMEM culture solution 1 mL. After the rats are sacrificed uniformly on the 28 th day, lung tissues are taken, pathological sections of the lung tissues are stained by hematoxylin-eosin (HE) and Masson to observe the lung inflammation and fibrosis, and the protein expression amounts of lung tissue transformation growth factor-beta 1 (TGF-beta 1), matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase tissue inhibitor-1 (TIMP-1) are detected by a Western blot method. As a result, first, MSCs were successfully isolated and cultured and identified. Secondly, the degree of the alveolitis and the pulmonary fibrosis of the model group is obviously aggravated compared with that of the normal control group, the degree of the alveolitis and the pulmonary fibrosis of the early-stage group treated by the MSCs is obviously lightened compared with that of the model group, and the difference between the degree of the alveolitis and the pulmonary fibrosis of the late-stage group treated by the MSCs and that of the model group has no statistical significance. Thirdly, the TGF-beta 1 and TIMP-1 protein expressions of the model group are obviously increased compared with the normal control group, the TGF-beta 1 and TIMP-1 protein expressions of the MSCs treatment group are obviously reduced compared with the model group, and the MMP-2 protein expression has no statistical significance in the difference among rats of each group. It was concluded that bone marrow MSCs could inhibit bleomycin-induced pulmonary fibrosis and that the intervention of MSCs given early in lung injury was more effective, probably by reducing the expression of TGF- β 1 protein and modulating the balance between MMP-2 and TIMP-1.

Treighur 29815 (treighur 29815, et al, influence of mesenchymal stem cells on bleomycin-induced pulmonary fibrosis in rats, china tuberculosis and journal of respiration, 2007, phase 09) a new method for treating pulmonary fibrosis was explored by observing influence of Mesenchymal Stem Cells (MSC) on bleomycin-induced pulmonary fibrosis animal models in rats. The method comprises the steps of in vitro separating and culturing bone marrow MSC of male 6-week-old SD rats. Randomly dividing 48 female SD rats into 6 groups, injecting 0.3ml of 5.0mg/kg of bleomycin into 1-5 groups through an air pipe, and injecting 0.2ml of MSC solution (the number of cells is 2.5 multiplied by 10^ 6) into male rats through tail veins on the 1 st day and the 7 th day of bleomycin injection respectively for the 1 st group and the 3 rd group; groups 2 and 4 were injected with 0.2ml of phosphate buffer via tail vein on day 1 and day 7 of bleomycin injection, respectively; group 5 as a model positive control, no other treatment was given after bleomycin injection; group 6 was used as a model negative control, and an equal amount of physiological saline 0.3ml was injected through the trachea in place of bleomycin, and no other treatment was given. The rats are sacrificed uniformly on the 28 th day of the experiment, and the lung tissues are left for pathological examination and hydroxyproline content determination; DNA of lung tissue was extracted, and sex-determining gene (sry gene) of male rat was detected by Polymerase Chain Reaction (PCR) -agarose electrophoresis to determine whether or not the foreign-administered MSC was present in lung tissue of female rat. As a result, the pathological changes of the lung of the rats after MSC intervention treatment on the 1 st day and the 7 th day of the bleomycin-induced lung injury are reduced compared with those of the control group, and the degree of pulmonary fibrosis is respectively scored as 1.0 +/-0.2, 2.5 +/-0.5, 1.6 +/-0.5 and 2.3 +/-0.8; the hydroxyproline content of lung tissues is respectively (83 +/-17) mu g/mg, (96 +/-20) mu g/mg and (123 +/-32) mu g/mg, and (127 +/-34) mu g/mg, and the effect of the MSC is more obvious when the MSC is administered on the 1 st day than the 7 th day of lung injury caused by bleomycin. PCR detection results show that sry gene can be detected in lung tissue of rats administered with MSC group on day 1 of bleomycin induced lung injury. It was concluded that exogenously administered MSCs could reduce the development of pulmonary fibrosis with better efficacy of MSC intervention given early in lung injury.

In the Liuchen literature (Liuchen, et al, the influence of human umbilical cord blood stem cells on TNF-alpha and NO of rats with pulmonary fibrosis, China journal of Industrial medicine, 2012, stage 06) the influence of human umbilical cord blood mesenchymal stem cells on rat pulmonary fibrosis and TNF-alpha and NO is explored. The method comprises the steps of randomly dividing 35 clean-grade healthy male SD rats into 15 bleomycin groups (P group), 15 stem cell treatment groups (M group) and 5 negative control groups (N group), and culturing the second-generation human umbilical blood mesenchymal stem cells to the fourth generation; the P group and the M group are respectively injected with bleomycin through the trachea to prepare a pulmonary fibrosis model, the N group is injected with physiological saline with the same amount, and the 5-bromo-2-deoxyuridine (Brdu) labeled stem cells are immediately injected through the rat tail vein after the M group is modeled. The N groups of rats were all sacrificed on day 7 after molding, and the P and M groups of rats were sacrificed on days 7, 14, and 28 after molding, respectively. Lung tissues are taken for HE and Masson staining, and the expression conditions of TNF-alpha and NO in alveolar lavage fluid are detected by an ELISA method. As a result, labeled stem cells were observed in lung tissue on days 7, 14, and 28 in group M; through observation after HE and Masson staining, compared with the group N, the alveolitis is most obvious in the group P at 7d, the pulmonary fibrosis degree of the group 28d is the heaviest, and the alveolitis and fibrosis degree of the group M are reduced compared with those of the group P; compared with the N group, the TNF-alpha and NO levels of the rats in the P group are obviously increased and reach a peak at 7d, the TNF-alpha and NO levels of the rats in the M group are obviously lower than those of the rats in the P group in each time period, and the difference between the rats has statistical significance. The conclusion is that the human umbilical cord blood mesenchymal stem cells can be planted in lung tissues, and alveolitis and pulmonary fibrosis can be effectively relieved in the early stage of pulmonary fibrosis; inhibiting the expression of TNF-alpha and NO may be the action mechanism.

The inventor of the invention utilizes a perfusion method to separate and culture the mesenchymal stem cells from the placenta, and obtains the mesenchymal stem cells with high purity. However, after perfusion, a large amount of stem cells still remain in the placenta tissue and cannot be effectively separated. Therefore, it is considered that the mesenchymal stem cells cannot be obtained to the maximum extent by the perfusion method.

The invention discloses a method for separating a large number of mesenchymal stem cells from placenta, and the method is used for preserving the mesenchymal stem cells of the placenta and establishing a placenta stem cell bank. Based on the summary of the conventional separation and culture of the mesenchymal stem cells, the inventor successfully separates and obtains a large amount of mesenchymal stem cells from the placenta by utilizing a mixed digestion of a plurality of tissue digestive enzymes and combining an adherence culture method. The mesenchymal stem cells obtained by the method have high purity and large quantity, have the same biological characteristics as the mesenchymal stem cells of the bone marrow, and can be differentiated into osteoblasts, chondrocytes, adipocytes, endothelial cells, nerve cells and the like. Because stem cells in the placenta are more immature than adult stem cells and rich in content, the placenta stem cells have wide application prospects in clinic, the mesenchymal stem cells are cryopreserved like cord blood by using a conventional cell cryopreservation method, a placenta stem cell bank is established, and a foundation is laid for the further research and clinical treatment of the stem cells.

Because the cord blood contains abundant hematopoietic stem cells, people establish a cord blood bank to store the cord blood hematopoietic stem cells which are an important biological resource, and a treatment means is provided for various blood system diseases and immune system diseases. Similarly, the placenta mesenchymal stem cells are used as a more important stem cell resource, and are frozen in liquid nitrogen at the deep low temperature of-196 ℃ for long-term storage by using a conventional cell freezing storage method, so that a placenta stem cell bank is established, and seeds are preserved for the treatment of stem cells in the future.

Particularly, the method can obtain primary cells with extremely high mesenchymal stem cell purity in the P0 generation, the expression of CD73 of the primary cells is more than 60 percent, the expression of CD45 of the primary cells is not carried out, and the content of the mesenchymal stem cells in the primary cells reaches 60 to 70 percent.

The technical effect of the method of the invention is obvious. For example, the invention selects a full-term placenta sample, cuts 15g of tissue at a specific position of a placenta lobule, digests the tissue by using a mixed enzyme system, obtains cells, and then purifies the cells to obtain a group of relatively pure mesenchymal stem cells (the expression of CD73 is more than 60 percent, and the expression of CD45 is not carried out), wherein the number of the obtained cells per gram of the tissue can reach 2.5 multiplied by 10⁷And the yield is stable, and the sample specificity is greatly reduced. The primary mesenchymal stem cells are subjected to recovery culture after cryopreservation, and are cultured by using a classical complete culture medium formula, so that more spindle-type adherent cells can be microscopically detected in about 4 days, the cell fusion rate reaches 70-80% in 10 days, and the cells can be transferred to P1 generations. After continuous passage to P5 generation, carrying out experiments such as flow type phenotype identification, cell cycle detection, induced differentiation and the like, wherein the results show that the cells of P1-P5 generation are mesenchymal stem cells, the positive expression (CD73, CD90 and CD105) is more than 98 percent, and the negative expression (CD34, CD45, CD19, CD11b and HLA-DR) is less than 2 percent; the G2 stage cells of the P5 generation cells are less than 1 percent, have strong proliferation capacity and do not enter the division stage; the ability to differentiate into osteoblasts, adipoblasts and chondroblasts under the stimulation of a specific induction medium.

The method is simple to operate, convenient and practical, can obtain a large amount of mesenchymal stem cells, has good differentiation performance, and has the capacity of differentiating osteoblasts, adipocytes, chondrocytes, endothelial cells, nerve cells and other cells. Comparison with existing methods: at present, MSC is mainly obtained by extracting donor bone marrow by an operation method or separating placenta by a perfusion method and carrying out adherent culture. The method has the advantages of low cell number, and infection possibility in marrow extraction and after marrow extraction. The invention successfully separates and obtains a large amount of mesenchymal stem cells with higher purity from the placenta, and establishes a placental stem cell bank by using the method to store the stem cells with great application prospect. The method is simple and easy to implement, and because the placenta is the same as cord blood, the cell components are more immature, the source is wide, and the method is convenient and easy to obtain, the method has wide prospect in the clinical application of stem cells.

Drawings

FIG. 1: the flow type phenotype identification result of the primary cell obtained by the invention is shown in the figure.

FIG. 2: micrographs of samples during passage P0.

FIG. 3: micrographs of samples during passage P5.

FIG. 4: the samples were phenotyped at P5.

FIG. 5: DNA content-cell number relationship of the sample P5 generation cells.

FIG. 6: the induced differentiation test of the P5 generation cells shows that the cells have the capacity of differentiating to osteogenic, adipogenic and chondrogenic cells.

Detailed Description

The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.

Example 1, placental whole cell treatment:

1. preparing a mixed synthase digestive solution: 22ml of HBSS (Hank' S balanced salt solution) containing calcium and magnesium ions, 0.4ml of Liberase Roche MNP-S enzyme (purchased from Sibao, for example, cat # 5578582001) and 0.7ml of DNA I enzyme were transferred into a 50ml centrifuge tube, and zinc chloride (added at a concentration of 0.2g/L, 0.25g/L or 0.3g/L) was added thereto, mixed, and preheated at 37 ℃ for 20min or more. The Hank's balanced salt solution consists of: 8.0g/L NaCl, 0.4g/L KCl, 0.1g/L MgSO4 & 7H2O, 0.1g/L MgCl2 & 6H2O, 0.06g/L Na2HPO4 & 2H2O, 0.06g/L KH2PO4, 1.0g/L glucose, 0.14g/L CaCl2, 0.35 g/L3, 0.2g/L phenol red, hydrochloric acid or sodium hydroxide to adjust the pH to 7.4.

2. Preparation of placental leaflets: the placenta is taken out from the collection bag and placed in a white porcelain dish, the blood stasis of the placenta is removed after the tissue cleaning solution is washed, and a small amount of placenta lobule tissue (about 20 g) is cut and placed in a steel cup. The tissue is washed twice with tissue washing solution (0.9% physiological saline + double antibody (double antibody is streptomycin, content 1%)), and after soaking for 5min, 15g + -1 g of better tissue is weighed into a 100mm glass dish.

3. Removal of blood cells: adding 10ml tissue cleaning solution, and cutting leaflets to 0.2cm³About the size, 100ml of tissue cleaning fluid is added and stirred evenly, then the mixture is filtered by a 300-mesh filter screen, and then the mixture is cleaned twice by the tissue cleaning fluid (the leaflet tissue is moved into a steel cup each time, 100ml of tissue cleaning fluid is added and stirred evenly, and then the mixture is filtered by a 300-mesh filter screen).

4. Mixed enzyme digestion and termination: adding the cleaned leaflet tissue into preheated 23ml of mixed enzyme digestive juice, fully and uniformly mixing, and then oscillating and digesting for 30min by a shaking table at 37 ℃ and 100 rpm. After digestion, the tissue fluid +2ml FBS was terminated.

5. Collecting primary cells:

adding 50ml of tissue cleaning fluid to dilute and mix the tissue fluid uniformly, filtering by a 300-mesh sieve, collecting cell fluid, washing the digested tissue twice (50 ml of tissue cleaning fluid is used each time), merging the filtrate into 1 250ml of centrifuge tubes, and centrifuging at 1500rpm for 8min (acceleration 9 and deceleration 7);

removing supernatant, adding appropriate amount of tissue washing solution, resuspending and supplementing to 200ml, centrifuging at 1500rpm for 8min (acceleration 9, deceleration 7);

removing supernatant, adding DMEM-F12 into the cell sediment, resuspending the cell sediment to 30ml, filtering with a 100um filter screen, and then washing the filter screen with 10ml DMEM-F12 to obtain 40ml cell suspension as primary cells; 1ml of suspension is taken and used for a sysmex hematology analyzer to count cells, and the primary cells are high in purity and have the mesenchymal stem cell content of about 60-70 percent through determination.

6. Freezing and storing primary cells:

the frozen stock solution is prepared on site, and the formula of the frozen stock solution is as follows: 65% DMEM-F12, 15% Human Serum Albumin (HSA), 20% DMSO, e.g., WAK brand DMSO, ready for use;

centrifuging the cell suspension at 1800rpm for 10min (acceleration 9 and deceleration 7), collecting cell precipitate and supernatant 5ml (sample of supernatant 10 ml), re-suspending, slowly adding the prepared frozen stock solution while shaking;

the cell suspension was dispensed into 9 2ml cryopreserved tubes, 1.5ml per tube (pre-cooled in a programmed cooling box). The remaining cell suspension + the retained sample supernatant are used for sterile detection;

and (4) performing program cooling by using a program cooling instrument, transferring the cells into a liquid nitrogen storage tank, and performing cryopreservation on the obtained primary cells.

Through the whole placenta cell processing process of the embodiment 1, a placenta sample of full term is selected, 15g of tissue at a specific position of a placenta lobule is cut, the placenta sample is digested by the mixed enzyme digestive fluid system, the cells are obtained and then purified, and a group of relatively pure primary mesenchymal stem cells (the expression of CD73 is more than 60 percent, and the expression of CD45 is not carried out) can be obtained, the content of the mesenchymal stem cells in the primary mesenchymal stem cells reaches 60 to 70 percent, and the number of the primary cells obtained by each gram of the placenta lobule tissue can reach (2.4 to 2.8) multiplied by 10⁷Moreover, the yield is stable, and the sample specificity is greatly reduced. However, when the mixed enzyme digestive fluid is not added with the zinc chloride, the content of the mesenchymal stem cells in the primary cells is less than 38 percent, and is usually in the range of 31-38 percent, and the number of the primary cells obtained per gram of the placenta lobule tissue is less than 5 multiplied by 10⁵A plurality of; in addition, the present inventors found that the number of primary cells obtained per gram of placental lobule tissue was less than 2X 10 when the primary cell preparation was performed with reference to other prior art techniques ⁶1/10 or less of the method of the present invention. According to the invention, the whole cell treatment of the placenta can efficiently obtain primary mesenchymal stem cells, and a good foundation is laid for the subsequent culture of the mesenchymal stem cells with high medical value.

For example, in this example, the cell yield was very stable after placental tissue treatment, and typical data for some experiments are shown in table 1.

Table 1: cell yield of primary cells from placental tissue

Date of treatment	Sample registration number	Tissue volume (g)	Number of cells (. times.10)⁸)	Cell yield (10)⁸/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, the flow type phenotype identification result of the primary cells obtained after the placenta treatment shows that the expression of CD73 is up to more than 60%, and CD45 is not expressed, which indicates that the primary cells are a group of relatively pure mesenchymal stem cells and do not contain blood cells. The flow phenotyping results are shown in FIG. 1.

Example 2 recovery and subculture of Primary cells

1. Cell recovery:

taking 2 tubes of frozen cells, quickly thawing at 37 ℃, transferring the cells to a 15ml centrifuge tube, and adding 8ml of complete culture base for resuscitating; as not otherwise specified, complete medium as used herein is DMEM-F12 medium containing 10% FBS;

centrifuging at 1200rpm for 5min (acceleration 9, deceleration 7), removing supernatant, and adding 5ml complete culture medium for resuspension; inoculating each tube of cells into 1T 75 culture bottle, supplementing complete culture medium to 30ml, and culturing in CO2 incubator (37 deg.C, 5% CO2, saturated humidity); performing total liquid change every 3-4 days with complete culture medium, recovering for 12 days, and counting according to clone formation condition until cell density is not less than 3000 cells/cm²The following passages can be performed;

2. cell passage: washing recovered P0 generation cells with PBS, adding 2ml pancreatin for 2-5min until most of cells are dropped, adding 5ml complete culture medium to stop digestion, transferring cells into a centrifuge tube, centrifuging at 1400rpm for 5min (addingSpeed 9, deceleration 7), discarding supernatant, adding 5ml of complete culture medium for resuspension, counting and inoculating to a culture flask, wherein the cell density is 8000-12000 cells/cm²Culturing in a CO2 incubator (37 ℃, 5% CO2 and saturated humidity) until the cell density reaches over 90% (usually culturing for about 5 days), and completing cell passage from P0 generation to P1 generation;

the passage operations from the generation P0 to the generation P1 are sequentially repeated to perform cell passages from the generation P1 to the generation P2, from the generation P2 to the generation P3, from the generation P3 to the generation P4 and from the generation P4 to the generation P5 respectively, so that the mesenchymal stem cells of each generation are obtained.

In this example, the seeding density was about 5X 10⁵cells/cm²And a plurality of adherent cells are arranged in the field of microscopic examination for 4 days after inoculation and are spindle-shaped. The strain can be transferred to P1 generation 10 days after inoculation. The cells grow fast and are full, and the cells are in spindle shapes. During passage from P0 to P1, the cell counts for some experiments are shown schematically in table 2 below. The micrograph of the PS162279 sample during passaging is shown in FIG. 2.

Table 2: cell count results during passage from passage P0 to passage P1

Sample registration number	P0-P1	P0 count
			PS162311	D10	8*10⁵(ADAM)
PS162319	D10	1.4*10⁶(ADAM)
			PS162279	D9	1.5*10⁶(ADAM)

In addition, in the process of inoculating and passaging P1-P5, the seeds are usually cultured for 4-5 days, harvested and passaged to the next generation. An exemplary micrograph of the PS162279 sample during passage P5 is shown in figure 3.

In this experiment, the flow phenotype identification of P1-P5 generation was carried out, the results showed that the positive expression of CD73, CD90 and CD105 was > 98%, and CD34, CD45, CD19 and HLA-DR were identified, and the results are shown in Table 3, which proved that the cells isolated and cultured in placenta were mesenchymal stem cells and had high purity.

Table 3: flow phenotyping results of P1-P5 generation cells

Exemplary, the P5 representative flow phenotypic identification results of the PS162279 sample are shown in fig. 4.

In addition, the growth cycle of some samples was measured for P5 generation cells, and the results showed < 1% G2 phase cells and > 10% S phase cells, demonstrating that these cells are highly proliferative and do not enter the division phase, and the specific results are shown in Table 4.

Table 4: growth cycle assay results for P5 generation cells

Sample registration number	Stage GO/G1	Stage S	Stage G2/M
				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, the DNA content-cell number relationship was plotted for PS162311-P5 cells, and typical results are shown in FIG. 5.

Example 3 biological characterization of placental MSCs

The biological characteristics of the placenta mesenchymal stem cells are identified by referring to the methods of issued patents CN102676451A [0062] to [0089], and the results show that the MSC separated by the method has the capacity of differentiating into osteoblasts, adipocytes and chondrocytes, thereby confirming that the MSC obtained by the method has the characteristics of stem cells.

For example, the P5 generation cells were illustratively tested for induced differentiation and showed the ability to differentiate into osteogenic, adipogenic and chondrogenic cells. A typical micrograph of adipogenic, osteogenic, and chondrogenic differentiation is shown in fig. 6.

Example 4 effectiveness of placental mesenchymal stem cells in treating SSc

More than 2/3 of SSc patients have lung involvement, most commonly pulmonary interstitial fibrosis, which is the main cause of death. Therefore, the classical bleomycin-induced pulmonary interstitial fibrosis mouse model was used as an animal model for preclinical studies to investigate the effectiveness of placental mesenchymal stem cells in treating SSc.

Reference is made to the document of threo-red (threo-red, et al. long-term stability of the mouse pulmonary fibrosis model induced by intraperitoneal bleomycin. study of tissue engineering in china, 2017, vol 21, stage 4, tests conducted 512-519). Female C57BL/6 mice in 6-8 weeks were treated with bleomycin 35mg/kg in 200. mu.L saline, i.p. for 2 injections per week for 8 consecutive injections. After the 8 th intraperitoneal injection, the pulmonary alveolitis and pulmonary fibrosis scores gradually increase from 2 weeks, reach the peak in 6-8 weeks, and still do not obviously decrease after lasting for 10 weeks, thereby completing the modeling of the bleomycin pulmonary fibrosis.

In the trial, 6-8 week female C57BL/6 mice were randomly divided into 4 groups, each: group 1-control (n ═ 20): modeling the normal animals without bleomycin in a simulated mode, and injecting physiological saline into the abdominal cavity 2 times a week for 8 times; group 2-pulmonary fibrosis model group (n ═ 20): bleomycin pulmonary fibrosis modeling animals; group 3-placental MSC treatment group a (n ═ 20): after successful modeling of the bleomycin pulmonary fibrosis, namely 10 weeks after the first bleomycin injection, tail vein injection of human placenta MSC preparation a is carried out, 2.5 multiplied by 10^5 MSC cells are injected each time, 1 time per week, and 2 times in total; group 4-placental MSC treatment group b (n ═ 20): after successful modeling of bleomycin pulmonary fibrosis, namely 10 weeks after the first bleomycin injection, tail vein injection of human placenta MSC preparation b is carried out, 2.5 multiplied by 10^5 MSC cells are injected each time, 1 time per week, and 2 times in total.

Groups

1 and 2 were given MSC in the other two groups simultaneously with an intravenous injection of an equal volume of saline.

In the present experiment, the placental mesenchymal stem cell injection preparation was prepared as follows: transferring the mesenchymal stem cells obtained by the cell passage of the step 2 in the example 2 (in the example, the PS162279 sample is used for P4 generation) into a centrifuge tube, centrifuging for 5min at 1500rpm, discarding supernatant, adding 0.9% sodium chloride solution for resuspension, and preparing a cell preparation with the cell concentration of 1-3 x 10^6 placental mesenchymal stem cells/ml; wherein, the 0.9% sodium chloride solution is additionally added with magnesium citrate and phospholipid (magnesium ion concentration is 2.5mmol/L, phospholipid concentration is 0.2mg/ml, phospholipid is from injection grade soybean, and the group added with magnesium citrate and phospholipid is marked as PD-MSC group a, namely the group 3) or not added with magnesium citrate and phospholipid (the group not added with magnesium citrate and phospholipid is marked as PD-MSC group b, namely the group 4).

Groups of 6 mice were sacrificed at 7, 14 and 28 days after the second tail vein placental MSC injection, and tested.

(1) Pathological changes of the lung

After the transplantation, 7 days, 14 days and 28 days, each group of mice (6 mice in each group) is sacrificed, lung tissues of the mice are fixed in 10% neutral formaldehyde solution for 24 hours, acetaldehyde is dehydrated, paraffin is embedded, the thickness of tissue sections is 5um, hematoxylin-eosin staining is carried out, and the alveolar structure is observed under a microscope. The results show that: the lung tissue of group 1 animals was normal; in the group 2, on the 7 th, 14 th and 28 th days, the lung tissues of the mice have obvious inflammatory cell infiltration, fibroblast foci and normal alveolar structure deformation; inflammatory cell infiltration is obviously less than that of the group 27 days, 14 days and 28 days after MSC is injected into tail vein of the group 3, and the alveolar structure is basically maintained in a normal state; at 7, 14 and 28 days after MSC tail vein injection in group 4, inflammatory cell infiltration was less but not significantly different than in group 2, and significantly more than in group 3, with a deformed alveolar structure.

(2) ELISA for detection of IL-1, IL-6, TNF-alpha and TGF-beta protein levels in Lung tissue

Freezing mouse lung tissue, cracking by protease inhibitor-containing lysis buffer, centrifuging, and collecting supernatant. ELISA was performed to determine the amount of IL-1, IL-6, TNF-. alpha.and TGF-. beta.proteins (weight of cytokines per unit weight of lung tissue) in mouse lung tissue lysates. ELISA plates were tested at 450nm and the concentration readings for the proteins were calculated from the standard curve. The results show that: bleomycin caused a significant increase in the expression of cytokines (IL-1, IL-6, TNF- α, TGF- β) in lung tissue that were able to mediate inflammation and fibrosis (p <0.01) compared to healthy mice at 14 days post-MSC injection, whereas group 3 given MSC expressed significantly lower amounts of these factors relative to group 2 (p <0.01 or p < 0.05). The specific results are shown in the following table:

group of	Group 1	Group 2	Group 3	Group 4
					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 the table, group 2p <0.05, p <0.01 compared to group 1; group 3 and 4 # p <0.05, # # p <0.01, compared to group 2.

(3) Determination of collagen content in mouse lung tissue

Hydroxyproline (HYP) is a non-essential amino acid, one of the main components of collagen tissue, and is a unique amino acid in collagen. The content change of the lung tissue collagen of the mice is evaluated by measuring the content of Hydroxyproline (Hydroxyproline) in the lung tissue. Frozen mouse lung tissue was taken, hydrolyzed in HCl, and at 558nm, absorbance of each group of samples was measured and hydroxyproline content was calculated. The results show that: the bleomycin-induced pulmonary fibrosis has obvious collagen deposition and obviously increased Hydroxyproline (HYP) content in 7 days, 14 days and 28 days. Group 3 given placental MSCs had a significant effect of attenuating collagen deposition (p < 0.01). The specific results are shown in the following table:

survey day	Group	1	Group 2	Group 3	Group 4
						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) Assessment of matrix metalloproteinase Activity

Matrix metalloproteinase activity in frozen lung tissue was assessed quantitatively by densitometry and gelatinase activity. Protein content (15 mg/sample) was loaded onto a gel containing 7.5% acrylamide and 1mg/ml gelatin. And evaluating changes in MMP-2, MMP-9, and MMP-13. The results show that: the enzyme activity of each mouse in the control group was set to 1, and the enzyme activity value of each group was calculated by densitometry quantitative calibration. The specific results are shown in the following table:

enzyme activity	Group	1	Group 2	Group 3	Group 4
						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 the table, group 3 and 4 # p <0.05, # # p <0.01, compared to group 2.

The above results confirm the effectiveness of the cell preparation of the present invention for SSc, and the results also show that the effect of PD-MSC group a is significantly better than the PD-MSC group b results.

Example 5 establishment of placental stem cell Bank

1. Detection of cell viability: the number of viable cells before and after cryopreservation was counted using trypan blue staining.

2. Detection of cell contamination: and (3) detecting whether the cells are polluted by fungi and bacteria by using a small amount of cell culture. And (3) detecting whether the cells are infected by hepatitis B two-and-two, hepatitis C, AIDS, cytomegalovirus, EB virus, syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM, EBV-IgA and TRUST by utilizing a pathogenic method.

3. Detection of genetic diseases: and detecting whether the frozen cells have genetic diseases or not by using a molecular genetics method.

4. HLA-ABC/DR match: cells were tested for HLA-ABC/DR phenotype and recorded.

5. Investigation of cell origin: the details of the fetus and its parents are recorded and recorded on the record.

6. Establishing a placental stem cell database: after normal placental stem cells are preserved, a database of placental stem cells is established, which includes the first six data, and associations with cryopreserved cells are established.

Claims

1. Use of a cell preparation for the preparation of a medicament for the treatment and/or prevention of systemic sclerosis; the cell preparation is a cell suspension prepared by suspending the placenta mesenchymal stem cells in 0.9% sodium chloride solution; the cell concentration in the cell preparation is 1-10 multiplied by 10⁶Individual cells/ml; magnesium citrate and phospholipid are also added into the 0.9% sodium chloride solution, wherein the magnesium citrate is added in an amount that the concentration of magnesium ions is 2.5mmol/L and the concentration of phospholipid is 0.2 mg/ml; the cell preparation is prepared by a method comprising: and transferring the mesenchymal stem cells obtained by cell passage to a centrifuge tube, centrifuging, discarding supernatant, adding 0.9% sodium chloride solution for resuspension, and thus obtaining the cell preparation.

2. Use according to claim 1, wherein the cell concentration in the cell preparation is 1-5 x 10⁶Individual cells/ml.

3. Use according to claim 1, wherein the cell concentration in the cell preparation is 1-3 x 10⁶Individual cells/ml.

4. Use according to claim 1, the phospholipids being of injection grade soy origin.

5. Use according to claim 1, wherein the mesenchymal stem cells are prepared by a method comprising the steps of:

(2) mixed enzyme digestion and termination: adding the cleaned leaflet tissue into 15-30 ml of mixed enzyme digestive juice preheated at 37 ℃, fully mixing uniformly, then digesting for 30min by shaking at 37 ℃ and 100rpm in a shaking table, and after digestion is finished, adding 2ml of FBS into the tissue juice to stop digestion;

(3) collecting primary cells: adding 50ml of tissue cleaning fluid into the tissue fluid obtained in the last step, uniformly mixing, filtering by a 300-mesh sieve, and collecting cell fluid; washing the digested tissue twice repeatedly, combining the filtrates of the two times into a centrifuge tube, and centrifuging at 1500rpm for 8 min; removing supernatant, adding appropriate amount of tissue washing solution, resuspending and supplementing to 200ml, and centrifuging at 1500rpm for 8 min; removing supernatant, adding DMEM-F12 into the cell sediment, resuspending the cell sediment to 30ml, filtering with a 100um filter screen, and then washing the filter screen with 10ml DMEM-F12 to obtain 40ml cell suspension as primary cells;

(4) freezing and storing primary cells: centrifuging the cell suspension at 1800rpm for 10min, collecting cell precipitate and lower solution 5ml, re-suspending, slowly adding frozen solution 10ml while shaking; subpackaging the obtained cell suspension into 9 freezing tubes of 2ml, each tube of 1.5ml, placing in a precooled program cooling box, performing program cooling by using a program cooling instrument, and transferring the cells into a liquid nitrogen storage tank for freezing;

(5) cell recovery: 2 tubes of the cells were taken out of the frozen stock,quickly thawing at 37 ℃, transferring the cells to a 15ml centrifuge tube, adding 8ml of complete culture base point drop for resuscitation; centrifuging at 1200rpm for 5min, removing supernatant, and adding 5ml complete culture medium for resuspension; each tube of cells was inoculated into 1T 75 flask, supplemented with complete medium to 30ml, CO was added₂Incubator, 37 ℃ C., 5% CO₂Culturing in saturated humidity; performing total liquid change every 3-4 days with complete culture medium, recovering for 12 days, and counting according to clone formation condition until cell density is not less than 3000 cells/cm²The following passages can be performed;

(6) cell passage: washing P0 generation cells with PBS, adding 2ml pancreatin for 2-5min until most of the cells fall off, adding 5ml complete culture medium to stop digestion, transferring the cells into a centrifuge tube, centrifuging at 1400rpm for 5min, discarding supernatant, adding 5ml complete culture medium for resuspension, counting, and inoculating to a culture bottle, wherein the cell density is 8000-12000 cells/cm²Placing CO in₂Incubator, 37 ℃ C., 5% CO₂Culturing in saturated humidity until the cell density reaches over 90 percent, and completing cell passage from P0 generation to P1 generation; repeating the operations in sequence to perform cell passage from P1 generation to P2 generation, P2 generation to P3 generation, P3 generation to P4 generation and P4 generation to P5 generation respectively to obtain the mesenchymal stem cells of each generation.

6. Use according to claim 5, wherein the tissue wash is 0.9% saline containing 1% double antibody.

7. The use according to claim 5, wherein the mixed enzyme digest comprises: 15-30 volumes of Hank' S balanced salt solution, 0.2-0.6 volumes of Liberase MNP-S enzyme and 0.2-2 volumes of DNA I-type enzyme.

8. Use according to claim 5, the formulation of the frozen stock solution being: 65% DMEM-F12, 15% Human Serum Albumin (HSA), 20% DMSO.

9. The use according to claim 5, wherein in the preparation of mesenchymal stem cells, further comprising:

(7) aiming at the placenta mesenchymal stem cells obtained in the step (6), detecting at least one item of the following items: cell viability, cell contamination, genetic disease, HLA-ABC/DR match;

(8) freezing and storing the passage-generated placenta mesenchymal stem cells obtained in the step (6) in liquid nitrogen; and

10. The use according to claim 5, wherein the cell purity of the obtained placental mesenchymal stem cells is greater than 90%.

11. The use according to claim 5, wherein said placental mesenchymal stem cells have a cell purity of greater than 95% after more than 3 passages.

12. Use according to claim 5, the Hank's balanced salt solution consisting of: 8.0g/L NaCl, 0.4g/L KCl, 0.1g/L MgSO₄·7H₂O, 0.1g/L MgCl₂·6H₂O, 0.06g/L Na₂HPO₄·2H₂O, 0.06g/L KH₂PO₄1.0g/L glucose, 0.14g/L CaCl₂0.35g/L NaHCO₃0.2g/L phenol red, hydrochloric acid or sodium hydroxide to adjust the pH to 7.4.

13. The use according to claim 5, wherein the mixed enzyme digestive juice comprises Hank' S balanced salt solution, Liberase MNP-S enzyme, DNA I type enzyme, and 0.2-0.3 g/L zinc chloride.

14. A cell preparation which is a cell suspension prepared by suspending placental mesenchymal stem cells in a 0.9% sodium chloride solution; the cell concentration in the cell preparation is 1-10 multiplied by 10⁶Individual cells/ml; magnesium citrate and phospholipid are also added into the 0.9% sodium chloride solution, wherein the magnesium citrate is added in an amount that the concentration of magnesium ions is 2.5mmol/L and the concentration of phospholipid is 0.2 mg/ml; the cell preparation is prepared by a method comprising: passage of cellsAnd transferring the mesenchymal stem cells into a centrifuge tube, centrifuging, discarding supernatant, adding 0.9% sodium chloride solution for resuspension, and preparing the cell preparation.

15. The cell preparation according to claim 14, wherein the cell concentration is 1 to 5X 10⁶Individual cells/ml.

16. A cell preparation according to claim 14, wherein said mesenchymal stem cells are prepared by a method according to claim 5.