CN108030791A - A kind of placenta multipotential stem cell preparation is preparing the application in treating acute lung injury medicine - Google Patents
A kind of placenta multipotential stem cell preparation is preparing the application in treating acute lung injury medicine Download PDFInfo
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- CN108030791A CN108030791A CN201711373750.3A CN201711373750A CN108030791A CN 108030791 A CN108030791 A CN 108030791A CN 201711373750 A CN201711373750 A CN 201711373750A CN 108030791 A CN108030791 A CN 108030791A
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
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Abstract
The invention discloses a kind of placenta multipotential stem cell preparation to prepare the application in treating acute lung injury medicine, the placenta multipotential stem cell derives from neonate placenta tissue fetus face part, for the fibroblast-like cells of adherent growth, CD73, CD90 and CD105 are expressed, does not express CD14, CD19, CD34, CD45 and HLA DR.In the present invention, placenta multipotential stem cell obtains convenient, abundance, and placenta multipotential stem cell preparation is prepared simply, and infusion techniques are easy, easy to operate, reduce acute lung injury degree and improve lung function significant effect, there is important clinical meaning and wide application prospect.
Description
(1) technical field
The present invention relates to acute lung injury therapy field, and more particularly to placenta multipotential stem cell is in acute lung injury treatment
Purposes and preparation method thereof.
(2) background technology
Acute lung injury (ALI) is mainly sucked by hydrochloric acid in gastric juice, infected, wound, fat embolism, suction toxic gas etc. cause,
Infection is most common reason.No matter the ALI caused by which kind of reason, inflammatory reaction has all played main function in its pathological change.
Although substantial amounts of research attempts to illustrate the pathogenesis of ALI, do not find particularly effective treatment means still so far.Mesh
Before, mainly clinically still maintain very high based on the treatment of supportive treatment, drug therapy and assisted ventilation in the treatment of ALI
Case fatality rate, about maintain 30%~40%, therefore develop effective ALI medicines and means are very urgent.
In recent years, flourishing for stem-cell research has brought new approaches and hope for organizational project and disease treatment.Grind
Study carefully and show there is abundant multipotential stem cell in human placenta, can be by enzyme digestion or tissue block adherent method from neonate's tire
Separate and obtain in disk tissue.Placenta multipotential stem cell has the general phenotypic characteristic of mescenchymal stem cell, and possesses Multidirectional Differentiation
Potential, while also have the advantages that abundance, convenient material drawing, in-vitro multiplication ability are strong, it is in tissue damage reparation and disease
A kind of cell of the great potential using values of biomedical sector such as treatment.
(3) content of the invention
The object of the present invention is to provide a kind of placenta multipotential stem cell preparation to prepare the purposes in treating ALI medicines, i.e.,
Acute lung injury degree can be reduced using cell suspension preparation made of placenta multipotential stem cell, improves lung function, be placenta
Multipotential stem cell provides a kind of new application.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention is：
The present invention provides a kind of placenta multipotential stem cell preparation answering in treatment acute lung injury (ALI) medicine is prepared
With.
Further, the placenta multipotential stem cell preparation is prepared as follows：Will be competent thin more than the placenta of adhere-wall culture
Born of the same parents are digested with 0.25% trypsase-EDTA digestive juices, after brine, then are suspended with physiological saline, are tire
Disk multipotential stem cell preparation.
Further, the placenta multipotential stem cell be adherent growth fibroblast-like cells, express CD73, CD90 and
CD105, does not express CD14, CD19, CD34, CD45 and HLA-DR.Placenta multipotential stem cell derives from neonate's placenta tissue tire
Youngster face part, is separated by enzyme digestion combination adhere-wall culture, and it is spare for cell to collect 3-5.
Further, cell concentration is (2~6) × 10 in the placenta multipotential stem cell preparation7A/ml.
Further, the medicine is treatment acute lung diseases reason damage or the medicine for the treatment of pulmonary function injury.
The drug dose is calculated as (8~24) × 10 with placenta multipotential stem cell number7A/kg weight, venoclysis.Institute
State cell suspension Infusion Time be modeling after 6~24 it is small when.
Compared with prior art, beneficial effect of the present invention is mainly reflected in：The present invention is acute using lipopolysaccharides LPS inductions
Injury of lungs mouse model, proves to significantly improve in acute lung injury by being injected intravenously placenta multipotential stem cell preparation first
The pulmonary function injury such as lung injury scoring and blood oxygen saturation and oxygenation index.
(4) illustrate
Fig. 1 is each group lung tissue structure and its Injury score figure；A, lung tissue HE are dyed；B, lung injury scoring knot
Fruit；Wherein Control, ALI and PP represent respectively normal group, ALI model groups, ALI model placenta multipotential stem cell injection groups.**
Represent placenta multipotential stem cell injection group with the poor heteropole of ALI model groups significantly (P ﹤ 0.01).
Fig. 2 is each group lung function index testing result figure；Wherein Control, ALI and PP represent normal group, ALI moulds respectively
Type group, ALI model placenta multipotential stem cell injection groups.* placenta multipotential stem cell injection group and ALI model group comparing differences are represented
Significantly (P ﹤ 0.05), * * represent placenta multipotential stem cell injection group with the poor heteropole of ALI model groups significantly (P ﹤ 0.01).
(5) embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
Embodiment 1：It is prepared by Mouse model of acute lung injury
Mouse injection 250 μ l, 1% yellow Jackets (PBS preparations) anaesthetized, along neck among cut off skin and muscle
Layer, exposure tracheae, with 1ml syringes, centripetal direction is pierced among two ring of tracheae, injects the LPS (PBS preparations) of 2.5mg/ml,
Dosage is 10mg/kg weight, at once erects mouse after the completion of injection and rotates so that solution is uniformly distributed lung both sides, suture
Afterwards, mouse normally feeds in an aseptic environment.
Embodiment 2：Placenta multipotential stem cell preparation
(1) placenta multipotential stem cell is separately cultured identifies with streaming
Placenta multipotential stem cell is separately cultured：With physiological saline to placenta surface clean for several times to remove extravasated blood and mucus etc.
Impurity, then Quick disinfection is carried out to placenta surface with 75% ethanol water of volumetric concentration, after with physiological saline wash remaining second off
Alcoholic solution；Clean the placenta finished and take fetus covering weave into clean centrifuge tube, and shred into rotten shape；Add the enzyme of 2 times of volumes
Solution (the DNA enzymatic I of Type I collagen enzyme+concentration 40IU/mL of concentration 300IU/mL, normal saline), digests 1 at 37 DEG C
Hour；70 μm of strainer filterings of digestion product remove tissue residue therein, obtain cell suspension；By cell suspension 1.077g/
The Ficoll of ml carries out density gradient centrifugation, separates monocyte, the mononuclearcell being separated to is washed 3 with physiological saline
It is secondary；With α-MEM nutrient solutions (addition volumetric concentration be 1% penicillin, streptomysin, amphotericin B and glutamine, and
Volumetric concentration is 15% hyclone, and α-MEM nutrient solutions are purchased from Invitrogen companies) suspension cell, with 1 × 106/cm2Density
It is seeded to 75cm2In plastic cell culture bottle, be placed in 37 DEG C, saturated humidity, containing 5%CO2Cultivated in incubator, topple over after 48h and remove
Non- attached cell is gone, changes fresh medium, changes liquid within then every 2~3 days, when cell length to 90% is converged, had digestive transfer culture, P3~
5 generation cells are collected spare.
Placenta multipotential stem cell streaming is identified：Taking P3, quantity is about 1.0 × 10 for placenta multipotential stem cell6, it is uniform respectively
9 pipes are distributed into, is resuspended to 200 μ l, washed 2 times with PBS after centrifugation.1 pipe is stayed to do blank after centrifugation, phenotypic markers are by often pipe one
Mark be added (antibody be respectively mouse anti human CD14-FITC, CD19-PE, CD29-FITC, CD34-PE, CD45-PE,
HLA-DR-FITC, CD73-PE, CD90-PE and CD105-PE).Darkroom is incubated 30min, is washed 2 times after centrifugation, after being then centrifuged for
200 μ l PBS are added to be resuspended, flow cytometer measure.
1 placenta multipotential stem cell flow cytometer detection result of table
(2) preparation of placenta multipotential stem cell preparation
Take the 4th generation of step (1) to grow to 90% or so the placenta multipotential stem cell converged, 0.25% trypsase-EDTA to disappear
Change, after brine 2 times, suspended with physiological saline and adjust concentration to 4 × 107A/ml, is placenta multipotential stem cell
Preparation.
Embodiment 3：Placenta multipotential stem cell preparation infusion reduces injury of lungs degree, improves lung function
(1) chmice acute injury of lungs (ALI) model established using 1 the method for embodiment, is tested as 3 groups：Group 1 is just
Normal group, group 2 are ALI model groups, group 3 is ALI model placenta multipotential stem cell preparation injection groups.3 groups of 6h after modeling, by 8 ×
107A/kg body weight doses are transfused the placenta multipotential stem cell preparation of the preparation of embodiment 2 by tail vein.After injection 90h sample into
Row pathologic state colony and blood gas analysis detection, action effect of the analysis placenta multipotential stem cell to ALI.
(2) pathologic state colony detects：Lung tissue is taken, 4 degree of fixations in 4% paraformaldehyde is placed in and overnight, then carries out stone
Wax is cut into slices and HE dyeing, and micro- sem observation is taken pictures, and passes through stethemia, empsyxis, inflammatory cell infiltration, alveolar wall thickness etc.
Aspect carries out lung pathology Injury score calculating.
(3) blood gas analysis detects：Mouse is noted after injection 250 μ l, 1% yellow Jackets (PBS preparations) anesthesia with 1ml
Emitter (anti-freezing of 1mg/ml heparin sodiums) extracts 500 μ l or so arterial blood from abdominal aorta, is detected by blood gas analyzer, point
Analyse blood oxygen saturation and oxygenation index.
The result shows that group 2 shows typical acute injury of lungs pathological characters, alveolar structure destruction bleeding, alveolar wall are obvious
Thicken, have inflammatory cell infiltration, placenta multipotential stem cell preparation injection group mouse lung tissue structural damage degree substantially reduces, table
Now for alveolar wall is thinning, alveolar structure improves, inflammatory cell infiltration is reduced (as shown in A in Fig. 1), lung injury scoring
(lung injury scores) the results show placenta multipotential stem cell preparation injection group, which improves the pathology of ALI, to be had significantly
Difference (as shown in B in Fig. 1).In addition, blood gas analysis detection shows, the injection of placenta multipotential stem cell preparation can significantly improve blood
Oxygen saturation and oxygenation index, improve lung function (as shown in Figure 2).
Finally, it should also be noted that it is listed above be only the present invention several specific embodiments.Obviously, this hair
It is bright to be not limited to above example, there can also be many deformations.Those of ordinary skill in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Claims (5)
1. a kind of placenta multipotential stem cell preparation is preparing the application in treating acute lung injury medicine.
2. application as claimed in claim 1, it is characterised in that the placenta multipotential stem cell preparation is prepared as follows：Will
The placenta multipotential stem cell of adhere-wall culture is digested with 0.25% trypsase-EDTA digestive juices, after brine, then
Suspended with physiological saline, be placenta multipotential stem cell preparation.
3. application as claimed in claim 2, it is characterised in that the placenta multipotential stem cell is adherent growth into fiber-like
Cell, expresses CD73, CD90 and CD105, does not express CD14, CD19, CD34, CD45 and HLA-DR.
4. application as claimed in claim 2, it is characterised in that in the placenta multipotential stem cell preparation cell concentration for (2~
6)×107A/ml.
5. application as claimed in claim 1, it is characterised in that the medicine is treatment acute lung diseases reason damage or treatment lung work(
The medicine that can be damaged.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711373750.3A CN108030791A (en) | 2017-12-19 | 2017-12-19 | A kind of placenta multipotential stem cell preparation is preparing the application in treating acute lung injury medicine |
| PCT/CN2018/099147 WO2019119819A1 (en) | 2017-12-19 | 2018-08-07 | Application of placental pluripotent stem cell preparation in preparing drug for treating acute lung injury |
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| CN201711373750.3A CN108030791A (en) | 2017-12-19 | 2017-12-19 | A kind of placenta multipotential stem cell preparation is preparing the application in treating acute lung injury medicine |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109646457A (en) * | 2018-12-20 | 2019-04-19 | 杭州易文赛生物技术有限公司 | A kind of application of umbilical cord mesenchymal stem cells in preparation treatment acute lung injury drug |
| WO2019119819A1 (en) * | 2017-12-19 | 2019-06-27 | 杭州易文赛生物技术有限公司 | Application of placental pluripotent stem cell preparation in preparing drug for treating acute lung injury |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105769910A (en) * | 2016-04-15 | 2016-07-20 | 遵义医学院附属医院 | Application of human amniotic mesenchymal stem cells |
| CN106038597A (en) * | 2016-05-27 | 2016-10-26 | 遵义医学院附属医院 | Application of mesenchyma stem cells to preparation of acute lung injury treating preparation |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3223830A4 (en) * | 2014-11-24 | 2018-06-06 | Cytostormrx LLC | Encapsulated stem cells for the treatment of inflammatory disease |
| CN108030791A (en) * | 2017-12-19 | 2018-05-15 | 杭州易文赛生物技术有限公司 | A kind of placenta multipotential stem cell preparation is preparing the application in treating acute lung injury medicine |
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2017
- 2017-12-19 CN CN201711373750.3A patent/CN108030791A/en active Pending
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2018
- 2018-08-07 WO PCT/CN2018/099147 patent/WO2019119819A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105769910A (en) * | 2016-04-15 | 2016-07-20 | 遵义医学院附属医院 | Application of human amniotic mesenchymal stem cells |
| CN106038597A (en) * | 2016-05-27 | 2016-10-26 | 遵义医学院附属医院 | Application of mesenchyma stem cells to preparation of acute lung injury treating preparation |
Non-Patent Citations (2)
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| 崔晴晴: ""胎盘间充质干细胞在油酸型急性肺损伤中的炎症调节作用研究"", 《中国优秀硕士学位论文全文数据库·医药卫生科技辑》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019119819A1 (en) * | 2017-12-19 | 2019-06-27 | 杭州易文赛生物技术有限公司 | Application of placental pluripotent stem cell preparation in preparing drug for treating acute lung injury |
| CN109646457A (en) * | 2018-12-20 | 2019-04-19 | 杭州易文赛生物技术有限公司 | A kind of application of umbilical cord mesenchymal stem cells in preparation treatment acute lung injury drug |
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| WO2019119819A1 (en) | 2019-06-27 |
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