CN110055214A - Ginsenoside 20 (R) -25-OH-Rg3Promote the purposes of umbilical cord mesenchymal stem cells in-vitro multiplication - Google Patents
Ginsenoside 20 (R) -25-OH-Rg3Promote the purposes of umbilical cord mesenchymal stem cells in-vitro multiplication Download PDFInfo
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- CN110055214A CN110055214A CN201910365658.5A CN201910365658A CN110055214A CN 110055214 A CN110055214 A CN 110055214A CN 201910365658 A CN201910365658 A CN 201910365658A CN 110055214 A CN110055214 A CN 110055214A
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Abstract
The invention discloses ginsenoside 20 (R) -25-OH-Rg3Promote the purposes of umbilical cord mesenchymal stem cells in-vitro multiplication.It is a discovery of the invention that 20 (R) -25-OH-Rg3Although 20 (R)-Rg are weaker than to the proliferation of umbilical cord mesenchymal stem cells3, but 20 (R) -25-OH-Rg3It will not influence its phenotype while promoting umbilical cord mesenchymal stem cells proliferation, be able to maintain that its cell purity and stem cell properties, can be studied for umbilical cord mesenchymal stem cells and more seed cells are provided.
Description
Technical field
The invention belongs to stem cell fields, and in particular to ginsenoside 20 (R) -25-OH-Rg3Promote umbilical cord mesenchyma dry
The purposes of cell proliferation in vitro.
Background technique
Mescenchymal stem cell is also present in its hetero-organization of human body, such as fat, bleeding of the umbilicus, navel in addition to being present in marrow
Band, placental villi, gum and parodontium etc..Wherein, human umbilical cord mesenchymal stem cells (hUCMSCs) because source is wide, materials are convenient,
Immunogenicity is low, becomes the main force in stem-cell therapy to pregnant woman fetus no pain and not by numerous advantages such as ethics disputes
Army.
Umbilical cord mesenchymal stem cells research, it is necessary first to be exactly sufficient amount of umbilical cord mesenchymal stem cells.Between umbilical cord
Although mesenchymal stem cells proliferation rates in vitro with higher itself, it is not able to satisfy research and clinical needs.Therefore, promote
The research of umbilical cord mesenchymal stem cells amplification in vitro is of great significance.
The study found that ginsenoside 20 (R)-Rg3There is proliferation to a variety of mescenchymal stem cells.But 20
(R)-Rg3While promoting mescenchymal stem cell proliferation, it can also promote its differentiation, reduce stem cell properties, researching value
It reduces.
Therefore, it is necessary to develop the rush proliferation that can promote umbilical cord mesenchymal stem cells proliferation and significantly reduce its stemness
Ingredient.
Ginsenoside 20 (R) -25-OH-Rg3For ginsenoside 20 (R)-Rg3A kind of derivative, (the ginseng soap such as Yang Jiamei
Glycosides 20 (R) -25-OH-Rg3Isolate and purify and Structural Identification, Dalian Polytechnic University's journal, 03 phase in 2016) be disclosed one kind
Prepare ginsenoside 20 (R) -25-OH-Rg3Method.Have not yet to see ginsenoside 20 (R) -25-OH-Rg3Between promotion umbilical cord
The research of mesenchymal stem cell proliferation.
Summary of the invention
The purpose of the present invention is to overcome the deficiencies of prior art and provide ginsenoside 20 (R) -25-OH-Rg3Between promotion umbilical cord
The purposes of mesenchymal stem cells in-vitro multiplication, ginsenoside 20 (R) -25-OH-Rg3For ginsenoside 20 (R)-Rg3A kind of derivative
Object can maintain its stem cell properties while promoting umbilical cord mesenchymal stem cells proliferation, overcome 20 (R)-Rg3Lack
It falls into.
The object of the invention is achieved through the following technical solutions:
Ginsenoside 20 (R) -25-OH-Rg3For promoting the purposes of promotion umbilical cord mesenchymal stem cells in-vitro multiplication.
Ginsenoside 20 (R) -25-OH-Rg3It is used to prepare the use for promoting the drug of umbilical cord mesenchymal stem cells in-vitro multiplication
On the way.
Ginsenoside 20 (R) -25-OH-Rg3It is used to prepare the culture solution for promoting umbilical cord mesenchymal stem cells in-vitro multiplication
Purposes.
Control group, 20 (R)-Rg3Group and 20 (R) -25-OH-Rg3The growth curve of group hUCMSCs is as shown in Figure 1, from Fig. 1
It can be seen that 20 (R)-Rg3、20(R)-25-OH-Rg3The proliferation of hUCMSCs can be remarkably promoted, wherein 20 (R)-Rg3Than
20(R)-25-OH-Rg3Rush proliferation activity it is stronger.
Control group, 20 (R)-Rg3Group and 20 (R) -25-OH-Rg3The positive rate of group hUCMSCs marker is as shown in table 2, from
Table 2 is not expressed and is made as can be seen that control group hUCMSCs high expression hUCMSCs specific surface marker CD73, CD90 and CD105
The surface markers CD34 and human leucocyte common antigen CD45 of hemocytoblast, meet the phenotypic characteristic of mescenchymal stem cell；With
Control group is compared, 20 (R) -25-OH-Rg3Group hUCMSCs also high expression hUCMSCs specific surface marker CD73, CD90 and
CD105, and positive rate and control group be without significant difference, at the same do not express candidate stem cell surface markers CD34 and the mankind it is white thin
Born of the same parents' common antigen CD45；But compared with the control group, 20 (R)-Rg3Group hUCMSCs specific surface marker CD73, CD90 and
The positive rate of CD105 significantly reduces, and illustrates 20 (R)-Rg3There is apparent differentiation in group hUCMSCs, and hUCMSCs purity is significant
It reduces, this also complies with 20 (R)-Rg in the prior art3There is the report for promoting differentiation to mescenchymal stem cell.Fig. 2 is each group
HUCMSCs specific surface marker's CD73, CD90 and CD105 positive rate compares, and Fig. 3 is 20 (R) -25-OH-Rg3The streaming of group
Detection figure.
Advantageous effects:
It is a discovery of the invention that 20 (R) -25-OH-Rg3Although 20 are weaker than to the proliferation of umbilical cord mesenchymal stem cells
(R)-Rg3, but 20 (R) -25-OH-Rg3It will not influence its phenotype while promoting umbilical cord mesenchymal stem cells proliferation, it can
Its cell purity and stem cell properties are maintained, can be studied for umbilical cord mesenchymal stem cells and more seed cells are provided.
Detailed description of the invention
Fig. 1 is control group, 20 (R)-Rg3Group and 20 (R) -25-OH-Rg3The growth curve of group hUCMSCs；
Fig. 2 is each group hUCMSCs specific surface marker CD73, CD90 and CD105 positive rate；
Fig. 3 is 20 (R) -25-OH-Rg3The flow cytometer detection figure of group.
Specific embodiment
Essentiality content of the present invention is introduced below with reference to specific, but is not limited the scope of protection of the present invention with this.
One, experimental material
Umbilical cord derives from the puerpera of health full term production, and contributor signs informed consent form, it is known that and agree to be used for scientific research
Experiment.
Fetal calf serum, DMEM culture solution are purchased from Sigma-Aldrich, and 0.25% trypsase is purchased from the green skies.
20(R)-Rg3、20(R)-25-OH-Rg3Purity is not less than 98%.
The mouse antihuman CD 34 antibody of anti-human monoclonal antibody CD45, CD73, CD90, CD105 and FITC label of the mouse of PE label is purchased from
U.S. company BD；The anti-human PE/FITC-IgG1 of mouse is purchased from BioLegend.
Two, experimental method
1, hUCMSCs is separately cultured
According to literature method, umbilical cord is cut off, is cleaned for several times with PBS, is taken out huatong plastic, shred, with containing 0.02%
0.25% trypsin digestion of EDTA is added DMEM culture solution and terminates digestion to liquid muddiness, with 200 mesh net filtrations,
1800r/min is centrifuged 10min, discards supernatant liquid, leaves bottom cell, and the DMEM culture solution that 10% fetal calf serum is added is blown
It is even, it is then inoculated in sterile petri dish, is placed in 37 DEG C, volume fraction 5%CO2Incubator culture.It is changed liquid 1 time every 3d.
It is passed on after cell density reaches 80% in the ratio of 1:2.
2, hUCMSCs grouping and proliferative capacity measurement
It takes the 3rd generation human umbilical cord mesenchymal stem cells to carry out the measurement of ability of cell proliferation, is disappeared with 0.25% trypsase
Change human umbilical cord mesenchymal stem cells, supernatant is removed after centrifugation, the DMEM culture solution containing 10% fetal calf serum, which is added, is resuspended cell, adjusts
Whole cell concentration is to 1 × 105/ mL is inoculated in 48 porocyte culture plates, every hole 0.5mL cell suspension.It is grouped as follows at random:
Control group: with the DMEM culture solution culture containing 10%FBS；
20(R)-Rg3Group: with containing 10%FBS and 200nM 20 (R)-Rg3DMEM culture solution culture；
20(R)-25-OH-Rg3Group: with containing 10%FBS and 200nM 20 (R) -25-OH-Rg3DMEM culture solution culture；
After inoculated and cultured 4h, culture solution is replaced according to above-mentioned grouping, is placed in 37 DEG C, volume fraction 5%CO2Incubator training
It supports, every 3d is replaced culture medium 1 time.3 hole cells were taken respectively at the 3rd, 6,9,12 day, were counted after digestion with cell counter,
It is the longitudinal axis by horizontal axis, cell quantity of incubation time, draws the growth curve of each group human umbilical cord mesenchymal stem cells.
3, flow cytometry hUCMSCs purity
Take the 12nd day hUCMSCs of each group in " hUCMSCs grouping and proliferative capacity measurement ", digestion be resuspended cell to 1 ×
106/ mL, being fixed on volume fraction is after 4 DEG C are stayed overnight, to carry out cell surface marker for flow cytometer in 70% cold ethyl alcohol
Measurement.The mouse antihuman CD 34 that cell is marked with mouse anti-human monoclonal antibody CD45, CD73, CD90, CD105 and FITC of PE label respectively
Antibody response places 30min in 4 DEG C of darkroom.Using the anti-human PE/FITC-IgG1 of mouse as Isotype control.
Three, experimental result
1, the proliferative capacity of each group hUCMSCs
Control group, 20 (R)-Rg3Group and 20 (R) -25-OH-Rg3The growth curve of group hUCMSCs is as shown in Figure 1, from Fig. 1
It can be seen that 20 (R)-Rg3、20(R)-25-OH-Rg3The proliferation of hUCMSCs can be remarkably promoted, wherein 20 (R)-Rg3Than
20(R)-25-OH-Rg3Rush proliferation activity it is stronger.Table 1 is the cell number of each group different time points.
The cell number of 1 each group different time points of table
2, each group hUCMSCs purity
Control group, 20 (R)-Rg3Group and 20 (R) -25-OH-Rg3The positive rate of group hUCMSCs marker is as shown in table 2, from
Table 2 is not expressed and is made as can be seen that control group hUCMSCs high expression hUCMSCs specific surface marker CD73, CD90 and CD105
The surface markers CD34 and human leucocyte common antigen CD45 of hemocytoblast, meet the phenotypic characteristic of mescenchymal stem cell；With
Control group is compared, 20 (R) -25-OH-Rg3Group hUCMSCs also high expression hUCMSCs specific surface marker CD73, CD90 and
CD105, and positive rate and control group be without significant difference, at the same do not express candidate stem cell surface markers CD34 and the mankind it is white thin
Born of the same parents' common antigen CD45；But compared with the control group, 20 (R)-Rg3Group hUCMSCs specific surface marker CD73, CD90 and
The positive rate of CD105 significantly reduces, and illustrates 20 (R)-Rg3There is apparent differentiation in group hUCMSCs, and hUCMSCs purity is significant
It reduces, this also complies with 20 (R)-Rg in the prior art3There is the report for promoting differentiation to mescenchymal stem cell.Fig. 2 is each group
HUCMSCs specific surface marker's CD73, CD90 and CD105 positive rate compares, and Fig. 3 is 20 (R) -25-OH-Rg3The streaming of group
Detection figure.
The positive rate (%) of 2 each group hUCMSCs marker of table
| Group | CD73 | CD90 | CD105 | CD34 | CD45 |
| Control group | 99.05 | 99.23 | 99.19 | 0.00 | 0.62 |
| 20(R)-Rg3Group | 52.60 | 45.69 | 49.78 | 1.94 | 3.77 |
| 20(R)-25-OH-Rg3Group | 96.76 | 98.37 | 97.02 | 0.00 | 0.85 |
The above results show 20 (R) -25-OH-Rg3Although 20 are weaker than to the proliferation of umbilical cord mesenchymal stem cells
(R)-Rg3, but 20 (R) -25-OH-Rg3It will not influence its phenotype while promoting umbilical cord mesenchymal stem cells proliferation, it can
Its cell purity and stem cell properties are maintained, can be studied for umbilical cord mesenchymal stem cells and more seed cells are provided.
The effect of above-described embodiment is specifically to introduce essentiality content of the invention, but should not be by protection model of the invention
It encloses and is confined to above-mentioned specific embodiment.
Claims (3)
- Ginsenoside 20 1. (R) -25-OH-Rg3For promoting the purposes of promotion umbilical cord mesenchymal stem cells in-vitro multiplication.
- Ginsenoside 20 2. (R) -25-OH-Rg3It is used to prepare the purposes for promoting the drug of umbilical cord mesenchymal stem cells in-vitro multiplication.
- Ginsenoside 20 3. (R) -25-OH-Rg3It is used to prepare the use for promoting the culture solution of umbilical cord mesenchymal stem cells in-vitro multiplication On the way.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111662868A (en) * | 2020-07-20 | 2020-09-15 | 淮安泰凯睿医药科技有限公司 | CXCR4 agonist and application thereof in-vitro culture of umbilical cord mesenchymal stem cells |
| CN113046306A (en) * | 2021-03-12 | 2021-06-29 | 广东东阳光药业有限公司 | Culture method of pluripotent stem cells |
| CN113930396A (en) * | 2020-06-29 | 2022-01-14 | 华子昂 | Method for enhanced removal of damaged stem cell seeds by using p53 |
-
2019
- 2019-05-02 CN CN201910365658.5A patent/CN110055214B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| 杨佳美等: "人参皂苷20(R)-25-OH-Rg3的分离纯化及结构鉴定", 《大连工业大学学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113930396A (en) * | 2020-06-29 | 2022-01-14 | 华子昂 | Method for enhanced removal of damaged stem cell seeds by using p53 |
| CN111662868A (en) * | 2020-07-20 | 2020-09-15 | 淮安泰凯睿医药科技有限公司 | CXCR4 agonist and application thereof in-vitro culture of umbilical cord mesenchymal stem cells |
| CN113046306A (en) * | 2021-03-12 | 2021-06-29 | 广东东阳光药业有限公司 | Culture method of pluripotent stem cells |
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