CN109966500A - Liquid is protected containing polysaccharides and its copes with the application method in internal Pathologic niche improving mescenchymal stem cell - Google Patents
Liquid is protected containing polysaccharides and its copes with the application method in internal Pathologic niche improving mescenchymal stem cell Download PDFInfo
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Abstract
Liquid is protected containing polysaccharides and its is improving the application method in the internal complicated Pathologic niche of mescenchymal stem cell reply.This method uses separation, culture, the identification of mescenchymal stem cell；The protection liquid containing polysaccharides is identified and is protected liquid ingredient, proportion using effect；Protect the methods of liquid mechanism of action step; using natural drug extract polysaccharides as main component; substitute the methods of existing gene editing or chemicals processing; that is: intervene mescenchymal stem cell using pure natural substance; so that it is preferably adapted to complicated Pathologic niche in vivo, improves survival ability and treatment potentiality.The present invention protects liquid to can significantly promote the survival rate of mescenchymal stem cell Pathologic niche in vivo, promotes therapeutic effect.
Description
Technical field
The present invention relates to wolfberry fruit extracts in the new application of biomedicine field, and it is more to contain fructus lycii more particularly, to one kind
Sugar protection liquid and its application method in the reply of raising mescenchymal stem cell in vivo complicated Pathologic niche.
Background technique
Mescenchymal stem cell (mesenchymal stem cells, MSCs) is that one kind is from a wealth of sources, is derived from embryonic development
Mesoblastic multipotency precursor in early days has self-renewing and the potential to three all cell type differentiations of germinal layer.Base
In self-renewing, multi-lineage potential and immunoloregulation function so that MSCs is compared with traditional treatment means, in tissue repair and
Immunological regulation etc. has unrivaled treatment advantage.It is including diabetes, cardiovascular disease that clinical research, which has proven to MSCs,
Subversive treatment is shown in the treatment of numerous diseases such as disease, autoimmune disease, mental disorder, hepatic failure, cancer
Effect has very strong clinical Transformation Potential.
Although MSCs shows powerful therapeutic effect in basis and clinical research, when MSCs enters patient's body
Afterwards, the harsh Pathologic niche built by elements such as blood oxygen missing, free radical, inflammation, active amino acids, causes MSCs moving
It is faced with the change of dead threat and function in a couple of days after plant, seriously affects therapeutic effect.Therefore, seek a kind of method,
Break the factor of limitation MSCs curative effect, improves survival ability of the MSCs in Pathologic niche, maintain every biology of MSCs
Function probes into regulatory mechanism and clinical meaning that may be present, is key scientific problems urgently to be resolved.
Currently, for survival rate, proliferative capacity, differentiation potential and paracrine water in raising MSCs in vivo Pathologic niche
It is flat, mainly it is achieved by the methods of genetic modification, bioactive molecule pretreatment, culture environment pretreatment.However, existing
Technical method, gene editing or chemicals intervention on the one hand carried out to cell, influence cell that can be different degrees of is normal
Physiology and metabolic activity, it is possible to change the original biological characteristics of cell and the potentiality to tumour cell vicious transformation,
There are unknown safety problems, seriously affect the clinical conversion of mescenchymal stem cell；On the other hand, there is limitation in intervention effect
Property, i.e. the condition just for a kind of internal Pathologic niche can not give the disease that mescenchymal stem cell copes with internal complexity comprehensively
Manage the ability of microenvironment.
Therefore, there is an urgent need in the art to develop it is a kind of safely and effectively protect liquid and be suitable for enhancing mescenchymal stem cell support
Pathologic niche in antibody.
Summary of the invention
In view of the deficiencies of the prior art, it is an object of the present invention to provide one kind containing polysaccharides protection liquid and its between raising
Application in the internal complicated Pathologic niche of mesenchymal stem cells reply.
Polysaccharides (lycium barbarum polysaccharide, LBP) is from more points of the medicinal Solanaceae of Ningxia characteristic
A kind of water-soluble polysaccharide extracted in branch shrub plant fructus lycii.Studies have shown that LBP is in anti-oxidant, immunological regulation, anti-aging, rush
Important biological action is played into radiation and the recovery of chemotherapy etc..To diabetes, Cranial nerve injury as birth trauma reparation, arthritis and
The diseases such as antitumor have potential therapeutic effect.
Polysaccharides belongs to natural plant extracts, through long-term application and research confirmation with very strong safety and extensively
The biological function of spectrum.The present invention sufficiently confirms that polysaccharides as main component, adapts in vivo again improving mescenchymal stem cell
Play important biological function in miscellaneous harsh Pathologic niche, compared with art methods, safety, in terms of
With unrivaled advantage.
Technical scheme is as follows: containing polysaccharides protection liquid and its improving mescenchymal stem cell reply disease in vivo
Manage the application method in microenvironment.
Liquid constituent and proportion are protected containing polysaccharides are as follows: polysaccharides 20-100g, human serum albumins 20-50g,
Mentioned component is pressed necessary requirement by Catergen 00-250mg, glutathione 80-100 mg, the phosphate buffer 1L of PH=7.2
It is mixed evenly and obtains this preparation.
Coping with the application method in internal Pathologic niche in raising mescenchymal stem cell containing polysaccharides protection liquid includes
Following steps:
1, separation, culture, the identification of mescenchymal stem cell
(1) separation and free serum culture of mescenchymal stem cell:
The placenta tissue for collecting health full term puerpera, takes the tissue of about 0.5 cm thickness of placenta side, is cut into 1 mm3Fragment, use
After HBSS is sufficiently rinsed, the Collagenase A and 1 μ g/mL DNase I of 1 g/L, 37 DEG C of water-bath digestion 2.0 are added
h.It collects the cell in supernatant and is transferred to serum free medium (STEMCELL Technologies), 37 DEG C, 5% carbon dioxide
It is cultivated in incubator.Liquid is changed for the first time after 24 h, when cell 80% ~ 90% merges, is passed on 1: 3 ratio.
(2) identification of mescenchymal stem cell specific markers:
Mescenchymal stem cell is prepared into individual cells suspension, according to specification, Xiang Jiayou 1 × 106The fluidic cell of a cell
Pipe is separately added into mAb:IgG2a-FITC, IgG1-PE, CD14-FITC, CD34-FITC, CD45- of fluorophor label
FITC, CD73-PE, CD90-FITC, CD105-PE, HLA-DR-FITC and isotype control Ab, until final volume is 100 μ L；It keeps away
Light is stored at room temperature 20 min, after PBS rinsing, Flow cytometry.
(3) Derived from Mesenchymal Stem Cells potential is identified:
1 × 10 is pressed respectively5A/hole inoculation mescenchymal stem cell converges to about 80% in 6 orifice plates, when cell is grown, and discards former culture
Base is separately added into fat cell induction liquid (containing 10% 1 nmol of FBS/0. 2 mmol of L dexamethasone/L Indomethacin 0.
5 mmol/L 3-isobutyl-1-methylxanthine and 10 mg/L insulin DMEM in high glucose culture medium) and skeletonization it is thin
Born of the same parents induce liquid (to contain 10% 1 nmol of FBS0./10 mmol of L dexamethasone/L-sodium glycero-phosphate and 0. 5 mmol
The DMEM in high glucose of/L ascorbic acid), it cultivates in the cell incubator of 37 DEG C of 5% CO2 saturated humidity and changes 2 not good liquors weekly,
It after culture 2~3 weeks, is dyed respectively with oil red O and alizarin red agent, inverted microscope observation.
2, the protection liquid containing polysaccharides is identified and is protected liquid ingredient, proportion using effect
(1) protection liquid handles mescenchymal stem cell:
The mescenchymal stem cell for choosing 2nd generation normal growth, after trypsin digestion, with 6000-8000 cell/cm2It connects
Kind is in culture dish.When cell grows to the degrees of fusion of 60-70%, add in cell culture fluid according to the dilution ratio of 1:100
Enter to protect liquid, after continuing culture for 24 hours, with trypsin digestion and collects cell, for use.
The ingredient of the protection liquid containing polysaccharides, proportion are as follows: phosphate buffer (PH=7.2) 1L；Polysaccharides 50g；Human serum
Albumin 50g；Catergen 50mg；100 mg of glutathione.
(2) animal packet, prepare mouse pulmonary fibrosis model and mescenchymal stem cell injection:
Male 6 week old C57BL/6J mouse 40 are taken, bleomycin group, mescenchymal stem cell treatment group and protection are randomly divided into
Liquid intervention mescenchymal stem cell group, negative control group, every group each 10.Through 10 g of intraperitoneal injection/L yellow Jackets anesthesia, win
Bleomycin group, mescenchymal stem cell treatment group and protection liquid intervene 50 μ L of injection in mescenchymal stem cell group difference transtracheal and win Lay
Mycin (1 g/L) prepares pulmonary fibrosis model, injects 50 μ L PBS in negative control group transtracheal.It utilizes
The Dil Stain of ThermoFisher marks mescenchymal stem cell, and 200 μ L of tail vein injection contains 5 × 105The PBS of a cell is molten
Liquid intervenes the pulmonary fibrosis model mouse of mescenchymal stem cell group to mescenchymal stem cell treatment group and protection liquid.
(3) the effect of protection liquid influence mescenchymal stem cell treatment pulmonary fibrosis, is assessed:
5 mouse of the 7th day each experimental group after modeling are taken, lungs frozen section is prepared, lung tissue is observed in the micro- border of fluorescence co-focusing
The quantity and distribution situation of middle mescenchymal stem cell determine protection liquid in mescenchymal stem cell to pulmonary fibrosis resistant Pathologic niche
In effect；5 mouse of the 21st day each experimental group after modeling are taken, the paraffin section of each group mouse lung is prepared, is dyed through HE, root
Pulmonary alveolitis and pulmonary fibrosis degree are determined according to Ashcroft methods of marking.Meanwhile Masson dyeing is carried out, observe lung tissue
The deposition of middle collagen.
(4) protection liquid improves the proliferative capacity of mescenchymal stem cell:
The mescenchymal stem cell for choosing 2nd generation normal growth is inoculated in 96 after trypsin digestion with 1000 cells/wells
Orifice plate, meanwhile, protection liquid is added according to the ratio of 1:100.Using CCK8 detection for 24 hours, the increasing of 48h, 72h, 96h and 120h cell
Situation is grown, using the cell of unprotected liquid as control group.
(5) protection liquid improves viability of the mescenchymal stem cell in pulmonary fibrosis Pathologic niche
Mouse pulmonary fibrosis model is prepared using bleomycin, collects the 7th day after modeling bronchoalveolar lavage fluid (BALF).By pancreas egg
Mescenchymal stem cell after white enzymic digestion is inoculated in 96 orifice plates with 2000 cells/wells, meanwhile, it is added according to the ratio of 1:100
Protect liquid and BALF.The proliferative conditions that cell after culture 72h is detected using CCK8, using the cell of unprotected liquid as control group.
(6) protection liquid inhibits apoptosis of the mescenchymal stem cell in pulmonary fibrosis Pathologic niche
Mescenchymal stem cell after trypsin digestion is inoculated in 35mm culture dish, when cell grows to 60-70%, is pressed
According to the method for 4.2.5, handle cell using protection liquid and BALF, for 24 hours after, utilize Annexin V & Propidium Iodide
(PI) the apoptosis situation of apoptosis kit detection cell, using the cell of unprotected liquid as control group.
3, liquid mechanism of action is protected:
(1) protection liquid influences the generation of mescenchymal stem cell autophagy in pulmonary fibrosis model
Through 10 g of intraperitoneal injection/L yellow Jackets anesthesia, 50 μ L bleomycin (1 g/L) of injection prepare lung in transtracheal
Fibrosis model, using mRFP-GFP-LC3 adenovirus infection mescenchymal stem cell, the 3rd day tail vein injection is normal after modeling
With protection liquid pretreatment Human plactnta source MSCs for 24 hours, it is total that lungs frozen section for 24 hours, fluorescence after cell infusion are prepared respectively
The case where focusing the mescenchymal stem cell dotted aggregation of appearance intracellular in micro- border observation lung tissue.
(2) generation of mescenchymal stem cell autophagy in liquid that modulates fibrosis Pathologic niche is protected
Mouse pulmonary fibrosis model is prepared using bleomycin, collects the 7th day after modeling bronchoalveolar lavage fluid (BALF).According to 1:
Above-mentioned BALF is added after pre-processing Human plactnta derived mesenchymal stem cell for 24 hours with protection liquid in 100 ratio, and it is total to extract cell
Protein, western trace detect the expression of autophagy marker molecule LC3I/II and Beclin1.Utilize mRFP-GFP-LC3
Adenovirus infection mescenchymal stem cell handles cell, cell climbing sheet, fluorescence co-focusing with protection liquid and BALF according to the method described above
Dotted aggregation in micro- border observation cell determines the generation of autophagy and the carry out situation of autophagy damp (autophagic flux).
(3) identification of Nrf2/ARE signal path regulating and controlling effect: certainly according to mescenchymal stem cell in aforementioned Pathologic niche
The method for generation processing mescenchymal stem cell bitten, extracts total cellular protein, western trace detects Nrf2 and its downstream albumen
The expression of DELTA rHO-1 (heme oxygenase-1, HO-1).Determine Nrf2/ARE signal path and protection liquid
Regulate and control the correlation of mescenchymal stem cell autophagy in pulmonary fibrosis Pathologic niche.
The present invention has following remarkable result:
The present invention is substituted at existing gene editing or chemicals using natural drug extract polysaccharides as main component
The methods of reason, it may be assumed that intervene mescenchymal stem cell using pure natural substance, it is made preferably to adapt to complicated Pathologic niche in vivo,
Improve survival ability and treatment potentiality.Said preparation can significantly promote the survival rate of mescenchymal stem cell Pathologic niche in vivo,
Promote therapeutic effect.It is embodied in the following aspects:
1, polysaccharides belongs to food and medicament dual-purpose plant, and by long-term numerous studies and application, safety has been obtained sufficiently
Certainly；
2, the genetic modification of the invention not being related to cell, signal path chemical induction and other improper physiology means is dry
In advance；
3, first passage experiment in vitro confirmation of the present invention invents protection liquid and has raising mescenchymal stem cell proliferative capacity, inhibits
The effect of cell ageing；
4, the biological function that there is protection liquid raising mescenchymal stem cell to resist complicated Pathologic niche in vivo, the present invention are invented
It is confirmed for the first time by zoopery；
5, application of the protection liquid in mescenchymal stem cell is invented, it is easy to operate, timeliness is good.
Detailed description of the invention
Fig. 1 is the morphological observation figure of free serum culture Human plactnta derived mesenchymal stem cell of the present invention；
Fig. 2 is Flow cytometry free serum culture hfPMSC surface marker schematic diagram of the present invention；
Fig. 3 is that free serum culture Human plactnta fetal origin MSCs differentiation potential of the present invention analyzes schematic diagram；
Fig. 4 is that present invention protection liquid promotes Human plactnta derived mesenchymal stem cell to inhibit pulmonary fibrosis schematic diagram；
Fig. 5 is that present invention protection liquid improves PMSCs to the inhibiting effect schematic diagram of mouse pulmonary fibrosis model collagen deposition；
Fig. 6 is the survival rate schematic diagram that present invention protection liquid improves PMSCs in pulmonary fibrosis mice model；
Fig. 7 is the proliferative capacity schematic diagram that present invention protection liquid improves Human plactnta mescenchymal stem cell；
Fig. 8 is that present invention protection liquid enhances viability signal of the Human plactnta mescenchymal stem cell in pulmonary fibrosis Pathologic niche
Figure；
Fig. 9 is the apoptosis signal that present invention protection liquid significantly inhibits Human plactnta mescenchymal stem cell in pulmonary fibrosis Pathologic niche
Figure；
Figure 10 is the generation schematic diagram that present invention protection liquid improves fPMSCs autophagy in pulmonary fibrosis mice model；
Figure 11 is the generation schematic diagram that present invention protection liquid raises PMSCs autophagy in pulmonary fibrosis Pathologic niche；
Figure 12 is the Nrf2/ARE signal path schematic diagram that present invention protection liquid raises PMSCs in pulmonary fibrosis Pathologic niche.
Specific embodiment
Technical solution of the present invention is clearer, is readily appreciated that in order to make, specific to the present invention below in conjunction with Detailed description of the invention
Embodiment is further described.
Liquid constituent and proportion are protected containing polysaccharides are as follows: polysaccharides 20-100g, human serum albumins 20-50g,
Mentioned component is pressed necessary requirement by vitamin C 200-250mg, glutathione 80-100 mg, the phosphate buffer 1L of PH=7.2
Filtering is mixed evenly and obtains this preparation.
The application method in the internal complicated Pathologic niche of mescenchymal stem cell reply is being improved containing polysaccharides protection liquid
Specific steps are as shown in the picture:
As shown in Figure 1, collecting the placenta tissue of health full term puerpera, the tissue of about 0.5 cm thickness of placenta side is taken, is cut into 1
mm3Fragment, after sufficiently being rinsed with HBSS, be added 1 g/L Collagenase A and 1 μ g/mL DNase I, 37
DEG C water-bath digests 2.0 h.It collects the cell in supernatant and is transferred to serum free medium (STEMCELL Technologies),
It 37 DEG C, cultivates in 5% carbon dioxide incubator.Liquid is changed for the first time after 24 h, when cell 80% ~ 90% merges, with 1: 3 ratio
Example passage.3rd generation Human plactnta derived mesenchymal stem cell of serum free medium culture, form is uniform, it is adherent, be vortexed in shuttle shape
Formula growth, meets typical mescenchymal stem cell form (40 ×).
As shown in Fig. 2, mescenchymal stem cell is prepared into individual cells suspension, according to specification, Xiang Jiayou 1 × 106It is a
The fluidic cell pipe of cell is separately added into mAb:IgG2a-FITC, IgG1-PE, CD14-FITC, CD34- of fluorophor label
FITC, CD45-FITC, CD73-PE, CD90-FITC, CD105-PE, HLA-DR-FITC and isotype control Ab, until final volume
For 100 μ L；It is protected from light, is stored at room temperature 20 min, after PBS rinsing, Flow cytometry.The hfPMSC of free serum culture is
CD73, CD90 and CD105 positive cell, and hematopoietic cell marker molecule CD14, CD34, CD45 and HLA-DR are negative, and have
The surface markers of typical mescenchymal stem cell.
As shown in figure 3, pressing 1 × 10 respectively5A/hole inoculation mescenchymal stem cell is in 6 orifice plates, when cell growth converges to about
80%, former culture medium is discarded, is separately added into fat cell induction liquid (containing 10% 1 nmol of FBS/0. 2 mmol of L dexamethasone
0. 5 mmol of/L Indomethacin/L 3-isobutyl-1-methylxanthine and 10 mg/L insulin DMEM in high glucose culture medium)
And osteoblast induction liquid (contains 10% 1 nmol of FBS0./10 mmol of L dexamethasone/L-sodium glycero-phosphate
With 0. 5 mmol/L ascorbic acid DMEM in high glucose), it cultivates in the cell incubator of 37 DEG C of 5% CO2 saturated humidity
It changes 2 not good liquors weekly to be dyed with oil red O and alizarin red agent respectively after culture 2~3 weeks, inverted microscope observation.
Human plactnta fetus side derived cell can be respectively formed the calcium of the fat drips (A) for being colored as shiny red and red after induction
Tubercle deposit (B) prompts separated cell to have multi-lineage potential, meets the characteristic of mescenchymal stem cell.
As shown in figure 4, protection liquid promotes Human plactnta derived mesenchymal stem cell to inhibit pulmonary fibrosis.It is normal to choose 2nd generation
The mescenchymal stem cell of growth, after trypsin digestion, with 6000-8000 cell/cm2It is inoculated in culture dish.To cell
When growing to the degrees of fusion of 60-70%, protection liquid is added in cell culture fluid according to the dilution ratio of 1:100, continues to cultivate
After for 24 hours, with trypsin digestion and cell is collected, for use.
Diagram are as follows: A.HE staining analysis bleomycin handles Human plactnta derived mesenchymal stem cell and protection after mouse 21d
The therapeutic effect of mescenchymal stem cell after liquid processing handles the mouse of 21d as control group (40 using normal and bleomycin
×)；B. the Ashscroft score of fibrosis analyzes (n=5) (p < 0.05 *).The results show that protection liquid fills between being remarkably improved
The effect of matter stem cell improvement pulmonary fibrosis.
In figure: Control indicates normal mouse；Bleo indicates bleomycin processing；PMSCs indicates to fill between Human plactnta source
Matter stem cell；FPMSCs indicates Human plactnta source MSCs after protection liquid processing.
As shown in figure 5, protection liquid improves PMSCs to the inhibiting effect of mouse pulmonary fibrosis model collagen deposition.A. Ma Sen
After staining analysis bleomycin handles Human plactnta source Human plactnta derived mesenchymal stem cell after mouse 21d, and protection liquid processing
Influence of the mescenchymal stem cell to its collagen deposition handles the mouse of 21d as control group using normal and bleomycin
(400 ×)；B. the quantitative analysis (n=5) (p < 0.05 *) of lungs collagen content.The result shows that protection liquid remarkably promotes mesenchyma
The inhibiting effect that stem cell generates collagen in pathological tissue.
Note: Control: normal mouse；Bleo: bleomycin processing；PMSCs: Human plactnta derived mesenchymal stem cell；
PMSCsLBP: Human plactnta derived mesenchymal stem cell after protection liquid processing.
As shown in fig. 6, preparing mouse pulmonary fibrosis model using bleomycin, the 7th day after modeling alveolar wass is collected
Liquid (BALF).By the mescenchymal stem cell after trypsin digestion, 96 orifice plates are inoculated in 2000 cells/wells, meanwhile, according to
Protection liquid and BALF is added in the ratio of 1:100.The proliferative conditions of cell after culture 72h are detected, using CCK8 with unprotected liquid
Cell be control group.The results show that protecting liquid in the Pathologic niche that pulmonary fibrosis model mouse bronchoalveolar lavage fluid is built
It is obviously improved the proliferative capacity of mescenchymal stem cell.
In figure, the normal PMSCs of A.；B. protection liquid pre-processes PMSCs.PMSCs is handled using Dil Stain, it will be normal
MSCs prepares the 3rd day pulmonary fibrosis mice mould with the MSCs after protection liquid pretreatment for 24 hours, tail vein injection to bleomycin
Type takes after 5d lung tissue to prepare frozen section, and fluorescence microscope mescenchymal stem cell is in lung tissue after DAPI dyes mounting
In there are situation (red-label) (40 ×).PMSCs is Human plactnta derived mesenchymal stem cell.The result shows that at protection liquid
Mescenchymal stem cell after reason still has the survival ability for improving mescenchymal stem cell in Pathological pattern animal body.
As shown in fig. 7, the mescenchymal stem cell of 2nd generation normal growth is chosen, it is thin with 1000 after trypsin digestion
Born of the same parents/hole is inoculated in 96 orifice plates, meanwhile, protection liquid is added according to the ratio of 1:100.Using CCK8 detection for 24 hours, 48h, 72h, 96h
With the proliferative conditions of 120h cell, using the cell of unprotected liquid as control group.The result shows that protection liquid can be more preferable in vitro
Ground maintains the long-term cultivation of mescenchymal stem cell, keeps higher proliferative capacity.
As shown in figure 8, viability of the protection liquid enhancing Human plactnta mescenchymal stem cell in pulmonary fibrosis Pathologic niche
Schematic diagram prepares mouse pulmonary fibrosis model using bleomycin, collects the 7th day after modeling bronchoalveolar lavage fluid (BALF).It will
Mescenchymal stem cell after trypsin digestion is inoculated in 96 orifice plates with 2000 cells/wells, meanwhile, according to the ratio of 1:100
Protection liquid and BALF is added.The proliferative conditions of cell after culture 72h are detected using CCK8, are control with the cell of unprotected liquid
Group.It is filled the results show that protection liquid is added and can significantly improve Human plactnta mescenchymal stem cell in pulmonary fibrosis model mouse alveolar
Survival rate in washing.
As shown in figure 9, protection liquid significantly inhibits the apoptosis of Human plactnta mescenchymal stem cell in pulmonary fibrosis Pathologic niche
Schematic diagram.Mescenchymal stem cell after trypsin digestion is inoculated in 35mm culture dish, when cell grows to 60-70%,
Cell is handled using protection liquid and BALF.It is shown in figure: mouse pulmonary fibrosis model is prepared using bleomycin, after collecting modeling
7th day bronchoalveolar lavage fluid (BALF).By Human plactnta mescenchymal stem cell kind in 35mm culture dish, reach to cell fusion degree
When 60-70%, protection liquid and/or BALF is added according to the ratio of 1:100.Utilize Dead Cell Apoptosis Kit with
Cell after the detection culture for 24 hours of 488 & Propidium Iodide (PI) kit of Annexin V Alexa Fluor
Apoptosis situation, using the cell of unprotected liquid as control group.The results show that pulmonary fibrosis model mouse alveolar wass can induce people
Apoptosis occurs for placenta mesenchyma stem cell, but protection liquid can significantly inhibit the apoptosis of cell.
As shown in Figure 10, protection liquid improves the generation schematic diagram of fPMSCs autophagy in pulmonary fibrosis mice model.In figure: A.
Normal PMSCs；B. protection liquid pre-processes PMSCs.Using mRFP-GFP-LC3 adenovirus infection PMSCs, by normal PMSCs and warp
Protect PMSCs after liquid pretreatment for 24 hours, tail vein injection to bleomycin prepares the 7th day pulmonary fibrosis mice model, for 24 hours after
Take lung tissue to prepare frozen section, DAPI dye after mounting fluorescence microscope PMSCs autophagy there is a situation where and degree (point
Shape aggregation) (400 ×).The results show that protection liquid can significantly raise the hair of PMSCs autophagy in pulmonary fibrosis mice model
It is raw.
As shown in figure 11, protection liquid raises the generation schematic diagram of PMSCs autophagy in pulmonary fibrosis Pathologic niche.Through abdominal cavity
10 g/L yellow Jackets anesthesia is injected, 50 μ L bleomycin (1 g/L) of injection prepare pulmonary fibrosis model in transtracheal,
Using mRFP-GFP-LC3 adenovirus infection mescenchymal stem cell, the 3rd day tail vein injection is normally after modeling and protection liquid is pre-
Processing Human plactnta source MSCs for 24 hours prepares lungs frozen section for 24 hours, the micro- border of fluorescence co-focusing after cell infusion respectively
The case where observing the mescenchymal stem cell dotted aggregation of appearance intracellular in lung tissue.In figure: A. protects liquid pretreatment PMSCs for 24 hours,
The 7th day bronchoalveolar lavage fluid of pulmonary fibrosis mice model is added and continues culture for 24 hours, western blot detects PMSCs autophagy mark point
The expression of sub- Beclin1 and LC3I/II；B. in quantitative analysis A Beclin1 and LC3-II expression (p < 0.05 *).As a result
Show to protect liquid that can raise PMSCs autophagy in in-vitro simulated pulmonary fibrosis microenvironment, protection liquid is prompted to reach by regulating and controlling autophagy
Improve the effect of PMSCs reply pulmonary fibrosis Pathologic niche.
As shown in figure 12, the present invention protects the Nrf2/ARE signal of PMSCs in liquid up-regulation pulmonary fibrosis Pathologic niche logical
Road schematic diagram.
In figure: A. protects liquid pretreatment PMSCs for 24 hours, and the 7th day bronchoalveolar lavage fluid of pulmonary fibrosis mice model is added and continues
For 24 hours, western blot detects the expression of Nrf2 and HO-1 in PMSCs for culture；B. Nrf2 and HO-1 in quantitative analysis A
It expresses (p < 0.05 *).The results show that protection liquid can raise the table of PMSCs Nrf2 and HO-1 in pulmonary fibrosis Pathologic niche
It reaches, Nrf2 signal path is prompted to influence to play important regulating and controlling effect in PMSCs biological function in protection liquid.
The present invention using natural drug extract polysaccharides as protection liquid main component, substitute existing gene editing or
The methods of chemicals processing makes it preferably adapt to complexity in vivo by intervening mescenchymal stem cell using pure natural substance
Pathologic niche improves survival ability and treatment potentiality.
Claims (2)
1. protecting liquid containing polysaccharides, which is characterized in that the constituent and proportion of the protection liquid are as follows: polysaccharides 20-100g,
Human serum albumins 20-50g, Catergen 00-250mg, glutathione 80-100 mg, the phosphate buffer 1L of PH=7.2 will
Mentioned component is mixed evenly filtering by necessary requirement and obtains this preparation.
2. coping with the application method in internal Pathologic niche, feature in raising mescenchymal stem cell containing polysaccharides protection liquid
Be, the application method the following steps are included:
(1) separation, culture, the identification of mescenchymal stem cell:
1. the separation and free serum culture of mescenchymal stem cell:
The placenta tissue for collecting health full term puerpera, takes the tissue of about 0.5 cm thickness of placenta side, is cut into 1 mm3Fragment, use
After HBSS is sufficiently rinsed, the Collagenase A and 1 μ g/mL DNase I of 1 g/L, 37 DEG C of water-bath digestion 2.0 are added
H collects the cell in supernatant and is transferred to serum free medium (STEMCELL Technologies), 37 DEG C, 5% carbon dioxide
It is cultivated in incubator, changes liquid for the first time after 24 h, when cell 80% ~ 90% merges, passed on 1: 3 ratio；
2. the identification of mescenchymal stem cell specific markers:
Mescenchymal stem cell is prepared into individual cells suspension, according to specification, Xiang Jiayou 1 × 106The fluidic cell of a cell
Pipe is separately added into mAb:IgG2a-FITC, IgG1-PE, CD14-FITC, CD34-FITC, CD45- of fluorophor label
FITC, CD73-PE, CD90-FITC, CD105-PE, HLA-DR-FITC and isotype control Ab, until final volume is 100 μ L；It keeps away
Light is stored at room temperature 20 min, after PBS rinsing, Flow cytometry；
3. Derived from Mesenchymal Stem Cells potential is identified:
1 × 10 is pressed respectively5A/hole inoculation mescenchymal stem cell converges to about 80% in 6 orifice plates, when cell is grown, and discards former culture
Base is separately added into fat cell induction liquid and osteoblast induction liquid, the cell culture of 37 DEG C of 5% CO2 saturated humidity
It is cultivated in case and changes 2 not good liquors weekly, after culture 2~3 weeks, dyed respectively with oil red O and alizarin red agent, inverted microscope observation；
(2) the protection liquid containing polysaccharides is identified and is protected liquid ingredient, proportion using effect:
1. liquid is protected to handle mescenchymal stem cell:
The mescenchymal stem cell for choosing 2nd generation normal growth, after trypsin digestion, with 6000-8000 cell/cm2Inoculation
In culture dish.When cell grows to the degrees of fusion of 60-70%, it is added in cell culture fluid according to the dilution ratio of 1:100
Liquid is protected, after continuing culture for 24 hours, with trypsin digestion and collects cell, for use；
2. animal packet prepares mouse pulmonary fibrosis model and mescenchymal stem cell injection:
Male 6 week old C57BL/6J mouse 40 are taken, bleomycin group, mescenchymal stem cell treatment group and protection are randomly divided into
Liquid intervention mescenchymal stem cell group, negative control group, every group each 10.Through 10 g of intraperitoneal injection/L yellow Jackets anesthesia, win
Bleomycin group, mescenchymal stem cell treatment group and protection liquid intervene 50 μ L of injection in mescenchymal stem cell group difference transtracheal and win Lay
Mycin (1 g/L) prepares pulmonary fibrosis model, injects 50 μ L PBS in negative control group transtracheal, utilizes
The Dil Stain of ThermoFisher marks mescenchymal stem cell, and 200 μ L of tail vein injection contains 5 × 105The PBS of a cell is molten
Liquid intervenes the pulmonary fibrosis model mouse of mescenchymal stem cell group to mescenchymal stem cell treatment group and protection liquid；
It is assessed 3. protection liquid influences the effect of mescenchymal stem cell treatment pulmonary fibrosis:
5 mouse of the 7th day each experimental group after modeling are taken, lungs frozen section is prepared, lung tissue is observed in the micro- border of fluorescence co-focusing
The quantity and distribution situation of middle mescenchymal stem cell determine protection liquid in mescenchymal stem cell to pulmonary fibrosis resistant Pathologic niche
In effect, take 5 mouse of the 21st day each experimental group after modeling, prepare the paraffin section of each group mouse lung, dyed through HE, root
Pulmonary alveolitis and pulmonary fibrosis degree are determined according to Ashcroft methods of marking, meanwhile, Masson dyeing is carried out, lung tissue is observed
The deposition of middle collagen；
4. liquid is protected to improve the proliferative capacity of mescenchymal stem cell:
The mescenchymal stem cell for choosing 2nd generation normal growth is inoculated in 96 after trypsin digestion with 1000 cells/wells
Orifice plate, meanwhile, protection liquid is added according to the ratio of 1:100.Using CCK8 detection for 24 hours, the increasing of 48h, 72h, 96h and 120h cell
Situation is grown, using the cell of unprotected liquid as control group；
5. liquid is protected to improve viability of the mescenchymal stem cell in pulmonary fibrosis Pathologic niche:
Mouse pulmonary fibrosis model is prepared using bleomycin, the 7th day after modeling bronchoalveolar lavage fluid (BALF) is collected, by pancreas egg
Mescenchymal stem cell after white enzymic digestion is inoculated in 96 orifice plates with 2000 cells/wells, meanwhile, it is added according to the ratio of 1:100
Liquid and BALF are protected, the proliferative conditions of cell after culture 72h are detected using CCK8, using the cell of unprotected liquid as control group；
6. liquid is protected to inhibit apoptosis of the mescenchymal stem cell in pulmonary fibrosis Pathologic niche:
Mescenchymal stem cell after trypsin digestion is inoculated in 35mm culture dish, when cell grows to 60-70%, is pressed
According to the method for 4.2.5, handle cell using protection liquid and BALF, for 24 hours after, utilize Annexin V & Propidium Iodide
(PI) the apoptosis situation of apoptosis kit detection cell, using the cell of unprotected liquid as control group；
(3) liquid mechanism of action is protected:
1. liquid is protected to influence the generation of mescenchymal stem cell autophagy in pulmonary fibrosis model:
Through 10 g of intraperitoneal injection/L yellow Jackets anesthesia, 50 μ L bleomycin (1 g/L) of injection prepare lung in transtracheal
Fibrosis model, using mRFP-GFP-LC3 adenovirus infection mescenchymal stem cell, the 3rd day tail vein injection is normal after modeling
With protection liquid pretreatment Human plactnta source MSCs for 24 hours, it is total that lungs frozen section for 24 hours, fluorescence after cell infusion are prepared respectively
The case where focusing the mescenchymal stem cell dotted aggregation of appearance intracellular in micro- border observation lung tissue；
2. protecting the generation of mescenchymal stem cell autophagy in liquid that modulates fibrosis Pathologic niche:
Mouse pulmonary fibrosis model is prepared using bleomycin, collects the 7th day after modeling bronchoalveolar lavage fluid (BALF), according to 1:
Above-mentioned BALF is added after pre-processing Human plactnta derived mesenchymal stem cell for 24 hours with protection liquid in 100 ratio, and it is total to extract cell
Protein, western trace detect the expression of autophagy marker molecule LC3I/II and Beclin1, utilize mRFP-GFP-LC3
Adenovirus infection mescenchymal stem cell handles cell, cell climbing sheet, fluorescence co-focusing with protection liquid and BALF according to the method described above
Dotted aggregation in micro- border observation cell determines the generation of autophagy and the carry out situation of autophagy damp (autophagic flux)；
3. the identification of Nrf2/ARE signal path regulating and controlling effect: according to the hair of mescenchymal stem cell autophagy in aforementioned Pathologic niche
Generation method handles mescenchymal stem cell, extracts total cellular protein, and western trace detects Nrf2 and its downstream albumen ferroheme
Oxygenase -1(heme oxygenase-1, HO-1) expression, determine Nrf2/ARE signal path and protection liquid regulation lung
The correlation of mescenchymal stem cell autophagy in fibrillatable pathological microenvironment.
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