CN108379277A - Application of the rhodioside in the drug for preparing treatment diabetic wounds ulcer - Google Patents
Application of the rhodioside in the drug for preparing treatment diabetic wounds ulcer Download PDFInfo
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- CN108379277A CN108379277A CN201810152377.7A CN201810152377A CN108379277A CN 108379277 A CN108379277 A CN 108379277A CN 201810152377 A CN201810152377 A CN 201810152377A CN 108379277 A CN108379277 A CN 108379277A
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- stem cell
- rhodioside
- mescenchymal stem
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- A61K31/7032—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a polyol, i.e. compounds having two or more free or esterified hydroxy groups, including the hydroxy group involved in the glycosidic linkage, e.g. monoglucosyldiacylglycerides, lactobionic acid, gangliosides
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
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Abstract
The invention discloses the drug therapy applications that rhodioside enhances mescenchymal stem cell；A kind of inhibitor of active oxygen, it is characterised in that：The active ingredient of the inhibitor is rhodioside；The drug that growth of mesenchymal stem cells inhibits under the conditions of a kind of improvement height is sugared, it is characterised in that：The active ingredient of drug is rhodioside；A kind of accelerating agent of expression and the migration of mescenchymal stem cell, it is characterised in that：The active ingredient of the accelerating agent is rhodioside；Under the conditions of high sugar, the rhodioside promotes the HO 1, FGF2 in mescenchymal stem cell, the mRNA expressions of HGF；Under the conditions of high sugar, the protein expression of HO 1, FGF2, HGF in the rhodioside promotion mescenchymal stem cell；Application of the rhodioside in the drug for preparing treatment diabetic wounds ulcer.
Description
Technical field
The present invention relates to drug fields, and specifically rhodioside enhancing is in the drug for preparing treatment diabetic wounds ulcer
Application.
Background technology
Diabetic wounds ulcer is one of complication of diabetes.Since the wound-healing abilities of diabetic are very poor,
Diabetic foot is easy to happen ulcer, and amputation can be caused when serious.In recent years, mescenchymal stem cell (mesenchymal
Stem cells, MSCs) transplanting be considered as treat diabetic wounds ulcer new strategy.Diabetic's wound-healing abilities
Impaired is existence, increasing so as to cause cell since high saccharide ring border seriously compromises the ability that the various factors are secreted in cell expression
It grows, migrate, raising the decline of the abilities such as other cells；In addition, a large amount of active oxygen caused by high saccharide ring border and high saccharide ring border
The proliferative capacity of cell seriously is reduced, while promoting Apoptosis, seriously reduces the viability of cell.
Mescenchymal stem cell (MSCs) can actively be migrated to damage location with itself and wound tissue is promoted to repair, between this is
Mesenchymal stem cells (MSCs) are used for the basic theory of cell therapy.But the survival ability of MSCs and the tolerance to ambient enviroment
Ability is poor；Especially under high saccharide ring border, proliferation, survival ability and the transfer ability of MSCs are badly damaged, this is greatly
Repair ability of the MSCs cell transplantations to tissue is limited, is a skill in application MSCs cell transplantation for diabetes wound ulcers
Art difficulty and bottleneck.
Therefore, existence and the tolerance for how promoting mescenchymal stem cell (MSCs) cell, to promote diabetes
The wound healing of wound ulcer becomes problem in the urgent need to address in MSCs transplantation treatment methods.
Rhodioside is the main component of rhodiola, and the structural formula of rhodioside is as shown in formula one.Studies have shown that red
Red-spotted stonecrop glycosides can enhance adaptability of the body to High aititude and anoxia condition.
However, inhibition and promotion mescenchymal stem cell (MSCs) of the rhodioside to mescenchymal stem cell (MSCs) apoptosis
Proliferation and shift function have not been reported；Can rhodioside promote the wound healing of diabetic wounds ulcer also unclear.
Invention content
Present invention aim to address problems of the prior art, open rhodioside is preparing treatment diabetes wound
Application in the drug of canker sore.
To realize the present invention purpose and the technical solution adopted is that：Rhodioside is in enhancing mescenchymal stem cell cell viability
Application.
A kind of inhibitor of active oxygen, it is characterised in that：The active ingredient of the inhibitor is rhodioside.
The drug of mescenchymal stem cell cell viability under the conditions of a kind of improvement height is sugared, it is characterised in that：The activity of the drug
Ingredient is rhodioside.
The drug of mescenchymal stem cell cell viability under the conditions of a kind of improvement height is sugared, it is characterised in that：The drug effectively changes
The proliferation of mescenchymal stem cell under the conditions of kind high sugar.
The drug of mescenchymal stem cell cell viability under the conditions of a kind of improvement height is sugared, it is characterised in that：The drug effectively drops
The apoptosis of mescenchymal stem cell under the conditions of low high sugar.
A kind of accelerating agent promoting mescenchymal stem cell expression growth factor, it is characterised in that：The accelerating agent is red scape
Its glycosides.
A kind of accelerating agent promoting mescenchymal stem cell migration, it is characterised in that：The accelerating agent is rhodioside
A kind of accelerating agent promoting mescenchymal stem cell expression growth factor, it is characterised in that：It is described under the conditions of high sugar
Rhodioside promotes the HO-1 in mescenchymal stem cell, the expression of FGF2, HGF.
A kind of accelerating agent promoting mescenchymal stem cell migration, it is characterised in that：Under the conditions of high sugar, the rhodioside
Promote the HO-1 in mescenchymal stem cell, the expression of FGF2, HGF.
Application of the rhodioside in the drug for preparing treatment diabetic wounds ulcer
It is worth noting that：The present invention has shown that the sugared environmental nuisance mesenchyma of the height caused by diabetes is dry thin by research
Born of the same parents are proliferated and promote Apoptosis (reducing cell viability), while also seriously damaging mescenchymal stem cell secreting function.However
Rhodioside can remarkably promote MSCs expression and secrete various growth factors, to promote cell Proliferation, the drop of mescenchymal stem cell
The apoptosis of low tone mesenchymal stem cells effectively enhances the survival ability after mesenchymal stem cell transplantation, finally promotes it to diabetes
The function of wound ulcer wound reparation.This will more effectively improve mesenchymal stem cell transplantation and burst in treatment clinical diabetes wound
Effect when ulcer.
Present invention demonstrates that rhodioside can improve under the conditions of high sugar mescenchymal stem cell expression secretion growth factor and thin
Born of the same parents are proliferated, and inhibit the apoptosis of mescenchymal stem cell under the conditions of high sugar, and diabetes patient's wound is treated to improve mescenchymal stem cell
The therapeutic effect of healing.
Description of the drawings
Fig. 1 is active oxygen (ROS) test experience figure in embodiment 1；
Fig. 2 is proliferation experiment (MTT) lab diagram of mescenchymal stem cell (MSCs) in embodiment 2；
Fig. 3 is the apoptosis lab diagram of mescenchymal stem cell (MSCs) in embodiment 3；
Fig. 4 is the migration lab diagram of mescenchymal stem cell (MSCs) in embodiment 4；
Fig. 5 is the migration lab diagram of mescenchymal stem cell (MSCs) in embodiment 5；
Fig. 6 is the HO-1 in mescenchymal stem cell, the mrna expression amount determination experiment figure of FGF2, HGF in embodiment 6；
Fig. 7 is the protein expression result figure of HO-1, FGF2, HGF in mescenchymal stem cell in embodiment 7；
Fig. 8 is diabetic mice therapeutic wound healing design sketch in embodiment 8；
Fig. 9 is the evaluation statistical results chart of diabetic mice wound reparation in embodiment 9.
Specific implementation mode
With reference to embodiment, the invention will be further described, but should not be construed the above-mentioned subject area of the present invention only
It is limited to following embodiments.Without departing from the idea case in the present invention described above, according to ordinary skill knowledge and used
With means, various replacements and change are made, should all include within the scope of the present invention.
The inventors discovered that rhodioside, which can heal to diabetes mice foot wounds, has good therapeutic effect.This
The degree of diabetes in invention does not have any restrictions, can be cercinoma prophase pathologic change, slight, moderate or serious diabetes.
The source of mescenchymal stem cell in the present invention does not have any restriction, can be purchase, can also be extraction.
Those skilled in the art it would be appreciated that, the extraction of mescenchymal stem cell can be according to conventional standard extracting mode.Transplanting institute
The mescenchymal stem cell used can be with the same individual in source, can also the different individual in source；And transplanting used between fill
Matter stem cell can be and be transplanted the same species of individual, can also be the mescenchymal stem cell of different plant species.
Diabetes in the present invention include type 1 diabetes, diabetes B and prediabetes.Type 1 diabetes and 2 types
Fasting blood-glucose >=7.0mmol/L of diabetes, the fasting blood-glucoses of prediabetes (pre-diabetes) more than 6.1mmol/L and
Less than 7.0mmol/L." high sugar " described in this specification refers to the sugared pathological conditions of height of diabetic.
In addition, those skilled in the art it would be appreciated that, the diabetic mice wound model in the embodiment of the present invention be
The back of diabetic mice manufactures a certain size wound, and the fasting blood-glucose of diabetic mouse model used >=
16.7mmol/L, the significantly larger than fasting blood-glucose (i.e. >=7.0mmol/L) of diabetes standard, are serious diabetic mices.Cause
This is it is found that the diabetes mouse in the embodiment of the present invention suffers from serious diabetes.Degree (i.e. blood glucose based on diabetes
Height) be inversely proportional with abilities such as tissue repair, wound healings, those skilled in the art it would be appreciated that, the aftermentioned present invention
Effect other than there is good therapeutic effect to severe diabetes mellitus wound ulcer, to wound-healing abilities stronger early periods
Lesion, slight or moderate diabetic wounds ulcer can play better therapeutic effect.
In the present invention, the medicines of diabetes includes rhodioside as active constituent.Rhodiola root in the present invention
Glycosides is the compound of structural formula such as formula 1.The source of rhodioside can be from sachalin rhodiola rhizome, rhodiola, the red scape of rose
It, extract in the crassulaceae plants such as Xizang Rhodiola root, can also be to pass through chemical synthesis.There is no limit for the purity of rhodioside,
Preferably 80% or more, more preferably 90% or more, and then preferably 95% or more, it is further preferably 98% or more.
Cell viability in the present invention refers to that cell is survived the ability of proliferation in certain circumstances, refers in particular to cell in spy
Determine the apoptosis under environment and proliferative conditions.
The active constituent of drug in the present invention is rhodioside.In addition to this, the drug in the present invention also may include one
Kind or multiple auxiliary materials.Auxiliary material does not limit, such as solvent, isotonic agent, excipient, pH regulators, antioxidant, disintegrant, tune
The auxiliary material commonly used in the art such as taste agent, fragrance, preservative agent.
It can be enumerated as solvent：Distilled water for injection, physiological saline, vegetable oil, it is propylene glycol, polyethylene glycol, ethyl alcohol, sweet
The alcohols etc. of oil etc.
It can be enumerated as isotonic agent：The isotonic agent commonly used in the art such as sorbierite, sodium chloride, glucose.
It can be enumerated as excipient：Lactose, mannitol, glucose, microcrystalline cellulose, starch etc..
It can be enumerated as pH regulators：Hydrochloric acid, citric acid, sodium hydroxide, Strong oxdiative potassium, sodium bicarbonate, phosphoric acid hydrogen two
Sodium etc..
It can be enumerated as antioxidant：Sodium sulfite, sodium hydrogensulfite, ascorbic acid etc..
It can be enumerated as disintegrant：Potato starch.
It can be enumerated as flavoring agent：The sweeteners such as sucrose, simple syrup, etc..
It can be enumerated as fragrance：Peppermint oil, orange oil etc..
It can be enumerated as preservative agent：The preservative agent commonly used in the art such as parabens, sorbic acid and its salt.
Administering mode in the present invention can be transplanted again after handling mescenchymal stem cell with rhodioside in advance,
Can also be that mesenchymal stem cell transplantation to wound is injected into rhodioside again, or it is mescenchymal stem cell and rhodioside is same
When be injected into wound.
Accelerating agent is the expression for promoting gene or the medicament of secretion level, may include that transcription, translation, protein is promoted to close
At, protein stability and the medicament of secretion.
Growth factor in the present invention includes the factor for promoting cells survival and the factor for promoting cell Proliferation, is preferably blood
Endothelial tube growth factor A (vascular endothelial growth factor A, VEGF-A), Desmocyte growth factor
2 (fibroblast growth factor 2, FGF2) of son, angiogenin (angiopoietin 1, ANG1), ferroheme oxygen
Synthase 1 (heme oxygenase, HO-1), Platelet-derived growth factor BB (platelet-derived growth
Factor BB, PDGF-BB) and hepatocyte growth factor (hepatocyte growth factor, HGF), it is preferably VEGF-
A, FGF2, HO-1 and HGF are preferably further FGF2, HO-1 and HGF.
Signified " expression " includes the expression of mRNA and/or protein in the present invention.
Embodiment 1：
Mouse mesenchymal cell cell line (MSCs) is purchased from American Type Culture Collection
(ATCC) company.
The culture of mescenchymal stem cell cell line Dulbecco ' s modified Eagle medium basic (Gibco,
Life Technologies, Grand Island, NY) in culture medium, 10% fetal calf serum is added in the medium.
Experiment for high sugar, medium culture of the cell containing 25mM glucose.
Control be by cell culture in 5.5mM dextrose culture-mediums (low sugar culture medium).
Active oxygen test experience is carried out, is included the following steps：
1) by mescenchymal stem cell MSCs kinds to 48 orifice plates, 7000/hole；
2) cell MSCs is used into low sugar (5.5mM) respectively, high sugar (25mM), high sugar+rhodioside (Salidroside,
Rhodioside is dissolved in PBS by Sa, and it is 100 μ g/ml to make its ultimate density in the medium) processing is for 24 hours；
3) production of ROS in ROS kit detection cells, specific steps is used to be operated according to kit specification.
As shown in Figure 1, as active oxygen test experience figure；It can be seen from the figure that rhodioside effectively inhibits ROS to generate
Effect.
Embodiment 2：
MTT colorimetric tests are carried out, are followed the steps below：
1) inoculating cell (100,000/hole) in six orifice plates；
2) low sugar (5.5mM), high sugared (25mM) and sugared (the 25mM)+rhodioside (100 μ g/ml) of height is used to combine three kinds of items
Part co-cultures mescenchymal stem cell (MSCs) 24 hours respectively；
3) it is inoculated in the later mescenchymal stem cell (MSCs) to 96 orifice plates of above-mentioned three kinds of processing with identical cell quantity,
Using Cell Counting Kit-8 kits (Dojindo, Kumamoto, Japan) detection different time points (0h, for 24 hours,
The cell quantity of mescenchymal stem cell (MSCs) 48h).
As shown in Fig. 2, as MTT colorometric assays figure；It can be seen from the figure that rhodioside can significantly improve high sugar
Under the conditions of mescenchymal stem cell (MSCs) proliferative capacity, * * p<0.01.
Embodiment 3：
Cell apoptosis assay is as follows：
1) inoculating cell (150,000/hole) in 6cm plates；
2) low sugar (5.5mM), high sugared (25mM) and sugared (the 25mM)+rhodioside (100 μ g/ml) of height is used to combine three kinds of items
Part co-cultures mescenchymal stem cell (MSCs) 24 hours respectively；
3) low sugar that renews respectively or high glucose medium culture 24 hours；
4) and then cell is collected, is grasped according to apoptosis kit (Annexin V-FITC kits, NeoBioscience)
Make.
As shown in Fig. 2, as apoptosis experiment statistics result；It can be seen from the figure that rhodioside can significantly inhibit height
The apoptosis of mescenchymal stem cell (MSCs) under the conditions of sugar, * * p<0.01.
Embodiment 4：
The migration experiment for carrying out mescenchymal stem cell (MSCs), includes the following steps：
1) inoculating cell in six orifice plates, 100,000/hole；
2) experimental group high sugar+rhodioside combined treatment (the final concentration of 100 μ g/ml of rhodioside), control group use etc.
The low sugar (5.5mM) of volume or high sugared (25mM) processing；
3) different condition treated three groups of cells are several on the cells transwell upper layer with identical cell,
The cells transwell lower layer is separately added into low sugar and high glucose medium, is put into hypoxemia box (Anaeropouch Box, 0.1%O2,
Mitsubishi Gas Chemical, Tokyo, Japan) hypoxemia culture for 24 hours, with crystal violet (Beyotime, China) dye move
The cell for moving on to the cells transwell lower layer, observation of taking pictures, and counting statistics fill between moving to lower layer from cell upper layer respectively
The cell number of matter stem cell (MSCs).
As shown in figure 4, the as migration lab diagram of mescenchymal stem cell (MSCs)；As can be seen from the figure rhodioside is aobvious
Write the migration of enhancing mescenchymal stem cell (MSCs).
Embodiment 5：
When observing different time, the migration situation of mescenchymal stem cell (MSCs) includes the following steps：
1) mescenchymal stem cell (MSCs) is seeded in six orifice plates (150,000/hole)；
2) low sugar is used respectively, and high sugared and height sugar+rhodioside combines three kinds of condition processing cells, and (cell quantity reaches for 24 hours
90%)；
3) straight line of an equal in width is drawn in culture plate bottom with micro pipette tips, taken pictures respectively in scuffing processing 0h and 48h
Observe the migration situation of mescenchymal stem cell (MSCs).
As shown in figure 5, the as migration lab diagram of mescenchymal stem cell (MSCs)；As can be seen from the figure rhodioside is aobvious
Write the migration for promoting mescenchymal stem cell (MSCs).
Embodiment 6：
HO-1 in mescenchymal stem cell, FGF2, the mrna expression amount determination experiment of HGF, include the following steps：
1) mescenchymal stem cell (MSCs) is seeded in six orifice plates, 100,000/hole；
2) after handling mescenchymal stem cell respectively with low sugar, high sugar and height sugar+condition of rhodioside three, mRNA is added and carries
It takes reagent TRIZOL to collect in processing cell to the centrifuge tube of 1.5mL and extracts mRNA；
3) real-time quantitative PCR (qPCR) is used to measure the mRNA of HO-1, FGF2, HGF in mescenchymal stem cell under different condition
Expression quantity；
Fig. 6 is the HO-1 in mescenchymal stem cell, and the mrna expression amount determination experiment figure of FGF2, HGF are red as shown in Figure 6
Red-spotted stonecrop glycosides dramatically increases under the conditions of high sugar HO-1, the mrna expression amount of FGF2, HGF, * * P in mescenchymal stem cell<0.01.
HO-1 in mescenchymal stem cell, FGF2, the mrna expression amount assay method of HGF：
RT-PCR
TAKARA-PrimeScript RT Reagent Kit with gDNA Eraser(Code No.RR047A)
Reverse transcription reaction is as shown in table 1；
Table 1
| Reagent | Usage amount |
| PrimeScript RT Enzyme Mix Ⅰ | 1.0μL |
| RT Prime Mix*4 | 1.0μL |
| 5*PrimeScript Buffer 2 | 4.0μL |
| RNase Free DH2O | 4.0μL |
| The reaction solution of step 1 | 10.0μL |
| Total | 20.0μL |
System completion is placed on above Bio-Rad PCR looms, and reaction condition is as follows：
37 DEG C of 15min, 85 DEG C of 5sec, 4 DEG C of preservations
10 times are diluted after obtaining cDNA；Sample after dilution is used for doing qPCR reactions.Shown in reaction system such as table 2；
Table 2
| Reagent | Dosage |
| SYBR | 5.0μL |
| PCR Forward Primer(10mM) | 0.4μL |
| PCR Reverse Primer(10mM) | 0.4μL |
| RT reaction solutions | 2.5μL |
| DH2O | 1.7μL |
| Total | 10μL |
QPCR response procedures：
1 50.0℃for 2min；
2 95.0℃for 10min；
3 95.0℃for 15sec；
4 60.0℃for 35sec；
5 GO TO 3.40times；
6 95.0℃for 15sec；
7 60.0℃for 1min；
8 Melting Curve 65.0 to 95.0,increment 0.5℃。
QPCR relevant primer sequences are as shown in table 3；
Table 3
Embodiment 7：
Rhodioside remarkably promotes the protein expression of HO-1, FGF2, HGF in mescenchymal stem cell under the conditions of high sugar；
The protein expression assay for carrying out HO-1, FGF2, HGF in mescenchymal stem cell, includes the following steps：
1) mescenchymal stem cell (MSCs) is seeded in 6cm tissue culture plates, 150,000/hole；
2) mesenchyma is handled respectively with three low sugar culture medium, high glucose medium and high glucose medium+rhodioside conditions
After stem cell, protein lysate (RIPA+PMSF+Protease inhibitor cocktail, the whole operation prepared is added
Carry out on ice, prevent protein degradation), Protein Extraction processing will be carried out in supernatant collection to centrifuge tube pipe, carry out
Western Blotting detect protein expression level, including following operating procedure：
1. preparing sample solution
The cell sample handled well is washed with PBS, outwells the egg for after remaining PBS, being added and preparing in tissue culture plate
White lysate collects cell sample using cell scraper, and places 30min on ice, and high-speed low temperature centrifugation 20min (12000r, 4
DEG C), supernatant, as total protein sample are drawn, BCA working solutions (BCA reagent As are used：BCA reagents B=50:1, Beyotime) and
After microplate reader (BioTek) measures protein sample concentration, total protein sample and sample-loading buffer (SDS-PAGE albumen loading buffers
Liquid, 5 ×, Beyotime) according to 4:1 ratio is mixed, and 5min is boiled for 100 DEG C in metal bath.The sample measured according to experiment
Concentration keeps the applied sample amount of total protein consistent (40 μ g), calculates the loading volume of each sample.
2. the preparation of PAGE gel
A. the glass plate of the drying standby gel of cleaning and wiping, after clamping glass plate using shelf and detect and be not in leakage situation
Start glue.
B. according to following 12% separation gel of system configurations lower layer：
Separation gel is added as needed and prepares required each component and mixing (being eventually adding TEMED and APS), uses liquid relief
The mixed liquor of separation gel is quickly added in the gap between glass plate (solution speed in filling is fast) by rifle, to be separated
Glue stops filling separation gel when being added to appropriate position, carrying out fluid-tight to separation gel using distilled water flattens separation gel (when fluid-tight
It is noted that filling should be slow, it otherwise will appear phenomenon of the separation gel not on a horizontal plane).
C. after separation gel solidifies, distilled water is outwelled, filter paper is used in combination to blot.
D. the upper layer for preparing 5% concentrates glue
Concentration glue is added as needed and prepares required each component and mixing, using liquid-transfering gun quickly by the mixed of separation gel
It closes liquid to be added in the gap between glass plate, wants quick insertion to correspond to required comb after the completion of filling, and prevent the production of bubble
It is raw.
3. loading and electrophoresis
Experimental facilities is assembled according to operating instruction, and gel is fixed on the electrode, is put into electrophoresis tank, takes out
1 × running buffer are added into electrophoresis tank, and are rinsed to each hole using syringe for comb.Use microsyringe
Identical matter is added after protein quantification result calculates before in loading standard albumen Marker and protein sample, protein sample
Amount.
Engaging means power supply, on upper layer, glue compresses protein sample using 60V voltages, when protein sample compression is into a line
When, it converts voltage and continues to carry out electrophoresis in separation into 120V, samples is waited to stop gel electrophoresis, and transferring film close to separation gel bottom.
4. transferring film
A. gel is first taken out, concentration glue part is prescinded, according to the size of protein band to be detected and maker size requirements,
Retain the separation gel part needed, cuts glue and separation gel is immersed into preprepared 1 × Trans Buffer buffer solutions after the completion
In, while the size of glue is measured, pvdf membrane of corresponding size is cut, film is first impregnated to complete to activate, so in methanol solution
Pvdf membrane is transferred in 1 × Trans Buffer buffer solutions afterwards, film is made to be balanced in buffer solution.
Testing protein is transferred to total on pvdf membrane from gel and is similar to " sandwich biscuits ", the black transferring film face of clip
Filter paper is located at lowest level on pad, is secondly separation gel, pvdf membrane, filter paper is later the white transferring film face of clip, works as whole process
By clamp after end of operation, in operation, it is careful not to make between various pieces with the presence of bubble, otherwise gel
In albumen can by bubble obstruct so that transferring film is failed, clip will be immersed in 1 × Trans Buffer in entire During migration and protect
Hold moistening.
Clip is put into slot after the completion of operation, to make clip black to black, and white can enter red, otherwise albumen
To in electrophoresis tank rather than on pvdf membrane, entire clip structure is put into electrophoresis tank after the completion of operation, because of meeting in During migration
High heat is generated, is carried out so can place an ice bag on vacant one side of slot and be placed in ice basin, or directly in 4 DEG C of conditions
Lower transferring film.Power on, selects 200mA electric currents, transferring film 120min.
B. after the completion of transferring film, can also be led to by observing albumen marker to determine whether being transferred on pvdf membrane
It crosses and Ponceaux dyeing liquor method is added dropwise on film to confirm, if albumen Marker is clear or Ponceaux dyeing liquor dyes
Later protein band is clear, then illustrates transferring film success, need cleaning that can just carry out subsequent step after Ponceaux dyeing.
5. immune response and development
A. it closes：It after the completion of transferring film, is marked in the front of film according to Marker, first rinses film surface with TBST
Pvdf membrane taking-up is placed in advance prepared 5% protein blocking liquid, in room temperature by the surfactants such as SDS after removing TBST
Under the conditions of closing 2 hours is shaken on shaking table at a slow speed.
B. the incubation of primary antibody：Primary antibody is diluted to suitable concentration with TBST buffer solutions, antibody is incubated with film under the conditions of 4 DEG C
It educates overnight.Pvdf membrane is cleaned three times with 1 × TBST after the completion of being incubated, 10 minutes every time.
C. the incubation of secondary antibody：The corresponding secondary antibody used is proportionally diluted with TBST, is incubated on shaking table at room temperature
90 minutes.It is cleaned three times with 1 × TBST after the completion of being incubated, 10 minutes every time.
| Antibody | Product number | Maker | Experiment | Dilution |
| Goat antirabbit lgG | ZB2301 | ZSGB-BIO | Western Blotting | 1/10000 |
| Goat anti-mouse lgG | ZB2305 | ZSGB-BIO | Western Blotting | 1/10000 |
6. ECL chemiluminescence chromogenic reactions
A.ECL developing solutions (ECA working solutions A：ECA working solutions B=1：1, photo-biological Technology Co., Ltd. is preserved in Chongqing) match
System：By reagent A and reagent B according to isometric mixing, it is kept in dark place for use.
B. pvdf membrane egg front is come into full contact with developing solution and is reacted, put and carry out development imaging in imaging systems.
C. the colour developing situation of protein band, statistics and analysis data are observed.
As shown in fig. 7, the protein expression result figure of HO-1, FGF2, HGF as in mescenchymal stem cell.
Embodiment 8：
By the wound reparative experiment of mescenchymal stem cell (MSCs) cell transplantation for diabetes mouse, include the following steps：
PBS：Physiological phosphate buffer control group；
MSCs：Injection 6 × 106The monotherapy group of a/ml mescenchymal stem cells (MSCs)；
MSCs+ rhodiosides：Injection 6 × 106A/ml mescenchymal stem cells (MSCs) and rhodioside (100 μ g/ml)
Combined therapy group.
As shown in figure 8, as diabetic mice therapeutic wound healing design sketch.
Embodiment 9：
1. the foundation of diabetic mouse model
C57BL/6J mouse (6 weeks, male) purchase (being purchased from Military Medical Univ No.3, P.L.A) is returned after a week
Mouse blood sugar is measured, blood glucose is measured after being fed 4 weeks with following high lipid foods, the feelings of prediabetes (pre-diabetes) occurs
After condition, streptozotocin (STZ) is continuously injected five days by muscle, and the amount of penetrating is 50mg/kg, is continued high lipid food and is fed after a week
Mouse blood sugar is measured, blood glucose is used as experiment in next step higher than the selected of 16.7mmol/L.Herein, it should be noted that general blood
Sugar has been thought suffering from diabetes for 7 or more, but the model of the present invention selectes the mouse that blood glucose is 16.7 or more.
The formula of high lipid food：15% lard
10% yolk
10% white sugar
65% normal diet
Wherein, normal diet, yolk, lard, white sugar are provided by great Ping hospitals of Third Military Medical University, and by medical university of third army
The level grounds Xue great hospital produces high lipid food.
The therapeutic effect of diabetic mice wound healing is tested 2. rhodioside mescenchymal stem cell (MSCs) is transplanted
Using above-mentioned diabetic mouse model, appropriate phenobarbital (80mg/kg) anesthetized mice is used.
Dorsal body setae loses hair or feathers, and the disinfection of iodine method, operative site is back right side and left side, and a diameter of 1 is formed using sterile scissors
Centimetre skin wound.
Wound formed first day, respectively 4 injection location 0.4ml phosphate buffer solutions (PBS) of wound circumference,
6×106A/ml mescenchymal stem cells (MSCs) or 6 × 106A/ml mescenchymal stem cells (MSCs)+rhodioside combines (red scape
Its glycosides concentration 100g/ml indicates to handle later mescenchymal stem cell with the rhodioside of 100g/ml here), it is noting respectively
Observation is measured after penetrating to diabetic mice back wound area within 0 day, 3 days, 7 days and 14 days to take pictures, with the make rate of wound
Indicate wound healing repairing effect：Wound healing rate (%)=((wound area of 0 day wound area after -14 days)/0 day
Wound area) × 100%.
The experimental results showed that mescenchymal stem cell (MSCs) and the combined therapy group of rhodioside are more dry than independent mesenchyma thin
Born of the same parents (MSCs) group has significant wound healing repairing and treating effect.
As shown in figure 9,14 days after injection, filled with the control group of injection physiological phosphate buffer (PBS) and individually between injection
Matter stem cell (MSCs) treatment group compares, and injection mescenchymal stem cell (MSCs) and the diabetes of rhodioside combined therapy group are small
Mouse wound healing repairing and treating most pronounced effects, this demonstrate rhodioside, to can effectively improve mescenchymal stem cell (MSCs) right
In the therapeutic effect of diabetes wound healing, * * p<0.01.
In addition, as shown in figure 9, according to data statistics as a result, mescenchymal stem cell (MSCs) and rhodioside group relative to
Control PBS and MSCs shows good wound-healing abilities (* * p in Fig. 9<0.01), i.e. controlling for diabetes wound healing
Treatment has good effect.
Sequence table
<110>University Of Chongqing
<120>Application of the rhodioside in the drug for preparing treatment diabetic wounds ulcer
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<170> SIPOSequenceListing 1.0
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Claims (10)
1. rhodioside is in the application of enhancing mescenchymal stem cell cell viability.
2. a kind of inhibitor of active oxygen, it is characterised in that：The active ingredient of the inhibitor is rhodioside.
3. the drug of mescenchymal stem cell cell viability under the conditions of a kind of high sugar of raising, it is characterised in that：The drug it is effective at
It is divided into rhodioside.
4. the drug of mescenchymal stem cell cell viability, feature under the conditions of the high sugar of a kind of raising according to claim 3
It is：The drug is effectively improved the proliferation of mescenchymal stem cell under the conditions of high sugar.
5. the drug of mescenchymal stem cell cell viability, feature under the conditions of the high sugar of a kind of raising according to claim 3
It is：The drug effectively reduces the apoptosis of mescenchymal stem cell under the conditions of high sugar.
6. a kind of accelerating agent promoting mescenchymal stem cell expression growth factor, it is characterised in that：The accelerating agent is rhodiola root
Glycosides.
7. a kind of accelerating agent promoting mescenchymal stem cell migration, it is characterised in that：The accelerating agent is rhodioside.
8. a kind of accelerating agent promoting mescenchymal stem cell expression growth factor according to claim 6, it is characterised in that：
Under the conditions of high sugar, the rhodioside promotes the HO-1 in mescenchymal stem cell, the expression of FGF2, HGF.
9. a kind of accelerating agent promoting mescenchymal stem cell migration according to claim 7, it is characterised in that：In high sugared item
Under part, the rhodioside promotes the HO-1 in mescenchymal stem cell, the expression of FGF2, HGF.
10. application of the rhodioside in the drug for preparing treatment diabetic wounds ulcer.
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| CN201810152377.7A CN108379277A (en) | 2018-02-14 | 2018-02-14 | Application of the rhodioside in the drug for preparing treatment diabetic wounds ulcer |
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| CN201810152377.7A CN108379277A (en) | 2018-02-14 | 2018-02-14 | Application of the rhodioside in the drug for preparing treatment diabetic wounds ulcer |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109517872A (en) * | 2018-10-15 | 2019-03-26 | 雷桅 | Application of the rhodioside in protection Stem Cell Activity |
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Application publication date: 20180810 |