CN106880512B - People's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powder of HA modification and its preparation and application - Google Patents
People's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powder of HA modification and its preparation and application Download PDFInfo
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Abstract
The invention discloses a kind of people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders of HA modification, it by mass percentage include following components: people's umbilical cord MSC serum-free medium activated protein ingredient 5~12%, chitosan 20~65%, sodium tripolyphosphate 1~15%, hyaluronic acid 20~65%, water≤5%.Preparation method is, first the stem cell serum-free culture supernatant with various active cell factor is coated using CS nanoparticle preparation process, and then be lyophilized, then make the active amino on CS nanoparticle surface that covalent coupling occur with the carboxyl on the surface HA and react, product of the present invention is made.
Description
Technical field
The invention belongs to cell engineering field, the people's umbilical cord MSC serum-free medium CS for being related to a kind of HA modification is received
Grain of rice freeze-dried powder has further related to the preparation method of the freeze-dried powder.
Background technique
Stem cell beauty is the application in plastic surgery earliest, and the more stem cell type applied in plastic surgery
It is mescenchymal stem cell, epidermal stem cells, endothelial progenitor cell and PECTORAL LIMB SKELETON etc..
Traditionally so-called stem cell cosmetology, typically direct injection stem cell injection.And this injection is dry thin
The source of born of the same parents is mainly to extract from the embryo of miscarriage, so can inevitably cause immunological rejection, and is existed centainly
Ethics problem.Even with autologous stem cells, but exogenous albumen can be introduced in the culture amplification procedure of stem cell
(fetal calf serum), easily causes immunological rejection.
It is raw that human mesenchymal stem cell (mesenchymal stem cell, MSC) secretes basic fibroblast in culture
The long factor (bFGF), hEGF (EGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-i
(IGF-1) a variety of regenerated growth factors of promotion Skin Cell and including hepatocyte growth factor (HGF).These have biology
Active growth factor can effectively promote the metabolism of Skin Cell；Promote extracellular hyaluronic acid, glycoprotein etc. big
The synthesis and secretion of molecule, enhance the hydrophily of skin；Growth, differentiation and the reparation for promoting epidermal cell, make aging death
Cell is replenished in time；It can speed up cuticula reparation simultaneously, accelerate the new old cell of substrate to substitute, skin is made to return to young appearance
State.
Summary of the invention
The first purpose of the invention is to provide a kind of people's umbilical cord MSC serum-free medium CS nanoparticle freeze-dryings of HA modification
Powder solves the bottleneck that liquid stem cell culture supernatant storage life is limited, while solving cell in stem cell culture supernatant and growing
Factor active saves problem.
A second object of the present invention is to provide above-mentioned people's umbilical cord MSC serum-free cell culture medium CS nano-granule freeze-dried powders
Preparation method.
First technical solution of the present invention be, a kind of people's umbilical cord MSC serum-free medium CS of HA modification receives
Grain of rice freeze-dried powder includes following components: people's umbilical cord MSC serum-free medium activated protein ingredient 5~12% by mass percentage,
CS (chitosan) 20~65%, TPP (sodium tripolyphosphate) 1~15%, HA (hyaluronic acid) 20~65%, water≤5% are above each
The quality summation of component is 100%.
Second technical solution of the present invention is that people's umbilical cord MSC serum-free medium CS of above-mentioned HA modification receives
The preparation method of grain of rice freeze-dried powder, comprising the following steps:
Step 1, preparation load people's umbilical cord MSC serum-free medium CS nanoparticle；
People's umbilical cord MSC serum-free medium is added in CS acid solution, and TPP is added, is reacted at room temperature.By receiving after reaction
The centrifugation of grain of rice suspension, gained washing of precipitate, freeze-drying obtain load people's umbilical cord MSC serum-free medium CS nanoparticle.
Step 2, load people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powder of HA modification is prepared；
Carboxyl in HA is activated, load people's umbilical cord MSC serum-free medium CS nanoparticle is then added to work
It is reacted in HA after change, negates the nanosuspension after answering, be centrifuged, gained washing of precipitate and being freeze-dried is modified to get HA
People's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powder.
Wherein, in step 1 people's umbilical cord MSC serum-free medium the preparation method comprises the following steps: neonatal umbilical cord is taken, by intravenous blood
Mark washes clean；Epidermis, two arteries and a vein blood vessel are removed, tissue is shredded, the DMEM/F12 containing FBS is added
Culture medium culture obtains primary human umbilical cord mesenchymal stem cells；When cell fusion degree is 80%-90%, trypsin digestion
Cell secondary culture, takes passage cell, continues to cultivate, and as cell fusion degree 80%-90%, abandons the culture supernatant containing FBS,
PBS washs cell, and the DMEM/F12 culture medium that serum-free is added continues culture 72 hours, collects serum-free medium.
Chitosan (CS) is called chitosan, soluble chitin and poly glucosamine etc., the entitled β-of chemistry
(1-4) -2-amino-2-deoxy-D-Glucose is product obtained from deacetylate after chitin is handled with concentrated base.Shell is poly-
Sugar is rare alkaline polysaccharide in natural polymer, and not soluble in water and organic solvent dissolves in the diluted acid of pH < 6.5, in diluted acid
In, Glucoamino is converted into R-NH3 +, form polycation gel solution.Chitosan have excellent biodegradability,
Less toxic, good biocompatibility, therefore it is highly suitable for biological medicine and cosmetic industry.
The present invention prepares CS nanoparticle using ionic cross-linking, using the sodium tripolyphosphate (TPP) having no adverse reaction to CS
It carries out ion induction gelation and forms nanoparticle.Under stiring, TPP solution is added in the acid solution of CS, by negatively charged
Phosphate anion and CS strand on positively charged protonated amino intramolecular and intermolecular cross-linking gelation, Bian Kexun occurs
Fast-growing is at nanoparticle.
The macromolecule polysaccharide that hyaluronic acid (HA) is made of D-Glucose aldehydic acid and N-acetyl-glucosamine is optimal guarantor
One of wet ingredient.Under certain pH value condition, hyaluronic acid is negatively charged, and CS nanoparticle is positively charged, between the two can
It is enough attracted each other by electrostatic force, makes CS nanoparticle that there is good moisture-keeping function.
The features of the present invention also characterized in that:
Preferably, the mass ratio of activated protein is 2:1~10:1 in CS and people's umbilical cord MSC serum-free medium in step 1,
The mass ratio of CS and TPP is 2:1~5:1, and the molecular weight of CS is 2-30 ten thousand.
Preferably, the acid in CS acid solution described in step 1 for dissolving CS be formic acid, acetic acid, propionic acid, lactic acid, malic acid,
Citric acid, ascorbic acid, oxalic acid, succinic acid, malonic acid, fatty acid, pyruvic acid, glutaric acid, tartaric acid, aspartic acid, bigcatkin willow
One or more of acid or glycolic.
Preferably, parameter of noncentricity in step 1 are as follows: 2~8 DEG C of centrifuging temperature, 10~60min of centrifugation time, centrifugal force 1.0 ×
103~1.0 × 105g。
Preferably, the molecular weight of HA is 8-30Kda, HA and CS nanometers of people's umbilical cord MSC serum-free medium of load in step 2
The mass ratio of grain is 1:1~5:1.
Preferably, the activated carboxylic method of HA is in step 2, sub- using 1- ethyl -3- (3- dimethylamino-propyl)-carbon two
Amine (EDC) and n-hydroxysuccinimide (NHS) activate the carboxyl in HA, then adjust pH to 7.5,37 DEG C of incubations.
Preferably, freeze-drying parameter is equal in step 1 and step 2 are as follows: ± 2 DEG C of -60 DEG C of pre-freezing temperature, pre-freeze time 6h with
On, cryogenic temperature -50~-56 DEG C, vacuum degree≤80mT, freeze-drying time 24-36h.
The beneficial effects of the present invention are:
(1) the stem cell culture supernatant used in the present invention is free serum culture supernatant, reduces stem cell for beauty
Present in immunological rejection.
(2) use CS nanoparticle preparation process by the stem cell serum-free culture supernatant with various active cell factor
Cladding, and then be lyophilized, the shelf-life is 3 years, much higher than the shelf-life of existing human mesenchymal stem cell, solves supernatant
The short problem of storage life.
(3) active amino on CS nanoparticle surface is reacted with the carboxyl on the surface HA generation covalent coupling, and HA is made and modifies CS
Nanoparticle not only solves the problem of CS nanoparticle stability difference in this way, while it is efficient in CS nanoparticle to introduce cosmetics
Moisturizer HA.
(4) CS used in the present invention as a kind of natural alkaline polysaccharide there is good biocompatibility and biology can drop
Xie Xing, safety is higher, low in cost, prepares nanoparticle using CS, and reaction condition is mild, simple process, at low cost.
Detailed description of the invention
Fig. 1 is the preparation method process of people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powder of HA of the present invention modification
Figure；
Fig. 2 MSC-CS-NPs and HA-MSC-CS-NPs 48h Flow cytometry result；
Fig. 3 MSC-CS-NPs and HA-MSC-CS-NPs 72h Flow cytometry result；
Fig. 4 MSC-CS-NPs and HA-MSC-CS-NPs 48h and 72h normal live cells number accounts for total number of cells percentage；
Fig. 5 MSC-CS-NPs and HA-MSC-CS-NPs 48h and 72h viable apoptotic cell number accounts for total number of cells percentage.
Specific embodiment
Present invention will be described in further detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited to
These embodiments.
Embodiment 1
A kind of people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powder of HA modification is prepared, referring to Fig.1, method is such as
Under:
Step (1), prepares human umbilical cord mesenchymal stem cells serum-free medium
Human umbilical cord mesenchymal stem cells extract: under aseptic condition, 5~10cm of neonatal umbilical cord taken, sets umbilical cord preserving fluid, 4
DEG C storage (time be no more than 8 hours).It is rinsed with D-Hanks liquid until intravenously without bloodstain.Remove epidermis, two arteries
And a vein blood vessel, tissue shear is broken to about 1mm3DMEM culture medium (containing dual anti-) culture containing 10%FBS is added in/block
About one week, condition of culture are 37 DEG C, saturated humidity, 5%CO2, primary human umbilical cord mesenchymal stem cells can be obtained.
Collect serum-free medium: when cell fusion degree 80%, trypsin digestion and cell secondary culture takes the thin of 6 generations
Born of the same parents continue to cultivate.When cell fusion degree 80%, the culture supernatant containing 10%FBS is abandoned, PBS (pH=7.0) washs cell three times,
The DMEM culture medium that serum-free is added continues culture 72 hours, collects serum-free medium.
Step (2), ionic cross-linking preparation load people's umbilical cord MSC serum-free medium CS nanoparticle
The CS powder 25mg that molecular weight is 20,000 is weighed, is dissolved in the 25mL acetum that mass fraction is 1%, is made into matter
The CS solution that score is 0.1% is measured, then ultrasound removing bubble crosses 0.45 μm of water system filter membrane, obtains clarifying bright CS solution.
2.5mL people's umbilical cord MSC serum-free medium that activated protein concentration is 998 μ g/mL is slowly dropped in CS solution, is stirred
10min obtains mixed liquor.0.2% TPP solution 5mL is added dropwise in above-mentioned mixed liquor, 30min is reacted at room temperature.Wherein CS with
The mass ratio of TPP is 2.5:1, and the mass ratio of activated protein is 10:1 in CS and people's umbilical cord MSC serum-free medium.
By the nanoparticle suspension high speed centrifugation after reaction, 4 DEG C of centrifuging temperature, centrifugation time 10min, centrifugal force 1.0 ×
105g.Centrifugation gained precipitating is washed with distilled water 3 times, is then freeze-dried, is freeze-dried parameter are as follows: pre-freezing temperature -60
DEG C ± 2 DEG C, pre-freeze time 6h or more, cryogenic temperature -50~-56 DEG C, vacuum degree≤80mT, freeze-drying time is for 24 hours.After freeze-drying
People's umbilical cord MSC serum-free medium CS nanoparticle must be loaded.
Step (3), HA activation；
The HA aqueous solution 50mL that the HA concentration that preparation molecular weight is 8KDa is 0.05%, is added 1- ethyl -3- (3- diformazan ammonia
Base propyl)-carbodiimide (EDC) 137.5mg, n-hydroxysuccinimide (NHS) 165mg is added after it is completely dissolved,
Being adjusted to pH with 0.1M sodium hydroxide is 7.5,37 DEG C of incubation 1h.
Step (4) prepares load people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powder of HA modification
After load people's umbilical cord MSC serum-free medium CS nanoparticle that step (2) obtains is added to step (3) activation
In HA, the mass ratio that feeds intake of HA and CS nanoparticle is 1:1.Be stirred at room temperature reaction 2h, negate the nanosuspension after answering, at a high speed from
The heart, parameter of noncentricity are as follows: 4 DEG C of centrifuging temperature, centrifugation time 30min, centrifugal force 1.0 × 104g.Gained precipitating is washed with distilled water
It 10 times, is then freeze-dried, is freeze-dried parameter are as follows: ± 2 DEG C of -60 DEG C of pre-freezing temperature, pre-freeze time 6h or more, freezing temperature
- 50~-56 DEG C of degree, vacuum degree≤80mT, freeze-drying time is for 24 hours.Up to people's umbilical cord MSC serum-free training of HA modification after freeze-drying
Nutrient solution CS nano-granule freeze-dried powder.
The people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powder for the HA modification that the present embodiment obtains is clear by measuring
It washes in CS nanoparticle liquid and the content of TPP determines the content for the TPP being crosslinked in centrifuged supernatant, detection method is using light splitting
The inventory of photometry (GB/T13171-1997), TPP is 10mg, and being crosslinked is 6.4mg.Percentage bound by detecting HA obtains
Know, the Percentage bound of HA and CS nanoparticle is 36.0%, wherein human umbilical cord mesenchymal stem cells serum-free medium activated protein at
Divide 5.5%CS55.2%, TPP14.1%, HA21.9%, water 3.3%.
Embodiment 2
A kind of people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powder of HA modification is prepared, method is as follows:
Step (1) prepares human umbilical cord mesenchymal stem cells serum-free medium, and specific method is the same as embodiment 1.It is wherein different
Cell fusion degree is 85% when place is to collect serum-free medium.
Step (2), ionic cross-linking preparation load people's umbilical cord MSC serum-free medium CS nanoparticle
The CS powder 20mg that molecular weight is 200,000 is weighed, is dissolved in the 10mL citric acid solution that mass fraction is 4.0%, matches
The CS solution for being 0.2% at mass fraction, ultrasound removing bubble, then crosses 0.45 μm of water system filter membrane, obtains clarifying bright CS
Solution.9.9mL people's umbilical cord MSC serum-free medium that activated protein concentration is 1015 μ g/mL is slowly dropped in CS solution,
20min is stirred, mixed liquor is obtained.0.2% TPP solution 2.5mL is added dropwise in above-mentioned mixed liquor, 30min is reacted at room temperature.Wherein
The mass ratio of CS and TPP is 4:1, and the mass ratio of activated protein is 2:1 in CS and people's umbilical cord MSC serum-free medium.
By the nanoparticle suspension high speed centrifugation after reaction, 4 DEG C of centrifuging temperature, centrifugation time 30min, centrifugal force 1.0 ×
104g.Centrifugation gained precipitating is washed with distilled water 5 times, is then freeze-dried, is freeze-dried parameter are as follows: pre-freezing temperature -60
DEG C ± 2 DEG C, pre-freeze time 6h or more, cryogenic temperature -50~-56 DEG C, vacuum degree≤80mT, freeze-drying time 30h.After freeze-drying
People's umbilical cord MSC serum-free medium CS nanoparticle must be loaded.
Step (3), HA activation；
Preparing molecular weight is the HA aqueous solution 100mL that 15KDaHA concentration is 0.14%, and 1- ethyl -3- (3- diformazan ammonia is added
Base propyl)-carbodiimide (EDC) 700mg, n-hydroxysuccinimide (NHS) 840mg is added after it is completely dissolved, and is used
It is 7.5,37 DEG C of incubation 5h that 0.1M sodium hydroxide, which is adjusted to pH,.
Step (4) prepares load people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powder of HA modification
After load people's umbilical cord MSC serum-free medium CS nanoparticle that step (2) obtains is added to step (3) activation
In HA, the feed ratio of HA and CS nanoparticle is 4.7:1.Be stirred at room temperature reaction 10h, negate the nanosuspension after answering, at a high speed from
The heart, parameter of noncentricity are as follows: 4 DEG C of centrifuging temperature, centrifugation time 60min, centrifugal force 1.0 × 103g.Gained precipitating is washed with distilled water 3
It is secondary, it is then freeze-dried, is freeze-dried parameter are as follows: ± 2 DEG C of -60 DEG C of pre-freezing temperature, pre-freeze time 6h or more, freezing temperature
- 50~-56 DEG C of degree, vacuum degree≤80mT, freeze-drying time 30h.Up to people's umbilical cord MSC serum-free training of HA modification after freeze-drying
Nutrient solution CS nano-granule freeze-dried powder.
The people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powder for the HA modification that the present embodiment obtains is clear by measuring
It washes in CS nanoparticle liquid and the content of TPP determines the content for the TPP being crosslinked in centrifuged supernatant, detection method is using light splitting
The inventory of photometry (GB/T13171-1997), TPP is 5mg, and being crosslinked is 3.3mg.Detection HA Percentage bound learn, HA
Percentage bound with CS nanoparticle is 42.3%, wherein human umbilical cord mesenchymal stem cells serum-free medium activated protein ingredient
10.8%, CS 21.3%, TPP3.5%, HA63.2%, water 1.3%.
Embodiment 3
A kind of people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powder of HA modification is prepared, method is as follows:
Step (1) prepares human umbilical cord mesenchymal stem cells serum-free medium, and specific method is the same as embodiment 1.It is wherein different
Cell fusion degree is 90% when place is to collect serum-free medium.
Step (2), ionic cross-linking preparation load people's umbilical cord MSC serum-free medium CS nanoparticle
The CS powder 50mg that molecular weight is 300,000 is weighed, is dissolved in the 10mL tartaric acid solution that mass fraction is 0.3%, matches
The CS solution for being 0.5% at mass fraction, ultrasound removing bubble, then crosses 0.45 μm of water system filter membrane, obtains clarifying bright CS
Solution.10.5mL people's umbilical cord MSC serum-free medium that activated protein concentration is 950 μ g/mL is slowly dropped in CS solution,
30min is stirred, mixed liquor is obtained.0.18% TPP solution 5.5mL is added dropwise in above-mentioned mixed liquor, 30min is reacted at room temperature.Its
The mass ratio of middle CS and TPP is 5:1, and the mass ratio of activated protein is 5:1 in CS and people's umbilical cord MSC serum-free medium.
By the nanoparticle suspension high speed centrifugation after reaction, 4 DEG C of centrifuging temperature, centrifugation time 60min, centrifugal force 1.0 ×
103g.Centrifugation gained precipitating is washed with distilled water 10 times, is then freeze-dried, is freeze-dried parameter are as follows: pre-freezing temperature -60
DEG C ± 2 DEG C, pre-freeze time 6h or more, cryogenic temperature -50~-56 DEG C, vacuum degree≤80mT, freeze-drying time 36h.After freeze-drying
People's umbilical cord MSC serum-free medium CS nanoparticle must be loaded.
Step (3), HA activation；
Preparing molecular weight is the HA aqueous solution 150mL that 30KDaHA concentration is 0.1%, and 1- ethyl -3- (3- diformazan ammonia is added
Base propyl)-carbodiimide (EDC) 750mg, n-hydroxysuccinimide (NHS) 900mg is added after it is completely dissolved, and is used
It is 7.5,37 DEG C of incubation 10h that 0.1M sodium hydroxide, which is adjusted to pH,.
Step (4) prepares load people's umbilical cord MSC serum-free medium chitosan nanoparticle freeze-dried powder of HA modification
After load people's umbilical cord MSC serum-free medium CS nanoparticle that step (2) obtains is added to step (3) activation
In HA, the feed ratio of HA and CS nanoparticle is 2.5:1.Be stirred at room temperature reaction for 24 hours, negate the nanosuspension after answering, at a high speed from
The heart, parameter of noncentricity are as follows: 4 DEG C of centrifuging temperature, centrifugation time 10min, centrifugal force 1.0 × 105g.Gained precipitating is washed with distilled water 6
It is secondary, it is then freeze-dried, is freeze-dried parameter are as follows: ± 2 DEG C of -60 DEG C of pre-freezing temperature, pre-freeze time 6h or more, freezing temperature
- 50~-56 DEG C of degree, vacuum degree≤80mT, freeze-drying time 36h.Up to people's umbilical cord MSC serum-free training of HA modification after freeze-drying
Nutrient solution CS nano-granule freeze-dried powder.
The people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powder for the HA modification that the present embodiment obtains is clear by measuring
It washes in CS nanoparticle liquid and the content of TPP determines the content for the TPP being crosslinked in centrifuged supernatant, detection method is using light splitting
The inventory of photometry (GB/T13171-1997), TPP is 10mg, and being crosslinked is 6.8mg.Detection HA Percentage bound learn,
The Percentage bound of HA and CS nanoparticle is 28.6%, wherein human umbilical cord mesenchymal stem cells serum-free medium activated protein ingredient
8.8%, CS44.1%, TPP6.0%, HA37.8%, water 3.3%.
The properties of people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powder of HA modification of the invention are examined
It surveys, sample sets are the HA-MSC-CS-NPs that embodiment 1 is prepared, and control group, which uses, does not have to the MSC-CS-NPs that HA is modified,
Other technological parameters are identical with embodiment 1.
(1) partial size and particle diameter distribution
1 MSC-CS-NPs and HA-MSC-CS-NPs particle diameter distribution of table and PDI
As it can be seen from table 1 the MSC-CS-NPs partial size slightly increase tendency after HA is modified, but PDI index is obvious
Reduce (PDI is polydispersity index, and size indicates the width of particle diameter distribution), illustrates the particle diameter distribution ratio of HA-MSC-CS-NPs
Control group is more uniform, and system also tends to stablize.
(2) flow cytometer investigates increasing of the people's umbilical cord MSC free serum culture CS nano-granule freeze-dried powder of HA modification to cell
Grow power.
It uses l cell L929 for target cell, compares control group --- people's umbilical cord MSC serum-free medium CS
Nano-granule freeze-dried powder (MSC-CS-NPs), people's umbilical cord MSC serum-free medium CS nanoparticle freeze-drying of sample sets --- HA modification
Influence of the powder (HA-MSC-CS-NPs) in culture 48h and 72h, to L929 cell Proliferation vigor.
As shown in Figure 2,3, on the scatter plot of bivariate flow cytometer, left lower quadrant was represented for X-axis and Y-axis institute's generation
The parameter of table is in cell, that is, normal live cells of double-negative reaction, and right lower quadrant representative is positive for parameter representated by X-axis
Reaction, the cell, that is, viable apoptotic cell negative for parameter representated by Y-axis, right upper quadrant are represented for X-axis and Y
Parameter representated by axis is in the cell, that is, late apoptic and dead cell of double sun reactions, and left upper quadrant representative is for representated by Y-axis
Parameter be positive, the cell, that is, fragment negative for parameter representated by X-axis and damaging cells.
As shown in Figure 4,5, compared with control group MSC-CS-NPs, 48h, normal work are thin after the addition by HA-MSC-CS-NPs
Born of the same parents' number is the 92.51% of sum, and control group is only 88.95%, is higher by 3.56 percentage points than control group；And viable apoptotic cell
Number is total 4.00%, control group 8.30%, 4.30 percentage points out lower than control group.
Compared with control group MSC-CS-NPs, 72h, normal live cells number are total to HA-MSC-CS-NPs after the addition
96.13%, control group is only 93.05%, is higher by 3.08 percentage points than control group；And viable apoptotic cell number is total
2.38%, control group 3.87%, 1.49 percentage points out lower than control group.
It can be seen that introduce in MSC-CS-NPs after HA from this result and be compared with unmodified nanoparticle, not only improved
Promotion cel l proliferation, while also having the function of delaying Apoptosis.
Claims (8)
1. the preparation method of hyaluronic acid decorated people's umbilical cord MSC serum-free medium chitosan nanoparticle freeze-dried powder, described
People's umbilical cord MSC serum-free medium chitosan nanoparticle freeze-dried powder of bright matter acid modification, includes following components by mass percentage:
People's umbilical cord MSC serum-free medium activated protein ingredient 5~12%, chitosan 20~65%, sodium tripolyphosphate 1~15%, thoroughly
The quality summation of bright matter acid 20~65%, water≤5%, the above components is 100%；
The following steps are included:
Step 1, preparation load people's umbilical cord MSC serum-free medium chitosan nanoparticle；
People's umbilical cord MSC serum-free medium is added in chitosan acid solution, and sodium tripolyphosphate is added, is reacted at room temperature；It will be anti-
Nanoparticle suspension centrifugation after answering, gained washing of precipitate, freeze-drying obtain load people's umbilical cord MSC serum-free medium shell
Glycan nanoparticle；
Step 2, hyaluronic acid decorated load people's umbilical cord MSC serum-free medium chitosan nanoparticle freeze-dried powder is prepared；
Carboxyl in hyaluronic acid is activated, then adds load people's umbilical cord MSC serum-free medium chitosan nanoparticle
Entering and reacted in the hyaluronic acid to after activation, negate the nanosuspension after answering, is centrifuged, gained washing of precipitate is simultaneously freeze-dried,
Up to hyaluronic acid decorated people's umbilical cord MSC serum-free medium chitosan nanoparticle freeze-dried powder.
2. hyaluronic acid decorated people's umbilical cord MSC serum-free medium chitosan nanoparticle freeze-drying according to claim 1
The preparation method of powder, which is characterized in that the MSC serum-free medium of people's umbilical cord described in step 1 the preparation method comprises the following steps: taking newborn
Umbilical cord, by intravenous bloodstain washes clean；Epidermis, two arteries and a vein blood vessel are removed, tissue is shredded, is added
DMEM/F12 culture medium culture containing FBS, obtains primary human umbilical cord mesenchymal stem cells；When cell fusion degree is 80%-90%
When, trypsin digestion and cell secondary culture takes passage cell, continues to cultivate, and as cell fusion degree 80%-90%, abandoning contains
The culture supernatant of FBS, PBS wash cell, and the DMEM/F12 culture medium that serum-free is added continues culture 72 hours, collect serum-free
Culture solution.
3. hyaluronic acid decorated people's umbilical cord MSC serum-free medium chitosan nanoparticle freeze-drying according to claim 1
The preparation method of powder, which is characterized in that activated protein in chitosan described in step 1 and people's umbilical cord MSC serum-free medium
Mass ratio is 2:1~10:1, and the mass ratio of chitosan and sodium tripolyphosphate is 2:1~5:1, and the molecular weight of chitosan is 2-30
Ten thousand.
4. hyaluronic acid decorated people's umbilical cord MSC serum-free medium chitosan nanoparticle freeze-drying according to claim 1
The preparation method of powder, which is characterized in that parameter of noncentricity described in step 1 are as follows: 2~8 DEG C of centrifuging temperature, centrifugation time 10~
60min, centrifugal force 1.0 × 103~1.0 × 105g。
5. hyaluronic acid decorated people's umbilical cord MSC serum-free medium chitosan nanoparticle freeze-drying according to claim 1
The preparation method of powder, which is characterized in that the molecular weight of hyaluronic acid described in step 2 is 8-30Kda, hyaluronic acid and load people's navel
Mass ratio with MSC serum-free medium chitosan nanoparticle is 1:1~5:1.
6. hyaluronic acid decorated people's umbilical cord MSC serum-free medium chitosan nanoparticle freeze-drying according to claim 1
The preparation method of powder, which is characterized in that the activated carboxylic method of hyaluronic acid described in step 2 is, using 1- ethyl -3- (3-
Dimethylamino-propyl)-carbodiimide and n-hydroxysuccinimide activate the carboxyl in hyaluronic acid, then adjust pH
It is incubated for 7.5,37 DEG C.
7. hyaluronic acid decorated people's umbilical cord MSC serum-free medium chitosan nanoparticle freeze-drying according to claim 1
The preparation method of powder, which is characterized in that freeze-drying parameter described in step 1 and step 2 is equal are as follows: -60 DEG C of pre-freezing temperature ± 2
DEG C, pre-freeze time 6h or more, cryogenic temperature -50~-56 DEG C, vacuum degree≤80mT, freeze-drying time 24-36h.
8. hyaluronic acid decorated people's umbilical cord MSC serum-free medium chitosan nanoparticle freeze-drying according to claim 1
The preparation method of powder, which is characterized in that the acid in chitosan acid solution described in step 1 for dissolving chitosan be formic acid, acetic acid,
Propionic acid, lactic acid, malic acid, citric acid, ascorbic acid, oxalic acid, succinic acid, malonic acid, pyruvic acid, glutaric acid, tartaric acid, asparagus fern
One or more of propylhomoserin, salicylic acid or glycolic.
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| CN1694903A (en) * | 2002-07-26 | 2005-11-09 | 株式会社Lg生命科学 | Hyaluronic acid derivative gel and method for preparing the same |
| CN104906073A (en) * | 2015-06-10 | 2015-09-16 | 青岛大学附属医院 | Preparation method of chitosan quaternary ammonium salt hyaluronic acid nanogel coated with basic fibroblast growth factors |
| CN104958251A (en) * | 2015-06-10 | 2015-10-07 | 杨甫进 | Preparation method of hyaluronic acid nanogel |
| CN106176563A (en) * | 2016-07-26 | 2016-12-07 | 西安艾尔菲生物科技有限公司 | People's umbilical cord lipidosome freeze-dried powder of MSC serum-free medium and preparation thereof and application |
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| CN1694903A (en) * | 2002-07-26 | 2005-11-09 | 株式会社Lg生命科学 | Hyaluronic acid derivative gel and method for preparing the same |
| CN104906073A (en) * | 2015-06-10 | 2015-09-16 | 青岛大学附属医院 | Preparation method of chitosan quaternary ammonium salt hyaluronic acid nanogel coated with basic fibroblast growth factors |
| CN104958251A (en) * | 2015-06-10 | 2015-10-07 | 杨甫进 | Preparation method of hyaluronic acid nanogel |
| CN106176563A (en) * | 2016-07-26 | 2016-12-07 | 西安艾尔菲生物科技有限公司 | People's umbilical cord lipidosome freeze-dried powder of MSC serum-free medium and preparation thereof and application |
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