CN108949676A - A kind of ginsenoside Rg1's autologous fat stem cell extracorporeal culturing method - Google Patents
A kind of ginsenoside Rg1's autologous fat stem cell extracorporeal culturing method Download PDFInfo
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Abstract
The present invention provides a kind of ginsenoside Rg1's autologous fat stem cell extracorporeal culturing method, includes the following steps: to take people's abdominal adipose tissue；People's abdominal adipose tissue is digested using collagenase type I, obtains digestion tissue；Primary cell separation and inoculated and cultured are carried out to the digestion tissue, obtain stem cell；It takes the stem cell to be added into induced medium, carries out secondary culture；It wherein, include ginsenoside Rg1 in culture medium；At 6-8 days through trypsin digestion, secondary secondary culture is carried out；It takes third generation fat mesenchymal stem cell to be cultivated, harvests ginsenoside Rg1's autologous fat stem cell.Ginsenoside Rg1's autologous fat stem cell prepared by method provided by the present invention is greatly improved the proliferation rate of stem cell, and differentiation capability is strong.
Description
Technical field
The present invention relates to technical field of stem cell culture, specifically, it is dry thin to be related to a kind of ginsenoside Rg1's autologous fat
Born of the same parents' extracorporeal culturing method.
Background technique
Stem cell (stem cell) is a kind of multipotential cell with the of self-replication capacity (self-renewing).?
Under certain condition, it can be divided into multiple functions cell.The stage of development according to locating for stem cell be divided into embryonic stem cell and
Adult stem cell.It is divided into three classes according to the potentiality of development of stem cell: myeloid-lymphoid stem cell, multipotential stem cell and unipotent stem cell.It is dry
Cell is a kind of inabundant differentiation, and still immature cell has the potential function for regenerating various histoorgans and human body, medicine
Boundary is known as " general-purpose cell ".
Fat stem cell (adipose-derived stem cells, ADSCs), ADSC pluripotent cell are in recent years from rouge
Isolated a kind of stem cell with multi-lineage potential in fat tissue.The repair function of main recovery organization cell, promotees
Into the regeneration of cell, while restoring young face, physical function is also fully improved, and is effectively improved inferior health, early ageing etc.
Disease is really effective against aging from inside to outside.
Currently, fat stem cell prepared by traditional preparation methods, differentiation capability is poor, has a single function, cell proliferation rate
It is low.
Summary of the invention
In view of this, providing a kind of ginseng the technical problem to be solved in the present invention is that overcome the deficiencies of existing technologies
Saponin(e Rg1 autologous fat stem cell extracorporeal culturing method, includes the following steps:
Take people's abdominal adipose tissue；
People's abdominal adipose tissue is digested using collagenase type I, obtains digestion tissue；
Primary cell separation and inoculated and cultured are carried out to the digestion tissue, obtain stem cell；
It takes the stem cell to be added into induced medium, carries out secondary culture；Also,
To being changed liquid 1 time after culture solution 24 hours of secondary culture, change within every 3 days liquid 1 time later；Wherein, in the culture medium
Including ginsenoside Rg1；
When cell growth fusion reaches the first default percentage within 6-8 days, through trypsin digestion, secondary passage training is carried out
It supports；
It takes and is cultivated by the third generation fat mesenchymal stem cell of secondary secondary culture, growing to culture bottle bottom
When the area of the second default percentage, ginsenoside Rg1's autologous fat stem cell is harvested.
Further, after described " taking people's abdominal adipose tissue ", further includes:
People's abdominal adipose tissue is rinsed using phosphate-buffered salt.
Further, the sampling amount of people's abdominal adipose tissue is 20mL.
Further, the concentration of ginsenoside Rg1 is 10 in the culture medium-6mol/L-10-8mol/L。
Further, the described first default percentage is 80%-90%.
Further, the described second default percentage is 70%-80%.
Further, the concentration of the trypsase is 0.20%-0.28%.
Further, described " when the growth fusion of 6-8 days cells reaches the first default percentage, through trypsin digestion,
Carry out secondary secondary culture " after, further includes:
By observing primary in secondary secondary culture and passage fat mesenchymal stem cell shape under inverted phase contrast microscope
State and upgrowth situation.
A kind of ginsenoside Rg1's autologous fat stem cell extracorporeal culturing method provided by the invention, by people's abdomen rouge
Fat tissue is digested, is separated, is inoculated with and cultivated, and includes two in the induced medium of ginsenoside Rg1 by addition
Secondary secondary culture, so that the third generation fat mesenchymal stem cell after secondary secondary culture passes through culture, to obtain ginseng
Saponin(e Rg1 autologous fat stem cell.Ginsenoside Rg1's autologous fat stem cell can be significantly prepared by method provided by the present invention
The proliferation rate of stem cell is improved, differentiation capability is strong and also has ginsenoside in addition to the possessed function of fat stem cell itself
The relieving fatigue of Rg1, anti-aging, improve the effects of learning and memory, protection cardiovascular function, immunoregulation effect.
Specific embodiment
In order to illustrate more clearly of technical solution of the present invention, combined with specific embodiments below to claim of the invention
It is described in further detail, it is to be understood that illustrate only certain embodiments of the present invention below, therefore be not to be seen as
It is the restriction to range, the modification of anyone limited times made within the scope of the invention as claimed, still in power of the invention
Within sharp claimed range.
The present invention provides a kind of ginsenoside Rg1's autologous fat stem cell extracorporeal culturing methods, include the following steps:
S1 takes people's abdominal adipose tissue；
S2 digests people's abdominal adipose tissue using collagenase type I, obtains digestion tissue；
S3 carries out primary cell separation and inoculated and cultured to the digestion tissue, obtains stem cell；
S4 takes the stem cell to be added into induced medium, carries out secondary culture；Also,
To being changed liquid 1 time after culture solution 24 hours of secondary culture, change within every 3 days liquid 1 time later；Wherein, in the culture medium
Including ginsenoside Rg1；
S5, through trypsin digestion, carries out secondary biography when cell growth fusion reaches the first default percentage within 6-8 days
It is commissioned to train feeding；
S6 takes and is cultivated by the third generation fat mesenchymal stem cell of secondary secondary culture, growing to culture bottle
When the area of the default percentage of the second of bottom, ginsenoside Rg1's autologous fat stem cell is harvested.
Above-mentioned, people's stomach fat group is preferably the extra adipose tissue of normal adults abdomen.
It is above-mentioned, it should be noted that mescenchymal stem cell is one kind of multipotential stem cell, the potential with Multidirectional Differentiation,
In vitro induce under conditions of can be divided into the Various Tissues such as muscle, nerve, bone, cartilage, fat, cardiac muscle and class hepatic tissue and
Cell.Still there is proliferation replication capacity after Long Term Passages, be a kind of seed cell of ideal histoorgan engineering.At present
Clinically also there are more and more people to be applied to the reparation and immune system of histoorgan using mescenchymal stem cell.
Ginsenoside (Ginsenoside) is a kind of steroid compound, triterpenoid saponin.It is primarily present in Panax medicinal material
In.Ginsenoside is considered to be the active constituent in ginseng, thus becomes the target of research.Because ginsenoside affects multiple
Metabolic pathway, so its efficiency is also complicated, and the monomer component of various ginsenosides is difficult to separate.
It should be noted that ginsenoside all has similar basic structure, all contain by 30 carbon atom arrangements at four
The gonane steroids core of a ring.They are divided into two groups according to the difference of glycosyl framework: dammarane type and oleanane type.It reaches
Ma alkanes type includes two classes: panoxadiol type-A type, glycosides are 20 (S)-protopanoxadiols originally.Most ginsenosides is contained,
Such as ginsenoside Rb1, Rb2, Rb3, Rc, Rd, Rg3, Rh2 and glycosyl PD；Panaxatriol type-Type B, glycosides are 20 (S)-protoplasts originally
Join triol.Contain ginsenoside Re, Rg1, Rg2, Rh1 and glycosyl PT.
Oleanane type: oleanolic acid type-c-type, glycosides are oleanolic acid originally.
Total saposins not haemolysis, A type anti-hemolysis and Type B, c-type haemolysis.
Wherein, ginsenoside Rg1 have can rapid recovery fatigue, improve learning and memory, delay senescence, there is excited maincenter
Effects on neural system inhibits platelet aggregation effect.
It is above-mentioned, in the step of S1 takes people's abdominal adipose tissue, may include:
S11, shearing obtain the extra abdominal adipose tissue of adult healthy, are placed in sterile bag, and being sent into laboratory prevents dirt
Dye；
S12 takes out abdominal adipose tissue in superclean bench, with 75% alcohol bubble 2min (50-100ml centrifugation
Pipe).
S13, it is complete with ethanol postincubation, it moves into be equipped in PBS (phosphate buffer) or the container of physiological saline rapidly and impregnate
Cleaning 2 times, each 2min.
A kind of ginsenoside Rg1's autologous fat stem cell extracorporeal culturing method provided by the invention, by people's abdomen rouge
Fat tissue is digested, is separated, is inoculated with and cultivated, and includes two in the induced medium of ginsenoside Rg1 by addition
Secondary secondary culture, so that the third generation fat mesenchymal stem cell after secondary secondary culture passes through culture, to obtain ginseng
Saponin(e Rg1 autologous fat stem cell.Ginsenoside Rg1's autologous fat stem cell can be significantly prepared by method provided by the present invention
The proliferation rate of stem cell is improved, differentiation capability is strong and also has ginsenoside in addition to the possessed function of fat stem cell itself
The relieving fatigue of Rg1, anti-aging, improve the effects of learning and memory, protection cardiovascular function, immunoregulation effect.
Further, after the S1 " taking people's abdominal adipose tissue ", further includes:
S7 is rinsed people's abdominal adipose tissue using phosphate-buffered salt.
Further, the sampling amount of people's abdominal adipose tissue is 20mL.
Further, the concentration of ginsenoside Rg1 is 10 in the culture medium-6mol/L-10-8mol/L。
Further, the described first default percentage is 80%-90%.
Further, the described second default percentage is 70%-80%.
Further, the concentration of the trypsase is 0.20%-0.28%.
Preferably, the concentration of the trypsase is 0.25%.
Further, the S5 " when cell growth fusion reaches the first default percentage within 6-8 days, disappears through trypsase
Change, carry out secondary secondary culture " after, further includes:
S8, by observing primary in secondary secondary culture and passage fat mesenchymal stem cell under inverted phase contrast microscope
Form and upgrowth situation.
In addition, currently, the domestic comparison extracted to general ginsenoside is more, but extraction ginsenoside monomer is less, because
Technical difficulty is relatively high, and the enterprise that the current country has production ginsenoside monomer condition is few, so the cost of monomer saponin
The cost of especially ginsenoside Rg1 is very high, and the purity of commercially available ginsenoside Rg1 cell culture grade is not achieved can not
Adapt to stem cell culture, later period high-volume is given in the culture for existing for stem cell of impurity, detection and using certain interference is caused
Production and research and development cause huge cost, to solve this problem, in the present invention before step S1, provide a kind of ginsenoside
The extracting method of Rg1, comprising:
1, the dry root 50Kg for taking ginseng, is crushed by pulverizer, is crossed 20-40 mesh screen, is obtained the raw material that feeds intake；
2, the raw material soaking that will feed intake is sufficiently stirred 20 points in each 1 hour therebetween 5 hours in 70% ethyl alcohol of normal temperature and pressure
Clock；
3, after impregnating, the immersion medical fluid of feed intake raw material and 70% ethyl alcohol is passed through together in high-pressure microwave extract equipment and is carried out
High-pressure microwave extracts, power 200-500W, and extraction time is 1-1.5 hours, after extraction, filtrate is obtained by filtration, then the dregs of a decoction are added
70% ethyl alcohol for entering 6 times is carried out second extraction 1 hour by high-pressure microwave extract equipment, filtrate twice is merged after filtering；
4, the filtrate of merging is concentrated under the conditions of reduced vacuum, then when concentration reaches 1.10-1.30, stops concentration, is soaked
Cream；
5, by the medicinal extract by belt drying method, under vacuum conditions and temperature setting is 70-85 DEG C, carries out belt
Ginsenoside Rg1's extract powder is obtained after dry, pulverize sieving；
6, ginsenoside Rg1's extract powder is dissolved in a small amount of ethyl alcohol, and by high speed adverse current chromatogram HSCCC into
Row purifies for the first time, and configuration extractant is ethyl acetate-n-butanol-water=30:10:1, after extractant is crossed liquid standing,
It inputs in HSCCC, wavelength is set as 540nm, carries out high speed abstraction purification, obtains refined solution；
7, it by after refined solution concentration, is purified by sephadex lh-20, LH-20 is molecular sieve, and solvent is
Methanol, flow velocity 2-5mL/min obtain molecular sieve filtration liquid；
8, the molecular sieve filtration liquid is separated, wavelength 540nm is obtained by preparation liquid phase using reversed silica gel
To ginsenoside Rg1's monomer.
In the present invention, crude separation, method are carried out using feed intake raw material of the high-pressure microwave extract equipment to the dry root of ginseng
Simple speed is fast, and recovery rate is high；And the loss for greatly reducing effective content in medical fluid is dried by belt drying method；
And then purifies and separates are carried out using high speed adverse current chromatogram, sephadex and high performance liquid chromatography, to finally obtain ginseng
Saponin(e Rg1 monomer, purity reach 98% or more, purity is high, and method is simple, reduces time cost, consumptive material less using solvent
Cost and cost of labor, and in preparing autologous fat stem cell make by oneself ginsenoside Rg1's monomer, greatly reduce raw material at
This.
Ginsenoside Rg1's autologous fat stem cell extracorporeal culturing method employed in embodiment 1-3 includes the following:
1, the dry root 50Kg for taking ginseng, is crushed by pulverizer, is crossed 20-40 mesh screen, is obtained the raw material that feeds intake；
2, the raw material soaking that will feed intake is sufficiently stirred 20 points in each 1 hour therebetween 5 hours in 70% ethyl alcohol of normal temperature and pressure
Clock；
3, after impregnating, the immersion medical fluid of feed intake raw material and 70% ethyl alcohol is passed through together in high-pressure microwave extract equipment and is carried out
High-pressure microwave extracts, power 200-500W, and extraction time is 1-1.5 hours, after extraction, filtrate is obtained by filtration, then the dregs of a decoction are added
70% ethyl alcohol for entering 6 times is carried out second extraction 1 hour by high-pressure microwave extract equipment, filtrate twice is merged after filtering；
4, the filtrate of merging is concentrated under the conditions of reduced vacuum, then when concentration reaches 1.10-1.30, stops concentration, is soaked
Cream；
5, by the medicinal extract by belt drying method, under vacuum conditions and temperature setting is 70-85 DEG C, carries out belt
Ginsenoside Rg1's extract powder is obtained after dry, pulverize sieving；
6, ginsenoside Rg1's extract powder is dissolved in a small amount of ethyl alcohol, and by high speed adverse current chromatogram HSCCC into
Row purifies for the first time, and configuration extractant is ethyl acetate-n-butanol-water=30:10:1, after extractant is crossed liquid standing,
It inputs in HSCCC, wavelength is set as 540nm, carries out high speed abstraction purification, obtains refined solution；
7, it by after refined solution concentration, is purified by sephadex lh-20, LH-20 is molecular sieve, and solvent is
Methanol, flow velocity 2-5mL/min obtain molecular sieve filtration liquid；
8, the molecular sieve filtration liquid is separated, wavelength 540nm is obtained by preparation liquid phase using reversed silica gel
To ginsenoside Rg1's monomer；
9, health adult supernumerary's abdominal adipose tissue is taken, comprising:
9.1, shearing obtains the extra abdominal adipose tissue 20mL of adult healthy, is placed in sterile bag, and it is anti-to be sent into laboratory
Only pollute；
9.2, in superclean bench, abdominal adipose tissue is taken out, with 75% alcohol bubble 2min (50-100ml centrifugation
Pipe)；
9.3 is complete with ethanol postincubation, moves into rapidly clear equipped with impregnating in PBS (phosphate buffer) or the container of physiological saline
It washes 2 times, people's abdominal adipose tissue can be obtained in each 2min；
10, soaking flushing is carried out using phosphate-buffered salt again to people's abdominal adipose tissue；
11, people's abdominal adipose tissue is digested using collagenase type I, obtains digestion tissue；
12, primary cell separation and inoculated and cultured are carried out to the digestion tissue, obtains stem cell；
13, it takes the stem cell to be added into induced medium, carries out secondary culture；Also,
To being changed liquid 1 time after culture solution 24 hours of secondary culture, change within every 3 days liquid 1 time later；Wherein, in the culture medium
Including ginsenoside Rg1, concentration 10-6mol/L-10-8mol/L；
14, when cell growth fusion reaches the first default percentage within 6-8 days, through trypsin digestion, secondary biography is carried out
It is commissioned to train feeding；
15, by observing primary in secondary secondary culture and passage fat mesenchymal stem cell under inverted phase contrast microscope
Form and upgrowth situation；
16, it takes and is cultivated by the third generation fat mesenchymal stem cell of secondary secondary culture, growing to culture bottle
When the area of the default percentage of the second of bottom, ginsenoside Rg1's autologous fat stem cell is harvested.
Ginsenoside Rg1's autologous fat stem cell extracorporeal culturing method parameter setting in 1 embodiment 1-3 of table
Embodiment 1:
The present embodiment uses the step in such as above-mentioned ginsenoside Rg1's autologous fat stem cell extracorporeal culturing method, also,
It is prepared using the parameter setting in such as table 1.
Embodiment 2:
The present embodiment uses the step in such as above-mentioned ginsenoside Rg1's autologous fat stem cell extracorporeal culturing method, also,
It is prepared using the parameter setting in such as table 1.
Embodiment 3:
The present embodiment uses the step in such as above-mentioned ginsenoside Rg1's autologous fat stem cell extracorporeal culturing method, also,
It is prepared using the parameter setting in such as table 1.
Proliferation rate comparative experiments:
Experimental principle: MTT analytic approach is with living cells metabolin reducing agent 3- (4,5)-dimethylthiahiazo (- z-
Y1 based on) -3,5-di-phenytetrazoliumromide, MTT thiazolyl blue.MTT is yellow compound, is a kind of receiving
Hydrionic dyestuff may act on the respiratory chain in living cells mitochondria, under the action of succinate dehydrogenase and cromoci
Tetrazolium ring opening, generate blue formazan crystallization, formazan crystallization production quantity only with number of viable cells at
Direct ratio (succinate dehydrogenase disappears in dead cell, cannot restore MTT).The formazan crystallization that reduction generates can contain 50%
N,N-Dimethylformamide and 20% ten dimethyl sodium sulfonates (pH 4.7) MTT lysate in dissolve, utilize microplate reader
The optical density OD value at 490nm is measured, to reflect number of viable cells.It can also be dissolved with DMSO.
Reagent: 10% tire calf serum, MTT solution, DMSO；
Instrument, consumptive material: 96 orifice plates, centrifuge, enzyme linked immunological monitor；
Laboratory sample: the commercially available fat stem cell of control sample 1-, the medulla mesenchyma of control sample 2- ginsenoside Rd induction are dry
Preparation-obtained ginsenoside Rg1's autologous fat stem cell in cell, embodiment 1-3；
Experimental procedure:
1, inoculating cell
It is made into individual cells suspension with culture solution is obtained containing 10% tire calf serum, (is divided with 1000-10000, every hole cell
Different laboratory samples is not added, each sample does at least two secondary orifices) it is inoculated into 96 orifice plates, every pore volume 200ul.
2, cell is cultivated
With general condition of culture, cultivate 3-5 days.
3, colour generation
After culture 3-5 days, every hole adds MTT solution (5mg/ml is matched with PBS) 20ul. to continue to be incubated for 4 hours, terminates culture,
Careful inhale abandons culture supernatant in hole, inhales again after needing to be centrifuged for suspension cell and abandons culture supernatant in hole.Every hole adds 150ul
DMSO vibrates 10 minutes, melts crystal sufficiently.
4, colorimetric
490nm wavelength is selected, each hole absorbance value is measured on enzyme linked immunological monitor, records result.
The cell proliferation rate result of table 2 embodiment 1-3 and control sample 1-2
| Proliferation rate % | |
| Embodiment 1 | 151.22 |
| Embodiment 2 | 155.28 |
| Embodiment 3 | 161.17 |
| Control sample 1 | 101.31 |
| Control sample 2 | 138.87 |
Experimental result:
Cell proliferation rate is measured to embodiment 1-3 and check sample 1-2 by mtt assay.Wherein, embodiment 1-3 exists
Reach 10-6mol/L-10-8Mol/L concentration Rg1 stimulates the proliferation times of lower stem cell to be higher by 50% or more than check sample 1.
And the cell proliferation rate of embodiment 1-3 is above check sample 2, it was demonstrated that utilizes ginsenoside Rg1 provided in the present invention
Ginsenoside Rg1's autologous fat stem cell prepared by autologous fat stem cell extracorporeal culturing method has very high cell Proliferation
Rate.
Rg1 also has osteoinductive activity simultaneously, can be in early promotion autologous fat mescenchymal stem cell to Osteoblast Differentiation.
It should be appreciated that although this specification is described in terms of embodiments, but not each embodiment only includes one
A independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should will say
As a whole, the technical solution in each embodiment may also be suitably combined to form those skilled in the art can for bright book
With the other embodiments of understanding.
The Applicant declares that the present invention can only for of the invention by the series of detailed descriptions listed above
Row embodiment illustrates, but the present invention is not limited to the above detailed process equipment and process flow.And i.e. not
Mean that the present invention should rely on above-mentioned detailed process equipment and process flow and could implement.Person of ordinary skill in the field answers
This is illustrated, any improvement in the present invention, the addition of equivalence replacement and auxiliary element to each raw material of product of the present invention, specific side
The selection etc. of formula, all of which fall within the scope of protection and disclosure of the present invention.
Claims (8)
1. a kind of ginsenoside Rg1's autologous fat stem cell extracorporeal culturing method characterized by comprising
Take people's abdominal adipose tissue；
People's abdominal adipose tissue is digested using collagenase type I, obtains digestion tissue；
Primary cell separation and inoculated and cultured are carried out to the digestion tissue, obtain stem cell；
It takes the stem cell to be added into induced medium, carries out secondary culture；Also,
To being changed liquid 1 time after culture solution 24 hours of secondary culture, change within every 3 days liquid 1 time later；Wherein, include in the culture medium
Ginsenoside Rg1；
When cell growth fusion reaches the first default percentage within 6-8 days, through trypsin digestion, secondary secondary culture is carried out；
It takes and is cultivated by the third generation fat mesenchymal stem cell of secondary secondary culture, growing to the second of culture bottle bottom
When the area of default percentage, ginsenoside Rg1's autologous fat stem cell is harvested.
2. ginsenoside Rg1's autologous fat stem cell extracorporeal culturing method as described in claim 1, which is characterized in that described " to take
After people's abdominal adipose tissue ", further includes:
People's abdominal adipose tissue is rinsed using phosphate-buffered salt.
3. ginsenoside Rg1's autologous fat stem cell extracorporeal culturing method as described in claim 1, which is characterized in that the people
The sampling amount of abdominal adipose tissue is 20mL.
4. ginsenoside Rg1's autologous fat stem cell extracorporeal culturing method as described in claim 1, which is characterized in that the training
The concentration for supporting ginsenoside Rg1 in base is 10-6mol/L-10-8mol/L。
5. ginsenoside Rg1's autologous fat stem cell extracorporeal culturing method as described in claim 1, which is characterized in that described
One default percentage is 80%-90%.
6. ginsenoside Rg1's autologous fat stem cell extracorporeal culturing method as described in claim 1, which is characterized in that described
Two default percentage are 70%-80%.
7. ginsenoside Rg1's autologous fat stem cell extracorporeal culturing method as described in claim 1, which is characterized in that the pancreas
The concentration of protease is 0.20%-0.28%.
8. ginsenoside Rg1's autologous fat stem cell extracorporeal culturing method as described in claim 1, which is characterized in that described "
When cell growth fusion reaches the first default percentage within 6-8 days, through trypsin digestion, secondary secondary culture is carried out " after, also
Include:
By under inverted phase contrast microscope observe in secondary secondary culture it is primary and passage fat mesenchymal stem cell form and
Upgrowth situation.
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| CN114191413A (en) * | 2021-12-14 | 2022-03-18 | 浙江大学 | Application of metal-organic ligand framework ZIF-67 modified hollow vanadium dioxide core-membrane composite structure drug carrier |
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