CN107988152A - The separation method of oxidation-resistant active ingredient in human umbilical cord mesenchymal stem cells nutrient solution - Google Patents
The separation method of oxidation-resistant active ingredient in human umbilical cord mesenchymal stem cells nutrient solution Download PDFInfo
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Abstract
The present invention provides a kind of method that the active ingredient with anti-oxidation efficacy is isolated in nutrient solution from human umbilical cord mesenchymal stem cells.The preparation of the active ingredient is carried out as steps described below：During normal Cesarean operation or natural labor, umbilical cord is taken under aseptic condition, blood vessel and mucous membrane are removed after cleaning and sterilizing is carried out to umbilical cord, umbilical cord tissue is shredded and is put into blake bottle and cultivate.Take the logarithm the human umbilical cord mesenchymal stem cells nutrient solution of phase growth, carry out centrifuging super rate using the ultra-filtration centrifuge tube of different size, the final isolate for obtaining the molecular weight with anti-oxidation efficacy in 10KDa~30KDa.This partial separation can freeze and freeze-dried powder is made, to facilitate storage to use.Human umbilical cord mesenchymal stem cells nutrient solution isolate provided by the invention has good antioxidation activity, it can help the infringement of cells against free radicals, isolate comes from human body autologous tissue, it is safe, it is expected to be applied in the products such as medicine, health products, skin care item and other daily chemicals.Separation method provided by the present invention is simple and practicable, of low cost, non-environmental-pollution, is adapted to industrialized production.
Description
Technical field
Separated the present invention relates to one kind from human umbilical cord mesenchymal stem cells nutrient solution prepare a kind of molecular weight for 10KDa~
The method with oxidation-resistant active ingredient between 30KDa, belongs to cell and bioengineering field.
Background technology
Antioxidant can weaken or dispel infringement of the free radical to human body, or protect food to become from oxidative damage
Matter.Scientific investigations showed that cancer, aging or Other diseases are mostly relevant with the generation of excessive free radicals.Study it is anti-oxidant can
Effectively to overcome harm caused by it, so anti-oxidant by medicine, health products, skin care item manufacturing enterprise and other daily-use chemical industries
Manufacturing enterprise is classified as one of R＆D direction, and one of most important feature demand in market.Chemical synthesis antioxidant, does not have such as
Propyl galate, dibutyl hydroxy toluene etc., DMEM hydantoins etc., it is cheap, it is widely used in food, daily use chemicals etc.
In industry.But the antioxidant of chemical synthesis has different degrees of toxic side effect, damage human body that can be in various degree
Liver, kidney and other organs, thus its additive amount in the product is strictly controlled by various countries.Come from natural in animal vegetable tissue
Antioxidant will effectively solve the problems, such as the toxic side effect of chemical synthesis antioxidant.
Mescenchymal stem cell（MSC）It is current laboratory and the most Adult multipotent stem cells of clinical research, in Hematopoietic Stem
There is important application in cell transplantation, graft versus host disease(GVH disease) etc..Derived from bone marrow MSC can be divided into according to source（BMSC）, fat
Source MSC and umbilical cord source MSC（hUCMSCs).Most study is the most widely BMSC, but due to when forefathers' bone at present
Marrow source BMSC levels are extremely low and higher there are viral infection rate, cell proliferation and differentiation potential decline with the increase at age and
Puncturing materials is an invasion, has wound, operation easy to pollute, and limitation is brought to its wide clinical application.Research find from
The hUCMSCs isolated in people's umbilical cord, BMSC is superior in terms of cell content, multiplication capacity, is repeatedly passed on amplification and is remained to protect
Vigorous function is held, is not expressed or low expression immunological rejection mark of correlation, immunogenicity is lower than BMSC, and convenient material drawing, no ethics
Dispute is learned, is presently considered to be ideal seed cell.
HUCMSCs cells when carrying out cell culture using adherent method, stablize, and opportunities for contamination is few by cell biological characteristics,
Research shows iuntercellular within 12 generations without multiplication capacity difference, and multiplication capacity is stronger, can be secreted in incubation a variety of
Growth factor, such as epithelial cell growth factor（EGF), fibroblast growth factor（bFGF）, conversion growth factor（TGF）
Deng.
It is contemplated that using anti-oxidation function to be oriented to, research exponential phase people's navel Mesenchymal stem cell nutrient solution has
Separated with component and to it, for the ingredient origin in tissue, safe, antioxygenic property is good, is expected to be applied to medicine
In product, health products and skin care item.People's navel mescenchymal stem cell can produce largely discarded nutrient solution, this hair in incubation
The bright utilization rate that can also improve these spent medias, reduces the cost of processing spent media, mitigates spent media to ring
The pollution in border, the application for improving people's navel Mesenchymal stem cell nutrient solution, have certain market economy benefit.
The content of the invention
The present invention only with centrifugal ultrafiltration method to the component of exponential phase people's navel Mesenchymal stem cell nutrient solution into
Row separation, and active ingredient contained in variant fraction is studied, it turns out that, filled between exponential phase people's navel
Component of the molecular weight between 10KDa~30KDa in matter stem cell medium has excellent anti-oxidation function, it can be effective
Remove DPPH free radicals, hydroxyl radical free radical and ultra-oxygen anion free radical.This method separating technology is simple, and separation costs are cheap,
Isolate is expected to be applied in the daily chemicals such as medicine, health products, skin care item, is prepared into the product of diversified forms.
A kind of point of exponential phase people's navel Mesenchymal stem cell nutrient solution with antioxidation activity provided by the invention
It is as follows from thing, preparation process.
Step 1：The preparation of human umbilical cord mesenchymal stem cells.
（1）The preservation of umbilical cord：During normal Cesarean operation or natural labor, umbilical cord is taken under aseptic condition, umbilical cord is stored
In preserving in liquid, stored for future use at 4 DEG C.
（2）The cleaning of umbilical cord：It is put into from taking-up umbilical cord in liquid is preserved in sterilization container, adds 40 ml~70 of physiological saline
Ml, concussion rinsing 20s~30s, is transferred in another sterilization container；The ml metronidazoles of 40 ml~70 are added into the sterilization container
Parenteral solution soaks 5~7 minutes, is then cleaned umbilical cord once again with the ml of 40 ml of physiological saline~70, after the completion of umbilical cord cleaning,
It is placed in disposable sterile petri dish.
（3）The preparation of human umbilical cord mesenchymal stem cells：Cut off the blood vessel and mucous membrane in umbilical cord with tweezers and scissors, and by its
Meat gruel shape is cut into, meat gruel shape tissue is transferred in centrifuge tube and adds appropriate phosphate buffer, in 1000 rpm~1500
5 min are centrifuged under rpm to wash 1 time, remove supernatant, after adding the ml of nutrient solution 10 ml~15, divide tissue suspension equally addition
Into two blake bottles, blake bottle is placed in 37 DEG C, is cultivated 3~5 days in 5% CO2gas incubator.
（4）The secondary culture of human umbilical cord mesenchymal stem cells：Reject culture medium, adds Tryple, is immediately placed in incubator,
After digesting 8min, treat that cell all comes off, add terminate liquid, that is, culture medium, dispel cell, under the rpm of 1000 rpm~1500
Centrifuge 5 min；Supernatant discarding, adds new culture medium, and blake bottle is placed in 37 DEG C, 3~5 are cultivated in 5% CO2gas incubator
My god.
Step 2：The separation of oxidation-resistant active ingredient.
（1）The collection and storage of exponential phase human umbilical cord mesenchymal stem cells nutrient solution：Between collection logarithmic phase people's umbilical cord
Mesenchymal stem cells nutrient solution, saves backup under the conditions of 1 DEG C~5 DEG C.
（2）The separation and screening of anti-oxidant active ingredient：By logarithmic phase human umbilical cord mesenchymal stem cells nutrient solution using not
The super filter tube of same specification carries out centrifuging super rate, in 1 DEG C~5 DEG C, the variant fraction of ultrafiltration collection under conditions of 2000g~5000g
Anti-oxidant experiment is carried out, assesses the antioxidant effect of variant fraction, final molecular weight of the acquisition with anti-oxidation efficacy is
Isolate between 10KDa-30KDa.Component with anti-oxidation efficacy is dispensed, is freezed spare.
Step 3：The measure of isolate antioxidation activity.
（1）The measure of scavenging ability of DPPH free radical.
Isolate freeze-dried powder is taken to be configured to the sample solution of various concentrations, removing energy of the measure isolate to DPPH free radicals
Power.Concretely comprise the following steps：The sample solution of 2 mL various concentrations gradients is separately added into centrifuge tube, then is separately added into 2 mLDPPH
Solution（95% ethanol is prepared, 0.1 mmol/L）, lucifuge reaction 30 minutes after mixing.Light absorption value A is measured at 517nm.On
State step and be added without DPPH, 2 mL95% ethanol, the constant data measured A1 of remaining step are added into each test tube.To centrifuge tube
2 mL purified waters of middle addition and 2 mLDPPH solution, remaining step is constant, measures light absorption value A0.Using purified water as blank, each
Sample does 3 parallel tests, is averaged, calculation formula is as follows：
DPPH free radical scavenging activities=[1- (A-A1)/A0] × 100%.
（2）The measure of hydroxyl radical free radical Scavenging activity.
Isolate freeze-dried powder is taken to be configured to the sample solution of various concentrations, removing energy of the measure isolate to hydroxyl radical free radical
Power.Experimental procedure is as follows：The Phen solution that 0.5 mL is prepared is separately added into the centrifuge tube of 10 mL, 1 mL phosphoric acid delays
Fliud flushing, the sample solution of 0.5 mL various concentrations, adds 0.5 mL0.75 mmol/LFeSO4Solution, H is added after mixing2O2
0.5 mL of solution, measures absorbance A x at 536nm；Sample solution in above-mentioned steps is changed to distilled water, remaining is constant, measure
Absorbance Af；By H in above-mentioned steps2O2Solution is changed to distilled water, the constant measure absorbance A of remaining step0；It is added without H2O2, its
Remaining step is constant, measure absorbance A s.Using purified water as blank, each sample does 3 parallel tests, is averaged, and calculates public
Formula is as follows：
Hydroxyl radical free radical clearance rate=（As-Ax）/(A0-Af) ×100%。
（3）The measure of ultra-oxygen anion free radical Scavenging activity.
Using assay NBT photoreduction.Take isolate freeze-dried powder to be configured to the sample solution of various concentrations, take 0.1 mL
Sample solution adds the Tris-Hcl buffer solutions of 0.1 mol/LpH8.2 of 2.8 mL, replaces sample molten with 0.1 mL purified waters
Liquid is as blank control.Concussion mixes mixed solution, and the thing of 0.1 mL 3mmol/L is added after ten minutes in 25 DEG C of water-bath insulations
The pyrogallol solution first preheated in 25 DEG C of water-baths, mixes rapidly and starts timing, A is measured at 325nm, every 30s
A325 is read, terminates after 4.5 minutes, is returned to zero with blank control.The regression equation that absorbance changes over time is done, its slope is neighbour
Benzenetriol autoxidation speed V, calculates inhibiting rate of the sample to superoxide anion according to the following formula：
Inhibiting rate（%）=（VControl-VSample）/VControl×100。
Oxidation-resistant active ingredient in human umbilical cord mesenchymal stem cells nutrient solution provided by the invention is to DPPH free radicals, hydroxyl
Base free radical and ultra-oxygen anion free radical have good scavenging action, and separating technology is simple, and easily operated, cost is low
It is honest and clean.
Brief description of the drawings
Fig. 1 is the measurement result of isolate scavenging ability of DPPH free radical.
Fig. 2 is the measurement result that isolate removes hydroxyl radical free radical ability.
Fig. 3 is that isolate removes the measurement result that ultra-oxygen anion free radical is removed.
Embodiment
Below in conjunction with attached drawing embodiment, the present invention is described in further detail.
The separation method of oxidation-resistant active ingredient, preparation process are as follows in human umbilical cord mesenchymal stem cells nutrient solution.
Step 1：The preparation of human umbilical cord mesenchymal stem cells.
（1）The preservation of umbilical cord：During normal Cesarean operation or natural labor, umbilical cord is taken under aseptic condition, umbilical cord is stored
In preserving in liquid, stored for future use at 4 DEG C.
（2）The cleaning of umbilical cord：It is put into from taking-up umbilical cord in liquid is preserved in sterilization container, adds 60 ml of physiological saline, concussion
25s is rinsed, is transferred in another sterilization container；Add 60 Metronidazule injections into the sterilization container to soak 5 minutes, Ran Houyong
60 ml of physiological saline cleans umbilical cord once again, after the completion of umbilical cord cleaning, is placed in disposable sterile petri dish.
（3）The preparation of human umbilical cord mesenchymal stem cells：Cut off the blood vessel and mucous membrane in umbilical cord with tweezers and scissors, and by its
Meat gruel shape is cut into, meat gruel shape tissue is transferred in centrifuge tube and adds appropriate phosphate buffer, 5 are centrifuged under 1500 rpm
Min is washed 1 time, removes supernatant, after adding 10 ml of nutrient solution, tissue suspension is divided equally and is added in two blake bottles, will be trained
Foster bottle is placed in 37 DEG C, cultivates 3~5 days in 5% CO2gas incubator.
（4）The secondary culture of human umbilical cord mesenchymal stem cells：Reject culture medium, adds Tryple, is immediately placed in incubator,
After digesting 8min, treat that cell all comes off, add terminate liquid, that is, culture medium, dispel cell, 5 min are centrifuged under 1500 rpm；
Supernatant discarding, adds new culture medium, and blake bottle is placed in 37 DEG C, is cultivated 3~5 days in 5% CO2gas incubator.
Step 2：The separation of oxidation-resistant active ingredient.
（1）The collection and storage of logarithmic phase human umbilical cord mesenchymal stem cells nutrient solution：Collect logarithmic phase human umbilical cord mesenchymal
Stem cell medium, saves backup under the conditions of 4 DEG C.
（2）The separation and screening of anti-oxidant active ingredient：By logarithmic phase human umbilical cord mesenchymal stem cells nutrient solution using not
The super filter tube of same specification carries out centrifuging super rate, and in 4 DEG C, the variant fraction of ultrafiltration collection carries out anti-oxidant reality under conditions of 3000g
Test, assess the antioxidant effect of variant fraction, it is final to obtain the molecular weight with anti-oxidation efficacy between 10KDa-30KDa
Isolate.Component with anti-oxidation efficacy is dispensed, is freezed spare.
Step 3：The measure of isolate antioxidation activity.
（1）The measure of scavenging ability of DPPH free radical.
Isolate freeze-dried powder is taken to be configured to the sample solution of various concentrations, removing energy of the measure isolate to DPPH free radicals
Power.Concretely comprise the following steps：The sample solution of 2 mL various concentrations gradients is separately added into centrifuge tube, then is separately added into 2 mLDPPH
Solution（95% ethanol is prepared, 0.1 mmol/L）, lucifuge reaction 30 minutes after mixing.Light absorption value A is measured at 517nm.On
State step and be added without DPPH, 2 mL95% ethanol, the constant data measured A1 of remaining step are added into each test tube.To centrifuge tube
2 mL purified waters of middle addition and 2 mLDPPH solution, remaining step is constant, measures light absorption value A0.Using purified water as blank, each
Sample does 3 parallel tests, is averaged, calculation formula is as follows：
DPPH free radical scavenging activities=[1- (A-A1)/A0] × 100%
The result is shown in Figure 1.
（2）The measure of hydroxyl radical free radical Scavenging activity.
Isolate freeze-dried powder is taken to be configured to the sample solution of various concentrations, removing energy of the measure isolate to hydroxyl radical free radical
Power.Experimental procedure is as follows：The Phen solution that 0.5 mL is prepared is separately added into the centrifuge tube of 10 mL, 1 mL phosphoric acid delays
Fliud flushing, the sample solution of 0.5 mL various concentrations, adds 0.5 mL0.75 mmol/LFeSO4Solution, H is added after mixing2O2
0.5 mL of solution, measures absorbance A x at 536nm；Sample solution in above-mentioned steps is changed to distilled water, remaining is constant, measure
Absorbance Af；By H in above-mentioned steps2O2Solution is changed to distilled water, the constant measure absorbance A of remaining step0；It is added without H2O2, its
Remaining step is constant, measure absorbance A s.Using purified water as blank, each sample does 3 parallel tests, is averaged, and calculates public
Formula is as follows：
Hydroxyl radical free radical clearance rate=（As-Ax）/(A0-Af) ×100%
The result is shown in Fig. 2.
（3）The measure of ultra-oxygen anion free radical Scavenging activity.
Using assay NBT photoreduction.Take isolate freeze-dried powder to be configured to the sample solution of various concentrations, take 0.1 mL
Sample solution adds the Tris-Hcl buffer solutions of 0.1 mol/LpH8.2 of 2.8 mL, replaces sample molten with 0.1 mL purified waters
Liquid is as blank control.Concussion mixes mixed solution, and the thing of 0.1 mL 3mmol/L is added after ten minutes in 25 DEG C of water-bath insulations
The pyrogallol solution first preheated in 25 DEG C of water-baths, mixes rapidly and starts timing, A is measured at 325nm, every 30s
A325 is read, terminates after 4.5 minutes, is returned to zero with blank control.The regression equation that absorbance changes over time is done, its slope is neighbour
Benzenetriol autoxidation speed V, calculates inhibiting rate of the sample to superoxide anion according to the following formula：
Inhibiting rate（%）=（VControl-VSample）/VControl×100
The result is shown in Fig. 3.
The human umbilical cord mesenchymal stem cells nutrient solution middle-molecular-weihydroxyethyl of exponential phase exists it can be seen from Fig. 1, Fig. 2 and Fig. 3
Component between 10KDa~30KDa has very strong antioxidation activity, it is cloudy to DPPH free radicals, hydroxyl radical free radical and super oxygen
Ion radical has good scavenging action.
Claims (4)
1. separation prepares the active ingredient with anti-oxidation efficacy from human umbilical cord mesenchymal stem cells nutrient solution, its method includes
Following steps：
Step 1：The preparation of human umbilical cord mesenchymal stem cells：
（1）The preservation of umbilical cord：During normal Cesarean operation or natural labor, umbilical cord is taken under aseptic condition, umbilical cord is deposited in into guarantor
In liquid storage, stored for future use at 4 DEG C；
（2）The cleaning of umbilical cord：Umbilical cord is taken out in liquid it is put into sterilization container from preserving, adds the ml of physiological saline 40 ml~70,
Concussion rinsing 20s~30s, is transferred in another sterilization container；The ml metronidazoles of 40 ml~70 note is added into the sterilization container
Penetrate liquid to soak 5~7 minutes, then cleaned umbilical cord once again with the ml of 40 ml of physiological saline~70, will after the completion of umbilical cord cleaning
It is placed in disposable sterile petri dish；
（3）The preparation of human umbilical cord mesenchymal stem cells：The blood vessel and mucous membrane in umbilical cord are cut off with tweezers and scissors, and is cut into
Meat gruel shape, meat gruel shape tissue is transferred in centrifuge tube and adds appropriate phosphate buffer, under the rpm of 1000 rpm~1500
Centrifuge 5 min to wash 1 time, remove supernatant, after adding the ml of nutrient solution 10 ml~15, tissue suspension is divided equally and is added to two
In blake bottle, blake bottle is placed in 37 DEG C, is cultivated 3~5 days in 5% CO2gas incubator；
（4）The secondary culture of human umbilical cord mesenchymal stem cells：Reject culture medium, adds Tryple, is immediately placed in incubator, digests
After 8min, treat that cell all comes off, add terminate liquid, that is, culture medium, dispel cell, 5 are centrifuged under the rpm of 1000 rpm~1500
min；Supernatant discarding, adds new culture medium, and blake bottle is placed in 37 DEG C, is cultivated 3~5 days in 5% CO2gas incubator；
Step 2：The separation of oxidation-resistant active ingredient：
（1）The collection and storage of logarithmic phase human umbilical cord mesenchymal stem cells nutrient solution：Logarithmic phase human umbilical cord mesenchymal is collected to do carefully
Born of the same parents' nutrient solution, saves backup under the conditions of 1 DEG C~5 DEG C；
（2）The separation and screening of anti-oxidant active ingredient：Logarithmic phase human umbilical cord mesenchymal stem cells nutrient solution is used into different rule
The super filter tube of lattice carries out centrifuging super rate, and in 1 DEG C~5 DEG C, the variant fraction of ultrafiltration collection carries out under conditions of 2000g~5000g
Anti-oxidant experiment, assesses the antioxidant effect of variant fraction, and final molecular weight of the acquisition with anti-oxidation efficacy is 10KDa-
Isolate between 30KDa.
2. separation is prepared with anti-oxidation efficacy in the nutrient solution according to claim 1 from human umbilical cord mesenchymal stem cells
Active ingredient, it is characterised in that in step 2, using centrifugal ultrafiltration method, in 1 DEG C~5 DEG C, surpass under conditions of 2000g~5000g
Active ingredient with anti-oxidation efficacy of the molecular weight between 10KDa-30KDa is collected in filter.
3. separation is prepared with anti-oxidation efficacy in the nutrient solution according to claim 1 from human umbilical cord mesenchymal stem cells
Active ingredient, it is characterised in that further include step 3：By in the slave human umbilical cord mesenchymal stem cells nutrient solution obtained in step 2
Component stoste with anti-oxidation efficacy packing prepared by separation is lyophilized, obtains point of the human cord blood serum with anti-oxidation efficacy
From the freeze-dried powder of thing.
4. separation prepares the activity with anti-oxidation efficacy in the slave human umbilical cord mesenchymal stem cells nutrient solution described in claim 1
Application of the component in medicine, health products, skin care item and other daily chemicals.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110974770A (en) * | 2019-12-26 | 2020-04-10 | 苏州克劳丽化妆品有限公司 | Anti-aging composition, preparation and preparation method |
| CN111728982A (en) * | 2019-03-22 | 2020-10-02 | 宣捷细胞生物制药股份有限公司 | Composition for anti-aging |
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