CN108823157A - Crocin II and albiflorin are promoting Marrow Mesenchymal Stem Cells In Vitro proliferation and are inhibiting the application in replicative senescence - Google Patents
Crocin II and albiflorin are promoting Marrow Mesenchymal Stem Cells In Vitro proliferation and are inhibiting the application in replicative senescence Download PDFInfo
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Abstract
The invention discloses crocins II and albiflorin to promote Marrow Mesenchymal Stem Cells In Vitro proliferation and inhibit the application in replicative senescence, and the crocin II and albiflorin coexist.Crocin II and albiflorin, which have, promotes Marrow Mesenchymal Stem Cells In Vitro proliferation function, while also having the function of inhibiting replicative senescence, provides enough stem cells for clinical treatment.Wherein crocin II and albiflorin are the crocin II and albiflorin (content >=98%) of commercially available high-purity, can also be prepared according to a conventional method.Its advantage is that：Crocin II and albiflorin can promote Proliferation of Bone Mesenchymal Stem Cells and can inhibit replicative senescence, enough cells are provided for the organizational projects such as treatment bone defect, cartilage defect, hepatic fibrosis-renal tubular ectasia syndrome, myocardial infarction, pulmonary fibrosis and diabetes and stem-cell therapy, while reducing immunity of organism rejection.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to crocin II and albiflorin are promoting in vitro culture bone
Application in bone marrow-drived mesenchymal stem proliferation and inhibition replicative senescence.
Background technique
Mesenchymal stem cell (MSC) be used as a kind of cellular type drug, bone defect, cartilage defect, hepatic fibrosis-renal tubular ectasia syndrome,
It is had broad application prospects in the diseases such as myocardial infarction, pulmonary fibrosis and diabetes.But in commercialized production process,
The production scale of MSC is often difficult to scale up.Wherein, growth rate is slow, aging causes stemness loss to be two in a replication process
Big main cause.
MSC source is in the mesoderm and ectoderm of mesoderm growing early stage, and content is extremely low in marrow, and growth rate is slow.In this way
Characteristic make it in vivo, on the one hand can react in time to outer signals, to determine the development side of cell
To --- proliferation or differentiation；On the other hand it can alleviate the harm of gene mutation bring and risk, make the discovery of cell having time simultaneously
Correct the mistake in proliferating cycle.However, in commercialized stem cell production process, on the one hand this characteristic limits life
On the other hand production scale also extends the production cycle.
Aging is the normal physiological phenomena in life entity development process, and MSC is in vitro in incubation, in mitosis 20
Hayflick phenomenon, i.e. replicative senescence phenomenon can occur after to 40 generations.The phenomenon is along with cellular morphology by original at fibre
Hypertrophyization, the relevant beta galactosidase increased activity of aging, differentiation capability loss and cell Proliferation occur for the spindle figure for tieing up sample
It stagnates.In addition, the pressure of replicability also results in the continuous accumulation of DNA damage in cellular senescence process, some researches show that due to multiple
The inducible telomeric dna of duplication pressure caused by the stopping of fork is made to damage.During MSC replication in vitro, telomeric dna
Length is constantly shortened with the speed of 30-120bp/PD.Meanwhile replicative senescence can also cause the cell factor of MSC release, life
The long factor and protease change, and significant become occurs for the microenvironment of the cell and tissue that are acted on so as to cause these albumen
Change.Replicative senescence has become one of maximum bottleneck in MSC clinical application field, limits MSC in commercialization and industrialization
Large-scale production.
Therefore, it finds and discovery can speed up MSC amplification rate and degree, inhibiting the ingredient of its replicative senescence is to accelerate
With the committed step for expanding stem cell clinical application.It is raw using growth factor etc. is added in MSC culture environment both at home and abroad at present
Object macromolecular is to promote the proliferation of stem cell and inhibit replicative senescence.But the recombinant proteins low output such as growth factor, cost
It is high, and biological half-life is short, it is difficult to it is widely used.Therefore it is badly in need of finding alternative active constituent for accelerating to do
The proliferation and inhibition replicative senescence of cell, to accelerate its clinical application and industrialization.
Chinese herbaceous peony (Paeonia lactiflora Pall) is Ranunculaceae perennial herb plant, originates in the southeast
It is sub-.First recorded in《Sheng Nong's herbal classic》, calling it, " bitter is flat, main perverse trend abdominal pain, removes blood-arthralgia, breaks hard product fever and chills, and hernia lump in the abdomen, analgesic, benefit are small
Just, QI invigorating, raw Chuan Gu and hills ".Northern and Southern Dynasties' period, Chinese herbaceous peony have red, Bai Zhifen, until today distinguishes Radix Paeoniae Alba, radix paeoniae rubra standard
For the processing method of peeling, boiling, the two function is slightly distinguished, the main fever and chills of radix paeoniae rubra, diuresis, and the soft liver analgesic of Radix Paeoniae Alba, blood-nourishing
Yin is held back, Chinese herbaceous peony is often used as medicine in the form of Radix Paeoniae Alba or two kinds of radix paeoniae rubra, is played a role.Modern studies have found that Chinese herbaceous peony makees with a variety of pharmacology
With：Analgesia, anti-spasm, calmness, activating microcirculation and removing stasis medicinal, anti-inflammatory, treatment gynaecological disease, enhancing gastrointestinal function etc..
Chinese herbaceous peony ingredient includes glycoside, terpene, flavonoids, tannin class, volatile oil, phenols and carbohydrate etc., albiflorin
It is the active constituent of Chinese herbaceous peony index, entitled 9- ((Benzoyloxy) methyl) -1- (beta-D- of chemistry
Glucopyranosyloxy) -4-hydroxy-6-methyl-7-oxatricyclononan-8- one, white powder.By
Currently, the effect for Chinese herbaceous peony and its contained active material in terms of promoting MSC proliferation and differentiation, has no comprehensive article and grinds
Study carefully achievement.
Safflower is the dry flower of composite family safflower plant safflower, first recorded in《Kaibao Bencao》." taste is acrid in flavour and warm in nature, nontoxic.Main product
Bruise lockjaw afterwards, not to the utmost, colic pain fails before being carrying out extravesated blood, and wine boils clothes in abdomen.Also blood under main disease due to noxious agents produced by various parasites." to the Ming Dynasty《This grass product is converged
Essence is wanted》It records：" main worry is smouldered, unhappy not dissipate, stimulating appetite, and long term usage grows lower member, pleases color." safflower is generally believed in Chinese medicine
Acrid flavour, warm-natured, return heart and Liver Channel, energy blood circulation, removing blood stasis and acesodyne.And it is extremely modern, 2010 editions《People's Republic of China's medicine
Allusion quotation》West safflower is recorded to cure mainly as " activating microcirculation and removing stasis medicinal, removing pattogenic heat from the blood and toxic material from the body, resolving stagnation for tranquilization.For Jing Bi Disorder lump in the abdomen, postpartum stasis, epidemic heat syndrome hair
Spot, melancholy ruffian is bored, and palpitation with fear is gone mad."
The chemical component of west safflower has flavonoids, carbene class, alkaloids, quinoid chalcones, lignanoids, spermidine
Class, alkyl diol class, organic acid, steroid class, non-tetraterpenes etc., wherein crocin II is the active constituent of its index,
Entitled 1- [(2E, 4E, 6E, 8E, 10E, 12E, the 14E)-b-D-glucopyranosyl-2,6,11,15- of chemistry
tetramethyl-2,4,6,8,10,12, 14-hexadecaheptaenedioate]6-O-b-D-glucopyranosyl-
B-D-Glucopyranose, red crystals.Crocin II is proved to Ca2+ overloading, hypoglycemic lipid-lowering effect, anti-atherogenic
Hardening, protect liver, antitumor, treatment coronary heart disease effect.But so far, influence of the crocin II to stem cell replicative senescence
It has not been reported.
II chemical structure of crocin is：
Albiflorin chemical structure is：
Summary of the invention
In view of the above deficiencies, the present invention provides crocin II and albiflorin in promotion in vitro culture medulla mesenchyma
Stem cells hyperplasia and inhibit the application in stem cell replicative senescence, which will be for the external expansion of mesenchymal stem cell
Increasing, stemness maintain and subsequent applications will provide important guarantee, provides enough stem cells for clinical treatment and scientific research, has
There are important economic value and social effect.
The technical solution adopted in the present invention is as follows：Crocin II and albiflorin are between promoting in vitro culture marrow
Application in mesenchymal stem cell proliferation and inhibition replicative senescence, the crocin II and albiflorin coexist.
Further, the mass ratio of the crocin II and albiflorin in culture solution is 1：10-10：Between 1,
It is significant applied to source of mouse Proliferation of Bone Mesenchymal Stem Cells.
Further, the mass ratio of the crocin II and albiflorin in culture solution is preferably 1：4-2：1 it
Between.
Further, the mass ratio of the crocin II and albiflorin in culture solution is 1：10-10：Between 1,
It is significant applied to source of people Proliferation of Bone Mesenchymal Stem Cells.
Further, the mass ratio of the crocin II and albiflorin in culture solution is preferably 1：2-3：1 it
Between.
Further, the mass ratio of the crocin II and albiflorin in culture solution is 1：20-20：Between 1,
It is significant applied to inhibition source of people mesenchymal stem cell replicative senescence.
Further, the mass ratio of the crocin II and albiflorin in culture solution is preferably 3：7-5：2 it
Between.
Further, the crocin II is the crocin II of commercially available high-purity, purity >=98%, in the Chinese herbaceous peony
Ester glycosides is the albiflorin of commercially available high-purity, purity >=98%.
The beneficial effects of the invention are as follows：Crocin II and albiflorin promote Proliferation of Bone Mesenchymal Stem Cells, also
It can inhibit replicative senescence, can be treatment bone defect, cartilage defect, hepatic fibrosis-renal tubular ectasia syndrome, myocardial infarction, pulmonary fibrosis and glycosuria
The organizational projects such as disease and stem-cell therapy provide enough cells, while reducing immunity of organism rejection.
Detailed description of the invention
Fig. 1 is crocin II and albiflorin in vitro culture source of mouse Proliferation of Bone Mesenchymal Stem Cells CCK8 experiment knot
Fruit；
Fig. 2 is crocin II and albiflorin in vitro culture source of people Proliferation of Bone Mesenchymal Stem Cells CCK8 experiment knot
Fruit；
Fig. 3 is the cell ageing β-half of crocin II and albiflorin in vitro culture source of people mesenchymal stem cell
Lactoside enzyme dyeing light microscopic is taken pictures experimental result；
Fig. 4 is the cell ageing β-half of crocin II and albiflorin in vitro culture source of people mesenchymal stem cell
Lactoside enzyme dyeing cell number statistical result.
Specific embodiment
The invention will be further described with reference to embodiments.
1. MATERIALS METHODS：
1.1 reagent DMEM low sugar culture solutions (Gibco BRL, the U.S.)；Fetal calf serum (FBS, Gibco BRL, the U.S.)；
0.25 wt.% pancreatin (contains 0.02wt.%EDTA, Gibco BRL, the U.S.)；One Streptomycin Solution of penicillin (Gibco BRL,
The U.S.)；Cell Counting Kit-8 (the green skies Bioisystech Co., Ltd in Shanghai, China)；Cell ageing beta galactose
Glycosides enzyme staining reagent kit (the green skies Bioisystech Co., Ltd in Shanghai, China)；
1.2 electronics balances (AL204, Mettler Toledo, Switzerland)；Full-automatic microplate reader (ELX800, BioTek
Instruments Inc., the U.S.)；Carbon dioxide cell incubator (HF90, Health Force, China)；Biohazard Safety Equipment
(Type A2, Beijing Dong Lianhaer instrument manufacturing Co., Ltd, China)；Ultrapure water instrument (Milli-Q, Millipore Co., beauty
State)；
1.3BMSCs is separately cultured the male SD rat for taking 3 week old, and cervical dislocation is put to death, and impregnates 5min with alcohol and disappear
Poison removes hind leg skin and muscle with operating scissors in super-clean bench, takes out shin bone and femur, carefully cuts bone both ends, cruelly
After undisguised pulp cavity, the DMEM low sugar culture solution repeated flushing ossis of the fetal calf serum containing 10% is drawn until bone with syringe
Appearance bleaches.After the obtained culture solution containing myeloid tissue is crossed 200 mesh cell sieves, 1200rpm is centrifuged 5min, discards supernatant
Liquid is added culture solution resuspension and is placed in culture dish, and in 37 DEG C and 5%CO2It is cultivated in environment, every other day changes a not good liquor.When
The BMSCs of had digestive transfer culture when cell density reaches 80%-90%, the second generation to the 5th generation is used for subsequent experimental.
1.4HMSCs culture takes P0For HMSCs, with 10% fetal calf serum DMEM low sugar culture solution at 37 DEG C and 5%
CO2It is cultivated in environment, changes a not good liquor every three days.The had digestive transfer culture when cell density reaches 80%-90%, 2nd generation is extremely
The HMSCs in the 5th generation is used for proliferation experiment, and the HMSCs in the 20th generation is used for cell ageing beta galactosidase Coloration experiment
The experiment of 1.5 proliferation experiments is divided into two groups, respectively BMSCs group and HMSCs group.Wherein it is divided into blank group, right for every group
According to group and administration group.Through filtration sterilization after crocin II and albiflorin dissolution in administration group, carried out in 10 kinds of ratios
Mixing, is divided into combination 1,2,3,4,5,6,7,8,9,10, selected combination quality proportional region is 1：10-10：Between 1.
The BMSCs and HMSCs for taking the third generation, respectively with 5 × 103The density kind in/hole is on 96 orifice plates.Culture 24 hours,
After cell is adherent, the crocin II and albiflorin mixed liquor of 10 kinds of different components are separately added into 96 orifice plates, often
5 holes of a component.After dosing for 24 hours, operated according to CCK8 kit, in 450nm wavelength microplate reader testing result.Proliferation rate=
ODAdministration group-ODBlank group/ODControl group-ODBlank group
1.6 cell ageing beta galactosidase Coloration experiment experimental setup control groups and administration group.West safflower in administration group
Through filtration sterilization after glycosides II and albiflorin dissolution, mixed in 6 kinds of ratios, selected portfolio ratio range is 1：20-
20：Between 1, it is divided into combination 1,2,3,4,5,6.
The HMSCs for taking for the 20th generation, respectively with 2 × 104The density kind in/hole is on 24 orifice plates.Culture 24 hours is pasted to cell
After wall, the crocin II and albiflorin mixed liquor of 6 kinds of different proportions, each combination 3 are separately added into 24 orifice plates
Hole.After dosing for 24 hours, cell culture fluid is absorbed, is washed 1 time with PBS or HBSS, the dyeing of 250ul beta galactosidase is added and fixes
Liquid, room temperature fix 15 minutes.Cell fixer is absorbed, is washed cell 3 times, every time 3 minutes with PBS or HBSS.Absorb PBS or
1 milliliter of dyeing working fluid is added in HBSS, every hole.The preparation method of dyeing working fluid is dyed with reference to cell ageing beta galactosidase
Kit specification.37 DEG C of overnight incubations, sealing 24 orifice plates with parafilm or preservative film prevents from evaporating.Ordinary optical microscope
Lower observation.The different visuals field is randomly selected, every hole counts 300 cells, calculates the ratio of senile cell.
1.7 statistical analysis experimental datas use ' x ± Sd are indicated, carry out statistical procedures analysis using Origin2017 software,
It is examined between two groups using t, one-way analysis of variance is used between multiple groups, works as P<Think there is statistical difference when 0.05.
2. result：
The ratio of 2.1BMSCs cell growth condition and CCK8 result crocin II and albiflorin in culture solution
1： 10-10：Between 1, preferred proportion range is in combination 6 (1：4-2：1) between, it is applied to source of mouse mesenchymal stem cell
It is proliferated significant (see figure 1).
The ratio of 2.2HMSCs cell growth condition and CCK8 result crocin II and albiflorin in culture solution
1： 10-10：Between 1, preferred proportion range is in combination 3 (1：2-3：1) between, it is applied to source of people mesenchymal stem cell
It is proliferated significant (as shown in Figure 2).
2.3HMSCs cell ageing beta galactosidase Coloration experiment result crocin II and albiflorin are being cultivated
Ratio in liquid is 1：20-20：Between 1, preferred proportion range is in combination 2 (3：7-5：2) between, in light microscopic observation, culture
The beta galactosidase positive cell rate of HMSCs reduce significant, positive cell number is reduced, and there is significant copy-resistant to decline
Old effect (see shown in Fig. 3, Fig. 4).
Claims (8)
1. crocin II and albiflorin are promoting Marrow Mesenchymal Stem Cells In Vitro proliferation and replicability are inhibited to decline
Application in old, which is characterized in that the crocin II and albiflorin coexist.
2. applying according to claim 1, which is characterized in that the crocin II and albiflorin are in culture solution
Mass ratio is 1：10-10：Between 1, it is significant to be applied to source of mouse Proliferation of Bone Mesenchymal Stem Cells.
3. applying according to claim 2, which is characterized in that the crocin II and albiflorin are in culture solution
Mass ratio is preferably 1：4-2：Between 1.
4. applying according to claim 1, which is characterized in that the crocin II and albiflorin are in culture solution
Mass ratio is 1：10-10：Between 1, it is significant to be applied to source of people Proliferation of Bone Mesenchymal Stem Cells.
5. applying according to claim 4, which is characterized in that the crocin II and albiflorin are in culture solution
Mass ratio is preferably 1：2-3：Between 1.
6. applying according to claim 1, which is characterized in that the crocin II and albiflorin are in culture solution
Mass ratio is 1：20-20：Between 1, it is applied to inhibit source of people mesenchymal stem cell replicative senescence significant.
7. applying according to claim 6, which is characterized in that the crocin II and albiflorin are in culture solution
Mass ratio is preferably 3：7-5：Between 2.
8. any one of -7 application according to claim 1, which is characterized in that the crocin II is the west of commercially available high-purity
Carthamic acid II, purity >=98%, the albiflorin are the albiflorin of commercially available high-purity, purity >=98%.
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Cited By (2)
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| CN108841781A (en) * | 2018-06-13 | 2018-11-20 | 延边大学 | Crocin is improving the application in porcine oocytes oxidation resistance and cleavage rates |
| CN116585301A (en) * | 2023-07-18 | 2023-08-15 | 青岛瑞源细胞生物科技开发有限公司 | Preparation for improving stem cell therapeutic capacity |
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| CN116585301B (en) * | 2023-07-18 | 2023-09-22 | 青岛瑞源细胞生物科技开发有限公司 | Preparation for improving stem cell therapeutic capacity |
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