CN106038596A - Stem cell preparation capable of resisting hepatic fibrosis and preparation method of stem cell preparation - Google Patents

Stem cell preparation capable of resisting hepatic fibrosis and preparation method of stem cell preparation Download PDF

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CN106038596A
CN106038596A CN201610356758.8A CN201610356758A CN106038596A CN 106038596 A CN106038596 A CN 106038596A CN 201610356758 A CN201610356758 A CN 201610356758A CN 106038596 A CN106038596 A CN 106038596A
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stem cell
cell
hepatic fibrosis
medicine
vitamin
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曾宪卓
鲁菲
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ISTEM REGENERATIVE MEDICINE SCI-TECH Co Ltd
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ISTEM REGENERATIVE MEDICINE SCI-TECH Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/07—Retinol compounds, e.g. vitamin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin

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Abstract

The invention discloses a stem cell preparation capable of resisting hepatic fibrosis and a preparation method of the stem cell preparation. The stem cell preparation capable of resisting hepatic fibrosis is prepared from mesenchymal stem cells, vitamin A, cortex dictamni lactone and PBS buffer solution. In the stem cell preparation capable of resisting hepatic fibrosis, the concentration of the mesenchymal stem cells is (1-5)x10<7>/ml, the concentration of vitamin A is 20-100 ng/ml, and the concentration of the cortex dictamni lactone is 20-100 mg/ml. With the adoption of the stem cell preparation capable of resisting hepatic fibrosis, the hepatic inflammation can be effectively alleviated, the hepatic fibrosis can be reversed, and the recovery of the liver function is promoted; the multiplication of hepatic stellate cells can be effectively inhibited, and thus the formation of the hepatic fibrosis is inhibited.

Description

Stem cell medicine of anti-hepatic fibrosis and preparation method thereof
Technical field
The present invention relates to stem cell and field of tissue engineering technology, particularly relate to a kind of anti-hepatic fibrosis stem cell medicine and Preparation method.
Background technology
Hepatic fibrosis is the preliminary stage of liver cirrhosis, specifically refers to by connective tissue in liver caused by various virulence factors abnormal Hypertrophy, causes the pathological process of diffusivity extracellular matrix over-deposit in liver；Liver cirrhosis is then that excess fibrosis makes liver Atrophy is hardening caused.
In hepatic fibrosis process, the activation of hepatic stellate cell is critical events.Mostly the extracellular matrixs such as collagen fiber are Produced by the myofibroblast of Hepatic Stellate Cell Activation.The hepatic injury that many causes of disease cause can cause liver starlike carefully The activation of born of the same parents (Hepatic Stellate Cell, HSC) be i.e. changed into by silent oscillation HSC have proliferative, become fibroid With inotropic myofibroblast (i.e. activated form HSC).HSC activation is an effective and reproducible process, greatly Cause can be divided into two stages: initiating stage and sustained period.The initiating stage occurs making HSC to cytokine and other local thorns The sharp factor has the gene expression of reactivity and quick character mutation；Sustained period i.e. by paracrine or autocrine stimulation and Extracellular matrix is rebuild, and amplifies the character mutation of HSC further, maintains its state of activation, promote fiber to generate.But, clinical On there is no the definite effective anti-fibrosis medicine suppressing hepatic stellate cell, hepatic fibrosis still lacks effective treatment means.
Summary of the invention
Present invention is primarily targeted at the stem cell medicine that a kind of anti-hepatic fibrosis is provided, it is intended to suppression hepatic stellate cell And treat hepatic fibrosis.
For achieving the above object, the present invention provides the stem cell medicine of a kind of anti-hepatic fibrosis, described stem cell medicine bag Include:
Mescenchymal stem cell；Cortex Dictamni lactone；Vitamin A；Cell preparation solution；In described stem cell medicine, institute The concentration stating mescenchymal stem cell is (1～5) × 107Individual/ml, the concentration of described Cortex Dictamni lactone is 20～100mg/ml, institute The concentration stating vitamin A is 20～100ng/ml.
Preferably, the concentration of described mescenchymal stem cell is (2～4) × 107Individual/ml.
Preferably, the concentration of described Cortex Dictamni lactone is 40～80mg/ml.
Preferably, described Cortex Dictamni lactone includes ketone and obakulactone.
Preferably, the concentration of described vitamin A is 40～80ng/ml.
Preferably, described mescenchymal stem cell is human umbilical cord mesenchymal stem cells.
Preferably, described cell preparation solution is PBS.
The present invention also provides for the preparation method of the stem cell medicine of a kind of above-mentioned anti-hepatic fibrosis, comprises the following steps that
Obtain mescenchymal stem cell；
Described mescenchymal stem cell, vitamin A, Cortex Dictamni lactone are added in cell preparation solution, and prepares dry thin Born of the same parents' preparation.
Preferably, described by described mescenchymal stem cell, vitamin A, Cortex Dictamni lactone addition cell preparation solution Step in,
First described vitamin A is added in described cell preparation solution.
Preferably, the step of described acquisition mescenchymal stem cell, including:
Obtaining and process umbilical cord tissue, being single cell suspension with the digestion of II Collagenase Type, described II Collagen Type VI is removed in washing Enzyme remains, and described single cell suspension is seeded in the DMEM/F12 culture medium containing hyclone cultivation, it is thus achieved that become threadiness thin Born of the same parents, when described one-tenth fibrous cell reaches 70～80% fusion, carry out digesting, passing on, and it are dry to collect the 3rd～5 generation mesenchymes Cell.
Technical solution of the present invention, by by mescenchymal stem cell, vitamin A, Cortex Dictamni lactone and cell preparation solution It is mixed to prepare the stem cell medicine of anti-hepatic fibrosis, and then this stem cell medicine can effectively alleviate inflammation degree, reverse Hepatic fibrosis, promotes liver function recovery, and suppresses the propagation of hepatic stellate cell to suppress hepatic fibrosis to be formed, it is possible to treatment liver is hard Change；Wherein, Cortex Dictamni lactone can affect the function of hepatic stellate cell and suppress it to breed, and this Cortex Dictamni lactone can be with Mescenchymal stem cell is collaborative and plays the effect that is obviously improved hepatic fibrosis；Vitamin A can promote derived mesenchymal stem cells in vitro Fast breeding, and keep undifferentiated state and multipotency in mescenchymal stem cell long-term cultivation.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing In having technology to describe, the required accompanying drawing used is briefly described, it should be apparent that, the accompanying drawing in describing below is only this Some embodiments of invention, for those of ordinary skill in the art, on the premise of not paying creative work, it is also possible to Other accompanying drawing is obtained according to the content shown in these accompanying drawings.
Fig. 1 is the growth of mescenchymal stem cell prepared by the embodiment of the present invention 1, comparative example 1C, embodiment 2, comparative example 2C Curve chart.
Detailed description of the invention
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete Describe, it is clear that described embodiment is only a part of embodiment of the present invention rather than whole embodiments wholely.Base Embodiment in the present invention, those of ordinary skill in the art obtained under not making creative work premise all its His embodiment, broadly falls into the scope of protection of the invention.
The present invention provides the stem cell medicine of a kind of anti-hepatic fibrosis, and this stem cell medicine includes that raw material is as follows:
Mescenchymal stem cell, vitamin A, Cortex Dictamni lactone and cell preparation solution；After mixing so that this stem cell system In agent, the concentration of this mescenchymal stem cell is (1～5) × 107Individual/ml, the concentration of this Cortex Dictamni lactone is 20～100mg/ml, The concentration of this vitamin A is 20～100ng/ml.In other words, in the stem cell medicine of 1ml, mescenchymal stem cell have (1～ 5)×107Individual, vitamin A is 20～100ng, and Cortex Dictamni lactone is 20～100mg.It should be noted that according to this area skill The general knowledge of art personnel, the concentration of mescenchymal stem cell in the stem cell medicine of anti-hepatic fibrosis of the present invention, the concentration of vitamin A and The concentration of Cortex Dictamni lactone is all only concentration when stem cell medicine has just configured, owing to mescenchymal stem cell is active, It is thus impossible to be interpreted as the concentration after configuration placement a period of time.
The stem cell medicine of anti-hepatic fibrosis of the present invention can effectively alleviate inflammation degree, reverses hepatic fibrosis, promotees Enter liver function recovery, and suppress the propagation of hepatic stellate cell to suppress hepatic fibrosis to be formed, it is possible to treatment liver cirrhosis；Wherein, in vain Cortex Dictamni lactone can affect the function of hepatic stellate cell and suppress it to breed, and this Cortex Dictamni lactone can be dry with mesenchyme thin Born of the same parents are collaborative and play the effect that is obviously improved hepatic fibrosis；Vitamin A can promote derived mesenchymal stem cells in vitro fast breeding, and Keep undifferentiated state and multipotency in mescenchymal stem cell long-term cultivation.
It should be noted that mescenchymal stem cell is derived from, growth is the most mesoblastic has height self-renewal capacity With the pluripotent stem cell of multi-lineage potential, it is widely present in the Various Tissues such as umbilical cord, bone marrow, amplification can be cultivated in vitro, And hepatocyte, neurocyte, osteoblast, chondrocyte, muscle cell, adipose cell etc. can be divided under given conditions； Specifically, mescenchymal stem cell of the present invention can derive from umbilical cord, bone marrow, umbilical blood, whole body connective tissue or organ interstitial.Shaggy-fruited dittany Skin is common a kind of Chinese crude drug, and the active component in Cortex Dictamni extract includes lactone compound and alkaloids chemical combination Thing, and Cortex Dictamni lactone of the present invention is i.e. the lactone compound in Cortex Dictamni extract.Cell preparation solution of the present invention is only The medium of the survival of stem cell is provided between offer to be met, can be specifically culture medium or buffer etc., as cell system The consumption of agent solution can be allocated according to the actual requirements, is the Normal practice of those skilled in the art, does not do superfluous at this State.
Further, the concentration of this mescenchymal stem cell is (2～4) × 107Individual/ml.
When concentration is (2～4) × 107Individual/ml time, can be that the hepatic tissue of damaged provides sufficient stem cell, and then Repairing the hepatic tissue of damage, the effect that this mescenchymal stem cell improves hepatic fibrosis is more significantly, and fibrosis Effect becomes apparent from.
Further, the concentration of this Cortex Dictamni lactone is 40～80mg/ml.
When concentration is 40～80mg/ml, this Cortex Dictamni lactone affects the function of hepatic stellate cell and suppresses it to breed Effect is obvious, is better protected from the further fibrosis of hepatic tissue of damaged.
Yet further, this Cortex Dictamni lactone includes ketone and obakulactone.
More preferable relative to the effect of other lactones, this ketone and the obakulactone propagation to controlling hepatic stellate cell, effect Become apparent from.
Further, the concentration of this vitamin A is 40～80ng/ml.
When concentration is 40～80ng/ml, vitamin A has significant proliferation function to mescenchymal stem cell, keeps simultaneously The undifferentiated state of mescenchymal stem cell and multipotency, prevent the mescenchymal stem cell in stem cell medicine from breaking up, and Impure, and then cause curative effect to reduce.
The present invention also provides for the preparation method of the stem cell medicine of a kind of above-mentioned anti-hepatic fibrosis, and this preparation method includes step Rapid as follows:
S1, acquisition mescenchymal stem cell；
S2, this mescenchymal stem cell, vitamin A, Cortex Dictamni lactone are added in cell preparation solution, and prepare dry thin Born of the same parents' preparation.
The preparation method of the stem cell medicine of anti-hepatic fibrosis of the present invention is simple, efficient, only need to be by vitamin A, mesenchyme Stem cell and Cortex Dictamni lactone add in cell preparation solution and are configured to corresponding concentration.
It should be noted that this mescenchymal stem cell can be obtained by excised cotyledon, it is also possible to by purchasing business The mescenchymal stem cell of product obtains.
Further, step S1 specifically includes:
Obtaining and process umbilical cord tissue, being single cell suspension with the digestion of II Collagenase Type, it is residual that II Collagenase Type is removed in washing Stay, single cell suspension is seeded in the DMEM/F12 culture medium containing hyclone cultivation, it is thus achieved that become fibrous cell, treat fibroblast Dimension shape cell reach 70～80% merge time, carry out digesting, passing on, reach purification and increase expand mescenchymal stem cell purpose, and Collect the 3rd～5 generation mescenchymal stem cells.
Further, in step S2, the addition cell preparation of vitamin A, mescenchymal stem cell and Cortex Dictamni lactone is with molten The sequencing of liquid is: vitamin A, mescenchymal stem cell, Cortex Dictamni lactone；Or, vitamin A, Cortex Dictamni lactone, mesenchyme Stem cell.
Owing to vitamin A adds in PBS prior to mescenchymal stem cell, mesenchyme can be efficiently avoid dry thin Born of the same parents break up at cell preparation solution, thus ensure mescenchymal stem cell purity in stem cell medicine.
Now by embodiment, stem cell medicine of anti-hepatic fibrosis of the present invention and preparation method thereof is further explained, with Describe its technical scheme and the technique effect brought in detail.
Embodiment 1
One, human umbilical cord mesenchymal stem cells is obtained
(1), obtain and process human umbilical tissue
Obtain in vitro human umbilical tissue, about 4～6cm length, after aseptically pumping Cord blood, be placed in PBS buffering Preserve in liquid, at 4 DEG C, and process in 4h；
The human umbilical tissue of about 4～6cm length is put in super-clean bench, removes arteriovenous and adventitia, use PBS (KH2PO40.2g, KCl 0.2g, NaCl 8.0g, Na2HPO41.15g constant volume 1L, PH=7.4) rinse for the first time several times, It is cut into 1cm segment, more repeatedly rinses to be eliminated as much as blood with same PBS, be finally cut into meat paste shape tissue；
(2), isolated and purified
By meat paste shape tissue move into 50mL centrifuge tube, add 5mL, through 37 DEG C preheating, mass fraction be 0.1% II type Collagenase (100ml PBS dissolves 0.1g collagenase), puts into vibration digestion 180min in 37 DEG C of shaking baths, is digested to Single cell suspension；
Then, adding 30mL DMEM/F12, repeatedly blow and beat, cell is sieved through filter, collects filtrate, and washs removal II type glue Protoenzyme remains；
Filtrate is centrifuged 10min with the rotating speed of 1000 turns/min, abandons supernatant, by the single cell suspension that is centrifugation down by 1 ×106The density of/mL is inoculated in the culture bottle of the DMEM/F12 culture medium containing 10%FBS (hyclone), moves into incubator In cultivate, next day changes liquid first, and later every 3d changes liquid 1 time, and is trained into fibrous cell；
When reaching 70～80% fusion as fibrous cell, prepare with trypsin trypsin solution: PBS buffers Liquid 200mL, trypsin 0.5g, ethylenediaminetetraacetic acid 0.04g) incomplete digestion carries out passing on purification, passes on algebraically labelling and divide Not Wei P1, P2, P3 etc., P3 passes on for rear catapepsis, finally, collect P3～P5 thin for human umbilical cord mesenchymal stem cells Born of the same parents are frozen.
Two, stem cell medicine is prepared
P3～P5 that above-mentioned steps is prepared for human umbilical cord mesenchymal stem cells recover, standby after, first by 400ng tie up Raw element A adds in 10ml PBS, then by 2 × 108Individual human umbilical cord mesenchymal stem cells adds in PBS, After 400mg Cortex Dictamni lactone (ketone+obakulactone) is added in PBS.
Comparative example 1A (human umbilical cord mesenchymal stem cells+vitamin A)
Embodiment 1 is prepared human umbilical cord mesenchymal stem cells recovery, standby after, first by 400ng vitamin A add In 10ml PBS, then by 2 × 108Individual human umbilical cord mesenchymal stem cells adds in PBS, prepares contrast system Agent.
Comparative example 1B (Cortex Dictamni lactone+vitamin A)
400ng vitamin A is added in 10ml PBS, then by 400mg Cortex Dictamni lactone (in ketone+Cortex Phellodendri Ester) add in PBS, prepare Comparative formulation.
Comparative example 1C (human umbilical cord mesenchymal stem cells+Cortex Dictamni lactone)
Embodiment 1 is prepared human umbilical cord mesenchymal stem cells recovery, standby after, by 2 × 108Fill between individual's umbilical cord Matter stem cell adds in 10ml PBS, and 400mg Cortex Dictamni lactone (ketone+obakulactone) then adds PBS buffering In liquid, prepare Comparative formulation.
Embodiment 2
One, human umbilical cord mesenchymal stem cells is obtained
Same as in Example 1.
Two, stem cell medicine is prepared
Embodiment 1 is prepared human umbilical cord mesenchymal stem cells recovery, standby after, first by 800ng vitamin A add In 10ml PBS, then by 4 × 108Individual human umbilical cord mesenchymal stem cells adds in PBS, finally by 800mg Cortex Dictamni lactone (ketone+obakulactone) adds in PBS.
Comparative example 2A (human umbilical cord mesenchymal stem cells+vitamin A)
Embodiment 1 is prepared human umbilical cord mesenchymal stem cells recovery, standby after, first by 800ng vitamin A add In 10ml PBS, then by 4 × 108Individual human umbilical cord mesenchymal stem cells adds in PBS, prepares contrast system Agent.
Comparative example 2B (Cortex Dictamni lactone+vitamin A)
800ng vitamin A is added in 10ml PBS, then by 800mg Cortex Dictamni lactone (in ketone+Cortex Phellodendri Ester) add in PBS, prepare Comparative formulation.
Comparative example 2C (human umbilical cord mesenchymal stem cells+Cortex Dictamni lactone)
Embodiment 1 is prepared human umbilical cord mesenchymal stem cells recovery, standby after, by 4 × 108Fill between individual's umbilical cord Matter stem cell adds in 10ml PBS, and 800mg Cortex Dictamni lactone (ketone+obakulactone) then adds PBS buffering In liquid.
Checking example 1
OD (optical density, the optical density) pH-value determination pH of mescenchymal stem cell:
Taking 5 piece of 96 orifice plate, every piece of 96 orifice plates are with 5 × 103In the living cells suspension that embodiment 1 is prepared by the density in/hole Cell is respectively inoculated in 5 holes, and the cell in living cells suspension embodiment 2 prepared equally is respectively inoculated in other 5 holes, Simple stem cell suspension, as comparison, is inoculated in 5 holes；It is placed in 37 DEG C, 5%CO2Incubator is cultivated, embodiment hole and right Liquid is changed once in every three days in ratio hole.Hereafter the every day same time, randomly draw a plate, measure OD value.During measurement, every hole adds MTT Solution (5mg/ml) 20 μ L, continues at 37 DEG C, 5%CO2After cultivating 6 hours in incubator, careful suction abandons liquid in hole, in every hole Add 150 μ L DMSO (dimethyl sulfoxide).96 orifice plates are placed on enzyme-linked immunosorbent assay instrument concussion 20 minutes, use double wave Long algoscopy, enzyme-linked immunoassay instrument sets detection wavelength 492nm.Reference wavelength 630nm, measures OD value, as shown in table 1 below, Table 1 is the OD value that three groups of stem cell suspensions measure when different number of days.
Table 1
According to table 1, along with the increase of natural law, the OD value of embodiment 1 and embodiment 2 is increasing trend, and the highest OD value in matched group, it is clear that the human umbilical cord mesenchymal stem cells in the stem cell medicine of anti-hepatic fibrosis of the present invention relative to Matched group keeps higher activity, and cell survival rate is high.
Checking example 2
Clinical stem cell injection Experiment on therapy:
The patient of this checking example is selected from inpatient, altogether 35 example, wherein posthepatitic cirrhosis 18 example, and blood sucker liver is hard Change 7 examples, Alcoholic and reason liver cirrhosis 10 example thereof.35 example liver cirrhosis patients are randomly divided into 7 groups, often organize 5 people, packet and often group Treatment as follows:
Group 1 is matched group, uses conventional medicine to treat；
The stem cell medicine of group 2 employing embodiment 1 preparation carries out injection for curing；
Stem cell medicine prepared by group 3 employing comparative example 1A carries out injection for curing；
Stem cell medicine prepared by group 4 employing comparative example 1B carries out injection for curing；
The stem cell medicine of group 5 employing embodiment 2 preparation carries out injection for curing；
Stem cell medicine prepared by group 6 employing comparative example 2A carries out injection for curing；
Stem cell medicine prepared by group 7 employing comparative example 2B carries out injection for curing；
The patient of group 2～7, with human umbilical cord mesenchymal stem cells injection for curing 3 times, is separated by one-month period every time, after treatment, Patient's spirit takes a turn for the better, and appetite increases, and ascites disappears, and serology biochemical indicator is obviously improved, and detects the serological index of patient (meansigma methods): ALT (alanine aminotransferase), AST (glutamic oxaloacetic transaminase, GOT), ALB (serum albumin), TBIL (total bilirubin), PT (prothrombin time), PT% (Liver disease), PT INR (prothrombin time international normalized ratio), AFP (alpha-fetoprotein)；Referring to such as table 2 below, this table 2 is the parameter of the serological index recorded after above-mentioned 7 groups of patient.
Table 2
From upper table 2, comparative example 1A (human umbilical cord mesenchymal stem cells+vitamin A) and comparative example 1B (Cortex Dictamni lactone + vitamin A), comparative example 2A (human umbilical cord mesenchymal stem cells+vitamin A) and comparative example 2B (Cortex Dictamni lactone+vitamin A) Liver cirrhosis is respectively provided with curative effect, all can improve liver cirrhosis, thus prove: mescenchymal stem cell and Cortex Dictamni lactone all can be controlled respectively Treat liver cirrhosis, improve fibrosis；Stem cell medicine prepared by Example 1 and Example 2 of the present invention can make liver cirrhosis patient Serological index recovers normal, close can be even up to the effect cured, and, the effect of embodiment 1 is far superior to contrast The effect sum of example 1A and comparative example 1B, the effect of same embodiment 2 is far superior to comparative example 2A and the effect of comparative example 2B generation Really sum, therefore, the liver cirrhosis that hepatic fibrosis is caused by the Cortex Dictamni lactone of stem cell medicine of the present invention and mescenchymal stem cell Treatment play synergism, be used individually hepatic fibrosis relative to Cortex Dictamni lactone or mescenchymal stem cell, this effect is the most excellent In both sums.
Checking example 3
The drafting of the growth curve of human umbilical cord mesenchymal stem cells:
Use mtt assay to measure cell growth curve, respectively Example 1, comparative example 1C, embodiment 2, comparative example 2C dry Cell preparation, is inoculated in 96 orifice plates respectively with the density of 2500 cells/well and cultivates, and is cultivating 1,2,3,4,5,6 respectively, Illustrate each group of cell is carried out MTT detection by test kit during 7d.Add MTT solution 10 μ L/ hole, hatch 4h, add for 37 DEG C Formanzan liquid 100 μ L/ hole, hatches to formanzan for 37 DEG C and is completely dissolved, on enzyme-linked immunosorbent assay instrument at wavelength 570nm Detecting optical density (OD) value in each hole, draw cell growth curve according to the absorbance recorded, often group sets 6 hole comparisons.Cell With 1 × 106After/mL density is inoculated in 24 orifice plates cultivation 24h,
Trypsin digestion is used to calculate the cell population doublings time (populationdoubling time, PDT), in order to Lower equation is tried to achieve: PDT=t × [lg2/ (lgNt lgNo)], and in formula, t represents incubation time, and it is (thin that No represents counting first When born of the same parents cultivate 24h) cell number that obtains, Nt represents the cell number after cultivating the t time.
Refer to the growth curve that Fig. 1, Fig. 1 are four groups of human umbilical cord mesenchymal stem cells.Shown by growth curve result, 4 Group cell all starts fast breeding after the incubation period of 1～2d and enters exponential phase (3～6d), progresses into after 6d Plateau.Embodiment 1 and embodiment 2 higher than other two groups of comparative examples, have significance poor in 1～5d inner cell multiplication rate Different.Embodiment 1 and embodiment 2 cell doubling time the most substantially shorten, and embodiment 1 cell proliferation is slightly slower than embodiment 2；Therefore, Vitamin A can remarkably promote stem cells hyperplasia, and keeps stem cell undifferentiated.
Checking example 4
Hepatic stellate cell CyclinD1 and P27 protein expression Western blot detection:
(1), cultured rat hepatic stellate cells and fibroblastic cultivation: be inoculated in after cell recovery in plastic culture bottle, in 37 DEG C, volume fraction be the CO of 5%2Cellar culture in saturated humidity incubator, culture fluid is for being 10% tire cattle containing volume fraction The L-DMEM of serum, every 2d change liquid 1 time.
(2), packet transaction:
Embodiment 1 group (human umbilical cord mesenchymal stem cells+Cortex Dictamni lactone):
The stem cell medicine of embodiment 1 preparation co-cultures by 1: 1 with hepatic stellate cell；
Comparative example 1A group (human umbilical cord mesenchymal stem cells):
Comparative formulation prepared by comparative example 1A and hepatic stellate cell co-culture by 1: 1；
Matched group:
Fibroblast and hepatic stellate cell co-culture by 1: 1.
(3) immunostaining
With cell pyrolysis liquid extract day part hepatic stellate cell total protein, Coomassie brilliant blue colorimetric method for determining protein content, Applied sample amount is 80 μ g, albumen row 15%SDS-PAGE gel electrophoresis, PVDF transferring film, blocking non-specific.Add an anti-mouse to resist CyclinD1, p27 monoclonal antibody (1: 500 dilution), 4 DEG C overnight, add horseradish peroxidase-labeled two anti-carry out miscellaneous Hand over.ECL luminous agent 1～5min, exposes, develops, fixing.Digital image-forming is analyzed systems soft ware and is analyzed result, with purpose The gray level ratio of albumen/GAPDH represents relative destination protein level.
(4) statistical analysis:
Application SPSS 13.0 software carries out statistical disposition, and measurement data represents with x ± s, and P < 0.05 has significance for difference Meaning.Statistical result is as shown in table 3 below and table 4, and table 3 is group hepatic stellate cell CyclinD1 protein expression each after co-culturing, table 4 For group hepatic stellate cell p27 protein expression each after co-culturing.
Table 3 (x ± s, n=6, A)
Group 24h 48h 72h
Embodiment 1 group 0.22±0.07 0.10±0.01 0.04±0.01
Comparative example 1A group 0.65±0.09 0.43±0.09 0.11±0.06
Matched group 0.87±0.10a 0.92±0.13a 0.84±0.12a
In table 3, co-culturing 24h, the CyclinD1 protein expression of embodiment 1 group and comparative example 1A group starts to lower, and implements 1 group of CyclinD1 expressing quantity of example is substantially less than comparative example 1A group, and when co-culturing 72h, expression is substantially less than matched group (P <0.01)。
Table 4 (x ± s, n=6, A)
Group 24h 48h 72h
Embodiment 1 group 1.64±0.05 1.81±0.09 1.84±0.10
Comparative example 1A group 0.82±0.07 1.03±0.02 0.96±0.06
Matched group 0.19±0.02a 0.18±0.02a 0.18±0.03a
In table 4, co-culturing 24h, the p27 protein expression relatively matched group of embodiment 1 group and comparative example 1A group significantly raises (P < 0.01), the most persistently high expressed state, and 1 group of p27 expressing quantity of embodiment are significantly higher than comparative example 1A group.
Theoretical foundation: cell proliferation is to be realized by running of cell cycle, and cell cycle regulating refers to various regulation and control The factor passes through activation and the inactivation of self, makes cell start and complete cell cycle critical event, and ensures these events in due order Sequence is normally carried out.Its molecular basis includes cyclin, cyclin dependent kinases and cyclin Dependent protein kinase enzyme inhibitor.The check point of G0/G1 phase～S phase is that intraor extracellular signal is through transmitting, integrate, being pooled to cell Core, cell proliferation carries out the key point regulated and controled, G1 phase cyclin cyclinD1 etc. regulate and control.Cells containing sequences is by limiting point Enter the S phase from the G1 phase and be largely dependent on the accumulation of CyclinD1 in the G1 phase.It is compound that CyclinD1 can be combined formation with CDK , there is phosphorylation, thus promote the expression of some gene in thing under the kinase whose mediation of CDK, the expression product of these genes can promote Make cell pass through G1 phase～S period regulation point, make cell enter autonomous division program.On the contrary, the expression of retardance Cyclin D1, can Make cell can not enter the S phase from the G1 phase.P27 is a member of CKI family, is mainly combined with cyclin and plays cyclinCDK Inhibitory action.P27 has two aspects to the inhibitory action of CDK, on the one hand can suppress the CDK having been incorporated into cyclin and being activated Activity, on the other hand can also suppress the activation process of CDK, the final transformation suppressing cell cycle G1 → S.
Therefore, understanding in conjunction with above-mentioned table 3 and table 4, the CyclinD1 protein expression of comparative example 1A group reduces, P27 albumen Substantially increasing, difference has significant compared with matched group, and the mescenchymal stem cell suppression to hepatic stellate cell proliferation is described One of mechanism be by lowering cyclin CyclinD1, raise the expression of P27 albumen, making cell cycle arrest in G0/ The G1 phase, thus play the effect of suppression hepatic stellate cell proliferation.The CyclinD1 protein expression of embodiment 1 group reduces, P27 egg Bai Mingxian increases, and difference has significant compared with comparative example 1A group, makes Cortex Dictamni lactone clear and uses same mechanism significantly to press down Hepatic stellate cell proliferation processed.
The foregoing is only the preferred embodiments of the present invention, not thereby limit the scope of the claims of the present invention, every at this Under the inventive concept of invention, utilize the equivalent transformation that description of the invention and accompanying drawing content are made, or directly/indirectly it is used in it The technical field that he is correlated with is included in the scope of patent protection of the present invention.

Claims (10)

1. the stem cell medicine of an anti-hepatic fibrosis, it is characterised in that including:
Mescenchymal stem cell；
Cortex Dictamni lactone；
Vitamin A；
Cell preparation solution；
In described stem cell medicine, the concentration of described mescenchymal stem cell is (1～5) × 107Individual/ml, described Cortex Dictamni lactone Concentration be 20～100mg/ml, the concentration of described vitamin A is 20～100ng/ml.
2. the stem cell medicine of anti-hepatic fibrosis as claimed in claim 1, it is characterised in that described mescenchymal stem cell dense Degree is (2～4) × 107Individual/ml.
3. the stem cell medicine of anti-hepatic fibrosis as claimed in claim 1, it is characterised in that the concentration of described Cortex Dictamni lactone It is 40～80mg/ml.
4. the stem cell medicine of anti-hepatic fibrosis as claimed in claim 3, it is characterised in that described Cortex Dictamni lactone includes Ketone and obakulactone.
5. the stem cell medicine of anti-hepatic fibrosis as claimed in claim 1, it is characterised in that the concentration of described vitamin A is 40～80ng/ml.
6. the stem cell medicine of the anti-hepatic fibrosis as described in claim 1 to 5 any one, it is characterised in that fill between described Matter stem cell is human umbilical cord mesenchymal stem cells.
7. the stem cell medicine of the anti-hepatic fibrosis as described in claim 1 to 5 any one, it is characterised in that described cell Preparation solution is PBS.
8. the preparation method of the stem cell medicine of an anti-hepatic fibrosis as claimed in claim 1, it is characterised in that include step Rapid as follows:
Obtain mescenchymal stem cell；
Described mescenchymal stem cell, vitamin A, Cortex Dictamni lactone are added in cell preparation solution, and the stem cell system of preparing Agent.
9. the preparation method of the stem cell medicine of anti-hepatic fibrosis as claimed in claim 8, it is characterised in that described by described Mescenchymal stem cell, vitamin A, Cortex Dictamni lactone add in the step in cell preparation solution,
First described vitamin A is added in described cell preparation solution.
10. the preparation method of the stem cell medicine of anti-hepatic fibrosis as claimed in claim 8, it is characterised in that described acquisition The step of mescenchymal stem cell, including:
Obtaining and process umbilical cord tissue, being single cell suspension with the digestion of II Collagenase Type, it is residual that described II Collagenase Type is removed in washing Stay, described single cell suspension is seeded in the DMEM/F12 culture medium containing hyclone cultivation, it is thus achieved that become fibrous cell, treat Described one-tenth fibrous cell reach 70～80% merge time, carry out digesting, passing on, and collect the 3rd～5 generation mescenchymal stem cells.
CN201610356758.8A 2016-05-26 2016-05-26 Stem cell preparation capable of resisting hepatic fibrosis and preparation method of stem cell preparation Pending CN106038596A (en)

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