CN108841784A - A kind of nucleus pulposus cell anti-aging anti-apoptotic culture medium containing Astragaloside IV - Google Patents
A kind of nucleus pulposus cell anti-aging anti-apoptotic culture medium containing Astragaloside IV Download PDFInfo
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Abstract
The present invention relates to disc tissue engineering and regeneration field, in particular to one kind contains Astragaloside IV, and especially the culture medium of factor-containing is not used for nucleus pulposus cell culture, plays the application of anti-aging and Anti-G value.
Description
Technical field
The present invention relates to disc tissue engineering and regeneration field, in particular to one kind contains Astragaloside IV, especially not
The culture medium of factor-containing is used for nucleus pulposus cell culture, plays the application of anti-aging and Anti-G value.
Background technique
Interverbebral disc is mainly made of nucleus pulposus, fibrous ring and cartilage endplate.Nucleus pulposus is the highest tissue of water content in interverbebral disc,
The extracellular matrix of nucleus pulposus cell secretion can form jelly spline structure, and nucleus pulposus is allowed to bear the various pressure of centrum.
Under certain pathologic conditions, regression can occur for interverbebral disc, and then lead to low back pain.It there is no for intervertebral disc degeneration and effectively control at present
Treatment means construct artificial intervertebral disk using organizational project means and heteroplastic transplantation intervertebral disc cells are that emerging interverbebral disc moves back
Become effective therapeutic modality.
Building organizational project interverbebral disc or the transplanting of allosome intervertebral disc cells require to use seed cell, and especially nucleus pulposus is thin
Born of the same parents.Traditional nucleus pulposus cell condition of culture is all to use high glucose medium, because high sugar culture environment facilitates cell and quickly expands
Increase；But high glucose medium can cause nucleus pulposus cell aging and apoptosis simultaneously, and the nucleus pulposus cell of aging, apoptosis is further being answered
With repairing effect can be seriously affected in the process.Therefore, it is necessary to develop effective method to prevent in nucleus pulposus cell incubation
Aging and apoptosis；But it there is no the culture medium of anti-nucleus pulposus cell apoptosis, aging at present.
Chinese patent CN106754683A discloses a kind of the anti-ageing without differentiation amplification of people's umbilical cord/fat mesenchymal stem cell
Old culture medium.The culture medium is applied to people's umbilical cord/fat mesenchymal stem cell, and nucleus pulposus cell is with entirely different with stem cell
Biological characteristics and function, therefore the culture medium may not apply to the external anti-aging of nucleus pulposus cell, apoptosis culture；In addition, should
Medium component contains chemical small molecule in 11 kinds of cell factors such as TGF-beta and lipoic acid etc. 9, complicated component, price
It is expensive.
Summary of the invention
The present invention proposes one kind (Astragaloside IV, AG-IV) containing Astragaloside IV, especially not factor-containing
The culture medium of nucleus pulposus cell anti-aging anti-apoptotic solves and easily draws for cultivating the high glucose medium of nucleus pulposus cell in the prior art
The problem of playing nucleus pulposus cell aging, apoptosis.
The present invention provides a kind of culture medium, and the culture medium includes high glycoform basal medium and Astragaloside IV.
Preferably, the high glycoform basal medium is liquid；
Preferably, the high glycoform basal medium be selected from DMEM culture medium, F12 culture medium, DMEM/F12 culture medium or
1640 culture medium of RPMI；
Preferably, the concentration of the Astragaloside IV in the medium is 0.01-10 μM；
It is furthermore preferred that the concentration of the Astragaloside IV in the medium is 1-10 μM；
Preferably, the culture medium not factor-containing.
Further, described the present invention also provides a kind of method of TERT expression and telomere length for increasing nucleus pulposus cell
Method includes by the processing of nucleus pulposus cell Astragaloside IV solution；
Preferably, the nucleus pulposus cell shortens because high saccharide ring border causes TERT expression to reduce with telomere length；
Preferably, the concentration of the Astragaloside IV solution is 0.01-10 μM；
It is furthermore preferred that the concentration of the Astragaloside IV solution is 1-10 μM；
Compared with prior art, beneficial effect of the present invention is mainly reflected in：The present invention provides one kind to contain Astragaloside IV, special
It is not free from the nucleus pulposus cell culture medium of cell factor, solves in the prior art for cultivating the high glucose medium of nucleus pulposus cell
The problem of easily causing nucleus pulposus cell aging, apoptosis, it is therefore prevented that the aging of nucleus pulposus cell and apoptosis in incubation, and be effectively improved
The growth conditions of nucleus pulposus cell.
Detailed description of the invention
Fig. 1 is high concentration glucose influence nucleus pulposus cell (NP cell) survival rate figure, wherein：
Fig. 1 a is that the Cell counting Kit (CCK-8) after being handled nucleus pulposus cell 24 hours with the glucose of various concentration is examined
Survey result.
Fig. 1 b is the Cell counting Kit -8 (CCK-8) after being handled nucleus pulposus cell 48 hours with the glucose of various concentration
Testing result.
Fig. 1 c is the Cell counting Kit -8 (CCK-8) after being handled nucleus pulposus cell 72 hours with the glucose of various concentration
Testing result.
Fig. 1 d is the Cell counting Kit -8 (CCK-8) after being handled nucleus pulposus cell 96 hours with the glucose of various concentration
Testing result.* p < 0.05, * * p < 0.01, * * * p < 0.001, * * * * p < 0.0001
Fig. 2 is influence diagram of the high concentration glucose to NP cellular telomerase reverse transcriptase (TERT) expression and telomere length,
Wherein：
Fig. 2 a is that western blot detects control group 5.5mM and 12.5,25,50,100 and 200mM high concentration grape
The protein expression of TERT in sugared group.And uses α-tubulin albumen to compare as load and carry out ribbon density standardization.
Fig. 2 b is to measure control group 5.5mM and 12.5,25,50,100 and 200mM high concentration grape by RT-qPCR
The mRNA expression of TERT in sugared group, wherein β-actin is as house-keeping gene for standardizing.
Fig. 2 c is to assess relative telomere length by using qPCR method, and wherein 36B4 is for standardized single copy base
Cause.Telomere length is checked using standard method Cawthon.* p < 0.05, * * p < 0.01, * * * p < 0.001, * * * * p <
0.0001。
Fig. 3 is influence diagram of the high concentration glucose to nucleus pulposus cell apoptosis and aging, wherein：
Fig. 3 a is that western blot detects apoptosis correlation egg after being handled nucleus pulposus cell 48 hours with the glucose of various concentration
The protein content of white C-C3, BAX and BCL-2 are simultaneously quantitative.
Fig. 3 b is FCM analysis FITC-Annexin- after being handled nucleus pulposus cell 48 hours with the glucose of various concentration
V。
Fig. 3 c is that aging correlation SA- β-gal dye is carried out after being handled nucleus pulposus cell 48 hours with the glucose of various concentration
Color：Original magnification × 100, scale bar：50μm.
Fig. 3 d is the protein with the aging GAP-associated protein GAP p16 of the nucleus pulposus cell of the glucose processing 48 hours of various concentration
Content western blot is detected and is quantified.Data in figure indicate mean+SD, aobvious between treatment group and control group
It writes difference and is expressed as * p < 0.05, * * p < 0.01, * * * p < 0.001, * * * * p < 0.0001.
Fig. 4 is cytotoxicity detection figure of the Astragaloside IV to nucleus pulposus cell, wherein：Astragaloside IV is detected with CCK8 method
The influence of (Astragaloside IV, AG-IV) to nucleus pulposus cell vigor.The value presented is mean+SD, experiment
It is repeated 3 times.* p < 0.05 compared with the control group, * * p < 0.01, * * * p < 0.001, * * * * p < 0.0001.
Fig. 5 is influence diagram of the Astragaloside IV to high sugared condition nucleus pulposus cell vigor, wherein：
Fig. 5 a shows that AG-IV significantly improves high sugared (high glucose, HG) item by the cell viability measured of CCK 8
The cell viability of nucleus pulposus cell under part.
Fig. 5 b is the morphological result of high sugar stimulation cell after AG-IV processing.High sugar treatment conditions：25mM concentration of glucose
Stimulate cell for 24 hours；Scale bar：100μm.* p < 0.05, * * p < 0.01, * * * p < 0.001, * * * * p < 0.0001
Fig. 6 is shadow of the Astragaloside IV to cellular telomerase reverse transcriptase (TERT) expression and telomere length under the conditions of high sugar
It rings, wherein：
Fig. 6 a is that western blot detects Astragaloside IV to the work of the protein expression of nucleus pulposus cell TERT under the conditions of high sugar
With.Use α-tubulin as load control and ribbon density standardization.
Fig. 6 b is that Astragaloside IV is detected by RT-qPCR to the work of the mRNA expression of nucleus pulposus cell TERT under the conditions of high sugar
With using β-Actin to be standardized as house-keeping gene.
Fig. 6 c is the relative telomere length by qPCR method assessment Astragaloside IV to nucleus pulposus cell under the conditions of high sugar, wherein
36B4 is for standardized single copy gene.Data in figure indicate mean+SD, between treatment group and control group
Significant difference be expressed as * p < 0.05, * * p < 0.01, * * * p < 0.001, * * * * p < 0.0001
Fig. 7：Influence of the Astragaloside IV to nucleus pulposus cell apoptosis and aging under the conditions of high sugar, wherein：
Fig. 7 a is that western blot detects Astragaloside IV to nucleus pulposus cell apoptosis-related protein C-C3, BAX under the conditions of high sugar
With the protein content of BCL-2.
Fig. 7 b is result of the FCM analysis Astragaloside IV to the FITC-Annexin-V of nucleus pulposus cell under the conditions of high sugar.
Fig. 7 c is that western blot detects Astragaloside IV to the egg of nucleus pulposus cell aging GAP-associated protein GAP p16 under the conditions of high sugar
White matter content.
Fig. 7 d is effect of the SA- β-gal dyeing detection Astragaloside IV to nucleus pulposus cell aging under the conditions of high sugar.Original amplification
Multiple × 100, scale bar：50μm.Data in figure indicate mean+SD, the significance difference between treatment group and control group
It is different to be expressed as * p < 0.05, * * p < 0.01, * * * p < 0.001, * * * * p < 0.0001
Specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This：
Embodiment 1
Nucleus pulposus cell separation and culture
Gel tissue (nucleus pulposus) is collected from the tail intervertebral disc of the SD rat children mouse of any gender of 2-3 week old.NP is organized
It is digested 4 hours in 0.2%II Collagenase Type (Sigma) in 37 DEG C.After being washed twice with PBS, the tissue of digestion is transferred to
Contain 15% fetal calf serum (FBS；Gibco, Invitrogen, Grand Island, NY), 1g/L (5.5mM) glucose and 1%
Antibiotic (100 units/ml penicillin and 100 μ g/ml streptomysins) DMEM (Gibco, Invitrogen, Grand Island,
NY) in culture medium, it is placed in 37 DEG C of 5%CO2In incubator.After mixing, by cell with 0.25% trypsase-EDTA
It passes on after (Gibco, Invitrogen) trypsin digestion and is implanted into 10cm culture plate again with density appropriate.We
Use first three for cell in experiment.
Embodiment 2
Cell administration scheme
D-Glucose (Sigma Aldrich) is added in DMEM (low sugar), sterile 0.45 μm of pvdf membrane is then passed through
Filter (Merck Millipore) filtering, 500mM concentration of glucose are used for subsequent experimental as stock solution.Then by stoste
It is diluted in complete medium to obtain the glucose (5.5-200mM) of required concentration.With the glucose of various dose and time
Handle cell.In order to study influence of the AG-IV to experimental group, they are dissolved in dimethyl sulfoxide (DMSO) with the concentration of 500mM
In, (final DMSO concentration is then suitably diluted with cell culture medium<1%).All experiments are at least repeated 3 times.
Embodiment 3
Cell viability measurement
By NP cell inoculation in 96 orifice plates (5000 cells/wells), incubate 48 hours.After adherent, contain 0% by displacement
Culture medium 12 hours of FBS keep cell hungry.Then, with ever-increasing concentration of glucose and the DMEM of time (15%FBS)
It handles NP cell or increases drug concentration by different time sections.Then after washing cell with PBS, every hole is added 100 μ l and contains 10 μ
The DMEM of l CCK8.Then plate is incubated about 1 hour.(the CCK- of Cell counting Kit -8 is used according to the scheme of kit
8) viability of solution (CCK-8, Dojindo Laboratories, Japan) measurement cell.It is surveyed using microplate reader in 450nm
The optical density of metering-orifice.
Embodiment 4
Western blot analysis
Cell 1mM PMSF (phenylmethanesulfonyl fluoride, Beyotime) is cracked on ice-cold RIPA.Pass through BCA albumen
The protein concentration of matter assay kit (Beyotime) measurement sample.In 12-15% sodium dodecyl sulfate-polypropylene amide
The sample containing 40 protein isolate matter is separated in gel electrophoresis (SDS-PAGE), and is transferred to polyvinylidene fluoride difluoride film
(Millipore, USA) is then closed with 5% skim milk or BSA.Later, with p16 (1：500), (1 C-C3：1000), BAX
(1：1000), (1 Bcl-2：1000), α-tubulin (1：And TERT (1 1000)：1000) primary antibody, 4 DEG C overnight, then and respectively
From secondary antibody be incubated at room temperature 2 hours.Finally, using 3.0 software of Image Lab (Bio-Rad) to the intensity of these bands
Quantified.
Embodiment 5
PCR analysis
Total RNAs extraction object is obtained using TRIzol reagent (Invitrogen), and synthesizes cDNA using extract total serum IgE
(TaKaRa, Japan).QPCR is carried out using SYBR Green system (Bio-Rad, Hercules, USA).The amplification of cDNA sample
It is carried out on CFX96 real-time PCR system (Bio-Rad).Step 1：95 DEG C are denaturalized 3 minutes.Step 2：95 DEG C 5 seconds, 60 DEG C 45 seconds,
Carry out 40 circulations.Last melting curve analysis：65 DEG C, then 95 DEG C and 5 DEG C, every time 5 seconds.The relative quantification needle of target gene
β-actin gene is standardized, and using 2^- Δ Δ Cq method that target gene is corresponding to from each of control sample
Target gene is compared.Table 1 lists the primer of TERT and β-actin.
DNA is extracted with the DNeasy kit from Qiagen (Hilden, Germany).In the scheme using manufacturer from thin
After born of the same parents extract DNA, qPCR then is carried out using identical scheme as described above and data are analyzed.Table 2 list telomere and
The primer of 36B4.
1 β-actin of table and TERT primer sequence
2 telomere of table and 36B4 primer sequence
Embodiment 6
SA- β-gal dyeing
By cell inoculation 48 hours in 6 holes or 12 orifice plates.Then twice by cells rinsed with PBS, use at room temperature
0.2% glutaraldehyde fixes 10 minutes.Then by cell with X-gal staining solution the stained over night at pH 6.0.It uses within second day
It is inverted Olympus IX71 microscope and captures image, and quantify SA- β-gal, the percentage of positive cell is for statisticalling analyze.
Embodiment 7
Annexin-V FITC and propidium iodide flow cytometry
As described in the scheme of manufacturer, with Annexin-V apoptosis detection kit (B&D, Franklin Lake, NJ,
USA Apoptosis) is detected.In short, cell is inoculated with 2 × 10^5 cell/mL density in 6 orifice plates and is carried out corresponding
Processing.Later, the about 10^6 cell from each hole is collected, washs and is resuspended in containing 5 μ L Annexin-VFITC and 5
It is acted in the dark 30 minutes in μ L propidium iodide (PI) buffer.Using flow cytometer (BECKMAN COULTER, Brea,
CA, USA) analysis cell.Total apoptosis rate of cell is calculated as the percentage for the ratio observed in bottom right and right upper quadrant.
Above embodiments, all experiments are at least in triplicate.As a result it is expressed as mean+SD.As a result statistical
Analysis is handled using SPSS statistical software program 20.0.Data are analyzed by one-way analysis of variance (ANOVA), then
Tukey is carried out to examine to compare control group and treatment group.P value<0.05 is considered to have statistical significance.
Embodiment 8
The cell activity of high concentration glucose inhibition nucleus pulposus cell
In order to study the effect in the cytotoxicity of primary nucleus pulposus cell middle and high concentration glucose induction, we are with different doses
The glucose of amount and time handle NPs.Using (CCK8) measuring method of Cell counting Kit -8 various concentration (5.5,12.5,
25,50,100 and 200mM) under measurement glucose to cytotoxic effect 24-96 hours of NP.As shown in Figure 1, high sugar is thin to NP
Born of the same parents have apparent cytotoxic effect, especially (Fig. 1) (P in high dose<0.05-0.0001).
Embodiment 9
High concentration glucose inhibits nucleus pulposus cell TERT expression and telomere length
Nucleus pulposus cell different glucose (5.5,12.5,25,50,100 and 200mM) passes through after handling 48 hours
The TERT that western blot detects.As shown in Figure 2 a, TERT is significant with concentration dependant manner in all concentration of glucose
Reduce (P<0.05-0.0001).Meanwhile as shown in Figure 2 b, RT-qPCR as the result is shown TERT mRNA express also with concentration dependant
Property mode declines (P<0.0005).In addition, as shown in Figure 2 c, the telomere length detected by using the qPCR method of Cawthon
Also (P is declined with concentration dependant manner<0.05-0.0005).These statistics indicate that, high concentration glucose can reduce TERT expression
With shortening telomere length.
Embodiment 10
High concentration glucose promotes apoptosis and the aging of nucleus pulposus cell
After being handled nucleus pulposus cell 48 hours with the glucose of various concentration (5.5,12.5,25,50,100 and 200mM), lead to
Western blotting detection apoptosis-related protein is crossed, i.e.,：C-C3, BAX and Bcl-2.As shown in Figure 3a, C-C3 and BAX protein expression
Increase, and Bcl-2 significantly reduces (P with the concentration dependant manner of concentration of glucose<0.05-0.0001).Meanwhile such as Fig. 3 b institute
Show, (P is dramatically increased with concentration dependant manner by the FITC percentage that Annexin-V is detected<0.0005).Result above mentions
Show, high concentration glucose can promote nucleus pulposus cell apoptosis.
Cell β-gal stained positive rate increases (P after β-gal dyeing display high concentration glucose processing in Fig. 3 c<0.005-
0.001)；In addition, the expression of the p-16 as aging marker also increases (P with concentration dependant manner in Fig. 3 d<
0.005-0.0001).Result above prompt, high concentration glucose can promote nucleus pulposus cell aging.
Embodiment 11
Cytotoxic effect of the Astragaloside IV to nucleus pulposus cell
We handle nucleus pulposus cell with the AG-IV of various dose, and are existed using Cell counting Kit (CCK8) measuring method
AG-IV is measured under various concentration (0.01,0.1,0.5,1,3,5,10,50 and 100 μM) to the toxic effect of nucleus pulposus cell.Such as
Shown in Fig. 4, there are significant cytotoxic effects (Fig. 4) for high dose (i.e. 50 and 100 μM) of the drug on NP cell (respectively
P<0.0005 and 0.05).
Embodiment 12
Astragaloside IV improves cell viability under the conditions of high sugar
Cell viability is measured after HG is handled 48 hours using Cell counting Kit (CCK8) measuring method.Such as Fig. 5 a institute
Show, there are significant differences between untreated fish group (DMSO HG) and processing group (AG-IV).After being handled with HG, cell viability decline,
But AG-IV shows significant protective effect (Fig. 5 a) (P to the HG cell death induced<0.05-0.01).
In addition, the size and shape of NP cell is different, when being handled with HG (25mM), cytoplasm and organelle seem coarse；
But after having added AG-IV, they can reverse this phenomenon to a certain extent.(Fig. 5 b).
Embodiment 13
Astragaloside IV can increase TERT expression and the telomere length of NP cell under the conditions of high sugar
In order to study the effect that AG-IV generates TERT under the conditions of high sugar, high glucose (HG) concentration of 25mM is handled
NP cell handled 24 hours with the AG-IV of 1,3,5 and 10 μM of concentration.As shown in FIG. 6 a, TERT expression is aobvious under the conditions of HG
Writing reduces, and TERT expression restores (P in medication therapy groups<0.05-0.01).Meanwhile as shown in Figure 6 b, in RT-qPCR
The mRNA expression of TERT is significantly reduced under the conditions of HG, and is dramatically increased in drug-treated group, especially dense at 1 and 3 μM
(P under degree<0.05).In addition, as fig. 6 c, also being shown under the conditions of HG by using the telomere length that Cawthon method measures
Writing reduces, and dramatically increases in AG-IV effect group, (the P especially under 1 and 3 μM of concentration<0.05).The above tables of data
Bright, AG-IV is shielded in the cellular damage that HG is induced by increasing TERT expression and extending telomere length.
Embodiment 14
Apoptosis and aging under the conditions of the high sugar of Astragaloside IV inhibition
It is in order to study effect of the AG-IV to Apoptosis and aging under the conditions of HG, the high glucose (HG) of 25mM is dense
The NP cell of processing is spent to handle 24 hours with 1,3,5 and 10 μM of concentration AG-IV.Fig. 7 a be shown under the conditions of simple HG C-C3 and
BAX protein increases and Bcl-2 is significantly reduced, and the expression of the above albumen is significantly reversed after AG-IV effect.Medication therapy groups and list
Pure HG treatment group compares, and has significant difference (P<0.01-0.001).Meanwhile as shown in Figure 7b, it is detected by Annexin-V
Percentage of cerebral apoptosis dramatically increased in the case where simple HG, and percentage in drug-treated group reduces (P<0.05-
0.01).In addition, in figure 7 c, the expression of the p-16 as aging marker dramatically increases under conditions of simple HG, and drug
Expression in processing group reduces (P<0.0005).β-gal dyeing in Fig. 7 d also shows identical phenomenon (P<0.001).More than
The results show that Astragaloside IV can significantly inhibit apoptosis and the aging of the nucleus pulposus cell under the conditions of high sugar.
The present invention is described in detail above, but the invention is not restricted to the embodiments described.It is all according to the present invention
Equivalent change or modification made by Spirit Essence, should be covered by the scope of protection of the present invention.
Sequence table
<110>Attached second hospital of Wenzhou Medical University, attached Yu Ying children's hospital of Wenzhou Medical University
<120>A kind of nucleus pulposus cell anti-aging anti-apoptotic culture medium containing Astragaloside IV not factor-containing
<130> 2018
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Claims (10)
1. a kind of culture medium, the culture medium includes high glycoform basal medium and Astragaloside IV.
2. culture medium as described in claim 1, the high glycoform basal medium is liquid.
3. culture medium as claimed in claim 1 or 2, the high glycoform basal medium is selected from DMEM culture medium, F12 is cultivated
1640 culture medium of base, DMEM/F12 culture medium or RPMI.
4. the culture medium as described in claim 1-3, the concentration of the Astragaloside IV in the medium is 0.01-10 μM.
5. culture medium as claimed in claim 4, the concentration of the Astragaloside IV in the medium is 1-10 μM.
6. culture medium as claimed in claims 1-5, the culture medium not factor-containing.
7. a kind of method for the TERT expression and telomere length for increasing nucleus pulposus cell, the method includes by nucleus pulposus cell Radix Astragali
The processing of first glycosides solution.
8. the method for claim 7, the nucleus pulposus cell leads to TERT expression reduction and telomere length because of high saccharide ring border
Shorten.
9. the method for claim 7, the concentration of the Astragaloside IV solution is 0.01-10 μM.
10. the method for claim 7, the concentration of the Astragaloside IV solution is 1-10 μM.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104762364A (en) * | 2015-04-13 | 2015-07-08 | 安徽农业大学 | Application method of astragalus polysaccharide in antagonism of dairy cow mammary epithelial cells apoptosis |
| CN106047801A (en) * | 2016-05-30 | 2016-10-26 | 深圳大学 | Nucleus pulposus cell separating and purifying method |
| CN107308497A (en) * | 2017-06-09 | 2017-11-03 | 浙江大学 | The structure of the active microcarrier of nucleus pulposus cell source property |
| WO2018033227A1 (en) * | 2016-08-17 | 2018-02-22 | Orthogen Ag | Anti-ageing pharmaceutical preparation |
| CN108186659A (en) * | 2018-03-20 | 2018-06-22 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | Application of the Astragaloside IV in preparing treatment and delaying degeneration of intervertebral disc's drug |
-
2018
- 2018-07-17 CN CN201810783088.7A patent/CN108841784A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104762364A (en) * | 2015-04-13 | 2015-07-08 | 安徽农业大学 | Application method of astragalus polysaccharide in antagonism of dairy cow mammary epithelial cells apoptosis |
| CN106047801A (en) * | 2016-05-30 | 2016-10-26 | 深圳大学 | Nucleus pulposus cell separating and purifying method |
| WO2018033227A1 (en) * | 2016-08-17 | 2018-02-22 | Orthogen Ag | Anti-ageing pharmaceutical preparation |
| CN107308497A (en) * | 2017-06-09 | 2017-11-03 | 浙江大学 | The structure of the active microcarrier of nucleus pulposus cell source property |
| CN108186659A (en) * | 2018-03-20 | 2018-06-22 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | Application of the Astragaloside IV in preparing treatment and delaying degeneration of intervertebral disc's drug |
Non-Patent Citations (5)
| Title |
|---|
| JAE-GWAN KONG等: "Effect of high glucose on stress-induced senescence of nucleus pulposus cells of adult rats", 《 ASIAN SPINE J》 * |
| 冯仲锴等: "黄芪甲苷通过SIRT1/PGC-1α通路促进退变的髓核细胞增殖", 《西安交通大学学报(医学版)》 * |
| 张存鑫等: "胰酶联合Ⅱ型胶原酶分离培养髓核细胞：培养基中葡萄糖浓度的选择", 《中国组织工程研究》 * |
| 徐维佳等: "黄芪甲苷对高糖诱导的肾小管上皮细胞损伤的保护作用", 《中国中西医结合肾病杂志》 * |
| 杨庆铭: "《骨科学》", 31 July 2008 * |
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