CN109609447A - The preparation of native copper leachate promotes the application of mesenchymal stem cells differentiation drug - Google Patents
The preparation of native copper leachate promotes the application of mesenchymal stem cells differentiation drug Download PDFInfo
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- CN109609447A CN109609447A CN201910118984.6A CN201910118984A CN109609447A CN 109609447 A CN109609447 A CN 109609447A CN 201910118984 A CN201910118984 A CN 201910118984A CN 109609447 A CN109609447 A CN 109609447A
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- mesenchymal stem
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- thiobacillus ferrooxidans
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- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 title claims abstract description 39
- 229910052802 copper Inorganic materials 0.000 title claims abstract description 39
- 239000010949 copper Substances 0.000 title claims abstract description 39
- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 29
- 230000004069 differentiation Effects 0.000 title claims abstract description 13
- 239000003814 drug Substances 0.000 title claims abstract description 12
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 229940079593 drug Drugs 0.000 title description 3
- 241000605222 Acidithiobacillus ferrooxidans Species 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims description 12
- 238000002386 leaching Methods 0.000 claims description 9
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 8
- 239000012530 fluid Substances 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- 235000015097 nutrients Nutrition 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 230000000694 effects Effects 0.000 abstract description 16
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 abstract description 15
- 239000011575 calcium Substances 0.000 abstract description 15
- 229910052791 calcium Inorganic materials 0.000 abstract description 15
- 230000011164 ossification Effects 0.000 abstract description 15
- 238000000338 in vitro Methods 0.000 abstract description 4
- 210000001185 bone marrow Anatomy 0.000 abstract description 3
- 210000004271 bone marrow stromal cell Anatomy 0.000 abstract description 3
- 230000008021 deposition Effects 0.000 abstract description 2
- 230000002188 osteogenic effect Effects 0.000 abstract 1
- 230000001737 promoting effect Effects 0.000 abstract 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 22
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 22
- 210000004027 cell Anatomy 0.000 description 10
- 210000000988 bone and bone Anatomy 0.000 description 7
- 230000001744 histochemical effect Effects 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 6
- 229910052683 pyrite Inorganic materials 0.000 description 6
- 239000011028 pyrite Substances 0.000 description 6
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 description 5
- 229960005309 estradiol Drugs 0.000 description 5
- 229930182833 estradiol Natural products 0.000 description 5
- NUHSROFQTUXZQQ-UHFFFAOYSA-N isopentenyl diphosphate Chemical compound CC(=C)CCO[P@](O)(=O)OP(O)(O)=O NUHSROFQTUXZQQ-UHFFFAOYSA-N 0.000 description 5
- 210000000963 osteoblast Anatomy 0.000 description 5
- 239000008363 phosphate buffer Substances 0.000 description 5
- 238000004043 dyeing Methods 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 241000700159 Rattus Species 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 239000000835 fiber Substances 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 210000000130 stem cell Anatomy 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- RGCKGOZRHPZPFP-UHFFFAOYSA-N alizarin Chemical compound C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000002449 bone cell Anatomy 0.000 description 2
- 159000000007 calcium salts Chemical class 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 210000002997 osteoclast Anatomy 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 238000013139 quantization Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 208000006386 Bone Resorption Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 208000003098 Ganglion Cysts Diseases 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 208000005400 Synovial Cyst Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000033558 biomineral tissue development Effects 0.000 description 1
- 210000002805 bone matrix Anatomy 0.000 description 1
- 230000024279 bone resorption Effects 0.000 description 1
- 230000002308 calcification Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 description 1
- 229960001008 heparin sodium Drugs 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 210000004409 osteocyte Anatomy 0.000 description 1
- 230000000865 phosphorylative effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000001044 red dye Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- AVPCPPOOQICIRJ-UHFFFAOYSA-L sodium glycerol 2-phosphate Chemical compound [Na+].[Na+].OCC(CO)OP([O-])([O-])=O AVPCPPOOQICIRJ-UHFFFAOYSA-L 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 230000005082 stem growth Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
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- C12N2500/72—Undefined extracts from bacteria
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1353—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from bone marrow mesenchymal stem cells (BM-MSC)
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- Bioinformatics & Cheminformatics (AREA)
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- Rheumatology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of native copper Thiobacillus ferrooxidans BY3 leachates to promote mesenchymal stem cell at the application in ossification differentiation medicament in preparation, the present invention is had studied native copper Thiobacillus ferrooxidans BY3 leachate and is acted on during promoting the differentiation of mesenchymal stem cell osteogenic using rat bone marrow mesenchymal stem cells as effective object.The result shows that native copper Thiobacillus ferrooxidans BY3 leachate can be improved the ALP activity of Marrow Mesenchymal Stem Cells In Vitro, promote the calcium deposition of mesenchymal stem cell, and then promotes breaking up at ossification for mesenchymal stem cell.
Description
Technical field
The present invention relates to a kind of native copper Thiobacillus ferrooxidans BY3 leachates to promote marrow in preparation
Mescenchymal stem cell belongs to biomedicine field at the application in ossification differentiation medicament.
Background technique
Osteoporosis is a kind of systemic and systematic skeletal diseases, since skeletal metabolism dynamic equilibrium is by broken
Bad, bon e formation is less than bone resorption and causes.Skeletal metabolism process needs the maintenance of many functioning cells, to keep the dynamic of bone
Balance, wherein osteoclast and osteoblast are main functioning cells during this.Osteoblast is in skeleton-forming process
Middle secretion calcium salt and bone matrix, to form new bone, the osteoclast then secreting acidic substance in skeleton-forming process is played the part of
The role of bone absorption.
Rat bone marrow mesenchymal stem cells (Rats bone marrow stromal stem cells, rBMSCs), are to deposit
It is that there is the stem cell for being divided into a variety of potential such as osteoblast, osteocyte, the performer in skeleton-forming process in marrow
Extremely important effect, the osteoblast with lasting update lay the foundation for the formation of bone.In long-term clinical application mistake
It is found in journey, Chinese medicine othopedics medication native copper promotes union obvious effect, however used at present is processed with classical way
Native copper solubility is small in water, bioavilability is low, medicinal brings very big problem to clinical.The present invention passes through native copper
Nature copper biochemical lixivium made from the method for Bioleaching increases the leaching of effective element in native copper, and dissolution rate is good, raw
Object availability is high, can promote breaking up at ossification for mesenchymal stem cell.
Summary of the invention
The purpose of the present invention is to provide a kind of native copper leachates to promote mesenchymal stem cell at ossification in preparation
Application in differentiation medicament, the native copper leachate are the biologies of native copper Thiobacillus ferrooxidans BY3
Leachate, leaching method the following steps are included:
(1) native copper is weighed, is placed in container, is added 200 times of 0K fluid nutrient medium into container, and with sulfuric acid tune pH
To 1.60；
(2) the Thiobacillus ferrooxidans BY3 that inoculation volume fraction is 10%, in 25 DEG C, 130r/min
It leaches 15 days；
(3) it after leaching, stands, collects supernatant, adjust pH value of solution to neutrality, native copper is obtained after degerming
Thiobacillus ferrooxidans BY3 leachate.
Present invention employs Thiobacillus ferrooxidans BY3 to be leached to native copper, increases certainly
The leaching of effective element in right copper.Experimental result shows, native copper Thiobacillus ferrooxidans BY3 leachate energy
The activity for improving the ALP (alkaline phosphatase) of Marrow Mesenchymal Stem Cells In Vitro, promotes the calcium of mesenchymal stem cell
Mineralization, and then promote breaking up at ossification for mesenchymal stem cell.
Detailed description of the invention
Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.
Fig. 1 is native copper Thiobacillus ferrooxidans BY3 leachate on the active influence of rBMSCs ALP
Result figure.Wherein Figure 1A is ALP histochemical stain figure, and Figure 1B is ALP Activity determination result figure, and Fig. 1 C and Fig. 1 D are ALP group
Result figure of the weave chemistry stained area after the analysis of IPP.6.0 software.
Fig. 2 is influence result of the native copper Thiobacillus ferrooxidans BY3 leachate to rBMSCs mineralising
Figure.Wherein Fig. 2A is calcium scoring colored graph；Fig. 2 B is the calcium scoring area quantization figure obtained with IPP.6.0 scanning analysis；
Fig. 2 C is that calcium scoring dyes qualification result figure under microscope.
Biomaterial
Thiobacillus ferrooxidans BY3 (Thiobacillus ferrooxidans BY3) was preserved on September 22nd, 2003
China typical culture collection center, deposit number are CCTCC NO:M 203071, preservation title: Thiobacillus ferrooxidans BY3
(Thiobacillus ferrooxidans BY3)。
Specific embodiment
Specific embodiment presented below is to realize native copper Thiobacillus ferrooxidans of the present invention
BY3 leachate promotes mesenchymal stem cell at the application in ossification differentiation medicament in preparation, but is not limited to following embodiment.
The preparation of 1 native copper Thiobacillus ferrooxidans BY3 leachate of embodiment
0.5g native copper is weighed, milled 200 mesh obtains powder.In clean 250mL conical flask, after sterilizing is added
0K fluid nutrient medium 100mL, 5M sulfuric acid tune pH to 1.60, after pH stablize after, inoculation volume fraction be 10%
Thiobacillus ferrooxidans BY3 is leached 15 days in 25 DEG C, 130r/min.5M sulfuric acid tune is used in experimentation
PH value is saved, evaporated moisture is supplemented with distilled water.Shaking flask stops after leaching 15 days, stands overnight, and collects supernatant, adjusts molten
Liquid pH is spare after crossing the degerming of 0.22m filter membrane to neutrality.
The culture of 2 mesenchymal stem cell of embodiment
The SD rat (purchased from Gansu university of TCM animal experimental center) of 120-150g or so is taken, de- neck is used after putting to death
75% alcohol disinfecting 10min removes bone formation, then has DMEM/F12 culture solution with suction (containing heparin sodium 500U/mL)
Syringe extracts the marrow in ossis out, and piping and druming dissipates cell by piping and druming as far as possible repeatedly, and the sieving of 150 mesh of cell suspension is adjusted
Ganglion cell's concentration is 1.0 × 107Cells/mL is inoculated in 90mm Tissue Culture Dish, with containing 10% fetal calf serum DMEM/F12
Culture solution culture cell.Condition of culture is 37 DEG C, 5%CO2It is cultivated under conditions of saturated humidity, replaces primary culture every 3d
Liquid is rinsed 2 times with sterile PBS (phosphate buffer) before replacing culture solution, is passed when cell reaches 70~80% fusion
In generation, is inoculated with when reaching for 3 generation.
Embodiment 3ALP determination of activity
Indicator index of the ALP as osteoblast maturation and mineralising is a kind of glycoprotein being prevalent on cell membrane,
Energy hydrolytic phosphatide is that bone cells mineralising forms bone offer material base.The present embodiment is using ALP activity as index, evaluation
Natural copper biochemical lixivium is to mesenchymal stem cell at the influence of ossification differentiation.
3.1 grouping
Native copper 0K+A.f+N-pyrite (1:100) group, 0K+A.f+A-pyrite (1:10000) group are set up, it is positive right
According to group and negative control group.
0K+A.f+N-pyrite (1:100) group is using inartificial nature copper as raw medicine, using the leaching method in embodiment 1
The native copper leachate of preparation, and the α-MEM culture medium for being 1:100 with volume ratio is diluted.
0K+A.f+A-pyrite (1:10000) group is to calcine native copper as raw medicine, using the leaching side in embodiment 1
The native copper leachate of method preparation, and the α-MEM culture medium for being 1:10000 with volume ratio is diluted.
Positive controls (Estradiol): 10 configured with α-MEM culture medium-8The estradiol of mol/l.
Negative control group (Control): α-MEM culture medium.
3.2ALP Activity determination
The mesenchymal stem cell for reaching for 3 generations is inoculated into 96 orifice plates, cell concentration 4000cell/mL, to bone
When bone marrow-drived mesenchymal stem growth reaches 70-80% fusion, with above-mentioned grouping test sample respectively to mesenchymal stem cell
It carries out into ossification Fiber differentiation and (also contains 50mg/L phosphorylating ascorbic acid, 10mmol/L sodium β-glycerophosphate and 1 in culture solution
×10-8Dexamethasone), with ALP Activity Assay Kit, (purchase builds up biological work in Nanjing when culture is to the 3rd, 6,9,12d
Cheng company) measurement ALP activity.
3.3ALP histochemical stain
The mesenchymal stem cell for reaching for 3 generations is inoculated in 60mm Tissue Culture Dish, with above-mentioned grouping test sample
Mesenchymal stem cell is carried out at ossification Fiber differentiation respectively.Culture solution is discarded when culture is to 12d, it is solid with formalin
After determining 30s, fixer is discarded, after PBS gently rinses 3 times, addition ALP histochemical stain liquid, static 30min of room temperature or so,
Start purple dot occur, when microscopically observation purple dot no longer increases, stops dyeing, discard dye liquor, gently rinse.It is aobvious
Film recording under micro mirror using the full ware of camera as a result, taken pictures simultaneously.Data result analysis: Ipp (Image-Pro is used after photograph
Plus 6.0) gray analysis software scans ALP pigmented section area, using formula: culture dish area × (ALP pigmented section is swept
Retouch the scan area of ware in area/photo) conversion ALP stained area.
3.4 result
Native copper Thiobacillus ferrooxidans BY3 leachate handles mesenchymal stem cell to 12d
Shi Jinhang ALP activity analysis and ALP histochemical stain, the result is shown in Figure 1: 0K+A.f+A-pyrite and 0K+A.f+N-pyrite
Group can significantly improve the ALP activity of mesenchymal stem cell, increase ALP histochemical stain quantity and area (P < 0.05
Or P < 0.01), it is identical as the effect of positive controls estradiol.Wherein Figure 1A is ALP histochemical stain figure, Figure 1B ALP
Activity determination result figure, Fig. 1 C and Fig. 1 D are ALP histochemical stain area result figure after the analysis of IPP.6.0 software.
The dyeing of 4 calcium scoring of embodiment
Calcium scoring dyes a kind of method identified as in vitro culture bone cells mineralising, and the present embodiment passes through calcification knot
The method for saving dyeing, evaluation nature copper biochemical lixivium is to mesenchymal stem cell at the influence of ossification differentiation.
4.1 method
3rd generation mesenchymal stem cell is inoculated into the culture dish of 60mm and continues to cultivate to when 70%~80% fusion
Random grouping, group carry out into ossification to mesenchymal stem cell respectively with embodiment 3, with the test sample in different grouping
Fiber differentiation.Microscopically observation cell when culture is to 12~14d or so, cell secretes a large amount of calcium salts and is simultaneously deposited on cell at this time
Outside, it is dyed with alizarin red calcium scoring, abandons culture solution, PBS is rinsed 3 times, and the fixed 10min of 10% formaldehyde, PBS rinsing 3 times is added
Alizarin red dye liquor, 37 DEG C of water-bath 30min, flowing water, which gently rinses, removes loose colour, microscopically observation identification, while camera is taken pictures note
As a result, area statistics use IPP 6.0, calcium scoring area is calculated in scan area for record.
4.2 result
This experiment calcium scoring mineralising coloration result is shown in Fig. 2: 0K+A.f+A-pyrite and 0K+A.f+N-pyrite group energy
The area (P < 0.05 or P < 0.01) of calcium scoring is enough significantly improved, it is identical as the effect of positive controls estradiol.Wherein scheme
2A is calcium scoring colored graph；Fig. 2 B is the calcium scoring area quantization figure obtained with IPP.6.0 scanning analysis；Fig. 2 C is feminine gender
Qualification result figure under control group and 0K+A.f+A-pyrite group mesenchymal stem cell calcium scoring dyeing microscope.
The present invention has studied native copper Thiobacillus ferrooxidans using estradiol as positive control medicine
BY3 leachate is in promotion mesenchymal stem cell at the effect in ossification atomization, the results showed that native copper
Thiobacillus ferrooxidans BY3 leachate can be improved the work of the ALP of Marrow Mesenchymal Stem Cells In Vitro
Property, promote the calcium deposition of mesenchymal stem cell, and then promote breaking up at ossification for mesenchymal stem cell.
Claims (2)
1. native copper Thiobacillus ferrooxidans BY3 leachate promotes mesenchymal stem cell skeletonization in preparation
Change the application in differentiation medicament.
2. native copper Thiobacillus ferrooxidans BY3 leachate as described in claim 1, which is characterized in that
The leaching method of the native copper Thiobacillus ferrooxidans BY3 leachate the following steps are included:
(1) native copper is weighed, is placed in container, 200 times of 0K fluid nutrient medium is added into container, and extremely with sulfuric acid tune pH
1.60；
(2) the Thiobacillus ferrooxidans BY3 that inoculation volume fraction is 10% is leached in 25 DEG C, 130r/min
15 days；
(3) it after leaching, stands, collects supernatant, adjust pH value of solution to neutrality, native copper is obtained after degerming
Thiobacillusferrooxidans BY3 leachate.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101264103A (en) * | 2008-04-14 | 2008-09-17 | 李红玉 | Application of native copper native copper microbial extracting liquid in preparing medicaments for treating bone fracture |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101264103A (en) * | 2008-04-14 | 2008-09-17 | 李红玉 | Application of native copper native copper microbial extracting liquid in preparing medicaments for treating bone fracture |
Non-Patent Citations (2)
| Title |
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Application publication date: 20190412 |