CN106421758A - Stem cell preparation as well as preparation method and application thereof - Google Patents
Stem cell preparation as well as preparation method and application thereof Download PDFInfo
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- CN106421758A CN106421758A CN201610878195.9A CN201610878195A CN106421758A CN 106421758 A CN106421758 A CN 106421758A CN 201610878195 A CN201610878195 A CN 201610878195A CN 106421758 A CN106421758 A CN 106421758A
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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
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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
- A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
Abstract
The invention relates to the field of biotherapy, and discloses a stem cell preparation as well as a preparation method and an application thereof. The stem cell preparation has a good curative effect on diabetic foot. The stem cell preparation disclosed by the invention comprises adipose-derived stem cells, fibroblast and fibronectin. The preparation method of the stem cell preparation disclosed by the invention comprises a step of mixing the adipose-derived stem cells, the fibroblast and the fibronectin with a solvent, so that the stem cell preparation is prepared. The invention also discloses the application of the stem cell preparation or the stem cell preparation prepared by virtue of the preparation method in preparing medicines for treating the diabetic foot.
Description
Technical field
The present invention relates to biological therapy field, particularly to a kind of stem cell medicine and its preparation method and application.
Background technology
Diabetes (diabetes mellitus, the DM) incidence of disease worldwide rises, it has also become continue angiocardiopathy
And the 3rd big NCD after tumour.Diabetes are a kind of serious complication of diabetes, are diabetic's causes
One of residual or even lethal major reason.Diabetes refer to that diabetic foot makes lower limb protect work(due to DPN
Can go down, big blood vessel and microangiopathies make the not enough disease shape causing microcirculation disorder and ulcer and gangrene occurring of arterial perfusion
State.1972, Catterall first proposed the three big factors that diabetes (diabeticfoot, DF) are fallen ill：Vascular occlusion
Property ischemic, infection and DPN.Wherein, vascular lesion is a key factor.It is not only and leads to DM patients feet to feel
Dye Etiological, and be also impact diabetic foot ulcers (diabetic footulcer, DFU) prognosis most important because
Element.
The result of study of Loomans etc. shows, diabetic in the state of metabolic disorder, endothelial progenitor cell
The quantity of (endothelial progenitor cell, EPC) reduces and functional obstacle, affects its propagation, sticks and blood vessel
Generate.The hyperinsulinemia of diabetes result in the propagation of endothelial progenitor cell and the attenuating of survival ability, therefore, in sugar
Under some diseases states such as urine disease, the quantity of endothelial progenitor cell and function two aspect all decrease.Blood vessel endothelium ancestral is thin
The functional status of born of the same parents leads to revascularization reduced capability extremely, and this possibly causes the basis of diabetic angiopathy, simultaneously
It is one of the reason such disease prognosis are poor, treatment is difficult.
Further, since diabetic's body is continuously in the nonenzymatic glycosylation state of hyperglycaemia and protein, cause fat generation
Thank to disorder.Blood is made to be continuously in highly viscous, hypercoagulative state, therefore patient's artery of lower extremity is easier to vessel wall thickening, pipe
Chamber is narrow to wait vascular lesion, and then leads to different degrees of microcirculation disorder, and lower limb blood supply gradually decreases, finally generation group
Knit damage, thus leading to the complication such as diabetes to produce.
DF is insensitive to various treatment methods at present, and therapeutic effect is not good, prognosis mala, and pathology deterioration is easily caused amputation.
The traditional therapy of DF has drug therapy, vascular bypass, partial smearing Chinese medicine and PCI, but operation and PCI
The selection of patient is required higher, have strict indication, lead to many self-conditions not good enough or to be associated with important organ seriously sick
Become and be not resistant to bypass surgery and the middle-older patient of PCI can only select drug therapy, and these treatment methods is remote
Phase effect is all undesirable and can not tackle the problem at its root, and a lot of patients finally cannot avoid the misfortune of amputation.Therefore, various countries
Researcher is constantly exploring and is searching out the therapeutic scheme of more preferable non-drug, to improve the treatment of patient with diabetic feet with this
Effect.
Stem cell is the multipotential cell that a class has self-replacation function power, under certain condition, can be divided into many
Plant the cell of function.In recent years, stem cells technology develops rapidly, its basic research in treating diabetes and clinical practice
Carried out rapidly, stem-cell research brings new hope to the treatment of diabetes.
Fat stem cell is a kind of stem cell of Multidirectional Differentiation, and find it can be divided into fat, bone, cartilage, muscle,
The cell set type such as blood vessel endothelium, liver, pancreas, nerve, has the features such as easily obtain, easily expand, be difficult aging.In recent years, in a large number
Research show that fat stem cell can produce and secrete the cell factor (as VEGF, HGF, bFGF etc.) of multiple vascularization promotings,
And take part in the vascularization process that tissue damage is repaired, and there is Hematopoiesis Support affect, Anti-G value, chemotaxis etc..
Fibroblast is the host cell composition in dermis of skin, it with itself secretion collagenous fibres, elastomer
And matrix components have together constituted with the main body of corium.The major function of fibroblast is：Synthesis and secretion collagenous fibres, elasticity
Fiber, Matrix protein material and some growth factors, have important function to the elasticity and toughness maintaining skin, and fiber is female thin
The minimizing of born of the same parents is also the major reason causing wrinkle to produce.
Fibronectin is also referred to as fibronectin (Fibronectin, FN), a kind of macromolecule glycoprotein, has multiple lifes
Thing function.Result of study both domestic and external proves in a large number, and conservative is very strong during evolution for FN molecule, in various animal body fluids
FN there is very close structure, property and biological function, thus the FN of separate sources can be substituted for each other use.FN master
Function is wanted to be mediated cell adhesion, the fibronectin of purifying can strengthen the adhesion of cell adhesion and cell and matrix.By viscous
, fibronectin can adjust the shape of cell and the tissue of cytoskeleton by cellular signal transduction pathways, promote cell paving
Exhibition.In embryogenesis, fibronectin is necessary for the migration of many cell types and differentiation.In wound repair
In, fibronectin is also important.In blood clot forming process, fibronectin promotes blood platelet to be attached to vascular injury portion
Position.
It is undesirable, because of its disease that the treatment of diabetes presently mainly controls blood sugar to improve the effect of conservative treatments such as microcirculation
Change feature is that distal vessels efferent tract is poor, even if therefore putting up a bridge, intervening all difficult leading to.Therefore, research and development a kind of diabetes are had good
The stem cell medicine of good curative effect is those skilled in the art's technical issues that need to address.
Content of the invention
In view of this, the invention discloses a kind of stem cell medicine and its preparation method and application, described stem cell medicine
There is good curative effect to diabetes.
The invention discloses a kind of stem cell medicine, including：Fat stem cell, fibroblast and fibronectin；
The content of described fat stem cell is 4 × 106～1 × 108Individual/ml；
The content of described fibroblast is 2 × 106～5 × 107Individual/ml；
The content of described fibronectin is 0.4～1.5 μ g/ml.
Preferably, described stem cell medicine includes：Fat stem cell, fibroblast and fibronectin；
The content of described fat stem cell is 8 × 106Individual/ml；
The content of described fibroblast is 5 × 106Individual/ml；
The content of described fibronectin is 0.5 μ g/ml.
The invention discloses the preparation method of above-mentioned stem cell medicine, comprise the following steps：
Described fat stem cell, described fibroblast, described fibronectin and solvent are mixed, described stem cell is obtained
Preparation.
Preferably, described solvent is physiological saline.
Preferably, the preparation method of described fat stem cell comprises the following steps：
A1), aseptically extract adipose tissue；
A2), aseptically clean adipose tissue with cleaning fluid, with eye scissors, adipose tissue is cut into 0.2-
0.3cm3Fritter, then cleaned with D-Hank ' s liquid；
A3), use NTx enzymic digestion fat lump 20min, adipocyte is collected by centrifugation；
A4), with 5000/cm2Density by step a3) adipocyte collected carries out inoculated and cultured, after culture three days
Again with 5000/cm after cell is digested with 0.25% pancreatin2Density continue inoculated and cultured；
A5), collect culture to the fat stem cell of the third generation, the outstanding of described fat stem cell is obtained with physiological saline is resuspended
Liquid.
Preferably, step 1) adipose tissue that extracts is adipose tissue under skin of abdomen.Subcutaneous abdomen position is selected to enter
The extraction of row adipose tissue was both conveniently drawn materials, and did not affect the life and health of individuality.The position extracting adipose tissue is not limited to
Subcutaneous tissue of abdomen, also may be selected other position and carries out adipose tissue extraction.
Preferably, step 2) described in cleaning fluid be D-Hank ' s liquid.
Preferably, the preparation method of described fibroblast comprises the following steps：
B1), aseptically take skin histology block；
B2), it is cut into small pieces with eye scissors after cleaning skin histology block, then be carried out；
B3), with NTx enzyme to step b2) the skin histology block that obtains carries out low-temperature digestion overnight, and transfer in second day is put
37 DEG C complete to digesting；
B4), add nutrient solution, after centrifugation, remove supernatant, add after nutrient solution mixes and cultivated, change a not good liquor within every 3 days；
Pass the second generation when first generation cell reaches 80% degree of converging；Pass the 3rd when second generation cell reaches 80% degree of converging
Generation；When third generation cell reaches 85% degree of converging, collect the cell of culture, described fiber mother is obtained with physiological saline is resuspended
The suspension of cell.
Preferably, step b3) described in low temperature be 4 DEG C.
The invention also discloses the stem cell medicine that described stem cell medicine and described preparation method are obtained is in treatment glycosuria
Application in foot disease.
In stem cell medicine disclosed by the invention, fibroblast can promote the healing of wound, and fat stem cell is not only
Repair function can be played at downright bad end, and the microenvironment of patient's body is had some improvement.Fibronectin can strengthen
Cell adhesion and cellular matrix adhesion, promote blood platelet to be attached to vascular injury position, reach the effect of reparation.
The present invention establishes the rat animal model of diabetes, public to stem cell medicine disclosed by the invention and the present invention
The stem cell medicine that the preparation method opened is obtained has carried out animal experiment, and the result of animal experiment shows disclosed by the invention doing carefully
The stem cell medicine that born of the same parents' preparation and preparation method disclosed by the invention are obtained has good curative effect to diabetes.
In sum, the stem cell medicine that stem cell medicine disclosed by the invention and preparation method disclosed by the invention are obtained
Have the advantages that：Stem cell medicine disclosed by the invention has good curative effect, energy to diabetes to diabetes
Enough mitigate the misery of patient, meet the patient of each progress extent of disease, be that the treatment of diabetes is laid a good foundation；And this
Bright disclosed preparation method is drawn materials easily, using popularization and application.
Specific embodiment
The invention discloses a kind of stem cell medicine and its preparation method and application, this stem cell medicine is to diabetes tool
There is good curative effect.Those skilled in the art can use for reference present disclosure, be suitably modified technological parameter and realize.Specifically
, all similar replacements and change apparent to those skilled in the art, they be considered as including
The present invention.The method of the present invention and application be described by preferred embodiment, related personnel substantially can without departing from
In present invention, spirit and scope, method described herein and application are modified or suitably change and combine, to realize
With application the technology of the present invention.
Composition of the present invention and preparation are commercially available or self-control source.
Fibronectin is purchased from Shanghai Jiang Lai bio tech ltd
With reference to embodiment, the present invention is expanded on further.
The preparation of embodiment 1 fat stem cell
1st, aseptically, take the 3mL adipose tissue under autologous rat belly healthy skin, be placed in 5mL centrifuge tube；
2nd, D-Hank ' 3 times adipose tissues of s liquid eccentric cleaning are aseptically used, centrifugal condition is 4 DEG C, 200g, centrifugation
Time is 10min；With eye scissors, adipose tissue is cut into 0.2-0.3cm after being centrifuged3Fritter, more clear with D-Hank ' s liquid
Wash 2 times；
3rd, NTx enzymic digestion fat lump 20min, 400g centrifugation 10min is used to collect fat stem cell；
4th, with 5000/cm2Density by the fat stem cell being collected by centrifugation be seeded to six orifice plates culture, often cultivate three days
Cell is digested again with 5000/cm with 0.25% pancreatin2Density inoculated and cultured；
5th, with 0.25% collected by trypsinisation the 3rd fat subsitutes stem cell, with physiological saline resuspended one-tenth 1.5mL cell suspension,
It is placed in 4 DEG C of preservations stand-by.
The preparation of embodiment 2 fibroblast
1st, aseptically anesthetized rat, the position depilatory cream selecting rat flatter removes fine hair, with the hand that sterilizes
1 × 1cm cut by art scissors2Autologous skin tissue block, is placed in 50mL centrifuge tube；
2nd, use D-Hank ' 2 times skin histology blocks of s cleaning fluid eccentric cleaning, be cut into 1-2mm with eye scissors2Fritter, then
Plus cleaning fluid cleans 3 times；
3rd, put 4 DEG C of digestion overnight with NTx enzyme, turn within second day and be displaced to 37 DEG C of digestion completely；
4th, add culture medium (DMEM+10%FBS) after digestion finishes, abandon supernatant after centrifugation, add culture medium, after mixing
Transfer to six orifice plates cultures, every 3d changes a not good liquor, and Real Time Observation cell state making a record；
5th, passed on when primary cell reaches 70% degree of converging；Pass when first generation cell reaches 80% degree of converging
The second generation；Pass the third generation when second generation cell reaches 80% degree of converging；When third generation cell reaches 85% degree of converging,
Use 0.25% trypsin digestion cell, 400g is centrifuged 10min；Abandon supernatant after centrifugation, add physiological saline re-suspended cell, 400g again
Centrifugation 10min；Then abandon supernatant again and add physiological saline re-suspended cell, 400g is centrifuged 10min, abandons supernatant；By collect 5
×106Individual cell is resuspended with 1.5mL physiological saline, is placed in 4 DEG C of preservations stand-by.
Embodiment 3 prepares stem cell medicine
The amount that fibronectin is 0.5 μ g/mL with concentration adds in 1.0mL physiological saline, makes Fibronectin solution.Past
Add the fat stem cell preparing and the fibroblast preparing in the Fibronectin solution preparing, make fat stem cell
Content be 8 × 106Individual/ml, the content making fibroblast is 5 × 106Individual/ml.Mix, be transferred in 5mL syringe and treat
With prepared stem cell medicine.
Embodiment 4 prepares stem cell medicine
The amount that fibronectin is 0.4 μ g/mL with concentration adds in 1.0mL physiological saline, makes Fibronectin solution.Past
Add the fat stem cell preparing and the fibroblast preparing in the Fibronectin solution preparing, make fat stem cell
Content be 4 × 106Individual/ml, the content making fibroblast is 5 × 107Individual/ml.Mix, be transferred in 5mL syringe and treat
With prepared stem cell medicine.
Embodiment 5 prepares stem cell medicine
The amount that fibronectin is 1.5 μ g/mL with concentration adds in 1.0mL physiological saline, makes Fibronectin solution.Past
Add the fat stem cell preparing and the fibroblast preparing in the Fibronectin solution preparing, make fat stem cell
Content be 1 × 108Individual/ml, the content making fibroblast is 2 × 106Individual/ml.Mix, be transferred in 5mL syringe and treat
With prepared stem cell medicine.
Embodiment 6 sets up animal model
1st, set up diabetes model
Take healthy female male Wistar rat 30.It is randomly divided into control group (n=5) and model group (n=25).High sugar is high
After fat diet 4 weeks, fasting 5 days (can't help water), weigh in.Model group presses the freshly prepared chain of 50mg/kg lumbar injection
Urea helps rhzomorph, and control group gives the citric acid-sodium citrate buffer solution of lumbar injection and model group Isodose.Survey morning after 5 days
Play fasting blood-glucose (fastingplasmaglucose, FPG), with 14.0mmol/L for becoming mould standard.And to inject for the first time be
Starting point, fasting blood-glucose from the 5th, 7,10,15 and 25 days measurement mornings.Blood sugar person not up to standard inject again according to blood sugar level
Streptozotocin (25mg/kg or 20mg/kg).
2nd, set up diabetes model
1) diabetes are become mould rat to carry out abdomen with 10% chloraldurate with the concentration of 0.03mL/kg together with control rats
Chamber is anaesthetized；
2) cut a stringer otch at the left side ligamentum inguinale of rat, separate sarcolemma layer and muscle layer；Amplify in operation
Femoral artery, vein and its branch, ligation detachment is exposed under mirror；
3) layer-by-layer suture hypodermis and skin, puts back to mouse cage after observation rat items vital sign is steady；Postoperative all
Rat gives penicillin 30,000 units/sky, continues 3 days to prevent to infect.
3rd, diabetes and diabetes model index determining
1) blood sugar injects Streptozotocin as starting point with first time, the 5th day, the 7th day, the 14th day and the 28th day after injection
With cut tail method measurement morning from fasting blood-glucose；
2) body weight, amount of drinking water, urine volume, body weight is the mensure of single sample body weight；Wherein, amount of drinking water is that each drinking bottle is total
Body amount of drinking water, urine volume is to visually observe bigness scale；
3) diabetic foot ulcer or gangrene are visually observed, respectively the general work of the ischemic limb of observing and nursing group and control group
Emotionally condition, skin color, ulcer or gangrenous time of occurrence and its accumulative position.
4th, result
1) blood sugar level：After 5 days, blood sugar all has different degrees of liter to 25 rat injection Streptozotocins of model group
Height, the wherein FPG of 23 rats>15.7mmol/l, and have many drink, diuresis, the symptom such as eat more, become thin.The blood sugar of control group exists
It is 4.53 ± 0.47mmol/L before injection Streptozotocin, injection Streptozotocin is 4.6 ± 0.32mmol/L after 5 days, no bright
Significant difference is different；Model group blood sugar is increased to 17.34+4.72mmol/ by 4.47+0.27mmol/L after injecting Streptozotocin 5 days
L, and control group same time point front with injection is compared variant substantially (P<0.05).
2) body weight：, after 7 days, the body weight of control rats is by 214.5 ± 14.21 increases before injecting for injection Streptozotocin
To 215.5 ± 15.46, no significant difference (P>0.05)；Injection Streptozotocin after 7 days, the body weight of model group rats is by injecting
Front 215.24 ± 16.03 drop to 148.47 ± 10.05, inject before Streptozotocin with test group and same time experimental tests
The body weight of group rat has compared notable difference (P<0.05).
3) after carrying out femoral artery ligation, rats eating amount significantly reduces, left hind limitation of activity.After 5 days, mental status have
Improved, but the energy of ischemic hindlimb is decreased obviously.Manufacture about 16cm with eye scissors in the lower side skin of Ligation of artery respectively2
The surface of a wound, diabetes model set up complete.
The therapeutic effect checking of embodiment 7 stem cell medicine
Choose FPG in embodiment 6 model group>In 23 rats of 15.7mmol/L 20 are only used as subjects, wherein
10 is blank control group, and 10 is therapeutic test group.The rat of therapeutic test group uses the stem cell medicine of embodiment 3 preparation
Injected, blank control group is injected using the physiological saline of equivalent.Aseptically, disinfect the skin around the surface of a wound
Skin, carries out the dry thin of uniform point-like intramuscular injection embodiment 3 preparation respectively around the surface of a wound is subcutaneous to therapeutic test group and blank control group
Born of the same parents preparation 4mL and physiological saline 4mL.Complete intramuscular injection in 1h, 10 days wound healing situations are observed in continuous timing, and carry out the surface of a wound
Record.
As shown in table 1, the surface of a wound average area of therapeutic test group rat constantly reducing result of the test treat for 10 days, has
The trend of recovery from illness.The surface of a wound average area of blank control group rat does not only reduce, and the area that festers on the contrary increases continuous.Say
The stem cell medicine of bright embodiment 3 preparation has good curative effect to treatment diabetes.Stem cell with embodiment 4 and 5 preparation
Preparation repeats above-mentioned test, obtains similar conclusion.
The surface of a wound average area change of table 1 each group rat
The above is only the preferred embodiment of the present invention it is noted that ordinary skill people for the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of stem cell medicine is it is characterised in that include：Fat stem cell, fibroblast and fibronectin；
The content of described fat stem cell is 4 × 106～1 × 108Individual/ml；
The content of described fibroblast is 2 × 106～5 × 107Individual/ml；
The content of described fibronectin is 0.4～1.5 μ g/ml.
2. stem cell medicine according to claim 1 is it is characterised in that include：Fat stem cell, fibroblast and fibre
Even albumen；
The content of described fat stem cell is 8 × 106Individual/ml；
The content of described fibroblast is 5 × 106Individual/ml；
The content of described fibronectin is 0.5 μ g/ml.
3. the preparation method of the stem cell medicine described in claim 1 or 2 is it is characterised in that comprise the following steps：
Described fat stem cell, described fibroblast, described fibronectin and solvent are mixed, described stem cell system is obtained
Agent.
4. preparation method according to claim 3 is it is characterised in that described solvent is physiological saline.
5. preparation method according to claim 3 it is characterised in that the preparation method of described fat stem cell include following
Step：
A1), extract adipose tissue；
A2), clean described adipose tissue with cleaning fluid, with eye scissors, described adipose tissue is cut into 0.2-0.3cm3Fat
Block, then cleaned with cleaning fluid；
A3), use NTx enzymic digestion fat lump 20min, fat stem cell is collected by centrifugation；
A4), with 5000/cm2Density by step a3) fat stem cell collected carries out inoculated and cultured, culture will be thin after three days
Born of the same parents digested with 0.25% pancreatin after again with 5000/cm2Density continue inoculated and cultured；
A5), collect culture to the fat stem cell of the third generation, with the resuspended suspension that described fat stem cell is obtained of physiological saline.
6. preparation method according to claim 5 is it is characterised in that step 1) adipose tissue that extracts is under skin of abdomen
Adipose tissue.
7. preparation method according to claim 5 is it is characterised in that step 2) described in cleaning fluid be D-Hank ' s liquid.
8. preparation method according to claim 3 it is characterised in that the preparation method of described fibroblast include following
Step：
B1), aseptically take skin histology block；
B2), it is cut into small pieces with eye scissors after cleaning skin histology block, then be carried out；
B3), with NTx enzyme to step b2) the skin histology block that obtains carries out low-temperature digestion overnight, and turn within second day and be displaced to 37
DEG C to digesting completely；
B4), add nutrient solution, after centrifugation, remove supernatant, add after nutrient solution mixes and cultivated, change a not good liquor within every 3 days；When
Generation cell reaches biography second generation during 80% degree of converging；Pass the third generation when second generation cell reaches 80% degree of converging；When
When third generation cell reaches 85% degree of converging, collect the cell of culture, described fibroblast is obtained with physiological saline is resuspended
Suspension.
9. preparation method according to claim 6 is it is characterised in that step b3) described in low temperature be 4 DEG C.
10. stem cell medicine described in claim 1 or 2 and prepared the doing carefully of the preparation method described in any one of claim 3 to 7
Application in the biologic product of preparation treatment diabetes for born of the same parents' preparation.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109939224A (en) * | 2019-04-24 | 2019-06-28 | 四川驰鼎盛通生物科技有限公司 | A kind of biological agent and preparation method thereof for repairing skin injury |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109939224A (en) * | 2019-04-24 | 2019-06-28 | 四川驰鼎盛通生物科技有限公司 | A kind of biological agent and preparation method thereof for repairing skin injury |
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Application publication date: 20170222 |