CN107496455A - A kind of injection for treating degenerative osteoarthropathy and preparation method thereof - Google Patents
A kind of injection for treating degenerative osteoarthropathy and preparation method thereof Download PDFInfo
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Abstract
A kind of injection for treating degenerative osteoarthropathy and preparation method thereof, the injection is mainly made up of umbilical cord mesenchymal stem cells, the content of cell is 40,000,000 in every 100 milliliters of injections, and the treatment of injection prepared by the present invention for osteoarthropathy takes effect immediately, curative effect is lasting, patient satisfaction is higher.
Description
Technical field
The present invention relates to a kind of method for the injection and its preparation for treating degenerative osteoarthropathy.
Background technology
Degenerative osteoarthropathy, i.e. retrogression arthritis, hypertrophic arthritis, senescent arthritis etc., be it is a kind of by
In Articular cartilage degeneration, cause articular cartilage integrity violations and joint margins subcartilaginous osseous lamella lesion, then cause to close
Save one group of chronic degenerative joint disease of sings and symptoms.It is soft including spur, osteoproliferation, bowlegs, cervical spondylopathy, kneecap
Change, lumbar vertebra disease etc..Counted according to WHO, in the crowd of more than 50 years old, the incidence of disease of osteoarthritis is 50%, in more than 55 years old crowd
The incidence of disease is 80%.The incidence of disease is up to 60%-70% in China over-65s crowd.Predicted according to WHO, to Chinese bone in 2015
Patient is up to 1.5 hundred million.Osteoarthritis turns into the first big disease for causing the mankind disabled, is referred to as " not dead cancer ".Move back
Row osteoarthropathy can be divided into primary and the class of Secondary cases two：Primary degenerative osteoarthropathy is occurred in person in middle and old age, without bright
True whole body or local inducement, there is certain relation with heredity and physical factors, Secondary cases degenerative osteoarthropathy can betide
Person between twenty and fifty, secondary to wound, inflammation, joint instability, chronic accumulation strain or congenital disorders repeatedly etc..Cardinal symptom is
Arthralgia and tenderness, weight-bearing joints and both hands are most easily involved.Treatment mainly includes physical treatment at present：Massage, massage, pin
Moxibustion, infrared ray, electro-ionic osmosis etc.；Operative treatment：Suitable for joint severe deformities person；Symptomatic treatment：Non-steroidal drug；Ammonia sugar is treated
Method：Damaged cartilage is repaired, mitigates pain etc..Above treatment method curative effect is undesirable, and some therapy side effects are very big.
Mescenchymal stem cell (mesenchymal stem cells, MSCs) is derived from mesoderm growing early stage mesoderm and outer embryo
A kind of multipotential stem cell of layer, initially finds in marrow, is received much concern because it has the characteristics that：(1) Multidirectional Differentiation,
Induction can be divided into fat, bone, cartilage, tendon, muscle, ligament, nerve, liver, cardiac muscle, endothelium under body inside/outside specified conditions
Deng Various Tissues cell, can still have Multidirectional Differentiation ability after continuous passage and freezing；(2) immunoregulation, MSCs expression MHC-
I quasi-molecules, the costimulatory molecules such as CD80, CD86, HLA-DR are not expressed, mixed lymphocyte reaction (MLP) can be suppressed in vitro, in vivo
Inducing immune tolerance, available for the graft versus host disease(GVH disease) after the rejection and HSCT after organ transplant；(3)
Self-replacation, MSCs can carry out self-replacation as stem cell, amplification in vitro, may be used as the stem cell platform of gene therapy.
MSCs multi-sources are in Adult Human Bone Marrow at present, but the MSCs cell quantities of adult origin, differentiation capability can be with the ages
Growth and decline, and have many non-controllable risks, and clinical materials are complicated, damaging big, research is found, MSCs is in human fetal
It is widely present with more tissues after life, but umbilical cord source is most extensive, convenient material drawing, it is dry thin without influence, human umbilical cord mesenchymal on donor
Born of the same parents are the preferable seed cells of organizational project.Because, except having convenient material drawing, source is wide for it, quantity is sufficient, and multiplication capacity is strong, long
Phase passage does not change, and secretion has the characteristics that the extracellular matrix being suitably constructed.In addition have further the advantage that：1st, umbilical cord is gathered
There is no any harm and damage to puerpera and neonate；2nd, ancestral cells are more original, there is stronger Proliferation, Differentiation ability；3rd, exempt from
Epidemic disease cell is more inmature, and functional activity is low, and immunologic function is not mature enough, is kept silent state in one kind, will not trigger immune response
And cause graft versus host disease(GVH disease)；4th, tumour cell is not had, the infection of virus and pathogenic microorganism and propagation probability are also relative
It is relatively low；5th, it is not related to society, ethics and legal more disputes.In recent years, domestic and foreign scholars are done to human umbilical cord mesenchymal
Cell is studied and achieves certain achievement.Umbilical cord mesenchymal stem cells are expected to substitute mesenchymal stem cells MSCs to turn into tissue
The more preferable research object of engineering.It is enough have differentiation potential cell be organizational project prerequisite, with novel biomaterial
With the appearance and development of bioreactor, a large amount of amplifications of seed cell in vitro are possibly realized.This can fundamentally be solved
The few problem of seed cell quantity in organizational project.
The content of the invention
It is a primary object of the present invention to provide a kind of injection for treating degenerative osteoarthropathy and preparation method thereof, with
Overcome the shortcomings of existing treatment method, taken effect immediately it is an object of the invention to provide one kind, curative effect persistently, patient satisfaction more
The injection of high treatment degenerative osteoarthropathy.It is a further object to provide the preparation method of this injection.
To achieve the above object, the present invention uses following technical scheme：
A kind of preparation method for the injection for treating degenerative osteoarthropathy, it is characterised in that this method includes following step
Suddenly：
Step 1, the preparation of injection human umbilical cord mesenchymal stem cells
A, full-term pregnancy is taken, the neonatal umbilical cord of the healthy puerpera of free from infection, clean with PBS by it on inspection,
Shred, add collagenase digesting, the addition of the clostridiopetidase A is 1-4g/L, and digestion temperature is 37 DEG C, and digestion time is 30 points
Clock -90 minutes, it is therefore preferable to 60 minutes；
B, add 10-30g/L pancreatin to re-digest, digestion temperature is 37 DEG C, and digestion time is -90 minutes 30 minutes, excellent
Selection of land is 30 minutes；
C, after not digesting umbilical cord tissue with 200 eye mesh screen filtration step B, add PBS and neutralize, digestive juice is 1 with PBS ratios：
10-1：15, it is therefore preferable to 1：12；
D, by the liquid in step C after neutralization carry out centrifugation obtain cell, centrifugal rotational speed be 1000 revs/min extremely
3000 revs/min, centrifugation time is 10 minutes to 20 minutes；The cell obtained by centrifugation is inoculated in culture dish and carries out cell
Culture, full dose changes liquid after 4 days, discards non-attached cell, and half amount changes liquid 2 times weekly later；
Step 2, the preparation of injection
The cultured cell of process obtained by step 1 is collected, treatment degeneration bone is made in addition cell solution
The injection of arthropathy；
2nd, a kind of preparation method for the injection for treating degenerative osteoarthropathy as claimed in claim 1, its feature exist
In the injection mescenchymal stem cell content be 40,000,000.
3rd, a kind of preparation method for the injection for treating degenerative osteoarthropathy as claimed in claim 1, its feature exist
The size shredded described in step 1 A is that tissue block size is 0.05mm-2mm, it is therefore preferable to 1.5mm；The clostridiopetidase A
Addition is 2g/L.
4th, a kind of preparation method for the injection for treating degenerative osteoarthropathy as claimed in claim 1, its feature exist
The addition of pancreatin is 25g/L in step 1 B.
5th, a kind of preparation method for the injection for treating degenerative osteoarthropathy as claimed in claim 1, its feature exist
Centrifugal rotational speed is preferably 2000 revs/min in step 1 D, is centrifuged 10 minutes.
6th, a kind of preparation method for the injection for treating degenerative osteoarthropathy as claimed in claim 1, its feature exist
The culture medium of culture dish used is serum free medium in step 1 D, skin growth factor in the serum free medium and into
The content of fibroblast growth factor is respectively 2 nanograms/milliliters to 5 nanograms/milliliters, preferably 4 nanograms/milliliters, in serum free medium
Hydrocortisone content be 0.2 to 2 mcg/ml, preferably 1 mcg/ml, the content of heparin is 5 to 20 micrograms/milli
Rise, preferably 15 mcg/mls.
7th, a kind of preparation method for the injection for treating degenerative osteoarthropathy as claimed in claim 1, its feature exist
Cell solution used is physiological saline, ringer's solution, Multiple electrolytes injection or base fluid in step 2.
8th, a kind of injection for treating degenerative osteoarthropathy, it is characterised in that such as claim is used using the injection
Method described in 1-4 is prepared.
Beneficial effect：
The injection be it is a kind of take effect immediately, curative effect persistently, patient satisfaction it is higher treatment degenerative osteoarthropathy
Injection.
Brief description of the drawings
Fig. 1 is the successful umbilical cord mesenchymal stem cells morphology photo under 100 times of light microscopes of culture；
Fig. 2 is the umbilical cord mesenchymal stem cells injection of the treatment degenerative osteoarthropathy of the specific embodiment of the present invention
The packaging photo of agent.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.
Illustrate the preparation method of the injection for the treatment of degenerative osteoarthropathy provided by the present invention in detail further below,
But the present invention is not therefore subject to any restriction.(1) full-term pregnancy is taken, on inspection the neonate of the healthy puerpera of free from infection
Umbilical cord, it is put into containing in dual anti-tissue preserration liquid, is handled after acquisition in 24 hours；(2) umbilical cord both sides are wiped out to be damaged
The umbilical cord tissue of wound, and umbilical cord periphery and umbilical vein inner chamber are cleaned repeatedly containing dual anti-PBS；(3) by the umbilical cord tissue after processing
Shredded with operating scissors, tissue block size is 0.05mm-2mm, it is therefore preferable to 1.5mm；(4) 2g/L clostridiopetidase As are added to be digested,
It is 37 DEG C to digest temperature, and digestion time is -90 minutes 30 minutes, it is therefore preferable to 60 minutes；(5) digestion of 25g/L pancreatin is added, is disappeared
It is 37 DEG C to change temperature, and digestion time is -90 minutes 30 minutes, it is therefore preferable to 30 minutes；(6) do not digested with 200 mesh sieve net filtrations
After umbilical cord tissue, add PBS and neutralize, digestive juice is 1 with PBS ratios：10-1：15, it is therefore preferable to 1：12；(7) centrifugation obtains thin
Born of the same parents, centrifugal rotational speed are 1000 revs/min to 3000 revs/min, and centrifugation time is 10 minutes to 20 minutes, it is therefore preferable to 2000
Rev/min, centrifuge 10 minutes；(8) it is inoculated in culture dish and carries out cell culture, full dose changes liquid after 4 days, discards non-attached cell,
Half amount changes liquid 2 times weekly later, and used medium is serum free medium, adds epidermal growth factor, fibroblast growth factor,
Content is respectively 2 nanograms/milliliters to 5 nanograms/milliliters, preferably 4 nanograms/milliliters, adds hydrocortisone and heparin, the hydrogenation
Content of the cortisone in nutrient solution can be 0.2 to 2 mcg/ml, preferably 1 mcg/ml；The heparin is in nutrient solution
In content can be 5 to 20 mcg/mls, preferably 15 mcg/mls.Above-mentioned nutrient solution and growth factor and active material
Material can be selected from a company or multiple companies (Cascade companies, Sigma companies, the Hyclne companies in such as U.S.)
Commercially available prod；(9) cultured enough cells are collected, detected, be prepared into injection human mesenchymal stem cell note
Liquid is penetrated, cell solution used is physiological saline, ringer's solution, Multiple electrolytes injection or base fluid, it is therefore preferable to compound electricity
Solve matter parenteral solution.Cell content per 100ml Multiple electrolytes injections is 20,000,000 to 80,000,000, it is therefore preferable to
40000000；(10) being transfused to the umbilical cord mesenchymal stem cells parenteral solution prepared in 8 hours to 24 hours needs to treat
Degenerative osteoarthropathy patient, it is therefore preferable to be transfused in 12 hours；(11) observation and assessment after being treated, bag
Include and carry out curative effect evaluation (with reference to clinic), side reaction observation.
The preparation method for the base fluid that the cell solution is selected comprises the following steps：
(1), Sodium Hyaluronate is dissolved in deionized water to concentration is made is 1-2wt% aqueous solution of sodium hyaluronate, collagen
Albumen is dissolved in deionized water that concentration is made is 3-5wt% collagen aqueous solutions, chitosan be dissolved in deionized water be made it is dense
Spend for 0.1-2wt% chitin sugar aqueous solutions；
(2), cross-linking reaction
By aqueous solution of sodium hyaluronate, collagen aqueous solution, chitin sugar aqueous solution mix, add chitosan, calcium chloride,
Glucosamine Sulphate and PBCA (Sodium Hyaluronate, collagen, chitosan, chitosan, chlorination
The mass ratio of calcium, Glucosamine Sulphate and PBCA is 1-2：3-5：0.1-2：1-3：0.5-1：1-4：
0.5-1), suspension is obtained by ultrasonic disperse 15-40min, 0.01-0.03wt% Geniposides and 0.5- is added after disperseing
1wt% polyvinyl alcohol carries out cross-linking reaction, is again stirring for 3-8h and obtains the solution by crosslinking；
(3), obtaining pH value by the solution addition 0.2-0.8wt% sodium chloride and pH adjusting agent that are crosslinked in step 2 is
5-9 mixed liquors；
(4), the preparation of nano-powder
The mixed liquor that step 3 is obtained is freeze-dried, and the freeze-drying is 0.5~0.1Pa at -60~-70 DEG C
Under the conditions of carry out vacuum freeze drying, obtain particle diameter be 10-1000nm nano particle；
Obtained nano-powder is reached into 5-100nm with the high-pressure homogeneous particle diameter made, and 10-50nm particle accounting is
More than 60%, it is described it is high-pressure homogeneous during homogenization pressure be 100 to 1500bar；Homogenisation cycle during high-pressure homogeneous
For 50-1000 circulation；
(5), the preparation of base fluid
After nano particle, antibacterial activator and the deionized water that step 4 is obtained are sufficiently mixed, carried out using cobalt -60
Irradiation sterilization obtains base fluid；The mass ratio of the nano particle, antibacterial activator and deionized water is 1-5： 0.01-0.1：
100-150；The irradiation sterilization of cobalt -60 intensity is 10~30kGy；Irradiation time is 10~30 minutes.
The relative molecular mass of Sodium Hyaluronate described in step (1) is 1.0 × 106~2.0 × 106Da。
PH adjusting agent described in step (3) is selected from sodium hydroxide, citric acid, sodium dihydrogen phosphate, disodium hydrogen phosphate etc.；Institute
The more excellent pH value of obtained mixed liquor is 6-8, most preferably 6.5-7.0.
The preferred 300-1000bar of homogenization pressure in step (4) mesohigh homogenizing process；Homogeneous during high-pressure homogeneous
Circulation is preferably 100 to 600 circulations.
Antibacterial activator described in step (5) is double (n- to chlorobenzene by bromination dodecyl dimethyl hexadecyldimethyl benzyl ammonium and 1,6-
Biguanides) hexane is using mass ratio as 1-2:0.5 is mixed to get；The irradiation sterilization of cobalt -60 intensity is preferably 20~25kGy；Irradiation
Time is preferably 15~20 minutes.
The injection for the treatment of degenerative osteoarthropathy provided by the present invention is further described with reference to embodiment
And preparation method thereof.
Embodiment 1：
Cell is separately cultured
(1) umbilical cord tissue after processing is shredded with operating scissors, tissue block size is 0.05mm；(2) 2g/L collagens are added
Enzyme is digested, and digestion temperature is 37 DEG C, and digestion time is 30 minutes；(3) digestion of 25g/L pancreatin is added, digestion temperature is 37
DEG C, digestion time is that digestion time is 30 minutes；(4) after not digesting umbilical cord tissue with 200 mesh sieve net filtrations, add PBS and neutralize,
Digestive juice is 1 with PBS：10；(5) centrifugation obtains cell, and centrifugal rotational speed is 1000 revs/min, and centrifugation time is 10 minutes；(6)
It is inoculated in culture dish and carries out cell culture, full dose changes liquid after 4 days, discards non-attached cell, and half amount changes liquid 2 times weekly later, institute
It is serum free medium with culture medium, adds epidermal growth factor, fibroblast growth factor, content is 2 nanograms/milliliters, is added
Hydrocortisone and heparin, content of the hydrocortisone in nutrient solution are 0.2 mcg/ml；Heparin containing in nutrient solution
Measure as 5 mcg/mls；(7) cultured enough cells are collected, detected, it is dry thin to be prepared into injection human mesenchyme
Born of the same parents' parenteral solution, cell solution used are physiological saline.Cell content per 100ml physiological saline is 20,000,000；(8) will prepare
Good umbilical cord mesenchymal stem cells parenteral solution was transfused to the degenerative osteoarthropathy patient for needing to treat in 8 hours；(9) carry out
Observation and assessment after treatment, including curative effect evaluation (with reference to clinic) is carried out, side reaction observation.
Embodiment 2：
(1) umbilical cord tissue after processing is shredded with operating scissors, tissue block size is 1.5mm；(2) 2g/L clostridiopetidase As are added
Digested, digestion temperature is 37 DEG C, and digestion time is 60 minutes；(3) digestion of 25g/L pancreatin is added, digestion temperature is 37
DEG C, digestion time is that digestion time is 30 minutes；(4) after not digesting umbilical cord tissue with 200 mesh sieve net filtrations, add PBS and neutralize,
Digestive juice is 1 with PBS：12；(5) centrifugation obtains cell, and centrifugal rotational speed is 2000 revs/min, and centrifugation time is 15 minutes；(6)
It is inoculated in culture dish and carries out cell culture, full dose changes liquid after 4 days, discards non-attached cell, and half amount changes liquid 2 times weekly later, institute
It is serum free medium with culture medium, adds epidermal growth factor, fibroblast growth factor, content is 4 nanograms/milliliters, is added
Hydrocortisone and heparin, content of the hydrocortisone in nutrient solution are 1 mcg/ml；Content of the heparin in nutrient solution
For 15 mcg/mls；(7) cultured enough cells are collected, detect, be prepared into injection human mesenchymal stem cell
Parenteral solution, cell solution used are ringer's solution.Cell content per 100ml ringer's solutions is 30,000,000；(8) will prepare
Umbilical cord mesenchymal stem cells parenteral solution be transfused in 12 hours and need the degenerative osteoarthropathy patient that treats；(9) carry out
Observation and assessment after treatment, including curative effect evaluation (with reference to clinic) is carried out, side reaction observation.
Embodiment 3：
(1) umbilical cord tissue after processing is shredded with operating scissors, tissue block size is 2mm；(2) 2g/L clostridiopetidase As are added
Digested, digestion temperature is 37 DEG C, and digestion time is 90 minutes；(3) digestion of 25g/L pancreatin is added, digestion temperature is 37 DEG C,
Digestion time is that digestion time is 90 minutes；(4) after not digesting umbilical cord tissue with 200 mesh sieve net filtrations, add PBS and neutralize, disappear
It is 1 to change liquid with PBS：15；(5) centrifugation obtains cell, and centrifugal rotational speed is 3000 revs/min, and centrifugation time is 20 minutes；(6) connect
Kind carries out cell culture in culture dish, and full dose changes liquid after 4 days, discards non-attached cell, and half amount changes liquid 2 times weekly later, used
Culture medium is serum free medium, adds epidermal growth factor, fibroblast growth factor, and content is 5 nanograms/milliliters, adds hydrogen
It is 2 mcg/mls to change cortisone and heparin, content of the hydrocortisone in nutrient solution；The heparin containing in nutrient solution
Measure as 20 mcg/mls；(7) cultured enough cells are collected, detected, it is dry thin to be prepared into injection human mesenchyme
Born of the same parents' parenteral solution, cell solution used are Multiple electrolytes injection.Often the cell content of 100ml Multiple electrolytes injections is
40000000；(8) the umbilical cord mesenchymal stem cells parenteral solution prepared was transfused to the degeneration for needing to treat in 24 hours
Osteoarthropathy patient；(9) observation and assessment after being treated, including curative effect evaluation (with reference to clinic) is carried out, side reaction observation.
Embodiment 4：
(1) umbilical cord tissue after processing is shredded with operating scissors, tissue block size is 2mm；(2) 2g/L clostridiopetidase As are added
Digested, digestion temperature is 37 DEG C, and digestion time is 90 minutes；(3) digestion of 25g/L pancreatin is added, digestion temperature is 37 DEG C,
Digestion time is that digestion time is 90 minutes；(4) after not digesting umbilical cord tissue with 200 mesh sieve net filtrations, add PBS and neutralize, disappear
It is 1 to change liquid with PBS：15；(5) centrifugation obtains cell, and centrifugal rotational speed is 3000 revs/min, and centrifugation time is 20 minutes；(6) connect
Kind carries out cell culture in culture dish, and full dose changes liquid after 4 days, discards non-attached cell, and half amount changes liquid 2 times weekly later, used
Culture medium is serum free medium, adds epidermal growth factor, fibroblast growth factor, and content is 5 nanograms/milliliters, adds hydrogen
It is 2 mcg/mls to change cortisone and heparin, content of the hydrocortisone in nutrient solution；The heparin containing in nutrient solution
Measure as 20 mcg/mls；(7) cultured enough cells are collected, detected, it is dry thin to be prepared into injection human mesenchyme
Born of the same parents' parenteral solution, cell solution used are base fluid.Cell content per 100ml Multiple electrolytes injections is 40,000,000；(8) will
The umbilical cord mesenchymal stem cells parenteral solution prepared was transfused to the degenerative osteoarthropathy patient for needing to treat in 24 hours；
(9) observation and assessment after being treated, including curative effect evaluation (with reference to clinic) is carried out, side reaction observation.
The preparation method of base fluid comprises the following steps：
(1), Sodium Hyaluronate is dissolved in deionized water to concentration is made is 1-2wt% aqueous solution of sodium hyaluronate, collagen
Albumen is dissolved in deionized water that concentration is made is 3-5wt% collagen aqueous solutions, chitosan be dissolved in deionized water be made it is dense
Spend for 0.1-2wt% chitin sugar aqueous solutions；
(2), cross-linking reaction
By aqueous solution of sodium hyaluronate, collagen aqueous solution, chitin sugar aqueous solution mix, add chitosan, calcium chloride,
Glucosamine Sulphate and PBCA (Sodium Hyaluronate, collagen, chitosan, chitosan, chlorination
The mass ratio of calcium, Glucosamine Sulphate and PBCA is 1-2：3-5：0.1-2：1-3：0.5-1：1-4：
0.5-1), suspension is obtained by ultrasonic disperse 15-40min, 0.01-0.03wt% Geniposides and 0.5- is added after disperseing
1wt% polyvinyl alcohol carries out cross-linking reaction, is again stirring for 3-8h and obtains the solution by crosslinking；
(3), obtaining pH value by the solution addition 0.2-0.8wt% sodium chloride and pH adjusting agent that are crosslinked in step 2 is
5-9 mixed liquors；
(4), the preparation of nano-powder
The mixed liquor that step 3 is obtained is freeze-dried, and the freeze-drying is 0.5~0.1Pa at -60~-70 DEG C
Under the conditions of carry out vacuum freeze drying, obtain particle diameter be 10-1000nm nano particle；
Obtained nano-powder is reached into 5-100nm with the high-pressure homogeneous particle diameter made, and 10-50nm particle accounting is
More than 60%, it is described it is high-pressure homogeneous during homogenization pressure be 100 to 1500bar；Homogenisation cycle during high-pressure homogeneous
For 50-1000 circulation；
(5), the preparation of base fluid
After nano particle, antibacterial activator and the deionized water that step 4 is obtained are sufficiently mixed, carried out using cobalt -60
Irradiation sterilization obtains base fluid；The mass ratio of the nano particle, antibacterial activator and deionized water is 1-5： 0.01-0.1：
100-150；The irradiation sterilization of cobalt -60 intensity is 10~30kGy；Irradiation time is 10~30 minutes.
The relative molecular mass of Sodium Hyaluronate described in step (1) is 1.0 × 106~2.0 × 106Da。
PH adjusting agent described in step (3) is selected from sodium hydroxide, citric acid, sodium dihydrogen phosphate, disodium hydrogen phosphate etc.；Institute
The more excellent pH value of obtained mixed liquor is 6-8, most preferably 6.5-7.0.
The preferred 300-1000bar of homogenization pressure in step (4) mesohigh homogenizing process；Homogeneous during high-pressure homogeneous
Circulation is preferably 100 to 600 circulations.
Antibacterial activator described in step (5) is double (n- to chlorobenzene by bromination dodecyl dimethyl hexadecyldimethyl benzyl ammonium and 1,6-
Biguanides) hexane is using mass ratio as 1-2:0.5 is mixed to get；The irradiation sterilization of cobalt -60 intensity is preferably 20~25kGy；Irradiation
Time is preferably 15~20 minutes.
Using homemade base fluid the injection is had the following advantages that：
1st, use chitosan in base fluid, one side chitosan per se with positive electricity, by and nano particle combination, pass through
Both interactions, form smaller and more fine and close structure, are easy to cell endocytic to form phagocytic vacuole, are more favorable for the bone in later stage
The treatment of arthropathy；, the similitude in the structure glycosaminoglycan structure in the structure and articular cartilage of chitosan, is enhanced in addition
Biocompatibility of the system in joint cavity injection；
2nd, chitosan is employed in the present invention, because chitosan has certain viscosity, the nano injection liquid of preparation combines
Umbilical cord mesenchymal stem cells can be enriched with joint injury, and the purpose of enrichment can be effective with Saving cortilage, two for one
Anti-inflammatory agent be wrapped in the inside, there is slow releasing function, promote the rehabilitation of arthropathy.Drug effect is compared to existing treatment of joint disease
Parenteral solution action time is longer, lasting medicine.
3rd, using being employed in homemade base fluid, high-pressure homogeneous progress is further to reduce grain diameter so that in joint
What is be enriched with is even closer；
4th, all raw materials of the embodiment have synergy between each other, and similar former material is learnt by many experiments
Material can not obtain having stronger self-bone grafting energy with identical technique effect of the present invention, the injection prepared by the present invention
Power, the parenteral solution of preparation use suitable for articular cavity, and stability is good, degree of safety is high, and plasticity is strong, good biocompatibility.
Embodiment 5：
The primary cell that morphological observation uses this method to separate with cell growth cycle is how adherent in 24 hours
Growth, after cultivating one week, visible cell is in fusiformis under inverted microscope, polygonal, is the fusiformis that form is homogeneous after two weeks, parallel
Arrangement or swirl shape growth, in the generation of continuous passage 20, cellular morphology is without significant change.Fig. 1 is the cell arrangement photo of passage one week
And cell growth curve, Fig. 2 are that the cell growth in 20 generations of passage arranges photo.Cell density reaches 80%-90% within the 10-15 days,
Cell arrangement has certain directionality, in swirling, radial arrangement, can carry out passage amplification cultivation.Cell is given birth to after passage
Long rapid, growth curve is S-shaped, and the 1-2 days are the latent laundering period after inoculation, bred since the 3rd day cell and into pair
In number growth period, peak within the 5th, 6 day.Cell still has stronger multiplication capacity after multiple passage.
Embodiment 6：
Clinical treatment case Liu, man, 50 years old, bilateral femur is diagnosed as in November, 2014 in General Hospital of Beijing Military Command
Head necrosis, degenerative osteoarthropathy, existing symptom be both legs pain, on foot when pain exacerbation, crutch need to be helped to walk, in 2015 3
Receive the treatment of umbilical cord mesenchymal stem cells injection through General Hospital of Beijing Military Command over month, each vein inputs this cell infusion agent
100ml, it is totally lost in 20 minutes.Feel that arthralgia substantially mitigates within second day after use, be used in conjunction three times, monthly, every time one
Bag (100ml adhesive tapes mesenchymal stem cell injection), pain symptom disappear, it is not necessary to help crutch to walk.
It should be understood that these embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.In addition, it is to be understood that
After the content of the invention lectured has been read, those skilled in the art can make various changes or modifications to the present invention, these
The equivalent form of value equally falls within the application appended claims limited range.
Claims (8)
1. a kind of preparation method for the injection for treating degenerative osteoarthropathy, it is characterised in that this method comprises the following steps：
Step 1, the preparation of injection human umbilical cord mesenchymal stem cells
A, full-term pregnancy is taken, the neonatal umbilical cord of the healthy puerpera of free from infection, clean with PBS by it on inspection, shreds,
Collagenase digesting is added, the addition of the clostridiopetidase A is 1-4g/L, and digestion temperature is 37 DEG C, and digestion time is 30 minutes -90
Minute, it is therefore preferable to 60 minutes；
B, add 10-30g/L pancreatin to re-digest, digestion temperature is 37 DEG C, and digestion time is -90 minutes 30 minutes, preferably
For 30 minutes；
C, after not digesting umbilical cord tissue with 200 eye mesh screen filtration step B, add PBS and neutralize, digestive juice is 1 with PBS ratios：10-1：
15, it is therefore preferable to 1：12；
D, the liquid in step C after neutralization is subjected to centrifugation and obtains cell, centrifugal rotational speed is 1000 revs/min to 3000
Rev/min, centrifugation time is 10 minutes to 20 minutes；The cell obtained by centrifugation is inoculated in culture dish and carries out cell training
Support, full dose changes liquid after 4 days, discards non-attached cell, and half amount changes liquid 2 times weekly later；
Step 2, the preparation of injection
The cultured cell of process obtained by step 1 is collected, treatment degeneration Bones and joints are made in addition cell solution
The injection of disease.
A kind of 2. preparation method for the injection for treating degenerative osteoarthropathy as claimed in claim 1, it is characterised in that institute
The mescenchymal stem cell content for stating injection is 40,000,000.
A kind of 3. preparation method for the injection for treating degenerative osteoarthropathy as claimed in claim 1, it is characterised in that step
The size shredded described in a rapid A is that tissue block size is 0.05mm-2mm, it is therefore preferable to 1.5mm；The addition of the clostridiopetidase A
Measure as 2g/L.
A kind of 4. preparation method for the injection for treating degenerative osteoarthropathy as claimed in claim 1, it is characterised in that step
The addition of pancreatin is 25g/L in a rapid B.
A kind of 5. preparation method for the injection for treating degenerative osteoarthropathy as claimed in claim 1, it is characterised in that step
Centrifugal rotational speed is preferably 2000 revs/min in a rapid D, is centrifuged 10 minutes.
A kind of 6. preparation method for the injection for treating degenerative osteoarthropathy as claimed in claim 1, it is characterised in that step
The culture medium of culture dish used is serum free medium in a rapid D, skin growth factor in the serum free medium and into fiber
The content of growth factor is respectively 2 nanograms/milliliters to 5 nanograms/milliliters, preferably 4 nanograms/milliliters, the hydrogen in serum free medium
It is 0.2 to 2 mcg/ml, preferably 1 mcg/ml to change cortisone content, and the content of heparin is 5 to 20 mcg/mls, excellent
Elect 15 mcg/mls as.
A kind of 7. preparation method for the injection for treating degenerative osteoarthropathy as claimed in claim 1, it is characterised in that step
Cell solution used is physiological saline, ringer's solution, Multiple electrolytes injection or base fluid in rapid two.
8. a kind of injection for treating degenerative osteoarthropathy, it is characterised in that such as claim 1-4 is used using the injection
Described method is prepared.
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Cited By (1)
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