CN103525890A - Preparation method of human placenta collagen - Google Patents
Preparation method of human placenta collagen Download PDFInfo
- Publication number
- CN103525890A CN103525890A CN201310448097.8A CN201310448097A CN103525890A CN 103525890 A CN103525890 A CN 103525890A CN 201310448097 A CN201310448097 A CN 201310448097A CN 103525890 A CN103525890 A CN 103525890A
- Authority
- CN
- China
- Prior art keywords
- neutral salt
- collagen
- type
- stirring
- standing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to a preparation method of human placenta collagen. According to the method, human placenta tissues serving as raw materials are subjected to pretreatment, enzymatic digestion and fractional salting-out sequentially to respectively obtain at least one of human I type, III type, IV type and V type medicinal collagen materials which maintain the natural structures. With the method, the preparation period of the human placenta I type, III type, IV type and V type collagen raw materials is greatly shortened, the preparation efficiency is improved, the yield is increased, and the product quality is stable. With the method, collagen with specific type can be extracted according to the actual requirement, and the human I type, III type, IV type and V type collagen materials with the natural structures can be obtained simultaneously, so that the human placenta tissue resources are utilized completely.
Description
Technical field
The present invention relates to that a kind of to take the Healthy People placenta obtaining after normal labor be raw material, preparation keeps the method for people's placenta I type, III type, IV type and the collagen type v material of natural structure, belongs to the preparation field of bio-medical material.
Background technology
Natural collagen is as a species specific structural protein, and its characteristics and advantages is extensively familiar with, and further becomes one of important source material of bio-medical material.Known at least 26 kinds of collagen-types in human body at present, each is all that the structure function of collagen has increased specific function.Collagen content in mammalian placenta, particularly people's placenta is very abundant, contains I type, III type, IV type and collagen type v simultaneously, is therefore a kind of very valuable starting material of preparing natural medical collagen.
End user's placenta is various natural medical collagen prepared by raw material, do not carry any known people's communicable disease, overcome heterogenous animal tissues such as using ox, pig or organ and prepared the risk of virus pollution that collagen may exist or potential zoonosis for raw material, so human placental collagen has higher security.
About take people's placenta as the patent of people's placenta I type, III type, IV type and collagen type v that raw material preparation has natural structure relative less with report.The domestic research of carrying out a small amount of human placental collagen of short-term in earlier 1990s, mainly represent that achievement has: Liu Bingci be take people's placenta as raw material, through gastric pepsin digestion, after saltouing, obtain precipitate A, and further acid-salt extracts centrifugal rear acquisition precipitate B, two kinds of precipitations are mixed in varing proportions and added after tethelin and lignocaine for filling miniature pitting skin injury (Chinese invention patent, publication number: CN1081379)
Up to the present, there is no documents and materials take people's placenta and has the report of people I type, III type, IV type and the collagen type v material of natural structure as raw material preparation.Therefore, the Healthy People placenta tissue of take is prepared natural structure people I type, III type, IV type and the V-type medical collagen material with stability as main raw material within the shorter cycle, has clear meaning.
Summary of the invention
By the comprehensive analysis of prior art is found to have the following disadvantages in prior art:
1. in people's placenta, contain I type, III type, IV type and collagen type v, but the multipotency of prior art extracts the collagen of three types, can not obtain the collagenic material of Four types simultaneously, abundant not to the human placenta utilization of resources;
2. existing collagen extraction technology cycle is tediously long, need more than 15 days, complex steps, thereby make extraction efficiency and productive rate comparatively low, the key link that affects quality product in technique is difficult to control, easily cause quality product stable not, finally affect effect and the value of collagen and collagen base biological medical material.
The object of the present invention is to provide a kind of preparation method of human placental collagen, obviously shorten the cycle of extracting collagen, obtain high-quality human placental collagen.
The present invention is achieved through the following technical solutions:
A preparation method for human placental collagen, take human placenta as raw material, carries out successively pre-treatment, enzymic digestion, salt fractionation, obtains respectively at least one in people I type, III type, IV type and the V-type medical collagen material that keeps natural structure.
By described pre-treatment step to placenta tissue clean, broken and remove major impurity;
In described enzymatic digestion stage, adopt the end peptide of stomach en-in collegen filament and the specific site between helical region to interrupt peptide chain, thereby being dissolved in Digestive system, collagen is separated with other structural constituents.
By described salt fractionation, dissimilar collagen is carried out to separation, purification, to obtain the collagen of particular type again.
As a kind of optional mode, described human placenta is the placenta tissue fresh or that preserve through cryogenic freezing that pre-puerpera has carried out spreading disease and obtained after screening normal labor before childbirth, therefore there is no the viral pathogens of known pollution placenta tissue, thereby make gained collagen there is good biological safety, be conducive to its application smoothly such as in fields such as tissue engineering bracket, cell culture vector, pathological study and drug developments.Described placenta tissue can be selected from the arbitrary portion of placenta, as amnion, chorion or complete placenta.
As a kind of optional mode, described pre-treatment step specifically comprises: placenta tissue is fully cleaned and is cut into small pieces, centrifugal removal supernatant liquor after low-temperature homogenate, precipitate with deionized water is centrifugal again after cleaning, repeatedly clean centrifugal 2-4 time transparent to supernatant liquor; Precipitation with pH be 7.2-7.4, the concentration that contains 1.0-20.0 mmol/L metal chelating agent, the 0.5-3.0mol/L neutral salt pre-treatment damping fluid that is 0.01-1.0mol/L soaks and slow stirring 2-4h, stirring velocity can not be too fast, with liquid level, slightly rotates and be advisable; Centrifugal rear gained precipitation again with pre-treatment buffer solution for cleaning once and carry out centrifugal, get precipitate standby.By this step, can remove more thoroughly impurity, obtain the higher mixture of collagen content, and consuming time short, efficiency is high.
Described metal chelating agent can be at least one in aminocarboxylate (as edetate, diethylenetriamine pentacarboxylic acid salt etc.) and hydroxycarboxylate's (as sodium tartrate, Sunmorl N 60S).The various metallic impurity that adopt described metal chelating agent to eliminate to contain in raw material, also remove various other active biomolecules simultaneously.
Described pre-treatment damping fluid can be in Tutofusin tris-hydrochloric acid, Sodium phosphate dibasic-citric acid, Sodium phosphate dibasic-SODIUM PHOSPHATE, MONOBASIC, potassium primary phosphate-sodium hydrate buffer solution a kind of.Adopt described damping fluid stablizing of maintenance system pH value on the one hand, can also play the effect of cleaning foreign protein simultaneously, wherein the best results of Tutofusin tris-hydrochloric acid cleaning foreign protein.
As a kind of optional mode, described enzymic digestion is that pretreated human placenta is soaked in Digestive system, described Digestive system is that stomach en-(vigor >=250U/mg) (also can be taked other weak acid with acetic acid, as citric acid) mixing solutions, its pH is 2.2-2.8, pepsin concn is 0.5-5g/L, and acetic acid (HAC) concentration is 0.2-0.8mol/L; The 5-10 that described Digestive system quality is human placenta solid weight doubly, slowly stirs under cold condition continuously, and stirring velocity can not be too fast, with liquid level, slightly rotates and be advisable; After enzymic digestion 6-30h, it is standby that supernatant liquor is got in centrifugation.Adopt above-mentioned enzymic digestion method, digestible degree is moderate, products therefrom steady quality, and with respect to existing human placental collagen extraction process, greatly shortened digestion time, improved efficiency.
As a kind of optional mode, described salt fractionation is operating as the collagen-type according to required extraction, chooses corresponding step combination from following salt fractionation step:
1) with acetic acid regulate sample after enzymic digestion to pH be 2.5-2.8, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 0.5-1.2mol/L, after neutral salt dissolves completely, standing 2-10h carries out centrifugally again, and gained precipitation is labeled as CD-A, and supernatant liquor is labeled as QY-A;
2) described precipitate C D-A is used after 0.5mol/L acetate dissolution, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 0.3-0.8mol/L, after neutral salt dissolves completely, standing 2-10h carries out centrifugally again, and gained precipitation is labeled as CD-B, and supernatant liquor is labeled as QY-B;
3) in described clear liquid QY-B, according to the final concentration of 2.0-4.0mol/L, add while stirring the neutral salt particle of porphyrize, after neutral salt dissolves completely, standing 2-10h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-C;
4) described precipitate C D-C is dissolved in after 0.2-0.5mol/L acetum and adjusts pH to 7.2-7.5 with sodium hydroxide solution, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 1.2-2.5mol/L, after neutral salt dissolves completely, standing 2-10h carries out centrifugal again, abandon supernatant liquor, gained precipitation is labeled as CD-D;
5) described precipitate C D-D is dissolved in after 0.5mol/L acetum, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 0.3-0.8mol/L, after neutral salt dissolves completely, standing 2-10h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-E;
6) described precipitate C D-E is dissolved in to 0.2-0.5mol/L acetum, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 1.2-2.5mol/L, after dissolving completely, neutral salt adjusts pH to 7.2-7.5 with sodium hydroxide solution, after standing 2-10h, carry out centrifugal, precipitation is dissolved in after 0.5mol/L acetum again, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 0.3-0.8mol/L, after neutral salt dissolves completely, standing 2-10h carries out centrifugal again, abandon supernatant liquor, gained precipitation is proceeded to dialysis band fully dialyses to deionized water, products therefrom is people's placenta type i collagen material after lyophilize,
7) described CD-B is dissolved in after 0.2-0.5mol/L acetum and adjusts pH to 7.2-7.5 with sodium hydroxide solution, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 0.9-2.0mol/L, after neutral salt dissolves completely, standing 2-10h carries out centrifugal again, abandon supernatant liquor, gained precipitation is labeled as CD-F;
8) described precipitate C D-F is dissolved in after 0.5mol/L acetum, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 0.3-0.8mol/L, after neutral salt dissolves completely, standing 2-10h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-G;
9) described precipitate C D-G is dissolved in after 0.2-0.5mol/L acetum, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 0.9-2.0mol/L, after dissolving completely, neutral salt adjusts pH to 7.2-7.5 with sodium hydroxide solution, after standing 2-10h, carry out centrifugal, precipitation is dissolved in after 0.5mol/L acetum again, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 0.3-0.8mol/L, after neutral salt dissolves completely, standing 2-10h carries out centrifugal again, abandon supernatant liquor, gained precipitation is proceeded to dialysis band fully dialyses to deionized water, products therefrom is people's placenta III Collagen Type VI material after lyophilize,
10) in described QY-A, according to the final concentration of 0.9-2.0mol/L, add while stirring the neutral salt particle of porphyrize, after neutral salt dissolves completely, standing 2-10h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-H;
11) described precipitate C D-H is dissolved in after 0.2-0.5mol/L acetum and adjusts pH to 7.2-7.5 with sodium hydroxide solution, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 1.8-4.5mol/L, after neutral salt dissolves completely, standing 2-10h carries out centrifugal again, abandon supernatant liquor, gained precipitation is labeled as CD-I;
12) described precipitate C D-I is dissolved in after 0.2-0.5mol/L acetum, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 0.6-1.9mol/L, after neutral salt dissolves completely, standing 2-10h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-J;
13) described in general, stating precipitate C D-J is dissolved in after 0.2-0.5mol/L acetum, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 1.8-4.5mol/L, after dissolving completely, neutral salt adjusts pH to 7.2-7.5 with sodium hydroxide solution, after standing 2-10h, carry out centrifugal, precipitation is dissolved in after 0.2-0.5mol/L acetum again, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 0.6-1.9mol/L, after neutral salt dissolves completely, standing 2-10h carries out centrifugal again, abandon supernatant liquor, gained precipitation is dissolved in to 0.05mol/L Tris-HCl, 0.1mol/L NaCl, pH be the damping fluid of 7.2-7.5 and same damping fluid is fully dialysed after carry out centrifugation, gained precipitation is labeled as CD-K, supernatant liquor is labeled as QY-C,
14) described clear liquid QY-C is proceeded to dialysis band deionized water is fully dialysed, products therefrom is people's placenta IV Collagen Type VI material after lyophilize;
15) described in general, state precipitate C D-K, be dissolved in after 0.2-0.5mol/L acetum, proceeding to dialysis band deionized water is fully dialysed, products therefrom is people's placenta collagen type v material after lyophilize.
This technological operation is flexible, can select corresponding operation steps according to the type of required extraction collagen, for example: while only needing to extract type i collagen, by described step 1) to step 6), operate successively; While only needing to extract III Collagen Type VI, select described step 1), step 2) and step 7) to step 9) operate successively; While only needing to extract IV Collagen Type VI, select described step 1), step 10) to step 14) to operate successively; While only needing to extract collagen type v, select described step 1), step 10) to step 13) and step 15) to operate successively; As extracted multiple collagen-type by corresponding step combination, by step 1) to step 15), operate, can from placenta tissue, isolate the collagen (I type, III type, IV type and V-type) of Four types the same period.
In people's placenta I type, III type, IV type or the collagen type v that adopts the method for the invention to prepare, comprise intrafascicular at least one of protofibril shape collagen, microfibrous collagen, particulate state fiber, collegen filament and collegen filament.The collagen of gained has farthest retained the original triple helix structure of natural collagen.
Neutral salt described in the present invention is at least one in sodium-chlor, ammonium sulfate, sodium sulfate, Trisodium Citrate and ammonium phosphate.
The present invention also provides a kind of collagen extracting from human placenta, and described collagen adopts the method for the invention to be prepared from, and the collagen of gained is for keeping a kind of in people I type, III type, IV type or the V-type medical collagen material of natural structure.In described collagen, comprise intrafascicular at least one of protofibril shape collagen, microfibrous collagen, particulate state fiber, collegen filament and collegen filament, farthest retained the original triple helix structure of natural collagen.Described collagen purity is high, and biological safety is high, through simply disinfecting biomedicine fields such as can be used for tissue engineering bracket, cell culture vector, pathological study and drug development, for example, as cell culture vector and surface modifying material; Main raw material as tissue engineering bracket material; Or further compound other material, be applied to other field of biomedical material.
Disclosed all features in this specification sheets, or the step in disclosed all methods or process, except mutually exclusive feature and/or step, all can combine by any way.
Beneficial effect of the present invention:
1, placental collagen preparation method of the present invention has significantly shortened the preparation cycle of people's placenta I type, III type, IV type and collagen type v material, has improved preparation efficiency and productive rate, makes the quality of product also more stable simultaneously.
2, adopt the method for the invention can shift to an earlier date according to actual needs the collagen of particular type, can also obtain people I type, III type, IV type and the collagen type v material with natural structure the same period, make full use of human placenta resource.
Accompanying drawing explanation
Fig. 1 is preparation technology's schema of the present invention;
Fig. 2 is differential scanning calorimeter (DSC) the detected result figure of each Collagen Type VI of preparing in embodiment of the present invention, in figure 1 be I type, 3 for III type, 4 for IV type, 5 be collagen type v.
Embodiment
Preparation technology's flow process of the present invention is: take human placenta as raw material, carry out successively pre-treatment, enzymic digestion, salt fractionation.
Wherein pre-treatment step comprises placenta tissue is fully cleaned and is cut into small pieces, centrifugal removal supernatant liquor after low-temperature homogenate, and precipitate with deionized water is cleaned centrifugal transparent to supernatant liquor repeatedly; Precipitation with the pre-treatment damping fluid that contains metal chelating agent and neutral salt, soak and slowly stir after centrifugal, gained precipitation again with pre-treatment buffer solution for cleaning once and carry out centrifugal, get precipitate standby; Enzymatic digestion stage comprises pretreated human placenta is soaked in Digestive system, under cold condition, slowly stirs continuously, and after enzymic digestion 6-30h, it is standby that supernatant liquor is got in centrifugation; Described salt fractionation is the difference of utilizing collagen without the type solubleness in the neutral salt solution of different concns, through collagen mixture is dissolved repeatedly with precipitation in the salts solution of different concns, dissimilar collagen is carried out to separated, purification, to obtain the collagen of particular type.
Following classified as several most preferred embodiments of the present invention, it should be understood that these embodiment, only for the object of illustration, never limit the scope of the invention.
embodiment 1
Extract according to the following steps collagen:
(1) whole person's placenta of selecting fresh process disease screening normal labor to obtain;
(2) placenta tissue is fully cleaned and is cut into small pieces, centrifugal removal supernatant liquor after low-temperature homogenate, precipitate with deionized water is centrifugal again after cleaning, repeatedly clean centrifugal 4 times transparent to supernatant liquor; Precipitation with pH be 7.2-7.4, Tutofusin tris-hydrochloric acid (Tris-HCl) damping fluid of the 0.5mol/L that contains 10 mmol/L sodium ethylene diamine tetracetates (Na2EDTA), 1.0 mol/L NaCl soaks and slow stirring 4h, stirring velocity can not be too fast, with liquid level, slightly rotates and be advisable; After stirring completes, carry out centrifugally, centrifugal rear gained precipitation again by Tris-HCl buffer solution for cleaning once and carry out centrifugally, finally weigh and as the foundation of follow-up measurement by the centrifugal precipitation obtaining;
(3) the final precipitation obtaining in step (2) is soaked in Digestive system, described Digestive system is pH 2.8, acetic acid (HAC) solution of the 0.5mol/L that stomach en-(vigor >=250U/mg) concentration is 5g/L, described Digestive system quality is 5 times of precipitation weight, under cold condition, slowly stir continuously, stirring velocity can not be too fast, with liquid level, slightly rotates and be advisable; After enzymic digestion 12h, it is standby that supernatant liquor is got in centrifugation.
(4) according to following steps, carry out salt fractionation operation:
1) with acetic acid regulate sample after enzymic digestion to pH be 2.8, according to the final concentration of 0.8mol/L, add while stirring the NaCl particle of porphyrize, after NaCl dissolves completely, standing 4h carries out centrifugally again, gained precipitation is labeled as CD-A, supernatant liquor is labeled as QY-A;
2) by described precipitate C D-A with after 0.5mol/L acetate dissolution, according to the final concentration of 0.7mol/L, add while stirring the NaCl particle of porphyrize, after NaCl dissolves completely, standing 4h carries out centrifugally again, gained precipitation is labeled as CD-B, supernatant liquor is labeled as QY-B;
3) in described clear liquid QY-B, according to the final concentration of 4.0mol/L, add while stirring the NaCl particle of porphyrize, after NaCl dissolves completely, standing 8h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-C;
4) described precipitate C D-C is dissolved in after 0.2mol/L acetum and adjusts pH to 7.4 with sodium hydroxide solution, the NaCl particle that adds while stirring porphyrize according to the final concentration of 2.5mol/L, after NaCl dissolves completely, standing 4h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-D;
5) described precipitate C D-D is dissolved in after 0.5mol/L acetum, adds while stirring the NaCl particle of porphyrize according to the final concentration of 0.7mol/L, after NaCl dissolves completely, standing 4h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-E;
6) described precipitate C D-E is dissolved in to 0.2mol/L acetum, the NaCl particle that adds while stirring porphyrize according to the final concentration of 2.5mol/L, after dissolving completely, NaCl adjusts pH to 7.4 with sodium hydroxide solution, after standing 4h, carry out centrifugal, precipitation is dissolved in after 0.5mol/L acetum again, the NaCl particle that adds while stirring porphyrize according to the final concentration of 0.7mol/L, after NaCl dissolves completely, standing 4h carries out centrifugal again, abandon supernatant liquor, gained precipitation is dissolved in and proceeds to dialysis band after 0.2mol/L acetum deionized water is fully dialysed, products therefrom is people's placenta type i collagen material after lyophilize,
7) described CD-B is dissolved in after 0.2mol/L acetum and adjusts pH to 7.4 with sodium hydroxide solution, the NaCl particle that adds while stirring porphyrize according to the final concentration of 2.0mol/L, after NaCl dissolves completely, standing 2h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-F;
8) described precipitate C D-F is dissolved in after 0.5mol/L acetum, adds while stirring the NaCl particle of porphyrize according to the final concentration of 0.7mol/L, after NaCl dissolves completely, standing 4h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-G;
9) described precipitate C D-G is dissolved in after 0.2mol/L acetum, the NaCl particle that adds while stirring porphyrize according to the final concentration of 2.0mol/L, after dissolving completely, NaCl adjusts pH to 7.4 with sodium hydroxide solution, after standing 2h, carry out centrifugal, precipitation is dissolved in after 0.5mol/L acetum again, the NaCl particle that adds while stirring porphyrize according to the final concentration of 0.7mol/L, after NaCl dissolves completely, standing 2h carries out centrifugal again, abandon supernatant liquor, gained precipitation is dissolved in and proceeds to dialysis band after 0.2mol/L acetum deionized water is fully dialysed, products therefrom is people's placenta III Collagen Type VI material after lyophilize,
10) in described QY-A, according to the final concentration of 2.0mol/L, add while stirring the NaCl particle of porphyrize, after NaCl dissolves completely, standing 4h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-H;
11) described precipitate C D-H is dissolved in after 0.2mol/L acetum and adjusts pH to 7.4 with sodium hydroxide solution, the NaCl particle that adds while stirring porphyrize according to the final concentration of 4.5mol/L, after NaCl dissolves completely, standing 4h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-I;
12) described precipitate C D-I is dissolved in after 0.5mol/L acetum, adds while stirring the NaCl particle of porphyrize according to the final concentration of 1.2mol/L, after NaCl dissolves completely, standing 2h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-J;
13) described in general, stating precipitate C D-J is dissolved in after 0.2mol/L acetum, the NaCl particle that adds while stirring porphyrize according to the final concentration of 4.5mol/L, after dissolving completely, NaCl adjusts pH to 7.4 with sodium hydroxide solution, after standing 4h, carry out centrifugal, precipitation is dissolved in after 0.5mol/L acetum again, the NaCl particle that adds while stirring porphyrize according to the final concentration of 1.2mol/L, after NaCl dissolves completely, standing 2h carries out centrifugal again, abandon supernatant liquor, gained precipitation is dissolved in to 0.05mol/L Tris-HCl, 0.1mol/L NaCl, pH be the damping fluid of 7.2-7.5 and same damping fluid is fully dialysed after carry out centrifugation, gained precipitation is labeled as CD-K, supernatant liquor is labeled as QY-C,
14) described clear liquid QY-C is proceeded to dialysis band deionized water is fully dialysed, products therefrom is people's placenta IV Collagen Type VI material after lyophilize;
15) described in general, state precipitate C D-K, be dissolved in after 0.2mol/L acetum, proceeding to dialysis band deionized water is fully dialysed, products therefrom is people's placenta collagen type v material after lyophilize.
Adopt above-mentioned technology preparation to have people I type, III type, IV type and the collagen type v material of natural structure, the cycle foreshortens to 5-10 days, and the efficiency and the productive rate that have improved preparation are higher.The raw material that the above-mentioned medical collagen material obtaining can be used as biomedical material is further processed into other forms of collagen base biological medical material, also can adopt ion-exchange chromatography isochromatic spectrum method to be further purified, obtain more high purity and the more concentrated various medical collagen material of molecular weight distribution simultaneously.In view of different collagen unique characteristic and performance separately, the medical collagen material that obtains somatotype can be brought into play Biological characteristics and advantage separately, meet different biomedical material research and the demand of clinical application, further improve the added value of collagenic material.
Extract according to the following steps collagen:
(1) whole person's placenta of selecting fresh process disease screening normal labor to obtain;
(2) placenta tissue is fully cleaned and is cut into small pieces, centrifugal removal supernatant liquor after low-temperature homogenate, precipitate with deionized water is centrifugal again after cleaning, repeatedly clean centrifugal 4 times transparent to supernatant liquor; Precipitation is 7.2, contains 20 mmol/L sodium ethylene diamine tetracetate (Na with pH
2eDTA), Tutofusin tris-hydrochloric acid (Tris-HCl) damping fluid of the 0.05mol/L of 2.0 mol/L NaCl soaks and slowly stirs 4h, stirring velocity can not be too fast, with liquid level, slightly rotates and be advisable; After stirring completes, carry out centrifugally, centrifugal rear gained precipitation again by Tris-HCl buffer solution for cleaning once and carry out centrifugally, finally weigh and as the foundation of follow-up measurement by the centrifugal precipitation obtaining;
(3) the final precipitation obtaining in step (2) is soaked in Digestive system, described Digestive system is pH 2.5, acetic acid (HAC) solution of the 0.8mol/L that stomach en-(vigor >=250U/mg) concentration is 1g/L, described Digestive system quality is 10 times of precipitation weight, under cold condition, slowly stir continuously, stirring velocity can not be too fast, with liquid level, slightly rotates and be advisable; After enzymic digestion 24h, it is standby that supernatant liquor is got in centrifugation.
(4) according to following steps, carry out salt fractionation operation:
1) with acetic acid regulate sample after enzymic digestion to pH be 2.5, according to the final concentration of 0.6mol/L, add while stirring (the NH of porphyrize
4)
2sO
4particle, treats (NH
4)
2sO
4after dissolving completely, standing 4h carries out centrifugally again, and gained precipitation is labeled as CD-A, and supernatant liquor is labeled as QY-A;
2) described precipitate C D-A is used after 0.5mol/L acetate dissolution, according to the final concentration of 0.4mol/L, add while stirring (the NH of porphyrize
4)
2sO
4particle, treats (NH
4)
2sO
4after dissolving completely, standing 4h carries out centrifugally again, and gained precipitation is labeled as CD-B, and supernatant liquor is labeled as QY-B;
3), in described clear liquid QY-B, according to the final concentration of 2.0mol/L, add while stirring (the NH of porphyrize
4)
2sO
4particle, treats (NH
4)
2sO
4after dissolving completely, standing 4h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-C;
4) described precipitate C D-C is dissolved in after 0.5mol/L acetum and adjusts pH to 7.5 with sodium hydroxide solution, according to the final concentration of 1.2mol/L, add while stirring (the NH of porphyrize
4)
2sO
4particle, treats (NH
4)
2sO
4after dissolving completely, standing 4h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-D;
5) described precipitate C D-D is dissolved in after 0.2mol/L acetum, according to the final concentration of 0.3mol/L, adds while stirring (the NH of porphyrize
4)
2sO
4particle, treats (NH
4)
2sO
4after dissolving completely, standing 4h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-E;
6) described precipitate C D-E is dissolved in to 0.5mol/L acetum, according to the final concentration of 1.2mol/L, adds while stirring (the NH of porphyrize
4)
2sO
4particle, treats (NH
4)
2sO
4after dissolving completely, with sodium hydroxide solution, adjust pH to 7.5, after standing 2h, carry out centrifugally, then precipitation is dissolved in after 0.5mol/L acetum, according to the final concentration of 0.3mol/L, add while stirring (the NH of porphyrize
4)
2sO
4particle, treats (NH
4)
2sO
4after dissolving completely, standing 2h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is dissolved in and proceeds to dialysis band after 0.2mol/L acetum deionized water is fully dialysed, and products therefrom is people's placenta type i collagen material after lyophilize;
7) described CD-B is dissolved in after 0.2mol/L acetum and adjusts pH to 7.5 with sodium hydroxide solution, according to the final concentration of 0.9mol/L, add while stirring (the NH of porphyrize
4)
2sO
4particle, treats (NH
4)
2sO
4after dissolving completely, standing 4h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-F;
8) described precipitate C D-F is dissolved in after 0.5mol/L acetum, according to the final concentration of 0.35mol/L, adds while stirring (the NH of porphyrize
4)
2sO
4particle, treats (NH
4)
2sO
4after dissolving completely, standing 4h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-G;
9) described precipitate C D-G is dissolved in after 0.2mol/L acetum, according to the final concentration of 0.9mol/L, adds while stirring (the NH of porphyrize
4)
2sO
4particle, treats (NH
4)
2sO
4after dissolving completely, with sodium hydroxide solution, adjust pH to 7.5, after standing 2h, carry out centrifugally, then precipitation is dissolved in after 0.5mol/L acetum, according to the final concentration of 0.3mol/L, add while stirring (the NH of porphyrize
4)
2sO
4particle, treats (NH
4)
2sO
4after dissolving completely, standing 2h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is dissolved in and proceeds to dialysis band after 0.2mol/L acetum deionized water is fully dialysed, and products therefrom is people's placenta III Collagen Type VI material after lyophilize;
10), in described QY-A, according to the final concentration of 0.9mol/L, add while stirring (the NH of porphyrize
4)
2sO
4particle, treats (NH
4)
2sO
4after dissolving completely, standing 4h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-H;
11) described precipitate C D-H is dissolved in after 0.5mol/L acetum and adjusts pH to 7.5 with sodium hydroxide solution, according to the final concentration of 2.0mol/L, add while stirring (the NH of porphyrize
4)
2sO
4particle, treats (NH
4)
2sO
4after dissolving completely, standing 2h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-I;
12) described precipitate C D-I is dissolved in after 0.5mol/L acetum, according to the final concentration of 0.6mol/L, adds while stirring (the NH of porphyrize
4)
2sO
4particle, treats (NH
4)
2sO
4after dissolving completely, standing 2h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-J;
13) described in general, state precipitate C D-J and be dissolved in after 0.2mol/L acetum, according to the final concentration of 2.0mol/L, add while stirring (the NH of porphyrize
4)
2sO
4particle, treats (NH
4)
2sO
4after dissolving completely, with sodium hydroxide solution, adjust pH to 7.5, after standing 2h, carry out centrifugally, then precipitation is dissolved in after 0.2mol/L acetum, according to the final concentration of 0.6mol/L, add while stirring (the NH of porphyrize
4)
2sO
4particle, treats (NH
4)
2sO
4after dissolving completely, standing 2h carries out centrifugal again, abandon supernatant liquor, gained precipitation be dissolved in to 0.05mol/L Tris-HCl, 0.1mol/L NaCl, the damping fluid that pH is 7.2-7.5 and same damping fluid is fully dialysed after carry out centrifugation, gained precipitation is labeled as CD-K, and supernatant liquor is labeled as QY-C;
14) described clear liquid QY-C is proceeded to dialysis band deionized water is fully dialysed, products therefrom is people's placenta IV Collagen Type VI material after lyophilize;
15) described in general, state precipitate C D-K, be dissolved in after 0.2mol/L acetum, proceeding to dialysis band deionized water is fully dialysed, products therefrom is people's placenta collagen type v material after lyophilize.
embodiment 3
Extract according to the following steps collagen:
(1) select the process disease screening of freezing preservation groups of people's placenta tissue that normal labor obtains;
(2) placenta tissue is fully cleaned and is cut into small pieces, centrifugal removal supernatant liquor after low-temperature homogenate, precipitate with deionized water is centrifugal again after cleaning, repeatedly clean centrifugal 3 times transparent to supernatant liquor; Precipitation with pH be 7.2, the Sodium phosphate dibasic of the 0.2mol/L that contains 5 mmol/L Sunmorl N 60Ss, 1.0 mol/L NaCl-phosphate sodium dihydrogen buffer solution soaks and slow stirring 2h, stirring velocity can not be too fast, with liquid level, slightly rotates and be advisable; After stirring completes, carry out centrifugally, centrifugal rear gained precipitation is cleaned once with phosphate buffered saline buffer and is carried out centrifugally, and the last centrifugal precipitation obtaining is weighed and as the foundation of follow-up measurement;
(3) the final precipitation obtaining in step (2) is soaked in Digestive system, described Digestive system is pH 2.2, acetic acid (HAC) solution of the 0.8mol/L that stomach en-(vigor >=250U/mg) concentration is 2g/L, described Digestive system quality is 8 times of precipitation weight, under cold condition, slowly stir continuously, stirring velocity can not be too fast, with liquid level, slightly rotates and be advisable; After enzymic digestion 30h, it is standby that supernatant liquor is got in centrifugation.
(4) according to following steps, carry out salt fractionation operation:
1) with acetic acid regulate sample after enzymic digestion to pH be 2.5, according to the final concentration of 0.5mol/L, add while stirring the NaCl particle of porphyrize, after NaCl dissolves completely, standing 4h carries out centrifugally again, gained precipitation is labeled as CD-A, supernatant liquor is labeled as QY-A;
2) by described precipitate C D-A with after 0.5mol/L acetate dissolution, according to the final concentration of 0.7mol/L, add while stirring the NaCl particle of porphyrize, after NaCl dissolves completely, standing 4h carries out centrifugally again, gained precipitation is labeled as CD-B, supernatant liquor is labeled as QY-B;
3) in described clear liquid QY-B, according to the final concentration of 4.0mol/L, add while stirring the NaCl particle of porphyrize, after NaCl dissolves completely, standing 8h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-C;
4) described precipitate C D-C is dissolved in after 0.2mol/L acetum and adjusts pH to 7.4 with sodium hydroxide solution, the NaCl particle that adds while stirring porphyrize according to the final concentration of 2.5mol/L, after NaCl dissolves completely, standing 4h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-D;
5) described precipitate C D-D is dissolved in after 0.5mol/L acetum, adds while stirring the NaCl particle of porphyrize according to the final concentration of 0.7mol/L, after NaCl dissolves completely, standing 4h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-E;
6) described precipitate C D-E is dissolved in to 0.2mol/L acetum, the NaCl particle that adds while stirring porphyrize according to the final concentration of 2.5mol/L, after dissolving completely, NaCl adjusts pH to 7.2 with sodium hydroxide solution, after standing 2h, carry out centrifugal, precipitation is dissolved in after 0.5mol/L acetum again, the NaCl particle that adds while stirring porphyrize according to the final concentration of 0.8mol/L, after NaCl dissolves completely, standing 2h carries out centrifugal again, abandon supernatant liquor, gained precipitation is dissolved in and proceeds to dialysis band after 0.2mol/L acetum deionized water is fully dialysed, products therefrom is people's placenta type i collagen material after lyophilize,
7) described CD-B is dissolved in after 0.2mol/L acetum and adjusts pH to 7.2 with sodium hydroxide solution, according to the final concentration of 1.0mol/L, add while stirring (the NH of porphyrize
4)
2sO
4particle, treats (NH
4)
2sO
4after dissolving completely, standing 4h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-F;
8) described precipitate C D-F is dissolved in after 0.5mol/L acetum, according to the final concentration of 0.4mol/L, adds while stirring (the NH of porphyrize
4)
2sO
4particle, treats (NH
4)
2sO
4after dissolving completely, standing 4h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-G;
9) described precipitate C D-G is dissolved in after 0.5mol/L acetum, according to the final concentration of 1.0mol/L, adds while stirring (the NH of porphyrize
4)
2sO
4particle, treats (NH
4)
2sO
4after dissolving completely, with sodium hydroxide solution, adjust pH to 7.5, after standing 2h, carry out centrifugally, then precipitation is dissolved in after 0.5mol/L acetum, according to the final concentration of 0.4mol/L, add while stirring (the NH of porphyrize
4)
2sO
4particle, treats (NH
4)
2sO
4after dissolving completely, standing 2h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is dissolved in and proceeds to dialysis band after 0.2mol/L acetum deionized water is fully dialysed, and products therefrom is people's placenta III Collagen Type VI material after lyophilize;
10) in described QY-A, according to the final concentration of 0.9mol/L, add while stirring the ammonium phosphate granules of porphyrize, after ammonium phosphate dissolves completely, standing 2h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-H;
11) described precipitate C D-H is dissolved in after 0.2mol/L acetum and adjusts pH to 7.5 with sodium hydroxide solution, the ammonium phosphate granules that adds while stirring porphyrize according to the final concentration of 1.8mol/L, after ammonium phosphate dissolves completely, standing 4h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-I;
12) described precipitate C D-I is dissolved in after 0.5mol/L acetum, adds while stirring the ammonium phosphate granules of porphyrize according to the final concentration of 0.6mol/L, after ammonium phosphate dissolves completely, standing 2h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-J;
13) described in general, stating precipitate C D-J is dissolved in after 0.2mol/L acetum, the ammonium phosphate granules that adds while stirring porphyrize according to the final concentration of 1.8mol/L, after dissolving completely, ammonium phosphate adjusts pH to 7.5 with sodium hydroxide solution, after standing 2h, carry out centrifugal, precipitation is dissolved in after 0.2mol/L acetum again, the ammonium phosphate granules that adds while stirring porphyrize according to the final concentration of 0.6mol/L, after ammonium phosphate dissolves completely, standing 2h carries out centrifugal again, abandon supernatant liquor, gained precipitation is dissolved in to 0.05mol/L Tris-HCl, 0.1mol/L NaCl, pH be 7.5 damping fluid and same damping fluid is fully dialysed after carry out centrifugation, gained precipitation is labeled as CD-K, supernatant liquor is labeled as QY-C,
14) described clear liquid QY-C is proceeded to dialysis band deionized water is fully dialysed, products therefrom is people's placenta IV Collagen Type VI material after lyophilize;
15) described in general, state precipitate C D-K, be dissolved in after 0.2mol/L acetum, proceeding to dialysis band deionized water is fully dialysed, products therefrom is people's placenta collagen type v material after lyophilize.
Adopt differential scanning calorimeter (DSC) to detect each Collagen Type VI of gained in the various embodiments described above, result as shown in Figure 2, as can be seen from the figure the DSC curve of the I type of gained, III type, IV type and collagen type v material to go out peak position substantially identical with the typical curve of corresponding collagen, the collagenic material of successfully having prepared I type, III type, IV type and this Four types of V-type by the method for the invention is described.
The foregoing is only the preferred embodiments of the present invention, is only illustrative for the purpose of the present invention, and nonrestrictive; Those of ordinary skills understand, and in the spirit and scope that limit, can carry out many changes to it in the claims in the present invention, revise, and even equivalence change, but all will fall into protection scope of the present invention.
Claims (10)
1. the preparation method of a human placental collagen, it is characterized in that, take human placenta as raw material, carry out successively pre-treatment, enzymic digestion, salt fractionation, obtain respectively at least one in people I type, III type, IV type and the V-type medical collagen material that keeps natural structure.
2. the preparation method of human placental collagen according to claim 1, is characterized in that, described human placenta is the placenta tissue fresh or that preserve through cryogenic freezing that pre-puerpera has carried out spreading disease and obtained after screening normal labor before childbirth.
3. the preparation method of human placental collagen according to claim 1, it is characterized in that, described pre-treatment is that placenta tissue is fully cleaned and is cut into small pieces, centrifugal removal supernatant liquor after low-temperature homogenate, precipitate with deionized water is centrifugal again after cleaning, repeatedly clean centrifugal 2-4 time transparent to supernatant liquor; Precipitation with pH be 7.2-7.4, the concentration that contains 1.0-20.0 mmol/L metal chelating agent, the 0.5-3.0mol/L neutral salt pre-treatment damping fluid that is 0.01-1.0mol/L soaks and slow stirring 2-4h, stirring velocity can not be too fast, with liquid level, slightly rotates and be advisable; Centrifugal rear gained precipitation again with pre-treatment buffer solution for cleaning once and carry out centrifugal, get precipitate standby.
4. human placental collagen preparation method according to claim 1, it is characterized in that: described enzymic digestion is that pretreated human placenta is soaked in Digestive system, described Digestive system is pH 2.2-2.8, pepsin concn is 0.2-0.8mol/L acetic acid (HAC) solution of 0.5-5g/L, described peptic activity of stomach >=250U/mg; The 5-10 that described Digestive system quality is human placenta solid weight doubly, slowly stirs under cold condition continuously, and stirring velocity can not be too fast, with liquid level, slightly rotates and be advisable; After enzymic digestion 6-30h, it is standby that supernatant liquor is got in centrifugation.
5. human placental collagen preparation method according to claim 1, is characterized in that, described salt fractionation is operating as the collagen-type according to required extraction, chooses corresponding step combination from following salt fractionation step:
1) with acetic acid regulate sample after enzymic digestion to pH be 2.5-2.8, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 0.5-1.2mol/L, after neutral salt dissolves completely, standing 2-10h carries out centrifugally again, and gained precipitation is labeled as CD-A, and supernatant liquor is labeled as QY-A;
2) described precipitate C D-A is used after 0.2-0.5mol/L acetate dissolution, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 0.3-0.8mol/L, after neutral salt dissolves completely, standing 2-10h carries out centrifugally again, and gained precipitation is labeled as CD-B, and supernatant liquor is labeled as QY-B;
3) in described clear liquid QY-B, according to the final concentration of 2.0-4.0mol/L, add while stirring the neutral salt particle of porphyrize, after neutral salt dissolves completely, standing 2-10h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-C;
4) described precipitate C D-C is dissolved in after 0.2-0.5mol/L acetum and adjusts pH to 7.2-7.5 with sodium hydroxide solution, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 1.2-2.5mol/L, after neutral salt dissolves completely, standing 2-10h carries out centrifugal again, abandon supernatant liquor, gained precipitation is labeled as CD-D;
5) described precipitate C D-D is dissolved in after 0.5mol/L acetum, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 0.3-0.8mol/L, after neutral salt dissolves completely, standing 2-10h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-E;
6) described precipitate C D-E is dissolved in to 0.2-0.5mol/L acetum, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 1.2-2.5mol/L, after dissolving completely, neutral salt adjusts pH to 7.2-7.5 with sodium hydroxide solution, after standing 2-10h, carry out centrifugal, precipitation is dissolved in after 0.5mol/L acetum again, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 0.3-0.8mol/L, after neutral salt dissolves completely, standing 2-10h carries out centrifugal again, abandon supernatant liquor, gained precipitation is dissolved in and proceeds to dialysis band after 0.2-0.5mol/L acetum deionized water is fully dialysed, products therefrom is people's placenta type i collagen material after lyophilize,
7) described CD-B is dissolved in after 0.2-0.5mol/L acetum and adjusts pH to 7.2-7.5 with sodium hydroxide solution, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 0.9-2.0mol/L, after neutral salt dissolves completely, standing 2-10h carries out centrifugal again, abandon supernatant liquor, gained precipitation is labeled as CD-F;
8) described precipitate C D-F is dissolved in after 0.5mol/L acetum, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 0.3-0.8mol/L, after neutral salt dissolves completely, standing 2-10h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-G;
9) described precipitate C D-G is dissolved in after 0.2-0.5mol/L acetum, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 0.9-2.0mol/L, after dissolving completely, neutral salt adjusts pH to 7.2-7.5 with sodium hydroxide solution, after standing 2-10h, carry out centrifugal, precipitation is dissolved in after 0.5mol/L acetum again, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 0.3-0.8mol/L, after neutral salt dissolves completely, standing 2-10h carries out centrifugal again, abandon supernatant liquor, gained precipitation is dissolved in to 0.2-0.5mol/L acetum to be proceeded to dialysis band deionized water is fully dialysed, products therefrom is people's placenta III Collagen Type VI material after lyophilize,
10) in described QY-A, according to the final concentration of 0.9-2.0mol/L, add while stirring the neutral salt particle of porphyrize, after neutral salt dissolves completely, standing 2-10h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-H;
11) described precipitate C D-H is dissolved in after 0.2-0.5mol/L acetum and adjusts pH to 7.2-7.5 with sodium hydroxide solution, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 1.8-4.5mol/L, after neutral salt dissolves completely, standing 2-10h carries out centrifugal again, abandon supernatant liquor, gained precipitation is labeled as CD-I;
12) described precipitate C D-I is dissolved in after 0.2-0.5mol/L acetum, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 0.6-1.9mol/L, after neutral salt dissolves completely, standing 2-10h carries out centrifugally again, abandons supernatant liquor, and gained precipitation is labeled as CD-J;
13) described in general, stating precipitate C D-J is dissolved in after 0.2-0.5mol/L acetum, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 1.8-4.5mol/L, after dissolving completely, neutral salt adjusts pH to 7.2-7.5 with sodium hydroxide solution, after standing 2-10h, carry out centrifugal, precipitation is dissolved in after 0.2-0.5mol/L acetum again, the neutral salt particle that adds while stirring porphyrize according to the final concentration of 0.6-1.9mol/L, after neutral salt dissolves completely, standing 2-10h carries out centrifugal again, abandon supernatant liquor, gained precipitation is dissolved in to 0.05mol/L Tris-HCl, 0.1mol/L NaCl, pH be the damping fluid of 7.2-7.5 and same damping fluid is fully dialysed after carry out centrifugation, gained precipitation is labeled as CD-K, supernatant liquor is labeled as QY-C,
14) described clear liquid QY-C is proceeded to dialysis band deionized water is fully dialysed, products therefrom is people's placenta IV Collagen Type VI material after lyophilize;
15) described in general, state precipitate C D-K, be dissolved in after 0.2-0.5mol/L acetum, proceeding to dialysis band deionized water is fully dialysed, products therefrom is people's placenta collagen type v material after lyophilize.
6. according to the human placental collagen preparation method described in any one in claim 1-5, it is characterized in that, in described people's placenta I type, III type, IV type or collagen type v, comprise intrafascicular at least one of protofibril shape collagen, microfibrous collagen, particulate state fiber, collegen filament and collegen filament.
7. human placental collagen preparation method according to claim 3, is characterized in that, described metal chelating agent is at least one in aminocarboxylate and hydroxycarboxylate.
8. according to the human placental collagen preparation method described in claim 3 or 5, it is characterized in that, described neutral salt is at least one in sodium-chlor, ammonium sulfate, sodium sulfate, Trisodium Citrate and ammonium phosphate.
9. human placental collagen preparation method according to claim 3, it is characterized in that, described pre-treatment damping fluid is a kind of in Tutofusin tris-hydrochloric acid, Sodium phosphate dibasic-citric acid, Sodium phosphate dibasic-SODIUM PHOSPHATE, MONOBASIC, potassium primary phosphate-sodium hydrate buffer solution.
10. the collagen extracting from human placenta, it is characterized in that, described collagen is prepared from according to method described in any one in claim 1-9, and the collagen of gained is for keeping a kind of in people I type, III type, IV type or the V-type medical collagen material of natural structure.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310448097.8A CN103525890B (en) | 2013-09-27 | 2013-09-27 | A kind of preparation method of human placental collagen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310448097.8A CN103525890B (en) | 2013-09-27 | 2013-09-27 | A kind of preparation method of human placental collagen |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103525890A true CN103525890A (en) | 2014-01-22 |
| CN103525890B CN103525890B (en) | 2016-08-10 |
Family
ID=49928237
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310448097.8A Active CN103525890B (en) | 2013-09-27 | 2013-09-27 | A kind of preparation method of human placental collagen |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103525890B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104120162A (en) * | 2014-07-23 | 2014-10-29 | 成都清科生物科技有限公司 | Preparation method of water soluble active collagen and epidermis repair growth factor liquid |
| CN104940100A (en) * | 2015-06-19 | 2015-09-30 | 成都清科生物科技有限公司 | Human placenta extract, method for preparing the same and application |
| CN104195205B (en) * | 2014-04-24 | 2019-08-16 | 吉林金梓源生物科技股份有限公司 | Method and prepared high activity collagen peptide and application thereof from animal placenta preparation high activity collagen peptide |
| CN111592592A (en) * | 2020-03-02 | 2020-08-28 | 内蒙古林宁医疗科技有限公司 | Selective extraction preparation method and application of different types of collagen of human placenta |
| CN113402599A (en) * | 2021-05-18 | 2021-09-17 | 铜仁市泛特尔生物技术有限公司 | Optimization method for extracting and purifying type I collagen from human placenta |
| CN115572328A (en) * | 2022-07-24 | 2023-01-06 | 胶原蛋白(武汉)生物科技有限公司 | Preparation method of medical grade III type collagen and III type collagen |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1081379A (en) * | 1992-07-29 | 1994-02-02 | 中国预防医学科学院劳动卫生与职业病研究所 | Human placental collagen and preparation method thereof |
-
2013
- 2013-09-27 CN CN201310448097.8A patent/CN103525890B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1081379A (en) * | 1992-07-29 | 1994-02-02 | 中国预防医学科学院劳动卫生与职业病研究所 | Human placental collagen and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 姚敏杰等: "人胎盘III、IV、V型胶原的提取与纯化", 《空军医高专学报》 * |
| 朱明华等: "人胎盘中I、III、IV型胶原的提取", 《生物化学与生物物理进展》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104195205B (en) * | 2014-04-24 | 2019-08-16 | 吉林金梓源生物科技股份有限公司 | Method and prepared high activity collagen peptide and application thereof from animal placenta preparation high activity collagen peptide |
| CN104120162A (en) * | 2014-07-23 | 2014-10-29 | 成都清科生物科技有限公司 | Preparation method of water soluble active collagen and epidermis repair growth factor liquid |
| CN104940100A (en) * | 2015-06-19 | 2015-09-30 | 成都清科生物科技有限公司 | Human placenta extract, method for preparing the same and application |
| CN111592592A (en) * | 2020-03-02 | 2020-08-28 | 内蒙古林宁医疗科技有限公司 | Selective extraction preparation method and application of different types of collagen of human placenta |
| CN113402599A (en) * | 2021-05-18 | 2021-09-17 | 铜仁市泛特尔生物技术有限公司 | Optimization method for extracting and purifying type I collagen from human placenta |
| CN115572328A (en) * | 2022-07-24 | 2023-01-06 | 胶原蛋白(武汉)生物科技有限公司 | Preparation method of medical grade III type collagen and III type collagen |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103525890B (en) | 2016-08-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103525890B (en) | A kind of preparation method of human placental collagen | |
| CN100569091C (en) | A kind of preparation method of collagen of red fish skin in deep sea | |
| CN103184262B (en) | Method for extracting and purifying pepsin-soluble collagens of black sea cucumber in East China Sea | |
| CN100381528C (en) | Preparation method of undenatured fish scale collagen | |
| CN103756974A (en) | Porcine epidemic diarrhea virus, and culture method and application thereof | |
| CN102276716B (en) | Method for extracting unmodified natural collagen from bullfrog skin | |
| CN102676619A (en) | Comprehensive utilization process for marine fish skins | |
| CN109735521B (en) | The crystallization extracting method of urase in a kind of maljoe | |
| CN109527270A (en) | Leech specific nutrient induces the extracting method of liquid and natural hirudin | |
| CN103408787B (en) | Sponge material of collagen from freshwater fish and preparation method thereof | |
| CN107095312A (en) | A kind of krill polypeptide formulations with reducing blood lipid ability and preparation method thereof | |
| CN106032546A (en) | Method for extracting medical material-based collagen from fish scales | |
| CN104774895B (en) | The method for preparing wood frog collagen | |
| CN1486745A (en) | Stem cell prepn for treating tissue ischemia disease and its prepn process | |
| CN105085605B (en) | A kind of extracting method of rice albumin and globulin mixed protein | |
| CN102936611A (en) | Collagen extracted from black butterflyfish skins with scales and application of collagen | |
| CN104292348B (en) | A kind of method that polysaccharide and albumen are synchronously extracted in the processing byproduct from pleurotus eryngii | |
| CN109486825A (en) | Sepiella maindroni Spef1 and application thereof | |
| CN109320379A (en) | A kind of phosphorus ammonium preparation method containing alginic acid synergistic carrier suitable for basic soil | |
| CN108531536A (en) | The preparation method and applications of Shelled Turtle Trionyx Sinensis collagen peptide | |
| CN110036950A (en) | A kind of artificial hybridization breeding method of sea-farming rainbow trout | |
| CN109295042A (en) | A kind of preparation method and applications of natural Sipunculus nudus plasmin | |
| CN110305758A (en) | A kind of production method that wizened bacterium is steep in wine | |
| CN110241074A (en) | A kind of preparation method of Novel goat sperm in vitro agonist | |
| CN109222110A (en) | A kind of antifatigue soluble granule and preparation method of the polypeptide dry powder containing Goat Placenta |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |