CN103525890B - A kind of preparation method of human placental collagen - Google Patents
A kind of preparation method of human placental collagen Download PDFInfo
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Abstract
The present invention relates to the preparation method of a kind of human placental collagen, described method is with human placenta as raw material, carry out pretreatment, enzymic digestion, salt fractionation successively, obtain at least one kept in people's I type of natural structure, type III, IV type and V-type medical collagen material respectively.The method has been greatly shortened Human plactnta I type, type III, IV type and the manufacturing cycle of collagen type v material, improve preparation efficiency and productivity, the quality simultaneously making product is the most stable, described method can also extract certain types of collagen according to actual needs, people's I type, type III, IV type and the collagen type v material with natural structure can also be obtained the same period, make full use of human placenta resource.
Description
Technical field
The present invention relates to a kind of Healthy People Placenta Hominis to obtain after normal labor as raw material, preparation keeps Human plactnta I type, type III, IV type and the method for collagen type v material of natural structure, belongs to the preparation field of bio-medical material.
Background technology
Natural collagen is extensively recognized as a species specific structural protein, its feature and advantage, and one of important source material becoming bio-medical material further.At present known at least 26 kinds of collagen-types in human body, each of which is that the structure function of collagen adds specific function.Collagen content in mammalian placenta, particularly Human plactnta is the abundantest, contains I type, type III, IV type and collagen type v simultaneously, is therefore the most valuable a kind of raw material preparing natural medical collagen.
Using Human plactnta is various natural medical collagen prepared by raw material, do not carry any of people's infectious disease, overcoming and using the heterogenous animal tissue such as cattle, pig or organ is that raw material prepares collagen virus that may be present pollution or the risk of potential zoonosis, and therefore human placental collagen has higher safety.
For raw material preparation about with Human plactnta there is Human plactnta I type, type III, IV type and the patent of collagen type v of natural structure and report relatively fewer.The domestic research carrying out a small amount of human placental collagen of short-term in earlier 1990s, main representative achievement has: Liu Bingci is with Human plactnta as raw material, through pepsin digestion, saltout after obtain precipitate A, and obtain precipitate B after ackd salt extraction is centrifugal further, it is used for filling miniature pitting skin injury (Chinese invention patent, publication number: CN1081379) after two kinds of precipitations are mixed and added into growth hormone and lignocaine in varing proportions
Up to the present, there is no documents and materials and there is with Human plactnta for raw material preparation people's I type, type III, IV type and the report of collagen type v material of natural structure.Therefore, within the shorter cycle, prepare natural structure people's I type, type III, IV type and the V-type medical collagen material with stability with Healthy People placenta tissue for primary raw material, there is clear meaning.
Summary of the invention
By finding prior art has the disadvantage that to comprehensive analysis of prior art
1. containing I type, type III, IV type and collagen type v in Human plactnta, but prior art multipotency extracts the collagen of three types, it is impossible to obtain the collagen-based materials of four types simultaneously, the most abundant to the human placenta utilization of resources；
The most existing collagen extraction techniques cycle is tediously long, need more than 15 days, complex steps, so that extraction efficiency and productivity are the lowest, the key link affecting product quality in technique is difficult to control to, easily cause product quality to be not sufficiently stable, finally affect application effect and the value of collagen and collagen base biological medical material.
It is an object of the invention to provide the preparation method of a kind of human placental collagen, hence it is evident that shorten the cycle extracting collagen, obtain high-quality human placental collagen.
The present invention is achieved through the following technical solutions:
The preparation method of a kind of human placental collagen, with human placenta as raw material, carries out pretreatment, enzymic digestion, salt fractionation successively, obtains at least one kept in people's I type of natural structure, type III, IV type and V-type medical collagen material respectively.
By described pre-treatment step, placenta tissue is carried out, crushes and remove major impurity；
In described enzymatic digestion stage, use the specific site between pepsin end peptide and helical region in collagen fiber to interrupt peptide chain, make collagen be dissolved in Digestive system thus be separated with other component of organization.
Again by described salt fractionation, different types of collagen is separated, purifies, to obtain certain types of collagen.
Alternatively, described human placenta is that pre-puerpera carries out the placenta tissue that is fresh or that preserve through freezing screening and obtaining after normal labor that can spread disease before childbirth, therefore there is no the viral pathogens of known pollution placenta tissue, so that gained collagen has 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 Hominis, such as amniotic membrane, chorion or complete Placenta Hominis.
Alternatively, described pre-treatment step specifically includes: is fully cleaned by placenta tissue and is cut into small pieces, centrifugal segregation supernatant after low-temperature homogenate, and precipitate with deionized water is centrifuged after cleaning again, repeatedly clean centrifugal 2-4 time transparent to supernatant；Precipitation pH is 7.2-7.4, the pre-treatment buffer that concentration is 0.01-1.0mol/L containing 1.0-20.0 mmol/L metal chelating agent, 0.5-3.0mol/L neutral salt soaks and be slowly stirred 2-4h, and mixing speed can not be too fast, slightly rotates with liquid level and is advisable；Centrifuged pellet cleans once with pre-treatment buffer and is centrifuged, and takes precipitation standby.Impurity can be removed more thoroughly, it is thus achieved that the mixture that collagen content is higher by this step, and the shortest, efficiency is high.
Described metal chelating agent can be at least one in aminocarboxylate (such as edetate, diethylenetriamine pentacarboxylic acid salt etc.) and hydroxycarboxylate's (such as sodium tartrate, sodium gluconate).Use described metal chelating agent can preferably eliminate the various metal impurities contained in raw material, also remove other active biomolecule various simultaneously.
Described pre-treatment buffer can be the one in trishydroxymethylaminomethane-hydrochloric acid, disodium hydrogen phosphate-citric acid, disodium hydrogen phosphate-sodium dihydrogen phosphate, potassium dihydrogen phosphate-sodium hydrate buffer solution.Use stablizing of described buffer on the one hand maintenance system pH value, the effect of cleaning foreign protein, the wherein best results of trishydroxymethylaminomethane-hydrochloric acid cleaning foreign protein can also be played simultaneously.
Alternatively, described enzymic digestion is to be soaked in Digestive system by pretreated human placenta, described Digestive system is that pepsin (vigor >=250U/mg) (also can take other weak acid with acetic acid, such as citric acid) mixed solution, 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；Described Digestive system quality is 5-10 times of human placenta solid weight, is slowly stirred continuously under cryogenic conditions, and mixing speed can not be too fast, slightly rotates with liquid level and is advisable；After enzymic digestion 6-30h, it is standby that centrifugation takes supernatant.Using above-mentioned enzymic digestion method, digestible degree is moderate, products therefrom steady quality, and relative to existing human placental collagen extraction process, substantially reduces digestion time, improve efficiency.
Alternatively, the required collagen-type extracted according to the operation of described salt fractionation, from following salt fractionation step, choose corresponding step combine:
1) regulating the sample after enzymic digestion with acetic acid is 2.5-2.8 to pH, finely ground neutral salt granule is added while stirring according to the final concentration of 0.5-1.2mol/L, standing 2-10h after neutral salt is completely dissolved to be centrifuged, gained precipitation is labeled as CD-A, and supernatant is labeled as QY-A again；
2) by after described precipitate C D-A 0.5mol/L acetate dissolution, finely ground neutral salt granule is added while stirring according to the final concentration of 0.3-0.8mol/L, standing 2-10h after neutral salt is completely dissolved to be centrifuged, gained precipitation is labeled as CD-B, and supernatant is labeled as QY-B again；
3) in described clear liquid QY-B, adding finely ground neutral salt granule while stirring according to the final concentration of 2.0-4.0mol/L, stand 2-10h and be centrifuged after neutral salt is completely dissolved, abandon supernatant, gained precipitation is labeled as CD-C；
4) pH to 7.2-7.5 is adjusted with sodium hydroxide solution after described precipitate C D-C is dissolved in 0.2-0.5mol/L acetum, finely ground neutral salt granule is added while stirring according to the final concentration of 1.2-2.5mol/L, after neutral salt is completely dissolved, stand 2-10h be centrifuged again, abandoning supernatant, gained precipitation is labeled as CD-D；
5) after described precipitate C D-D being dissolved in 0.5mol/L acetum, adding finely ground neutral salt granule while stirring according to the final concentration of 0.3-0.8mol/L, stand 2-10h and be centrifuged after neutral salt is completely dissolved, abandon supernatant, gained precipitation is labeled as CD-E；
6) described precipitate C D-E is dissolved in 0.2-0.5mol/L acetum, finely ground neutral salt granule is added while stirring according to the final concentration of 1.2-2.5mol/L, pH to 7.2-7.5 is adjusted with sodium hydroxide solution after neutral salt is completely dissolved, it is centrifuged after standing 2-10h, after again precipitation being dissolved in 0.5mol/L acetum, finely ground neutral salt granule is added while stirring according to the final concentration of 0.3-0.8mol/L, after neutral salt is completely dissolved, stand 2-10h be centrifuged again, abandon supernatant, gained precipitation is proceeded to dialysis band deionized water is fully dialysed, Human plactnta type i collagen material it is after products therefrom is freeze-dried；
7) pH to 7.2-7.5 is adjusted with sodium hydroxide solution after described CD-B is dissolved in 0.2-0.5mol/L acetum, finely ground neutral salt granule is added while stirring according to the final concentration of 0.9-2.0mol/L, after neutral salt is completely dissolved, stand 2-10h be centrifuged again, abandoning supernatant, gained precipitation is labeled as CD-F；
8) after described precipitate C D-F being dissolved in 0.5mol/L acetum, adding finely ground neutral salt granule while stirring according to the final concentration of 0.3-0.8mol/L, stand 2-10h and be centrifuged after neutral salt is completely dissolved, abandon supernatant, gained precipitation is labeled as CD-G；
9) after described precipitate C D-G being dissolved in 0.2-0.5mol/L acetum, finely ground neutral salt granule is added while stirring according to the final concentration of 0.9-2.0mol/L, pH to 7.2-7.5 is adjusted with sodium hydroxide solution after neutral salt is completely dissolved, it is centrifuged after standing 2-10h, after again precipitation being dissolved in 0.5mol/L acetum, finely ground neutral salt granule is added while stirring according to the final concentration of 0.3-0.8mol/L, after neutral salt is completely dissolved, stand 2-10h be centrifuged again, abandon supernatant, gained precipitation is proceeded to dialysis band deionized water is fully dialysed, Human plactnta type III collagen-based materials it is after products therefrom is freeze-dried；
10) in described QY-A, adding finely ground neutral salt granule while stirring according to the final concentration of 0.9-2.0mol/L, stand 2-10h and be centrifuged after neutral salt is completely dissolved, abandon supernatant, gained precipitation is labeled as CD-H；
11) pH to 7.2-7.5 is adjusted with sodium hydroxide solution after described precipitate C D-H is dissolved in 0.2-0.5mol/L acetum, finely ground neutral salt granule is added while stirring according to the final concentration of 1.8-4.5mol/L, after neutral salt is completely dissolved, stand 2-10h be centrifuged again, abandoning supernatant, gained precipitation is labeled as CD-I；
12) after described precipitate C D-I being dissolved in 0.2-0.5mol/L acetum, finely ground neutral salt granule is added while stirring according to the final concentration of 0.6-1.9mol/L, standing 2-10h after neutral salt is completely dissolved to be centrifuged again, abandon supernatant, gained precipitation is labeled as CD-J；
null13) state after precipitate C D-J is dissolved in 0.2-0.5mol/L acetum by described，Finely ground neutral salt granule is added while stirring according to the final concentration of 1.8-4.5mol/L，PH to 7.2-7.5 is adjusted with sodium hydroxide solution after neutral salt is completely dissolved，It is centrifuged after standing 2-10h，After again precipitation being dissolved in 0.2-0.5mol/L acetum，Finely ground neutral salt granule is added while stirring according to the final concentration of 0.6-1.9mol/L，After neutral salt is completely dissolved, stand 2-10h be centrifuged again，Abandon supernatant，Gained precipitation is dissolved in 0.05mol/L Tris-HCl、0.1mol/L NaCl，PH is the buffer of 7.2-7.5 and carries out centrifugation after fully dialysing same buffer，Gained precipitation is labeled as CD-K，Supernatant is labeled as QY-C；
14) described clear liquid QY-C proceeds to dialysis band deionized water is fully dialysed, after products therefrom is freeze-dried, be Human plactnta IV Collagen Type VI material；
15) state precipitate C D-K by described, after being dissolved in 0.2-0.5mol/L acetum, proceed to dialysis band and deionized water is fully dialysed, after products therefrom is freeze-dried, be Human plactnta collagen type v material.
This technological operation is flexible, can select corresponding operating procedure, such as: when only needing to extract type i collagen, then operate successively to step 6) by described step 1) according to the type of required extraction collagen；Only need extract type III collagen time, then select described step 1), step 2) and step 7) operate successively to step 9)；When only needing to extract IV Collagen Type VI, then described step 1), step 10) is selected to operate successively to step 14)；When only needing to extract collagen type v, then described step 1), step 10) is selected to operate successively to step 13) and step 15)；Then corresponding step is combined as extracted multiple collagen-type, operate by step 1) to step 15), from placenta tissue, the collagen (I type, type III, IV type and V-type) of four types can be isolated the same period.
Human plactnta I type, type III, IV type or collagen type v prepared by employing the method for the invention comprise at least one in fibril shape collagen, microfibrous collagen, graininess fiber, collagen fiber and collagenous fiber bundle.The collagen of gained farthest remains natural collagen original triple helix structure.
Heretofore described neutral salt is at least one in sodium chloride, ammonium sulfate, sodium sulfate, sodium citrate and ammonium phosphate.
Present invention also offers a kind of collagen extracted from human placenta, described collagen uses the method for the invention to be prepared from, and the collagen of gained is to keep the one in people's I type of natural structure, type III, IV type or V-type medical collagen material.Described collagen comprises at least one in fibril shape collagen, microfibrous collagen, graininess fiber, collagen fiber and collagenous fiber bundle, farthest remains natural collagen original triple helix structure.Described collagen purity is high, and biological safety is high, through simply disinfecting biomedicine fields such as i.e. can be used for tissue engineering bracket, cell culture vector, pathological study and drug development, such as cell culture vector and surface modifying material；Primary raw material as tissue engineering bracket material；Or it is combined other material further, it is applied to other field of biomedical material.
All features disclosed in this specification, or disclosed all methods or during step, in addition to mutually exclusive feature and/or step, all can combine by any way.
Beneficial effects of the present invention:
1, placental collagen preparation method of the present invention has been greatly shortened Human plactnta I type, type III, IV type and the manufacturing cycle of collagen type v material, improves preparation efficiency and productivity, and the quality simultaneously making product is the most stable.
2, the method for the invention is used can to shift to an earlier date certain types of collagen according to actual needs, it is also possible to the same period obtains people's I type, type III, IV type and the collagen type v material with natural structure, makes full use of human placenta resource.
Accompanying drawing explanation
Fig. 1 is the preparation technology flow chart of the present invention；
Fig. 2 is differential scanning calorimeter (DSC) the testing result figure of each Collagen Type VI of preparation in embodiment of the present invention, 1 be I type, 3 is type III, 4 is IV type, 5 is collagen type v in figure.
Detailed description of the invention
The preparation technology flow process of the present invention is: with human placenta as raw material, carries out pretreatment, enzymic digestion, salt fractionation successively.
Wherein pre-treatment step includes fully being cleaned by placenta tissue and being cut into small pieces, centrifugal segregation supernatant after low-temperature homogenate, precipitate with deionized water repeatedly clean be centrifuged to supernatant transparent；Precipitation is centrifugal after soaking with the pre-treatment buffer containing metal chelating agent and neutral salt and be slowly stirred, and gained precipitation is cleaned once with pre-treatment buffer and is centrifuged, and takes precipitation standby；Enzymatic digestion stage includes being soaked in Digestive system pretreated human placenta, is slowly stirred continuously under cryogenic conditions, and after enzymic digestion 6-30h, it is standby that centrifugation takes supernatant；Described salt fractionation is the difference of the dissolubility utilizing the collagen without type in the neutral salt solution of variable concentrations, different types of collagen is separated, purifies through making collagen mixture dissolve repeatedly in the saline solution of variable concentrations and precipitate, to obtain certain types of collagen.
Several most preferred embodiments for the present invention set forth below, it should be understood that these embodiments are only used for the purpose of illustration, are never limited in protection scope of the present invention.
Embodiment
1
Extract collagen according to the following steps:
(1) the fresh whole person's Placenta Hominis obtained through disease screening normal labor is selected；
(2) placenta tissue fully cleaned and be cut into small pieces, centrifugal segregation supernatant after low-temperature homogenate, precipitate with deionized water is centrifuged after cleaning again, repeatedly clean centrifugal 4 times transparent to supernatant；Precipitation pH is 7.2-7.4, trishydroxymethylaminomethane-hydrochloric acid (Tris-HCl) buffer containing 10 mmol/L sodium ethylene diamine tetracetates (Na2EDTA), the 0.5mol/L of 1.0 mol/L NaCl soaks and be slowly stirred 4h, mixing speed can not be too fast, slightly rotates with liquid level and is advisable；Being centrifuged after having stirred, centrifuged pellet once and is centrifuged by Tris-HCl buffer solution for cleaning again, and finally the centrifugal precipitation obtained is weighed and as the foundation of follow-up measurement；
(3) precipitation obtained final in step (2) is soaked in Digestive system, described Digestive system is pH 2.8, pepsin (vigor >=250U/mg) concentration is acetic acid (HAC) solution of the 0.5mol/L of 5g/L, described Digestive system quality is 5 times of Sediment weight, it is slowly stirred continuously under cryogenic conditions, mixing speed can not be too fast, slightly rotates with liquid level and is advisable；After enzymic digestion 12h, it is standby that centrifugation takes supernatant.
(4) carry out salt fractionation according to following steps to operate:
1) regulating the sample after enzymic digestion with acetic acid is 2.8 to pH, adds finely ground NaCl granule while stirring according to the final concentration of 0.8mol/L, stands 4h and be centrifuged after NaCl is completely dissolved, and gained precipitation is labeled as CD-A, and supernatant is labeled as QY-A；
2) by after described precipitate C D-A 0.5mol/L acetate dissolution, adding finely ground NaCl granule while stirring according to the final concentration of 0.7mol/L, stand 4h and be centrifuged after NaCl is completely dissolved, gained precipitation is labeled as CD-B, and supernatant is labeled as QY-B；
3) in described clear liquid QY-B, adding finely ground NaCl granule while stirring according to the final concentration of 4.0mol/L, stand 8h and be centrifuged after NaCl is completely dissolved, abandon supernatant, gained precipitation is labeled as CD-C；
4) pH to 7.4 is adjusted with sodium hydroxide solution after described precipitate C D-C is dissolved in 0.2mol/L acetum, finely ground NaCl granule is added while stirring according to the final concentration of 2.5mol/L, standing 4h after NaCl is completely dissolved to be centrifuged again, abandon supernatant, gained precipitation is labeled as CD-D；
5) after described precipitate C D-D being dissolved in 0.5mol/L acetum, adding finely ground NaCl granule while stirring according to the final concentration of 0.7mol/L, stand 4h and be centrifuged after NaCl is completely dissolved, abandon supernatant, gained precipitation is labeled as CD-E；
6) described precipitate C D-E is dissolved in 0.2mol/L acetum, finely ground NaCl granule is added while stirring according to the final concentration of 2.5mol/L, pH to 7.4 is adjusted with sodium hydroxide solution after NaCl is completely dissolved, it is centrifuged after standing 4h, after again precipitation being dissolved in 0.5mol/L acetum, finely ground NaCl granule is added while stirring according to the final concentration of 0.7mol/L, after NaCl is completely dissolved, stand 4h be centrifuged again, abandon supernatant, proceed to dialysis band after gained precipitation is dissolved in 0.2mol/L acetum deionized water is fully dialysed, Human plactnta type i collagen material it is after products therefrom is freeze-dried；
7) pH to 7.4 is adjusted with sodium hydroxide solution after described CD-B is dissolved in 0.2mol/L acetum, finely ground NaCl granule is added while stirring according to the final concentration of 2.0mol/L, standing 2h after NaCl is completely dissolved to be centrifuged again, abandon supernatant, gained precipitation is labeled as CD-F；
8) after described precipitate C D-F being dissolved in 0.5mol/L acetum, adding finely ground NaCl granule while stirring according to the final concentration of 0.7mol/L, stand 4h and be centrifuged after NaCl is completely dissolved, abandon supernatant, gained precipitation is labeled as CD-G；
9) after described precipitate C D-G being dissolved in 0.2mol/L acetum, finely ground NaCl granule is added while stirring according to the final concentration of 2.0mol/L, pH to 7.4 is adjusted with sodium hydroxide solution after NaCl is completely dissolved, it is centrifuged after standing 2h, after again precipitation being dissolved in 0.5mol/L acetum, finely ground NaCl granule is added while stirring according to the final concentration of 0.7mol/L, after NaCl is completely dissolved, stand 2h be centrifuged again, abandon supernatant, proceed to dialysis band after gained precipitation is dissolved in 0.2mol/L acetum deionized water is fully dialysed, Human plactnta type III collagen-based materials it is after products therefrom is freeze-dried；
10) in described QY-A, adding finely ground NaCl granule while stirring according to the final concentration of 2.0mol/L, stand 4h and be centrifuged after NaCl is completely dissolved, abandon supernatant, gained precipitation is labeled as CD-H；
11) pH to 7.4 is adjusted with sodium hydroxide solution after described precipitate C D-H is dissolved in 0.2mol/L acetum, finely ground NaCl granule is added while stirring according to the final concentration of 4.5mol/L, standing 4h after NaCl is completely dissolved to be centrifuged again, abandon supernatant, gained precipitation is labeled as CD-I；
12) after described precipitate C D-I being dissolved in 0.5mol/L acetum, adding finely ground NaCl granule while stirring according to the final concentration of 1.2mol/L, stand 2h and be centrifuged after NaCl is completely dissolved, abandon supernatant, gained precipitation is labeled as CD-J；
null13) state after precipitate C D-J is dissolved in 0.2mol/L acetum by described，Finely ground NaCl granule is added while stirring according to the final concentration of 4.5mol/L，PH to 7.4 is adjusted with sodium hydroxide solution after NaCl is completely dissolved，It is centrifuged after standing 4h，After again precipitation being dissolved in 0.5mol/L acetum，Finely ground NaCl granule is added while stirring according to the final concentration of 1.2mol/L，After NaCl is completely dissolved, stand 2h be centrifuged again，Abandon supernatant，Gained precipitation is dissolved in 0.05mol/L Tris-HCl、0.1mol/L NaCl，PH is the buffer of 7.2-7.5 and carries out centrifugation after fully dialysing same buffer，Gained precipitation is labeled as CD-K，Supernatant is labeled as QY-C；
14) described clear liquid QY-C proceeds to dialysis band deionized water is fully dialysed, after products therefrom is freeze-dried, be Human plactnta IV Collagen Type VI material；
15) state precipitate C D-K by described, after being dissolved in 0.2mol/L acetum, proceed to dialysis band and deionized water is fully dialysed, after products therefrom is freeze-dried, be Human plactnta collagen type v material.
Using the preparation of above-mentioned technology to have people's I type, type III, IV type and the collagen type v material of natural structure, cycle time was to 5-10 days, and the efficiency and the productivity that improve preparation are higher.The raw material that the above-mentioned medical collagen material obtained can be used as biomedical material is further processed into the collagen base biological medical material of other forms, ion exchange chromatography isochromatic spectrum method can also be used to be further purified, it is thus achieved that the various medical collagen material that higher purity and molecular weight distribution are more concentrated simultaneously.The feature each unique in view of different collagen and performance, it is thus achieved that the medical collagen material of typing can play respective Biological characteristics and advantage, meets the demand of different biomedical material research and clinical practice, improve the added value of collagen-based materials further.
Embodiment
2
Extract collagen according to the following steps:
(1) the fresh whole person's Placenta Hominis obtained through disease screening normal labor is selected；
(2) placenta tissue fully cleaned and be cut into small pieces, centrifugal segregation supernatant after low-temperature homogenate, precipitate with deionized water is centrifuged after cleaning again, repeatedly clean centrifugal 4 times transparent to supernatant；Precipitation pH is 7.2, containing 20 mmol/L sodium ethylene diamine tetracetate (Na2EDTA), trishydroxymethylaminomethane-hydrochloric acid (Tris-HCl) buffer of the 0.05mol/L of 2.0 mol/L NaCl soak and be slowly stirred 4h, mixing speed can not be too fast, slightly rotates with liquid level and is advisable；Being centrifuged after having stirred, centrifuged pellet once and is centrifuged by Tris-HCl buffer solution for cleaning again, and finally the centrifugal precipitation obtained is weighed and as the foundation of follow-up measurement；
(3) precipitation obtained final in step (2) is soaked in Digestive system, described Digestive system is pH 2.5, pepsin (vigor >=250U/mg) concentration is acetic acid (HAC) solution of the 0.8mol/L of 1g/L, described Digestive system quality is 10 times of Sediment weight, it is slowly stirred continuously under cryogenic conditions, mixing speed can not be too fast, slightly rotates with liquid level and is advisable；After enzymic digestion 24h, it is standby that centrifugation takes supernatant.
(4) carry out salt fractionation according to following steps to operate:
1) regulating the sample after enzymic digestion with acetic acid is 2.5 to pH, adds finely ground (NH while stirring according to the final concentration of 0.6mol/L4)2SO4Granule, treats (NH4)2SO4Standing 4h after being completely dissolved to be centrifuged, gained precipitation is labeled as CD-A, and supernatant is labeled as QY-A again；
2) by after described precipitate C D-A 0.5mol/L acetate dissolution, finely ground (NH is added while stirring according to the final concentration of 0.4mol/L4)2SO4Granule, treats (NH4)2SO4Standing 4h after being completely dissolved to be centrifuged, gained precipitation is labeled as CD-B, and supernatant is labeled as QY-B again；
3) in described clear liquid QY-B, finely ground (NH is added while stirring according to the final concentration of 2.0mol/L4)2SO4Granule, treats (NH4)2SO4Standing 4h after being completely dissolved to be centrifuged again, abandon supernatant, gained precipitation is labeled as CD-C；
4) adjust pH to 7.5 with sodium hydroxide solution after described precipitate C D-C is dissolved in 0.5mol/L acetum, add finely ground (NH while stirring according to the final concentration of 1.2mol/L4)2SO4Granule, treats (NH4)2SO4Standing 4h after being completely dissolved to be centrifuged again, abandon supernatant, gained precipitation is labeled as CD-D；
5), after described precipitate C D-D being dissolved in 0.2mol/L acetum, finely ground (NH is added while stirring according to the final concentration of 0.3mol/L4)2SO4Granule, treats (NH4)2SO4Standing 4h after being completely dissolved to be centrifuged again, abandon supernatant, gained precipitation is labeled as CD-E；
6) described precipitate C D-E is dissolved in 0.5mol/L acetum, adds finely ground (NH while stirring according to the final concentration of 1.2mol/L4)2SO4Granule, treats (NH4)2SO4Adjust pH to 7.5 with sodium hydroxide solution after being completely dissolved, stand after 2h and be centrifuged, then after precipitation is dissolved in 0.5mol/L acetum, add finely ground (NH while stirring according to the final concentration of 0.3mol/L4)2SO4Granule, treats (NH4)2SO4Stand 2h after being completely dissolved to be centrifuged again, abandon supernatant, proceed to dialysis band after gained precipitation is dissolved in 0.2mol/L acetum and deionized water is fully dialysed, after products therefrom is freeze-dried, be Human plactnta type i collagen material；
7) adjust pH to 7.5 with sodium hydroxide solution after described CD-B is dissolved in 0.2mol/L acetum, add finely ground (NH while stirring according to the final concentration of 0.9mol/L4)2SO4Granule, treats (NH4)2SO4Standing 4h after being completely dissolved to be centrifuged again, abandon supernatant, gained precipitation is labeled as CD-F；
8), after described precipitate C D-F being dissolved in 0.5mol/L acetum, finely ground (NH is added while stirring according to the final concentration of 0.35mol/L4)2SO4Granule, treats (NH4)2SO4Standing 4h after being completely dissolved to be centrifuged again, abandon supernatant, gained precipitation is labeled as CD-G；
9), after described precipitate C D-G being dissolved in 0.2mol/L acetum, finely ground (NH is added while stirring according to the final concentration of 0.9mol/L4)2SO4Granule, treats (NH4)2SO4Adjust pH to 7.5 with sodium hydroxide solution after being completely dissolved, stand after 2h and be centrifuged, then after precipitation is dissolved in 0.5mol/L acetum, add finely ground (NH while stirring according to the final concentration of 0.3mol/L4)2SO4Granule, treats (NH4)2SO4Stand 2h after being completely dissolved to be centrifuged again, abandon supernatant, proceed to dialysis band after gained precipitation is dissolved in 0.2mol/L acetum and deionized water is fully dialysed, after products therefrom is freeze-dried, be Human plactnta type III collagen-based materials；
10) in described QY-A, finely ground (NH is added while stirring according to the final concentration of 0.9mol/L4)2SO4Granule, treats (NH4)2SO4Standing 4h after being completely dissolved to be centrifuged again, abandon supernatant, gained precipitation is labeled as CD-H；
11) adjust pH to 7.5 with sodium hydroxide solution after described precipitate C D-H is dissolved in 0.5mol/L acetum, add finely ground (NH while stirring according to the final concentration of 2.0mol/L4)2SO4Granule, treats (NH4)2SO4Standing 2h after being completely dissolved to be centrifuged again, abandon supernatant, gained precipitation is labeled as CD-I；
12), after described precipitate C D-I being dissolved in 0.5mol/L acetum, finely ground (NH is added while stirring according to the final concentration of 0.6mol/L4)2SO4Granule, treats (NH4)2SO4Standing 2h after being completely dissolved to be centrifuged again, abandon supernatant, gained precipitation is labeled as CD-J；
13) state after precipitate C D-J is dissolved in 0.2mol/L acetum by described, add finely ground (NH while stirring according to the final concentration of 2.0mol/L4)2SO4Granule, treats (NH4)2SO4Adjust pH to 7.5 with sodium hydroxide solution after being completely dissolved, stand after 2h and be centrifuged, then after precipitation is dissolved in 0.2mol/L acetum, add finely ground (NH while stirring according to the final concentration of 0.6mol/L4)2SO4Granule, treats (NH4)2SO4Stand 2h after being completely dissolved to be centrifuged again, abandoning supernatant, gained precipitation is dissolved in 0.05mol/L Tris-HCl, 0.1mol/L NaCl, pH is the buffer of 7.2-7.5 and carries out centrifugation after fully dialysing same buffer, gained precipitation is labeled as CD-K, and supernatant is labeled as QY-C；
14) described clear liquid QY-C proceeds to dialysis band deionized water is fully dialysed, after products therefrom is freeze-dried, be Human plactnta IV Collagen Type VI material；
15) state precipitate C D-K by described, after being dissolved in 0.2mol/L acetum, proceed to dialysis band and deionized water is fully dialysed, after products therefrom is freeze-dried, be Human plactnta collagen type v material.
Embodiment
3
Extract collagen according to the following steps:
(1) the groups of people's placenta tissue obtained through disease screening normal labor of freezen protective is selected；
(2) placenta tissue fully cleaned and be cut into small pieces, centrifugal segregation supernatant after low-temperature homogenate, precipitate with deionized water is centrifuged after cleaning again, repeatedly clean centrifugal 3 times transparent to supernatant；Precipitation pH is 7.2, disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution containing 5 mmol/L sodium gluconates, the 0.2mol/L of 1.0 mol/L NaCl soaks and be slowly stirred 2h, and mixing speed can not be too fast, slightly rotates with liquid level and is advisable；Being centrifuged after having stirred, centrifuged pellet cleans once and be centrifuged with phosphate buffer again, and finally the centrifugal precipitation obtained is weighed and as the foundation of follow-up measurement；
(3) precipitation obtained final in step (2) is soaked in Digestive system, described Digestive system is pH 2.2, pepsin (vigor >=250U/mg) concentration is acetic acid (HAC) solution of the 0.8mol/L of 2g/L, described Digestive system quality is 8 times of Sediment weight, it is slowly stirred continuously under cryogenic conditions, mixing speed can not be too fast, slightly rotates with liquid level and is advisable；After enzymic digestion 30h, it is standby that centrifugation takes supernatant.
(4) carry out salt fractionation according to following steps to operate:
1) regulating the sample after enzymic digestion with acetic acid is 2.5 to pH, adds finely ground NaCl granule while stirring according to the final concentration of 0.5mol/L, stands 4h and be centrifuged after NaCl is completely dissolved, and gained precipitation is labeled as CD-A, and supernatant is labeled as QY-A；
2) by after described precipitate C D-A 0.5mol/L acetate dissolution, adding finely ground NaCl granule while stirring according to the final concentration of 0.7mol/L, stand 4h and be centrifuged after NaCl is completely dissolved, gained precipitation is labeled as CD-B, and supernatant is labeled as QY-B；
3) in described clear liquid QY-B, adding finely ground NaCl granule while stirring according to the final concentration of 4.0mol/L, stand 8h and be centrifuged after NaCl is completely dissolved, abandon supernatant, gained precipitation is labeled as CD-C；
4) pH to 7.4 is adjusted with sodium hydroxide solution after described precipitate C D-C is dissolved in 0.2mol/L acetum, finely ground NaCl granule is added while stirring according to the final concentration of 2.5mol/L, standing 4h after NaCl is completely dissolved to be centrifuged again, abandon supernatant, gained precipitation is labeled as CD-D；
5) after described precipitate C D-D being dissolved in 0.5mol/L acetum, adding finely ground NaCl granule while stirring according to the final concentration of 0.7mol/L, stand 4h and be centrifuged after NaCl is completely dissolved, abandon supernatant, gained precipitation is labeled as CD-E；
6) described precipitate C D-E is dissolved in 0.2mol/L acetum, finely ground NaCl granule is added while stirring according to the final concentration of 2.5mol/L, pH to 7.2 is adjusted with sodium hydroxide solution after NaCl is completely dissolved, it is centrifuged after standing 2h, after again precipitation being dissolved in 0.5mol/L acetum, finely ground NaCl granule is added while stirring according to the final concentration of 0.8mol/L, after NaCl is completely dissolved, stand 2h be centrifuged again, abandon supernatant, proceed to dialysis band after gained precipitation is dissolved in 0.2mol/L acetum deionized water is fully dialysed, Human plactnta type i collagen material it is after products therefrom is freeze-dried；
7) adjust pH to 7.2 with sodium hydroxide solution after described CD-B is dissolved in 0.2mol/L acetum, add finely ground (NH while stirring according to the final concentration of 1.0mol/L4)2SO4Granule, treats (NH4)2SO4Standing 4h after being completely dissolved to be centrifuged again, abandon supernatant, gained precipitation is labeled as CD-F；
8), after described precipitate C D-F being dissolved in 0.5mol/L acetum, finely ground (NH is added while stirring according to the final concentration of 0.4mol/L4)2SO4Granule, treats (NH4)2SO4Standing 4h after being completely dissolved to be centrifuged again, abandon supernatant, gained precipitation is labeled as CD-G；
9), after described precipitate C D-G being dissolved in 0.5mol/L acetum, finely ground (NH is added while stirring according to the final concentration of 1.0mol/L4)2SO4Granule, treats (NH4)2SO4Adjust pH to 7.5 with sodium hydroxide solution after being completely dissolved, stand after 2h and be centrifuged, then after precipitation is dissolved in 0.5mol/L acetum, add finely ground (NH while stirring according to the final concentration of 0.4mol/L4)2SO4Granule, treats (NH4)2SO4Stand 2h after being completely dissolved to be centrifuged again, abandon supernatant, proceed to dialysis band after gained precipitation is dissolved in 0.2mol/L acetum and deionized water is fully dialysed, after products therefrom is freeze-dried, be Human plactnta type III collagen-based materials；
10) in described QY-A, adding finely ground ammonium phosphate granules while stirring according to the final concentration of 0.9mol/L, stand 2h and be centrifuged after ammonium phosphate is completely dissolved, abandon supernatant, gained precipitation is labeled as CD-H；
11) pH to 7.5 is adjusted with sodium hydroxide solution after described precipitate C D-H is dissolved in 0.2mol/L acetum, finely ground ammonium phosphate granules is added while stirring according to the final concentration of 1.8mol/L, standing 4h after ammonium phosphate is completely dissolved to be centrifuged again, abandon supernatant, gained precipitation is labeled as CD-I；
12) after described precipitate C D-I being dissolved in 0.5mol/L acetum, adding finely ground ammonium phosphate granules while stirring according to the final concentration of 0.6mol/L, stand 2h and be centrifuged after ammonium phosphate is completely dissolved, abandon supernatant, gained precipitation is labeled as CD-J；
null13) state after precipitate C D-J is dissolved in 0.2mol/L acetum by described，Finely ground ammonium phosphate granules is added while stirring according to the final concentration of 1.8mol/L，PH to 7.5 is adjusted with sodium hydroxide solution after ammonium phosphate is completely dissolved，It is centrifuged after standing 2h，After again precipitation being dissolved in 0.2mol/L acetum，Finely ground ammonium phosphate granules is added while stirring according to the final concentration of 0.6mol/L，After ammonium phosphate is completely dissolved, stand 2h be centrifuged again，Abandon supernatant，Gained precipitation is dissolved in 0.05mol/L Tris-HCl、0.1mol/L NaCl，PH is the buffer of 7.5 and carries out centrifugation after fully dialysing same buffer，Gained precipitation is labeled as CD-K，Supernatant is labeled as QY-C；
14) described clear liquid QY-C proceeds to dialysis band deionized water is fully dialysed, after products therefrom is freeze-dried, be Human plactnta IV Collagen Type VI material；
15) state precipitate C D-K by described, after being dissolved in 0.2mol/L acetum, proceed to dialysis band and deionized water is fully dialysed, after products therefrom is freeze-dried, be Human plactnta collagen type v material.
Use differential scanning calorimeter (DSC) that each Collagen Type VI of gained in the various embodiments described above is detected, result is as shown in Figure 2, as can be seen from the figure the DSC curve of the I type of gained, type III, IV type and collagen type v material to go out peak position the most identical with the standard curve of corresponding collagen, illustrate to be successfully prepared I type, type III, IV type and the collagen-based materials of V-type these four type by the method for the invention.
The foregoing is only the preferred embodiments of the present invention, be merely illustrative for the purpose of the present invention, and nonrestrictive；Those of ordinary skill in the art understand, it can be carried out many changes, amendment, even equivalence change, but fall within protection scope of the present invention in the spirit and scope that the claims in the present invention are limited.
Claims (9)
1. the preparation method of a human placental collagen, it is characterised in that with human placenta as raw material, carries out pretreatment, enzymic digestion, salt fractionation successively, obtains at least one kept in people's I type of natural structure, type III, IV type and V-type medical collagen material respectively；Wherein said pre-treatment step specifically includes: is fully cleaned by placenta tissue and is cut into small pieces, centrifugal segregation supernatant after low-temperature homogenate, and precipitate with deionized water is centrifuged after cleaning again, repeatedly clean centrifugal 2-4 time transparent to supernatant；Precipitation pH is 7.2-7.4, the pre-treatment buffer that concentration is 0.01-1.0 mol/L containing 1.0-20.0 mmol/L metal chelating agent, 0.5-3.0 mol/L neutral salt soaks and is slowly stirred 2-4h；Centrifuged pellet cleans once with pre-treatment buffer and is centrifuged, and takes precipitation standby；Described enzymic digestion is to be soaked in Digestive system by pretreated human placenta, described Digestive system is the mixed solution of pepsin and acetic acid, and its pH is 2.2-2.8, and pepsin concn is 0.5-5g/L, enzyme activity >=250U/mg, acetate concentration is 0.2-0.8mol/L；Described Digestive system quality is 5-10 times of human placenta solid weight, the required collagen-type extracted according to the operation of described salt fractionation, chooses corresponding step and combine from following salt fractionation step:
1) regulating the sample after enzymic digestion with acetic acid is 2.5-2.8 to pH, finely ground neutral salt granule is added while stirring according to the final concentration of 0.5-1.2mol/L, standing 2-10h after neutral salt is completely dissolved to be centrifuged, gained precipitation is labeled as CD-A, and supernatant is labeled as QY-A again；
2) by after described precipitate C D-A 0.2-0.5mol/L acetate dissolution, finely ground neutral salt granule is added while stirring according to the final concentration of 0.3-0.8mol/L, standing 2-10h after neutral salt is completely dissolved to be centrifuged, gained precipitation is labeled as CD-B, and supernatant is labeled as QY-B again；
3) in described clear liquid QY-B, adding finely ground neutral salt granule while stirring according to the final concentration of 2.0-4.0mol/L, stand 2-10h and be centrifuged after neutral salt is completely dissolved, abandon supernatant, gained precipitation is labeled as CD-C；
4) pH to 7.2-7.5 is adjusted with sodium hydroxide solution after described precipitate C D-C is dissolved in 0.2-0.5mol/L acetum, finely ground neutral salt granule is added while stirring according to the final concentration of 1.2-2.5mol/L, after neutral salt is completely dissolved, stand 2-10h be centrifuged again, abandoning supernatant, gained precipitation is labeled as CD-D；
5) after described precipitate C D-D being dissolved in 0.5mol/L acetum, adding finely ground neutral salt granule while stirring according to the final concentration of 0.3-0.8mol/L, stand 2-10h and be centrifuged after neutral salt is completely dissolved, abandon supernatant, gained precipitation is labeled as CD-E；
6) described precipitate C D-E is dissolved in 0.2-0.5mol/L acetum, finely ground neutral salt granule is added while stirring according to the final concentration of 1.2-2.5mol/L, pH to 7.2-7.5 is adjusted with sodium hydroxide solution after neutral salt is completely dissolved, it is centrifuged after standing 2-10h, after again precipitation being dissolved in 0.5mol/L acetum, finely ground neutral salt granule is added while stirring according to the final concentration of 0.3-0.8mol/L, after neutral salt is completely dissolved, stand 2-10h be centrifuged again, abandon supernatant, proceed to dialysis band after gained precipitation is dissolved in 0.2-0.5mol/L acetum deionized water is fully dialysed, Human plactnta type i collagen material it is after products therefrom is freeze-dried；
7) pH to 7.2-7.5 is adjusted with sodium hydroxide solution after described CD-B is dissolved in 0.2-0.5mol/L acetum, finely ground neutral salt granule is added while stirring according to the final concentration of 0.9-2.0mol/L, after neutral salt is completely dissolved, stand 2-10h be centrifuged again, abandoning supernatant, gained precipitation is labeled as CD-F；
8) after described precipitate C D-F being dissolved in 0.5mol/L acetum, adding finely ground neutral salt granule while stirring according to the final concentration of 0.3-0.8mol/L, stand 2-10h and be centrifuged after neutral salt is completely dissolved, abandon supernatant, gained precipitation is labeled as CD-G；
9) after described precipitate C D-G being dissolved in 0.2-0.5mol/L acetum, finely ground neutral salt granule is added while stirring according to the final concentration of 0.9-2.0mol/L, pH to 7.2-7.5 is adjusted with sodium hydroxide solution after neutral salt is completely dissolved, it is centrifuged after standing 2-10h, after again precipitation being dissolved in 0.5mol/L acetum, finely ground neutral salt granule is added while stirring according to the final concentration of 0.3-0.8mol/L, after neutral salt is completely dissolved, stand 2-10h be centrifuged again, abandon supernatant, by gained precipitation be dissolved in 0.2-0.5mol/L acetum proceed to dialyse band deionized water is fully dialysed, Human plactnta type III collagen-based materials it is after products therefrom is freeze-dried；
10) in described QY-A, adding finely ground neutral salt granule while stirring according to the final concentration of 0.9-2.0mol/L, stand 2-10h and be centrifuged after neutral salt is completely dissolved, abandon supernatant, gained precipitation is labeled as CD-H；
11) pH to 7.2-7.5 is adjusted with sodium hydroxide solution after described precipitate C D-H is dissolved in 0.2-0.5mol/L acetum, finely ground neutral salt granule is added while stirring according to the final concentration of 1.8-4.5mol/L, after neutral salt is completely dissolved, stand 2-10h be centrifuged again, abandoning supernatant, gained precipitation is labeled as CD-I；
12) after described precipitate C D-I being dissolved in 0.2-0.5mol/L acetum, finely ground neutral salt granule is added while stirring according to the final concentration of 0.6-1.9mol/L, standing 2-10h after neutral salt is completely dissolved to be centrifuged again, abandon supernatant, gained precipitation is labeled as CD-J；
null13) after described precipitate C D-J being dissolved in 0.2-0.5mol/L acetum，Finely ground neutral salt granule is added while stirring according to the final concentration of 1.8-4.5mol/L，PH to 7.2-7.5 is adjusted with sodium hydroxide solution after neutral salt is completely dissolved，It is centrifuged after standing 2-10h，After again precipitation being dissolved in 0.2-0.5mol/L acetum，Finely ground neutral salt granule is added while stirring according to the final concentration of 0.6-1.9mol/L，After neutral salt is completely dissolved, stand 2-10h be centrifuged again，Abandon supernatant，Gained precipitation is dissolved in 0.05mol/L Tris-HCl、0.1mol/L NaCl，PH is the buffer of 7.2-7.5 and carries out centrifugation after fully dialysing same buffer，Gained precipitation is labeled as CD-K，Supernatant is labeled as QY-C；
14) described clear liquid QY-C proceeds to dialysis band deionized water is fully dialysed, after products therefrom is freeze-dried, be Human plactnta IV Collagen Type VI material；
15), by described precipitate C D-K, after being dissolved in 0.2-0.5mol/L acetum, proceed to dialysis band and deionized water is fully dialysed, after products therefrom is freeze-dried, be Human plactnta collagen type v material.
The preparation method of human placental collagen the most according to claim 1, it is characterised in that described human placenta is that pre-puerpera carries out the placenta tissue that is fresh or that preserve through freezing screening and obtaining after normal labor that can spread disease before childbirth.
The preparation method of human placental collagen the most according to claim 1, it is characterised in that described in be slowly stirred step and slightly rotate with liquid level and be as the criterion.
The preparation method of human placental collagen the most according to claim 1, it is characterized in that: described enzymic digestion is to be soaked in Digestive system by pretreated human placenta, 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；Described Digestive system quality is 5-10 times of human placenta solid weight, is slowly stirred continuously under cryogenic conditions, and mixing speed can not be too fast, slightly rotates with liquid level and is advisable；After enzymic digestion 6-30h, it is standby that centrifugation takes supernatant.
5. according to the preparation method of human placental collagen any one of in claim 1-4, it is characterized in that, described Human plactnta I type, type III, IV type or collagen type v comprises at least one in fibril shape collagen, microfibrous collagen, graininess fiber, collagen fiber and collagenous fiber bundle.
The preparation method of human placental collagen the most according to claim 1, it is characterised in that described metal chelating agent is at least one in aminocarboxylate and hydroxycarboxylate.
The preparation method of human placental collagen the most according to claim 1, it is characterised in that described neutral salt is at least one in sodium chloride, ammonium sulfate, sodium sulfate, sodium citrate and ammonium phosphate.
The preparation method of human placental collagen the most according to claim 1, it is characterized in that, described pre-treatment buffer is the one in trishydroxymethylaminomethane-hydrochloric acid, disodium hydrogen phosphate-citric acid, disodium hydrogen phosphate-sodium dihydrogen phosphate, potassium dihydrogen phosphate-sodium hydrate buffer solution.
9. according to the preparation method of human placental collagen any one of in claim 1-4, it is characterized in that, the Human plactnta I type, type III, IV type or the collagen type v that prepare remain natural collagen original triple helix structure, it is possible to meet medical adhesive raw-material application requirement.
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| 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 |
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