CN106701674A - Purification and extraction process of resuscitation-promoting factors capable of increasing survival rate of transplanted adipocytes or filled adipocytes - Google Patents
Purification and extraction process of resuscitation-promoting factors capable of increasing survival rate of transplanted adipocytes or filled adipocytes Download PDFInfo
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Abstract
The invention discloses a purification and extraction process of resuscitation-promoting factors capable of increasing survival rate of transplanted adipocytes or filled adipocytes. The method comprises the following steps: (a) collection of a whole blood sample; (b) isolation of the whole blood sample; (c) fermentation of the whole blood sample; (d) freezing of a supernatant of a fermentation broth; (e) resuscitation of whole blood sample cells; (f) purification and extraction of the resuscitation-promoting factors: the liquid after standing is added to a centrifugal tube and placed in a centrifuge for secondary centrifugation, the liquid after centrifugation is left to stand in a constant-temperature environment, plasma on the upper layer of the centrifugal tube is discarded, a new plasma sample is added, so that precipitated platelets suspend again, and the resuscitation-promoting factors are obtained. PRP (platelet rich plasma) required for injection of autologous fat can be well extracted, the extraction efficiency of PRP from cells is greatly improved, the vitality of autologous fat after injection can be guaranteed, so that the autologous fat injection surgery is more secure and reliable, and the purification and extraction process is worthy of promotion.
Description
Technical field
The present invention relates to cell extraction techniques field, and in particular to one kind improves transplantability fat cell or filled type fat
The purification of the resurrection factor of fat cell survival rate and extraction process.
Background technology
Autologous fat draws unnecessary subcutaneous fat cells from some positions of human body itself, then the mixture by suctioning out
Purified treatment, injection of medicine obtain composite fat granule, select complete Grainy fat tissue cell to be moved again by way of injection
Plant the position for oneself needing to carry out fatty filling, such as breast, face etc. are used to treat that chest is flat, both sides breast is not right
Title, the fine wrinkle of superficial, thin lip are grand into thick lips etc..But there is a certain degree of suction after many fat fillings at present
Receive, in order to ensure that best effect needs to be performed the operation again, the consumption of fat is excessive or injection is excessively concentrated, significant quantities of fat heap
Product can cause adiponecrosis, dissolving, absorb because of blood supply insufficiency, easily trigger infection, fibrosis or calcification, adiponecrosis etc. occur
Sequelae, after injection, survival rate is all relatively low for many autologous fats, can cause the counter productive of operation, and influence is performed the operation into
Fruit, it is necessary to improve the survival rate of autologous fat injection using PRP, therefore devise it is a kind of improve transplantability fat cell or
The purification of the resurrection factor of filled type fat cell survival rate and extraction process.
The content of the invention
For problem above, improve transplantability fat cell the invention provides one kind or filled type fat cell is survived
The purification of the resurrection factor of rate and extraction process, can be good at the PRP needed for extracting autologous fat injection, substantially increase
The efficiency of PRP is extracted from cell, the vigor after autologous fat injection is ensure that so that autologous fat injection operation is more pacified
It is complete reliable, it is worthy to be popularized.
To achieve these goals, the technical solution adopted by the present invention is as follows：One kind improve transplantability fat cell or
The purification of the resurrection factor of filled type fat cell survival rate and extraction process, the method are comprised the following steps：
（a）For the collection of whole blood sample：Collection adult's blood, is put into test tube, and 12-14min is centrifuged in centrifuge；Centrifugation
Blood adds anti-coagulants to stand 30min afterwards.
（b）The separation of whole blood sample：Whole blood sample after standing is divided into three obvious levels, and drawing supernatant blood plasma is used for
Extract platelet rich plasma；Add hydroxyethyl starch solution to be placed on shaking table fully to mix.
（c）The fermentation of whole blood sample：Whole blood sample is added in zymotic fluid, whole blood sample zymotic fluid is obtained, 10min is stirred
After stand, extract fermented liquid supernatant.
（d）Fermented liquid supernatant freezing processing：Fermented liquid supernatant is added into frozen stock solution, resuspended whole blood zymotic fluid, addition freezes
Container, is freezed using semiconductor refrigerating equipment, and temperature is gradually reduced, and is put into preservation in liquid nitrogen container.
（e）Whole blood sample cell recovery：The zymotic fluid for freezing is taken out from liquid nitrogen container, by physiological saline water-bath,
After zymotic fluid liquefies again, stewing process.
（f）Bring back to life the purification and extraction of the factor：Liquid after standing is added in centrifuge tube, be placed in centrifuge carry out it is secondary
Centrifugal treating, after the completion of centrifugation under isoperibol stewing process, discard the blood plasma on centrifuge tube upper strata, add new blood plasma sample
This so that the blood platelet of precipitation suspends again, obtains bringing back to life the factor.
As a kind of preferred technical scheme of the present invention, the step（a）In, centrifuge speed keeps 1000-
1100rpm so that whole blood sample initial gross separation, is easy to separate blood platelet with serum.
As a kind of preferred technical scheme of the present invention, the step（a）In, anti-coagulants uses ethylenediamine tetra-acetic acid, grass
Sour sodium, heparin and sodium citrate are mixed, and the anti-coagulants and whole blood sample ratio are 1:9~11.
As a kind of preferred technical scheme of the present invention, the step（b）In, solution stands at room temperature, and the superiors are
Containing hematoblastic plasma layer, intermediate layer is presented yellow, is buffy coat, and lowermost layer thickness is maximum, is red blood cell layer.
As a kind of preferred technical scheme of the present invention, the step（b）In, hydroxyethyl starch solution is per 100ml components
It is containing the 6g of HES 130/0.4 and sodium chloride 0.9g.
As a kind of preferred technical scheme of the present invention, the step（c）In, the ratio of zymotic fluid and whole blood sample is 1:
20 ~ 22, zymotic fluid uses nucleic acid zymotic fluid.
As a kind of preferred technical scheme of the present invention, the step（d）In, frozen stock solution mixes for calf serum with DMSO
Thing, and calf serum and DMSO ratios are 1:9, wherein DMSO concentration is 10%.
As a kind of preferred technical scheme of the present invention, the step（d）In, semiconductor refrigerating equipment temperature decrease rate
It is 8-12 DEG C/min.
As a kind of preferred technical scheme of the present invention, the step（e）In, concentration of physiological saline is 5%.
As a kind of preferred technical scheme of the present invention, the step（f）In, centrifuge speed keeps 800-1000rpm,
Thermostat temperature is 23-25 DEG C, it is ensured that hematoblastic to suspend again.
Beneficial effects of the present invention：
The present invention can be good at the PRP needed for extracting autologous fat injection, substantially increase the effect that PRP is extracted from cell
Rate, ensure that the vigor after autologous fat injection so that autologous fat injection operation is more safe and reliable, is worthy to be popularized.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Embodiment：
A kind of purification of resurrection factor for improving transplantability fat cell or filled type fat cell survival rate and extraction process,
The method is comprised the following steps：
（a）For the collection of whole blood sample：Collection adult's blood, is put into test tube, and 12-14min is centrifuged in centrifuge；Centrifugation
Blood adds anti-coagulants to stand 30min afterwards.
（b）The separation of whole blood sample：Whole blood sample after standing is divided into three obvious levels, and drawing supernatant blood plasma is used for
Extract platelet rich plasma；Add hydroxyethyl starch solution to be placed on shaking table fully to mix.
（c）The fermentation of whole blood sample：Whole blood sample is added in zymotic fluid, whole blood sample zymotic fluid is obtained, 10min is stirred
After stand, extract fermented liquid supernatant.
（d）Fermented liquid supernatant freezing processing：Fermented liquid supernatant is added into frozen stock solution, resuspended whole blood zymotic fluid, addition freezes
Container, is freezed using semiconductor refrigerating equipment, and temperature is gradually reduced, and is put into preservation in liquid nitrogen container.
（e）Whole blood sample cell recovery：The zymotic fluid for freezing is taken out from liquid nitrogen container, by physiological saline water-bath,
After zymotic fluid liquefies again, stewing process.
（f）Bring back to life the purification and extraction of the factor：Liquid after standing is added in centrifuge tube, be placed in centrifuge carry out it is secondary
Centrifugal treating, after the completion of centrifugation under isoperibol stewing process, discard the blood plasma on centrifuge tube upper strata, add new blood plasma sample
This so that the blood platelet of precipitation suspends again, obtains bringing back to life the factor.
Step（a）In, centrifuge speed keeps 1000-1100rpm so that whole blood sample initial gross separation, is easy to blood is small
Plate is separated with serum；Step（a）In, anti-coagulants mixes system with sodium citrate using ethylenediamine tetra-acetic acid, sodium oxalate, heparin
Into the anti-coagulants and whole blood sample ratio are 1:9~11；The step（b）In, solution stands at room temperature, the superiors be containing
Hematoblastic plasma layer, intermediate layer is presented yellow, is buffy coat, and lowermost layer thickness is maximum, is red blood cell layer；Step（b）In,
Hydroxyethyl starch solution is containing the 6g of HES 130/0.4 and sodium chloride 0.9g per 100ml components.
As a kind of preferred technical scheme of the present invention, the step（c）In, the ratio of zymotic fluid and whole blood sample is 1:
20 ~ 22, zymotic fluid uses nucleic acid zymotic fluid；Step（d）In, frozen stock solution is calf serum and DMSO mixtures, and small ox blood
It is 1 clearly with DMSO ratios:9, wherein DMSO concentration is 10%；Step（d）In, semiconductor refrigerating equipment temperature decrease rate is 8-
12℃/min；Step（e）In, concentration of physiological saline is 5%；Step（f）In, centrifuge speed keeps 800-1000rpm, constant temperature
Temperature is 23-25 DEG C, it is ensured that hematoblastic to suspend again.
Survival rate how is improved for autologous fat injection at present, mainly there are these methods, one, attentional selection suction
Method, as far as possible using empty needle liposuction, the method that negative pressure does not exceed suction of fat tissue in 0.05MPa operations directly affects operation
Result.Because in operation aspiration procedure, inevitably causing the damage and destruction of part cell.The degree of damage is big,
Living cells is fewer, and survival rate is also lower.Most scholars advocate to use empty needle liposuction, and its negative pressure is no more than 0.05MPa, so may be used
Reduce the destruction of cell.Such as with other atraumatic technique suction of fat, then suction pipe should all select the tubule of below 4mm be preferred.Due to
Whole operation is carried out in the subcutaneous fat deposit without big blood vessel and Substance P, so what is pumped out is all fat, is suffered from
Person not bleeding, no pain substantially.2nd, the thigh position fat research higher of selection lipolytic activity finds that human body is different as far as possible
The activity of the lipoprotein lipase of the adipose tissue at position has differences.It was found that the activity of the lipoprotein lipase of the adipose tissue at thigh position
Higher than other positions.The activity of LPL is high, is conducive to the regeneration of transplant fat cell to survive.The LPL activity of thigh and buttocks is most
Height, is followed successively by lower abdomen, upper abdomen and successively decreases.Deep fat distribution according to this research and these positions, therefore preferably in trunk
Lower half first-selection make the confession area of autologous fat chest enlarge fat transfer.3rd, tried one's best fat point multiple points dispersion during rich temple
The injection rich temporal principle of autologous fat is fatty multiple point dispersion injections to be dispersed in being knitted by district's groups.Each point
Injection rate is no more than 1ml, should avoid for substantial amounts of fat concentrating on a certain position, and massage is smoothed after injection.This method is due to fat
Small volume, position dispersion, implant blood fortune are easily set up, and are conducive to surviving for lipochondrion.4th, the purification process extraction of lipochondrion
Take high-quality fat and collect 4 degree of physiological saline cleaning filterings of lipochondrion application, wash the fat cell of most destroyed, fat drips and blood
Liquid.Purge process should be specifically noted that sterile working;Should also try one's best and reduce the lipochondrion resting period in vitro, to improve fat
Cells survival rate.
Wherein, experiment is by determining ribose and protein content in each pipe that automatic collector is collected, it can be deduced that
As a result zymotic fluid shows that what is afforded at first is capsular polysaccharide, most through the distribution curve after sepharose-4FF posts wash-out
What is afforded afterwards is protein and nucleic acid, because protein and nucleic acid are often below capsular polysaccharide, sepharose-4FF gels
The merging of PRP ribose in pipe is separated and collected according to two principles：First, the main component for merging pipe is ribose, secondly：Molecular weight
Meet ratios of the Kd less than 0.5 that Chinese Pharmacopoeia specifies and account for more than 50%, PRP cores are calculated using the common experimental technique in this area
The elution curve of sugar, corresponding elution volume is assured that capture range when calculating KD=O.5.Therefore sepharose-4FF
Protein, nucleic acid and ribose can be very easily separated, in the capsular polysaccharide collected at first, the pod membrane close to void volume is more
The molecular weight of sugar is than larger, and the molecular weight of centre position capsular polysaccharide out is smaller, and according to preceding described, molecular size range is
Sugar unit repeat number is different, and because capsular polysaccharide molecular weight is generally excessive, therefore molecular weight is big and small capsular polysaccharide immune
Originality difference is little.
Show by experiment, the resurrection factor that the present invention is produced, after autologous fat is injected, can be good at improving
The activity of autologous fat, the survival rate after autologous fat injection improves 60%, and is had no side effect for body.
Based on above-mentioned, it is an advantage of the current invention that needed for the present invention can be good at extracting autologous fat injection
PRP, substantially increases the efficiency that PRP is extracted from cell, ensure that the vigor after autologous fat injection so that autologous fat
Injection operation is more safe and reliable, is worthy to be popularized.
Presently preferred embodiments of the present invention is the foregoing is only, is not intended to limit the invention, it is all in essence of the invention
Any modification, equivalent and improvement made within god and principle etc., should be included within the scope of the present invention.
Claims (10)
1. a kind of purification of resurrections factor for improving transplantability fat cell or filled type fat cell survival rate and work is extracted
Skill, it is characterised in that the method is comprised the following steps：
（a）For the collection of whole blood sample：Collection adult's blood, is put into test tube, and 12-14min is centrifuged in centrifuge；Centrifugation
Blood adds anti-coagulants to stand 30min afterwards；
（b）The separation of whole blood sample：Whole blood sample after standing is divided into three obvious levels, and drawing supernatant blood plasma is used to extract
Platelet rich plasma；Add hydroxyethyl starch solution to be placed on shaking table fully to mix；
（c）The fermentation of whole blood sample：Whole blood sample is added in zymotic fluid, whole blood sample zymotic fluid is obtained, it is quiet after stirring 10min
Put, extract fermented liquid supernatant；
（d）Fermented liquid supernatant freezing processing：Fermented liquid supernatant is added into frozen stock solution, resuspended whole blood zymotic fluid, addition freezes container,
Freezed using semiconductor refrigerating equipment, temperature is gradually reduced, and be put into preservation in liquid nitrogen container；
（e）Whole blood sample cell recovery：The zymotic fluid for freezing is taken out from liquid nitrogen container, by physiological saline water-bath, in fermentation
After liquid liquefies again, stewing process；
（f）Bring back to life the purification and extraction of the factor：Liquid after standing is added in centrifuge tube, and being placed on carries out secondary centrifuging in centrifuge
Treatment, after the completion of centrifugation under isoperibol stewing process, discard the blood plasma on centrifuge tube upper strata, add new plasma sample, make
The blood platelet that must be precipitated suspends again, obtains bringing back to life the factor.
2. a kind of resurrection factor for improving transplantability fat cell or filled type fat cell survival rate according to claim 1
Purification and extraction process, it is characterised in that the step（a）In, centrifuge speed keeps 1000-1100rpm so that whole blood
Sample initial gross separation, is easy to separate blood platelet with serum.
3. a kind of resurrection factor for improving transplantability fat cell or filled type fat cell survival rate according to claim 1
Purification and extraction process, it is characterised in that the step（a）In, anti-coagulants uses ethylenediamine tetra-acetic acid, sodium oxalate, heparin
It is mixed with sodium citrate, the anti-coagulants and whole blood sample ratio are 1:9~11.
4. a kind of resurrection factor for improving transplantability fat cell or filled type fat cell survival rate according to claim 1
Purification and extraction process, it is characterised in that the step（b）In, solution stands at room temperature, and the superiors are containing hematoblastic
Plasma layer, intermediate layer is presented yellow, is buffy coat, and lowermost layer thickness is maximum, is red blood cell layer.
5. a kind of resurrection factor for improving transplantability fat cell or filled type fat cell survival rate according to claim 1
Purification and extraction process, it is characterised in that the step（b）In, hydroxyethyl starch solution is containing ethoxy per 100ml components
The 6g of starch 130/0.4 and sodium chloride 0.9g.
6. a kind of resurrection factor for improving transplantability fat cell or filled type fat cell survival rate according to claim 1
Purification and extraction process, it is characterised in that the step（c）In, the ratio of zymotic fluid and whole blood sample is 1:20 ~ 22, hair
Zymotic fluid uses nucleic acid zymotic fluid.
7. a kind of resurrection factor for improving transplantability fat cell or filled type fat cell survival rate according to claim 1
Purification and extraction process, it is characterised in that the step（d）In, frozen stock solution is calf serum and DMSO mixtures, and calf
Serum and DMSO ratios are 1:9, wherein DMSO concentration is 10%.
8. a kind of resurrection factor for improving transplantability fat cell or filled type fat cell survival rate according to claim 1
Purification and extraction process, it is characterised in that the step（d）In, semiconductor refrigerating equipment temperature decrease rate be 8-12 DEG C/
min。
9. a kind of resurrection factor for improving transplantability fat cell or filled type fat cell survival rate according to claim 1
Purification and extraction process, it is characterised in that the step（e）In, concentration of physiological saline is 5%.
10. a kind of resurrection for improving transplantability fat cell or filled type fat cell survival rate according to claim 1 because
The purification of son and extraction process, it is characterised in that the step（f）In, centrifuge speed keeps 800-1000rpm, constant temperature temperature
Spend is 23-25 DEG C, it is ensured that hematoblastic to suspend again.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108030977A (en) * | 2017-11-29 | 2018-05-15 | 祖秉毅 | A kind of method realized from donor local solids flux |
| CN111896340A (en) * | 2020-06-24 | 2020-11-06 | 四川大学华西医院 | Simple PBMC separation method for flow cytometry detection |
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