CN103083726A - Liver tissue cell removal solution - Google Patents
Liver tissue cell removal solution Download PDFInfo
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- CN103083726A CN103083726A CN2013100676113A CN201310067611A CN103083726A CN 103083726 A CN103083726 A CN 103083726A CN 2013100676113 A CN2013100676113 A CN 2013100676113A CN 201310067611 A CN201310067611 A CN 201310067611A CN 103083726 A CN103083726 A CN 103083726A
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Abstract
The invention discloses a liver tissue cell removal solution. The novel cell removal solution prepared from two components is introduced into a whole animal cell removal technology. The cell removal solution is prepared from 0.01 to 0.05 percent of recombinant tissue plasminogen activator (rt-PA) and 0.5 to 2 percent of tween-80 by using sterile normal saline. By using the strong thrombolytic effect of the rt-PA and the solubilization and red blood cell destruction effects of the tween-80, the liver tissue cell removal solution is relatively easy to prepare and implement, high in cell removal efficiency and low in requirement for warm ischemia time of the liver, and greatly improves the potential probability of a cell removal liver scaffold in clinical application.
Description
Technical field
The present invention relates to a kind of biological support, particularly a kind ofly can be used as that in liver tissue engineering, the liver organization of biologic bracket material takes off Cell sap.
Background technology
The hepatopathy in latter stage at end such as serious symptom liver failure and liver cirrhosis has had a strong impact on people's life quality, becomes one of the highest disease of whole world mortality rate.At present, liver transplantation is the unique effective Therapeutic Method of generally acknowledging.But the shortage of donor livers is restricted the popularization of this technology, makes a lot of patients lose best treatment opportunity.
The ultimate principle of tissue engineering integrated use biology and engineering, basic theories, basic fundamental and method, how research is built with bioactive histoorgan under vitro conditions, in order to the subject of the histoorgan that substitutes disease damage.Desirable tissue engineering tissue or organ should have vitality, can rebuild and reach the standard of permanent alternative disease damage tissue or organ on form, 26S Proteasome Structure and Function.The tissue engineering tissue organ is compared with repair mode in the past has multiple advantage: without the allosome rejection, good biocompatibility can overcome the problem of organ origin deficiency.
Build desirable engineered artificial liver, need three critical problems of research: the source of seed cell, timbering material and can provide bioreactor near physiological environment for the cytoskeleton complex.Think at present that the timbering material near physiological status is that full organ takes off cytoskeleton.It is to use various means, and Cell Components all in whole organ is removed, and keeps the biological support that organ peplos, original extracellular matrix and blood vessel network obtain.This support has the not available advantage of many man-made supports: keep the difficult problem that original blood vessel network has solved the artificial organ vascularization, keep extracellular matrix composition and 3-D solid structure and be beneficial to the cell adhesion growth, it constitutes the natural substrates material has good biocompatibility to overcome again the shortcoming of other natural substrates material simultaneously.
The cell means of taking off that adopt now are mainly to use two kinds of detergents: the SDS(sodium lauryl sulphate) and Triton-X 100[1, and 2].These two kinds of detergents (1%SDS or 1%Triton-X100) are arrived each position of full liver by the continuous vessel perfusion, thereby reach the purpose that full liver takes off cell.But existing method has following shortcoming:
The method that 1, must adopt live body to get tissue, if animal dead, the thrombosis that approximately after 10 minutes, in liver, blood vessel just forms extensively, fills the air, cause detergent to be difficult to arrive to take off the cell position, detergent only acts on the lipide component of surface of cell membrane, thereby it is not high to take off cell efficient, does not reach and takes off cell effect.
2, the method that adopts live body to get tissue, to having relatively high expectations for the liver source, if be applied to clinically, liver support donor source is restricted, and is difficult to promote, Clinical practicability is not strong.
Summary of the invention
The purpose of this invention is to provide a kind of liver organization and take off Cell sap, introduced recombined human tissue plasminogen activator and tween 80 these two kinds of compositions in taking off the composition of Cell sap.Its configuration is used easily, takes off cell efficient high, and is less demanding to the warm ischemia time of liver, greatly increased take off cell liver support may at the potential of clinical practice.
Particular content:
A kind of liver organization takes off Cell sap, is to introduce the novel Cell sap that takes off that a kind of two kinds of compositions are made in the full liver of animal takes off cell technology.This takes off Cell sap is by the 0.01%-0.05% recombined human rt-PA of tissue plasminogen activator and 0.5-2% tween 80, forms with the physiological saline solution configuration.
Recombined human tissue plasminogen activator was the clinical application for the dissolving Intravascular Thrombus originally, but because adopting gene recombination technology production, it can obtain in a large number, effect with selective dissolution thrombosis, do not affect fibrinolytic system in blood circulation, the thrombolytic process does not need the in vivo participation of the various factors, thereby its thrombus under vitro conditions is achieved.
Tween80 is a kind of Nonionic Detergents, can destroy cell membrane, and is particularly remarkable to erythrocytic destruction, but less on the impact of albumen, can strengthen the effect of thrombolytic in early stage, makes the vascular system of whole liver unobstructed, improves the later stage and takes off cell efficient.In addition, Tween80 is that a kind of solubilizing agent can improve by emulsification the dissolubility of recombined human tissue plasminogen activator, thereby indirectly improves its activity.
The present invention has introduced a kind of novel Cell sap that takes off that contains two kinds of compositions cleverly in original liver organization takes off cell technology, utilize the strong thrombolytic effect of recombined human tissue plasminogen activator (rt-PA) and tween 80 solubilisings, hematoclasis effect, make liver organization take off Cell sap and have the configuration use easily, take off cell efficient high, to the less demanding characteristic of the warm ischemia time of liver, greatly increased and taken off cell liver support in the potential possibility of clinical practice.
The specific embodiment
Embodiment 1:
A kind of liver organization takes off Cell sap, to introduce two kinds of novel Cell saps that take off that composition is made in the full liver of animal takes off cell technology, this takes off Cell sap is by 0.01% recombined human tissue plasminogen activator (rt-PA) and 2%tween80, configures with physiological saline solution.
Concrete operations:
1, full liver obtains:
After rat euthanasia, hara kiri longitudinally, exposed Rats liver.Proximal part caval vein, portal vein and Hepatic artery are put after sleeve pipe (BD Intima) labelling of 20G from disconnected, after separating falciform ligament and heart-shaped ligament crosscut, mobile liver, be exposed in the visual field postcava gently, ligation, separation distal end caval vein and any adnexa that adheres on liver.
2, taking off cell prepares:
(1) take off 37 ℃ of Cell saps (proportioning: 0.01% rt-PA, 2%tween 80) and take off Cell sap and inject through portal vein, folder closes Hepatic artery sleeve pipe and proximal part caval vein, keeps 30min.Then folder closes the Hepatic artery sleeve pipe, open proximal part caval vein, and the connection door vein pours into 4ml/min speed by portal vein with taking off Cell sap on peristaltic pump.
(2) perfusion contains the normal saline of 1%SDS.The connection door vein is on peristaltic pump, and open proximal part caval vein and Hepatic artery pour into 4ml/min speed by portal vein, continue 30min.
(3) perfusion contains the normal saline of 1%Triton-X100, and the perfusion volume is probably 50 times of full liver volume.At last, then take off Cell sap with what normal saline flushing did not flow to end.
3, the full liver of rat takes off cytoskeleton authentication method and result:
(1) general form learn to be observed: after taking off cell, the full liver of perusal rat takes off cell liver support and is white in color translucently, spongy, and outside peplos is intact, sees through after birth and can see in liver blood vessel network clearly.Under low power microscope, vascular tree is high-visible.
(2) microexamination:
Scanning electron microscopic observation:
Take off the substrate of cell liver support, during 8000 times of amplifications, can see that the collagen fabric screen distance keeps good, have between the aperture and be convenient to the connection that cell is creeped, adhered to.During 4000 times of amplifications, can see the fiber alignment of liver plate structure sample.
HE and three-color process are observed:
Take off the liver support after cell, after paraffin embedding, section, row HE dyeing and Trichrome dyeing, do not see cell residue, and the albumen of collagen scaffold keeps intact.As seen collagen scaffold network clearly after HE dyeing, but do not see cell.Three-color process dyeing is visible obtain support mainly formed by collagen protein and sugared Fibronectin.
DNA residues detection (reacting cells residual quantity):
Organize test kit (to be purchased from Qiagen Inc with DNeasy, Valencia, CA) take off the DNA composition of cell rami hepatici frame by the different rats of test kit operating instruction extracting (n=6) after, be DNA with ultraviolet spectrophotometer (U.S. Thermo) quantitative, DNA do not detected, and matched group (normal liver tissue) can detect DNA content.
Embodiment 2:
A kind of liver organization takes off Cell sap, to introduce two kinds of novel Cell saps that take off that composition is made in the full liver of animal takes off cell technology, this takes off Cell sap is by 0.02% recombined human tissue plasminogen activator (rt-PA) and 1%tween80, configures with physiological saline solution.
Concrete operations:
1, full liver obtains: identical with embodiment 1.
2, taking off cell prepares:
(1) take off 37 ℃ of Cell saps (proportioning: 0.02% rt-PA, 1%tween 80) and take off Cell sap and inject through portal vein, folder closes Hepatic artery sleeve pipe and proximal part caval vein, keeps 30min.Then folder closes the Hepatic artery sleeve pipe, open proximal part caval vein, and the connection door vein pours into 4ml/min speed by portal vein with taking off Cell sap on peristaltic pump.
(2) the post-processed method is identical with embodiment 1.
3, the full liver of rat takes off cytoskeleton authentication method and result:
(1) general form learn to be observed: after taking off cell, the full liver of perusal rat takes off cell liver support and is white in color translucently, spongy, and outside peplos is intact, sees through after birth and can see in liver blood vessel network clearly.Under low power microscope, vascular tree is high-visible.
(2) microexamination:
Scanning electron microscopic observation:
Take off the substrate of cell liver support, during 8000 times of amplifications, can see that the collagen fabric screen distance keeps good, have between the aperture and be convenient to the connection that cell is creeped, adhered to.During 4000 times of amplifications, can see the fiber alignment of liver plate structure sample.
HE and three-color process are observed:
Take off the liver support after cell, after paraffin embedding, section, row HE dyeing and Trichrome dyeing, do not see cell residue, and the albumen of collagen scaffold keeps intact.As seen collagen scaffold network clearly after HE dyeing, but do not see cell.Three-color process dyeing is visible obtain support mainly formed by collagen protein and sugared Fibronectin.
DNA residues detection (reacting cells residual quantity):
Organize test kit (to be purchased from Qiagen Inc with DNeasy, Valencia, CA) take off the DNA composition of cell rami hepatici frame by the different rats of test kit operating instruction extracting (n=6) after, be DNA with ultraviolet spectrophotometer (U.S. Thermo) quantitative, DNA do not detected, and matched group (normal liver tissue) can detect DNA content.
Embodiment 3:
A kind of liver organization takes off Cell sap, to introduce two kinds of novel Cell saps that take off that composition is made in the full liver of animal takes off cell technology, this takes off Cell sap is by 0.05% recombined human tissue plasminogen activator (rt-PA) and 0.5%tween80, configures with physiological saline solution.
Concrete operations:
1, full liver obtains: identical with embodiment 1.
2, taking off cell prepares:
(1) take off 37 ℃ of Cell saps (proportioning: 0.05% rt-PA, 0.5%tween 80) and take off Cell sap and inject through portal vein, folder closes Hepatic artery sleeve pipe and proximal part caval vein, keeps 30min.Then folder closes the Hepatic artery sleeve pipe, open proximal part caval vein, and the connection door vein pours into 4ml/min speed by portal vein with taking off Cell sap on peristaltic pump.
(2) the post-processed method is identical with embodiment 1.
3, the full liver of rat takes off cytoskeleton authentication method and result:
(1) general form learn to be observed: after taking off cell, the full liver of perusal rat takes off cell liver support and is white in color translucently, spongy, and outside peplos is intact, sees through after birth and can see in liver blood vessel network clearly.Under low power microscope, vascular tree is high-visible.
(2) microexamination:
Scanning electron microscopic observation:
Take off the substrate of cell liver support, 8000 * time of amplification, can see that the collagen fabric screen distance keeps good, have between the aperture and be convenient to the connection that cell is creeped, adhered to.Amplification 4000 * times the time, can see the fiber alignment of liver plate structure sample.
HE and three-color process are observed:
Take off the liver support after cell, after paraffin embedding, section, row HE dyeing and Trichrome dyeing, do not see cell residue, and the albumen of collagen scaffold keeps intact.As seen collagen scaffold network clearly after HE dyeing, but do not see cell.Three-color process dyeing is visible obtain support mainly formed by collagen protein and sugared Fibronectin.
DNA residues detection (reacting cells residual quantity):
Organize test kit (to be purchased from Qiagen Inc with DNeasy, Valencia, CA) take off the DNA composition of cell rami hepatici frame by the different rats of test kit operating instruction extracting (n=6) after, be DNA with ultraviolet spectrophotometer (U.S. Thermo) quantitative, DNA do not detected, and matched group (normal liver tissue) can detect DNA content.
Claims (1)
1. a liver organization takes off Cell sap, it is characterized in that: introduce a kind of novel Cell sap that takes off of being made by two kinds of compositions in full liver takes off cell technology, this takes off Cell sap is to swash agent rt-PA and 0.5-2% tween 80 by 0.01%-0.05% recombined human tissue plasmin, configures with physiological saline solution.
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| CN2013100676113A CN103083726A (en) | 2013-03-04 | 2013-03-04 | Liver tissue cell removal solution |
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| CN2013100676113A CN103083726A (en) | 2013-03-04 | 2013-03-04 | Liver tissue cell removal solution |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103690997A (en) * | 2013-12-05 | 2014-04-02 | 南方医科大学珠江医院 | Kit for preparing acellularized liver scaffold and application method of kit |
| CN115453021A (en) * | 2022-08-15 | 2022-12-09 | 首都医科大学附属北京友谊医院 | SDS (sodium dodecyl sulfate) decellularized solution and application thereof in decellularization treatment or identification of insoluble components of extracellular matrix |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1617738A (en) * | 2001-11-26 | 2005-05-18 | 杰南技术公司 | Catheter composition and uses thereof |
| CN1775189A (en) * | 2005-11-30 | 2006-05-24 | 烟台正海生物技术有限公司 | Acellular dermal matrix and its preparing method |
-
2013
- 2013-03-04 CN CN2013100676113A patent/CN103083726A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1617738A (en) * | 2001-11-26 | 2005-05-18 | 杰南技术公司 | Catheter composition and uses thereof |
| CN1775189A (en) * | 2005-11-30 | 2006-05-24 | 烟台正海生物技术有限公司 | Acellular dermal matrix and its preparing method |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103690997A (en) * | 2013-12-05 | 2014-04-02 | 南方医科大学珠江医院 | Kit for preparing acellularized liver scaffold and application method of kit |
| CN115453021A (en) * | 2022-08-15 | 2022-12-09 | 首都医科大学附属北京友谊医院 | SDS (sodium dodecyl sulfate) decellularized solution and application thereof in decellularization treatment or identification of insoluble components of extracellular matrix |
| CN115453021B (en) * | 2022-08-15 | 2023-11-14 | 首都医科大学附属北京友谊医院 | SDS decellularized solution and application thereof in decellularized treatment or identification of extracellular matrix insoluble components |
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Application publication date: 20130508 |