CN103751843B - Prepare the test kit of full liver biological support and full liver biological support preparation method - Google Patents
Prepare the test kit of full liver biological support and full liver biological support preparation method Download PDFInfo
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Abstract
The invention discloses a kind of test kit preparing full liver biological support, comprise infusion liquid I and infusion liquid II, wherein, infusion liquid I comprises 0.2g/L EDTA, 25000U/L heparin sodium and phosphate buffer, and infusion liquid II comprises 2.5g/L sodium lauryl sulphate.Utilize the infusion liquid of this test kit to prepare full liver biological support, be specially infusion liquid I to pour in vitro liver with the portal vein of the perfusion flow velocity of 15ml/min through in vitro liver, infusion liquid II is poured in vitro liver with the portal vein of the perfusion flow velocity of 3ml/min through in vitro liver.By mentioned reagent box and method, the time of contact of sodium lauryl sulphate and full liver biological support can be reduced, make the full liver biological support of preparation farthest retain liver cell epimatrix composition, and liver cell can be removed completely.
Description
Technical field
The present invention relates to liver tissue engineering field, particularly relate to a kind of prepare full liver biological support test kit and full liver biological support preparation method.
Background technology
At present, it is the unique effective method for the treatment of organs exhaustion that organ in situ transplants, and for the demand of suitable organ transplantation considerably beyond the quantity that can utilize organ, therefore, studies transplantable parenchymatous organ particularly important based on organizational project and regenerative medicine.
Utilize natural organa parenchymatosum to carry out decellularization process, just can obtain the intact stent that contain piping network, the method not only removes a large amount of cells and nuclearity material, also retains three-dimensional extra-cellular matrix structure.
The impact of decellularization method on biological support different at present has bigger difference, and impact shows as: the loss of Fibronectin and chitosan component, the destruction of collagenous network, and this all will change the Mechanics of Machinery characteristic of biological support.Need in existing decellularization process to use detergent, as Triton X-100 or sodium lauryl sulphate.Detergent goes the impact of the effectiveness of cell and extracellular matrix composition to have tissue specificity, is specially Triton X-100 and forms significantly destruction when removing cell to collagen component; Sodium lauryl sulphate goes to cause during cell the loss of collagen distortion in ligament structure and glycosaminoglycans composition.
Extracellular matrix components is synthesized and release by the cell that histoorgan is intrinsic usually, and keeps dynamic equilibrium with ambient, external environment.Because extracellular matrix is synthesized by the former host cell of each histoorgan, therefore, be a kind of desirable cell substrate in theory.How reducing the impact of cell detergent on liver organization biological characteristics, is build one of full liver biological support problem demanding prompt solution at present.
Summary of the invention
The object of this invention is to provide a kind of prepare full liver biological support test kit and full liver biological support preparation method, it can reduce the time of contact of sodium lauryl sulphate and full liver biological support, maximum reservation liver cell epimatrix composition, and liver cell can be removed completely.
For achieving the above object, the technical scheme that the present invention adopts is: provide a kind of test kit preparing full liver biological support, comprise infusion liquid I and infusion liquid II.
Wherein, infusion liquid I comprises 0.2g/L EDTA, 25000U/L heparin sodium and phosphate buffer; Infusion liquid II comprises 2.5g/L sodium lauryl sulphate.
Wherein, the PH=7-8 of phosphate buffer in infusion liquid I.The solvent of infusion liquid II is the deionized water of PH=7-8.
Wherein, the compound method of infusion liquid I is: it is in the phosphate buffer of 7-8 that 0.2g/L EDTA, 25000U/L heparin sodium is dissolved in pH value.
The compound method of infusion liquid II is: 2.5g/L sodium lauryl sulphate being dissolved in pH value is in the deionized water of 7-8.
For achieving the above object, the technical scheme that the present invention adopts is: provide a kind of full liver biological support preparation method, comprise the following steps.
First time perfusion: the infusion liquid I containing 0.2g/L EDTA, 25000U/L heparin sodium and phosphate buffer through the portal vein perfusion 500ml of in vitro liver, wherein, the rate of flooding of infusion liquid I is 15ml/min.The PH=7-8 of phosphate buffer in infusion liquid I.
Second time perfusion: after first time has poured into, contain the infusion liquid II of 2.5g/L sodium lauryl sulphate through the portal vein perfusion 500ml of in vitro liver, wherein, the rate of flooding of infusion liquid II is 3ml/min.The solvent of infusion liquid II is the deionized water of PH=7-8.
Third time perfusion, after second time has been poured into, with the portal vein perfusion 2000ml deionized water of the perfusion flow velocity of 15ml/min through in vitro liver, to remove infusion liquid II residual in vitro liver, and then obtains full liver biological support.
Wherein, first time perfusion, second time perfusion and third time perfusion are all by pouring in the mode of the mid-pipe of in vitro liver portal vein.
The invention has the beneficial effects as follows: the situation being different from prior art, the present invention comprises infusion liquid I and infusion liquid II for the preparation of the test kit of full liver biological support, wherein, infusion liquid I comprises 0.2g/L EDTA, 25000U/L heparin sodium and phosphate buffer, and infusion liquid II comprises 2.5g/L sodium lauryl sulphate.Utilize the infusion liquid of this test kit to prepare full liver biological support, be specially infusion liquid I to pour in vitro liver with the portal vein of the perfusion flow velocity of 15ml/min through in vitro liver, infusion liquid II is poured in vitro liver with the portal vein of the perfusion flow velocity of 3ml/min through in vitro liver.By the perfusion flow velocity of mentioned reagent box and infusion liquid II 3ml/min, the time of contact of sodium lauryl sulphate and full liver biological support can be reduced, make the full liver biological support of preparation farthest retain liver cell epimatrix composition, and liver cell can be removed completely.
Accompanying drawing explanation
Fig. 1 is the structure chart of full liver biological support prepared by the present invention;
Fig. 2 is the scattergram of laminin，LN after the full liver biological support immunofluorescence dyeing prepared of the present invention;
Fig. 3 is the scattergram of collagen protein IV after the full liver biological support immunofluorescence dyeing prepared of the present invention;
Fig. 4 is the scattergram of collagen protein I after the full liver biological support immunofluorescence dyeing prepared of the present invention;
Fig. 5 is the scattergram of fibronectin after the full liver biological support immunofluorescence dyeing prepared of the present invention;
Fig. 6 is the scattergram of elastin laminin after the full liver biological support immunofluorescence dyeing prepared of the present invention;
Fig. 7 is the electron-microscope scanning figure mono-of full liver biological support prepared by the present invention;
Fig. 8 is the electron-microscope scanning figure bis-of full liver biological support prepared by the present invention;
Fig. 9 is the comparison diagram of GAG content and normal rat liver in the full liver biological support prepared of the present invention;
Figure 10 is the comparison diagram of DNA content and normal rat liver in the full liver biological support prepared of the present invention.
Detailed description of the invention
Below in conjunction with drawings and Examples, the present invention is described in detail.
Embodiment 1
The experiment material of the present embodiment is the liver of rat.
The test kit that the present embodiment prepares the full liver biological support of rat comprises infusion liquid I and infusion liquid II.
Wherein, infusion liquid I comprises 0.2g/L EDTA, 25000U/L heparin sodium and phosphate buffer; Infusion liquid II comprises 2.5g/L sodium lauryl sulphate.
Wherein, the PH=7-8 of phosphate buffer in infusion liquid I.The solvent of infusion liquid II is the deionized water of PH=7-8.
In this enforcement, the compound method of infusion liquid I and infusion liquid II is as follows.
The preparation of infusion liquid I: getting 100mg EDTA and 2ml heparin sodium, to be dissolved in PH be in the 1*PBS liquid of 7.35, is settled to 500ml.Wherein, 2ml heparin sodium is containing the heparin sodium of 25000U.
The preparation of infusion liquid II: getting 1.25g sodium lauryl sulphate, to be dissolved in PH be in the deionized water of 7.35, is settled to 500ml.
The test kit of the present embodiment is utilized to prepare the method for rat full liver biological support as follows:
A. the acquisition of Isolated Rat Liver
At rats by intraperitoneal injection chloral hydrate with anesthetized rat, then realize whole body heparinization by 2ml heparin injections to rat body.Get abdominal transverse incision and appear portal vein, ligation branch of portal vein, main portal vein puts pipe, and 1
#silk thread is dual fixing.Isolate liver postcava, ligation from disconnected, whole operating process keeps action softly to ensure the integrity of acquisition liver.
B. utilize automatic pump trans-portal vein intubate to pour into 500ml infusion liquid I, rate of flooding is 15ml/min.
Infusion liquid I rinses the blood in liver, makes liver become khaki from redness.
The effect of infusion liquid I comprises: by EDTA chelating Ca
2+, connect with the desmosome of loosening between liver cell, make cell processes relatively easy; Can prevent the blood capillary in liver from forming thrombosis by the flushing of heparin sodium, infusion liquid is fully contacted with each liver cell.
C. utilize automatic pump trans-portal vein intubate to pour into 500ml infusion liquid II, rate of flooding is 3ml/min.
After perfusion 500ml infusion liquid II, rat liver becomes transparence from khaki.
The present embodiment selects the sodium lauryl sulphate of 2.5g/L and the rate of flooding of 3ml/min, intactly can not only remove liver cell, can also retain abundant liver cell epimatrix composition.Sodium lauryl sulphate goes cell agent as nonionic simultaneously, is easily gone out liver by deionized water, and does not residue in full liver biological support.
Wherein, infusion liquid II is selected to carry out cell with the perfusion flow velocity of 3ml/min, liver cell can not only be made fully to contact with infusion liquid II, the infusion time of infusion liquid II can also be shortened to 2 hours about 40 minutes, in this research work carried out at present in this field, the used time is the shortest substantially.The cell that goes of short time can be avoided producing adverse influence to full liver biological support.
D. automatic pump trans-portal vein intubate is utilized to pour into 2000ml deionized water, with the full liver support of abundant lavation, the infusion liquid II that eluting is residual.Wherein, rate of flooding is 15ml/min.
Above-mentioned steps A, step B, step C and step D carry out successively, and a perfusion step completes, then carry out next one perfusion step.
The full liver biological support prepared below by concrete method validation the present embodiment meets the needs of liver tissue engineering.
Refer to Fig. 1, Fig. 1 is the structure chart of full liver biological support prepared by the present invention, and as shown in Figure 1, the full liver biological support of rat presents white clear shape, does not have any khaki material to remain.
Refer to Fig. 2, Fig. 2 is the scattergram of laminin，LN after the full liver biological support immunofluorescence dyeing prepared of the present invention, and as shown by the arrows in Figure 2, laminin，LN (Laminin) is mainly distributed in the basement membrane of blood vessel.
Refer to Fig. 3, Fig. 3 is the scattergram of collagen protein IV after the full liver biological support immunofluorescence dyeing prepared of the present invention, as shown by the arrows in Figure 3, collagen protein IV(Collagen-IV) be mainly distributed in the basement membrane of blood vessel.
Refer to Fig. 4, Fig. 4 is the scattergram of collagen protein I after the full liver biological support immunofluorescence dyeing prepared of the present invention, as shown by the arrows in Figure 4, collagen protein I(Collagen-I) be mainly distributed between lobules of liver fibrous connective tissue.
Refer to Fig. 5, Fig. 5 is the scattergram of fibronectin after the full liver biological support immunofluorescence dyeing prepared of the present invention, and as shown by the arrows in Figure 5, fibronectin (Fibronection) is mainly distributed in the fibrous connective tissue between lobules of liver.
Refer to Fig. 6, Fig. 6 is the scattergram of elastin laminin after the full liver biological support immunofluorescence dyeing prepared of the present invention, and as shown by the arrows in Figure 6, elastin laminin (Elastin) is mainly distributed in the fibrous connective tissue between lobules of liver.
Refer to Fig. 7 and Fig. 8, Fig. 7 and Fig. 8 is respectively internal electrical scarnning mirror figure and the surface electrical scarnning mirror figure of full liver biological support prepared by the present invention.As shown in Figure 7, Figure 8, support tunicle is complete, and collagen fiber are neat and have no fracture at racks, are also shown in the existence of vascular system in the drawings.
Refer to Fig. 9, Fig. 9 is glycosaminoglycans (glycosaminoglycan in the full liver biological support prepared of the present invention, GAG) comparison diagram of content and rat normal liver (Native liver), as shown in Figure 9, in support, the content of GAG is 4.98 ± 0.78 μ g/mg, is about 53% of GAG content in normal liver (8.16 ± 0.28 μ g/mg).
Refer to Figure 10, Figure 10 is the comparison diagram of DNA content and normal rat liver in the full liver biological support prepared of the present invention, as shown in Figure 10, in support, residual DNA content is 49.15 ± 5.98ng/mg, be only about 1.5% of normal liver (2546.81 ± 526.39ng/mg), lower than the minimum flow (50ng/mg, dry weight) of residual DNA required by current liver biological support.
Wherein, the data in table 1 are utilized to draw the result of Fig. 9 and Figure 10.
Table 1
It is pointed out that the test kit of the present embodiment and utilize it to prepare the method for full liver biological support, also can be used for the full liver biological support preparing people or other animals, as: the full liver biological support such as corpse liver, Hepar Sus domestica or monkey liver.
In sum, the present invention accuracy controlling can not only remove in cell processes perfusion flow velocity for the preparation of the test kit of full liver biological support and using method thereof, also simplifies the process of removing cell, thus significantly shortens the time that liver perfusion goes required for cell.
Test kit of the present invention utilizes sodium lauryl sulphate can complete eluting cytoskeletal protein and Vimentin, and the rate of flooding of sodium lauryl sulphate and perfusion concentration can reduce the action time of itself and biological support, reduce the destruction to biological support structure and composition.Therefore the full liver biological support prepared of the present invention, while removing cell completely, can retain abundant liver cell epimatrix.
The foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every utilize description of the present invention and accompanying drawing content to do equivalent structure or equivalent flow process conversion; or be directly or indirectly used in other relevant technical fields, be all in like manner included in scope of patent protection of the present invention.
Claims (7)
1. prepare a test kit for full liver biological support, it is characterized in that, comprise infusion liquid I and infusion liquid II;
Described infusion liquid I comprises 0.2g/L EDTA, 25000U/L heparin sodium and phosphate buffer;
Described infusion liquid II comprises 2.5g/L sodium lauryl sulphate, and wherein, the rate of flooding of described infusion liquid II is 3ml/min.
2. test kit according to claim 1, is characterized in that, the pH=7-8 of phosphate buffer in described infusion liquid I.
3. test kit according to claim 2, is characterized in that, the solvent of described infusion liquid II is the deionized water of pH=7-8.
4. a full liver biological support preparation method, is characterized in that, comprise the following steps:
First time perfusion: the infusion liquid I containing 0.2g/L EDTA, 25000U/L heparin sodium and phosphate buffer through the portal vein perfusion 500ml of in vitro liver, wherein, the rate of flooding of described infusion liquid I is 15ml/min;
Second time perfusion: after described first time has poured into, contain the infusion liquid II of 2.5g/L sodium lauryl sulphate through the portal vein perfusion 500ml of described in vitro liver, wherein, the rate of flooding of described infusion liquid II is 3ml/min;
Third time perfusion, after described second time has been poured into, with the portal vein perfusion 2000ml deionized water of the perfusion flow velocity of 15ml/min through described in vitro liver, to remove infusion liquid II residual in described in vitro liver, and then obtains full liver biological support.
5. method according to claim 4, is characterized in that, the pH=7-8 of phosphate buffer in described infusion liquid I.
6. method according to claim 5, is characterized in that, the solvent of described infusion liquid II is the deionized water of pH=7-8.
7. method according to claim 6, is characterized in that, described first time perfusion, second time perfusion and third time perfusion are all by pouring in the mode of the mid-pipe of in vitro liver portal vein.
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| CN104288838B (en) * | 2014-10-22 | 2017-05-17 | 浙江大学医学院附属邵逸夫医院 | Kit for obtaining multiple-organ and tissue extracellular matrix and using method of kit |
| CN104353115B (en) * | 2014-10-29 | 2016-06-01 | 南通大学附属医院 | Take off the test kit of cytoskeleton, the preparation of support for pancreas and plant method for planting again |
| CN104841017B (en) * | 2015-05-28 | 2017-06-13 | 四川大学华西医院 | Decellularized liver biological scaffold with anticoagulant property and preparation method thereof |
| CN106267345B (en) * | 2016-08-04 | 2019-03-26 | 中国人民解放军第三军医大学第一附属医院 | A kind of preparation method of biologically active regeneration period organ acellular organism bracket and products thereof and application |
| CN107281551A (en) * | 2017-07-11 | 2017-10-24 | 温旭东 | The method that freezing method prepares liver biological support |
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| CN102448476A (en) * | 2009-03-31 | 2012-05-09 | 明尼苏达大学董事会 | Decellularization and recellularization of organs and tissues |
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| US20050249816A1 (en) * | 1999-12-29 | 2005-11-10 | Wake Forest University Health Services | Tissue engineered liver constructs |
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| CN102448476A (en) * | 2009-03-31 | 2012-05-09 | 明尼苏达大学董事会 | Decellularization and recellularization of organs and tissues |
| CN101564554A (en) * | 2009-06-03 | 2009-10-28 | 南方医科大学珠江医院 | Kit and method used for obtaining full liver stent |
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