CN104353115B - Take off the test kit of cytoskeleton, the preparation of support for pancreas and plant method for planting again - Google Patents
Take off the test kit of cytoskeleton, the preparation of support for pancreas and plant method for planting again Download PDFInfo
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- CN104353115B CN104353115B CN201410590216.8A CN201410590216A CN104353115B CN 104353115 B CN104353115 B CN 104353115B CN 201410590216 A CN201410590216 A CN 201410590216A CN 104353115 B CN104353115 B CN 104353115B
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Abstract
The present invention discloses a kind of for the test kit of the de-cytoskeleton of pancreas, the preparation of support and plant method for planting again, and test kit comprises perfusion liquid 1, perfusion liquid 2 and perfusion liquid 3; Does wherein perfusion liquid 1 comprise 0.25g/L? EDTA, 10000? U/L heparin sodium and PBS; Does perfusion liquid 2 comprise 1%? Triton-X? 100 and 0.1% ammoniacal liquor; Does perfusion liquid 3 comprise 0.01g/L? DNase I. Agent box expense of the present invention is low, cell de-except thoroughly, to be easy to remove washing composition, extracellular matrix and vascular system structure intact, be conducive to plantation again and the growth of external source and endogenous cell, and be conveniently transplanted to diabetic experimental animal.
Description
Technical field
The present invention relates to a kind of for the test kit of the de-cytoskeleton of pancreas, the preparation of support and plant method for planting again.
Background technology
Diabetes are impaired by defect of insulin secretion or its biological action, or both to have what cause concurrently take hyperglycemia as the metabolic disease of feature. By regulating diet, physical activity, oral antidiabetic drug and exogenous insulin injection can reduce the incidence of diabetes microvascular and great vessels pathology and related complication to a certain extent, but the effect of healing can not be reached, the organ injury that hyperglycemia is relevant is still the first cause of diabetic death.
�� cell has the function of excreting insulin, by pancreas or islet cell transplantation, is the methods for the treatment of that uniquely can play control glucose level steady in a long-term at present in type 1 diabetes patient. At present, desirable can be used for pancreas or the pancreas islet transplanted quantity not sufficient when, preparation can be very necessary for the organizational project pancreas transplanted. Pancreatic tissue engineering is the novelty approach building portable pancreas, but, owing to lacking suitable timbering material, usually constructed organizational project pancreas structure is simple, and blood vessel network improves not and can not reach the object of replacement therapy.
Go cell pancreas support as a kind of natural biological support, not only remain with cell adhesion, breed the extracellular matrix components relied on, comprising: protein, polysaccharide, somatomedin and cytokine; And the pipeline configuration required for remaining with high-density cell growth, physiological blood pressure can be maintained after being transplanted in body, it is possible to so that ancestor cell differentiates is organ specificity phenotype.
But, how to select suitable to remove cell reagent, reduce and go cell rests washing composition on the impact of pancreas biological characteristics, obtain the pancreas biological support being conducive to cell to plant again, be build portable pancreas biological support problem in the urgent need to address at present.
Summary of the invention
It is an object of the invention to provide a kind of prepare the test kit of pancreas acellular organism support, the preparation of pancreas Acellularized valve and again plantation obtain portable pancreas support method. Described test kit expense is low, cell de-except thoroughly, to be easy to remove washing composition, extracellular matrix and vascular system structure intact, be conducive to plantation again and the growth of external source and endogenous cell, and be conveniently transplanted to diabetic experimental animal.
The technical solution of the present invention is:
For a test kit for the de-cytoskeleton of pancreas, it is characterized in that: comprise perfusion liquid 1, perfusion liquid 2 and perfusion liquid 3; Wherein perfusion liquid 1 comprises 0.25g/LEDTA, 10000U/L heparin sodium and PBS; Perfusion liquid 2 comprises 1%Triton-X100 and 0.1% ammoniacal liquor; Perfusion liquid 3 comprises 0.01g/LDNase I;
Wherein, the PH=7-8 of PBS in perfusion liquid 1; The solvent of perfusion liquid 2 is the distilled water of PH=7-8; The solvent of perfusion liquid 3 is the distilled water of PH=7-8.
The compound method of perfusion liquid 1 is: be dissolved in the PBS of 1LPH=7-8 by 0.25gEDTA, 10000U heparin sodium;
The compound method of perfusion liquid 2 is: be dissolved in the deionized water of 1LPH=7-8 by 10mLTriton-X100 and 1mL ammoniacal liquor;
The compound method of perfusion liquid 3 is: be dissolved in the deionized water of 1LPH=7-8 by 0.01gDNase I.
A preparation method for the de-cytoskeleton of pancreas, is characterized in that: comprise the following steps:
(1) from body pancreas peristaltic pump trans-portal vein, mouse slowly being poured into 100ml perfusion liquid 1, control speed is 5ml/min; Perfusion liquid 1 rinses the blood in pancreas, pancreas is turned into faint yellow from incarnadine;
(2)-80 DEG C of refrigerators will be placed in from body pancreas, and be taken out after one day and melt under normal temperature;
(3) utilizing peristaltic pump trans-portal vein slowly to pour into 1L perfusion liquid 2, speed control is at 2ml/min; After perfusion 1L perfusion liquid 2, mice pancreatic turns into transparent shape from faint yellow;
(4) utilizing peristaltic pump trans-portal vein perfusion 200mlDNase I solution, rear continuation pours into 2L deionized water, to remove perfusion liquid remaining in body pancreas, obtains the de-cytoskeleton of full pancreas, and rate of flooding controls at 5ml/min.
The de-cytoskeleton of pancreas plant a method for planting again, it is characterized in that: comprise the following steps:
(1) pancreas biological support is adopted gamma-ray irradiation sterilizing;
(2) the de-cytoskeleton circumfusion 500ml in three-dimensional circular culture systems of full pancreas after sterilizing contains the DMEM high glucose medium of 10% foetal calf serum, and flow rate control is at 5ml/min;
(3) by the DMEM high glucose medium containing 10% foetal calf serum to 3ml resuspended after 1 �� 107 cell dissociation; Remaining needle in trans-portal vein injects 1ml cell suspension, and interval 20min re-injects 1ml, and interval 20min third time injects 1ml cell suspension; Pancreas support is left standstill 2h in the medium after completing by cell infusion, is beneficial to the subsides wall of cell;
(4) the remaining needle circumfusion substratum in trans-portal vein, flow rate control is at 4ml/min, and substratum is changed once every other day, Dual culture 5 days.
Agent box expense of the present invention is low, cell de-except thoroughly, to be easy to remove washing composition, extracellular matrix and vascular system structure intact, be conducive to plantation again and the growth of external source and endogenous cell, and be conveniently transplanted to diabetic experimental animal.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the invention will be further described.
Fig. 1 is the cardinal principle audio-visual picture of mice pancreatic Acellularized valve;
Fig. 2 is the HE colored graph before and after the de-cell of mice pancreatic and scanning electron microscope (SEM) photograph;
Wherein A and C is the HE dyeing before the de-cell of pancreas and scanning electron microscope (SEM) photograph; B and D is that pancreas dyes and scanning electron microscope (SEM) photograph via the HE of the pancreas biological support of preparation after the de-cell of method process of this patent.
Fig. 3 is that the de-cytoskeleton of mice pancreatic plants the HE colored graph after MIN-6 cell and immunohistochemical methods figure again.
Wherein (A) HE colored graph; (B) immunohistochemical methods figure.
Embodiment
The test kit that the present embodiment prepares the full pancreas biological support of mouse comprises perfusion liquid 1, perfusion liquid 2 and perfusion liquid 3.
Wherein, perfusion liquid 1 is the PBS of 0.25g/LEDTA, 10000U/L heparin sodium; Perfusion liquid 2 is 1%Triton-X100 and the mixed solution of 0.1% ammoniacal liquor, and perfusion liquid 3 is 0.01gDNase I.
Wherein, the PH=7.35 of phosphoric acid buffer in perfusion liquid 1; The PH=7.35 of solvent deionized water in perfusion liquid 2,3.
In this enforcement, the compound method of perfusion liquid 1, perfusion liquid 2 and perfusion liquid 3 is as follows:
The configuration of perfusion liquid 1: get 125mgEDTA and 0.4ml heparin sodium and be dissolved in the 1*PBS liquid that PH is 7.35, be settled to 500ml, wherein 0.4ml heparin sodium is containing the heparin sodium of 10000U.
The configuration of perfusion liquid 2: get 10mlTritonX-100 and 1ml ammoniacal liquor and be dissolved in the deionized water of PH=7.35, be settled to 1000ml.
The configuration of perfusion liquid 3: precise 10mgDNase I is dissolved in the deionized water of PH=7.35, is settled to 1000ml.
The application of described test kit in the preparation full pancreas biological support of mouse.
The test kit of the present embodiment is utilized to prepare the method for mouse full pancreas biological support as follows:
A. mouse is from the acquisition of body pancreas
Inject Chloral Hydrate with anesthetized mice at mouse peritoneal, then it is injected in mouse body by 1ml heparin to realize whole body heparinization. Trans-portal vein drives in the wrong direction intubate (blue remaining needle 22G), and No. 1 silk thread is dual fixing. By the big branch of the far-end of mesentery upper vein and splenetic artery and splenetic vein successively careful ligation to prevent seepage, finally by the weave construction careful separation of pancreas and surrounding, such as stomach, small intestine, spleen and mesentery tissue. In whole operating process, maintenance action is softly to ensure to obtain the integrity of pancreas.
B. utilizing peristaltic pump trans-portal vein slowly to pour into 100ml perfusion liquid 1, control speed is 5ml/min.
Perfusion liquid 1 rinses the blood in pancreas, pancreas is turned into faint yellow from incarnadine.
The effect of perfusion liquid 1 comprises: by EDTA chelating Ca2+, with the connection of loosen cell and cytoskeleton, makes follow-up de-cell processes comparatively easy. Can prevent the capillary blood vessel in pancreas from forming thrombus by the flushing of heparin sodium so that perfusion liquid can abundant and cells contacting.
C. will be placed in-80 DEG C of refrigerators from body pancreas, and be taken out after one day and melt under normal temperature.
D. utilizing peristaltic pump trans-portal vein slowly to pour into 1L perfusion liquid 2, speed control is at 2ml/min.
After perfusion 1L perfusion liquid 2, mice pancreatic turns into transparent shape from faint yellow.
E. utilizing peristaltic pump trans-portal vein perfusion 200mlDNase I solution, rear continuation pours into 2L deionized water, to remove perfusion liquid remaining in body pancreas, obtains the de-cytoskeleton of full pancreas, and rate of flooding controls at 5ml/min.
Above-mentioned steps A, B, C, D and E carry out successively, and a perfusion step completes, then carry out next perfusion step.
Pancreas biological support adopts gamma-ray irradiation sterilizing.
The de-cytoskeleton circumfusion 500ml in three-dimensional circular culture systems of full pancreas after sterilizing contains the DMEM high glucose medium of 10% foetal calf serum, and flow rate control is at 5ml/min.
By in the DMEM high glucose medium containing 10% foetal calf serum to 3ml resuspended after 1 �� 107 cell dissociation. Remaining needle in trans-portal vein injects 1ml cell suspension, and interval 20min re-injects 1ml, and interval 20min third time injects 1ml cell suspension. Pancreas support is left standstill 2h in the medium after completing by cell infusion, is beneficial to the subsides wall of cell.
Remaining needle circumfusion substratum in trans-portal vein, flow rate control is at 4ml/min, and substratum is changed once every other day, Dual culture 5 days.
The full pancreas biological support prepared below by concrete method validation the present embodiment meets the needs of pancreatic tissue engineering.
Referring to Fig. 2, wherein A and C is the HE dyeing before the de-cell of pancreas and scanning electron microscope (SEM) photograph; B and D is that pancreas dyes and scanning electron microscope (SEM) photograph via the HE of the pancreas biological support of preparation after the de-cell of method process of this patent.
Referring to Fig. 3, after the full pancreas biological support for being prepared by this patent method plants mouse MIN-6 cell, circumfusion is cultivated the HE after five days and is dyeed and immunohistochemical staining figure.
It is noted that the test kit of the present embodiment and utilize its method preparing full pancreas biological support, it is equally applicable to prepare the full pancreas biological support of other animals such as rat, cavy, pig.
The foregoing is only embodiments of the invention; not thereby the patent scope of the present invention is limited; every utilize specification sheets of the present invention and accompanying drawing content to do equivalent structure or equivalence flow process conversion; or directly or indirectly it is used in other relevant technical fields, all it is included in the scope of patent protection of the present invention with reason.
Claims (1)
1. a preparation method for the de-cytoskeleton of pancreas, is characterized in that: comprise the following steps:
(1) from body pancreas peristaltic pump trans-portal vein, mouse slowly being poured into 100ml perfusion liquid 1, control speed is 5ml/min; Perfusion liquid 1 rinses the blood in pancreas, pancreas is turned into faint yellow from incarnadine;
(2)-80 DEG C of refrigerators will be placed in from body pancreas, and be taken out after one day and melt under normal temperature;
(3) utilizing peristaltic pump trans-portal vein slowly to pour into 1L perfusion liquid 2, speed control is at 2ml/min; After perfusion 1L perfusion liquid 2, mice pancreatic turns into transparent shape from faint yellow;
(4) utilizing peristaltic pump trans-portal vein perfusion 200mlDNase I solution, rear continuation pours into 2L deionized water, to remove perfusion liquid remaining in body pancreas, obtains the de-cytoskeleton of full pancreas, and rate of flooding controls at 5ml/min;
Described perfusion liquid 1 comprises 0.25g/LEDTA, 10000U/L heparin sodium and PBS; Perfusion liquid 2 comprises 1%Triton-X100 and 0.1% ammoniacal liquor; DNase I is 0.01g/LDNase I, DNase I compound method: be dissolved in the deionized water of 1LpH=7-8 by 0.01gDNase I;
Wherein, the pH=7-8 of PBS in perfusion liquid 1;
The compound method of perfusion liquid 1 is: be dissolved in the PBS of 1LpH=7-8 by 0.25gEDTA, 10000U heparin sodium;
The compound method of perfusion liquid 2 is: be dissolved in the deionized water of 1LpH=7-8 by 10mLTriton-X100 and 1mL ammoniacal liquor.
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| CN106075583B (en) * | 2016-07-07 | 2020-07-14 | 中国人民解放军第二军医大学 | Method for preparing acellular matrix biological material by perfusion-pressure difference method |
| CN107412865A (en) * | 2016-10-27 | 2017-12-01 | 浙江保尔曼生物科技有限公司 | The decellularization kidney biological support and preparation method with anticoagulant functions of high intensity |
| CN107158463A (en) * | 2017-04-27 | 2017-09-15 | 汤礼军 | Preparation method and purposes for the pancreatic parenchymal hydrogel of repairing pancreas tissue |
| CN108079376A (en) * | 2018-02-11 | 2018-05-29 | 首都医科大学附属北京口腔医院 | One kind is for regenerated de- cell scaffold material of salivary gland and preparation method thereof |
| CN108295311A (en) * | 2018-03-07 | 2018-07-20 | 南京市第医院 | A kind of preparation method of temperature sensitive type extracellular matrix of kidney hydrogel |
| CN108841779A (en) * | 2018-06-11 | 2018-11-20 | 南通大学附属医院 | Pancreas specificity ECM is the application in insulin secretory cell promoting BMSCs proliferation, migration and directed differentiation |
| CN110694114A (en) * | 2019-10-31 | 2020-01-17 | 南通大学附属医院 | PRP-loaded pancreas acellular scaffold and preparation method thereof |
| CN110894492A (en) * | 2019-12-17 | 2020-03-20 | 南通大学附属医院 | Pancreatic cancer in-vitro 3D model construction method based on pancreatic acellular scaffold |
| CN114632188A (en) * | 2022-01-26 | 2022-06-17 | 北京大学第三医院（北京大学第三临床医学院） | Adipose tissue mass acellular stent with vascular pedicle and preparation method and application thereof |
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| IL139708A0 (en) * | 2000-11-15 | 2002-02-10 | Amiel Gilad | Process of decellularizing biological matrices and acellular biological matrices useful in tissue engineering |
| 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 |
| CN101829359B (en) * | 2010-04-16 | 2013-04-03 | 中国人民解放军第二军医大学 | Acellular ligament stent and seed cell compound culture method |
| CN103751843B (en) * | 2013-12-05 | 2015-09-16 | 南方医科大学珠江医院 | Prepare the test kit of full liver biological support and full liver biological support preparation method |
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