CN104353115A - Kit for pancreas decellularized scaffold and preparation and reseeding methods of scaffold - Google Patents
Kit for pancreas decellularized scaffold and preparation and reseeding methods of scaffold Download PDFInfo
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- CN104353115A CN104353115A CN201410590216.8A CN201410590216A CN104353115A CN 104353115 A CN104353115 A CN 104353115A CN 201410590216 A CN201410590216 A CN 201410590216A CN 104353115 A CN104353115 A CN 104353115A
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Abstract
The invention discloses a kit for a pancreas decellularized scaffold and preparation and reseeding methods of the scaffold. The kit comprises perfusion fluid 1, perfusion fluid 2 and perfusion fluid 3, wherein the perfusion fluid 1 comprises 0.25 g/L of EDTA (ethylene diamine tetraacetic acid), 10,000 U/L of heparin sodium and PBS (phosphate buffer solution); the perfusion fluid 2 comprises 1% Triton-X100 and 0.1% ammonia water; the perfusion fluid 3 comprises 0.01 g/L of DNase I (eoxyribonuclease I). The kit has the characteristics that the expense is low, cells are radically removed, a detergent is easily eliminated, the structures of extracellular matrixes and blood vascular systems are well preserved, the reseeding and growth of exogenous and endogenous cells are facilitated, and the exogenous and endogenous cells can be conveniently transplanted to diabetic experiment animals.
Description
Technical field
The present invention relates to and a kind ofly take off cytoskeletal test kit, the preparation of support and implantation methods again for pancreas.
Background technology
Diabetes be by defect of insulin secretion or its biological agent impaired, or both to have what cause concurrently take hyperglycemia as the metabolic disease of feature.By regulating diet, physical training, oral antidiabetic drug and exogenous insulin injection can reduce the incidence rate of diabetes microvascular and macroangiopathy and related complication to a certain extent, but the effect of healing can not be reached, the organ injury that hyperglycemia is correlated with remains the first cause of diabetic death.
β cell has the function of excreting insulin, by pancreas or islet cell transplantation, is in type 1 diabetes patient, uniquely to play the Therapeutic Method controlling blood sugar level steady in a long-term at present.At present, under the condition of the desirable lazy weight that can be used for pancreas or the islets of langerhans transplanted, preparation can be very necessary for the organizational project pancreas transplanted.Pancreatic tissue engineering is the novelty approach building transplantation 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 tryptase rami glandulares frame 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 remained with required for high-density cell growth, being transplanted to after in body and can maintaining physiological blood pressure, ancestor cell differentiates also can be made to be organ specificity phenotype.
But how to select suitable to remove cell reagent, reduce and go cell rests detergent on the impact of pancreas biological characteristics, obtaining the pancreas biological support being conducive to cell and planting, is build transplantation pancreas biological support problem in the urgent need to address at present.
Summary of the invention
The object of the present invention is to provide a kind of method prepared the test kit of pancreas acellular organism support, the preparation of pancreas Acellularized valve and plant acquisition transplantation pancreas support again.Described test kit expense is low, cell removes thoroughly, it is intact to be easy to remove detergent, extracellular matrix and vascular system structure, is conducive to plantation again and the growth of external source and endogenous cell, and is conveniently transplanted to diabetic experimental animal.
Technical solution of the present invention is:
A kind of pancreas that is used for takes off cytoskeletal test kit, it is characterized in that: comprise infusion liquid 1, infusion liquid 2 and infusion liquid 3; Wherein infusion liquid 1 comprises 0.25 g/L EDTA, 10000 U/L heparin sodiums and PBS buffer; Infusion liquid 2 comprises 1% Triton-X 100 and 0.1% ammonia; Infusion liquid 3 comprises 0.01 g/L DNase I;
Wherein, the PH=7-8 of PBS buffer in infusion liquid 1; The solvent of infusion liquid 2 is the distilled water of PH=7-8; The solvent of infusion liquid 3 is the distilled water of PH=7-8.
The compound method of infusion liquid 1 is: be dissolved in the PBS buffer of 1 L PH=7-8 by 0.25 g EDTA, 10000 U heparin sodiums;
The compound method of infusion liquid 2 is: 10 mL Triton-X 100 and 1mL ammonia are dissolved in the deionized water of 1 L PH=7-8;
The compound method of infusion liquid 3 is: be dissolved in by 0.01 g DNase I in the deionized water of 1 L PH=7-8.
A kind of pancreas takes off cytoskeletal preparation method, it is characterized in that: comprise the following steps:
(1) in vitro for mice pancreas peristaltic pump trans-portal vein is slowly poured into 100ml infusion liquid 1, control rate is 5 ml/min; Infusion liquid 1 rinses the blood in pancreas, pancreas is become faint yellow from pale red;
(2) in vitro pancreas is placed in-80 DEG C of refrigerators, is taken out after one day and melt under room temperature;
(3) utilize peristaltic pump trans-portal vein slowly to pour into 1L infusion liquid 2, speed controlling is at 2 ml/min; After pouring into 1 L infusion liquid 2, mice pancreatic becomes transparence from faint yellow;
(4) utilize peristaltic pump trans-portal vein to pour into 200 ml DNase I solution, 2 L deionized waters are poured in rear continuation, and to remove infusion liquid remaining in vitro pancreas, obtain full pancreas and take off cytoskeleton, rate of flooding controls at 5 ml/min.
A kind of pancreas takes off cytoskeletal implantation methods again, it is characterized in that: comprise the following steps:
(1) pancreas biological support is adopted gamma-ray irradiation sterilizing;
(2) the full pancreas after sterilizing takes off cytoskeleton circumfusion 500 ml in three-dimensional circular culture systems and contains the DMEM high glucose medium of 10% hyclone, and flow speed control is at 5 ml/min;
(3) contain in the DMEM high glucose medium of 10% hyclone by resuspended after 1 × 107 cell dissociation to 3 ml; Remaining needle in trans-portal vein injects 1 ml cell suspension, and interval 20 min re-injects 1 ml, and interval 20 min third time injects 1ml cell suspension; After cell infusion completes, pancreas support is rested on 2 h in culture medium, be beneficial to the adherent of cell;
(4) the remaining needle circumfusion culture medium in trans-portal vein, flow speed control is at 4 ml/min, and culture medium is changed once every other day, Dual culture 5 days.
Agent box expense of the present invention is low, cell removes thoroughly, it is intact to be easy to remove detergent, extracellular matrix and vascular system structure, is conducive to plantation again and the growth of external source and endogenous cell, and is 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 that mice pancreatic takes off HE colored graph before and after cell and scanning electron microscope (SEM) photograph;
Wherein A and C be before pancreas takes off cell HE dyeing and scanning electron microscope (SEM) photograph; The HE dyeing of the pancreas biological support that B and D is prepared after to be pancreas take off cell via the method process of this patent and scanning electron microscope (SEM) photograph.
Fig. 3 is that mice pancreatic takes off cytoskeleton and plants the HE colored graph after MIN-6 cell and SABC figure again.
Wherein (A) HE colored graph; (B) SABC figure.
Detailed description of the invention
The test kit that the present embodiment prepares the full pancreas biological support of mice comprises infusion liquid 1, infusion liquid 2 and infusion liquid 3.
Wherein, infusion liquid 1 is the PBS buffer of 0.25 g/L EDTA, 10000U/L heparin sodium; Infusion liquid 2 is the mixed liquor of 1% Triton-X 100 and 0.1% ammonia, and infusion liquid 3 is 0.01 g DNase I.
Wherein, the PH=7.35 of phosphate buffer in infusion liquid 1; The PH=7.35 of solvent deionized water in infusion liquid 2,3.
In this enforcement, the compound method of infusion liquid 1, infusion liquid 2 and infusion liquid 3 is as follows:
The configuration of infusion liquid 1: getting 125 mg EDTA and 0.4 ml heparin sodium, to be dissolved in PH be in the 1*PBS liquid of 7.35, and be settled to 500 ml, wherein 0.4 ml heparin sodium is containing the heparin sodium of 10000 U.
The configuration of infusion liquid 2: get 10 ml Triton X-100 and 1 ml ammonia is dissolved in the deionized water of PH=7.35, is settled to 1000 ml.
The configuration of infusion liquid 3: precise 10 mg DNase I is dissolved in the deionized water of PH=7.35, is settled to 1000 ml.
The application of described test kit in the full pancreas biological support of preparation mice.
The test kit of the present embodiment is utilized to prepare the method for mice full pancreas biological support as follows:
A. the acquisition of the in vitro pancreas of mice
At mouse peritoneal injection chloral hydrate with anesthetized mice, then realize whole body heparinization by 1 ml heparin injections to Mice Body.Trans-portal vein retrograde catheterization (blue remaining needle 22 G), No. 1 silk thread is dual fixing.By the large branch of the far-end of superior mesenteric vein and splenetic artery and splenetic vein successively careful ligation to prevent seepage, finally by the organizational structure careful separation of pancreas and surrounding, as stomach, small intestinal, spleen and mesentery tissue.Action is kept softly to ensure to obtain the integrity of pancreas in whole operating process.
B. utilize peristaltic pump trans-portal vein slowly to pour into 100 ml infusion liquid 1, control rate is 5 ml/min.
Infusion liquid 1 rinses the blood in pancreas, pancreas is become faint yellow from pale red.
The effect of infusion liquid 1 comprises: by EDTA chelating Ca2+, and to loosen, cell is connected with cytoskeletal, makes follow-up de-cell processes comparatively easy.Can prevent blood capillary in pancreas from forming thrombosis by the flushing of heparin sodium, make infusion liquid can fully and cells contacting.
C. in vitro pancreas is placed in-80 DEG C of refrigerators, is taken out after one day and melt under room temperature.
D. utilize peristaltic pump trans-portal vein slowly to pour into 1L infusion liquid 2, speed controlling is at 2 ml/min.
After pouring into 1 L infusion liquid 2, mice pancreatic becomes transparence from faint yellow.
E. utilize peristaltic pump trans-portal vein to pour into 200 ml DNase I solution, 2 L deionized waters are poured in rear continuation, and to remove infusion liquid remaining in vitro pancreas, obtain full pancreas and take off cytoskeleton, rate of flooding controls at 5 ml/min.
Above-mentioned steps A, B, C, D and E carry out successively, and a perfusion step completes, then carry out next one perfusion step.
Pancreas biological support adopts gamma-ray irradiation sterilizing.
Full pancreas after sterilizing takes off cytoskeleton circumfusion 500 ml in three-dimensional circular culture systems and contains the DMEM high glucose medium of 10% hyclone, and flow speed control is at 5 ml/min.
Contain resuspended after 1 × 107 cell dissociation in the DMEM high glucose medium of 10% hyclone to 3 ml.Remaining needle in trans-portal vein injects 1 ml cell suspension, and interval 20 min re-injects 1 ml, and interval 20 min third time injects 1ml cell suspension.After cell infusion completes, pancreas support is rested on 2 h in culture medium, be beneficial to the adherent of cell.
Remaining needle circumfusion culture medium in trans-portal vein, flow speed control is at 4 ml/min, and culture medium 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.
Refer to Fig. 2, wherein A and C be before pancreas takes off cell HE dyeing and scanning electron microscope (SEM) photograph; The HE dyeing of the pancreas biological support that B and D is prepared after to be pancreas take off cell via the method process of this patent and scanning electron microscope (SEM) photograph.
Refer to Fig. 3, for after the full pancreas biological support plantation mice MIN-6 cell prepared by this patent method, circumfusion is cultivated the HE after five days and is dyeed and immunohistochemical staining figure.
It is pointed out that the test kit of the present embodiment and utilize it to prepare the method for full pancreas biological support, being equally applicable to the full pancreas biological support preparing other animals such as rat, Cavia porcellus, pig.
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 (3)
1. take off a cytoskeletal test kit for pancreas, it is characterized in that: comprise infusion liquid 1, infusion liquid 2 and infusion liquid 3; Wherein infusion liquid 1 comprises 0.25 g/L EDTA, 10000 U/L heparin sodiums and PBS buffer; Infusion liquid 2 comprises 1% Triton-X 100 and 0.1% ammonia; Infusion liquid 3 comprises 0.01 g/L DNase I;
Wherein, the PH=7-8 of PBS buffer in infusion liquid 1; The solvent of infusion liquid 2 is the distilled water of PH=7-8; The solvent of infusion liquid 3 is the distilled water of PH=7-8.
The compound method of infusion liquid 1 is: be dissolved in the PBS buffer of 1 L PH=7-8 by 0.25 g EDTA, 10000 U heparin sodiums;
The compound method of infusion liquid 2 is: 10 mL Triton-X 100 and 1mL ammonia are dissolved in the deionized water of 1 L PH=7-8;
The compound method of infusion liquid 3 is: be dissolved in by 0.01 g DNase I in the deionized water of 1 L PH=7-8.
2. pancreas takes off a cytoskeletal preparation method, it is characterized in that: comprise the following steps:
(1) in vitro for mice pancreas peristaltic pump trans-portal vein is slowly poured into 100ml infusion liquid 1, control rate is 5 ml/min; Infusion liquid 1 rinses the blood in pancreas, pancreas is become faint yellow from pale red;
(2) in vitro pancreas is placed in-80 DEG C of refrigerators, is taken out after one day and melt under room temperature;
(3) utilize peristaltic pump trans-portal vein slowly to pour into 1L infusion liquid 2, speed controlling is at 2 ml/min; After pouring into 1 L infusion liquid 2, mice pancreatic becomes transparence from faint yellow;
(4) utilize peristaltic pump trans-portal vein to pour into 200 ml DNase I solution, 2 L deionized waters are poured in rear continuation, and to remove infusion liquid remaining in vitro pancreas, obtain full pancreas and take off cytoskeleton, rate of flooding controls at 5 ml/min.
3. pancreas takes off a cytoskeletal implantation methods again, it is characterized in that: comprise the following steps:
(1) pancreas biological support is adopted gamma-ray irradiation sterilizing;
(2) the full pancreas after sterilizing takes off cytoskeleton circumfusion 500 ml in three-dimensional circular culture systems and contains the DMEM high glucose medium of 10% hyclone, and flow speed control is at 5 ml/min;
(3) contain in the DMEM high glucose medium of 10% hyclone by resuspended after 1 × 107 cell dissociation to 3 ml; Remaining needle in trans-portal vein injects 1 ml cell suspension, and interval 20 min re-injects 1 ml, and interval 20 min third time injects 1ml cell suspension; After cell infusion completes, pancreas support is rested on 2 h in culture medium, be beneficial to the adherent of cell;
(4) the remaining needle circumfusion culture medium in trans-portal vein, flow speed control is at 4 ml/min, and culture medium is changed once every other day, Dual culture 5 days.
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Cited By (9)
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| CN106075583A (en) * | 2016-07-07 | 2016-11-09 | 中国人民解放军第二军医大学 | A kind of method using perfusion pressure reduction method to prepare acellular matrix biomaterial |
| CN107158463A (en) * | 2017-04-27 | 2017-09-15 | 汤礼军 | Preparation method and purposes for the pancreatic parenchymal hydrogel of repairing pancreas tissue |
| 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 |
| 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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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106075583A (en) * | 2016-07-07 | 2016-11-09 | 中国人民解放军第二军医大学 | A kind of method using perfusion pressure reduction method to prepare acellular matrix biomaterial |
| 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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