CN104826165A - Preparation method of acellular bionet and acellular bionet prepared by preparation method - Google Patents
Preparation method of acellular bionet and acellular bionet prepared by preparation method Download PDFInfo
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Abstract
The invention discloses a preparation method of a acellular bionet and the acellular bionet prepared by the preparation method. The preparation method of the acellular bionet comprises the step of acellular disposal by immersing omentum majus in a buffer solution containing lauryl sodium sulfate and DNA enzyme. The invention also discloses the acellular bionet prepared by the above method. By combining gradient elution and physical oscillation, the method has advantages as follows: conditions are mild; acellular efficiency of omentum majus is enhanced; and composition of omentum majus acellular matrix is retained to the maximum. The method also has advantages of simple operation and strong enforceability.
Description
Technical field
The present invention relates to organizational project and biology medical material technical field, the preparation method being specifically related to de-cell biological nethike embrane and the de-cell biological nethike embrane obtained by it.
Background technology
Extracellular matrix (extracellular matrix, ECM) be the complex composite thing of structural protein and functional protein, comprise multiple fibrin, as components such as collagen, fibronectin, laminin，LN, proteoglycans, its component is relatively conservative between different plant species.Extracellular matrix, as Growth of Cells support, plays an important role in cell adhesion, propagation and differentiation.In Tissue Engineering Study field, in order to better simulate the three-dimensional microenvironment of natural extracellular matrix, in recent years, research worker develops series obtains acellular matrix biologic bracket material technology and method by de-cell technology.
De-cell strategy mainly refers to and utilizes de-cell technology methods such as () physics, chemistry, enzymolysis to remove in tissue various cell component and hereditary material thus obtain natural biological timbering material.Acellular matrix material has many advantages relative to traditional biological timbering material, mainly comprises, 1) three dimensional structure of extracellular matrix retains complete; 2) natural extracellular matrix constituent is retained, and is beneficial to Growth of Cells and Function; 3) contain the cytokine in natural extracellular matrix and signaling molecule in de-cell scaffold material, the function of cell can be strengthened and promote biological function; 4) de-cell processes can reduce extracellular matrix immunogenicity, is beneficial to the therapeutic application of the tissue of heterologous source, extends the source of donor; 5) acellular matrix has biodegradability, does not cause inflammatory reaction.Just because of above advantage, the research of acellular matrix biologic bracket material obtains the extensive concern of domestic and international research worker in recent years and obtains serial breakthrough, is successfully applied to organizational project every field.At present, people can obtain acellular matrix timbering material from Various Tissues, as small intestinal, skin, blood vessel, cornea and the more complicated heart, lung, liver, kidney etc.
Omentum majus is the one of peritoneum, is one deck mucosa be present in higher mammal abdominal cavity, is the membranaceous tissue formed by connective tissue.Omentum majus connects the peritoneum of greater gastric curvature to transverse colon.Omentum majus is rich in extremely strong elasticity and the structure of very vascular, and these advantages make it be with a wide range of applications in organizational project and regenerative medicine field.At present, it is at the early-stage that omentum majus takes off cell correlational study, also there are problems urgently to be resolved hurrily.Organize due to the 26S Proteasome Structure and Function of omentum majus tissue and skin, blood vessel, valve etc. and there is bigger difference, traditional method for removing cells, as multigelation method, surfactant method, enzyme digestion etc. can not be applicable to the de-cell process of omentum majus tissue completely.And method step is loaded down with trivial details, cost is high, is not suitable for batch making.Therefore, need exploitation one at utmost can retain omentum majus extracellular matrix composition and composition at present, and method is simple, omentum majus method for removing cells with low cost.
Summary of the invention
The object of the present invention is to provide the omentum majus method for removing cells that a kind of operational approach is simple, with low cost.Utilize the method to obtain omentum majus acellular matrix and can retain omentum majus extracellular matrix composition and composition, there is good biocompatibility.
According to an aspect of the present invention, provide the preparation method of de-cell biological nethike embrane, it comprises and uses the buffer containing sodium lauryl sulphate and DNA enzymatic to soak the step that omentum majus carries out de-cell process.
According to a further aspect in the invention, provide the preparation method of de-cell biological nethike embrane, it is characterized in that, carry out according to following operating procedure:
(1) preparation of degreasant solution
Methanol, chloroform and ether are mixed with the ratio that volume ratio is 1: 1: 0.2-1;
(2) preparation of de-Cell sap
1) de-Cell sap I: take sodium lauryl sulphate and DNA enzymatic, be dissolved in phosphate buffer, wherein, the concentration of sodium lauryl sulphate in phosphate buffer is 1.5-3.5% weight ratio, the concentration of DNA enzymatic in phosphate buffer is 3500-4500U/L, pH is 7.2-7.4, filtration sterilization;
2) de-Cell sap II: take sodium lauryl sulphate and DNA enzymatic, be dissolved in phosphate buffer, wherein, the concentration of sodium lauryl sulphate in phosphate buffer is 0.6-1.5% weight ratio, the concentration of DNA enzymatic in PBS is 2500-3500U/L, pH is 7.2-7.4, filtration sterilization;
3) de-Cell sap III: take sodium lauryl sulphate and DNA enzymatic, be dissolved in phosphate buffer, wherein, the concentration of sodium lauryl sulphate in phosphate buffer is 0.1-0.6% weight ratio, the concentration of DNA enzymatic in PBS is 1500-2500U/L, pH is 7.2-7.4, filtration sterilization;
(3) omentum majus takes off cell process
1) get fresh omentum majus tissue, clean in phosphate buffer, the tissue concussion after cleaning be soaked in described degreasant solution and process, degreasant solution soak time is 18-30h, and concussion frequency is 150-250r/min;
2) the described omentum majus removing fat is organized in phosphate buffer and is cleaned, and concussion is soaked in described de-Cell sap I and processes, soak time is 4-12h, and concussion frequency is 100-200r/min;
3) be organized in phosphate buffer by the described omentum majus after de-Cell sap I process and clean, and shake to be soaked in de-Cell sap II and process, soak time is 4-12h, and concussion frequency is 100-200r/min;
4) be organized in phosphate buffer by the omentum majus after de-Cell sap II process and clean, and shake to be soaked in described de-Cell sap III and process, soak time is 4-12h, and concussion frequency is 100-200r/min;
5) by the described omentum majus tissue sterilizing after lyophilization after de-Cell sap III process.
Method employing gradient elution of the present invention shakes with physics the method combined and carries out de-cell process to omentum majus, compared with existing method for removing cells, method mild condition of the present invention, improve omentum majus and take off cell efficiency, farthest remain the composition of omentum majus acellular matrix simultaneously; This method also has simple to operate, that exploitativeness is strong advantage.
According to a further aspect in the invention, the de-cell biological nethike embrane obtained according to said method is provided.
Accompanying drawing explanation
Fig. 1 takes off cell biological nethike embrane type i collagen immunohistochemical staining × 200.
Fig. 2 takes off cell biological nethike embrane IV Collagen Type VI immunohistochemical staining × 200.
Fig. 3 takes off cell biological postretinal fiber and connects albumen epidemic disease histochemical stain × 200.
Fig. 4 takes off cell biological nethike embrane elastin laminin epidemic disease histochemical stain × 200.
The natural omentum majus tissue of Fig. 5 and the DNA content taken off in cell biological nethike embrane detect (* p < 0.01).
Detailed description of the invention
In this application, the implication of abbreviation is as follows:
SDS: sodium lauryl sulphate;
PBS: phosphate buffer;
DNA enzymatic: deoxyribonuclease;
Min: minute.
According to an aspect of the present invention, provide the preparation method of de-cell biological nethike embrane, it comprises and uses the buffer containing sodium lauryl sulphate and DNA enzymatic to soak the step that omentum majus carries out de-cell process.
In certain embodiments, also shake in the step of carrying out de-cell process.
In certain embodiments, the step of ungrease treatment is also comprised.In an exemplary embodiment, the step of ungrease treatment was carried out before the step of de-cell process.In certain preferred aspects, the step of this ungrease treatment uses that volume ratio is the methanol of 1: 1: 0.2-1, the mixed solution of chloroform and ether carries out.In some preferred embodiment, the volume ratio of methanol, chloroform and ether is 1: 1: 0.4-0.7.In the most preferred embodiment, the volume ratio of methanol, chloroform and ether is 1: 1: 0.5.
In certain embodiments, the step point gradient of de-cell process is carried out, and described gradient comprises at least the first gradient and the second gradient.
In certain preferred aspects, in the first gradient, the concentration of sodium lauryl sulphate is 1.5-3.5% weight ratio, and the concentration of DNA enzymatic is 3500-4500U/L, pH is 7.2-7.4.In some preferred embodiment, in the first gradient, the concentration of sodium lauryl sulphate is 1.5-2.5% weight ratio, and the concentration of DNA enzymatic is 3600-4400U/L.In some preferred embodiment, in the first gradient, the concentration of sodium lauryl sulphate is 1.8-2.2% weight ratio, and the concentration of DNA enzymatic is 3800-4200U/L.In the most preferred embodiment, in the first gradient, the concentration of sodium lauryl sulphate is 2.0% weight ratio, and the concentration of DNA enzymatic is 4000U/L.
In certain preferred aspects, in the second gradient, the concentration of sodium lauryl sulphate is 0.6-1.5% weight ratio, and the concentration of DNA enzymatic is 2500-3500U/L, pH is 7.2-7.4.In some preferred embodiment, in the second gradient, the concentration of sodium lauryl sulphate is 0.7-1.3% weight ratio, and the concentration of DNA enzymatic is 2600-3400U/L.In some preferred embodiment, in the second gradient, the concentration of sodium lauryl sulphate is 0.8-1.2% weight ratio, and the concentration of DNA enzymatic is 2800-3200U/L.In the most preferred embodiment, in the second gradient, the concentration of sodium lauryl sulphate is 1.0% weight ratio, and the concentration of DNA enzymatic is 3000U/L.
In certain preferred aspects, described gradient also comprises the 3rd gradient, and wherein in described 3rd gradient, the concentration of sodium lauryl sulphate is 0.1-0.6% weight ratio, and the concentration of DNA enzymatic is 1500-2500U/L, pH is 7.2-7.4.In some preferred embodiment, in the 3rd gradient, the concentration of sodium lauryl sulphate is 0.2-0.6% weight ratio, and the concentration of DNA enzymatic is 1600-2400U/L.In some preferred embodiment, in the 3rd gradient, the concentration of sodium lauryl sulphate is 0.4-0.6% weight ratio, and the concentration of DNA enzymatic is 1800-2200U/L.In the most preferred embodiment, in the 3rd gradient, the concentration of sodium lauryl sulphate is 0.5% weight ratio, and the concentration of DNA enzymatic is 2000U/L.
According to a further aspect in the invention, provide the preparation method of de-cell biological nethike embrane, it is characterized in that, carry out according to following operating procedure:
(1) preparation of degreasant solution
Methanol, chloroform and ether are mixed with the ratio that volume ratio is 1: 1: 0.2-1;
(2) preparation of de-Cell sap
1) de-Cell sap I: take sodium lauryl sulphate and DNA enzymatic, be dissolved in phosphate buffer, wherein, the concentration of sodium lauryl sulphate in phosphate buffer is 1.5-3.5% weight ratio, the concentration of DNA enzymatic in phosphate buffer is 3500-4500U/L, pH is 7.2-7.4, filtration sterilization;
2) de-Cell sap II: take sodium lauryl sulphate and DNA enzymatic, be dissolved in phosphate buffer, wherein, the concentration of sodium lauryl sulphate in phosphate buffer is 0.6-1.5% weight ratio, the concentration of DNA enzymatic in PBS is 2500-3500U/L, pH is 7.2-7.4, filtration sterilization;
3) de-Cell sap III: take sodium lauryl sulphate and DNA enzymatic, be dissolved in phosphate buffer, wherein, the concentration of sodium lauryl sulphate in phosphate buffer is 0.1-0.6% weight ratio, the concentration of DNA enzymatic in PBS is 1500-2500U/L, pH is 7.2-7.4, filtration sterilization;
(3) omentum majus takes off cell process
1) get fresh omentum majus tissue, clean in phosphate buffer, the tissue concussion after cleaning be soaked in described degreasant solution and process, degreasant solution soak time is 18-30h, and concussion frequency is 150-250r/min;
2) the described omentum majus removing fat is organized in phosphate buffer and is cleaned, and concussion is soaked in described de-Cell sap I and processes, soak time is 4-12h, and concussion frequency is 100-200r/min;
3) be organized in phosphate buffer by the described omentum majus after de-Cell sap I process and clean, and shake to be soaked in de-Cell sap II and process, soak time is 4-12h, and concussion frequency is 100-200r/min;
4) be organized in phosphate buffer by the omentum majus after de-Cell sap II process and clean, and shake to be soaked in described de-Cell sap III and process, soak time is 4-12h, and concussion frequency is 100-200r/min;
5) by the described omentum majus tissue sterilizing after lyophilization after de-Cell sap III process.
In certain embodiments, fresh omentum majus is tissue-derived in mammals such as pig, cattle, sheep.
In this application, omentum majus tissue is commercially available, can purchased from such as slaughterhouse.
According to a further aspect in the invention, the de-cell biological nethike embrane obtained according to said method is provided.
Now by following examples, the present invention is described in detail, but following examples are only illustratively, do not form any restriction to the present invention.
The omentum majus used in following examples takes off the preparation of cell process and the required reagent of qualification thereof:
1) PBS: take 8g NaCl, 0.2g KCl, 3.491g Na
2hPO
412H
2o, 0.2g KH
2pO
4, be dissolved in 1L ultra-pure water, adjust ph is 7.2-7.4,121 DEG C of high pressure steam sterilization 20min, 4 DEG C of preservations.
2) degreasant solution: 400mL methanol, 400mL chloroform and 200mL ether are mixed.
3) de-Cell sap I: take 20g SDS and be dissolved in PBS, add DNA enzymatic and standardize solution in 1L, make DNA enzymatic concentration reach 4000U/L, regulate pH to 7.2-7.4, after filtration sterilization, room temperature is preserved.
4) de-Cell sap II: take 10g SDS and be dissolved in PBS, add DNA enzymatic and standardize solution in 1L, make DNA enzymatic concentration reach 3000U/L, regulate pH to 7.2-7.4, after filtration sterilization, room temperature is preserved.
5) de-Cell sap III: take 5g SDS and be dissolved in PBS, add DNA enzymatic and standardize solution in 1L, make DNA enzymatic concentration reach 2000U/L, regulate pH to 7.2-7.4, after filtration sterilization, room temperature is preserved.
6) 4% paraformaldehyde fixative: the PBS solution 1L of heating 0.1M is to boiling, and add 40g paraformaldehyde, stirring and dissolving, adjust ph is 7.2-7.4, filtering and impurity removing matter, 4 DEG C of preservations.
7) the required antibody of extracellular matrix qualification of de-cell biological nethike embrane: type i collagen, IV Collagen Type VI, laminin，LN, elastin laminin antibody available from Sigma, use according to shop instruction, for the qualification of de-cell biological retinulae epimatrix.
The de-cell process of embodiment 1 miniature pig source omentum majus
Get fresh miniature pig peritoneum omentum majus tissue (purchased from slaughterhouse), clean 3 times in PBS.Tissue concussion after cleaning be soaked in 2L degreasant solution and process 24h, concussion frequency is 200r/min.Subsequently, be organized in PBS by the omentum majus removing fat and clean 3 times, and concussion is soaked in 1L takes off in Cell sap I and process 8h, concussion frequency is 150r/min.Subsequently, the omentum majus after de-Cell sap I process is organized in PBS and cleans 3 times, and concussion is soaked in 1L takes off in Cell sap II and process 8h, shake frequency 150r/min.Subsequently, be organized in PBS by the omentum majus after de-Cell sap II process and clean 3 times, and shake and be soaked in 1L and take off in Cell sap III and process 8h, concussion frequency is 150r/min.Finally, by the omentum majus tissue irradiation sterilization after lyophilization after de-Cell sap III process, room temperature is preserved.
Embodiment 2 takes off immunohistochemical staining and the qualification of cell biological nethike embrane
Use the paraformaldehyde solution of 4% to fix the de-cell biological nethike embrane of preparation in embodiment 1, Concentraton gradient dehydration of alcohol, paraffin embedding, preparing thick is degree 4 μm section.Section is placed on pathological tissue drift baking instrument and dries 2h.Subsequently, tissue slice is through the aquation of graded ethanol (concentration from high to low), and distilled water flushing is cut into slices.Add 3% hydrogen peroxide effect 10 minutes, remove endogenous peroxydase; PBS rinsing three times, each 5 minutes; Utilize antigen retrieval buffers to carry out Microwave method, after reparation, cool room temperature, PBS rinsing three times, each 5 minutes; 1% bovine serum albumin 37 DEG C closes 30 minutes; Drip anti-I type collagen (1: 200), anti-IV type collagen (1: 200), anti-fibronectin antibody (1: 200) and elastoresistance albumen primary antibodie respectively, 4 DEG C are spent the night.Clean 3 times with 0.01M PBS, drip goat anti-mouse two and resist, hatch 30 minutes for 37 DEG C, PBS cleans 3 times, each 5 minutes; Under lucifuge, DAB develops the color 10 minutes, and distilled water flushing is cut into slices; Gradient alcohol dehydration (concentration from low to high), dimethylbenzene is transparent, neutral gum mounting.Optical microphotograph Microscopic observation coloration result is also taken pictures.
Result is visible, the de-cell biological nethike embrane prepared by the present invention has typical network structure, and present type i collagen, IV Collagen Type VI, fibronectin and elastin laminin great expression, show that the de-cell biological nethike embrane prepared is after acellular process of the present invention and processing, the intact major protein component remaining natural extracellular matrix.In addition, various organization stained is showed no obvious cell component, shows that the present invention has well de-cell effect (Fig. 1-4).
Embodiment 3 takes off the DNA content test of cytoreticulum membrane matrix
Take the natural omentum majus tissue of about 30mg fast, after grinding, carry out extracting genome DNA according to " blood/cell/tissue genome DNA extracting reagent kit " (TIANGEN) operating procedure.Utilize same procedure to take the de-cell biological nethike embrane of about 30mg, extract and prepare DNA in material.D nucleic acid-protein analyzer (BIO-RAD) is utilized to measure DNA content.
Result is visible, compared with natural omentum majus tissue (335.78 ± 26.10 μ g/g), DNA content in de-cell biological nethike embrane significantly reduces (11.67 ± 8.76 μ g/g), show that, under method for removing cells process of the present invention, the cell DNA composition contained in natural omentum majus tissue is substantially eliminated (Fig. 5).
Claims (10)
1. a preparation method for de-cell biological nethike embrane, it comprises and uses the buffer containing sodium lauryl sulphate and DNA enzymatic to soak the step that omentum majus carries out de-cell process.
2. the method for claim 1, wherein also shakes in the step of carrying out de-cell process.
3. method as claimed in claim 1 or 2, it also comprises the step of ungrease treatment.
4. method as claimed in claim 3, the step use volume ratio of wherein said ungrease treatment is 1: 1: 0.2-1, the mixed solution of the methanol of preferably 1: 1: 0.4-0.7, more preferably 1: 1: 0.5, chloroform and ether carries out.
5. the method as described in claim arbitrary in Claims 1-4, the step point gradient of wherein said de-cell process is carried out, and described gradient comprises at least the first gradient and the second gradient.
6. method as claimed in claim 5, wherein:
In described first gradient, the concentration of sodium lauryl sulphate is 1.5-3.5% weight ratio, preferably 1.5-2.5% weight ratio, more preferably 1.8-2.2% weight ratio, most preferably 2.0% weight ratio, the concentration of DNA enzymatic is 3500-4500U/L, preferably 3600-4400U/L, more preferably 3800-4200U/L, most preferably 4000U/L, pH is 7.2-7.4, and
In described second gradient, the concentration of sodium lauryl sulphate is 0.6-1.5% weight ratio, preferably 0.7-1.3% weight ratio, more preferably 0.8-1.2% weight ratio, most preferably 1.0% weight ratio, the concentration of DNA enzymatic is 2500-3500U/L, preferably 2600-3400U/L, more preferably 2800-3200U/L, most preferably 3000U/L, pH are 7.2-7.4.
7. method as claimed in claim 5, wherein said gradient also comprises the 3rd gradient, wherein in described 3rd gradient, the concentration of sodium lauryl sulphate is 0.1-0.6% weight ratio, preferably 0.2-0.6% weight ratio, more preferably 0.4-0.6% weight ratio, most preferably 0.5% weight ratio, the concentration of DNA enzymatic is 1500-2500U/L, preferably 1600-2400U/L, more preferably 1800-2200U/L, most preferably 2000U/L, pH are 7.2-7.4.
8. a preparation method for de-cell biological nethike embrane, is characterized in that, carries out according to following operating procedure: the preparation of (1) degreasant solution
Methanol, chloroform and ether are mixed with the ratio that volume ratio is 1: 1: 0.2-1;
(2) preparation of de-Cell sap
1) de-Cell sap I: take sodium lauryl sulphate and DNA enzymatic, be dissolved in phosphate buffer, wherein, the concentration of sodium lauryl sulphate in phosphate buffer is 1.5-3.5% weight ratio, the concentration of DNA enzymatic in phosphate buffer is 3500-4500U/L, pH is 7.2-7.4, filtration sterilization;
2) de-Cell sap II: take sodium lauryl sulphate and DNA enzymatic, be dissolved in phosphate buffer, wherein, the concentration of sodium lauryl sulphate in phosphate buffer is 0.6-1.5% weight ratio, the concentration of DNA enzymatic in PBS is 2500-3500U/L, pH is 7.2-7.4, filtration sterilization;
3) de-Cell sap III: take sodium lauryl sulphate and DNA enzymatic, be dissolved in phosphate buffer, wherein, the concentration of sodium lauryl sulphate in phosphate buffer is 0.1-0.6% weight ratio, the concentration of DNA enzymatic in PBS is 1500-2500U/L, pH is 7.2-7.4, filtration sterilization;
(3) omentum majus takes off cell process
1) get fresh omentum majus tissue, clean in phosphate buffer, the tissue concussion after cleaning be soaked in described degreasant solution and process, degreasant solution soak time is 18-30h, and concussion frequency is 150-250r/min;
2) the described omentum majus removing fat is organized in phosphate buffer and is cleaned, and concussion is soaked in described de-Cell sap I and processes, soak time is 4-12h, and concussion frequency is 100-200r/min;
3) be organized in phosphate buffer by the described omentum majus after de-Cell sap I process and clean, and shake to be soaked in de-Cell sap II and process, soak time is 4-12h, and concussion frequency is 100-200r/min;
4) be organized in phosphate buffer by the omentum majus after de-Cell sap II process and clean, and shake to be soaked in described de-Cell sap III and process, soak time is 4-12h, and concussion frequency is 100-200r/min;
5) by the described omentum majus tissue sterilizing after lyophilization after de-Cell sap III process.
9. method as claimed in claim 8, it is characterized in that, described fresh omentum majus is tissue-derived in mammals such as pig, cattle, sheep.
10. the de-cell biological nethike embrane that the method according to claim arbitrary in claim 1-9 obtains.
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