CN117771434A - Urethral bioremediation material with gene editing function and preparation method thereof - Google Patents
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Abstract
The invention belongs to the technical field of medical materials, and particularly relates to a urethral bioremediation material with a gene editing function and a preparation method thereof. The invention relies on the ultrastructure and physicochemical properties of bacteria-like cellulose synthetic materials and the specific gene editing function of CRISPR Cas9 technology, and carries out efficient chemical combination on Cas9 protein, transRNA and biological materials by a chemical method so as to construct the functional repair material capable of editing cell-specific genes which are adhered and grown on the surface of the biological materials.
Description
Technical Field
The invention belongs to the technical field of medical materials, and particularly relates to a urethral bioremediation material with a gene editing function and a preparation method thereof.
Background
Urethral stricture is one of the more common and difficult diseases of urinary surgery, the incidence rate of which is 300-600/10 ten thousand, and also presents a continuously rising situation in China in the last decade. The urethral repair and reconstruction is very troublesome in clinical practical treatment due to the characteristics of high operation difficulty, high technical requirement, low success rate, more postoperative complications and the like. While in the clinical work of the near half century clinicians explore a variety of available autologous tissue repair and surgical protocols for treating patients, in many complex cases, existing treatment protocols still show significant limitations. The limitations of repair materials, complications of donor sites, and surgical complexity have all prompted rapid development of the concept of repairing the urethra with various biological or synthetic materials in basic and clinical studies.
At present, the three-dimensional space structure of the tissue to be repaired is highly fitted by using a composite material, and various requirements of promoting tissue regeneration, accelerating vascularization and the like become the main standard of the 'functional' design of a new generation of biological material. However, all biological materials are designed based on the mode set according to the requirement before being implanted into the body, and once the materials are implanted into the body, a series of accurate regulation and control of the materials for promoting the reconstruction, plasticity, regeneration and the like of tissues in the body cannot be realized due to the fact that the interaction mechanism between the body and the materials is not clear.
The CRISPR screening technology based on CRISPR gene editing principle provides theoretical feasibility for solving the above-mentioned aim. The CRISPR gene editing technique is a technique for precisely editing a cell target gene by RNA-guided Cas9 protein. In a specific procedure, cas9 nuclease protein site-directed cleavage of DNA upstream of a target gene by recognizing a protospacer adjacent motif (Protospacer Adjacent Motif, PAM) in the vicinity of the PAM under the direction of a guide RNA (sgRNA). Resulting in double-strand breaks (DSBs) of the target site DNA, a subsequently initiated Non-homologous end joining (Non-homologous end joining, NHEJ) repair mechanism introduces short deletions or insertions at the editing site, ultimately achieving the goal of consistent or modified function of a particular gene. Compared with the prior gene editing technology such as Talent or Zinc Finger, the CRISPR-Cas9 gene editing technology has the advantages of simpler operation, lower off-target effect and lower toxic side effect.
After the critical genes are determined theoretically through CRISPR screening technology, a proper delivery system needs to be established to carry sgRNA-Cas9 into cells so as to regulate and control target genes. Currently, in basic research, lentiviruses are mostly used as vectors to re-integrate sgrnas in host cells and to exert gene editing effects. In addition, physical electroporation or liposome plasmid transfection methods have been reported for delivery. However, none of the above methods is suitable for the purpose of modifying the existing materials to control the genes of the experimental materials themselves. And adopts ionic polymer to be covalently combined with Cas9 protein and sgRNA to form polymer-based CRISPR nano-particles. May become an effective solution to the above problems.
Disclosure of Invention
Aiming at the problems, the invention provides a urethral bioremediation material with a gene editing function and a preparation method thereof. The invention aims to provide a biological material with a gene editing function, so that in the process of repairing and reconstructing clinical urethral tissues, target genes implanted into cells of a nearby organism can be directionally edited after the biological material is implanted into the body, and the related functions of the genes are subjected to over-expression or expression silencing, so that the aim of promoting urethral tissue repair is finally realized.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention discloses a urethral bioremediation material with a gene editing function, which is a bacterial cellulose-based composite material taking porous gelatin as an osseous bracket and loaded with an active CRISPR-Cas9 gene editing component.
The invention also discloses a preparation method of the urethral biological repair material, which comprises the following steps:
(1) Preparation of three-dimensional composite biological material
The porous gelatin is used as a composite material of the bone scaffold, and a final three-dimensional composite biological material is constructed through chemical combination with bacterial cellulose;
(2) Construction of active CRISPR-Cas 9-loaded three-dimensional composite material
Reacting the polyether imide bPEI after being activated by sulfo-smcc with SpCas9 to obtain SpCas9-bPEI, incubating sgRNA with the SpCas9-bPEI to form a CRISPR nano complex, immersing the CRISPR nano complex with the three-dimensional composite biological material prepared in the step (1), and further performing freeze drying treatment for standby.
Further, in the preparation method of the present invention, the specific preparation steps of the step (1) are as follows: immersing the porous gelatin stent into ethanol for sterilization, then cleaning the porous gelatin stent with fermentation culture solution, and sucking the extruded excess fermentation culture solution with sterile filter paper; then slowly dripping the fermentation culture solution containing the strain on the porous gelatin bracket, and putting the porous gelatin bracket into a constant temperature incubator for fermentation treatment to obtain the double-layer bacterial cellulose-based three-dimensional composite biological material.
Further, in the preparation method of the present invention, the strain used in the step (1) is acetobacter xylinum 1.1812.
Further, in the preparation method of the invention, the fermentation culture solution is: glucose 5w/v%, peptone 0.5w/v%, citric acid 0.1w/v%, disodium hydrogen phosphate 0.2w/v%, potassium dihydrogen phosphate 0.1w/v%, yeast extract 0.5w/v%, sodium hydroxide to pH=5.0, and sterilizing at l2l℃under 0.1 MPa.
Further, in the preparation method of the invention, the condition of the fermentation treatment is that the fermentation treatment is carried out for 7 days at 30 ℃.
Further, in the preparation method, the porous gelatin scaffold is obtained by crosslinking and freeze-drying a 1-2wt% gelatin solution through glutaraldehyde.
Further, in the preparation method of the present invention, the specific preparation steps of the step (2) are as follows: activating bPEI by using sulfo-SMCC for 2-4h at room temperature, wherein the molar ratio of bPEI to sulfo-SMCC is 1 (10-20); the activated bPEI was reacted with SpCas9 in a molar ratio of (50-200): 1 followed by dialysis against buffer, the dialyzed reaction product was reacted with sgRNA in a molar ratio of 1: (1-3) incubating in deionized water at pH 6.5 at room temperature for 15-20min to form CRISPR nanocomposite; and (3) carrying out immersion treatment on the three-dimensional composite biological material obtained in the step (1) for 2-3h, and then carrying out further freeze drying treatment for standby.
Further, in the preparation method disclosed by the invention, bPEI is dialyzed by deionized water by using a reaction product activated by sulfo-SMCC, and the molecular weight cut-off is 500-1000.
Furthermore, in the preparation method disclosed by the invention, the molecular weight cut-off of the reaction product SpCas9-bPEI dialysis is 50000.
According to the invention, by means of the ultrastructure and physicochemical properties of the bacteria-like cellulose synthetic material and the specific gene editing function of the CRISPR Cas9 technology, the Cas9 protein, the TransRNA and the biological material are subjected to efficient chemical combination by a chemical method, so that a functional repair material capable of editing the cell specific gene which is attached and grown on the surface of the biological material is constructed, and the functional repair material can be used as a simple scaffold to promote the climbing growth of somatic cells and activate or inhibit the specific function of surrounding cells, thereby further playing the biological function of repairing the implant material and finally realizing the aim of rapidly repairing the urethral tissue defect by using the biological material. Therefore, the biological material with the gene editing function prepared by the invention can fully meet a great amount of clinical demands of clinicians engaged in urethral repair and reconstruction work.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention simulates the three-dimensional micro-nano structure of the normal urethra tissue, so that the three-dimensional micro-nano structure is closer to the normal tissue, and is suitable for repairing and reconstructing the urethra tissue.
2. The gene editing complex in the design of the invention can selectively adjust sgRNA according to the actual requirement of urethral repair, thereby infinitely expanding the functionality of the repair material.
3. The biological material designed by the invention can be preserved for a long time after freeze drying treatment, thereby being beneficial to commercialized storage, transportation and clinical application.
Drawings
FIG. 1 (A) is a schematic diagram of the structure of SpCas 9-bPEI; (B) is a schematic diagram of sgRNA-Cas9 nanoparticle assembly; (C) Is a schematic diagram of a three-dimensional composite material carrying sgRNA-Cas9 nano particles;
fig. 2 is a graph of fluorescence confocal effect of a sgRNA-Cas9-eGFP loaded composite material, wherein blue color shows a spatial structure of acetobacter xylinum material, and green color shows distribution positions and distribution amounts of the sgRNA-Cas 9-eGFP;
FIG. 3 is a graph of the functional detection results of gene regulation of the sgRNA-loaded (EGR 1) -Cas9-eGFP composite.
Detailed Description
The invention discloses a urethral bioremediation material with a gene editing function and a preparation method thereof, and a person skilled in the art can refer to the content of the urethral bioremediation material and properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
Examples
The invention discloses a urethral bioremediation material with a gene editing function, which comprises the following steps:
(1) Preparation of three-dimensional composite biological material
The invention adopts a composite material with porous gelatin as a bone scaffold, and constructs the final composite material through chemical combination with bacterial cellulose.
And (3) strain: acetobacter xylosojae (Gluconacetobacter xylinus) 1.1812; fermentation medium: glucose 5w/v%, peptone 0.5w/v%, citric acid 0.1w/v%, disodium hydrogen phosphate 0.2w/v%, potassium dihydrogen phosphate 0.1w/v%, yeast extract 0.5w/v%, sodium hydroxide to pH=5.0, and sterilized at l2l℃for 30 minutes under 0.1 MPa.
Porous gelatin scaffold: is obtained by freeze drying 2wt% gelatin solution and glutaraldehyde crosslinking.
The porous gelatin stent was immersed in 75wt% ethanol and sterilized at room temperature for 1 hour. Then, the fermentation broth was washed 3 times. The gelatin template was slowly squeezed with flat-headed forceps and the expressed broth was aspirated with sterile filter paper. Placing the porous gelatin stent into a square culture dish, taking 40mL of fermentation culture solution containing G.xylinus strains, slowly dripping the fermentation culture solution onto a gelatin template, placing the square culture dish into a constant temperature incubator, and fermenting and culturing at 30 ℃ for 7 days to obtain the double-layer bacterial cellulose-based tissue engineering urethral stent (BC-Gel/BC).
(2) Construction of active CRISPR-Cas 9-loaded three-dimensional composite material
1. To pure water, 16mg of bPEI and 5mg of sulfoo-SMCC were added and activated at 25℃for 3 hours, bPEI: the molar ratio of sulfo-SMCC is 1:10);
2. dialyzing the reacted solution with deionized water (with molecular weight cut-off of 500-1000) for 24 hours; and (5) freeze drying.
3. 2.1mg of sulfo-smcc activated bPEI was added, reacted with 2mg of SpCas9, in PBS with pH=6.9 at 4℃for 4h, the molar ratio of SpCas9 to bPEI was 1:100, the final product was dialyzed with buffer at 4℃for 24 hours, 1 change of fluid every 4 hours, molecular weight cut-off was 50000, and the dialyzed product was frozen in liquid nitrogen. (see FIG. 1)
4. sgRNA (EGR 1 sequence ACGCCCTTACGCTTGCCCAG PAM TGG) (1.8 μm) and SpCas9-Bpei (990 nM) prepared above were incubated in deionized water at pH 6.5 for 15 minutes at 25 ℃. A CRISPR nanocomposite was formed and immersed with the above composite for 2 hours. Further freeze-drying the mixture for standby. And meanwhile, the material is detected by a laser confocal microscope, and the result is shown in figure 2.
5. Culturing and collecting 1×10 7 MSC (mesenchymal stem cells) and suspended in 500. Mu.L DMEM, and inoculated in 1X 1cm 2 And (3) incubating the surface of the composite material in an incubator at 37 ℃ for 4 hours, and then adding 10mL of culture solution to form gas-liquid level culture. After 4 days, a piece of material cells are digested and Western blot is performed. The results are shown in FIG. 3.
Claims (10)
1. The urethral bioremediation material with the gene editing function is characterized in that the urethral bioremediation material is a bacterial cellulose-based composite material taking porous gelatin as an osseous scaffold and is loaded with an active CRISPR-Cas9 gene editing component.
2. A method of preparing a urethral bioremediation material according to claim 1, comprising the steps of:
(1) Preparation of three-dimensional composite biological material
The porous gelatin is used as a composite material of the bone scaffold, and a final three-dimensional composite biological material is constructed through chemical combination with bacterial cellulose;
(2) Construction of active CRISPR-Cas 9-loaded three-dimensional composite material
Reacting the sulfo-smcc activated bPEI with SpCas9 to obtain SpCas9-bPEI, incubating sgRNA with the SpCas9-bPEI to form a CRISPR nano-complex, immersing the CRISPR nano-complex with the three-dimensional composite biological material prepared in the step (1), and further performing freeze drying treatment for standby.
3. The preparation method according to claim 2, wherein the specific preparation step of step (1) is: immersing the porous gelatin stent into ethanol for sterilization, then cleaning the porous gelatin stent with fermentation culture solution, and sucking the extruded excess fermentation culture solution with sterile filter paper; then slowly dripping the fermentation culture solution containing the strain on the porous gelatin bracket, and putting the porous gelatin bracket into a constant temperature incubator for fermentation treatment to obtain the double-layer bacterial cellulose-based three-dimensional composite biological material.
4. The method according to claim 3, wherein the strain used in the step (1) is Acetobacter xylinum 1.1812.
5. A method of preparation according to claim 3, wherein the fermentation broth is: glucose 5w/v%, peptone 0.5w/v%, citric acid 0.1w/v%, disodium hydrogen phosphate 0.2w/v%, potassium dihydrogen phosphate 0.1w/v%, yeast extract 0.5w/v%, sodium hydroxide to pH=5.0, and sterilizing at l2l℃under 0.1 MPa.
6. The method according to claim 3, wherein the fermentation treatment is carried out at 30℃for 7 days.
7. A method of preparation according to claim 3, wherein the porous gelatin scaffold is obtained from a 1-2wt% gelatin solution by glutaraldehyde cross-linked freeze-drying.
8. The preparation method according to claim 2, wherein the specific preparation step of step (2) is: activating bPEI by using sulfo-SMCC for 2-4h at room temperature, wherein the molar ratio of bPEI to sulfo-SMCC is 1 (10-20); the activated bPEI was reacted with SpCas9 in a molar ratio of (50-200): 1 followed by dialysis against buffer, the dialyzed reaction product was reacted with sgRNA in a molar ratio of 1: (1-3) incubating in deionized water at pH 6.5 at room temperature for 15-20min to form CRISPR nanocomposite; and (3) carrying out immersion treatment on the three-dimensional composite biological material obtained in the step (1) for 2-3h, and then carrying out further freeze drying treatment for standby.
9. The method of claim 8 wherein the bPEI is dialyzed against deionized water using a sulfoo-SMCC activated reactant having a molecular weight cut-off of 500 to 1000.
10. The method of claim 8, wherein the reaction product SpCas9-bPEI dialyzed has a molecular weight cut-off of 50000.
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