CN114931665A - Application of hexa-type collagen alpha 2 subunit in nerve repair product - Google Patents

Abstract

The invention provides the application of the alpha 2 subunit of type 6 collagen in nerve repair products, improves the order of regenerated nerves, makes the regenerated nerve fibers morphologically closer to the normal nerve bundle structure, and reduces the mismatch between nerves and target organs In addition to better order promotion, the difference between the use of collagen type VI is that the COL6α2 subunit protein is prepared through genetic engineering, which reduces production costs and makes commercial applications more efficient. It has great possibility and can benefit more patients; in addition, nerve repair products including drugs, nerve grafts, hydrogels, etc. can be prepared by adding an effective amount of COL6α2 subunit protein, and a preparation method is provided.

Description

Application of Type VI Collagen α2 Subunit in Nerve Repair Products

technical field

The invention relates to the technical field of medicine, in particular to the application of six-type collagen α2 subunit (COL6 α2) in the preparation of axon-ordering hydrogel.

Background technique

Peripheral nerve injury is a common disabling disease. Various approaches, including end-to-end nerve suture, autologous nerve transplantation, electrical stimulation, and tissue engineering, have been shown to be effective in promoting the number and rate of regeneration of peripheral nerves [1-3]. However, the disorder of regenerated nerve fibers is still an important factor restricting the functional recovery of peripheral nerves [4-6].

In order to increase the order of regenerated axons, a common method is to introduce exogenous guiding signals in nerve transplantation [7,8]. Such exogenous guiding signals must satisfy a specific spatial distribution pattern (such as biochemical guiding molecules distributed in a concentration gradient or physical guiding structures arranged in parallel) in order to play the expected role [9-11]. Due to the small diameter and fine structure of peripheral nerve tissue, it is technically difficult to construct finely guided signals in nerve grafts.

A guiding signal-independent phenomenon of axonal self-organization provides a new avenue for orderly neural regeneration. It has been reported that axons regenerated in the peripheral nerve ECM microenvironment spontaneously form ordered nerve bundle structures [12]. Further research found that collagen type VI (COL6) in peripheral nerve ECM is the main functional molecule that triggers axonal self-organization. Relevant experimental results show that just by adding an appropriate concentration of COL6 to the homogeneous hydrogel, the regenerated axons can spontaneously form an ordered nerve bundle structure, which can reduce the regenerated nerve and the distal target. The mismatch rate of organs thus promotes the recovery of neurological function [13].

The above studies suggest that COL6 can effectively promote the order of regenerated axons, and the implementation method is simple. However, there are two major drawbacks in practical application. First, COL6 is a protein complex composed of COL6 α1, COL6 α2 and COL6 α3, and its biological assembly process is quite complicated and its molecular weight is huge (≈2000 kDa) [14]. It is impossible to achieve mass production; second, COL6 exhibits stronger immunogenicity than other collagens [15, 16], which further limits the feasibility of using xenogeneic or allogeneic COL6 purified protein to repair human nerves.

In general, intact macromolecular proteins are often difficult to become suitable drug candidates due to their easy degradation and denaturation, strong antigenicity, and high production costs.

SUMMARY OF THE INVENTION

In order to promote the self-organization and ordering of nerve regeneration, and at the same time solve the problems of high immunogenicity and difficult preparation of COL6, the inventors have found through experimental research that the main functional subunit of COL6, COL6 α2, can promote the ordering of axons. The COL6α2 subunit was prepared by genetic engineering, and its application in the hydrogel carrier endowed the conventional hydrogel material with a new function of promoting axonal ordering.

The present invention provides the application of type VI collagen α2 subunit in nerve repair products, and the effective concentration range of the type VI collagen α2 subunit is 2-100 μg/ml.

Further, the nerve repair products include: nerve grafts and hydrogels.

Further, the COL6α2 subunit is a human COL6α2 subunit.

Further, the preparation method of type VI collagen α2 subunit protein comprises:

1) Amplification: The human COL6 α2 ORF cloned plasmid with Myc-DDK tag was transformed into DH5α E. coli for amplification;

2) Transduction: Transduce the amplified plasmid into HEK293 cells to express the Myc-DDK-tagged COL6 α2 protein chain;

3) Lysis: 72 hours after transduction, cells were collected and lysed with a lysis buffer containing 150 mM NaCl, 50 mM Tris-HCL, 1% TritonX-100 and 5 mM EDTA;

4) Elution: The Myc-DDK-labeled COL6α2 protein chain was separated by Myc affinity gel, and then eluted with 0.15M glycine-hydrochloric acid buffer (pH 3.0);

5) Dialysis: Dialyze the COL6α2 eluate with deionized water;

6) Freeze-drying: perform freeze-drying on step 5), and collect COL6α2 freeze-dried powder, which is type 6 collagen α2 subunit protein.

Further, the preparation method of the hydrogel is as follows: dissolving the lyophilized powder of COL6α2 purified protein in physiological saline to prepare the original solution with a concentration of 1 mg/ml; placing a container containing the original solution of COL6α2 purified protein Put it on an ice box and mix it with the original solution of Matrigel with a concentration of 10 mg/mL in a ratio of 1:49 to form a hydrogel; leave the hydrogel at room temperature for 5 minutes to solidify naturally.

Further, the preparation method of the hydrogel is as follows: 20 μg/mL COL6α2 purified protein solution is mixed with 6.9 mg/mL thiolated hyaluronic acid, and 1.8 mg/mL polyethylene glycol is added to the mixed system (Diol) diacrylate cross-linking reaction for 15 minutes to solidify into gel.

Further, the preparation method of the nerve graft includes: mold forming, directional freeze-drying, gel perfusion, and solution soaking.

The beneficial effects of the present invention are:

1) By adding COL6α2 functional protein to the hydrogel, the hydrogel material can promote the self-organization and ordering of axons;

2) The COL6α2 subunit overcomes the deficiency that COL6 cannot be prepared by genetic engineering, and its immunogenicity is lower than that of COL6. Description of drawings:

Figure 1 is a schematic diagram of the structure of COL6α2 domain (A) and recombinant COL6α2 protein (B);

Figure 2 shows the purity of recombinant COL6α2 protein by protein silver staining;

Figure 3 shows the effect of different concentrations of COL6α2 hydrogel on promoting axonal bundling and ordering in vitro;

Figure 4 shows the effect of COL6α2-loaded hydrogel in promoting axonal bundling and ordering immunofluorescence in vitro;

Figure 5 shows the immunofluorescence effect of COL6α2-loaded hydrogel in repairing rat sciatic nerve defect;

Figure 6 shows the effect of subcutaneous injection of equal amounts of COL6α2 and COL6 on serum IgM concentration.

Detailed ways

Based on the research on the use of type 6 collagen to improve the order of regenerated nerves, in order to solve the problems of high immunogenicity and difficult preparation of type 6 collagen, it is not suitable for commercial application. Continuing in-depth research on type 6 collagen, it was found that COL6α2 subunit protein has a good function of promoting axonal ordering, and can be commercially prepared by genetic engineering; in addition, the role of COL6α2 subunit protein in nerve repair products is discussed. application, and prepared a nerve repair product with the function of promoting orderly regeneration of axons. The specific implementation is as follows:

Example 1: Preparation of COL6α2 subunit protein

1) Amplification: Self-constructed or commercialized human-derived COL6 α2 ORF clone plasmid with Myc-DDK tag (Cat. No. RC209476, OriGene) was transformed into DH5α E. coli for amplification;

2) Transduction: Transduce the amplified plasmid into HEK293 cells to express the Myc-DDK-tagged COL6 α2 protein chain;

3) Lysis: 72 hours after transduction, cells were collected and lysed with a lysis buffer containing 150 mM NaCl, 50 mM Tris-HCL, 1% TritonX-100 and 5 mM EDTA;

4) Elution: The Myc-DDK-labeled COL6 α2 protein chain was separated by Myc affinity gel, and then eluted with 0.15M glycine-hydrochloric acid buffer (pH 3.0), wherein the elution was detected by protein silver staining technique COL6 α2 purity in liquid (Figure 2)

5) Dialysis: Dialyze the COL6 α2 eluate with deionized water;

6) Freeze-drying: freeze-dry step 5), collect COL6 α2 freeze-dried powder and store it in a sterile, sealed and dry environment.

It should be noted that, from the application point of view, commercial recombinant human-derived COL6α2 purified protein can also be directly purchased, such as the product of OriGene Company, whose product number is TP309476.

Example 2: Preparation of hydrogel products containing COL6 α2 by non-covalent binding

The lyophilized powder of COL6α2 purified protein obtained in Example 1 was dissolved in physiological saline to prepare an original solution with a concentration of 1 mg/ml, and then mixed with the original solution of Matrigel with a concentration of 10 mg/mL in a ratio of 1:49. At this time, the concentration of COL6 α2 in the mixed hydrogel can be 20 μg/mL; the mixed hydrogel can be naturally solidified into a gel after being placed at room temperature for about 5 minutes. At this time, the COL6 α2 molecule and the Matrigel hydrogel molecule are non-covalent. combined with each other. To maintain the fluidity of the hydrogel, the entire procedure should be performed on an ice box.

Example 3: Preparation of hydrogel products containing COL6 α2 by chemical cross-linking

The COL6 α2 purified protein lyophilized powder obtained in Example 1 was mixed with other carrier hydrogel materials after being mixed with other carrier hydrogel materials. In this example, thiolated hyaluronic acid was selected, and then polyethylene glycol ( Diol) diacrylate (PEGDA) initiates the thiol cross-linking reaction between COL6α2 and thiolated hyaluronic acid molecules, wherein the concentration of COL6 α2 in the mixed system is 20 μg/mL, and the concentration of thiolated hyaluronic acid is 6.9 mg/mL mL; the PEGDA concentration was 1.8 mg/mL, and this reaction formed a solid hydrogel in 15 minutes at room temperature.

Embodiment 4:

The difference between this example and Example 2 is that the mixed hydrogel does not contain COL6α2.

Embodiment 5:

The difference between this example and Example 2 is that the concentration of COL6α2 in the mixed hydrogel is 2 μg/mL.

Embodiment 6:

The difference between this example and Example 2 is that the concentration of COL6α2 in the mixed hydrogel is 100 μg/mL.

The experimental results of Examples 2, 4, 5, and 6 are shown in Figure 3. The concentration of COL6α2 in the range of 2-100 μg/mL has the effect of promoting axons, but considering the cost and overall effect, the concentration of 20 μg/mL is preferred. .

Example 7: Preparation of COL6α2-enriched nerve grafts.

The nerve graft can be loaded with an effective concentration of COL6α2 in the material gap or pore of the multi-channel catheter by means of solution immersion or gel perfusion, so that the conventional nerve graft also has the effect of promoting the self-organization of nerves.

Example 8: In vitro experiments of COL6α2-containing hydrogels promoting regenerated nerve fiber bundles and ordering

The pregel solution obtained in Example 2 was applied to the bottom of the cell culture dish at 2 μg/cm ² , and the excess liquid was absorbed and placed at room temperature for 5-10 minutes to form a gel, and then the rat dorsal root ganglion (DRG) tissue block was planted in hydrogel-coated petri dishes. The formation of ordered nerve bundle structures was seen after 3 days of incubation in a 37 °C incubator. In the control group, the hydrogel plating without COL6 α2 was used, and the DRG axons showed a scattered and disordered structure (Figure 4).

Example 9: Repair of rat sciatic nerve injury by COL6α2 hydrogel

The COL6α2-rich hydrogel solution obtained in Example 2 was injected into a medical silicone tube with an inner diameter of 1 mm and a length of 8 mm through a syringe, and was placed in a 37 ° C incubator for 10 min. After it was completely gelled, the catheter was placed in a sterile stored in normal saline. Adult SD rats were routinely anesthetized, the sciatic nerve was exposed, the 8 mm nerve defect was excised, the nerve stump was bridged with a hydrogel-loaded nerve guide, and the muscles and skin were sutured in layers. The entire surgical procedure was performed aseptically. Routine feeding was performed after operation, and sciatic nerves were harvested by perfusion after 4 weeks, and the sections were stained for observation. As shown in Figure 5, the addition of COL6α2 to the hydrogel was able to significantly increase the order of regenerated axons.

Example ten: COL6α2 hydrogel immunogenicity detection

The dorsal skin of adult SD rats (two months old) was shaved and disinfected. The tail vein blood was collected as the sample of the 0-day group before immunization, and then 0.1 mL of COL6α2 protein solution with a concentration of 100 μg/mL was subcutaneously injected, and the control group was injected with the same amount of COL6 protein solution. The tail vein blood was collected on the 3rd and 7th days after injection. All the blood was allowed to stand at room temperature for 2 hours. After it was coagulated, the serum was collected by centrifugation. The serum was detected by enzyme-linked immunosorbent assay (ELISA) at different time points. Changes in the concentration of IgM. The results showed that subcutaneous injection of COL6 caused an increase in blood IgM, while the same amount of COL6α2 did not cause an increase in IgM (Figure 6).

The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art can still modify or equivalently replace the specific embodiments of the present invention. , and any modifications or equivalent replacements that do not depart from the spirit and scope of the present invention are all within the protection scope of the claims of the present invention for which the application is pending.

references:

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2. Gordon T, English AW. Strategies to promote peripheral nerve regeneration: electrical stimulation and/or exercise. Eur J Neurosci. 2016Feb;43(3):336-50. doi: 10.1111/ejn.13005.

3. Eggers R, de Winter F, Tannemaat MR, Malessy MJA, Verhaagen J. GDNFGene Therapy to Repair the Injured Peripheral Nerve. Front Bioeng Biotechnol.2020 Oct 30;8:583184. doi: 10.3389/fbioe.2020.583184.

4. Robinson GA, Madison RD. Motor neurons can preferentially reinnervate cutaneous pathways. Exp Neurol. 2004 Dec;190(2):407-13. doi:10.1016/j.expneurol.2004.08.007.

5. English AW. Enhancing axon regeneration in peripheral nerves also increases functionally inappropriate reinnervation of targets. J Comp Neurol. 2005 Oct 3;490(4):427-41. doi: 10.1002/cne.20678.

6.de Ruiter GC, Spinner RJ, Verhaagen J, Malessy MJ. Misdirection and guidance of regenerating axons after experimental nerve injury and repair. JNeurosurg. 2014 Feb;120(2):493-501. doi: 10.3171/2013.8.JNS122300.

7. Krick K, Tammia M, Martin R, Höke A, Mao HQ. Signaling cuepresentation and cell delivery to promote nerve regeneration. Curr OpinBiotechnol. 2011 Oct;22(5):741-6. doi: 10.1016/j.copbio. 2011.04.002.

8. Liu D, Mi D, Zhang T, Zhang Y, Yan J, Wang Y, Tan X, Yuan Y, YangY, Gu X, Hu W. Tubulation repair mitigates misdirection of regenerating motoraxons across a sciatic nerve gap in rats. Sci Rep. 2018 Feb 21;8(1):3443.doi: 10.1038/s41598-018-21652-y.

9. Handarmin, Tan GJ, Sundaray B, Marcy GT, Goh EL, Chew SY. Nanofibrous scaffold with incorporated protein gradient for directing neuriteoutgrowth. Drug Deliv Transl Res. 2011 Apr;1(2):147-60. doi: 10.1007/ s13346-011-0017-3.

10. Oh SH, Kang JG, Kim TH, Namgung U, Song KS, Jeon BH, Lee JH.Enhanced peripheral nerve regeneration through asymmetrically porous nerveguide conduit with nerve growth factor gradient. J Biomed Mater Res A. 2018Jan;106(1) :52-64. doi: 10.1002/jbm.a.36216.

11. Hsu RS, Chen PY, Fang JH, Chen YY, Chang CW, Lu YJ, Hu SH. Adaptable Microporous Hydrogels of Propagating NGF-Gradient by InjectableBuilding Blocks for Accelerated Axonal Outgrowth. Adv Sci (Weinh). 2019 Jul11;6( 16):1900520. doi: 10.1002/advs.201900520.

12. Zou JL, Liu S, Sun JH, Yang WH, Xu YW, Rao ZL, Jiang B, Zhu QT, Liu XL, Wu JL, Chang C, Mao HQ, Ling EA, Quan DP, Zeng YS. Peripheral nerve- derived matrix hydrogel promotes remyelination and inhibits synapseformation. Adv Funct Mater. 2018 Jan;28(13). doi: 10.1002/adfm.201705739.

13. Sun JH, Huang M, Fang Z, Li TX, Wu TT, Chen Y, Quan DP, Xu YY, Wang YM, Yang Y, Zou JL. Nerve bundle formation during the promotion of peripheral nerve regeneration: collagen VI-neural cell adhesion molecule 1interaction. Neural Regen Res. 2022 May;17(5):1023-33. doi: 10.4103/1673-5374.324861.

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Boeer U, Buettner FF, Klingenberg M, Antonopoulos GC, Meyer H, Haverich A, Wilhelmi M. Immunogenicity of intensively decellularized equinecarotid arteries is conferred by the extracellular matrix protein collagentype VI. PLoS One. 2014 Aug 26;9(8):e105964 . doi: 10.1371/journal.pone.0105964.

SEQUENCE LISTING

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<120> Application of Collagen VI α2 Subunit in Nerve Repair Products

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Claims (7)

1. The application of the type 6 collagen α2 subunit in nerve repair products, the added amount of the type 6 collagen α2 subunit is 2-100 μg/ml. 2 . The application of the type 6 collagen α2 subunit according to claim 1 in a nerve repair product, wherein the nerve repair product comprises: nerve graft and hydrogel. 3 . 3 . The application of the type 6 collagen α2 subunit according to claim 1 in a nerve repair product, wherein the COL6α2 subunit is a human-derived COL6α2 subunit. 4 . 4. the application of type 6 collagen α2 subunit according to claim 1 in nerve repair product, it is characterized in that, the preparation method of described type 6 collagen α2 subunit protein comprises: 1) Amplification: The human COL6α2 ORF cloned plasmid with Myc-DDK tag was transformed into DH5α Escherichia coli for amplification; 2) Transduction: Transduce the amplified plasmid into HEK293 cells to express the Myc-DDK-tagged COL6α2 protein chain; 3) Lysis: 72 hours after transduction, cells were collected and lysed with a lysis buffer containing 150 mM NaCl, 50 mM Tris-HCL, 1% Triton X-100 and 5 mM EDTA; 4) Elution: The Myc-DDK-labeled COL6 α2 protein chain was separated by Myc affinity gel, and then eluted with 0.15M glycine-hydrochloric acid buffer (pH 3.0); 5) Dialysis: Dialyze the COL6α2 eluate with deionized water; 6) Freeze-drying: freeze-dry step 5) and collect COL6α2 freeze-dried powder, which is type 6 collagen α2 subunit protein. 5. the application of type VI collagen α2 subunit in nerve repair product according to claim 2, is characterized in that, the preparation method of described hydrogel is: dissolving COL6α2 purified protein freeze-dried powder in physiological saline , prepare the original solution with a concentration of 1 mg/ml; place the container containing the original solution of COL6α2 purified protein on an ice box, and mix it with the original solution of Matrigel with a concentration of 10 mg/mL at a ratio of 1:49 to form water Gel; leave the hydrogel at room temperature for 5 minutes to solidify naturally. 6. The application of collagen VI α2 subunit in nerve repair products according to claim 2, wherein the preparation method of the hydrogel is: combining 20 μg/mL COL6α2 purified protein solution with 6.9 mg/mL of thiolated hyaluronic acid was mixed, and 1.8 mg/mL of polyethylene glycol (diol) diacrylate was added to the mixed system for cross-linking reaction for 15 minutes to solidify into a gel. 7 . The application of the type 6 collagen α2 subunit in a nerve repair product according to claim 2 , wherein the preparation method of the nerve graft comprises: gel perfusion and solution soaking. 8 .

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