CN115025235A

CN115025235A - Compound for treating facial nerve injury disease and preparation method and application thereof

Info

Publication number: CN115025235A
Application number: CN202210265914.5A
Authority: CN
Inventors: 林云锋; 李佳杰; 蔡潇潇
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2022-03-17
Filing date: 2022-03-17
Publication date: 2022-09-09
Anticipated expiration: 2042-03-17
Also published as: CN115025235B

Abstract

The invention provides a compound of tetrahedral framework nucleic acid and miR-22, which is more excellent in stability and treatment effectiveness than the compound of the tetrahedral framework nucleic acid and miR-22 reported in the prior art through unique viscous terminal linkage compounding, can promote the uptake of cells to miR-22, effectively improves the microenvironment after facial nerve injury, provides conditions for axon regeneration, and has a good treatment effect on facial nerve injury.

Description

Compound for treating facial nerve injury disease and preparation method and application thereof

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to a compound for treating facial nerve injury diseases, and a preparation method and application thereof.

Background

The facial nerve is the longest peripheral nerve of the human body in the bone canal, and the walking from the brain stem to the facial muscle tissue is complex and powerful, and governs the movement of maxillofacial muscles, secretion of glands, taste, hearing and the like. Most maxillofacial surgeries are centered on the facial nerve, which makes them very susceptible to iatrogenic injuries. Oral and maxillofacial surgery has been reported to account for 40% of the causes of injury, with temporomandibular joint replacement being the most common cause of iatrogenic facial nerve injury associated with maxillofacial surgery. The resection of other head and neck pathological changes accounts for 25%, the otorhinolaryngological operation accounts for 17%, the medical cosmetology operation accounts for 11%, and other operations account for 7%. If the injury is not repaired in time or the repair effect is not good, the injury will cause different degrees of hypofunction of the dominant effector muscle group, facial expression loss, abnormal lacrimal/salivary gland secretion and the like, and seriously affect the physical and mental health of the patient.

After facial nerve injury, the patient's recovery correlates well with the severity of the trauma or symptoms. If mechanical axons are excessively destroyed, the patient may require a longer recovery time with severe dysfunction and sequelae (e.g., numbness, tingling, burning or severe pain). Various therapeutic means, such as expressive muscle training, electrical stimulation, neural decompression, neural anastomosis, neural transplantation, etc., have been developed clinically according to the degree of injury. Despite the progress of microsurgery, it is still a clinical problem to obtain good repair effect after facial nerve injury, and the main disadvantage of current nerve repair is that complete recovery of nerve function cannot be guaranteed. Furthermore, in existing peripheral nerve regeneration studies, relatively few people are concerned with the facial nerve, which makes drugs that can effectively treat facial nerve regeneration deficient.

MicroRNAs (miRNAs) are a group of short, non-coding microRNA sequences that are widely expressed in the nervous system by binding to 3 untranslated regions (3-UTRs) of messenger RNA (mRNA) that regulate Gene expression at the transcriptional level and cellular response to extracellular stimuli, where miRNA-22-3p (miR-22, Gene ID: 407004) has been demonstrated to be a neuronal specific miRNAs expressed in synapses and is considered potentially valuable in neurorepair therapies due to their ability to promote primary sensory neuron regeneration. However, its clinical use is greatly limited due to its inherent instability.

DNA Tetrahedra (TDN), also called tetrahedral framework nucleic acids or tetrahedral framework nucleic acids (tFNAs), is a tetrahedral structure composed of 4 highly specific single-stranded DNAs by a simple annealing process based on the base complementary pairing principle. Due to the advantages of good biocompatibility, histocyte permeability, programmability and the like, the miRNAs are suitable therapeutic vectors for delivering the miRNAs into cells and improving the biological application rate of the miRNAs.

Patent application CN20211029324.0 discloses a complex of tetrahedral framework nucleic acid and miR-22 for the treatment of optic nerve injury. However, miR-22 is linked to 1 to 4 single strands constituting the tetrahedral framework nucleic acid through the deoxyribonucleotide sequence-TTTTT-single strand, and is linked to the vertices of the tetrahedral framework nucleic acid. However, the stability of the complex remains to be further improved. Particularly, compared with the optic nerve injury disease which can be directly administrated by adopting vitreous injection, the difficulty of facial nerve injury administration is that the medicine is fused with body fluid after entering the injury part, and serum and enzyme in the body fluid can directly influence the stability of the medicine, which has higher requirement on the stability of the medicine, so that the existing medicine carrying systems of tetrahedral framework nucleic acid and miR-22 are difficult to realize the effective treatment of the facial nerve injury. Therefore, a more stable and effective miRNA drug delivery system needs to be established for treating facial nerve injury diseases.

Disclosure of Invention

The invention aims to provide a compound for treating facial nerve injury diseases and a preparation method and application thereof.

The invention provides a compound for treating facial nerve injury diseases, which is formed by connecting tetrahedral framework nucleic acid and miR-22 through sticky ends, wherein the molar ratio of the tetrahedral framework nucleic acid to the miR-22 is 1 (1-4).

Further, the molar ratio of the tetrahedral framework nucleic acid to the miR-22 is 1: 1.

Further, the tetrahedral framework nucleic acid is formed by base complementary pairing of 4 single-stranded DNAs, at least one of which has a cohesive end sequence a connected to the 3 'end or the 5' end; the miR-22 is miR-22 with a cohesive end sequence b connected to the 3 'end or the 5' segment; the cohesive end sequence a is complementary or reverse complementary to the cohesive end sequence b.

Furthermore, the sequence of the sticky end sequence a is shown as SEQ ID NO.6, and the sequence of the sticky end sequence b is shown as SEQ ID NO. 7;

preferably, the cohesive end sequence a is linked to the single-stranded DNA by a linker sequence; more preferably, the linking sequence is TT.

Furthermore, one of the 4 single strands is connected with a cohesive end sequence a at the 5 'end, and the miR-22 is a miR-22 of which the cohesive end sequence b is connected with the 5' segment.

Furthermore, the sequences of the 4 single-stranded DNAs are respectively and independently selected from the sequences shown in SEQ ID No. 1-4 one by one; the sequence of the miR-22 is shown in SEQ ID NO. 5.

The invention also provides a preparation method of the compound, which comprises the following steps:

co-incubating nucleic acid with a tetrahedral framework and miR-22 at 20-30 ℃ for 20-40 min; the vertex of the tetrahedral framework nucleic acid is connected with a cohesive end sequence a, the miR-22 is connected with a cohesive end sequence b, and the cohesive end sequence a is complementary or reverse complementary to the cohesive end sequence b.

Further, 4 single-stranded DNAs of tetrahedral framework nucleic acid are placed at a temperature sufficient for denaturation and maintained for more than 10min, and then the temperature is reduced to 2-8 ℃ and maintained for more than 20 min; the 3 'end or 5' segment of at least one of the four single-stranded DNAs is connected with a cohesive end sequence a.

The invention also provides the application of the compound in the medicine for treating facial nerve injury diseases; preferably, the facial nerve injury disease comprises loss of facial expression, abnormal lacrimal/salivary gland secretion.

Further, the above-mentioned drugs are drugs that promote repair of facial nerves.

Experimental results show that the compound of the tetrahedral framework nucleic acid and the miR-22 is compounded through unique viscous terminal linkage, so that the compound of the tetrahedral framework nucleic acid and the miR-22, which is reported in the prior art, has more excellent stability and more excellent treatment effectiveness, can promote the uptake of cells to the miR-22, improve the microenvironment after facial nerve injury, provide conditions for axon regeneration, and has a good treatment effect on the facial nerve injury.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is (a) a gel of nucleic acids PAGE carrying different fluorophores (green: FAM, red: Cy5, combined: orange); (b) PAGE gel picture of nucleic acid after nucleic acid dye Gelred double color; (c) a nucleic acid PAGE gel picture carrying FAM green fluorescent group; (d) a nucleic acid PAGE gel map carrying a Cy5 red fluorophore; lane (1: miR-22-FAM, 2: cS3, 3: cS3-miR22-FAM, 4: tFNAs-Cy5, 5: ctFNAs-Cy5, 6: ctFNAs-miR22-FAM, 7: Cy5-ctFNAs-miR22-FAM, 8: Marker)

FIG. 2 is (a) TEM and AFM images of tFNAs; (b) TEM and AFM images of ctFNAs-miR 22.

FIG. 3 shows (a) the particle size and Zeta potential of tFNAs; (b) the grain size and Zeta potential of ctFNAs-miR 22; (c) statistical table of tFNAs and ctFNAs-miR22 particle size and potential.

FIG. 4 is the intake of ctFNAS-miR22 by RAW 264.7. (a) Flow cytometry detects rapid uptake of miR-22 and ctFNAS-miR22 by RAW264.7 within 3 hours; (b) cellular immunofluorescence was measured for the efficient uptake of miR-22 and ctFNAS-miR22 by RAW264.7 within 3 hours (Red: Cy5 fluorophore; Green: Coprinus comatus; blue: DAPI), scale: 5 μm

FIG. 5 is the uptake of ctFNAS-miR22 by SCs. (a) Flow cytometry detects rapid uptake of miR-22 and ctFNAS-miR22 by SCs within 12 hours; (b) carrying out cell immunofluorescence detection on SCs to efficiently take miR-22 and ctFNAS-miR22 within 12 hours; red: cy5 fluorophore; green: a cytoskeleton; blue, nuclei, scale: 5 μm.

FIG. 6 shows that ctFNAs-miR22 promotes proliferation and migration of SCs. (a) Detecting an SCs image in a proliferation stage by an EdU method; (b) statistical analysis of EdU; (c)24 hour invasion experimental images; (d) SCs vertical migration statistical analysis; (e)24 hour scratch test images; (f) statistical analysis of SCs horizontal migration. Statistical data are mean ± sd (n ═ 3), and statistical differences were indicated by one-way variance analysis, with P < 0.05, P < 0.01, P < 0.001, P < 0.0001.

FIG. 7 shows that ctFNAs-miR22 promotes macrophage migration by Schwann cells. (a) Macrophage scratch experimental images under different culture environments within 24 hours; (b) schematic diagram of co-culture scratch experiment; (c) statistical analysis of the horizontal migration of RAW264.7, the data were all means ± sd (n ═ 3), and statistical differences were indicated by one-way variance analysis, with P < 0.05, P < 0.01, P < 0.001, and P < 0.0001. (d)24 hour RAW264.7 vertically migrated images; (e) schematic diagram of co-culture invasion experiment.

FIG. 8 shows that ctFNAs-miR22 promotes the expression of anti-inflammatory factors in the microenvironment constructed by macrophages and Schwann cells after injury. (a) Schematic diagram of the effect of ctFNAs-miR22 on inflammatory factors in the microenvironment after injury; (b) ELISA detects TNF-alpha released by RAW264.7 for statistical analysis; (c) ELISA was used for the statistical analysis of IL-10 released from RAW 264.7. The statistics were all means ± standard deviations (n ═ 3), and statistical differences were indicated by one-way variance analysis, × P < 0.05, × P < 0.01, × P < 0.001, × P < 0.0001.

FIG. 9 shows that ctFNAs-miR22 promotes FGF-9 expression in the microenvironment constructed by macrophages and Schwann cells after injury. (d) Schematic representation of the effect of ctFNAs-miR22 on cytokines in the post-injury microenvironment; (e) ELISA assays for FGF-9 released by RSCs after different treatments in the co-culture environment. Mean ± standard deviation of statistical data (n ═ 3), corrected by one-way anova, Tukey test: p < 0.05, P < 0.01, P < 0.001, P < 0.0001, indicating a statistical difference.

FIG. 10 is the change in whisker movement at different times in rats after different treatments. (a) Video recordings of rat whisker movements. The movements of tentacle extension (1) and tentacle contraction (2) are recorded, on the operative (left) and control (right) sides, observing the amplitude and frequency of movement of the tentacles starting from the sagittal line (Fr-Occ). (b) Video-based whisker motion score statistics. Statistics were performed according to the Vibrissa scoring criteria, with data all mean ± sd (n ═ 5), corrected by one-way anova, Tukey test: p < 0.05, P < 0.01, P < 0.001, P < 0.0001, indicating a statistical difference.

Fig. 11 is the results of HE staining of facial nerve tissue at different times for different treated posterior nerves, scale: 20 μm.

Fig. 12 shows toluidine blue staining of facial nerve tissue at different times for different treated posterior nerves, scale: 50 μm.

Fig. 13 is an image of facial nerve tissue under transmission electron microscopy. (a) Electron microscope tissue images of normal facial nerve tissue (1) and nerve tissue (2) clamped behind; (b) electron microscopy histograms of the posterior nerves at different times with different treatments, scale: 5 μm.

FIG. 14 is (a) immunofluorescence detection of β -III Tubulin expression; red: β -III Tubulin, blue: nucleus, scale: 20 mu m; (b) histochemical staining examined the expression of MBP, scale: 20 μm.

Detailed Description

The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.

Example 1 Synthesis of a Complex of tetrahedral framework nucleic acid (tFNA) and miR-22 (ctFNAs-miR22)

1. Synthesis of

Four DNA single strands (S1, S2, cS3, S4, see Table 1 in sequence) were dissolved in TM Buffer (10mM Tris-HCl,50mM MgCl ₂ pH 8.0), the final concentration of the four DNA single strands is 1000nM, mixed well, heated rapidly to 95 ℃ for 10 minutes, then cooled rapidly to 4 ℃ for more than 20 minutes to obtain ctFNA; a novel DNA nanocomposite, ctFNAs-miR22, was synthesized by sticky-end based base-complementary pairing by incubating with cmiR-22 (sequence shown in Table 1) at room temperature for 30 minutes at a ratio of 1: 1.

2. Identification

ctFNA-miR22 was detected using PAGE electrophoresis; detecting the shape of ctFNA-miR22 by using a transmission electron microscope; and (3) detecting the zeta potential and the particle size of ctFNA-miR22 by using a nano-particle size potentiometer.

3. Identification results

Electrophoresis results show that the molecular weight of the band of ctFNA-miR22 is significantly higher than that of miR-22, single-stranded DNA and DNA tetrahedrons (tFNAs, ctFNAs), indicating that the single-stranded DNA is assembled together (FIG. 1).

The transmission electron microscopy revealed tetrahedral particles (FIG. 2), zeta potential and particle size as detected by a nano-particle size potentiometer (FIG. 3).

TABLE 1 nucleotide sequences to which the invention relates

Wherein Cy5 and FAM fluorescent labeling groups are used for tracing

The beneficial effects of the invention will be further described in the following experimental examples, wherein ctFNA-miR22 in the experimental examples is prepared by the method of example 1, and in part of experiments, for tracing, ctFNA-miR22 is prepared by replacing S1 with a single chain of 5 'segment modified Cy5 fluorophore, and/or ctFNA-miR22 is prepared by replacing cmiR-22 with a single chain of 3' segment modified FAM fluorophore.

The beneficial effects of the present invention are demonstrated by the following experimental examples.

Experimental example 1 uptake of ctFNAs-miR22 by SCs cells and RAW264.7 cells

1. Method of producing a composite material

In order to detect the capacity of taking ctFNAs-miR22 by SCs cells and RAW264.7 cells, the content and the location of the medicament in the SCs are observed after the ctFNAs-miR22 is incubated for a certain time by using flow cytometry and a cell immunofluorescence staining method. Grouping setting: control (SCs without any treatment), 250nM Cy5-miR-22, 250nM Cy5-ctFNAs-miR 22.

2. Results

As shown in FIGS. 4-5, 3h, the ctFNAs-miR22 was taken up by RAW264.7 cells up to 99.30%, while the amount of miR-22 taken up was only about 4.28%. 12h, about 66.20% of SCs cells were able to take up ctFNAs-miR22, whereas miR 22-taking cells were only about 5.94%. Observation under a laser confocal microscope shows that red fluorescence is miR22/ctFNAS-miR22 with a Cy5 fluorophore modified, green fluorescence is a cytoskeleton, and ctFNAS-miR22 after being absorbed in large quantity is uniformly distributed in cytoplasm in a scattered point shape, and only a small quantity of miR22 is absorbed by SCs/RAW264.7 at the moment. The results show that the ctFNAS-miR22 delivery system can help miR-22 to be quickly and efficiently taken by cells.

Experimental example 2, ctFNAs-miR22 regulates interaction between SCs and macrophages to improve microenvironment after injury

1. Method of producing a composite material

Co-culturing SCs with macrophages; grouping setting: control group (RAW 264.7/SCs without any treatment), LPS (1. mu.g/mL) injured group, LPS + miR-22(250nM) treated group, LPS + tFNAs (250nM) treated group, LPS + ctFNAs-miR22(250nM) treated group, were examined as follows:

(1) observing the influence of ctFNAs-miR22 on the proliferation and migration of SCs under a microscope;

(2) observing the influence of ctFNAs-miR22 mediated SCs on promoting macrophage migration under a microscope

(3) Detecting the secretion of cell factors in the co-culture environment by using an enzyme-labeling instrument;

(4) detecting the expression of Bax, caspase-3 and BCL-2 by using immunofluorescence and Western blot;

2. results

As shown in FIG. 6, ctFNAs-miR22 promotes the proliferation and migration of SCs. compared with ctFNA and single-chain miR22, ctFNA-miR22 can inhibit apoptosis caused by NMDA.

As shown in FIG. 7, ctFNAs-miR22 promotes macrophage migration by Schwann cells.

Fig. 8 shows that ctFNAs-miR22 promotes the expression of anti-inflammatory factors in the microenvironment constructed by macrophages and schwann cells after injury.

FIG. 9 shows that ctFNAs-miR22 promotes the expression of FGF-9 in the microenvironment constructed by macrophages and Schwann cells after injury.

The results of the experimental example show that ctFNAs-miR22 can enhance the interaction between SCs and macrophages, promote SCs-mediated M2 macrophage polarization, improve the microenvironment after injury and provide conditions for axon regeneration.

Experimental example 3, ctFNAs-miR22 promotes repair of rat facial nerve injury

1. The method comprises the following steps:

establishing a rat facial nerve clamp injury model 1) weighing the weight of each rat, diluting chloral hydrate in proportion, and performing intraperitoneal injection anesthesia.

2) Taking facial nerve as operation side, scraping hair around the back of left ear, and sterilizing with iodophor and 75% alcohol.

3) An incision is made at 0.5-1mm of the posterior margin of the left ear, the myofascium is separated, the mastoid process is exposed, and after finding the facial nerve, the facial nerve trunk is clamped by a straight-head hemostatic forceps perpendicular to the facial nerve forceps for 60 seconds. The jaws were then sutured with 3-0 silk suture adjacent the muscle marking the injury site. The surrounding skin was sutured with 3-0 nylon thread and the injured area was re-surfaced on the skin. After operation, antibiotics are added into drinking water of rats to prevent postoperative infection.

4) After 24 hours of model building, they were randomly assigned to 4 groups (6 rats per group): injury group (60 μ L physiological saline), miR-22 treatment group (250nM 60 μ L), tFNAs treatment group (250nM 60 μ L), ctFNAs-miR22 treatment group (250nM 60 μ L). The first injection was given 24 hours after modeling and every other day injections were taken for 3 weeks continuously. The control group was normal mice (3) and each rat in the remaining groups had no surgical operation on the right lateral nerve.

2. In vivo detection:

A) whisker tremor experiment in rats

B) Facial nerve HE staining

C) Toluidine blue staining

D) Transmission electron microscope

E) Immunohistochemistry

F) Immunofluorescence

2. Results

Rat whisker tremor experiment (fig. 10): palpus dyskinesia was observed 24 hours after the facial nerve forceps were injured, palpus on the operation side completely stopped moving, some rats also had facial paralysis such as eyelid dyskinesia, and healthy side palpus could move with high frequency and large amplitude. After the treatment of ctFNAs-miR22 for two weeks, the almost complete recovery of the movement amplitude of the surgical palpus can be observed until the movement frequency and amplitude of the surgical palpus return to normal in the third week.

Facial nerve HE staining (fig. 11): the normal control group has regular arrangement of nerve fiber tissues, compact structure and uniform shape and distribution of cell nuclei. After the forceps are injured, the nerve fiber structure is destroyed, and fracture, gap, edema, vacuole degeneration and the like occur. After two weeks of treatment with ctFNAs-miRNA22, the density of the fibers was significantly increased and the fiber arrangement was more compact and regular compared to the other treatment groups. After three weeks of treatment, the ctFNAs-miRNA22 group showed more pronounced fiber regeneration, and the fiber morphology approached that of the control group.

Toluidine blue staining (fig. 12): normal nerves are surrounded by intact perineurium, and dense myelin sheaths surround axons, which are intact in morphology and uniform in size. Whereas a large number of necrotic axons, demyelinating changes and cellular debris were visible until the third week after injury. After one week of treatment, repair-type SCs with abundant cytoplasm and new axons were observed in the treated group, especially in ctFNAs-miRNA22 treated group. In the second week, ctFNAs-miRNA22 showed a large number of progressively mature axons and myelin sheaths compared to the other treatment groups; in the third week, axons in ctFNAs-miRNA22 group became gradually abundant, the myelin sheaths covering the axons were denser, and the sizes and forms were more regular

Transmission electron microscope (fig. 13): in the ctFNAs-miRNA22 group, repair-type SCs containing a large amount of cytoplasm and new axons appeared in the nerve tissue from the first week compared to the other groups, and remyelination of SCs and new axons gradually matured in the second week until the nerve tissue structure approached the control group in the third week.

Immunohistochemistry and immunofluorescence (fig. 14): when the neuron marker protein beta-III Tubulin is detected, the beta-III Tubulin (red color) in each group is obviously reduced compared with that in a control group in the first week after the injury. In the second week, the expression of β -III Tubulin was significantly increased in the ctFNAs-miRNA22 group compared to the other groups, and this change was most significant in the third week and consistently better than in the tFNAs, miR-22 treated group. Similar changes were observed after detection of myelin marker protein MBP.

In conclusion, the invention provides the compound of the tetrahedral framework nucleic acid and the miR-22, which is more excellent in stability and treatment effectiveness than the compound of the tetrahedral framework nucleic acid and the miR-22 reported in the prior art through unique cohesive end linkage compounding, can effectively promote the uptake of cells to the miR-22, improve the microenvironment after facial nerve injury, provide conditions for axon regeneration, and has a good treatment effect on the facial nerve injury.

SEQUENCE LISTING

<110> Sichuan university

<120> a compound for treating facial nerve injury diseases, and a preparation method and application thereof

<130> GYKH1118-2022P0114940CC

<160> 9

<170> PatentIn version 3.5

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Claims

1. The compound for treating facial nerve injury diseases is characterized by being formed by connecting tetrahedral framework nucleic acid and miR-22 through cohesive ends, wherein the molar ratio of the tetrahedral framework nucleic acid to the miR-22 is 1 (1-4).

2. The complex of claim 1, wherein the molar ratio of tetrahedral framework nucleic acids and miR-22 is 1: 1.

3. The complex of claim 1, wherein the tetrahedral framework nucleic acid is formed from 4 single-stranded DNAs through base complementary pairing, at least one of the four single-stranded DNAs having a cohesive end sequence a attached to the 3 'end or the 5' end; the miR-22 is miR-22 with a cohesive end sequence b connected to the 3 'end or the 5' segment; the cohesive end sequence a is complementary or reverse complementary to the cohesive end sequence b.

4. The complex of claim 3, wherein the sequence of the cohesive end sequence a is shown as SEQ ID No.6, and the sequence of the cohesive end sequence b is shown as SEQ ID No. 7;

5. The complex of claim 3, wherein one of the 4 single strands is 5 'linked to a cohesive end sequence a, and the miR-22 is a miR-22 whose 5' segment is linked to a cohesive end sequence b.

6. The complex of any one of claims 3 to 5, wherein the sequences of the 4 single-stranded DNAs are independently selected from the sequences shown in SEQ ID nos. 1 to 4; the sequence of the miR-22 is shown in SEQ ID NO. 5.

7. A method for preparing a composite as claimed in any one of claims 1 to 6, comprising the steps of:

8. The method according to claim 7, wherein 4 single-stranded DNAs of tetrahedral framework nucleic acid are maintained at a temperature sufficient to denature them for 10min or more, and then the temperature is lowered to 2 to 8 ℃ for 20min or more; the 3 'end or 5' segment of at least one of the four single-stranded DNAs is connected with a cohesive end sequence a.

9. Use of a complex according to any one of claims 1 to 6 in a medicament for the treatment of a facial nerve injury disease; preferably, the facial nerve injury disease comprises loss of facial expression, abnormal lacrimal/salivary gland secretion.

10. The use of claim 9, wherein the medicament is a medicament for promoting repair of facial nerves.