CN112826920A

CN112826920A - Application of ID1/ID3 in inducing reprogramming of fibroblasts into Schwann cells to promote nerve regeneration

Info

Publication number: CN112826920A
Application number: CN202110093988.0A
Authority: CN
Inventors: 陈泽林; 史春梦; 王钰; 沈谷芳; 谭旭
Original assignee: Army Medical University
Current assignee: Army Medical University
Priority date: 2021-01-23
Filing date: 2021-01-23
Publication date: 2021-05-25

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to application of ID1 and ID3 in reprogramming fibroblasts into Schwann cells and promoting peripheral nerve regeneration, wherein the application uses the Schwann cells reprogrammed by ID1 or ID3 or virus vectors overexpressed by ID1 or ID3, can promote nerve regeneration after trauma, promote the healing of the trauma and improve the healing quality of the trauma, and is an effective way for solving the problem of difficult trauma caused by local nerve regeneration or peripheral neuropathy after trauma.

Description

Application of ID1/ID3 in inducing reprogramming of fibroblasts into Schwann cells to promote nerve regeneration

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to application of ID1/ID3 in inducing reprogramming of fibroblasts into Schwann cells to promote nerve regeneration.

Background

Wound repair is a complex biological process involving multiple ordered cellular biological processes such as re-epithelialization, angiogenesis, etc. The nerve branch has great influence on wound healing, and the previous research shows that pathological or trauma-caused nerve innervation disorder can cause the delay of wound healing and even cause non-healing. In addition, the recovery of pain sensation, temperature sensation and touch sensation of local tissues after trauma is of great importance to the improvement of the life quality of patients, so that the improvement of the local nerve repair after trauma has important clinical significance in treating the difficult-to-heal wound surface and improving the healing quality of the wound surface.

Previous studies have shown that schwann cells play an important role in the repair of peripheral nerve injury. After nerve injury, Schwann cells interact with various cells at and around the injured site and participate in important processes such as debris removal, nerve repair and nerve regeneration. In addition, Schwann cells secrete neurotrophic factors to promote nerve regeneration. The previous research shows that the peripheral nerve regeneration can be effectively promoted by transplanting exogenous Schwann cells after the peripheral nerve is damaged. However, the large number of schwann cells required for cell transplantation therapy has limitations in cell supply and matching, which may lead to immune rejection. Therefore, Schwann cells are generated by reprogramming adult somatic cells such as fibroblasts, so that the bottleneck of immune matching and cell source can be effectively broken through, and the adult somatic cells become a cell source with great prospect for clinical application.

The family of differentiation Inhibitors (IDs) is a group of evolutionarily conserved proteins. Four ID proteins are found in mammals, namely ID1 to ID 4. The expression of the ID protein is widely but widely varied in various cell types. Previous studies have found that the ID protein can promote cell cycle progression, accelerate cell migration, inhibit differentiation of different types of progenitor cells and reduce cell senescence, and thus the ID protein is involved in various pathophysiological processes in vivo such as neurogenesis, stem cell maintenance, angiogenesis, organ development, tumorigenesis and metastasis, energy metabolism, and the like. However, the role of the ID protein molecule in somatic cell reprogramming has not been reported.

Disclosure of Invention

An object of the invention is to provide an application of ID1/ID3 in inducing reprogramming of fibroblasts into Schwann cells to promote nerve regeneration, wherein the application uses Schwann cells reprogrammed by ID1 or ID3 or virus vectors overexpressed by ID1 or ID3 to promote nerve regeneration after trauma, promote wound healing and improve the quality of wound healing, and the application is an effective way for solving the problem of difficult trauma caused by local nerve regeneration or peripheral neuropathy after trauma.

The specific technical scheme is as follows:

the application of the differentiation inhibition factor in preparing the medicine for treating peripheral nerve injury is that the differentiation inhibition factor promotes nerve regeneration by inducing the reprogramming of fibroblast into Schwann cells.

The differentiation inhibitory factor is ID1 or/and ID 3.

The Schwann cell comprises ID1 or ID3 induction, or both ID1 and ID3 induction.

The fibroblast is an adult of human or murine origin, or an embryonic fibroblast.

The peripheral nerve injury is a post-traumatic local nerve injury.

The post-traumatic local nerve injury is peripheral nerve injury including sciatic nerve full-thickness dissection, local nerve injury caused by skin full-thickness injury, or tissue local nerve injury caused by other types of trauma.

The wound includes mechanical wound of skin and other tissues, or burn and composite wound of skin or other tissues.

ID1 and ID3 involved in the present invention include protein gene sequences encoding ID1 and ID3, recombinant vectors, recombinant microorganisms and the like. After the ID1 or ID3 gene is over-expressed in fibroblasts, the expression of Schwann cell markers s100 beta and Gfap in the fibroblasts can be improved, and the axon extension of dorsal root ganglion neurons can be promoted.

The fibroblasts contemplated by the present invention include, but are not limited to, primary fibroblasts isolated from adult tissues, and fibroblasts isolated during embryonic period are also within the scope of the present invention.

Wounds contemplated by the present invention include, but are not limited to, mechanical wounds of the skin, mechanical wounds of other tissues, or burns, composite wounds of the skin or other tissues, and the like are also within the scope of the present invention. After the fibroblasts are induced to be reprogrammed to Schwann cells by ID1 and ID3, the Schwann cells induced by transplantation can effectively promote local nerve regeneration after trauma, accelerate the healing speed of the trauma, reduce the collagen deposition of the wound surface and improve the healing quality of the wound surface.

Peripheral nerve injuries contemplated by the present invention include, but are not limited to, sciatic nerve injuries, post-traumatic localized nerve injuries. After the fibroblasts are induced to be reprogrammed to schwann cells by ID1 and ID3, the schwann cells induced by transplantation can effectively promote the regeneration of damaged peripheral nerves and relieve the atrophy of innervated muscles.

The invention also directly uses the virus vectors of ID1 and ID3 to the local part of the wound, can promote the nerve repair of the local part of the wound, and accelerate the healing speed of the wound, the re-epithelialization and the granulation formation after the wound.

On the basis of establishing a full-thickness skin injury wound (which can be healed through a normal wound repair process) and an in vitro fibroblast scratch injury model, the applicant proves that the expressions of ID1 and ID3 in fibroblasts after injury are obviously enhanced through a histopathology technology and a molecular biology technology.

Over-expressing ID1 or ID3 in fibroblasts, found that both ID1 and ID3 increased the proliferative and migratory capacity of fibroblasts, while also reprogramming the fibroblasts to schwann cells expressing Gfap and s100 β. After coculture of the induced Schwann cells and dorsal root ganglion neurons, the induced Schwann cells can better promote the axon growth of the neurons compared with non-induced fibroblasts.

The experiment of the applicant proves that after Schwann cells induced by ID1 or ID3 are locally transplanted to wound tissues after full-thickness skin injury, local nerve repair can be promoted, the wound healing speed can be accelerated, the collagen deposition of the wound is reduced, and the wound healing quality is improved; the ID1 or ID3 virus vector is directly injected into the ear wound part, and compared with a control virus vector, the transplantation of the ID1 or ID3 virus vector can promote the nerve repair of the wound part, accelerate the wound healing speed, and promote epithelialization and granulation after the wound; schwann cells induced by ID1 or ID3 are locally transplanted to the severed ends of a sciatic nerve full-thickness cutting model, and the Schwann cells induced by ID1 or ID3 are found to have better effects of promoting nerve regeneration and reducing muscular atrophy at nerve innervation compared with blank transplantation and control fibroblast transplantation.

Drawings

FIG. 1 shows the expression of fibroblasts ID1 and ID3 after trauma according to the invention;

FIG. 2 shows the induction of fibroblasts into Schwann cells by ID1 and ID3 according to the present invention;

FIG. 3 is a graph of the neurite outgrowth of dorsal root ganglion neurons promoted by Schwann cells induced by ID1 or ID3 according to the present invention;

FIG. 4 shows that Schwann cells induced by ID1 or ID3 promote local nerve regeneration and wound healing according to the invention;

FIG. 5 shows that Schwann cells induced by ID1 or ID3 promote sciatic nerve injury repair;

FIG. 6 is a graph showing that ID1 or ID3 lentiviral vectors of the present invention promote local nerve regeneration and wound healing.

Detailed Description

The present invention is described in detail below by way of examples, it should be noted that the examples are only for the purpose of further illustration, and are not to be construed as limiting the scope of the present invention, and that those skilled in the art may make insubstantial modifications and adaptations of the present invention based on the above disclosure.

The mice used in the invention are C57 mice, and the rats are SD rats, and are purchased from the experimental animal center of army and military medical university;

ID1, ID3, Tuj1, s100 beta antibody, available from Abcam,

antibody Gfap was purchased from Sanying, Wuhan;

fluorescent secondary antibody, HRP secondary antibody, double antibody and crystal violet staining solution are purchased from Biyuntian biotechnology limited company;

b27, L-glutamine, fetal bovine serum from Gibco;

4% paraformaldehyde and PBS were purchased from doctor DebBiotechnology GmbH;

chloral hydrate was purchased from Shanghai Biotechnology Ltd; collagenase type I from Worthington corporation;

pancreatin and DMEM medium were purchased from Hyclone,

Neurobasal^TMthe medium was purchased from Millpore.

Flow sorting was performed using FACS AriaIII from BD;

immunoblotting was performed using an electrophoresis apparatus from bella corporation;

image acquisition was performed using a DM3000 microscope from lycra;

immunoblotting, immunofluorescent staining of cells and tissues, colony-forming ability assays, HE staining and other non-well documented experimental procedures and conditions reference (Chen, Z., et al. (2019). "therapeutic fiber-selective near-isolated photothermal to control staining." therapeutics 9(23): 6797-6808).

In the present invention, commercially available biological reagents are used as reagents other than the above-mentioned reagents.

Example 1 expression of ID1 and ID3 in post-traumatic fibroblasts

Making a mouse skin full-layer excision injury model: selecting a C57 female mouse with the weight of about 20g and the week of 8-12, carrying out conventional skin preparation and unhairing one day before trauma, carrying out intraperitoneal injection anesthesia (5ml/kg) by using 1% chloral hydrate, slightly lifting the back skin of the mouse at the midline of the back of the mouse, and carrying out trauma induced by using a full-layer skin puncher with the diameter of about 1 cm.

Collecting specimens of 1 st, 3 th, 5 th, 7 th, 9 th, 12 th, 15 th and 20 th days of the wound surface of the whole skin defect of the mouse, fixing by 4% paraformaldehyde, embedding by paraffin, slicing by 5 mu m, carrying out immunohistochemical staining on the slices for ID1 and ID3 proteins, replacing primary antibodies by PBS (phosphate buffer solution) as a negative control group, and displaying the experimental results as shown in figures 1A and B, wherein the staining of the slices shows that ID1 and ID3 are expressed in the wound surface granulation tissue fibroblasts from 3 days.

In vitro fibroblast scratch wound model preparation: selecting human dermal fibroblasts (within 6 generations) subjected to tissue isolation culture, inoculating the human dermal fibroblasts into a 24-pore plate preset with a circular cell slide, and performing culture under the following conditions: DNEM + 10% fetal calf serum + double antibody, changing the liquid 1 time in a conventional way every 3 days, culturing for 2 days after the liquid is cultured to a full state, and drawing a transverse line along the diameter of a 24-pore plate by using a 200ul gun head to cause wound.

Cell slides at 6,12,24,36,48,60,72h post-wounding were collected, cytoimmunofluorescent stained with ID1 and ID3 in place of primary antibody in PBS as a negative control group, and the results of the experiment are shown in fig. 1C, D, and the staining shows that the expression of ID1 and ID3 in the scared post-traumatic fibroblasts was significantly enhanced at 12h post-wounding.

Cellular proteins at 24,36,48 and 72h after the wound are collected, immunoblotting is carried out, ID1 and ID3 expression is detected, experimental results are shown in a figure 1E, and the results show that the expression of ID1 and ID3 in fibroblasts after the wound is obviously enhanced, which indicates that the high expression of ID1 and ID3 in the fibroblasts plays an important role in wound repair.

Example 2 Induction of fibroblasts into Schwann cells by ID1 or ID3

Inoculating human and mouse dermal fibroblasts into a 24-well plate, respectively inoculating human and mouse lentiviral vectors Vector-mCherry, ID1-mCherry and ID3-mCherry which are constructed and purified by Jima gene biotechnology limited when the fusion degree of the cells reaches 40%, wherein the number of virus particles/cell number (MOI) is 100, after transfection and complete cell fusion, carrying out passage treatment, after the cells are amplified for 1 generation, sorting mChery control fibroblasts by flow cells, ID1 overexpression fibroblasts and ID3 overexpression fibroblasts.

Colony formation experiments tested the proliferative capacity of ID1 and ID3 overexpressing cells: human and mouse controls, ID1 overexpressing and ID3 overexpressing fibroblasts were plated at 1X 10³The number of cells/well was inoculated in 6-well plates, DMEM + 10% fetal bovine serum + double antibody was changed 1 time every 3 days, after 12 days of culture, fixed with 4% paraformaldehyde for 20min, cells were stained with crystal violet for 15min, and the number of colonies per group was counted under a microscope after rinsing with clear water. The experimental results are shown in fig. 2A, and the result shows that the number of colonies formed by fibroblasts over-expressed by ID1 and over-expressed by ID3 is obviously greater than that of the control cells, which suggests that ID1 and ID3 can enhance the self-renewal capacity of the fibroblasts.

Detection of Gfap, S100 β: control fibroblasts, namely the fibroblasts with over-expressed ID1 and over-expressed ID3 are inoculated to a cell slide, after 24 hours of culture, the cell is fixed for 20 minutes by 4% paraformaldehyde, cell immunofluorescence staining is carried out, PBS is used for replacing primary antibody, the cell is used as a negative control group, and the experimental results are shown in FIG. 2B, and the result shows that ID1 and ID3 can induce the fibroblasts to enhance the expression of Schwann cell markers Gfap and S100 beta.

Immunoblotting was used to detect Sox2, Nestin, Gfap, S100 β: after inoculating control fibroblasts, ID1 overexpressed and ID3 overexpressed fibroblasts, when the cell fusion degree reaches more than 90%, extracting whole cell protein, detecting the expression of Sox2, Nestin, Gfap and S100 beta in the control fibroblasts, ID1 overexpressed and ID3 overexpressed fibroblasts by an immunoblotting method, and finding that ID1 and ID3 can induce the fibroblasts to express Sox2, Nestin, Gfap and S100 beta, and combining the immunofluorescence staining result, the fact that ID1 and ID3 can induce the fibroblasts to be Schwann cells is suggested.

Example 3 Induction of Schwann cells by ID1 or ID3 promotes axonal growth in dorsal root ganglion neurons

Rat dorsal root ganglion neuron isolation: selecting a female SD mouse with the weight of about 200g for 8-12 weeks, breaking the neck, and dying, separating the dorsal root ganglion of the 4 th-5 th segment, applying collagenase I (0.25%) + 0.01% pancreatin, shaking the table at 37 ℃ for 40min, sieving the cell with a 75um cell sieve, and culturing the obtained cell in Neurobasal^TMMedium + 2% B27+ L-glutamine (2mM), and after 1 week of culture, the surviving cells were dorsal root ganglion neurons.

Co-culturing dorsal root ganglion neurons and induced schwann cells: using the flow sorted control of example 2, ID1 and ID3 over-expressed fibroblasts, at 1X 10⁴The number of cells/well is inoculated in a 24-well plate with a preset cell slide, and the culture conditions are as follows: DMEM + 10% fetal calf serum + double antibody, and after 24h, dorsal root ganglion neuron 5 x 10 is inoculated²Culturing in 24-well plate pre-inoculated into fibroblast under changed conditions^TMMedium + 2% B27+ L-glutamine (2mM), culturing for 72h, fixing with 4% paraformaldehyde for 20min, and refiningAnd (3) carrying out cellular immunofluorescence staining on a nerve axon marker Tuj1, replacing a primary antibody with PBS (phosphate buffer solution) to serve as a negative control group, selecting axons of 6-10 neurons in a 20-fold picture in each group, and calculating the average length of the axons of the neurons in each group by using Image J software. As a result, as shown in fig. 3, it was found that the axonal growth of dorsal root ganglion neurons co-cultured with schwann cells induced by ID1 or ID3 was significantly longer than that of dorsal root ganglion neurons co-cultured with control fibroblasts, suggesting that schwann cells induced by ID1 or ID3 significantly promoted neurite growth.

Example 4 Schwann cells induced by ID1 or ID3 promote nerve repair and wound healing in radiation-compounded wounded skin wounds

Manufacturing a radiation composite wound surface model: selecting C57 female mice with the weight of about 20g for 8-12 weeks, and performing conventional skin preparation and depilation one day before trauma. General-body 5Gy X-ray irradiation was given, followed by intraperitoneal injection of 1% chloral hydrate for anesthesia (5ml/kg), and the skin on the back of the mouse was gently lifted at the midline of the back of the mouse, and was subjected to wound using a full-thickness skin punch having a diameter of about 1 cm.

Schwann cells induced at 2X 10 using control fibroblasts obtained in example 2, ID1 or ID3⁶Each cell/mouse, 500ul volume of PBS, control fibroblasts, schwann cells induced by ID1 or ID3 were transplanted around the wound tissue and fed to the wounded mice. The sizes of the wound surfaces were photographed at 0,3,5,7,15 and 21 days after the trauma, and the healing rate and the scar area of the wound surfaces were calculated by using Image J software, and as a result, as shown in fig. 4A and B, it was found that schwann cells induced by ID1 or ID3 significantly promoted the healing of the wound surfaces and reduced the scar area.

Wound surface tissues 19 days after trauma are taken, and a nerve fiber marker Tuj1 in the wound surface tissues is stained through immunofluorescence staining, and the result is shown in FIG. 4C, and the result shows that the expression of Tuj1 in the wound surface tissues of Schwann cell transplantation induced by ID1 or ID3 is obviously higher than that in PBS transplantation and control fibroblast transplantation groups, and the suggestion that Schwann cells induced by ID1 or ID3 can obviously promote local nerve repair after trauma.

Example 5 stimulation of Schwann cells induced by ID1 or ID3 to repair sciatic nerve injury

Making a sciatic nerve injury model: selecting an SD female mouse with the weight of about 200g and the week of 8-12, carrying out intraperitoneal injection anesthesia (5ml/kg) by using 1% chloral hydrate, separating the sciatic nerve near the upper end of the femoral head, then carrying out full transection, suturing the two severed ends by using a silica gel catheter, and then connecting the two severed ends, wherein the distance between the two severed ends is 5 mm.

Schwann cells induced at 2X 10 using control fibroblasts obtained in example 2, ID1 or ID3⁶Each cell/rat, PBS in a volume of 200ul, control fibroblasts, schwann cells induced by ID1 or ID3 were transplanted into a silica gel catheter, and the muscle and skin were sutured layer by layer. Normal sciatic nerve fibers in the contralateral and neutral silica gel catheters were harvested 6 weeks after trauma. Half of the tissues were fixed with 4% paraformaldehyde for 72h, dehydrated, paraffin-embedded, sliced at 4um, HE-stained to measure the diameter of the nerve bundle, and the length of the diameter of the nerve bundle was calculated using Image J software, as shown in FIG. 5A, and it was found that Schwann cell transplantation induced by ID1 or ID3 significantly increased the diameter of the regenerated sciatic nerve.

Half of the tissues were frozen sectioned (10um thick), Tuj1 was immunofluorescent stained, and the relationship between transplanted cells and regenerated nerves was examined, and as a result, it was found in FIG. 5B that transplanted ID1 or ID3 induced more bundles of regenerated nerves in sciatic nerves of Schwann cells and the contact with transplanted cells was closer.

The gastrocnemius muscle on the side of injured sciatic nerve is taken, fixed by 4% paraformaldehyde for 72h, dehydrated, embedded in paraffin, sliced by 4um, and subjected to HE staining to detect the atrophy condition of the gastrocnemius muscle, and the result is shown in figure 5C, and the gastrocnemius muscle atrophy induced by transplanting ID1 or ID3 is obviously reduced compared with that of a control. The results indicate that Schwann cells induced by ID1 or ID3 can obviously promote sciatic nerve injury repair.

Example 6 ID1 or ID3 Lentiviral vectors promote local nerve repair and wound healing following ear injury

Ear wound model preparation: selecting a C57 female mouse with the weight of about 20g and the week of 8-12, carrying out intraperitoneal injection anesthesia (5ml/kg) by using 1% chloral hydrate, and punching a hole in the center of an ear by using an ear puncher with the diameter of 2mm to prepare an ear wound model.

The Vector-mCherry, ID1-mCherry and ID3-mCherry lentiviral vectors of example 2 were used in a volume of 20ulContaining 1X 10⁷U virus amount, local injection around ear wound, taking pictures of wound size 0,3,7,14,21 and 35 days after wound, calculating wound healing rate by using Image J software, and finding that ID1 or ID3 lentivirus vector can accelerate ear wound healing.

The method comprises the following steps of taking 35d wound tissue, fixing by 4% paraformaldehyde for 72h, dehydrating, embedding paraffin, slicing by 4um, and expressing by immunofluorescence staining Tuj1, wherein the result is shown in figure 6, and the ID1 and ID3 lentivirus transplantation can obviously promote local nerve repair and regeneration.

Claims

1. The application of the differentiation inhibition factor in preparing the medicine for treating peripheral nerve injury is that the differentiation inhibition factor promotes nerve regeneration by inducing the reprogramming of fibroblast into Schwann cells.

2. Use according to claim 1, characterized in that: the differentiation inhibitory factor is ID1 or/and ID 3.

3. Use according to claim 1, characterized in that: the Schwann cell comprises ID1 or ID3 induction, or both ID1 and ID3 induction.

4. Use according to claim 1, characterized in that: the fibroblast is an adult of human or murine origin, or an embryonic fibroblast.

5. Use according to claim 1, characterized in that: the peripheral nerve injury is a post-traumatic local nerve injury.

6. Use according to claim 5, characterized in that: the local nerve injury after trauma comprises sciatic nerve full-thickness cutting, local nerve injury caused by skin full-thickness injury, or tissue local nerve injury caused by other types of trauma.

7. Use according to claim 5, characterized in that: the wound includes mechanical wound of skin and other tissues, or burn and composite wound of skin or other tissues.