Construction method and application of humanized keloid mouse model

Abstract

The invention provides a construction method of a humanized keloid mouse model. The construction method comprises the following steps: constructing and preparing BAC plasmids; carrying out superovulation and fertilized egg collection on the laboratory mice; carrying out pronucleus microinjection on the fertilized ova; embryo transplantation after injection; performing genotype identification; breeding and genetic analysis; and verifying the construction result of the humanized immune system by using immunohistochemistry and immunofluorescence. According to the invention, peripheral blood mononuclear cells treated by sCD27 and skin (homologous cells and tissues) around keloids are transplanted into a CD70 gene overexpressed NSG-MHC-DKO immune deficiency mouse body for the first time, so that HLA rejection possibly caused by different sources of immune cells and tissues is avoided; the internal microenvironment of the keloid and the interaction between the keloid and an immune system are restored to the greatest extent, and the influence of immune factors on the occurrence and development of the keloid can be researched in vitro. It is found and verified for the first time that activation of the CD27-CD70 axis can promote generation and development of keloids, and a suitable animal model is provided for research and development of anti-scar drugs, especially immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of keloid animal models, and in particular to a method for constructing a humanized keloid mouse model and application thereof. Background Art

[0002] The development of new treatments requires an ideal preclinical model. The formation of keloids in existing animal models relies on induction or implantation of human keloids. However, due to the many physiological differences between humans and animals, scar tissue induced in animals is usually hypertrophic scars rather than keloids, so the model of implanting human keloid cells or tissues into nude mice is more credible. However, as the importance of immune factors in the occurrence and development of keloids is gradually revealed, "humanized" mice with human immune cells that reconstruct the immune system are more suitable for in vivo models of keloids, which makes it possible to study the interaction between the systemic immune system and local factors.

[0003] The nude mouse keloid model provided in the prior art lacks immune system support. The transplanted humanized keloid generally shrinks in size within 1 month, making it impossible to effectively observe the interaction between the immune system and local keloids. At the same time, the window period of various experiments is short, making it impossible to effectively observe the efficacy and mechanism of anti-scar drugs; at the same time, it is impossible to observe the impact of immune factors on the occurrence and development of keloids. Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for constructing a humanized keloid mouse model and its application to solve the problems raised in the above-mentioned background technology. The present invention restores the internal microenvironment of keloid and its interaction with the immune system to the greatest extent, and for the first time proposes and verifies that the activation of the CD70-27 axis can significantly promote the occurrence and development of keloid, providing sufficient experimental time for studying the effects of immune factors on the occurrence and development of keloid, and providing a suitable animal model for the development of anti-scar drugs, especially immunotherapy.

[0005] In order to achieve the above object, the present invention is implemented by the following technical scheme: a method for constructing a humanized keloid mouse model, comprising the following steps:

[0006] Step 1: Select plndigoBAC536 as the BAC plasmid vector, insert the loxp site into the promoter region of the hCD70 gene, and connect the plasmid vector and the target gene containing the loxp site with DNA ligase;

[0007] Step 2: Use a needle puller to prepare an injection needle and a fixed needle. The fixed needle has an outer diameter of 100-120um and an inner diameter of 20-30um. The needle is bent at a 15° angle for standby use. Use the injection needle to inject the diluted BAC plasmid into the male pronucleus of the embryo until the pronucleus swells. Finally, transfer the needle to M16 culture medium for culture.

[0008] Step 3: Embryo transfer: Use a 1mL syringe needle to make an incision on the front side of the ampulla, and then blow the embryo into the ampulla of the fallopian tube through this incision;

[0009] Step 4: Determine the genotype of the overexpressing mice;

[0010] Step 5. Use absolute quantitative PCR to detect the copy number of F2 mice of each strain to obtain the copy number concentration of CD70 in transgenic mice; use relative quantitative PCR to detect the expression of CD70 gene, and Western Blot to detect CD70 protein expression.

[0011] Step 6: Take CD70 overexpressing transgenic NSG-MHC-DKO mice, and transplant the skin tissue around human keloids on the left and right sides of their backs under anesthesia;

[0012] Step 7: collecting peripheral blood from patients with keloid, separating homologous peripheral blood mononuclear cells (PBMC) with lymphocyte separation fluid, adding soluble CD27 (sCD27), and injecting the PBMC into mice through the tail vein after CD27 treatment;

[0013] Step 8. Most of the PBMCs that proliferate in vivo are T cells (CD3+ positive), and all mononuclear cells are CD45+ positive. Two weeks after cell infusion, the proportion of CD3+CD45+ double-stained cells in the mouse peripheral blood is regularly observed by flow cytometry. When the proportion of CD3+CD45+ double-stained cells is ≥15%, humanized immune reconstruction is considered successful.

[0014] Furthermore, the BAC plasmid strain in step 1 is inoculated into 100 mL of chloramphenicol-resistant LB culture medium, cultured with shaking at 32° C. overnight for 16-20 h, and the BAC plasmid is extracted using a plasmid extraction kit, digested with restriction endonucleases, and identified by pulsed-field gel electrophoresis.

[0015] Furthermore, the correctly identified BAC plasmid was dialyzed and purified overnight in a dialysate (0.5 mL of 1 mol / LTris-HCL, 10 uL of 0.5 mmol / L EDTA and 1 mL of 5 mol / L NaCl made up to 50 mL with sterile water), and the BAC plasmid solution was diluted to 1-3 ng / uL for injection.

[0016] Furthermore, in step 2, 5 U of PMSG was intraperitoneally injected into 4-week-old NSG-MHC-DKO female mice on the first day. After feeding for 47 hours, 5 U of HCG was intraperitoneally injected into each mouse and the mice were caged together with the breeding male mice overnight.

[0017] Furthermore, on the fourth day, fertilized eggs were collected from the oviduct dilatation for microinjection. The diluted BAC plasmid was injected into the male pronucleus of the embryo using an injection needle until the pronucleus was enlarged, and then the embryo was transferred to M16 culture medium for culture.

[0018] Further, in step 3, 0.5d pseudopregnant ICR recipient mice were taken, anesthetized, back hair was removed, alcohol and iodine tincture were used for disinfection, the skin on the dorsal side of the spine was cut open, the muscle layer 0.5cm on both sides of the spine was cut open, and the fat pad was clamped with blunt forceps to pull out the attached ovary and ampulla of the fallopian tube; and after the embryo was insufflated, the muscle layer and skin were sutured respectively, and iodine tincture was used for disinfection. After 18d, the pregnancy and delivery of the surrogate mice were observed, and the ears of the mice born on day 7 were pierced and marked, and the integration of the hCD70 exogenous gene was identified.

[0019] Furthermore, in step 4, the CD70 transgenic mice are mated with mice expressing Cre recombinase to activate the recombination event between loxp sites, thereby achieving CD70 gene overexpression in the offspring mice. Genetic analysis.

[0020] Furthermore, in step eight, the changes in peripheral blood CD27 expression are detected, and a keloid skin transplantation model without immune system reconstruction is constructed as a control group. The changes in scar skin thickness and volume between the two groups are observed and compared. At the end of the study, the scar skin tissue on the back of the mice is collected, and the expression levels of FN, COL1, COL3, and α-SMA in the experimental group and the control group are compared by qRT-PCR and Western Blot; paraffin tissue sections are collected, and HE staining is used to compare the collagen arrangement morphology of scar skin tissue between the two groups, Sirius red staining is used to compare the collagen content, and immunohistochemistry and immunofluorescence staining are used to detect the expression of CD70 and CD27.

[0021] Furthermore, tissue sections of mouse liver, spleen, lung, kidney and scar skin, HE staining, skin immunohistochemistry and immunofluorescence staining with human-specific antibodies verified the successful reconstruction of the humanized immune system.

[0022] An application of the above construction method is to construct an ideal animal model and also to study the influence of immune factors on the occurrence and development of keloid.

[0023] Beneficial effects of the present invention:

[0024] 1. The present invention discovered and verified for the first time that the activation of the CD27-CD70 axis can promote the occurrence and development of keloids, providing a good animal model for keloid immunotherapy and other target therapies.

[0025] 2. The present invention combines bacterial artificial chromosomes with the Cre-loxp system to construct transgenic mice with overexpression of CD70, and simultaneously transplants homologous skin around keloids and PBMC treated with sCD27, which can observe the occurrence and development of keloids, better preserve the biological characteristics of keloids, extend the experimental window period of the model, and facilitate observation of drug efficacy. The homology of skin and cells avoids HLA-related rejection between transplants and immune cells.

[0026] 3. The present invention found that activation of the CD27-CD70 axis can promote the occurrence and development of keloids, verified the effect of CD70 overexpression on promoting fibrosis, and provided a good animal model for anti-fibrosis treatment of keloids. This model breaks through the bottleneck of the traditional nude mouse transplantation model lacking an immune system and the process of keloid occurrence, and provides an ideal animal model for exploring the influence of immunity on the occurrence and development of keloids. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A: Basic structure of loxP site; B: Design diagram of loxP site in CD70 gene sequence;

[0028] Figure 2 Figure 1 shows the human keloid transplantation model, including: NSG-MHC-DKO (top), B2m (middle), and nude mice (bottom);

[0029] Figure 3 is the curve of keloid volume change;

[0030] Figure 4 This is the ratio of hCD3+CD45+ double positive cells in mouse peripheral blood detected by flow cytometry;

[0031] Figure 5 This is a graph showing the changing trend of the proportion of hCD3+CD45+ double positive cells in the periphery of humanized mice;

[0032] Figure 6 This is an ELISA data diagram showing that the peripheral blood of humanized mice contains human cytokines;

[0033] Figure 7 This is a picture of a mouse keloid.

[0034] Figure 8 This is a picture of the new blood vessels around the transplanted scar in dissected mice;

[0035] Fig. 9HE sections of humanized mouse lungs and livers infiltrated with a large number of mononuclear cells relative to immunodeficient mice;

[0036] Fig.10 A large number of hCD45+ cells infiltrated the liver / spleen of humanized mice;

[0037] Fig.11 hCD3+CD45+ cells infiltrated into keloids transplanted into humanized mice;

[0038] Fig.12 HC indicates that the transplanted keloids in humanized mice are infiltrated with hCD3+CD45+ cells and the capsule around the scars is highly infiltrated with hCD8+T cells;

[0039] Fig.13 The diagram shows the construction of BAC-CD70 and the loxP site insertion;

[0040] Fig.14 Technical roadmap for CD70 gene overexpression mice;

[0041] Fig.15 Flow chart of constructing a humanized keloid mouse model by reinfusing sCD27-treated hPBMCs into CD70-overexpressing mice. DETAILED DESCRIPTION

[0042] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0043] See also Figures 1 to 15 , the present invention provides the following technical solutions:

[0044] 1. Construction and preparation of BAC plasmid: The complete coding sequence of CD70 gene was obtained by PCR amplification, loxp site was designed in the promoter region of CD70 gene, a restriction endonuclease site HindIII was introduced in the upstream primer, another restriction endonuclease site SnlI was introduced in the downstream primer, pIndigoBAC536 was selected as BAC vector, and the CD70 gene containing loxp site was inserted into the BAC vector using restriction endonuclease and DNA ligase. The BAC CD70 cloning vector was separated by ion column chromatography, and the cloning vector was separated from the backbone by restriction endonuclease to obtain linear BAC DNA molecules. Polyamine (PA) buffer was prepared, the components of which were: 10mmol / L Tris-HCl (pH 7.5), 0.1mmol / L EDTA, 30μmol / L spermine, 70mmol / L spermidine, and 100mmol / L NaCl. Dissolve the linear BAC CD70 in PA buffer filtered through an inorganic filter with a pore size of 0.02 μm. Be careful to avoid vortexing and violent shaking during the operation. Store the resuspended BAC solution at 4°C. Before preparing for microinjection, the concentration of the BAC solution was measured and calibrated at an absorbance of 260 nm using a NanoDrop spectrophotometer (product of Thermo Corporation, USA). The integrity of BAC-CD70 was determined by pulsed field electrophoresis (product of Bio-Rad, USA, model CHEF-DR).

[0045] 2. Superovulation and fertilized egg collection of experimental mice: 3-4 week old female donor mice (NSG-MHC-DKO and B-NDG B2m KO) were purchased one week before the experiment. After being raised in the experimental animal breeding barrier system for one week, 10 U of pregnant mare serum gonadotropin (PMSG) was injected. 46-50 hours later, 10 U of human chorionic gonadotropin (hCG) was injected. After hCG injection, the female mice were caged with male mice of the same strain that were raised in a single cage. The ovulation status of the female mice was checked on the morning of the second day after caged. The same ovulation-inducing drug dosage and injection interval were used for the NSG-MHC-DKO and B-NDG B2m KO female donor mice. The oviducts of the donor mice with plugs were selected and euthanized. The oviducts on both sides were cut off and rinsed in M2 embryo culture medium (product of Sigma, USA, item number M7167). The oviducts were placed in a 15mg / mL M2 hyaluronidase droplet, and the ovum was punctured with microtweezers to release the fertilized egg mass. After about 3 minutes, the eggs that had digested the cumulus cells were transferred to the M2 microdroplet with a mouth pipette, and after multiple rinses, they were placed in a 37°C, 5% CO2 incubator for culture. There were two backgrounds of mice used to collect fertilized eggs for microinjection: 6-8 week-old NSG-MHC-DKO / B-NDG B2m KO mice were taken, the weight was weighed and recorded, the amount of anesthetic (sodium pentobarbital) was calculated according to the weight, and the corresponding drugs were extracted for intraperitoneal injection anesthesia.

[0046] 3. Pronuclear microinjection of fertilized eggs: The egg-holding needle used for pronuclear microinjection was homemade (outer diameter 120 μm, inner diameter 50 μm), and the injection needle was purchased commercial injection needle (product of Eppendorf Company, USA, model Ⅱ). The micromanipulation system workstation includes a fluorescent inverted microscope (product of Japan Olympus, model IX71), a micromanipulator arm and a microinjection pump (all products of the American Eppendorf Company). Before each experiment, the prepared BAC CD70 solution was poured into the microinjection needle through a micropipette tip (American Eppendorf Microloader TM product, 0.5-20μL), and then the egg holding needle and injection needle were fixed on the micromanipulator arm. The culture dish containing the fertilized eggs was placed under the microscope, the positions of the egg holding needle and the injection needle were adjusted, and the fertilized eggs with clear male and female pronuclei were selected, and BAC CD70 was injected into one of the pronuclei (the injection was stopped when the nuclear membrane was obviously expanded). The injected embryos were placed in M2 microdrops, transferred to a 37°C, 5% CO2 incubator, and transplanted 0.5 h later, or placed in KSOM (Millipore, USA, product number M-107-D) microdrops, transferred to a 37°C, 5% CO2 incubator, cultured overnight until they developed to the 2-cell stage on the second day and then transplanted.

[0047] 4. Embryo transplantation after injection: Weigh sodium pentobarbital powder, prepare 1% sodium pentobarbital anesthetic with sterile water, and after fully dissolving, filter with a 0.22μm filter (purchased from Millipore, USA) for later use. The recipient ICR with 0.5 days of embolism (ICR female mice are caged with ligated male mice, and the embolism is checked on the morning of the second day, which is recorded as 0.5 days of embolism) is anesthetized by intraperitoneal injection of 1% sodium pentobarbital at a dose of 40-50mg / kg, and then placed under a stereo microscope (Olympus) to cut off a small area of ​​hair on the back side of the body, and disinfect the skin with 75% ethanol solution. Then make a longitudinal incision at the position equivalent to the last rib on the back, carefully clamp the fat pad with small tweezers, pull out the ovary, fallopian tube and uterus, clamp the fat pad with a fat clamp, and fix the ovary and fallopian tube. Find the fimbria of the fallopian tube under the dissecting microscope, and use Venus scissors to cut a small opening on the ovarian capsule to avoid large blood vessels. Carefully suck out 25 to 28 fertilized eggs injected that day or embryos that have developed to 2 cells after injection with a transplant needle, insert it into the fimbria of one side of the fallopian tube, and blow the embryos in. Put the fat pad, ovary and fallopian tube back into the abdominal cavity together, and suture the muscle layer and skin separately.

[0048] 5. Genotype identification: When the pups are 7-10 days old, the toes are clipped and numbered. During the clipping process, the pups are anesthetized with isoflurane. DNA is extracted from the toe tissue. PCR is used to identify whether the target gene is integrated into the mouse genome. Immunofluorescence staining is used to verify the positive mice.

[0049] 6. Breeding and genetic analysis: After knocking the Cre gene into the specific gene regulatory element sequence of the BAC clone, a BAC cloning vector was constructed, and the BAC vector was injected into the fertilized egg pronuclear method to construct a BAC transgenic Cre mouse model. The Cre male mice were mated with CD70 transgenic female mice to activate the recombination event between the loxP sites, thereby achieving overexpression of the CD70 gene in the offspring mice. The genotype of the overexpressed mice was determined by genetic analysis, and the effect of overexpression was evaluated.

[0050] 7. Make an incision of about 0.5 cm on the left and right sides of the back of the CD70 overexpressing mouse, transplant 2 grains of human keloid skin (with epidermis, about 5 mm in diameter), suture the incision and fix the bite guard, and keep the mouse in a single cage after recovery.

[0051] 8. One week later, peripheral blood mononuclear cells (PBMCs) from keloid patients with the same origin as the transplanted tissue were collected and treated with soluble CD27 (sCD27, 25.0 ng / ml, ReliaTech) and cultured in a cell culture incubator for 6 h. After counting, PBMCs mixed with sCD27 were injected into CD70 gene overexpressing mice through the caudal vein with a syringe. Each mouse was infused with 5-7*10 6 PBMC cells (scars from the same mouse and PBMCs were from the same patient).

[0052] 9. Two weeks later, about 1 ml of blood was collected from each mouse using the mouse submandibular blood sampling method. After lysing the red blood cells, the blood was stained with CD3 and CD45 flow cytometry antibodies. The proportion of CD3+CD45+ double-stained cells in the mouse peripheral blood was observed by flow cytometry. When the proportion of double-stained cells was ≥15%, humanized immune reconstruction was considered successful.

[0053] 10. Blood samples were collected dynamically every 2 weeks, and the changes in the proportion of double positive cells (CD3+CD45+) were observed by flow cytometry.

[0054] 11. After 80 days of model observation, scar tissue, liver, spleen, lung, and kidney sections were obtained, and immunohistochemistry and immunofluorescence were used to verify the successful construction of the humanized immune system.

[0055] The basic principles and main features of the present invention and the advantages of the present invention are shown and described above. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention.

[0056] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for constructing a humanized keloid mouse model, characterized in that: The following steps are involved: Step 1: Select plndigoBAC536 as the BAC plasmid vector, insert the loxp site into the promoter region of the hCD70 gene, and connect the plasmid vector and the target gene containing the loxp site with DNA ligase; Step 2: Use a needle puller to prepare an injection needle and a fixed needle. The fixed needle has an outer diameter of 100-120um and an inner diameter of 20-30um. The needle is bent at a 15° angle for standby use. Use the injection needle to inject the diluted BAC plasmid into the male pronucleus of the embryo until the pronucleus swells. Finally, transfer the needle to M16 culture medium for culture. Step 3: Embryo transfer: Use a 1mL syringe needle to make an incision on the front side of the ampulla, and then blow the embryo into the ampulla of the fallopian tube through this incision; Step 4: Determine the genotype of the overexpressing mice; Step 5: Use absolute quantitative PCR to detect the copy number of F2 mice of each strain to obtain the copy number concentration of CD70 in transgenic mice; use relative quantitative PCR to detect the expression of CD70 gene, and use Western Blot to detect the expression of CD70 protein; Step 6: Take CD70 overexpressing transgenic NSG-MHC-DKO mice, and transplant the skin tissue around human keloids on the left and right sides of their backs under anesthesia; Step 7: collecting peripheral blood from patients with keloid, separating homologous peripheral blood mononuclear cells (PBMC) with lymphocyte separation fluid, adding soluble CD27 (sCD27), and injecting the PBMC into mice through the tail vein after CD27 treatment; Step 8. Most of the PBMCs that proliferate in vivo are T cells (CD3+ positive), and all mononuclear cells are CD45+ positive. Two weeks after cell infusion, the proportion of CD3+CD45+ double-stained cells in the mouse peripheral blood is regularly observed by flow cytometry. When the proportion of CD3+CD45+ double-stained cells is ≥15%, humanized immune reconstruction is considered successful.

2. The method for constructing a humanized keloid mouse model according to claim 1, characterized in that: In the step 1, the BAC plasmid strain is inoculated into 100 mL of chloramphenicol-resistant LB culture medium, cultured overnight at 32° C. with shaking for 16-20 hours, and the BAC plasmid is extracted using a plasmid extraction kit, cut with restriction endonucleases, and identified by pulsed-field gel electrophoresis.

3. The method for constructing a humanized keloid mouse model according to claim 2, characterized in that: The correctly identified BAC plasmid was dialyzed and purified overnight in a dialysate (0.5 mL of 1 mol / LTris-HCL, 10 uL of 0.5 mmol / L EDTA and 1 mL of 5 mol / L NaCl made up to 50 mL with sterile water), and the BAC plasmid solution was diluted to 1-3 ng / uL for injection.

4. The method for constructing a humanized keloid mouse model according to claim 3, characterized in that: In the step 2, 5 U of PMSG was intraperitoneally injected into 4-week-old NSG-MHC-DKO female mice on the first day. After feeding for 47 hours, 5 U of HCG was intraperitoneally injected into each mouse and the mice were caged together with male mice overnight.

5. The method for constructing a humanized keloid mouse model according to claim 4, characterized in that: On the fourth day, fertilized eggs were collected from the dilated part of the oviduct for microinjection. The diluted BAC plasmid was injected into the male pronucleus of the embryo using an injection needle until the inside of the pronucleus was seen to swell. Finally, the eggs were transferred to M16 culture medium for culture.

6. The method for constructing a humanized keloid mouse model according to claim 1, characterized in that: In the step 3, a 0.5-day pseudo-pregnant ICR recipient mouse was taken, anesthetized, the back hair was removed, and the mouse was disinfected with alcohol and iodine tincture. The skin on the dorsal side of the spine was cut open, the muscle layer 0.5 cm on both sides of the spine was cut open, and the fat pad was clamped with blunt-tipped forceps to pull out the attached ovary and ampulla of the fallopian tube; and after the embryo was insufflated, the muscle layer and skin were sutured respectively, and the mouse was disinfected with iodine tincture. After 18 days, the pregnancy and delivery of the surrogate mouse was observed, and the 7-day-old mouse was marked with ear piercing, and the integration of the hCD70 exogenous gene was identified.

7. The method for constructing a humanized keloid mouse model according to claim 1, characterized in that: In step 4, the CD70 transgenic mice are mated with mice expressing Cre recombinase to activate the recombination event between the loxp sites, thereby achieving overexpression of the CD70 gene in the offspring mice.

8. The method for constructing a humanized keloid mouse model according to claim 1, characterized in that: In the step eight, the changes in peripheral blood CD27 expression are detected, a keloid skin transplantation model without immune system reconstruction is constructed as a control group, and the changes in scar skin thickness and volume between the two groups are observed and compared. At the end of the study, the scar skin tissue on the back of the mice is collected, and the expression levels of FN, COL1, COL3, and α-SMA between the experimental group and the control group are compared by qRT-PCR and Western Blot; paraffin tissue sections are collected, HE staining is used to compare the collagen arrangement morphology of scar skin tissue between the two groups, Sirius red staining is used to compare the collagen content, and immunohistochemistry and immunofluorescence staining are used to detect the expression of CD70 and CD27.

9. The method for constructing a humanized keloid mouse model according to claim 8, characterized in that: Mouse liver, spleen, lung, kidney and scar skin tissue sections, HE staining, skin immunohistochemistry and immunofluorescence staining with human-specific antibodies verified the successful reconstruction of the humanized immune system.

10. An application of the construction method according to claim 1, characterized in that: This construction method is used to construct an ideal animal model and is also used in research on the impact of immune factors on the occurrence and development of keloids.

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