System and method for increasing hair follicle tissue in-vitro preparation by using autologous hair follicle preserving fluid rich in arginine

Abstract

The invention belongs to the technical field of biomedicine, and discloses a preparation system and method for increasing hair follicle tissue in vitro by using an autologous hair follicle preserving fluid rich in arginine. According to the invention, the differential low expression of arginine in androgenetic alopecia patients is reported for the first time; according to the invention, androgenetic alopecia is taken as a research object to clarify the effect of arginine in the attack of androgenetic alopecia and the effect of arginine on hair growth, and it is found that the hair follicle activity can be further preserved and the success rate of hair transplantation can be increased by improving the hair follicle preserving fluid by increasing the concentration of arginine in vitro. Therefore, a new thought is provided for in-vitro preservation of the hair follicle tissue in the hair transplantation operation.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and in particular relates to an in vitro preparation system and method for increasing hair follicle tissue by using an autologous hair follicle preservation solution rich in arginine. Background Art

[0002] Androgenic alopecia is a common type of alopecia, with a prevalence rate of up to 74.8%, which brings a heavy psychological burden to patients and seriously impairs their quality of life. It is currently believed that androgen-mediated miniaturization of hair follicles is its main pathogenesis. Currently, traditional treatments for patients with androgenic alopecia are limited, and hair transplantation is an important supplementary means for patients whose hair growth is ineffective or cannot be restored by therapeutic drugs. In addition, hair transplantation is also widely used in other scarring and non-scarring alopecia, and has a large market demand. At present, large-scale hair transplantation (more than 3,500 hairs) has been widely used in the treatment of androgenic alopecia. After the donor hair is removed from the body, its survival state has a great impact on the success rate of hair transplantation, so in vitro hair preservation is crucial. At present, in addition to cryopreservation, there is no clear definition of the preservation environment. Therefore, exploring the preservation environment of hair follicles in hair transplantation, that is, the "formula" of local hair preservation solution, has important scientific and clinical significance.

[0003] In recent years, studies have found that the metabolic level of patients with androgenic alopecia changes. Metabolic disorder is one of the key factors in the onset of androgenic alopecia, and the research on amino acid metabolism in androgenic alopecia is still very limited. At present, arginine is often promoted as having the effect of promoting hair growth, but the specific mechanism has not yet been elucidated. Therefore, studying the relationship and mechanism between arginine and hair follicle growth is of great significance to the improvement of hair follicle preservation and culture technology.

[0004] Through the above analysis, the problems and defects of the prior art are as follows:

[0005] With the increasing demand for large-scale hair transplantation, the time consumption of hair transplantation surgery has greatly increased. How to improve the survival rate of hair follicles in vitro is crucial to the benefits of hair transplantation surgery. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a method for preparing hair follicle tissue in vitro by using an autologous hair follicle preservation solution rich in arginine.

[0007] The present invention is achieved by: a method for preparing an autologous hair follicle preservation solution rich in arginine to increase hair follicle tissue in vitro, comprising:

[0008] Step 1: Use metabolomics analysis to determine the expression of arginine in healthy people and patients with androgenic alopecia;

[0009] Step 2, by isolating human occipital hair follicles and depriving them of arginine in a hair follicle culture system, measuring the growth length of the hair follicles, observing the growth morphology of the hair follicles, and statistically analyzing the growth indexes and apoptosis indexes of the hair follicles to clarify the effect of arginine on hair growth;

[0010] Step 3, by re-depriving the arginine-derived hair follicle culture system and supplementing different concentrations of arginine, the growth length of the hair follicles was measured, the growth morphology of the hair follicles was observed, and the effects of different concentrations of arginine on the growth of human hair follicles were explored;

[0011] Step 4, by co-culturing arginine precursor amino acids with human hair follicles, measuring the growth length of human hair follicles, observing the growth morphology of hair follicles, and clarifying the effect of arginine precursor amino acids on human hair follicles;

[0012] Step 5, construct a mouse skin organoid model and observe the effects of arginine on the survival and morphology of mouse skin organoids.

[0013] Furthermore, the arginine precursor amino acids are: citrulline and aspartic acid.

[0014] Another object of the present invention is to provide an in vitro preparation system for increasing hair follicle tissue by using autologous hair follicle preservation solution rich in arginine, comprising:

[0015] An analysis module is used to clarify the expression of arginine in healthy people and patients with androgenetic alopecia through metabolomics analysis;

[0016] The measurement module is used to separate the human occipital hair follicles and deprive them of arginine in the hair follicle culture system, measure the growth length of the hair follicles, observe the growth morphology of the hair follicles, and count the growth indexes and apoptosis indexes of the hair follicles to clarify the effect of arginine on hair growth; supplement different concentrations of arginine in the hair follicle culture system deprived of arginine again, measure the growth length of the hair follicles, observe the growth morphology of the hair follicles, and explore the effect of different concentrations of arginine on the growth of human hair follicles; co-culture arginine precursor amino acids with human hair follicles to measure the growth length of human hair follicles, observe the growth morphology of the hair follicles, and clarify the effect of arginine precursor amino acids on human hair follicles;

[0017] The building block is used to construct a mouse skin organoid model and observe the effects of arginine on the survival and morphology of mouse skin organoids.

[0018] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0019] First, the present invention reports for the first time that arginine is differentially underexpressed in patients with androgenic alopecia; the present invention uses androgenic alopecia as a research object to illustrate the role of arginine in the pathogenesis of androgenic alopecia and its role in hair growth. The present invention finds that by increasing the concentration of arginine in vitro to improve the hair follicle preservation solution, the activity of hair follicles can be further preserved, thereby improving the success rate of hair transplantation. Therefore, the present invention provides a new idea for the in vitro preservation of hair follicle tissue in hair transplantation surgery.

[0020] The expected benefits and commercial value after the transformation of the technical solution of the present invention are as follows: the incidence of androgenic alopecia is extremely high, and the age of onset is tending to be younger, and the market demand for hair transplantation is also increasing. With the maturity and commercialization of hair transplantation technology, the application of large-area hair transplantation is becoming more and more extensive. For large-area hair transplantation, the time of hair follicles being out of body will be correspondingly prolonged. The present invention aims to provide a hair follicle preservation solution with increased arginine to increase the activity and survival rate of hair follicles after being out of body, which is of great significance to improving the survival rate of hair transplantation. The present invention can provide mature hair follicle preservation solution for private hair transplantation institutions and public hair transplantation teams, and has great commercial value and market value.

[0021] The technical solution of the present invention fills the technical gap in the industry at home and abroad: the pathogenesis of androgenic alopecia is unclear, and the current mainstream theory believes that the onset of androgenic alopecia is mainly related to androgen sensitivity. Clinically, it can be found that androgenic alopecia is easily associated with metabolic-related diseases such as obesity and hyperinsulinemia. On the one hand, the present invention innovatively clarifies the mechanism of amino acid metabolic disorder of androgenic alopecia from the metabolic aspect for the first time; on the other hand, it utilizes the characteristics of local arginine deficiency in androgenic alopecia to provide a new hair follicle preservation solution that can improve the survival rate of hair follicles in vitro.

[0022] The technical solution of the present invention overcomes technical prejudice: in the current hair transplantation process, after the hair follicles are extracted, they are mainly stored in physiological saline at low temperature. The hair follicle preservation solution on the market mainly adds growth factors, while the solution of the present invention mainly targets the characteristics of local hair follicle amino acid metabolism disorders in patients with androgenic alopecia, and adds the missing arginine to the hair follicle preservation solution to improve the survival rate of hair follicles.

[0023] Second, the technical solution of the present invention solves several key problems in hair follicle preservation and regeneration in the prior art through the arginine-rich autologous hair follicle preservation solution, and has achieved significant technical progress. The following are the key technical problems and technical progress of the present invention in industrial applications:

[0024] 1. Solved the problem of decreased activity of hair follicles during in vitro preservation

[0025] Existing hair follicle preservation methods cannot effectively maintain the activity of hair follicles during hair transplantation and other hair follicle regeneration treatments. Hair follicles are easily affected by the environment after being removed from the body, which causes their activity to decrease, affecting the survival rate after transplantation. The present invention uses arginine-rich hair follicle preservation fluid to provide the metabolic environment required for hair follicle growth during the preservation period, maintain their metabolic activity, and reduce the apoptosis of hair follicle cells. This technological advancement greatly improves the survival rate and regeneration ability of hair follicle transplantation, and significantly optimizes the success rate of hair transplantation.

[0026] 2. Improved the effect of in vitro culture and regeneration of hair follicles

[0027] In the prior art, hair follicle regeneration treatment for patients with androgenic alopecia is difficult, especially because the regeneration capacity of hair follicles after ex vivo treatment is limited. The present invention enhances the growth potential of hair follicles and significantly improves the proliferation and growth effect of hair follicles by supplementing arginine and its precursor amino acids in the culture system. Through the study of the effect of arginine on hair follicle growth, the key regulatory factor of hair follicle growth is found. This breakthrough method provides a new solution for the regeneration treatment of alopecia patients and greatly improves the effect of existing treatment methods.

[0028] 3. Reduce apoptosis and damage of hair follicle cells

[0029] During the preservation and culture of hair follicles, the existing preservation solution or culture system cannot effectively prevent the apoptosis or damage of hair follicle cells, which affects the regeneration ability of hair follicles. The present invention determines the role of arginine in hair follicles through metabolomics analysis, and further introduces different concentrations of arginine into the hair follicle culture system to achieve precise regulation of hair follicle growth and cell apoptosis. This technology effectively reduces the damage of hair follicle cells during the preservation and culture process, and significantly improves the regeneration ability and stability of hair follicles.

[0030] 4. Create a more physiologically relevant hair follicle preservation and culture environment

[0031] The present invention constructs a mouse skin organoid model to verify the role of arginine in hair follicle regeneration, and further optimizes the hair follicle culture environment by introducing amino acid precursors. Compared with the prior art, the present invention not only supplements arginine in the preservation solution, but also takes into account the complex metabolic needs of hair follicle growth, simulating an environment closer to physiological conditions, thereby improving the effect of hair follicle preservation and regeneration.

[0032] 5. Significant technological progress

[0033] The technical solution of the present invention has achieved significant progress in many aspects:

[0034] Improved preservation efficiency: In hair transplantation and regeneration treatment, the preservation time of hair follicles in vitro is significantly prolonged, the damage to hair follicles caused by environmental influences is reduced, and the survival rate of hair follicle transplantation is improved.

[0035] Enhanced regeneration ability: Through the supplementation of arginine and the optimization of the culture system, the growth rate and regeneration ability of hair follicles are significantly improved, especially for patients with androgenic alopecia, providing a more efficient treatment plan.

[0036] Wide application: The present invention is not only applicable to hair transplantation surgery, but also can be used for hair follicle regeneration treatment, providing effective treatment options for more baldness patients.

[0037] In summary, the present invention has achieved significant technological progress in the field of hair follicle preservation and regeneration, overcomes key difficulties in the prior art, and has broad industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The present invention provides a flowchart of a method for preparing hair follicle tissue in vitro using an autologous hair follicle preservation solution rich in arginine.

[0039] Figure 2 This is a structural block diagram of a system for preparing hair follicle tissue in vitro using an autologous hair follicle preservation solution rich in arginine provided by an embodiment of the present invention.

[0040] Figure 3 It is a workflow diagram provided by an embodiment of the present invention.

[0041] Figure 4 A principal component analysis of metabolomics of androgenic alopecia patients and healthy control patients provided by the embodiments of the present invention; B differential metabolites significantly upregulated / downregulated in the serum of androgenic alopecia patients; C Venn diagram obtained by the intersection of one-dimensional and multidimensional analysis results; D relative abundance analysis of metabolites in samples; E heat map of metabolites in samples.

[0042] Figure 5 A is the serum arginine level of a healthy control group and androgenic alopecia patients provided by the embodiments of the present invention; B is the total growth length (mm) of hair follicles after 6 days of arginine treatment (including deprivation of arginine and supplementation of arginine at different concentrations); D is the gross morphology of hair follicles after arginine treatment (including deprivation of arginine and supplementation of arginine at a specific concentration); E is the HE staining result of hair follicles after arginine deprivation treatment.

[0043] Figure 6 A is the immunostaining of the proliferation indicator Ki67 and the percentage of Ki67-positive cells in the hair follicles after arginine deprivation provided by the embodiments of the present invention; B is the immunostaining of the proliferation indicator PHH3 and the number of PHH3-positive cells in the hair follicles after arginine deprivation; C is the immunostaining of the apoptosis indicator Tunel and the number of Tunel-positive cells in the hair follicles after arginine deprivation.

[0044] Figure 7A is the morphological characteristics of mouse skin organoids after arginine treatment (including arginine deprivation and supplementation of a specific concentration of arginine) provided in an embodiment of the present invention; B is the HE staining result of mouse skin organoids after arginine treatment (including arginine deprivation and supplementation of a specific concentration of arginine); C is the K14, KI67, and VEMINTIN immunofluorescence staining image of mouse skin organoids after arginine deprivation.

[0045] Figure 8 A is the total growth length (mm) of hair follicles after 6 days of supplementation with aspartic acid and citrulline after arginine deprivation; B is the general morphology of hair follicles supplemented with aspartic acid and citrulline after arginine deprivation provided by the embodiments of the present invention. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0047] The method for preparing hair follicle tissue in vitro by using an autologous hair follicle preservation solution rich in arginine provided in the embodiments of the present invention combines metabolomics analysis, in vitro culture of hair follicles and construction of a mouse skin organoid model. The following is a detailed description and arrangement of each step of the method.

[0048] Step 1: Metabolomics analysis of arginine expression in healthy subjects and AGA patients

[0049] 1.1. Selection and inclusion of study subjects

[0050] The study included patients with androgenetic alopecia (AGA) and healthy controls, ensuring that the two groups were consistent in population attributes (age, gender, BMI, etc.) and screened through strict inclusion and exclusion criteria. AGA patients were diagnosed by dermatologists, and other influencing factors such as history of drug use and other types of hair loss were excluded. The healthy control population was required to have no history of long-term medication and systemic diseases to ensure that the differences in serum metabolites were attributed to AGA.

[0051] 1.2. Collection of peripheral blood

[0052] After the subjects signed the informed consent form, 5 mL of peripheral venous blood was collected and processed and stored according to standard procedures. Before blood collection, it was ensured that the subjects had not drunk alcohol or exercised strenuously within 24 hours. The samples were stored at 4°C in the short term and -80°C in the long term.

[0053] 1.3. Identification and screening of differential metabolites

[0054] Circulating serum of AGA patients and healthy controls was analyzed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) to screen out metabolites with significant differences. The analysis included multivariate statistical analysis such as principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), orthogonal partial least squares discriminant analysis (OPLS-DA), and univariate analysis such as t-test and Mann-Whitney-Wilcoxon analysis. The volcano plot threshold (p<0.05, Log2FC>0) was set to screen significant metabolites.

[0055] Step 2: Arginine-deprived hair follicle culture

[0056] By isolating human occipital hair follicles and observing the growth length, morphology and growth indicators of hair follicles in a hair follicle culture system deprived of arginine, the effect of arginine on hair follicle growth was clarified.

[0057] 1.4. In vitro hair follicle culture and growth index statistics

[0058] Hair follicles were obtained from the occipital region of AGA patients, separated and placed in 24-well plates for culture. After the hair follicles were balanced overnight in serum-free William'E medium, photos were taken regularly (0d, 2d, 4d, 6d) to record the growth length, morphological changes and hair cycle of the hair follicles. ImageJ software was used to count the length and morphological changes of the hair follicles.

[0059] 1.5. Arginine deprivation and supplementation

[0060] Arginine was supplemented in the arginine-deprived culture medium, and different concentrations of culture medium were prepared using L-arginine mother solution, and the pH was adjusted to 7.2-7.4. The growth changes of hair follicles after supplementation with different concentrations of arginine were observed to clarify its effect on hair follicle growth.

[0061] Step 3: Supplement hair follicle culture with different concentrations of arginine

[0062] By gradually supplementing different concentrations of arginine after arginine deprivation, the growth length and growth index of hair follicles were measured. Through microscopic observation and quantitative analysis, the effects of different concentrations of arginine on hair follicle growth were explored to determine the optimal concentration.

[0063] Step 4: Role of Arginine Precursor Amino Acids

[0064] By supplementing arginine precursor amino acids, culturing human hair follicles, measuring the growth length of hair follicles, observing the morphology of hair follicles, and further analyzing the effects of precursor amino acids on hair follicle growth.

[0065] 1.6. Human hair follicle cryosection and biomarker detection

[0066] Cell proliferation and apoptosis indicators were detected by frozen sections of hair follicles to clarify the regulatory effect of arginine on hair follicle cell activity.

[0067] Proliferation indicators: Ki67 and PHH3 immunofluorescence staining was used to detect the proliferation of hair follicle cells, and the positive cells were counted using ImageJ software.

[0068] Apoptosis indicators: TUNEL apoptosis cell detection kit was used to detect apoptotic cells, and cell apoptosis was observed under a fluorescence microscope.

[0069] 1.7. Hair follicle RNA extraction and RNA-Seq

[0070] RNA was extracted from hair follicles after arginine deprivation for RNA-Seq analysis to ensure RNA quality (OD260 / 280 value between 1.8-2.0) and detect changes in gene expression.

[0071] Step 5: Construction of mouse skin organoid model

[0072] By constructing a mouse skin organoid model, the effects of arginine on hair follicle growth and skin tissue survival were further verified.

[0073] 1.8. Mouse Skin Organoid Culture

[0074] Newborn mice within 24 hours were selected, epidermal and dermal cells were isolated, and inoculated into transwell chambers in 12-well plates for culture. The survival of organoids was observed after 1-2 days of culture, and the effects of arginine on the morphology and function of organoids were analyzed.

[0075] Through a series of detailed experimental steps, the present invention systematically studies the effects of arginine and its precursor amino acids on hair follicle growth and regeneration, from cell proliferation and apoptosis to the regulation of gene expression and overall hair follicle function. Combining in vitro hair follicle culture and mouse skin organoid model, a new method for effectively improving hair follicle activity and preservation is provided.

[0076] Example 1: Preservation and enhancement of hair follicle activity during hair transplantation

[0077] In hair transplantation surgery, the in vitro preservation of hair follicles is a key step. During the in vitro preservation period, hair follicles are susceptible to environmental stress, resulting in a decrease in growth ability. The arginine-rich preservation solution of the present invention significantly increases the survival rate and growth ability of hair follicles by increasing the metabolic activity of hair follicles. In specific applications, the arginine preservation solution can be used before and after surgery, and the extracted hair follicles are soaked in the preservation solution to maintain the healthy state of the hair follicles during transplantation. Through the role of arginine in hair follicle growth, it reduces the apoptosis rate of hair follicles, improves the transplantation effect, and ultimately improves the success rate of surgery and patient satisfaction.

[0078] Example 2: Hair follicle regeneration treatment for patients with androgenic alopecia

[0079] For patients with androgenic alopecia, arginine-rich hair follicle preservation solution can be used for hair follicle regeneration treatment. Through the hair follicle culture system provided by the present invention, the hair follicle tissue of the patient can be used clinically to supplement and culture arginine in vitro to promote hair follicle growth and reactivate the hair follicles. After a certain period of in vitro culture, the active hair follicles are re-transplanted to the bald area of ​​the patient to achieve hair regeneration treatment. This method not only improves the regeneration ability of hair follicles, but also provides new ideas and approaches for treating alopecia, which helps to reduce the dependence on drug treatment.

[0080] like Figure 1 As shown, the embodiment of the present invention provides an in vitro preparation method of increasing hair follicle tissue by using an autologous hair follicle preservation solution rich in arginine, comprising the following steps:

[0081] S101, to clarify the expression of arginine in healthy people and patients with androgenetic alopecia through metabolomics analysis;

[0082] S102, by isolating human occipital hair follicles and depriving them of arginine in a hair follicle culture system, measuring the growth length of hair follicles, observing the growth morphology of hair follicles, and statistically analyzing the growth indexes and apoptosis indexes of hair follicles, to clarify the effect of arginine on hair growth;

[0083] S103, by re-depriving the arginine-deprived hair follicle culture system, adding different concentrations of arginine, measuring the growth length of hair follicles, observing the growth morphology of hair follicles, and exploring the effects of different concentrations of arginine on the growth of human hair follicles;

[0084] S104, by co-culturing arginine precursor amino acids with human hair follicles, measuring the growth length of human hair follicles, observing the growth morphology of hair follicles, and clarifying the effect of arginine precursor amino acids on human hair follicles;

[0085] S105, construct a mouse skin organoid model and observe the effects of arginine on the survival and morphology of mouse skin organoids.

[0086] The arginine precursor amino acids provided in the embodiment of the present invention are: citrulline and aspartic acid.

[0087] The present invention provides a method for preparing an autologous hair follicle preservation solution rich in arginine. Through systematic experiments and analysis, the effect of arginine on the growth and survival of hair follicles is clarified, providing a theoretical basis and technical support for improving the success rate of hair follicle transplantation.

[0088] First, in step 1, metabolomics analysis was used to study the expression of arginine in healthy people and patients with androgenic alopecia. Metabolomics analysis can comprehensively reveal the metabolic differences of arginine in different groups. The results showed that the arginine level in healthy people was significantly higher than that in patients with androgenic alopecia, indicating that arginine plays an important role in hair follicle growth and hair health.

[0089] Next, step 2 further explored the specific effect of arginine on hair follicle growth through experiments. The researchers isolated hair follicles from the human occipital region and deprived them of arginine in the hair follicle culture system. By measuring the length of hair follicle growth, observing the growth morphology of hair follicles, and counting the growth and apoptosis indicators of hair follicles, they found that the lack of arginine significantly inhibited the growth of hair follicles and increased the apoptosis rate of hair follicles, further confirming the key role of arginine in promoting hair follicle growth.

[0090] In step 3, the effects of different concentrations of arginine on hair follicle growth were studied by re-supplementing different concentrations of arginine to the arginine-deprived hair follicle culture system. The results showed that appropriate arginine supplementation significantly promoted the growth and survival of hair follicles, but too high or too low concentrations had adverse effects on hair follicles, indicating that precise control of arginine concentration is crucial to the health of hair follicles.

[0091] Step 4 further explored the effect of arginine precursor amino acids on hair follicles. In the experiment, arginine precursor amino acids were co-cultured with human hair follicles, the growth length of the hair follicles was measured, and their growth morphology was observed. The study found that arginine precursor amino acids can be converted into arginine and promote hair follicle growth, which provides a theoretical basis for the development of autologous hair follicle preservation fluid rich in arginine.

[0092] In step 5, the researchers constructed a mouse skin organoid model to further verify the effects of arginine on hair follicles and skin tissue. By observing the effects of arginine on the survival rate and morphological changes of mouse skin organoids, they found that arginine significantly increased the survival rate of skin organoids and promoted the growth of hair follicles, which laid the foundation for further research on the application of arginine in hair transplantation.

[0093] In summary, the present invention has clarified the key role of arginine in promoting hair follicle growth through a series of experiments, and proposed a method for preparing an arginine-rich autologous hair follicle preservation solution using arginine and its precursor amino acids, which provides a new technical means for hair transplantation and is expected to improve the success rate of hair follicle transplantation and the effect of hair regeneration.

[0094] like Figure 2 As shown, an embodiment of the present invention provides an arginine-rich autologous hair follicle preservation solution to increase the in vitro preparation system of hair follicle tissue, comprising:

[0095] An analysis module is used to clarify the expression of arginine in healthy people and patients with androgenetic alopecia through metabolomics analysis;

[0096] The measurement module is used to separate the human occipital hair follicles and deprive them of arginine in the hair follicle culture system, measure the growth length of the hair follicles, observe the growth morphology of the hair follicles, and count the growth indexes and apoptosis indexes of the hair follicles to clarify the effect of arginine on hair growth; supplement different concentrations of arginine in the hair follicle culture system deprived of arginine again, measure the growth length of the hair follicles, observe the growth morphology of the hair follicles, and explore the effect of different concentrations of arginine on the growth of human hair follicles; co-culture arginine precursor amino acids with human hair follicles to measure the growth length of human hair follicles, observe the growth morphology of the hair follicles, and clarify the effect of arginine precursor amino acids on human hair follicles;

[0097] The building block is used to construct a mouse skin organoid model and observe the effects of arginine on the survival and morphology of mouse skin organoids.

[0098] The purpose of the present invention is to provide an application of arginine as an important component in a local hair preservation solution in hair transplantation, to supplement arginine to improve the survival rate of hair follicles in vitro for hair transplantation, so as to solve the problems raised in the above background technology.

[0099] To achieve the above object, the present invention provides the following technical solutions:

[0100] The application of arginine in hair follicle preservation in hair transplantation is firstly to clarify the expression of arginine in healthy people and patients with androgenic alopecia through metabolomics analysis;

[0101] By isolating human occipital hair follicles and depriving them of arginine in a hair follicle culture system, the length of hair follicle growth was measured, the growth morphology of hair follicles was observed, and the growth and apoptosis indicators of hair follicles were counted to clarify the effect of arginine on hair growth.

[0102] By re-depriving the arginine-deficient hair follicle culture system and supplementing different concentrations of arginine, the growth length of the hair follicles was measured, the growth morphology of the hair follicles was observed, and the effects of different concentrations of arginine on the growth of human hair follicles were explored.

[0103] By co-culturing arginine precursor amino acids (citrulline and aspartic acid) with human hair follicles, measuring the growth length of human hair follicles, observing the growth morphology of hair follicles, and clarifying the effects of arginine precursor amino acids on human hair follicles;

[0104] Finally, a mouse skin organoid model was constructed to observe the effects of arginine on the survival and morphology of mouse skin organoids.

[0105] The present invention collects serum from healthy control group and androgenic alopecia patients, detects the difference in the content of arginine in the serum between the two groups, and finds that there is a significant difference in amino acid metabolism between androgenic alopecia patients and the healthy control group, and the content of arginine in the serum of androgenic alopecia patients is significantly lower than that of the healthy control group (e.g. Figure 5 A), results indicate that arginine metabolism differs between patients with androgenetic alopecia and healthy controls.

[0106] The present invention obtains hair follicles from the occipital scalp of a patient undergoing hair transplant surgery, divides the hair follicle units into separate complete hair follicles under a microscope, deprives the patient of arginine in a culture system, and counts the growth lengths of the hair follicles at 0 days, 2 days, 4 days, and 6 days. The results show that the growth of the hair follicles is inhibited, as shown by a decrease in the growth length of the hair follicles, a thinning of the diameter of the newly formed hair shafts, and other manifestations similar to those of the catagen period (such as Figure 5 BD), HE staining results showed that as time went on, the diameter of the newly formed hair shaft of the arginine-deprived follicles became thinner, the hair matrix and DP gradually separated from the black hair shaft at the upper part of the follicle, and the newly formed hair shaft lost its pigment. The results showed that the growth of the hair follicle organ was significantly delayed after arginine stripping (such as Figure 5 E).

[0107] The present invention supplemented different concentrations of arginine in the arginine-deprived hair follicle culture system, and found that the phenotype of hair follicle growth inhibition was reversed. The results showed that the effect was arginine concentration-dependent (e.g. Figure 5 BD).

[0108] The present invention co-cultures arginine precursor amino acids (citrulline and aspartic acid) with human hair follicles, measures the length of human hair follicle growth, and observes the growth morphology of hair follicles to further clarify the effects of arginine precursor amino acids on human hair follicles. The results show that supplementation with aspartic acid and citrulline can increase the length of hair follicle growth and reverse the phenotype of hair follicle growth inhibition (such as Figure 6 A, B), Results indicate that arginine precursor amino acids favor hair follicle growth in in vitro human hair follicle organ culture.

[0109] The present invention performs frozen sectioning and immunofluorescence staining of proliferation indexes (KI67, pHH3) and apoptosis index (TUNEL) on hair follicles deprived of arginine in a hair follicle culture system at time points (6h, 12h, 1d, 2d, 4d) before the phenotype appears. The results show that when arginine is deprived for 2d and 4d, KI67 around the hair papilla cells decreases with statistical significance, and the degree of decrease in KI67 level increases with time (e.g. Figure 7 A), when deprived of arginine for 2 days and 4 days, pHH3 around dermal papilla cells decreased statistically (e.g. Figure 7B), when depriving of arginine for 1 h, 3 h, 6 h, 12 h, 24 h, 2 d, and 4 d, there was no statistically significant change in TUNEL levels (e.g. Figure 7 C, Figure 8 ), the results showed that arginine deprivation affects hair follicle growth by inhibiting hair matrix cells rather than promoting cell apoptosis.

[0110] Similarly, the present invention performs frozen sectioning and immunofluorescence staining on hair follicles that have been re-supplemented with arginine in a hair follicle culture system deprived of arginine.

[0111] The proliferation index (KI67) and apoptosis index (TUNEL) showed that when arginine was deprived for 2 days and 4 days, KI67 around the hair papilla cells had no statistically significant change compared with the healthy control group, but increased significantly compared with the arginine deprivation group (such as Figure 7 A).

[0112] The above results show that arginine can significantly affect the growth and morphology of hair follicles in vitro. Improving the hair follicle preservation solution by increasing the arginine concentration in vitro is more conducive to preserving the activity of hair follicles and improving the survival rate of hair transplantation.

[0113] See also Figure 3-Figure 6 , an embodiment provided by the present invention: application of arginine as a target in AGA;

[0114] Example 1, the present invention collects serum from healthy control group and androgenic alopecia patient, detects the difference in the content of arginine in the serum between the two groups, and finds that there is a significant difference in amino acid metabolism between androgenic alopecia patients and the healthy control group, and the content of arginine in the serum of androgenic alopecia patients is significantly lower than that of the healthy control group (such as Figure 5 A), results indicate that arginine metabolism differs between patients with androgenetic alopecia and healthy controls.

[0115] In Example 2, the present invention takes scalp hair follicles from the occipital part of a patient undergoing hair transplant surgery, divides the hair follicle units into separate complete hair follicles under a microscope, deprives the patient of arginine in a culture system, and counts the growth lengths of the hair follicles at 0 days, 2 days, 4 days, and 6 days. The results show that the growth of the hair follicles is inhibited, as shown by a decrease in the length of the hair follicles, a thinning of the diameter of the newly formed hair shafts, and other manifestations similar to those of the catagen period (e.g. Figure 5 BD), HE staining results showed that as time went on, the diameter of the newly formed hair shaft of the arginine-deprived follicles became thinner, the hair matrix and DP gradually separated from the black hair shaft at the upper part of the follicle, and the newly formed hair shaft lost its pigment. The results showed that the growth of the hair follicle organ was significantly delayed after arginine stripping (such as Figure 5 E).

[0116] Example 3: In the present invention, different concentrations of arginine were supplemented in the arginine-deprived hair follicle culture system, and the results showed that the phenotype of hair follicle growth inhibition was reversed. The results showed that the effect was arginine concentration-dependent (e.g. Figure 5 BD).

[0117] Example 4, the present invention co-cultures arginine precursor amino acids (citrulline and aspartic acid) with human hair follicles, measures the length of human hair follicle growth, and observes the growth morphology of hair follicles to further clarify the effects of arginine precursor amino acids on human hair follicles. The results show that supplementation with aspartic acid and citrulline can increase the length of hair follicle growth and reverse the phenotype of hair follicle growth inhibition (such as Figure 6 A, B), Results indicate that arginine precursor amino acids favor hair follicle growth in in vitro human hair follicle organ culture.

[0118] Example 5, the present invention performed frozen sectioning and immunofluorescence staining of proliferation indexes (KI67, pHH3) and apoptosis indexes (TUNEL) on the hair follicles deprived of arginine in the hair follicle culture system at time points before the phenotype appeared (6h, 12h, 1d, 2d, 4d). The results showed that when arginine was deprived for 2d and 4d, KI67 around the hair papilla cells showed a statistically significant decrease, and the degree of decrease in KI67 levels increased with time (such as Figure 7 A), when deprived of arginine for 2 days and 4 days, pHH3 around dermal papilla cells decreased statistically (e.g. Figure 7 B), when depriving of arginine for 1 h, 3 h, 6 h, 12 h, 24 h, 2 d, and 4 d, there was no statistically significant change in TUNEL levels (e.g. Figure 7 C, Figure 8 ), the results showed that arginine deprivation affects hair follicle growth by inhibiting hair matrix cells rather than promoting cell apoptosis. Similarly, the present invention performed frozen sectioning and immunofluorescence staining of proliferation index (KI67) and apoptosis index (TUNEL) on hair follicles that were re-supplemented with arginine in the arginine-deprived hair follicle culture system. The results showed that when arginine was deprived for 2d and 4d, the KI67 around the hair papilla cells had no statistically significant change compared with the healthy control group, but had a statistically significant increase compared with the arginine deprivation group (such as Figure 7 A).

[0119] It is obvious to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be regarded as exemplary and non-limiting in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes within the meaning and scope of the equivalent elements of the claims be included in the present invention. Any reference numeral in a claim should not be regarded as limiting the claim to which it relates.

[0120] The invention is mainly used in hair transplantation surgery and improves the survival rate of hair follicles by providing a hair follicle preservation solution rich in arginine.

[0121] The incidence of androgenic alopecia is extremely high, and the age of onset is tending to be younger. The demand for hair transplantation market is also increasing. With the maturity and commercialization of hair transplantation technology, the application of large-area hair transplantation is becoming more and more extensive. For large-area hair transplantation, the time of hair follicles out of body will be correspondingly prolonged. The present invention aims to provide a hair follicle preservation solution with increased arginine to improve the activity and survival rate of hair follicles after ex vivo treatment, which is of great significance to improving the survival rate of hair transplantation. The present invention can provide mature hair follicle preservation solution for private hair transplantation institutions and public hair transplantation teams, and has great commercial value and market value.

[0122] The pathogenesis of androgenic alopecia is unclear, and the current mainstream theory believes that the onset of androgenic alopecia is mainly related to androgen sensitivity. Clinically, it can be found that androgenic alopecia is easily associated with metabolic-related diseases such as obesity and hyperinsulinemia. On the one hand, the present invention innovatively clarifies the mechanism of amino acid metabolism disorder of androgenic alopecia from the metabolic aspect for the first time; on the other hand, it uses the characteristics of local arginine deficiency in androgenic alopecia to provide a new hair follicle preservation solution that can improve the survival rate of hair follicles in vitro. In the current hair transplantation process, after the hair follicles are extracted, they are mainly stored in physiological saline at low temperature. The hair follicle preservation solution on the market is mainly based on the addition of growth factors, while the present invention's solution mainly targets the characteristics of local hair follicle amino acid metabolism disorder in patients with androgenic alopecia, and adds the missing arginine to the hair follicle preservation solution to improve the survival rate of hair follicles.

[0123] First, at the tissue and organ level, arginine deprivation significantly inhibited hair follicle growth, while the addition of 0.5 mM arginine effectively restored and promoted hair follicle growth. Immunofluorescence staining results showed that after the addition of arginine, Ki67 expression in hair follicle keratinocytes increased, indicating that arginine can promote the proliferation of hair follicle keratinocytes, thereby promoting hair follicle growth.

[0124] Secondly, at the surgical level, arginine is added to the hair follicle preservation fluid during the hair transplant process and used for hair transplantation to recede the hairline and increase the density of hair on the top of the head. It can be seen that the hair follicle survival rate is high and the hair transplantation effect is good.

[0125] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. An arginine-rich system for hair follicle preservation and regeneration treatment, characterized in that: The system comprises: Metabolomics analysis module, used to analyze the arginine expression levels in healthy people and patients with androgenic alopecia (AGA), screen out related metabolites and perform differential analysis; The hair follicle separation module is used to separate the patient's occipital hair follicles and deprive them of arginine through an in vitro culture system to count the hair follicle growth length, apoptosis index and growth morphology; The arginine supplementation module is used to supplement different concentrations of arginine in the arginine-deprived culture system, observe the growth morphology and indicators of hair follicles, and explore its effects on hair follicle growth.

2. The arginine enrichment system according to claim 1, characterized in that The system also includes a data processing and statistics module for performing multivariate statistical analysis such as principal component analysis, partial least squares discriminant analysis (PLS-DA), orthogonal partial least squares discriminant analysis (OPLS-DA) on the metabolite data obtained from the metabolomics analysis, and screening for arginine metabolites related to hair growth; The system further includes a hair follicle culture system, which deprives arginine through serum-free culture medium, adds L-arginine mother solution into the culture medium, and supplements arginine at different concentrations to simulate the hair follicle growth environment to detect the effect of arginine on hair follicle growth.

3. The arginine enrichment system according to claim 1, characterized in that The system includes a hair follicle proliferation and apoptosis detection module, which is used to detect proliferation indicators (such as Ki67, PHH3) and apoptosis indicators (such as TUNEL) in hair follicles by immunofluorescence staining, and to perform statistical results in combination with fluorescence microscope photography and data analysis software; the system also includes an RNA extraction and RNA-Seq analysis module, which is used to extract RNA from hair follicles, perform gene expression analysis through RNA-Seq technology, and clarify the effect of arginine deprivation on hair follicle gene expression.

4. The arginine enrichment system according to claim 1, characterized in that The system includes a mouse skin organoid model construction module, which is used to separate epidermal and dermal cells of newborn mice by trypsin and collagenase digestion, and mix and inoculate them in a transwell chamber to culture the organoid model to observe the effect of arginine on the survival and morphology of mouse skin tissue.

5. A method for preparing hair follicle tissue in vitro by using an autologous hair follicle preservation solution rich in arginine, characterized in that: The following steps are involved: Step 1: Use metabolomics analysis to determine the expression of arginine in healthy people and patients with androgenic alopecia; Step 2, isolating human occipital hair follicles and depriving them of arginine in a hair follicle culture system, measuring the growth length of the hair follicles, observing the growth morphology of the hair follicles, and counting the growth indexes and apoptosis indexes of the hair follicles; Step 3, re-supplementing the arginine-deprived hair follicle culture system with different concentrations of arginine, measuring the growth length of the hair follicles, observing the growth morphology of the hair follicles, and exploring the effects of different concentrations of arginine on the growth of human hair follicles; Step 4, co-culturing the arginine precursor amino acid and human hair follicles, measuring the growth length of the hair follicles, observing the growth morphology of the hair follicles, and clarifying the effect of the arginine precursor amino acid on the growth of hair follicles.

6. The method for preparing hair follicle tissue in vitro by using an autologous hair follicle preservation solution rich in arginine according to claim 1, characterized in that: In step 1, patients with androgenetic alopecia (AGA) and sex- and age-matched healthy controls were included, and differential metabolites in circulating serum were detected by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), and multivariate and univariate statistical analyses were used to screen arginine-related metabolites.

7. The method for preparing hair follicle tissue in vitro by using an autologous hair follicle preservation solution rich in arginine according to claim 1, characterized in that: In step 2, the in vitro hair follicle culture was performed by using serum-free William'E medium, adding L-glutamine, bovine insulin, and hydrocortisone, and observing the growth morphology and index changes of the hair follicles after deprivation and supplementation of arginine.

8. The method for preparing hair follicle tissue in vitro by using an autologous hair follicle preservation solution rich in arginine according to claim 1, characterized in that: In step 5, a mouse skin organoid model was constructed, and the epidermal and dermal cells of newborn mice were separated by collagenase digestion and inoculated into a transwell chamber to observe the effects of arginine on the survival and morphological changes of mouse skin organoids.

9. The method for preparing hair follicle tissue in vitro by using an autologous hair follicle preservation solution rich in arginine as claimed in claim 1, characterized in that: The arginine precursor amino acids are: citrulline and aspartic acid.

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