CN113425741A - Preparation for treating dyschromatosis diseases and preparation method and application thereof - Google Patents
Preparation for treating dyschromatosis diseases and preparation method and application thereof Download PDFInfo
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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Abstract
The invention discloses a preparation for treating dyschromatosis diseases, and a preparation method and application thereof. The preparation is injected into the pigment disorder area to perform a series of evaluations of histopathological staining, immunohistochemical analysis, Fontana-Masson method melanin granule staining, immunohistochemical SP method detection inducible nitric oxide synthase content and the like by making an animal model of the guinea pig pigment disorder disease, and the preparation proves that the preparation can inhibit the pigment disorder, has reliable treatment effect and provides a new breakthrough for treating the pigment disorder disease.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a preparation for treating dyschromatosis and a preparation method and application thereof.
Background
The externally applied medicine for treating dyschromatosis is mainly used for inhibiting the proliferation and activity of melanocytes, inhibiting the formation and transportation of melanin granules, or destroying pigment granules so as to accelerate the metabolism and degradation of the melanin granules. However, the drug therapy has slow effect and poor curative effect, and particularly, the drug therapy has many adverse reactions, such as skin irritation, allergy and the like, and even may cause new pigment disorders.
The Q-switched laser technology with selective photothermal effect can be used for reducing the thermal injury to normal skin and improving the safety and the effectiveness of treatment when being used for matching laser treatment for some stubborn and intractable. However, the laser technology is a 'double-edged sword', and when the laser technology is used for treating the skin diseases with dyschromatosis, the Q-switched laser is particularly easy to cause new chromatism (commonly called 'spot reaction of laser') for Asians under the condition of conventional treatment energy or higher energy.
At present, the main active ingredients of most cosmetics or whitening agents are tyrosinase inhibitors, but the tyrosinase inhibitors have certain toxic and side effects and may cause certain influence on the health of human bodies. Therefore, research and search for a preparation for treating the pigment obstacle with lower toxicity and better whitening effect are the development trend in the field of treating the pigment obstacle diseases.
Disclosure of Invention
In order to make up for the defects of the prior art, the invention provides a preparation for treating the dyschromatosis disease, a preparation method and application thereof.
The technical problem of the invention is solved by the following technical scheme:
a preparation for treating dyschromatosis disease contains platelet rich plasma and adipose tissue derived mesenchymal stem cells as active ingredients.
Preferably, the formulation is an injectable formulation.
Preferably, the platelet rich plasma is platelet rich plasma activated by heparin; the adipose tissue-derived mesenchymal stem cells exist in a suspension consisting of normal saline and adipose tissue-derived mesenchymal stem cells, and 10 per milliliter of the suspension7Adipose tissue-derived mesenchymal stem cells; the preparation is prepared by uniformly mixing the platelet-rich plasma and the suspension according to the volume ratio of 1: 1.
Preferably, the platelet rich plasma is derived from human peripheral blood; the adipose tissue-derived mesenchymal stem cells are derived from the inner thigh of a human.
Preferably, the platelet rich plasma is prepared by the following method:
(1) taking human peripheral blood, centrifuging for the first time under aseptic condition, taking upper layer plasma, centrifuging the upper layer plasma for the second time, taking lower liquid, and blowing, beating and mixing to obtain suspension;
(2) and (2) under the aseptic condition, adding two units of heparin into each milliliter of suspension obtained in the step (1), uniformly mixing, centrifuging, and taking supernatant, wherein the supernatant is activated platelet-rich plasma.
Preferably, the suspension is prepared by the following method: resuscitating and culturing P3 generation cells of adipose tissue-derived mesenchymal stem cells derived from inner thigh of human body, collecting P4 generation cells, and adding physiological saline to make into 10 per ml7A suspension of individual cells.
Preferably, the first centrifugation in step (1) is centrifugation at 360g for 20min at room temperature; the second centrifugation is centrifugation at 500g for 10min at room temperature; the centrifugation in the step (2) is carried out at 4000rpm for 20 min; in the step (2), before centrifugation, the suspension mixed with heparin is placed in liquid nitrogen for 5min, then placed in a constant temperature water tank with the temperature of 37 ℃ for 5min, and the steps are repeated for 3 times.
A method for preparing a preparation for treating a dyschromatosis disease, comprising the steps of:
s1, preparing platelet rich plasma, comprising the following steps:
s1.1, taking peripheral blood of a human body, centrifuging the peripheral blood for the first time under an aseptic condition, taking upper plasma, centrifuging the upper plasma for the second time, taking lower liquid, and blowing, beating and uniformly mixing the lower liquid and the upper plasma to form suspension;
s1.2, under the aseptic condition, adding two units of heparin into each milliliter of suspension obtained in the step (1), uniformly mixing, centrifuging, and taking supernatant, wherein the supernatant is activated platelet-rich plasma;
s2, preparing a suspension containing adipose tissue-derived mesenchymal stem cells, comprising the following steps: recovering and culturing P3 generation cells of adipose tissue-derived mesenchymal stem cells derived from inner thigh of human body, collecting P4 generation cells, and adding physiological salineIs made into 10 per milliliter7A suspension of individual cells;
s3, uniformly mixing the activated platelet rich plasma and the suspension according to the volume ratio of 1:1 to obtain the preparation.
Preferably, the first centrifugation in step S1.1 is centrifugation at 360g at room temperature for 20min, and the second centrifugation is centrifugation at 500g at room temperature for 10 min; the centrifugation in step S1.2 is at 4000rpm for 20 min; in the step S1.2, before centrifugation, the suspension mixed with heparin is placed in liquid nitrogen for 5min, then placed in a 37 ° constant temperature water tank for 5min, and the above steps are repeated for 3 times.
The application of the preparation for treating the dyschromatosis diseases in preparing the medicines for treating the dyschromatosis diseases of the human body.
Compared with the prior art, the invention has the advantages that: the active ingredients of the preparation comprise Platelet-rich plasma (PRP) and Adipose tissue-derived mesenchymal stem cells (ADSCs), the preparation is injected into a pigment barrier area by manufacturing an animal model of guinea pig pigment disorder diseases, the PRP can provide nutrition for the ADSCs, and the ADSCs can replace aged dead cells, repair damaged cells and secrete cytokines. A series of evaluations of histopathological staining, immunohistochemical analysis, melanin granule staining by a Fontana-Masson method, detection of the content of inducible nitric oxide synthase by an immunohistochemical SP method and the like are carried out on the preparation, and the preparation provided by the embodiment of the invention is proved to be capable of inhibiting dyschromatosis, reliable in treatment effect and capable of providing a new breakthrough for treating dyschromatosis diseases.
Drawings
Fig. 1 is a schematic skin condition of an animal model of a guinea pig pigmentary disorder according to an embodiment of the present invention;
FIG. 2 is a schematic representation of the skin condition after four weeks of treatment with subcutaneous injections of a formulation of an embodiment of the present invention;
FIG. 3 is a graphical representation of the results of HE staining of skin in accordance with an embodiment of the present invention;
FIG. 4 is a schematic diagram showing the evaluation results of the synthesis of melanin granules according to the example of the present invention;
FIG. 5 is a schematic diagram showing the expression of iNOS in the skin in accordance with an embodiment of the present invention;
FIG. 6 is a technical roadmap of a formulation for pigmentary obstructive disease in accordance with an embodiment of the present invention.
Detailed Description
The invention will be further described with reference to the accompanying drawings and preferred embodiments. It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict.
The embodiment of the invention provides a preparation for treating dyschromatosis, and the active ingredients of the preparation comprise platelet-rich plasma and adipose tissue-derived mesenchymal stem cells.
In a preferred embodiment, the formulation is an injectable formulation.
In a preferred embodiment, the platelet rich plasma is platelet rich plasma activated by heparin; the adipose tissue-derived mesenchymal stem cells exist in a suspension consisting of normal saline and adipose tissue-derived mesenchymal stem cells, and 10 per milliliter of the suspension7Adipose tissue-derived mesenchymal stem cells; the preparation is prepared by uniformly mixing the platelet-rich plasma and the suspension according to the volume ratio of 1: 1.
In a preferred embodiment, the platelet rich plasma is derived from human peripheral blood; the adipose tissue-derived mesenchymal stem cells are derived from the inner thigh of a human.
In a preferred embodiment, the platelet rich plasma is prepared by the following method:
(1) taking human peripheral blood, centrifuging for the first time under aseptic condition, taking upper layer plasma, centrifuging the upper layer plasma for the second time, taking lower liquid, and blowing, beating and mixing to obtain suspension;
(2) and (2) under the aseptic condition, adding two units of heparin into each milliliter of suspension obtained in the step (1), uniformly mixing, centrifuging, and taking supernatant, wherein the supernatant is activated platelet-rich plasma.
In a preferred embodiment, the suspension is prepared by the following method: resuscitating and culturing P3 generation cells of adipose tissue-derived mesenchymal stem cells derived from inner thigh of human body, collecting P4 generation cells, and adding physiological saline to make into 10 per ml7A suspension of individual cells.
In a preferred embodiment, the first centrifugation in step (1) is centrifugation at 360g at room temperature for 20 min; the second centrifugation is centrifugation at 500g for 10min at room temperature; the centrifugation in the step (2) is carried out at 4000rpm for 20 min; in the step (2), before centrifugation, the suspension mixed with heparin is placed in liquid nitrogen for 5min, then placed in a constant temperature water tank with the temperature of 37 ℃ for 5min, and the steps are repeated for 3 times.
The embodiment of the invention also provides a preparation method of the preparation for the dyschromatosis diseases, which comprises the following steps:
s1, preparing platelet rich plasma, comprising the following steps:
s1.1, taking peripheral blood of a human body, centrifuging the peripheral blood for the first time under an aseptic condition, taking upper plasma, centrifuging the upper plasma for the second time, taking lower liquid, and blowing, beating and uniformly mixing the lower liquid and the upper plasma to form suspension;
s1.2, under the aseptic condition, adding two units of heparin into each milliliter of suspension obtained in the step (1), uniformly mixing, centrifuging, and taking supernatant, wherein the supernatant is activated platelet-rich plasma;
s2, preparing a suspension containing adipose tissue-derived mesenchymal stem cells, comprising the following steps: recovering and culturing P3 generation cells of adipose tissue-derived mesenchymal stem cells derived from inner thigh of human body, collecting P4 generation cells, and adding normal saline to make into 10 ml7A suspension of individual cells;
s3, uniformly mixing the activated platelet rich plasma and the suspension according to the volume ratio of 1:1 to obtain the preparation.
In a preferred embodiment, the first centrifugation in step S1.1 is centrifugation at 360g at room temperature for 20min, and the second centrifugation is centrifugation at 500g at room temperature for 10 min; the centrifugation in step S1.2 is at 4000rpm for 20 min; in the step S1.2, before centrifugation, the suspension mixed with heparin is placed in liquid nitrogen for 5min, then placed in a 37 ° constant temperature water tank for 5min, and the above steps are repeated for 3 times.
The embodiment of the invention also provides application of the preparation for treating the dyschromatosis diseases in preparation of a medicament for treating the dyschromatosis diseases.
In the embodiment of the present invention, the curative effect of the preparation on the dyschromatosis is verified by locally injecting the platelet rich plasma adipose tissue-derived mesenchymal stem cell preparation for treating the dyschromatosis, which is described in detail below (hereinafter,% means volume percent vol% unless otherwise specified).
Animal model for establishing guinea pig pigment obstacle disease by irradiating guinea pig back with 308nm ultraviolet therapeutic apparatus
All experimental animals used in this example were provided by southern university of medical laboratory animal center and were of the general grade of healthy male guinea pigs. 10 guinea pigs with larger back area were selected, aged 2-3 months and weighed between 329-410 g. Two areas were selected on the back of each experimental animal, one of which was irradiated with UV light and the other was used as a normal control. Irradiation was performed using a 308nm UV light treatment apparatus. The guinea pig back hair was shaved off with an electric razor, and the back skin was fully exposed with depilatory cream. Directly contacting therapeutic head of ultraviolet instrument with wavelength of 308nm with naked back skin of guinea pig, and irradiating with energy of 600mJ/cm each time2The frequency was 1 time per week for a total of 3 times per week and the total irradiation energy was 1800mJ/cm2. Redness of skin can be seen on the day of irradiation, erythema can be obvious on the next day, desquamation of skin can be seen in 3-5 days, pigmentation disorder can be seen in 5-7 days, and the pigmentation disorder reaches a peak after 3 weeks and is uniform and stable.
Preparation of platelet-rich plasma and adipose tissue-derived mesenchymal stem cell preparation
Taking 14ml of peripheral blood donated by a healthy volunteer, transferring the peripheral blood into a 15ml centrifuge tube under aseptic condition, centrifuging for 20min at room temperature of 360g, sucking upper plasma into a new centrifuge tube, centrifuging for 10min at room temperature of 500g, and blowing and mixing 1ml of the residual bottom to form suspension. Under the aseptic condition, adding two units of heparin into each milliliter of suspension, uniformly mixing, and subpackaging by using a freezing tube. The frozen tube is placed in liquid nitrogen for 5min and then placed in a constant temperature water tank of 37 ℃ for 5min, and the steps are repeated for 3 times. The frozen tube was replaced in a centrifuge tube and centrifuged at 4000rpm for 20min to obtain the supernatant, i.e., activated Platelet-rich plasma (PRP).
Adipose tissue-derived mesenchymal stem cells (ADSCs) are human body thigh medial Adipose tissue-derived mesenchymal stem cells frozen by applicant liquid nitrogen, P3 generation cells are taken out for resuscitation and culture, P4 generation cells are collected and prepared into 10 per milliliter by normal saline7A suspension of individual cells.
Taking activated platelet rich plasma and 10 per ml7The adipose tissue-derived mesenchymal stem cell suspensions are uniformly mixed according to the volume ratio of 1:1 to obtain the preparation used in the embodiment of the invention (in practical application, aiming at a subject, blood samples and adipose tissues of the subject can be collected to prepare the preparation so as to reduce the safety risk), in vivo, PRP can provide nutrition for ADSCs, ADSCs can replace aged dead cells, repair damaged cells and secrete cytokines, the preparation disclosed by the invention is lower in toxicity and better in whitening effect, and skin problems can be repaired and improved fundamentally.
Injection of pigmentary disorder area
The preparation of the second ingredient is injected into the pigment-deficient area of the first ingredient, and the same volume of physiological saline is injected into the skin of a control group. Covering a 1ml syringe with a 30g needle, injecting the mixture between the epidermis layer and the dermis layer, withdrawing the needle outwards, injecting the preparation simultaneously, carrying out a plain thread needle method, enabling the needle to form an angle of 15 degrees with the skin, uniformly mixing the preparation before use, and uniformly injecting 10 sites into the skin at the injection dose volume of 0.1ml per square centimeter.
Fourth, result analysis
Histological changes in the skin after injection of the formulation were individually analyzed by visual assessment, HE staining, Fontana-Masson method, and determination of Inducible Nitric Oxide Synthase (iNOS) content.
1. Evaluation by eye
NB-UVB guinea pigs were irradiated with bare skin on the back 1 time per week for a total of 3 times. Treatment groups the formulations were injected into the skin pigmentation disorder area after NB-UVB irradiation and skin pigmentation disorder was visually assessed 4 weeks after treatment. Skin pigmentation disorder scoring criteria were: the skin is not changed, namely the normal skin color is 0 point; the light brown is 1 point; medium brown is 2 points; dark brown was 3 points. After 3 times of NB-UVB irradiation, uniform and stable pigmentation disorder of guinea pig back skin was observed (as shown in FIG. 1, A is normal skin before NB-UVB irradiation, B is pigmented skin after NB-UVB irradiation, and C is comparison of pigmented skin with normal skin), and after 4 weeks of treatment, the preparation was photographed to significantly improve pigmentation disorder of guinea pig skin after treatment (as shown in FIG. 2, skin condition after four weeks of treatment by subcutaneous injection of the preparation).
2. Hematoxylin-eosin (HE) staining
The skin of the injected area was sampled and fixed in 10% formalin solution after marking. And after soaking for 48 hours, carrying out conventional tissue paraffin embedding and slicing on the specimen, and carrying out HE staining. Hematoxylin-eosin (HE) staining: the skin specimens of the control group and the experimental group were subjected to histopathological HE staining and observed for histological changes. FIG. 3 is a graph showing the results of HE staining of skin, wherein Panel A and Panel B are graphs showing the results of HE staining of normal skin at multiples of 10X and 40X, respectively; panels C and D are schematic representations of the results at multiples of 10X and 40X, respectively, after HE staining of pigmented skin; panels E and F are schematic representations of results at multiples of 10X and 40X, respectively, after HE staining of skin after treatment with the formulation. HE staining showed that the stratum granulosum, stratum spinosum and stratum corneum of the experimental group were thickened, infiltration of occasional inflammatory cells in the dermis, and epidermal melanocytes and melanocytes were increased, compared to the control group, and this morphological change of the tissue was consistent with skin pigmentation disorder caused by uv irradiation. After treatment, pigment particles are obviously reduced, and a small amount of melanin particles are scattered in a basal layer and a spinous layer; the change in pigment particles was evident in the blank control.
3. Evaluation of Melanin Synthesis Condition (Fontana-Masson method)
(1) Fixing guinea pig skin tissue with 10% neutral formaldehyde solution, embedding with paraffin, slicing paraffin, dewaxing to water, washing with distilled water, and adding silver ammonia solution in dark place.
(2) And dip-dyeing for 12-18h at room temperature in the dark.
(3) And (5) washing with distilled water.
(4) Soaking in 0.2% gold chloride solution for 2-5 min.
(5) Excess silver particles are washed away.
(6) Washing with distilled water for 2-3 min.
(7) The sections were fixed in 5% sodium thiosulfate solution for 5 min.
(8) Flushing with flowing water for 5 min.
(9) Counterstaining was performed for 1min using hematoxylin solution (prepared according to standard protocol).
(10) Differentiating with 1% ethanol solution of hydrochloric acid for 10-15 s.
(11) Ammonia (prepared according to standard protocols) returns blue for 10 s.
(12) Treated with eosin solution (prepared according to standard protocol) for 30-60 s.
(13) The sample was cleared by conventional dehydration with xylene addition.
(14) The gel was mounted on a neutral gum and observed under an inverted microscope.
Evaluation of melanin granule synthesis: melanin granules were stained by the Fontana-Masson method. The evaluation criteria were graded as 5: grade 1 is the occasional visible melanin granules in basal cells and spinous layers; the 2-stage visible discontinuous distribution of melanin is mainly concentrated on a basal layer; grade 3 is a continuous band-shaped distribution of melanin granules, mainly concentrated in basal cells and acanthosis; grade 4 is a continuous band of melanin granules concentrated within basal cells and in the stratum spinosum, and it is seen that more melanin caps appear within keratinocytes; grade 5 shows a dense distribution of melanin granules within the epidermis and a more prominent melanin cap. FIG. 4 is a graph showing the results of evaluation of melanin granule synthesis, wherein A and B are graphs showing the results of distribution of melanin granules in normal skin at multiples of 10X and 40X, respectively; panel C and panel D are schematic representations of the results of melanin granule distribution in pigmented skin at multiples of 10X and 40X, respectively; panels E and F are schematic representations of the results of melanin granule distribution at multiples of 10X and 40X, respectively, in skin treated with the formulation. The Fontana-Masson staining results show: F-M staining results show: in guinea pig normal skin, there are only a few melanin granules in the basal cells and the spinous layers of the epidermis. After ultraviolet irradiation, melanin is increased in the epidermis, a large number of melanin granules are continuously distributed in a band shape in basal cells and a spinous layer, and densely distributed melanin granules are distributed in a part of the whole epidermis. After treatment, the melanin is obviously reduced, and after 4 weeks of follow-up, the melanin does not have obvious recurrence and increase.
4. Immunohistochemical detection of Inducible Nitric Oxide Synthase (iNOS) content in guinea pig skin
(1) Dewaxing: the slices are left at room temperature for 60min (or baked in a 60-degree oven for at least 20min (the previous day may be baked for 3 h).
(2) Hydration: xylene for 5 min; xylene for 10 min; absolute ethyl alcohol for 3 min; 95% ethanol for 3 min.
(3) 85% ethanol for 3min, and tap water for 10 min; the samples were washed 3 times with PBS buffer for 5min each.
(4) Blocking endogenous peroxidase 3% H2O2(H2O2Formulated with methanol), incubated at room temperature for 5min, washed 3 times with PBS for 5min each.
(5) High pressure heat repairing of antigen (paraffin embedded tissue section for formalin fixation) placing into a staining jar containing EDTA (pH8.0) or sodium citrate Buffer (pH6.0) in boiling water in an autoclave, placing the glass slide into the staining jar, covering, and heating under high pressure. And (5) timing 5min after the air valve rotates to exhaust, closing the heat source, and washing for 5min by PBS 3 times after natural cooling.
(6) Antigen blocking: adding normal goat serum blocking solution dropwise, blocking at room temperature for 1h, sucking off the blocking solution after the time is up, adding diluted iNOS primary antibody (1:100) dropwise without washing, standing at room temperature for at least 2h or 37 ℃ for 1h or 4 ℃ overnight (after overnight at 4 ℃, room temperature is required to be re-warmed for 1h), and washing with PBS for 5min for 3 times.
(7) Adding a secondary antibody (goat anti-rabbit, 1:100) dropwise, standing at room temperature for 2h or 1h at 37 ℃, washing with PBS for 3 times and 5min respectively, (HRP anti-mouse IgG, diluent is immune non-fluorescent staining secondary antibody diluent, and the dilution concentration is 1: 50).
(8) DAB was developed for 3min (different antibody times, specifically observed under microscope).
(9) DAB color development: adding 1 drop of 50 μ LDAB solution A, solution B and solution C into 1mL of distilled water, mixing well for use, and preparing for use. Dripping 1-2 drops of DAB working solution on the slices, and developing at room temperature in a dark place. Observing the color development degree under a microscope, and controlling the color development time.
(10) Nuclear counterstaining: staining nuclei with hematoxylin solution for 5min, and washing with tap water.
(11) Sealing: and (5) sealing the neutral resin.
(12) Observing under an upright microscope.
Determination of the content of Inducible Nitric Oxide Synthase (iNOS): the immunohistochemical SP method is used for detecting the content and distribution of iNOS, and the iNOS content and distribution are used as indirect indexes to reflect the change of Nitric Oxide (NO). The blank control was made by replacing primary antibody with 0.01mol/LPBS, and positive cells were found to have brownish or yellowish particles in cytoplasm and cell membrane. Scoring of staining results: no dyeing is 0 min; the dark yellow is 1 point; light brown is 2 points; the dark brown-yellow color is 3 points. FIG. 5 is a schematic view showing the expression of iNOS in skin, in which graphs A and B are graphs showing the results of iNOS expression in normal skin at multiples of 10X and 40X, respectively; panel C and D are graphs showing the results of iNOS expression in pigmented skin at multiples of 10X and 40X, respectively; panels E and F are graphs showing the results of iNOS expression at multiples of 10X and 40X in skin treated with the formulations, respectively. The experimental results show that: staining positive cells were distributed throughout the epidermis except the stratum corneum and in the hair follicles, and iNOS was mainly distributed in the cytoplasm and membranes of epidermal cells. Compared with the control group, the expression of iNOS in the epidermis of the irradiated group is obviously increased, and the expression of iNOS in the experimental group after treatment is reduced.
In conclusion, a uniform and stable pigmentation disorder was formed in the dorsal skin of guinea pigs after NB-UVB irradiation. The preparation significantly improved the pigmentation disorder of guinea pig skin as seen by 4 weeks of photograph after completion of treatment, with no significant change in the control group. After the F-M staining visible ultraviolet irradiation, melanin particles in the model are increased remarkably, the melanin is reduced remarkably after the treatment, the result is P <0.05 after the rank sum test of the content of the melanin particles in a control group, the difference has statistical significance (table 1), and the immunohistochemical staining result indicates that the iNOS expression in the epidermis of the ultraviolet irradiation group is high, and the iNOS expression of an experimental group is reduced after the treatment. The iNOS content in the control group was checked for rank and found to be P <0.05, and the differences were statistically significant (see Table 1 below)
TABLE 1 Guinea pig skin pigmentation score, melanin content and Inducible Nitric Oxide Synthase (iNOS) content results
(median)
In this example, the preparation was injected into the pigment-deficient region to perform histopathological staining, immunohistochemical analysis, melanin granule staining by Fontana-Masson method, immunohistochemical SP method to detect the content of inducible nitric oxide synthase by creating an animal model of guinea pig pigmentary disorder, and the results showed that:
the skin of the guinea pig after being irradiated for 3 times by NB-UVB forms uniform and stable pigmentation disorder, and the pigmentation disorder of the skin of the guinea pig can be obviously improved after being treated by the preparation by photographing for 4 weeks after the treatment is finished, and the change of a control group is not obvious.
HE staining was visible: compared with the control group, the stratum granulosum, stratum spinosum and stratum corneum of the experimental group are thickened, the infiltration of inflammatory cells occasionally occurs in the dermis, and epidermal melanocytes and melanin granules are increased, and the change of the tissue morphology accords with the skin pigment disorder caused by ultraviolet irradiation. After treatment, pigment particles are obviously reduced, and a small amount of melanin particles are scattered in a basal layer and a spinous layer; the change in pigment particles was not apparent in the blank control.
Evaluation of melanin granule synthesis: F-M staining was seen in guinea pig normal skin, with only a few melanin granules in the basal cells and spinous layers of the epidermis. After ultraviolet irradiation, melanin is increased in the epidermis, a large number of melanin granules are continuously distributed in a band shape in basal cells and a spinous layer, and densely distributed melanin granules are distributed in a part of the whole epidermis. After treatment, the melanin is obviously reduced, and after 4 weeks of follow-up, the melanin does not have obvious recurrence and increase.
Immunohistochemical staining results show: staining positive cells were distributed throughout the epidermis except the stratum corneum and in the hair follicles, and iNOS was mainly distributed in the cytoplasm and membranes of epidermal cells. Compared with the control group, the expression of iNOS in the epidermis of the irradiated group is obviously increased, and the expression of iNOS in the experimental group after treatment is reduced.
According to the embodiment of the invention, 308nm excimer laser is adopted to irradiate the back skin of a brown yellow guinea pig, an animal model of the guinea pig pigmentary disorder disease is established, the preparation is prepared and is locally injected in a pigmentary disorder area, the treatment effect of the preparation on the guinea pig pigmentary disorder disease is analyzed and judged, and the preparation can provide support for treating the pigmentary disorder disease by inhibiting the pigmentary disorder clinically.
The background of the present invention may contain background information related to the problem or environment of the present invention and does not necessarily describe the prior art. Accordingly, the inclusion in the background section is not an admission of prior art by the applicant.
The foregoing is a more detailed description of the invention in connection with specific/preferred embodiments and is not intended to limit the practice of the invention to those descriptions. It will be apparent to those skilled in the art that various substitutions and modifications can be made to the described embodiments without departing from the spirit of the invention, and these substitutions and modifications should be considered to fall within the scope of the invention. In the description herein, references to the description of the term "one embodiment," "some embodiments," "preferred embodiments," "an example," "a specific example," or "some examples" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Various embodiments or examples and features of various embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction. Although embodiments of the present invention and their advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the scope of the claims.
Claims (10)
1. A formulation for use in a pigmentary disorder, comprising: the active ingredients of the preparation comprise platelet-rich plasma and adipose tissue-derived mesenchymal stem cells.
2. The formulation for use in pigmentary disorders as claimed in claim 1, wherein: the preparation is an injection preparation.
3. The formulation for use in pigmentary disorders as claimed in claim 1, wherein: the platelet rich plasma is activated by heparin; the adipose tissue-derived mesenchymal stem cells exist in a suspension consisting of normal saline and adipose tissue-derived mesenchymal stem cells, and 10 per milliliter of the suspension7Adipose tissue-derived mesenchymal stem cells; the preparation is prepared by uniformly mixing the platelet-rich plasma and the suspension according to the volume ratio of 1: 1.
4. The formulation for use in pigmentary disorders as claimed in claim 1, wherein: the platelet rich plasma is derived from human peripheral blood; the adipose tissue-derived mesenchymal stem cells are derived from the inner thigh of a human.
5. The formulation for use in pigmentary disorders as claimed in claim 4, wherein: the platelet-rich plasma is prepared by the following method:
(1) taking human peripheral blood, centrifuging for the first time under aseptic condition, taking upper layer plasma, centrifuging the upper layer plasma for the second time, taking lower liquid, and blowing, beating and mixing to obtain suspension;
(2) and (2) under the aseptic condition, adding two units of heparin into each milliliter of suspension obtained in the step (1), uniformly mixing, centrifuging, and taking supernatant, wherein the supernatant is activated platelet-rich plasma.
6. The formulation for use in pigmentary disorders as claimed in claim 3, wherein: the suspension is prepared by the following method:
resuscitating and culturing P3 generation cells of adipose tissue-derived mesenchymal stem cells derived from inner thigh of human body, collecting P4 generation cells, and adding physiological saline to make into 10 per ml7A suspension of individual cells.
7. The formulation for use in pigmentary disorders as claimed in claim 5, wherein: the first centrifugation in the step (1) is centrifugation at 360g at room temperature for 20 min; the second centrifugation is centrifugation at 500g for 10min at room temperature; the centrifugation in the step (2) is carried out at 4000rpm for 20 min; in the step (2), before centrifugation, the suspension mixed with heparin is placed in liquid nitrogen for 5min, then placed in a constant temperature water tank with the temperature of 37 ℃ for 5min, and the steps are repeated for 3 times.
8. A method for preparing a preparation for treating a pigmentary disorder disease, comprising the steps of:
s1, preparing platelet rich plasma, comprising the following steps:
s1.1, taking peripheral blood of a human body, centrifuging the peripheral blood for the first time under an aseptic condition, taking upper plasma, centrifuging the upper plasma for the second time, taking lower liquid, and blowing, beating and uniformly mixing the lower liquid and the upper plasma to form suspension;
s1.2, under the aseptic condition, adding two units of heparin into each milliliter of suspension obtained in the step (1), uniformly mixing, centrifuging, and taking supernatant, wherein the supernatant is activated platelet-rich plasma;
s2, preparing a suspension containing adipose tissue-derived mesenchymal stem cells, comprising the following stepsThe method comprises the following steps: recovering and culturing P3 generation cells of adipose tissue-derived mesenchymal stem cells derived from inner thigh of human body, collecting P4 generation cells, and adding normal saline to make into 10 ml7A suspension of individual cells;
s3, uniformly mixing the activated platelet rich plasma and the suspension according to the volume ratio of 1:1 to obtain the preparation.
9. The method of claim 8, wherein the first centrifugation in step S1.1 is centrifugation at 360g for 20min at room temperature and the second centrifugation is centrifugation at 500g for 10min at room temperature; the centrifugation in step S1.2 is at 4000rpm for 20 min; in the step S1.2, before centrifugation, the suspension mixed with heparin is placed in liquid nitrogen for 5min, then placed in a 37 ° constant temperature water tank for 5min, and the above steps are repeated for 3 times.
10. Use of the formulation for dyschromatosis in claim 1 for the manufacture of a medicament for treating dyschromatosis in a human.
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