CN117582402A - Gel containing leukocyte extract and preparation method and application thereof - Google Patents

Gel containing leukocyte extract and preparation method and application thereof Download PDF

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CN117582402A
CN117582402A CN202410071069.7A CN202410071069A CN117582402A CN 117582402 A CN117582402 A CN 117582402A CN 202410071069 A CN202410071069 A CN 202410071069A CN 117582402 A CN117582402 A CN 117582402A
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gel
extract
leukocyte extract
leukocyte
gel containing
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CN117582402B (en
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郭继彤
冯梁
周玉海
李树裕
赵昕宇
张茜
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Inner Mongolia Yihong Medical Research Co ltd
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Abstract

The invention provides a gel containing leukocyte extract, a preparation method and application thereof. The preparation raw materials of the gel containing the leukocyte extract comprise the following components in percentage by mass: 1-10% of leucocyte extract, 1-5% of panthenol, 0.5-2.5% of leuconostoc/radish root fermentation product filtrate, 0.5-2% of carnosine, 0.1-0.5% of natural heparinoids, 0.05-0.2% of allantoin, 1-15% of humectant, 0.5-2.5% of gel matrix, 0.01-0.05% of complexing agent and the balance of conditioned medium. The gel containing the leucocyte extract does not contain medicinal components, has a simple formula, has obvious curative effect on postherpetic pain, and has no recurrence; the gel disclosed by the invention has no damage to skin and extremely low sensitization.

Description

Gel containing leukocyte extract and preparation method and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to a gel containing a leukocyte extract, a preparation method and application thereof.
Background
Herpes Zoster (HZ) is an infectious disease caused by the reactivation of varicella zoster virus (varicella zoster virus, VZV) which is latent in the dorsal root ganglion or cranial ganglion of the spinal cord after primary infection of the human body. Clinically, papules and blisters distributed along the innervated area are accompanied by sustained cauterized, incised, and torn spontaneous pain. The disease can occur at any age, and the occurrence rate of the disease is obviously increased with the age, and the disease mainly affects middle-aged and elderly people. The national institute of herpes zoster medical science (2022 edition) reports that the annual incidence rate of herpes zoster of the general population is 0.3 to 0.5 percent, and the incidence rate of herpes zoster of people over 50 years is increased to 0.5 to 1.1 percent. The incidence of the herpes zoster in China is basically consistent with that of other countries and regions, the incidence of the herpes zoster of people over 50 years old is 0.3-0.6% each year, and the incidence of the herpes zoster of women is slightly higher than that of men for life, and can reach 0.4-0.8%.
Postherpetic neuralgia (postherpetic neuralgia, PHN) is the most common complication or sequelae of HZ, which is the most common post-infection neuropathic pain, and is also a complex neuropathic pain, the pathogenesis of which is not completely clear at present, once occurring, the occurrence of which often causes significant pain and heavy burden to patients and families thereof, and clinical treatment of PHN is difficult. PHN is the most common complication of HZ, and is generally defined as pain lasting 3 months or more after improvement of HZ skin lesions, and pain lasting 1 month or more after healing of rash, which is a more accepted statement in china, is defined as PHN. The national expert consensus (2016) for post-herpetic neuralgia diagnosis reports that about 9-34% of herpetic patients develop PHN. PHN has a similar incidence to that of shingles, and has a tendency to gradually increase with age, with about 65% of patients with shingles aged 60 years and older developing PHN, and up to 75% of those aged 70 years and older. Wherein 30-50% of post-herpetic neuralgia patients have pain lasting for more than 1 year, and part of the disease course can even reach 10 years.
At present, PHN patients can clinically shorten the pain time and the rash healing time of the patients through antiviral, glucocorticoid and analgesic drug treatment. However, the aged herpes zoster patients have low immunity and weak constitution, and the skin rash is easy to be complicated with persistent neuropathic pain after healing. At present, the treatment of PHN in China mainly comprises drug treatment, interventional treatment, physical treatment, psychological treatment and the like. The recommended first-line therapeutic drugs mainly include the calcium channel blocker pregabalin, tricyclic antidepressants, opioids, glucocorticoids, neurotensin, tramadol, botulinum toxin type a, 5% lidocaine gel patches, and the like. Pregabalin often causes dizziness, somnolence, ataxia, peripheral edema, and the like. In order to reduce adverse reactions caused by using the maximum dose of a single drug, multiple drug combination treatments are often required, and the combined drug administration modes, such as 5% lidocaine gel patch combined pregabalin, small-dose prednisone combined pregabalin and botulinum toxin type A combined pregabalin, are explored clinically at present, and although good clinical effects are obtained, adverse reactions can be reduced without increasing or even increasing, but side effects are still obvious. The intervention of the drug combination, while reducing side effects, is traumatic and increases the risk of causing nerve injury. And the combination of the medicaments and the physical therapy often causes physical injury and has unstable treatment effect. Because the pathogenesis of PHN is not clear, a treatment mode is difficult to obtain satisfactory curative effect at present, and although a combination of two or more methods can obtain better effect, the radical treatment problem of PHN can not be fundamentally solved.
Although the pathogenesis of PHN is not completely understood, studies have found that PHN onset may be associated with viral-induced nerve injury, peripheral neuritic response, and changes in immune cell subsets resulting from abnormal conduction function, central nervous sensitization, hypoimmunity, and even anxiety or depression. Many cytokines, such as tumor necrosis factor (Tumor Necrosis Factor, TNF) - α, interleukin (IL) IL-1, IL-6, IL-8, IL-10, etc. have also been shown to cause neuralgia. Indeed, HZ-induced nerve injury does lead to an increase in TNF- α, IL-1, suggesting that PHN is closely related to inflammatory factors.
In view of this, the present invention has been made.
Disclosure of Invention
It is an object of the present invention to provide a gel containing a leukocyte extract. The gel containing the leucocyte extract does not contain medicinal components, has a simple formula, and has a synergistic effect by matching the components, and has very obvious curative effect on postherpetic pain.
The second object of the present invention is to provide a method for preparing a gel containing leukocyte extract.
The invention also aims at the application of the gel containing the leucocyte extract in preparing products for treating postherpetic neuralgia. The gel containing the leukocyte extract can effectively treat PHN, and particularly can cure patients with postherpetic neuralgia for up to 11 years. Cure has been achieved for half a year so far, and no recurrence exists; and compared with other treatments, the gel provided by the invention has no damage to skin and extremely low sensitization.
In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:
in a first aspect, the invention provides a gel containing a leukocyte extract, wherein the gel containing the leukocyte extract is prepared from the following raw materials in percentage by mass:
1-10% of leucocyte extract, 1-5% of panthenol, 0.5-2.5% of leuconostoc/radish root fermentation product filtrate, 0.5-2% of carnosine, 0.1-0.5% of natural heparinoids, 0.05-0.2% of allantoin, 1-15% of humectant, 0.5-2.5% of gel matrix, 0.01-0.05% of complexing agent and the balance of conditioned medium.
The amount of the white blood cell extract to be added may be 1 to 10%, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc., based on 100% of the total mass of the raw materials for preparing the gel containing the white blood cell extract.
The amount of the white panthenol added is 1 to 5% based on 100% by mass of the total raw material for preparing the gel containing the white blood cell extract, and may be, for example, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc.
The amount of the leucocyte extract-containing gel fermentation product filtrate added is 0.5 to 2.5%, for example, 0.5%, 0.6%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.5%, etc., based on 100% of the total mass of the raw materials for preparing the leucocyte extract-containing gel.
The amount of carnosine added may be 0.5 to 2%, for example, 0.5%, 0.6%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2%, etc., based on 100% of the total mass of the raw materials for preparing the gel containing the leukocyte extract.
The natural heparinoids may be added in an amount of 0.1 to 0.5%, for example, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, etc., based on 100% of the total mass of the raw materials for preparing the gel containing the leukocyte extract.
The amount of allantoin added may be 0.05 to 0.2%, for example, 0.05%, 0.06%, 0.08%, 0.1%, 0.12%, 0.14%, 0.16%, 0.18%, 0.2%, etc., based on 100% of the total mass of the raw materials for preparing the gel containing the leukocyte extract.
The humectant may be added in an amount of 1 to 15% by weight, based on 100% by weight of the total raw material for preparing the leukocyte extract-containing gel, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% or the like.
The gel matrix may be added in an amount of 0.5 to 2.5%, for example, 0.5%, 0.6%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.5%, or the like, based on 100% by mass of the total raw materials for preparing the gel containing the leukocyte extract.
The complexing agent may be added in an amount of 0.01 to 0.05%, for example, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, etc., based on 100% by mass of the total raw material for preparing the gel containing the leukocyte extract.
Firstly, the leukocyte extract is added into the preparation raw materials of the gel, and the leukocyte extract is rich in various growth and cytokines, so that the effects of diminishing inflammation, easing pain, activating blood, removing toxin, repairing skin damage, promoting regeneration and the like of the gel are greatly improved, and the symptoms of nerve injury and peripheral nerve inflammatory reaction caused by viruses are effectively relieved. Thereby solving the radical problem of PHN more effectively.
Further, the leukocyte extract of the invention is preferably prepared from leukocyte extract extracted from the original patent of 'leukocyte extract, anti-wrinkle tightening cream and preparation method thereof' (application publication No. CN116948961A; application No. 2023112134006).
More specifically, the preparation method of the leukocyte extract comprises the following steps:
(1) And (3) collecting: collecting umbilical cord blood of horse or donkey after delivery;
(2) Separating: centrifuging umbilical cord blood to obtain a leukocyte supernatant; wherein, the parameters of the centrifugal treatment in the separation are as follows: the centrifugal force is 300-600 g, and the centrifugal time is 10-20 min; and the supernatant of the leucocyte layer is required to be frozen and stored at the temperature of-85 ℃ to-75 ℃;
(3) Virus detection: carrying out RT-PCR fluorescence and/or PCR fluorescence quantitative detection on the supernatant of the leucocyte layer; wherein the virus comprises type a 1 equine influenza virus, type 2 equine influenza virus, equine infectious anemia virus, equine herpes virus, equine arteritis virus, venezuelan equine encephalomyelitis virus, eastern western Ma Naosui inflammatory virus, foot and mouth disease virus, and african equine pestivirus;
(4) And (3) microorganism detection: endotoxin and bacteria culture are carried out on the supernatant of the leucocyte layer to detect microorganisms; wherein, the endotoxin detection adopts an endotoxin limulus test method; the bacteria culture detection adopts an agar plate culture method;
(5) Extracting: centrifuging the supernatant of the white blood cell layer qualified in virus detection and microorganism detection, and extracting to obtain a white blood cell extract; wherein, the standard of the qualified leukocyte supernatant is: the PCR detection is negative, the endotoxin detection is lower than 0.125 EU/mL, and the bacterial detection is not detected; the parameters of the centrifugal treatment in the extraction are as follows: the centrifugal force is 4500-5500 g, and the centrifugal time is 25-30 min; the supernatant fluid after centrifugal treatment is filtered by a filter membrane with the diameter of 0.45 mu m and a filter membrane with the diameter of 0.1 mu m in sequence; the leukocyte extract is frozen and preserved at the temperature of-85 ℃ to-75 ℃.
And besides the white blood cell extract is added as a main active ingredient, a plurality of nutritional ingredients are added, and various components are mutually matched, so that the curative effect on the postherpetic pain is further improved, the adverse reaction of the medicine is reduced, the treatment time of the herpes zoster is shortened, and the occurrence of the postherpetic pain is reduced.
Wherein, the skin moisture retention and skin protection film repair capability of panthenol promote epidermis regeneration; in addition, it can promote proliferation and epithelialization of fibroblast, promote wound healing, and induce erythema reduction and regeneration of elastic solid tissue. The Leuconostoc mesenteroides/radix Raphani fermentation product filtrate not only helps the formula to effectively control microorganisms, but also helps the skin to strengthen moisture retention, enhances the permeability of the gel, so that the gel is easier to absorb, deep moisturizes, nourishes and repairs the skin, and repairs damaged or nutrient-deficient cells. Carnosine is taken as a water-soluble dipeptide naturally existing in a human body, so that the cell repair and growth are promoted, and the anti-injury capability of the cell is enhanced; perfecting scalp barrier, and can remove free radical to prevent skin from being oxidized and damaged again. Natural heparinoids can act anti-inflammatory, antiproliferative, softening or smoothing scar tissue. Allantoin has effects in promoting cell growth, promoting wound healing, and softening keratin.
Preferably, the humectant is a combination of glycerin and propylene glycol with the mass ratio of (1-10): 1-5;
wherein, "1 to 10" may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.;
wherein "1 to 5" may be, for example, 1, 2, 3, 4, 5, etc.
Preferably, the gel matrix is a carbomer, preferably carbomer 980.
Preferably, the complexing agent is sodium ethylenediamine tetraacetate.
As a preferred embodiment of the invention, the preparation raw materials of the gel containing the leukocyte extract comprise the following components in percentage by mass:
1-10% of leucocyte extract, 1-5% of panthenol, 0.5-2.5% of leuconostoc/radish root fermentation product filtrate, 0.5-2% of carnosine, 0.1-0.5% of natural heparinoids, 0.05-0.2% of allantoin, 1-10% of glycerin, 1-5% of propylene glycol, 0.5-2.5% of carbomer 980, 0.01-0.05% of sodium ethylenediamine tetraacetate and the balance of conditioned medium.
Preferably, the conditioned medium is obtained by culturing mesenchymal cells in a medium containing a leukocyte extract.
Preferably, the culture medium is selected from the group consisting of DMEM/F12 medium, DMEM medium andα-any one of the MEM media.
Preferably, the amount of the leukocyte extract added to the culture solution is 5-15 wt%, for example, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, etc.
Preferably, the conditioned medium is prepared by the steps of:
(a) Mixing the leukocyte extract with the culture solution to obtain a culture solution containing the leukocyte extract;
(b) Mixing umbilical cord mesenchymal stem cells with a culture solution containing a leukocyte extract, centrifuging, and removing supernatant to obtain resuspended cells; then placing the resuspended cells in a culture solution containing a leukocyte extract for culturing;
(c) And (3) after the cells grow to 70-80% (such as 70%, 72%, 74%, 76%, 78%, 80% and the like) confluency, collecting supernatant and performing centrifugal treatment to obtain a conditioned medium.
Preferably, in the step (b), the centrifugal force of the centrifugal treatment is 1000-2000 g, for example, 1000 g, 1200 g, 1400 g, 1600 g, 1800 g, 2000 g, and the like, preferably 1500 g; the centrifugation time is 1 to 10 min, for example, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, etc., preferably 5 min.
Preferably, in step (b), the supernatant is discarded, and the resuspended cells are obtained and counted.
Preferably, in step (b), the culturing is performed in a T175 flask.
Preferably, in step (b), the cells are inoculated in an amount of 1X 10 3 ~1×10 5 Individual cells/cm 2 For example, it may be 1X 10 3 Individual cells/cm 2 、2×10 3 Individual cells/cm 2 、4×10 3 Individual cells/cm 2 、6×10 3 Individual cells/cm 2 、8×10 3 Individual cells/cm 2 、1×10 4 Individual cells/cm 2 、2×10 4 Individual cells/cm 2 、4×10 4 Individual cells/cm 2 、6×10 4 Individual cells/cm 2 、8×10 4 Individual cells/cm 2 、1×10 5 Individual cells/cm 2 Etc., preferably 1X 10 4 Individual cells/cm 2 。
Preferably, in the step (b), the temperature of the culture is 36 to 38 ℃, for example, 36 ℃, 36.5 ℃, 37 ℃, 37.5 ℃, 38 ℃, etc., preferably 37 ℃.
Preferably, in step (b), the cultivation is at 5% CO 2 In an incubator.
Preferably, in the step (c), the interval time for collecting the supernatant is 42-54 h, which may be, for example, 42 h, 43 h, 44 h, 45 h, 46 h, 47 h, 48 h, 49 h, 50 h, 51 h, 52 h, 53 h, 54 h, and preferably 48 h.
Preferably, in step (c), the centrifugation is specifically: and (3) centrifuging the collected supernatant in a centrifuge tube, and collecting the supernatant again to obtain the conditioned medium.
Preferably, in the step (c), the centrifugal force of the centrifugal treatment is 2000-4000 g, for example, 2000 g, 2200 g, 2400 g, 2600 g, 2800 g, 3000 g, 3200 g, 3400 g, 3600 g, 3800 g, 4000 g, etc., preferably 3000 g; the centrifugation time is 1 to 10 min, for example, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, etc., preferably 5 min.
Preferably, in step (c), the conditioned medium is filtered through a filter membrane having a pore size of 0.2. Mu.m.
As a preferred embodiment of the invention, the conditioned medium is prepared by the following steps:
(a) Mixing the leukocyte extract with the culture solution to obtain a culture solution containing 5-15wt% of the leukocyte extract;
(b) Firstly, adding umbilical cord mesenchymal stem cells into a culture solution containing a leukocyte extract, gently mixing, centrifuging for 1-10 min by using a centrifugal force of 1000-2000 g, discarding supernatant to obtain resuspended cells, and performing cell counting; adding culture solution containing leukocyte extract into culture flask, and mixing at a ratio of 1×10 3 ~1×10 5 Individual cells/cm 2 Inoculating into a culture flask, lightly mixing, and placing into a culture flask at 36-38deg.C and 5% CO 2 Culturing in an incubator;
(c) Collecting a supernatant culture solution every 42-54 h when the cells grow to 70-80% confluence, and changing the solution; centrifuging the collected supernatant culture solution for 1-10 min by using a centrifugal force of 2000-4000 g, and taking the supernatant; filtering the supernatant to obtain the conditioned medium.
In a second aspect, the present invention provides a method for preparing a gel comprising a leukocyte extract as described in the first aspect, said method comprising the steps of:
mixing the preparation raw materials of the gel containing the white blood cell extract according to the proportion, and then sequentially adjusting the pH and centrifuging to obtain the gel containing the white blood cell extract.
Preferably, the reagent used for adjusting the pH is 5-15 wt% (for example, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt% etc.) of aqueous sodium hydroxide solution.
Preferably, the pH is adjusted to a pH of 6.8 to 7.2, for example, 6.8, 6.9, 7.0, 7.1, 7.2, etc. can be used.
Preferably, the centrifugal force of the centrifugal treatment is 2000-4000 g, for example, 2000 g, 2200 g, 2400 g, 2600 g, 2800 g, 3000 g, 3200 g, 3400 g, 3600 g, 3800 g, 4000 g, and the like, preferably 3000 g; the centrifugation time is 5-20 min, for example, 5 min, 6 min, 8 min, 10 min, 12 min, 14 min, 16 min, 18 min, 20 min, etc.
As a preferred embodiment of the present invention, the preparation method of the gel containing leukocyte extract comprises the following steps:
(1) Mixing part of the conditioned medium with leukocyte extract, panthenol, leuconostoc mesenteroides/radix Raphani fermentation product filtrate, carnosine, natural heparinoids, allantoin, humectant, gel matrix, and complexing agent, adding the rest conditioned medium to 100%, and mixing;
(2) Adjusting the pH value of the mixed gel obtained in the step (1) to 6.8-7.2 by adopting an alkaline pH regulator, and uniformly mixing;
(3) And (3) centrifuging the mixed gel obtained in the step (2) for 5-20 min by using a centrifugal force of 2000-4000 g to obtain the gel containing the leukocyte extract.
In a third aspect, the present invention provides the use of a gel comprising a leukocyte extract as described in the first aspect for the preparation of a product for the treatment of postherpetic neuralgia.
Compared with the prior art, the invention has the following beneficial effects:
(1) The gel containing the leucocyte extract and used for treating postherpetic neuralgia does not contain medicinal components, has a simple formula and has very obvious curative effect on postherpetic neuralgia;
(2) Curing the patients with postherpetic neuralgia for up to 11 years. The gel has the advantages of no recurrence, no damage to skin and low sensitization compared with other treatments. The patient is unknowingly treated.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the comparison of the front and rear of a gel containing a leukocyte extract provided in example 1 of the present invention for a patient with herpes zoster on the right abdomen and right waist.
Fig. 2 is a graph showing the comparison of the front and rear of the gel containing the leukocyte extract provided in example 1 of the present invention for a patient with herpes zoster on the right back and right waist.
Fig. 3 is a graph showing a comparison of the front and rear of a gel containing a leukocyte extract provided in example 1 of the present invention for a patient having herpes zoster in the abdomen.
Fig. 4 is a graph showing a comparison of the front and rear of a gel containing a leukocyte extract provided in example 1 of the present invention for a patient with herpes zoster on the upper arm.
Detailed Description
Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms should be clear, however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or extraneous definition. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "include" and other forms is not limiting.
Generally, the nomenclature used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and the techniques thereof are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally well known in the art and are performed according to conventional methods as described in various general and more specific references cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to manufacturer's instructions, as commonly accomplished in the art, or as described herein. Nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques therefor, are those well known and commonly employed in the art.
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention is further illustrated by the following examples. The materials in the examples were prepared according to the existing methods or were directly commercially available unless otherwise specified.
Preparation example 1
The preparation example provides a preparation method of a leukocyte extract, which comprises the following steps:
(1) Collecting postpartum horse umbilical cord blood, and conveying the horse umbilical cord blood to a laboratory at room temperature;
(2) Centrifuging to separate supernatant containing leukocyte layer, and freeze preserving at-80deg.C;
the parameters of the centrifugal treatment are as follows: the centrifugal force is 300 g, and the centrifugal time is 10 min;
(3) Quantitative detection of equine influenza virus (type A1 and type 2), equine infectious anemia virus, equine herpesvirus, equine arteritis virus, venezuelan equine encephalomyelitis virus, eastern western Ma Naosui inflammatory virus, foot-and-mouth disease virus, african equine pestivirus;
(4) Detecting microbial contamination by endotoxin and bacterial culture; the endotoxin detection adopts an endotoxin limulus test method; the bacteria culture detection adopts an agar plate culture method;
(5) Thawing the non-polluted leukocyte supernatant, and separating and extracting leukocyte extract;
the standards for the clear leukocyte supernatant are: the PCR detection is negative, the endotoxin detection is lower than 0.125 EU/mL, and the bacterial detection is not detected;
the parameters of the centrifugal treatment are as follows: the centrifugal force is 5000 g, and the centrifugal time is 28 min;
(6) After sterilization by 0.45 μm and 0.1 μm suction filtration, the product was stored at-80℃for a long period of time.
Preparation example 2
The preparation example provides a conditional culture solution, which is prepared by the following steps:
(a) 10% of the leukocyte extract obtained in preparation example 1 was added to a DMEM/F12 (Gibco) culture solution, and the mixture was homogenized to obtain a culture solution containing the leukocyte extract.
(b) The umbilical cord mesenchymal stem cells thawed at 37℃were transferred to a 15 mL centrifuge tube to which 8 mL of the above-mentioned culture solution containing a leukocyte extract was added, gently mixed, and centrifuged at 1500 g for 5 min. The supernatant was discarded to give 2 mL resuspended cells, which were counted. Adding 20 mL culture solution containing leukocyte extract into T175 culture flask, and mixing at a ratio of 1×10 4 Individual cells/cm 2 Inoculating into culture flask, mixing with 5% CO at 37deg.C 2 Culturing in an incubator.
(c) When the cells grew to 70% confluence, the supernatant culture was harvested every 48, h, and changed. The collected supernatant culture was centrifuged at 3000 g for 5 min in a 50 mL centrifuge tube, and the supernatant was collected. Filtering the supernatant with 0.2 μm to obtain conditioned medium, and storing at-20deg.C.
Preparation example 3
The preparation example provides a conditional culture solution, which is prepared by the following steps:
(a) 10% of the white blood cell extract obtained in preparation example 1 was added to a DMEM (Gibco) medium, and the mixture was homogenized to obtain a medium containing the white blood cell extract.
(b) The umbilical cord mesenchymal stem cells thawed at 37℃were transferred to a 15 mL centrifuge tube to which 8 mL of the above-mentioned culture solution containing a leukocyte extract was added, gently mixed, and centrifuged at 1500 g for 5 min. The supernatant was discarded to give 2 mL resuspended cells, which were counted. Adding 20 mL culture solution containing leukocyte extract into T175 culture flask, and mixing at a ratio of 1×10 4 Individual cells/cm 2 Inoculating into culture flask, mixing with 5% CO at 37deg.C 2 Culturing in an incubator.
(c) When the cells grew to 75% confluence, the supernatant culture was harvested every 48, h, and changed. The collected supernatant culture was centrifuged at 3000 g for 5 min in a 50 mL centrifuge tube, and the supernatant was collected. Filtering the supernatant with 0.2 μm to obtain conditioned medium, and storing at-20deg.C.
Preparation example 4
The preparation example provides a conditional culture solution, which is prepared by the following steps:
(a) To the direction ofα10% of the leukocyte extract obtained in preparation example 1 was added to the MEM (Gibco) culture solution, and the mixture was homogenized to obtain a culture solution containing the leukocyte extract.
(b) The umbilical cord mesenchymal stem cells thawed at 37℃were transferred to a 15 mL centrifuge tube to which 8 mL of the above-mentioned culture solution containing a leukocyte extract was added, gently mixed, and centrifuged at 1500 g for 5 min. The supernatant was discarded to give 2 mL resuspended cells, which were counted. Adding 20 mL culture solution containing leukocyte extract into T175 culture flask, and mixing at a ratio of 1×10 4 Individual cells/cm 2 Inoculating into culture flask, mixing with 5% CO at 37deg.C 2 Culturing in an incubator.
(c) When the cells grew to 80% confluence, the supernatant culture was harvested every 48, h, and changed. The collected supernatant culture was centrifuged at 3000 g for 5 min in a 50 mL centrifuge tube, and the supernatant was collected. Filtering the supernatant with 0.2 μm filter membrane to obtain conditioned medium, and storing at-20deg.C.
Preparation example 5
The preparation example provides a conditional culture solution, which is prepared by the following steps:
(a) 10% of the leukocyte extract obtained in preparation example 1 was added to a culture medium of MSC SFM (Gibco), and the mixture was homogenized to obtain a culture medium containing the leukocyte extract.
(b) The umbilical cord mesenchymal stem cells thawed at 37℃were transferred to a 15 mL centrifuge tube to which 8 mL of the above-mentioned culture solution containing a leukocyte extract was added, gently mixed, and centrifuged at 1500 g for 5 min. The supernatant was discarded to give 2 mL resuspended cells, which were counted. Adding 20 mL culture solution containing leukocyte extract into T175 culture flask, and mixing at a ratio of 1×10 4 Individual cells/cm 2 Inoculating into culture flask, mixing with 5% CO at 37deg.C 2 Culturing in an incubator.
(c) When the cells grew to 80% confluence, the supernatant culture was harvested every 48, h, and changed. The collected supernatant culture was centrifuged at 3000 g for 5 min in a 50 mL centrifuge tube, and the supernatant was collected. Filtering the supernatant with 0.2 μm filter membrane to obtain conditioned medium, and storing at-20deg.C.
Example 1
The embodiment provides a gel containing a leukocyte extract, wherein the gel containing the leukocyte extract is prepared from the following raw materials in percentage by mass:
5% of leukocyte extract, 3% of panthenol, 1.5% of Leuconostoc mesenteroides/radish root fermentation product filtrate, 1% of carnosine, 0.3% of natural heparinoids, 0.1% of allantoin, 5% of medical glycerol, 3% of propylene glycol, 980% of medical sterile carbomer, 0.02% of sodium ethylenediamine tetraacetate and the balance of conditioned medium;
wherein the leukocyte extract is the leukocyte extract provided in preparation example 1; the conditioned medium was the conditioned medium provided in preparation example 2.
The gel containing the leukocyte extract in this example was prepared by the following steps:
(1) Adding the conditioned medium into a sterile 500 mL centrifuge tube, adding the leukocyte extract, panthenol, leuconostoc mesenteroides/radix Raphani fermentation product filtrate, carnosine, natural heparinoids, allantoin, glycerol, propylene glycol, carbomer 980, and sodium ethylenediamine tetraacetate according to the above ratio, and finally adding the conditioned medium to 500 mL, and mixing well.
(2) And (3) regulating the pH value of the mixed gel obtained in the step (1) to 7.0 by using 10 wt% NaOH which is subjected to filtration sterilization by a 0.2 mu m filter membrane, and uniformly mixing.
(3) Centrifuging the mixed gel obtained in the step (2) for 10 min at 3000 g, subpackaging in 15 mL light-tight acrylic vacuum bottles, and preserving at 4 ℃.
Example 2
The embodiment provides a gel containing a leukocyte extract, wherein the gel containing the leukocyte extract is prepared from the following raw materials in percentage by mass:
8% of leukocyte extract, 2% of panthenol, 2% of Leuconostoc mesenteroides/radish root fermentation product filtrate, 1.5% of carnosine, 0.2% of natural heparinoids, 0.08% of allantoin, 10% of medical glycerol, 2% of propylene glycol, 980% of medical sterile carbomer, 0.03% of sodium ethylenediamine tetraacetate and the balance of conditioned medium;
wherein the leukocyte extract is the leukocyte extract provided in preparation example 1; the conditioned medium was the conditioned medium provided in preparation example 3.
The gel containing the leukocyte extract in this example was prepared by the following steps:
(1) Adding the conditioned medium into a sterile 500 mL centrifuge tube, adding the leukocyte extract, panthenol, leuconostoc mesenteroides/radix Raphani fermentation product filtrate, carnosine, natural heparinoids, allantoin, glycerol, propylene glycol, carbomer 980, and sodium ethylenediamine tetraacetate according to the above ratio, and finally adding the conditioned medium to 500 mL, and mixing well.
(2) And (3) regulating the pH value of the mixed gel obtained in the step (1) to 7.0 by using 10 wt% NaOH which is subjected to filtration sterilization by a 0.2 mu m filter membrane, and uniformly mixing.
(3) Centrifuging the mixed gel obtained in the step (2) for 10 min at 3000 g, subpackaging in 15 mL light-tight acrylic vacuum bottles, and preserving at 4 ℃.
Example 3
The embodiment provides a gel containing a leukocyte extract, wherein the gel containing the leukocyte extract is prepared from the following raw materials in percentage by mass:
3% of leukocyte extract, 5% of panthenol, 2.5% of Leuconostoc mesenteroides/radish root fermentation product filtrate, 2% of carnosine, 0.5% of natural heparinoids, 0.2% of allantoin, 3% of medical glycerol, 5% of propylene glycol, 0.5% of medical sterile carbomer 980, 0.01% of sodium ethylenediamine tetraacetate and the balance of conditioned medium;
wherein the leukocyte extract is the leukocyte extract provided in preparation example 1; the conditioned medium was the conditioned medium provided in preparation example 4.
The gel containing the leukocyte extract in this example was prepared by the following steps:
(1) Adding the conditioned medium into a sterile 500 mL centrifuge tube, adding the leukocyte extract, panthenol, leuconostoc mesenteroides/radix Raphani fermentation product filtrate, carnosine, natural heparinoids, allantoin, glycerol, propylene glycol, carbomer 980, and sodium ethylenediamine tetraacetate according to the above ratio, and finally adding the conditioned medium to 500 mL, and mixing well.
(2) And (3) regulating the pH value of the mixed gel obtained in the step (1) to 7.0 by using 10 wt% NaOH which is subjected to filtration sterilization by a 0.2 mu m filter membrane, and uniformly mixing.
(3) Centrifuging the mixed gel obtained in the step (2) for 10 min at 3000 g, subpackaging in 15 mL light-tight acrylic vacuum bottles, and preserving at 4 ℃.
Example 4
The embodiment provides a gel containing a leukocyte extract, wherein the gel containing the leukocyte extract is prepared from the following raw materials in percentage by mass:
5% of leukocyte extract, 3% of panthenol, 1.5% of Leuconostoc mesenteroides/radish root fermentation product filtrate, 1% of carnosine, 0.3% of natural heparinoids, 0.1% of allantoin, 5% of medical glycerol, 3% of propylene glycol, 980% of medical sterile carbomer, 0.02% of sodium ethylenediamine tetraacetate and the balance of conditioned medium;
wherein the leukocyte extract is the leukocyte extract provided in preparation example 1; the conditioned medium was the conditioned medium provided in preparation example 5.
The gel containing the leukocyte extract in this example was prepared by the following steps:
(1) Adding the conditioned medium into a sterile 500 mL centrifuge tube, adding the leukocyte extract, panthenol, leuconostoc mesenteroides/radix Raphani fermentation product filtrate, carnosine, natural heparinoids, allantoin, glycerol, propylene glycol, carbomer 980, and sodium ethylenediamine tetraacetate according to the above ratio, and finally adding the conditioned medium to 500 mL, and mixing well.
(2) And (3) regulating the pH value of the mixed gel obtained in the step (1) to 7.0 by using 10 wt% NaOH which is subjected to filtration sterilization by a 0.2 mu m filter membrane, and uniformly mixing.
(3) Centrifuging the mixed gel obtained in the step (2) for 10 min at 3000 g, subpackaging in 15 mL light-tight acrylic vacuum bottles, and preserving at 4 ℃.
Comparative example 1
This comparative example provides a gel differing from example 1 in that the white blood cell extract was not added, the missing part was supplemented to 100% with conditioned medium, and the other component contents and preparation method were completely identical to those of example 1.
Comparative example 2
This comparative example provides a gel differing from example 1 in that the conditioned medium provided in preparation example 2 was replaced with an equal mass of MSC SFM (Gibco) medium, and the other component contents and preparation methods were identical to those of example 1.
Comparative example 3
This comparative example provides a gel differing from example 1 in that no panthenol is added and the glycerol content is increased to 8%, the other component contents and the preparation method are exactly the same as in example 1.
Comparative example 4
This comparative example provides a gel differing from example 1 in that no carnosine was added, the natural heparinoids increased to 0.8% and allantoin increased to 0.6%, and the other component contents and preparation methods were exactly the same as in example 1.
Comparative example 5
This comparative example provides a gel differing from example 1 in that the allantoin content was increased to 0.4% without the addition of natural heparinoids, and the other component contents and preparation methods were exactly the same as in example 1.
Comparative example 6
This comparative example provides a gel differing from example 1 in that the natural heparinoids content was increased to 0.4% without the addition of allantoin, and the other component contents and preparation methods were exactly the same as in example 1.
Test example 1
Test sample: the gels containing the leukocyte extract provided in examples 1 to 4, and the gels provided in comparative examples 1 to 6;
the testing method comprises the following steps: patients suffering from serious infection, blood system and nervous system diseases, allergic constitution, or gel allergy of the invention and mental diseases are excluded, volunteer patients meeting test conditions are selected, and informed consent is signed and the patients participate in the test. Patients who participated in the trial agreed to discontinue all topical, oral and injectable medications for treatment of postherpetic neuralgia. The pain area of the patient with postherpetic neuralgia is selected, and the wet tissue is used for wiping the pain area, and then gel is applied for treatment 2 times a day. The control group was selected from patients treated conventionally. The medicine is mecobalamin tablet 3 times daily, 0.5 per time mg.
Clinical test results: specific test results of the effective rate of the gel containing the leukocyte extract for treating PHN after herpes zoster are shown in table 1:
TABLE 1
Wherein, in the recovery, the effect, the invalidation and the total effective rate, the data before the brackets represent the number of examples, the numbers in the brackets represent the proportion of the number of people occupied by the project, and a and b represent the significant difference between the two in chi-square test (P < 0.05).
And (3) carrying out clinical treatment effect statistics: clinical treatment heals, namely pain feeling and accompanying symptoms completely disappear; has obvious effect, and obviously relieves pain and accompanying symptoms; ineffective, unrelieved pain sensation, or insignificant relief, and other symptoms are not ameliorated or aggravated. Total effective rate= (clinical cure+onset).
The time-to-use and time-to-use test results are shown in Table 2 below:
TABLE 2
In the test process, the gel is used by invalid patients for only 6-10 days, and the average time is 8.3 days. Whether these patients are effective after continued use is not yet definitive, and the present invention considers ineffective. The gel of the invention takes 46.06 days on average for curing PHN and 6.68 days on average for onset of action.
As shown in the test results of tables 1 and 2, the total effective rate of the gel containing the leukocyte extract is as high as 60-76%, the average time for curing is 46.06 days, and the average time for starting to take effect is 6.68 days. Therefore, the leucocyte extract-containing postherpetic neuralgia gel has remarkable curative effect on postherpetic pain, and various components are mutually matched, so that the curative effect on postherpetic pain is further improved, adverse reactions of medicines are reduced, the treatment time of the postherpetic pain is shortened, and the occurrence of postherpetic pain is reduced.
The data of comparative example 1 shows that the total effective rate of the gel without adding leukocyte extract is reduced and obvious, and the gel curing effect can be improved to a certain extent only by prolonging the gel using time, and the average effective time is extremely long.
The data of comparative examples 2 to 6 show that although the total effective rate of table 1 is not obvious compared with that of example 1, the data of table 2 show that the gel improves the effect of curing PHN by the gel to a certain extent on the basis of prolonging the time for using the gel, and the effective average time is relatively longer, so that the invention shows that various components are mutually matched, has a synergistic effect, can improve the treatment effect of herpes zoster, and further cures and shows the effective time.
Test example 2
Test sample: control group (mecobalamin control group); treatment group (gel containing leukocyte extract provided in example 1);
the testing method comprises the following steps: patients suffering from serious infection, blood system and nervous system diseases, allergic constitution, or gel allergy of the invention and mental diseases are excluded, volunteer patients meeting test conditions are selected, and informed consent is signed and the patients participate in the test. Patients who participated in the trial agreed to discontinue all topical, oral and injectable medications for treatment of postherpetic neuralgia. The pain area of the patient with postherpetic neuralgia is selected, and the wet tissue is used for wiping the pain area, and then gel is applied for treatment 2 times a day. And simultaneously counting the scoring results of the international Visual Analog Scale (VAS) before and after treatment.
The specific test results are shown in table 3 below:
TABLE 3 Table 3
As shown in Table 3, the gel containing the leucocyte extract for treating postherpetic neuralgia effectively treats PHN patients with age of 36-78 years, and average age of 58 years; the course of the disease is between 1 month and 11 years, and the average course is between 41.4 months. The treatment result shows that the traditional Chinese medicine composition generally takes effect for about one week, and is completely cured for 21-62 days.
In addition, fig. 1 to 4 are front and rear contrast charts of patients with herpes zoster at different positions, before and after treating PHN patients with the leukocyte extract-containing postherpetic neuralgia gel of the invention, as shown in fig. 1 to 4, the external application and direct administration of the herpes zoster on the skin surface are carried out, and after using the leukocyte extract-containing gel, the red marks at the positions where the herpes zoster appears almost disappear, thus the curative effect on postherpetic neuralgia is very remarkable.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims (10)

1. The gel containing the leukocyte extract is characterized by comprising the following raw materials in percentage by mass:
1-10% of leucocyte extract, 1-5% of panthenol, 0.5-2.5% of leuconostoc/radish root fermentation product filtrate, 0.5-2% of carnosine, 0.1-0.5% of natural heparinoids, 0.05-0.2% of allantoin, 1-15% of humectant, 0.5-2.5% of gel matrix, 0.01-0.05% of complexing agent and the balance of conditioned medium.
2. The leukocyte extract-containing gel according to claim 1, wherein said humectant is a combination of glycerin and propylene glycol in a mass ratio of (1-10): (1-5).
3. The leukocyte extract-containing gel according to claim 1, wherein said gel matrix is carbomer.
4. The leukocyte extract-containing gel according to claim 1, wherein said complexing agent is sodium ethylenediamine tetraacetate.
5. The gel containing a leukocyte extract according to claim 1, wherein said conditioned medium is obtained by culturing mesenchymal cells in a culture medium containing a leukocyte extract;
wherein the culture solution is selected from any one of DMEM/F12 culture medium, DMEM culture medium, alpha-MEM culture medium or MSC SFM culture medium;
wherein the addition amount of the white blood cell extract in the culture solution is 5-15 wt%.
6. The leukocyte extract-containing gel according to claim 1 or 5, wherein said conditioned medium is prepared by:
(a) Mixing the leukocyte extract with the culture solution to obtain a culture solution containing the leukocyte extract;
(b) Mixing umbilical cord mesenchymal stem cells with a culture solution containing a leukocyte extract, centrifuging, and removing supernatant to obtain resuspended cells; then placing the resuspended cells in a culture solution containing a leukocyte extract for culturing;
(c) And (5) after the cells grow to 70-80% confluence, collecting supernatant and carrying out centrifugal treatment to obtain a conditioned medium.
7. A method for preparing a gel containing a leukocyte extract according to any one of claims 1 to 6, characterized in that it comprises the steps of:
mixing the preparation raw materials of the gel containing the white blood cell extract according to the proportion, and then sequentially adjusting the pH and centrifuging to obtain the gel containing the white blood cell extract.
8. The method for preparing a gel containing leukocyte extract according to claim 7, wherein the reagent for adjusting pH is 5-15 wt% aqueous sodium hydroxide solution;
wherein, the pH is adjusted to 6.8-7.2.
9. The method for preparing a gel containing a leukocyte extract according to claim 7, wherein the centrifugal force of the centrifugal treatment is 2000 to 4000 g, and the time of the centrifugal treatment is 5 to 20 min.
10. Use of a leukocyte extract-containing gel according to any one of claims 1 to 6 in the preparation of a product for the treatment of postherpetic neuralgia.
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CN116870159A (en) * 2023-07-06 2023-10-13 周辉 A topical ointment for treating varicella and herpes zoster skin disease, and its preparation method
CN117159401A (en) * 2023-09-27 2023-12-05 广州美思生物技术有限公司 Scalp-activating, nourishing and protecting recombinant collagen composition and preparation method and application thereof

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