CN111548988B - Medical rinsing liquid and preparation method and application thereof - Google Patents

Medical rinsing liquid and preparation method and application thereof Download PDF

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CN111548988B
CN111548988B CN202010405909.0A CN202010405909A CN111548988B CN 111548988 B CN111548988 B CN 111548988B CN 202010405909 A CN202010405909 A CN 202010405909A CN 111548988 B CN111548988 B CN 111548988B
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CN111548988A (en
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王学庆
王楠
王嘉轩
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Jiangsu Zhihai Biotechnology Co ltd
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Abstract

The invention provides a medical rinsing liquid and a preparation method and application thereof, and belongs to the technical field of medical reagents. The medical rinsing liquid comprises glucose, vitamins, basic fibroblast growth factor, keratinocyte growth factor, hepatocyte growth factor, vascular endothelial growth factor, leptin and buffer; the concentrations of glucose, vitamins, basic fibroblast growth factor, keratinocyte growth factor, hepatocyte growth factor, vascular endothelial growth factor and leptin are 0.5-4 g/L, 0.002-0.02 g/L, 0.005-0.05 mg/L, 0.002-0.02 mg/L, 0.02-0.25 mg/L, 0.04-0.4 mg/L and 0.002-0.02 mg/L in sequence; the pH value of the medical rinsing liquid is 7.2-7.5. The medical rinsing liquid provided by the invention can effectively ensure the activity of cells or tissues.

Description

Medical rinsing liquid and preparation method and application thereof
Technical Field
The invention relates to the technical field of medical reagents, in particular to a medical rinsing liquid and a preparation method and application thereof.
Background
In the in vitro treatment of cells or tissues, it is generally necessary to wash them with a rinsing liquid in order to remove impurities on the basis of guaranteeing the activity of the cells or tissues, so that the subsequent related treatments are smoothly carried out. Therefore, the rinsing liquid has an important influence on the smooth progress of the in vitro treatment process of the cells or tissues.
At present, the fat particle injection autograft technique is widely applied to soft tissue filling and has the advantages of good biocompatibility, convenient acquisition, abundant sources, simple operation, good filling effect, low cost and the like. However, the absorption rate of the fat particles after the autologous transplantation is higher, the volume and weight of the transplanted fat particles are reduced by more than 50%, the effect after the fat transplantation is difficult to predict, and the non-viable cells are easy to liquefy, necrotic and infect, so that the survival rate of the fat transplantation is low.
The survival rate of fat transplantation is related to a plurality of factors, and the steps of obtaining fat tissues, treating in vitro, injecting and the like can have a certain influence on the survival rate of fat transplantation. Fat particles are usually obtained by negative pressure liposuction under the condition of swelling anesthesia in the autograft technique of fat particle injection, but at present, there is no consensus on how to treat fat cells, and when a part of plastic doctors wash fat cells, physiological saline (ringer's solution can be used for replacing physiological saline clinically) is adopted, so that the survival rate still needs to be improved.
Disclosure of Invention
The invention aims to provide a medical rinsing liquid, a preparation method and application thereof, and the medical rinsing liquid provided by the invention adopts the combined action of a plurality of growth factors and is matched with an energy substance and a buffer system, so that the activity of cells or tissues can be effectively ensured, and the survival rate of autologous fat transplantation can be effectively improved.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a medical rinsing liquid, which comprises glucose, vitamins, basic fibroblast growth factor, keratinocyte growth factor, hepatocyte growth factor, vascular endothelial growth factor, leptin and buffer solution;
wherein the concentration of glucose is 0.5-4 g/L, the concentration of vitamin is 0.002-0.02 g/L, the concentration of basic fibroblast growth factor is 0.005-0.05 mg/L, the concentration of keratinocyte growth factor is 0.002-0.02 mg/L, the concentration of hepatocyte growth factor is 0.02-0.25 mg/L, the concentration of vascular endothelial growth factor is 0.04-0.4 mg/L, and the concentration of leptin is 0.002-0.02 mg/L;
the solvent of the buffer solution is water;
the pH value of the medical rinsing liquid is 7.2-7.5.
Preferably, the concentration of glucose in the medical rinsing liquid is 0.5-2 g/L, the concentration of vitamin is 0.002-0.01 g/L, the concentration of basic fibroblast growth factor is 0.005-0.03 mg/L, the concentration of keratinocyte growth factor is 0.005-0.015 mg/L, the concentration of hepatocyte growth factor is 0.04-0.15 mg/L, the concentration of vascular endothelial growth factor is 0.08-0.2 mg/L, and the concentration of leptin is 0.005-0.015 mg/L.
Preferably, the concentration of glucose in the medical rinsing liquid is 1g/L, the concentration of vitamin is 0.002g/L, the concentration of basic fibroblast growth factor is 0.01mg/L, the concentration of keratinocyte growth factor is 0.01mg/L, the concentration of hepatocyte growth factor is 0.05mg/L, the concentration of vascular endothelial growth factor is 0.1mg/L, and the concentration of leptin is 0.01mg/L.
Preferably, the vitamins comprise vitamin B, vitamin C, vitamin K1 and vitamin E, and the mass ratio of the vitamin B to the vitamin C to the vitamin K1 to the vitamin E is 1:1:1:1.
Preferably, the buffer is a phosphate buffer, comprising NaCl, KCl, naHCO in composition 3 、Na 2 HPO 4 、KH 2 PO 4 And water; the pH value of the phosphate buffer solution is 7.2-7.5.
The invention provides a preparation method of the medical rinsing liquid, which comprises the following steps:
and mixing the components of the medical rinsing liquid, and then sterilizing to obtain the rinsing liquid.
The invention provides application of the medical rinsing liquid according to the scheme or the medical rinsing liquid prepared by the preparation method according to the scheme in cell or tissue culture, transfer, storage or rinsing.
Preferably, the cells comprise stem cells or adipocytes and the tissue comprises adipose tissue.
Preferably, the method of rinsing comprises the steps of:
and (3) mixing the medical rinsing liquid with the cells or tissues to be treated, rinsing, and removing liquid in the obtained system to finish rinsing of the cells or tissues.
Preferably, the rinsing times are 2-3 times, and the volume ratio of the medical rinsing liquid to the cells or tissues to be treated is 3-4:10 in the single rinsing process.
The invention provides a medical rinsing liquid, which comprises glucose, vitamins, basic fibroblast growth factor, keratinocyte growth factor, hepatocyte growth factor, vascular endothelial growth factor, leptin and buffer solution; wherein the concentration of glucose is 0.5-4 g/L, the concentration of vitamin is 0.002-0.02 g/L, the concentration of basic fibroblast growth factor is 0.005-0.05 mg/L, the concentration of keratinocyte growth factor is 0.002-0.02 mg/L, the concentration of hepatocyte growth factor is 0.02-0.25 mg/L, the concentration of vascular endothelial growth factor is 0.04-0.4 mg/L, and the concentration of leptin is 0.002-0.02 mg/L; the solvent of the buffer solution is water; the pH value of the medical rinsing liquid is 7.2-7.5. The medical rinse liquid provided by the invention adopts the combined action of a plurality of growth factors and is matched with an energy substance and a buffer system, so that the activity of cells or tissues can be effectively ensured, and the survival rate of autologous fat transplantation can be effectively improved. The results of the examples show that the rinsing liquid provided by the invention is used for rinsing the fat particles, and the cell proliferation rate is obviously higher than that of the fat particles rinsed by normal saline; compared with physiological saline containing 1% of double antibodies, the rinsing liquid provided by the invention is used for rinsing and culturing adipose tissues, and primary cells can be adhered to and stretched more quickly.
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FIG. 1 is a graph showing comparison of cell proliferation rates of adipocytes obtained in the experimental group and the control group of application example 1 after culturing under the same conditions;
FIG. 2 is a graph showing comparison of the growth states of cells after the adipose-derived stem cells of the experimental group and the control group of application example 2 were isolated and cultured under the same conditions for 3 days.
Detailed Description
The invention provides a medical rinsing liquid, which comprises glucose, vitamins, basic fibroblast growth factor, keratinocyte growth factor, hepatocyte growth factor, vascular endothelial growth factor, leptin and buffer solution; the solvent of the buffer solution is water; the pH value of the medical rinsing liquid is 7.2-7.5. In the invention, the concentration of glucose in the medical rinsing liquid is 0.5-4 g/L, the concentration of vitamin is 0.002-0.02 g/L, the concentration of alkaline fibroblast growth factor is 0.005-0.05 mg/L, the concentration of keratinocyte growth factor is 0.002-0.02 mg/L, the concentration of hepatocyte growth factor is 0.02-0.25 mg/L, the concentration of vascular endothelial growth factor is 0.04-0.4 mg/L, and the concentration of leptin is 0.002-0.02 mg/L; preferably, the concentration of glucose in the medical rinsing liquid is 0.5-2 g/L, the concentration of vitamin is 0.002-0.01 g/L, the concentration of basic fibroblast growth factor is 0.005-0.03 mg/L, the concentration of keratinocyte growth factor is 0.005-0.015 mg/L, the concentration of hepatocyte growth factor is 0.04-0.15 mg/L, the concentration of vascular endothelial growth factor is 0.08-0.2 mg/L, and the concentration of leptin is 0.005-0.015 mg/L; more preferably, the concentration of glucose in the medical rinse solution is 1g/L, the concentration of vitamin is 0.002g/L, the concentration of basic fibroblast growth factor is 0.01mg/L, the concentration of keratinocyte growth factor is 0.01mg/L, the concentration of hepatocyte growth factor is 0.05mg/L, the concentration of vascular endothelial growth factor is 0.1mg/L, and the concentration of leptin is 0.01mg/L.
In the present invention, the basic fibroblast growth factor, keratinocyte growth factor, hepatocyte growth factor, vascular endothelial growth factor and leptin may be natural growth factors (or leptin), or recombinant growth factors (or leptin); in embodiments of the invention, recombinant growth factors (or leptins) are particularly employed.
In the present invention, the vitamins preferably include vitamin B, vitamin C, vitamin K1 and vitamin E, and the mass ratio of the vitamin B, the vitamin C, the vitamin K1 and the vitamin E is preferably 1:1:1:1. In the present invention, the vitamins can function as cofactors (e.g., vitamin K and most of vitamin B) and biological antioxidants (e.g., vitamin C and vitamin E), which are beneficial for maintaining cell viability and stability.
In the present invention, the buffer is preferably a phosphate buffer, the pH of which is preferably 7.4, and the composition preferably includes NaCl, KCl, naHCO 3 、Na 2 HPO 4 、KH 2 PO 4 And water; the pH value of the phosphate buffer solution is preferably 7.2-7.5; in the present invention, more preferably, the concentration of NaCl in the medical rinse solution is 8g/L, the concentration of KCl is 0.4g/L, and the concentration of NaHCO 3 The concentration of (C) is 0.35g/L, na 2 HPO 4 Is 0.1g/L, KH 2 PO 4 Is 0.06g/L. In the present invention, the buffer can provide a necessary buffer environment for extraction and purification of cells, tissues, and the like. In the present invention, it is preferable to use a phosphate buffer solution, which can remove blood, anesthetic, fiber fragments, etc. mixed in cells or tissues when rinsing the cells or tissues; at the same time can improve the extraction from the bodyThe cell stability of the adipose cells and the mesenchymal stem cells, the integrity and the activity of the adipose cells are maintained, and the cell transplantation survival rate is improved.
In the present invention, the basic fibroblast growth factor (bFGF) is capable of promoting proliferation of preadipocytes and connective tissue, promoting a revascularization process, and facilitating survival of adipocytes; keratinocyte Growth Factor (KGF) and Vascular Endothelial Growth Factor (VEGF) can promote endothelial cell mitosis, promote nerve vessel regeneration around transplanted tissue, and increase survival rate of adipocyte; hepatocyte Growth Factor (HGF) has strong effects of promoting angiogenesis and establishing collateral circulation, and is beneficial to improving survival rate of autologous fat transplantation; leptin (Leptin) is secreted by fat cells, and can promote transformation of preadipocyte into mature fat cells, and vascular endothelial cells have Leptin receptor, and Leptin can promote proliferation of vascular endothelial cells, promote local vascular proliferation, increase local blood supply, and enhance vascular proliferation of transplanted tissue.
In the present invention, the glucose is used as an energy substance, and can provide a necessary nutritional environment for extraction and purification of adipocytes, tissues, and the like.
The invention adopts the combined action of a plurality of growth factors and is matched with an energy substance and a buffer system, thereby effectively improving the survival rate of autologous fat transplantation.
In the present invention, the water is preferably purified water.
The invention provides a preparation method of the medical rinsing liquid, which comprises the following steps: and mixing the components of the medical rinsing liquid, and then sterilizing to obtain the rinsing liquid.
In the invention, after the components of the medical rinsing liquid are mixed according to the proportion, the pH value of the obtained mixed liquid is 7.2-7.5, and HCl or NaOH can be further adopted to adjust the pH value of the mixed liquid according to the requirement. The sterilization is not particularly limited, and methods well known to those skilled in the art can be adopted; the invention preferably adopts filtration sterilization, in particular to realize sterilization by mixing the components of the medical rinse liquid and then passing through a 0.22 mu m filter membrane. After sterilization, the invention preferably carries out sub-packaging under the protection of helium gas (so as to avoid oxidation of unstable components) to obtain the rinsing liquid.
The invention provides application of the medical rinsing liquid in the technical scheme or the medical rinsing liquid prepared by the preparation method in the technical scheme in cell or tissue culture, transfer, storage or rinsing. The type of the cells or tissues is not particularly limited, and any cells or tissues which need to be subjected to the in vitro treatment (culture, transfer, storage or rinsing) can be specifically selected from adipocytes or stem cells, and the stem cells can be adipose stem cells or other stem cells, and the invention is not particularly limited thereto; the tissue may be adipose tissue. The medical rinsing liquid provided by the invention can be applied to fat transplantation operations of dermatology, plastic surgery and medical cosmetology, and can be particularly used for in vitro treatment of extracted fat cells and in vitro treatment of stem cells in the stem cell transplantation process.
In the present invention, the rinsing method preferably includes the steps of: and (3) mixing the medical rinsing liquid with the cells or tissues to be treated, rinsing, and removing liquid in the obtained system to finish rinsing of the cells or tissues. In the invention, the rinsing times are preferably 2-3 times, and the volume ratio of the medical rinsing liquid to the cells or tissues to be treated is preferably 3-4:10 in the single rinsing process. In the embodiment of the invention, specifically, cells or tissues to be treated are filled into an injector, the injector is used for sucking the medical rinsing liquid, the medical rinsing liquid is turned up and down for 3 to 5 times, and then the medical rinsing liquid is kept stand for 5 to 15 minutes to remove lower liquid, so that 1 rinsing is completed; rinsing is repeated for 1-2 times according to the above operation to obtain purified cells or tissues. The method provided by the invention is used for rinsing the cells or tissues, the loss rate of the cells or tissues after the whole rinsing process is finished is less than 5%, and the survival rate of the cells is high.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. 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.
Example 1
In this example, recombinations bFGF, KGF, HGF, VEGF and Leptin were purchased from R & D Systems, trade names and trade numbers were as follows:
recombinant bFGF: under the trade name Recombinant Human FGF basic, under the trade name 233-FB-025;
recombinant KGF: the trade name is Recombinant Human KGF/FGF-7Protein, and the trade name is 251-KG-050;
recombinant HGF: under the trade name Recombinant Human HGF Protein, trade name 294-HG-025;
recombinant VEGF: under the trade name Recombinant Human VEGF, under the trade name 293-VE-050
Recombinant Leptin: under the trade name Recombinant Human LeptinProtein, commercial number 398-LP-01M.
Weighing the following raw materials according to the following concentration: naCl is 8g/L, KCl is 0.4g/L, naHCO 3 0.35g/L Na 2 HPO 4 KH at 0.1g/L 2 PO 4 0.06g/L glucose 1g/L, 0.5mg/L vitamin B0.5 mg/L vitamin C0.5 mg/L vitamin E0.5 mg/L vitamin K1 0.5mg/L bFGF 0.01mg/L KGF 0.01mg/L HGF 0.05mg/L VEGF 0.1mg/L Leptin 0.01mg/L, balance purified water;
the preparation method comprises the following steps: mixing the above materials, adjusting pH to 7.4, filtering, sterilizing (filtering with 0.22 μm filter membrane), and packaging under helium protection to obtain the final product.
Application example 1
Rinsing of adipocytes
Experimental group: sucking under negative pressure by using a syringe to obtain fat particles, filling 50mL of fat particles into the syringe, sucking 15mL of the rinsing liquid prepared in the example 1 by using the syringe, turning up and down for 4 times, standing for 10min to remove the lower liquid, and completing rinsing for 1 time; rinsing was repeated 2 more times according to the above procedure to obtain purified fat particles.
Control group: the procedure was followed except that the rinse solution prepared in example 1 was replaced with physiological saline.
Taking purified fat particles of experimental group and control group, diluting with DMEM+10% FBS medium to cell number of 5×10 4 After 100. Mu.L of each well is inoculated into a 96-well plate per mL, pre-cultured for 24 hours, CCK-8 detection is carried out, specifically 10. Mu.L of CCK-8 solution is added into each well, incubation is carried out for 2 hours, the light absorption value at 450nm is measured, and the cell proliferation rate is calculated, and the result is shown in figure 1, wherein the "normal saline" corresponds to a control group and the "rinse liquid" corresponds to an experimental group in figure 1. As can be seen from FIG. 1, after the adipocytes were cultured under the same conditions by the rinse solution prepared in example 1, the cell proliferation rate was 135% (P) of the control group<0.01)。
Application example 2
Isolated culture of adipose-derived stem cells
Experimental group: fully sucking the adipose tissue blocks by using the rinsing liquid prepared in the example 1, removing connective tissues and small blood vessels on the adipose tissue blocks by using an ophthalmic scissors, rinsing the adipose tissue blocks for 3 times by using the rinsing liquid according to the method of the application example 1, placing the rinsed adipose tissue blocks on sterile gauze, and drying the adipose tissue blocks (particularly, airing the adipose tissue blocks in a sterile environment until no obvious rinsing liquid trace exists on the surface); adding pure calf serum to the dried adipose tissue block, and rapidly cutting the adipose tissue block into pieces of about 1mm 3 Is planted in a dispersion way in a small tissue block of 25cm 2 In the flask, the flask was cultured upside down in a carbon dioxide incubator at 37 ℃.
Control group: the procedure was followed except that the rinse solution prepared in example 1 was replaced with 1% diabody in physiological saline.
The cells after 3 days of culture in the experimental group and the control group were observed by a microscope, and the results are shown in FIG. 2, in which "physiological saline" corresponds to the control group and "rinse" corresponds to the experimental group in FIG. 2. As can be seen from fig. 2, the primary cells adhere to and spread more rapidly when the adipose tissues are cultured after rinsing with the rinsing liquid provided by the present invention, as compared with the control group. The continuous culture shows that the bottle bottom is paved by using primary cells rinsed by physiological saline containing 1% of double antibodies for 15 days, and the bottle bottom can be paved by using the primary cells rinsed by the rinsing liquid provided by the invention for 10 days. This shows that the rinsing liquid provided by the invention can promote the growth of the adipose-derived stem cells and maintain the activity of the adipose-derived stem cells.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. A medical rinsing liquid is characterized by comprising glucose, vitamins, basic fibroblast growth factor, keratinocyte growth factor, hepatocyte growth factor, vascular endothelial growth factor, leptin and buffer solution;
wherein the concentration of glucose is 0.5-4 g/L, the concentration of vitamin is 0.002-0.02 g/L, the concentration of basic fibroblast growth factor is 0.005-0.05 mg/L, the concentration of keratinocyte growth factor is 0.002-0.02 mg/L, the concentration of hepatocyte growth factor is 0.02-0.25 mg/L, the concentration of vascular endothelial growth factor is 0.04-0.4 mg/L, and the concentration of leptin is 0.002-0.02 mg/L;
the vitamin consists of vitamin B, vitamin C, vitamin K1 and vitamin E;
the solvent of the buffer solution is water; the buffer solution is phosphate buffer solution, and comprises NaCl, KCl, naHCO in composition 3 、Na 2 HPO 4 、KH 2 PO 4 And water;
the pH value of the medical rinsing liquid is 7.2-7.5.
2. The medical rinse solution according to claim 1, wherein the concentration of glucose in the medical rinse solution is 0.5 to 2g/L, the concentration of vitamins is 0.002 to 0.01g/L, the concentration of basic fibroblast growth factor is 0.005 to 0.03mg/L, the concentration of keratinocyte growth factor is 0.005 to 0.015mg/L, the concentration of hepatocyte growth factor is 0.04 to 0.15mg/L, the concentration of vascular endothelial growth factor is 0.08 to 0.2mg/L, and the concentration of leptin is 0.005 to 0.015mg/L.
3. The medical rinse solution according to claim 2, wherein the concentration of glucose in the medical rinse solution is 1g/L, the concentration of vitamins is 0.002g/L, the concentration of basic fibroblast growth factor is 0.01mg/L, the concentration of keratinocyte growth factor is 0.01mg/L, the concentration of hepatocyte growth factor is 0.05mg/L, the concentration of vascular endothelial growth factor is 0.1mg/L, and the concentration of leptin is 0.01mg/L.
4. A medical rinse solution according to any one of claims 1 to 3, wherein the mass ratio of vitamin B, vitamin C, vitamin K1 and vitamin E is 1:1:1:1.
5. the medical rinse solution of claim 1, wherein the phosphate buffer has a pH of 7.2 to 7.5.
6. The method for preparing the medical rinse liquid according to any one of claims 1 to 5, comprising the steps of:
and mixing the components of the medical rinsing liquid, and then sterilizing to obtain the rinsing liquid.
7. Use of a medical rinse solution according to any one of claims 1 to 5 or a medical rinse solution prepared by a method of preparation according to claim 6 for the culture, transfer, storage or rinsing of cells or tissues.
8. The use of claim 7, wherein the cells comprise stem cells or adipocytes and the tissue comprises adipose tissue.
9. Use according to claim 7 or 8, characterized in that the rinsing method comprises the following steps:
and (3) mixing the medical rinsing liquid with the cells or tissues to be treated, rinsing, and removing liquid in the obtained system to finish rinsing of the cells or tissues.
10. The use according to claim 9, wherein the number of rinses is 2-3, and the volume ratio of medical rinse liquid to cells or tissues to be treated is 3-4 during a single rinse: 10.
CN202010405909.0A 2020-05-14 2020-05-14 Medical rinsing liquid and preparation method and application thereof Active CN111548988B (en)

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