CN105255815A - Fibroblast cleaning solution and preparation method thereof - Google Patents
Fibroblast cleaning solution and preparation method thereof Download PDFInfo
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- CN105255815A CN105255815A CN201510726086.0A CN201510726086A CN105255815A CN 105255815 A CN105255815 A CN 105255815A CN 201510726086 A CN201510726086 A CN 201510726086A CN 105255815 A CN105255815 A CN 105255815A
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- amphotericin
- gentamicin
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Abstract
The invention relates to the technical field of cell cleaning solution, in particular to a fibroblast cleaning solution and a preparation method thereof2+And Mg2+The method has the advantages that the method does not influence the activity of digestive enzymes, the enzymolysis activity is good, the digestion time is short, the obtained fibroblasts are sufficient in quantity and high in survival rate, glucose contained in D-Hank's liquid can provide tissue nutrition in the tissue block cleaning process, better cell activity is kept to the culture stage, the method is favorable for cell growth, amphotericin B can effectively inhibit the growth of fungi, gentamicin has a strong inhibition effect on the growth of bacteria, the total consumption of antibiotics in cleaning liquid is reduced, the use of antibiotics is greatly reduced, meanwhile, the bacteria and fungi are strongly inhibited, and the cells are well protected.
Description
Technical field
The present invention relates to cell scavenging solution technical field, be specifically related to a kind of fibroblast scavenging solution and preparation method thereof.
Background technology
Fibroblast, also referred to as inoblast, is the main cellular of loose connective tissue, belongs to terminally differentiated cells, by the Derived from Mesenchymal Stem Cells of embryonic stage.Fibroblast does not substantially have differentiation function but splitting function is vigorous, can keep good fissiparity ability in vitro under culture condition.In skin, the major function of fibroblast is the extracellular matrixs such as secretion collagen protein, spandex, hyaluronic acid, and this collagen protein deriving from self repairs system once be formed, and its life animated period is 10-15.Fibroblast is the host cell composition in dermis of skin, and it and collegen filament, spandex fiber and the matrix components self secreted together constitute the main body of corium.The major function of fibroblast is: synthesis and secretion collegen filament, spandex fiber, Matrix protein material and some somatomedin, have vital role to the elasticity and toughness that maintain skin, the minimizing of fibroblast is also the major reason causing wrinkle to produce.
Along with the raising of people's living standard, require more and more higher, more focus on radiant to the face of self, at present, in esthetics shaping, Non-surgical treatment rapid development, the ratio occupied has exceeded 70%.Dermal filler has many types, and because fibroblast has the function of synthesis collagen protein, its utilization also just develops rapidly, so autologous fibroblast injection smoothing wrinkle becomes a kind of emerging methods for the treatment of.
Fibroblast separates by human skin tissue, because epidermis is exposed in air, parasitic multiple microorganism, will from skin histology separation and Culture fibroblast, whole separation and Culture process is needed to keep absolutesterility, so removing microorganism in process skin histology block process is vital step.
In prior art, many times the microorganisms such as bacterium can not be eliminated in skin histology block treating processes, cause cell in cultivation to be contaminated by bacterial, cultivate unsuccessfully; Be contaminated by bacterial to prevent cultured cells, usually in nutrient solution, a large amount of microbiotic is added, with bacteria growing inhibiting, in culturing process, a large amount of bacterial growth is there is not in this way although adopt, can cell once to clinical application, repressed bacterium is exactly the major hidden danger buried, and impacts the healthy of user, and security can not meet the requirement of people.
Summary of the invention
The present invention is directed to problems of the prior art; a kind of fibroblast scavenging solution is provided; it can not impact the activity of digestive ferment; enzymolysis vigor is good, digestion time is short; the fibroblast abundant amount obtained, motility rate is high, while greatly reducing antibiotic usage; to bacterium and fungi, there is very strong restraining effect, cell is played a good protection.
The present invention also provides a kind of preparation method of fibroblast scavenging solution.
The present invention is achieved through the following technical solutions this object:
A kind of fibroblast scavenging solution, the raw material of described scavenging solution comprises D-Hank ' s liquid, amphotericin B, gentamicin, and in described scavenging solution, the final concentration of amphotericin B is 0.2-10ug/mL, and the final concentration of gentamicin is 10-200U/mL.
As preferred scheme, in described scavenging solution, the final concentration of amphotericin B is 0.3-5ug/mL, and the final concentration of gentamicin is 15-100U/mL.
As further preferred scheme, it is characterized in that, in described scavenging solution, the final concentration of amphotericin B is 0.5ug/mL, and the final concentration of gentamicin is 20U/mL.
As preferably, the concentration of described amphotericin B is 0.2%, and the concentration of described gentamicin is 0.3%.
A preparation method for fibroblast scavenging solution, comprises the following steps:
Be the amphotericin B of 0.2% respectively by concentration, concentration be 0.3% gentamicin join in D-Hank ' s liquid, the final concentration of amphotericin B is made to be 0.2-10ug/mL, the final concentration of gentamicin is 10-200U/mL, mixes, obtained fibroblast scavenging solution.
Wherein, described D-Hank ' s liquid stores in advance under the condition of 4 DEG C.
Relative to prior art, beneficial effect of the present invention is: fibroblast scavenging solution of the present invention comprises D-Hank ' s liquid, amphotericin B, gentamicin, in described scavenging solution, the final concentration of amphotericin B is 0.2-10ug/mL, the final concentration of gentamicin is 10-200U/m, liquid based on D-Hank ' s liquid, not containing Ca
2+and Mg
2+, can not impact the activity of digestive ferment, enzymolysis vigor is good, digestion time is short, the fibroblast abundant amount obtained, motility rate is high, the glucose contained in D-Hank ' s liquid can provide histotrophic nutrition in tissue block cleaning process, keep better cell viability to cultivation stage, be conducive to Growth of Cells, amphotericin B can effectively grow by Antifungi, the growth of gentamicin to bacterium has very strong restraining effect, in scavenging solution, antibiotic dosage totally reduces, while greatly reducing antibiotic usage, to bacterium and fungi, there is very strong restraining effect, cell is played a good protection.
Accompanying drawing explanation
Fig. 1 is the cell growth state figure of control group.
Fig. 2 is the cell growth state figure of experimental group.
Embodiment
Describe the present invention below in conjunction with drawings and the specific embodiments.
Embodiment 1,
The present embodiment provides a kind of preparation method of fibroblast scavenging solution, comprises the following steps:
Be the amphotericin B of 0.2% respectively by concentration, concentration be 0.3% gentamicin join in D-Hank ' s liquid, the final concentration of amphotericin B is made to be 0.2-10ug/mL, the final concentration of gentamicin is 10-200U/mL, mixes, obtained fibroblast scavenging solution.
Wherein, described D-Hank ' s liquid stores in advance under the condition of 4 DEG C.
Embodiment 2.
The raw material of the fibroblast scavenging solution of the present embodiment comprises D-Hank ' s liquid, amphotericin B, gentamicin, and in described scavenging solution, the final concentration of amphotericin B is 0.2ug/mL, and the final concentration of gentamicin is 10U/mL.
Embodiment 3.
The raw material of the fibroblast scavenging solution of the present embodiment comprises D-Hank ' s liquid, amphotericin B, gentamicin, and in described scavenging solution, the final concentration of amphotericin B is 0.3ug/mL, and the final concentration of gentamicin is 15U/mL.
Embodiment 4.
The raw material of the fibroblast scavenging solution of the present embodiment comprises D-Hank ' s liquid, amphotericin B, gentamicin, and in described scavenging solution, the final concentration of amphotericin B is 5ug/mL, and the final concentration of gentamicin is 100U/mL.
Embodiment 5.
The raw material of the fibroblast scavenging solution of the present embodiment comprises D-Hank ' s liquid, amphotericin B, gentamicin, and in described scavenging solution, the final concentration of amphotericin B is 10ug/mL, and the final concentration of gentamicin is 200U/mL.
Embodiment 6.
The raw material of the fibroblast scavenging solution of the present embodiment comprises D-Hank ' s liquid, amphotericin B, gentamicin, and in described scavenging solution, the final concentration of amphotericin B is 0.5ug/mL, and the final concentration of gentamicin is 20U/mL.
Comparative example 1.
The present embodiment preparation regular growth scavenging solution, described regular growth scavenging solution comprises PBS, concentration be 0.2% penicillin and concentration be the Streptomycin sulphate of 0.3%.
The washing test of embodiment 7, fibroblast scavenging solution
1) choose male and female small white mouse each 5 (buying in Guangdong Medical Lab Animal Center), body weight is all at about 20g, and normal condition is raised one week, observes mouse skin situation at any time, in case there is tetter mouse;
2) put to death mouse by disconnected cervical approach, select the more smooth position depilatory cream of mouse to remove fine hair; Every mouse cuts 1 × 1cm with sterilizing surgical scissors respectively
3skin histology block 6 pieces, is divided into two groups at random, and one group is control group, and one group is experimental group, is placed in 50mL centrifuge tube;
3) experimental group is with the formulated fibroblast scavenging solution eccentric cleaning of embodiment 62 times, is cut into 1-2mm with eye scissors
2fritter, then add fibroblast scavenging solution and clean 3 times; Control group with the formulated regular growth scavenging solution of comparative example 1, with experimental group disposal methods tissue block;
4) 4 DEG C of digestion 3h are put with NTx enzyme, transfer puts 37 DEG C of digestion completely, transitional cell cultivates two days to six orifice plates, extract culture supernatant, bottle is detected to Mei Liai microbial cultivation instrument with injector to inject, cultivating 3 days, whether have bacterium, two groups of experimental results are as shown in table 1 if detecting culture supernatant:
Table 1 Bacteria Detection experimental result
Note: ▲: for without bacterial growth ★: for there being bacterial growth
As shown in Table 1: the scavenging solution cleaning skin histology block of experimental group does not have bacterial growth, and the microbial contamination rates such as bacterium are 0%; And control group in different number of days four groups have bacterial growth, contamination rate is 80%, and illustrative experiment group scavenging solution has very strong restraining effect to bacterium and fungi, removes bacterium comparatively control group Be very effective.
5) cellular form observation experiment
The discarded control group culture dish having had bacterial growth, choose and do not have the culture dish of bacterial growth to change liquid continuation cultivation by the 5th day, experimental group continues to change liquid equally and cultivates the 5th day, observe two groups of cell states, experimental result respectively as shown in Figure 1, 2, can be found out by Fig. 1,2, when cultivating the 5th day, experimental group Growth of Cells is good, and degree of converging reaches about 60%; Cellular control unit state is general, and rare numbers, illustrates that fibroblast scavenging solution of the present invention can play a good protection to cell.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (6)
1. a fibroblast scavenging solution, it is characterized in that, the raw material of described scavenging solution comprises D-Hank ' s liquid, amphotericin B, gentamicin, and in described scavenging solution, the final concentration of amphotericin B is 0.2-10ug/mL, and the final concentration of gentamicin is 10-200U/mL.
2. fibroblast scavenging solution according to claim 1, is characterized in that, in described scavenging solution, the final concentration of amphotericin B is 0.3-5ug/mL, and the final concentration of gentamicin is 15-100U/mL.
3. fibroblast scavenging solution according to claim 2, is characterized in that, in described scavenging solution, the final concentration of amphotericin B is 0.5ug/mL, and the final concentration of gentamicin is 20U/mL.
4. the fibroblast scavenging solution according to claims 1 to 3 any one, is characterized in that, the concentration of described amphotericin B is 0.2%, and the concentration of described gentamicin is 0.3%.
5. a preparation method for fibroblast scavenging solution, is characterized in that, comprises the following steps:
Be the amphotericin B of 0.2% respectively by concentration, concentration be 0.3% gentamicin join in D-Hank ' s liquid, the final concentration of amphotericin B is made to be 0.2-10ug/mL, the final concentration of gentamicin is 10-200U/mL, mixes, obtained fibroblast scavenging solution.
6. the preparation method of fibroblast scavenging solution according to claim 5, is characterized in that, described D-Hank ' s liquid stores in advance under the condition of 4 DEG C.
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| CN201510726086.0A CN105255815A (en) | 2015-10-29 | 2015-10-29 | Fibroblast cleaning solution and preparation method thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1074708A (en) * | 1993-02-22 | 1993-07-28 | 北京邮电医院 | The cultural method of homogeneity-heterobody skin cell |
| CN103667177A (en) * | 2013-12-10 | 2014-03-26 | 中山奈德生物科技有限公司 | Kit used for muscle fiber in vitro culturing |
| CN104083803A (en) * | 2013-04-01 | 2014-10-08 | 陕西佰傲再生医学有限公司 | Biomembrane for ocular surface restoration and preparation method thereof |
-
2015
- 2015-10-29 CN CN201510726086.0A patent/CN105255815A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1074708A (en) * | 1993-02-22 | 1993-07-28 | 北京邮电医院 | The cultural method of homogeneity-heterobody skin cell |
| CN104083803A (en) * | 2013-04-01 | 2014-10-08 | 陕西佰傲再生医学有限公司 | Biomembrane for ocular surface restoration and preparation method thereof |
| CN103667177A (en) * | 2013-12-10 | 2014-03-26 | 中山奈德生物科技有限公司 | Kit used for muscle fiber in vitro culturing |
Non-Patent Citations (2)
| Title |
|---|
| 胡晓红等: "一种新型的原代成纤维细胞分离培养方法", 《2013年全军烧伤外科学术年会论文集》 * |
| 陆凤花等: "水牛皮肤成纤维细胞的分离与体外培养", 《细胞生物学杂志》 * |
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