CN116411041A - Method for detecting sebum fusion and release based on linoleic acid induced human sebaceous gland cells - Google Patents
Method for detecting sebum fusion and release based on linoleic acid induced human sebaceous gland cells Download PDFInfo
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Abstract
The invention discloses a method for detecting sebum fusion and release based on linoleic acid induced human sebaceous gland cells, which takes TG as a biomarker to quantitatively detect skin sebum release, and takes TG, cell lipid droplets, PLIN2 and CIDEA to detect and evaluate skin sebum fusion and release. The invention adopts a method of inducing human sebaceous gland cell SZ95 by linoleic acid, simulates the whole process from sebum synthesis and sebum fusion to sebum release of the sebaceous gland cell, creatively constructs a detection method of a sebum release stage, takes the LA after inducing the sebaceous gland cell for a specific time as the beginning of the sebum release stage, adopts a sample to be detected for treatment at the beginning of the sebum release stage, quantitatively detects the content of a specific component TG of the sebum secreted by the cell into a culture supernatant as a secretion index at the ending time of the release stage, further evaluates the inhibition rate of the sebum release by the sample to be detected, and has a detection result which is consistent with the instant time control oil effect of a human body test.
Description
Technical Field
The invention relates to the technical field of histological detection, in particular to a method for detecting skin sebum fusion and release based on linoleic acid induced human sebaceous gland cells.
Background
The sebaceous gland is an important gland attached to the skin and is an important part for producing sebum, and the sebaceous gland cells are main cells of a sebaceous gland structure, so that the oil-out state of the skin can be improved and the skin health can be maintained by regulating the sebaceous gland cells and reducing the synthesis and secretion of sebum.
The human immortalized sebaceous gland cell line (SZ 95 cells) obtained by transfecting PBR-322 plasmid containing a Simian virus-40 coding region makes the human sebaceous gland cell mutated, has the basic function of normal sebaceous gland cells, and provides a good material for researching sebaceous gland related diseases such as acne and the like. Linoleic acid is an essential unsaturated fatty acid which can be absorbed and utilized by human cells, and after being combined with peroxisome proliferator-activated receptors, the linoleic acid promotes differentiation, proliferation and sebum synthesis of sebaceous glands by regulating genes related to lipid metabolism in peroxisomes, microsomes and mitochondria. Testosterone or dihydrotestosterone, melanogenesis-promoting agent, insulin-like growth factor 1, acetylcholine, etc., act on sebaceous gland cells, and can also bind to their associated receptors to induce sebum synthesis.
The method for testing the sebum synthesis of the sebaceous gland cells generally adopts nile red staining or oil red O staining, and mainly aims at the total lipid in the cells, firstly, the method has no specificity, the sebum and the cell lipid cannot be distinguished, secondly, the dye cannot be cleaned, and is easy to remain on a cell plate, so that the background is deepened, the quantitative detection is not easy, and most of the method only uses pictures for qualitative display. The test of sebum synthesis of sebaceous gland cells is suitable for early regulation of sebum synthesis, is relatively consistent with the human body test result of long-acting oil control of skin (inhibiting sebum synthesis for about 14-28 days), but is not suitable for evaluation of instant oil control effect (inhibiting sebum release within 6 hours) and short-acting oil control (reducing sebum fusion within 7 days). The existing oil control efficacy human body test selects at least 30 subjects to test the sebum content, has the characteristics of long test period, large test data difference and high cost, and the measured sebum content is sebum secreted from the skin surface and can not evaluate the sebum synthesized and stored in the pilo-sebaceous gland unit.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a method for detecting sebum fusion and release based on linoleic acid induced human sebaceous gland cells.
The invention aims at realizing the following technical scheme:
in a first aspect, the invention provides the use of a Triglyceride (TG) as a biomarker for quantitatively detecting the efficacy of inhibiting sebum release from skin.
In a second aspect, the invention provides the use of TG as a biomarker for screening or assessing substances having an efficacy of inhibiting sebum release from skin.
In a third aspect, the invention provides the use of a biomarker panel comprising TG, cell lipid droplets, PLIN2 (lipid droplet coat protein) and CIDEA in the Cidea protein family (cell de ath-reduction Dff45 like effector) for the detection and assessment of sebum fusion and release.
In a fourth aspect, the invention provides the use of a biomarker panel comprising triglycerides, PLIN2 and CIDEA for screening or assessing substances having an inhibitory effect on sebum fusion and release.
In a fifth aspect, the present invention provides a quantitative detection method for sebum release inhibition based on linoleic acid induced human sebaceous cells, comprising the steps of:
a1, culturing the human sebaceous gland cells, and treating the human sebaceous gland cells by adopting a medium containing linoleic acid;
a2, treating cells treated by linoleic acid by adopting a culture medium containing a sample to be tested and the linoleic acid as an experimental group; and the same treatment was performed with medium using only linoleic acid as a control group;
and A3, collecting culture medium supernatant of the experimental group and the control group treated in the step A2, detecting the TG content, and calculating to obtain the sebum release inhibition rate.
Preferably, in the step A1, the culture of the human sebaceous gland cells sequentially comprises the steps of cell resuscitation, cell passage and cell plating;
the cell resuscitation comprises the following steps: taking out the frozen human sebaceous gland cells, thawing, adding the thawed human sebaceous gland cells into a cell culture medium, centrifuging to remove supernatant, and adding the obtained cells into the cell culture medium for resuspension;
the steps of the cell passage are as follows: re-inoculating the cells subjected to the first inoculation culture for subculture, washing the cells and then digesting the cells when the growth density of the cells is 70-80%, collecting the obtained digested cells, centrifuging and re-suspending;
the steps of cell plating are: the resuspended cells were inoculated into well plates and incubated.
Preferably, in step A1, the human sebaceous gland cells are SZ95 cells.
Preferably, in the step of resuscitating cells, the first inoculation culture is performed in an amount of 2X 10 inoculations per 10cm cell culture dish 6 A cell;
in the step of passaging the cells, the inoculation amount for carrying out the subculture is 2 multiplied by 10 6 Individual cells/dish (10 cm cell culture dish).
The step of plating the cells to adjust the density to 1X 10 5 Per ml, and inoculated into 48-well plates at 200 μl per well; the incubation time is 16-24 h.
Preferably, in step A1, the mass concentration of linoleic acid in the medium containing linoleic acid is 0.002-0.004%; the treatment time of the linoleic acid is 4-12 days. In the previous experiments of the inventor, when the mass concentration of the linoleic acid is too low, the secretion of grease is slow, so that the culture is required to be carried out for a long time, and the differentiation is weak; too high a concentration of linoleic acid by mass results in easy cell death. When the treatment time is too short, floating grease cannot be observed in the culture medium, which indicates that the stage of sebum release is not reached yet, and the difference of detection indexes is reduced; when the treatment time is too long, sebum is released much, and the sample effect may not be exhibited. So that too long or too short a treatment time affects the sensitivity of the test.
In the step A2, the mass concentration of the linoleic acid in the culture medium containing the sample to be detected and the linoleic acid is 0.002-0.004%; the treatment time is 2 to 4 days.
More preferably, the mass concentration of linoleic acid in the medium containing linoleic acid is 0.003%; the treatment time of the linoleic acid is 6 days, and the liquid is changed 1 time every 3 days;
in the step A2, the mass concentration of the linoleic acid in the culture medium containing the sample to be detected and the linoleic acid is 0.003%; the treatment time was 3 days.
The invention improves the current linoleic acid induction mode, adopts the method of inducing lipid synthesis first to form lipid fusion, and then observes the whole process of sebum secretion of sebaceous gland cells and release to a culture medium. Sebum is oily semi-fluid mixture containing multiple lipids, and comprises TG, fatty acids, phospholipids, cholesterol, etc. The inhibition effect of the substance to be tested on sebum release was determined by testing the content of TG, the main ingredient in sebum, in the cell culture supernatant.
In a sixth aspect, the invention provides a method for detecting sebum fusion and release based on linoleic acid induced human sebaceous gland cells, comprising quantitatively detecting TG content and detecting expression levels of PLIN2 and CIDEA genes;
the method for quantitatively detecting the TG content to react sebum fusion comprises the following steps:
b1, after culturing the human sebaceous gland cells, adopting a culture medium containing a sample to be tested and linoleic acid to treat the human sebaceous gland cells as an experimental group; and the same treatment was performed with medium using only linoleic acid as a control group;
b2, collecting culture medium supernatant of the experimental group and the control group treated in the step B1, detecting the TG content, and calculating to obtain the inhibition rate of sebum fusion.
Preferably, in the step B1, the mass concentration of linoleic acid in the medium containing linoleic acid is 0.002-0.004%, more preferably 0.003%; the linoleic acid treatment time is 4 to 12 days, more preferably 3 days; the liquid was changed 1 time every 3 days.
Preferably, the step of detecting the expression level of PLIN2 and CIDEA genes is as follows:
c1, after culturing the human sebaceous gland cells, treating the human sebaceous gland cells by adopting a culture medium containing linoleic acid and a sample to be tested;
and C2, collecting samples after 24-72h of treatment, cleaning, adding a lysate for lysis, performing RNA extraction and RT-PCR on the obtained cell lysate, and then detecting the expression level of PLIN2 and CIDEA genes.
Preferably, in the step C1, the mass concentration of linoleic acid in the medium containing linoleic acid and the sample to be tested is 0.002-0.004%, more preferably 0.003%.
The invention improves the current linoleic acid induction mode, adopts the method of inducing lipid synthesis first, then promotes lipid fusion by regulating and controlling PLIN2 and CIDEA gene expression quantity, and observes the variation of TG content when small lipid drops are fused into large lipid drops.
Compared with the prior art, the invention has the following beneficial effects:
1) The invention adopts the method of inducing human sebaceous cell SZ95 by linoleic acid, simulates the whole process of sebaceous cell from sebum synthesis and sebum fusion to sebum release, innovatively constructs a detection method of sebum release stage, takes the sebaceous cell induced by specific concentration LA for a specific time as the beginning of sebum release stage, and determines the release stage of sebum induced by D6-D9 days when the LA concentration is 0.003%. The method comprises the steps of adopting sample treatment at the initial time of sebum release, quantitatively detecting the content of a specific sebum component TG secreted by cells into culture supernatant at the end time of the release phase as a secretion index, and further evaluating the inhibition rate of the to-be-detected sample on sebum release.
2) The present invention shows that the trend of the results of the two methods is consistent by comparing with the existing human body test method (see the present invention examples 4 and 5). Thus, the results of the inhibition of sebum release measured using the method of the present invention can be used to evaluate the instant timed oil effect.
Drawings
Other features, objects and advantages of the present invention will become more apparent upon reading of the detailed description of non-limiting embodiments, given with reference to the accompanying drawings in which:
FIG. 1 is a photomicrograph (top panel) and an oil red O-stained photograph (bottom panel) of the linoleic acid group (0.003% LA) cells of example 1 at D1, D6, D9 and D12;
FIG. 2 shows the TG content results at D1, D6 and D9 of the negative control group and linoleic acid group in the cell lysate (intracellular) and culture supernatant (extracellular) of example 1; wherein, the bar graph is from left to right in proper order: control-intracellular, 0.003% la-intracellular, control-extracellular, 0.003% la-extracellular;
FIG. 3 shows the results of the expression level of PLIN2 genes in the negative control group and linoleic acid group in example 1;
FIG. 4 shows the results of the expression level of CIDEA genes in the negative control group and the linoleic acid group in example 1;
FIG. 5 is a photomicrograph of the results of the D9 extracellular lipid droplets of each of the experimental groups of example 2;
FIG. 6 shows the results of the intracellular (cell lysate) and extracellular (cell supernatant) TG content at D9 for each experimental group of example 2;
FIG. 7 shows the results of PLIN2 gene expression levels measured at D1 and D3 in each experimental group in example 2; a negative control group, a linoleic acid group, a 0.5% sample group and a 1% sample group are sequentially arranged from left to right;
FIG. 8 shows the results of CIDEA gene expression levels measured at D1 and D3 in each experimental group in example 2; a negative control group, a linoleic acid group, a 0.5% sample group and a 1% sample group are sequentially arranged from left to right;
FIG. 9 is a graph showing the results of the change in sebum content of the skin before and after use of each of the experimental groups of example 4;
FIG. 10 is a photograph of the cell microscopic photograph of each experimental group at D6 in example 5;
FIG. 11 is a photograph of a cell microscope at D6 (upper panel) and a photograph of oil red O staining (lower panel) for each experimental group in example 6.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications could be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present invention.
The words "preferred," "more preferred," and the like in the present disclosure refer to embodiments of the present disclosure that may provide certain benefits in some instances. However, other embodiments may be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.
It should be understood that all numbers expressing, for example, amounts of ingredients used in the specification and claims, except in any operating example or otherwise indicated, are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
Example 1
The embodiment provides a method for detecting sebum fusion and release based on linoleic acid induced human sebaceous gland cells, which comprises the following specific steps:
1. TG quantitative detection
1. Cell source: human sebaceous gland cells. Human immortalized sebaceous gland cell line (SZ 95), which is a proprietary cell, was obtained by mutating human sebaceous gland cells by transfection of the PBR-322 plasmid containing the coding region of Simian virus-40, which has been used by the company.
2. Composition of cell culture medium: DMEM/F12 (1:1) medium+10% Fetal Bovine Serum (FBS) +9ng/ml hEGF+3ng/ml hKGF+1% diabody (P/S).
3. Cell resuscitation: taking frozen SZ95 cells out of liquid nitrogen, rapidly placing in a water bath at 37deg.C, thawing, adding into preheated cell culture medium prepared in advance, centrifuging at room temperature for 5min at 1000 rpm, discarding supernatant, re-suspending cells with culture medium, inoculating 2M (million ) SZ95 cells per 10cm cell culture dish, shaking, and standing at 37deg.C under 5% CO 2 Culturing in a cell culture incubator.
4. Cell passage: subculture (2M (million) cells were seeded per 10cm cell culture dish) was performed with a liquid change treatment every two days, and when cells were grown to a density of 70% -80%, the following treatments were performed:
a) Firstly, washing with 10ml of PBS, and discarding the PBS;
b) Digesting with 3-5 mL of preheated 0.25% pancreatin at 37 ℃ for 6-10 minutes at 37 ℃, and stopping the digestion process after observing that the cells fall and adding 5mL of cell culture medium;
c) The digested cells were collected in a centrifuge tube, the heart was separated at 1000 rpm for 5min, and the cells were counted after being resuspended.
5. Cell plating: the resuspended cells were adjusted to a cell density of 1 x 10 5 Per ml, 200. Mu.L of each well was inoculated into a 48-well plate, and placed at 37℃with 5% CO 2 Incubation was performed for 24h in an incubator.
6. Linoleic acid treatment: observing the attached SZ95 cell plate obtained after 24h incubation under a microscope, and then beginning linoleic acid treatment, namely diluting the linoleic acid to 0.003% concentration by using a cell culture medium, wherein the cell culture medium contains linoleic acidThe oleic acid cell culture medium was applied to wells corresponding to the linoleic acid group (LA group) in the well plate at 400 μl of replacement fluid per well, at least 6 wells per group, and 400 μl of replacement fluid per well was used as a negative control group. The 48-well plate was then placed at 37℃with 5% CO 2 Incubation in incubator. The time point of LA stimulation was D0, and the same linoleic acid treatment as described above was performed at D3, D6, D9, and D12, with each 72h of liquid change.
7. Sample collection processing and detection: and respectively photographing cells under a microscope at D1, D6, D9 and D12 after LA treatment, and observing whether oil drops exist in a cell culture solution. At the same time, culture supernatants from the cell plates were collected. Then, 0.5% oil red O staining solution is added into a part of cells, the cells are stained for 10min at room temperature, and after PBS (phosphate buffered saline) is washed, the synthesis and fusion of sebum in the cells are observed under a microscope.
And (3) carrying out lysis on the other part of cells after being diluted by Nonidet P40 for 20 times, carrying out room temperature lysis on 200 mu L of lysate in each hole for 10-20 min, transferring the collected cell lysate into an EP tube, incubating in a water bath at 80 ℃ for 2-5 min, cooling to room temperature, repeating for 2 times, and fully dissolving TG. The collected culture supernatant (extracellular) and cell lysate (intracellular) were subjected to TG content detection using the MAK266 kit, and specific detection methods were referred to the instructions of the MAK266 kit.
8. Detection result: the photomicrographs of the LA groups at different treatment times (D1, D6, D9, D12) are shown in fig. 1. As can be seen from FIG. 1, the formation of droplets (red) was observed in the cells at D1 under LA stimulation, and the synthesis of large amounts of droplets was still observed in the cells at D6, with partial droplet fusion becoming larger and small amounts of droplet release also observed in the cell culture supernatant. In the case of D9, the presence of a large number of lipid droplets in the culture supernatant was observed, and cell death increased, indicating that the cells had entered the collapse release stage and that the results of the oil red O staining were inaccurate. D12 causes massive cell death, and a relatively complete cell contour cannot be observed, which is not suitable for staining detection.
FIG. 2 shows the results of the measurement of the intracellular and extracellular TG contents of the LA group at different treatment times (D1, D6, D9), and as can be seen from FIG. 2, TG is synthesized in large amounts in the cells under the stimulation of LA at D1, and the formation of lipid droplets and the TG content are hardly detected in the extracellular region. D6, TG starts to be secreted into the cell culture supernatant, the content of TG can be detected extracellularly and formation of lipid droplets is observed, and the content of TG in the cells appears to decrease due to fusion of lipid droplets and release of lipid droplets. D9, TG was secreted in large amounts into the cell culture supernatant, indicating the release of large amounts of lipid droplets and cell necrosis.
From the above experimental results, it was finally determined that: when LA treatment was used at a concentration of 0.003%, D6-D9 days were the sebum release phase, and this phase was therefore selected as the treatment time for subsequent quantitative detection of sebum release.
2. Quantitative detection of PLIN2 and CIDEA
1. Cell source: human sebaceous gland cells. Human immortalized sebaceous gland cell line (SZ 95), which is a proprietary cell, was obtained by mutating human sebaceous gland cells by transfection of the PBR-322 plasmid containing the coding region of Simian virus-40, which has been used by the company.
2. Composition of cell culture medium: DMEM/F12 (1:1) medium+10% Fetal Bovine Serum (FBS) +9ng/ml hEGF+3ng/ml hKGF+1% double antibody (P/S)
3. Cell resuscitation: taking frozen SZ95 cells out of liquid nitrogen, rapidly placing in a water bath at 37deg.C, thawing, adding into preheated cell culture medium prepared in advance, centrifuging at room temperature for 5min at 1000 rpm, discarding supernatant, re-suspending cells with culture medium, inoculating 2M (million ) SZ95 cells per 10cm cell culture dish, shaking, and standing at 37deg.C under 5% CO 2 Culturing in a cell culture incubator.
4. Cell passage: subculturing is carried out according to the liquid exchange treatment carried out every two days, and when the cells grow to 70-80% density, the following treatment is carried out:
a) Firstly, washing with 10ml of PBS, and discarding the PBS;
b) Digesting with 3-5 ml of preheated 0.25% pancreatin at 37 ℃ for 6-10 minutes at 37 ℃, and stopping the digestion process after observing that the cells fall and adding 5ml of cell culture medium;
c) The digested cells were collected in a centrifuge tube, the heart was separated at 1000 rpm for 5min, and the cells were counted after being resuspended.
5. Cell plating: the resuspended cells were adjusted to a cell density of 1 x 10 5 1ml of each well was inoculated into 6-well plates and placed at 37℃with 5% CO 2 Incubating for 16-24 h in an incubator.
6. Linoleic acid treatment: the attached SZ95 cell plates obtained after 24h of incubation are observed under a microscope, and then linoleic acid treatment is started, specifically, the linoleic acid is diluted to 0.003% concentration by using a cell culture medium, the cell culture medium containing the linoleic acid is added into holes corresponding to linoleic acid groups (0.003% LA groups) in the pore plates by 2mL of liquid exchange per hole, at least 3 holes are formed in each group, and 2mL of liquid exchange per hole is used as a negative control group. Then the 6-well plate was placed at 37℃with 5% CO 2 Incubation in incubator.
7. Sample collection processing and detection: respectively collecting samples of cells after incubation for 6h, 24h (D1), 48h (D2), 72h (D3) and 96h (D4), washing with PBS for one time, adding 150 mu L of lysate RL (containing DTT) into each well, standing for 1min, and lightly blowing to ensure sufficient lysis; freezing the obtained cell lysate at-80 ℃, and then carrying out RNA extraction and RT-PCR by adopting the conventional method; then, PLIN2 and CIDEA gene expression detection is carried out by using a kit, and 2 is adopted -ΔΔCT The relative expression fold was calculated by the method to analyze the rising amplitude of gene expression.
8. Detection result: as shown in FIGS. 3 and 4, the relative expression fold of PLIN2 gene was significantly increased 6h after treatment after LA stimulation, and the higher expression levels were maintained at D1 to D4, and the maximum increase was reached at D3. The relative expression fold of CIDEA gene is obviously up-regulated at D3, which shows that the expression quantity of the two genes can be up-regulated under LA stimulation, so that the lipid fusion and lipid release processes can be accelerated.
From the above experimental results, it was finally determined that: d3 was most different and the effect of assessing sebum fusion and release was more pronounced by detecting the relative expression of the PLIN2 and CIDEA genes at 72h incubation.
Example 2
The embodiment provides a method for detecting sebum fusion and release based on linoleic acid induced human sebaceous gland cells, which is used for screening or evaluating substances with the effect of inhibiting skin sebum release, and comprises the following specific steps:
1. cell source: step 1 of quantitative detection of TG in example 1.
2. Composition of cell culture medium: step 2 of quantitative detection of TG in example 1.
3. Cell resuscitation: step 3 of quantitative detection of TG in example 1.
4. Cell passage: step 4 of quantitative detection of TG in example 1.
5. Cell plating: step 5 of quantitative detection of TG in example 1.
6. Linoleic acid treatment: the attached SZ95 cell plates obtained after 24h of incubation are observed under a microscope, and then linoleic acid treatment is started, specifically, linoleic acid is diluted to 0.003% concentration by using a cell culture medium, the cell culture medium containing the linoleic acid is added into holes corresponding to a linoleic acid group (LA group) in an orifice plate in a mode of 400 mu L of liquid exchange per hole, at least 6 holes are formed in each group, and 400 mu L of cell culture medium liquid exchange per hole is used as a negative control group. The 48-well plate was then placed at 37℃with 5% CO 2 Incubation in incubator. The time point of LA stimulation was D0, and the incubation was performed for D6 days with each 72h of fluid change.
7. Sample treatment: sample 1 (Jiayanjing, a raw material product developed by Shanghai Jia Kai organism Co., ltd.) was diluted to 0.006% concentration with a cell culture medium to 1% concentration as a medium containing sample 1, and sample 2 (quercetin, main component 1 of Baimai seed extract), sample 3 (5' -O-rhamnosyluridine, main component 2 of Baimai seed extract) were diluted to 200 μg/mL concentrations with a cell culture medium as a medium containing sample 2 and a medium containing sample 3, respectively. After incubation for D6 days with linoleic acid treatment, each experimental group was changed according to the medium shown in Table 1, and then a 48-well plate was placed at 37℃with 5% CO 2 Incubation was continued for 3 days in the incubator (D9).
TABLE 1
8. Photographing by a cell microscope and detecting the TG content: cell lysates and culture supernatants were collected according to the method of example 1. Lipid droplets in each cell culture supernatant were photographed under a microscope. And TG content detection was performed on cell lysates and culture supernatants of the negative control group, linoleic acid group and sample 1 group, and culture supernatants of sample 2 and sample 3 group.
The lipid droplet content of the cell culture supernatants under D9 microscope photographing for each experimental group is shown in fig. 5. After LA stimulation, lipid droplets secreted into the supernatant were significantly increased, and the cell profile was unclear, and necrosis (linoleic acid group) occurred. When the samples 1 are synchronously treated, lipid drops in the supernatant can be obviously reduced, and the cell morphology is more complete (the samples 1 group). Sample 2 did not significantly improve cell morphology and lipid droplets in the supernatant (sample 2 group), and sample 3 exhibited some improvement in cell morphology (sample 3 group). By combining these test results, sample 1 can reduce sebum fusion and sebum release by controlling the expression levels of PLIN2 and CIDEA genes, sample 3 can reduce sebum release to some extent, and sample 2 has no strong effect on sebum release. The method can distinguish samples with different effects on sebum release, and can also be used for sebum fusion and mechanism verification of sebum release.
The OD value results and TG content (average value) measured on D9 days for the negative control group, linoleic acid group and sample 1 group are shown in table 2 and fig. 6. The TG content (average value) and the inhibition rate results measured in the sample 2 group and the sample 3 group are shown in table 3. The inhibition rate is calculated by the following steps: (linoleic acid group content value-sample group content value)/linoleic acid group content value 100%. In the cell lysate, the TG content of the linoleic acid group was significantly higher than that of the negative control group, indicating that after treatment of D9 with linoleic acid, the sebum of the cells was synthesized in large amounts and the sebum thus synthesized was released from the cells, so that the TG content was also detected in the supernatant of the cells to be significantly higher than that of the negative control group.
In the supernatant, the TG content of sample 1 was significantly lower than that of linoleic acid group, indicating that sample 1 at a concentration of 0.5% had significant inhibition of sebum release from cells, and the inhibition rate reached 40.80%. Whereas the results of sample 2 group showed no inhibition; the sample 3 group has a certain inhibition effect, but the inhibition rate is only 13.85%.
TABLE 2
TABLE 3 Table 3
PLIN2 and CIDEA detection: the linoleic acid was diluted to 0.006% concentration with cell culture medium as medium containing 0.006% la, and the sample (Jiayanjing, a raw material product developed by Shanghai Jia Kai organism Co., ltd.) was diluted to 1% and 2% concentration with cell culture medium as medium containing sample. The adherent SZ95 cells obtained after incubation for 24 hours by the same method as in steps 1 to 5 of quantitative detection of PLIN2 and CIDEA in example 1 were subjected to liquid exchange (D0) in the culture medium shown in Table 4 for each experimental group cell well plate, and then the 6 well plate was placed at 37℃and 5% CO 2 Incubate to D1, D3 in incubator. Cells were collected by the same method as in example 1, and the gene expression amounts of PLIN2 and CIDEA were measured.
TABLE 4 Table 4
The relative fold expression of PLIN2 and CIDEA measured at D1 and D3 for each experimental group is shown in FIGS. 7 and 8. After LA stimulation, the relative fold expression of both PLIN2 and CIDEA genes showed an increase, with a greater increase in magnitude at D3 (linoleic acid group). And under the treatment of 0.5% and 1% of samples (0.5% of sample group and 1% of sample group), the expression quantity of the two genes can be obviously reduced.
Example 3
The embodiment provides a method for detecting the oil control efficiency in human clinic at the moment, which is used for comparing the change of the sebum content of the facial skin before and after the use of a sample 1 (Jiayanjing), a sample 2 (Quercetin) and a sample 3 (5' -O-rhamnosyluridine) for different times to clarify the consistency of the oil control efficiency in the moment of a product to be detected and a cell test result.
Skin sebum content was tested using a Sebumeter skin sebum test probe in the multifunctional skin test system MPA6 from CK, germany. The Sebumeter method is based on photometer principle, and uses a special extinction adhesive tape with the thickness of 0.1mm, which can become a semitransparent adhesive tape after absorbing sebum on human skin, the light transmission amount can be changed, the more the absorbed sebum is, the larger the light transmission amount is, and the sebum content of the skin can be measured.
The test recipe composition and test packet information are shown in table 5 below:
TABLE 5
The method comprises the following specific steps:
1. the screening age is between 18 and 55 years old, the sebum secretion of the facial skin is more vigorous, and the sebum secretion exceeds 120 mug/cm within 8 hours after cleaning 2 Is involved in the test;
2. the method comprises the steps of cleaning the face by using alkaline soap on the test day, wiping water by using dry facial tissues, balancing for 20-30 min in a static sitting state in a test environment (a constant temperature and humidity area, a temperature and humidity range of 20-22 ℃ and 40-60%), and preventing water and beverage from being drunk during balancing;
3. dividing the forehead area of 20 subjects into left, middle and right three sides according to the central position, wherein the interval between the groups is at least 0.5cm, the left and right two sides are randomly divided into a sample 1 group, a sample 2 group and a sample 3 group, and the middle is a matrix control group. Measuring initial sebum content by using a Sebumeter probe, smearing a sample once by using a latex finger sleeve according to the dosage of 100 mu l in each area, starting timing after the smeared sample is completely dried, and measuring sebum content again after 3 hours respectively;
4. for sebum content, the rate of change of the sample was calculated using the following formula. Significant differences were calculated using the paired TTest approach, where P <0.05 and P <0.01. The greater the rate of change, the more effective the timed oil.
a) Detection change value Δ=detection value After use -detection value Before use
b) Change rate (%) = (1-detection change value) Sample group Detecting a change value Control group )×100％
5. Test results: as shown in table 6, the sebum level of the matrix control group was maintained at a higher level after 3 hours, indicating that the sebum level of the skin was continuously increased after cleansing. In the group 1, the sebum content is obviously reduced by 26.4 percent and the sebum content is obviously reduced by 13.5 percent when being treated by the sample 3, but the sebum content is basically not influenced when being treated by the sample 2, compared with the matrix control group, after 3 hours of use. By detecting the change in sebum content secreted from the skin surface 3 hours after cleansing, the change in sebum content released and stored in the pilosebaceous unit duct can be reflected, and the result shows a relatively consistent trend with the results of sebum release from sebaceous cells induced by linoleic acid.
TABLE 6
Example 4
The embodiment provides a method for detecting the long-acting oil control effect of human clinic, which is to compare the change of the sebum content of the facial skin before and after the use for 28 days by comparing the essence containing 5% of sample 1 (Jiayanjing) for 28 days, clarify the long-acting oil control effect of a product to be detected and recheck the test result of sebum fusion on cells.
Formulation composition information of test essence S containing 5% of sample 1, test essence C (matrix control) containing no test sample is as follows in table 7:
TABLE 7
The method comprises the following specific steps:
1. subject screening: the screening age is between 18 and 55 years old, the sebum secretion of the facial skin is more vigorous, and the sebum secretion exceeds 120 mug/cm within 8 hours after cleaning 2 Is involved in the test;
2. the test was performed using a half-face control, designed as a control, random test. The test period is 28 days, the test time is 0 th, 14 th and 28 th days when the test sample is used by the test subjects in the morning and evening respectively;
3. test scheme: the left and right faces are respectively smeared with matrix (essence C) or sample (essence S), and the left and right faces are simultaneously subjected to comparative analysis before and after use. The basic values of the left and right faces of the subject before using the sample and sebum contents at different time points are detected.
4. The face is cleaned by clean water on the test day, the water is wiped by a dry facial tissue, the test environment is balanced for 20-30 min by sitting still, and water and beverage cannot be drunk during the balancing period. Dividing the forehead area into a left side and a right side according to the central position, wherein the interval between the groups is at least 0.5cm. Measuring the initial sebum content of each of the three points on the left and right sides by adopting a Sebumeter probe, starting timing after all the tests are completed, and measuring the sebum content again after 3 hours respectively;
5. testing according to step 4 at 14 days and 28 days, respectively;
6. for sebum content, the rate of change of the sample was calculated using the following formula. Significant differences were calculated using the paired TTest approach, where P <0.05 and P <0.01. The larger the change rate is, the stronger the long-acting oil control effect is.
Change rate (%) = (1-detection change value) Sample group Detecting a change value Control group )×100％
7. Test results: as can be seen from fig. 9, before use (D0T 0h ) MeasuringThe skin sebum content values of the test essence C and the essence S are not obviously different, and after the 5% jia yan-containing clean essence S14 and 28 days (D14 and D28) are used, compared with the test essence C (control group), the skin sebum content is obviously reduced after cleaning for 0h and 3h, the reduction rates of 0h respectively reach 14.2% and 19.0%, and the reduction rates of 3h respectively reach 9.4% and 10.9%. By detecting the sebum content changes secreted by the skin surface for 0h and 3h after cleaning, the sebum content changes caused by sebum synthesis and sebum release can be respectively reflected, and the results are consistent with the trend of the results of the sebaceous gland cells induced by linoleic acid.
Example 5
The present example provides a method for detecting sebum fusion and release by different stimulators acting on human sebaceous gland cells to verify the stimulatory effect of linoleic acid, comprising the following specific steps:
1. cell source: step 1 of quantitative detection of TG in example 1.
2. Composition of cell culture medium: step 2 of quantitative detection of TG in example 1.
3. Cell resuscitation: step 3 of quantitative detection of TG in example 1.
4. Cell passage: step 4 of quantitative detection of TG in example 1.
5. Cell microscope photographing and oil red O staining detection: the resuspended cells were adjusted to a cell density of 1 x 10 5 Per ml, 200. Mu.L of each well was inoculated into a 48-well plate, and placed at 37℃with 5% CO 2 Incubation was performed for 24h in an incubator. Linoleic acid, dihydrotestosterone, melanotropin, insulin-like growth factor 1, acetylcholine were diluted to 0.003%, 20. Mu.g/mL, 100. Mu.g/mL, 1mg/mL, respectively, with cell culture medium as media containing different stimulators. mu.L of cell plate was added to each well, and the mixture was subjected to 5% CO at 37 ℃ 2 Incubation was continued in the incubator with 1 change of fluid every 3 days. The lipid droplets in the cell culture supernatant were photographed under a microscope at each liquid change, and at the time of treatment to day 6, the medium was discarded, 0.5% of oil red O staining solution was added, and after 10min of staining at room temperature, 40% of isopropanol was washed 2 times, and photographed under a microscope.
6. Detection result: at the time of treatment to D6, no formation of lipid droplets was found in the culture supernatants of each treatment group except for the linoleic acid group. The intracellular lipid is stained by oil red O staining, as shown in figure 10, under the treatment of linoleic acid, the cells can be induced to produce a large amount of lipid (red oil drops), while under the action of other several stimulators, except for the dihydrotestosterone, the cells can be promoted to produce a small amount of lipid, and other stimulators (melanogenesis promoting hormone, insulin-like growth factor 1 and acetylcholine) have no influence on the production of the cell lipid.
Example 6
The embodiment provides a method for detecting sebum fusion and release by acting linoleic acid with different concentrations on human sebaceous gland cells, which is used for verifying the influence of the concentration of the linoleic acid on the cells, and comprises the following specific steps:
1. cell source: step 1 of quantitative detection of TG in example 1.
2. Composition of cell culture medium: step 2 of quantitative detection of TG in example 1.
3. Cell resuscitation: step 3 of quantitative detection of TG in example 1.
4. Cell passage: step 4 of quantitative detection of TG in example 1.
5. Cell microscope photographing and oil red O staining detection: adjusting the cell density of the digested cells to 1 x 10 5 Per ml, 200. Mu.L of each well was inoculated into a 48-well plate, and placed at 37℃with 5% CO 2 Incubation was performed for 24h in an incubator. Linoleic acid was diluted to 0.001%, 0.003%, 0.006% concentration with cell culture medium as medium containing different concentrations of the stimulus. mu.L of cell plate was added to each well, and the mixture was subjected to 5% CO at 37 ℃ 2 Incubation was continued in the incubator with 1 change of fluid every 3 days. The lipid droplets in the cell culture supernatant were photographed under a microscope at each liquid change, and at the time of treatment to day 6, the medium was discarded, 0.5% of oil red O staining solution was added, and after 10min of staining at room temperature, 40% of isopropanol was washed 2 times, and photographed under a microscope.
6. Detection result: as shown in FIG. 11, lipid droplet formation was observed in the culture supernatants of the 0.003% and 0.006% linoleic acid groups at the time of treatment to D6, but the cells died largely at the concentration of 0.006%, which was detrimental to subsequent further treatment and detection. The intracellular lipid is dyed by adopting oil red O dyeing, and under the treatment of linoleic acid with the concentration of 0.001%, a large amount of synthetic lipid (red oil drops) can be induced by cells, but the shape of the lipid drops is more uniform and the diameter is smaller; with linoleic acid at a concentration of 0.006%, the lipid droplets were larger in diameter, but the quantitative analysis was inaccurate due to massive cell death. Therefore, the linoleic acid with the concentration of 0.003 percent not only can maintain the cell morphology, is beneficial to subsequent continuous treatment and various detection, but also can promote the synthesis of lipid, the fusion of lipid droplets and the release of lipid droplets, is suitable for the evaluation of different growth stages of sebaceous gland cells and different stages of lipid synthesis, and is also suitable for the evaluation of different types of samples.
There are many ways in which the invention may be practiced, and what has been described above is merely a preferred embodiment of the invention. It should be noted that the above examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that modifications may be made without departing from the principles of the invention, and such modifications are intended to be within the scope of the invention.
Claims (10)
1. Use of triglycerides as biomarkers for quantitative detection of sebum release from skin.
2. Use of triglycerides as biomarkers for screening or evaluating substances having an inhibitory effect on sebum release from skin.
3. Use of a biomarker panel for detecting and assessing sebum fusion and release from skin, wherein the biomarker panel comprises triglycerides, cell lipid droplets, PLIN2 and CIDEA.
4. Use of a biomarker panel for screening or evaluating a substance having an effect of inhibiting sebum fusion and release from skin, wherein the biomarker panel comprises triglycerides, cell lipid droplets, PLIN2 and CIDEA.
5. A quantitative detection method for inhibiting sebum release based on linoleic acid induced human sebaceous gland cells, which is characterized by comprising the following steps:
a1, culturing the human sebaceous gland cells, and treating the human sebaceous gland cells by adopting a medium containing linoleic acid;
a2, treating cells treated by linoleic acid by adopting a culture medium containing a sample to be tested and the linoleic acid as an experimental group; and the same treatment was performed with medium using only linoleic acid as a control group;
and A3, collecting culture medium supernatant of the experimental group and the control group treated in the step A2, detecting triglyceride content, and calculating to obtain the inhibition rate of sebum release.
6. The method for quantitative determination of skin sebum release inhibition according to claim 5, wherein in step A1, the culture of human sebaceous gland cells includes the steps of cell resuscitation, cell passage and cell plating in this order;
the cell resuscitation comprises the following steps: taking out the frozen human sebaceous gland cells, thawing, adding the thawed human sebaceous gland cells into a cell culture medium, centrifuging to remove supernatant, adding the obtained cells into the cell culture medium for resuspension, and then performing first inoculation culture;
the steps of the cell passage are as follows: re-inoculating the cells subjected to the first inoculation culture for subculture, washing the cells and then digesting the cells when the growth density of the cells is 70-80%, collecting the obtained digested cells, centrifuging and re-suspending;
the steps of cell plating are: the resuspended cells were inoculated into well plates and incubated.
7. The quantitative determination method for inhibiting skin sebum release according to claim 5, wherein in the step A1, the mass concentration of linoleic acid in the medium containing linoleic acid is 0.002-0.004%; the treatment time of the linoleic acid is 4-12 days;
in the step A2, the mass concentration of the linoleic acid in the culture medium containing the sample to be detected and the linoleic acid is 0.002-0.004%; the treatment time is 2 to 4 days.
8. A method for detecting sebum fusion and release based on linoleic acid induced human sebaceous gland cells, which is characterized by comprising the steps of quantitatively detecting triglyceride content and detecting the expression level of PLIN2 and CIDEA genes;
the step of quantitatively detecting the inhibition rate of sebum release according to any one of claims 5 to 7.
9. The method for detecting sebum fusion and release according to claim 8, wherein the step of detecting the expression level of PLIN2, CIDEA genes is as follows:
b1, culturing the human sebaceous gland cells, and then adopting a culture medium containing linoleic acid and a sample to be tested to treat the human sebaceous gland cells;
b2, collecting samples after 24-72h of treatment, adding a lysate for lysis after cleaning, performing RNA extraction and RT-PCR on the obtained cell lysate, and then detecting the expression level of PLIN2 and CIDEA genes.
10. The method for detecting sebum fusion and release according to claim 9, wherein in the step B1, the mass concentration of linoleic acid in the medium containing linoleic acid and the sample to be detected is 0.002 to 0.004%.
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| CN117517635B (en) * | 2023-11-24 | 2024-05-10 | 广州伽能生物科技有限公司 | Comprehensive oil control effect judging method for skin oil control scheme |
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