CN111077295A - Method for detecting whether to-be-detected compound regulates and controls growth of lipid droplets - Google Patents
Method for detecting whether to-be-detected compound regulates and controls growth of lipid droplets Download PDFInfo
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Abstract
The invention discloses a method for detecting whether a compound to be detected regulates and controls the growth of lipid droplets. The invention provides application of a substance for detecting the proportion of large fat droplets in preparing a compound product for detecting or screening whether a compound to be detected is a compound for regulating and controlling the growth of fat droplets; the present invention allows for the size of lipid droplets to be increased by stable expression of the Cidec protein in immortalized cells, thereby obtaining a lipid droplet phenotype similar to adipocytes. The small molecular compound with the lipid droplet size is screened by utilizing the characteristic that the immortalized cells are easy to screen in a large scale and combining the added lipid droplet phenotype. Such a screening scale can be made very large. Meanwhile, a full-automatic lipid drop recognition algorithm is used, the lipid drop form is recognized automatically in a large scale, and the form of the lipid drop can be represented quickly by using three lipid drop indexes.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for detecting whether a compound to be detected regulates the growth of lipid droplets.
Background
With the improvement of living conditions, the number of obese people worldwide has increased, and by 2016, 19 hundred million people worldwide have overweight, of which 6.5 hundred million people are obese. Overweight and obesity bring about a variety of other metabolic diseases such as diabetes, fatty liver, cardiovascular diseases, etc., and lipid droplets are important as intracellular organelles storing neutral lipids for maintaining the stability of lipid metabolism and the balance of energy metabolism.
Adipose tissue of the body is the main energy storage site, and due to the high energy density of neutral lipids, the excess energy in the body is generally stored in the form of neutral lipids in the fat droplets of adipose tissue. Because of their hydrophobic nature, large amounts of neutral lipids cause lipotoxicity in cells, which store them in special organelle lipid droplets. The lipid drop is used as a special energy storage organelle, and can adjust the storage and decomposition of neutral lipid according to the energy requirement of the organism so as to maintain the energy balance of the organism. Adipose tissue is divided into two types, white adipose tissue and brown adipose tissue, respectively. White adipose tissue is primarily responsible for energy storage and is characterized by a single large lipid droplet occupying the major space of the cell. While brown adipose tissue is responsible for maintaining body temperature through adaptive heat generation under cold stimulation conditions. Under cold stimulation conditions, brown adipose tissue converts chemical energy to thermal energy via uncoupling of UPC1 in mitochondria to maintain body temperature. Brown adipose tissue has a large number of lipid droplets and a smaller volume than white adipose tissue, while having many mitochondria. In most other cells, lipid droplets are also present, but are smaller in size. In non-alcoholic fatty liver, larger lipid droplets are also formed due to excessive lipid storage and in hepatocytes. The proteins required for the formation of large lipid droplets are expressed in fatty liver such as Plin1 and Cidec, among others.
The growth and development of lipid droplets is critical to the energy balance of the body. If the neutral lipid in the lipid droplets is accumulated excessively, non-alcoholic fatty liver, obesity, etc. may result. Furthermore, proliferation and metastasis of tumor cells are also associated with the growth of lipid droplets. If the lipid droplets are not regulated effectively, ectopic storage of fat may result, leading to physical problems such as insulin resistance. Therefore, the lipid drop growth regulation is taken as a treatment target, and the small molecular drug capable of regulating the lipid drop growth is searched, so that the method is a feasible direction for treating metabolic diseases.
CIDE (cell death-inducing DEF45-like effector) family proteins include Cidea, Cideb, Cidec, and are mainly involved in the relevant regulation of cellular lipid metabolism. Deletion of the Cidea protein can make mice resistant to obesity induced by high fat diet and remarkably improve insulin appetency. In the liver of obese mice (ob/ob), Cidea expression is increased, and silencing Cidea can alleviate fatty liver. Cideb knockout mice also display a lean phenotype and increased insulin sensitivity. Mice with Cidec knockout had significantly reduced white adipose tissue and exhibited many and small lipid droplets. The mitochondrial activity of white adipocytes increases the rate of lipid hydrolysis and increases the fatty acid oxidation activity. Similarly, deletion of Cidec can make mice resistant to obesity induced by high fat diet, and increase insulin sensitivity.
Cell biology shows that Cidec and the CIDE family of proteins it belongs to can mediate lipid droplet fusion between lipid droplets by forming LDCS (lipid droplet conjugation site). The neutral lipid inside the small lipid droplet is infused into the large lipid droplet, resulting in the fusion of the large lipid droplet with the small lipid droplet, forming a larger lipid droplet. It is believed that the small radius of the small lipid droplet is small and the internal pressure is high due to the surface tension, so that the neutral lipid flows in a single direction under the action of the pressure difference. This mechanism of lipid droplet fusion is critical for adipocyte formation of large lipid droplets, and in the absence of Cidec, white adipocytes cannot form single large lipid droplets, while mice also exhibit a lean phenotype.
Previous small molecule screens for adipocyte modulation have generally employed adipocyte cells that induce differentiation. But the differentiation induction time of adipocytes is longer (8 days). Moreover, after the cells are differentiated, the sizes of lipid droplets of the cells are greatly different from one another, so that the phenotype screening is disturbed. Expression of a fat-related gene such as PPARr in immortalized cells has also been performed to screen for regulation of this gene-related protein. Although such cells express genes such as PPARr, they do not form lipid droplets having characteristics of adipocytes, and therefore, information on lipid droplets that directly affect the lipid droplets is not obtained.
Disclosure of Invention
It is an object of the present invention to provide the use of a substance for detecting the proportion of large lipid droplets.
The substance for detecting the proportion of large fat drops is applied to preparing and detecting or screening whether a compound to be detected is a compound product for regulating and controlling the growth of fat drops;
or the application of the substance for detecting the large fat drop proportion in detecting or screening whether the compound to be detected is a compound for regulating the growth of fat drops;
the substances for detecting the proportion of the large fat droplets comprise Hela cells expressing Cidec, a high content microscope and image analysis software Harmony;
the Cidec Hela expressing cell is a cell obtained by introducing CDS of a Cidec protein into a Hela cell;
the parameters (scripts) of the image analysis software Harmony are as follows:
1Input Image:
Stack processing: Maximum project
Flatfield correction:none
2Find Nuclei：
Channel: DAPI；
ROI Population: None
ROI Region of none
Method: B
Output Population:Nuclei
3 Select Cell Region:
Population: Nuclei
Method: Resize Region
Region Type: Nucleus Region
Outer Border -128％
Inner Border 88％
Output Region: cyt0sol region
4 Find Spots:
Channel: Alexa 488
ROI Population: Nuclei
ROI Region Cyt0sol region
Method B
Output Population: LDs
5 Calculate Morphology Properties:
Population LDs
Region: Spot
Mehod: Standard
Output Properties: LD properties
6 Select Population:
Population: LDs
Method Filter by property
Filter F1
LD properties Area um^2>2
Filter F2
LD properties roundness>0.9
Output Population: LD_large
7Select Population(2):
Population: LDs
Method: Filter by Property
Filter F1
LD properties<＝ 2
LD properties>0.05
Output Population: LDs small
8Define Results:
1)Method: Formula Output Formula:a/(a+b)
Population Type: objects
Variable A LD_Large_LD properties_area(um^2) ,Sum
Variable B LDs small_LD properties area(um^2),Sum
Output Name Large LD area ratio (big fat drop ratio)
2)Method: Formula Output Formula:(a+b)/c
Population Type: objects
Variable A LD_Large_LD Number of objects
Variable B LDs small_LD Number of objects
Variable C Nuclei-Number of objects
Output Name LD number (average number of fat droplets)
3)Method: Formula Output Formula:(a+b)/c
Population Type: objects
Variable A LD_Large_LD properties_area(um^2) ,Sum
Variable B LDs LDs small_LD properties area(um^2),Sum
Variable C Nuclei-Number of objects
Output Name: lipid average area
The large lipid droplet proportion is the total area of the large lipid droplets to the total area of all the lipid droplets.
In the above application, the substance for detecting the proportion of the large fat droplets further comprises a readable carrier which is described in the following standard;
the criteria are as follows:
if the proportion of cell large lipid droplets after treatment of a certain small molecule compound to be detected meets the following condition A or B:
A. after a certain small molecular compound to be detected is treated, the proportion of large cell lipid droplets is more than or equal to 0.7, and then the small molecular compound to be detected promotes the growth of lipid droplets and/or promotes the fusion of lipid droplets and/or increases the size of lipid droplets;
B. after a certain small molecular compound to be detected is treated, the proportion of large lipid droplets of cells is less than or equal to 0.3, and then the small molecular compound to be detected inhibits the growth of lipid droplets and/or inhibits the fusion of lipid droplets and/or reduces the size of lipid droplets;
if the condition is not met, the micromolecule compound to be detected does not regulate and control the growth of lipid droplets, does not regulate and control the fusion of the lipid droplets and does not regulate and control the size of the lipid droplets;
or, the regulation of lipid droplet growth is promoting lipid droplet growth or inhibiting lipid droplet growth.
The application of the substance for detecting the proportion of the large lipid droplets in the preparation of the compound for detecting or screening whether the compound to be detected is a lipid droplet fusion compound product is also within the protection scope of the invention;
or the application of the substance for detecting the proportion of the large lipid droplets in detecting or screening whether the compound to be detected is the lipid droplet fusion regulating compound is also within the protection scope of the invention.
In the application, the regulation of lipid droplet fusion is to promote lipid droplet fusion or inhibit lipid droplet fusion.
The application of the substance for detecting the proportion of the large lipid droplets in the preparation of a compound product for detecting or screening whether a compound to be detected regulates and controls the size of the large lipid droplets is also within the protection scope of the invention;
or the application of the substance for detecting the proportion of the large lipid droplets in detecting or screening whether the compound to be detected regulates and controls the size of the lipid droplets is also within the protection scope of the invention.
In the above application, the adjusting of the size of the lipid droplet is to increase the size of the lipid droplet or decrease the size of the lipid droplet.
It is another object of the invention to provide a product.
The product provided by the invention is a substance for detecting the proportion of large fat drops;
the substances for detecting the proportion of the large fat droplets comprise Hela cells expressing Cidec, a high content microscope and image analysis software Harmony;
the Cidec Hela expressing cell is a cell obtained by introducing CDS of a Cidec protein into a Hela cell; the parameters of the image analysis software Harmony are as follows:
1Input Image:
Stack processing: Maximum project
Flatfield correction:none
2Find Nuclei：
Channel: DAPI；
ROI Population: None
ROI Region of none
Method: B
Output Population:Nuclei
3Select Cell Region:
Population: Nuclei
Method: Resize Region
Region Type: Nucleus Region
Outer Border -128％
Inner Border 88％
Output Region: cyt0sol region
4Find Spots:
Channel: Alexa 488
ROI Population: Nuclei
ROI Region Cyt0sol region
Method B
Output Population: LDs
5Calculate Morphology Properties:
Population LDs
Region: Spot
Mehod: Standard
Output Properties: LD properties
6Select Population:
Population: LDs
Method Filter by property
Filter F1
LD properties Area um^2>2
Filter F2
LD properties roundness>0.9
Output Population: LD_large
7Select Population(2):
Population: LDs
Method: Filter by Property
Filter F1
LD properties<＝ 2
LD properties>0.05
Output Population: LDs small
8Define Results:
1)Method: Formula Output Formula:a/(a+b)
Population Type: objects
Variable A LD_Large_LD properties_area(um^2) ,Sum
Variable B LDs small_LD properties area(um^2),Sum
Output Name Large LD area ratio (big fat drop ratio)
2)Method: Formula Output Formula:(a+b)/c
Population Type: objects
Variable A LD_Large_LD Number of objects
Variable B LDs small_LD Number of objects
Variable C Nuclei-Number of objects
Output Name LD number (average number of fat droplets)
3)Method: Formula Output Formula:(a+b)/c
Population Type: objects
Variable A LD_Large_LD properties_area(um^2) ,Sum
Variable B LDs LDs small_LD properties area(um^2),Sum
Variable C Nuclei-Number of objects
Output Name: lipid average area
The large lipid droplet proportion is the total area of the large lipid droplets to the total area of all the lipid droplets.
In the product, the substance for detecting the proportion of the large fat droplets further comprises a readable carrier which is described in the following standard;
the criteria are as follows:
if the proportion of cell large lipid droplets after treatment of a certain small molecule compound to be detected meets the following condition A or B:
A. after a certain small molecular compound to be detected is treated, the proportion of large cell lipid droplets is more than or equal to 0.7, and then the small molecular compound to be detected promotes the growth of lipid droplets and/or promotes the fusion of lipid droplets and/or increases the size of lipid droplets;
B. after a certain small molecular compound to be detected is treated, the proportion of large lipid droplets of cells is less than or equal to 0.3, and then the small molecular compound to be detected inhibits the growth of lipid droplets and/or inhibits the fusion of lipid droplets and/or reduces the size of lipid droplets;
if the condition is not met, the micromolecule compound to be detected does not regulate and control the growth of lipid droplets, does not regulate and control the fusion of the lipid droplets and does not regulate and control the size of the lipid droplets;
or, the product has the following functions:
1) detecting or screening whether the compound to be detected is a compound for regulating lipid droplet growth;
2) detecting or screening whether the compound to be detected is a regulation lipid droplet fusion compound;
3) detecting or screening whether the compound to be detected regulates and controls the size of the lipid droplets.
The 3 rd object of the present invention is to provide a method for detecting or screening whether a test compound modulates the growth of lipid droplets.
The method provided by the invention comprises the following steps:
1) using a compound to be detected to carry out drug adding treatment and dyeing treatment on the Cidec Hela cells to obtain drug-added and dyed cells;
2) detecting the cells after the drug is added and dyed by using a high content microscope to obtain image information of all lipid droplets;
3) carrying out Harmony software analysis on the image information of all lipid droplets to obtain the proportion of large lipid droplets expressing Cidec Hela cells after treatment of each small molecular compound to be detected;
if the proportion of cell large lipid droplets after treatment of a certain small molecule compound to be detected meets the following condition A or B:
A. after a certain small molecular compound to be detected is treated, the proportion of large cell lipid droplets is more than or equal to 0.7, and then the small molecular compound to be detected promotes the growth of lipid droplets and/or promotes the fusion of lipid droplets and/or increases the size of lipid droplets;
B. after a certain small molecular compound to be detected is treated, the proportion of large lipid droplets of cells is less than or equal to 0.3, and then the small molecular compound to be detected inhibits the growth of lipid droplets and/or inhibits the fusion of lipid droplets and/or reduces the size of lipid droplets;
if the condition is not met, the micromolecule compound to be detected does not regulate and control the growth of the lipid droplets, does not regulate and control the fusion of the lipid droplets and does not regulate and control the size of the lipid droplets.
The above-mentioned lipid droplet size is embodied in particular by the lipid droplet diameter.
The experiment of the invention proves that the invention,
1) immortalized HeLa cells were taken to obtain a large lipid droplet phenotype in HeLa cells similar to adipocytes by expressing the key protein for lipid droplet fusion, Cidec. And because a stable expression cell line system is adopted, the lipid droplets in the cells are distributed more uniformly, so that the difference between the cells is reduced, and the statistics of lipid droplet shapes is facilitated. Meanwhile, the HeLa cells have good cell consistency, easy culture conditions and short experimental period, and can be beneficial to large-scale screening.
2) The lipid droplet morphology is represented by adopting three parameters, namely the proportion of large lipid droplets, the average lipid droplet number and the average lipid droplet area through a full-automatic high-precision lipid droplet identification algorithm, so that the understanding of the influence of small molecules on the lipid droplets is enhanced; the large lipid droplet proportion and the lipid droplet number can characterize the fusion capacity of the lipid droplets; the number of lipid droplets and the area of lipid droplets are related to the neutral lipid content in the cells. The influence of the drug on the cells can be comprehensively known through the parameter analysis of the three lipid droplet forms. The reliability of the index of the large fat drop proportion is found to be the highest through an actual screening experiment. Therefore, the screening is carried out by taking the compound as a main index, and the other two are taken as reference indexes.
3) Through screening of Sigma LOPAC (1280) small molecule library, drugs showing black (large lipid droplet ratio of about 0.2) or white (large lipid droplet ratio of about 0.8) in heatmap with large lipid droplet ratio were selected. After elimination of the drug causing significant cell death; 8 small molecules had a clear effect on lipid droplets, with only 1 of the lipid droplet phenotype increased and 7 of the lipid droplet phenotype decreased. Because Ouabain and Dihydrouabain in the lipid droplet phenotype are both cardiac glycoside inhibitors acting on Na/K-ATPase. Two inhibitors with the same target point suggest that the inhibitor can be effective on lipid droplets, so that another cardiac glycoside inhibitor Digoxin (Digoxin) which is relatively easy to obtain is selected for testing, and the inhibitor has the same lipid droplet inhibition effect. Further experiments on Digoxin show that the expression level of Cidec protein is inhibited by Digoxin, and Digoxin also inhibits the expression level of other proteins such as GM130, so that the effect on lipid droplets is probably achieved by inhibiting the expression of Cidec.
In conclusion, Heila cells stably expressing Cidec were established as a model of cellular obesity and small molecules causing lipid droplet size changes could be screened by characterizing lipid droplet morphology. The establishment of the three indexes facilitates the understanding of the effect of the medicine on the cells. The strategy of artificially forming large lipid droplets in cells by utilizing the property of lipid droplet fusion and then observing the morphological change of the lipid droplets can effectively perform related screening on mammalian cells. Of course, drug screening in hela cells also requires subsequent confirmation of small molecule specificity and confirmation of function in adipocytes. The range of a drug library used for screening small molecule drugs can be expanded continuously.
The present invention allows for the size of lipid droplets to be increased by stable expression of the Cidec protein in immortalized cells, thereby obtaining a lipid droplet phenotype similar to adipocytes. The small molecular compound with the lipid droplet size is screened by utilizing the characteristic that the immortalized cells are easy to screen in a large scale and combining the added lipid droplet phenotype. Such a screening scale can be made very large. Meanwhile, a full-automatic lipid drop recognition algorithm is used, the lipid drop form is recognized automatically in a large scale, and the form of the lipid drop can be represented quickly by using three lipid drop indexes.
Drawings
FIG. 1 shows that stable expression of Cidec in HeLa cells increases lipid droplet size.
FIG. 2 shows that the lipid exchange capacity of Cidec in stable cell lines is the same as that in transiently expressing cells.
FIG. 3 is a control group of four experiments prepared to establish screening conditions.
FIG. 4 is a graph depicting average lipid droplet area for neutral lipid content.
FIG. 5 is a large lipid droplet ratio characterizing the fusion ability of lipid droplets.
FIG. 6 shows that the average lipid droplet number can also represent the lipid droplet fusion capacity.
Figure 7 is a distribution of sample holes in a high content screen.
FIG. 8 is a graph showing the results of one 384-well plate experiment.
FIG. 9 shows the Z-factor results for each lipid droplet parameter.
FIG. 10 is a heatmap of the effect of small molecule drugs on the proportion of large lipid droplets in cells.
FIG. 11 shows the screened phenotypic drugs.
FIG. 12 is a graph of the effect of cardiac glycosides on lipid droplet size.
FIG. 13 shows that digoxin causes a decrease in Cidec protein expression.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 detection method establishment and parameter selection for whether to control lipid droplet growth by test Compound
Selection of parameters required by detection method for detecting whether to-be-detected compound regulates and controls growth of lipid droplets
1. Construction of HeLa cell line stably expressing Cidec
The CDS sequence of murine Cidec was recombined into the genome of HeLa cells using the PiggyBac Transposon System as follows:
the recombinant plasmid is obtained by replacing the CDS sequence (sequence 1) of Cidec of a mouse source between Pac1 and Xma1 sites of a PiggyBac vector (System biosciences, PB 513B-1).
The recombinant plasmid and a Super PiggyBac transpose Expression Vector (System biosciences, PB210PA-1) are co-transfected into HeLa cells (ATCC, CCL-2), and a HeLa cell line stably expressing Cidec is obtained by separating monoclonal cells.
The adopted Super PiggyBac Expression Vector expresses Super PiggyBacTransposase, and a Cidec gene can be inserted into a TTAA site of a HeLa cell genome, so that stable Expression of the Cidec gene in a HeLa cell is realized, and a HeLa cell line for stably expressing the Cidec is obtained by separating a monoclonal cell.
2. Cell dosing and staining
The screening is performed in 384-well plates as follows:
digestion: the Cidec-stably expressing HeLa cell line obtained in1 above, grown in 10cm culture dishes, was first digested with Trypsin (AMRESCO T0458-250G), and the cells were plated in 384-well microplates (CellCarrier) using 405Washer Dispenser (BioTek)TMUltra 384-well microplates, PerkinElmer, USA), 40ul per well volume;
cell dosing: after 24 hours of culture, the small molecule compounds to be tested were added to the corresponding wells using Echo 550Liquid Handler (labctye, San Jose, USA), then 20ul DMEM containing 600uM OA (oleic acid) was supplemented per well to reach a final concentration of 200uM OA, after 18 hours of incubation, the cells were washed three times with PBS, then fixed for 15min with 4% paraformaldehyde, and then washed three times with PBS;
dyeing: staining with PBS containing Bodipy (1ug/ml, Life Science) and Hoechst 33342(4ug/ml, Sigma-Aldrich) for 15 minutes, washing three times with PBS; obtaining the HeLa cell line which stably expresses Cidec after being treated by adding medicine.
Each of the above steps was performed using 405Washer Dispenser (BioTek).
3. Acquisition of cellular lipid droplet images
The lipid droplets are not distributed on the same focal plane of the microscope, so the cells are subjected to image acquisition by adopting a layer scanning mode, which specifically comprises the following steps:
the drug-treated stably Cidec-expressing HeLa cell line (Cidec) obtained in 2 above was examined using an Opera Phenix High-Content Screening System (PerkinELmer, USA) High-Content microscope, and images of 5 positions were collected for each well using a 60X (NA1.15) water mirror, and 30 layers were taken for each position to obtain image information of all lipid droplets. The 488 channel is responsible for acquiring images of Bodipy-stained lipid droplets, and the 405 channel is responsible for acquiring images of Hoechst-stained nuclei.
A wild-type HeLa cell line (wild-type) was used as a control.
Results are shown in FIG. 1, scale, 20 μm, 5 μm, smaller lipid droplets for wild-type HeLa cell line (wild-type) (a), which did not increase the size of the lipid droplets more than 2.5 μm (diameter) even when incubated with Oleic Acid (OA), because there was no CIDE family protein expression in the wild-type HeLa cell line, and there was no reason for the lipid droplet fusion process; while the size of the lipid droplets of the HeLa cell line (Cidec) stably expressing Cidec was significantly increased compared to wild type and the number of lipid droplets was reduced.
To test the fusion capacity of Cidec in HeLa cells, a lipid exchange rate test was performed on HeLa cell lines (Cidec) stably expressing Cidec. Control HeLa cell line transiently expressing Cidec.
The detection method of the above-mentioned lipid exchange rate is described in the following documents J.Gong, Z.Sun, L.Wu, W.xu, N.Schieber, D.Xu, G.Shui, H.Yang, R.G.Parton, P.Li, Fsp27 proteins lipid drop below and transfer at lipid drop below, J CellBiol,195(2011) 953-gel 963; the specific method comprises the following steps: the method comprises the following steps of (1) enabling cells to grow in a living cell dish (MatTek, P35G-0-20-C) in an adherent mode, adding a fluorescent dye (Invitrogen, Bodipy 558/568C12), adding OA, changing into a common culture medium after 17 hours, observing under a microscope to find a pair of lipid droplets, quenching red fluorescence carried by small lipid droplets in the lipid droplets by using an FRAP mode, and recording changes of fluorescence reduction in the large lipid droplets and fluorescence increase in the small lipid droplets; the exchange rate of lipid droplets is calculated according to a formula, which is described in the literature.
The above HeLa cell line transiently expressing Cidec: HeLa cells were transfected with 0.5. mu.g of pepFN 1-Cidec plasmid (described in J.Gong, Z.Sun, L.Wu, W.xu, N.Schieber, D.xu, G.Shui, H.Yang, R.G.Parton, P.Li, Fsp27 promoter lipid droplet growth by lipid droplet exchange and transfer at lipid droplet contacts, J Cell Biol,195(2011) 953-963) using Lipofectamine 2000(Thermofisher, 11668027), and then 4h later the same fluorochrome and OA were added and the same lipid droplet pair was searched for lipid exchange rate experiments 17h after transfection.
The results of the lipid exchange rate test are shown in fig. 2, (a) the lipid exchange rate test is used for testing the lipid fusion capacity of Cidec in the hela cells, the transient expression cell test is used for testing 18 pairs of lipid droplets, the stable expression cell test is used for testing 23 pairs of lipid droplets, the sizes of the lipid droplet pairs selected in the two groups are similar, the error line represents the standard deviation, and the student's t test is adopted, so that NS has no statistical difference; (b) comparing the difference in protein levels between transient and stable expression of Cidec, transient expression of Cidec-flag as a positive control; it can be seen that the HeLa cell line stably expressing Cidec has the same lipid droplet fusion capacity as cells transiently expressing Cidec; the level of Cidec protein in HeLa cell lines stably expressing Cidec is lower than that in transiently expressing cells.
4. Analysis of the cell image yields the proportion of large lipid droplets, the average number of lipid droplets within the cell and the average lipid droplet area within the cell
The image information of all lipid droplets of the drug-treated HeLa cell line (Cidec) stably expressing Cidec obtained in the above 3 was subjected to cell image analysis by using Harmony (Perkinelmer, USA) software carried by a high content microscope, and the specific software parameters were as follows:
the settings made on Harmony are as follows:
1 Input Image:
Stack processing:Maximum project
Flatfield correction:none
2 Find Nuclei：
Channel:DAPI；
ROI Population:None
ROI Region of none
Method:B
Output Population:Nuclei
3 Select Cell Region:
Population:Nuclei
Method:Resize Region
Region Type:Nucleus Region
Outer Border-128％
Inner Border 88％
Output Region:cyt0sol region
4 Find Spots:
Channel:Alexa 488
ROI Population:Nuclei
ROI Region Cyt0sol region
Method B
Output Population:LDs
5 Calculate Morphology Properties:
Population LDs
Region:Spot
Mehod:Standard
Output Properties:LD properties
6 Select Population:
Population:LDs
Method Filter by property
Filter F1
LD properties Area um^2>2
Filter F2
LD properties roundness>0.9
Output Population:LD_large
7 Select Population(2):
Population:LDs
Method:Filter by Property
Filter F1
LD properties<＝2
LD properties>0.05
Output Population:LDs small
8Define Results:
1)Method:Formula Output Formula:a/(a+b)
Population Type:objects
Variable A LD_Large_LD properties_area(um^2),Sum
Variable B LDs small_LD properties area(um^2),Sum
Output Name Large LD area ratio (big fat drop ratio)
2)Method:Formula Output Formula:(a+b)/c
Population Type:objects
Variable A LD_Large_LD Number of objects
Variable B LDs small_LD Number of objects
Variable C Nuclei-Number of objects
Output Name LD number (average number of fat droplets)
3)Method:Formula Output Formula:(a+b)/c
Population Type:objects
Variable A LD_Large_LD properties_area(um^2),Sum
Variable B LDs LDs small_LD properties area(um^2),Sum
Variable C Nuclei-Number of objects
Output Name: lipid average area
Obtaining the large lipid drop proportion, the average intracellular lipid drop number and the average intracellular lipid drop area of all lipid drops of the HeLa cell line (Cidec) which stably expresses the Cidec after the drug adding treatment.
The ratio of large lipid droplets, the average number of intracellular lipid droplets and the average area of intracellular lipid droplets are taken as detection parameters, and the detection parameters have the following significances:
second, establishing a method for screening the size of lipid droplets
1. Establishment of four control groups
Wild type hela cell group: the HeLa cell line was cultured in a medium for 2 days to obtain cells.
Expression of Cidec hela cell group: and (3) culturing the constructed HeLa cell line stably expressing Cidec in a culture medium for 2 days to obtain cells.
HeLa cell group expressing Cidec-KKRA with fusion loss of function mutation: and (3) culturing the HeLa cell line expressing the fusion function deletion mutant Cidec-KKRA in a culture medium for 2 days to obtain cells.
Addition of BFA-treated Cidec hela cell group: and (3) culturing the stably-expressing Cidec Hela cells constructed in the previous step in a culture medium containing 200ng/ml BFA for 1 day to obtain cells.
The HeLa cell expressing the Cidec-KKRA with the fusion function deletion mutation is obtained by introducing a Cidec-KKRA mutation gene into a HeLa cell line through a recombinant vector; wherein, the recombinant vector is a plasmid obtained by replacing a Cidec-KKRA mutant gene (sequence 2) between Pac1 and Xma1 sites of a PiggyBac vector.
The 4 control groups were tested by the methods of 2 and 3 in the above-mentioned group, wherein the drug to be tested was Digoxin (Selleck, S4290).
The microscopic results are shown in FIG. 3, (a) wild-type Hela cells, lipid droplet size is small; (b) cidec expressing Hela cell, lipid droplet size is large; (c) HeLa cells expressing the Cidec-KKRA mutation have small lipid droplet size; (d) heila cells expressing Cidec treated with medium containing 200ng/ml BFA, with small lipid droplet size; scale, 20 μm, 5 μm; it can be seen that wild type Hela cells are of the small lipid droplet phenotype, Hela cells expressing Cidec are of the large lipid droplet phenotype, and mutants of Cidec-KKRA deprive Cidec of the ability to fuse lipid droplets are of the small lipid droplet phenotype. BFA treatment also lost Cidec fusion, which is also a lipid droplet phenotype; indicating that HeLa cells expressing Cidec increase lipid droplet size.
2. Detection of
1) Detection of average lipid droplet area
The group of HeLa cells expressing Cidec was subdivided into 2 groups as follows:
OA +: the HeLa cell line stably expressing Cidec constructed in the above-mentioned one was cultured in OA medium containing 200. mu.mol concentration for 1 day to obtain cells.
OA-: and (3) culturing the HeLa cell line which is constructed in the step one and stably expresses Cidec in a culture medium for 1 day to obtain cells.
The 2 groups were tested by methods 2, 3 and 4 in the first group, wherein the drug to be tested was Digoxin (Selleck, S4290).
The mean lipid droplet area characterizes the neutral lipid content in the cells. To test the reliability of this parameter, the effect of the addition of oleic acid on this parameter was compared; since the cells store oleic acid synthesized neutral lipids in lipid droplets in media containing oleic acid, the neutral lipid content of the cells increases.
The results of the average lipid droplet area are shown in FIG. 4, and it can be seen that the neutral lipid content increased after OA was added to the medium expressing Cidec Hela cells, and the average lipid droplet area increased three times, corresponding to the expected change in the neutral lipid content of the cells. Error bars represent standard deviations, tested by student's t,. p < 0.05. The above results indicate that this parameter, the mean lipid droplet area, can characterize the neutral lipid content in the cells.
2) Fat drop ratio detection
And (3) detecting 4 control groups in the group 1 by using the methods 2, 3 and 4 in the first step, wherein the drug to be detected is digoxin.
The results of large lipid droplet ratios are shown in FIG. 5, which shows that wild-type cells do not have lipid droplet fusion ability, cells expressing Cidec-KKRA do not have lipid droplet fusion ability, BFA inhibits the fusion ability of Cidec, and the large lipid droplet ratios are reduced in all three conditions compared with cells expressing Cidec; indicating that the large lipid droplet ratio can characterize this difference in lipid droplet fusion capacity. Error bars represent standard deviations, tested using student's t,. p < 0.01.
3) Average lipid droplet number detection
And (3) detecting 4 control groups in the group 1 by using the methods 2, 3 and 4 in the first step, wherein the drug to be detected is digoxin.
The mean lipid droplet number characterizes the condition of lipid droplet fusion.
As a result, as shown in FIG. 6, the number of lipid droplets in the process of lipid droplet fusion is reduced, so the average number of lipid droplets can also characterize the condition of lipid droplet fusion, and this parameter is influenced by other factors such as the generation condition of lipid droplets. Therefore, in the case of BFA treatment, the average number of lipid droplets is reduced although the ability to fuse lipid droplets is hindered, and the generation of lipid droplets may be affected. Cidec-KKRA only blocks fusion of lipid droplets so that the number of lipid droplets is higher than in cells expressing Cidec as in wild type. Error bars represent standard deviations, NS, no statistical difference using student's t test,. p <0.05,. p < 0.01.
Thirdly, the LOPAC (1280) micromolecule drug library of sigma is used for screening drug experiments influencing the growth of lipid droplets
And a LOPAC (1280) micromolecule drug library of sigma is selected for screening, so that micromolecule drugs influencing the growth of lipid droplets are expected to be found, and the reliability of a screening strategy is verified.
Since the working range of the high content microscope for 384-well plates does not include the outermost two layers, each screening is limited to 240 wells in the interior, and the specific method is as follows:
1. adding medicine and dyeing
Adding medicine: after the HeLa cells stably expressing Cidec prepared above were plated in 384-well plates, after 24h of adherence, each test compound in the small molecule drug library was added to the corresponding well using Echo 550Liquid Handler (LABCYTE, San Jose, USA), then 20ul of DMEM containing 600uM OA (oleic acid) was supplemented to each well to make the final concentration of OA 200uM, after 18h of incubation, the cells were washed three times with PBS, then fixed for 15min with 4% paraformaldehyde, and then washed three times with PBS.
Dyeing: the cells were stained with PBS containing Bodipy (1ug/ml, Life Science) and Hoechst 33342(4ug/ml, Sigma-Aldrich) for 15 minutes and washed three times with PBS. Each of the above steps was performed using 405Washer Dispenser (BioTek).
Media containing DMSO and OA served as negative controls for large lipid droplets.
Two positive controls with reduced lipid droplets were culture medium containing DMSO without OA, and culture medium containing BFA and OA, respectively.
The distribution of sample pores in the high content screen is shown in figure 7. The middle two columns are used for placing cells of a control group, and the rest red areas are experimental groups added with small molecule drugs. The outermost two layers of the 384-well plate could not be tested, so the drug test samples were placed in red frames, the middle two columns were placed with negative controls (left, blue frames) and positive controls (right, green frames), and the remaining wells were the experimental groups. (a) The culture medium contains DMSO and OA, and cells form a negative control of large lipid droplets; (b) positive control 1, which contained no OA in the medium and had slightly smaller lipid droplets than (a); (c) medium contained BFA and OA, positive control 2 with small and numerous lipid droplets in the cells. (b) And (c) alternately arranged. Scale, 20 μm.
2. Acquisition of cellular lipid droplet images
The method is the same as 3 in the first step.
3. Analysis of the cell image yields the proportion of large lipid droplets, the average number of lipid droplets within the cell and the average lipid droplet area within the cell
The method is the same as 4 in the first step.
The results are shown in FIG. 8, where the X-axis is the proportion of large lipid droplets in the sample, the Y-axis is the location of the well where the sample is located, and the Z-axis is the number of lipid droplets. The colors in the figure represent the average lipid droplet area. The projection of the sample on the XZ plane is represented by a small blue dot. The orange dotted line circles the positive control 2 without OA medium. Circled within the light blue dashed line is BFA-treated positive control 1. Cell death at position H6. It can be seen that most drugs have little effect on lipid droplets in cells, and their parameters are concentrated in a narrow range. The positive control group in the experiment can be obviously displayed in the population, which indicates that the control is very stable. The average number of lipid droplets and the average lipid droplet area are sensitive to whether OA is added, while the large lipid droplet fraction is sensitive to BFA addition.
Through a Z-factor test for detecting the reliability of the parameters, the reliability of the three parameters is different. The Z-factor is highest for large lipid droplet ratios. This may be related to the nature of the positive control, BFA has good inhibition of Cidec fusion without significant effect on lipid content, so the average lipid droplet area reliability under BFA is not high. The addition of OA has a significant effect on lipid content, which is used as a positive control with a higher Z-factor value for the average lipid droplet area. The Z-factor values for the average lipid droplet numbers were low, and it was likely that no suitable positive control could be found.
Therefore, the reliability of the index of the large lipid droplet ratio is found to be the highest through actual screening experiments, the large lipid droplet ratio is used as a main standard for screening, and the other two lipid droplet parameters are only used as reference standards for lipid droplet morphology.
To compare the effectiveness of the individual lipid droplet parameters, the Z-factor was compared under two positive controls (red for BFA and OA containing media and green for DMSO only) and negative controls for OA only media (FIG. 9). The results of the three tests, error bars represent standard deviations.
Experiment for screening drugs influencing lipid drop growth by cardiac glycoside drugs
Screening was performed using a small molecule library of Sigma LOPAC (1280) with known active sites.
The detection method obtains the ratio of large lipid droplets of the HeLa cell line which stably expresses Cidec and is treated by each medicament according to the ratio of 2-4 in the first step.
A heatmap was prepared using the large lipid droplet ratio as a screening standard, and drugs corresponding to black and white positions in the heatmap of each 384-well plate were used as the screened drugs. Since some drugs are lethal, the drug candidates that cause massive cell death are removed, resulting in the drugs in the red boxes in FIG. 10.
The results of the large lipid droplet ratio for each drug treatment are indicated by color, and the two central columns of each plate are the negative control group and the positive control group, respectively. The red box is the selected hit.
The corresponding parameter values for the drugs obtained from figure 10. The left y-axis represents the value of the mean lipid droplet number and also the value of the mean lipid droplet area (in μm)2) The right y-axis represents the large fat droplet ratio. The corresponding drug names are shown in table 1.
Cidec expressing Heila cells were each dosed with 10. mu.M of the drugs shown in Table 1: (a) DMSO, (b) dihydrouabain, (c) Ouabain, (d) Digoxin. Scale, 20 μm, 5 μm.
TABLE 1 screening of small molecule drugs affecting lipid droplet growth
Under the same experimental conditions, the expression level of the protein such as Cidec is checked, and digoxin is found to cause the expression level of Cidec and GM130 to be reduced.
Then, another two indexes of each candidate drug are researched, and the 8 candidate drugs do not influence the total content of lipid droplets or even slightly increase the total content of lipid droplets when the drugs for inhibiting lipid droplet fusion. The positive control drug BFA is also present in a small molecule drug library and is found to affect lipid droplet fusion in the screening, which indicates that the screening accuracy is high. In the screened candidate drugs, it is found that two cardiac glycoside drugs, Ouabain and Dihydroouabain, are small molecules acting on a sodium-potassium ion pump, and both can inhibit fusion of lipid droplets. This suggests that cardiac glycosides may influence the lipid droplet fusion process (fig. 11).
In summary, a method for detecting whether a compound to be detected regulates the growth of lipid droplets, or regulates the fusion of lipid droplets, or regulates the size of lipid droplets is established, which specifically comprises the following steps:
1) adding medicine for dyeing
After the HeLa cells expressing Cidec are paved on a 384-well plate, after 24 hours of adherence, small molecule compounds to be detected are respectively added into corresponding wells, then 20ul of DMEM containing 600uM OA (oleic acid) is supplemented into each well to enable the OA final concentration to reach 200uM, after 18 hours of incubation, the cells are washed three times by PBS, then 4% paraformaldehyde is fixed for 15 minutes, and then the cells are washed three times by PBS.
The cells were stained with PBS containing Bodipy (1ug/ml, Life Science) and Hoechst 33342(4ug/ml, Sigma-Aldrich) for 15 minutes and washed three times with PBS.
Each of the above steps was performed using 405Washer Dispenser (BioTek).
2) Acquisition of cellular lipid droplet images
Detecting the cells treated in the step 1) by using a high content microscope (the method is the same as the step 2) to obtain image information of all lipid droplets;
3) analysis of cell images to obtain large lipid droplet ratios
Analyzing the image information of all lipid droplets obtained in the step 2) by using Harmony software to obtain the ratio of large lipid droplets expressing Cidec Hela cells after treatment of each small molecular compound to be detected;
if the proportion of cell large lipid droplets after treatment of a certain small molecule compound to be detected meets the following conditions:
A. after a certain small molecular compound to be detected is treated, the proportion of large cell lipid droplets is more than or equal to 0.7, and then the small molecular compound to be detected promotes the growth of lipid droplets and/or promotes the fusion of lipid droplets and/or increases the size of lipid droplets;
or, the ratio of cell large lipid droplets after treatment of a certain small molecular compound to be detected is less than or equal to 0.3, and the small molecular compound to be detected inhibits the growth of lipid droplets and/or inhibits the fusion of lipid droplets and/or reduces the size of lipid droplets;
if the condition is not met, the micromolecule compound to be detected does not regulate and control the growth of the lipid droplets, does not regulate and control the fusion of the lipid droplets and does not regulate and control the size of the lipid droplets.
Example 2 detection of whether test Compounds are related to lipid droplet growth control
According to the method for detecting whether the compound to be detected regulates the growth of lipid droplets, or regulates the fusion of lipid droplets or regulates the size of lipid droplets, which is established in the embodiment 1, the Dihydrouabain, Ouabain and digoxin are respectively used as the compound to be detected, and the proportion of the large lipid droplets after the Cidec Hela cells are expressed is detected.
The high content microscopy results are shown in fig. 12; a is Hela-Cidec cells plus control DMSO, b is Dihydrouabain, C is Ouabain, and d is Digoxin.
The proportion of large lipid droplets in cells after the treatment of Dihydroouabain was 0.21, the average lipid droplet area in cells after the treatment of Dihydroouabain was 85, and the average lipid droplet number in cells after the treatment of Dihydroouabain was 76.
The proportion of large lipid droplets in cells after Ouabain treatment was 0.18, the average lipid droplet area in cells after Ouabain treatment was 82, and the average lipid droplet number in cells after Ouabain treatment was 76.
The ratio of intracellular lipid droplets after digoxin treatment was 0.26, the average lipid droplet area in cells after digoxin treatment was 75, and the average lipid droplet number in cells after digoxin treatment was 56.
It can be seen that digoxin has the ability to reduce lipid droplet size.
Since digoxin drugs are readily available, their effect on Cidec protein expression was examined and was found to affect Cidec expression and also to reduce expression of protein GM130 on the golgi (figure 13). This suggests that these drugs may be broadly inhibitory to protein expression, thus producing the lipid droplet phenotype. This also suggests that the results obtained from the screening will require more experiments to confirm whether the small molecule drug found specifically acts only on the lipid droplet growth regulatory pathway.
Sequence listing
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Claims (9)
1. The application of the substance for detecting the large fat drop proportion in preparing a compound product for detecting or screening whether a compound to be detected is a compound for regulating and controlling the growth of fat drops;
or the application of the substance for detecting the large fat drop proportion in detecting or screening whether the compound to be detected is a compound for regulating the growth of fat drops;
the substances for detecting the proportion of the large fat droplets comprise Hela cells expressing Cidec, a high content microscope and image analysis software Harmony;
the Cidec Hela expressing cell is a cell obtained by introducing CDS of a Cidec protein into a Hela cell;
the large lipid droplet proportion is the total area of the large lipid droplets to the total area of all the lipid droplets.
2. Use according to claim 1, characterized in that: the substance for detecting the proportion of the large fat droplets also comprises a readable carrier which is described as the following standard;
the criteria are as follows:
if the proportion of cell large lipid droplets after treatment of a certain small molecule compound to be detected meets the following condition A or B:
A. after a certain small molecular compound to be detected is treated, the proportion of large cell lipid droplets is more than or equal to 0.7, and then the small molecular compound to be detected promotes the growth of lipid droplets and/or promotes the fusion of lipid droplets and/or increases the size of lipid droplets;
B. after a certain small molecular compound to be detected is treated, the proportion of large lipid droplets of cells is less than or equal to 0.3, and then the small molecular compound to be detected inhibits the growth of lipid droplets and/or inhibits the fusion of lipid droplets and/or reduces the size of lipid droplets;
if the condition is not met, the micromolecule compound to be detected does not regulate and control the growth of lipid droplets, does not regulate and control the fusion of the lipid droplets and does not regulate and control the size of the lipid droplets;
or, the regulation of lipid droplet growth is promoting lipid droplet growth or inhibiting lipid droplet growth.
3. The use of a substance for detecting the proportion of large lipid droplets as claimed in claim 1 or 2 in the preparation of a product for detecting or screening whether a test compound is a fusion compound for regulating lipid droplets;
or the use according to claim 1 or 2, wherein said substance for detecting the proportion of large lipid droplets is used for detecting or screening whether a test compound is a compound for modulating lipid droplet fusion.
4. Use according to claim 3, characterized in that: the regulation lipid droplet fusion is to promote lipid droplet fusion or inhibit lipid droplet fusion.
5. The use of the substance for detecting the proportion of large lipid droplets in the use according to claim 1 or 2 for preparing a compound product for detecting or screening whether a test compound regulates and controls the size of lipid droplets;
or the use according to claim 1 or 2, for detecting the proportion of large lipid droplets in a test or screening test compound for modulating lipid droplet size.
6. Use according to claim 5, characterized in that: the adjusting of the size of the lipid droplet is to increase the size of the lipid droplet or decrease the size of the lipid droplet.
7. A product is used for detecting the proportion of large fat droplets;
the substances for detecting the proportion of the large fat droplets comprise Hela cells expressing Cidec, a high content microscope and image analysis software Harmony;
the Cidec Hela expressing cell is a cell obtained by introducing CDS of a Cidec protein into a Hela cell;
the large lipid droplet proportion is the total area of the large lipid droplets to the total area of all the lipid droplets.
8. The product of claim 7, wherein:
the substance for detecting the proportion of the large fat droplets also comprises a readable carrier which is described as the following standard;
the criteria are as follows:
if the proportion of cell large lipid droplets after treatment of a certain small molecule compound to be detected meets the following condition A or B:
A. after a certain small molecular compound to be detected is treated, the proportion of large cell lipid droplets is more than or equal to 0.7, and then the small molecular compound to be detected promotes the growth of lipid droplets and/or promotes the fusion of lipid droplets and/or increases the size of lipid droplets;
B. after a certain small molecular compound to be detected is treated, the proportion of large lipid droplets of cells is less than or equal to 0.3, and then the small molecular compound to be detected inhibits the growth of lipid droplets and/or inhibits the fusion of lipid droplets and/or reduces the size of lipid droplets;
if the condition is not met, the micromolecule compound to be detected does not regulate and control the growth of lipid droplets, does not regulate and control the fusion of the lipid droplets and does not regulate and control the size of the lipid droplets;
or, the product has the following functions:
1) detecting or screening whether the compound to be detected is a compound for regulating lipid droplet growth;
2) detecting or screening whether the compound to be detected is a regulation lipid droplet fusion compound;
3) detecting or screening whether the compound to be detected regulates and controls the size of the lipid droplets.
9. A method for detecting or screening whether a compound to be detected regulates the growth of lipid droplets comprises the following steps:
1) using a compound to be detected to carry out drug adding treatment and dyeing treatment on the Cidec Hela cells to obtain drug-added and dyed cells;
2) detecting the cells after the drug is added and dyed by using a high content microscope to obtain image information of all lipid droplets;
3) carrying out Harmony software analysis on the image information of all lipid droplets to obtain the proportion of large lipid droplets expressing Cidec Hela cells after treatment of each small molecular compound to be detected;
if the proportion of cell large lipid droplets after treatment of a certain small molecule compound to be detected meets the following condition A or B:
A. after a certain small molecular compound to be detected is treated, the proportion of large cell lipid droplets is more than or equal to 0.7, and then the small molecular compound to be detected promotes the growth of lipid droplets and/or promotes the fusion of lipid droplets and/or increases the size of lipid droplets;
B. after a certain small molecular compound to be detected is treated, the proportion of large lipid droplets of cells is less than or equal to 0.3, and then the small molecular compound to be detected inhibits the growth of lipid droplets and/or inhibits the fusion of lipid droplets and/or reduces the size of lipid droplets;
if the condition is not met, the micromolecule compound to be detected does not regulate and control the growth of the lipid droplets, does not regulate and control the fusion of the lipid droplets and does not regulate and control the size of the lipid droplets.
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