Stable transfection cell strain for evaluating fibroblast autophagy flux as well as construction method and application of stable transfection cell strain

Abstract

The invention discloses a stably transfected cell strain for evaluating fibroblast autophagy flux and a construction method of the stably transfected cell strain. The construction method comprises the following step: infecting fibroblasts by using HBAD (Hepatitis B Antigen)-red fluorescent protein-EGFP-LC3 autophagy double-labeled adenovirus. The construction method can be combined with a super-resolution microscope to dynamically observe the autophagy intensity change in the primary fibroblasts through an imaging method.

Description

Technical Field

[0001] The present application relates to the technical field of cell biology, and in particular to a stable cell line for evaluating autophagic flux in fibroblasts, and a construction method and application thereof. Background Art

[0002] Autophagy is a highly conserved cellular metabolic process, whereby the cell's own structures, through the lysosome mechanism, transport damaged organelles, misfolded proteins, and other macromolecules to the lysosome for degradation and reuse. It is widely present in eukaryotic cells. Autophagy primarily consists of four phases: phagophore formation, autophagosome formation, autolysosome formation, and autolysosomal degradation. The formation and degradation of autolysosomes are considered the terminal stages of autophagy, and at this stage, autophagy is generally considered irreversible.

[0003] Autophagic flux is a dynamic process in which autophagy occurs continuously within cells. The absence of any link in the autophagic flux will hinder the functional implementation of the autophagic process. Therefore, autophagic flux is considered to be the "gold standard" for cellular autophagy detection.

[0004] Currently, the main methods for detecting autophagy include transmission electron microscopy and LC3 fluorescent labeling. Transmission electron microscopy is the most traditional method, capable of monitoring the complete autophagy process. LC3 fluorescent labeling uses a fluorescent tag protein to label the marker protein LC3 on the inner membrane of the autophagosome, and combines this with fluorescence microscopy to quantitatively analyze autophagy. However, these methods are associated with experimental costs and time constraints. Summary of the Invention

[0005] Based on this, the present application at least provides a stable cell line and a construction method thereof for evaluating the autophagic flow of fibroblasts.

[0006] In a first aspect of the present application, a method for constructing a stable cell line for evaluating autophagic flux in fibroblasts is provided, which comprises infecting fibroblasts with a HBAD-red fluorescent protein-EGFP-LC3 autophagy double-labeled adenovirus.

[0007] In a second aspect of the present application, a stable cell line for evaluating autophagic flux in fibroblasts is provided, which is constructed using the construction method described in the first aspect.

[0008] In a third aspect of the present application, a method for evaluating autophagic flux in fibroblasts is provided, the method comprising:

[0009] Cell climbing and nuclear staining: implanting a cover glass into a multi-well plate, culturing the stably transfected cell line described in the second aspect in the multi-well plate, performing nuclear staining on the cultured cells, and continuing to culture after nuclear staining;

[0010] Autophagy flow photography: Use a super-resolution microscope or confocal microscope to excite EGFP and red fluorescent protein fluorescence, and evaluate the cell autophagy flow through fluorescence staining results; wherein, the fluorescence staining results include the number of red bright spots.

[0011] This application proposes a method for constructing a red fluorescent protein-EGFP-LC3 adenovirus-stable cell line based on fibroblasts, which can be combined with super-resolution microscopy to dynamically observe changes in autophagy intensity in fibroblasts through imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the technical solutions in the implementation methods and examples of this application and to more completely understand the application and its beneficial effects, the following briefly introduces the drawings required for the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present application.

[0013] Figure 1 These are primary fibroblasts infected with adenovirus in one embodiment of the present application.

[0014] Figure 2 This is a comparison of the autophagic flux of senescent fibroblasts prepared by UVA irradiation and normal cells in one embodiment of the present application.

[0015] Figure 3 This is a schematic diagram of the results of cell staining in one embodiment of the present application; the left side is the β-gal staining result with p=5; the right side is the β-gal staining result of fibroblasts with p=40. DETAILED DESCRIPTION

[0016] To facilitate understanding of the present application, a more comprehensive description of the present application will be provided below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present application. However, the present application may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present application.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0018] In this application, unless otherwise specified, "one or more" refers to any one of the listed items or any combination of the listed items. Similarly, "one or more" and other similar expressions that refer to "one or more" are also understood in the same way unless otherwise specified.

[0019] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0020] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method", etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0021] In this application, the terms "further," "further," "particularly," "for example," "such as," "example," and "for example" are used for descriptive purposes to indicate that the preceding and following technical solutions are related in terms of the content covered, but should not be construed as limiting the preceding technical solution or the scope of protection of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it is understood that A is not limited to B.

[0022] In this application, the terms "optionally," "optional," and "optional" mean optional, that is, they refer to either option selected from the two parallel options of "yes" or "no." If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "optional" is independent. Unless otherwise specified, the descriptions "optionally include," "optionally include," etc. in this application, taking "optionally include" as an example, mean "may include or not include."

[0023] As used herein, the terms "comprising," "including," and "include" are synonymous and are inclusive or open-ended, not excluding additional, unrecited members or features. Examples of members or features include materials or components, structures, elements, and instruments. Non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, and states.

[0024] In this application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0025] In this application, exemplary descriptions such as "in some embodiments (or examples)" and "in one embodiment (or example)" may include but are not limited to the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0026] In this application, the terms "first," "second," "third," "fourth," etc. in "the first aspect," "the second aspect," "the third aspect," "the fourth aspect," etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor should they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc. serve only for the purpose of non-exhaustive enumeration and description, and should be understood not to constitute a closed-ended limitation on quantity.

[0027] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values in the numerical interval is considered to be continuous and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval only refers to an integer in the numerical interval, including the two endpoint integers of the numerical range and each integer between the two endpoints, is equivalent to directly enumerating each integer. When multiple numerical ranges are provided to describe a feature or characteristic, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0028] In this application, if a method flow involves multiple steps, unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in an order other than the order described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages do not necessarily need to be completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating, or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0029] In a first aspect of the present application, a method for constructing a stable cell line for evaluating autophagic flux in fibroblasts is provided.

[0030] In some embodiments, a method for constructing a stable cell line for evaluating autophagic flux in fibroblasts comprises infecting fibroblasts with a HBAD-red fluorescent protein-EGFP-LC3 autophagy double-labeled adenovirus.

[0031] This construction method can be combined with super-resolution microscopy to dynamically observe changes in autophagy intensity in fibroblasts through imaging methods.

[0032] The principle of dynamic observation includes at least the following: EGFP and red fluorescent protein expressed in a red fluorescent protein-EGFP-LC3 tandem fluorescent protein adenovirus are used to mark and track LC3B. The weakening of EGFP indicates the fusion of lysosomes and autophagosomes to form autolysosomes (because EGFP fluorescent protein is sensitive to acid, EGFP fluorescence is quenched after the fusion of autophagosomes and lysosomes, and only red fluorescence can be detected at this time).

[0033] It is well known that observing autophagic flux in fibroblasts presents significant technical challenges compared to other cell types, primarily due to their unique biological properties and close interaction with the extracellular matrix (ECM). Specifically, on the one hand, the more complex cellular structure and metabolism of fibroblasts, for example, their high secretory activity interferes with signal detection, and spatial competition between autophagosomes and organelles increases the difficulty of observing autophagic flux. On the other hand, the dynamic influence of the ECM microenvironment, such as the permeability limitations of 3D culture models and interference with bidirectional ECM-autophagy regulation, is also present. Furthermore, the heterogeneity and low throughput of autophagic flux dynamics, such as differences in transition rates between autophagic stages and heterogeneity of cell subpopulations, are also present. Finally, functional verification is complex.

[0034] For example, when comparing the autophagic flux observations in fibroblasts with those in epithelial / cancer cells, it was found that:

[0035] 1. Autophagosome generation rate: Fibroblasts are slower (require long-term starvation induction), while epithelial cells / cancer cells are faster (high basal autophagy level);

[0036] 2. ECM interference: Fibroblasts secrete high levels of ECM, leading to background noise; whereas epithelial cells / cancer cells secrete low levels of ECM, resulting in clear signals.

[0037] 3. Mechanical sensitivity: Fibroblasts are highly dependent on ECM stiffness regulation, while epithelial cells / cancer cells respond less strongly to mechanical signals;

[0038] 4. Lysosomal activity: Fibroblasts have high degradation efficiency and p62 changes are not significant; while epithelial cells / cancer cells have a moderate degradation rate and p62 dynamics are obvious.

[0039] The inventors creatively constructed a stably transfected cell line, which can be used to evaluate the autophagic flux of fibroblasts and can be combined with super-resolution microscopy to achieve dynamic observation through imaging methods.

[0040] Unless otherwise specified, the term "stably transfected cell line" in this application refers to a cell population in which exogenous DNA (such as plasmids or viral vectors) has been stably integrated into the host cell genome through genetic engineering techniques, resulting in long-term, sustained expression of the target gene. After screening and amplification, such cell lines can transmit the exogenous gene to daughter cells during cell division, creating a genetically stable experimental model.

[0041] Unless otherwise specified, the term "autophagic flux" in this application refers to the dynamic flow of the entire cellular autophagy process, encompassing the complete chain of autophagosome formation, substrate encapsulation, autophagosome-lysosome fusion (forming autolysosomes), and content degradation. Essentially, it is a dynamic equilibrium process in which cells degrade damaged organelles, misfolded proteins, or pathogens through the autophagic pathway, emphasizing the "flux" of autophagic activity rather than a single stage.

[0042] In some embodiments, the construction method comprises:

[0043] S100 (cell plating): culture of primary fibroblasts with p≤40;

[0044] S200 (adenovirus infection): Fibroblasts were infected with HBAD-red fluorescent protein-EGFP-LC3 autophagy double-labeled adenovirus and then cultured;

[0045] S300 (cell screening): Add puromycin and continue culturing to screen the positive cells that have been successfully infected.

[0046] In some embodiments, in step S100, primary fibroblasts with p=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 are cultured.

[0047] Primary cells refer to cells obtained by isolating human tissues. Unlike immortalized cell lines, human primary fibroblasts (or human primary fibroblasts) can be propagated for about 50 generations after being isolated and prepared in vitro. Cells with p≤40 are considered to be fibroblasts that do not show signs of aging.

[0048] In some embodiments, the seeding density of cells in step S100 (cell plating) is 3×10 5 / hole up to 7×10 5 For example, in some embodiments, the cell density is 3×10 5 / hole, 3.5×10 5 / hole, 4×10 5 / hole, 4.5×10 5 / hole, 5×10 5 / hole, 5.5×10 5 / hole, 6×10 5 / hole, or a value or range between any two values.

[0049] In some embodiments, the culturing conditions in step S100 include culturing in complete medium at 37° C. and 5% CO 2 .

[0050] In some embodiments, the culturing time in step S100 is 16 hours to 30 hours. For example, in some embodiments, the culturing time is 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, or a range or value between any two values.

[0051] In some embodiments, in step S200, the culture medium used for culturing includes DMEM medium.

[0052] In some embodiments, in step S200, the titer of HBAD-red fluorescent protein-EGFP-LC3 autophagy double-labeled adenovirus is 5×10 8 ~2×10 9 For example, in some embodiments, the autophagy double-labeled adenovirus titer is 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 1.5×10 9 , 2×10 9 , or a value or range between any two values.

[0053] In the present application, the red fluorescent protein may be mCherry. Unless otherwise specified, the term "mCherry" in the present application refers to a red fluorescent protein that can be observed under a microscope.

[0054] Unless otherwise specified, the term "LC3" in this application refers to microtubule-associated protein 1A / 1B-light chain 3, which is involved in regulating and mediating autophagy. Specifically, it refers to LC3B, with the protein number NP_073729.

[0055] The term "HBAD-Red Fluorescent Protein-EGFP-LC3" refers to a fusion of two fluorescent proteins, Red Fluorescent Protein and EGFP, with LC3. Under normal conditions, autophagy activity in cells is low, and Red Fluorescent Protein-EGFP-LC3 is primarily present in the cytoplasm. When cells initiate autophagy, LC3-I converts to LC3-II and binds to the autophagosome membrane. Because EGFP is sensitive to acidic conditions, it gradually loses fluorescence in the acidic environment of the autophagosome, while Red Fluorescent Protein is less sensitive to acidic environments and maintains red fluorescence. By observing the fluorescence expression of Red Fluorescent Protein-EGFP-LC3, it is possible to determine whether the cell is undergoing autophagy.

[0056] HBAD is a promoter region, typically derived from viral or cellular genes, that drives the expression of subsequent genes. It can enhance the efficiency of gene expression in specific cell types or conditions.

[0057] The mechanism of this experiment is to utilize the pH-sensitive characteristic of GFP. In normal cells, red and green are expressed evenly. However, when entering the lysosomal step of the autophagy process, the pH changes, the GFP fluorescence is quenched, and only red fluorescence remains.

[0058] In some embodiments, HBAD-red fluorescent protein-EGFP-LC3 is HBAD-mcherry-EGFP-LC3. For example, HBAD-mcherry-EGFP-LC3 can be purchased from Hanbio Biotechnology.

[0059] In some embodiments, during the adenoviral infection step, DMEM medium is added 4 to 8 hours after infection and culture is continued for 36 to 48 hours. For example, the infection time can be 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, or a range or value between any two values.

[0060] In some embodiments, the culture is continued after supplementing with DMEM medium for 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, or a range or value between any two values.

[0061] The second aspect of the present application provides a stably transfected cell line for evaluating autophagic flux in fibroblasts, which is constructed using the construction method described in the first aspect.

[0062] Without wishing to be bound by any theory, it is believed that successfully transfected cells can be excited by 488 nm and 584 nm lasers, and can exhibit green and red fluorescence after excitation.

[0063] The third aspect of the present application provides use of the stably transfected cell line of the second aspect in evaluating autophagic flux in fibroblasts.

[0064] A fourth aspect of the present application provides a method for evaluating autophagic flux in fibroblasts.

[0065] In some embodiments, a method of assessing autophagic flux in fibroblasts is provided, the method comprising:

[0066] Cell climbing and nuclear staining: implanting a cover glass into a multi-well plate, adding the stably transfected cell line described in the second aspect into the multi-well plate and culturing the cultured cells, performing nuclear staining on the cultured cells, and continuing to culture after nuclear staining;

[0067] Autophagy flow photography: EGFP and mCherry fluorescence were stimulated, and the cell autophagy flow was evaluated by fluorescence staining results; wherein, the fluorescence staining results included the number of red bright spots.

[0068] Unless otherwise specified, the term "nuclear staining" in this application refers to the selective staining of cell nuclei by a specific staining method to make them clearly visible under a microscope. For example, Hoechst staining can be used to stain cells, which usually does not cause cell death after staining and can continue to be cultured.

[0069] In some embodiments, in the steps of cell climbing and nuclear staining, the concentration of the stably transfected cell line is 2×10 5 / hole~4×10 5 / well. For example, the concentration of the stably transfected cell line is 2×10 5 / hole, 2.5×10 5 / hole, 3×10 5 / hole, 3.5×10 5 / hole, 4×10 5 / hole, or a range or value between any two values.

[0070] In some embodiments, in the steps of cell climbing and nuclear staining, the cell culture time before nuclear staining is 8 hours to 24 hours, and the cell culture time after nuclear staining is 16 hours to 48 hours. Exemplarily, the cell culture time can be 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or a range or value between any two values.

[0071] In some embodiments, the culture is continued for 10 to 40 minutes after nuclear staining. Exemplarily, the culture is continued for 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 minutes, or any range or value between any two values.

[0072] In some embodiments, in the steps of cell slicing and nuclear staining, Hochest is used for nuclear staining. The nuclear staining time can be a conventional time in the art, such as 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, or a range or value between any two values.

[0073] Some examples are provided below.

[0074] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and may also be based on the experimental manuals or conventional conditions in this area, or on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0075] Example 1 Comparison of autophagic flux between UVA-irradiated senescent fibroblasts and normal fibroblasts

[0076] (1) Cell plating

[0077] The present invention is divided into negative control group (NC group), experimental group and positive control group (PC group). One day before the formal experiment, primary fibroblasts with p = 20 were selected and cultured at 3 × 10 5 The cells were planted in a six-well plate in advance, 2 mL of complete culture medium was added to each well, and then cultured in an incubator at 37°C and 5% CO2 for 24 hours.

[0078] (2) Adenovirus infection

[0079] According to the growth rate of fibroblasts, the cell density of the six-well plate was estimated to be 6×10 5 / well. Use a pipette to completely remove the culture medium in the six-well plate, wash twice with 200 μL of room temperature PBS, add 970 μL of DMEM complete culture medium to each well of the six-well plate, and then use a pipette to add 30 μL of HBAD-mcherry-EGFP-LC3 adenovirus working solution to each group of cells. Return the six-well plate to the incubator for culture. The titer of HBAD-mcherry-EGFP-LC3 adenovirus (purchased from Hanheng Bio) is 1×10 10 When used, 20 μL of adenovirus stock solution was added to 180 μL of complete culture medium for dilution. The adenovirus titer after dilution was 1×10 9 , calculated according to the formula (amount of virus added per well (μL) = MOI × number of cells / virus titer (PFU / mL) × 1000).

[0080] Six hours after infection, 1 mL of fresh DMEM complete medium was added to each well of the six-well plate, and the cells were cultured in an incubator for another 48 hours.

[0081] (3) Positive cell screening

[0082] 48 hours after adenovirus infection, 1 μg / mL of puromycin was added to each well of the six-well plate and cultured in an incubator for 72 hours to screen out positive cells successfully infected with adenovirus. The cells were then observed under a fluorescence microscope to confirm the positive rate.

[0083] (4) Quercetin treatment

[0084] The coverslips were placed in 24-well plates, and the cells in the six-well plates were then digested with trypsin. The cell suspension was then diluted to 20% (3 × 10 5 PCs were seeded in a 24-well plate at a density of 100 cells / well. Then, quercetin was added to the PC group at a final concentration of 300 nM, and the cells were returned to the incubator for overnight culture.

[0085] (5) Irradiation and nuclear staining

[0086] Three non-experimental 24-well plates were placed in the marked grid of the UVA irradiator to serve as backing plates. The 24-well plates were then placed sequentially on the backing plates of the UVA irradiator. The experimental and PC groups were irradiated according to the irradiation time, with a total irradiation of 30 joules. After irradiation, Hochest was diluted 1:1000 in PBS and added to the cells for staining for 10 minutes before incubation for an additional 24 hours.

[0087] (6) Super-resolution microscopy photography

[0088] After irradiation, use tweezers to carefully remove the coverslip from the 24-well plate, cover it on a glass slide, and then take pictures under a super-resolution microscope with fluorescence excitation intensities of 488 nm and 584 nm.

[0089] Primary fibroblast cell lines co-expressing mCherry and EGFP fluorescence were obtained by infection with HBAD-mcherry-EGFP-LC3 adenovirus and imaged using a fluorescence microscope. Figure 1 It was shown that 48 hours after HBAD-mcherry-EGFP-LC3 infection, significant red and green fluorescence could be observed under a fluorescence microscope, proving that the infection of this batch of experimental adenovirus was successful and the infection efficiency was high.

[0090] Figure 2 The results showed that compared with the NC group, the intensity of autophagic flux in the UVA-prepared senescent fibroblast model was significantly increased (the number of red bright spots), and fibroblasts treated with quercetin overnight in advance could resist the senescent effects of UVA irradiation.

[0091] According to the test results of fibroblasts of different generations, the results showed that from the p=40 generation, fibroblasts showed obvious senescence phenotype, which was characterized by a significant increase in the β-gal content in the cells. Figure 3 shown.

[0092] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0093] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the patent in this application shall be determined by the appended claims, and the specification and drawings shall serve to interpret the claims.

Claims

1. A method for constructing a stable cell line for evaluating autophagic flux in fibroblasts, characterized in that: It involves infecting fibroblasts with adenovirus encoding HBAD, red fluorescent protein, EGFP, and LC3 autophagy.

2. The construction method according to claim 1, wherein The construction method comprises: Cell plating: Culture primary fibroblasts with p≤40; Adenovirus infection: The cultured fibroblasts were infected with HBAD-red fluorescent protein-EGFP-LC3 autophagy double-labeled adenovirus and then continued to be cultured; Cell screening: Add puromycin and continue culturing to screen positive cells that have been successfully infected.

3. The construction method according to claim 2, wherein: Step 1: Cell plating meets one or more of the following conditions: A1) The seeding density of cells was 3×10 5 / hole up to 7×10 5 / hole; A2) Culture conditions include incubation in complete medium at 37°C and 5% CO2; A3) The culture time is 16 to 30 hours.

4. The construction method according to claim 2, wherein: The adenovirus infection step meets one or more of the following conditions B1) to B3): B1) The culture medium used includes DMEM medium; B2) The titer of HBAD-red fluorescent protein-EGFP-LC3 autophagy double-labeled adenovirus was 5×10 8 ~2×10 9 ; B3) 4 to 8 hours after infection, add DMEM medium and continue culturing for 36 to 48 hours.

5. A stable cell line for evaluating autophagic flux in fibroblasts, characterized in that: It is constructed using the construction method according to any one of claims 1 to 4. Use of the stably transfected cell line according to claim 5 in evaluating autophagic flux in fibroblasts.

7. A method for evaluating autophagic flux in fibroblasts, characterized in that The method comprises: Cell climbing and nuclear staining: implanting a cover glass into a multi-well plate, adding the stably transfected cell line according to claim 5 into the multi-well plate and culturing the cells, performing nuclear staining on the cultured cells, and continuing to culture after nuclear staining; Autophagy flow photography: EGFP and red fluorescent protein fluorescence are stimulated, and the cell autophagy flow is evaluated by the fluorescence staining results; wherein, the fluorescence staining results include the number of red bright spots.

8. The method according to claim 7, wherein Step 2: In cell culture and nuclear staining, the concentration of stably transfected cell lines is 2×10 5 / hole~4×10 5 / hole.

9. The method according to claim 7, wherein In the steps of cell climbing and nuclear staining, the cell culture time before nuclear staining is 8 hours to 24 hours, and the cell culture time after nuclear staining is 10 minutes to 40 minutes.

10. The method according to any one of claims 7 to 9, wherein In the steps of cell climbing and nuclear staining, Hochest was used for nuclear staining.

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