A method for constructing a dry eye cell model based on mitochondrial dysfunction

Abstract

The present invention relates to a method for constructing a dry eye cell model based on mitochondrial dysfunction, which is characterized in that the human corneal epithelial cells cultured in vitro are treated by a method of targeted knockout or knockdown of the expression level of TFAM in human corneal epithelial cells, and then a dry eye cell model induced by mitochondrial dysfunction is constructed in a directional manner. The present invention uses targeted knockout / knockdown of the expression of TFAM in human corneal epithelial cells to establish a dry eye cell model that can better and more realistically reflect mitochondrial dysfunction-induced dry eye cell model, which has the characteristics of high model construction success rate, stable model characteristics, and no generation of non-target cell models, and is conducive to the study of the mechanism of downstream mitochondrial dysfunction in the occurrence and development of dry eye or horizontal comparison between treatment schemes.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for constructing a dry eye cell model based on mitochondrial dysfunction. Background Art

[0002] As one of the most common ocular surface diseases, dry eye can not only cause eye discomfort, but also cause blindness in severe cases, seriously affecting the quality of life of patients. With the widespread use of electronic devices and changes in people's lifestyles, work intensity, and living environments, the prevalence of dry eye in the world's population is 5-50%. Because dry eye is affected by many factors, there is still a lack of effective treatment methods. The pathogenesis of dry eye is complex, and the role of mitochondrial dysfunction in the occurrence and development of dry eye has attracted more and more attention from researchers. Existing dry eye cell models often use hypertonic treatment of human corneal epithelial cells. For example, an article reported that different concentrations of benzalkonium chloride were used to act on corneal epithelial cells cultured in vitro, and the changes in cell morphology were observed under a light microscope, and cell activity and apoptosis were detected to prepare a dry eye corneal epithelial cell injury model. The properties of this model constructed based on hypertonic treatment of human corneal epithelial cells are not stable. The properties of the dry eye cell models obtained from different batches, different concentrations and treatment times are different, and even the properties of the dry eye cell models obtained from the same treatment batch are different. This also limits the horizontal comparison of research conclusions on treatment options based on dry eye cell models. High concentrations can easily lead to a large number of cell apoptosis, resulting in a low success rate for model construction, a waste of time, reagent materials and labor costs, while low concentrations may not lead to significant mitochondrial dysfunction in cells.

[0003] In addition, although changes in mitochondrial function may be observed in the hypertonic dry eye cell model, the effects of hypertonic treatment on corneal epithelial cells are multifaceted, complex, and comprehensive, and are likely to produce many unnecessary cell characteristics that may interfere with subsequent research, and cannot truly reflect the pathological changes in dry eye caused by corneal epithelial cell mitochondrial dysfunction and be used to evaluate the therapeutic effects of related programs. Therefore, there is currently a lack of a method for efficiently and accurately constructing a dry eye cell model with mitochondrial dysfunction. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the above shortcomings and deficiencies of the prior art, the present invention provides a method for constructing a dry eye cell model based on mitochondrial dysfunction, which uses targeted knockout / knockdown of the expression of TFAM in human corneal epithelial cells to establish a dry eye cell model that can better and more realistically reflect mitochondrial dysfunction. The method of the present invention has a high success rate in model construction, more stable model traits / characteristics, no non-target cell traits, and the cell model has significant mitochondrial dysfunction, which is conducive to the study of the mechanism of downstream mitochondrial dysfunction in the occurrence and development of dry eye or horizontal comparison between treatment schemes.

[0006] (II) Technical solution

[0007] The technical solution of the present invention is as follows:

[0008] A method for constructing a dry eye cell model based on mitochondrial dysfunction, which uses a method of targeted knockout or knockdown of TFAM expression levels in human corneal epithelial cells to treat human corneal epithelial cells cultured in vitro, and then directionally constructs a dry eye cell model induced by mitochondrial dysfunction.

[0009] According to a preferred embodiment of the present invention, a method for targeted knockout or knockdown of TFAM expression level in human corneal epithelial cells comprises treating human corneal epithelial cells cultured in vitro with at least one of siRNA, Vector-based shRNA, CRISPR / Cas9 editing system, transcription activator-like effector nuclease technology TALEN, and zinc finger nuclease technology Zinc finger nuclease for targeted knockout or knockdown of TFAM gene in cells.

[0010] According to a preferred embodiment of the present invention, the siRNA targeting the knockout or knockdown of the TFAM gene in cells is obtained by combining the following four siRNAs in equal amounts according to a molar ratio of 1:1:1:1:

[0011] The nucleic acid sequence of siRNA-1 is: CGGAGUGGCAGGUAUAUAA;

[0012] The nucleic acid sequence of siRNA-2 is: CCAAGAAGCUAAGGGUGAU;

[0013] The nucleic acid sequence of siRNA-3 is: GAAAUCAUGGACAAACAUU;

[0014] The nucleic acid sequence of siRNA-4 is: GAAGAAUUGCCCAGCGUUG.

[0015] According to a preferred embodiment of the present invention, the construction method comprises: using siRNA that targets the knockout or knockdown of the TFAM gene in cells to disperse in siRNA Buffer, and then adding buffer and Reagent to obtain a transfection mixture, and use the transfection mixture to treat human corneal epithelial cells cultured in vitro.

[0016] According to a preferred embodiment of the present invention, in the treatment solution, the total concentration of siRNA is 25-50 nmol / L, preferably 25 nmol / L.

[0017] According to a preferred embodiment of the present invention, the following steps are included:

[0018] (1) Human corneal epithelial cells were digested with 0.25% trypsin at 37°C and counted. The cells were resuspended in complete medium containing antibiotics and 450 μL of the cell suspension was added to each well of a 24-well plate to make the number of cells per well 1×10 5 ; Incubate cells overnight at 37°C, 5% CO2;

[0019] (2) Using a transfection mixture containing 25-50 nmol / L siRNA, culture human corneal epithelial cells at 37° C. and 5% CO 2 for 24-48 hours.

[0020] According to a preferred embodiment of the present invention, after transfection, the following operations are also included: using Western blot and immunofluorescence to detect the expression level of TFAM protein in cells, using CCK8 to detect cell activity, using TMRM to detect mitochondrial membrane potential, using quantitative PCR to detect mtDNA copy number, using Seahorse energy metabolism method to analyze mitochondrial function, and using real-time quantitative RT-PCR and immunofluorescence to detect the expression level of inflammatory factors.

[0021] (III) Beneficial effects

[0022] The present invention can accurately / efficiently and directionally construct a dry eye cell model with obvious mitochondrial functional damage characteristics by knocking down the expression of TFAM in human corneal epithelial cells. Compared with the existing method for constructing dry eye cell models, the method of the present invention is simple and rapid, and the constructed cell model has stable properties or characteristics, and is not prone to produce multifaceted, complex, unnecessary, and even cell properties that may interfere with subsequent research. The present invention is more suitable for the study of the mechanism of mitochondrial dysfunction in the occurrence and development of dry eye.

[0023] The cell model construction method of the present invention can retain the cell physiological characteristics caused by non-mitochondrial dysfunction to the maximum extent, has a high success rate in cell model construction, and saves time and effort. The application of the present invention includes: constructing a dry eye cell model that can more realistically reflect mitochondrial dysfunction-induced dry eye cell models, so as to facilitate theoretical research on mitochondrial dysfunction-induced dry eye cell models and the development of new drugs / treatment plans. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 In Example 1, the knockdown efficiency of TFAM in human corneal epithelial cells cultured in vitro was measured by treating the human corneal epithelial cells with siRNA.

[0025] Figure 2 These are the results of the effect of knocking down TFAM in human corneal epithelial cells cultured in vitro on the activity of human corneal epithelial cells.

[0026] Figure 3 The results show the effects of knocking down TFAM in human corneal epithelial cells cultured in vitro on mitochondrial membrane potential, mtDNA copy number and mitochondrial function of human corneal epithelial cells.

[0027] Figure 4 These are the results of the effect of knocking down TFAM in human corneal epithelial cells cultured in vitro on the expression levels of inflammatory factors in human corneal epithelial cells. DETAILED DESCRIPTION

[0028] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation modes in conjunction with the accompanying drawings.

[0029] Recent studies have found that mtDNA damage exists in the ocular surface tissue of dry eye mouse models. mtDNA is a double-stranded circular DNA molecule that exists in a condensed DNA-protein complex called a nucleoid and contains 13 protein subunits, 2 rRNAs, and 22 tRNAs responsible for encoding the mitochondrial electron transport chain complex. Mitochondrial function is highly dependent on functional mtDNA, and the maintenance of its stability is mainly mediated by TFAM. TFAM is a packaging protein required for mtDNA replication and transcription. Under pathological conditions, the destruction of TFAM can lead to mtDNA loss, resulting in mitochondrial functional defects or mitochondrial dysfunction.

[0030] Based on the new findings of the above research, the present invention uses a method of targeted knockout or knockdown of the expression level of TFAM in human corneal epithelial cells to treat human corneal epithelial cells cultured in vitro, and then constructs a mitochondrial dysfunction-induced dry eye cell model. The method of knocking out or knocking down the expression level of TFAM in human corneal epithelial cells includes but is not limited to any gene editing method or genetic modification engineering method currently known, for example, the method can use at least one of siRNA targeting the TFAM gene, Vector-based shRNA, CRISPR / Cas9 editing system, transcription activator-like effector nuclease technology TALEN, and zinc finger nuclease technology Zinc finger nuclease.

[0031] The following is a description of the present invention in conjunction with specific embodiments.

[0032] Example 1

[0033] The specific operation method of this embodiment is as follows:

[0034] (1) Designing and chemically synthesizing siRNAs. The siRNAs used in this example are obtained by combining the following four siRNAs in equal amounts according to a molar ratio of 1:1:1:1:

[0035] siRNA-1: CGGAGUGGCAGGUAUAUAA, see SEQ ID NO.1;

[0036] siRNA-2: CCAAGAAGCUAAGGGUGAU, see SEQ ID NO. 2;

[0037] siRNA-3: GAAAUCAUGGACAAACAUU, see SEQ ID NO. 3;

[0038] siRNA-4: GAAGAAUUGCCCAGCGUUG, see SEQ ID NO.4.

[0039] (2) Dissolve 5 nmol siRNA dry powder in 250 μL 1× siRNA Buffer, mix well with a pipette, and place it on a shaker for 30 min at room temperature to obtain a 20 μM siRNA stock solution, which is then stored at -20°C for experimental use.

[0040] (3) In vitro culture of human corneal epithelial cells: Digest the cells with 0.25% trypsin at 37°C and count them; resuspend the cells in complete medium containing antibiotics and add 450 μL of cell suspension to each well of a 24-well plate so that the number of cells in each well is 1×10 5 ; Incubate cells overnight at 37°C, 5% CO2.

[0041] (4) Set the final concentration of siRNA to 25nM and 50nM respectively. Take the transfection of cells in two wells of a 24-well plate as an example. In order to calculate the loss during the pipetting process, multiply the volume of reagents required in the transfection experiment by 2.5. The preparation of transfection solution and transfection method are as follows:

[0042] ① Prepare 250nM siRNA solution in tube a: take 1.56μL 20μM siRNA stock solution and add 123.44μL buffer, vortex for 10 seconds, and centrifuge briefly;

[0043] ② Prepare 500nM siRNA solution in tube b: Take 3.13μL 20μM siRNA stock solution and add 121.87μL buffer, vortex for 10 seconds and centrifuge briefly.

[0044] ③ Add 5 μL to tube a and tube b respectively Reagent, vortex for 1s, and incubate at room temperature for 10min; take 52μL of the prepared transfection mixture in tube a and tube b respectively (due to pipetting loss, 50μL is actually added), add them to the wells (450μL of cell suspension has been pre-added) to obtain a final siRNA concentration of 25nM and 50nM, and place the 24-well plate at 37°C, 5% CO2 for 24-48h before subsequent experiments.

[0045] (5) Identification of knockdown efficiency: 24 hours after transfection, the RNA level expression of the target gene TFAM was detected by real-time quantitative RT-PCR, and the optimal final concentration of siRNA was determined; 48 hours after transfection, the protein level expression was detected by Western blot and immunofluorescence, and the protein level expression of TFAM was detected by immunofluorescence.

[0046] like Figure 1 As shown in (A), the RNA expression of TFAM was detected by real-time quantitative RT-PCR. The results showed that the knockdown efficiency of siRNA with a final concentration of 25nM and 50nM was 92% and 94%, respectively, but there was no statistical difference. Therefore, 25nM was selected as the optimal final concentration of siRNA in subsequent experiments. Figure 1 As shown in (B), the protein expression of TFAM was detected by Western blot. Figure 1 As shown in (C), Figure (B) was further analyzed using ImageJ analysis, showing that the knockdown efficiency of TFAM protein level was approximately 50%.

[0047] like Figure 1As shown in (D), the protein expression of TFAM in the 25 nM transfection group was detected by immunofluorescence. Figure 1 As shown in (E), further analysis of Figure (D) using ImageJ analysis showed that the TFAM fluorescence intensity in the knockdown group was significantly reduced, indicating that its TFAM protein level was significantly reduced. The scale bar in the figure is 25μm.

[0048] (5) Identification of cell activity, mitochondrial function, and inflammatory levels: 24 hours after transfection, CCK8 was used to detect cell activity; 48 hours after transfection, mitochondrial membrane potential was detected by TMRM, mtDNA copy number was detected by quantitative PCR, and mitochondrial function was analyzed by Seahorse energy metabolism method; real-time quantitative RT-PCR and immunofluorescence were used to detect the expression levels of inflammatory factors.

[0049] The nucleotide sequences of the Control siRNAs used in the experiment are as follows:

[0050] A mixture of UGGUUUACAUGUCGACUAA, UGGUUUACAUGUUGUGUGA, UGGUUUACAUGUUUUCUGA, and UGGUUUACAUGUUUUCCUA in a molar ratio of 1:1:1:1. The method for preparing the transfection mixture is as described in step (4) of this example, and the final concentration is 25 nM.

[0051] like Figure 2 As shown in the figure, CCK8 was used to detect cell activity, and the results showed that knocking down the expression of TFAM in human corneal epithelial cells by siRNA did not affect the activity of the cells. Compared with the hypertonic induced dry eye cell model method, this can improve the accuracy and customization of cell modeling, and the modeling process does not affect cell activity. Therefore, when used for the study of dry eye pathological mechanisms or treatment plans, accurate research or evaluation results can be obtained, avoiding the modeling process causing a decrease in cell activity and interfering with subsequent research conclusions.

[0052] like Figure 3 As shown, 48 h after transfection, the changes in mitochondrial membrane potential were detected using the fluorescent probe labeled mitochondrial dye TMRM (as shown in Figure 3 A), ImageJ analysis ( Figure 3 B) shows that the TMRM fluorescence intensity in the knockdown group was significantly decreased, indicating that its mitochondrial membrane potential was significantly reduced (mitochondrial functional damage). The scale bar in the figure is 10 μm.

[0053] See also Figure 3As shown in Figure C, real-time fluorescence quantitative PCR was used to detect changes in mtDNA copy number. The results showed that the copy number of mtDNA (including mt-CYB, mt-COX1, mt-COX2, and mt-ATP6) in the knockdown group was significantly reduced. The copy number of mt-CYB decreased by 50%, the copy number of mt-COX1 decreased by 40%, the copy number of mt-COX2 decreased by 20%, and the copy number of mt-ATP6 decreased by 30%. This shows that by targeting and knocking down TFAM in human corneal epithelial cells, the copy number of mtDNA in cells can indeed be significantly damaged. Figure 3 (D) and (F) , Seahorse energy metabolism method was used to evaluate mitochondrial function. The results showed that mitochondrial function was impaired in the knockdown group, and ATP production (ATP production) and maximum respiration (Maximal respiration) were significantly reduced. The scale bar in the figure is 25μm.

[0054] See also Figure 4 As shown in (A), 24 hours after transfection, the expression of inflammatory factors in cells was detected by real-time quantitative RT-PCR. The results showed that the RNA levels of TNF-α and IL-6 in the knockdown group were significantly upregulated; the inflammatory factor TNF-α in the model cells increased by more than 70% compared with the control group, and IL-6 increased by more than 65% compared with the control group. Figure 4 As shown in (B), 48 h after transfection, the protein level of TNF-α in cells was detected by immunofluorescence and further analyzed by ImageJ. Figure 4 (C) shows that the fluorescence intensity of TNF-α in the knockdown group increased significantly, indicating that its protein level was significantly upregulated. The scale bar in the figure is 10 μm. The above data show that the dry eye cell model was successfully constructed.

[0055] Combined with the above experimental data, it can be seen that by targeting and knocking down TFAM in human corneal epithelial cells, obvious mitochondrial dysfunction can be induced in the cells, and the expression of inflammatory factors (TNF-α, IL-6) is significantly upregulated. These results show that knocking down the expression of TFAM in human corneal epithelial cells can establish a dry eye cell model that can better and more realistically reflect mitochondrial dysfunction-induced dry eye cells.

[0056] It should be noted that, although the above embodiments only use siRNAs to illustrate the scheme of the present invention, this is not a limitation on the specific construction method of the dry eye cell model induced by mitochondrial dysfunction. The present invention mainly provides a biological method to efficiently, accurately and stably construct a dry eye model that can reflect mitochondrial dysfunction. It can maximize the retention of cell characteristics caused by other non-mitochondrial dysfunction of human corneal epithelial cells, has the advantages of stable model construction characteristics, no non-target cell model characteristics, and no reduction in cell activity, which is very conducive to the study of the mechanism of downstream mitochondrial dysfunction in the occurrence and development of dry eye or horizontal comparison between treatment schemes.

[0057] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing a dry eye cell model based on mitochondrial dysfunction, characterized in that: The method uses a method of targeted knockout or knockdown of TFAM expression level in human corneal epithelial cells to treat human corneal epithelial cells cultured in vitro, thereby directionally constructing a dry eye cell model induced by mitochondrial dysfunction; the method of targeted knockout or knockdown of TFAM expression level in human corneal epithelial cells includes using siRNA that targets the knockout or knockdown of TFAM gene in cells; The siRNA targeting the knockout or knockdown of the TFAM gene in cells is obtained by combining the following four siRNAs in equal amounts at a molar ratio of 1:1:1:1: The nucleic acid sequence of siRNA-1 is: CGGAGUGGCAGGUAUAUAA; The nucleic acid sequence of siRNA-2 is: CCAAGAAGCUAAGGGUGAU; The nucleic acid sequence of siRNA-3 is: GAAAUCAUGGACAAACAUU; the nucleic acid sequence of siRNA-4 is: GAAGAAUUGCCCAGCGUUG.

2. The construction method according to claim 1, characterized in that: The construction method comprises: dispersing siRNA targeting the knockout or knockdown of TFAM gene in cells in siRNA Buffer, and then adding buffer and Reagent to obtain a transfection mixture, and use the transfection mixture to treat human corneal epithelial cells cultured in vitro.

3. The construction method according to claim 2, characterized in that: In the treatment solution, the total concentration of siRNA is 25-50 nmol / L.

4. The construction method according to claim 2, characterized in that: The steps include: (1) Human corneal epithelial cells were digested with 0.25% trypsin at 37°C and counted; the cells were resuspended in complete medium containing antibiotics, and 450 μL of the cell suspension was added to each well of a 24-well plate so that the number of cells in each well was 1×105; the cells were incubated overnight at 37°C and 5% CO2; (2) Using a transfection mixture containing 25-50 nmol / L siRNA, culture human corneal epithelial cells at 37° C. and 5% CO 2 for 24-48 hours.

5. The construction method according to claim 4, characterized in that: After transfection, the following operations were also included: Western blot and immunofluorescence were used to detect the expression of TFAM in cells, CCK8 was used to detect cell activity, TMRM was used to detect mitochondrial membrane potential, quantitative PCR was used to detect mtDNA copy number, Seahorse energy metabolism method was used to analyze mitochondrial function, and real-time quantitative RT-PCR and immunofluorescence were used to detect the expression levels of inflammatory factors.

Images