PINK1 inducible expression cell line of porcine umbilical vein endothelial cells

Abstract

The invention discloses a PINK1 inducible expression cell line of a porcine umbilical vein endothelial cell and a construction method and application thereof, a stable cell line with controllable PINK1 expression level is constructed by introducing a porcine PINK1 gene into the porcine umbilical vein endothelial cell and combining with an inducible expression system taking doxycycline as an inducer. The cell line is in a low-expression or non-expression state under the condition that an inducer is not added, and dose-dependent induced expression can be realized after doxycycline is added. Experimental results show that the induced expression of PINK1 can regulate and control the expression of angiogenesis related genes, activate mitochondrial autophagy related pathways under oxidative stress conditions, and influence the expression of cell proliferation and apoptosis related proteins at the same time. The cell line is clear in construction method and good in stability, can be used as an in-vitro model for researching angiogenesis, oxidative stress and related regulation and control mechanisms of porcine vascular endothelial cells, and has good practical value.

Description

Technical Field

[0001] This invention relates to an endothelial cell line that can be inducibly expressed, specifically a porcine umbilical vein endothelial cell line that can be inducibly expressed by PINK1, belonging to the field of molecular cell biology. Background Technology

[0002] China is the world's largest producer of pigs, accounting for 48% of the global market. However, according to the "2024-2029 China Pig Farming and Slaughtering Industry Market Panorama Survey and Development Prospect Forecast Report," the PSY (Pigs per Sow per Year) for sows in my country in 2024 was 25.05, while Denmark's was 34.14, indicating a significant gap. Low sow productivity is a bottleneck restricting the development of my country's pig farming industry. The number of live piglets per litter and the birth weight of piglets directly determine sow productivity. Uteroplacental efficiency in sows is a crucial determinant of embryonic survival and growth in early pregnancy. Placental angiogenesis plays a vital role in increasing uteroplacental efficiency, promoting embryonic growth and development, and improving sow litter size. Insufficient placental angiogenesis affects the efficiency of placental blood flow, oxygen, and nutrient transport, and is a major cause of high fetal mortality and intrauterine growth retardation (IUGR). Therefore, a key scientific issue for improving sow productivity is promoting placental angiogenesis.

[0003] VEGF / VEGFR2-mediated signaling pathways regulate the proliferation, migration, and lumen formation of vascular endothelial cells, playing a crucial role in placental angiogenesis and directly impacting embryonic nutrient supply and survival. Porcine placenta is a diffuse epithelial placenta, and embryonic nutrient acquisition primarily depends on the exchange of substances between the uterus and placental blood vessels. Studies have shown that VEGF expression and vascular density in porcine placenta increase with gestational age and are significantly positively correlated with fetal weight and placental efficiency. VEGF deficiency leads to fetal developmental arrest, further clarifying that promoting placental angiogenesis is key to improving sow reproductive efficiency. Early studies reported that increased PINK1 / BRPK expression is positively correlated with cancer cell metastatic potential and invasiveness. Further research revealed that tumor cell invasiveness is driven by PINK1, a process dependent on Rab27-mediated exocytosis of mtDNA-containing extracellular vesicles. Since tumor cell metastasis and invasiveness are closely related to angiogenesis, this suggests that PINK1 may be involved in the regulation of angiogenesis. In our previous study, using porcine placenta with intrauterine growth retardation (IUGR) as a research model, we found decreased PINK1 expression, reduced expression of the angiogenic factor VEGF, and increased expression of matrix metalloproteinase inhibitors TIMP1 and TIMP2, suggesting that PINK1 is involved in the regulation of angiogenesis in porcine placenta. Although PINK1 has been shown to affect cell function in other cell types, such as tumor cells and stem cells, by regulating cell metabolism and proliferation, its function is specific across different cell types. Therefore, the role of PINK1 in porcine umbilical vein endothelial cells remains unknown, necessitating the establishment of a targeted research model.

[0004] Currently, there are no reported studies on constructing PINK1-inducible expression cell lines in porcine umbilical vein endothelial cells. Porcine placental development includes the construction of the placental vascular network, such as angiogenesis and endothelial cell proliferation, migration, and lumen formation. Constructing a PINK1-inducible expression line can clarify how PINK1-mediated mitophagy activates and clears damaged mitochondria, how it affects angiogenesis, and how it maintains endothelial cell homeostasis and vascular function. Furthermore, placental development is a highly dynamic process; traditional stable overexpression may lead to persistently high PINK1 expression, which does not conform to its physiological regulatory mechanism. Using a doxycycline-inducible system to construct an induced expression line allows for artificial control of the relative expression level of PINK1, more accurately simulating PINK1 expression in angiogenesis under specific conditions, and thus accurately analyzing the protective mechanism under specific conditions. Additionally, pigs are animal models for cardiovascular adaptation studies, and their vascular physiology, size, and certain pathological responses are more similar to humans. Constructing such an induced expression cell line can provide a pathophysiologically relevant cell model comparable to the human placenta, offering another perspective and approach to the pathological mechanisms of related diseases.

[0005] In the existing technology, there have been studies that have constructed cell models with stable overexpression or inducible expression of PINK1 in common human or mouse cells, such as tumor cells, embryonic kidney cells or stem cells, by regulating the expression of PINK1 through stable transfection or tetracycline induction system, for the purpose of studying biological processes such as mitophagy, cell metabolism regulation or cell invasion.

[0006] However, existing technologies mainly focus on non-porcine cells or non-vascular endothelial cell types, and the research conclusions obtained are difficult to directly reflect the physiological regulatory characteristics of porcine vascular endothelial cells, especially porcine placental-associated vascular endothelial cells. At the same time, the existing technologies mentioned above do not involve the construction of stable and dose-controllable PINK1-inducible expression cell lines in porcine umbilical vein vascular endothelial cells, nor do they reveal the regulatory role of PINK1 on angiogenesis-related functions in porcine vascular endothelial cells.

[0007] In addition, in angiogenesis-related research, existing techniques typically use human vascular endothelial cells as the research object, focusing on the VEGF / VEGFR2 signaling pathway to analyze the processes of vascular endothelial cell proliferation, migration, and lumen formation, in order to elucidate the molecular mechanisms of angiogenesis.

[0008] However, the above studies have mostly focused on the classical angiogenesis signaling pathway itself, lacking in-depth research on the role of key factors related to mitochondrial quality control in vascular endothelial cells; in particular, the existing technology has not established a controllable induced expression model of PINK1 in porcine umbilical vein vascular endothelial cells, nor has it revealed the mechanism by which PINK1 regulates the expression of angiogenesis-related genes and cell function in porcine vascular endothelial cells.

[0009] In summary, current technologies lack a stable cell model that can precisely regulate PINK1 expression levels in porcine umbilical vein endothelial cells, and also fail to systematically elucidate the mechanism of action of PINK1 in porcine vascular endothelial cell angiogenesis and oxidative stress response.

[0010] Therefore, by constructing a porcine umbilical vein endothelial cell line that induces PINK1 expression, precise regulation of PINK1 expression can be achieved, which can further elucidate the role and regulatory mechanism of PINK1 in regulating porcine placental angiogenesis and provide a reference method for the subsequent establishment of porcine cell lines that induce PINK1 expression. Summary of the Invention

[0011] The purpose of this invention is to provide a PINK1-inducible expression cell line of porcine umbilical vein endothelial cells, and to disclose its construction method and application.

[0012] The present invention achieves the above objectives through the following technical solutions:

[0013] A PINK1-inducible expression cell line of porcine umbilical vein endothelial cells, wherein the cell line incorporates an induction expression system using doxycycline as an inducer, the induction expression system comprising:

[0014] (1) PINK1 gene expression cassette regulated by the TetIIP promoter,

[0015] (2) The PINK1 gene is derived from pigs.

[0016] (3) The PINK1 gene is fused with the tag protein for expression.

[0017] (4) The PINK1 gene was in a low-expression or non-expression state without the addition of an inducer.

[0018] (5) Under the condition of adding doxycycline, the PINK1 gene was induced to express in a dose-dependent manner.

[0019] The cell line is derived from porcine umbilical vein endothelial cells.

[0020] Preferably, the PINK1 gene is expressed as a fusion protein with a 3×FLAG tag.

[0021] Preferably, the inducible expression system further includes a TetR transcriptional repression element and a puromycin selection element.

[0022] Preferably, after treatment with doxycycline for 12–48 hours, the expression level of the PINK1 protein significantly increased, while the control group without doxycycline showed no expression.

[0023] This invention also discloses a method for constructing a PINK1-inducible expression cell line of porcine umbilical vein endothelial cells, comprising the following steps:

[0024] (1) Construct an inducible expression vector for the porcine PINK1 gene regulated by the TetIIP promoter;

[0025] (2) The inducible expression vector was introduced into porcine umbilical vein endothelial cells via a viral vector;

[0026] (3) Cells stably integrating the inducible expression vector were obtained through resistance screening;

[0027] (4) By adding doxycycline to induce the expression of the PINK1 gene, a cell line that can be induced to express the gene was obtained.

[0028] Preferably, the concentration of doxycycline used is 0.1–50 μg / mL.

[0029] Preferably, the resistance screening uses puromycin at a concentration of 2–4 μg / mL.

[0030] The present invention also discloses the application of a PINK1-inducible expression cell line of porcine umbilical vein endothelial cells.

[0031] Preferably, the study includes the application of the cell line in regulating the expression of angiogenesis-related genes in vascular endothelial cells and in regulating mitochondrial autophagy, proliferation, or apoptosis in vascular endothelial cells under oxidative stress.

[0032] Preferably, the application is implemented by detecting changes in the expression of VEGFR2, MMP2, TIMP2, Parkin, p62, LC3B, PCNA, or CytC.

[0033] The beneficial effects of this invention are:

[0034] Compared with the prior art, the present invention has the following advantages:

[0035] 1. The first stable cell line in porcine umbilical vein endothelial cells with induced PINK1 expression was constructed, filling the gap in existing technologies for the lack of a research model for the precise regulation of PINK1 in porcine vascular endothelial cells. Existing studies mostly focus on human tumor cells or stem cells, which are difficult to directly reflect the physiological characteristics of porcine placental vascular endothelial cells; the cell line constructed in this invention has clear species and cell type specificity, which is more in line with the research needs of porcine placental angiogenesis.

[0036] 2. The use of an inducible expression system enables controllable regulation of PINK1 expression levels, overcoming the problem that traditional stable overexpression methods do not conform to physiological regulatory principles. Through doxycycline induction, PINK1 expression levels can be precisely regulated under different time and dosage conditions, avoiding interference with cellular homeostasis caused by sustained high expression, thus making experimental results more physiologically relevant and interpretable.

[0037] 3. This study reveals for the first time that upregulation of PINK1 expression in porcine umbilical vein endothelial cells can regulate the expression of multiple angiogenesis-related genes. Experimental results show that after inducing PINK1 expression in porcine umbilical vein endothelial cells, its regulation of angiogenesis-related genes exhibits selective and condition-dependent characteristics. Specifically, without affecting HIF1α and VEGF expression, it significantly reduces the expression of VEGFR2 and MMP2, and further inhibits TIMP2 expression under oxidative stress. This regulatory spectrum has not been reported in existing technologies and represents a technical effect that would be difficult for those skilled in the art to anticipate.

[0038] 4. The constructed cell line can stably activate the PINK1-mediated mitophagy pathway under oxidative stress, providing a reliable model for studying the stress response mechanism of vascular endothelial cells. Under H2O2 treatment, the expression of autophagy-related proteins such as Parkin, p62, and LC3B in this cell line is significantly enhanced, indicating that this model can be used to systematically study the intrinsic relationship between oxidative stress, mitophagy, and vascular function.

[0039] 5. This cell line can be used to simultaneously study the regulation of proliferation and apoptosis in vascular endothelial cells, expanding the research dimensions of single-cell models. After inducing PINK1 expression, the expression of cell proliferation-related protein PCNA increased, while the expression of apoptosis-related protein CytC decreased, indicating that this model is not only suitable for angiogenesis research, but also for comprehensive analysis of endothelial cell survival status and functional homeostasis.

[0040] 6. This invention provides a stable, reproducible, and in vitro experimental animal cell engineering model with good practicality and promotional value. The cell line construction method is well-defined, the operating conditions are controllable, and the resulting cell line maintains stable induced expression characteristics during continuous passage, making it suitable for widespread use in basic research and related applied research. Attached Figure Description

[0041] Figure 1 This is a diagram of the induced expression vector used in this invention and study.

[0042] Figure 2 This diagram illustrates the construction and validation of a PINK1-induced monoclonal cell line according to the present invention.

[0043] Figure 3 This is a verification diagram of the PINK1-induced expression cell line of this invention.

[0044] Figure 4 This is a diagram illustrating the gene level verification of PINK1-induced expression in cells according to the present invention.

[0045] Figure 5 This is a diagram showing the cell viability detection and Western blot detection of mitochondrial autophagy protein expression in this invention; Figure 5 A is a graph showing the changes in cell viability detected by the CCK8 assay after treating cells with 0 and 10 μg / ml doxycycline (DOX) for 6 h, 12 h, 24 h, and 48 h. Figure 5 B refers to the method used in this invention, where 0 and 10 μg / ml DOX were used for 12 h, and 0.3 mM H2O2 was used for 12 h. The expression of mitochondrial autophagy proteins was detected by Western blot.

[0046] Figure 6This is a diagram illustrating the quantitative PCR detection of angiogenesis gene expression in cells according to the present invention. Figure 6 (A) represents HIF1a gene expression; Figure 6 (B) represents VEGF gene expression; Figure 6 (C) represents VEGFR2 gene expression; Figure 6 (D) represents MMP2 gene expression; Figure 6 (E) represents TIMP2 gene expression.

[0047] Figure 7 This is a Western blot diagram showing the expression of proteins involved in the proliferation and apoptosis of SUVEC cells, as presented in this invention. Detailed Implementation

[0048] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0049] Example 1: Method for constructing a PINK1-inducible expression cell line from porcine umbilical vein endothelial cells

[0050] 1.1 Cell Culture

[0051] Porcine umbilical vein endothelial cells (SUVECs) were cultured in DMEM high glucose medium (11965118, Gibco), 10% fetal bovine serum (10099141C), and 1% penicillin and streptomycin (15140122).

[0052] 1.2 Vector Construction and Lentiviral Packaging

[0053] The porcine gene PINK1 (XM_021095478) and vector (GV308) were selected. The vector element sequence is: TetIIP-MCS-3FLAG-Ubi-TetR-IRES-Puromycin, vector size: 12362bp. The vector contains the TetIIP promoter (2713-2991), 3FLAG (3028-3105), and TetR (4372-5073), thus it can be used for 3×FLAG-PINK1 fusion protein expression. Vector map: http: / / www.genechem.com.cn / service / index.php?ac=gene&at=vector_search&keyword=GV308. The insert fragment is located in MCS, and the target gene PINK1 (97601-1)-p1 sequence is: AACCGTCAGATCGCACCGGTCGCCACCATGGTGGTGCGACAGG.

[0054] The inducible expression plasmid was constructed using the GV308 (TetIIP-MCS-3FLAG-Ubi-TetR-IRES-Puromycin) vector from Gilead Sciences. 293T cells were cultured at a ratio of 2*102 6 Seeds were placed in 10cm cell culture dishes and cultured overnight. Two sterile centrifuge tubes, A and B, were taken. In tube A, 5 μg of the target plasmid, 1.8 μg of the packaging plasmid PMD2.G, and 3.2 μg of PAX2 plasmid were added, along with 500 μL of OPTI-MEM (31985070). The mixture was gently pipetted and incubated for 5 min. Then, in tube B, 20 μL of transfection reagent (EZTrans cell transfection reagent, Shanghai Liji Biotechnology Co., Ltd.) and 500 μL of OPTI-MEM were added and gently pipetted and mixed. The liquid from tube A was then added to tube B, mixed, and incubated at room temperature for 15 min. The above liquid was then added to 293T cells, and the cells were cultured for another 48 h. The cell supernatant was obtained by centrifugation and filtered through a 0.45 μm filter membrane.

[0055] 1.3 Construction of cell lines for inducible expression

[0056] Porcine umbilical vein endothelial (SUVEC) cells were seeded at a density of 2*10⁵ in 6-well plates and cultured overnight. 300 μL of viral particles and 8 μg / mL Polybrene (40804ES76, Shanghai Yisheng Biotechnology Co., Ltd.) were added for infection. After 48 h of infection, 2 μg / mL puromycin (ST551, Beyotime Biotechnology) was added for continuous screening for 3-7 days to obtain single clones. Single clones were randomly selected, and Western blot was used to verify the PINK1-induced expression effect, thus constructing a PINK1-induced expression cell line.

[0057] In the above embodiments, an induction expression system using doxycycline as an inducer was introduced into the cell line, the induction expression system comprising:

[0058] (1) PINK1 gene expression cassette regulated by the TetIIP promoter,

[0059] (2) The PINK1 gene is derived from pigs.

[0060] (3) The PINK1 gene is fused with the tag protein for expression.

[0061] (4) The PINK1 gene was in a low-expression or non-expression state without the addition of an inducer.

[0062] (5) Under the condition of adding doxycycline, the PINK1 gene was induced to express in a dose-dependent manner.

[0063] The cell line is derived from porcine umbilical vein endothelial cells.

[0064] In the above embodiments, the PINK1 gene is expressed as a fusion protein with a 3×FLAG tag.

[0065] The inducible expression system also includes a TetR transcriptional repression element and a puromycin selection element.

[0066] Example 2: Monoclonal Cell Validation

[0067] After cell construction, the supernatant was removed, and the cells were washed twice with PBS. 1 mL of RIPA protein lysis buffer (KGP703, Jiangsu Kaiji Biotechnology Co., Ltd.), containing a protease inhibitor (Roche 05892791001), was added to each dish. Cells were scraped from the wells using a cell scraper, transferred to centrifuge tubes, placed on ice, and lysed for 30 min. The cells were then centrifuged at 12000 g for 30 min, and the supernatant was collected. The antibodies used in the experiment are shown in Table 1-1 below.

[0068] Table 1-1 Antibody Sources and Dilution Ratios

[0069]

[0070] The criteria for successful cell line construction are as follows: If the cell line is successfully constructed, PINK1 / Flag protein will be highly expressed in monoclonal cells treated with doxycycline (DOX) for 48 hours. PINK1 / Flag protein will not be expressed in the group without DOX, and PINK1 / Flag expression will show a dose-dependent effect with the concentration of DOX added.

[0071] Monoclonal cells were treated with 1 μg / ml DOX for 48 h, and the expression of PINK1 and Flag proteins was detected by Western blot. Figure 2 We selected three monoclonal cell lines and treated them with 1 μg / ml DOX for 48 h. Total protein was extracted, and PINK1 and Flag proteins were detected by Western blot. The results showed that 1 μg / ml DOX treatment significantly increased the expression of PINK1 and Flag proteins in monoclonal cell lines #2 and #3, while no significant expression was observed in cell line #1 after DOX treatment. This suggests that cell lines #2 and #3 may have been successfully constructed, while cell line #1 was not successfully constructed.

[0072] The results showed that after treatment with doxycycline for 12–48 hours, the expression level of the PINK1 protein increased significantly, while the control group without doxycycline did not express it.

[0073] To further verify the successful construction of the cell line, we selected the #2 induced expression cell line for subsequent experiments. We treated the #2 monoclonal cell line with different concentrations of DOX (μg / ml) for 48 h, and Western blot was used to detect the expression of PINK1 and Flag proteins. The results showed that Flag protein was not expressed in the group without DOX. After adding different doses of DOX, Flag protein expression generally showed an increasing trend with increasing DOX concentration, indicating that the induced expression cell line was successfully constructed. We chose 10 μg / ml DOX treatment for subsequent experiments.

[0074] Example 3 Gene Expression Assay

[0075] After the cells were constructed, they were washed twice with PBS, and 700 μL of TRK lysis buffer was added. RNA was extracted using an RNA extraction kit (R6841-01, Omega). The concentration of extracted RNA was measured using a NanoDrop-100; generally, an A260 / 280 ratio between 1.8 and 2.0 indicates good RNA purity. The RNA was then reverse transcribed into cDNA using a reverse transcription kit (R323-01, Nanjing Novizan Biotechnology Co., Ltd.). Primer sequences are shown in Table 1-2 below. Primers were synthesized using Primer 3 Input software and were synthesized by Invitrogen (Shanghai).

[0076] Table 1-2 Primer sequences used in this experiment

[0077]

[0078] The quantitative PCR reaction system is shown in Table 1-3. Each sample was replicated in triplicate. The reaction conditions were as follows: Step 1: 95°C for 30 s; Step 2: 95°C for 5 s, 60°C for 34 s, repeated 40 times; Step 3: 95°C for 15 s, 60°C for 60 s, 95°C for 15 s. The quantitative PCR results were obtained using 2... -(ΔΔCt) Method, ΔΔCt = (Ct) Target – Ct β-actin Processing group – (Ct) Target –Ct β-actin In the control group, the relative expression levels of genes in the test samples were calculated.

[0079] Table 1-3 Quantitative PCR System

[0080]

[0081] Cells were treated with 1 μg / ml, 10 μg / ml DOX and 0.3 mM H2O2 for 12 h. Western blot was used to detect the expression of PINK1 and Flag proteins. The gene expression levels showed that compared with the control group, the treatment with 1 μg / ml and 10 μg / ml DOX significantly increased PINK1 expression (nearly 120-fold, P<0.05). Under the condition of 0.3 mM H2O2 treatment, the PINK1 gene expression was further increased by the treatment with 10 μg / ml DOX (P<0.05).

[0082] This case demonstrates that, at the endothelial cell gene level, induced expression cell lines significantly enhance the expression of the target gene.

[0083] Example 4: Application of PINK1-induced SUVEC cell lines in the regulation of mitophagy

[0084] like Figure 5 As shown in Figure A, PINK1-induced expression cells were treated with 0 and 10 μg / ml DOX for 6 h, 12 h, 24 h, and 48 h, and cell viability changes were detected using the CCK8 assay. Figure 5 Results showed that adding DOX for different durations had no effect on SUVEC cell viability.

[0085] Figure 5 Cells were treated with 0 and 10 μg / ml DOX for 12 h to induce PINK1 expression, and simultaneously treated with 0.3 mM H2O2 for 12 h. Western blot analysis was used to detect the expression of mitophagy proteins. Figure 5Results B showed that, without DOX treatment, Flag and PINK1 proteins were not expressed after H2O2 treatment. Under 10 μg / ml DOX treatment, the expression of Flag, PINK1, parkin, p62, and LC3B proteins further increased, indicating that H2O2 activates the PINK1-mediated mitophagy pathway. This case demonstrates that induced expression cell lines are an important tool for studying oxidative stress-mediated endothelial cell autophagy.

[0086] Example 5: Application of PINK1-induced SUVEC cell line in the regulation of angiogenesis gene expression

[0087] Figure 6 PINK1-induced expression cells were treated with 0 and 10 μg / ml DOX for 12 h, and simultaneously treated with 0.3 mM H2O2 for 12 h. The expression of angiogenesis genes was detected by quantitative PCR. Figure 6 The results showed that, under control and H2O2 treatment, induced expression of PINK1 had no effect on HIF1α and VEGF gene expression (P>0.05). Under control and 0.3 mM H2O2 treatment, induced expression of PINK1 significantly reduced VEGFR2 gene expression (P<0.05). Induced expression of PINK1 reduced MMP2 gene expression (P<0.05p), with no significant difference between the two groups under H2O2 treatment (P>0.05). Induced expression of PINK1 did not alter TIMP2 gene expression, but under H2O2 treatment, induced expression of PINK1 significantly reduced TIMP2 expression (P<0.05). This case demonstrates that induced expression cell lines are an important tool for studying the expression of endothelial cell angiogenesis-related genes.

[0088] Example 6: Application of PINK1-induced SUVEC cell lines in endothelial cell proliferation and apoptosis

[0089] Figure 7 PINK1-induced expression cells were treated with 0 and 10 μg / ml DOX for 12 h, and simultaneously treated with 0.3 mM H2O2 for 12 h. The expression of cell proliferation and apoptosis proteins was detected by quantitative Western blot. Figure 7 The results showed that under normal culture conditions, treatment with 10 μg / ml DOX increased the expression of the proliferation protein PCNA and decreased the expression of the apoptosis protein CytC. However, under H2O2 treatment, treatment with 10 μg / ml DOX decreased the expression of both PCNA and CytC. This case demonstrates that induced expression cell lines are an important tool for studying oxidative stress-induced endothelial cell proliferation and apoptosis.

[0090] In summary, this experiment successfully constructed a stable porcine umbilical vein endothelial cell line capable of inducibly expressing PINK1. This cell line has the advantage of controllable expression levels and can be used for subsequent research. Treatment with 10 μg / ml DOX at different time points had no effect on SUVEC cell viability. Induced expression of PINK1 affected the expression of angiogenesis genes in vascular endothelial cells, increased the expression of mitochondrial autophagy proteins in SUVECs, and decreased the expression of apoptosis proteins. These results suggest that this cell line can be used to study the regulatory mechanisms and roles of oxidative stress and angiogenesis in porcine vascular endothelial cells.

[0091] The cell line constructed in this invention can be used as an in vitro research model to study the regulatory mechanisms of porcine vascular endothelial cells under angiogenesis and oxidative stress conditions by detecting changes in the expression of angiogenesis-related genes VEGFR2, MMP2, and TIMP2, as well as mitophagy and cell function-related proteins Parkin, p62, LC3B, PCNA, and CytC.

[0092] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0093] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A PINK1-inducible expression cell line of porcine umbilical vein endothelial cells, characterized in that, The cell line was introduced with an induction expression system using doxycycline as an inducer, the induction expression system comprising: (1) PINK1 gene expression cassette regulated by the TetIIP promoter, (2) The PINK1 gene is derived from pigs. (3) The PINK1 gene is fused with the tag protein for expression. (4) The PINK1 gene was in a low-expression or non-expression state without the addition of an inducer. (5) Under the condition of adding doxycycline, the PINK1 gene was induced to express in a dose-dependent manner. The cell line is derived from porcine umbilical vein endothelial cells.

2. The PINK1-inducible expression cell line of porcine umbilical vein endothelial cells according to claim 1, characterized in that, The PINK1 gene is expressed as a fusion protein with a 3×FLAG tag.

3. The PINK1-inducible expression cell line of porcine umbilical vein endothelial cells according to claim 1, characterized in that, The inducible expression system also includes a TetR transcriptional repression element and a puromycin selection element.

4. The PINK1-inducible expression cell line of porcine umbilical vein endothelial cells according to claim 1, characterized in that, After treatment with doxycycline for 12–48 hours, the expression level of the PINK1 protein increased significantly, while the control group without doxycycline showed no expression.

5. A method for constructing a PINK1-inducible expression cell line of porcine umbilical vein endothelial cells, characterized in that, Includes the following steps: (1) Construct an inducible expression vector for the porcine PINK1 gene regulated by the TetIIP promoter; (2) The inducible expression vector was introduced into porcine umbilical vein endothelial cells via a viral vector; (3) Cells stably integrating the inducible expression vector were obtained through resistance screening; (4) By adding doxycycline to induce the expression of the PINK1 gene, a cell line that can be induced to express the gene was obtained.

6. The method for constructing a PINK1-inducible expression cell line of porcine umbilical vein endothelial cells according to claim 5, characterized in that, The concentration of doxycycline used is 0.1–50 μg / mL.

7. The method for constructing a PINK1-inducible expression cell line of porcine umbilical vein endothelial cells according to claim 5, characterized in that, The resistance screening used puromycin at a concentration of 2–4 μg / mL.

8. Application of a PINK1-inducible expression cell line in porcine umbilical vein endothelial cells.

9. The application of the PINK1-inducible expression cell line of porcine umbilical vein endothelial cells according to claim 8, characterized in that, This includes studying the application of the cell line in regulating the expression of angiogenesis-related genes in vascular endothelial cells, as well as its application in regulating mitochondrial autophagy, proliferation, or apoptosis in vascular endothelial cells under oxidative stress.

10. The application of a PINK1-inducible expression cell line of porcine umbilical vein endothelial cells according to claim 8 or claim 9, characterized in that, The application is preferably achieved by detecting changes in the expression of VEGFR2, MMP2, TIMP2, Parkin, p62, LC3B, PCNA, or CytC.

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