Co-culture system construction method based on TGM2 targeted regulation of mesenchymal stem cells and co-culture system

Abstract

The invention relates to the technical field of cell biology and biological medicine, and particularly discloses a co-culture system construction method based on TGM2 targeted regulation of mesenchymal stem cells and a co-culture system prepared by the method. The method comprises the following steps: respectively preparing human umbilical cord mesenchymal stem cells, lung adenocarcinoma cells A549 and lung squamous carcinoma cells SW1573 in a logarithmic phase; mixing A549 and SW1573 cells according to a ratio of 1: 1 to obtain mixed lung cancer cells; and mixing the hUC-MSC and the mixed lung cancer cells according to a quantitative ratio of 5: 1 to 13: 1, inoculating the mixture into a culture container containing a culture medium, and culturing for 3-9 days under the conditions of 37 DEG C and 5% CO2 to obtain the co-culture system. The method does not need to carry out gene modification on hUC-MSC, and can effectively inhibit proliferation, migration and invasion of lung cancer cells and induce the lung cancer cells to generate cell cycle arrest and apoptosis by optimizing the co-culture proportion and time and utilizing natural communication between cells.

Description

Technical Field

[0001] This invention relates to the fields of cell biology and biomedicine, and in particular to a method for constructing a co-culture system for mesenchymal stem cells based on TGM2-targeted regulation and the co-culture system itself. Background Technology

[0002] Lung cancer is the leading cause of cancer-related morbidity and mortality worldwide, with approximately 2.5 million new cases and 1.8 million deaths annually, resulting in a heavy disease burden. Clinical treatment primarily involves surgery combined with radiotherapy and chemotherapy; however, the recurrence and metastasis rate after surgery reaches 30%-50%, which is the core reason for poor postoperative outcomes.

[0003] Current treatments face four major bottlenecks: ① Cancer-associated fibroblasts (CAFs) proliferate and secrete collagen in the tumor microenvironment, forming a fibrotic barrier that hinders drug penetration and immune cell infiltration; ② Regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) accumulate, secreting IL-10 and TGF-β to construct an immunosuppressive microenvironment, suppressing cytotoxic T cell (CTL) function; ③ Lung cancer cells develop resistance to chemotherapeutic agents such as cisplatin and paclitaxel by activating ABC transporters and enhancing DNA repair, leading to decreased efficacy; ④ Radiotherapy and chemotherapy damage alveolar epithelial and vascular endothelial cells, disrupting lung structure and even inducing inflammation that promotes tumor regeneration. Transglutaminase 2 (TGM2) is a key hub in lung cancer progression, highly expressed in cancer tissues: on the one hand, it activates the PI3K / AKT pathway, upregulating Cyclin D1 and Bcl-2 to promote proliferation and inhibit apoptosis; on the other hand, it regulates Snail and Twist to promote epithelial-mesenchymal transition (EMT), enhancing invasion, metastasis, and chemotherapeutic resistance. Therefore, TGM2 is an important target, but existing interventions have limitations: small molecule inhibitors have insufficient specificity and high off-target toxicity; gene editing, although precise, is technically complex, has high barriers to clinical translation, and is expensive.

[0004] Human umbilical cord mesenchymal stem cells (hUC-MSCs) are readily available (umbilical cord tissue, free from ethical controversies), have low immunogenicity (do not express HLA-DR), and high safety. They also possess tumor chemotaxis and cell communication capabilities, making them a promising candidate. However, current applications are limited: while gene modification (such as viral transfection) to enhance antitumor activity is common, it increases costs and risks; when natural hUC-MSCs are used alone, their ability to target and regulate tumor pathways is weak, their effects are poor, and their mechanisms are unclear.

[0005] In summary, current technologies lack a method for co-culturing hUC-MSCs with lung cancer cells to regulate the TGM2-PI3K / AKT pathway through natural cell communication, and there is a lack of research on targeted anti-tumor effects through autocrine / paracrine secretion without genetic modification. Therefore, constructing a co-culture system with specific ratios to precisely regulate tumor pathways through cell interaction is an urgent need to overcome bottlenecks and develop novel and safe agents. Summary of the Invention

[0006] The technical solution of this invention to solve the above-mentioned technical problems is to provide a method for constructing a co-culture system based on TGM2-targeted regulation of mesenchymal stem cells, comprising the following steps: (1) hUC-MSCs, A549 cells and SW1573 cells in the logarithmic growth phase were prepared respectively; (2) Mix A549 cells and SW1573 cells at a ratio of 1:1 to obtain mixed lung cancer cells; (3) The hUC-MSCs obtained in step (1) and the mixed lung cancer cells obtained in step (2) are mixed in a ratio of 5:1 to 13:1, seeded into a culture container, culture medium is added, and cultured at 37°C and 5% CO2 for 3 to 9 days to obtain the co-culture system.

[0007] Further, in step (1), the hUC-MSC is a 3rd to 5th generation cell that has been isolated and cultured in vitro.

[0008] Further, in step (1), the separation and culture steps of hUC-MSC include: taking umbilical cord tissue, removing blood vessels and outer membrane, cutting it into small pieces, digesting it with collagenase I, collecting the digested cells for culture, and using DMEM / F12 medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin.

[0009] Further, in step (1), the A549 cells and SW1573 cells are obtained by cryopreservation and cultured in DMEM / F12 medium containing 10% fetal bovine serum.

[0010] Further, in step (3), the culture medium is DMEM / F12 culture medium containing 10% fetal bovine serum by volume.

[0011] Furthermore, in step (3), the culture medium is replaced with fresh medium every 2 days during the culture period.

[0012] The present invention also proposes a co-culture system prepared by the method described above.

[0013] The purpose of this invention is to provide a co-culture system of hUC-MSCs and lung cancer cells. This system does not require genetic modification of hUC-MSCs. By optimizing the co-culture ratio and time of hUC-MSCs and lung cancer cells, it utilizes natural intercellular communication mechanisms to downregulate TGM2 expression and inhibit the PI3K / AKT pathway, achieving a multi-dimensional anti-lung cancer effect through apoptosis induction, cell cycle arrest, and EMT inhibition. Another objective of this invention is to provide a method for constructing this co-culture system and its application in anti-lung cancer therapy, providing a novel, safe, and highly effective live cell preparation for lung cancer treatment. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0015] Figure 1 The images show the morphological changes observed under a microscope during co-culture (A549 cells: a is normal, bd are morphological changes at 3, 6 and 9 days respectively; SW1573 colon cancer cells: e is normal, fh are morphological changes at 3, 6 and 9 days respectively). Figure 2 This is a diagram from an osteogenic induction differentiation experiment. Figure 3 Image for RT-qPCR detection of high expression; Figure 4 This is a CCK-8 detection image; Figure 5 This image shows the inhibited growth and morphological changes in both types of lung cancer cells. Figure 6 Diagram of a clone formation experiment; Figure 7 A graph showing the correlation between the invasive and migratory abilities of tumor cells and their malignancy. Figure 8 A diagram illustrating EMT-related biomarkers; Figure 9 This is a flow cytometry analysis result; Figure 10 A graph showing the expression of apoptosis-related proteins (Bcl-2, Cleaved-caspase-9 / 3) after co-culture, as detected by Western blot. Figure 11 To analyze the cell cycle distribution after co-culture using flow cytometry; Figure 12A graph showing the expression of cycle-related proteins (Cyclin D1 and P21) as detected by Western blot. Figure 13 The TGM2 knockdown efficiency was verified by RT-qPCR and Western blot. Figure 14 The graph shows the TGM2 overexpression efficiency for validation by RT-qPCR and Western blot. Detailed Implementation

[0016] This invention proposes a method for constructing a co-culture system based on TGM2-targeted regulation of mesenchymal stem cells, aiming to provide novel, safe, and efficient live cell preparations for lung cancer treatment.

[0017] The following specific embodiments will illustrate the method for constructing a co-culture system based on TGM2-targeted regulation of mesenchymal stem cells proposed in this invention: Example 1: In this embodiment, a method for constructing a co-culture system for TGM2-targeted regulation of mesenchymal stem cells includes the following steps: (1) hUC-MSCs, A549 cells and SW1573 cells in the logarithmic growth phase were prepared respectively; (2) Mix A549 cells and SW1573 cells at a ratio of 1:1 to obtain mixed lung cancer cells; (3) The hUC-MSCs obtained in step (1) and the mixed lung cancer cells obtained in step (2) are mixed in a ratio of 5:1 to 13:1, seeded into a culture container, culture medium is added, and cultured at 37°C and 5% CO2 for 3 to 9 days to obtain the co-culture system.

[0018] Specifically, the isolation and culture of hUC-MSCs: umbilical cord tissue was collected, blood vessels and outer membrane were removed, and the tissue was minced. The cells were digested with 0.2% collagenase I at 37°C for 1 hour. After centrifugation, the cells were collected and seeded into DMEM / F12 medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin. The cells were cultured in a 37°C, 5% CO2 incubator, with the medium changed every 3 days, until the 3rd-5th generation for use. Lung cancer cell resuscitation and culture: Frozen A549 and SW1573 cells were resuscitated separately and seeded in DMEM / F12 medium containing 10% fetal bovine serum. The cells were cultured at 37°C in a 5% CO2 incubator. When the cell confluence reached 80%, the cells were passaged, and cells in the logarithmic growth phase were collected for later use. Co-culture system construction: Passages 3-5 of hUC-MSCs, logarithmic growth phase A549 cells, and SW1573 cells were digested and counted separately. Lung cancer cells were mixed at a ratio of A549 cells:SW1573 cells = 1:1. hUC-MSCs and the mixed lung cancer cells were then seeded into culture plates at ratios of 13:1, 12:1, or 5:1. DMEM / F12 medium was added, and the plates were incubated at 37°C in a 5% CO2 incubator for 3-9 days, with the medium replaced every 2 days to obtain the co-culture system.

[0019] Further, in step (1), the hUC-MSC is a 3rd to 5th generation cell that has been isolated and cultured in vitro.

[0020] Further, in step (1), the separation and culture steps of hUC-MSC include: taking umbilical cord tissue, removing blood vessels and outer membrane, cutting it into small pieces, digesting it with collagenase I, collecting the digested cells for culture, and using DMEM / F12 medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin.

[0021] Further, in step (1), the A549 cells and SW1573 cells are obtained by cryopreservation and cultured in DMEM / F12 medium containing 10% fetal bovine serum.

[0022] Further, in step (3), the culture medium is DMEM / F12 culture medium containing 10% fetal bovine serum by volume.

[0023] Furthermore, in step (3), the culture medium is replaced with fresh medium every 2 days during the culture period.

[0024] Experimental verification: Umbilical cord mesenchymal stem cells (hUC-MSCs) were successfully isolated and identified from the umbilical cord. Cells were observed to migrate out on day 6 of culture, and reached 80% confluence on day 9. Passaging culture showed that at 30% confluence, cells extended and produced pseudopodia-like structures; above 80% confluence, cells exhibited uniform morphology, arranging themselves in a whorled or radial pattern. Figure 1 Osteogenic induction differentiation experiments confirmed its multi-directional differentiation potential. Figure 2RT-qPCR detected high expression of specific positive markers CD73, CD90, and CD105, while negative markers CD45, CD34, CD31, and embryonic stem cell markers OCT4 and SOX2 were not expressed. Figure 3 This confirms the successful acquisition of hUC-MSCs.

[0025] Based on this, the effects of hUC-MSCs on the proliferation of lung adenocarcinoma A549 and lung squamous cell carcinoma SW1573 cells were systematically studied. Morphological changes observed under an inverted microscope on days 3, 6, and 9 of co-culture showed that the growth of both lung cancer cell types was inhibited and morphological changes were observed. Figure 5 CCK-8 assay showed that the cell proliferation activity in the co-culture group was significantly lower than that in the monoculture group in a time-dependent manner. Figure 4 The colony formation experiment further confirmed that the number of colonies decreased significantly with prolonged co-culture time. Figure 6 The above results clearly demonstrate that co-culture of hUC-MSCs can significantly inhibit the proliferation of A549 and SW1573 cells.

[0026] Figure 1 hUC-MSCs can be seen crawling out on days 6-9 of primary culture. When the confluence of passaged cells is 30%, they extend and produce pseudopodia. When the confluence is above 80%, the cells are uniform in morphology and arranged in a whorled or radial pattern. Figure 2 Identification of osteogenic induction differentiation; Figure 3 RT-qPCR analysis showed that hUC-MSCs highly expressed positive markers CD73, CD90, and CD105, but did not express negative markers CD45, CD34, CD31, OCT4, and SOX2. Figures 4-6 After hUC-MSCs were co-cultured with lung cancer cells, cell viability decreased in a time-dependent manner as detected by CCK-8 assay, cell morphology changed, and clonogenic ability was significantly reduced. p<0.05, p<0.01, p<0.001, p<0.0001.

[0027] Figure 3 The left image shows the detection of positive biomarkers (CD73, CD90, CD105), indicating that hUC-MSCs highly express mesenchymal stem cell-specific positive biomarkers. The middle image shows the detection of negative biomarkers (CD34, CD45, CD31), indicating that hUC-MSCs do not express hematopoietic cell and endothelial cell-specific biomarkers, and their purity is acceptable. The right image shows the detection of embryonic stem cell biomarkers (OCT4, SOX2), indicating that hUC-MSCs do not express embryonic stem cell pluripotency biomarkers, which is consistent with the molecular characteristics of adult mesenchymal stem cells (non-embryonic stem cells).

[0028] co-culture of hUC-MSCs inhibits the invasion and migration of lung cancer cells: The invasive and migratory ability of tumor cells is related to their malignancy. Figure 7 To further investigate the mechanism, we examined the expression changes of EMT-related biomarkers. The results showed that after co-culturing hUC-MSCs, the expression of some mesenchymal biomarkers was downregulated, while the expression of epithelial biomarkers rebounded, suggesting that the EMT process may be affected to some extent. Figure 8 Preliminary data suggest that hUC-MSCs may inhibit lung cancer cell migration by regulating EMT-related pathways. These results indicate that hUC-MSCs can inhibit lung cancer cell invasion and metastasis in vitro by regulating the EMT process. Flow cytometry analysis showed that co-culture of hUC-MSCs induced lung cancer cell apoptosis in a time-dependent manner; the apoptosis rate of both A549 and SW1573 cells gradually increased with prolonged co-culture time. Figure 9 ).

[0029] Figures 7 to 9 The effects of hUC-MSCs on the migration, invasion, and apoptosis of A549 and SW1573 cells were investigated. Cell migration and invasion abilities of hUC-MSCs co-cultured with A549 and SW1573 cells were assessed (scale bar = 100 μm). Western blotting was used to analyze the expression trends of EMT-related proteins. Flow cytometry was used to observe cell apoptosis after co-culture. Statistical significance: p < 0.05, p < 0.01, p < 0.001. p<0.0001, ns indicates no statistical difference.

[0030] hUC-MSCs co-culture induces lung cancer cell apoptosis: Apoptosis protein assays showed activation of Cleaved-caspase-9 / 3 and downregulation of Bcl-2. Figure 10 This suggests that apoptosis is mediated via the mitochondrial pathway. Co-culture of hUC-MSCs significantly increased the proportion of A549 and SW1573 cells in G1 phase: after 3, 6, and 9 days of co-culture, the proportion of A549 cells increased from 31.10% to 43.26%, 54.78%, and 62.04%, respectively, while the proportion of SW1573 cells increased from 39.68% to 44.17%, 60.52%, and 67.01%, respectively. Figure 11 Mechanistic studies show that cyclin D1 is downregulated and P21 is upregulated. Figure 12 This suggests that it induces G1 phase arrest by regulating cyclin D1 / P21. Transfection efficiency verification showed that TGM2 expression in the control group (Si-NC / OE-NC) was not significantly changed, while TGM2 expression in the Si-TGM2 and OE-TGM2 groups was significantly downregulated and upregulated, respectively. Figure 13 , Figure 14A549 / SW1573 cell models with TGM2 knockdown and overexpression were successfully constructed.

[0031] Figures 10 to 14 This study investigated the effects of hUC-MSCs on lung cancer cell apoptosis and cell cycle, and validated TGM2 gene manipulation. TGM2 expression affected lung cancer cell proliferation: overexpression significantly enhanced proliferation. CCK-8 assays showed that TGM2 knockdown reduced the viability of A549 and SW1573 cells to 39.8% and 46.1%, respectively (p<0.001), while overexpression increased them to 179.3% and 173.3%, respectively (p<0.001). Colony formation assays further confirmed a positive correlation between cell colony formation ability and TGM2 expression levels. Transwell assays showed that TGM2 knockdown significantly decreased the invasion and migration abilities of A549 and SW1573 cells, while overexpression significantly enhanced them. These results confirm that the proliferation, migration, and invasion abilities of lung cancer cells are directly related to TGM2 overexpression.

[0032] Example 2: A co-culture system, prepared by the method described above, specifically comprises human umbilical cord mesenchymal stem cells (hUC-MSCs) and lung cancer cells cultured in a specific ratio; the lung cancer cells are A549 cells (human lung adenocarcinoma cells) and SW1573 cells (human lung squamous cell carcinoma cells), with a ratio of A549 cells to SW1573 cells of 1:1; the co-culture ratio of hUC-MSCs with lung cancer cells (A549 cells and SW1573 cells) is 13:1, 12:1, or 5:1 (cell ratio); the culture time of the co-culture system is 3-9 days, and the culture conditions are 37°C, 5% CO2, and DMEM / F12 medium containing 10% fetal bovine serum.

[0033] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for constructing a co-culture system for mesenchymal stem cells based on TGM2-targeted regulation, characterized in that, Includes the following steps: (1) hUC-MSCs, A549 cells and SW1573 cells in the logarithmic growth phase were prepared respectively; (2) Mix A549 cells and SW1573 cells at a ratio of 1:1 to obtain mixed lung cancer cells; (3) The hUC-MSCs obtained in step (1) and the mixed lung cancer cells obtained in step (2) are mixed in a ratio of 5:1 to 13:1, seeded into a culture container, culture medium is added, and cultured at 37°C and 5% CO2 for 3 to 9 days to obtain the co-culture system.

2. The method according to claim 1, characterized in that, In step (1), the hUC-MSCs are cells from the 3rd to 5th generation after in vitro isolation and culture.

3. The method according to claim 1, characterized in that, In step (1), the separation and culture steps of hUC-MSC include: taking umbilical cord tissue, removing blood vessels and outer membrane, cutting it into small pieces, digesting it with collagenase I, collecting the digested cells for culture, and using DMEM / F12 medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin.

4. The method according to claim 1, characterized in that, In step (1), the A549 cells and SW1573 cells were obtained by cryopreservation and cultured in DMEM / F12 medium containing 10% fetal bovine serum.

5. The method according to claim 1, characterized in that, In step (3), the culture medium is DMEM / F12 medium containing 10% fetal bovine serum by volume.

6. The method according to claim 1, characterized in that, In step (3), the culture medium is replaced with fresh medium every 2 days during the culture period.

7. A co-culture system, prepared by the co-culture system construction method based on TGM2-targeted regulation of mesenchymal stem cells as described in any one of claims 1 to 6.

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