Rumen fibroblast line as well as construction method, culture method and application thereof

Abstract

The invention discloses a rumen fibroblast line as well as a construction method, a culture method and application thereof, the rumen fibroblast line G8 is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: C2025332, the rumen fibroblast line G8 has a typical fibroblast-like form and can be stably passaged, and the rumen fibroblast line G8 can be used as a rumen fibroblast cell line. The cell culture medium also has the characteristic of highly expressing fibroblast marker genes and the function of tolerating and utilizing rumen core metabolite butyric acid, and the proliferation rate and metabolic activity of the cell culture medium under the condition that 15mmol / L butyric acid is used as a main energy source are obviously superior to those of glucose culture; according to the construction method of the rumen fibroblast line, the steps of function screening and molecular identification are introduced, so that the identity authenticity of the constructed cell line can be ensured; the culture method of the rumen fibroblast line is simple and effective, culture medium materials are easy to obtain, and culture conditions are easy to realize; the rumen fibroblast line can be used for rumen in-vitro research, and is beneficial to clarification of a mechanism for regulating rumen interstitial metabolism and tissue repair by butyric acid.

Description

TECHNICAL FIELD

[0001] The present application relates to a fibroblast cell line and its construction method, culture method and application, in particular to a rumen fibroblast cell line and its construction method, culture method and application. BACKGROUND

[0002] Rumen is a unique organ of ruminants, which is an important place for digestion, absorption and metabolism of nutrients. Rumen health is particularly important in various growth and production cycles, and is the core of maintaining the high production performance of ruminants. Rumen tissue is a complex functional unit composed of stratified squamous epithelium and interstitial tissue below it. As the supporting structure of rumen tissue, fibroblasts in the interstitial tissue not only synthesize extracellular matrix and maintain papilla structure, but also participate in tissue repair and communicate with epithelial cells through secreted factors to jointly regulate rumen development and homeostasis. Therefore, in-depth study of fibroblasts is crucial for a comprehensive understanding of rumen physiology and pathological mechanisms.

[0003] Butyric acid is one of the short-chain fatty acids produced in the rumen. Studies have shown that butyric acid may indirectly affect the remodeling of extracellular matrix and the process of tissue repair by regulating the activity of fibroblasts. The existing rumen fibroblast cell line has many defects: 1. The proliferation ability of primary rumen fibroblasts is limited, and they usually enter the growth arrest phase at the 5th passage, showing stop proliferation, aging and apoptosis, which cannot meet the long-term and stable experimental research needs; 2. Poor tolerance to core metabolite butyric acid, difficult to maintain stable proliferation in culture system containing butyric acid, often prematurely entering the growth inhibition state, unable to reproduce the physiological regulation mediated by butyric acid in vivo, making it difficult to in-depth study the specific mechanism of butyric acid on rumen fibroblast function in vitro. SUMMARY

[0004] The purpose of the present application is to provide a rumen fibroblast cell line that can be stably passaged, retain the characteristics of rumen fibroblasts, and tolerate and utilize butyric acid, as well as its construction method, culture method and application.

[0005] Technical scheme: The rumen fibroblast cell line (G8) has a preservation number of CCTCC NO: C2025332, which was preserved in the China Center for Type Culture Collection on October 31, 2025. The Latin name is Bos taurus.

[0006] The construction method of the rumen fibroblast cell line comprises the following steps: Step one, collect rumen epithelial tissue, digest and collect and culture cells; Step two, immortalize the cells obtained in step one; Step three, monoclonal amplification of the immortalized cells obtained in step two; Step 4: Screen for cell lines that can utilize butyrate from the monoclonal cell lines obtained in Step 3. Step 5: Detect the expression of fibroblast marker genes and rumen epithelial cell marker genes in the cell lines obtained in Step 4.

[0007] In step four, the screening method preferably uses butyrate medium to culture cell lines; the butyrate medium is preferably sugar-free DMEM medium containing 10-20 mmol / L sodium butyrate; the screening criteria are preferably cell morphology and proliferation rate.

[0008] In step five, the fibroblast marker gene is preferably the collagen gene COL1A1, COL1A2, or COL3A1; the rumen epithelial cell marker gene is preferably the basal cell marker KRT14 or the ketogenic key enzyme HMGCS2.

[0009] In step one, the rumen tissue is preferably taken from cattle, such as Holstein cows.

[0010] The rumen fibroblast cell line was cultured using sugar-free DMEM medium containing 10-20 mmol / L sodium butyrate at 37°C and 5% CO2.

[0011] The rumen fibroblast cell line described in this invention can be used for in vitro rumen research.

[0012] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: First, the rumen fibroblast cell line (G8) exhibits typical fibroblast-like morphology and can be stably passaged; it also shows high expression of fibroblast marker genes (COL1A1, COL1A2, and COL3A1), indicating its active extracellular matrix synthesis and remodeling function; it also demonstrates excellent tolerance to and utilization of the rumen core metabolite butyrate. Under culture conditions with 15 mmol / L butyrate as the main carbon source, the G8 cell line exhibits a significant proliferative effect: after 24 hours of culture, the cell metabolic activity is approximately 1.5 times higher than that of the glucose group; after 72 hours of culture, its cell number significantly exceeds that of the 25 mmol / L glucose treatment group. The rumen fibroblast cell line (G8) is a favorable model for rumen research, providing a reliable in vitro platform for directly studying the regulatory effect of butyrate on rumen fibroblasts. Second, the method for constructing the rumen fibroblast cell line introduces functional screening and molecular identification steps, which can ensure the authenticity of the identity of the constructed cell line, avoid data misinterpretation due to cell contamination, and provide experimental materials with verifiable identities for rumen interstitial research. Third, the culture method for the rumen fibroblast cell line is simple and effective, the culture medium materials are easy to obtain, and the culture conditions are easy to achieve; Fourth, the rumen fibroblast cell line can be applied to in vitro rumen studies, which is beneficial for clarifying the mechanism by which butyrate regulates rumen interstitial metabolism and tissue repair. Attached Figure Description

[0013] Figure 1 Cell morphology diagram of mixed cells obtained from rumen epithelial tissue after immortalization treatment (scale bar = 100 μm). Figure 2 Morphological images of rumen epithelial cells from nine monoclonal Holstein dairy cow strains (scale bar = 100 μm). Figure 3 A comparison of the morphology of F3 cell lines under different carbon source culture conditions (scale bar = 100 μm). Figure 4 A comparison of the morphology of F9 cell lines under different carbon source culture conditions (scale bar = 100 μm). Figure 5 A comparison of the morphology of G8 cell line under different carbon source culture conditions (scale bar = 100 μm). Figure 6 A comparison of the morphology of E7 cell lines under different carbon source culture conditions (scale bar = 100 μm). Figure 7 A comparison of the morphology of F8 cell lines under different carbon source culture conditions (scale bar = 100 μm). Figure 8 A comparison of the morphology of E6 cell line under different carbon source culture conditions (scale bar = 100 μm). Figure 9 A comparison of the morphology of C9 cell lines under different carbon source culture conditions (scale bar = 100 μm). Figure 10 A comparison of the morphology of B7 cell line under different carbon source culture conditions (scale bar = 100 μm). Figure 11 A comparison of the morphology of E10 cell line under different carbon source culture conditions (scale bar = 100 μm). Figure 12 The graph shows the growth rate of G8 cells cultured with 25 mmol / L glucose and 15 mmol / L butyric acid, respectively. Figure 13 The effect of butyric acid on the proliferation activity of G8 cells; Figure 14 The expression of KRT14 and HMGCS2 gene mRNA in G8; Figure 15 This refers to the mRNA expression of rumen fibroblast marker genes in G8 cells. Detailed Implementation

[0014] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0015] Example 1

[0016] This embodiment provides a rumen fibroblast cell line (G8), which was deposited at the China Center for Type Culture Collection on October 31, 2025, and will be deposited at the China Center for Type Culture Collection on October 31, 2025.

[0017] Example 2

[0018] This embodiment provides a method for constructing the rumen fibroblast cell line (G8) described in Example 1, and the steps are as follows: Step 1: Collect rumen epithelial tissue, digest it, and then collect and culture the cells. Tissue sampling: Rumen tissue was collected aseptically from healthy 4-week-old Holstein dairy cows (sample source: Animal Room, Nanjing Agricultural University) immediately after slaughter. The tissue was placed in pre-cooled D-Hanks balanced salt solution containing 6% penicillin-streptomycin and rinsed until no visible impurities were visible on the surface. Tissue from the post-cecal groove of the rumen was harvested and immersed in pre-cooled DMEM medium containing 2% penicillin-streptomycin at 4°C for later use.

[0019] Tissue preparation: Under aseptic conditions, repeatedly wash the tissue with pre-cooled D-Hanks solution containing 2% penicillin-streptomycin until the solution is clear. Carefully remove fat, blood vessels, serous layers, and muscle layers from the tissue using ophthalmic scissors and forceps.

[0020] Digestion: The purified rumen tissue was cut into pieces approximately 1 mm in size. 3 Place the small pieces in a 50 mL centrifuge tube, add 10 mL of 0.25% trypsin digestion solution, and shake to digest in a 37°C water bath.

[0021] Termination of digestion and cell collection: Aspirate the digestion fluid containing cell clusters and add complete DMEM medium (basal medium is DMEM, supplemented with 10% FBS and 1% penicillin-streptomycin) containing 10% fetal bovine serum (FBS) to terminate digestion. Repeat this digestion-termination process three times to maximize cell harvest. Combine all digestion fluids, filter using a 70 μm pore size cell sieve, and collect the filtrate.

[0022] Cell washing and culture: Centrifuge the filtrate at 1000 rpm for 3 minutes and discard the supernatant. Resuspend the cell pellet in PBS containing 2x penicillin-streptomycin, and repeat the centrifugation washing three times. Finally, resuspend the cells in complete culture medium at 3×10⁻⁶ cells / mL. 6Cells were seeded at a density of 10 cm⁻¹ cells / mL in 10 cm cell culture dishes and cultured at 37°C in a 5% CO₂ incubator. After 1 hour of culture, the culture medium was replaced to remove unattached cells, and the cells were cultured until they confluent.

[0023] Step two: Immortification treatment of the cells obtained in step one: Virus packaging: Resuscitate and culture 293FT cells to 85%-90% confluence, and transfect the plasmid system containing the SV40T gene into 293FT cells for lentiviral packaging according to the instructions of the lentiviral packaging kit.

[0024] Virus collection and concentration: Cell culture supernatant was collected every 24 hours after transfection, for a total of 3 times. The collected virus solution was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was filtered through a 0.45 μm filter membrane for sterilization, aliquoted, and stored at -80℃.

[0025] Viral infection: The primary rumen mixed cells obtained in step one were passaged into 6 cm culture dishes. When the cell density reached 50% confluence, the experimental group was replaced with complete culture medium containing 20% ​​volume-concentrated lentivirus, while the control group was replaced with an equal volume of fresh complete culture medium. The cells were cultured at 37°C and 5% CO2 for 12 hours.

[0026] Resistance selection: 12 hours after infection, the culture medium for both groups of cells was replaced with normal complete culture medium. When the cells reached 90% confluence, they were passaged, and after cell attachment, blastidin S (BSD) was added to the culture medium at a final concentration of 10 μg / mL for selection. The BSD-containing medium was replaced every 48 hours until all cells in the control group died. Selection was then stopped, and the experimental group cells were cultured again to obtain immortalized cell lines (see...). Figure 1 This includes a variety of mixed cells such as rumen epithelial cells and rumen fibroblasts.

[0027] Step 3: Perform monoclonal expansion on the immortalized cells obtained in Step 2: Limiting dilution: The immortalized cell line obtained in step two was serially diluted with complete culture medium to adjust the cell density to 0.5 cells / well. The cells were then seeded into 96-well plates, with 100 μL of cell suspension containing less than 1 cell added to each well. The 96-well plates were then incubated statically at 37°C and 5% CO2 for 7-10 days.

[0028] Monoclonal labeling and observation: The cell growth in each well was observed daily using an inverted microscope. Wells containing only a single cell and no other cells in the surrounding area were labeled.

[0029] Monoclonal expansion: When the cells in the labeled wells reached 90% confluence, they were digested with 0.25% trypsin and sequentially passaged into culture containers with larger well diameters (in order: 48-well plate → 24-well plate → 12-well plate → 6-well plate → 6cm culture dish → 10cm culture dish). Each passage was performed only after the cells had grown to 90% confluence. Through this process, nine monoclonal cell lines were finally obtained (see...). Figure 2 ).

[0030] Step four: Screen for cell lines that can utilize butyrate from the monoclonal cell lines obtained in step three: Functional screening: The nine monoclonal cell lines obtained in step three were seeded into 6-well plates at a density of 102. 5 Cells per well. After cell attachment, the experimental group was replaced with sugar-free DMEM medium supplemented with 15 mmol / L sodium butyrate as a carbon source, while the control group was replaced with sugar-free DMEM medium supplemented with 25 mmol / L glucose as a carbon source. Cells were cultured at 37℃ and 5% CO2 for 0, 24, 48, and 72 hours, respectively. Cell morphology and proliferation were observed and recorded using an inverted microscope (see [link to documentation]). Figures 3-11 G8 cell lines that exhibited good morphology and rapid proliferation in butyric acid medium were selected for subsequent studies.

[0031] The growth curve of the G8 cell line was measured (using a hemocytometer) and its proliferation activity was detected by the MTT assay to verify its butyrate utilization capacity. Results are shown below. Figure 12 , 13 Under culture conditions with 15 mmol / L butyrate as the sole carbon source, the G8 cell line exhibited a significant proliferative effect, with its metabolic activity increasing by approximately 1.5 times after 24 hours of culture compared to the glucose group; and after 72 hours of culture, its cell number significantly exceeded that of the 25 mmol / L glucose treatment group. This effect demonstrates that the G8 cell line not only tolerates high concentrations of butyrate but can also efficiently convert it into an energy source that promotes its own proliferation.

[0032] Step 5: Detect the expression of fibroblast marker genes and rumen epithelial cell marker genes in the cell lines obtained in Step 4: RNA extraction: Total RNA was extracted from G8 cells and fresh Holstein bovine rumen epithelial tissue (as a control) using the TRIzol method. After assessing RNA purity and integrity, cDNA was synthesized using a reverse transcription kit.

[0033] cDNA synthesis: 1 μg of total RNA was reverse transcribed into cDNA using the HiScript® II Q RT SuperMix for qPCR kit.

[0034] RT-qPCR: Using this cDNA as a template, the expression levels of fibroblast marker genes (COL1A1, COL1A2, COL3A1) and rumen epithelial cell marker genes (basal cell marker KRT14, ketogenic enzyme HMGCS2) were detected by real-time quantitative PCR using the AceQ qPCR SYBR Green Master Mix kit with POLG2R as an internal control gene.

[0035] The primer sequences used are shown in Table 1.

[0036] .

[0037] See results Figure 14 , 15 The expression of COL1A1, COL1A2, and COL3A1 genes in G8 cells was significantly higher than that in rumen epithelial tissue controls, while the HMGCS2 gene was not expressed or expressed at very low levels, thus identifying the G8 cell line as originating from rumen fibroblasts. Combined with its fibroblast-like morphology, G8 was ultimately determined to be a Holstein dairy cow rumen fibroblast cell line capable of utilizing butyrate.

[0038] Example 3

[0039] This embodiment provides a method for culturing the rumen fibroblast cell line (G8) described in Example 1: using sugar-free DMEM medium containing 10-20 mmol / L sodium butyrate, and culturing at 37°C and 5% CO2.

[0040] Example 4

[0041] This embodiment provides the application of the rumen fibroblast cell line (G8) described in Example 1 in in vitro rumen studies.

Claims

1. A rumen fibroblast cell line, characterized in that, The accession number is CCTCC NO:C2025332, and it was deposited at the China Center for Type Culture Collection on October 31, 2025.

2. The method for constructing the rumen fibroblast cell line according to claim 1, characterized in that, Includes the following steps: Step 1: Collect rumen epithelial tissue, digest it, and then collect and culture the cells. Step two: Immortification treatment is performed on the cells obtained in step one; Step 3: Perform monoclonal expansion on the immortalized cells obtained in Step 2; Step 4: Screen for cell lines that can utilize butyrate from the monoclonal cell lines obtained in Step 3. Step 5: Detect the expression of fibroblast marker genes and rumen epithelial cell marker genes in the cell lines obtained in Step 4.

3. The construction method according to claim 2, characterized in that, In step four, the screening method involves culturing cell lines in butyric acid medium.

4. The construction method according to claim 3, characterized in that, The butyric acid medium is a sugar-free DMEM medium containing 10-20 mmol / L sodium butyrate.

5. The construction method according to claim 2, characterized in that, In step four, the screening criteria are cell morphology and proliferation rate.

6. The construction method according to claim 2, characterized in that, In step five, the fibroblast marker gene is the collagen gene COL1A1, COL1A2, or COL3A1.

7. The construction method according to claim 2, characterized in that, In step five, the rumen epithelial cell marker gene is either the basal cell marker KRT14 or the ketogenic key enzyme HMGCS2.

8. The construction method according to claim 2, characterized in that, In step one, the rumen tissue is taken from a cow.

9. The method for culturing the rumen fibroblast cell line according to claim 1, characterized in that, Cultured in sugar-free DMEM medium containing 10-20 mmol / L sodium butyrate at 37°C and 5% CO2.

10. The application of the rumen fibroblast cell line of claim 1 in in vitro rumen studies.

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