Immortalized livestock precursor fat cell line and preparation method thereof

Abstract

The invention provides an immortalized livestock precursor fat cell line and a preparation method thereof, and belongs to the technical field of biology. The invention provides a preparation method of an immortalized livestock precursor adipocyte line, which takes adipose tissues of livestock larvae as a cell source, the cell source contains a small amount of mature adipocytes, but a main cell population is precursor adipocytes, so that the purity of a starting material for subsequent immortalization is ensured; after immortalized induction, the monoclonal cell strain is screened by using a limited dilution method, the immortalized monoclonal cell strain still has the capability of being differentiated into mature adipocytes and forming lipid droplets after induced differentiation treatment, and the immortalized cell line can keep normal cell morphology and overcome replicative senescence. The immortalized cell strain disclosed by the invention can be efficiently differentiated into fat cells with mature functions after being induced, and has normal lipid synthesis and storage capabilities.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an immortalized livestock precursor fat cell line and its preparation method. Background Technology

[0002] As an important energy storage site, adipose tissue in mammals plays a crucial role in maintaining the body's energy balance through the interaction and synergistic effects of numerous internal factors and external signals. In livestock, adipose tissue is mainly divided into four categories: subcutaneous fat, visceral fat, intramuscular fat, and intramuscular fat. In sheep production, while the accumulation of intramuscular fat improves the tenderness and flavor of mutton and enhances meat quality, excessive fat deposition in other parts of the body, especially in the tail, reduces the quality of livestock products and increases feeding costs. Therefore, understanding the patterns and molecular mechanisms of fat formation, regulating fat deposition in animals, and improving the quality of mutton products are of great significance in sheep production.

[0003] Fat deposition in animals involves both the continuous synthesis and accumulation of fat by mature adipocytes and the continuous proliferation, differentiation, and maturation of precursor adipocytes. Therefore, precursor adipocytes, as the precursor cells of mature adipocytes, are ideal cell models for studying the laws and molecular mechanisms of adipogenesis. In vitro culture of precursor adipocytes allows for the reproduction of the entire process of proliferation and differentiation from precursor adipocytes to mature adipocytes. It also facilitates the observation of the influence of various factors on this process, enabling the exploration of the molecular mechanisms of adipogenesis at the cellular level, which is beneficial for understanding and mastering the laws of fat deposition in animals. Although primary culture systems for precursor adipocytes in livestock such as pigs, cattle, and sheep have been established, primary precursor adipocytes isolated from mammalian adipose tissue have limited proliferative capacity and cannot be continuously passaged in vitro. Multiple passages not only lead to the loss of in vivo morphology and function and changes in chromosome ploidy, but also cause primary precursor adipocytes to lose their ability to differentiate into mature adipocytes, thus affecting research on the molecular mechanisms of adipogenesis. Furthermore, due to individual differences, primary preadipocytes isolated from different animal individuals exhibit variations in genetic background between different batches, making it difficult to obtain stable and reliable results. Therefore, establishing immortalized livestock preadipocyte lines will facilitate research on the molecular mechanisms of fat deposition and other biological functions in livestock at the cellular level. Summary of the Invention

[0004] This invention provides an immortalized livestock precursor adipocyte line and its preparation method. The immortalized cell line can be efficiently differentiated into functionally mature adipocytes after induction, and has normal lipid synthesis and storage capabilities.

[0005] The present invention provides a method for preparing an immortalized livestock pre-adipocyte cell line, comprising the following steps: (1) extracting adipose tissue from livestock larvae, filtering the cell suspension produced by digestion with type I collagenase, collecting the system with a thickness of less than 40 μm, centrifuging and collecting the precipitate; (2) The precipitate collected in step (1) is inoculated into a culture dish containing 10% FBS DMEM complete medium and cultured until it reaches 60-70% confluence. Immortification induction is then performed to obtain the immortalized livestock precursor adipocyte line.

[0006] In a preferred embodiment of the present invention, the livestock mentioned in step (1) includes sheep.

[0007] In a preferred embodiment of the present invention, step (1) includes extracting adipose tissue from a lamb on the 80th day of gestation, wherein the adipose tissue is extracted from the tail.

[0008] In a preferred embodiment of the present invention, the method for inducing immortalization in step (2) includes lentiviral infection.

[0009] In a preferred embodiment of the present invention, the lentivirus includes hTERT and / or SV40T.

[0010] In a preferred embodiment of the present invention, the lentivirus is used to inoculate cells into a cell culture medium containing puromycin for screening of resistant cells. Immortalized positive cells with resistance are screened, and then the cells are passaged to obtain the immortalized livestock preadipocyte line.

[0011] In a preferred embodiment of the present invention, the culture medium for inducing differentiation is based on DMEM medium and further includes 10% FBS, 0.5mM IBMX, 1μM DEX, 5~10 mg / L INS, 20μM RSG and 1.5μg / mL puromycin.

[0012] The present invention also provides an immortalized livestock preadipocyte line prepared using the above-described preparation method.

[0013] The present invention also provides an immortalized precursor adipocyte monoclonal prepared based on the above-mentioned immortalized livestock precursor adipocyte line.

[0014] The present invention also provides a method for screening the above-mentioned immortalized preadipocyte monoclonals, comprising digesting the above-mentioned immortalized livestock preadipocyte line with trypsin and resuspending it into a single-cell suspension, and then screening the immortalized preadipocyte monoclonals by limiting dilution method.

[0015] Beneficial Effects: This invention provides a method for preparing immortalized livestock precursor adipocyte lines, using adipose tissue from young livestock as the cell source. While the cell source contains a small number of mature adipocytes, the main cell population consists of precursor adipocytes, ensuring the purity of the starting material for subsequent immortalization. After immortalization induction, monoclonal cell lines are obtained through limiting dilution screening. These immortalized monoclonal cell lines, after induction and differentiation treatment, still possess the ability to differentiate into mature adipocytes and form lipid droplets. Furthermore, the immortalized cell lines maintain normal cell morphology and overcome replicative senescence. The immortalized cell lines of this invention can efficiently differentiate into functionally mature adipocytes after induction, possessing normal lipid synthesis and storage capabilities. Attached Figure Description

[0016] Figure 1 The image shows the morphology of preadipocytes in the tail of a Kazakh sheep (magnification 100×). In the image, A represents the cell morphology when the cell confluence is 30-40%; B represents the cell morphology when the cell confluence is 100%; the arrows indicate mature adipocytes. Figure 2 Comparison of different adipogenic induction differentiation methods for sheep preadipocytes (magnification 100×), in the figure A: Group A; B: Group B; C: Group C; D: Group D; E: Control group; Figure 3 The image shows the morphology and lipid droplet formation ability of precursor adipocytes after different immortalization treatments (magnification 100×). In the figure, A: cell morphology observation; B: Oil Red O staining. Figure 4 Morphology of three monoclonal precursor adipocytes (magnification 100×). Figure 5 Senescence detection of three monoclonal precursor adipocytes (magnification 100×). Figure 6 The karyotypes of three monoclonal precursor adipocytes; Figure 7 Oil Red O staining of three monoclonal precursor adipocytes after induced differentiation (magnification 100×). Figure 8 This study aimed to detect the expression of genes related to adipogenic differentiation after induction of three monoclonal precursor adipocytes. Detailed Implementation

[0017] The present invention provides a method for preparing an immortalized livestock pre-adipocyte cell line, comprising the following steps: (1) extracting adipose tissue from livestock larvae, filtering the cell suspension produced by digestion with type I collagenase, collecting the system with a thickness of less than 40 μm, centrifuging and collecting the precipitate; (2) The precipitate collected in step (1) is inoculated into a culture dish containing 10% FBS DMEM complete medium and cultured until it reaches 60-70% confluence. Immortification induction is then performed to obtain the immortalized livestock precursor adipocyte line.

[0018] This invention extracts adipose tissue from young livestock. There is no specific limitation on the type of livestock; for example, a sheep is used in one embodiment, but this should not be considered the entire scope of protection of this invention. In this embodiment, the young sheep may be a sheep on its 80th day of gestation, from which adipose tissue is extracted, specifically from the tail.

[0019] This invention utilizes 1×D-Hanks solution (containing penicillin and streptomycin) to wash collected tail adipose tissue. Subsequently, blood vessels and muscle tissue on the surface of the adipose tissue are removed in a clean bench, and the tissue is minced. The minced tissue is then digested using type I collagenase at 37 °C for 30 min. Digestion is terminated by adding an equal proportion of complete culture medium, and the tissue fluid is collected. This tissue fluid collection process can be repeated three times to obtain a cell suspension.

[0020] In this invention, the cell suspension obtained from digestion is filtered to collect cell clusters with a particle size of less than 40 μm. For example, in the embodiment, the cells can be filtered through 70 μm and 40 μm nylon sieves in sequence. The filtrate is then collected and centrifuged at 800 r / min for 5 min. The supernatant is discarded, and the cell pellet is resuspended and washed with DMEM complete medium containing 10% FBS. The pellet is then centrifuged at 800 r / min for 5 min, and the supernatant is discarded.

[0021] This invention utilizes complete medium containing 10% FBS and DMEM to resuspend cell pellets, which are then seeded into culture dishes and cultured at 37°C in a 5% CO2 incubator. Immortification induction is performed when cells adhere to the culture dish and reach 60-70% confluence. The immortalization induction method of this invention includes lentiviral infection, where the lentivirus includes hTERT and / or SV40T. Examples of this invention compare infection using hTERT and SV40T alone versus infection using a 1:1 volume ratio mixture of hTERT and SV40T. The results show that the combined lentiviral infection method is more effective.

[0022] In this embodiment of the invention, the hTERT lentivirus construction method includes the following steps: constructing the hTERT (Gene ID: 7015) recombinant lentivirus plasmid according to the PLEX-MCS lentivirus packaging instructions, and co-transfecting it with the packaging plasmids (psPAX2 and pMD2.G) into 293T cells via calcium phosphate transfection. After 36 hours, the cell supernatant is collected and filtered through a 0.45 μm filter (i.e., the lentivirus infection solution). The filtered supernatant can be directly used to infect target cells. The SV40T lentivirus of this invention was purchased from Hanheng Biotechnology Co., Ltd., and the construction method includes the following steps: constructing the SV40T recombinant lentivirus plasmid according to the pHBLV-CMV-MCS-3flag-EF1-puro lentivirus packaging instructions, and using a three-plasmid lentivirus system to transfect the SV40T (Taxonomy ID: 1891767) recombinant lentivirus plasmid and the packaging plasmids (psPAX2 and pMD2.G) via Lipofiter. TM The transfection reagent was used to transfect 293T cells. Viral supernatant was collected at 48 and 72 h after transfection, and after centrifugation and purification, it could be directly used to infect target cells.

[0023] The present invention does not specifically limit the method of lentivirus infection; conventional lentivirus infection methods in the art can be used to infect cells.

[0024] This invention utilizes the lentivirus infection followed by inoculation into a cell culture medium containing puromycin for resistant cell selection. Immortalized positive cells with resistance are selected, and then passaged, plated, and induced to differentiate, yielding the immortalized livestock preadipocyte line. The differentiation-inducing medium of this invention is based on DMEM medium and further includes 10% FBS, 0.5 mM IBMX, 1 μM DEX, 5–10 mg / L INS, 20 μM RSG, and 1.5 μg / mL puromycin.

[0025] The present invention also provides an immortalized livestock preadipocyte line prepared using the above-described preparation method.

[0026] The immortalized livestock preadipocyte line described in this invention is similar to the primary preadipocytes, still spindle-shaped, and still has the ability to differentiate into mature adipocytes and form lipid droplets after induction, which is consistent with the characteristics of immortalized cells.

[0027] The present invention also provides an immortalized precursor adipocyte monoclonal prepared based on the above-mentioned immortalized livestock precursor adipocyte line.

[0028] The present invention also provides a method for screening the above-mentioned immortalized preadipocyte monoclonals, comprising digesting the above-mentioned immortalized livestock preadipocyte line with trypsin and resuspending it into a single-cell suspension, and then screening the immortalized preadipocyte monoclonals by limiting dilution method.

[0029] The present invention does not impose any special limitations on the specific operation method of the limiting dilution method; it can be operated using conventional methods in the field.

[0030] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of an immortalized livestock preadipocyte line and its preparation method provided by the present invention, should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1 1. Isolation and culture of preadipocytes from the tail of Kazakh sheep The adipose tissue used to isolate the prefat cells from sheep tails was derived from the fetus of a Kazakh sheep at day 80 of gestation (E80). The collected tail adipose tissue was washed three times in 1×D-Hanks solution (containing penicillin and streptomycin), and then, in a laminar flow hood, blood vessels and muscle tissue on the surface of the adipose tissue were removed using ophthalmic scissors and forceps, and the tissue was minced to 1 mm. 3 The fragments were mixed with 1 mg / ml type I collagenase at a 1:1 ratio, digested at 37 ℃ for 30 min, then removed and a complete culture medium of equal proportion was added to stop the digestion. The tissue fluid was collected. The above steps were repeated 3 times.

[0032] The completely digested cell suspension was filtered sequentially through 70 μm and 40 μm nylon sieves into 50 ml centrifuge tubes. The tubes were centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the cell pellet was resuspended in DMEM complete medium containing 10% FBS. The cell pellet was then centrifuged at 800 rpm for 5 min, and the supernatant was discarded. The cell pellet was resuspended in DMEM complete medium containing 10% FBS and seeded into culture dishes. The cells were incubated at 37 ℃ in a 5% CO2 incubator. Figure 1 As shown, when the isolated and cultured primary preadipocytes grow to approximately 30-40% confluence, the cell morphology exhibits an irregular spindle shape. Figure 1 (A) As cells proliferate to full confluence (100% confluence), the cells are tightly packed together, pressing against each other, and the cell morphology tends to be uniform, exhibiting a flowing appearance. Figure 1 (B). Notably, very few cells in the initially isolated cell population were observed to contain tiny lipid droplets in their cytoplasm. Figure 1 (As indicated by the arrow) This indicates that the cell population contains a small number of mature adipocytes, but the main cell population consists of precursor adipocytes, ensuring the purity of the starting material for subsequent immortalization experiments.

[0033] 2. Induction and differentiation of sheep precursor adipocytes and Oil Red O staining To determine the optimal conditions for inducing differentiation, four groups of induction culture media, as shown in Table 1, were set up for comparison, with insulin (INS) concentration and rosiglitazone (RSG) as key factors. Cells that did not undergo induction treatment served as the control group.

[0034] Preadipocytes in the logarithmic growth phase were seeded into 6-well plates. When the cell density reached 80%, the induction differentiation medium was changed and marked as day 0 (d). The cells were cultured until day 9 (d), with the medium changed every 3 days. After the induction differentiation treatment, the cells were stained with Oil Red O to observe the induction differentiation of the preadipocytes.

[0035] First, fix the induced differentiated cells with 4% neutral paraformaldehyde solution for 30 min, wash with 60% isopropanol, then incubate with 60% Oil Red O working solution at room temperature for 10 min, wash with 60% isopropanol and distilled water to remove excess staining solution, and take pictures under an inverted digital microscope.

[0036] like Figure 2 As shown, under different induction conditions, the group containing 20 μM RSG had significantly higher intracellular lipid droplet accumulation than the group without RSG. Furthermore, in the RSG-containing groups, high concentrations of INS (10 mg / L) further enhanced lipid accumulation. Based on Oil Red O staining results, the lipid droplet formation in each group was compared, and group A was determined to have the most significant induction differentiation effect, thus being identified as the optimal induction protocol. Group A culture medium was subsequently used for induction differentiation.

[0037] Table 1 Differentiation induction culture medium

[0038] 3. Immortality treatment of sheep primary preadipocytes The isolated preadipocytes were plated into 6-well cell culture plates. Once the cells adhered and reached 60-70% confluence, they were infected with three immortalization treatments: hTERT, SV40T, and a 1:1 mixture of hTERT and SV40T lentiviruses. Forty-eight hours after infection, the cells were transferred to 10cm cell culture plates, and cell culture medium containing 1.5 μg / mL puromycin was added for selection of resistant cells. The puromycin-containing cell culture medium was changed every 3-4 days. Immortalized positive cells with resistance were obtained when no further cell death occurred in the culture plate. Subsequent cell passages, plated cells, and induction of differentiation were all performed with puromycin-containing medium.

[0039] Immortification of sheep primary preadipocytes using three methods—hTERT, SV40T, and hTERT+SV40T—revealed that the morphology of the immortalized preadipocytes was similar to that of the primary preadipocytes, still exhibiting a spindle shape. Figure 3 (A). Induction of differentiation into immortalized sheep preadipocytes was performed using three different treatment methods, and it was found that the preadipocytes still possessed the ability to differentiate into mature adipocytes and form lipid droplets. Figure 3 Among the cells treated with hTERT+SV40T for immortalization, those treated with hTERT+SV40T showed the best ability to differentiate into mature adipocytes and form lipid droplets.

[0040] 4. Immortalized precursor adipocyte monoclonal screening (limiting dilution method) Cells in the logarithmic growth phase and in good condition after hTERT+SV40T immortalization were digested with 0.05% trypsin and resuspended into a single-cell suspension. Based on cell counting results, the cell concentration was adjusted to 10 cells / mL using complete medium containing 10% FBS. Ten 96-well plates were prepared, and 100 μL of cell suspension was added to each well. The plates were then incubated at 37 ℃ with 5% CO2 and saturated humidity for 3–5 days. Each well was then carefully observed under an inverted microscope. Wells with only one cell colony (i.e., monoclonal wells) were marked. When the cells in the marked monoclonal wells reached approximately 30–50% confluence at the bottom of the well, they were digested with trypsin and resuspended. The cells were then transferred sequentially to 24-well plates, 6-well plates, and T25 culture flasks to gradually scale up the culture.

[0041] 5. Identification of immortalized monoclonal precursor adipocyte lines (1) Cell morphology observation Fifty-four monoclonal cell lines were isolated from immortalized cells using the limiting dilution method. After continuous passage and morphological observation, three cell lines (numbered 14, 20, and 47) were selected as the most stable and exhibited the strongest proliferative activity for further research. Figure 4 As shown, these three cell lines still exhibit the typical spindle shape consistent with primary preadipocytes, proving that they maintained normal cell morphology after immortalization.

[0042] (2) Detection of senescence in immortalized monoclonal preadipocytes (β-galactosidase method) Monoclonal preadipocytes were seeded in 6-well plates. When the cell density reached 80%, the cell culture medium was discarded, and the cells were washed twice with PBS. 1 ml of β-galactosidase staining fixative was added, and the cells were fixed at room temperature for 15 min. The cells were then washed three times with PBS, 2 min each time. 1 ml of β-galactosidase staining working solution was added, and the plates were incubated at 37 ℃ for 14 h. The staining solution was discarded, and 2 ml of PBS was added to cover the cells. The cells were observed and photographed under a light microscope.

[0043] Results of β-galactosidase senescence staining identification are as follows: Figure 5 As shown, no senescence-related β-galactosidase activity was detected in any of the three monoclonal precursor adipocytes (i.e., no blue precipitate was generated), proving that the cell line has successfully overcome replicative senescence and is consistent with the characteristics of immortalized cells.

[0044] (3) Chromosome karyotype analysis of immortalized monoclonal precursor adipocytes Monoclonal preadipocytes were seeded in 6-well plates. When the cell density reached 80%, the medium was replaced with complete medium containing 0.2 μg / ml colchicine, and incubated at 37 ℃ for 4–6 h. Cells were digested and collected, washed 1–2 times with PBS, centrifuged at 800 rpm for 5 min, and the supernatant was discarded.

[0045] Hypotonic treatment: Resuspend cells in 0.5% KCl solution and incubate in a 37 ℃ water bath for 30 min, inverting and mixing once every 10 min during the incubation period.

[0046] Pre-fixation: Add 1 ml of freshly prepared and pre-cooled fixative (methanol:acetic acid = 3:1) along the tube wall, gently pipette to mix, incubate at room temperature for 10 min, centrifuge at 1000 rpm for 5 min, and discard the supernatant.

[0047] Fixation: Add 1 ml of fixative, mix by pipetting, then add to 8 ml of fixative. Incubate at room temperature for 10 min, centrifuge at 1000 rpm for 5 min, discard the supernatant, and keep about 500 μl of fixative.

[0048] Slide preparation: Gradually drip the glass slides to a pre-cooled temperature of -20 ℃.

[0049] Stain with Giemsa staining solution for 5 minutes, rinse with tap water for 10-30 seconds, air dry naturally, mount with resin, and photograph.

[0050] Karyotype analysis of three monoclonal precursor adipocytes yielded the following results: Figure 6 As shown, most cells have a chromosome number of 54. Their karyotype is consistent with sheep chromosome atlases, indicating that the cultured cells have not undergone significant chromosome variation.

[0051] (4) Verification of induced differentiation ability The differentiation capacity of the three immortalized monoclonal precursor adipocyte lines was verified. After culturing under the optimal induction conditions for 9 days, Oil Red O staining was performed for identification. Results are as follows: Figure 7 As shown, all three cell lines exhibited highly efficient differentiation capabilities, with a large number of lipid droplets of varying sizes accumulating in the cytoplasm. This indicates that the immortalized cell lines of the present invention can efficiently differentiate into functionally mature adipocytes after induction, possessing normal lipid synthesis and storage capabilities.

[0052] To further verify the differentiation capacity of the immortalized monoclonal precursor adipocyte line at the molecular level, cells were cultured under the optimal induction conditions until day 9. Cellular RNA was extracted and reverse transcribed into cDNA. Using the cDNA as a template, qRT-PCR was performed (qRT-PCR primers are shown in Table 2). The results showed ( Figure 8 Following induced differentiation, the relative mRNA expression levels of adipogenic differentiation-related genes CEBPα, PPARγ, LPL, and FABP4 in the three cell lines showed a significant increase compared to pre-differentiation levels. This indicates that the immortalized cell lines of this invention successfully activated the core adipogenic differentiation program at the gene expression level after induced differentiation, confirming their mature cellular function at the transcriptional level. This is corroborated by Oil Red O staining results, fully demonstrating that the cell lines possess complete and stable adipocyte differentiation capabilities.

[0053] Table 2 Primers for qRT-PCR amplification of genes related to adipogenesis differentiation.

[0054] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing an immortalized livestock precursor adipocyte cell line, characterized in that, The steps include: (1) extracting adipose tissue from young livestock, filtering the cell suspension produced by digestion with type I collagenase, collecting the system with a thickness of less than 40 μm, centrifuging and collecting the precipitate; (2) The precipitate collected in step (1) is inoculated into a culture dish containing 10% FBS DMEM complete medium and cultured until it reaches 60-70% confluence. Immortification induction is then performed to obtain the immortalized livestock precursor adipocyte line.

2. The preparation method according to claim 1, characterized in that, The livestock mentioned in step (1) include sheep.

3. The preparation method according to claim 2, characterized in that, Step (1) includes extracting adipose tissue from a lamb on the 80th day of gestation, the adipose tissue being extracted from the tail.

4. The preparation method according to claim 1, characterized in that, The method for inducing immortalization in step (2) includes lentiviral infection.

5. The preparation method according to claim 4, characterized in that, The lentiviruses include hTERT and / or SV40T.

6. The preparation method according to claim 4 or 5, characterized in that, After infecting cells with the lentivirus, the cells are inoculated into cell culture medium containing puromycin for screening of resistant cells. Immortalized positive cells with resistance are screened, and then the cells are passaged to obtain the immortalized livestock preadipocyte line.

7. The preparation method according to claim 6, characterized in that, The culture medium for inducing differentiation is based on DMEM medium and also includes 10% FBS, 0.5mM IBMX, 1μM DEX, 5~10 mg / L INS, 20μM RSG and 1.5μg / mL puromycin.

8. An immortalized livestock preadipocyte line prepared by the preparation method according to any one of claims 1 to 7.

9. Immortalized preadipocyte monoclonal cells prepared based on the immortalized livestock preadipocyte line of claim 8.

10. The method for screening immortalized precursor adipocyte monoclonal cells according to claim 9, characterized in that, This includes digesting the immortalized livestock preadipocyte line of claim 8 with trypsin and resuspending it into a single-cell suspension, and then using a limiting dilution method to screen for the immortalized preadipocyte monoclonals.

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