NL2032736B1 - Immortalized yak rumen fibroblast cell line and construction and application thereof - Google Patents

Abstract

The invention discloses an immortalized yak rumen fibroblast cell line and construction and application thereof. Trypsin and dispase are adopted, to digest yak rumen epithelial tissues to obtain primary yak rumen fibroblast cells, lentivirus carrying SV4OT is 5 adopted to infect the primary fibroblast cells, and then purine toxin is used to screen out immortalized yak rumen fibroblast cell lines stably transferred with SV4OT genes and preserve the strains. The cell line is preserved, in China Center for Type Culture Collection with the preservation number of CCTCC lO NO.C2021253. The establishment of the cell line solves the defects of difficult culture and limited, passage times of the primary rumen fibroblast, enriches the yak source cell line library, fills in the blank of yak fibroblast related research and test materials, and also provides a cell model for inducing and 15 differentiating the yak fibroblast line into yak fat cells, osteoblasts and the like. (+ Fig.)

Description

P1517 /NLpd

IMMORTALIZED YAK RUMEN FIBROBLAST CELL LINE AND CONSTRUCTION AND

APPLICATION THEREOF

Technical field

The invention relates to a cell line and a construction meth- od thereof, in particular to an immortalized yak rumen fibroblast cell line and construction and application thereof.

Background art

The yak is a kind of bovine animal living on the cold plateau above 3 kilometers above sea level. The yak is a unique and pre- cious genetic resource in China. Almost all yaks are distributed throughout the Qinghai-Tibet Plateau, especially in Gansu, Qinghai and Tibet. According to the Records of Chinese Livestock and Poul- try Breeds, there are currently 12 officially recognized domestic yak breeds in China, including Jiulong yak and Maiwa yak in Si- chuan, Tianzhu white yak and Gannan yak in Gansu, Bali yak, Jiali yak and four-step yak in Tibet, Huanhu yak, plateau yaks and long- bodied yaks in Qinghai, Bazhou yakin Xinjiang and Zhongdian yakin

Yunnan. Yak is not only the main economic source and pillar of life for local herdsmen, but also closely related to local cul- ture, religion and social life. Because of its special biological characteristics, economic characteristics and distribution charac- teristics, whether in nutrition, breeding or genetics, there are many studies on animal production of yak, but the reports on these studies at the cellular level are very limited. Yak-related cell lines are also extremely limited. Based on the reality of germplasm conservation in China, it is also of great significance to construct cell lines of important yak populations to conserve their genetic resources.

Fibroblasts are the most common multifunctional cells in con- nective tissue and an important component of mesenchymal tissue.

They usually remain in a resting state and only acquire temporary activity during tissue remodeling and repair after tissue damage, after which they die or return to a dormant state. It is known that one of the main functions of fibroblasts is to synthesize collagen and other extracellular matrix, which plays an important role in the process of tissue and organ fibrosis. Rumen is the most important organ in the digestive system of ruminants, and it is also an important part of the body's immune system. The gastric wall is composed of four layers: mucosa, submucosa, muscularis and adventitia, with the distribution of nerves, blood vessels and lymphatic vessels. Fibroblasts were mainly distributed in the lam- ina propria, submucosa and adventitia. Fibroblasts are highly het- erogeneous and are essential for maintaining tissue homeostasis.

The origin of fibroblasts is very important and valuable for the study of invasion and metastasis, organ growth, angiogenesis, ma- trix remodeling, immune regulation and so on. Fibroblasts are large and well-defined, most of them are prominent spindle-shaped or star-shaped flat structures, with regular oval nuclei and large and obvious nucleoli. Under the electron microscope, there were abundant rough endoplasmic reticulum, free ribosomes and Golgi complexes in the cytoplasm of fibroblasts, which indicated that fibroblasts had the function of synthesizing and secreting pro- teins.

The isolation and culture of fibroblasts are mainly used to study the aging of cells, the damage of various exogenous factors to cells, the malignant transformation of cells in vitro, and some congenital metabolic abnormalities and enzyme defects. At present, there are some studies on different organs and tissues of differ- ent species, especially some fibroblast culture has been widely used in basic medicine and clinical medicine research, but there is no outstanding report on yak related fibroblast cell line. Alt- hough the primary cells isolated and cultured are relatively ma- ture, they have great defects, and the limited life generation will also restrict the research progress of their in vitro experi- ments. There are few reports on the analysis of the functions of yak related organs at the level of yak rumen fibroblasts. The main reason is that the technology of cell isolation and culture of yak species is not yet mature. Human skin fibroblasts can be trans- formed into pluripotent stem cells, such as adipocytes, endotheli- al cells, chondrocytes, tenocytes and neural cells, after treated with different transcription factors. However, the mechanism or potential of proliferation, function and differentiation of vak rumen fibroblast cell line is still unclear. The use of primary cultured cells for research can ensure that it is not affected by other factors such as hemodynamics in vivo, and can also control other factors through drug treatment, which provides a basis for the study of various physiological and pathological processes.

Cell immortalization is expected to fundamentally solve the problem of yak donor shortage, which is a hot research topic in the world. In order to make the normal primary cells have the characteristics of in vitro culture and proliferation, a retrovi- ral vector was constructed to transduce the immortalized gene into the cells, and the immortalized primary cells could still maintain their physiological and biochemical characteristics. Immortaliza- tion is defined as a process in which diploid cells cultured in vitro escape from the crisis of proliferation and aging spontane- ously or under the influence of external factors and have the ability of unlimited proliferation. Under spontaneous conditions, the probability of immortalization of diploid cells is very small.

Therefore, in order to meet the needs of research, scholars intro- duce exogenous immortalization gene into cells through plasmid transfection and other methods, and obtain cell lines with stable expression of immortalization gene after screening, so as to achieve cell immortalization.

SV40T is a commonly used target gene for cell immortalization and transfection in vitro. SV40 virus belongs to the family

Papovaviridae, genus Polyomavirus, and its genome consists of 5224bp of double-stranded circular DNA, which encodes two trans- forming proteins, large T antigen and small T antigen, before vi- ral DNA replication. Tag is a phosphorylated protein, which plays a decisive role in cell transformation and is widely used in the establishment of transgenic animal tumor models. SV40T gene can affect telomere length and activate telomerase activity. Telomeres are composed of guanine-rich DNA repeats and associated proteins at the 3 'end of eukaryotic chromosomes. The activation of te- lomerase and the stability of telomere length are closely related to cellular replicative senescence and immortalization. The acti-

vation of telomerase can keep the length of telomere stable, and then keep the chromosome stable, which mediates cell immortaliza- tion. We successfully immortalized yak rumen fibroblasts by lenti- virus-mediated method, and confirmed the existence of immortalized gene SV40T and its expression product by gene and protein level detection. In vitro experiments confirmed the functional stability of the cell line, which can be used for subsequent research.

Summary of the invention

The invention aims to provide an immortalized yak rumen fi- broblast cell line as well as construction and application there- of. The establishment of the cell line solves the defects of diffi- cult culture and limited subculture times of primary rumen fibro- blasts, enriches a yak source cell line library, and can provide main raw materials for scientific research in the related fields of life science, vaccine production and the like.

In order to achieve the purpose, the invention provides an immortalized yak rumen fibroblast cell line, which is preserved in

China Center for Type Culture Collection with the preservation number of CCTCC NO. C2021253.

The invention also provides a method for constructing an im- mortalized yak rumen fibroblast cell line, which comprises the following steps:

S1. Sample preprocessing yak rumen epithelial tissue is cleaned by washing liquid containing antibiotic and cut into piec- es;

S2. Enzymatic digestion and culture treating the tissue with digestive enzyme, collecting the upper layer of digestive juice and terminating digestion, centrifuging and collecting the cell precipitate, dispersing the cell precipitate with complete culture medium and inoculating into a culture flask for conventional cell culture; , placing the enzyme-digested tissues in a culture flask at intervals, and adding a complete culture medium for culture;

Carrying out subculture when the concentration is between 70%~80%, digesting for 1~2min by using a trypsin-EDTA solution, centrifu- gally collecting cell sediment after the digestion is stopped, and then resuspending the cell sediment by using a complete culture medium containing basic fibroblast growth factors to obtain cells which are fibroblasts; 83. Lentivirus infection when the passage fibroblasts grew to 40% -50%, the lentivirus was diluted with polybrene and complete 5 medium containing 10 ng/mL basic fibroblast growth factor, and added to the cells for infection overnight, and the medium was changed every 2 days for continucus culture for 4 days.

S4. Cell line screening when the cells grew to 20% ~ 30%, puromycin was added for screening, and the complete medium con- taining puromycin was replaced every two days for continuous cul- ture for 4 days.

S5. Expanded culture and passage the selected cells were sub- cultured for more than 20 generations, and the obtained cell line was the yak rumen fibroblast cell line.

The complete medium contained basal medium, 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin.

Wherein the reagents are sterile reagents, and the operations are conventional sterile operations.

Preferably, the above digestive enzyme is a mixture of 2% trypsin and dispase in a volume ratio of 10:1.

Preferably, the basal medium is RPMI 1640 or DMEM/F-12.

Preferably, the lentivirus is a lentiviral vector comprising

SV40T.

The application of the cell line provided by the invention in the field of life science.

Preferably, the cell line is useful for vaccine research and production.

The invention relates to an immortalized yak rumen fibroblast cell line as well as construction and application thereof, which solves the problems of difficult culture of primary rumen fibro- blasts, limited passage times and the like, and has the following advantages:

The construction method of the cell line is simple and con- venient to operate, the constructed cell line is stable and relia- ble, the yak source cell line library is enriched, the blank of yak fibroblast related research and test materials is filled, and a cell model is provided for yak fibroblast cells to be induced and differentiated into yak fat cells, osteoblasts and the like,

It can also provide the main raw materials for scientific research in life science related fields and vaccine production.

According to the invention, a plurality of multiplicity of infection (MOI) is set and the purine toxin concentration is ex- plored in the exploration of the optimal dose of the lentivirus infection multiplicity and the optimal purine toxin screening con- centration to screen out the last survival cells, so that the blindness of the test is reduced, and the test failure caused by the lentivirus infection multiplicity with a certain concentration or inappropriate purine toxin concentration is prevented.

The yak fibroblast cell line constructed by the invention has strong proliferation capability, presents the form of fibroblasts, and still maintains the morphological characteristics and growth characteristics of the fibroblasts after more than 30 generations of cell passage.

Brief description of the drawings

Fig. 1 is a microscopic view of primary cells of yak rumen fibroblasts (200X).

Fig. 2 is a microscopic view of an immortalized yak rumen fi- broblast cell line (200X).

Fig. 3 is a microscopic view (200 times) of beta - galactosidase stained immortalized yak rumen fibroblast cell line.

Fig. 4 is a graph of immunofluorescence identification re- sults of an immortalized yak rumen fibroblast cell line.

Fig. 5 is a graph of growth curve results of an immortalized yak rumen fibroblast cell line.

Detailed description of the invention

The technical solutions in the embodiments of the present in- vention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all of them. Based on the embodi- ments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work are with-

in the scope of the present invention.

Primary Reagent

RPMI-1640 medium (containing L-glutamine and HEPES), fetal bovine serum, dispase (Gibco); PBS, penicillin, streptomycin, trypsin-EDTA solution (0.25%: 0.02%) (Solebo); bFGF (RD); Chlor- hexidine Puromycin, Puromycin, Polybrene (Sigma); SV40T lentivirus packaging (Yunzhou Biology); CCK-8 (AbMole Company); vimentin (Vi- mentin) primary antibody, SV40T primary antibody (abcam), Triton- 100, 4% paraformaldehyde, goat serum, CY3-labeled goat anti-mouse

IgG (H+L), DAPI staining solution, cell senescence $B-galactosidase staining kit (Beyotime); Other reagents are conventional reagents in the laboratory. «

Example 1 (1) Primary culture: The yak rumen epithelial tissue was stripped of fat, blood vessels, muscle and other tissues, washed twice with PBS (containing 400 U/mL penicillin and 400 U/mL strep- tomycin), placed in chlorhexidine solution for 5 minutes, and then washed with PBS. Clean rumen epithelial tissues of yaks were cut into 50mL centrifuge tubes, cut into pieces as much as possible with sterile surgical scissors, digested with 2% trypsin and dis- pase at a volume ratio of 10:1 for 30min, and the upper digestive

Juice was collected and cultured in RPMI 1640 complete medium (containing 10% fetal calf serum, 100 U/mL penicillin and 100 U/mL streptomycin), the cell suspension was centrifuged at 1000 rpm/5 min, the cell pellet was collected, and PBS (containing 100 U/ml penicillin and 100 u/mL streptomycin) was added to gently blow and homogenize the cell pellet with a pipette for washing twice. Then

RPMI 1640 complete medium (containing 10% fetal bovine serum, 100

U/mL penicillin, 100 U/mL streptomycin and 10 ng/mL basic fibro- blast growth factor) was added, and the cells were gently pipetted with a pipette to form a cell suspension, which was inoculated in- to a cell culture flask. The cells were incubated in a cell incu- bator at 37°C(21% O:-5% CO:). After 50 min of incubation, the upper layer of the culture medium in the flask was removed and replaced with a new complete medium. At the same time, RPMI 1640 complete culture medium (containing 10% fetal bovine serum, 100 U/mL peni- cillin and 100 U/mL streptomycin) was added to the trypsin-

digested tissue blocks to scak the tissue blocks. The tissue blocks were placed in a culture dish with ophthalmic forceps. The distance between the tissue blocks was about 5 mm to ensure that cells crawled out of the surrounding tissue blocks. Gently cover the tissue blocks with sterile coverslips, place them in a cell incubator for 5 H, and then add a few drops of complete culture medium around each tissue block to continue the culture to prevent the tissue blocks from floating. After 2 days, 8 mL of culture me- dium was supplemented to continue the culture, and the culture me- dium was changed every 3 days. When about 80% of the cells cul- tured by trypsin digestion method in the culture flask or the cells from the tissue block cover the bottom of the culture dish, observe the results under the microscope as shown in Figure 1, perform the first passage, pour out the original culture medium, wash and discard it once with PBS solution, add 0.25% trypsin to digest the cells for 1~2min, When The fibroblasts showed cytoplas- mic retraction, sphericity and increased intercellular space, trypsin was removed and RPMI 1640 complete medium (containing 10% fetal bovine serum, 100 U/mL penicillin, 100 U/mL streptomycin and 10 ng/mL basic fibroblast growth factor) was added to stop diges- tion. Use the pipette to blow from one side of the bottom of the

Petri dish to the other side in order, gently, to avoid foam as much as possible. Prepare the detached cells into cell suspension, centrifuge the cell suspension at 1000rpm/5min, collect the cell sediment, add the complete culture medium and mix well to form cell suspension, and subculture at the density of one bottle of cells to three bottles of cells. At this time, most of the cells obtained are fibroblasts, and the cells not detached in the cul- ture dish are mainly epithelial cells. (2) SV40T lentivirus-infected primary yak rumen fibroblast cell line: After primary cells were cultured to a cell density of 50% in a six-well plate, the lentivirus was incubated with 8 mg/L polybrene and RPMI 1640 complete medium (containing 10% fetal bo- vine serum, 100 U/mL penicillin, 100 U/mL of streptomycin and 10 ng/mL of basic fibroblast growth factor), and the lentivirus was diluted into dilutions with multiplicity of infection (MOI) of 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 and added to each well. Af-

ter overnight culture, the medium was changed and the culture was continued for 4 days. When the cells grew to 20% -30%, puromycin was added for screening. Concentration gradients of 0, 0.5, 1, 2, 4, 5, 6 and 7g/mL were set, and 2 duplicate wells were set for each gradient. The growth status of the cells was observed and recorded, and the new purine toxin medium was changed every two days to continue screening. After 26h of continuous culture, the surviving cells were immortalized yak rumen fibroblasts. The cell line was successfully screened when the multiplicity of infection of lentivirus was 60 and the concentration of purine toxin was 0.5g/mL. The Immortalized yak rumen fibroblast cell line was ob- tained by routine culture in RPMI 1640 complete medium {containing 10% fetal bovine serum, 100 U/mL penicillin, 100 U/mL streptomycin and 10 ng/mL basic fibroblast growth factor) for 20 passages. The cell line is preserved in China Center for Type Culture Collection on October 14, 2021 with the preservation number of CCTCC NO.

C2021253.

Experimental Example 1 Cell Senescence Beta-Galactosidase

Staining Kit Detection

After the 30th generation of fibroblast cell line was inocu- lated into a six-well plate for cell growth and fusion, the cell culture medium was removed by aspiration, washed once with PBS or

HBSS, added with 1 ml of B-galactosidase staining fixative, and fixed at room temperature for 15 minutes. The cell fixative was removed by aspiration, and the cells were washed three times with

PBS or HBSS for 3 minutes each time. The PBS or HBSS was aspirat- ed, and 1 mL of staining solution was added to each well. Refer to

Table 1 below for the preparation method of dyeing working solu- tion.

Table 1 eme |e 93041 solution C

Incubate overnight at 37°C, and seal the 6-well plate with sealing film or plastic wrap to prevent evaporation. Among them,

incubation at 37°C can not be carried out in a carbon dioxide incu- bator, but should be observed under a common optical microscope.

If the count cannot be observed in time, the staining solution can be removed, and 2 ml of PBS can be added, which can be stored at 4°C for several days, or after the sealing solution is added. It can be stored for a long time at 4°C.

The cell senescence beta-galactosidase staining kit takes X-

Gal as a substrate and generates a dark blue product under the ca- talysis of senescence-specific beta-galactosidase. O that cells or tissues expressing beta-galactosidase that turn blue can be easily observed under an optical microscope. The immortalized yak rumen immortalized cell line was observed under the microscope, as shown in Figure 3. The results showed that the cell line had no obvious dark blue.

Experimental Example 2 Immunofluorescence identification of vimentin and SV40T protein expression

After the fifth generation of fibroblast cell line was made into cell suspension, it was inoculated into a 6-well cell culture plate at the density of 1 x 10°/mL after counting, and a cell sheet was placed in each well. When the cells grew and fused to 80%, the culture medium was removed, and the cells were washed with sterile

PBS for 3 times, 3 minutes each time. Cells were fixed with 4% paraformaldehyde for 15 min and washed with PBS for 3 times, 3 min each time. Cells were permeated with 0.5% Triton-100X at room tem- perature for 20 min and washed with PBS for 3 times, each time for 3 min. Cells were blocked with 5% goat serum at room temperature for 30 min and washed with PBS for 3 min each time. The primary antibody of SV40T protein was added to 6-well plates at a dilution of 1:50 and incubated overnight at 4°C; the cells were washed three times with PBS for 3 min each time, and then the secondary anti- body labeled with CY3 fluorescence was added and incubated at room temperature for 2 H in the dark, and the cells were washed three times with PBS for 3 min each time; The cells were washed with PBS for 3 times (3 min each time) after 5 min incubation with DAPI in the dark, and the cell slides were mounted with mounting solution containing anti-fluorescence guencher , and then observe the col-

lected image under the fluorescence microscope. The results are shown in Figure 4. A and D in the figure are the immunofluores- cence results of antigen molecules SV40T and vimentin respective- ly, and B and E in the figure are the DAPI immunofluorescence re- sults corresponding to A and D respectively. C and F in the figure are the results of immunofluorescence of antigen SV40T and vi- mentin and immunofluorescence Merge of DAPI respectively, and the scale in the figure is 100 um. The results showed that vimentin was highly expressed, indicating that this cell line was a fibro- blast cell line, and SV40T fluorescence was positive, indicating that SV40T gene was successfully transferred into primary fibro- blasts.

Experimental example 3 determination of growth curve of im- mortalize yak rumen fibroblast cell links

Fibroblast cell line of the 10th passage was made into cell suspension and inoculated into 96-well cell culture plate at the density of 1 x 10%/mL after counting, 6 replicates were set, 6 wells were taken out for determination each time, CCK-8 of 10% culture medium was added into each well, and OD value was deter- mined at 450 nm of microplate reader after 2 hours. The 10-day cell cycle was determined. The culture time was taken as the hori- zontal axis, and the average OD value of 6-well cells per day was taken as the vertical axis. The growth curve of the cell line showed an "3" curve, which was in line with the general cell growth law, and went through the incubation period, the logarith- mic growth period and the plateau period. The results are shown in

Figure 5.

While the present invention has been described in detail with respect to the preferred embodiments, it should be understood that the above description should not be taken as limiting the inven- tion. Various modifications and alternatives to the present inven- tion will become apparent to those skilled in the art upon reading the foregoing disclosure. Accordingly, the scope of the present invention is defined by the appended claims.

Claims (7)

CONCLUSIONS 1. Immortalized yak rumen fibroblast cell line, characterized in that the cell line is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NC.C2021253. A method of constructing an immortalized yak rumen fibroblast cell line according to claim 1, characterized by comprising the following steps: S1. Sample pre-processing: yak rumen epithelial tissue is cleaned by washing liquid with antibiotic and cut into pieces; S2. Enzymatic digestion and culture: treatment of the tissue with a digestive enzyme, collection of the upper layer of digestive juice and termination of the digestion, centrifugation and collection of the cell pellet, dispersion of the cell pellet with a complete culture medium and seeding in a culture flask for a conventional cell culture; intermittently placing the enzyme-digested tissues in a culture flask and adding a complete culture medium for the culture; performing subculture when the concentration is between 70%~80%, digestion for 1~2min using trypsin-EDTA solution, centrifugal collection of cell sediment after digestion is stopped, and then resuspending the cell sediment using a complete culture medium containing basic fibroblast growth factors to obtain cells that are fibroblasts; S3. Lentivirus infection: when the passage fibroblasts grew to 40%-50%, the lentivirus was diluted with polybrene and complete medium containing 10 ng/mL basic fibroblast growth factor and added to the cells for overnight infection, and the medium was refreshed every 2 days for continuous culture for 4 days gene. S4. Cell line screening: When the cells grew to 20% ~ 30%, puromycin was added for screening, and the entire medium containing puromycin was replaced every two days for continuous culture for 4 days. S5. Extensive culture and passage: the selected cells were subcultured for more than 20 generations, and the cell line obtained was a yak rumen fibroblast cell line; wherein the complete medium comprises a basal medium, 10% fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin. 3. The construction method according to claim 2, wherein the digestive enzyme is a mixture of 2% trypsin and dispase in a volume ratio of 10:1. The method of construction according to claim 2, wherein the basal medium is RPMI 1640 or DMEM/F-12. The construction method of claim 2, wherein the lentivirus is an SV40T lentivirus vector. 6. Application of the cell line according to claim 1 in the field of life science. Use according to claim 6, characterized in that it comprises the research and production of vaccines.