CN114058592B - Immortalized yak rumen epithelial cell line and construction method thereof - Google Patents
Immortalized yak rumen epithelial cell line and construction method thereof Download PDFInfo
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Abstract
The invention discloses an immortalized yak rumen epithelial cell line and a construction method thereof, wherein the preservation number of the cell line is as follows: cctccc No. C2021245, classified under the name: immortalized yak rumen epithelial cell line SV40T-YREC-hTERT. The invention cultures the primary rumen epithelial cells by adhering the tissue digested by the primary yak rumen epithelial papilla I collagenase, takes the cells as host cells, uses slow viruses carrying SV40T and hTERT genes to infect the cells, obtains an immortalized yak rumen epithelial cell line after screening and expanding culture, and solves the defects of difficult culture and limited passage times of the primary rumen epithelial cells by establishing the cell line, and enriches yak source cell line resources; the cell model can stably passage, can keep the morphological characteristics of primary rumen epithelial cells and has normal functions, and provides a useful in vitro cell model for researching rumen epithelial nutrition digestion, absorption and metabolism, a cell signal path mechanism and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an immortalized yak rumen epithelial cell line and a construction method thereof.
Background
The yaks are special beef breeds which can adapt to special severe natural environments in Qinghai-Tibet plateau areas in the world, are important living and production data of local herd survival, and are main prop industries for the pasture areas to become lean and rich. The Chinese is a large country for breeding yaks in the world, and accounts for more than 92% of the total amount of the world yak breeding, and is mainly distributed in Tibet, qinghai, sichuan, gansu, yunnan, xinjiang and other places in China. Basic researches and application basic researches related to genetic breeding, nutrition, feed, epidemic disease prevention and control and the like of yaks are in world leading level in China. So far, the research on rumen is more and more at home and abroad, and the regulation mechanism in the epithelial cells of the rumen is also attracting attention. The greatest feature of ruminants in physiological structure, unlike monogastric animals, is that ruminants have a complex gastric structure in which the rumen is most important. The rumen epithelium is an important barrier between the environment in the rumen and the environment of a host organism, the rumen epithelium is a main part of the rumen, a large number of epithelial papillae extend into the inner cavity from the surface of the rumen, the surface area for absorbing volatile fatty acids and electrolytes is greatly increased, and nutrient substances absorbed by the rumen epithelium are essential for the whole animal energy metabolism. Yaks are a dominant species of livestock that live in the special geographical environment of Qinghai-Tibet plateau throughout the year, and their rumen metabolism has many specificities compared to other ruminants. However, in the current research, the research on the yak rumen epithelial cells is less, and a stable source rumen epithelial cell line is not established, so that the research on the yak rumen epithelial cells is restricted, and the technical shortage in the related field affects the development of more molecular biology fields. At present, primary cells or cell lines derived from yaks are deficient, and it is important to obtain more cell lines derived from yaks and enrich the regulation and control mechanism of cell molecules related to yaks.
Most normal cells are considered to have limited ability to divide and to enter an aging state after failing to divide. At this point the cells are still viable, but the gene and protein expression profile of the cells is greatly altered. Senescent cells cannot re-induce cell division under some conventional stimuli, and the cell cycle distribution of senescent cells is also more specific, unlike some injury-induced cell dormancy, and unlike the case of cell growth contact inhibition, senescent cells generally become larger in volume. The research shows that the primary yak rumen epithelial cells can not be stored for a long time after in vitro culture, and the increase of the passage times has serious damage to the morphology and physiological functions of the primary cells. At the same time, the limited number of isolated tissues severely limits the molecular expression mechanism of exogenous nutrients at the cellular level.
The Chinese patent with publication number of CN107881197A discloses an immortalized cow rumen epithelial cell line and a construction method thereof, wherein the construction method comprises the following steps: step a), cutting, cleaning and digesting the collected cow rumen epithelial tissue in a culture medium, and performing primary culture to obtain rumen epithelial cells; step b), incubating the rumen epithelial cells in the step a) with a virus solution carrying SV40T antigen genes to obtain infected rumen epithelial cells; step c) further culturing the rumen epithelial cells infected in the step b) to obtain an immortalized cow rumen epithelial cell line. Although the immortalized cow rumen epithelial cell line provides a test cell model for the study of the physiological regulation and nutrition absorption mechanism of the cow rumen, the culture method is simple and has high growth speed, and the construction method can obtain the BRECs which have physiological functions and can be continuously passed, but the immortalized cow rumen epithelial cell line cannot be cultured by the method due to the obvious difference of cow and yak species. Through searching, the establishment of an immortalized yak rumen epithelial cell line has not been reported so far. Therefore, an immortalized yak rumen epithelial cell line capable of stable passage is urgently needed to be established, and the method has important significance for basic research of yaks.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide an immortalized yak rumen epithelial cell line which has complete cell line functions and can maintain the original characteristics of yak rumen epithelial cells;
the invention also aims to provide a method for constructing the immortalized yak rumen epithelial cell line, which is simple in operation and high in immortalization success rate.
The aim of the invention is achieved by the following technical scheme: an immortalized yak rumen epithelial cell line deposited under accession number: CCTCC No. c2021245, class designation: immortalized yak rumen epithelial cell line SV40T-YREC-hTERT. The preservation date is: 2021, 10 months and 14 days, the preservation unit is: china center for type culture collection, with the preservation addresses: university of martial arts in chinese.
Further, the yak is an adult yak.
A method for constructing an immortalized yak rumen epithelial cell line, comprising the following steps:
s1, sample pretreatment: collecting rumen epithelial tissues of yaks, cleaning, shearing rumen papillae, adding Hanks solution containing antibiotics, and cleaning;
s2, enzyme digestion and culture: adding type I collagenase into cleaned rumen papilla, oscillating at 37deg.C for 30min, placing the rumen papilla digested with the enzyme in modified DMEMF/12 cell complete culture medium for 4-8min, spreading the rumen papilla with small amount of culture medium in rat tail collagen coated culture dish, dispersing the tissue block at intervals, inverting the culture dish at 37deg.C, and 5% CO 2 Culturing in an incubator until the proliferation of cells climbing out around the rumen papilla is fused to 50%, removing tissue blocks and cleaning the cells; wherein the modified complete medium of the DMEMF/12 cells is the complete medium of the DMEMF/12 cells, and further comprises 200U/ml penicillin, 0.2mg/ml streptomycin, 100 mug/ml gentamicin and 10% bovine serum;
s3, purifying: adding EDTA solution of trypsin into the cells washed in the step S2, and placing at 37deg.C and 5% CO 2 After digesting the treated cells in the incubator for 2min, stopping digestion, and cleaning to obtain purified yak rumen epithelial cells;
s4, virus infection: placing purified yak rumen epithelial cells in a cell complete culture medium to grow until 70% of cells are fused, adding slow virus infected cells carrying SV40T and hTERT genes overnight, continuously culturing for 4d, adding purine toxins, continuously culturing for 96h, and screening cell lines; wherein, the cell complete culture medium is: RPMI-1640+10% FBS+10ng/mL EGF+2ng/mL bFGF+2ng/mL IGF-1+0.5ug/mL hydrocortisone+1% penicillin-streptomycin;
s5, performing expansion culture and passage: and (3) performing expansion culture on the screened cell line until the cell line grows and covers a culture bottle by 90%, adding an EDTA solution of trypsin to digest the cells until the cells retract and become round, and stopping digestion to obtain primary yak rumen epithelial cells, wherein the primary cells are passaged to more than 20 generations to obtain the immortalized yak rumen epithelial cell line.
Further, the antibiotics in step S1 are penicillin, streptomycin, gentamicin and amphotericin B.
Further, in the step S2, the concentration of the type I collagenase is 0.1%, and the volume ratio of the type I collagenase to the rumen papilla is 5:1.
Further, the content of trypsin in the EDTA solution of trypsin in step S3 and step S5 was 0.25%, and the content of EDTA was 0.02%.
Further, the lentivirus in step S4 also contains 5ug/mL polybrene for enhancing lentivirus infection efficiency.
Further, the retroviral vector of SV40T in step S4 is pGMLV-SV40T-PURO, and the retroviral vector of hTERT is mammalian gene expression lentiviral vector pLV [ Exp ] -PURO-EF1A > hTERT.
Further, the immortalized yak rumen epithelial cells in the step S5 are stored in a frozen manner, the frozen solution is a mixed solution of a complete culture medium, FBS and DMSO, and the volume ratio is as follows: complete medium FBS, DMSO=7:2:1, wherein the complete medium is RPMI-1640+10% FBS+10ng/mL EGF+2ng/mL bFGF+2ng/mL IGF-1+0.5ug/mL hydrocortisone+1% penicillin-streptomycin.
The invention has the following advantages:
(1) The invention uses I type collagen to digest the rumen epithelial nipple of the yak and then combines the tissue block adherence method to successfully separate the rumen epithelial cells of the yak, compared with the tissue block culture method, the culture period is reduced, the cell proliferation form is good, the cell activity is stronger, the cost is saved by the enzyme digestion tissue block culture method, the test time is reduced, the success rate of primary cell culture is improved, the yak rumen epithelial cells with stable growth are obtained by the method, and the method for separating and culturing the rumen epithelial cells of the yak is enriched;
(2) According to the invention, the slow virus carrying SV40T and hTERT genes is used for infecting primary yak rumen epithelial cells, and an immortalized yak rumen epithelial cell line is obtained after screening and culturing the purine toxins;
(3) The immortalized yak rumen epithelial cell line constructed by the invention can be stably passaged, and has no aging state, and the cell morphology accords with the primary rumen epithelial cell morphology;
(4) The invention improves the success rate of the test and increases the success rate of the conversion of primary cells into cell lines by optimizing the components of the complete culture medium in the process of infecting cells by slow viruses, and the culture medium is more suitable for the growth of immortalized yak rumen epithelial cell lines, has higher proliferation speed and better cell state, can obtain more cells in a shorter time, contains sufficient nutrient substances, and meets the substances and energy required by biochemical reactions such as synthesis of new cells, cell metabolism and the like.
(5) The cell line established by the method solves the defects of difficult culture and limited passage times of the primary rumen epithelial cells, enriches the resources of the current yak source cell line, can keep the morphological characteristics and normal functions of the primary rumen epithelial cells, and provides a useful in vitro cell model for application and research of the nutrition digestion, absorption and metabolism of the rumen epithelium, the cell signal path mechanism and the like.
Drawings
FIG. 1 is a diagram showing the isolation and culture process of the rumen epithelial cells of yaks according to the present invention, wherein A: rumen epithelial tissue used to culture primary cells; b: rumen papilla cut from rumen epithelial tissue; c: the cleaned rumen papilla; d:0.1% type i collagenase digests the rumen papilla; e: after digestion, the rumen papilla tissue blocks were plated on 10cm dishes.
FIG. 2 is a graph showing morphological characteristics of yak rumen epithelial cells cultured by the combination of enzyme digestion and tissue mass culture method of the present invention; in the figure, a: morphology of epithelial cells climbing around the tissue mass (100 x); b: the epithelial cells were observed to be in the form of typical cobblestone-like epithelial cells (200X).
FIG. 3 is a diagram of SV40T and hTERT lentiviral vectors, where A: SV40T; b: hTERT.
FIG. 4 is a morphological characterization of an immortalized yak rumen epithelial cell line following expanded culture by purine toxin screening according to the invention; in the figure, a: immortalizing the rumen epithelial cells of the 2 nd generation yak; b: immortalized 15 th generation yak rumen epithelial cells.
FIG. 5 is a graph of immunofluorescence identification of primary yak rumen epithelial cells and immortalized yak rumen epithelial cell line keratin 18 of the invention, wherein A: primary yak rumen epithelial cells; b: immortalized yak rumen epithelial cell line.
FIG. 6 is a graph of the growth curve of the generation 10 immortalized yak rumen epithelial cell line according to the present invention.
FIG. 7 is a graph showing qRT-PCR identification of SV40T and hTERT genes in primary yak rumen epithelial cells of generation 1 and immortalized yak rumen epithelial cell lines of generation 10.
FIG. 8 is a diagram of the detection of the third generation primary yak rumen epithelial cells and the 30 th generation immortalized yak rumen epithelial cells of the invention by using the cell senescence beta-galactosidase staining kit, wherein A: a 3 rd generation primary yak rumen epithelial cell; b: generation 30 immortalized yak rumen epithelial cell line.
FIG. 9 is a graph of chromosome karyotype analysis of a 20 th generation immortalized yak rumen epithelial cell line according to the present invention.
Detailed Description
The invention will be further described with reference to the accompanying drawings and examples, to which the scope of the invention is not limited:
the main reagents used in the following experiments were: RPMI-1640 medium, DMEM/F12 medium, fetal bovine serum, 0.25% trypsin-0.02% EDTA (Gibco); EGF, bFGF, IGF-1 (RD Co.); collagenase type i (MP); puromycin, polybrene, hydrocortisone Hydrocortisone (Sigma); SV40T and hTERT lentiviral packages (Yun Zhou organisms); penicillin, streptomycin, gentamicin, amphotericin B, hank's, PBS, colchicine, giemsa, methanol, rat tail collagen (Solarbio corporation); CCK-8 (AbMobile Co.); the Cytokeratin 18 antibody was purchased from Santa cruz company; triton-100, 4% paraformaldehyde, goat serum, FITC-labeled goat anti-mouse IgG (H+L), CY 3-labeled goat anti-mouse IgG (H+L), DAPI staining solution, and cell aging beta-galactosidaseStaining kit (Beyotime corporation); molPureCell RNAKit cultured cell RNA extraction kit and Hifair TM II1st Strand cDNA Synthesis Super Mix for qPCR reverse transcription kit, hieffUNICONUniversal Blue qPCR SYBR Green Master Mix kit (YESEN Co.). The other reagents are all laboratory routine reagents.
Example 1: a method for constructing an immortalized yak rumen epithelial cell line, comprising the following steps:
s1, sample pretreatment: collecting rumen epithelial tissue of healthy adult yaks in a slaughterhouse of Sichuan province, placing a self-sealing bag containing Hanks solution of antibiotics (200U/ml penicillin, 0.2mg/ml streptomycin, 100 mug/ml gentamicin and 1 mug/ml amphotericin B) on ice for carrying back to a laboratory, shearing rumen papillae in a sterile super clean bench, adding Hanks solution of antibiotics (200U/ml penicillin, 0.2mg/ml streptomycin, 100 mug/ml gentamicin and 1 mug/ml amphotericin B) for cleaning for 4-5 times, and 3-5 minutes each time;
s2, enzyme digestion and culture: 0.1% type I collagenase was added in a volume ratio of 5:1 adding the rumen papilla into a 250ml glass bottle filled with rumen papilla, placing the glass bottle into a constant temperature air bath oscillation box at 37 ℃ for 30min, discarding supernatant, cleaning the glass bottle once by using Hanks solution, and placing the rumen papilla into a modified DMEMF/12 complete cell culture medium for 5min, wherein the modified DMEMF/12 complete cell culture medium is the complete cell culture medium of the DMEMF/12, and further comprises 200U/ml penicillin, 0.2mg/ml streptomycin, 100 mug/ml gentamicin and 10% fetal bovine serum;
evenly spreading the digested rumen headband with a small amount of culture medium at 1ug/cm 2 The rat tail collagen coated 10cm culture dish, the tissue blocks were dispersed at 5mm intervals, and the inverted culture dish was placed at 37℃with 5% CO 2 After about 3-4 hours in the incubator, observing whether the water content of the tissue blocks is evaporated and reduced to enable the tissue blocks to tightly adhere to the bottom, if the water content is more and the inversion time is prolonged, then taking out the culture dish, slowly adding 5mL of modified DMEMF/12 complete culture medium into the bottom of the culture dish, covering each tissue small block with the culture medium, keeping the culture dish still as much as possible, and supplementing the modified DMEMF/12 complete culture medium to 10mL after 24 hours of culture, wherein each culture dish is replaced by one culture mediumChanging the primary liquid for five days; observing morphological change of cells, removing tissue blocks when proliferation of cells climbing out around the digested rumen papilla is fused to 50%, and washing the cells by PBS;
s3, purifying: adding 2mL of 0.25% trypsin-0.02% EDTA into the washed cells in step S2, slightly shaking the culture dish to spread the digestion solution over the bottom of the dish, and placing into 37 ℃ and 5% CO 2 Digesting the treated cells in the incubator for 2min, beating the side wall of the cell culture dish for a few minutes, rapidly adding an improved DMEMF/12 complete medium to stop digestion, beating the shaking culture dish to remove fibroblasts as much as possible, washing the cells twice by PBS, and after purifying the cells, adding the improved DMEMF/12 complete medium to continue culturing to obtain purified yak rumen epithelial cells;
s4, virus infection: placing purified yak rumen epithelial cells in a cell complete culture medium to grow to 70% of cells for fusion, adding slow virus MOI=80 (containing 5ug/mL polybrene to enhance slow virus infection efficiency) carrying SV40T and hTERT genes to infect the cells overnight, changing liquid every 2 days, continuously culturing for 4 days, observing the surviving cell state in the whole process, adding 1ug/mL purine toxin, continuously culturing for 96 hours, screening cell lines, expanding and culturing the surviving cell lines, changing liquid and passaging until the cell morphology is gradually stable; wherein, the cell complete culture medium is: RPMI-1640+10% FBS+10ng/mL EGF+2ng/mLbFGF+2ng/mL IGF-1+0.5ug/mL hydrocortisone+1% penicillin-streptomycin;
the retroviral vector of SV40T is pGMLV-SV40T-PURO, and the retroviral vector of hTERT is a mammalian gene expression lentiviral vector pLV [ Exp ] -PURO-EF1A > hTERT;
s5, performing expansion culture and passage: expanding and culturing the screened cell line until the cell line covers a culture bottle by 90%, washing the cell by using a PBS solution, digesting the cell by using 0.25% trypsin-0.02% EDTA, lightly blowing the cell to fall off when the cell is retracted and rounded by using an inverted microscope, adding a complete cell culture medium to stop digestion, wherein the digestion time is about 5min, and centrifuging at 1200rpm/min for 5min, so as to carry out passage or freezing, wherein the freezing solution is a mixed solution of the complete cell culture medium, FBS and DMSO, and the volume ratio is as follows: complete medium comprises FBS, DMSO=7:2:1, wherein the complete medium comprises RPMI-1640+10% FBS+10ng/mL EGF+2ng/mL bFGF+2ng/mL IGF-1+0.5ug/mL hydrocortisone+1% penicillin-streptomycin; and (5) carrying out passage on the cells to more than 20 generations to obtain the immortalized yak rumen epithelial cell line.
The separation and culture process of the yak rumen epithelial cells is shown in figure 1, and morphological characteristics of the yak rumen epithelial cells cultured by the combination of enzyme digestion and tissue mass culture are shown in figure 2; SV40T and hTERT lentiviral vectors are shown in FIG. 3; morphological features of the immortalized yak rumen epithelial cell line after expanded culture with the purine toxins screen are shown in fig. 4.
Example 2: identification of first generation primary yak rumen epithelial cells and 5 generation immortalized yak rumen epithelial cell lines by using keratin Cytokeratin 18 immunofluorescence
Rumen epithelial cells were 1X 10 6 The individual cells/mL are inoculated in a 6-hole plate, the growth condition of the cells is observed after the cells are stably attached, when the cells are uniformly grown, gaps are reserved among the cells, the cells are not connected into slices, a culture dish is taken out, precooled PBS buffer solution is added, and the culture dish is put on a shaking table for cleaning for 3 times for 10min each time. Adding pre-cooled 4% paraformaldehyde fixing solution for 15min, removing the fixing solution, adding pre-cooled PBS, and cleaning on a shaker for 3 times each for 10min. Cells were treated with pre-chilled 0.1% Triton X100 for 10min, dialysates removed, pre-chilled PBS was added and washed 3 times for 10min each time on a shaker. 5% BSA was added and blocked at room temperature for 30min. The blocking solution was removed and diluted 500. Mu.L of primary anti-CK 18 antibody (1:50) was added to each well overnight at 4 ℃. Removing primary antibody, adding pre-cooled PBS, and washing on a shaker for 10min 3 times each time. Adding diluted fluorescent secondary antibody (1:200) in dark place, shaking at 37 ℃ for 1h, removing secondary antibody, adding precooled PBS, and cleaning on a shaking table for 3 times for 10min each time. Adding diluted DAPI to completely cover cells, keeping away from light for 5min, removing staining solution, adding precooled PBS, and cleaning on a shaker for 3 times each for 10min. The results of the observation under a fluorescence inversion microscope and photographing are shown in FIG. 5.
As can be seen from fig. 5: the keratin 18 of all cells showed fluorescence, the cell nucleus was stained blue, the protein was expressed, the cell purity was high, indicating that both the primary cells and the cell lines were epithelial cell-like cells.
Example 3: cell proliferation growth curve
Taking 10 th generation of yak rumen epithelial cells with good growth state, digesting the cells to prepare cell suspension, and mixing the cells at a ratio of 5×10 3 The density of each mL is inoculated in a 96-well plate for culture, 6 replicates are arranged in each plate, 10 plates are inoculated at 37 ℃ and 5% CO 2 Culturing in a saturated humidity cell incubator. Only one plate was removed daily from day 2 and 10% CCK8 solution was added to each well and after 2 hours of standing, the OD at 450nm was measured with a microplate reader. The growth curve of the cells was plotted on the abscissa and the average OD value on the ordinate, as shown in fig. 6. The result shows that the cell proliferation and growth curve presents an S shape, and accords with the growth rule of epithelial cells.
Example 4: expression of first-generation primary yak rumen epithelial cells and first-generation immortalized yak rumen epithelial cell lines
Taking first-generation primary yak rumen epithelial cells and 10-generation immortalized yak rumen epithelial cell lines to obtain 1×10 cells 6 Inoculating each cell/mL into a 6-well plate, setting three repetitions, after growing the cells for 24 hours, washing the cells twice with PBS, and extracting total RNA of the cells by using a MolPureCell RNAKit culture cell RNA extraction kit according to the operation instructions of the kit, wherein Hifair is a kit TM II1st Strand cDNA Synthesis Super Mix for qPCR reverse transcription was performed to reverse transcribe not less than 1. Mu.g of total cellular RNA in a reaction volume of 10. Mu.l. Hieff
Universal Blue qPCR SYBR Green Master Mix kit RT-PCR was performed. The sequences of the primers used for PCR in the test are shown in Table 1, and the primers were synthesized by Shanghai.
Table 1: primer sequence information
The qRT-PCR identification of SV40T and hTERT genes shows the expression patterns of the 1st generation primary yak rumen epithelial cells and the 10 th generation immortalized yak rumen epithelial cell lines in FIG. 7, and the results show that the immortalized cell lines stably and highly express the SV40T and hTERT genes, and further demonstrate that the success of the lentivirus infection of the primary cells.
Example 5: beta-galactosidase staining experiments
Inoculating the 3 rd generation primary yak rumen epithelial cells and the 30 th generation immortalized yak rumen epithelial cell lines into a 6-hole plate, observing the growth condition of cells after the cells are stably attached, and detecting according to the operation instruction of the cell aging beta-galactosidase staining kit when the growth density reaches 90%. The detection results are shown in fig. 8: primary cells are transmitted to the 3 rd generation, the cell state is abnormal, the cell senescence morphology is irregular, the senescence cells are usually large in volume, and cells expressing beta-galactosidase which turn blue are easily observed under an optical microscope. The absence of a distinct dark blue color observed in the immortalized yak rumen epithelial cell line indicates that the cell line does not senesce, and the cell line is stably passaged, has high cell activity and stable proliferation state.
Example 6: chromosome nuclear analysis of generation 20 immortalized yak rumen epithelial cell line
Generation 20 immortalized yak rumen epithelial cell line at 1×10 6 Inoculating individual cells/mL into a culture flask, culturing for 72h, adding colchicine into the culture flask when culturing for 68h, and culturing in an incubator for 2-4h until the final concentration of colchicine in the culture flask reaches 0.1-0.2ug/mL, so that cell division is stopped in the metaphase. The following treatments were then carried out:
collecting cells: the culture was blown and mixed evenly and transferred to a 10ml glass centrifuge tube and centrifuged at 1800rpm for 6min.
Hypotonic treatment: the supernatant was discarded, 8ml of 0.075mol/L potassium chloride pre-warmed at 37℃was added, and the mixture was gently stirred with a pipette and subjected to hypotonic treatment at 37℃for 20min.
Pre-fixing: 1ml of freshly prepared fixative (methanol: glacial acetic acid=3:1) was added, gently mixed and centrifuged at 1800rpm for 6min.
Fixing: the supernatant was discarded, 8ml of the fresh fixative was added, gently mixed, left to stand at room temperature for 20min, and centrifuged at 1800rpm for 6min. The supernatant was discarded, and the fixation was repeated once, and centrifuged at 1800rpm for 6min. Discarding the supernatant, adding a proper amount of fresh fixing solution according to the amount of sediment, and lightly mixing to prepare the frosted suspension.
And (3) tabletting: taking 1-2 drops of the suspension, dripping the suspension onto a clean glass slide stained with ice water or dried, blowing off, passing fire, and air drying. The sample is placed in a 37 ℃ incubator for 3-4 days or baked for 2-3 hours at 70 ℃ for aging treatment so as to prepare the banding analysis.
Dyeing: the Giemsa stock solution (9:1) is diluted by phosphate buffer with pH of 6.8, 1 piece of the prepared chromosome specimen is taken for dyeing for 15-20 min, washed by water and dried by air.
And (3) observation: selecting metaphase with good chromosome dispersion under a low power microscope, and then converting the metaphase into an oil microscope for observation. If the prepared slide specimen needs to be preserved for a long time, the slide specimen can be sealed by a sealing agent to isolate the slide specimen from external contact.
Photographing: placing the prepared sample sheet under an objective lens, and adjusting the microscope; observing with a low-power mirror to find out metaphase of cell division, selecting metaphase with good dispersion, amplifying and counting.
Chromosome number statistics: a number of well-dispersed dividing cells can be used for counting.
Recording and measuring and calculating morphological characteristics.
Chromosome karyotype analysis system: video TesT.Karyo chromosome karyotype analysis System.
The results are shown in fig. 9, and as can be seen from fig. 9: the immortalized yak rumen epithelial cell line shows diploid characteristics of yak species, is not mutated into polyploid, and the chromosome number of cells after immortalization is obviously increased, which is consistent with the condition that primary cells infected by slow viruses are converted into immortalized cells, and belongs to the normal phenomenon.
Comparative example 1:
cutting and cleaning rumen papilla, directly placing the rumen papilla into a cell complete culture medium for 5min, uniformly spreading small tissue blocks with a small amount of culture medium in a 10cm culture dish, dispersing the tissue blocks at intervals of 5mm, placing an inverted culture bottle in an incubator for about 3-4h, observing whether the water content of the tissue blocks is evaporated and reduced to enable the tissue blocks to tightly adhere to the bottom, if the water content is more, prolonging the inversion time, then taking out the culture bottle, slowly adding 2mL of cell complete culture medium on the side surface of the bottom of the culture bottle, carefully placing the culture bottle vertically, keeping the culture bottle still as much as possible, supplementing 10mL of culture medium after culturing for 24h, and changing liquid every five days. Morphological changes of the cells were observed. The time for climbing out the epithelial cells around the rumen epithelial tissue of the yak by the tissue block culture method at least needs more than two weeks, the cell density in a culture dish can reach 80% approximately in one month, and the cell density for climbing out around the tissue block can reach 80% by combining the enzyme digestion with the tissue block culture method for two weeks.
Comparative example 2:
when primary yak rumen epithelial cells are infected only by lentiviruses carrying SV40T virus genes, the success rate of cell transformation into an immortalized cell line is very low, the primary cells die, the immortalized cell line with stable passage cannot be selected, and the cell morphology is completely different from the epithelial cell morphology, so that the cell line establishment fails. Meanwhile, two slow viruses carrying SV40T and hTERT genes are used for infecting primary yak rumen epithelial cells, the success rate of cell transformation into an immortalized cell line is high, the cell line is continuously passaged in vitro, the cell line is vigorous, and the proliferation speed of the cell line is high.
Comparative example 3:
cells were not stable in growth when cultured using only the complete medium DMEMF/12 (200U/mL penicillin, 0.2mg/mL streptomycin, 100. Mu.g/mL gentamicin, 10% fetal bovine serum) during transformation of immortalized cell lines with lentiviruses, the complete medium was optimized for normal growth, high cell proliferation rate, high cell viability, stable passage to maintain primary yak rumen epithelial cell morphology using RPMI-1640+10% FBS+10ng/mL EGF+2ng/mL bFGF+2ng/mL IGF-1+0.5ug/mL hydrocortisone.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art who is skilled in the art to which the present invention pertains will appreciate that the technical scheme and the inventive concept according to the present invention are equally substituted or changed within the scope of the present invention.
SEQUENCE LISTING
<110> Sichuan university of agriculture
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Claims (6)
1. The construction method of the immortalized yak rumen epithelial cell line is characterized by comprising the following steps of:
s1, sample pretreatment: collecting rumen epithelial tissue of adult yaks, cleaning, shearing rumen papilla, adding Hanks solution containing antibiotics, and cleaning;
s2, enzyme digestion and culture: adding type I collagenase into cleaned rumen papilla, oscillating at 37deg.C for 30min, placing the rumen papilla digested with the enzyme in modified DMEMF/12 cell complete culture medium for 4-8min, spreading the rumen papilla with small amount of culture medium in rat tail collagen coated culture dish, dispersing the tissue block at intervals, inverting the culture dish at 37deg.C, and 5% CO 2 Culturing in an incubator until the proliferation of cells climbing out around the rumen papilla is fused to 50%, removing tissue blocks and cleaning the cells; wherein the modified complete medium of the DMEMF/12 cells is the complete medium of the DMEMF/12 cells, and further comprises 200U/ml penicillin, 0.2mg/ml streptomycin, 100 mug/ml gentamicin and 10% bovine serum; wherein the concentration of the type I collagenase is 0.1%, and the volume ratio of the type I collagenase to the rumen papilla is 5:1;
s3, purifying: adding EDTA solution of trypsin into the cells washed in the step S2, and placing at 37deg.C and 5% CO 2 After digesting the treated cells in the incubator for 2min, stopping digestion, and cleaning to obtain purified yak rumen epithelial cells;
s4, virus infection: placing purified yak rumen epithelial cells in a cell complete culture medium to grow until 70% of cells are fused, adding slow virus infected cells carrying SV40T and hTERT genes overnight, continuously culturing for 4d, adding purine toxins, continuously culturing for 96h, and screening cell lines; wherein, the cell complete culture medium is: RPMI-1640+10% FBS+10ng/mL EGF+2ng/mL bFGF+2ng/mL IGF-1+0.5ug/mL hydrocortisone+1% penicillin-streptomycin;
s5, performing expansion culture and passage: and (3) performing expansion culture on the screened cell line until the cell line grows and covers a culture bottle by 90%, adding an EDTA solution of trypsin to digest the cells until the cells retract and become round, and stopping digestion to obtain primary yak rumen epithelial cells, wherein the primary cells are passaged to more than 20 generations to obtain the immortalized yak rumen epithelial cell line.
2. The method for constructing an immortalized yak rumen epithelial cell line according to claim 1, wherein the antibiotics in step S1 are penicillin, streptomycin, gentamicin and amphotericin B.
3. The method for constructing an immortalized yak rumen epithelial cell line according to claim 1, wherein the trypsin in the EDTA solution in step S3 and step S5 has a trypsin content of 0.25% and EDTA content of 0.02%.
4. The method of claim 1, wherein in step S4, the lentivirus further comprises 5ug/mL of polybrene for enhancing the efficiency of lentivirus infection.
5. The method according to claim 1, wherein in the step S4, the SV40T retrovirus vector is pGMLV-SV40T-PURO, and the hTERT retrovirus vector is a mammalian gene expression lentivirus vector pLV [ Exp ] -PURO-EF1A > hTERT.
6. The method for constructing an immortalized yak rumen epithelial cell line according to claim 1, wherein the immortalized yak rumen epithelial cell line in step S5 is stored in a frozen state, the frozen solution is a mixed solution of complete medium, FBS and DMSO, and the volume ratio is: complete medium FBS, DMSO=7:2:1, wherein the complete medium is RPMI-1640+10% FBS+10ng/mL EGF+2ng/mL bFGF+2ng/mL IGF-1+0.5ug/mL hydrocortisone+1% penicillin-streptomycin.
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