CN114058592A - 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 cell line has a preservation number of: CCTCC No. c2021245, categorically named: the immortalized yak rumen epithelial cell line SV 40T-YREC-hTERT. The invention relates to a method for preparing immortalized yak rumen epithelial cell line, which comprises the steps of carrying out adherent culture on a tissue digested by collagenase I on primary yak rumen epithelial papilla to obtain primary rumen epithelial cells, using the cells as host cells, infecting the cells by using lentiviruses carrying SV40T and hTERT genes, and obtaining the immortalized yak rumen epithelial cell line after screening and expanding culture, wherein the establishment of the cell line solves the defects that the primary rumen epithelial cells are difficult to culture and have limited passage times, and enriches yak source cell line resources; the rumen epithelial cell culture medium can be stably passaged, can keep the morphological characteristics and normal functions of primary rumen epithelial cells, and provides a useful in-vitro cell model for researching nutrient digestion, absorption and metabolism of rumen epithelial cells, cell signal pathway mechanisms 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 yak is a special cattle species which can adapt to the special and harsh natural environment of the Qinghai-Tibet plateau in the world, is an important life and production data on which local herdsmen live, and is also a main supporting industry for poached and enriched. China is a world-wide yak breeding big country, accounts for more than 92% of the total yak breeding amount in the world, and is mainly distributed in Tibet, Qinghai, Sichuan, Gansu, Yunnan, Xinjiang and other places in China. The method relates to basic research and application basic research of genetic breeding, nutrition and feed, epidemic disease prevention and control and the like of yaks, and China is at the leading level in the world. So far, the research on rumen at home and abroad is more and more, and the regulation mechanism in rumen epithelial cells is also concerned. The biggest characteristic of ruminants that are physiologically structurally different from monogastric animals is that ruminants have a regurgitation structure, where the rumen is most important. Rumen epithelial tissue is an important barrier between the environment in rumen and the environment of host organism, rumen epithelial tissue is the main part of rumen, a large number of epithelial papilla extend into the inner cavity from the surface of rumen, the surface area for absorbing volatile fatty acid and electrolyte is greatly increased, and nutrient substances absorbed by the rumen epithelial tissue are indispensable for the energy metabolism of the whole animal. The rumen metabolism of yaks, which are a dominant stock species living in the special regional environment of Qinghai-Tibet plateau throughout the year, has many specificities compared with other ruminants. However, in the current research, the research on the rumen epithelial cells of yaks is less, and a stable source rumen epithelial cell line is not established yet, so that the research on the rumen epithelial cells of yaks is restricted, and the technology shortage in the related field influences the development of more molecular biology fields. At present, primary cells or cell lines from yaks are deficient, and cell lines from more yaks are obtained to enrich cell molecule regulation mechanisms related to yaks.
Most normal cells are considered to have only a limited ability to divide and enter a senescent state after failing to divide. The cells are still viable at this point, but the expression profile of the genes and proteins of the cells is greatly altered. Senescent cells are unable to re-induce cell division under some conventional stimuli and the cell cycle distribution of senescent cells is also distinctive, unlike some injury-induced cell dormancy, and unlike the case of contact inhibition of cell growth, senescent cells generally become voluminous. Researches show that the primary yak rumen epithelial cells can not be subjected to long-term passage storage even in vitro culture, and the increase of the passage times has serious damage effect on the form and physiological functions of the primary cells. Meanwhile, the limited number of isolated tissues severely limits the molecular expression mechanism of exogenous nutrients on the cellular level.
Chinese patent publication No. 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 collected rumen epithelial tissues of dairy cows in a culture medium, cleaning and digesting the tissues, and then carrying out primary culture to obtain rumen epithelial cells; step b) incubating the rumen epithelial cells obtained in the step a) with virus liquid 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 cow rumen physiological regulation and nutrition absorption mechanism research, the culture method is simple and the growth speed is high, BRECs which have physiological functions and can be continuously passaged can be obtained by the construction method, but due to the obvious difference of cow and yak species, the immortalized yak rumen epithelial cell line can not be cultured by the method. Through retrieval, no report is found about the establishment of the immortalized yak rumen epithelial cell line. Therefore, an immortalized yak rumen epithelial cell line capable of being stably passaged needs to be established urgently, 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 function and can maintain the original characteristics of yak rumen epithelial cells;
the invention also aims to provide a method for constructing an immortalized yak rumen epithelial cell line, which has simple operation method and high immortalization success rate.
The purpose of the invention is realized by the following technical scheme: an immortalized yak rumen epithelial cell line, wherein the preservation number of the immortalized yak rumen epithelial cell line is as follows: CCTCC No. c2021245, categorically named: the immortalized yak rumen epithelial cell line SV 40T-YREC-hTERT. The preservation date is as follows: at 14/10/2021, the preservation unit is: china center for type culture Collection, the collection addresses are: wuhan university in Wuhan, China.
Furthermore, the yak is an adult yak.
A construction method of an immortalized yak rumen epithelial cell line comprises the following steps:
s1, sample pretreatment: collecting rumen epithelial tissue of yaks, cleaning, cutting rumen papilla, adding Hanks solution containing antibiotics, and cleaning;
s2, enzyme digestion and culture: adding collagenase type I into cleaned rumen papilla, oscillating at 37 deg.C in constant temperature air bath for 30min, placing enzyme-digested rumen papilla in improved DMEMF/12 cell complete culture medium for 4-8min, uniformly spreading digested rumen papilla with small amount of culture medium in culture dish coated with collagen protein from rat tail, dispersing tissue blocks at intervals, inverting culture dish at 37 deg.C and 5% CO for 30min2Culturing in an incubator until the cell climbing out around the rumen papilla is proliferated and fused to 50%, removing tissue blocks and cleaning cells; wherein the improved DMEMF/12 cell complete culture medium is that the DMEMF/12 cell complete culture medium also contains 200U/ml penicillin, 0.2mg/ml streptomycin, 100 mu g/ml gentamicin and 10% fetal calf serum;
s3, purification: adding EDTA solution of trypsin into the cells washed in step S2, placing at 37 deg.C and 5% CO2Digesting the cells in an incubator for 2min, terminating digestion, and cleaning to obtain purified cellsYak rumen epithelial cells;
s4, virus infection: placing the purified yak rumen epithelial cells in a cell complete culture medium to grow until 70% of the cells are fused, adding lentivirus carrying SV40T and hTERT genes to infect the cells overnight, continuously culturing for 4 days, adding purine toxin, continuously culturing for 96 hours, and screening cell lines; wherein, the complete cell 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, expanding culture and passage: and (3) expanding and culturing the screened cell line until the cell line grows to cover 90% of a culture bottle, adding EDTA (ethylene diamine tetraacetic acid) solution of trypsin to digest the cells, stopping digestion until the cells retract and become round, obtaining primary yak rumen epithelial cells, and passaging the primary cells to more than 20 generations to obtain the immortalized yak rumen epithelial cell line.
Further, the antibiotics described in step S1 are penicillin, streptomycin, gentamicin, and amphotericin B.
Further, in step S2, the collagenase type I is present at a concentration of 0.1%, and the volume ratio of the collagenase type I to the rumen papilla is 5: 1.
Further, the content of trypsin in the EDTA solution of trypsin in steps S3 and S5 is 0.25%, and the content of EDTA is 0.02%.
Further, the lentivirus described in step S4 further contains polybrene at 5ug/mL for enhancing the infection efficiency of lentivirus.
Further, the retroviral vector of SV40T in step S4 is pGMLV-SV40T-PURO, and the retroviral vector of hTERT is a mammalian gene expression lentiviral vector pLV [ Exp ] -PURO-EF1A > hTERT.
Further, the immortalized yak rumen epithelial cells in step S5 are preserved in a frozen manner, wherein the frozen solution is a mixed solution of a complete culture medium, FBS and DMSO, and the volume ratio is: the 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.
The invention has the following advantages:
(1) according to the method, the yak rumen epithelial cells are successfully separated by combining a tissue block adherence method after the yak rumen epithelial papilla is digested by the collagen I, compared with a tissue block culture method, the culture period is shortened, the cell proliferation form is good, the cell activity is strong, the cost is saved by an enzyme digestion tissue block culture method, the test time is shortened, 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 yak rumen epithelial cells is enriched;
(2) according to the invention, lentiviruses carrying SV40T and hTERT genes are used for infecting primary yak rumen epithelial cells, and an immortalized yak rumen epithelial cell line is obtained after screening and culturing purine toxins;
(3) the immortalized yak rumen epithelial cell line constructed by the invention can be stably passaged without an 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 primary cells converted into the cell line by optimizing the components of the complete culture medium in the process of infecting the cells by the slow virus, and the culture medium is more suitable for the growth of the rumen epithelial cell line of the immortalized yak, has higher proliferation speed of the cell line and better cell state, can obtain more cells in a shorter time, contains sufficient nutrient substances and meets the requirements of the synthesis of new cells, the cell metabolism and other biochemical reactions.
(5) The cell line established by the method of the invention solves the defects of difficult culture and limited passage times of primary rumen epithelial cells, enriches the resources of the existing yak source cell line, and the immortalized yak rumen epithelial cell line can maintain the morphological characteristics and normal functions of the primary rumen epithelial cells through stable passage, thereby providing a useful in vitro cell model for application and research of nutrient digestion, absorption and metabolism of the rumen epithelial cells, cell signal access mechanisms and the like.
Drawings
Fig. 1 is a diagram of a separation and culture process of rumen epithelial cells of yaks, wherein A: culturing rumen epithelial tissue for primary cells; b: rumen papilla harvested from rumen epithelial tissue; c: a washed rumen papilla; d: 0.1% collagenase type i digests rumen papillae; e: after digestion, the tumor stomach papilla tissue block is spread in a 10cm culture dish.
FIG. 2 is a diagram of morphological characteristics of rumen epithelial cells of yaks cultured by enzyme digestion combined with a tissue block culture method; in the figure, A: epithelial cell morphology features (100x) crawled out around the tissue mass; b: the epithelial cells were observed to assume a typical cobblestone paving stone-like epithelial cell morphology (200X).
FIG. 3 is a diagram of SV40T and hTERT lentiviral vectors, in which A: SV 40T; b: hTERT.
FIG. 4 is a morphological feature diagram of immortalized yak rumen epithelial cell line after screening and expanded culture of puroxin of the invention; in the figure, A: immortalizing rumen epithelial cells of 2 nd generation yaks; b: immortalizing the rumen epithelial cells of the 15 th generation yaks.
Fig. 5 is an immunofluorescence identification chart of keratin 18 of primary yak rumen epithelial cells and immortalized yak rumen epithelial cell lines, wherein A: primary yak rumen epithelial cells; b: immortalized yak rumen epithelial cell line.
FIG. 6 is a graph of the growth curve determination of 10 th generation immortalized yak rumen epithelial cell line.
FIG. 7 is a qRT-PCR identification expression diagram of SV40T and hTERT genes in 1st generation primary yak rumen epithelial cells and 10 th generation immortalized yak rumen epithelial cell lines.
Fig. 8 is a diagram of a cell senescence beta-galactosidase staining kit detecting rumen epithelial cells of primary yaks of 3 rd generation and immortalized yaks of 30 th generation of the present invention, wherein, a: 3 rd generation primary yak rumen epithelial cells; b: and (3) immortalizing a yak rumen epithelial cell line at the 30 th generation.
FIG. 9 is a chromosome karyotype analysis chart of the 20 th generation immortalized yak rumen epithelial cell line of the present invention.
Detailed Description
The invention is further described with reference to the following figures and examples, without limiting the scope of the invention to the following:
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 company); puromycin, Polybrene, hydrocortisone (Sigma); SV40T and hTERT lentiviral packaging (nephela organisms); penicillin, streptomycin, gentamicin, amphotericin B, Hank's, PBS, colchicine, Giemsa stain, methanol, rat tail collagen (Solarbio corporation); CCK-8(AbMole Corp.); keratin Cytokeratin 18 antibody was purchased from Santa cruz; 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, cell senescence β -galactosidase staining kit (Beyotime corporation); MolPureCelRNAKit culture cell RNA extraction kit and HifairTMII1st Strand cDNA Synthesis Super Mix for qPCR reverse transcription kit, HieffUNICON Universal Blue qPCR SYBR Green Master Mix kit (YESEN Co.). The other reagents are laboratory conventional reagents.
Example 1: a construction method of an immortalized yak rumen epithelial cell line comprises the following steps:
s1, sample pretreatment: collecting rumen epithelial tissues of healthy adult yaks in a slaughterhouse of Sichuan province, putting a self-sealing bag of Hanks solution containing antibiotics (200U/ml penicillin, 0.2mg/ml streptomycin, 100 mu g/ml gentamicin and 1 mu g/ml amphotericin B) into an ice bag, taking the self-sealing bag back to a laboratory, shearing rumen nipples in a sterile super clean bench, adding Hanks solution containing antibiotics (200U/ml penicillin, 0.2mg/ml streptomycin, 100 mu g/ml gentamicin and 1 mu g/ml amphotericin B) and cleaning for 4-5 times, wherein each time lasts for 3-5 minutes;
s2, enzyme digestion and culture: 0.1% collagenase type i in a volume ratio of 5:1 adding the rumen papilla into a 250ml glass bottle filled with the rumen papilla, placing the glass bottle into a constant-temperature air bath shaking box at 37 ℃ for 30min, then removing supernatant, washing the glass bottle once by using Hanks solution, and placing the rumen papilla in a modified DMEMF/12 cell complete culture medium for 5min, wherein the modified DMEMF/12 cell complete culture medium is DMEMF/12 cell complete culture medium which also contains 200U/ml penicillin, 0.2mg/ml streptomycin, 100 mu g/ml gentamicin and 10% fetal calf serum;
evenly spreading the digested rumen papilla with a small amount of culture medium at 1ug/cm2In a 10cm culture dish coated with rat tail collagen, the tissue block spacing was dispersed by 5mm, and the culture dish was placed upside down at 37 deg.C and 5% CO2Observing whether the water of the tissue blocks is evaporated and reduced to ensure that the tissue blocks can be tightly attached to the bottom after approximately 3-4 hours in the incubator, if the water is more, prolonging the inversion time, taking out the culture dish, slowly adding 5mL of improved DMEMF/12 cell complete culture medium to the bottom of the culture dish, covering each tissue block with the culture medium, keeping the culture dish as static as possible, adding the improved DMEMF/12 cell complete culture medium to 10mL after 24 hours of culture, and changing the liquid once every five days; observing the morphological change of the cells, removing tissue blocks when the cell crawled out around the rumen papilla after digestion is proliferated and fused to 50%, and washing the cells by PBS;
s3, purification: adding 2mL of 0.25% trypsin-0.02% EDTA into the cells washed in the step S2, gently shaking the culture dish to make the digestive juice spread on the bottom of the dish, placing the dish at 37 ℃ and 5% CO2Digesting the cells in an incubator for 2min, beating the side wall of a cell culture dish for several times, quickly adding an improved DMEMF/12 cell complete culture medium to terminate digestion, beating and shaking the culture dish to remove fibroblasts as much as possible, washing the cells twice with PBS, purifying the cells, and adding the improved DMEMF/12 cell complete culture medium to continue culturing to obtain purified rumen epithelial cells of the yak;
s4, virus infection: placing purified yak rumen epithelial cells in a cell complete culture medium to grow until 70% of the cells are fused, adding lentivirus MOI (MoI) carrying SV40T and hTERT genes (containing 5ug/mL polybrene to enhance lentivirus infection efficiency) to infect the cells overnight, changing the liquid every 2 days, continuously culturing for 4 days, observing the state of the surviving cells in the whole process, adding 1ug/mL puromycin to continuously culture for 96 hours to screen cell lines, carrying out expanded culture on the surviving cell lines, and carrying out liquid change passage until the cell forms are gradually stabilized; wherein, the complete cell 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, expanding culture and passage: expanding and culturing the screened cell line to 90% of a covered culture bottle, washing the cells by using a PBS solution, digesting the cells by using 0.25% trypsin-0.02% EDTA, observing the cells by using an inverted microscope, slightly blowing the cells to fall off when the cells retract and become round, adding a cell complete culture medium to terminate digestion, wherein the digestion time is about 5min, the cell suspension is 1200rpm/min, centrifuging for 5min, and carrying out passage or cryopreservation, wherein the cryopreservation solution is a mixed solution of the complete culture medium, FBS and DMSO, and the volume ratio is as follows: the complete culture medium is FBS (DMSO) ═ 7:2:1, wherein the 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; and (5) the cells are passaged to more than 20 generations to obtain an immortalized yak rumen epithelial cell line.
The separation and culture process of the rumen epithelial cells of the yaks is shown in figure 1, and the morphological characteristics of the rumen epithelial cells of the yaks cultured by enzyme digestion combined with a tissue block culture method are shown in figure 2; SV40T and hTERT lentiviral vectors are shown in FIG. 3; the morphological characteristics of the immortalized yak rumen epithelial cell line after purine toxin screening and expanded culture are shown in figure 4.
Example 2: identification of 1st generation primary yak rumen epithelial cells and 5 th generation immortalized yak rumen epithelial cell line by keratin Cytokeratin 18 immunofluorescence
1 × 10 rumen epithelial cells6Inoculating each cell/mL in 6-well plate, observing cell growth condition after cell is stably attached to wall, and taking out when cell growth is uniform and gaps are left between cells and cells are not connected into slicesThe petri dish was added with pre-cooled PBS buffer and washed on a shaker for 10min 3 times. Adding pre-cooled 4% paraformaldehyde stationary liquid for 15min, removing the stationary liquid, adding pre-cooled PBS, and washing on shaking table for 10min each time for 3 times. Adding pre-cooled 0.1% Triton X100 to treat cells for 10min, removing dialysate, adding pre-cooled PBS, and washing on shaker for 10min each time for 3 times. 5% BSA was added and blocked at room temperature for 30 min. The blocking solution was removed and 500. mu.L of a diluted primary anti-CK 18 antibody (1:50) was added to each well overnight in a refrigerator at 4 ℃. After removing the primary antibody, add pre-cooled PBS, and wash on a shaker for 10min each time for 3 times. Adding diluted fluorescent secondary antibody (1: 200) in dark, shaking table at 37 deg.C for 1h, removing secondary antibody, adding precooled PBS, and washing on shaking table for 10min each time for 3 times. Adding diluted DAPI to completely cover the cells, keeping away from light for 5min, removing staining solution, adding precooled PBS, and washing on shaking table for 10min each time for 3 times. The observation and photographing were carried out under a fluorescence inverted microscope, and the results of the identification are shown in FIG. 5.
As can be seen from fig. 5: keratin 18 of all cells shows fluorescence, the cell nucleus is dyed blue, protein is expressed, and the cell purity is high, which indicates that the cultured primary cells and cell lines are epithelial cell-like cells.
Example 3: cell proliferation growth curve
Collecting 10 th generation of rumen epithelial cells of yak with good growth state, digesting the cells to obtain cell suspension, and making the cell suspension at 5 × 103The cells were cultured in 96-well plates at a density of one/mL, 6 replicates per plate were inoculated into 10 plates at 37 ℃ in 5% CO2Culturing in a cell culture box with saturated humidity. From day 2, only one plate was removed daily, 10% CCK8 solution was added to each well, and after standing for 2 hours, OD at 450nm was measured using a microplate reader. The growth curve of the cells was plotted with the culture time on the abscissa and the mean value of the OD values on the ordinate, as shown in FIG. 6. The result shows that the cell proliferation growth curve is S-shaped and accords with the growth rule of epithelial cells.
Example 4: expression of 1st generation primary yak rumen epithelial cells and 10 th generation immortalized yak rumen epithelial cell line
Taking the 1st generation primary yak rumen epithelial cells and the 10 th generation permanentBiochemical yak rumen epithelial cell line 1 × 106Inoculating each cell/mL into a 6-well plate, setting three times of repetition, after the cells grow for 24 hours, washing the cells twice by PBS, and extracting total RNA and Hifair of the cells by using a MolPureCel RNAKit culture cell RNA extraction kit according to the kit operation instructionTMII1st Strand cDNA Synthesis Super Mix for qPCR reverse transcription reaction, in 10 u l reaction volume reverse transcription of not less than 1u g cell total RNA. Hieff
RT-PCR was performed using the Universal Blue qPCR SYBR Green Master Mix kit. The sequences of the primers used in the PCR experiments are shown in Table 1, and the primers were synthesized in Shanghai.
Table 1: primer sequence information
qRT-PCR identifies the expression graphs of SV40T and hTERT genes in the rumen epithelial cells of primary yaks of the 1st generation and the immortalized rumen epithelial cell lines of immortalized yaks of the 10 th generation as shown in figure 7, and the result shows that the immortalized cell lines stably and highly express SV40T and hTERT genes, and further shows that the lentiviruses successfully infect the primary cells.
Example 5: staining experiment with beta-galactosidase
Inoculating 3 rd generation primary yak rumen epithelial cells and 30 th generation immortalized yak rumen epithelial cell lines in a 6-well plate, observing the growth condition of the cells after the cells are stably attached to the wall, and detecting according to the operation instruction of a cell senescence beta-galactosidase staining kit when the growth density reaches 90%. The detection results are shown in fig. 8: primary cells were transmitted to passage 3, the cell state was abnormal, the senescent morphology of the cells was irregular, the senescent cells generally became large in size, and β -galactosidase expressing cells that turned blue were readily observed under an optical microscope. And no obvious dark blue is observed in the immortalized yak rumen epithelial cell line, which indicates that the cell line does not have aging, the cell line is stably passaged, the cell activity is high, and the proliferation state is stable.
Example 6: karyotype analysis of 20 th generation immortalized yak rumen epithelial cell line
The 20 th generation immortalized yak rumen epithelial cell line is 1 × 106Inoculating each cell/mL into a culture bottle, culturing for 72h, adding colchicine into the culture bottle when the culture is 68h, wherein the final concentration of the colchicine in the culture bottle reaches 0.1-0.2ug/mL, and continuously culturing for 2-4h in an incubator to stop cell division at a metaphase phase. Then the following treatments were carried out:
collecting cells: the culture was blown up and mixed well, transferred to a 10ml glass centrifuge tube and centrifuged at 1800rpm for 6 min.
Hypotonic treatment: discarding the supernatant, adding 8ml of 0.075mol/L potassium chloride preheated at 37 ℃, gently blowing and stirring by a suction pipe, and performing hypotonic treatment at 37 ℃ for 20 min.
Pre-fixing: 1ml of freshly prepared fixative (methanol: glacial acetic acid 3:1) was added, mixed gently and centrifuged at 1800rpm for 6 min.
Fixing: and (3) removing the supernatant, adding 8ml of the fresh stationary liquid, gently mixing uniformly, standing and fixing at room temperature for 20min, and centrifuging at 1800rpm for 6 min. Discard the supernatant, repeat the fixation once, centrifuge at 1800rpm for 6 min. And (4) discarding the supernatant, adding a proper amount of fresh fixing solution according to the amount of the precipitate, and lightly mixing uniformly to prepare frosty suspension.
Tabletting: dropping 1-2 drops of the suspension onto a clean glass slide stained with ice water or dried, blowing, passing fire, and air-drying. And (3) placing the film in an incubator at 37 ℃ for 3-4 days or baking the film at 70 ℃ for 2-3 hours for aging treatment for band display analysis.
Dyeing: diluting the Giemsa stock solution (9: 1) by using pH6.8 phosphoric acid buffer, taking 1 prepared chromosome specimen slice, dyeing for 15-20 min, washing with water, and drying in air.
And (4) observation: the metaphase with good chromosome dispersion is selected under a low power microscope, and then the oil lens observation is switched. The prepared slide specimen may be mounted with a mounting agent to isolate it from external contact if it is to be stored for a prolonged period of time.
And (3) photographing: placing the prepared specimen slice under an objective lens, and adjusting the microscope; observing with low power lens to find metaphase of cell division, selecting well dispersed metaphase, amplifying, and counting.
Counting the number of chromosomes: a certain number of dividing cells with well dispersed chromosomes can be counted.
And recording morphological characteristics and measuring and calculating.
Karyotyping system: video TesT. Karyo chromosome karyotype analysis System.
As a result, as shown in fig. 9, it can be seen from fig. 9 that: the immortalized yak rumen epithelial cell line shows the diploid characteristics of yak species, does not mutate into polyploids, and obviously increases the chromosome number of cells after immortalization, which is consistent with the condition that primary cells are infected by lentivirus and transformed into immortalized cells, and belongs to the normal phenomenon.
Comparative example 1:
cutting and cleaning sheared rumen papilla, directly placing the rumen papilla into a complete cell culture medium for 5min, uniformly paving small tissue blocks with a small amount of the 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 evaporation of the tissue blocks is reduced to ensure that the tissue blocks can be tightly attached to the bottom, if the water content is more, prolonging the inversion time, taking out the culture bottle, slowly and lightly adding 2mL of the complete cell culture medium into the side face of the bottom of the culture bottle, carefully placing the culture bottle upright, keeping the culture bottle as static as possible, supplementing the culture medium for 10mL after 24h of culture, and changing the liquid once every five days. The cells were observed for morphological changes. The time for climbing out 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 climbing out around the tissue block can reach 80% by combining enzyme digestion with the two-week time of the tissue block culture method.
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 screened out, the cell morphology is completely different from the epithelial cell morphology, and the cell line establishment fails. Meanwhile, two lentiviruses carrying SV40T and hTERT genes are used for infecting primary yak rumen epithelial cells, the success rate of converting the cells into immortalized cell lines is high, the cell lines are continuously passaged in vitro, the cell lines are vigorous, and the proliferation speed of the cell lines is high.
Comparative example 3:
when only a complete culture medium DMEMF/12(200U/mL penicillin, 0.2mg/mL streptomycin, 100 mu g/mL gentamicin and 10% fetal bovine serum) is used for culturing cells in the process of infecting primary cells by using lentiviruses to transform an immortalized cell line, the growth state of the cells is unstable, the complete culture medium is optimized, the cell line is in a normal growth state, the cell proliferation speed is high, the cell activity is strong, and the shape of primary yak rumen epithelial cells is maintained through stable passage.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can substitute or change the technical solution of the present invention and the inventive concept within the technical scope of the present invention.
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Claims (9)
1. An immortalized yak rumen epithelial cell line, which is characterized in that the preservation number of the immortalized yak rumen epithelial cell line is as follows: CCTCC number C2021245, named as: the immortalized yak rumen epithelial cell line SV 40T-YREC-hTERT.
2. The rumen epithelial cell line of an immortalized yak according to claim 1, wherein said yak is an adult yak.
3. The method for constructing the immortalized yak rumen epithelial cell line according to claim 1 or 2, which comprises the following steps:
s1, sample pretreatment: collecting rumen epithelial tissue of yaks, cleaning, cutting rumen papilla, adding Hanks solution containing antibiotics, and cleaning;
s2, enzyme digestion and culture: adding collagenase type I into cleaned rumen papilla, oscillating at 37 deg.C in constant temperature air bath for 30min, placing enzyme-digested rumen papilla in improved DMEMF/12 cell complete culture medium for 4-8min, uniformly spreading digested rumen papilla with small amount of culture medium in culture dish coated with collagen protein from rat tail, dispersing tissue blocks at intervals, inverting culture dish at 37 deg.C and 5% CO for 30min2Culturing in an incubator until the cell climbing out around the rumen papilla is proliferated and fused to 50%, removing tissue blocks and cleaning cells; wherein the improved DMEMF/12 cell complete culture medium is that the DMEMF/12 cell complete culture medium also contains 200U/ml penicillin, 0.2mg/ml streptomycin, 100 mug/ml gentamicin and 10% fetal calf serum;
s3, purification: adding EDTA solution of trypsin into the cells washed in step S2, placing at 37 deg.C and 5% CO2Digesting the cells in an incubator for 2min, stopping digestion, and cleaning to obtain purified rumen epithelial cells of yaks;
s4. viral infection: placing the purified yak rumen epithelial cells in a cell complete culture medium to grow until the cells are 70% fused, adding lentiviruses carrying SV40T and hTERT genes to infect the cells overnight, continuously culturing for 4 days, adding purine toxin, continuously culturing for 96 hours, and screening cell lines; wherein, the complete cell 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, expanding culture and passage: and (3) expanding and culturing the screened cell line until the cell line grows to cover 90% of a culture bottle, adding EDTA (ethylene diamine tetraacetic acid) solution of trypsin to digest the cells, stopping digestion until the cells retract and become round, obtaining primary yak rumen epithelial cells, and passaging the primary cells to more than 20 generations to obtain the immortalized yak rumen epithelial cell line.
4. The method for constructing an immortalized yak rumen epithelial cell line according to claim 3, wherein the antibiotics in step S1 are penicillin, streptomycin, gentamicin and amphotericin B.
5. The method for constructing an immortalized yak rumen epithelial cell line according to claim 3, wherein the collagenase type I is present in a concentration of 0.1% in step S2, and the volume ratio of collagenase type I to rumen papilla is 5: 1.
6. The method for constructing an immortalized yak rumen epithelial cell line according to claim 3, wherein the content of trypsin in the EDTA solution of trypsin in steps S3 and S5 is 0.25%, and the content of EDTA is 0.02%.
7. The method for constructing an immortalized yak rumen epithelial cell line according to claim 3, wherein the lentivirus further comprises polybrene 5ug/mL in step S4 for enhancing the infection efficiency of the lentivirus.
8. The method for constructing an immortalized yak rumen epithelial cell line according to claim 3, wherein the retroviral vector of SV40T in step S4 is pGMLV-SV40T-PURO, and the retroviral vector of hTERT is mammalian gene expression lentivirus vector pLV [ Exp ] -Puro-EF1A > hTERT.
9. The method for constructing the immortalized yak rumen epithelial cell line according to claim 3, wherein the immortalized yak rumen epithelial cell line in step S5 is preserved in a frozen manner, wherein the frozen solution is a mixture of complete culture medium, FBS and DMSO, and the volume ratio is as follows: 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, and the total medium is DMSO =7:2: 1.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112458040A (en) * | 2020-12-23 | 2021-03-09 | 四川农业大学 | Isolation culture method of rumen epithelial cells of adult yaks |
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101886060A (en) * | 2010-07-20 | 2010-11-17 | 东北农业大学 | Methods for in vitro isolation and culture and induced differentiation of bovine muscle satellite cells |
| CN103289950A (en) * | 2013-06-28 | 2013-09-11 | 浙江大学 | Methods for in vitro separation, culture, preservation and recovery of milk cow omasum epithelial cells |
| CN105238741A (en) * | 2015-11-14 | 2016-01-13 | 河南农业大学 | Complete medium for culturing ruminal epithelium cells of dairy cows and application thereof |
| CN107881197A (en) * | 2017-11-17 | 2018-04-06 | 扬州大学 | One kind immortalizes cow rumen epithelial cell line and its construction method |
| CN108373994A (en) * | 2018-01-26 | 2018-08-07 | 河北医科大学第四医院 | The tissue block method's original cuiture and identification method of a kind of gastric and esophageal engaging portion smooth muscle cell |
| CN109762789A (en) * | 2019-02-25 | 2019-05-17 | 浙江大学 | It is a kind of immortalize sheep cud epithelial cell line foundation and application method |
| CN112458040A (en) * | 2020-12-23 | 2021-03-09 | 四川农业大学 | Isolation culture method of rumen epithelial cells of adult yaks |
| LU502505B1 (en) * | 2021-11-16 | 2023-01-16 | Univ Sichuan Agricultural | Immortalized yak rumen epithelial cell line and construction method thereof |
-
2021
- 2021-11-16 CN CN202111366012.2A patent/CN114058592B/en active Active
-
2022
- 2022-07-14 LU LU502505A patent/LU502505B1/en active IP Right Grant
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101886060A (en) * | 2010-07-20 | 2010-11-17 | 东北农业大学 | Methods for in vitro isolation and culture and induced differentiation of bovine muscle satellite cells |
| CN103289950A (en) * | 2013-06-28 | 2013-09-11 | 浙江大学 | Methods for in vitro separation, culture, preservation and recovery of milk cow omasum epithelial cells |
| CN105238741A (en) * | 2015-11-14 | 2016-01-13 | 河南农业大学 | Complete medium for culturing ruminal epithelium cells of dairy cows and application thereof |
| CN107881197A (en) * | 2017-11-17 | 2018-04-06 | 扬州大学 | One kind immortalizes cow rumen epithelial cell line and its construction method |
| CN108373994A (en) * | 2018-01-26 | 2018-08-07 | 河北医科大学第四医院 | The tissue block method's original cuiture and identification method of a kind of gastric and esophageal engaging portion smooth muscle cell |
| CN109762789A (en) * | 2019-02-25 | 2019-05-17 | 浙江大学 | It is a kind of immortalize sheep cud epithelial cell line foundation and application method |
| CN112458040A (en) * | 2020-12-23 | 2021-03-09 | 四川农业大学 | Isolation culture method of rumen epithelial cells of adult yaks |
| LU502505B1 (en) * | 2021-11-16 | 2023-01-16 | Univ Sichuan Agricultural | Immortalized yak rumen epithelial cell line and construction method thereof |
Non-Patent Citations (4)
| Title |
|---|
| JUNMEI WANG 等: "Estallishment of Immortalized Yak Ruminal Epithelial Cell Lines by Lentivirus-Mediated SV40T and hTERT Gene Transduction", pages 1 - 17 * |
| P.GALFI 等: "Culture of epithelial cells from bovine ruminal mucosa", vol. 4, no. 4, pages 295 - 300 * |
| 上官陶 等: "表达hTERT及SV40T永生化奶牛乳腺上皮细胞系的建立", vol. 45, pages 1 - 4 * |
| 刘思乐 等: "不同培养方法对山羊瘤胃上皮细胞生长及角蛋白18表达量的影响", vol. 28, no. 4, pages 1225 - 1232 * |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112458040A (en) * | 2020-12-23 | 2021-03-09 | 四川农业大学 | Isolation culture method of rumen epithelial cells of adult yaks |
| CN112458040B (en) * | 2020-12-23 | 2022-07-05 | 四川农业大学 | Isolation culture method of rumen epithelial cells of adult yaks |
| CN114934072A (en) * | 2022-05-18 | 2022-08-23 | 华中科技大学同济医学院附属协和医院 | Immortalization construction method of human heart valve mesenchymal cell line |
| CN114934072B (en) * | 2022-05-18 | 2023-10-20 | 华中科技大学同济医学院附属协和医院 | Immortalized construction method of human heart valve mesenchymal cell line |
| CN114891734A (en) * | 2022-06-02 | 2022-08-12 | 四川农业大学 | Immortalized yak rumen fibroblast line and construction and application thereof |
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| CN115011549A (en) * | 2022-07-12 | 2022-09-06 | 上海交通大学医学院附属仁济医院 | Gallbladder epithelial cell line establishing method, gallbladder epithelial cell line and application thereof |
| NL2032736A (en) * | 2022-08-11 | 2022-09-20 | Univ Sichuan Agricultural | Immortalized yak rumen fibroblast cell line and construction and application thereof |
| CN115316340A (en) * | 2022-08-12 | 2022-11-11 | 山东大学 | Method, device, equipment and medium for fattening dzo based on biochemical indexes |
| CN115316340B (en) * | 2022-08-12 | 2023-08-18 | 山东大学 | Yak dzo fattening method, device, equipment and medium based on biochemical indexes |
| CN115896032A (en) * | 2022-09-20 | 2023-04-04 | 甘肃农业大学 | Yak ovary cumulus cell line and construction method and application thereof |
| CN115896032B (en) * | 2022-09-20 | 2023-08-29 | 甘肃农业大学 | Yak ovary cumulus cell line and construction method and application thereof |
| CN117503800A (en) * | 2024-01-04 | 2024-02-06 | 北京益华生物科技有限公司 | Gastric mucosa epithelial cell extract and preparation method and application thereof |
| CN117503800B (en) * | 2024-01-04 | 2024-04-05 | 北京益华生物科技有限公司 | Gastric mucosa epithelial cell extract and preparation method and application thereof |
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| CN114058592B (en) | 2023-07-04 |
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