CN109628404A - The construction method and purposes of Preadipocyte immortalized cell line under pigskin - Google Patents
The construction method and purposes of Preadipocyte immortalized cell line under pigskin Download PDFInfo
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Abstract
The invention discloses a kind of construction methods of Preadipocyte immortalized cell line under pigskin, comprising the following steps: obtains primary pig subcutaneous fat cells；Cell is cultivated, when it is 60%~70% that cell, which grows to density, transfects pBABE-puro-hTERT plasmid；The DMEM Screening of Media for containing 30% serum of 4 μ g/mL puromycins is used after transfecting 48 ± 2h；It is that DMEM culture medium is cultivated that liquid is changed after 48 ± 2h of screening；When cell forms cell clone, it will be cultivated under the digestion of resulting cell clone, until Preadipocyte model under identification being immortalized pigskin.The purposes of Preadipocyte immortalized cell line is under pigskin: being proliferated for Preadipocyte, or the research of differentiation polyester mechanism, Regulation Mechanism.
Description
Technical field
The present invention constructs rouge under the pigskin for being overexpressed Telomerase retroviral gene by transfecting the method for Telomerase plasmid
Fat precursor, being immortalized cell line belong to the application technology of biology and modern agricultural technology.
Background technique
Adipose tissue plays important role in the energetic supersession of animal body, is important storage and energy supply
Place.Some Adipocyte Factors such as adiponectin, leptin that adipose tissue is secreted as endocrine organ, to generations such as obesity, diabetes
It thanks to disease to play an important role.Wherein, fat deposition then depend mainly on proliferation, differentiation and the polyester of Preadipocyte at
It is ripe, directly influence glycolipid metabolism and the health of the mankind.On the other hand, from the angle of husbandry sector, subcutaneous fat deposits
The quality of pork and the utilization efficiency of feed are directly affected, economic benefit is reduced.Therefore, research fat deposition seems especially
Urgently.And by establishing Preadipocyte in-vitro culture model under pigskin, explore the differentiation of pig PECTORAL LIMB SKELETON, proliferation, polyester
Mechanism and regulatory mechanism, formation and Regulating Lipid Metabolism to adipose tissue is understood in depth, so be fat deposition and meat
Matter forms regulation and provides theoretical foundation.Therefore, it establishes precursor in-vitro culture model and then probes into Adipocyte Differentiation polyester
All there is important scientific meaning in fields such as medicine, life science, Animal Sciences.
Life science is often related to two class cells at present, and one kind is primary cell, and another kind is for immortalized cells or carefully
Born of the same parents system.Currently, the primary Preadipocyte culture of pig is unfavorable for passing on, moreover, separation Primary adipocyte requires to slaughter every time
3-7 age in days piglet is killed, the success rate of experimental cost and experiment is greatly improved.But have no at present porcine adipocyte cell line or
Immortalized cells.Reverse transcriptase of telomere (TERT) can extend telomere (its cellular replication ability of the telomere of shortening is limited), thus
Enhance the proliferative capacity of cell in vitro, it can be used to building immortalized cell line.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of buildings of Preadipocyte immortalized cell line under pigskin
The purposes of method and the cell line.
It is constructed in order to solve the above technical problem, the present invention provides a kind of stable transfection hTERT and immortalizes pig subcutaneous fat
The method (construction method of Preadipocyte immortalized cell line under pigskin) of precursor, comprising the following steps:
1) primary pig subcutaneous fat cells (Pigs Inoculated subcutaneous fat precursor), are obtained；
2) cell, is cultivated, when it is 60%~70% that cell, which grows to density, transfects pBABE-puro-hTERT plasmid；
3) the DMEM Screening of Media for containing 30% serum of 4 μ g/mL puromycins is used after, transfecting 48 ± 2h；
4) it is that DMEM culture medium (that is, common complete medium) is cultivated that liquid, is changed after 48 ± 2h of screening；
5), when cell forms cell clone, resulting cell clone is digested and is cultivated (passage to new 12
Cultivated in orifice plate), until (Preadipocyte immortalizes Preadipocyte model under pigskin under identification being immortalized pigskin
Cell line).
In the step 5), by repeatedly passing on, identifying, to obtain immortalizing Preadipocyte model under pigskin.
As the improvement of the construction method of Preadipocyte immortalized cell line under pigskin of the invention, the step 5)
Using the half-quantitative detection of hTERT gene expression:
The specific primer of hTERT are as follows:
hTERT-F GACCTCCATCAGAGCCAGTC
hTERT-R GTTCTTGGCTTTCAGGATGG；
Select the cell line for having hTERT gene expression.
As the improvement of the construction method of Preadipocyte immortalized cell line under pigskin of the invention, fluorescent quantitation is used
The expression of PCR detection proliferation marker gene DHFR and KI67；Fluorescence quantification PCR primer is respectively as follows:
DHRF-F:GGTGATTATGGGTAGGAAGACCT
DHRF-R:ATTCTGGCTGCTCAGTAAGTTTT
Ki67-F:GCACCAGGCTTTACGGAAG
Ki67-R:ATTTTAGCCACTTCTGACTTTCG。
The present invention goes back while providing the purposes of Preadipocyte immortalized cell line under above-mentioned pigskin: before fat
Somatic cell proliferation, or the research of differentiation polyester mechanism, Regulation Mechanism.
Preadipocyte has following purposes under the resulting immortalization pigskin of the present invention: immortalizing fat precursors under pigskin
Cell is important in vitro study model, can be used for Preadipocyte proliferation, differentiation polyester mechanism, Regulation Mechanism etc.
Research；The separation number of piglet Primary adipocyte can be reduced simultaneously, and is suitable for carrying out the more and relevant reality of fat cell
It tests, application prospect is extensive.
The present invention has following technical advantage: stablize expression hTERT in the immortalization Preadipocyte established, and
HTERT increased activity, slows down the rate that cell enters Clonal aging, cell can repeatedly pass on and be able to maintain preferable proliferation,
Differentiation characteristic can be widely used for carrying out correlative study.
The present invention reverses on the basis of isolated pig primary subcutaneous fat precursor, through transfection containing human telomerase
The plasmid of enzyme (hTERT) gene is recorded, and Preadipocyte In Vitro under the pigskin for being overexpressed hTERT can be stablized by antibiotic-screening,
Preadipocyte immortalized cell line enhances with telomerase activation under the pigskin that the present invention constructs, and slows down cell and enters clone
Property aging rate the characteristics of, the ability that finally makes Preadipocyte under pigskin obtain infinite multiplication.The cell of this immortalization
System can be used to carry out the researchs such as lipocyte proliferation under pigskin, differentiation polyester, nutrition regulation, and it is thin to be important pork fat precursor
The outer research model of cell space, has very extensive application prospect.
Detailed description of the invention
Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.
Fig. 1 is plasmid map used.
Fig. 2 is that the steady of screening formation turns cell clone cellular morphology electron microscope picture；A figure --- 40 times；B figure -- 100
Times.
Fig. 3 is the nucleic acid electrophoresis figure of hTERT expression in 12 single cell clones that half-quantitative detection is screened.
Fig. 4 be the 8th generation immortalize pigskin under Preadipocyte In Vitro passage after 1 day to 6 days cellular morphology electron microscope picture.
Fig. 5 be screening obtain surely turn clone cell secondary culture 4 (p4), 10 (p10), 30 (p30) for when hTERT express
Nucleic acid electrophoresis figure.
Fig. 6 is MTT detection immortalized cells proliferation results.In the 9th generation of PAC--, immortalizes Preadipocyte under pigskin,
Preadipocyte under the common pigskin of Con-.
Fig. 7 is violet staining detection immortalized cells proliferation results.Preadipocyte under the primary pigskin of A--, B--
In 10 generations, immortalized Preadipocyte under pigskin.
Fig. 8 is Immunofluorescence test immortalized cells proliferation results.Cell used is the 12nd generation immortalized cells.A--
DAPI coloration result；B-- is proliferated antibody KI67 immunofluorescence dyeing result；C--Merge result.
Fig. 9 is cell Proliferation marker gene DHFR expression fluorogenic quantitative detection result.Rouge under the 10th generation of CON-- primary pigskin
Fat cell；In the 10th generation of PAC--, immortalizes Preadipocyte under pigskin.
Figure 10 is cell Proliferation marker gene Ki67 expression fluorogenic quantitative detection result.Under the 10th generation of CON-- primary pigskin
Preadipocyte；In the 10th generation of PAC--, immortalizes Preadipocyte under pigskin.
Figure 11 is oil red coloration result after immortalized cells differentiation.
Figure 12 is immortalized cells polyester differentiation after ripening fat marker gene expression fluorogenic quantitative detection result.
Specific embodiment
(1) it separates, cultivate Preadipocyte under primary pigskin
1. separating subcutaneus adipose tissue under aseptic condition, PBS (containing penicillin and streptomysin) rinsing three times, is cut in PBS
At 1-2mm3Tissue block.PBS (containing penicillin and streptomysin) the preparation method comprises the following steps: every 100ml PBS buffer solution (pH 7.2-
7.4) penicillin and streptomysin of final concentration of 100U/ml are added in.
2. 37 DEG C of water-baths of digestive juice about 15ml containing 0.15% Type I collagen enzyme are added in 10g tissue block digests 70min-
90min。
The formula of digestive juice are as follows: 0.15g Type I collagen enzyme is added in 100ml digestive juice, obtains containing 0.15% Type I collagen enzyme
Digestive juice；Type I collagen enzyme is for example purchased from Gibco company.
3. after the completion of digestion, the isometric DMEM complete medium (DMEM+10% fetal calf serum) of digestive juice being added and terminates
Digestion, then through filtered through gauze, 100 mesh screen filtrations, 1000rpm centrifugation 5min, abandoning supernatant.
4. being resuspended obtained by step 3 with DMEM complete culture solution (DMEM+10% fetal calf serum) 1ml containing 10% fetal calf serum
Precipitating.
5. by the obtained cell inoculation of step 4 in 10cm culture dish, sets 37 DEG C, cultivates in 5%CO2 incubator.It obtains former
For pig subcutaneous fat cells (the primary subcutaneous fat precursor of pig).Then change within every 2 days liquid 1 time.It is used for DMEM culture to change liquid
Liquid, dosage is the same as step 3.
(2) Preadipocyte culture model under pigskin is immortalized to establish
1. being grown to 80% converging state (about 3-4d) to the primary subcutaneous fat precursor of above-mentioned isolated pig, PBS drift
After washing 1 time, with 0.25% trypsase (about 1ml), 37 DEG C of digestion 1-3min, after terminating digestion with complete medium, 1000rpm
It is centrifuged 5min, PBS is rinsed 3 times, and cell is resuspended with DMEM complete culture solution 1ml.
2. cell is then reached 6 porocyte culture plates, cultivated under the conditions of 37 DEG C, 5%CO2, and make cell about 70%
When fusion rate (cell density, about second day), tested for next liposome transfection.
3. liposome transfection: transfecting pBABE-puro-hTERT plasmid (Fig. 1) using Lipofectamine 2000；
4. with the DMEM Screening of Media of 30% serum containing 4 μ g/mL puromycins after transfecting 48h；
5, culture medium changes common complete medium into and is cultivated after screening 48h；It changes within every 2 days liquid 1 time.
6. after screening and culturing 4-6 days, there is the monoclonal cell cluster (Fig. 2) of Preadipocyte under pigskin.
7. obtained cell monoclonal carries out immortalized cells identification after digesting, expanding culture.
(3) Preadipocyte morphological observation and identification under pigskin are immortalized
1.hTERT expression identification: the monoclonal cell strain that (two) are obtained, 12 plants of picking are transferred to after pancreatin digests
12 porocyte culture plates cultures, then expand culture, and carry out half-quantitative detection (the sxemiquantitative mirror of hTERT gene expression
It is fixed to be carried out according to conventional RT-PCR method), the specific primer of the hTERT of design are as follows:
hTERT-F GACCTCCATCAGAGCCAGTC
hTERT-R GTTCTTGGCTTTCAGGATGG
PCR system are as follows: 2X T5 PCR Mix:10 μ L, hTERT-F:0.5 μ L, hTERT-R:0.5 μ L, cDNA:1 μ L,
H2O:3μL；
PCR response procedures are as follows: (1) 95 DEG C of initial denaturation 3min；(2) 95 DEG C of denaturation 10s, 60 DEG C of annealing and extend 10s, and totally 40
A circulation；(3) 72 DEG C of 2min, 4 DEG C of preservations.95 DEG C of 3min, 95 DEG C of 10s, 60 DEG C of 10s, 72 DEG C of 15s (35 circulations), 72 DEG C
2min, 4 DEG C of preservations.
PCR product length is 262bp, finds there is hTERT gene in addition to the 10th plant in 1-12 monoclonal cell strain
It expresses (Fig. 3).It is mainly cultivated with remaining cell in addition to the 10th plant of cell strain in follow-up study, condition of culture 37
DEG C, 5%CO2Incubator culture.
2. immortalized cells Morphological observation: the cell strain of above-mentioned identification expression hTERT carries out secondary culture, takes the
In 8 generations, immortalized Preadipocyte under pigskin and carry out morphologic observation, and discovery cellular morphology is uniform, visible afterwards for 24 hours to have a large amount of shuttle
Type cell is adherent (Fig. 4 A), and cell starts extensional deformation after 48h, and most cellular morphologies are in spindle shape or star, and minority is in not advise
Then triangle (Fig. 4-B)；Cell Proliferation is rapid after 72h, and shuttle-type and irregular type cell increase significantly (Fig. 4 C)；It is the 4-5 days thin
Born of the same parents' quantity increased significantly, and about reach 80%, can be passed on (Fig. 4 D, E)；Cellular morphology elongation in 6th day, in differentiation due (figure
4F)。
3. Preadipocyte hTERT detection of expression is identified under the pigskin immortalized: fat precursors under the pigskin of immortalization
After cellular morphology is similar with Preadipocyte form under the primary pigskin of earlier generations, however primary Preadipocyte passed for 10 generations
It senesces, cytoplasm (cell volume) increases.In order to determine the activity for immortalizing Preadipocyte under pigskin, pass through semidefinite
Amount PCR has detected the expression of hTERT in the immortalized cells of different algebra (4,10,30 generation), PCR reaction system and condition
The result shows that, hTERT gene (Fig. 5) is expressed in the 4th, 10,30 generation immortalized cells with 1., it was demonstrated that passage is forever more than 30 generations
Still there is the hTERT gene of overexpression in biochemical Preadipocyte.
(4) proliferation activity of Preadipocyte under the pigskin of immortalization is studied:
Mainly pass through the experiments mirror such as MTT experiment, violet staining and immunofluorescence Ki67, proliferation marker gene detection of expression
Surely the proliferation activity for the immortalization Zhu's subcutaneous fat precursor established.
1.MTT experimental identification method is as follows: the 9th generation 1X10 is inoculated in 96 orifice plates4Immortalization pigskin under fat precursors
Preadipocyte under cell and primary pigskin is cultivated in the incubator of 37 DEG C and 5% carbon dioxide.At 0,2,4,6,8 day
With MTT detect cell proliferative conditions.MTT detection is as follows: configured MTT working solution (the 5mg/ml PBS of 50 μ L is added in every hole
With), 37 DEG C of incubation 4h make MTT be reduced to formazan；Culture is terminated, careful inhale abandons culture supernatant in hole, supernatant be sucked out, often
The DMSO of 150 μ L is added in hole, and shaking table shakes 10 minutes, goes out to detect light absorption value in 490nm wavelength.Record is as a result, be cross with the time
Coordinate, being converted into number of cells according to light absorption value is that ordinate draws cell growth curve.The result shows that the increasing of immortalized cells
It grows and is significantly higher than control cell (Fig. 6).
2. violet staining identification of cell enrichment procedure is as follows: being inoculated with 1X10 in 6 orifice plates4Primary pork fat precursor it is thin
Born of the same parents and Preadipocyte under pigskin is immortalized, after cultivating 3-5 days in the incubator of 37 DEG C and 5% carbon dioxide, by cell
It is washed 1-2 times with PBS, the fixed 15min of 4% paraformaldehyde is added, is then dyed with 0.1% crystal violet working solution (PBS preparation)
15min, then observation shooting is carried out after washing three times with PBS.As a result as shown in fig. 7, compared with primary cell, violet staining is visible
Preadipocyte is more under immortalization pigskin, shows immortalized cells multiplication rate faster.
3. immunofluorescence dyeing identification of cell enrichment procedure is as follows: the 10th generation being immortalized Preadipocyte under pigskin and is used
PBS washes a cell, and the fixed 15min of 4% paraformaldehyde is added, and PBS is washed three times, and 0.1M glycine is added and is stored at room temperature 10min,
PBS is washed three times, and the confining liquid that every 200 μ L of hole is added is stored at room temperature 60min, and 4 DEG C of addition primary antibody Ki67 (confining liquid dilution) is overnight；
It is washed three times after second day abandoning primary antibody with PBS, secondary antibody is added and HOCHST (PBS dilution) is protected from light room temperature dyeing 45-60min, PBS is washed
Three times, fluorescence shooting is carried out, is stored one week in 4 DEG C.Ki67 is a kind of marker protein of cell Proliferation, the cell table of the KI67 positive
Clear-cells is in vegetative state.Show that Preadipocyte has preferable proliferative capacity under the pigskin of immortalization in Fig. 8.
4. fluorescent quantitation identification of cell enrichment procedure is as follows: after cell is passed on (the 20th generation) culture, with Trizol reagent
Total serum IgE is extracted, with the expression of fluorescence quantitative PCR detection proliferation marker gene DHFR and KI67.Fluorescence quantification PCR primer point
Not are as follows:
DHRF-F:GGTGATTATGGGTAGGAAGACCT
DHRF-R:ATTCTGGCTGCTCAGTAAGTTTT
Ki67-F:GCACCAGGCTTTACGGAAG
Ki67-R:ATTTTAGCCACTTCTGACTTTCG
PCR amplification system: 5 μ L, Forward primer (10 μm of ol/L) of SYBR Rox (2 ×) 0.2 μ L, Reverse
Primer (10 μm of ol/L) 0.2 μ L, 1 μ L, dd H of DNA profiling23.6 10 μ L of μ L, Total of O.
Amplification condition: (1) 95 DEG C of initial denaturation 10s；(2) 95 DEG C of denaturation 5s, 60 DEG C are annealed and extend 30s and acquire fluorescence letter
Number, totally 40 recycle.The primer sequence of amplification DHFR, ki67 gene is respectively as follows:
Fluorogenic quantitative detection the result shows that, compared with primary control cell, immortalize under pigskin in Preadipocyte
DHFR (Fig. 9) and KI67 (Figure 10) expression significantly improves, and the immortalized cells is further prompted to have stronger proliferative capacity.
(5), the differentiation for immortalizing pig subcutaneous fat cells is studied
The immortalized cells for passing on for 20 generations are inoculated on 12 orifice plates, by cell adipogenic induction culture after merging completely
Base induction differentiation, examines it to break up polyester ability.Induced medium composition are as follows: be added 50nmol/L's in complete medium
The Rosiglitazone of the IMBX and 100nmol/L of the Dex of Insulin, 100nmol/L, 0.25mmol/L.Induction is denoted as 0 in first day
It, induced medium induces 4 days, then with differential medium (Insulin of 50nmol/L is added in DMEM complete culture solution)
Induction 4 days.After induction differentiation 8 days, numerous small fat drips start aggregation fusion, and volume increases, and form big fat drips in the cell, so
It is identified by oil red O stain.
Oil red O stain identification: configuration oil red O first stores liquid, and it is molten that 0.5g oil red is dissolved in 100mL isopropanol progress ultrasound
Solution is put in brown bottle after filter paper filtering and is saved.Oil red O working solution be then it is ready-to-use, in 6mL storage liquid and 4mL water ratio mix
Even standing 10min, uses after being filtered with filter paper.After cell in six orifice plates is washed one time with PBS, 4% paraformaldehyde is added
Fixed 20min, every hole adds the working solution of 1.5mL to dye 20min after discarding paraformaldehyde, washes two with 60% isopropanol after dyeing
Time, it saves and takes pictures in 60% glycerol, the immortalized cells after differentiation are after oil red O stain, it is seen that the mature rouge of crocus
Fat cell (Figure 11).According to Figure 11, it can be seen that producing fat drips after adipogenic induction culture medium is added.Show the immortalized cell line
For adipose cell lines.
(6), it is expressed using Preadipocyte research differentiation associated gene under immortalization pigskin
After Preadipocyte induction differentiation under the immortalization pigskin after 20 generations of passage, identified by fluorogenic quantitative detection
The expression of maturation fat marker gene PPAR γ, C/EBP a, AdipoQ.Fluorescent quantitation primer is as follows:
PPARγ-F TGTGGACCTGTCGGTGATG
PPARγ-R TGGAGTGGAAATGCTGGAGA
C/EBPa-F GGTGGACAAGAACAGCAACG
C/EBPa-R TCACTGGTCAGCTCCAGCAC
AdipoQ-F ACGGTCTACTTGAAGGATGTGA
AdipoQ-R TCCAGATAGAGGAGCACAGAG
Fluorescent quantitation system and program are as above.According to Figure 12, it can be seen that polyester related gene PPAR- γ, C/EBP after differentiation
β and AdipoQ has and its significantly increases before relatively breaking up, show the immortalized cell line can as differentiation polyester gene expression,
The cell model of the correlative studys such as lipid-metabolism regulation.
The above list is only a few specific embodiments of the present invention for finally, it should also be noted that.Obviously, this hair
Bright to be not limited to above embodiments, acceptable there are many deformations.Those skilled in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
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Claims (4)
1. the construction method of Preadipocyte immortalized cell line under pigskin, it is characterized in that the following steps are included:
1) primary pig subcutaneous fat cells, are obtained；
2) cell, is cultivated, when it is 60%~70% that cell, which grows to density, transfects pBABE-puro-hTERT plasmid；
3) the DMEM Screening of Media for containing 30% serum of 4 μ g/mL puromycins is used after, transfecting 48 ± 2h；
4) it is that DMEM culture medium is cultivated that liquid, is changed after 48 ± 2h of screening；
5) it, when cell forms cell clone, will be cultivated under the digestion of resulting cell clone, until being immortalized of identification
Preadipocyte model under pigskin.
2. the construction method of Preadipocyte immortalized cell line under pigskin according to claim 1, characterized in that
The step 5) uses the half-quantitative detection of hTERT gene expression:
The specific primer of hTERT are as follows:
hTERT-F GACCTCCATCAGAGCCAGTC
hTERT-R GTTCTTGGCTTTCAGGATGG；
Select the cell line for having hTERT gene expression.
3. the construction method of Preadipocyte immortalized cell line under pigskin according to claim 1, it is characterized in that: with
The expression of fluorescence quantitative PCR detection proliferation marker gene DHFR and KI67；Fluorescence quantification PCR primer is respectively as follows:
DHRF-F:GGTGATTATGGGTAGGAAGACCT
DHRF-R:ATTCTGGCTGCTCAGTAAGTTTT
Ki67-F:GCACCAGGCTTTACGGAAG
Ki67-R:ATTTTAGCCACTTCTGACTTTCG。
4. the purposes of Preadipocyte immortalized cell line under the pigskin as described in claims 1 to 3 is any, it is characterized in that:
It is proliferated for Preadipocyte, or the research of differentiation polyester mechanism, Regulation Mechanism.
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