Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of employing exogenous human telomerase reverse transcriptase (hTERT) transfected with human nucleus pulposus cell activated end granzyme is provided and induces the method for preparing immortalized human intervertebral disc nucleus pulposus cell system, to make up immortal human nucleus pulposus cell system truly, for the pathology mechanism of further studying the degenerative disc disease provides the standard cell lines strain, for the integral body level of preventing and treating that improves the degenerated discopathy provides new thinking.
Purpose of the present invention is achieved by the following technical programs:
The preparation method of a kind of immortalized human intervertebral disc nucleus pulposus cell system provided by the invention induces being immortalized human disc nucleus pulposus cell system with exogenous human telomerase reverse transcriptase transfected with human nucleus pulposus cell activated end granzyme.
The nucleotide sequence of described exogenous human telomerase reverse transcriptase is shown in sequence in the sequence table 1.
The aminoacid sequence of described exogenous human telomerase reverse transcriptase is shown in sequence in the sequence table 2.
Exogenous human telomerase reverse transcriptase of the present invention can obtain in accordance with the following methods:
Extract total RNA from liver cancer tissue, Oligo-dT is primer, and take total RNA as template, reverse transcription obtains cDNA under the effect of avian ThermoScript II; Then carry out PCR reaction amplifying target genes, obtain goal gene PCR product through reclaiming purifying, and be connected acquisition recombinant chou T-hTERT with carrier T-Easy.
Wherein, the primer of described PCR reaction employing is:
RT-1:5 '-tggaaggagttcgatgccgcgcgctccccgctg-3 ' adds the Xmn1 restriction enzyme site
RT-2:5 '-caccctcgaggtgagacgctcgg-3 ' band Xho1 restriction enzyme site
RT-3:5 '-acctcgagggtgaaggcactgttca-3 ' band Xho1 restriction enzyme site
RT-4:5 '-cgctcgagctagtccaggatggtcttgaagtct-3 ' band Xho1 restriction enzyme site;
And with RT-1 and RT-2, RT-3 and RT-4 primer, carry out respectively the PCR reaction.
Recombinant chou T-hTERT of the present invention is by being built into pGFP-hTERT eukaryon expression plasmid transfected with human nucleus pulposus cell, and the construction process of described pGFP-hTERT eukaryon expression plasmid is as follows:
A) described recombinant chou T-hTERT connects with carrier pAAV-MCS plasmid DNA, and transforms DH5 α competence colibacillus, from the LB flat board that contains kantlex, and the primary election positive colony, and enzyme is cut the pAAV-hTERT plasmid of identifying acquisition;
B) digestion connects pAAV-hTERT plasmid and pGFP plasmid DNA, and transforms DH5 α competence colibacillus, from the LB flat board that contains kantlex, and the primary election positive colony, and enzyme cuts evaluation, is built into the pGFP-hTERT eukaryon expression plasmid.
The present invention has following beneficial effect:
(1) adopt the method for hTERT transfected with human nucleus pulposus cell activated end granzyme to make up immortalization nucleus pulposus cell system, M1 phase and M2 phase have been passed through, the immortalized cells that obtains is the same with germline stem cell, it is immortalization truly, for the pathology mechanism of further studying the degenerative disc disease provides the standard cell lines strain, for the integral body level of preventing and treating that improves the degenerated discopathy provides new thinking.
(2) immortalized cells of hTERT mediation is normal cell, and non-transformed cell, has normal growth rate, caryogram, presents contact inhibition and anchor and the securities such as dependence, does not have carinogenicity and soft-agar cloning and forms ability.
(3) transfection hTERT nucleus pulposus cell speed of growth compared with normal nucleus pulposus cell is fast, the nucleus pulposus cell of transfection hTERT can successfully pass more than 20 generations, compare with normal nucleus pulposus cell 1st generation, in the cell and extracellular fluid II Collagen Type VI, the equal no significant difference of glycosaminoglycan gene expression amount, the experiment of nude mouse tumorigenesis shows that nucleus pulposus cell is without tumorigenicity.
Embodiment
Fig. 1ï½Figure 18 shows that embodiment of immortalized human intervertebral disc nucleus pulposus cell system preparation method of the present invention, its techniqueflow as shown in Figure 1, the structure by goal gene sequencing, goal gene eukaryon expression plasmid in goal gene clone, the eukaryon expression plasmid, transfection hTERT are to people's nucleus pulposus cell and carry out the step such as detection of expression and realize.The present embodiment is by making up exogenous human telomerase reverse transcriptase (hTERT) restructuring green fluorescence expression vector, the success transfection normal nucleus pulposus cell activated end granzyme and in cell stably express, thereby being immortalized human disc nucleus pulposus cell system, and its biological characteristics and security measured.Concrete grammar is as follows:
One, the separation of normal nucleus pulposus cell, cultivates and go down to posterity
Normal nucleus pulposus: from the capable orthopedic patient of 3 routine scoliosis, wherein 2 examples 13 years old, 1 example 15 years old can be seen the tremelloid nucleus pulposus of fibrous ring and center of periphery white.Soaked intervertebral disk 10 minutes with containing pair physiological saline of anti-(green grass or young crops-Streptomycin sulphates), gently nucleus pulposus is separated from intervertebral disk with curet, two antibiosis reason saline soaks wash about 3ï½4 times, until without till the obvious bloodstain.With eye scissors nucleus pulposus is cut into 1 * 1 * 1mm size, the normal disc nucleus pulposus is put into the 100ml beaker that fills 10ml 2%II Collagenase Type, magnetic stirrer approximately 60 minutes.After organizing fully dissolving, centrifugal 10 minutes of 1000r/min, sucking-off supernatant liquor, dispel gently cell with the DMEM substratum 1ml that contains 10% foetal calf serum, be drawn in the 50ml culturing bottle, add the DMEM substratum 6ï½8ml of 10% foetal calf serum, be statically placed in 37 â, saturated humidity, 5%CO 2Cultivated 3 days in the incubator.Inverted microscope observation of cell adherent growth situation is changed liquid every other day after 3 days.When Growth of Cells to 90% merges state, with trysinization, go down to posterity.
Two, make up the pGFP-hTERT eukaryon expression plasmid
1, material
The RNA test kit is purchased from TaKaRa, little upgrading grain test kit, glue reclaim test kit, restriction enzyme, T4DNA ligase enzyme, Taq polysaccharase all available from MBI company, western blot test kit is purchased from GEHealthcare company, primary antibodie hTERT is available from abcam company, and two anti-mouse-HRP are available from doctor's moral company.
2, design of primers
RT-1:5 '-tggaaggagttcgatgccgcgcgctccccgctg-3 ' adds the Xmn1 restriction enzyme site
RT-2:5 '-caccctcgaggtgagacgctcgg-3 ' band Xho1 restriction enzyme site
RT-3:5 '-acctcgagggtgaaggcactgttca-3 ' band Xho1 restriction enzyme site
RT-4:5 '-cgctcgagctagtccaggatggtcttgaagtct-3 ' band Xho1 restriction enzyme site
3, goal gene clone
Extract test kit with RNA and press the total RNA of test kit explanation extraction from liver cancer tissue, Oligo-dT is primer, and take total RNA as template, reverse transcription 1h under avian ThermoScript II (M-MULV) effect obtains cDNA; Then, with RT-1 and RT-2, RT-3 and RT-4 primer, carry out respectively the PCR reaction, amplifying target genes.The PCR product separates, identifies through 1% agarose gel electrophoresis, confirms that amplified production is 1.9kb and 1.4kb (seeing Fig. 2), and is consistent with expected results.Press PhastGel and reclaim test kit specification sheets method recovery purified pcr product, obtain goal gene PCR product, and be connected acquisition recombinant chou T-hTERT with carrier T-Easy.
4, goal gene sequencing in the eukaryon expression plasmid
Extract plasmid from recombinant chou T-hTERT, select the recombinant chou through preliminary evaluation, adopt two-way assay method order-checking, its nucleotide sequence is shown in sequence in the sequence table 1, and aminoacid sequence is shown in sequence in the sequence table 2.With DNASIS software the sequence that the gene order surveyed and aminoacid sequence and GenBank announce is carried out homology relatively.
5, the structure of goal gene eukaryon expression plasmid
A) use Xmn1 and Xho1 respectively complete digestion T-hTERT and carrier pAAV-MCS plasmid DNA, 1% agarose gel electrophoresis separates, and recovery purifying target DNA fragment, then connect, and conversion DH5 α competence colibacillus, from the LB flat board that contains kantlex, the primary election positive colony, and enzyme is cut evaluation pAAV-hTERT plasmid.
B) use Not I complete digestion pAAV-hTERT plasmid and pGFP plasmid DNA, goal gene pAAV-hTERT expression cassette and pEGFP eukaryon expression plasmid are cut from glue, purifying reclaims, then connect and transform DH5 α competence colibacillus, from the LB flat board that contains kantlex, the primary election positive colony, and the BamH1 enzyme cuts evaluation, is built into the pGFP-hTERT eukaryon expression plasmid.Enzyme is cut qualification result as shown in Figure 3, confirms goal gene at 3kb, and is consistent with expected results.
Three, transfection pEGFP-hTERT is to normal nucleus pulposus cell and detection of expression thereof
Extract pEGFP-hTERT with the BIOMIGA plasmid extraction kit, cell transfecting is undertaken by reagent Lipofect-amine 2000 process specificationss.Transfection pEGFP-hTERT is to normal nucleus pulposus cell 24h, and gene green fluorescent protein transfection efficiency is seen Fig. 4.After changing liquid, the nutrient solution that adds G418 (250 μ g/mL) screens, and changes liquid 1 time in per 2 days.Carry out passage by preceding method when treating cell near 90% fusion state, immortalization nucleus pulposus cell the 20th generation fluorescence photo of acquisition as shown in Figure 5.
During transfection, transfection empty carrier pEGFP group is set makes empty plasmid transfection control group detection expression.
1. take Oligo-dT as reverse transcriptase primer, take total RNA of extracting as template, special reverse transcription goes out cDNA under AMV catalysis.With RT-3 and the RT-4 primer amplification goal gene of hTERT, set reaction conditions, carry out the PCR reaction.Product is electrophoresis in 1.5% sepharose (containing ethidium bromide 5 μ g/ μ L), observes under the gel imaging system and takes a picture.As shown in Figure 6, confirm the purpose product at 1.5kb through RT-PCR behind the plasmid pEGFP-hTERT transfectional cell, quote RT-3 and the RT-4 pair of primers is finished, confidential reference items are at 370bp, consistent with expection.
2. western blot, 10%SDS-PAGE glue loading, voltage 90V, constant voltage electrophoresis 120min, electrophoretic transfer is to the pvdf membrane of 0.45um; Add primary antibodie hTERT (1: 500), two anti-mouse-HRP (1: 3000) wash film, develop with the ECL reagent react and analyze, and the result as shown in Figure 7.
Four, the biological characteristics of normal nucleus pulposus cell and immortalization nucleus pulposus cell detects contrast
1, XTT measures the nucleus pulposus cell growth curve
Prepare cell suspension by aforementioned passage method when treating cell near 90% fusion state, counting is rear with 2 * 10 4/ ml inoculates 96 well culture plates, and the total liquid measure in every hole is 200 μ l, is designated as 0d with inoculation time, and every 24h adds 5mg/L XTT solution 20 μ l.After continuing to cultivate 4h, suck gently nutrient solution, add the DMSO liquid of 150 μ l, vibration shakes up 10min, and wavelength 490nm detects the OD value on the microplate reader, and 6 multiple holes are set, and averages.Take incubation time as transverse axis, the OD value is made graphic representation for the longitudinal axis.Get the 20th generation of normal nucleus pulposus cell 1st generation, immortalization nucleus pulposus cell and do 7 days cell growth curves and measure, the result as shown in Figure 8, the immortalization nucleus pulposus cell was since the 4th day, multiplication rate is apparently higher than normal nucleus pulposus cell.
2, the relative growth factor gene detects in the cell
The mrna expression level of II Collagen Type VI, type i collagen, glycosaminoglycan, BMP-2, IGF-1, TGF-β, BMP-4, PDGF, bFGF in the normal nucleus pulposus cell 1st generation of quantitative fluorescent PCR (SYBR Green) technology for detection and immortalization nucleus pulposus cell the 20th generation cell.Detected result is seen Fig. 9, wherein:
1 expression Type I collagen
2 expression Type II collagen
3 expression Aggrecan
4 expression PDGF
5 expression IGF-1
6 expression TGF-β
7 expression bFGF
8 expression BMP-2
9 expression BMP-4
* represent pï¼0.05, H-β-actin=1
The result shows, II Collagen Type VI in the cell, the glycosaminoglycan gene is expressed in the immortalization nucleus pulposus cell with normally the nucleus pulposus cell 1st generation is similar; IGF-1, TGF-B gene are expressed the compared with normal nucleus pulposus cell and have been raised 2ï½3 times in the immortalization nucleus pulposus cell.
3, extracellular fluid II Collagen Type VI, glycosaminoglycan detect (normal nucleus pulposus cell 1st generation and immortalization nucleus pulposus cell the 20th generation)
(1) extracellular fluid II Collagen Type VI ELISA detects
Application of sample: establish respectively blank well, standard orifice, testing sample hole.Blank well adds sample diluent 100ul, and remaining hole adds respectively standard substance or testing sample 100ul; 37 â of epiphragma incubation reaction 120 minutes; Discard liquid, dry, need not wash.Every hole adds detects solution 100ul; 37 â of epiphragma incubation reaction 60 minutes; Wash plate 3 times, soaked 1ï½2 minute at every turn; Every hole adds detects solution 100ul; 37 â of epiphragma incubation reaction 60 minutes; Wash plate 5 times, soaked 1ï½2 minute at every turn; Every hole adds substrate solution 90ul, 37 â of lucifuge colour developings (in 30 minutes, there is the blue look of obvious gradient in front 3ï½4 holes of the visible standard substance of naked eyes this moment, and rear 3ï½4 hole gradients are not obvious, can stop); Sequentially every hole adds stop bath 50ul, and termination reaction is sequentially measured the optical density(OD) (OD value) in each hole at the 450nm wavelength with microplate reader, detect.The results are shown in Table 1 and Figure 10.
(2) extracellular fluid glycosaminoglycan content detection
A) nitrite ion (PH3.0): with DMMB16mg, glycine 3.04g, NaCl 2.37g and 0.1M HCl 5.0ml place in the same container, add distilled water and are settled to 1000ml.Above stock solution is after filtration sterilization all, and used container is processed through 50% nitric acid dousing.
B) Chondroitin Sulfate A typical curve
Prepare by the following method each concentration C hondroitin Sulfate A:
1000ug/ml:100mgChondroitin Sulfate A adds distilled water and is settled to 100ml, then prepares successively the Chondroitin Sulfate A solution of 500ug/ml, 250ug/ml, 125ug/ml, 62.5ug/ml, 31.25ug/ml, 15.6ug/ml, 7.8ug/ml., 3.9ug/ml.
Measure optical density(OD) (OD) under the A525, the drawing standard curve.
C) glycosaminoglycan assay
Get the 100ul cell conditioned medium, add developer 2.5ml, when 15s, put into spectrophotometer behind the mixing, measure optical density value (OD) under the A525.According to Chondroitin Sulfate A typical curve institute is surveyed the OD value and be scaled sample glycosaminoglycan content, the results are shown in Table 1 and Figure 11.
II Collagen Type VI and glycosaminoglycan expression amount (μ g/L) in the table 1 nucleus pulposus cell epimatrix
Can find out from table 1 and Figure 10, Figure 11, normal nucleus pulposus cell 1st generation and the 20th generation of immortalization nucleus pulposus cell, the synthetic not notable difference of both II Collagen Type VIs, normally nucleus pulposus cell is 29.89 ± 3.18, the immortalization nucleus pulposus cell is 33.49 ± 3.94.Glycosaminoglycan synthesizes does not have notable difference yet, and normal nucleus pulposus cell is 41.55 ± 2.80 μ g/L, and the immortalization nucleus pulposus cell is 47.96 ± 6.74 μ g/L.
The detected result of relative growth factor gene and extracellular fluid II Collagen Type VI, glycosaminoglycan shows in the above-mentioned cell, NP cell proliferation is accelerated behind the transfection hTERT, passed 20 generation cell have not yet to see agingly, II Collagen Type VI in the cell, glycosaminoglycan genetic expression are similar with normal nucleus pulposus cell 1st generation; Extracellular fluid II Collagen Type VI, glycosaminoglycan and normal nucleus pulposus cell 1st generation are similar.And 4ï½5 generations increment of the NP cell of untransfected hTERT biography has been stagnated substantially.The cell that surpassed for 20 generations that goes down to posterity is called as immortalized cells, and the immortalization nucleus pulposus cell behind the transfection hTERT shows as contact inhibition when passing 30 generations to 50 generation, and cell proliferation stops substantially.
4, NP cell Collagen-I, the dyeing of Collagen-II Immunofluorescence
Use thing: Anti-Collagen-I:Calbiochem (USA), Anti-Collagen-II:Calbiochem (USA), CY3: doctor's moral biotechnology company limited (Wuhan)
Qualitative analysis is carried out in method: Collagen-I, the dyeing of Collagen-II Immunofluorescence.Get the 20th generation the immortalization nucleus pulposus cell with 1 * 10 4/ ml is inoculated on the disinfection cap slide that the surface scribbles poly-lysine, and every inoculation 2ml is in 37 â, 5%CO 2, cultivate after 24 hours under 95% air, saturated humidity condition, the collecting cell creep plate is washed 3 times with the phosphate buffered saline buffer (PBS) of 0.02mmol/L, each 5min, 4% Paraformaldehyde 96 stationary liquid is 30min fixedly, with distillation washing 4 times, at every turn 5min termination.Use respectively 30%H 2O 210 parts of immersions of 1 part+pure methyl alcohol 10min blocking-up endogenous peroxidase activity, 0.3%Triton X-100 soaks 15min broken cell film, nonimmune lowlenthal serum sealing, add respectively the anti-human Collagen-I of monoclonal mouse, 1: 200 (dilution of 1% bovine serum albumin) 100ul of Collagen-II by the experiment grouping, put the wet box of 4 â of refrigerators and spend the night; 35 ± 1 â of incubator rewarming 30min, 0.02mmol/L add respectively sheep anti-mouse igg or the goat anti-rabbit igg 100ul of 1: 50 CY3 mark after the abundant rinsing of PBS by antibody sources, 35 ± 1 â of wet box lucifuge effect 30min of incubator, 0.02mmol/L the rinsing of PBS lucifuge is ended, the DAPI mounting, fluorescence microscope system, digital color ccd video camera and microimage analysing system are adopted figure.
As shown in figure 12, just transfection hTERT soon, the nucleus pulposus cell epimatrix dyes take the II Collagen Type VI as main; Pass after 20 generations, have not yet to see type i collagen and express (seeing Figure 13).And type i collagen had replaced II Collagen Type VI (seeing Figure 14, Figure 15) after normally nucleus pulposus cell passed for 4 generations.Immortalization nucleus pulposus cell the 20th generation type i collagen immunofluorescence dyeing is negative.
6, experimentation on animals
10 nude mouses are divided into 2 groups at random, 5 every group, are divided into experimental group and Normal group.Experimental group is with 1 * 10 7Individual/ml immortalization nucleus pulposus cell is planted under the right omoplate butt of nude mice, every some 0.2ml.Control group is planted normal nucleus pulposus cell under the right omoplate butt of nude mouse.1,2,3,4 weeks observed after the plantation, and nude mouse becomes the knurl situation as follows:
Experimental group: during 1 week, 1 * 1cm appears in the place at the right omoplate injection of nude mouse immortalization nucleus pulposus cell 2Large tag, matter is hard, mobility can.After 2 weeks, swollen thing slightly reduces when all than 1, approximately 1 * 0.5cm 2Size.Swollen thing obviously reduces after 3 weeks, approximately 0.3 * 0.3cm 2Size, matter is hard.After 4 weeks, swollen thing basic absorption under the right omoplate butt of nude mouse, about 0.1 * 0.1cm 2Size.
Control group: naked eyes generate there are no swollen thing.
Draw materials and carry out histopathologic examination.
7, histopathologic examination
After drawing materials, with the sample volume fraction be 4% neutral formalin liquid-solid fixed after, the decalcification of 100g/L formic acid, step by step ethanol dehydration â paraffin embedding â section â conventional hematoxylin-eosin staining â microscopically is observed situation and is made film, and observes its feature.
1st, 2,3,4 all histopathology studies show that: formed cell mass (seeing Figure 16) under the right omoplate butt of experimental group nude mouse; As shown in figure 17, there is red necrotic tissue in central authorities, have neutrophil leucocyte to invade profit on every side, and inflammatory cell forms cell packing structure, visible more fiber-like parent cell, and visible scavenger cell has no the cell atypia, generates without tumour.The control group naked eyes have no swollen thing and form, but after the section of drawing materials, and see still that cell mass forms (seeing Figure 18), inflammatory cell is invaded profit etc., are showed no the cell atypia.
In sum, the present embodiment constructs hTERT restructuring green fluorescence expression vector, the normal nucleus pulposus cell of successful transfection and stably express in cell.Transfection hTERT nucleus pulposus cell speed of growth compared with normal nucleus pulposus cell is fast, the cell of transfection hTERT can successfully pass more than 20 generations, compare with normal nucleus pulposus cell 1st generation, II Collagen Type VI, glycosaminoglycan gene expression amount no significant difference in the cell, extracellular fluid II Collagen Type VI, glycosaminoglycan be no significant difference also; The experiment of nude mouse tumorigenesis shows that the immortalization nucleus pulposus cell is without tumorigenicity.Show and use hTERT transfection nucleus pulposus cell successfully to make up immortalization nucleus pulposus cell system.
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