CN1731151A - Method for adipose-derived adult stem cell labeling by using exogenous green fluorescent protein (GFP) - Google Patents
Method for adipose-derived adult stem cell labeling by using exogenous green fluorescent protein (GFP) Download PDFInfo
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- CN1731151A CN1731151A CN 200510021580 CN200510021580A CN1731151A CN 1731151 A CN1731151 A CN 1731151A CN 200510021580 CN200510021580 CN 200510021580 CN 200510021580 A CN200510021580 A CN 200510021580A CN 1731151 A CN1731151 A CN 1731151A
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Abstract
The invention relates to a method for using an extrinsic green luminescence protein to label fat body stem cell. The method uses adenovirus vectors infection extrinsic green luminescence protein to get into the fat body stem cell of small mouse to displace original green luminescence protein GM small mouse fat body stem cell to label. It manifests that the Balb/c small mouse fat body stem cell of adenovirus vectors infection extrinsic green luminescence protein and the original green luminescence protein GM small mouse fat body stem cell have the same growth, value adding and diversity from morphology, mRNA and protein expressing level.
Description
One, technical field:
The present invention relates to a kind of new method of utilizing exogenous green fluorescent protein (GFP) (GFP) to carry out adipose-derived adult stem cell labeling, more particularly, relate to a kind of exogenous green fluorescent protein (GFP) that utilizes and be transfected in the Balb/c mouse adipose-derived adult stem cell, substitute the new method that endogenous gfp transgene mouse adipose-derived adult stem cell carries out cell marking by adenovirus vector.
Two, background technology:
Stem cell can be divided into embryonic stem cell and adult stem cell according to its source, and the research of embryonic stem cell is owing to be subjected to the constraint of ethics, ethnic idea and the influence of potential allosome rejection, and its application prospect is not very optimistic.
Adult stem cell is to be present in people and the mammiferous mature tissue organ, has self and certain multipotency differentiation potential, is in the intermediate between totipotent cell and the mature cell.Under field conditions (factors), adult stem cell tends to be divided into the various cells of place tissue, is beneficial to keep the eubolism of body; Induce down in specific external condition, a kind of adult stem cell of tissue can " laterally break up " functioning cell that becomes its hetero-organization, participates in the injury repair of tissue; The classical notion of " tissue specificity stem cell can only directed differentiation " has been rewritten in the discovery of " laterally differentiation " potential theoretically, for the cytology treatment of numerous disease provides new thinking and prospect.The research of adult stem cell is after major technological breakthroughs such as transgenic animals, cloned animal, human embryonic stem cell line foundation, the another field of obtaining bigger progress in the life science.Because adult stem cell has and can be used for the regulation and control of in vitro study and cell development differentiation associated gene in plurality of advantages such as manipulation in vitro; Explore cell differentiation, the molecular mechanism that tissue or organ form etc., thereby for generation, growth at cellular level research life, the fundamental mechanism of inquiring into vital movement at molecular level is significant.
Recently studies have shown that, really the adipose-derived adult stem cell that exists some to have multidirectional differentiation potential in the lipid substrate, this adipose-derived adult stem cell have the ability to a plurality of direction differentiation such as adipocyte, Gegenbaur's cell, chondroblast, myocyte and neurocyte.And adipose-derived adult stem cell has stable population doublings rate, have cytothesis and self potential and immune compatibility simultaneously, in genetic manipulation, be easy to infect adenovirus, slow virus, retrovirus, can become the desirable target cell of this viroid carrier.
Current research also shows, adipose-derived adult stem cell has wide material sources, draws materials conveniently, the stem cell amount is big, be easy to advantage such as separation and purification, is the potential maximum dry cell bank of human body.Now about the research of adipose-derived adult stem cell comprise mainly that cell separation is identified, differentiated result under the different condition and the research of differentiation mechanism.How disclosing the multidirectional molecular mechanism of inducing differentiation of adipose-derived adult stem cell from signal conduction and gene regulation angle is a important topic the stem-cell research, and in mechanism research for the multidirectional differentiation capability of adipose-derived adult stem cell, search out one efficiently, cheap, be convenient to the inside and outside trace labelling of observing of living cell body and just seem particularly important.What gain public acceptance now is to adopt the adipose-derived adult stem cell of gfp transgene mouse to carry out the method for cell marking.This cell has the endogenous green fluorescent protein, can be under fluorescent microscope vital movements such as the growth of observation of cell, propagation, differentiation intuitively, be present optimal adipose-derived adult stem cell labeling method.
But owing to cultivation, the breeding condition of gfp transgene mouse are very difficult, yet there are no this type of mouse at home breeds, also only there is a few experiments chamber to have abroad and produces the condition of breeding, but price is extremely expensive, every about 2000 dollars of the market prices of gfp transgene mouse, and the supply of goods and limited.Therefore, can't be applied to research on a large scale, hinder the research extensively and profoundly of adipose-derived adult stem cell inside and outside greatly.So the cell marking problem about adipose-derived adult stem cell never obtains desirable solution, also be one of main bottleneck of restricting current adipose-derived adult stem cell further investigation.
Green fluorescent protein is a kind of novel reporter molecules that is used to detect cellular gene expression and albumen location, do not need reaction substrate can produce fluorescence, and stable in properties, free of toxic effects can be carried out the real-time positioning of living cells and be observed (R. Y. Tsien .Annu Rev Biochem during as cell marking, 1998.67:509-544.), therefore be better than labeling method (D.A.Persons, Nat Med, 1998.4 (10): 1201-1205.) such as dye well Lac-Z such as DAPI, DIL.In the gene transfection technology, the most frequently used reporter gene mainly contains Lac-Z gene and green fluorescence protein gene.Couffinhal (T.Couffinhal, Hum Gene Ther, 1997.8 (8): 929-934) etc. discovery is used Lac-Z and is measured gene transfering efficiency as reporter gene, X-gal as the method for chromogenic substrate and can cause the result seriously on the low side, and it is former because the relevant enzyme protein active has been subjected to restriction of factors such as space structure and reaction conditions.Green fluorescent protein can directly be launched green fluorescence under excitation light irradiation, therefore avoided the problem of enzyme and catalytic activity, thereby reduced experimental error, has strengthened the susceptibility of experiment, and can make the separation that is labeled cell and measure process automation.
In numerous gene transfer vector instruments, (Adenovirus is a kind of high efficiency gene transfer vector Ad) to adenovirus, is widely used in importing foreign gene to mammalian cell.The molecule of adenovirus own is stable, intramolecular rearrangement does not take place in genome, pathological lesion to target cell is less, does not integrate with host genome, does not have genotoxicity, the foreign gene that inserts is also more stable, both can infect the cell of division stage, can infect the cell of repose period again, be better than retrovirus and other non-virus carrier transfer method, therefore, the present invention adopts adenovirus to make the transfer vector of exogenous green fluorescent protein (GFP) gene.
Three, summary of the invention:
At existing defective in the cell marking method of existing adipose-derived adult stem cell, the objective of the invention is to make up a kind of exogenous green fluorescent protein (GFP) (GFP) that utilizes is transfected in the Balb/c mouse adipose-derived adult stem cell by adenovirus vector, substitute endogenous gfp transgene mouse adipose-derived adult stem cell, and it is with low cost, easy and simple to handle, the new method of the adipose-derived adult stem cell labeling that the exogenous green fluorescent protein (GFP) transfer efficiency is high, this method can be applied to the adipose-derived adult stem cell labeling technology on a large scale, thereby promotes and promote the research extensively and profoundly of adipose-derived adult stem cell.
The technical scheme that purpose of the present invention is made of following measure realizes:
The present invention utilizes exogenous green fluorescent protein (GFP) to carry out the method for adipose-derived adult stem cell labeling, comprises the separating of Balb/c mouse and gfp transgene mouse adipose-derived adult stem cell, cultivation; Carry the preparation of the adenovirus vector (Ad-GFP) of green fluorescence protein gene; In Balb/c mouse adipose-derived adult stem cell, substitute the adipose-derived adult stem cell of endogenous gfp transgene mouse by adenovirus vector transfection green fluorescence protein gene; And from morphology, immunocytochemistry, growth, the increment of two kinds of adipose-derived adult stem cells, the similarity of differentiation are identified and analyzed in aspects such as mRNA and protein expression level.
In such scheme, the separating of Balb/c mouse and gfp transgene mouse adipose-derived adult stem cell, incubation: get Balb/c mouse and gfp transgene mouse, under conventional aseptic condition, cut the groin subcutaneous fat, after shredding, removing processing such as blood, filtration, adding and fatty isopyknic mass percent are 0.075% type i collagen enzyme in adipose tissue, process digestion separates adipose-derived adult stem cell α-MEM medium culture to 2～10 weeks that the back obtains, and promptly gets two kinds of adipose-derived adult stem cells.
In such scheme, carry the preparation of green fluorescence protein gene adenovirus vector:, transform homologous recombination bacterium BJ5183 cell jointly with conventional electroporation with viral skeleton plasmid with shuttle plasmid restriction enzyme I linearization for enzyme restriction; Transformed bacteria is inoculated in the LB culture plate that contains kanamycins and screens, and choosing colony extracts recombined adhenovirus green fluorescence protein gene adenovirus vector; Utilize 293 cells to carry out the amplification of green fluorescence protein gene adenovirus vector.
In such scheme, by adenovirus vector transfection green fluorescence protein gene in Balb/c mouse adipose-derived adult stem cell: the viral liquid and the third generation adipose-derived adult stem cell Mixed culture that will contain the adenovirus vector of green fluorescence protein gene are spent the night, the screening positive clone cell purification promptly obtains the adipose-derived adult stem cell of the Balb/c mouse of transfection exogenous green fluorescent protein (GFP) gene.
The present invention has the following advantages and good effect:
1, the present invention passes through the adenovirus vector transfection in Balb/c mouse adipose-derived adult stem cell with exogenous green fluorescent protein (GFP), its result is from morphology, immunocytochemistry, and mRNA and protein expression level etc. has proved that fully Balb/c mouse adipose-derived adult stem cell and gfp transgene mouse adipose-derived adult stem cell have growth about the same, propagation, differentiation characteristic.
2, why the present invention adopts into chondrocyte induction, be in order to prove in the Balb/c mouse adipose-derived adult stem cell of cell differentiation procedure transfer green colouring fluorescin and the similarity between the gfp transgene mouse adipose-derived adult stem cell, thereby illustrate fully can be with the transfection of cheapness the Balb/c mouse adipose-derived adult stem cell of green fluorescent protein substitute expensive gfp transgene mouse adipose-derived adult stem cell, carry out the research of adipose-derived adult stem cell inside and outside; The Balb/c mouse is dirt cheap, 2 yuans/only; And about 2000 dollars of transgenic mice/only; The structure first of green fluorescence protein gene adenovirus vector approximately needs 3000 Renminbi, and the reagent average price that each transfection is consumed is about 100 yuans; The research cost of adipose-derived adult stem cell is descended greatly.
3, the green fluorescence protein gene adenovirus vector of the present invention's structure can infinitely increase and use, and is with low cost, and domestic ordinary laboratory all can be finished.
4, the present invention found a kind of efficiently, cheap adipose-derived adult stem cell labeling method, establish experiment and learn the basis for furtheing investigate its differentiation mechanism; Compared adipose-derived adult stem cell atomization, the similarity of interior exogenous egfp expression from mRNA and protein expression level success first at home and abroad; This method also provides strong cell tracker technology for the inside and outside research of adipose-derived adult stem cell, will promote the further investigation of adipose-derived adult stem cell greatly.
Four, description of drawings
Fig. 1 is the adenovirus vector (Ad-GFP) of green fluorescence protein gene, with the synoptic diagram of Pac I restriction enzyme digestion and electrophoresis evaluation.
Among Fig. 1, M is λ DNA/Hind III marker; 1 is Ad-GFP (Pac I); 2 is Ad-GFP; The result confirms successfully to make up the adenovirus vector that carries green fluorescence protein gene.
Fig. 2 is Ad-GFP transfection Balb/c mouse adipose-derived adult stem cell and morphology HE dyeing relatively the synoptic diagram of GFP transgenic mice adipose-derived adult stem cell when 2 weeks and 10 weeks.
Among Fig. 2, (A) Ad-GFP transfection Balb/c mouse adipose-derived adult stem cell HE dyeing during 2 weeks, (B) Ad-GFP transfection Balb/c mouse adipose-derived adult stem cell HE dyeing during 10 weeks, (C) transgenic mice adipose-derived adult stem cell HE dyeing when 2 weeks, (D) GFP transgenic mice adipose-derived adult stem cell HE dyeing when 10 weeks; The morphology performance of the two kind adipose-derived adult stem cells of HE dyeing prompting when inducing back 2 weeks (A), (C) and inducing back 10 weeks (B), (D) is almost completely identical.
Fig. 3 is an Ad-GFP transfection Balb/c mouse adipose-derived adult stem cell and GFP transgenic mice adipose-derived adult stem cell result of GFP expression under Aggrecan immunocytochemical stain and the fluorescent microscope when becoming 2 weeks of chondrocyte induction and 10 weeks.
Among Fig. 3, the GFP-transfected gene of adenovirus vector is in Balb/c mouse adipose-derived adult stem cell, and after passing through into chondrocyte induction, not only Aggrecan highly expresses (A), same highly express (B) of GFP; And along with the prolongation of induction time, the expression of Aggrecan and GFP (C) and (D) weaken gradually;
The adipose-derived adult stem cell of GFP transgenic mice passes through into giving expression to Aggrecan (E) behind the chondrocyte induction equally, same highly express (F) of GFP; And along with the prolongation of time, (G), (H) also appear reducing gradually in These parameters, and the performance of Balb/c mouse adipose-derived adult stem cell in inducing the proliferation and differentiation process of this and Ad-GFP transfection is just the same.
Fig. 4 be Ad-GFP transfection Balb/c mouse adipose-derived adult stem cell with GFP transgenic mice adipose-derived adult stem cell when becoming 2 weeks of chondrocyte induction, 6 weeks and 10 weeks, become the Rt-PCR of cartilage index of correlation to detect.
Among Fig. 4, two kinds of adipose-derived adult stem cells break up the Rt-PCR testing result of back relevant identified gene Aggecan, SOX9, Col-I, Col-II, Col-X to the cartilage cell.Ad-GFP transfection Balb/c mouse adipose-derived adult stem cell (A) and GFP transgenic mice (B) fit like a glove in expression time, the intensity of passing through into before and after the chondrocyte induction; Proved absolutely that from the mRNA level the alternative cell that the Balb/c mouse adipose-derived adult stem cell of AD-GFP transfection can become GFP transgenic mice adipose-derived adult stem cell fully carries out cell tracker research.
Fig. 5 be Ad-GFP transfection Balb/c mouse adipose-derived adult stem cell with GFP transgenic mice adipose-derived adult stem cell when becoming 2 weeks of chondrocyte induction, 6 weeks and 10 weeks, become the Western blot of cartilage index of correlation to detect.
Among Fig. 5, the Balb/c mouse adipose-derived adult stem cell (A) that Western blot has detected the Ad-GFP transfection is passing through behind chondrocyte induction with GFP transgenic mice adipose-derived adult stem cell (B), the quantitative expression of Col-I and Col-II collagen, the result confirms that the two protein expression fits like a glove; Thereby illustrated that from protein level the Balb/c mouse adipose-derived adult stem cell of Ad-GFP transfection can become the alternative cell of the adipose-derived adult stem cell of GFP transgenic mice, to carry out relevant cell tracker of adipose-derived adult stem cell and inside and outside research.
Five, embodiment
The present invention is further illustrated with embodiment below.
Embodiment
Used main material and reagent in the embodiment of the invention:
Material therefor:
4-6 week Balb/c male mice (Sichuan University's Experimental Animal Center provides); Gfp transgene mouse (Huaxi Hospital Attached to Sichuan Univ ophthalmology and hereditary disease laboratory provide)
Agents useful for same:
α-MEM nutrient culture media, pancreas enzyme-EDTA, HEPES (U.S. Gibco company);
Hyclone (FBS) (U.S. Hyclone company);
Type i collagen enzyme, bovine serum albumin(BSA) (BSA), kerbs-Lin Geshi bicarbonate saline solution (KRB) ascorbic acid, insulin, TGF-β 1 (U.S. Sigma company);
Shuttle plasmid pAdTrack-CMV, viral skeleton plasmid pAdEasy-1, Escherichia coli BJ5183 and DH5 α, human embryo kidney (HEK) 293 cell lines (Huaxi Hospital Attached to Sichuan Univ ophthalmology and hereditary disease laboratory provide);
Trypsase (U.S. Promeaga company);
The anti-mouse Aggrecan of rabbit associated antibodies (Wuhan doctor's moral company);
Trizol RNA extracts kit (U.S. Gibco company);
Dna molecular amount, Taq enzyme, dNTP, PCR kit (Lithuania MBI company).
Used instrument and equipment:
CO
2Incubator (U.S. Forma Scientific company);
Tissue Culture Flask (U.S. Corning company);
Super quiet worktable (Suzhou purifying apparatus instrument factory);
The IX70 type is inverted and is differed fluorescent microscope (Japanese OLYMPUS company);
Stereoscopic dissecting microscope (optical instrument factory, Chongqing);
Flow cytometer (U.S. Coulter company);
Supercentrifuge (Japanese Sanyo company);
Electrophoresis apparatus (U.S. BIO-RAD company).
1, the separating of Balb/c mouse adipose-derived adult stem cell and gfp transgene mouse adipose-derived adult stem cell, incubation:
Get 32 of 4-6 week Balb/c male mices, 1% yellow Jackets (30mg/Kg) intraperitoneal injection of anesthesia cuts the about 1mL of groin subcutaneous fat under the conventional aseptic condition, be dipped in the physiological saline; Machinery is cut apart and tissue digestion, in superclean bench fat is shredded, and removes blood with the KRB flushing of fatty two volumes; After 100 mesh filter screens filtered, adding and fatty isopyknic mass percent were 0.075% type i collagen enzyme in the adipose tissue, and 37 ℃ of constant temperature stir 60min, and 200 mesh filter screen eliminations do not digest piece of tissue completely; (cell mass suspends with α-MEM low-speed centrifugal again for 300g * 5min), collecting cell group, and the centrifugal 3 * 5min of 300g again is with abundant removal clostridiopetidase A; Adipose-derived adult stem cell suspends with α-MEM nutrient culture media (including 10% hyclone), is inoculated in 25cm
2In the culture flask, insert temperature and be 37 ℃, volume fraction and be 5% CO
2Cultivate in the incubator, every other day is changed liquid.As cellular incubation 7～10d, cell can reach 80% and merge, and the nutrient culture media in the culture flask that inclines adds D-Hanks damping fluid 5ml cleaning hypsokinesis and goes repeated washing 3 times.Add 0.25% trypsase 1ml digestion, 2～3min afterwards, microscopically is observed and to be seen that cell begins to occur cell space and shrinks to add and contain blood serum medium 2ml and stop digestion, blow and beat attached cell repeatedly with nutrient culture media in the suction pipe absorption culture flask, to 80% above cell detachment, piping and druming is prepared into single cell suspension repeatedly then.Carry out after the cell count according to 1 * 10
4The density of/ml is inoculated in the plastic culture bottle, is labeled as the first generation on the culture flask, and the back 2h that goes down to posterity promptly changes liquid once, changed liquid once in per three days later on, treat that attached cell is merged each other and repeat aforesaid operations when 80% converges, the amplification of going down to posterity repeatedly, be labeled as second, third, the 4th generation etc.In 2～10 weeks of cellular incubation, promptly obtain the adipose-derived adult stem cell of Balb/c mouse and gfp transgene mouse.
2, carry the preparation of green fluorescence protein gene adenovirus vector:
With shuttle plasmid (pAdTrack-CMV) restriction enzyme Pme I linearization for enzyme restriction, transform homologous recombination bacterium BJ5183 cell jointly with viral skeleton plasmid (pAdEasy-1) conventional electroporation under 2.5kv, 2000hms, 25 μ F conditions; Transformed bacteria is inoculated in the screening of spending the night in the LB culture plate that contains kanamycins, and choosing colony extracts recombined adhenovirus green fluorescence protein gene adenovirus vector, cuts the identification mark segment with Pac I enzyme, identifies that the back electroporation transforms DH5 α bacterium and increases in a large number.With 293 cells with 2 * 10
8The density of/L is inoculated in 25cm
2In the culture flask, when cell grows to 80% fusion behind the 24h, 4 μ g are inoculated in 293 cell culture mediums through the green fluorescence protein gene adenovirus vector of Pac I linearization for enzyme restriction, remove nutrient solution behind the 6h, add α-MEM nutrient culture media, collecting cell and nutrient culture media when treating cytopathies such as cell appearance circle contracts, comes off, multigelation is 3 times then, centrifuging and taking supernatant results virus, through conventional cesium chloride density gradient centrifugation purifying ,-70 ℃ of temperature in hyclone dilution back are preserved standby.
3, pass through adenovirus vector transfection green fluorescence protein gene in Balb/c mouse adipose-derived adult stem cell:
The viral liquid and the Balb/c mouse third generation adipose-derived adult stem cell Mixed culture that will contain the adenovirus vector of green fluorescence protein gene are spent the night, the screening positive clone cell carries out purifying, promptly obtains the adipose-derived adult stem cell of the Balb/c mouse of transfection exogenous green fluorescent protein (GFP) gene.
4, induce the Balb/c mouse adipose-derived adult stem cell of Adenovirus Transfection green fluorescent protein and gfp transgene mouse adipose-derived adult stem cell to break up and evaluation to chondroblast:
Spend the night with Ad-GFP virus liquid and Balb/c mouse the 3rd fat subsitutes adult stem cell Mixed culture, scrape with aseptic cell scraper and to get the positive colony cell and carry out purifying amplification back as experimental group, get gfp transgene mouse adipose-derived adult stem cell the 3rd generation cell and organize in contrast, all with 2 * 10
7The density of individual/L is inoculated in 24 orifice plates, adds into chondrocyte induction liquid (ascorbic acid, the 10 μ g/L TGF-β that contain 6.25mg/L insulin, 0.05mmol/L
1And contain the α-MEM nutrient culture media of 10% hyclone) cultivate; Cell in 24 orifice plates 2,6, the 10w collecting cell carries out HE dyeing, Aggrecan immunocytochemical stain, Rt-PCR and Western Blot detection.
1) two kinds of mouse adipose-derived adult stem cells induce back morphology and immunocytochemistry to identify:
Morphology after two kinds of mouse adipose-derived adult stem cells break up is observed by inverted microscope.Treat that cell reaches 80% when merging, take out cell climbing sheet, the PBS washing, 4% paraformaldehyde is fixed, and simply clean the back to immerse volume fraction be 15min among 0.3% the TritonX-100, distilled water flushing, volume fraction is 0.03 H
2O
2Incubated at room 10min, PBS soaks 5min, be to seal 30min under 10% the lowlenthal serum room temperature with volume fraction again, the serum deprivation that inclines, dripping respectively with bSA (BSA) dilution is that 1: 1000 the anti-mouse Aggrecan of rabbit antibody (one resists) temperature is spent the night for 4 ℃; 37 ℃ of rewarming 1h, PBS washes 3 times * 5min, drip 1: 100 biotin labeled goat-anti rabbit (two is anti-) again, hatch 30min for 37 ℃, again 3 times * 5min of PBS flushing, drip 1: the 100ABC compound is hatched 30min for 37 ℃, also with 3 times * 5min of PBS flushing, DAB develops the color, and tap water fully washes, mounting places under the optical microscope and observes.
2) RT-PCR detects the cartilage index of correlation:
1. total RNA extracts:
The extraction of total RNA is extracted kit instructions extraction step according to the TRIZOL RNA of Gibco company and is extracted two groups of total RNA of adipose-derived adult stem cell, gets total RNA of blank group adipose-derived adult stem cell simultaneously, and experimental technique is identical with experimental group with condition.Step is as follows:
A) centrifugal behind the D-Hanks liquid centrifuge washing cell 2 times, supernatant inclines.
B) add 1ml Trizol Reagent, suction pipe washes centrifuge tube repeatedly, and cell is dissolved fully.
C) the Trizol Reagent that will be dissolved with cell transfers in the EP pipe of 1.5mlDEPC processing, and 4 ℃, 12000 * centrifugal 10min moves to the EP pipe that DEPC handles with supernatant, hatches 5min under the room temperature.
D) add the 0.2ml chloroform in the supernatant, thermal agitation fully behind the mixing, is hatched 3min under the room temperature, and 4 ℃, the centrifugal 15min of 12000 * g.Liquid is divided into three layers in the EP pipe, the upper strata water, middle mutually with lower floor phenol～chloroform mutually.Lower floor's phenol～chloroform places 4 ℃ of refrigerators to wait until the extraction total protein mutually.
E) the careful water that contains RNA of drawing is transferred in the EP pipe that another DEPC handles, and adds the 0.5ml isopropyl alcohol, leaves standstill incubated at room 10min, 4 ℃, the centrifugal 10min of 12000 * g behind the mixing.Supernatant is discarded, and pipe end precipitation is RNA.
F) add the 75% ethanol 1ml that DEPC handled, put upside down washing EP tube wall gently, carefully discard ethanol, clean twice, 4 ℃ of RNA, the centrifugal 5min of 7500 * g removes supernatant, air dry RNA precipitation in superclean bench.50 μ lDEPC handle water-soluble separating, and 5 μ l make optical density and the ratio thereof of 1% agarose gel electrophoresis and ultraviolet light spectrophotometric determination 260nm, 280nm.Measure RNA concentration and purity thereof, be splined on 1.2% agarose gel electrophoresis, the ethidium bromide colour developing, whether the uviol lamp observation has or not RNA and RNA to degrade.With the RNA sample retention in-80 OC.If total RNA purity of extracting is not enough, then need be further purified.
2. reverse transcription:
Promptly 2 weeks, 6 weeks and 10 all two kinds of total RNA of adipose-derived adult stem cell that extract carry out reverse transcription with different time after becoming chondrocyte induction, this reverse transcription product is as the pcr amplification template, adopt the existing One-Step RT-PCR kit of MBI company, on the PTC-2000PCR instrument, carry out reverse transcription to specifications
3. design of primers:
β-actin 5’-ACTCTTCCAGCCTTCCTTCC-3’
5’-ACTCGTCATACTCCTGCTTGC-3’
SOX9 5’-GAACGCACATCAAGACGGAG-3’
5’-TCTCGTTGATTTCGCTGCTC-3’
Aggrecan 5’-GCAGAGACGCATCTAGAAATTG-3’
5’-GGTAATTGCAGGGAACATCATT-3’
Col-I 5’-CATCTCCCCTTCGTTTTTGA-3’
5’-CTGTGGAGGAGGGTTTCAGA-3’
Col-II 5’-TTCAGCTATGGAGATGACAATC-3’
5’-AGAGTCCTAGAGTGACTGAG-3’
Col-X 5’-CACCAGGCATTCCAGGATTCC-3’
5’-AGGTTTGTTGGTCTGATAGCTC-3’
4. PCR reaction:
Above-mentioned reverse transcription product is carried out pcr amplification, and reaction product is carried out the observation of 2% agarose gel electrophoresis and is taken a picture, and the pcr amplification operation steps is as follows: 0 ℃ of following solution of following premix is in the PCR reaction tube:
Reagent application of sample amount
10×buffer(Mg
2+free) 3μl
dNTPs 0.75μl
Taq enzyme 0.25 μ l
MgCl
2 3μl
ddH
2O 19μl
----------------------------------------
Cumulative volume 30 μ l
Each different time points i.e. 8 reaction tubes that amount to of two groups of cells in 0 week, 2 weeks, 6 weeks and 10 weeks adds to put after the mineral oil 30 μ l sealing and carries out amplified reaction in the pcr amplification instrument.The pcr amplification condition:
a)94℃?2min
B) 94 ℃ of 10sec, 55 ℃ of 30sec, 72 ℃ of 1min, 35 circulations
c)72℃?5min
D) 4 ℃ of insulations
Reaction product is got 8 μ l make 1.5% conventional agarose gel electrophoresis, observe and photograph.
3) extract albumen (Western Blot)
Get the phenol-chloroform phase solution of keeping somewhere in the total RNA step of said extracted, add the 1.5ml isopropyl alcohol, leave standstill 10min to room temperature behind the mixing, 12000 commentaries on classics/min are centrifugal, drawing supernatant washs 3 times with the guanidine hydrochloride ethanolic solution, drying precipitated after the abandoning supernatant, use the 1%SDS dissolution precipitation, get supernatant and carry out electrophoretic separation through the 10%SDS-PAGE gel, change film through electricity, after the sealing of 5% skimmed milk power, resist (1: 400) under 37 ℃, to hatch 45min more with goat-anti Col II and Col I, after 1 * TBST cleans, the goat anti-rabbit igg that adds horseradish peroxidase-labeled in 37 ℃ of reaction 45min down, develops the color with DAB after washing film, take pictures and gray scale scanning, by the expression of cell ColII and Col I in the Western Blot method half-quantitative detection co-incubation different time group.
Result of the present invention:
(1) evaluation of recombinant adenoviral vector Ad-GFP:
Ad-GFP cuts the back with Pac I enzyme and obtains the adenoviral gene group segment of 30kb and replication origin (ori) and the kalamycin resistance encoding gene segment of 3.0kb, confirms to have obtained recombined adhenovirus (Fig. 1).
(2) the adipose-derived adult stem cell morphological observation of Ad-GFP transfection Balb/c mouse adipose-derived adult stem cell and GFP transgenic mice:
Two kinds of former situations of supporting of being commissioned to train of adipose-derived adult stem cell are similar: visible a large amount of round cell in the cell suspension that the former foster digestion of being commissioned to train is obtained, and contain a spot of cell fragment; Inoculation back 6～24h, visible cell begins adherent, and cell is fusiformis, and cell space is less, single nuclear; Inoculation back 24～48h, attached cell quantity increases, and a few cell is the growth of colony sample, and cell is fusiformis, irregular shape etc., and cell volume increases gradually; Inoculation back 72h, the attached cell One's name is legion, part is assembled agglomerating, and cell is typical fibroblast sample growth; Cultivate 7～10d, cell can reach 80% and merge, and cell is grown in flakes, and cell is in strip, fusiformis and a small amount of polygonal sexual cell.
16h can see egfp expression behind the Ad-GFP infecting mouse fat stem cell, increases gradually later on, and week back expression tends towards stability.
(3) two kinds of adipose-derived adult stem cell cells become cartilage phenotype to identify after the directional induction of one-tenth cartilage:
Through becoming continuously chondrocyte induction to cultivate 14d, two kinds of fat stem cells have become polygon by the spindle shape in former generation, and cell volume obviously increases and stretches out pseudopodium, and the secretion (Fig. 2) of the extracellular polysaccharide in the similar cartilaginous tissue is arranged around the HE dyeing showed cell.Immunocytochemical stain can be observed the formation (Fig. 3) that there is aggrecan in the intensive place of cell.
(4) two kinds of fat stem cell processes induce the RT-PCR of the one-tenth cartilage related gene of back expression to detect:
SOX9, Aggrecan, Col-I, Col-II, Col-X are the relevant specific genes of cartilage.In the present invention, carrying endogenous still is that the adipose-derived adult stem cell of exogenous green fluorescent protein (GFP) has all highly been expressed these several genes after inducing.And not said gene expression (Fig. 4) in negative control group; Western blot result confirms endogenous and ectogenous fat adult stem cell secretion Col-I, the Col-II (Fig. 5) after inducing; The adipose-derived adult stem cell that no matter carries endogenous and exogenous green fluorescent protein (GFP) is in inducing process, the morphology of cell, immunocytochemistry, mRNA level, protein level all show the consistance of height, proved absolutely that the adipose-derived adult stem cell that can substitute the gfp transgene mouse fully with the Balb/c mouse adipose-derived adult stem cell of adenovirus vector transfection green fluorescent protein carries out cell marking, tracking, thereby carried out the further investigation of adipose-derived adult stem cell inside and outside.
Claims (4)
1, a kind of method of utilizing exogenous green fluorescent protein (GFP) (GFP) to carry out adipose-derived adult stem cell labeling is characterized in that comprising the separating of Balb/c mouse and gfp transgene mouse adipose-derived adult stem cell, cultivation; Carry the preparation of the adenovirus vector (Ad-GFP) of green fluorescence protein gene; By adenovirus vector transfection green fluorescence protein gene in Balb/c mouse adipose-derived adult stem cell, substitute the adipose-derived adult stem cell of endogenous gfp transgene mouse: and from morphology, immunocytochemistry, growth, the increment of two kinds of adipose-derived adult stem cells, the similarity of differentiation are identified and analyzed in aspects such as mPNA and protein expression level.
2, in accordance with the method for claim 1, it is characterized in that separating of Balb/c mouse and gfp transgene mouse adipose-derived adult stem cell, incubation: get Balb/c mouse and gfp transgene mouse, under conventional aseptic condition, cut the groin subcutaneous fat, through shredding, remove blood, after the filtration treatment, adding and fatty isopyknic mass percent are 0.075% type i collagen enzyme in adipose tissue, process digestion separates adipose-derived adult stem cell a-MEM medium culture to 2～10 weeks that the back obtains, and promptly gets two kinds of mouse adipose-derived adult stem cells.
3, in accordance with the method for claim 1, it is characterized in that carrying the preparation of green fluorescence protein gene adenovirus vector:, transform homologous recombination bacterium BJ5183 cell jointly with conventional electroporation with viral skeleton plasmid with shuttle plasmid restriction enzyme I linearization for enzyme restriction; Transformed bacteria is inoculated in the LB culture plate that contains kanamycins and screens, and choosing colony extracts recombined adhenovirus green fluorescence protein gene adenovirus vector; Utilize 293 cells to carry out the amplification of green fluorescence protein gene adenovirus vector.
4, in accordance with the method for claim 1, it is characterized in that by adenovirus vector transfection green fluorescence protein gene in Balb/c mouse adipose-derived adult stem cell: the viral liquid and the third generation adipose-derived adult stem cell Mixed culture that will contain the adenovirus vector of green fluorescence protein gene are spent the night, the screening positive clone cell purification promptly obtains the adipose-derived adult stem cell of the Balb/c mouse of transfection exogenous green fluorescent protein (GFP) gene.
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| CN103439500A (en) * | 2013-08-27 | 2013-12-11 | 陕西科技大学 | Visible tracking and detecting method for protease in leather treatment process |
| CN103920189A (en) * | 2014-04-24 | 2014-07-16 | 刘毅 | Method of constructing engineering fat |
| CN106075585A (en) * | 2016-07-08 | 2016-11-09 | 福建师范大学 | A kind of preparation method based on artificial tendon scaffold materials microstructure through engineering approaches artificial tendon graft |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1348012A (en) * | 2000-10-11 | 2002-05-08 | 中国科学院水生生物研究所 | Double fluorescent screening process for fixed point integration of foreign gene |
| IL156410A0 (en) * | 2000-12-27 | 2004-01-04 | Axiogenesis Ag | System for the cell-specific and development-specific selection of differentiating embryonic stem cells, adult stem cells and embryonic germline cells |
| AU2003293319A1 (en) * | 2002-09-27 | 2004-05-04 | Cardion Ag | Expression vectors for selection from stem cells of differentiated cardiomyocytes |
| WO2005026335A2 (en) * | 2003-09-15 | 2005-03-24 | Ramot At Tel Aviv University Ltd. | Insulin-producing bone marrow derived cells and methods of generating and using same |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103091294A (en) * | 2013-01-21 | 2013-05-08 | 中南民族大学 | Cell marking detection method applied to motion trail tracking of bone marrow stem cell |
| CN103439500A (en) * | 2013-08-27 | 2013-12-11 | 陕西科技大学 | Visible tracking and detecting method for protease in leather treatment process |
| CN103439500B (en) * | 2013-08-27 | 2015-01-28 | 陕西科技大学 | Visible tracking and detecting method for protease in leather treatment process |
| CN103920189A (en) * | 2014-04-24 | 2014-07-16 | 刘毅 | Method of constructing engineering fat |
| CN106075585A (en) * | 2016-07-08 | 2016-11-09 | 福建师范大学 | A kind of preparation method based on artificial tendon scaffold materials microstructure through engineering approaches artificial tendon graft |
| CN106075585B (en) * | 2016-07-08 | 2019-06-21 | 福建师范大学 | A kind of preparation method based on artificial tendon scaffold materials microstructure engineering artificial tendon graft |
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| CN100420936C (en) | 2008-09-24 |
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