CN106520836A

CN106520836A - Method for constructing rat bone mesenchymal stem cells (BMSCs) cell strain for expressing c-met protein through regulation of doxycycline

Info

Publication number: CN106520836A
Application number: CN201611020740.7A
Authority: CN
Inventors: 朱传龙; 李毓雯; 王坤; 李军; 金柯; 岳明; 朱甜甜
Original assignee: Jiangsu Province Hospital
Current assignee: Jiangsu Nahua Biotechnology Co ltd
Priority date: 2016-11-14
Filing date: 2016-11-14
Publication date: 2017-03-22
Anticipated expiration: 2036-11-14
Also published as: CN106520836B

Abstract

The invention discloses a method for constructing a rat bone mesenchymal stem cells (BMSCs) cell strain for expressing c-met protein through regulation of doxycycline. The method comprises the steps that an rtTA gene lentiviral expression vector is constructed and subjected to transfection into BMSCs so that a BMSCs cell strain (a Tet-On Advanced BMSCs cell strain) for stably and efficiently expressing rtTA protein can be obtained; then, a Len-TRE min CMV-c-met/ALB-GFP lentiviral vector is constructed and subjected to transfection into the Tet-On Advanced BMSCs, and a flow cytometry is used for screening GFP positive BMSCs; and finally the GFP (+) Tet-on-c-met BMSCs cell strain is obtained. The doxycycline can be added into the obtained BMSCs cell strain to induce the obtained BMSCs to express the c-met protein, and the method has important significance for study on blood doping of the cell strain for treating hepatic failures, the directional homing and differentiation capacities of the BMSCs along with the HGF concentration and the dose-effect relationship between the capacities and the HGF/c-met axis expression level.

Description

Build the rat bone marrow mesenchymal stem cellses of doxycycline regulating and expressing c-met albumen The method of cell strain

Technical field

A kind of the present invention relates to technical field of cell biology, more particularly to structure doxycycline regulating and expressing c-met albumen Rat bone marrow mesenchymal stem cellses cell strain method.

Background technology

Gene therapy is that the exogenous gene with preventing and treating potential is thin by the related organization that suitable carrier is transferred to patient Intracellular, and appropriate expression is obtained, with the purpose for reaching preventing and treating or mitigating disease.About gene therapy liver failure research we Do a lot of work, and detailed overview has been carried out to its present Research and prospect.Gene therapy at present has two main paths, i.e., In vivo and ex vivo.In vivo approach is referred to and for exogenous gene to be assemblied in specific eukaryotic expression vector (virus type Or non-viral type) on, it is introduced directly in human body, because limiting its clinical application range the problems such as transfection efficiency and safety； Ex vivo approach is referred to and for the carrier containing exogenous gene to import human body itself or variant cell, Jing vitro cell expansions in vitro Feed back internal afterwards, this kind of method is combined with stem cell transplantation the stem cell transplantation after genetic modification.Mediated gene transfer Carrier is generally divided into viral vector and non-virus carrier, and current viral vector has higher transfection efficiency, in stem cell transplantation Using most in research.But as after street virus vectors into cells, destination gene expression amount and expression time are difficult to control to.

Gossen etc. constructs gene of eucaryote cell controllability expression system using prokaryotic gene controlling element earliest, i.e., Tet systems.The system is made up of two parts：1) Tet reactive components (tetracycline responsive element, TRE), by antibacterial tetracycline operator DNA sequence (Tet operator DNA sequence, TetO) and the startup of 7 copies Son composition；2) transcriptional activators for merging, tTA (tet-controlled transcriptional activator) or rtTA(reverse tet-controlled transcriptional activator).In tTA and rtTA TetR compositions with TetO sequences are combined, and play transcriptional activity by VP16.TTA is tied with above-mentioned reactive component when its inducible factor is not present Close, start genes of interest transcription；After adding inducible factor, inducible factor is combined with tTA makes which come off from TRE, terminates purpose Genetic transcription.And the model of action of rtTA with tTA's conversely, start the transcription of genes of interest when inducible factor is added, when luring After inducement is removed from system, the transcription of genes of interest is closed.Both systems can in the presence of exogenous inducing factors The expression or closing of manual control genes of interest, therefore it is hereinafter referred to as Tet-off/Tet-on systems.At present using most The Tet-on advanced doxycycline regulator control systems that Tet-on systems are provided for Clontech companies, the system and other bases Because regulator control system is compared, its controllability and destination gene expression efficiency have greatly raising.But Tet-on advanced systems The controllability of system is mainly reflected in the control of destination gene expression time, for the position or cell category expressed lack control, Therefore in genetic modification stem-cell therapy targeting control research field also existing defects.Have not yet to see with regard to building how western ring Element regulation and control c-met protein expressions, the research of the rat bone marrow mesenchymal stem cellses strain of ALB promoteres control GFP expression and report.

The content of the invention

For the problems referred to above, the present invention provides a kind of rat bone that can pass through doxycycline regulating and expressing c-met albumen The method of bone marrow-drived mesenchymal stem cell strain, goes back to the nest and divides along HGF concentration orientation during for studying the cell strain treatment liver failure The research of the dose-effect relationship between change ability, and the ability and HGF/c-met axle expressions, the present invention are obtained by 's：

A kind of method of the rat bone marrow mesenchymal stem cellses cell strain for building doxycycline regulating and expressing c-met albumen, Characterized in that, comprising the following steps that：

1st, 4 week old SPF level SD male rats are chosen, extracts and cultivate BMSCs；

2nd, Tet-On Advanced BMSCs cell strains are set up：

2.1 respectively with SEQ ID NO.1 and SEQ ID NO.2 as upstream and downstream primer, with pTet-On dvanced Vector carriers are template, enter performing PCR amplification, obtain genes of interest CMV-rtTA；

2.2, with restricted enzyme BamHI, AgeI enzyme action slow virus carrier GV211, react 3h, digestion products in 37 DEG C As linearized vector DNA；

Enzyme action system：2 μ l of 10 × CutSmart Buffer, 5 μ l, 1 μ g/ μ L slow virus carriers GV211, the limit of 10U/ μ l Property restriction endonuclease BamHI, AgeI enzyme 1 μ l, ddH processed₂O complements to 50 μ l；

2.3 prepare linearized vector DNA and genes of interest recombining reaction system, react 30min in 37 DEG C, obtain slow viruss Expression vector GV211-CMV-rtTA；

2.4 add 20 μ g of GV211-CMV-rtTA, pHelper1.0 15 μ g, pHelper2.0 10 μ g in centrifuge tube, Complemented to 2000 transfection reagents of Invitrogen Lipofectamine 15min is incubated under 1ml, room temperature；Then instill cell During density is 70%～80% 293T cells, in 37 DEG C, 5%CO₂Cell culture incubator culture 6h, discard culture medium, add Containing 10% serum low sugar culture medium, in 37 DEG C, containing 5%CO₂Cell culture incubator continue culture 48～72h；

293T cell supernatants after the culture of 2.5 collection step 2.4, in 4 DEG C, 4000g is centrifuged 10min；Take supernatant with 0.45 μm of filter is filtered, and regathers filtrate in 4 DEG C, and 25000rpm is centrifuged 2h；Supernatant discarded, adds virus to preserve liquid, that is, obtains Len-CMV-rtTA slow viruss；

2.6 are inoculated in the BMSCs of exponential phase in 96 orifice plates, add 4～5 × 10 per hole⁴Individual cell and 100 μ l contain 10% serum low sugar culture medium, when cell fusion degree reaches 30% or so, discards culture medium, adds 10 μ l steps in each hole 2.5 virus titers for obtaining are 2 × 10⁸The Len-CMV-rtTA slow viruss of TU/ml, 10 μ l final concentration of 5 μ g/ml Ploybrene strengthen liquid and 80 μ l containing 10% serum low sugar culture medium, in 37 DEG C, 5%CO₂Cell culture incubator culture；

After infection 12h, culture medium is discarded, add per hole 100 μ l to contain 10% serum low sugar culture medium；After infection 72h, again To change liquid containing 10% serum low sugar culture medium, and the puro of final concentration of 9 μ g/ml is added to carry out drug screening to every hole, Viable cells are Tet-On Advanced BMSCs；

3rd, GFP (+) Tet-on-c-met BMSCs cell strains are set up：

3.1 synthetic gene sequence TRE-minCMV promoter-c-met-ALB promoter-GFP；

3.2 prepare linearized vector DNA and genes of interest recombining reaction system：5 × CE II Buffer, 2 μ l, step 2.2 concentration for obtaining are 2.5 μ l of 800ng/ μ L linearized vectors DNA, TRE- of the concentration that step 3.1 is obtained for 800ng/ μ L 1 μ l of minCMV promoter-c-met-ALB promoteres-GFP, 1 μ l of Exnase II, ddH₂O complements to 10 μ l；

Reaction system is placed in and 30min is reacted in 37 DEG C, obtain Lentiviral

GV211-TRE minCMV-c-met/ALB-GFP；

Recombining reaction system：

3.3 add in centrifuge tube 20 μ g of GV211-TRE minCMV-c-met/ALB-GFP, pHelper1.0 15 μ g, 10 μ g of pHelper2.0, complement to 1ml with 2000 transfection reagents of Invitrogen Lipofectamine, are incubated under room temperature 15min；Then instill during cell density is 70%～80% 293T cells, in 37 DEG C, 5%CO₂6h is cultivated in incubator, is abandoned Culture medium is gone, is added containing 10% serum low sugar culture medium, in 37 DEG C, containing 5%CO₂Continue culture 48-72h in incubator；3.4 receive 293T cell supernatants after collection step 3.3 culture, in 4 DEG C, 4000g is centrifuged 10min；Supernatant is taken with 0.45 μm of filter mistake Filter, regathers filtrate in 4 DEG C, and 25000rpm is centrifuged 2h；Supernatant discarded, adds virus to preserve liquid, that is, obtain slow viruss Len- TRE minCMV-c-met/ALB-GFP, its virus titer are 2 × 10⁸TU/ml；

3.5 are inoculated in the Tet-On Advanced BMSCs of exponential phase in 96 orifice plates, per hole add 4～5 × 10⁴Individual cell and 100 μ l contain 10% serum low sugar culture medium, when cell fusion degree reaches 30% or so, discard culture medium, to 10 μ l Len-TRE minCMV-c-met/ALB-GFP slow viruss, the final concentration of 5 μ g/ml of 10 μ l is added in each hole Ploybrene strengthen liquid and 80 μ l containing 10% serum low sugar culture medium, in 37 DEG C, 5%CO₂Cell culture incubator culture；

After infection 12h, culture medium is discarded, add per hole 100 μ l to contain 10% serum low sugar culture medium；After infection 72h, again Liquid is changed containing 10% serum low sugar culture medium, and add the puro of final concentration of 9 μ g/ml to every hole；By fluidic cell Instrument screens GFP positive BMSCs cells, that is, obtain GFP (+) Tet-on-c-met BMSCs cell strains；

Wherein, the 10% serum low sugar culture medium compound method that contains is：By quality very in terms of, by 89% DMEM/ Low culture medium, 10% hyclone and 1% Pen .- Strep solution mixing after obtain.

Further, the rat marrow mesenchyme for building doxycycline regulating and expressing c-met albumen as described herein is dry thin In the method for born of the same parents' cell strain, the cell density is that 70%～80% 293T cells are obtained by：24h before transfection, uses The 293T cells of trypsinization exponential phase, to adjust cell density as 5x10 containing 10% serum low sugar culture medium⁶Carefully Born of the same parents/15ml, are inoculated in the Tissue Culture Dish of a diameter of 10cm, in 37 DEG C, containing 5%CO₂Incubator in culture, treat that cell is close Can be used to transfect when degree is up to 70%～80%；And 2h replaces medium to serum-free medium before transfection.

Further, the rat marrow mesenchyme for building doxycycline regulating and expressing c-met albumen as described herein is dry thin In the method for born of the same parents' cell strain, enter performing PCR amplification described in step 2.1 and refer to：

PCR reaction systems：The 4 μ l of dNTP Mix of 5 × PS Buffer, 10 μ l, 2.5mM each, 10 μM of forward primer 1 μ l, 10 μM of 1 μ l of downstream primer, the 1 μ l of template of 10ng/ μ L, 0.5 μ l of PrimeSTAR HS DNA polymerase, ddH₂O complements to 50 μ l；

PCR reaction conditions：98 DEG C of preheating 5min；98 DEG C of 10s, 55 DEG C of 10s, 72 DEG C of 90s, totally 30 circulations；4 DEG C of extensions.

Further, the rat marrow mesenchyme for building doxycycline regulating and expressing c-met albumen as described herein is dry thin In the method for born of the same parents' cell strain, linearized vector DNA is prepared described in step 2.2 and genes of interest recombining reaction system is：5×CE II Buffer2 μ l, concentration are 2.5 μ l of 800ng/ μ L linearized vectors DNA, genes of interest CMV- of the concentration for 700ng/ μ L 1 μ l of 1 μ l of rtTA, Exnase II, with ddH₂O complements to 10 μ l.

The present invention will be transformed on the Tet-on expression systems basis that Clontech companies provide, by TRE- MinCMV regulation and control c-met and ALB promoter regulations GFP expressed sequences be implemented in same plasmid (plasmid be Tet-on The reaction plasmid of system), and lentiviral particle is packaged as, to set up the c-met expression systems of doxycycline control, obtained Synthetic gene sequence TRE-minCMV promoter-c-met-ALB promoter-GFP, the sequence contain two promoteres TRE- MinCMV and ALB, regulates and controls the expression of c-met and GFP respectively；TRE-minCMV promoteres receive under rtTA albumen existence conditions The regulation and control of doxycycline, you can induce c-met expression with by doxycycline, and ALB promoteres start for hepatocyte specificity Son, expresses to be conducive to filtering out to be divided into hepatocellular bone marrow BMSCs, therefore the system by promoter regulation GFP With by ALB promoter regulations GFP in the function with quasi-liver cell differentiation potential BMSCs targeted expression.In the present invention, two Individual promoter plays its respective action, goes out GFP (+) BMSCs using the screening system and feeds back interior therapeutic liver failure model, grinds Study carefully BMSCs to go back to the nest and differentiation capability along HGF concentration orientation, and the dose-effect between the ability and HGF/c-met axle expressions Relation；Interior therapeutic liver failure is fed back for the cell strain is studied, BMSCs goes back to the nest and differentiation capability along HGF concentration orientation, and Dose-effect relationship between the ability and HGF/c-met axle expressions is significant.

Description of the drawings

Fig. 1 is gene order TRE-minCMV promoter-c-met-ALB promoter-GFP structural representations.

Fig. 2 is by flow cytometer GFP (+) Tet-on-c-met BMSCs mirror of the screening with hepatocyte differentiation potentiality Inspection figure.

Fig. 3 is the expression of results schematic diagram that Real-time PCR methods detect c-met mRNA.

Fig. 4 is the electrophoresis result schematic diagram that Western-blot methods detect c-met albumen.

Fig. 5 is the expression of results schematic diagram that Western-blot methods detect c-met albumen.

Specific embodiment

Below in conjunction with the accompanying drawings, technical solution of the present invention is described in further detail.

Following examples are related to experiment material and are originated with instrument:

4 week old SPF level SD male rats are purchased from Nanjing Medical University's pharmaceutical experiment animal center；

293T cells are given by No.1 Attached Hospital, Nanjing Medical Univ infection Bing Ke professors Li Jun；

Hyclone is purchased from Corning companies；

Low DMEM are purchased from Hyclone companies；

PTet-On dvanced Vector carriers, In-Fusion^TMPCR Cloning Kit are purchased from Clotech companies；

Slow virus carrier GV211 (1 μ g/ μ L), gene order TRE-minCMV promoter-c-met-ALB promoter-GFP Synthesized by Shanghai JiKai Gene Chemical Technology Co., Ltd；

Slow virus carrier GV211, virus packaging helper plasmid (Helper1.0, Helper2.0) are purchased from triumphant purchased from Shanghai Ji Chemical gene Technology Co., Ltd.；

1kp DNA ladder Marker are purchased from Fermentas companies；

250bp DNA ladder Marker are purchased from JaRa company；

Taq polymerase are purchased from SinoBio companies；

DNTP is purchased from Takara；

Restricted enzyme is purchased from NEB companies；

Plasmid extracting Kit are purchased from promega；

Puromycin is purchased from amresco companies；

Agarose gel DNA QIAquick Gel Extraction Kits are purchased from Tiangeng biochemical corp；

Serum-free medium, PBS are purchased from Hyclone companies；

Virus preserves liquid and is purchased from Shanghai JiKai Gene Chemical Technology Co., Ltd；

Trypsin is purchased from Sigma companies；

PCR instrument is purchased from Applied Biosystems companies；

Positive clone sequencings are completed by U.S. season biotechnology；

Voltage stabilizing DNA electrophresis apparatuses are purchased from BioRad companies；

Gel imaging instrument is purchased from Tian Neng companies.

Culture medium source/preparation method that embodiment is related to：

Containing 10% serum low sugar culture medium prescription：By quality very in terms of, by the DMEM/Low of 89% (mass percent) Culture medium (purchased from Hyclone companies), the hyclone (purchased from Corning companies) of 10% (mass percent) and 1% (quality Percent) Pen .- Strep solution (purchased from green skies company, 100 × dual anti-) mixing after obtain；

The DNA sequence that following examples are related to：

SEQ ID NO.1:5’-GGATCC CTTGACATGC TCCCCGGGTA-3’；

SEQ ID NO.2:5’-ACCGGT TTACCCGGGG AGCATGTCAA-3’；

SEQ ID NO.3 (ALB initiating sequences, 237bp)：

SEQ ID NO.4 (Pmin CMV sequences, 60bp)：

taggcgtgta cggtgggagg cctatataag cagagctcgt ttagtgaacc gtcagatcgc 60

SEQ ID NO.5 (TRE sequences, 250bp)：

SEQ ID NO.6 (GFP sequences, 720bp)：

The extraction and culture of 1 rat bone marrow mesenchymal stem cellses of embodiment (BMSCs)

4 week old SPF level SD male rats are chosen, cervical dislocation is put to death, and takes out tibia and femur, remove the dry epiphysis in two ends End, with 5 milliliters of syringes absorption normal saline flushing medullary cavity in 50 milliliters of centrifuge tubes, 1000rpm centrifugal elutriation liquid 10min Afterwards, hanging heart bottom of the tube precipitation is rushed containing 10% serum low sugar culture medium using 3ml, liquid is added to into the lymph of rat containing 3ml thin In 15 milliliters of centrifuge tubes of born of the same parents' separating liquid, after 2000rpm centrifugation 20min, third layer tunica albuginea layer in centrifuge tube is drawn, and is added to In centrifuge tube containing its 4 times of mL normal salines, 1000rpm centrifugation 10min, the outstanding bottom of the punching containing 10% serum low sugar culture medium Precipitation be inoculated in culture dish, and cell is put into into 37 DEG C, containing 5%CO₂Culture in incubator.

The foundation of 2 Tet-On Advanced BMSCs cell strains of embodiment

1st, build Lentiviral GV211-CMV-rtTA

1.1 respectively with SEQ ID NO.1 and SEQ ID NO.2 as upstream and downstream primer, with pTet-On advanced Vector carriers are template, enter performing PCR reaction, and as shown in table 1, reaction condition is as shown in table 2 for reaction system；Reactant is mixed Gently piping and druming is mixed afterwards, is placed in amplifying target genes rtTA and upstream CMV promoter in PCR instrument, and is reclaimed with test kit, sequencing Correct amplified production is genes of interest CMV-rtTA.

1 PCR reaction systems of table

2 PCR reaction conditions of table

3 slow virus carrier GV211 enzyme action systems of table

Reagent	Volume (μ l)
		ddH₂O	42
10×CutSmart Buffer	5
		Slow virus carrier GV211 (1 μ g/ μ L)	2
BamHI, Age I enzymes (10U/ μ l)	1
		Total	50

1.2 according to table 3, prepares 50 μ l enzyme action systems, sequentially adds various reagents by 3 order of table, gently blown with pipettor Beat and mix, be placed in 37 DEG C of reaction 3h, to obtain the digestion products of slow virus carrier GV211, to obtain the load of the linearisation after enzyme action Body DNA；

4 recombinant slow virus expression vector GV211-CMV-rtTA systems of table

Reagent	Volume (μ l)
		ddH₂O	3.5
5×CE II Buffer	2
		Linearized vector DNA after enzyme action	2.5
Genes of interest CMV-rtTA	1
		Exnase^TM II	1
Total	10

1.3 are produced with the genes of interest amplification that the linearized vector DNA after the enzyme action of step 1.2 acquisition and step 1.1 are obtained Thing prepares reaction system as shown in table 4 in ice-water bath, gently blows and beats mixing with pipettor, keeps away a gram generation bubble, in 37 DEG C Reaction 30min, obtains the Lentiviral GV211- for being formed that recombinated by linearized vector and genes of interest CMV-rtTA CMV-rtTA。

2nd, rtTA gene recombinaton slow viruss packaging, concentration and purification

2.1 transfection before 24h, be purchased from Gibco companies with trypsin) digestion exponential phase 293T cells, to contain 10% serum low sugar culture medium adjusts cell density about 5 × 10⁶Cell/15ml, is re-seeded into a diameter of 10cm cell culture In ware, 37 DEG C, 5%CO₂Culture in cell culture incubator；Can be used to transfect after 24h when cell density is up to 70%～80%；And Before transfection, 2h is replaced by serum-free medium；

20 μ g of GV211-CMV-rtTA, pHelper1.0 15 μ for adding step 1 to obtain in 2.2 to one sterile centrifugation tubes 10 μ g of g, pHelper2.0, complement to 1ml with 2000 transfection reagents of Invitrogen Lipofectamine, incubation at room temperature 15min；Then instill during cell density is 70%～80% 293T cells, in 37 DEG C, 5%CO₂Cell culture incubator culture 6h, then discards culture medium, adds containing 10% serum low sugar culture medium, in 37 DEG C, containing 5%CO₂Cell culture incubator continue training Foster 48-72h；

The 293T cell supernatants of 48h (being 0h to count during transfection) after 2.3 collection transfections；In 4 DEG C, 4000g is centrifuged 10min, removes cell debriss；Again with 0.45 μm of filter filtering supernatant in the ultracentrifugation pipe of 40ml；Then distinguish trim Sample, the ultracentrifugation pipe with vial supernatant is put into Beckman supercentrifuges one by one, is arranged parameter of noncentricity and is 25000rpm, centrifugation time are 2h, and centrifuging temperature control is at 4 DEG C；After centrifugation terminates, supernatant discarded is removed as far as possible and remains in pipe Liquid on wall, adds virus to preserve liquid, that is, obtain rtTA gene slow viruss；

2.4 gene slow viruss titer determinations：Using fluorescence spectrometry virus titer, the previous day is determined, it is adherent using 293T Plating cells, 96 orifice plates, per hole 4 × 10⁴Individual cell；Prepare 7 aseptic EP pipes, numbering 1-7, often adds 90 μ l in pipe respectively Serum-free medium, takes 10 μ l of rtTA gene slow viruss liquid stock solution to be determined and is added in No. 1 pipe, after mixing, take 10 μ l and add Enter in No. 2 pipes, continue identical operation until the 7th pipe；Cell hole needed for choosing, discards 90 μ l culture medium of each hole, successively The viral solution in 7 pipes that 90 μ l have diluted, incubator culture is added after 4 days, to observe luciferase expression situation, fluorecyte Number is reduced with the increase of extension rate, according to formula：Virus titer=fluorecyte number/virus stock solution used amount, you can calculate this Virus titer is 2 × 10⁸TU/ml。

2.5 determine puromycin screening concentration：Take the logarithm the rat bone marrow mesenchymal stem cellses (BMSCs) of trophophase, use After twice of 2ml PBSs, 2ml trypsinization 2min are added, after basis of microscopic observation BMSCs all digests, Add 2ml to terminate digestion containing 10% serum low sugar culture medium, and add it in 15ml sterile centrifugation tubes, 1000rpm centrifugations 5min, supernatant discarded add the outstanding bottom precipitation of 6ml punchings containing 10% serum low sugar culture medium, and are inoculated in 6 orifice plates.Treat After each hole cell is covered with, discard former culture medium, gradient add purine-containing mycin containing 10% serum low sugar culture medium 2ml, each hole Puromycin concentration is respectively 0,6,7,8,9,10 μ g/ml；Then in 37 DEG C, 5%CO₂Cell culture incubator culture；Cell is per two It is changed once containing respective concentration puromycin containing 10% serum low sugar culture medium, and daily each hole cell growth status is entered Row observation；The puromycin least concentration of whole cells in respective aperture can be killed within 4 days, final drug screening is dense in the present embodiment Spend for 9 μ g/ml.

3rd, rtTA gene recombinaton slow virus carrier transfected into rat BMSCs and transfection after puromycin screening

3.1 day before transfection, the BMSCs in exponential phase is inoculated in 96 orifice plates, adds 4～5 × 10 per hole⁴ Individual cell (inoculum density is covered with for cell and is advisable for 3～4 days) and 100 μ l contain 10% serum low sugar culture medium, are put into cell culture Cultivate in case；When cell fusion degree reaches 25%-35%, former culture medium is discarded, carry out turning with infection multiplicity (MOI=100) Dye, i.e., add in each hole 10 μ l steps 2.3 obtain rtTA gene slow viruss (virus titer be 2 × 10⁸TU/ml)、10μl The ploybrene of final concentration of 5 μ g/ml strengthen liquid and 80 μ l containing 10% serum low sugar culture medium, in 37 DEG C, containing 5%CO₂Training Cultivate in foster case；

After 3.2 infection 12h, culture medium is discarded, add per hole 100 μ l to contain 10% serum low sugar culture medium；In transfection 48h Afterwards, you can use fluorescence microscope transfected condition；After infection 72h, again to change liquid containing 10% serum low sugar culture medium, and to The puro of final concentration of 9 μ g/ml is added per hole, and the drug screening cycle is one week, and the observing effect under fluorescence microscope, After basis of microscopic observation cell fluorescence positive rate up to after 100%, drug screening concentration is maintained into 1.8 μ g/ml, that is, is stablized The rat bone marrow mesenchymal stem cellses cell strain (i.e. Tet-On Advanced BMSCs) of high efficient expression rtTA albumen.

The foundation of 3 GFP (+) Tet-on-c-met BMSCs cell strains of embodiment

1st, slow viruss Len-TRE minCMV-c-met/ALB-GFP are built

1.1 transfer to Shanghai JiKai Gene Chemical Technology Co., Ltd synthetic gene sequence TRE-minCMV promoter-c- Met-ALB promoter-GFP, its structure as shown in figure 1, ALB initiating sequences (237bp) are as shown in SEQ ID NO.3, minCMV Promoter sequence (60bp) as shown in SEQ ID NO.4, TRE sequences (250bp) as shown in SEQ ID NO.5, GFP sequences (720bp) as shown in SEQ ID NO.6；

1.2 prepare linearized vector DNA and genes of interest recombining reaction system：5 × CE II Buffer, 2 μ l, embodiment Linearized vector DNA (concentration 800ng/ μ L) 2.5 μ l, TRE-minCMV promoter-c- after the enzyme action that 2 steps 1.2 are obtained Met-ALB promoter-GFP (concentration 800ng/ μ L) 1 μ l, 1 μ l of Exnase II, ddH₂O complements to 10 μ l；

By above-mentioned system as 37 DEG C of reaction 30min, that is, obtain Lentiviral GV211-TRE

minCMV-c-met/ALB-GFP；

1.3 add above-mentioned GV211-TRE minCMV-c-met/ALB-GFP 20 μ g, pHelper1.0 in centrifuge tube 15 μ g, pHelper2.0 10 μ g, complement to 1ml with 2000 transfection reagents of Invitrogen Lipofectamine, under room temperature Incubation 15min；Then instill during cell density is 70%～80% 293T cells, in 37 DEG C, 5%CO₂Cultivate in incubator 6h, then discards former culture medium, adds containing 10% serum low sugar culture medium, in 37 DEG C, containing 5%CO₂Continue culture in incubator 48-72h；

1.4 collect above-mentioned 293T cell supernatants, and in 4 DEG C, 4000g is centrifuged 10min；Supernatant is taken with 0.45 μm of filter mistake Filter, collects filtrate in 4 DEG C, and 25000rpm is centrifuged 2h；Supernatant discarded, adds virus to preserve liquid, that is, obtain slow viruss Len-TRE (virus titer is 2 × 10 to minCMV-c-met/ALB-GFP⁸TU/ml)；

2nd, slow viruss Len-TRE minCMV-c-met/ALB-GFP transfections Tet-On Advanced BMSCs cell strains

2.1 day before transfection, the Tet-On Advanced BMSCs in exponential phase are inoculated in 96 orifice plates, 4～5 × 10 are added per hole⁴Individual cell (inoculum density is covered with for cell and is advisable for 3～4 days) and 100 μ l are trained containing 10% serum low sugar Foster base, cultivates in being put into cell culture incubator；When cell fusion degree reaches 25%-35%, former culture medium is discarded, with infection multiplicity (MOI=100) transfected, i.e., in each hole, add 10 μ l steps 1.4 to obtain slow viruss Len-TRE minCMV-c-met/ ALB-GFP, the ploybrene of the final concentration of 5 μ g/ml of 10 μ l strengthen liquid and 80 μ l containing 10% serum low sugar culture medium, in 37 DEG C, containing 5%CO₂Cultivate in incubator；

After 2.2 infection 12h, culture medium is discarded, add per hole 100 μ l to contain 10% serum low sugar culture medium；In transfection 48h Afterwards, you can use fluorescence microscope transfected condition；After infection 72h, again to change liquid containing 10% serum low sugar culture medium, and to The puro of final concentration of 9 μ g/ml is added per hole, and the drug screening cycle is one week, and the observing effect under fluorescence microscope, After basis of microscopic observation cell fluorescence positive rate up to after 100%, drug screening concentration is maintained into 1.8 μ g/ml, that is, obtains western ring (i.e. GFP (+) Tet-on-c-met BMSCs are thin for the rat bone marrow mesenchymal stem cellses cell strain of plain regulating and expressing c-met albumen Born of the same parents' strain).

Embodiment 4

1st, GFP (+) Tet-on-c-met BMSCs of the flow cytometer screening with hepatocyte differentiation potentiality

Experimental group：GFP (+) the Tet-on-c-met BMSCs that embodiment 2 is obtained；

Matched group：BMSCs without genetic modification；(experimental technique list of references：Xiao LK,et al.Stem Cells Int.2016；2016:8906945).

As shown in Fig. 2 wherein, Fig. 2A is to observe GFP (+) Tet-on-c-met BMSCs under simple microscope to experimental result Form schematic diagram, it is seen that the cell growth is good；Fig. 2 B are observation portion GFP (+) Tet-on-c-met under fluorescence microscope BMSCs, it is seen which can express GFP；Fig. 2 C are matched group GFP positive expression cell number；Fig. 2 D are that experimental group GFP positive expressions are thin Born of the same parents.

Experimental group obtains the positive expression of GFP, shows that GFP (+) BMSCs accounts for total cell number using flowcytometric results About 57%.In illustrating the BMSCs for obtain stable transfection pALB-GFP plasmids, about 57% cell possesses the function of expressing ALB, that is, have It is potential to be divided into hepatocellular ability, on the other hand also illustrate that about 43% cell lacks that ALB promoteres play a role intracellular Environment, thus it is speculated that may not possess the potentiality of differentiation quasi-liver cell.

2nd, c-met is expressed by doxycycline external evoked GFP (+) Tet-on-c-met BMSCs cell strains

GFP (+) Tet-on-c-met BMSCs cell strain In vitro culture, adds in the low sugar culture medium which contains 10% serum Variable concentrations doxycycline (respectively 0.01,0.1,1,10,100ng/ml), and cell is put into into 37 DEG C, containing 5%CO₂Training Cell is collected after culture 12h in foster case.

Expression (the list of references of c-met mRNA is detected by Real-time PCR methods：Zhu X,et al.Cell Death Dis.2016；7(6):E2239), as a result as shown in Figure 3, it is seen that, c-met mRNA expressions and doxycycline Concentration is related, i.e., with the increase (0.1,1,10ng/ml) of doxycycline concentration, expression is stepped up, 10ng/ml with The c-met expression no significant differences of the doxycycline concentration induction of 100ng/ml, in Fig. 3, P ＜ 0.01 refer to that two groups have Significant difference.

Expression (the inspection list of references of c-met albumen is detected by Western-blot methods：Zhu X,et al.Cell Death Dis.2016；7(6):E2239), as a result as shown in figure 4, with the increase of doxycycline concentration, in Fig. 4, from a left side to Right 1-5 be respectively cell sequentially add 0.01,0.1,1,10, after 100ng/ml doxycycline, the detection of Western-blot methods C-met expressing quantities are also stepped up.

Fig. 5 is the expression schematic diagram that Western-blot methods detect c-met albumen, it is seen that with doxycycline concentration Increase (0.1,1,10ng/ml), c-met expressing quantities are stepped up, the doxycycline induction c-met expression of 10ng/ml Amount reaches top level, as a result illustrates that the optimal doxycycline induced concentration of c-met gene high expressions is 10ng/ml.

Concrete application approach of the present invention is a lot, and the above is only the preferred embodiment of the present invention, it is noted that for For those skilled in the art, under the premise without departing from the principles of the invention, some improvement can also be made, this A little improvement also should be regarded as protection scope of the present invention.

SEQUENCE LISTING

<110>Jiangsu Prov. People's Hospital

<120>The method for building the rat bone marrow mesenchymal stem cellses cell strain of doxycycline regulating and expressing c-met albumen

<130> 6

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 26

<212> DNA

<213>Synthetic

<400> 1

ggatcccttg acatgctccc cgggta 26

<210> 2

<211> 26

<212> DNA

<213>Synthetic

<400> 2

accggtttac ccggggagca tgtcaa 26

<210> 3

<211> 237

<212> DNA

<213>Synthetic

<400> 3

caaagtcttg tgcatggggg tgggggtggg gttagagggg aacagctcca gatggcaaac 60

atacgcaagg gatttagtca aacaactttt tggcaaagat ggtatgattt tgtaatgggg 120

taggaaccaa ttgaaatgcg aggtaagtat ggttaatgat ctacagttat tggttaaaga 180

agtatattag agcgagtctt tctgcacaca cgatcacctt tcctatcaac cccacta 237

<210> 4

<211> 60

<212> DNA

<213>Synthetic

<400> 4

taggcgtgta cggtgggagg cctatataag cagagctcgt ttagtgaacc gtcagatcgc 60

<210> 5

<211> 250

<212> DNA

<213>Synthetic

<400> 5

cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct atcagtgata 60

gagaacgatg tcgagtttac tccctatcag tgatagagaa cgtatgtcga gtttactccc 120

tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag aacgtatgtc 180

gagtttatcc ctatcagtga tagagaacgt atgtcgagtt tactccctat cagtgataga 240

gaacgtatgt 250

<210> 6

<211> 720

<212> DNA

<213>Synthetic

<400> 6

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720

Claims

1. it is a kind of build doxycycline regulating and expressing c-met albumen rat bone marrow mesenchymal stem cellses cell strain method, its It is characterised by, comprises the following steps that：

2nd, Tet-On Advanced BMSCs cell strains are set up：

2.1 respectively with SEQ ID NO.1 and SEQ ID NO.2 as upstream and downstream primer, are carried with pTet-On dvanced Vector Body is template, enters performing PCR amplification, obtains genes of interest CMV-rtTA；

2.2, with restricted enzyme BamHI, AgeI enzyme action slow virus carrier GV211, react 3 h in 37 DEG C, and digestion products are For linearized vector DNA；

Enzyme action system：2 l of 10 × CutSmart Buffer, 5 l, 1 g/ L slow virus carriers GV211, the restriction of 10 U/ l Property restriction endonuclease BamHI, AgeI enzyme 1 l, ddH₂O complements to 50 l；

2.3 prepare linearized vector DNA and genes of interest recombining reaction system, react 30 min in 37 DEG C, obtain slow viruss Expression vector GV211-CMV-rtTA；

2.4 add 20 μ g of GV211-CMV-rtTA, pHelper1.0 15 μ g, pHelper2.0 10 μ g in centrifuge tube, with 2000 transfection reagents of Invitrogen Lipofectamine to be complemented to and be incubated 15min under 1ml, room temperature；Then instill cell close Spend in the 293T cells for 70%～80%, in 37 DEG C, 5% CO₂Cell culture incubator culture 6h, discard culture medium, add containing 10% Serum low sugar culture medium, in 37 DEG C, containing 5% CO₂Cell culture incubator continue culture 48～72h；

293T cell supernatants after the culture of 2.5 collection step 2.4, in 4 DEG C, 4000 g are centrifuged 10 min；Take supernatant with 0.45 μm of filter is filtered, and regathers filtrate in 4 DEG C, and 25000 rpm are centrifuged 2h；Supernatant discarded, adds virus to preserve liquid, that is, obtains Obtain Len-CMV-rtTA slow viruss；

2.6 are inoculated in the BMSCs of exponential phase in 96 orifice plates, add 4～5 × 10 per hole⁴Individual cell and 100 μ l contain 10% Serum low sugar culture medium, when cell fusion degree reaches 30% or so, discards culture medium, adds 10 μ l steps 2.5 to obtain in each hole The virus titer for obtaining is 2 × 10⁸The Len-CMV-rtTA slow viruss of TU/ml, the ploybrene of the final concentration of 5 μ g/ml of 10 μ l increase Strong liquid and 80 μ l containing 10% serum low sugar culture medium, in 37 DEG C, 5% CO₂Cell culture incubator culture；

After infection 12h, culture medium is discarded, add per hole 100 μ l to contain 10% serum low sugar culture medium；After infection 72h, again containing 10% serum low sugar culture medium changes liquid, and adds the puro of final concentration of 9 μ g/ml to carry out drug screening to every hole, survives thin Born of the same parents are Tet-On Advanced BMSCs；

3rd, set up GFP（+）Tet-on-c-met BMSCs cell strains：

3.1 synthetic gene sequence TRE-minCMV promoter-c-met-ALB promoter-GFP；

3.2 prepare linearized vector DNA and genes of interest recombining reaction system：

5 × CE II Buffer, 2 l, step 2.2 obtain concentration be 2.5 l of 800ng/ L linearized vectors DNA, step TRE-minCMV promoter-c-met-ALB promoter-GFP 1 l, Exnase II of 3.1 concentration for obtaining for 800ng/ L 1 l, ddH₂O complements to 10 l；

Reaction system is placed in and 30 min is reacted in 37 DEG C, obtain Lentiviral

GV211-TRE minCMV-c-met/ALB-GFP；

3.3 add in centrifuge tube 20 μ g of GV211-TRE minCMV-c-met/ALB-GFP, pHelper1.0 15 μ g, 10 μ g of pHelper2.0, complement to 1ml with 2000 transfection reagents of Invitrogen Lipofectamine, are incubated under room temperature 15min；Then instill during cell density is 70%～80% 293T cells, in 37 DEG C, 5% CO₂6h is cultivated in incubator, is abandoned Culture medium is gone, is added containing 10% serum low sugar culture medium, in 37 DEG C, containing 5% CO₂Continue culture 48-72h in incubator；

293T cell supernatants after the culture of 3.4 collection step 3.3, in 4 DEG C, 4000 g are centrifuged 10 min；Take supernatant with 0.45 μm of filter is filtered, and regathers filtrate in 4 DEG C, and 25000 rpm are centrifuged 2h；Supernatant discarded, adds virus to preserve liquid, i.e., Obtain slow viruss Len-TRE minCMV-c-met/ALB-GFP；

3.5 are inoculated in the Tet-On Advanced BMSCs of exponential phase in 96 orifice plates, add 4～5 × 10 per hole⁴It is individual Cell and 100 μ l contain 10% serum low sugar culture medium, when cell fusion degree reaches 30% or so, discard culture medium, in each hole The ploybrene of 10 μ l Len-TRE minCMV-c-met/ALB-GFP slow viruss, the final concentration of 5 μ g/ml of 10 μ l is added to strengthen Liquid and 80 μ l containing 10% serum low sugar culture medium, in 37 DEG C, 5% CO₂Cell culture incubator culture；

After infection 12h, culture medium is discarded, add per hole 100 μ l to contain 10% serum low sugar culture medium；After infection 72h, again containing 10% serum low sugar culture medium changes liquid, and adds the puro of final concentration of 9 μ g/ml to every hole；Screened by flow cytometer GFP positive BMSCs cells, that is, obtain GFP（+）Tet-on-c-met BMSCs cell strains；

Wherein, the 10% serum low sugar culture medium compound method that contains is：By quality very in terms of, by 89% DMEM/Low cultivate Base, 10% hyclone and 1% Pen .- Strep solution mixing after obtain.

2. the rat bone marrow mesenchymal stem cellses for building doxycycline regulating and expressing c-met albumen according to claim 1 are thin The method of born of the same parents' strain, it is characterised in that the cell density is that 70%～80% 293T cells are obtained by：Transfection front 24 H, with the 293T cells of trypsinization exponential phase, to adjust cell density as 5x10 containing 10% serum low sugar culture medium⁶ Cell/15 ml, is inoculated in the Tissue Culture Dish of a diameter of 10cm, in 37 DEG C, containing 5% CO₂Incubator in culture, treat thin Can be used to transfect when born of the same parents' density is up to 70%～80%；And 2h replaces medium to serum-free medium before transfection.

3. it is according to claim 1 or claim 2 build doxycycline regulating and expressing c-met albumen rat bone marrow mesenchymal stem cellses The method of cell strain, it is characterised in that enter performing PCR amplification described in step 2.1 and refer to：

PCR reaction systems：The 4 μ l of dNTP Mix of 5 × PS Buffer, 10 μ l, 2.5 mM each, the forward primer of 10 M 0.5 μ l of the 1 μ l of template of the 1 μ l of downstream primer of 1 μ l, 10 M, 10 ng/ L, PrimeSTAR HS DNA polymerase, ddH₂O complements to 50 μ l；

PCR reaction conditions：98 DEG C of preheating 5min；98 DEG C of 10s, 55 DEG C of 10s, 72 DEG C of 90s, totally 30 circulations；4 DEG C are prolonged Stretch.

4. the rat bone marrow mesenchymal stem cellses for building doxycycline regulating and expressing c-met albumen according to claim 3 are thin The method of born of the same parents' strain, it is characterised in that prepare linearized vector DNA described in step 2.3 and genes of interest recombining reaction system is： 5 2 l of × CE II Buffer, concentration are 2.5 l of 800ng/ L linearized vectors DNA, and concentration is the purpose base of 700ng/ L Because of 1 l of CMV-rtTA, 1 l of Exnase II, with ddH₂O complements to 10 l.

5. the rat bone marrow mesenchymal stem cellses for building doxycycline regulating and expressing c-met albumen according to claim 4 are thin The method of born of the same parents' strain, it is characterised in that step 3.4 obtains the virus titer of slow viruss Len-TRE minCMV-c-met/ALB-GFP For 2 × 10⁸TU/ml。