CN102250841A - Recoverable immortalized rat bone marrow mesenchyme stem cell as well as preparation method and application thereof - Google Patents
Recoverable immortalized rat bone marrow mesenchyme stem cell as well as preparation method and application thereof Download PDFInfo
- Publication number
- CN102250841A CN102250841A CN2011101837490A CN201110183749A CN102250841A CN 102250841 A CN102250841 A CN 102250841A CN 2011101837490 A CN2011101837490 A CN 2011101837490A CN 201110183749 A CN201110183749 A CN 201110183749A CN 102250841 A CN102250841 A CN 102250841A
- Authority
- CN
- China
- Prior art keywords
- cell
- immortalization
- reversibility
- bone marrow
- rat
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Abstract
The invention discloses a recoverable immortalized rat bone marrow mesenchyme stem cell. The cell carries an SV40T (Simian Virus 40T) gene and a hygromycin resistance gene and two ends of the SV40T gene also have orthokinetic Loxp sites. The preparation method of the recoverable immortalized rat bone marrow mesenchyme stem cell comprises the following steps of: co-transfecting an HEK293 (Human Embryo Kidney 293) cell by SSR69 (Simple Sequence Repeat 69) plasmids and pAmhpo plasmids and screening by the hygromycin to obtain the recoverable immortalized rat bone marrow mesenchyme stem cell with hygromycin resistance; the cell has the same biological property with a primary rat and also has the advantages of strong external multiplication capacity, stable biological property, capability of being recovered to a previous state, good biological safety and the like; and the cell provided by the invention can be used as a seed cell induced and differentiated by nerves and a seed cell for treating hypoxicischemic brain damage of a newly-born rat.
Description
Technical field
The present invention relates to the cell engineering field, be specifically related to a kind of reversibility immortalized cells, also relate to the preparation method and the application of this reversibility immortalized cells.
Background technology
Mesenchymal stem cells MSCs (Mesenchymal Stem Cells, MSCs) be to be present in the stem cell that a class in the tissues such as marrow, peripheral blood and bleeding of the umbilicus has self and strides the germinal layer differentiation potential, have the in-vitro separation of being easy to, cultivation and amplification, import easily foreign gene, can particular organization settle down or go back to the nest, characteristics such as reduced immunogenicity and multidirectional differentiation potential.MSCs can be divided into neural like cell under specific external evoked environment, and experimentation on animals shows also that MSCs transplants survive and also can be divided into the neural like cell of function in central nervous system, for the treatment of nervous system disorders has brought new hope.
Though MSCs has powerful proliferation potential in vivo, division growth repeatedly, but in the in-vitro separation culturing process, owing to lost neurohumoral adjusting and intercellular interaction, live in the relatively stable environment that lacks running balance, the cells survival time is short, and multiplication capacity is limited and sex change easily takes place.Therefore, obtaining in-vitro multiplication MSCs good and that have a normal proterties is the important leverage of research of MSCs inside and outside and clinical application.
Summary of the invention
In view of this, one of purpose of the present invention is to provide a kind of reversibility immortalization rat MSCs, can be in the external cultivation of going down to posterity for a long time, strong and the proterties of multiplication capacity does not change, can also easily be returned to the preceding state of immortalization, biological safety is good, can overcome that the external survival time of former generation rat MSCs is short, multiplication capacity is limited and the shortcoming of sex change easily takes place.
For achieving the above object, reversibility immortalization rat MSCs provided by the invention carries SV40T (being simian virus 40 large T antigens) gene and hygromycin gene, and the two ends of described SV40T gene also have Loxp site in the same way.
The SV40T gene is imported rat MSCs, and the important regulating and controlling point of adjustable ganglion cell in the cycle makes cell enter the vegetative state of immortalization, and becoming can be at the external immortalized cell line that goes down to posterity for a long time and cultivate.Respectively add 1 Loxp site at the two ends of SV40T gene and make the direction in 2 Loxp sites identical, the characteristic of utilizing the Cre-LoxP recombinase system is (if 2 LoxP sites are positioned on 1 DNA chain and direction is identical, the Cre recombinase can effectively excise 2 sequences between the LoxP site), then the SV40T gene can be knocked out by Cre recombinase orientation, thereby can make the cell of immortalization be returned to the preceding state of immortalization.
Two of purpose of the present invention is to provide the preparation method of described reversibility immortalization rat MSCs, and is easy and simple to handle, quick.
For achieving the above object, the preparation method of reversibility immortalization rat MSCs provided by the invention may further comprise the steps:
A. with retrovirus expression plasmid SSR69 and packaging plasmid pAmhpo cotransfection HEK293 cell, obtain recombinant retrovirus;
B. with step a gained recombinant retrovirus infected rats MSCs, screen, promptly get reversibility immortalization rat MSCs with hygromycin resistance with Totomycin.
Contain SV40T gene and hygromycin gene among the retrovirus expression plasmid SSR69, there is Loxp site in the same way at the two ends of described SV40T gene.With step a gained recombinant retrovirus infected rats MSCs, use the hygromycin selection positive colony, promptly get reversibility immortalization rat MSCs with hygromycin resistance.Preferably, described step b infects the 3rd generation rat MSCs with step a gained recombinant retrovirus, and infecting the back was 2 weeks of hygromycin selection of 4 μ g/ml with concentration on the 3rd day, promptly got the reversibility immortalization rat MSCs with hygromycin resistance.
Three of purpose of the present invention is to provide the application of described reversibility immortalization rat MSCs.
The experiment in vivo and vitro result shows, reversibility immortalization rat MSCs of the present invention can break up by neuralward unit like cell external, be implanted into and improve HIBI (HIBD) in the body, therefore, reversibility immortalization rat MSCs of the present invention can be used as into the seed cell application of the seed cell and the treatment neonate rat HIBD of nerve-inducing differentiation.
Beneficial effect of the present invention is: the present invention imports rat MSCs by the SV40T gene that the LoxP site-specific is modified, made up the reversibility immortalized cell line, this cell strain has the same biological characteristics with former generation rat MSCs, it is strong also to have the in-vitro multiplication ability, biological character is stable, can be returned to original state, characteristics such as biological safety is good, can replace former generation rat MSCs to carry out the experimental study of transplantation treatment nervous system disorders in external Neural Differentiation and the body, therefore, the present invention provides important cell instrument for above-mentioned research.
Description of drawings
Fig. 1 is the 3rd generation rat MSCs light microscopic figure (A, 100 *) and H.E colored graph (B, 100 *).
Fig. 2 is the fluidic cell detection figure of the 3rd generation rat MSCs surface marker CD34, CD45, CD90 and CD106.
Fig. 3 is reversibility immortalization rat MSCs hygromycin selection figure.
Fig. 4 is the 10th generation reversibility immortalization rat MSCs light microscopic figure (A, 100 *) and H.E colored graph (B, 100 *).
Fig. 5 is the fluidic cell detection figure of the 10th generation reversibility immortalization rat MSCs surface marker CD34, CD45, CD90 and CD106.
Fig. 6 is the reversible western blot figure that knocks out of the SV40T gene of reversibility immortalization rat MSCs.
Fig. 7 is the reversible cell growth curve figure that knocks out front and back of the SV40T gene of reversibility immortalization rat MSCs.
Fig. 8 is 24 hours a light microscopic comparison diagram of the external nerve-inducing differentiation of reversibility immortalization rat MSCs and former generation rat MSCs.
Fig. 9 is the Western blot comparison diagram of neural correlating markings NSE in reversibility immortalization rat MSCs and the external nerve-inducing atomization of former generation rat MSCs.
Figure 10 is the Real-time PCR comparison diagram of 24 hours neural correlating markings Nestin of the external nerve-inducing of reversibility immortalization rat MSCs and former generation rat MSCs differentiation, NSE, MAP-2, GDNF.
Figure 11 is that the immunofluorescence of 24 hours neural correlating markings Nestin of the external nerve-inducing of reversibility immortalization rat MSCs and former generation rat MSCs differentiation, NSE, MAP-2, GDNF detects comparison diagram.
Figure 12 is the resting membrane electric potential comparison diagram of 24 hours neural like cells of the external nerve-inducing differentiation of reversibility immortalization rat MSCs and former generation rat MSCs.
Figure 13 is that the Morris water maze of transplantation treatment neonate rat HIBD in reversibility immortalization rat MSCs and the former generation rat MSCs body arrives platform time comparison diagram.
Figure 14 is that the Morris water maze of transplantation treatment neonate rat HIBD in reversibility immortalization rat MSCs and the former generation rat MSCs body arrives platform number of times comparison diagram.
The former generation rat MSCs of " MSCs " expression that occurs in the above-mentioned accompanying drawing, " IMSCs " represents reversibility immortalization rat MSCs of the present invention, " IMSCs Cre T Ag " expression is knocked out the reversibility immortalization rat MSCs of SV40T gene by the Cre recombinase.
Embodiment
In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is described in further detail below in conjunction with accompanying drawing.
SSR69 plasmid that uses in the preferred embodiment and pAmhpo plasmid are presented by professor He Tongchuan of Chicago University.
One, separation and Culture and the evaluation of former generation rat MSCs
Get 4 ~ 8 week of the SPF level SD rat in age (available from Medical University Of Chongqing's animal center) of body weight 80 ~ 150g, the cervical vertebra dislocation is put to death, and aseptic condition takes out femur and shin bone down, goes out marrow with the DMEM/F12 substratum, and centrifugal removal supernatant and fat are by every bottle 3 * 10
6Individual cell inoculation is at 25cm
2The plastic culture bottle in, be 37 ℃, CO with the DMEM/F12 perfect medium that contains 10% foetal calf serum in temperature
2Volume fraction is to cultivate under 5% the condition, hypsokinesis in 24 hours goes culture supernatant to reach not attached cell, adds fresh culture and continues to cultivate, 1 fresh culture of replacing in per afterwards 3 ~ 4 days, mirror is observed down, as seen MSCs is the cell colony sample growth that is dispersed in, and the cellular form of colony central authorities is round, and the cell at colony edge is flat fusiformis, cultivating 4 ~ 5 days cell quantities obviously increases, colony enlarges gradually, and at the bottom of 6 ~ 7 days cells were paved with bottle substantially, the form homogeneous was the growth of fusiformis whirlpool shape.When cell grows to 80% when converging, use TrypLE
TMExpress digestion is centrifugal, go down to posterity by the concentration of 1:2 or 1:3, the passage cell uniform distribution, growth rapidly, at the bottom of can being paved with in about 3 ~ 4 days bottle, cell is fusiformis more, whirlpool shape growth (Fig. 1).
Flow cytometry is identified the cell surface marker of rat MSCs: get near the 3rd generation MSCs that merges, use TrypLE
TMAfter the Express digestion, use the PBS of pH7.0 to wash 3 times, make 1 * 10
6The single cell suspension of/ml.Get 100 μ l cell suspensions, 5 pipes: a pipe adds IgG for standard control
1-FITC monoclonal antibody; The b pipe adds the CD34-FITC monoclonal antibody; The c pipe adds the CD45-PE monoclonal antibody; The d pipe adds the CD90-PE monoclonal antibody; The e pipe adds the CD106-PE monoclonal antibody; Room temperature reaction 30 minutes, flow cytometer (FACSCanto
TMII system, BD Biosciences) detects, CellQuest Pro software (BD Biosciences) analyzes, the result shows: this cell expressing CD90 (99.99%) and CD106 (99.91%), express CD34 (3.51%) and CD45 (2.23%) hardly (Fig. 2), meet the biological characteristics of rat MSCs.
Two, carry the retroviral structure of SV40T gene
With the HEK293 cell inoculation in 25cm
2Culturing bottle, treat that degrees of fusion is to carry out liposome transfection at 60 ~ 70% o'clock: unparalleled anti-DMEM substratum 250 μ l mix with SSR69 plasmid 10 μ l, pAmpho plasmid 5 μ l, Lipfectamine 2,000 15 μ l and serum-free, incubated at room 10 minutes makes the transfection mixed solution; 293 cells of cultivating are washed 2 times with the unparalleled anti-DMEM substratum of serum-free, added the unparalleled anti-DMEM substratum 2.5ml of serum-free, placed 10 minutes for 37 ℃, add above-mentioned transfection mixed solution again, mixing is replaced by DMEM perfect medium 6ml after 4 ~ 6 hours.Collect 293 cells and supernatant in 24 hours after the transfection, promptly contain the retrovirus of the packaged SV40T of carrying gene in this supernatant.
Three, the structure of reversibility immortalization rat MSCs, screening and evaluation
Get 293 cells and supernatant (containing the retrovirus that carries the SV40T gene) of collection, the ratio that adds 2mg/ml polybrene 5 μ l in every ml supernatant adds polybrene, is the filter filtration of 0.45 μ m with the aperture, makes the retrovirus nutrient solution.Former generation rat MSCs is removed outmoded substratum, with above-mentioned retrovirus nutrient solution overnight incubation, be replaced by fresh culture again and continue to cultivate, the former generation rat MSCs that setting is not simultaneously infected is cell in contrast.In infecting the back the 3rd day, the Totomycin that adds final concentration and be 4 μ g/ml in cell culture fluid screens, and the screening concentration of keeping Totomycin simultaneously of going down to posterity when cell density is 90 ~ 95% is constant.Screened the 4th day, cell begins obvious poor growth, the original polygon form of lost cell, and most cells slowly become spindle shape, and can not be adherent in going down to posterity, and are dead gradually; Screen about 2 weeks, control cells is all dead, through only 5% survival of cell of retroviral infection processing; Keeping the constant continuation of screening concentration of Totomycin afterwards cultivates, survivaling cell begins to recover the normal speed of growth and state (Fig. 3), back cell growth and the form of going down to posterity do not have considerable change (Fig. 4), then obtains the MSCs of hygromycin resistance, is reversibility immortalization rat MSCs yet.
Flow cytometry is identified the surface marker of reversibility immortalization rat MSCs: get near the 10th generation reversibility immortalization rat MSCs that merges, use TrypLE
TMAfter the Express digestion, use the PBS of pH7.0 to wash 3 times, make 1 * 10
6The single cell suspension of/ml.Get 100 μ l cell suspensions, 5 pipes: a pipe adds IgG for standard control
1-FITC monoclonal antibody; The b pipe adds the CD34-FITC monoclonal antibody; The c pipe adds the CD45-PE monoclonal antibody; The d pipe adds the CD90-PE monoclonal antibody; The e pipe adds the CD106-PE monoclonal antibody; Room temperature reaction 30 minutes, flow cytometer (FACSCanto
TMII system, BD Biosciences) detects, CellQuest Pro software (BD Biosciences) analyzes, the result shows: this cell expressing CD90 (99.84%) and CD106 (99.45%), express CD34 (1.32%) and CD45 (4.27%) hardly (Fig. 5), compare indifference with former generation rat MSCs.
Four, the reversible of SV40T gene of reversibility immortalization rat MSCs knocks out
Reversibility immortalization rat MSCs is pressed 1 * 10
5/ ml is inoculated in 6 orifice plates, and overnight incubation adds Cre recombinase adenovirus, and the blank group is set simultaneously, and with former generation rat MSCs as negative control.Extract the total protein of respectively organizing cell after 48 hours, BCA detects protein concentration, get sample on the equal protein (is confidential reference items with β-actin), carry out 10% polyacrylamide gel electrophoresis, (the electrotransfer time is respectively SV40T protein 90 minute to the 80V electrotransfer to pvdf membrane again, β-actin 40 minutes), sealed film 1 hour with 5% skimmed milk, adding one respectively resists: mouse anti rat SV40T and mouse anti rat β-actin (all pressing the 1:200 dilution with PBS), 4 ℃ of overnight incubation, wash film 3 times with TBST, the goat-anti mouse two anti-(press 1:500 with PBS dilutes) that adds the HRP mark again, incubated at room 1 hour is carried out the ECL luminous detection after washing film once more.The result shows: former generation rat MSCs does not express SV40T albumen, reversibility immortalization rat MSCs expresses a large amount of SV40T albumen, and Cre recombinase adenovirus infection reversibility immortalization rat MSCs is after 48 hours, the proteic expression of SV40T is returned to the expression level (Fig. 6) of former generation rat MSCs, and the SV40T gene of prompting reversibility immortalization rat MSCs can be knocked out by the Cre recombinase.
Reversibility immortalization rat MSCs is pressed 1 * 10
5/ ml is inoculated in 6 orifice plates, and overnight incubation adds Cre recombinase adenovirus, and the blank group is set simultaneously, and with former generation rat MSCs as negative control.Respectively at cultivating the 0th, 1,2,3,4, the 5 day counting cells in back, draw cell growth curve: treat that counting cells is after trysinization, PBS washing, add nutrient solution 1ml, cell suspension is made in piping and druming, get 100 μ l cell suspensions in the 1.7ml centrifuge tube, add 0.4% trypan blue dye liquor, 100 μ l, the piping and druming mixing, get 10 μ l after 3 ~ 5 minutes and drip to tally, put microscopically and observe counting.The result shows: the growth curve of reversibility immortalization rat MSCs is S-type, propagation obviously faster than former generation rat MSCs (
p＜0.05), and after the Cre recombinase knocked out the SV40T gene, ability of cell proliferation was returned to the state (Fig. 7) of former generation rat MSCs.
Five, reversibility immortalization rat MSCs replaces former generation rat MSCs to carry out the experimental study of external nerve-inducing differentiation
Reversibility immortalization rat MSCs and former generation rat MSCs are pressed 3 * 10 respectively
4/ ml is inoculated in 24 orifice plates, cultivate with the DMEM/F12 perfect medium that contains 10% foetal calf serum, reject perfect medium after 24 hours, with HBSS washed cell 2 times, add serum-free MNM nerve-inducing liquid 0.5ml and carry out external nerve-inducing differentiation, in different time points at inverted microscope (TE2000-S, Nikon, Japan) morphocytology that dynamic observes under before and after inducing changes, the neural like cell differentiation efficiency of Taking Pictures recording.The result shows: induced 30 minutes, former generation rat MSCs and reversibility immortalization rat MSCs see that all big and flat cell space shrinks; Induced 60 minutes, the cell refractivity strengthens, and shrinks the cell space that becomes multistage property gradually, and many thin digitations are arranged; Induced 3 ~ 24 hours, cell is typical neuron morphology, and the cell that has has the projection of a plurality of length, and the projection of a plurality of neural like cells can extend to form netted (Fig. 8) mutually.The neural like cell differentiation efficiency of former generation rat MSCs and reversibility immortalization rat MSCs is respectively 51.4% and 57.6%, no significant difference.
Western blot detects the expression of reversibility immortalization rat MSCs and former generation rat MSCs neural correlating markings NSE in external nerve-inducing atomization: reversibility immortalization rat MSCs and former generation rat MSCs are carried out external nerve-inducing differentiation by preceding method, respectively at inducing the 0th, 6,12,18, extracted total protein of cell in 24 hours, get sample on the equal protein (is confidential reference items with β-actin), carry out 10% polyacrylamide gel electrophoresis, electrotransfer is to pvdf membrane again, sealed film 1 hour with 5% skimmed milk, add one respectively and resist: anti-NSE (pressing the 1:200 dilution) and anti-β-actin (pressing the 1:500 dilution), 4 ℃ of overnight incubation with PBS with PBS, wash film, two anti-(press 1:500 with PBS dilutes) that add the HRP mark again, incubated at room 1 ~ 2 hour is carried out the ECL luminous detection after washing film.The result shows that inductive rat MSCs of former generation does not express NSE, induces to begin to express NSE in back 6 hours, and strengthens gradually in 24 hours; The NSE of reversibility immortalization rat MSCs expresses and is lower than former generation rat MSCs slightly, but expression trend unanimity (Fig. 9).
Real-time PCR detects reversibility immortalization rat MSCs and the former generation rat MSCs expression at 24 hours neural correlating markings Nestin of external nerve-inducing differentiation, NSE, MAP-2, GDNF: reversibility immortalization rat MSCs and former generation rat MSCs are pressed the external nerve-inducing of preceding method broke up 24 hours, extract the total mRNA of cell, reverse transcription becomes the cDNA template, adopts 5 pairs of Auele Specific Primers (Nestin:SEQ ID No.1 ~ 2 more respectively; NSE:SEQ ID No.3 ~ 4; MAP-2:SEQ ID No.5 ~ 6; GDNF:SEQ ID No.7 ~ 8; Confidential reference items β-actin:SEQ ID No.9 ~ 10) carries out real-time PCR, detect the expression abundance of each neural sign; The PCR reaction system is: cDNA template 2 μ l, and each 0.5 μ l of the upstream and downstream primer of 10 μ mol/L, 2.5 * RealMasterMix/20 * SYBR solution 9 μ l adds ultrapure water to 20 μ l; The PCR loop parameter is: 94 ℃ 2 minutes, 94 ℃ 20 seconds, 56 ℃ 20 seconds, 72 ℃ 20 seconds, totally 39 circulations.The result shows: the external nerve-inducing differentiation of reversibility immortalization rat MSCs and former generation rat MSCs is after 24 hours, and the expression of Nestin, NSE, MAP-2, GDNF does not have significant difference (Figure 10).
Immunofluorescence detects reversibility immortalization rat MSCs and 24 hours neural correlating markings Nestin of the external nerve-inducing differentiation of former generation rat MSCs, NSE, MAP-2, the expression of GDNF: reversibility immortalization rat MSCs and former generation rat MSCs are pressed the external nerve-inducing differentiation of preceding method 24 hours, substratum is abandoned in suction, methyl alcohol-20 ℃ is fixed 15 minutes, PBS washing 3 times, 5% fetal bovine serum albumin sealing 30 minutes, PBS washing 3 times, adding one respectively resists: anti-NSE (pressing the 1:500 dilution with PBS), anti-Nestin (pressing the 1:200 dilution) with PBS, anti-MAP-2 (pressing the 1:500 dilution) and anti-GDNF (pressing the 1:300 dilution) with PBS with PBS, 4 ℃ of overnight incubation, PBS washing 3 times, adding corresponding two again resists, incubated at room 2 hours, PBS washing, DAPI redyed karyon 10 minutes, observed under the fluorescent microscope and took pictures.The result shows: former generation rat MSCs and reversibility immortalization rat MSCs without the MNM nerve-inducing there is no tangible luciferase expression, after MNM induces 24 hours, all visible significantly NSE of gained neuron cell and Nestin express (green fluorescence) at endochylema, MAP-2 and GDNF express (red fluorescence) at endochylema and aixs cylinder, and the neural correlating markings of two kinds of cells is expressed no significant difference (Figure 11).
Full cell patch pincers detect the resting membrane electric potential of reversibility immortalization rat MSCs and 24 hours neural like cells of the external nerve-inducing differentiation of former generation rat MSCs: former generation rat MSCs and reversibility immortalization rat MSCs are inoculated in respectively on the Tissue Culture Dish that diameter is 35mm, press the external nerve-inducing differentiation of preceding method 24 hours, it is strong to choose refractivity, have two or more long projections, the cell space neural like cell of circle detects, simultaneously with without the former generation rat MSCs of MNM nerve-inducing and reversibility immortalization rat MSCs (control) in contrast: microelectrode external diameter 1.2mm, internal diameter 0.5mm dashes and irritates liquid in the electrode; Resistance is 2 ~ 5M, sample frequency 10kHz, and low-pass filtering is 1kHz; Adopt the high resistance sealing technology, the negative pressure punching forms cell and clamps down on; Stimulate granting and signals collecting with AXOPATCH 200B amplifier well-established law, write down full cell resting membrane electric potential.The result shows: be respectively-4.825 ± 1.121mV and-5.057 ± 1.115mV without the former generation rat MSCs of MNM nerve-inducing and the resting membrane electric potential of reversibility immortalization rat MSCs, after MNM induces differentiation, the resting membrane electric potential of the neural like cell of gained is respectively-36.875 ± 8.132mV and-39.286 ± 6.849 mV (Figure 12), and two kinds of resting potential of ventricular cells do not have significant difference.
Six, reversibility immortalization rat MSCs replaces former generation rat MSCs to carry out the experimental study of transplantation treatment neonate rat HIBD in the body
Make up neonate rat HIBD model: get 7 age in days newborn SD rat, with reference to the Rice method, free ligation left common carotid artery is sewed up the incision, in 3 hours rearmounted encloses containers, and logical nitrogen (keeping the volume fraction of oxygen is 8 ~ 9%) 2.5 hours.After the modelling 1 day, set up following three groups to carry out transplanting in the body: 1. PBS control group; 2. former generation rat MSCs transplantation group; 3. reversibility immortalization rat MSCs transplantation group; After MSCs made cell suspension, with microsyringe injection model mouse abdominal cavity, every 100 μ l (contained MSCs 1 ~ 2 * 10
6).After the transplanting, three groups of mouse are put back in the former feeding environment feed, do not give immunosuppressor.Simultaneously, organize (control) in contrast with normal newborn SD rat.
The Morris water maze laboratory detects learning and memory function: three groups of experimental mouse were carried out water maze laboratory in 28 days at the HIBD postoperative.Adopt MWM water maze system (SLY-WMS 2.0): diameter is the round pool of 94cm, depth of water 40cm, 25 ± 1 ℃ of water temperatures; With the pond mid point is the center of circle, and the pond is divided into four quadrants (I, II, III and IV quadrant); Put the platform of a 10cm * 38cm from pool wall 20cm place.The 1st day is visual training, platform is higher than labyrinth liquid level 0.5cm, platform central authorities indicate with bright-coloured penoncel, in different quadrants experimental mouse is put into the labyrinth at random, write down animal automatically by computer and arrive the platform required time, if animal is not found platform in setting-up time (60 seconds) from place of entry, computer stops to follow the tracks of and be 60 seconds writing time, simultaneously animal is placed on the platform and stopped 10 seconds, train each 30 minutes at interval altogether 5 times; The 2nd ~ 5 day is non-visual training, platform is lower than labyrinth liquid level 2.0cm, from different quadrants experimental mouse is put into the labyrinth according to the 1st day order, does not find the time record of platform and animal training mode with the 1st day in setting-up time, train each 60 minutes at interval every day 6 times.Removed platform for the platform exploration detects on the 6th day, and from the right side quadrant of platform place quadrant experimental mouse was put into the labyrinth, the record experimental mouse finds number of times and each required time of platform position at the appointed time.The result shows: compare with normal newborn rat, the learning and memory function of HIBD neonate rat descends, and the PBS control group finds the number of times of position of platform obviously to reduce, find the time of position of platform obviously to prolong (
P＜0.05), former generation rat MSCs transplantation group and reversibility immortalization rat MSCs transplantation group find the position of platform inferior number average significantly more than the PBS control group, find the time of position of platform all significantly to shorten (
p＜0.05), no significant difference between two MSCs transplantation group (
p0.05), illustrating that MSCs transplants the learning and memory function that can improve the HIBD neonate rat, reversibility immortalization rat MSCs can substitute transplantation treatment neonate rat HIBD (Figure 13 ~ 14) in the former generation rat MSCs body.
Explanation is at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although by invention has been described with reference to the preferred embodiments of the present invention, but those of ordinary skill in the art is to be understood that, can make various changes to it in the form and details, and the spirit and scope of the present invention that do not depart from appended claims and limited.
＜110〉Children's Hospital Attached to Chongqing Medical Univ.
＜120〉reversibility immortalization rat bone marrow mesenchymal stem cells and its production and application
<160>?10
<210>?1
<211>?18
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉be used to detect the PCR upstream primer of Nestin gene
<400>?1
gggcaagtgg?aacgtaga 18
<210>?2
<211>?18
<212>?DNA
＜213〉artificial sequence
＜220〉be used to detect the PCR downstream primer of Nestin gene
<400>?2
tcccaccgct?gttgattt 18
<210>?3
<211>?19
<212>?DNA
＜213〉artificial sequence
＜220〉be used to detect the PCR upstream primer of NSE gene
<400>?3
ctgtttgctg?ctcaaggtc 19
<210>?4
<211>?18
<212>?DNA
＜213〉artificial sequence
＜220〉be used to detect the PCR downstream primer of NSE gene
<400>?4
tcccactacg?aggtctgc 18
<210>?5
<211>?21
<212>?DNA
＜213〉artificial sequence
＜220〉be used to detect the PCR upstream primer of MAP-2 gene
<400>?5
gtatcaggag?acagggagga?g 21
<210>?6
<211>?21
<212>?DNA
＜213〉artificial sequence
＜220〉be used to detect the PCR downstream primer of MAP-2 gene
<400>?6
ggggtagtag?gtgttgaggt?g 21
<210>?7
<211>?18
<212>?DNA
＜213〉artificial sequence
＜220〉be used to detect the PCR upstream primer of gdnf gene
<400>?7
cactgacttg?ggtttggg 18
<210>?8
<211>?22
<212>?DNA
＜213〉artificial sequence
＜220〉be used to detect the PCR downstream primer of gdnf gene
<400>?8
tcacttgtta?gccttctact?tc 22
<210>?9
<211>?20
<212>?DNA
＜213〉artificial sequence
＜220〉be used to detect the PCR upstream primer of β-actin gene
<400>?9
tttgagacct?tcaacacccc 20
<210>?10
<211>?17
<212>?DNA
＜213〉artificial sequence
＜220〉be used to detect the PCR downstream primer of β-actin gene
<400>?10
ggatggcatg?agggagc 17
Claims (5)
1. reversibility immortalization rat bone marrow mesenchymal stem cells is characterized in that, carries SV40T gene and hygromycin gene, and the two ends of described SV40T gene also have Loxp site in the same way.
2. the preparation method of the described reversibility immortalization of claim 1 rat bone marrow mesenchymal stem cells is characterized in that, may further comprise the steps:
A. with retrovirus expression plasmid SSR69 and packaging plasmid pAmhpo cotransfection HEK293 cell, obtain recombinant retrovirus;
B. with step a gained recombinant retrovirus infected rats mesenchymal stem cells MSCs, screen, promptly get reversibility immortalization rat bone marrow mesenchymal stem cells with hygromycin resistance with Totomycin.
3. the preparation method of reversibility immortalization rat bone marrow mesenchymal stem cells according to claim 2, it is characterized in that, described step b be with step a gained recombinant retrovirus infect the 3rd generation rat bone marrow mesenchymal stem cells, infecting the back was 2 weeks of hygromycin selection of 4 μ g/ml with concentration on the 3rd day, promptly got the reversibility immortalization rat bone marrow mesenchymal stem cells with hygromycin resistance.
4. the described reversibility immortalization of claim 1 rat bone marrow mesenchymal stem cells is as the application of the seed cell that becomes the nerve-inducing differentiation.
5. the described reversibility immortalization of claim 1 rat bone marrow mesenchymal stem cells is as the application of the seed cell of treatment HIBI.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101837490A CN102250841A (en) | 2011-07-01 | 2011-07-01 | Recoverable immortalized rat bone marrow mesenchyme stem cell as well as preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101837490A CN102250841A (en) | 2011-07-01 | 2011-07-01 | Recoverable immortalized rat bone marrow mesenchyme stem cell as well as preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102250841A true CN102250841A (en) | 2011-11-23 |
Family
ID=44978412
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011101837490A Pending CN102250841A (en) | 2011-07-01 | 2011-07-01 | Recoverable immortalized rat bone marrow mesenchyme stem cell as well as preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102250841A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103865877A (en) * | 2014-03-11 | 2014-06-18 | 中国人民解放军第三军医大学第二附属医院 | Preparation method of immortalized human cartilage endplate stem cell line and use of immortalized human cartilage endplate stem cell line |
| CN104450620A (en) * | 2014-06-09 | 2015-03-25 | 重庆医科大学附属儿童医院 | Recoverable immortalized hepatic cell line carrying double suicide genes and establishing method of recoverable immortalized hepatic cell line |
| CN105969735A (en) * | 2016-05-11 | 2016-09-28 | 中国医学科学院血液病医院（血液学研究所） | Ischemia hypoxia-resistant human mesenchymal stem cells, and preparation method and application |
| CN114752626A (en) * | 2022-03-16 | 2022-07-15 | 重庆医科大学附属儿童医院 | Reversible immortalized II-type alveolar epithelial cell and construction and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5449614A (en) * | 1988-09-01 | 1995-09-12 | Whitehead Institue For Biomedical Research | Recombinant retroviruses with amphotropic and ecotropic host ranges |
| US6645763B2 (en) * | 2001-10-12 | 2003-11-11 | Naoya Kobayashi | Immortalized bone marrow mesenchymal stem cell |
-
2011
- 2011-07-01 CN CN2011101837490A patent/CN102250841A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5449614A (en) * | 1988-09-01 | 1995-09-12 | Whitehead Institue For Biomedical Research | Recombinant retroviruses with amphotropic and ecotropic host ranges |
| US6645763B2 (en) * | 2001-10-12 | 2003-11-11 | Naoya Kobayashi | Immortalized bone marrow mesenchymal stem cell |
Non-Patent Citations (3)
| Title |
|---|
| 《Int J Oncol》 20031031 Nishioka K et al. Immortalization of bone marrow-derived mesenchymal stem cells by removable simian virus 40T antigen gene: analysis of the abilitu to support expansion of cord blood hematopoietic progenitor cells 第23卷, 第4期 * |
| NISHIOKA K ET AL.: "Immortalization of bone marrow-derived mesenchymal stem cells by removable simian virus 40T antigen gene: analysis of the abilitu to support expansion of cord blood hematopoietic progenitor cells", 《INT J ONCOL》 * |
| 洪小杨等: "BMSCs鼠脑移植对HIBD新生鼠脑血管再生的影响", 《广东医学》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103865877A (en) * | 2014-03-11 | 2014-06-18 | 中国人民解放军第三军医大学第二附属医院 | Preparation method of immortalized human cartilage endplate stem cell line and use of immortalized human cartilage endplate stem cell line |
| CN103865877B (en) * | 2014-03-11 | 2016-08-31 | 中国人民解放军第三军医大学第二附属医院 | The preparation method and its usage of immortal human cartilage endplate stem line |
| CN104450620A (en) * | 2014-06-09 | 2015-03-25 | 重庆医科大学附属儿童医院 | Recoverable immortalized hepatic cell line carrying double suicide genes and establishing method of recoverable immortalized hepatic cell line |
| CN104450620B (en) * | 2014-06-09 | 2017-03-01 | 重庆医科大学附属儿童医院 | A kind of replied immortalized hepatocyte strain carrying double independent variable and its construction method |
| CN105969735A (en) * | 2016-05-11 | 2016-09-28 | 中国医学科学院血液病医院（血液学研究所） | Ischemia hypoxia-resistant human mesenchymal stem cells, and preparation method and application |
| CN105969735B (en) * | 2016-05-11 | 2019-07-23 | 中国医学科学院血液病医院（血液学研究所） | A kind of human mesenchymal stem cell and preparation method and application of resistance to hypoxic-ischemic |
| CN114752626A (en) * | 2022-03-16 | 2022-07-15 | 重庆医科大学附属儿童医院 | Reversible immortalized II-type alveolar epithelial cell and construction and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102127522B (en) | Human umbilical mesenchymal stem cell and preparation method thereof | |
| CN1860222A (en) | Stem cells for clinical and commercial uses | |
| CN106801032B (en) | Construction method of human amniotic epithelial stem cell bank | |
| CN103667349B (en) | Method for efficiently acquiring inductive pluripotent stem cells (iPSCs) | |
| CN102367435A (en) | Preparation of human platelet-rich plasma and application of same in isolation and culture of human mesenchymal stem cells | |
| CN103881971B (en) | Culture medium and culture method for culturing and/or amplifying mesenchymal stem cells | |
| CN109988746A (en) | A kind of mescenchymal stem cell adipogenic induction differentiation method | |
| CN102732586A (en) | Method for culturing mesenchymal stem cell secretin | |
| KR20190112186A (en) | Method and system for regenerating hair using 3D organoid system of hair follicle stem cells | |
| CN105087474A (en) | Culture method of deciduous tooth pulp stem cells | |
| CN110475855A (en) | For the method that cell prepares dental pulp stem cell derived from pulp tissue | |
| CN105238738A (en) | Isolated culture method of piglet myocardial fibroblasts | |
| CN102250841A (en) | Recoverable immortalized rat bone marrow mesenchyme stem cell as well as preparation method and application thereof | |
| CN107287156A (en) | A kind of isolated culture method of fat mesenchymal stem cell and its application | |
| CN102703387A (en) | Astrocyte separating and cultivating method | |
| CN102755667A (en) | Preparation method and application of tissue-engineering human tooth root implantation material | |
| CN108373990B (en) | Culture medium for inducing differentiation from human amniotic epithelial stem cells to melanocytes and method thereof | |
| CN1221660C (en) | Method for exosomatic separation and amplification of filled stem cells between human marrow and funic blood and directional induction toward nerve cells | |
| CN107287158A (en) | The method of effective acquisition mescenchymal stem cell from mouse dense bone | |
| CN102533647B (en) | Method for inducing neural differentiation of stem cells | |
| CN105062960B (en) | Primary isolation and culture method of periodontal ligament stem cells | |
| CN106032529A (en) | Method for in-vitro separation, amplification and induced differentiation of placenta-derived mesenchymal stem cells | |
| CN107858322B (en) | Method for establishing primary hippocampal cell culture system | |
| CN109439617A (en) | A kind of stem cell serum-free culture medium and preparation method thereof | |
| CN105255821B (en) | Method for culturing periodontal ligament stem cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20111123 |