CN107304452A - BK channel proteins are preparing the purposes during osteoblastic proliferation intervenes medicine extremely - Google Patents
BK channel proteins are preparing the purposes during osteoblastic proliferation intervenes medicine extremely Download PDFInfo
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Abstract
The invention belongs to field of biological pharmacy, it is related to BK channel proteins and is preparing the purposes of the purposes during Gegenbaur's cell intervenes medicine, especially BK channel proteins in osteoblastic proliferation disdifferentiation intervention medicine is being prepared.The present invention establishes the Relationship Model of large conductance calcium activated potassium channel (also known as BK passages) knockout and osteoblasts in vitro and the function of reduction Gegenbaur's cell, simulates the function of the pathological state and Gegenbaur's cell of Gegenbaur's cell in bone related disease；Experimental result shows that BK knocks out propagation and mineralising after clone and is decreased obviously, and the overall expression of osteoblast differentiation marker gene is significantly lower than wild type Gegenbaur's cell.As a result show, BK passages are the important target spots for the treatment for adjusting osteoblastic proliferation and bone related disease, described BK passages can be used for preparing Gegenbaur's cell intervention medicine, be used especially for preparing osteoblastic proliferation disdifferentiation intervention medicine.
Description
Technical field
The invention belongs to field of biological pharmacy, it is related to BK channel proteins and is preparing the purposes during Gegenbaur's cell intervenes medicine, especially
It is that BK channel proteins are preparing the purposes during osteoblastic proliferation disdifferentiation intervenes medicine.
Background technology
Prior art discloses BK channel proteins large conductance calcium activated potassium channel (BK), also known as MaxiK/KCa1.1 are logical
Road, by KCNMA1 gene codes and in most cells and wide expression in tissue；The uniqueness of BK channel proteins
Being in can be adjusted in it by membrane voltage and local intracellular Ca2+.On BK channel proteins in excitable cells
Research is existing a lot, and its effect in non-excitable tissue such as osteocyte, tumour cell and immunocyte is rarely reported.
BK passages are present in human osteosarcoma cell's (MG-63, Saos-2 and G292 cell) and bone precursor cells, and (C1 is thin
Born of the same parents).There are some researches show, non-specific potassium inhibitor triethylammonium tetrakis (TEA) cell quantity can be increased at low concentrations and
Cell quantity can be reduced under high concentration；Importantly, osteoporosis, long bone density is presented in the mouse of BK gene knockouts
Reduce, the increase of girder hole, this demonstrate the important function of the BK passages in osteocyte.However, in Gegenbaur's cell
The effect of BK passages is unclear.Knocked out although Zhang is found that in mesenchymal stem cells MSCs by shRNA
KCNMA1 genes can suppress cell propagation and skeletonization, but the knockout that mediated rnai is led is incomplete, is only weakened
Gene function.Preferable genetic analysis method is thoroughly to eliminate gene function by the change of genetic code (knockout).
ZFN (Zinc finger nuclease) appearance, TALEN (activating transcription factor sample effector nuclease) and rule
The short palindrome in cluster interval repeats (CRISPR)-Casenzymes technologies in recent years in eukaryotic and the target of experimental animal
Extensive use in genomic modification, TALE is found in phytopathogen xanthomonassp, and TALEN is by TALE
Albumen and FokI histone-nuclease fusions are built-up.TALE albumen has the variable bis-amino acid residue (RVD) of repetition, this
Determine the specificity (NI=A, HD=C, NG=T, NN=G or A) that nucleotides is combined；It is different by assembling
TALE modules, researcher can for any nucleotide sequence and control endogenous gene expression.In addition, FokI
Two pairs of nuclease domain fusion C- ends synthesis TALEs can provide effective DNA sequence dna specific endonucleases
Activity.Under normal circumstances, two TALEN formation One function FokI dimerization of recognizable object region or so two-arm
Body, and cause the DNA double chain in target area to be broken (DSB), cause non-homologous end joining reparation or homologous recombination
Repair, so as to cause the mismatch of nucleotides, insert, or missing, and the introducing that functioning gene is knocked out.
The basis studied based on prior art about Gegenbaur's cell in the latent effect of bon e formation and bone related disease, the application
Inventor intend providing the effects of BK channel proteins in Gegenbaur's cell, more particularly to BK channel proteins for
Prepare the purposes in osteoblastic proliferation disdifferentiation intervention medicine.
The prior art relevant with the present invention has：
1.Bentzen,B.H.,Olesen,S.P.,Ronn,L.C.and Grunnet,M.(2014)BK channel activators and their
therapeutic perspectives.Frontiers in physiology 5,389.
2.Chen,J.,Zhang,W.,Lin,J.,Wang,F.,Wu,M.,Chen,C.,Zheng,Y.,Peng,X.,Li,J.and Yuan,
Z.(2014)An efficient antiviral strategy for targeting hepatitis B virus genome using transcription
activator-like effector nucleases.Molecular therapy:the journal of the American Society of Gene
Therapy 22,303-311.
3.Ding,Q.,Lee,Y.K.,Schaefer,E.A.,Peters,D.T.,Veres,A.,Kim,K.,Kuperwasser,N.,Motola,
D.L.,Meissner,T.B.,Hendriks,W.T.,et al.(2013)A TALEN genome-editing system for
generating human stem cell-based disease models.Cell stem cell 12,238-251.
4.Zhang,Y.Y.,Yue,J.,Che,H.,Sun,H.Y.,Tse,H.F.and Li,G.R.(2014)BKCa and hEag1
channels regulate cell proliferation and differentiation in human bone marrow-derived
mesenchymal stem cells.Journal of cellular physiology 229,202-212.
5.Zhao Wang,J.L.,Huang Huang,Gancheng Wang,Mingjun Jiang,Shenyi Yin,Changhong Sun,,
Hanshuo Zhang,Fengfeng Zhuang,Jianzhong Jeff Xi(2012)An Integrated Chip for the
High-Throughput Synthesis of Transcription Activator-like Effectors.Angew Chem Int Ed 51,,
8505-8508.。
The content of the invention
The purposes during Gegenbaur's cell intervenes medicine is being prepared it is an object of the invention to provide BK channel proteins, especially
BK channel proteins are preparing the purposes during osteoblastic proliferation disdifferentiation intervenes medicine.
Some important physiological functions are participated in based on BK, such as nervous excitation tissue, heart and muscle, and in bone tissue,
Gegenbaur's cell BK passages report is rarely seen.The present invention establish large conductance calcium activated potassium channel (also known as BK channel proteins or
BK passages) knock out and osteoblasts in vitro with reduce Gegenbaur's cell function Relationship Model, using the modeling into
The pathological state of osteocyte and the research for carrying out Gegenbaur's cell function in bone related disease；Use activating transcription factor sample
Effect nuclease (TALEN) technology is knocked out to rat ROS17/2.8 Gegenbaur's cell BK passages, and detects BK
Cell line volume propagation mineralising is knocked out, the effect of Gegenbaur's cell BK passages is observed, experimental result is shown, BK knocks out clone
Propagation and mineralising are decreased obviously afterwards, and the overall expression of osteoblast differentiation marker gene is significantly lower than wild type Gegenbaur's cell.
The present invention devises KCNMA1 (NM_031828) gene target on rat osteoblast ROS17/28
TALENs, and it has been successfully set up BK knockout cell lines；The knockout cell line is presented compared with wild-type cell
Low propagation and differentiation capability, as a result show, BK passages are the treatments for adjusting osteoblastic proliferation and bone related disease
Important target spot, described BK passages can be used for preparing Gegenbaur's cell intervention medicine, be used especially for preparing Gegenbaur's cell increasing
Grow disdifferentiation and intervene medicine.
In the present invention, BK knocks out the instrument that cell can be studied as function of osteoblast.
In the present invention, the experiment comprised the following steps：
(1) cell culture and reagent；
(2) TALENs design and structure；
(3) PCR amplifications and DNA sequence analysis sequence；
(4) foundation of gene knockout clone；
(5) analysis of mRNA expression；
(6) immunofluorescence dyeing；
(7) mineralising；
(8) alizarin red S is dyed；
(9) CCK8 is determined；
(10) statistical analysis.
Experimental result is shown：
(1) BK knocks out the structure for being cloned in ROS17/2.8 cell lines
In order to set up BK gene clonings in ROS17/2.8 cells, TALENs is transferred to ROS17/2.8 in the application experiment
And puromycin progress medicine sieve is added, remaining cell is diluted into unicellular culture, monoclonal is grown into 96 orifice plates；
KCNMA1 gene knockouts clone is screened with immunofluorescence microscopy and DNA sequence dna；Gene sequencing is shown in target spot
There is overlap peak appearance between position and afterwards, show that TALEN is targetted successfully；Immunofluorescence analysis shows that BK albumen exists
All clones are wholly absent；
(2) influence of the BK gene knockouts to ROS17/28 cell proliferation and differentiations
The influence bred using CCK-8 cell proliferation experiment research KCNMA1 gene knockouts to ROS17/2.8 cells,
As a result show, KO cell masses proliferation rate is significantly lower than wild-type cell group (as shown in Figure 1)；
By comparing the differentiation of wild type and gene cloning Mineral nodules formation research Gegenbaur's cell, as a result show (such as Fig. 2
It is shown) ROS17/2.8 cells BK knock out after mineralization ability be remarkably decreased；Proceed skeletonization transcription factor and skeletonization
The expression experiment of the mark of cell differentiation, includes Runx2 expression BGP (OCN), osteopontin (OPN),
With real-time fluorescent quantitative RT-PCR method analysis in ROS17/2.8 cell KCNMA1 gene knockouts, as a result show,
Runx2 mRNA expression, OCN and OPN are substantially reduced compared with wild type, show the differentiation of Gegenbaur's cell
Ability declines.
Test result indicates that, BK passages can as regulation osteoblastic proliferation and bone related disease treatment important target
Point, described BK passages can be used for preparing Gegenbaur's cell intervention medicine, be used especially for preparing proliferation and differentiation of osteoblasts
It is abnormal to intervene medicine.
Brief description of the drawings
Fig. 1：ROS17/2.8 cell lines BK knocks out the foundation of clone, wherein,
A) DNA sequence figure, overlap peak is shown between target position and afterwards；
B the BK of) immunofluorescence dyeing, wild type (WT) and KCNMA1 genes (KO) knocks out clone, scale
The μ of chi=5.
Fig. 2:The influence of KCNMA1 gene pairs osteoblast differentiations, wherein,
A) formed in KCNMA1 Knockout cells and wild-type cell Mineral nodules, cell culture 10 days after,
Mineralising alizarin red S is dyed；
B) in KCNMA1 Knockout cells and the osteoblast marker mRNA of wild-type cell expression, into
Bone transcription factor mRNA expression (Runx2), osteoblast marker (BGP (OCN), osteopontin (OPN))
Real time RT-PCR analysis, average ± standard deviation, n=3, P ＜ 0.05.
Embodiment
Embodiment 1
1st, material and method
(1) cell culture and reagent
ROS17/2.8 cells (Johns Hopkins), cell is in Ham ' s F-12 nutritional blends culture mediums (Sigma adrich)
Middle culture, wherein containing sodium acid carbonate, is aided with the carbon dioxide that 10% tire ox E serum (FBS, Gibco) is in 5%
In the incubator of 95% air, every two days Secondary Cultures, when density reaches 90%, cell trypsase-EDTA
Digest and be inoculated in 6 well culture plates；
The anti-BK polyclonal antibodies (AP107) of rat Alomone, E.Z.N.A.Plasmid Mini Kit are purchased from
Yeasen biologies (Shanghai)；Genome DNA extracting reagent kit is biological (Shanghai) purchased from Tiangen；
(2) TALENs design and structure
TALEN is pressed to be built by SiDanSai biotechnologys with the detailed description of the Fast TALETM kits assembled
(SiDanSai, China), the TALEN left arms and right arm of KCNMA1 targetings are in website
(https://tale-nt.cac.cornell.edu/) design, PCR (PCR) amplification progress aggregation TALEN, choosing
12 of each arm are taken to clone and use E.Z.N.A.Plasmid Mini Kit extract TALEN plasmids, and the plasmid of purifying is used
Simultaneously (Shanghai) is sequenced by SanGon Biotech in PstI and BamHI digestions；
(3) PCR amplifications and DNA sequence analysis sequence
Genome DNA extracting reagent kit extracts genomic DNA, enters performing PCR amplification,
Forward primer is 5 '-ATATCCACGCGAACCATCTCAGCCT-3 ',
- the GGCCGTAGCCACCAATAACCCTACA-3 ' of reverse primer 5 ',
PCR amplifications are at CFX96-PCR instrument (Bio-Rad laboratories Hercules, CA) operation, 95 DEG C, initially
Activate as 30 seconds, 42 circulation denaturation (95 °, 5 seconds) are annealed (60 DEG C, 34 seconds), extend (72 DEG C, 30
Second)；PCR primer is analyzed by SanGonBiotech (Shanghai)；
(4) gene knockout clone is set up
Clone is knocked out in order to build BK, by TALEN plasmid transfections to ROS17/2.8 cells, then after transfection
Reinstate within second day 4 μ g/ml puromycins be administered 3 days, medicine sieve after remaining clone cell recover growth 1 week, formation
Single cell clone group；Cell clone group sets up KCNMA1 by immunofluorescence dyeing and DNA sequence analysis screening
The cell line of gene knockout；
(5) immunofluorescence dyeing
BK knocks out clone and wild-type cell is seeded in cell ware, and 70% when converging degree, 4% paraformaldehyde of cell
15 minutes are fixed, after PBS is rinsed, cell yong0.1% Triton X-100 dialyse 10 minutes, and 3% ox blood is pure
Albumen (BSA) room temperature P is closed 1 hour, BK protein antibodies (1:200, Abcam, Britain) 4 DEG C cultivate all night,
It is incubated 1 hour under fluorescence secondary antibody AlexaFluor647 antibody (Jackson ImmunoResearch, the U.S.) room temperature condition,
Image is caught using Leica TCS SP5 confocal systems focusing microscope (Leica, Wetzlar, Germany)；
(6) mineralising
ROS17/2.8 cells are inoculated in 48 orifice plates.After density reaches 90% or so, by sodium β-glycerophosphate (10mM)
It is added to L-AA phosphate (50 μ g/mL) in culture medium as Mineralized Culture base, every three days of Mineralized Culture base
Change once, after 10 days, cell is washed with PBS prepares Alizarin red staining twice；
(7) alizarin red S dyeing μ
Cell is handled with 4% paraformaldehyde, and 10min is fixed at room temperature, then is gently washed three times with PBS, is added per ware
Plus the alizarin red Ss of 500 μ L 0.1% (Yeasen, Shanghai) room temperature dyeing 15min；After dyestuff is absorbed, cell distilled water
500 μ L wash three times and vibrate 5min；
(8) CCK8 is determined
BK Knockout cells are inoculated in 96 orifice plates that concentration is 2000 cells/well plates, the 2nd after inoculation, and 3,4,
5,7 and 9 days, 10 μ L CCK8 solution are added, 37 DEG C are cultivated 2 hours, with ELIASA (TECAN infinite m200
Pro, Switzerland) absorbance is determined at wavelength 450nm；
The structure result that BK knockouts are cloned in ROS17/2.8 cell lines is shown, in order to set up BK in ROS17/2.8 cells
Gene cloning, is transferred to ROS17/2.8 by TALENs and adds puromycin progress medicine sieve, remaining cell is diluted into
Unicellular culture, monoclonal is grown into 96 orifice plates；KCNMA1 gene knockout clone immunofluorescence microscopies and
DNA sequence dna is screened, and gene sequencing is shown between target position and has overlap peak appearance afterwards, shows TALEN
Target successfully；Immunofluorescence analysis shows that BK albumen is wholly absent in all clones；
Influence result of the BK gene knockouts to ROS17/28 cell proliferation and differentiations is shown, breeds real using CCK-8 cells
Checking reality KCNMA1 gene knockouts have an impact to ROS17/2.8 cells propagation, and KO cell mass proliferation rates are significantly lower than
Wild-type cell group (as shown in Figure 1)；By comparing wild type and gene cloning Mineral nodules formation research Gegenbaur's cell
Differentiation, as a result as shown in Fig. 2 ROS17/2.8 cells BK knock out after mineralization ability be remarkably decreased；Further grind
Study carefully the expression of the mark of skeletonization transcription factor and osteoblast differentiation, include Runx2 expression BGP (OCN), bone
Pontin protein (OPN), with real-time fluorescent quantitative RT-PCR method analysis in ROS17/2.8 cell KCNMA1 bases
Because knocking out, as a result show, Runx2 mRNA expression, OCN and OPN are substantially reduced compared with wild type,
Show that the differentiation capability of Gegenbaur's cell declines.
Claims (5)
1.BK channel proteins are preparing the purposes during Gegenbaur's cell intervenes medicine.
2.BK channel proteins are preparing the purposes during osteoblastic proliferation disdifferentiation intervenes medicine.
3.BK channel proteins are for preparing the use during the therapy target of regulation osteoblastic proliferation and bone related disease intervenes medicine
On the way.
4. the purposes as described in claim 1 or 2 or 3, it is characterised in that BK knocks out propagation and mineralising after clone and declined.
5. the purposes as described in claim 1 or 2 or 3, it is characterised in that BK knocks out osteoblast differentiation mark after clone
The overall expression of gene is less than wild type Gegenbaur's cell.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109701024A (en) * | 2019-03-04 | 2019-05-03 | 复旦大学 | The new application of BK channel opener |
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| CN102242125A (en) * | 2011-04-28 | 2011-11-16 | 上海大学 | BK channel blocker gene, recombinant vector thereof and cloning method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2004037813A1 (en) * | 2002-10-25 | 2004-05-06 | Merck Frosst Canada & Co. | Pyrrolidin-2-on derivatives as ep4 receptor agonists |
| CN102242125A (en) * | 2011-04-28 | 2011-11-16 | 上海大学 | BK channel blocker gene, recombinant vector thereof and cloning method thereof |
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| BO LI: ""The role of BK channel in cellular proliferation and differentiation in human osteoblast and osteoblast-like cells"", 《A THESIS PRESENTED TO THE CARDIFF SCHOOL OF PHARMACY AND PHARMACEUTICAL SCIENCE OF CARDIFF UNIVERSITY》 * |
| ZHANG YY1 ET AL: ""BKCa and hEag1 channels regulate cell proliferation and differentiation in human bone marrow-derived mesenchymal stem cells"", 《J CELL PHYSIOL》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109701024A (en) * | 2019-03-04 | 2019-05-03 | 复旦大学 | The new application of BK channel opener |
| CN109701024B (en) * | 2019-03-04 | 2020-12-11 | 复旦大学 | New application of BK channel opener |
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