CN102433297A - Method for extending telomeres of adult pluripotent stem cells - Google Patents
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Abstract
The present invention is in the field of biomedicine and is especially the method of extending telomeres of adult multipotent stem cells, and the individual's senility is caused by shortening the telomeres of multipotent adult stem cells, so that the functions of the aged tissue and organ in the individual may be reversed to the level of young people by extending the telomeres of multipotent adult stem cells to the level of young people. The selective temporary inhibition of the expression of one gene in ATPX or DAXX or the activity of protein can induce the homologous recombination of telomeres of various stem cells to extend the telomeres, and the simultaneous inhibition of two genes is not needed, so that the method has the effects of simplifying operation and saving cost.
Description
Technical field
The invention belongs to biomedical sector, particularly relate to the method for extending adult multipotential stem cell telomere.
Background technology
Multipotent adult stem cells, be in individuality one type can self and multidirectionally be divided into multiple histiocytic stem cell, like hemopoietic stem cell, Miu Si cell (Muse cells) etc.
One, individuality is made up of stem cell and non-stem cell
Organ-tissue in the individuality is made up of stem cell and non-stem cell; Non-stem cell is one type of functioning cell in the organ-tissue; Usually can not self, because of causing death when reducing, reasons such as telomere shortening, dna mutation can replenish through breaking up by stem cell; The death that stem cell is caused because of reasons such as cytodifferentiation, dna mutations also can replenish through improving the self rate.
All somatocyte that comprise the cerebral neuron cell; The cell of every day average nearly 1% can fall because of death takes place the rubbish accumulation in dna mutation, telomere shortening, the cell or the like problem; But also have the cell of equivalent newborn simultaneously, what lean on is exactly self and these two kinds of functions of multidirectional differentiation of adult stem cell.
Two, the size of proliferation potential of stem cell and multidirectional differentiation potential maybe be relevant with telomere length
In marrow; Be that the Miu Si cell (the multidirectional differentiation potential of Miu Si cell almost can compare favourably with embryonic stem cell) that belongs to mescenchymal stem cell is compared with other mescenchymal stem cell equally; Multidirectional differentiation potential is just different; The Miu Si cell can be divided into more eurypalynous cell than other mescenchymal stem cell, and what is the reason that causes this multidirectional differentiation potential difference? It is cell inherent differentiation difference? Still because the difference of telomere length?
Hemopoietic stem cell can be divided into two subgroups, a long-term reconstruct potential with height, and the long-term reconstruct potential that another subgroup is lower, the hemopoietic stem cell with long-term reconstruct potential of height has come to light and has had the suitable telomerase activation of cancer cells.But symmetry and imparity division repeatedly takes place in adult stem cell in self and atomization, can cause telomere to shorten, along with the telomere of stem cell is shorter and shorter, the activity of Telomerase also can be more and more lower (people such as Allsopp, 1992; 1995; People such as Vaziri, 1994; People such as Hiyama, 1995; Hodes, 1999), secular reconstruct potential will be lost; Because marrow mainly is to live to the long adult stem cell of telomere; Besides old and feeble adult stem cell divides multidirectionalization potential lower; Therefore; Adult stem cell old and feeble in marrow can be eliminated naturally, for example, is exactly the hemopoietic stem cell that belongs to old and feeble at the hemopoietic stem cell in blood of drifting about; Gronthos (2003) imports telomerase catalytic subunit to people's mescenchymal stem cell, passage 80 times, and multidirectional differentiation potential can preserve for 40 generations, and did not import the control group of telomerase catalytic subunit, and multidirectional differentiation potential can only remain to for the 18th generation; The enzymoprivic mescenchymal stem cell of telomere can not be divided into fat and chondrocyte.But; These are according to the algebraically that keeps telomerase activation can prolong the multidirectional differentiation potential of adult stem cell just is described; And the illustrative adult stem cell that lost multidirectional differentiation potential not can recover multidirectional differentiation potential through extending to telomere certain-length.
The instruction information realization sequencing that is recorded in the program carrier is read, just must make program carrier displacement constantly.Therefore, make the procedure operation that is stored in the computingmachine in the hard disk, a phonomoter will be set in the hard disk drive, the power that is produced by phonomoter drives the hard disk rotation.As a same reason, genetic program is operationalized, promptly realize the sequential expression of gene, also need a kind of power to drive.Old and feeble " life cycle driven by program theory " thought, telomere is exactly the phonomoter that drives the genetic program running.Therefore; The cell of same differentiation type, because the difference of telomere length or telomerase activation, the spectrum of gene expression amount is also different; Therefore the same novel cell that produces with cytodifferentiation has appearred; This phenomenon is a kind of content unreal " pseudo-differentiation ", and according to this inference, the differentiation type of human body cell has not just had kind more than 200.Again according to this inference, in marrow, the mescenchymal stem cell of difference in functionality, the difference between them possibly only be the difference of telomere length.
I think in sum, and for a kind of stem cell, the size of multidirectional differentiation potential is relevant with telomere length, and promptly telomere is long more, and the cell type that is divided into is just many more, otherwise the cell type that is divided into is just few more.In view of the above, as long as telomere extends to certain-length, genetic program will reverse, and the multidirectional differentiation that forfeiture is fallen is dived and also can be replied, and the general mescenchymal stem cell in the marrow just can be converted into the Miu Si cell.If this conjecture obtains proof, it is more valuable to make induced multi-potent stem cells (iPS cell) than invention, is the achievement of Nobel's grade of prizes.
Three, the shortening of adult stem cell telomere is to cause individual old and feeble first cause
Along with the increase of frequency dividing cell, the self ability of adult stem cell also can weaken gradually.And the telomere shortening is to cause the self potential of adult stem cell and multidirectional differentiation potential that the basic reason of reduction takes place.I think in sum; Damaged tissue regeneration obstruction is not to be the quantity not sufficient of adult stem cell in the individuality of individual aging course and aging; But aging has taken place in adult stem cell itself, the deficiency of soma cell telomere length, individual old and feeble reason that Here it is.For this reason, manufacturing genetic background multipotent adult stem cells consistent with the patient, that telomere is long is individual aging of treatment and the optimal path that improves the repair of damaged tissues rate.
Four, the adult stem cell telomere is long more, transplants consumption and can lack more
People's sperm telomere length is 15kb, and made embryonic stem cell (ES cell and iPS cell telomere length only have 8～12kb people such as () Amit, only the suitable telomere length of grownup's adult stem cell.Telomere is long more, and cell fission algebraically is just many more, and proliferative ability is just vigorous more, and therefore, the stem cell population of transplanting usefulness just can lack more, and people is formed by the zygote proliferation and differentiation that a telomere reaches 14～15kb.Therefore, in theory, if the telomere length of adult stem cell can reach 14kb, it is just enough that people transplants an adult stem cell.
The technology of five, extending the adult stem cell telomere is inquired into
Scientists is found some tumour cells to be arranged and do not rely on the length that Telomerase is kept telomere in recent years, they this phenomenon is referred to as " telomere prolongation replacement mechanism " (alternative lengthening of telomeres, ALT).One piece of one piece of report that is published on " Nature Cell Biology " magazine: " telomere of ovocyte and spermatogonium is shorter than somatocyte; but can increase considerably through the reduction division telomere length, find that in the early stage division embryo of the mouse that Telomerase lacks telomere also can extend unexpectedly, in mature oocyte and cleavage stage embryo; telomerase activation reduces; telomere length but increases considerably, and recovers telomerase activation afterwards in the blastocyst again, telomere extension is simultaneously but slowed down.Scientists is reached a conclusion for this reason, and when thinking the spilting of an egg in early days, it possibly be because homologous recombination is machine-processed that telomere prolongs, and since blastula stage, Telomerase only is a length of keeping the telomere of having confirmed ".
This can explain the increase along with telomere length, and Telomerase extends telomere meeting more and more difficult, wants to extend the telomere of stem cell, can not depend merely on Telomerase, and can only adopt the homologous recombination technique of telomeric dna.Stem cell contains Telomerase though Here it is; The stem cell telomere is extended, and can only delay the reason of the shortening speed of telomere at most, and these are over more than 10 years; Scientist adopts Telomerase to extend the reason of telomere failure; This can explain that also major part contains the cancer cells of high reactivity Telomerase simultaneously, the reason that telomere length is also shorter than normal somatic cell.
People such as Meeker from the Johns Hopkins University have resolved telomere and have prolonged replacement mechanism in some special tumour cells recently, and they find that the telomere that two transgenations and these tumours do not rely on Telomerase keeps closely related." science " (Science) on the magazine that be published on June 30th, 2011 that this achievement in research is online.
Meeker and his colleague have studied 41 patients' endocrine tumor of pancreas cell; Be presented at the fluorescent dyeing of selectively targeted telomere and all have " gathering in a large number of telomeric dna " in 25 neoplasmic tissue sample, the intensity of each phosphor dot approximately is 100 times of telomeric dna in the normal cell.Attention: the telomere length of end of chromosome does not increase by 100 times; But outer telomere Tumor-necrosis factor glycoproteins DNA (the extra-chromosomal telomeres repat DNA of karyomit(e); ECTR DNA), be free in a large number outside the endonuclear karyomit(e) with the nucleosome form.However, telomere is still grown than normal cell, for example, leans on the negative WI-38-2RA cell of Telomerase of ALT, and the telomere mean length is 21.2kb.
In these 25 samples, there are 19 samples to include ATRX or DAXX transgenation.Though wherein 6 samples are not found ATRX or DAXX transgenation, yet these tumour cells all do not have ATRX and DAXX genetic expression.And substitute and both do not found transgenation in all the other 16 samples that prolong mark lacking telomere, and ATRX and DAXX genetic expression are normal.
ATRX is chromatin reorganization apysase in the SNF-2 family, once in a thalassemia---and the chain syndrome of the sluggish X of intelligence is furtherd investigate, and having identified ATRX has the sudden change above 100 kinds to come to light can to influence proteins encoded, but still the retaining part activity.Discoveries such as Jiao, phase shift mutation and nonsense mutation can cause ATPX and the proteic disappearance fully of DAXX, and this occurs in most endocrine tumor of pancreas.
Antisense oligonucleotide (asONs) can combine inhibition of gene expression with base complementrity paired mode with specific DNA or RNA; RNA disturbs (RNA interference; RNAi) be meant importing and endogenous mRNA coding region homologous double-stranded RNA (double stranded RNA in cell; DsRNA) time, dsRNA decomposes the small molecules interference RNA (siRNA) that produces in cell antisense strand and multiple nucleicacidase have formed reticent mixture (RISC) and have caused the reticent phenomenon of genetic expression, and this phenomenon occurs in post-transcriptional level; Have specificity and scale effect; And consumption is few, effect lasting (the existing special software of at present a lot of companies designs siRNA, and produces and sell various siRNA).
Summary of the invention
The purpose of this invention is to provide the method for extending adult multipotential stem cell telomere, its characteristic comprises:
Scheme one step:
1, ATRX or the proteic antibody of DAXX (these two kinds of antibody commercializations, used antibody can be passed through the active part that enzyme downcuts), or in the multipotent adult stem cells of the asONs of ATRX or DAXX gene or siRNA importing cultivation;
2, be placed on multipotent adult stem cells in the nutrient solution then and cultivate for some time, make telomere obtain extending;
3, described antibody or asONs whenever will add once between 2 hours, because antibody or antisense oligonucleotide can be fallen by cell degradation, promptly had the transformation period.
4, also telomerase catalytic subunit gene (hTRT) can in multipotent adult stem cells, be imported simultaneously, to increase the extension efficient of telomere;
Scheme two steps:
1, from the JEG-3 of no Telomerase, isolate ECTR DNA, process for extracting is referring to the extraction Mitochondrial DNA, or purchase is with artificial synthetic ECTR DNA;
2, import to ECTR DNA in the multipotent adult stem cells of cultivation;
3, also can in multipotent adult stem cells, import hTRT simultaneously, to increase the extension efficient of telomere;
4, be placed on multipotent adult stem cells in the nutrient solution then and cultivate for some time, make telomere obtain extending.
The present invention has simple to operate, and technology is ready-made, and cost is low.
Embodiment
In the face of doing, the present invention describes in further detail down.
Extend the method for adult multipotential stem cell telomere, its characteristic comprises:
Scheme one step:
1, with methods such as electroporation, liposome or microinjections ATRX or the proteic antibody of DAXX, or the asONs of ATRX or DAXX gene or siRNA import in the multipotent adult stem cells of cultivating;
2, be placed on multipotent adult stem cells in the nutrient solution then and cultivate for some time, can confirm incubation time by telomere length as required, made telomere obtain extending to a week in general 3 days;
3, described antibody or asONs whenever will add once between 2 hours, because antibody or antisense oligonucleotide can be fallen by cell degradation, promptly had the transformation period.
4, also can in multipotent adult stem cells, import hTRT simultaneously, to increase the extension efficient of telomere;
Scheme two steps:
1, from the JEG-3 of no Telomerase, isolate ECTR DNA, process for extracting is referring to the extraction Mitochondrial DNA, or purchase is with artificial synthetic ECTR DNA;
2, import to ECTR DNA in the multipotent adult stem cells of cultivation with methods such as electroporation, liposome or microinjections, ECTR DNA consumption is 50～100 times of normal cell telomeric dna;
3, also can in multipotent adult stem cells, import hTRT simultaneously, to increase the extension efficient of telomere;
4, be placed on multipotent adult stem cells in the nutrient solution then and cultivate for some time, be generally 2 to 4 days, make telomere obtain extending.
ATPX can form " ATPX-DAXX " complex body and be combined on heterochromatin zone and the telomere with DAXX, and will cause some oncogene expression to be suppressed with the extension of telomere after the combination being obstructed.And the situation that ATPX and DAXX transgenation take place in same tumour simultaneously also not to be found.I think in view of the above; As long as optionally temporarily suppress the telomere generation homologous recombination that an expression of gene or proteic activity among ATPX or the DAXX just can be induced various stem cells; And need not to suppress simultaneously two genes, have the effect that simplifies the operation and practice thrift cost.
Suppress cancer suppressor gene p16 and can slow down telomere shortening speed, and not rising (virgin smooth monarch etc.) of telomerase activation; Make human epithelial cell's immortalization, promptly telomerase activation will be arranged, also will make cancer suppressor gene inactivations such as Rb and p16, INK4 (people such as Kjyone, 1998); Also contain Telomerase in sperm and the ovum; Usually the cancer cells that contains the high reactivity Telomerase, telomere length is also shorter than normal somatocyte, and therefore, single is can not extend telomere with Telomerase.But the HeLa cell telomere that contains the high reactivity Telomerase but reaches 14kb, and the method that telomere is extended in this explanation is to adopt (cocktail) of compound system; The human mesenchymal stem cell of cultivating ECTR DNA will occur in the 7th generation of going down to posterity, and along with the increase of propagation algebraically, the quantity of ECTR DNA also more and more people such as (, 2010) Zhao Qiang.Prompting; ECTRDNA can with Telomerase together or have a collaborative effect of extending telomere; If the homologous recombination process has the participation of Telomerase, the effect of extending telomere can be better, perhaps can activate with telomere the activated end granzyme time and extend other relevant expression of gene.The function of Telomerase is that handle becomes the telomere repeat sequence dna fragmentation with telomere repeat sequence DNA complementary RNA rt, and then is connected to telomere reiterated DNA sequences fragment on the telomeric sequence of end of chromosome.Therefore, Telomerase not only plays the effect of reversed transcriptive enzyme, but also might play the effect of ligase enzyme, or some factor actives that connect telomeric dnas are raise.
In sum, the present invention assists the adult multipotential stem cell to extend telomere with importing the telomerase catalytic subunit gene.
Antibody of the present invention, asONs or siRNA, hTRT etc. consumption and method of use all be ready-made, need not here to give unnecessary details in detail, please check pertinent literature.
Claims (2)
1. extend the method for adult multipotential stem cell telomere; Its characteristic comprises: scheme one step: 1, with methods such as electroporation, liposome or microinjections ATRX or the proteic antibody of DAXX, or the asONs of ATRX or DAXX gene or siRNA import in the multipotent adult stem cells of cultivating; 2, be placed on multipotent adult stem cells in the nutrient solution then and cultivate for some time, can confirm incubation time by telomere length as required, made telomere obtain extending to a week in general 3 days; 3, described antibody or asONs whenever will add once between 2 hours, because there is the transformation period; Scheme two steps: 1, from the JEG-3 of no Telomerase, isolate ECTRDNA, or with artificial synthetic ECTR DNA; 2, import to ECTR DNA in the multipotent adult stem cells of cultivation with methods such as electroporation, liposome or microinjections, ECTR DNA consumption is 50～100 times of normal cell telomeric dna; 3, also can in multipotent adult stem cells, import hTRT simultaneously, to increase the extension efficient of telomere; 4, be placed on multipotent adult stem cells in the nutrient solution then and cultivate for some time, make telomere obtain extending.
2. the method for extension adult multipotential stem cell telomere according to claim 1 is characterized in that also can in multipotent adult stem cells, importing hTRT simultaneously, to increase the extension efficient of telomere.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103585618A (en) * | 2013-10-31 | 2014-02-19 | 中山大学 | Application of death-domain associated protein (DAXX) in preparation of tumor cell regulation agent |
| CN105164269A (en) * | 2013-02-22 | 2015-12-16 | 里兰斯坦福初级大学理事会 | Compounds, compositions, methods and kits related to telomere extension |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105164269A (en) * | 2013-02-22 | 2015-12-16 | 里兰斯坦福初级大学理事会 | Compounds, compositions, methods and kits related to telomere extension |
| CN103585618A (en) * | 2013-10-31 | 2014-02-19 | 中山大学 | Application of death-domain associated protein (DAXX) in preparation of tumor cell regulation agent |
| CN103585618B (en) * | 2013-10-31 | 2016-04-06 | 中山大学 | Telomere binding protein DAXX is preparing the application in tumor cell adjusting control agent |
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