CN108004200A - A kind of mitochondria transfer liquid and preparation method thereof - Google Patents
A kind of mitochondria transfer liquid and preparation method thereof Download PDFInfo
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- CN108004200A CN108004200A CN201711257531.9A CN201711257531A CN108004200A CN 108004200 A CN108004200 A CN 108004200A CN 201711257531 A CN201711257531 A CN 201711257531A CN 108004200 A CN108004200 A CN 108004200A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N2500/00—Specific components of cell culture medium
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
Abstract
The present invention provides a kind of mitochondria transfer liquid and preparation method thereof.The mitochondria shifts liquid not calcium ions and animal blood serum, can keep mitochondria activity, while does not cause live body to produce rejection, allergic reaction, additionally it is possible to meets the needs of mitochondria transfer, can use in vivo, in vitro.The preparation method of the mitochondria transfer liquid, it is easy to operate, it is adapted to large-scale promotion to use.
Description
Technical field
The invention belongs to cell or organelle treatment fluid technical field, and in particular to it is a kind of efficiently mitochondria transfer liquid and its
Preparation method.
Background technology
Mitochondria is the organelle that energy is provided for cell, it has a set of DNA (mtDNA) of itself, passes through mother's
Ovum passes to the next generation.The mitochondrial mutations occurred in ovum may cause maternal family's property disease.This mutation can be led
Cause the problem of serious, it is impacted it is most of be the high organ of energy requirement, such as brain, muscle, heart are related to a series of
Extensive Disease in Infants hereditary disease.Intramitochondrial DNA sequence dna can also develop and mutation is produced in aging course, and this new hair is prominent
Become and only occur with patient, without occurring with its breeder mother.Current therapeutic scheme is extremely limited, and patient can only lean on medicine
Short-term relief symptom, can not cure.Since the mitochondrial DNA of most of mitochondrial diseases is with Disease in Infants heredity by ooecium matter
Mode enter of future generation, therefore doctor usually suggests the women with a certain amount of Mitochondrial DNA Mutation without fertility.Therefore,
Mitochondrial disease brings painful and unfortunate to family【Lightowlers,R.N.,Taylor,R.W.&Turnbull,
D.M.Mutations causing mitochondrial disease:What is new and what challenges
remainScience 349,1494-9(2015).】.
It is conservative to estimate in the natural growing process that growing process is intervened and optimized not over genetic decoding technology
Count every 5000 live body babies and have a people and suffer from mitochondrial disease.Mitochondrial disease can it is abnormal by nucleus DNA or
Mitochondrial DNA causes.The disease related with the gene order of human mitochondrial shares 178 kinds at present, there is 1363 genes
The progress of mitochondria normal activity is participated in, participates in the generation of disease.It has been found that there are 3711 gene order changes to cause
The generation of mitochondrial disease【Shen,L.et al.MSeqDR:A Centralized Knowledge Repository and
Bioinformatics Web Resource to Facilitate Genomic Investigations in
Mitochondrial Disease.Hum Mutat 37,540-8(2016).】
The appearance of genetic decoding technology provides a cost-effective method for quick and precisely identification mitochondrial disease,
Possibility is provided for precisely treatment.Either from the mutation of the exception of mitochondrial DNA, or nuclear DNA, gene sequence
It is the method using gene correction that disease caused by the change of row, which is considered most efficient method,.But mitochondrial genomes is different
Matter, the diversity of Clinical signs and the gene that the difficulty in exogenous genetic material importing mitochondria is caused to mitochondrial gene disorder
Treatment becomes abnormal difficult【Taylor,R.W.&Turnbull,D.M.Mitochondrial DNA mutations in
human disease.Nat Rev Genet 6,389-402(2005).】.At present, as caused by changing Mitochondrial gene sequence
The therapeutic scheme of disease mainly has different position gene representation and mitochondria displacement method.In different position gene representation, in nucleus
Interior expression mitochondrial protein, this protein in cell nuclear expression partly can recover chondriogen and cause disease to reach extremely
To therapeutic effect【Taylor,R.W.&Turnbull,D.M.Mitochondrial DNA mutations in human
disease.Nat Rev Genet 6,389-402(2005).】.Mitochondria is contributed to be used for replacing mother by auxiliary procreation technology
Entrained by parent, the defective mitochondria of gene, so that eliminate offspring causes disease because mother's mitochondria in itself is obtained
Disease【Amato,P.,Tachibana,M.,Sparman,M.&Mitalipov,S.Three-parent in vitro
fertilization:gene replacement for the prevention of inherited mitochondrial
diseases.Fertil Steril 101,31-5(2014).】, legitimacy of the Britain by legislative confirmation using the technology
【Dimond,R.&Stephens,N.Three persons,three genetic contributors,three parents:
Mitochondrial donation,genetic parenting and the immutable grammar of the'
three x x'.Health(London),1363459316689380(2017).】.But this technology can only be used for not giving birth to
Clear and definite mitochondrial gene disorder when educating, for avoiding chondriogen defect mother fertility disorders offspring, it is impossible to for treating
The embryo bred or the adult with mitochondrial disease.
When carrying out the transfer of mitochondria, most common method is direct hatching method.In this approach, freshly prepd line
Plastochondria is suspended using the culture medium of Fresh, then carries out hatching training with the culture medium containing mitochondrial and culture cell
Support, so that mitochondria is entered in culture cell.In cell culture medium, can generally there are abundant calcium ion and animal blood
Clearly；The presence of calcium ion can damage the activity of mitochondria, so as to reduce the global transfer efficiency of mitochondria；And serum because into
Divide excessively complexity, if used in living animal experiment, human injection's liquid, serious rejection, allergic reaction can be caused.It is comprehensive
On, there is no also one kind to keep mitochondria activity in the prior art, moreover it is possible to not cause live body to produce rejection, anaphylactoid line
Plastochondria shifts liquid.
The content of the invention
It is existing in the prior art in order to solve the problems, such as, the present invention provides one kind transfer liquid and preparation method thereof, it is particularly
A kind of mitochondria transfer liquid and preparation method thereof.The mitochondria shifts liquid not calcium ions and animal blood serum, can keep line
Mitochondrial activity, at the same do not cause live body produce rejection, allergic reaction, additionally it is possible to meet mitochondria transfer needs, can in vivo,
It is external to use.
It should be noted that the mitochondria transfer liquid can be used not only for transfer mitochondria, additionally it is possible to for shifting other
Organelle, plastid, lysosome, vacuole, may also be used for progress cell and are merged with intercellular.
The object of the present invention is to provide a kind of mitochondria to shift liquid.
Another object of the present invention is to provide a kind of preparation method of the mitochondria transfer liquid.
Mitochondria transfer liquid according to the present invention, the mitochondria transfer liquid include following components：Buffer, isotonic agent,
Calcium chelating agent, directional couple accelerating agent, mitochondria activity accelerating agent and mitochondria infiltration lane inhibitor.
Mitochondria transfer liquid according to the present invention, wherein, the buffer solution is HEPES buffer solution, the HEPES buffer solution
The concentration of middle HEPES is 10-30mmol/L.
HEPES is the abbreviation of 4- hydroxyethyl piperazineethanesulfonic acids.
Mitochondria transfer liquid according to the present invention, wherein, the isotonic solution is sucrose solution, and the concentration of the sucrose is
0.8-1.2mol/L。
Mitochondria transfer liquid according to the present invention, wherein, the directional couple accelerating agent is
RETWWETWWTEWSQPRRRRKV- mercaptoethylmaines, the concentration of the RETWWETWWTEWSQPRRRRKV- mercaptoethylmaines is 40-
60mmol/L。
Mitochondria transfer liquid according to the present invention, wherein, the calcium chelating agent is EGTA, and the concentration of the EGTA is 0.3-
0.7mmol/L。
Mitochondria transfer liquid according to the present invention, wherein, the mitochondria activity accelerating agent include glutamic acid, maleic acid,
Butanedioic acid and deoxyadenosine diphosphate；The concentration of the glutamic acid is 3-7mmol/L, the concentration of maleic acid is 3-7mmol/L, amber
The concentration of amber acid is 3-7mmol/L and the concentration of deoxyadenosine diphosphate is 0.5-1.5mmol/L.
Mitochondria transfer liquid according to the present invention, wherein, the mitochondria infiltration lane inhibitor is Ruo360, Ruo360
Chemical name be called (μ-oxo) bis (trans-formatotetramine ruthenium), be one kind by near-linear
, combined by two ruthenium atoms by oxygen bridge, with the complex bound complex compound of amino.The concentration of the Ruo360 is 0.05-
0.15mmol/L。
Mitochondria transfer liquid according to the present invention, wherein, the buffer solution is HEPES buffer solution, and the isotonic solution is sugarcane
Sugar juice, the directional couple accelerating agent are RETWWETWWTEWSQPRRRRKV- mercaptoethylmaines, and the calcium chelating agent is EGTA,
The mitochondria activity accelerating agent includes glutamic acid, maleic acid, butanedioic acid and deoxyadenosine diphosphate, and the mitochondria infiltration is logical
Road inhibitor is Ruo360；
The concentration of HEPES is 20mmol/L in the HEPES buffer solution, and the concentration of the sucrose is 0.8-1.2mol/L,
The concentration of the RETWWETWWTEWSQPRRRRKV- mercaptoethylmaines is 50mmol/L, and the concentration of the EGTA is 0.5mmol/L,
The concentration of the glutamic acid is 5mmol/L, and the concentration of maleic acid is 5mmol/L, and the concentration of butanedioic acid is 5mmol/L and deoxidation gland
The concentration of glycosides diphosphonic acid is 1mmol/L, and the concentration of the Ruo360 is 0.1mmol/L.
Concentrations above, is that each component adds mixed ultimate density.
The preparation method of the mitochondria transfer liquid, the preparation method comprise the following steps according to the present invention：
A part of water is first used, after all components are dissolved, adjusts pH value to 7.0-7.4, and adds the water of remainder,
Stir evenly.
The preparation method of mitochondria transfer liquid according to the present invention, wherein, using hydrochloric acid or sodium hydroxide solution by pH
Value is adjusted to 7.2；Stir evenly, be placed in 4 DEG C of environment and preserve after then being sterilized using the membrane filtration of 0.25um.
Beneficial effects of the present invention are：
1st, calcium ion and animal blood serum are not contained in the mitochondria transfer liquid, while (EGTA is chelated using calcium chelating agent
Agent) residual calcium ion in fixed environment so as to reduce the infringement of calcium ion mitochondria, increases the transfer of effective mitochondria.
2nd, due to the stability and low irritability of mitochondria transfer liquid, it can in vitro test and be adopted with experiment in vivo
With same transfer liquid, the uniformity of preclinical study, clinical data can be increased, accelerate mitochondria transfer and controlled in gene
Treat, the examination ＆ approval in gene correction, research and development cost is greatly lowered.
3rd, the infiltration lane of mitochondria, under the action of calcium ion, the cooling mechanism of meeting activation wire grain, causes mitochondria to damage
Wound.Mitochondria infiltration lane inhibitor (i.e. mitochondrial calcium uptake inhibitor, Ruo360) is used in the mitochondria transfer liquid, is led to
The calcium pickup for suppressing mitochondria is crossed, increases the transfer efficiency of mitochondria.
4th, with the addition of in the mitochondria transfer liquid mitochondria activity accelerating agent (glutamic acid, maleic acid, butanedioic acid and
ADP), by increasing Metabolism of Mitochondria intermediate, the stability and activity of mitochondria key enzyme are increased.
5th, directional couple accelerating agent is with the addition of in mitochondria transfer liquid, uses the protein and peptide of engineer can be with
Mitochondria is anchored on cell by intermolecular force, increases the endocytosis of mitochondria, so as to increase substantially line grain
The transfer efficiency of body.
6th, the preparation method of the mitochondria transfer liquid, it is easy to operate, it is adapted to large-scale promotion to use.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is attached drawing needed in technology description to be briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, without creative efforts, can be with
Other attached drawings are obtained according to these attached drawings.
Fig. 1 is the consumption rate line chart using cell obtained by the sugared nutrient solution transfer mitochondrias of DMEM high；
Fig. 2 is the consumption rate line chart using cell obtained by HEPES transfer liquid transfer mitochondrias；
Fig. 3 is the consumption rate line chart using cell obtained by EGTA transfer liquid transfer mitochondrias；
Fig. 4 is the consumption rate line chart using cell obtained by coupling agent transfer liquid transfer mitochondria；
Fig. 5 is the consumption rate line chart using cell obtained by metabolic intermediate transfer liquid transfer mitochondria；
Fig. 6 is the consumption rate line chart using cell completely obtained by transfer liquid transfer mitochondria.
Embodiment
To make the object, technical solutions and advantages of the present invention clearer, technical scheme will be carried out below
Detailed description.Obviously, described embodiment is only part of the embodiment of the present invention, instead of all the embodiments.Base
Embodiment in the present invention, those of ordinary skill in the art are obtained all on the premise of creative work is not made
Other embodiment, belongs to the scope that the present invention is protected.
In the present invention, if not refering in particular to, all parts, percentage are unit of weight, and all equipment and raw material etc. are equal
It is commercially available or the industry is common.Method in following embodiments, is the routine of this area unless otherwise instructed
Method.
Embodiment 1
A kind of mitochondria shifts liquid, and the combination of the mitochondria transfer liquid and dosage are as shown in table 1：
Table 1：Mitochondria shifts the component list of liquid
Cumulative volume：1L；Wherein, JXNANO- coupling agents are RETWWETWWTEWSQPRRRRKV- mercaptoethylmaines.
The preparation method of mitochondria transfer liquid is：
The measuring cup of 1L is taken, adds 600ml ultra-pure waters, each component of the mitochondria transfer liquid is taken respectively, is added in order
In ultra-pure water, added in agitation, until all dissolvings.PH value is adjusted to 7.0 with 1M hydrochloric acid or NaOH, continues to add ultra-pure water
To 950ml, pH value is adjusted to pH value to 7.0 with 1M hydrochloric acid or NaOH, addition ultra-pure water uses the filter membrane mistake of 0.25um to 1 liter
Filter sterilization, it is spare to be placed on 4 degree of refrigerators.
Embodiment 2
A kind of mitochondria shifts liquid, and the combination of the mitochondria transfer liquid and dosage are as shown in table 2：
Table 2：Mitochondria shifts the component list of liquid
Cumulative volume：1L；Wherein, JXNANO- coupling agents are RETWWETWWTEWSQPRRRRKV- mercaptoethylmaines.
The preparation method of mitochondria transfer liquid is：
The measuring cup of 1L is taken, adds 600ml ultra-pure waters, each component of the mitochondria transfer liquid is taken respectively, is added in order
In ultra-pure water, added in agitation, until all dissolvings.PH value is adjusted to 7.4 with 1M hydrochloric acid or NaOH, continues to add ultra-pure water
To 950ml, pH value is adjusted to pH value to 7.4 with 1M hydrochloric acid or NaOH, addition ultra-pure water uses the filter membrane mistake of 0.25um to 1 liter
Filter sterilization, it is spare to be placed on 4 degree of refrigerators.
Embodiment 3
A kind of mitochondria shifts liquid, and the combination of the mitochondria transfer liquid and dosage are as shown in table 3：
Table 3：Mitochondria shifts the component list of liquid
Cumulative volume：1L；Wherein, JXNANO- coupling agents are RETWWETWWTEWSQPRRRRKV- mercaptoethylmaines.
The preparation method of mitochondria transfer liquid is：
The measuring cup of 1L is taken, adds 600ml ultra-pure waters, each component of the mitochondria transfer liquid is taken respectively, is added in order
In ultra-pure water, added in agitation, until all dissolvings.PH value is adjusted to 7.2 with 1M hydrochloric acid or NaOH, continues to add ultra-pure water
To 950ml, pH value is adjusted to pH value to 7.2 with 1M hydrochloric acid or NaOH, addition ultra-pure water uses the filter membrane mistake of 0.25um to 1 liter
Filter sterilization, it is spare to be placed on 4 degree of refrigerators.
Experimental example
This experimental example is that the mitochondria obtained using embodiment 3 is shifted liquid and do not add the transfer liquid work of various components
For contrast.
With DMEM high glucose mediums (Glutmax 4.5g/l, 10% calf serum, 100mg/ml Sodium Pyruvates, 50mg/ml
Uracil and antibiotic, antimycin (being purchased from Thermo Fisher), straight through in 3 centimetres of culture dish, culture M0HeLa is thin
Born of the same parents remove culture medium, wash culture dish with no calcium PBS solution, add the mitochondrial suspension of Fresh to 80% density
2ml, 109A/milliliter.Temperature is incubated 2 hours at room temperature, adds 2ml complete mediums.Continue culture 24 it is small when.Into line grain
The detection of body consumption rate is compared.
Using the cell of different particulate bodies transfer liquid transfer and not into the cell of line grain transfer, using 0.5 tryptose
Enzyme EDTA solution digestions prepare 3 × 106 suspension of cell, and suspension is using DMEM high sugar cultures, wherein containing 10% small ox blood
Clearly, 100mg/ml Sodium Pyruvates, 50mg/ml uracils and antibiotic-antimycin.Cell liquid is placed in oxytherm cells,
Consumption rate of every 10 minutes measure, each sample measure 6 times, and the consumption rate curve of cell is drawn according to testing result.It is vertical to sit
Mark represents consumption rate, and unit is pmol/min, and abscissa represents the time, unit (axis of ordinates, unit pmol/ are used as using minute
Min, axis of abscissas：min).As shown in figures 1 to 6, experimental data shows experimental result, the group not shifted by mitochondria, consumption
Oxygen rate is baseline values, DMEM high sugar nutrient solutions and the basis transfer liquidus plastochondria transfer efficiency prepared by HEPES quite (Fig. 1,
Fig. 2), add coupling agent, metabolic intermediate and EGTA can individually effectively improve mitochondria transfer efficiency (Fig. 3,4,
5).But three kinds of components at the same time in the presence of, the transfer effect of mitochondria is optimal, reaches highest level, thin with normal wild type HeLa
The consumption rate of born of the same parents is close to (Fig. 6).Illustrate that the effect for the complete mitochondria transfer liquid that embodiment 3 obtains is best.
The above description is merely a specific embodiment, but protection scope of the present invention is not limited thereto, any
Those familiar with the art the invention discloses technical scope in, change or replacement can be readily occurred in, should all be contained
Cover within protection scope of the present invention.Therefore, protection scope of the present invention should be based on the protection scope of the described claims.
Claims (10)
1. a kind of mitochondria shifts liquid, it is characterised in that the mitochondria transfer liquid includes following components：Buffer, isotonic agent,
Calcium chelating agent, directional couple accelerating agent, mitochondria activity accelerating agent and mitochondria infiltration lane inhibitor.
2. mitochondria according to claim 1 shifts liquid, it is characterised in that the buffer solution is HEPES buffer solution, described
The concentration of HEPES is 10-30mmol/L in HEPES buffer solution.
3. mitochondria according to claim 1 shifts liquid, it is characterised in that the isotonic solution is sucrose solution, the sugarcane
The concentration of sugar is 0.8-1.2mol/L.
4. mitochondria according to claim 1 shifts liquid, it is characterised in that the directional couple accelerating agent is
RETWWETWWTEWSQPRRRRKV- mercaptoethylmaines, the concentration of the RETWWETWWTEWSQPRRRRKV- mercaptoethylmaines is 40-
60mmol/L。
5. mitochondria according to claim 1 shifts liquid, it is characterised in that the calcium chelating agent is EGTA, the EGTA
Concentration be 0.3-0.7mmol/L.
6. mitochondria according to claim 1 shifts liquid, it is characterised in that the mitochondria activity accelerating agent includes paddy ammonia
Acid, maleic acid, butanedioic acid and deoxyadenosine diphosphate；The concentration of the glutamic acid is 3-7mmol/L, the concentration of maleic acid is 3-
7mmol/L, the concentration of butanedioic acid is 3-7mmol/L and the concentration of deoxyadenosine diphosphate is 0.5-1.5mmol/L.
7. mitochondria according to claim 1 shifts liquid, it is characterised in that the mitochondria infiltration lane inhibitor is
The concentration of Ruo360, the Ruo360 are 0.05-0.15mmol/L.
8. mitochondria according to claim 1 shifts liquid, it is characterised in that the buffer solution is HEPES buffer solution, described
Isotonic solution is sucrose solution, and the directional couple accelerating agent is RETWWETWWTEWSQPRRRRKV- mercaptoethylmaines, the calcium chela
Mixture is EGTA, and the mitochondria activity accelerating agent includes glutamic acid, maleic acid, butanedioic acid and deoxyadenosine diphosphate, described
Mitochondria infiltration lane inhibitor is Ruo360；
The concentration of HEPES is 20mmol/L in the HEPES buffer solution, and the concentration of the sucrose is 0.8-1.2mol/L, described
The concentration of RETWWETWWTEWSQPRRRRKV- mercaptoethylmaines is 50mmol/L, and the concentration of the EGTA is 0.5mmol/L, described
The concentration of glutamic acid is 5mmol/L, and the concentration of maleic acid is 5mmol/L, and the concentration of butanedioic acid is 5mmol/L and desoxyadenossine two
The concentration of phosphoric acid is 1mmol/L, and the concentration of the Ruo360 is 0.1mmol/L.
9. the preparation method of any mitochondria transfer liquid of claim 1-8, it is characterised in that the preparation method include with
Lower step：
A part of water is first used, after all components are dissolved, adjusts pH value to 7.0-7.4, and adds the water of remainder, is stirred
Uniformly.
10. preparation method according to claim 9, it is characterised in that using hydrochloric acid or sodium hydroxide solution by pH value tune
To 7.2；Stir evenly, be placed in 4 DEG C of environment and preserve after then being sterilized using the membrane filtration of 0.25um.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101381741A (en) * | 2008-10-31 | 2009-03-11 | 中国科学技术大学 | Polypeptides vector for gene transport of eukaryotic cell and derivates |
| CN106350479A (en) * | 2011-04-14 | 2017-01-25 | 通用医疗公司 | Composition and method for energy transfer of autologous germline mitochondria |
-
2017
- 2017-12-01 CN CN201711257531.9A patent/CN108004200A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101381741A (en) * | 2008-10-31 | 2009-03-11 | 中国科学技术大学 | Polypeptides vector for gene transport of eukaryotic cell and derivates |
| CN106350479A (en) * | 2011-04-14 | 2017-01-25 | 通用医疗公司 | Composition and method for energy transfer of autologous germline mitochondria |
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| Title |
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| AKIHIRO MASUZAWA: "Transplantation of autologously derived mitochondria protects the heart from ischemia-reperfusion injury", 《AM J PHYSIOL HEART CIRC PHYSIOL》 * |
| JUI-CHIH CHANG: "Treatment of human cells derived from MERRF syndrome by peptide-mediated mitochondrial delivery", 《CYTOTHERAPY》 * |
| 余元勋: "《中国分子医学系列丛书 中国分子心脏病学》", 31 August 2015, 安徽科学技术出版社 * |
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