CN106701669A - Mesenchymal stem cell for clinical treatment as well as preparation method and application thereof - Google Patents
Mesenchymal stem cell for clinical treatment as well as preparation method and application thereof Download PDFInfo
- Publication number
- CN106701669A CN106701669A CN201510459332.0A CN201510459332A CN106701669A CN 106701669 A CN106701669 A CN 106701669A CN 201510459332 A CN201510459332 A CN 201510459332A CN 106701669 A CN106701669 A CN 106701669A
- Authority
- CN
- China
- Prior art keywords
- stem cell
- mescenchymal stem
- cell
- tissue block
- tissue
- 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
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a mesenchymal stem cell for clinical treatment as well as a preparation method and application thereof. The method comprises the following steps: (1) culturing multiple mesenchymal stem cell-containing tissue blocks in a first culture dish so as to obtain mesenchymal stem cells climbing out of the multiple tissue blocks, and selecting optimized tissue blocks from the multiple mesenchymal stem cell-containing tissue blocks; (2) culturing the optimized tissue blocks obtained in the step (1) in a second culture dish so as to obtain the mesenchymal stem cells climbing out of the optimized tissue blocks, wherein a condition for selecting the optimized tissue blocks in the step (1) comprises at least one of the following conditions: (a) no endothelial cells climb out; (b) the mesenchymal stem cells climb out; (c) the mesenchymal stem cells climb out at the highest speed. According to the method, the utilization rate of cord tissues is high, and the yield of the cells is greatly increased; furthermore, the obtained mesenchymal stem cells is high in purity and high in activity and can meet the requirement of clinical application.
Description
Technical field
The present invention relates to bioengineering field, in particular it relates to the preparation method of cell, uses more particularly, to clinical treatment
Mescenchymal stem cell and its production and use.
Background technology
Mescenchymal stem cell (Mesenchymal stem cells, MSC) is the important member of stem cell line, from development
The mesoderm and ectoderm of early stage, belong to multipotential stem cell, are distributed widely in various groups of marrow in animal body, liver, fat etc.
In knitting.MSCs has powerful self-renewal capacity and multi-lineage potential, still has after continuous passage culture and freezen protective
There is multi-lineage potential, can be divided into fat, bone, cartilage, muscle, flesh in vivo or in vitro under specific inductive condition
The Various Tissues cell such as tendon, ligament, nerve, liver, cardiac muscle, endothelium, is that the renewable source that fields of implantation has a extensive future is thin
Born of the same parents.Meanwhile, MSCs is easy to purifies and separates, non-immunogenicity, and without ethics problem so that basic research on it and
Its clinical practice flourishes.
Human umbilical cord mesenchymal stem cells (HUMSCs), are the special embryo's mucoid connective tissues in umbilical cord between arteriovenous
The isolated stroma cell of Wal Tong Shi glue (Whartonps Jelly).Human umbilical cord mesenchymal stem cells are in cell content, increasing
Grow ability aspect and be better than bone marrow MSCs, and immunogenicity is lower than bone marrow MSCs, and with convenient material drawing, to puerpera and newly
Raw youngster does not have any infringement, does not have tumour cell, and the infection of virus and pathogenic microorganism and propagation probability are relatively low,
The advantages of being disputed on without ethics, therefore, increasingly paid close attention to by research workers.
However, the clinical practice of umbilical cord mesenchymal stem cells need further exploitation.
At present, MSCs, selection of the enzyme digestion in enzyme are generally obtained from umbilical cord tissue using enzyme digestion or plant block method
With there is no unified standard in digestion time, more using clostridiopetidase A, make the collagenous fibres and extracellular protease between animal tissue cell
Solution, so as to obtain individual cells.But collagenase digesting can excessively cause the change of cell surface some living materials and then influence thin
Born of the same parents' vigor, has damaging action to cell.There are certain requirement, and umbilical cord group to be digested to the time of enzymic digestion umbilical cord tissue
Knit and be not a system for complete and homogeneous, it is difficult to ensure that the uniformity of all cell dissociation degree, therefore be difficult to hold optimal
Digestion time, can influence the quality and quantity of gained cell, and then influence later research and use.Separately due to existing clostridiopetidase A
From pluck, using enzyme digestion when being separately cultured, there is the pollution of animal derived protein and animal sources pathogen, increase
The risk of clinical practice stem cell is added.Compared with collagenase method, planting block method can avoid above mentioned problem, and tissue damage is small, umbilical cord
Tissue utilization rate is high, easily obtains the preferable cell of survival.Because cell of moving out is not affected by infringement, and in early stage block secretion in a organized way
Nutriment support, therefore be easier to survival.And it is existing plant block method it is although simple to operate, but by mechanically actuated formed
Tissue block quality is uneven, and adherent ability is also not quite similar, and can so largely effect on cultivation cycle；And it is thin often to have endothelium
The residual of born of the same parents, influences the quality and purity of primary cell；Passed on for cell to P0 simultaneously after, primary tissue block is lost completely
Abandon, the yield of cell is subject to very big restriction.Importantly, operation is very low to the utilization rate of umbilical cord tissue above, and it is
One greatly wastes.
Therefore, the preparation method of mescenchymal stem cell is further improved.
The content of the invention
It is contemplated that at least solving one of technical problem present in prior art.Therefore, it is an object of the present invention to carry
Go out that a kind of umbilical cord tissue utilization rate is high, cell yield is high and active good, purity clinical treatment mescenchymal stem cell high, together
When propose purposes of the mescenchymal stem cell in medicine is prepared.
Thus, according to the first aspect of the invention, the invention provides a kind of method for preparing clinical treatment mescenchymal stem cell.
Embodiments in accordance with the present invention, the method includes：
(1) tissue block of the multiple containing mescenchymal stem cell is cultivated in the first culture dish, to obtain from described many
The mescenchymal stem cell climbed out of in individual tissue block, and optimization group is selected from the multiple tissue block containing mescenchymal stem cell
Knit block；And
(2) the optimizing tissue block obtained in step (1) is cultivated in the second culture dish, to obtain from described excellent
Change the mescenchymal stem cell that tissue block is climbed out of,
Wherein, the condition of optimizing tissue block is selected in step (1) including at least one following：
A () climbs out of without endothelial cell；
B () has mescenchymal stem cell to climb out of；
C () mescenchymal stem cell is most climbed out of soon.
Inventor has surprisingly found that, with it has been reported that mescenchymal stem cell preparation method compared with, between clinical grade umbilical cord of the invention
Mesenchymal stem cells preparation method：Utilization rate to umbilical cord tissue is high, and especially, the yield of cell is greatly improved, and can further be expired
The big demand of sufficient clinical practice amount；And the clinical treatment for preparing is high with mescenchymal stem cell purity, cell state is good, living
Property it is high, differentiation capability is strong, disclosure satisfy that the functional quality requirement of clinical practice.
In addition, the method for preparing clinical treatment mescenchymal stem cell according to the above embodiment of the present invention, can also have as follows
Additional technical characteristic：
Embodiments in accordance with the present invention, the tissue block containing mescenchymal stem cell derives from umbilical cord tissue.
Other embodiments of the invention, the tissue block containing mescenchymal stem cell is obtained through the following steps：
Reject the arteria umbilicalis and umbilical vein of umbilical cord tissue；And Wal Tong Shi glue is separated from the umbilical cord tissue, wherein, the Wal
Tong Shi glue constitutes the tissue block containing mescenchymal stem cell.Thus, the mescenchymal stem cell tissue of the umbilical cord tissue for obtaining
Purity is high, and endothelial cell content is few.
Embodiments in accordance with the present invention, after step (2), further include：(3) by the optimizing tissue block the 3rd
Cultivated in culture dish, the P0 climbed out of from the optimizing tissue block can be obtained after 5-7 days for mescenchymal stem cell.Thus,
This can obtain more P0 for mescenchymal stem cell, and the utilization rate to umbilical cord tissue is high.
Embodiments in accordance with the present invention, repeat step (3) at least one times, preferably 2~5 times.Thus, by repeating to cultivate,
The multiple utilization of optimizing tissue block is realized, substantial amounts of primary (i.e. P0 generations) mescenchymal stem cell can be obtained, to umbilical cord group
The utilization rate knitted is significantly improved.
Embodiments in accordance with the present invention, the P0 obtained in collection step (1)~(3) carries out passage training for mescenchymal stem cell
Support.It is possible thereby to obtain the mescenchymal stem cell in substantial amounts of P3-P5 generations such that it is able to while meet clinical treatment mesenchyma doing
The requirement of cell by cell algebraical sum quantity.
Embodiments in accordance with the present invention, the Secondary Culture uses serum free medium.Some specific examples of the invention,
The Secondary Culture uses serum free medium (Sciencell), the pollution without any animal derived protein and animal sources pathogen.
Thus, cell proliferation rate is fast, and activity is high.
Embodiments in accordance with the present invention, Secondary Culture, the time interval of the passage are carried out by the P0 for mescenchymal stem cell
It is 2-4 days, and using resulting P3~P5 for mescenchymal stem cell as clinical treatment mescenchymal stem cell.Thus, lead to
Crossing the passage of cell can quickly obtain the low algebraically mescenchymal stem cell of clinical number of levels.
Embodiments in accordance with the present invention, are cultivated the tissue block in step (1)~(3) using serum free medium.
Some specific examples of the invention, the culture medium is serum free medium (Sciencell), without any animal derived protein
With the pollution of animal sources pathogen.Thus, the mescenchymal stem cell state for climbing out of from tissue block is good, and multiplication capacity is strong, and vigor is high.
According to the second aspect of the invention, the invention provides a kind of mescenchymal stem cell.The mescenchymal stem cell is by preceding
What the method for preparing clinical treatment mescenchymal stem cell described in face was obtained.Inventor has surprisingly found that, with it has been reported that face
Bed level umbilical cord mesenchymal stem cells preparation method is compared：The clinical treatment prepared using the method for the present invention is dry thin with mesenchyma
Born of the same parents' purity is high, and cell state is good, and activity is high, and differentiation capability is strong, and stem cell index is good, with clinical value very high.
According to the third aspect of the invention we, the purposes the invention provides foregoing mescenchymal stem cell in medicine is prepared,
The medicine is used to treat tissue damage.Inventor has surprisingly found that, using the clinic of foregoing method of the present invention preparation
Treatment mescenchymal stem cell, cell purity is high, and activity is good, and differentiation capability is strong, and stem cell index is good, can be effective for system
The medicine for the treatment of tissue damage is ready for use on, and tissue damage therapeutic effect is protruded.
According to the fourth aspect of the invention, the purposes the invention provides foregoing mescenchymal stem cell in medicine is prepared,
The medicine is used to treat Alzheimer disease.Inventor is had surprisingly found that, is prepared using the foregoing method of the present invention
Clinical treatment mescenchymal stem cell, cell purity is high, and activity is good, and differentiation capability is strong, and stem cell index is good, can be effective
In the medicine for preparing treatment Alzheimer disease, the medicine has for each index of Alzheimer disease pathologic and significantly changes
It is kind.
Additional aspect of the invention and advantage will be set forth in part in the description, and partly will from the following description become bright
It is aobvious, or recognized by practice of the invention.
Brief description of the drawings
Of the invention above-mentioned and/or additional aspect and advantage be will be apparent from description of the accompanying drawings below to embodiment is combined and
It is readily appreciated that, wherein：
Fig. 1 shown according to one embodiment of the invention, two kinds of form pictures of the microexamination of mescenchymal stem cell,
Wherein,
Figure 1A shows the cellular morphology picture using the conventional primary mescenchymal stem cell for planting the preparation of block method,
Figure 1B shows the cellular morphology of the primary mescenchymal stem cell obtained through the 4th repetition culture using the method for the present invention
Picture；
Fig. 2 shows that according to one embodiment of the invention the optimizing tissue block in embodiment 1 obtained when the 4th repetition is cultivated
Primary mescenchymal stem cell Secondary Culture to the 3rd generation (P3) cell surface marker expression picture；
Fig. 3 shows that according to one embodiment of the invention the optimizing tissue block in embodiment 1 obtained when the 4th repetition is cultivated
Primary mescenchymal stem cell Secondary Culture to the mesenchymal cell markers and epithelial cell marker expression during the 3rd generation (P3)
Immunochemistry fluorescent method testing result；
Fig. 4 shows that according to one embodiment of the invention the optimizing tissue block in embodiment 1 obtained when the 4th repetition is cultivated
Primary mescenchymal stem cell Secondary Culture to during the 3rd generation (P3) into fat, skeletonization and the picture into cartilage differentiation situation；
Fig. 5 shows that according to one embodiment of the invention the optimizing tissue block in embodiment 1 obtained when the 4th repetition is cultivated
Primary mescenchymal stem cell Secondary Culture to karyotyping result figure during the 15th generation (P15)；
Fig. 6 shown according to one embodiment of the invention, the mescenchymal stem cell obtained in embodiment 1 be used for control mice group and
The water maze interpretation of result figure of MSC treatment groups；
Fig. 7 shown according to one embodiment of the invention, the mescenchymal stem cell obtained in embodiment 1 be used for control mice group and
MSC treatment groups cerebral hippocampus position and cortical sites Alzheimer disease related molecular marker thing expression of results analysis chart；And
Fig. 8 shown according to one embodiment of the invention, the mescenchymal stem cell obtained in embodiment 1 be used for control mice group and
Cerebral hippocampus area of MSC treatment groups and cortex neural stem cell key point thing Nestin protein expression interpretation of result figures.
Specific embodiment
Embodiments of the invention are described below in detail.Embodiment below with reference to Description of Drawings is exemplary, is only used for solution
The present invention is released, and is not considered as limiting the invention.
It should be noted that term " first ", " second " are only used for describing purpose, and it is not intended that indicating or implying relative
Importance or the implicit quantity for indicating indicated technical characteristic.Thus, " first " is defined, the feature of " second " can be with
Express or implicitly include one or more this feature.Further, in the description of the invention, unless otherwise indicated,
" multiple " is meant that two or more.
According to the first aspect of the invention, the invention provides a kind of method for preparing clinical treatment mescenchymal stem cell.According to
Embodiments of the invention, the method includes：
(1) tissue block of the multiple containing mescenchymal stem cell is cultivated in the first culture dish, to obtain from described many
The mescenchymal stem cell climbed out of in individual tissue block, and optimization group is selected from the multiple tissue block containing mescenchymal stem cell
Knit block；And
(2) the optimizing tissue block obtained in step (1) is cultivated in the second culture dish, to obtain from described excellent
Change the mescenchymal stem cell that tissue block is climbed out of,
Wherein, the condition of optimizing tissue block is selected in step (1) including at least one following：
A () climbs out of without endothelial cell；
B () has mescenchymal stem cell to climb out of；
C () mescenchymal stem cell is most climbed out of soon.
, wherein it is desired to explanation, compared with common mescenchymal stem cell, clinical treatment mescenchymal stem cell is general right
Than larger, the requirement of purity and activity to cell is higher, and dryness index is normal, i.e., cell is external for the demand of cell
Differentiation capability is confirmed, and generally clinical mescenchymal stem cell is the mescenchymal stem cell in P3-P5 generations, it is therefore desirable to improved
The cell yield of primary mescenchymal stem cell tissue, obtains primary mescenchymal stem cell as much as possible, i.e. P0 is dry for mesenchyma
Cell.Cultural method of the invention, selects, and be transferred to second by the tissue block for quickly climbing out of mescenchymal stem cell
Culture dish carries out continuation culture, can get high-purity, the primary mescenchymal stem cell without endothelial cell pollution, such that it is able to aobvious
Write the activity and purity for improving mescenchymal stem cell.
Inventor has surprisingly found that, with it has been reported that mescenchymal stem cell preparation method compared with, between clinical grade umbilical cord of the invention
Mesenchymal stem cells preparation method：Utilization rate to umbilical cord tissue is high, and especially, the yield of cell is greatly improved, and can further be expired
The big demand of sufficient clinical practice amount；And the clinical treatment for preparing is high with mescenchymal stem cell purity, cell state is good, living
Property it is high, differentiation capability is strong, disclosure satisfy that the functional quality requirement of clinical practice.
Embodiments in accordance with the present invention, the source of the tissue block containing mescenchymal stem cell is not particularly limited, can be by city
Sell directly purchase, it is also possible to obtained from umbilical cord tissue.Embodiments in accordance with the present invention, the group containing mescenchymal stem cell
Block is knitted from umbilical cord tissue.Wherein, the method for the tissue block containing mescenchymal stem cell is not obtained from umbilical cord tissue not yet
It is particularly limited, some specific examples of the invention, the tissue block containing mescenchymal stem cell is through the following steps
Obtain：Reject the arteria umbilicalis and umbilical vein of umbilical cord tissue；And Wal Tong Shi glue is separated from the umbilical cord tissue, wherein,
The Wal Tong Shi glue constitutes the tissue block containing mescenchymal stem cell.Thus, the mesenchyma of the umbilical cord tissue for obtaining is dry thin
The purity of born of the same parents' tissue is high, and endothelial cell content is few.
Embodiments in accordance with the present invention, after step (2), further include：(3) by the optimizing tissue block the 3rd
Cultivated in culture dish, the P0 climbed out of from the optimizing tissue block can be obtained after 5-7 days for mescenchymal stem cell.Thus,
This can obtain more P0 for mescenchymal stem cell, and the utilization rate to umbilical cord tissue is high.
It should be noted that the mescenchymal stem cell climbed out of from each tissue block obtained in step (1), and step (2)
The mescenchymal stem cell climbed out of from optimizing tissue block obtained in~(3), is P0 for mescenchymal stem cell.
Embodiments in accordance with the present invention, repeat step (3) at least one times, preferably 2~5 times.Thus, by repeating to cultivate,
The multiple utilization of optimizing tissue block is realized, substantial amounts of primary mescenchymal stem cell can be obtained, the utilization rate to umbilical cord tissue shows
Write and improve.
Embodiments in accordance with the present invention, the P0 obtained in collection step (1)~(3) carries out passage training for mescenchymal stem cell
Support.Thus, it is possible to obtain the mescenchymal stem cell in substantial amounts of P3-P5 generations such that it is able to while meeting clinical treatment mesenchyma
Requirement of the stem cell to cell algebraical sum quantity.
Embodiments in accordance with the present invention, the kinds of culture medium for carrying out Secondary Culture is not particularly limited, as long as can be effectively real
Existing mescenchymal stem cell passage, it is ensured that cell proliferation rate, keeps the Stem Cell Activity of passage cell.It is of the invention
Some embodiments, the Secondary Culture uses serum free medium.Some specific examples of the invention, the Secondary Culture
Using serum free medium (Sciencell), the pollution without any animal derived protein and animal sources pathogen.Thus, cell increases
Grow speed fast, activity is high, and can keep good Stem Cell Activity.And any animal is free of in above-mentioned serum free medium
The serum and antibiotic in source, can effectively reduce heterologous protein, mycoplasma in Secondary Culture etc. and pollute.
Embodiments in accordance with the present invention, Secondary Culture, the time interval of the passage are carried out by the P0 for mescenchymal stem cell
It is 2-4 days, and using resulting P3~P5 for mescenchymal stem cell as clinical treatment mescenchymal stem cell.Thus, lead to
Crossing the passage of cell can quickly obtain the low algebraically mescenchymal stem cell of clinical number of levels.
Embodiments in accordance with the present invention, are used for the kinds of culture medium cultivated tissue block not by spy in step (1)~(3)
Do not limit, if mescenchymal stem cell can be effectively facilitated climb out of, and the mescenchymal stem cell state for climbing out of is good, multiplication capacity is strong,
Vigor is high.Some embodiments of the invention, using serum free medium to described group in step (1)~(3)
Block is knitted to be cultivated.Some specific examples of the invention, the culture medium is serum free medium (Sciencell), nothing
The pollution of any animal derived protein and animal sources pathogen.Thus, the mescenchymal stem cell state for climbing out of from tissue block is good, propagation
Ability is strong, and vigor is high.
According to another aspect of the present invention, stem cell is filled the invention provides an inter-species.The mescenchymal stem cell is by above
What the described method for preparing clinical treatment mescenchymal stem cell was obtained.Inventor has surprisingly found that, with it has been reported that clinic
Level umbilical cord mesenchymal stem cells preparation method is compared：The clinical treatment mescenchymal stem cell prepared using the method for the present invention
Purity is high, and cell state is good, and activity is high, and differentiation capability is strong, and stem cell index is good, with clinical value very high.
, wherein it is desired to, directly can be used for for mescenchymal stem cell to treat tissue damage or Alzheimer disease by explanation,
The medicine prepared with mescenchymal stem cell can be used to treat tissue damage or Alzheimer disease.
Thus, according to another aspect of the invention, the invention provides foregoing mescenchymal stem cell in medicine is prepared
Purposes, the medicine is used to treat tissue damage.Inventor has surprisingly found that, using the clinical treatment of foregoing method preparation
With mescenchymal stem cell, cell purity is high, and activity is good, and differentiation capability is strong, and stem cell index is good, can be used effective for preparing
In the medicine for the treatment of tissue damage, and tissue damage therapeutic effect is protruded.
In accordance with a further aspect of the present invention, the purposes the invention provides foregoing mescenchymal stem cell in medicine is prepared,
The medicine can be used for treating Alzheimer disease.Inventor has surprisingly found that the clinic prepared using foregoing method is controlled
Treatment mescenchymal stem cell, cell purity is high, and activity is good, and differentiation capability is strong, and stem cell index is good, can be effective for preparing
The medicine of Alzheimer disease is treated, the medicine has for each index of Alzheimer disease pathologic significantly to be improved.
Wherein, some specific embodiments of the invention, in purposes of the invention, the mescenchymal stem cell for being used is
3 generations to the 5th generation.Thus, cell is active good, and purity is high, and differentiation capability is strong, and stem cell index is good, so that, clinic is controlled
Therapeutic effect is good.Inventor's discovery, is substantially better than what is obtained by other means by the therapeutic effect using the mescenchymal stem cell
Mescenchymal stem cell.
The term " administration " for being used herein refers to and for the material of scheduled volume to introduce patient by certain suitable mode.This hair
Bright mescenchymal stem cell can be administered by any common approach, as long as it can reach expected tissue.What is be administered is each
Kind of mode can be orally, local, the nasal cavity it is contemplated that including peritonaeum, vein, muscle is subcutaneous, cortex, lung and directly
Intestines, but the invention is not restricted to administering mode that these have been illustrated.However, due to be administered orally when, peptide is digested, and orally gives
The active component of the composition of medicine should be coated or be formulated to prevent it to be degraded in stomach.Preferably, combination of the invention
Thing can be administered with ejection preparation.Additionally, pharmaceutical composition of the invention can use the spy that active component is sent to target cell
Determine apparatus to be administered.
The administration frequency and dosage of pharmaceutical composition of the invention can be determined by multiple correlative factors, and the factor includes will quilt
The order of severity of the disease type for the treatment of, method of administration, patient age, sex, body weight and disease and as active component
Drug type.Some embodiments of the invention, daily dose can be divided into 1 dose, 2 doses or multi-agent of suitable form, with whole
With 1 time, 2 times or multiple dosing in the individual time period, as long as reaching therapeutically effective amount.
Term " therapeutically effective amount " refers to the amount that compound is enough to significantly improve some symptoms related to disease or illness, namely
The amount of therapeutic effect is provided to give illness and dosage regimen.For example, in treatment of alzheimer, reducing, preventing, prolonging
The medicine or compound of any symptom of slow, suppression or retardance disease or illness should be therapeutically effective.The medicine of therapeutically effective amount
Thing or compound do not need cure diseases or illness, but will be that disease or illness provide treatment so that the disease of individuality or illness
Breaking-out is delayed, prevents or prevents, or the symptom of disease or illness is alleviated, or the time limit of disease or illness is changed,
Such as disease or illness become not serious, or accelerate rehabilitation.
Term " treatment " is used to refer to the desired pharmacology of acquisition and/or physiologic effect.The effect is just prevented wholly or in part
Can be preventative for disease or its symptom, and/or just partially or completely ill-effect caused by cure diseases and/or disease
For can be curative." treatment " used herein covers the disease of mammal, particularly people, including：A () exists
It is easily ill still not yet to make a definite diagnosis prevention disease (such as preventing Alzheimer disease) or illness generation in the individuality fallen ill；B () presses down
Disease processed, for example, block disease development；Or (c) alleviates disease, such as mitigate the symptom related to disease.It is used herein " to control
Treat " to cover and give individuality by medicine or compound to treat, cure, alleviate, improve, mitigate or suppress appointing for the disease of individuality
What medication, including but not limited to gives individuality in need by the medicine containing mescenchymal stem cell described herein.
Embodiments in accordance with the present invention, mescenchymal stem cell of the invention or pharmaceutical composition can be with conventional treatments and/or treatments
Method is combined and uses, or can be used separately with conventional treatments and/or therapy.When mescenchymal stem cell of the invention or medicine
When being administered in using the conjoint therapy with other medicines, they can sequentially or simultaneously give individuality to compositions.Or,
Pharmaceutical composition of the invention can include mescenchymal stem cell of the invention, pharmaceutically acceptable carrier or pharmaceutically acceptable
The combination of excipient and other curatives known in the art or preventive medicine.
The solution of the present invention is explained below in conjunction with embodiment.
It will be understood to those of skill in the art that the following examples are merely to illustrate the present invention, and should not be regarded as limiting of the invention
Scope.Unreceipted particular technique or condition in embodiment, according to the technology or condition described by document in the art (for example
Write with reference to J. Pehanorm Brookers etc., what Huang Peitang etc. was translated《Molecular Cloning:A Laboratory guide》, the third edition, Science Press) or
Carried out according to product description.Agents useful for same or the unreceipted production firm person of instrument, be can by city available from conventional produce
Product, for example, can purchase from Gibco companies.
Embodiment 1
With conventional plant block method, as control, (specific method can be found in：Nekanti U,Mohanty L,et al.Optimization
and scale-up of Wharton’s jelly-derived mesenchymal stem cells for clinical
applications.Stem Cell Res.2010；5(3):244-254., by referring to be incorporated by herein), root
According to the method for preparing clinical treatment mescenchymal stem cell of the invention, mescenchymal stem cell is prepared according to following steps：
First, the umbilical cord tissue of XX hospital neonates is collected, will be without hepatitis B, plum with the physiological saline containing penicillin and streptomysin
Poison, the healthy neonatal umbilical cord tissue of HIV infection are fully cleaned, and remove blood stains；By umbilical cord scissors into the uniform segment of length,
Mechanical Method separation, blunt separation China Tong Shi glue are carried out, while removing arteria umbilicalis and umbilical vein；The magnificent Tong Shi glue peeled off uniformly is cut
Broken, wherein the magnificent Tong Shi glue for shredding is the tissue block comprising mescenchymal stem cell.
Then, tissue block of the multiple of above-mentioned acquisition comprising mescenchymal stem cell is inoculated in culture dish, using free serum culture
Base (is the MSC serum free medium MSCM-SF of Sciencell companies of U.S. production, article No.：7511) it is resuspended, it is placed in body
Fraction is 5% CO2Saturated humidity incubator culture 7-10 days, microscopy is picked out and most climb out of soon MSCs (i.e. mesenchyma is dry thin
Born of the same parents) and the tissue block that is climbed out of without endothelial cell --- i.e. optimizing tissue block.
Next, the optimizing tissue block that above-mentioned screening is obtained is transferred in new culture dish to continue to cultivate, and collect former culture dish
The mescenchymal stem cell (i.e. P0 is for mescenchymal stem cell) climbed out of from tissue block carries out Secondary Culture.Wherein, optimizing tissue block
Culture still use above-mentioned serum free medium, the P0 climbed out of from the optimizing tissue block can be obtained after 5-7 days for mesenchyma
Stem cell.
Further, then during the optimizing tissue block is gone into new culture dish cultivated (equally using above-mentioned free serum culture
Base), to obtain the mescenchymal stem cell climbed out of from the optimizing tissue block.
Next, repeat the above steps 2-5 times, to make full use of the optimizing tissue block, obtain from optimizing tissue it is fast in climb
The mescenchymal stem cell for going out.
The P0 climbed out of from tissue block in culture every time is collected for mescenchymal stem cell, Secondary Culture is carried out into new culture dish, its
In, the Secondary Culture uses above-mentioned serum free medium, and per 2-4 days, passage once, can obtain substantial amounts of using the method
High purity raw is for MSCs.
Contrast finds that the relatively control (i.e. conventional to plant block method) in terms of yield improves 20-50 to the method for the present invention for cell in P0
Times, clinic cell algebraically is passaged to this yield, can series increase acquisition with a collection of cell, be fully able to one people's of satisfaction
Repeatedly treat or treated while how personal.And compared with the control, this method is optimized to the condition of culture of primary cell,
Mescenchymal stem cell is cultivated in serum free medium, can obtain good amplification ability, it is often more important that, without appoint
What animal-derived sera can reduce the pollution such as heterologous protein, mycoplasma with antibiotic.Carried out using above-mentioned serum free medium
The culture of mescenchymal stem cell, the state of cell is good, and multiplication capacity is fast, and vigor is high.
Embodiment 2
The primary mescenchymal stem cell that the optimizing tissue block that inventor is chosen in embodiment 1 obtained when the 4th repetition is cultivated enters
Row morphologic detection and surface molecular label flow cytometer detection, to ensure the dirt without endothelial cell and other hematopoietic cells
Dye, it is ensured that the purity of cell；Immunochemistry fluorescence, Analytical Chemical Experiment and cell karyotyping detection are carried out, to ensure what is obtained
The quality of primary mescenchymal stem cell.It is specific as follows：
1st, cellular morphology detection
The primary mescenchymal stem cell for for the optimizing tissue block in embodiment 1 obtained when the 4th is repeated and cultivated (is utilized
Prepared by the method for the present invention), and the mescenchymal stem cell of synchronous with method of the present invention control acquisition carries out Morphology observation,
Result is shown in Fig. 1.As shown in figure 1, figure A shows that the primary MSC appearance for utilizing control " conventional to plant block method " to obtain is obvious
Endothelial cell；And scheme B and show the optimizing tissue block using the method for the present invention " optimizing tissue block method " preparation through the 4th weight
The primary MSC obtained when cultivating again, without endothelial cell, form is homogeneous, in swirl shape adherent growth.Thus, show using this
The purity and cytoactive of mescenchymal stem cell prepared by the method culture of invention are substantially better than and are obtained using conventional block method (control) of planting
The mescenchymal stem cell for obtaining.
2nd, flow cytomery cell surface marker
Surface mark is carried out for mescenchymal stem cell to the P3 obtained using the method for the present invention in embodiment 1 using flow cytometer
Will detects that the wherein P3 is that the optimizing tissue block that will be prepared by the method for the present invention is repeated through the 4th for mescenchymal stem cell
The primary mescenchymal stem cell that culture is obtained, carries out what Secondary Culture to P3 generations obtained.Detection method is specific as follows：
(1) optimizing tissue block is repeated to cultivate the primary MSC passages culture for obtaining to the cell in P3 generations through the 4th, is disappeared
Single cell suspension is made after change, is counted, by 4 × 105Individual often pipe is dispensed to centrifuge tube.
(2) cell after centrifugation is washed once with PBS, 300g centrifugation 10min abandon supernatant.
(3) 100 microlitres of PBS re-suspended cells, add primary antibody CD3-FITC, CD29-PE, CD73-PE, CD166-PE,
CD105-FITC, CD90-PE, CD14-FITC, CD45-PE, HLA-DR-PE, CD38-FITC, CD31-APC, CD34-PE,
And two controls are set, wherein a pipe is straight labeling antibody PE-isotype blanks, another Guan Weizhi labeling antibodies FITC-isotype
Blank, then in 4 DEG C of shaking table lucifuge reaction 40min.
Wherein, the addition of each primary antibody is as follows：
(4) reacted cell is cleaned 3 times with PBS, each 300g is centrifuged 10min, 1% paraformaldehyde is fixed, 4 DEG C
Lower placement, then carries out flow cytomery (detection is finished within a week), and testing result is shown in Fig. 2.Fig. 2 shows carefully
The surface marker expression of born of the same parents.As shown in Fig. 2 cell phenotype is homogeneous, cell expression mesenchymal cell markers CD73, CD90,
CD105, CD29, CD44, CD166, hematopoietic cell markers CD3, CD14, CD34, CD38 and endothelial cell marker are not expressed
CD31, and human leucocyte antigen (HLA) HLA-DR is not expressed, " international cell therapy association " is complied fully with mescenchymal stem cell
The standard that surface marker specifies.
3rd, immunofluorescence staining detection cell surface marker
Dyeing observation is carried out to mescenchymal stem cell by immunofluorescence dyeing detection method, specific method is as follows：
(1) by embodiment 1 by the method for the present invention prepare optimizing tissue block through the 4th repeat culture obtain it is primary
Mescenchymal stem cell, Secondary Culture to P3 generations is with 5.0 × 103Individual/hole is inoculated in 24 orifice plates, and the culture medium in 24 orifice plates is abandoned
Go, washed with PBS 1~2 time.
(2) it is fixed：15min is fixed with 4% paraformaldehyde room temperature of Fresh (2 weeks in), is cooled to after room temperature with containing
The PBS of 0.05%Tween-20 is washed 2 times.
(3) rupture of membranes：Normal temperature adds 0.1%triton-x-100 ruptures of membranes 10 minutes, and PBS is washed 2 times.
(4) close：Closed 30 minutes with confining liquid (10% lowlenthal serum) room temperature, suck confining liquid.
(5) primary antibody is incubated：Primary antibody dilutes (1 with confining liquid:200), 4 DEG C of overnight incubations, PBS is washed, 3 × 5min.Wherein
Primary antibody includes following：Mouse anti human a-SMA (Sigma, 1:800), mouse anti human E-cadherin (BD, 1:100) it is, small
The anti-human N-cadherin of mouse (BD, 1:100), rabbit-anti people CK18 (Santa cruz, 1:50).
(6) secondary antibody is incubated：Add the IgG secondary antibodies (1 of TRITC/FITC marks:100) reaction of room temperature lucifuge 30min, PBS
Wash, 3 × 5min.Wherein secondary antibody includes following：The goat anti-rabbit igg of rhodamine (TRITC) mark (give birth to by Beijing Zhong Shan Golden Bridge
Thing Technology Co., Ltd., 1:100), the goat anti-mouse IgG of fluorescein isothiocynate (FITC) mark (give birth to by Beijing Zhong Shan Golden Bridge
Thing Technology Co., Ltd., 1:100).
(7) addition room temperature reaction 2min, PBS are washed, and 3 × 5min, third time PBS is stayed in hole, is placed under fluorescence microscope
Observation, observation result is shown in Fig. 3.
From the figure 3, it may be seen that mescenchymal stem cell expression mesenchymal cell markers α-SMA and N-cadherin, do not express epithelial cell
Mark CK18, E-cadherin.Illustrate that the mescenchymal stem cell prepared using method of the present invention culture is that typical interstitial is thin
Born of the same parents.
4th, immunochemistry fluoroscopic examination cell differentiation
Primary mescenchymal stem cell (the i.e. embodiment 1 of culture is repeated to the 4th in embodiment 1 by immunochemistry fluorescent method
In by the method for the present invention prepare optimizing tissue block repeatedlys cultivate the primary mescenchymal stem cell for obtaining through the 4th) pass on train
Supporting to the cell in P3 generations carries out differentiation capability (including mescenchymal stem cell into Adipose Differentiation, Osteoblast Differentiation and into cartilage differentiation)
Detection, it is specific as follows：
4.1 inducing mesenchymal stem cells are into Adipose Differentiation
4.1.1 induction differentiation
Above-mentioned P3 is carried out into induction differentiation for mescenchymal stem cell, specific method is：With 1.0 × 104Individual/hole Zhong Yu24 holes
Plate, after culture 24h, 2 groups is divided into by cell, and one group is induction differentiation group, and culture medium is replaced by into fatty inducing culture,
Another group is control group, and the culture medium for using is DMEM-HG (i.e. DMEM high glucose mediums)+10%FBS, carries out continuation culture,
Wherein, the composition of fatty inducing culture is：DMEM-HG culture mediums (i.e. DMEM high glucose mediums), 10%FBS, 10-6M
Dexamethasone (DEX), 0.5mM IBMX (3-isobutyl-1-methylxanthine), 60 micromoles per liter indocins and 5 micrograms/ml
Insulin, changes liquid once in every three days, coinduction 2 weeks.
4.1.2 identify
Adipose Differentiation is identified by oil red O stain, specific method is as follows：It is quiet by oil red O and the 4ml distilled water of 6ml 0.5%
Put 8min, filter paper filtering.Inducing cell is removed into culture medium, PBS is washed once, add 4% paraformaldehyde to fix 30min.With
Distillation washing 3 times, oil red O stain 20min, distilled water is washed 3 times again, Microscopic observation, in cellular morphology such as Fig. 4 into fat
Shown in differentiation figure, there are obvious fat drips in induction group, and oil red O stain is positive；And control group, without fat drips, oil red O stain is in
The positive, illustrates that cell has the strong ability to Adipocyte Differentiation.
4.2 inducing mesenchymal stem cell Osteoblast Differentiations
4.2.1 induction differentiation
By above-mentioned P3 for mescenchymal stem cell with 5.0 × 103Be divided into for cell in after 24 orifice plates, culture 24h by individual/hole kind
2 groups, one group is induction differentiation group, and culture medium is replaced by into Osteogenic Induction Medium, and another group is control group, the culture of use
Base is DMEM-HG+10%FBS, carries out continuation culture, wherein, the composition of Osteogenic Induction Medium is：DMEM in high glucose culture medium,
10%FBS, 10-7M dexamethasone (DEX), 50mM vitamin Cs and 10 micromoles per liters β-phosphoglycerol, change liquid in every three days,
Coinduction 5 weeks.
4.2.2 identify
Identify the Osteoblast Differentiation situation of mescenchymal stem cell respectively by two methods of Alizarin red staining and cma staining, specific side
Method is as follows：
(1) Alizarin red staining
Culture medium will be removed through the osteogenic induction mescenchymal stem cell of 2-3 weeks, 1 × PBS washings are fixed with 4% paraformaldehyde
30min, then, the cell after fixation is washed 2 times with 1 × PBS, and 5min is dyeed using Alizarin Red, then 1 ×
PBS is washed 3 times, and light Microscopic observation is taken pictures, and cellular morphology as shown in the Osteoblast Differentiation Alizarin red staining figure in Fig. 4, break up by induction
Group Alizarin red staining is positive；And control group is negative, illustrate that cell has the strong ability to osteoblast differentiation.
(2) cma staining
After osteogenic induction 4~5 weeks, after cell removes culture medium, PBS is washed one time, adds 4% paraformaldehyde to fix 20min,
Deionization is washed 3 times, each 5min, adds 5%AgNO3The aqueous solution, in reacting 1h under high light, deionized water thoroughly cleaning,
5%Na2S2O3.5H2The O aqueous solution processes 1min, to remove unnecessary silver, fully washing.Light Microscopic observation is taken pictures, cellular morphology
As shown in the Osteoblast Differentiation cma staining figure in Fig. 4.Induction differentiation group is dyed it can be seen that obvious bone tubercle, and compares
Group is negative, and illustrates that cell has the strong ability to osteoblast differentiation.
4.3 inducing mesenchymal stem cells are into cartilage differentiation
4.3.1 induction differentiation
By above-mentioned P3 for mescenchymal stem cell with 3.0 × 105It is individual to be laid on 15ml plastics point bottom test tube, 220g low-speed centrifugal 8min,
Cell is formed micelle, cell be divided into 2 groups, one group is induction differentiation group, and culture medium is replaced by into chondrocyte induction culture medium,
Another group is control group, and the culture medium for using is DMEM-HG (i.e. DMEM high glucose mediums)+10%FBS, carries out continuation culture,
Wherein, the composition of chondrocyte induction culture medium is：DMEM in high glucose, 2 × 10-7M dexamethasone (DEX), 50 micrograms/ml vitamins
C, 1 × ITS (Insulin-Transferrin-selenium additive) (I2521), 10mM Sodium Pyruvates, 1 × P/S (i.e. penicillin/chains
Mycin), 50 micrograms/ml proline and 10ng/ml TGF-βs 3, change within every 4 days liquid, coinduction 5 weeks.
4.3.2 identify
It should be noted that the micelle that MSC is formed can not be cultivated normally, only the micelle through inducing liquid culture just can be with one
It is straight to keep micelle form.After induction is finished, the cell through inducing is carried out into FFPE and section.Paraffin section is through dimethylbenzene 5
Min, dimethylbenzene 5min, absolute ethyl alcohol 1min, 95% ethanol 1min, 85% ethanol 1min, 75% ethanol 1min, running water
2min, distillation washing 2min are washed, it is special through the blue dyeing identification cartilages of HE (Hematoxylin-eosin dyeing) and A Erxin after dewaxing
Observed under specific proteoglycans, microscope, cellular morphology as shown in Fig. 4 into cartilage differentiation figure, " control group " not
It is negative through the blue dyeing of the A Erxin of inducing cell, the cell for forming cartilage differentiation can be clearly visible cartilage through A Erxinlan dyeing
Specific proteins glycan is positive, and illustrates that cell has the strong ability to Chondrocyte Differentiation.
Wherein, HE is morphological observation, illustrate induction after tissue in be still complete eucaryotic cell structure.And A Erxin indigo plants are pins
To the specific staining of cartilage specificity proteoglycans, being fully able to the cell after explanation differentiation has the characteristic of cartilage cell.
The above-mentioned mescenchymal stem cell to acquisition is broken up and is broken up the result of identification, shows what is obtained by the method for the present invention
Derived from Mesenchymal Stem Cells ability is strong, with the characteristic broken up to fat, skeletonization and cartilage direction.
5th, cell karyotyping
The primary mescenchymal stem cell that the 4th in embodiment 1 repeats culture ((is passed through into the method for the present invention i.e. in embodiment 1
The optimizing tissue block of acquisition repeatedly cultivates the P0 for obtaining through the 4th for mescenchymal stem cell)) Secondary Culture is carried out, according to cell
State, every 3 days passage once, when cell culture is to 15 generation, chromosome karyotype analysis are carried out to cell, as a result as scheme
Shown in 5, G- band karyotyping no abnormality seens are completely the same with normal karyotype, are male's normal karyotype.
In sum, mescenchymal stem cell preparation method effect is significant of the invention, can obtain substantial amounts of MSCs, and simply
Easy, low cost, security is good, has value very high for exploitation MSC various applications clinically.
Experimental study of the umbilical cord mesenchymal stem cells intervention of embodiment 3 to quick brain aging mouse cognition dysfunction
1. material
1.1 research objects
Senescence-accelerated Mouse's SAMP820 is only (male).
1.2 human umbilical cord mesenchymal stem cells are obtained by the method described in embodiment 1.
1.3 laboratory apparatus and equipment
The composition of Morris water mazes (Morris water maze)：Water maze is by round pool, platform and record system three
Part constitutes.Pool diameter 150cm, 50cm high, are arbitrarily divided into four quadrants (northeast, the southeast, southwest and northwests by pond
Quadrant).Experiment daily starts, pond water filling 30cm depths, and adds 1 jin of milk powder, makes water into opaque milky, and water temperature is protected
Hold at 21 DEG C ± 1 DEG C or so.There is abundant space object of reference (door, lamp, tables and chairs, camera and experimenter etc.) in pond surrounding,
And position keeps constant, so that mouse is set to platform.Cylindrical bar diameter 6cm, 29cm high, are placed in any quadrant center,
Plane is not in underwater 1cm.Camera is placed at the top 1.5m in pond center, automatic data collection animal swimming image, is received
Collection signal directly inputs computer, is automatically analyzed and is processed by image automatic data collection and analysis system, including the escape of animal is hidden
Phase, swimming path, the residence time of different quadrant, the length of initial angle and swimming route, search strategy and in, tour around outward
Swimming is apart from parameters such as percentages.
2. method
2.1 animal packets
Mouse is randomly divided into control group (injecting normal saline), MSC treatment groups, and every group 10, the dosage of MSC is 5 × 106
HUC-MSCs/, intraperitoneal injection.Model group and blank control group give isometric physiological saline.Laggard every trade is within 10 weeks
Learn test.
2.2 Behavior tests
Mainly include that two parts are tested in orientation navigation experiment and space exploration
Orientation navigation is tested
Last 5 days, daily by mouse towards pool wall respectively from 4 place of entry be put into water in 1 time, record it and search out and be hidden in
The time (escape latency) of water surface lower platform.
Space exploration is tested
It is to remove platform after orientation navigation experiment, after randomly selecting a place of entry, mouse is put into water from the place of entry successively
Chi Zhong, records its swimming track within a certain period of time, investigates memory of the mouse to original platform.
2.3 data processings
All data mean ± standard deviation (x ± S), represents, the data that orientation navigation experiment is obtained use duplicate measurements number
According to two-way analysis of variance (two-way ANOVA).Compare between space exploration experiment sample and use one-way analysis of variance
(one-way ANOVA).Inspection level positioning P ＜ 0.05 have conspicuousness for difference.Result is as shown in Figure 6.
Fig. 6 .A are the escape tracks of mouse in water maze laboratory；Fig. 6 .B are the 3rd, 4,5 days mouse in orientation navigation experiment
Escape latency；The 6th day in Fig. 6 .C space explorations experiment, mouse wears platform number of times.As seen from Figure 6, MSC is added
Treatment, collection data were analyzed since the 3rd day, it is possible to find the escape of the mouse after the 3rd, 4,5 days in Data Collection
There is obvious footpath tropism the escape track that incubation period has the trend of obvious shortening, MSC treatments group mouse compared to PBS injection groups
And purpose.It can also be seen that the platform number of times of wearing for the treatment of group mouse increases in space exploration experiment.Result above shows, MSC
Really play the role of to improve model mice spatial memory.
The umbilical cord mesenchymal stem cells intervention of embodiment 4 influences on Alzheimer disease related molecular marker thing in quick brain aging mouse
The material of research 1.
1.1 research objects
Senescence-accelerated Mouse's SAMP812 is only (male).
1.2 human umbilical cord mesenchymal stem cells are obtained by the method described in embodiment 1.
2. method
2.1 samples prepare：
2.1.1 drawn materials for SABC：After last 1 Morris water maze test 24h, 1% amobarbital of mouse
Sodium 50mg/kg intraperitoneal injections, open chest exposure heart, through left ventricular cannulation to sustainer, while cutting off the right heart immediately after anesthesia
Ear, quick filling physiological saline bleaches to liver and changes perfusion liquid for 4% paraformaldehyde, and infusion time about 30min is shown in mouse
Tail, four limbs are stiff to be considered as satisfaction；Broken end takes brain after perfusion is finished, in 4% paraformaldehyde solution of input overnight, after dehydration,
FFPE.
2.1.2 cerebral cortex and hippocampal protein are extracted：After last 1 Morris water maze test 24h, mouse is with 1% penta
Barbital sodium 50mg/kg intraperitoneal injections, open chest exposure heart, through left ventricular cannulation to sustainer, while cutting immediately after anesthesia
Right auricle of heart is opened, quick filling physiological saline breaks end after bleaching to liver and takes brain.Hippocampus is separated with reference to mouse brain stereotaxic atlas
And cerebral cortex.Respectively as EP pipes in, often pipe adds 500 microlitres of Ripa+1%PI to be placed in 4 DEG C of cracking overnight.Then 3000
Turn/be centrifuged 20 minutes, draw supernatant and simultaneously measure concentration with spectrophotometer, be placed in -80 DEG C of preservations.
2.2 detections
2.2.1 SABC：Mouse brain paraffin specimen is taken, 15min is fixed with 4% paraformaldehyde, PBS washes 2min × 3
It is secondary.Again plus the fresh configuration 0.5%H of distilled water2O2, room temperature 30min is with inactivating endogenous catalase.Dioxygen washes 2min × 3
It is secondary.Normal Goat Serum, room temperature 20min is added dropwise.Surplus liquid is got rid of, is not washed.Primary antibody BACE (1: 800) is added dropwise respectively,
Primary antibody GSK-3 β (1: 800), primary antibody P-tau (1: 800), negative control is made with PBS instead of primary antibody, is incubated at 37 DEG C
1h, PBS wash 2min × 3 time.The anti-igg of biotinylation two (goat antirabbit) 37 DEG C of 30min are added dropwise.PBS washes 2min × 3
It is secondary.SABC, 20~37 DEG C of 20min is added dropwise.PBS washes 5min × 4 time.DAB develops the color.Distilled water cyclic washing.Dehydration,
It is transparent, mounting.Sample is in basis of microscopic observation result.
2.2.2 protein immunoblot：The albumen of hippocampus is taken, electrophoresis is carried out, transferring film, closing, primary antibody is incubated, and secondary antibody is incubated
Developed afterwards.
Result as shown in fig. 7,
Fig. 7 .A are the P-Tau expression in mouse hippocampus and cerebral cortex；Fig. 7 .B are in mouse hippocampus and cerebral cortex
GSK-3 β are expressed；Fig. 7 .C are the expression of the BACE in mouse hippocampus；Fig. 7 .D are protein immunoblot result, mouse hippocampus
APP and P-tau protein expressions in area.As seen from Figure 7, through MSC treat quick brain aging mouse in A β
The related BACE1 rate-limiting enzymes of senile plaque expelling are formed, causes the GSK-3 β of A beta peptide aggregations all to decline substantially, cause neurofibril to twine
The P-tau albumen of knot also occurs in that decline in the quick brain aging mouse treated by MSC, and the result of protein immunoblot
Display that the APP protein expressions of the quick brain aging mouse only treated with PBS are treated mouse group and increased compared to MSC, and through MSC
The downward of P-tau albumen, shows that the treatment of MSC can be alleviated to a certain extent and causes A Er in the quick brain aging mouse for the treatment of
Topmost two pathological characters of Ci Haimo diseases.
The umbilical cord mesenchymal stem cells intervention of experimental example 5 improves the crucial mark of NSC of hippocampus and cortex in quick brain aging mouse
Will is expressed
1. material
1.1 research objects
Senescence-accelerated Mouse's SAMP820 is only (male).
1.2 human umbilical cord mesenchymal stem cells are obtained by the method described in embodiment 1.
2. method
2.1 samples prepare：
2.1.1 drawn materials for SABC：After last 1 Morris water maze test 24h, 1% amobarbital of mouse
Sodium 50mg/kg intraperitoneal injections, open chest exposure heart, through left ventricular cannulation to sustainer, while cutting off the right heart immediately after anesthesia
Ear, quick filling physiological saline bleaches to liver and changes perfusion liquid for 4% paraformaldehyde, and infusion time about 30min is shown in mouse
Tail, four limbs are stiff to be considered as satisfaction；Broken end takes brain after perfusion is finished, in 4% paraformaldehyde solution of input overnight, after dehydration,
FFPE.
2.1.2 cerebral cortex and hippocampal protein are extracted：After last 1 Morris water maze test 24h, mouse is with 1% penta
Barbital sodium 50mg/kg intraperitoneal injections, open chest exposure heart, through left ventricular cannulation to sustainer, while cutting immediately after anesthesia
Right auricle of heart is opened, quick filling physiological saline breaks end after bleaching to liver and takes brain.Hippocampus is separated with reference to mouse brain stereotaxic atlas
And cerebral cortex.Respectively as EP pipes in, often pipe adds 500 microlitres of Ripa+1%PI to be placed in 4 DEG C of cracking overnight.Then 3000
Turn/be centrifuged 20 minutes, draw supernatant and simultaneously measure concentration with spectrophotometer, be placed in -80 DEG C of preservations.
2.2 detection methods
2.2.1 SABC：Hippocampus of mice area paraffin specimen is taken, 15min is fixed with 4% paraformaldehyde, PBS washes 2min × 3
It is secondary.Again plus the fresh configuration 0.5%H of distilled water2O2, room temperature 30min is with inactivating endogenous catalase.Dioxygen washes 2min × 3
It is secondary.Normal Goat Serum, room temperature 20min is added dropwise.Surplus liquid is got rid of, is not washed.Primary antibody Nestin (1: 800) is added dropwise,
Negative control is made instead of primary antibody with PBS, 1h is incubated at 37 DEG C, PBS washes 2min × 3 time.The anti-igg of biotinylation two is added dropwise
(goat antirabbit) 37 DEG C of 30min.PBS washes 2min × 3 time.SABC, 20~37 DEG C of 20min is added dropwise.PBS washes 5min × 4
It is secondary.DAB develops the color.Distilled water cyclic washing.Dehydration, transparent, mounting.Sample is in basis of microscopic observation result.
2.2.2 protein immunoblot：Hippocampus or corticocerebral albumen are taken, electrophoresis, transferring film, closing, primary antibody is carried out
It is incubated, secondary antibody is developed after being incubated.
Result is as shown in figure 8, Fig. 8 .A display comparisons treatment control group, MSC treatment groups Nestin in hippocampus and cerebral cortex
Positive cell number increases, and cell is intensive, and cell space is loose, stretches out the different projection of different in size, thickness；
Fig. 8 .B display comparisons treat control group, and MSC treatment groups promote nerve Nestin positive in hippocampus and cerebral cortex
The propagation of stem cell, it is seen that cell is in oval, triangle or fusiformis, endochylema is in brown color, and karyon is blueness；
Fig. 8 .C show protein immunoblot result, and compared to treatment control group, MSC treatments can improve hippocampus and corticocerebral
Nestin protein expressions
To sum up, as seen from Figure 8, the cerebral cortex of the quick brain aging mouse treated through MSC and Nestin in hippocampus
Positive cell number increases, and cell is intensive, and displays that nestin to the protein immunoblot result of cerebral cortex and hippocampus
The rise of albumen, showing the treatment of MSC can promote the activation of induced nerve stem cells to a certain extent.
The problem that conventional method acquisition umbilical cord mesenchymal stem cells are primarily present has：First, quantity is very limited, because passing
System method obtains that cell yield is relatively low, while mescenchymal stem cell has the limitation of algebraically in itself, and (generally 3-5 is alternative in clinic
Treatment), this causes that stem cell its quantity in actual application turns into the primary problem for solving.Secondly, quality problems, because
The cell purity and homogeneity obtained for conventional method are still present deficiency, can also influence the maintenance of its dryness；Along with quantity not
Foot and can not meet every time treatment needed for cell number so as to cause treatment DeGrain even without effect, for example, in order to
Ensure to be transfused sufficient amount of cell every time, the umbilical cord mesenchymal stem cells product of same individual infusion separate sources often occur,
Homogeneity and stability between product it cannot be guaranteed that, can so directly affect stabilization and the assessment of therapeutic effect, different of infusion
The umbilical cord mesenchymal stem cells in body source also increase many unknown treatment hidden danger.In addition, the cell that is obtained of conventional method by
Can also increase more financial burden in above mentioned problem.And pass through we method obtain umbilical cord mesenchymal stem cells be directed to should
Clinical grade umbilical cord mesenchymal stem cells, its outstanding feature is that the yield of cell is high, and 8-10 is at least improved compared with conventional method
Times, it is often more important that, cytoactive is high, and purity is high, and differentiation capability is strong, and stability and homogeneity are greatly improved, that is to say, that
The raising of quality and quantity is most important for clinical demand.In addition, can also substantially reduce cost in itself using the method for optimization
And financial burden.Therefore, the umbilical cord mesenchymal stem cells for being obtained by our method are for clinical practice compared with conventional method phase
Than with obviously advantage.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specific example ",
Or the description of " some examples " etc. means to combine specific features, structure, material or feature bag that the embodiment or example are described
It is contained at least one embodiment of the invention or example.In this manual, the schematic representation to above-mentioned term not necessarily refers to
Be identical embodiment or example.And, the specific features of description, structure, material or feature can be at any one
Or combined in an appropriate manner in multiple embodiments or example.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that：This is not being departed from
Can these embodiments be carried out with various changes, modification, replacement and modification in the case of the principle and objective of invention, it is of the invention
Scope is limited by claim and its equivalent.
Claims (10)
1. a kind of method for preparing clinical treatment mescenchymal stem cell, it is characterised in that including：
(1) tissue block of the multiple containing mescenchymal stem cell is cultivated in the first culture dish, to obtain from described many
The mescenchymal stem cell climbed out of in individual tissue block, and optimization group is selected from the multiple tissue block containing mescenchymal stem cell
Knit block；And
(2) the optimizing tissue block obtained in step (1) is cultivated in the second culture dish, to obtain from described excellent
Change the mescenchymal stem cell that tissue block is climbed out of,
Wherein, the condition of optimizing tissue block is selected in step (1) including at least one following：
A () climbs out of without endothelial cell；
B () has mescenchymal stem cell to climb out of；
C () mescenchymal stem cell is most climbed out of soon.
2. method according to claim 1, it is characterised in that the tissue block containing mescenchymal stem cell is derived from
Umbilical cord tissue.
3. method according to claim 2, it is characterised in that the tissue block containing mescenchymal stem cell is to pass through
What the following steps were obtained：
Reject the arteria umbilicalis and umbilical vein of umbilical cord tissue；And
Wal Tong Shi glue is separated from the umbilical cord tissue,
Wherein, the Wal Tong Shi glue constitutes the tissue block containing mescenchymal stem cell.
4. method according to claim 1, it is characterised in that after step (2), further include：
(3) the optimizing tissue block is cultivated in the 3rd culture dish, can be obtained after 5-7 days from the optimizing tissue block
The P0 for climbing out of is for mescenchymal stem cell.
5. method according to claim 4, it is characterised in that repeat step (3) at least one times, preferably 2~5 times,
Optionally, the P0 obtained in collection step (1)~(3) carries out Secondary Culture for mescenchymal stem cell.
6. method according to claim 5, it is characterised in that the Secondary Culture uses serum free medium,
Optionally, the Secondary Culture uses serum free medium (Sciencell), without any animal derived protein and animal sources disease
The pollution of the original,
Optionally, the P0 is carried out into Secondary Culture for mescenchymal stem cell, the time interval of the passage is 2-4 days, and
And using resulting P3~P5 for mescenchymal stem cell as clinical treatment mescenchymal stem cell.
7. method according to claim 1, it is characterised in that use serum free medium in step (1)~(3)
Tissue block is cultivated,
Optionally, the culture medium is serum free medium (Sciencell), without any animal derived protein and animal sources pathogen
Pollution.
8. an inter-species fills stem cell, and the mescenchymal stem cell is obtained by the method described in claim any one of 1-8.
9. purposes of the mescenchymal stem cell described in claim 8 in medicine is prepared, the medicine is used to treat tissue damage.
10. purposes of the mescenchymal stem cell described in claim 8 in medicine is prepared, the medicine is used to treat alzheimer '
Silent disease.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510459332.0A CN106701669A (en) | 2015-07-29 | 2015-07-29 | Mesenchymal stem cell for clinical treatment as well as preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510459332.0A CN106701669A (en) | 2015-07-29 | 2015-07-29 | Mesenchymal stem cell for clinical treatment as well as preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106701669A true CN106701669A (en) | 2017-05-24 |
Family
ID=58901669
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510459332.0A Pending CN106701669A (en) | 2015-07-29 | 2015-07-29 | Mesenchymal stem cell for clinical treatment as well as preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106701669A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106381281A (en) * | 2016-11-01 | 2017-02-08 | 浙江译美生物科技有限公司 | Culture method of embryonic stem cells |
| CN108220225A (en) * | 2018-01-23 | 2018-06-29 | 中国药科大学 | Mescenchymal stem cell conditioned medium is based on protective effect of the p38-MAPK signal paths to damage of human umbilical vein endothelial |
| CN108220229A (en) * | 2017-12-18 | 2018-06-29 | 北京康亿瑞生物科技有限公司 | A kind of preparation method for improving umbilical cord derived mesenchymal stem cell primary cell yield |
| CN111728982A (en) * | 2019-03-22 | 2020-10-02 | 宣捷细胞生物制药股份有限公司 | Composition for anti-aging |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009030092A1 (en) * | 2007-09-05 | 2009-03-12 | Institute Of Basic Medical Sciences Chinese Academy Of Medical Sciences | Culture medium and method for in vitro culturing human adult primary mesenchymal stem cells on a large scale, primary mesenchymal stem cells obtained by the method, the uses thereof |
-
2015
- 2015-07-29 CN CN201510459332.0A patent/CN106701669A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009030092A1 (en) * | 2007-09-05 | 2009-03-12 | Institute Of Basic Medical Sciences Chinese Academy Of Medical Sciences | Culture medium and method for in vitro culturing human adult primary mesenchymal stem cells on a large scale, primary mesenchymal stem cells obtained by the method, the uses thereof |
Non-Patent Citations (4)
| Title |
|---|
| ZHENGQI CHANG ET AL.: "Umbilical Cord Wharton’s Jelly Repeated Culture System: A New Device and Method for Obtaining Abundant Mesenchymal Stem Cells for Bone Tissue Engineering", 《PLOS ONE》 * |
| 刘晓峰 等: "干细胞治疗阿尔茨海默病的现状及未来", 《中国组织工程研究》 * |
| 李艳琪 等: "人脐带源间充质干细胞分离培养方法的改进", 《中国组织工程研究》 * |
| 高彦琳 等: "改良原代培养体系提高人脐带间充质干细胞的产量", 《中国组织工程研究》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106381281A (en) * | 2016-11-01 | 2017-02-08 | 浙江译美生物科技有限公司 | Culture method of embryonic stem cells |
| CN108220229A (en) * | 2017-12-18 | 2018-06-29 | 北京康亿瑞生物科技有限公司 | A kind of preparation method for improving umbilical cord derived mesenchymal stem cell primary cell yield |
| CN108220229B (en) * | 2017-12-18 | 2021-05-04 | 北京康亿瑞生物科技有限公司 | Preparation method for improving output of umbilical cord source mesenchymal stem cell primary cells |
| CN108220225A (en) * | 2018-01-23 | 2018-06-29 | 中国药科大学 | Mescenchymal stem cell conditioned medium is based on protective effect of the p38-MAPK signal paths to damage of human umbilical vein endothelial |
| CN111728982A (en) * | 2019-03-22 | 2020-10-02 | 宣捷细胞生物制药股份有限公司 | Composition for anti-aging |
| CN111728982B (en) * | 2019-03-22 | 2022-05-03 | 宣捷细胞生物制药股份有限公司 | Composition for anti-aging |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104666344B (en) | Application of the mescenchymal stem cell excretion body in the pharmaceutical preparation of preparation treatment pulmonary fibrosis | |
| CN101748096B (en) | Sub totipotential stem cell and preparation method and application thereof | |
| CN105861430A (en) | Exosome, preparing method of exosome and application of exosome in preparing medicine or preparation for treating sepsis | |
| WO2018103406A1 (en) | Neural stem cell injection for treating brain damage diseases and preparation method and use method thereof | |
| US11821005B2 (en) | Umbilical cord mesenchymal stem cells (MSCs) and culture method and application thereof | |
| CN106434557A (en) | Method for preparing CD34 positive cells from umbilical cord mesenchymal stem cells | |
| CN106701669A (en) | Mesenchymal stem cell for clinical treatment as well as preparation method and application thereof | |
| CN109893541B (en) | Application of exosome derived from menstrual blood stem cells in preparation of medicine for treating intrauterine adhesion | |
| CN105779383A (en) | Preparation method and application of adipose-derived stem cell-hydrogel three-dimensional cultivation system | |
| CN103484432A (en) | Induction medium for inducing neural stem/progenitor cells to be differentiated into oligodendrocyte precursor cells and induction method and application thereof | |
| CN108865986A (en) | For repairing articular cartilage damage/defect mescenchymal stem cell preparation and its preparation method and application | |
| CN109106726A (en) | A kind of anti-aging stem cell composition and its application | |
| CN108066750A (en) | Stem cell and its secretion are used to treat the new application of skin burn | |
| CN104232570B (en) | Set up the method and its application of monoclonal mescenchymal stem cell | |
| CN1486745A (en) | Stem cell prepn for treating tissue ischemia disease and its prepn process | |
| CN108619169A (en) | A kind of mesenchymal stem cell injection and preparation method for treating cerebral arterial thrombosis | |
| Liang et al. | Perfusable adipose decellularized extracellular matrix biological scaffold co-recellularized with adipose-derived stem cells and L6 promotes functional skeletal muscle regeneration following volumetric muscle loss | |
| CN102703380B (en) | Sub-totipotent stem cell, preparation method and application thereof | |
| CN106062184A (en) | Odontogenic stem cells and use of genetically modified odontogenic stem cells | |
| CN108066749A (en) | Purposes of the Stem Cell Activity factor in skin injury drug | |
| CN108159045B (en) | application of robinine A and derivatives thereof in preparation of medicines for preventing or treating central nervous system diseases | |
| WO2019119819A1 (en) | Application of placental pluripotent stem cell preparation in preparing drug for treating acute lung injury | |
| CN109939128A (en) | A kind of neural stem cell preparation and preparation method thereof | |
| KR20050086606A (en) | Method of organ regeneration | |
| CN105176924B (en) | A kind of regenerating bone or cartilage stem cell medicine and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170524 |