A kind of sustained-release micro-spheres and preparation method of the body of excretion containing mescenchymal stem cellTechnical field
The present invention relates to the sustained release in biological stem cells technology field more particularly to a kind of excretion containing mescenchymal stem cell body is micro-Ball and preparation method.
Background technique
Uterus is most important for all one's life of women, once but endometrial impairment, it is existing it will cause a series of consequencesSome endometrium reparation difficulty are big, and treatment time is longer, brings great puzzlement to many women.
Mescenchymal stem cell (Mesenchymal Stem Cells, MSC) mesenchymal cell belongs to multipotential stem cell, hasMultidirectional Differentiation, hematopoiesis support and promote stem cell implantation, the multiple functions such as immunoregulation, is primarily present in endometrium, fatIn marrow；Excretion body is the vesicles that diameter secreted by cell is 30~150nm, includes albumen relevant from cellThe biomolecule such as matter and nucleotide, play an important role between cell communication.
Excretion body is the atom membrane vesicle of cell secretion, and compared with mescenchymal stem cell, excretion body volume is smaller, complicatedProperty it is lower, be readily produced and store, matter stem cell excretion body such as protein and RNA is present in excretion rich in bioactive moleculeMany protein in body belong to enzyme, have catalytic activity, but existing excretion body medicament is difficult to continuous action, causesLasting medicine power is lower, thus how to extend excretion body treatment endometrial impairment become those skilled in the art urgentlyIt solves the problems, such as.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art, provide a kind of dry containing mesenchymaThe sustained-release micro-spheres and preparation method of cell excretion body constantly discharge the excretion body of stem cell secretion using sustained-release micro-spheres, effectivelyExtend the action time of excretion body, thus provide safely, effectively, it is easy to use treatment endometrial impairment drug.
As shown, the present invention is achieved by the following technical programs: a kind of body of excretion containing mescenchymal stem cellSustained-release micro-spheres, by volume parts ratio, including 1-4 parts of mescenchymal stem cell excretion liquid solutions, 92-98 parts of poly lactic-co-glycolic acidsCopolymer solution and 1-4 parts of poly-vinyl alcohol solutions, by mass fraction ratio, mescenchymal stem cell excretion liquid solution includes 10-25Body freeze-dried powder and 1-4 parts of distilled water are especially secreted, by mass parts ratio, poly lactide-glycolide acid solution includes 10-25 parts of poly lactide-glycolide acids and 75-90 parts of dichloromethane solutions, the excretion body freeze-dried powder pass through endometriumThe culture processing of mescenchymal stem cell starvation is made, and the time of starvation culture is 12-24 hours, and self endometrium mesenchyma is dry thinBorn of the same parents extract from the mescenchymal stem cell of people's endometrium, are cultivated to obtain according to the method normally cultivated, incubation time 48-72 hours.
According to the above technical scheme, it is preferable that sustained-release micro-spheres are placed in -20 DEG C of preservations.
According to the above technical scheme, it is preferable that it is mainly used for treating endometrial impairment.
A kind of preparation method of the sustained-release micro-spheres of the body of excretion containing mescenchymal stem cell, comprising the following steps:
(1) culture of self endometrium mescenchymal stem cell: mescenchymal stem cell is isolated from endometrium, with nothingPhenol red DMEM/DF12 and volume by volume concentration are that 10% fetal calf serum does complete medium, be put into T75 Tissue Culture Flask 37 DEG C,5%5-90 parts of dichloromethane solutions, poly-vinyl alcohol solution concentration are 1%-2%, and excretion body freeze-dried powder passes through mescenchymal stem cellExcretion body is process, and mescenchymal stem cell excretion body passes through self CO2Under the conditions of cultivated；
(2) preparation of mescenchymal stem cell excretion body freeze-dried powder: reach 70%-80% to cell extent of growth, use lifeIt manages salt water to clean cell 2 times, carries out hungry culture 12-24 hours with Multiple electrolytes injection, reach to cell extent of growthAfter 90%-95%, cell culture supernatant is collected, and carry out to mescenchymal stem cell excretion body using excretion body extracts kitIt extracts, human serum albumins is added according to mass volume ratio 2-4%, uniformly mixed post-processing obtains excretion body freeze-dried powder；
(3) it the preparation of mescenchymal stem cell excretion liquid solution: chooses 10-25 and especially secretes body freeze-dried powder, and be dissolved in1-4 parts of distilled water, stir evenly obtained；
(4) preparation of poly lactide-glycolide acid solution: mass fraction ratio is pressed, 10-25 parts of polylactic acid-glycolics are weighedAcetic acid copolymer and 75-90 parts of dichloromethane solutions, are then dissolved in methylene chloride for poly lactide-glycolide acidIn solution, stir evenly obtained；
(5) preparation of sustained-release micro-spheres: press volume parts ratio, successively choose 1-4 parts of mescenchymal stem cell excretion liquid solutions,92-98 parts of poly lactide-glycolide acid solution and 1-4 parts of poly-vinyl alcohol solutions are put into mixing arrangement, are stirred evenlyIt is made.
The beneficial effects of the present invention are: mescenchymal stem cell excretion body is mixed into sustained-release micro-spheres, so that excretion bodyActivity and function are saved and are utilized, and the time survived can be longer, is able to extend excretion body function and effect, effectivelyRepair endometrial impairment.
Detailed description of the invention
Fig. 1 is the excretion body quantity figure that different cultural methods extract；
Fig. 2 is the gene expression difference figure of three kinds of cell factors of different experiments group endometrium；
Fig. 3 is animal uterus inner film injury area statistics table.
Specific embodiment
In order to make those skilled in the art more fully understand technical solution of the present invention, with reference to the accompanying drawing and mostThe present invention is described in further detail for good embodiment.
Embodiment 1:
The present invention provides a kind of sustained-release micro-spheres of body of excretion containing mescenchymal stem cell, by volume parts ratio, including between 1 partMesenchymal stem cells excretion liquid solution, 98 parts of poly lactide-glycolide acid solution and 1 part of poly-vinyl alcohol solution, by qualityScore ratio, mescenchymal stem cell excretion liquid solution includes that 10-25 especially secretes body freeze-dried powder and 1-4 parts of distilled water, by mass partsNumber ratio, poly lactide-glycolide acid solution includes 10-25 parts of poly lactide-glycolide acids and 75-90 parts of dichlorosDichloromethane, excretion body freeze-dried powder are made by the culture processing of endometrium mescenchymal stem cell starvation, the time of starvation cultureIt is 12-24 hours, self endometrium mescenchymal stem cell extracts from the mescenchymal stem cell of people's endometrium, according to normal trainingFeeding method is cultivated to obtain, and incubation time is 48-72 hours.
Embodiment 2:
The present invention provides a kind of sustained-release micro-spheres of body of excretion containing mescenchymal stem cell, by volume parts ratio, including between 4 partsMesenchymal stem cells excretion liquid solution, 92 parts of poly lactide-glycolide acid solution and 4 parts of poly-vinyl alcohol solutions, by qualityScore ratio, mescenchymal stem cell excretion liquid solution includes that 10-25 especially secretes body freeze-dried powder and 1-4 parts of distilled water, by mass partsNumber ratio, poly lactide-glycolide acid solution includes 10-25 parts of poly lactide-glycolide acids and 75-90 parts of dichlorosDichloromethane, excretion body freeze-dried powder are made by the culture processing of endometrium mescenchymal stem cell starvation, the time of starvation cultureIt is 12-24 hours, self endometrium mescenchymal stem cell extracts from the mescenchymal stem cell of people's endometrium, according to normal trainingFeeding method is cultivated to obtain, and incubation time is 48-72 hours.
Embodiment 3:
The present invention provides a kind of sustained-release micro-spheres of body of excretion containing mescenchymal stem cell, by volume parts ratio, including between 2 partsMesenchymal stem cells excretion liquid solution, 96 parts of poly lactide-glycolide acid solution and 2 parts of poly-vinyl alcohol solutions, by qualityScore ratio, mescenchymal stem cell excretion liquid solution includes that 10-25 especially secretes body freeze-dried powder and 1-4 parts of distilled water, by mass partsNumber ratio, poly lactide-glycolide acid solution includes 10-25 parts of poly lactide-glycolide acids and 75-90 parts of dichlorosDichloromethane, excretion body freeze-dried powder are made by the culture processing of endometrium mescenchymal stem cell starvation, the time of starvation cultureIt is 12-24 hours, self endometrium mescenchymal stem cell extracts from the mescenchymal stem cell of people's endometrium, according to normal trainingFeeding method is cultivated to obtain, and incubation time is 48-72 hours.
According to above-described embodiment, it is preferable that sustained-release micro-spheres are placed in -20 DEG C of preservations.
According to above-described embodiment, it is preferable that it is mainly used for treating endometrial impairment.
A kind of preparation method of the sustained-release micro-spheres of the body of excretion containing mescenchymal stem cell, comprising the following steps:
(1) culture of self endometrium mescenchymal stem cell: mescenchymal stem cell is isolated from endometrium, with nothingPhenol red DMEM/DF12 and volume by volume concentration are that 10% fetal calf serum does complete medium, be put into T75 Tissue Culture Flask 37 DEG C,5%5-90 parts of dichloromethane solutions, poly-vinyl alcohol solution concentration are 1%-2%, and excretion body freeze-dried powder passes through mescenchymal stem cellExcretion body is process, and mescenchymal stem cell excretion body passes through self CO2Under the conditions of cultivated；
(2) preparation of mescenchymal stem cell excretion body freeze-dried powder: reach 70%-80% to cell extent of growth, use lifeIt manages salt water to clean cell 2 times, carries out hungry culture 12-24 hours with Multiple electrolytes injection, reach to cell extent of growthAfter 90%-95%, cell culture supernatant is collected, and carry out to mescenchymal stem cell excretion body using excretion body extracts kitIt extracts, human serum albumins is added according to mass volume ratio 2-4%, uniformly mixed post-processing obtains excretion body freeze-dried powder；
(3) it the preparation of mescenchymal stem cell excretion liquid solution: chooses 10-25 and especially secretes body freeze-dried powder, and be dissolved in1-4 parts of distilled water, stir evenly obtained；
(4) preparation of poly lactide-glycolide acid solution: mass fraction ratio is pressed, 10-25 parts of polylactic acid-glycolics are weighedAcetic acid copolymer and 75-90 parts of dichloromethane solutions, are then dissolved in methylene chloride for poly lactide-glycolide acidIn solution, stir evenly obtained；
(5) preparation of sustained-release micro-spheres: press volume parts ratio, successively choose 1-4 parts of mescenchymal stem cell excretion liquid solutions,92-98 parts of poly lactide-glycolide acid solution and 1-4 parts of poly-vinyl alcohol solutions are put into mixing arrangement, are stirred evenlyIt is made.
The beneficial effects of the present invention are: mescenchymal stem cell excretion body is mixed into sustained-release micro-spheres, so that excretion bodyActivity and function are saved and are utilized, and the time survived can be longer, is able to extend excretion body function and effect, effectivelyRepair endometrial impairment.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the artFor member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answeredIt is considered as protection scope of the present invention.