CN109929799A - Human umbilical cord mesenchymal stem cells excretion body and its preparation method and application - Google Patents
Human umbilical cord mesenchymal stem cells excretion body and its preparation method and application Download PDFInfo
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Abstract
Human umbilical cord mesenchymal stem cells excretion body and its preparation method and application, preparation method is the following steps are included: acquire human umbilical tissue, and extract and obtain umbilical cord mesenchymal stem cells；Obtained umbilical cord mesenchymal stem cells carry out centrifugal treating, obtain excretion body to extract.HucMSC excretion body of the present invention can activate primordial follicle and promote newborn mice follicular development, and the mature oocyte obtained after follicular development is mature, quality is not affected by influence.HucMSC excretion body can improve Aged Mice reproductive function, and after aged rats ovary packet intracapsular injection excretion body, with public mouse post-coitum, experimental mice litter size is apparently higher than control group.
Description
Technical field
The invention belongs to excretion body fields, and in particular to a kind of umbilical cord mesenchymal stem cells source excretion body and its preparation side
Method and its purposes in primordial follicle Activation In Vitro.
Background technique
Excretion body is that a most commonly used subgroup is studied in extracellular vesica, it is by Pan and Johnstone in nineteen eighty-three
It finds for the first time.Excretion body is a kind of from early stage intracellular, the extracellular vesicles with membrane structure, its diameter between
Between 50-150nm.At present multiple studies have shown that excretion body can secreted by most cells, while can blood, urine
It is detected in most of body fluid such as liquid, cerebrospinal fluid, ascites, liquor folliculi, amniotic fluid, joint fluid.The surface and inside of excretion body contain
There are a large amount of protein, lipid, many kinds of substance such as nucleic acid, and the excretion body of different cell origins, each not phase of internal ingredient
Together.The excretion body in the source mescenchymal stem cell (MSC), since there is similar characteristic with derived cell itself, in wound
Repair, tissue reconstruction and cardiovascular disease, cerebral injury, inflammation etc. show potential treatment ability abundant, have extensive
Application prospect.Excretion body from mescenchymal stem cell is as a kind of emerging cell-free (cell-free) therapeutic modality, tool
There is lower immunogenicity, potential oncogenicity is not present, and compared with derived cell, half-life period is shorter in vivo, energy
It is enough preferably to be absorbed by recipient cell.In addition, excretion body can freeze spare after being singulated, do not reduce in a long time
Itself original bioactivity, later period are also used as the carrier targeting conveying of packaging medicine after the modes such as modification are handled
To privileged site.Therefore, excretion body clinical application safety is higher, and ethics risk is low, application potential and value with higher.
In female mammal body, ovarian follicle is the basic function unit for constituting ovary, by centrally located ovum and outside
Enclose one or more layers granular cell composition.According to the different growth and development stages of egg mother cell in ovarian follicle, original ovum can be divided into
Bubble, primary follicle, secondary follicle, chamber ovarian follicle, preovulatory follicle.Primordial follicle is the ovarian follicle library that quiescent condition is in ovary,
It being formed before birth or after birth in mammal, quantity is fixed, the primordial follicle about 400 in each ovary of people,
000 or so.Primordial follicle is not parked during Follicular growth, in primordial follicle library to be activated and enter growth period, this
A process is referred to as initial and recruits (initial recruiment).With the continuous progress initially raised, the number of primordial follicle
Amount gradually tails off, and when follicle number amount original in human ovarian is lower than 1000, ovarian follicle will not develop, and women is also into menopause
Phase.The age of meuopause of women was at 50 years old or so, however, under some pathological conditions, due to primordial follicle under-reserve in ovary
And women is enabled to enter menopause (before 40 years old) in advance to cause premature ovarian failure.Premature ovarian failure is a kind of very common infertile
Sterility, the disease incidence in Women of childbearing age is probably in 1-2%.Currently, about premature ovarian failure, there is no highly effective treatments
Method, most patient realize artificial menstrual cycle by hormone replacement therapy, need to meet fertility demand for ovum.Original ovum
The outer activation technique (IVA) of foam, is the emerging technology by ovary tissue extracorporeal treatment to activate wherein primordial follicle, for treatment
Premature ovarian failure patient is of great significance, be in the world it is first for primordial follicle propose therapeutic technique.2011, the world
The first IVA baby is born in Japan, and Chinese the first IVA baby due in 2015 indicates technology clinical application success.It is existing
The PI3K/mTOR signal path that IVA technology mainly passes through in signal path activator activation egg mother cell, which is realized, activates original ovum
The purpose of bubble.Whether has the function of activation primordial follicle about from human umbilical cord mesenchymal stem cells (HucMSC) excretion body
It then has not been reported, however, this research will have very important significance for the treatment of premature ovarian failure.
Summary of the invention
The technical issues of solution: the present invention provides a kind of human umbilical cord mesenchymal stem cells excretion body and preparation method thereof and answers
With excretion body of the invention is the excretion body of human umbilical cord mesenchymal stem cells, can be activated in the case where ovarian reserve reduces
Primordial follicle improves reproductive function.
A kind of technical solution: preparation method of excretion body, comprising the following steps: acquisition human umbilical tissue, and extract and obtain
Umbilical cord mesenchymal stem cells；Obtained umbilical cord mesenchymal stem cells carry out centrifugal treating, obtain excretion body to extract.
The preparation method of above-mentioned excretion body, step are as follows: the umbilical cord for removing artery and vein is cleaned, 1mm is cut into3Tissue
Block, evenly laid out in culture dish, 2mL culture medium is added in every ware, and placement is incubated at 37 DEG C, 5%CO2Incubator in 6 hours
Afterwards, 5mL culture medium is added into ware, changes the liquid once within every 2-3 days, when cell, which grows to convergence degree, reaches 70%-80%, uses
The digestion of 0.25%EDTA- pancreatin, 1:3 are passaged to P3 to P7 for cell, obtain excretion body.
When above-mentioned umbilical cord mesenchymal stem cells grow to convergence degree and reach 60%-70%, original fluid is sucked, it is slow with PBS
10mL conditioned medium, collection condition culture supernatant after culture 48 hours, under 4 DEG C of environment, being collected into is added in fliud flushing cleaning
Conditioned medium with 500g be centrifuged 10 minutes to remove cell, with 2000g be centrifuged 20 minutes it is broken to remove dead cell and cell
Piece is centrifuged 30 minutes with 10000g to remove big vesica and apoptotic body, is then divided with the relative centrifugal force of 120000g 90
Clock sucks supernatant, and after centrifugation bottom of the tube excretion body precipitating is resuspended with 1mL PBS, the liquid in all centrifuge tubes is mixed to same
In one centrifuge tube, PBS is added to liquid and is full of centrifuge tube, was sucked again with 120000g centrifugation 90 minutes with cleaning excretion body
Excretion body is resuspended with 150-200 μ L PBS for subsequent experimental use in layer liquid.
Human umbilical cord mesenchymal stem cells excretion body made from above-mentioned preparation method.
Application of the above-mentioned excretion body in non-diagnostic therapeutic purposes primordial follicle Activation In Vitro.
Above-mentioned excretion body is preparing the application in primordial follicle Activation In Vitro reagent.
Primordial follicle Activation In Vitro reagent, effective component are above-mentioned human umbilical cord mesenchymal stem cells excretion body.
The utility model has the advantages that HucMSC excretion body can activate primordial follicle and promote newborn mice follicular development, follicular development
The mature oocyte obtained after maturation, quality are not affected by influence.HucMSC excretion body can improve Aged Mice fertility function
Can, after aged rats ovary packet intracapsular injection excretion body, with public mouse post-coitum, experimental mice litter size is apparently higher than control group.
Detailed description of the invention
Fig. 1 human umbilical cord mesenchymal stem cells morphology and differentiation and identification figure.A:P5HucMSC cell morphology characteristic；
B: calcium tubercle Alizarin red staining；C: fat drips oil red O stain；D: cartilage acidic mucopolysaccharide A Li Xinlan dyeing.Scale=50 μm.
Fig. 2 human umbilical cord mesenchymal stem cells surface marker flow cyctometry qualification figure.A:HucMSC positive indication's object
CD44, CD73, CD90, CD105 expression；B:HucMSC negative markers CD11b, CD19, CD34, CD45, HLA-DQ/DR
Expression.
The characterization figure of Fig. 3 excretion body.A:Wester blotting detects excretion body positive protein Alix, Tsg101,
CD9, negative proteins Gm130, internal reference Protein G apdh；B: transmission electron microscope observing excretion volume morphing；C:NTA detects excretion body particle
Diameter distribution.
Fig. 4 HucMSC excretion body handles newborn mice ovary and activates primordial follicle result schematic diagram.A: ovary culture for 24 hours after
Foxo3a immunohistochemical staining；B: Foxo3a goes out the increase of core ratio after the processing of excretion body；C: rear p-rps6 is immune for 24 hours for ovary culture
Histochemical staining.PBS, PBS handle control group, Exo, excretion body processing group.Scale=50 μm.
Fig. 5 newborn mice ovary transplanted 14 days under kidney envelope after the processing of HucMSC excretion body after developmental state figure.A:
Ovary is transplanted after cultivating in pairs, ovarian morphology after 14 days, and processing group volume is greater than control group；B: ovarian morphology HE dyeing,
It can be seen that processing group developmental condition is more preferable；C: Follicles counting statistics analysis；D: ovary PCNA dyeing, it is seen that processing group ovary
And granulosa cell proliferation better off.A icon ruler=1mm, B figure, D icon ruler=50 μm.
Fig. 6 HUCMSC excretion structural reform is apt to Aged Mice reproductive function figure.A: control group and experimental group aged rats newborn mouse
It is counted with non-newborn mouse；B: Aged Mice mating test curve；C: Aged Mice takes ovum number to count.
Specific embodiment
Describe the present invention in detail below in conjunction with embodiment, but provided herein embodiment for illustration purposes only, and
It is not intended to limit the present invention.
The isolation and culture of embodiment 1:HucMSC
After removing about 15cm long umbilical cord, fresh umbilical cord tissue is placed in dual anti-containing 1% penicillin/streptomycin
It in PBS buffer solution, transports on ice into Biohazard Safety Equipment, with previous solu repeated flushing, until after without obvious remained blood, with normal
Surgical instrument mechanical stripping umbilical artery and vein are advised, again repeated flushing umbilical cord tissue.The umbilical cord ophthalmology that will be rinsed well
It cuts and is carefully cut into 1mm3The small tissue blocks of size or so, evenly laid out in diameter is in 10cm culture dish, and 2mL culture is added in every ware
Base, placement are incubated at 37 DEG C, 5%CO2Incubator in after 6 hours, into ware be added 5mL culture medium, change liquid one within every 2-3 days
It is secondary, it when cell, which grows to convergence degree, reaches 70%-80%, is digested with 0.25%EDTA- pancreatin, 1:3 passage.In P3 to P7 generation, is thin
Born of the same parents are for testing (Fig. 1: A).
Cell culture media formulations (50mL)
| Ingredient | Dosage |
| DME/F12 | 44.25mL |
| FBS | 5mL |
| It is dual anti- | 750μL |
The multidirectional induction of embodiment 2:HucMSC is broken up
When P5 reaches 70%-80% for cell culture to convergence degree, with 0.25%EDTA- pancreatin digest, carry out skeletonization and
, need to be by cell inoculation in 6 orifice plates when adipogenic induction Analytical Chemical Experiment, when break up at chondrocyte induction, cell is with suspension ball
Mode is incubated at 15mL centrifugation bottom of the tube.
Osteoinductive differentiation experiment: when cell, which grows to convergence degree, reaches 70%-80%, original culture medium is sucked, more
It changes self-bone grafting differential medium into, induces to specifications, change the liquid once within every 2-3 days, the fixed cell of 4%PFA is used after 21 days
Afterwards, observation (Fig. 1: B) is dyed with alizarin red dye liquor；
Adipogenic induction Analytical Chemical Experiment: when cell growth convergence degree reaches 100%, original culture medium is sucked, rouge is replaced with
Inductive differentiation medium induces to specifications, changes the liquid once within every 2-3 days, after fixing cell using 4%PFA after 21 days, oil red
O dye liquor dyeing observation (Fig. 1: C)；
At chondrocyte induction Analytical Chemical Experiment: when cell growth convergence degree reaches 90%-100%, with 0.25%EDTA- pancreatin
After digestion, cell is placed in 15mL centrifugation bottom of the tube in a manner of suspension ball, sucks original culture medium, is replaced with chondrocyte induction differentiation
Culture medium changes the liquid once to specifications, fixes cell ball after 28 days, after embedded section to be drained off, use A Lixin for every 2 days
Blue dye liquor dyeing observation (Fig. 1: D).
As it can be seen that isolated HucMSC have good skeletonization, at rouge, at cartilage differentiation ability.
The flow cyctometry of embodiment 3:HucMSC detects
P5 is for cell, when cell, which grows to convergence degree, reaches 70%-80%, is digested with 0.25%EDTA- pancreatin, then
2 times are cleaned with PBS and is centrifuged and is resuspended cell, calculate dosage by antibody specification, CD19, CD34, the CD44 of FITC label is added,
The CD105 antibody of the CD11b of CD45, HLA-DQ/DR antibody and PE label, CD73, CD90 antibody, APC label are protected from light incubation, and 15
PI is added after minute to be contaminated altogether to mark dead cell, 400g is used after contaminating 15 minutes altogether, 4 DEG C are centrifuged the unbonded antibody of 5min removal,
Cell is resuspended in flow cytometer detection pipe with 200 μ L PBS, upper machine testing.The P5 as shown in Fig. 2: A is positive for cell expression HucMSC
Marker CD44, CD73, CD90, CD105 do not express HucMSC negative markers CD11b, CD19, CD34, CD45, HLA-DQ/
DR (Fig. 2: B).
The extraction and identification of embodiment 4:HucMSC excretion body
When cell, which grows to convergence degree, reaches 60%-70%, original fluid is sucked, is carefully cleaned carefully with PBS buffer solution
Born of the same parents 3 times, 10mL conditioned medium, collection condition culture supernatant after culture 48 hours is added.Then the CMC model being collected into
Base 500g is centrifuged 10 minutes to remove cell, is centrifuged 20 minutes with 2000g to remove dead cell and cell fragment, uses 10000g
Then 0.22 μm of sterile filters of gained culture supernatant are filtered, temporarily with removing big vesica and apoptotic body etc. within centrifugation 30 minutes
When deposit in 4 DEG C.When ultracentrifugation, the conditioned medium handled is carefully transferred in ultracentrifugation pipe, after trim, is used
The relative centrifugal force of 120000g 90 minutes, sucks supernatant, with 1mL PBS be resuspended centrifuge tube bottom precipitation after, by it is all from
Liquid in heart pipe is mixed into same centrifuge tube, and PBS is added to liquid and is full of centrifuge tube, is centrifuged 90 points again with 120000g
Clock sucks supernatant liquid to clean excretion body, above each with 150-200 μ L PBS resuspension excretion body for subsequent experimental use
Step centrifugation carries out under 4 DEG C of environment.
Conditioned cell culture medium formula (50mL)
| Ingredient | Dosage |
| DME/F12 | 44.25mL |
| FBS | 5mL |
| It is dual anti- | 750μL |
Embodiment 5: the detection (Nanosight) of excretion body nanometer particle size
It is operated using nano particle trace analysis instrument according to machine specification using the technology of nano-particle trace analysis,
Upper machine operation, the particle size distribution range and number of particles of test sample after the excretion body sample that need to be detected is diluted 50-100 times.
Such as Fig. 3: C, particle diameter detected is the diameter range that 62.7-146.1nm meets excretion body, Average particle density 2.7 ×
1010particles/mL, albumen 0.79mg/mL.
Embodiment 6: transmission electron microscope observing excretion body
The excretion body precipitating that ultracentrifugation is obtained is resuspended with the glutaraldehyde solution of 100 μ L 2.5%, and 4 DEG C of fixations are stayed overnight,
Drawing 20 μ L samples, to be placed in clean copper online, liquid is carefully blotted with dust-free paper, after drying at room temperature 1-2 minutes, then with 2%
Uranium acetate dyes 2 minutes at room temperature, then in drying at room temperature, then copper mesh is carefully transferred in ultrapure water droplet, 2 points
Zhong Hou carefully sucks liquid with dust-free paper, observes machine on sample after waiting drying at room temperature.As shown in Fig. 3: B, under transmission electron microscope
Isolated vesica has the structure of typical excretion body round aecidioid, and diameter is 100nm or so.
Embodiment 7: excretion body fluorescent marker
It is careful to draw upper layer culture medium after ultracentrifugation, using PKH67 fluorescent labeling reagent box, excretion body is precipitated into weight
It is suspended from 0.5mL Diluent C.Meanwhile 4 μ LPKH67 dye liquors being added in 0.5mL Diluent C, the two is mixed,
After room temperature is protected from light incubation 4min, it is rapidly added 1mL 1%BSA/PBS solution and stops staining reaction, be then transferred to sample super
In fast centrifuge tube, it is centrifuged 90 minutes with 120000g at 4 DEG C, carefully draws supernatant liquid, excretion body precipitating in bottom is resuspended in
In 200 μ L PBS, it is kept in dark place under the conditions of -20 DEG C spare.
Embodiment 8: immunohistochemical staining after the external excretion body culture of newborn mice ovary
Take newborn 3 days ICR female mice 15, put to death using freezing method, alcohol spray disinfectant is placed on diameter 10cm's
In culture dish.In the interior operation of cell culture, from suckling mouse back, costovertebral angle opens an osculum, takes out bilateral ovaries, is placed in L15 balance
In the droplet of liquid, the coating of ovary is stripped carefully under stereoscope to expose ovary tissue.By free ovary tissue clean
L15 equilibrium liquid in clean 2 times after, be randomly divided into 2 big groups, be randomly divided into 6 groups per big group, in pairs culture in 24 orifice plates in
Set in culture cell, 400 μ L of ovary tissue culture solution be added in every hole, be separately added into each group hole basic culture solution, PBS and
Excretion body.Ovary tissue, fixed embedded section are collected in culture afterwards for 24 hours.
As shown in Fig. 4: A, it is marked using the marker molecule Foxo3a that primordial follicle activates, excretion body processing group ovary
It can be seen that apparent Foxo3a goes out core situation.Ovarian follicle count results also indicate that the primordial follicle number of activation increased significantly (Fig. 4: B).This
Outside, it is dyed using PCNA, can also find that excretion body processing group proliferative cell increases (Fig. 4: C).As it can be seen that the processing of excretion body can swash
Primordial follicle living and the proliferation for promoting ovary inner cell.
Embodiment 9: newborn mice ovary kidney envelope after excretion body culture is transplanted
5-6 week old ICR female mice is chosen as receptor mouse, the Tribromoethyl alcohol of intraperitoneal injection 240mg/kg is anaesthetized to mouse without anti-
Ying Hou, shaving off back of mice waist side, nearby hair, alcohol disinfecting skin are horizontally placed on operating table, confirm kidney position with finger,
A 1cm long osculum is cut, subcutaneous tissue is carefully exposed, successively enters abdominal cavity, after finding kidney, is carefully drawn out kidney, it will
Kidney is stuck at incision site, tears an osculum on kidney envelope with sharp tweezer, the ovary of culture is carefully filled in wherein and solid
Positioning is set, and prevents ovary from leaking out.After the completion of Autotransplanted Ovary, adipose tissue finds itself ovary tissue of receptor mouse near kidney,
Ovary is cut off with the eye scissors after high-temperature heating disinfection, using high temperature blood coagulation, confirmation will expose tissue in vitro without after bleeding
It carefully also receives in vivo in order, layer-by-layer suture wound is recovered with mouse is placed on warm platform after iodine disinfection.It sends back in animal
Heart raising, every 1 day intraperitoneal injection 10IU FSH, and observes wound recovery situation.After transplanting 14 days, retrieving part mouse, two
Carbonoxide method is put to death, and kidney is carefully cut in exposure abdominal cavity, peels off kidney envelope, and the ovary of exposure transplanting is taken pictures under stereoscope
Afterwards, fixed embedded section, records Follicles number, and immunohistochemistry detects PCNA protein expression situation.
Such as Fig. 5: A, shown in B, C, the ovary of excretion body processing group obviously becomes larger, and morphological analysis and ovarian follicle count results are aobvious
Show, follicular development increases, and antral follicles number increased significantly.In addition, PCNA label also shows excretion body ovary processing group interior
Granulocyte proliferative conditions are good.As it can be seen that the processing of excretion body can promote the development of ovarian follicle.
Embodiment 10:HucMSC excretion structural reform is apt to Aged Mice reproductive function
30 50 week old Aged Mices are selected, 2 groups of carry out ovary packet intracapsular injections are randomly divided into.One group of injection PBS, one group
Excretion body is injected, carry out mating test is mated after 3 weeks.We have continuously monitored newborn's situation of 14 weeks each group mouse, and draw
Mate curve.(4 mouse) compared with the control group, experimental group farrowing mouse number increased significantly (9 mouse).It is bent by mating
The drafting of line, it is seen then that every mouse of experimental group averagely farrows 5.40, and control group average every is farrowed 2.13.Therefore, excretion
Body also plays a role (Fig. 6) in terms of improving Aged Mice reproductive function.
Claims (7)
1. a kind of preparation method of excretion body, it is characterised in that the following steps are included: acquisition human umbilical tissue, and extract and obtain navel
Band mescenchymal stem cell；Obtained umbilical cord mesenchymal stem cells carry out centrifugal treating, obtain excretion body to extract.
2. the preparation method of excretion body according to claim 1, it is characterised in that step are as follows: the navel of artery and vein will be removed
Band is cleaned, and 1mm is cut into3Tissue block, evenly laid out in culture dish, every ware is added 2mL culture medium, and placement is incubated at 37 DEG C, and 5%
CO2Incubator in after 6 hours, into ware be added 5mL culture medium, change the liquid once within every 2-3 days, reached when cell grows to convergence degree
It when to 70%-80%, is digested with 0.25%EDTA- pancreatin, 1:3 is passaged to P3 to P7 for cell, obtains excretion body.
3. the preparation method of excretion body according to claim 2, it is characterised in that the umbilical cord mesenchymal stem cells are grown to
When convergence degree reaches 60%-70%, original fluid is sucked, is cleaned with PBS buffer solution, 10mL conditioned medium is added, culture 48 is small
When after collection condition culture supernatant the conditioned medium being collected into 500g is centrifuged 10 minutes thin to remove under 4 DEG C of environment
Born of the same parents are centrifuged 20 minutes with 2000g to remove dead cell and cell fragment, are centrifuged 30 minutes with 10000g to remove big vesica and wither
Corpusculum is died, relative centrifugal force 90 minutes for then using 120000g suck supernatant, are resuspended outside centrifugation bottom of the tube with 1mL PBS
After secreting body precipitating, the liquid in all centrifuge tubes is mixed into same centrifuge tube, PBS is added to liquid full of centrifuge tube, then
It is secondary with 120000g centrifugation 90 minutes to clean excretion body, suck supernatant liquid, with 150-200 μ L PBS be resuspended excretion body for
Subsequent experimental uses.
4. human umbilical cord mesenchymal stem cells excretion body made from any preparation method of claim 1-3.
5. application of the excretion body described in claim 4 in non-diagnostic therapeutic purposes primordial follicle Activation In Vitro.
6. excretion body described in claim 4 is preparing the application in primordial follicle Activation In Vitro reagent.
7. primordial follicle Activation In Vitro reagent, it is characterised in that effective component is dry for human umbilical cord mesenchymal as claimed in claim 4
Cell excretion body.
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| WO2023040938A1 (en) * | 2021-09-15 | 2023-03-23 | 青岛卓云海智医疗科技有限公司 | Method for treating ilk signaling pathway related diseases using exosome derived from mesenchymal stem cells, and pharmaceutical composition |
| CN114134107A (en) * | 2021-11-29 | 2022-03-04 | 南京医科大学 | Artificial ovary participated by mesenchymal stem cells and preparation method and application thereof |
| CN114134107B (en) * | 2021-11-29 | 2024-01-16 | 南京医科大学 | Artificial ovary with mesenchymal stem cells participating, and preparation method and application thereof |
| CN115463215A (en) * | 2022-07-26 | 2022-12-13 | 苏州大学 | New use of HDAC9 and inhibitors thereof |
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