CN109692184A - A kind of menses stem cell medicine and its preparation method and application - Google Patents
A kind of menses stem cell medicine and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of menses stem cell medicine and its preparation method and application, the menses stem cell medicine of infertility caused by the middle severe Asherman's syndrom, it is by being formed through hemocytoblast, human serum albumin and normal saline, this is simple through hemocytoblast acquisition methods, it is not limited by ethics, and its cell viability is high, proliferative ability is strong, with multi-lineage potential, overall process uses free serum culture technology, safety is good, and preparation is easy, and cell culture result is made to realize better economy, repeatability and stability；Menses stem cell medicine infusion techniques of the invention are simple to operate, barrenness caused etc. using severe Asherman's syndrom in the treatment of menses mescenchymal stem cell, have very important clinical meaning and wide application prospect.
Description
Technical field
The invention belongs to biological products cells to be separately cultured/technical field of cell biology, and specifically a kind of menses are dry thin
Born of the same parents' preparation and its preparation method and application.
Background technique
Asherman's syndrom (Intrauterine adhesions, IUA) refers to due to improper palace transluminal operation and infection repeatedly
Etc. reasons cause basal layer it is impaired caused by intimal fibrosis, uterine wall partially or completely occlude, there is menstruation
Amount subtracts little or no amenorrhea, a series of infertile, clinical complications such as recurrent abortion.The method of clinical treatment IUA, such as hysteroscope at present
Lower separation of synechia, estrogen and progestogen, using barrier media etc., it is expected that repairing inner film injury, restore uterus normal morphology.However, passing
The therapeutic effect of system therapy centering severe IUA patient is poor.Severe Asherman's syndrom patient uterine form and inner membrance in being difficult to restore
Function.Therefore, new technology and therapy are sought, makes the remodeling of the internal film tissue of damage or lesion, restores its normal function, prevent
The treatment that centering severe IUA occurs again of adhesion is of great significance.The remoldability reflection of internal film tissue during menstruation and gestation
There are self-renewing and the stem cell of proliferation for endometrium.Excessive damage causes inner membrance stem cell population to endometrium repeatedly
It reduces even missing, function to be damaged, endometrium cannot be repaired and be regenerated completely, and IUA is finally caused.With to IUA pathology and
The further investigation of inner membrance regenerative system, discovery, which restricts regenerated two key factors of endometrium, is: the 1. function of endometrium
Layer and hypothallus stem cell population are less, and functional layer stem cell periodically falls off with menses, and impaired basal layer stem cell can not
Regeneration proliferation.2. certain molecules in intrauterine infection or inflammatory environment may inhibit the regeneration of impaired inner membrance, and promote
The generation and deposition of fibr tissue.With the further investigation to stem cell, stem cell answers regenerative medicine and organizational project
With more and more extensive.One side stem cell can migrate to impaired tissue and supplement, substitute or be divided into relevant functionality
Tissue, participates in the reconstruction of tissue.Another aspect stem cell secretes the multiple biological activities factor by paracrine mechanism, promotes part
Cell (including endogenous retinal stem cells) and revascularization.Meanwhile stem cell can also inhibit inflammatory reaction by it[5]With it is immune
Regulatory function reduces tissue damage, promotes to repair.
In recent years, the clinical research of stem-cell therapy IUA also illustrates its great desired potential applicability in clinical practice.Using bone
Severe IUA patient in marrow stem-cell therapy, patient's endometrium has before relatively treating and obviously thickens after discovery treatment, part menstruation
Recovery or naturally pregnancy or the Pregnancy Success after embryo transfer.The stem cell turned out and intrauterine are successfully separated from menstrual blood
The stem cell that film biopsy is extracted is in form and function and its similar, i.e. stem cell (the Menstrual in menstrual blood source
Blood-derived stromal cells, MenSCs).MenSCs has mesenchymal cell sample characteristic, expresses mesenchymal cell
Surface marker molecule CD73, CD90, CD105, CD44, but do not express hemopoietic stem cell surface molecule CD34, CD45,
CD133, and can induce to fat, skeletonization, cartilage, liver sample, nucleus pulposus sample, the differentiation of neuroglia like cell.Early period, animal was real
Test research shows that, MenSCs in terms of the diseases such as treatment progressive muscular dystrophy, myocardial infarction, limb ischemia and apoplexy
Achieve preferable effect.It is increasing at present about the clinical research of MenSCs.MenSCs treatment congestive heart failure,
Injury of lungs caused by cirrhosis, H7N9, serious limb ischemia, type-1 diabetes mellitus and the clinical application research of embryo transfer are
Progress in.
Other than local transplantation in uterine cavity, zoopery and clinical studies show are transfused through system (vein or artery) and do
Cell therapy IUA also obtains good curative effect.In IUA mouse model, venoclysis and uterus local transplantation human bone marrow-derived are dry
After cell, the stem cell of transplanting can be detected in Mouse Uterus, and the former stem cell homing rate is better than the latter.
Taylor etc. compares two different therapeutic modality stem cell migrations to the case where being damaged endometrium, and discovery passes through vein
Injection, stem cell can preferably move to injury region.Promoting impaired Morphological examination of endometrial tissue and functional rehabilitation is treatment
The target of Asherman's syndrom.During traditional treatment means such as hysteroscope synechiolysis, estrogen and progestogen and barrier media are difficult to restore
Severe Asherman's syndrom patient uterine form and inner membrance function.In recent years, become heat using the research of stem cells for damaged tissue repair
Point.Early period studies have shown that the mescenchymal stem cell of derived from bone marrow is shown in Asherman's syndrom animal model and clinical research
Effective therapeutic effect.However, the mescenchymal stem cell of materials derived from bone marrow has certain traumatic, application is by a fixed limit
System.The study found that the stem cell in menses source has proliferation and differentiation potential, and there is higher migration and revascularization energy
Power.
The mescenchymal stem cell characteristic that MenSCs has, and endometrium is derived from, it can preferably adapt to endometrium
Microenvironment, migration go back to the nest to inner film injury position, reparative regeneration damaged tissues.In addition, menses acquisition modes no pain and can
It obtains repeatedly.These characteristics of MenSCs make it become the treatment ideal stem cell of IUA.Currently, having reported menstrual blood source
The animal model of temper endometrial stem cells transplantation treatment severe Asherman's syndrom.And the effect in endometrial impairment reparation
Research.It is not directed in treatment the contents such as severe Asherman's syndrom is barrenness caused.Thus, it is expected to heavy in treatment as being applied to
The barrenness caused drug of Asherman's syndrom is spent, yet there are no relevant report up to now.
Summary of the invention
In view of the above-mentioned problems, the present invention provides a kind of through hemocytoblast in preparation treatment caused by severe Asherman's syndrom not
The application of the drug of pregnant disease.The cell survival rate is high, and cell quantity is more, and character is stablized, and the noninvasive easy acquisition of safety is easy amplification training
Support, the barrenness caused therapeutic effect of centering severe Asherman's syndrom is significant, significantly improves endometrium thickness, be conducive to the later period by
It is pregnant.
Technical solution provided by the invention: a kind of menses stem cell medicine, the menses stem cell medicine is done by menses
The physiological saline that cell, human serum albumin and mass fraction are 0.9% configures, and menses are dry in the menses stem cell medicine
The concentration of cell is 4.5-6.0*106A/ml, the mass concentration of human serum albumin is 5%, described to express through hemocytoblast height
CD73, CD90, CD105, CD44, low expression CD34, CD45, CD19 and HLA-DR.
A kind of application of menses stem cell medicine, the menses stem cell medicine is for being injected under basal layer
Three sites for being at least spaced apart from each other 1.5cm at 0.5cm, it is described for infertility caused by severe Asherman's syndrom in treating
The dosage of menses stem cell medicine is at least 1.0*107A/3ml.
A kind of preparation method of menses stem cell medicine, includes the following steps:
(1) Sterility patient for choosing severe Asherman's syndrom in Endometrial stem cell treatment, checks vagina, palace the moon before menstruation
Neck secretion, no bacterium, fungi, mycoplasma, choamydiae infection；Second day menstrual period iodophor disinfection vulva, vagina, use heparin sodium
Rinse menstrual cup is placed in intravaginal arched roof and clasps cervix, collects patient's menstrual period second day in 12 hours menses 3-
10ml；
(2) step (1) resulting menses are diluted in equal volume with PBS, by the blood after dilution along filling half
The centrifuge tube upper tube wall of the Ficoll lymphocyte separation medium of volume is slowly added to, and is centrifuged 30min, is drawn intermediate layer cell, is set
In the PBS solution of about 2 times of volumes, it is centrifuged 10min；Supernatant is abandoned, PBS is cleaned 2 times again, on the PBS after taking last time to be centrifuged
Clearly, bacterium, fungal detection are done；Leave and take cell precipitation；
(3) step (2) resulting cell precipitation is resuspended with complete medium, is transferred to a T25 culture bottle, is placed in dioxy
Change the incubator culture of carbon constant temperature and humidity, condition of culture is temperature: 37 DEG C, carbon dioxide content: 5%, rear PBS solution washing for 24 hours
The 1-2 non-attached cell of removing, attached cell are denoted as P0 generation, and addition 5ml complete medium continues to cultivate；
(4) every 3-5 days replacement culture mediums, had digestive transfer culture when cell fusion degree reaches 80%-90% abandons culture solution, PBS
0.25% pancreatin, 37 DEG C of incubation 2-3min are added in cleaning cell one time, and to cell rounding, gently patting body keeps MenSCs complete
Portion is split away off, and complete medium termination digestion is added, and collects cell, and 1000rpm, 5min centrifugation are collected and precipitated with fresh
Cell is resuspended to required cell concentration in complete culture solution, moves to new culture bottle, is placed in 37 DEG C, 5%CO2Cell incubator passes
It is commissioned to train feeding, repeats aforesaid operations for cell culture passages to P3-P5 generation and establish working cardial cell library after collection, freeze stand-by；
(5) it is with 0.9% physiological saline adjusting cell number after hemocytoblast thaws recovery by what step (4) were collected
4.5-6.0*106A/ml, adds human serum albumin, makes the mass concentration 5% of human serum albumin, and it is dry thin to be configured to menses
Born of the same parents' injection；
Any source of people or animal sources serum are not added in the complete medium.
Further, the complete medium is the serum that Lonza serum-free basal medium adds mass fraction 2%
The glutamine of substitute and mass fraction 1% configures, and is free of antibiotic.
Further, 1 × antibacterial-antifungal agent is added in the ratio of 1:100 in step (2) PBS
(Antibiotic-Antimycotic) three is anti-.
Further, the step (2) is if gained cell precipitation contains blood clot or dope, after filter screen filtration
Carry out subsequent use.
The present invention has the advantage that compared with the existing technology
(1) of the invention simple through hemocytoblast acquisition methods, it is not limited by ethics, and its cell viability is high, cell is deposited
Motility rate is high, and cell quantity is more, and character is stablized, and the noninvasive easy acquisition of safety is easy amplification cultivation, and proliferative ability is strong, has Multidirectional Differentiation
Potential, preparation are easy；
(2) method that overall process of the present invention separation preparation through hemocytoblast uses free serum culture, is not added with anyone
Source or animal sources serum, avoid with blood source reduction and serum price rise steadily and introduce animal sources cause a disease because
Sub- bring risk makes cell culture result have better safety, repeatability and stability, reduces production cost, mention
High cell culture quality；
(3) in menses stem cell medicine of the invention, human serum albumin is added, can be used as stabilizer and protective agent, maintain
The activity of cell can protect cell, provide nutrition, growth factor, relative to existing preparation, therapeutic effect is more preferably；
(4) menses stem cell medicine infusion techniques of the invention are simple to operate, controlled using menses mescenchymal stem cell
Severe Asherman's syndrom is barrenness caused etc. in treatment, has very important clinical meaning and wide application prospect.
The present invention is therefrom separately cultured the technical method of MenSCs in the menses of severe IUA patient and establishes clinical application grade
MenSCs work library；Using the local administration method of palace intraluminal grafting, become by observation patient menstrual's situation, endometrium thickness
Change, uterus shape and pregnancy outcome are to determine MenSCs therapeutic effect, optimal therapeutic dosage, approach and the course for the treatment of, reconstruction patients
Palace normal morphology promotes the recovery of endometrial thickness and function.
Detailed description of the invention
Fig. 1 is morphologic observation microscopy figure under the microscope of the invention through hemocytoblast；
Fig. 2 is surface marker of the Flow cytometry through hemocytoblast；Wherein Fig. 2-1 is CD73, and Fig. 2-2 is
CD90, Fig. 2-3 are CD105, and Fig. 2-4 is CD44, and Fig. 2-5 is CD45, and Fig. 2-6 is CD19, and Fig. 2-7 is CD34, and Fig. 2-8 is
HLA-DR；
Fig. 3 is that certain patient of the invention receives endometrium thickness ultrasound diagnosis figure before MenSCs is transplanted, intrauterine before transplanting
Film thickness is 3.3mm；
Fig. 4 is that certain patient of the invention receives endometrium thickness ultrasound diagnosis figure after MenSCs transplanting, intrauterine after transplanting
Film thickness is 6.5mm；
Fig. 5 is the statistical results chart that several patients of the invention receive MenSCs transplanting front and back endometrium thickness.
Specific embodiment
The present invention is described further with reference to the accompanying drawings and examples.
Stem cell method for separating and preparing and the application for present embodiments providing a kind of menstrual blood source, specifically include following step
It is rapid:
(a) consumptive material: Ficoll lymphocyte separation medium (GE healthcare), Lonza UItraCULTURE Serum-
Free culture medium (Lonza company), 0.25% pancreatin (Life technologies), serum substitute (PALL Life
Science), PBS (Gibco/Life technologies), CELLBANKER 2 (cell commercialization frozen stock solution) (ZENOAQ),
50ml centrifuge tube, 10ml centrifuge tube (Thermo Scientific), 5ml pipette, 25ml pipette, Pasteur's tubule, T25,
T75 culture bottle Tissue Culture Flask (CORNING), 5ml syringe (Shanghai Jinta Medical Equipment Co., Ltd.), antibacterial-is antimycotic
Agent (Antibiotic-Antimycotic) (Gibco), 0.9% sodium chloride injection (Shijiazhuang Siyao Co., Ltd).
(b) instrument: flow cytometer (Beckman), Biohazard Safety Equipment (power health), cell incubator (power health), at a high speed from
Scheming (Kubo field), inverted microscope (Jiangnan), ultra low temperature freezer (Haier), nitrogen storage tank (Thermo).
One, the collection of MenSCs with separate
1. checking vagina, cervical secretions, no bacterium, fungi, mycoplasma, choamydiae infection the moon before menstruation；Menstrual period second
Its iodophor disinfection vulva, vagina are placed in intravaginal arched roof and clasp cervix, collected 12 hours with heparin sodium rinse menstrual cup
The menses 3.5ml in second day interior patient's menstrual period, menses color are dark red.
2. by menses and PBS (containing 1 × antibiotic-antimycotic) isometric dilution, by the blood edge after dilution
Fill half volume the centrifuge tube upper tube wall of Ficoll lymphocyte separation medium be slowly added to, room temperature 900g, centrifugation
30min.Sterile Pasteur pipet slowly draws intermediate layer cell, is placed in the PBS solution of about 2 times of volumes, room temperature 300g, centrifugation
10min.Supernatant is abandoned, PBS is cleaned 2 times again, leaves and takes cell precipitation.PBS supernatant after taking last time to be centrifuged, does bacterium, fungi
Detection.If cell precipitation contains blood clot or dope, filter screen filtration is used.
Two, the culture and amplification of MenSCs
1. originally culture: cell precipitation is resuspended with complete medium, is transferred to a T25 culture bottle, is placed in carbon dioxide perseverance
Constant temperature and humidity incubator culture, condition of culture are temperature: 37 DEG C, carbon dioxide content: and 5%, rear PBS solution, which is washed 1-2 times, for 24 hours removes
Non- attached cell is gone, attached cell is denoted as P0 generation, and addition 5ml complete medium continues to cultivate, and bacterial fungus detection is feminine gender.
2. secondary culture and freezing and building library: every 3-5 days replacement culture mediums, cell fusion degree disappeared when reaching 80%-90%
Change passage, abandon culture solution, PBS is cleaned cell one time, and 0.25% pancreatin is added, covers all cells, 37 DEG C of incubation 3min, to thin
Born of the same parents are rounded, and gently patting body splits away off MenSCs all, and complete medium is added and terminates digestion, collects cell,
1000rpm, 5min centrifugation collect the fresh complete culture solution of precipitating and cell are resuspended to required cell concentration, moves to new culture
Bottle, is placed in 37 DEG C, 5%CO2Cell incubator secondary culture repeats aforesaid operations, and cell culture passages to P3-P5 generation are received
Working cardial cell library is established after collection, is frozen stand-by；
The embodiment of the present invention, the complete medium in the step are by 2% serum substitute of mass fraction, mass fraction
1% glutamine and Lonza basal medium configure, and are free of serum and antibiotic.
The embodiment of the present invention, the basal medium are Lonza UItraCULTURE Serum-free culture solution, are free of
Antibiotic.
According to an embodiment of the invention, culture described in the step is to expand bottle culture.
According to an embodiment of the invention, passage is passed on by 1:2-1:4 in the step.
Cell culture record
1, the 1st day through hemocytoblast culture, culture solution supernatant was limpid, adherent compared with many cells, was denoted as P0 for cell.PBS
Cleaning cell 1-2 times, removes non-attached cell, and addition 5ml culture medium continues to cultivate.Bacterial fungus detection: being feminine gender.
2, the 3rd day through hemocytoblast culture, cell is adherent to be increased, and discarded cell conditioned medium, and addition 5ml culture medium continues to train
It supports.
3, the 4th day through hemocytoblast culture, the agglomerating growth of cell, attached cell increased, takes pictures.
4, the 5th day through hemocytoblast culture, attached cell is digested, cell is collected and passage is seeded to 2 T25 cultures
Bottle, is denoted as P1 for cell.
5, the 8th day through hemocytoblast culture, liquid is changed.
6, the 9th day through hemocytoblast culture, cell density 80% digested attached cell, collects cell and passage connects
Kind is denoted as P2 for cell to 2 T75 culture bottles.
7, the 14th day through hemocytoblast culture, cell density 90%, the P2 that takes pictures was for cell.Vitellophag collects cell
And pass on and be seeded to 8 T75 culture bottles, P3 is denoted as cell.
Cell cryopreservation record
1, the 18th day through hemocytoblast culture, cell adherent 95% digested and collects cell, and last time is taken to clean
Bacterium, fungi, endotoxin, detection of mycoplasma are done clearly；Then cell count presses 6 × 106/ ml density is resuspended in cells frozen storing liquid.
Frozen stock solution containing cell is dispensed into the cryopreservation tube marked, cryopreservation tube places into program temperature reduction box, marks, and puts
Enter -80 DEG C of Temperature drop in refrigerator, is transferred in -196 DEG C of liquid nitrogen and saves after 24 hours.
Three, the detection and identification of MenSCs
1. cellular morphology observation and counting: long fusiform is presented through blood cell development in microscopically observation, at fiber-like, patch
Wall growth；Cell activity >=95%, cell quantity are about 1 × 107A/3ml.See Fig. 1, table 1
2. microbiologic inhibition tests: cell conditioned medium carries out bacterium, fungi, mycoplasma, endotoxin and human virus detection detection.
Once having detected microbial contamination, cell should abandon immediately.
1 menses dry cell mass testing result of table
3. the surface marker of MenSCs is identified: taking the good P3-5 of growth conditions for cell, cell dissociation is dispersed into
Individual cells, and be divided into 7 parts and be placed in streaming pipe, every part 1 × 106A cell is separately added into anti-with downflow system as required
Body: CD73, CD90, CD105, CD44, CD34, CD45, CD19, HLA-DR, room temperature be protected from light be incubated for 30min, as required loading and
Use flow cytometer.In triplicate, flow cytometer showed result is shown in Fig. 2 and table 2.
The identification of 2 cell phenotype of table
Flow cytometer showed discovery, menses stem cell surface marker CD73, CD90, CD105, CD44 positive expression rate >
90%, CD34, CD45, CD19, HLA-DR positive expression rate are < 2%.
4. MenSCs induction differentiation: taking the good cell of growth conditions, cell dissociation is dispersed into individual cells suspension, is pressed
1×106/ hole is uniformly layered on 6 orifice plates.Rouge, skeletonization are replaced with after cell is adherent, at chondrocyte induction culture medium.The training of replacement in every 3-4 days
Support base.Culture carried out oil red O (Oil Red O), alizarin red (Alizarin Red), A Li Xinlan (Alcian after 2-3 weeks respectively
Blue the differentiation potential of identification of M enSCs) is dyed.
Four, cellular replacement therapy and hormone therapy scheme
MenSCs transplanting: the menstrual period the 2nd day, patient is notified to prepare to feed back MenSCs.After the information for checking patient, by work
The cell recovery in library, 800g are centrifuged 10min, and physiological saline cleans 2 times, cell count, and cell is resuspended with physiological saline, is added
Human serum albumin adjusts concentration to 4.5-6 × 10 after counting6/ ml takes 3 1ml to be respectively charged into spare in 3 1ml syringes.
Patient takes lithotomy position, Iodophor routine disinfection vulva and vagina, and sterile gauze dries vagina, uterine neck and fornix vagina portion, before transplanting
Endometrium and removing uterine cavity intravascular coagulation block are scraped in slight scratching.Under abdominal B-scan ultrasonography guidance, reached through uterine neck insertion embryo transplantation tube
0.5cm under basal layer takes out tube core, connects 5ml dress physiological saline device, resistance has been injected in pressurization, then has been hinged with
The MenSCs suspension of 1ml slowly injects cell suspension 0.5ml, and it is remaining to continue injection while then slowly recalling grafts
0.5ml cell suspension until tip is exited outside endometrium.Other two site is chosen, repeated embryo transfer operates 2 times.Three shiftings
It plants site and is at least spaced 1.5cm, postoperative patient bending knee lies low 2 hours, and art medium sized vein is transfused 250ml physiological saline+phloroglucin
40mg inhibits uterine contraction.
Hormone therapy: the aglow 2mg of the sweet smell of patient began oral on the same day of feedback even served 14 days, simultaneously three times a day
Oral aspirin enteric coatel tablets, a 1000mg, once a day, clothes withdraw in the period.14 days and 19 days after transplanting stem cell
Ultrasonic examination endometrium thickness and form.If endometrium thickness < 7mm, patient gives fragrant logical pornographic movie 2mg and takes orally,
Three times a day, 14 days.After withdrawal bleeding, cyclic estrogen and progesterone treatment repeat three or three or more menstruation weeks
It after phase, checks the thickness of endometrium, such as falls flat, the second wheel cells transplantation treatment can be carried out.If ultrasonic
Wave checks that endometrium thickness > 7mm, patient give fragrant logical pornographic movie 2mg and take orally, three times a day, 14 days, after withdrawal bleeding
Third day continues oral aglow 2mg of sweet smell and takes 12 days ultrasonic examination endometrium thickness and form three times a day, if
Ultrasonic examination endometrium thickness > 7mm and endometrial morphology A shape or B shape continue oral fragrant aglow three days, a 2mg,
Three times a day, blood drawing looks into sex hormone estradiol in 200pg/ml, and progesterone is less than 1ng/ml, and progesterone converts endometrium, selects a time
Defrosting embryo transfer is given fragrant logical pornographic movie 2mg and is taken orally after transplanting, three times a day, vagina snow promise ketone 90mg, and once a day,
Routinely return institute test it is pregnant.
Five, typical case
The effect that the patient for meeting inclusion criteria to 4 carries out menses stem cell transplantation counts:
The inclusion criteria of clinical patients: a.20-43 year receive " test-tube baby " patient, fail for transplanting 2 times or more, 2 palaces
Hysteroscope turns out to be in uterine cavity, severe adhesion (according to European Gynecologic Endoscopy association IUA classification standard (ESGE))；B. traditional treatment
Endometrium thickness is still less than 6mm after means；C. informed consent form is signed, agrees to menses cellular replacement therapy.
1, certain is thanked, 33 years old, uterine cavity severe was adhered
5 Hysteroscope in Treatment art+artificial cycles, onset of ovulation inner film thickness 4.0mm before transplanting, 14 days B ultrasound of menstruation after treatment
6.4mm, hidden three line, blood flow is abundant, 19 days B ultrasound inner membrance 7.5mm after menstruation, hidden three line, and blood flow is abundant.
2, poplar, 40 years old, uterine cavity severe was adhered
3 Hysteroscope in Treatment art+artificial cycles, former onset of ovulation inner film thickness 4.4mm before treating, July 1, which examined to scrape, to be moved back
It plants.10 days B ultrasound 8mm of menstruation, boundary is clear, there is fluid in uterine, and blood flow is abundant, 14 days B ultrasound 8mm of menstruation, and blood flow is abundant.
3, Liang, 40 years old, severe uterine cavity was adhered
4 Hysteroscope in Treatment art+artificial cycles, former onset of ovulation inner film thickness 4.4mm before treating, July 24, which examined to scrape, to be moved back
It plants, the 14th day inner membrance 7.1mm of menstruation after treatment, hidden three line, blood flow is abundant.
4, Lee, 40 years old, severe uterine cavity was adhered
Before treatment: seeing Asherman's syndrom, cicatrization under patient's hysteroscope, internal film tissue is few.The super display inner film thickness of B
3.3mm, as shown in Figure 3.After treatment: 19 days after stem cell transplantation, B ultrasound shows inner film thickness 6.5mm, as shown in Figure 4.Embryo
After transplanting, the visible yolk bag of color ultrasound, plumule and the beating of heart pipe, bigeminal pregnancy.
Endometrium thickness before and after 3 stem cell transplantation of table
Before treating after vs treatment: difference has statistical significance (P < 0.05).
The foregoing is merely the detailed descriptions of specific embodiments of the present invention, do not limit the present invention with this, all at this
Made any modifications, equivalent replacements, and improvements etc. in the mentality of designing of invention, should be included in protection scope of the present invention it
It is interior.
Claims (6)
1. a kind of menses stem cell medicine, it is characterised in that: the menses stem cell medicine is by through hemocytoblast, the white egg of people's blood
The physiological saline that white and mass fraction is 0.9% configures, and the concentration through hemocytoblast is in the menses stem cell medicine
4.5-6.0*106A/ml, the mass concentration of human serum albumin is 5%, described to express CD73, CD90 through hemocytoblast height,
CD105, CD44, low expression CD34, CD45, CD19 and HLA-DR.
2. the application of menses stem cell medicine according to claim 1, it is characterised in that: the menses stem cell medicine is used
In three sites for being at least spaced apart from each other 1.5cm being injected under basal layer at 0.5cm, for severe palace in treating
The dosage of infertility caused by chamber adhesion, the menses stem cell medicine is at least 1.0*107A/3ml.
3. a kind of preparation method of menses stem cell medicine, it is characterised in that include the following steps:
(1) Sterility patient for choosing severe Asherman's syndrom in Endometrial stem cell treatment, checks vagina, uterine neck point the moon before menstruation
Secretion, no bacterium, fungi, mycoplasma, choamydiae infection；Second day menstrual period iodophor disinfection vulva, vagina, with heparin sodium rinse
Menstrual cup is placed in intravaginal arched roof and clasps cervix, collects patient's menstrual period second day in 12 hours menses 3-10ml；
(2) step (1) resulting menses are diluted in equal volume with PBS, by the blood after dilution along filling half volume
The centrifuge tube upper tube wall of Ficoll lymphocyte separation medium be slowly added to, be centrifuged 30min, draw intermediate layer cell, be placed in 2 times
In the PBS solution of volume, it is centrifuged 10min；Supernatant is abandoned, PBS is cleaned 2 times again, and the PBS supernatant after taking last time to be centrifuged is run business into particular one
Bacterium, fungal detection；Leave and take cell precipitation；
(3) step (2) resulting cell precipitation is resuspended with complete medium, is transferred to a T25 culture bottle, be placed in carbon dioxide perseverance
Constant temperature and humidity incubator culture, condition of culture are temperature: 37 DEG C, carbon dioxide content: and 5%, rear PBS solution, which is washed 1-2 times, for 24 hours removes
Non- attached cell is gone, attached cell is denoted as P0 generation, and addition 5ml complete medium continues to cultivate；
(4) every 3-5 days replacement culture mediums, had digestive transfer culture when cell fusion degree reaches 80%-90% abandons culture solution, PBS cleaning
Cell one time, 0.25% pancreatin, 37 DEG C of incubation 2-3min are added, to cell rounding, gently patting body makes MenSCs all fall off
Get off, complete medium is added and terminates digestion, collect cell, 1000rpm, 5min centrifugation collect and precipitates fresh complete training
Cell is resuspended to required cell concentration in nutrient solution, moves to new culture bottle, is placed in 37 DEG C, 5%CO2Cell incubator secondary culture,
It repeats aforesaid operations cell culture passages to P3-P5 generation are established into working cardial cell library after collection, freeze stand-by；
It (5) is 4.5- with 0.9% physiological saline adjusting cell number after hemocytoblast thaws recovery by step (4) collection
6.0*106A/ml, adds human serum albumin, makes the mass concentration 5% of human serum albumin, is configured to infuse through hemocytoblast
Penetrate liquid；
Any source of people or animal sources serum are not added in the complete medium.
4. the preparation method of menses stem cell medicine according to claim 3, it is characterised in that: the complete medium is
Lonza serum-free basal medium adds the glutamine configuration of the serum substitute and mass fraction 1% of mass fraction 2%
It forms, is free of antibiotic.
5. the preparation method of menses stem cell medicine according to claim 3, it is characterised in that: in step (2) PBS
It is anti-that 1 × antibacterial-antifungal agent (Antibiotic-Antimycotic) three is added in the ratio of 1:100.
6. the preparation method of menses stem cell medicine according to claim 3, it is characterised in that: obtained by the step (2)
If cell precipitation contains blood clot or dope, with carrying out subsequent use after filter screen filtration.
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