CN104958319A - Mesenchymal stem cell and cytokine preparation having treatment effects on premature ovarian failures and perimenopausal syndromes, and preparing method for preparation - Google Patents
Mesenchymal stem cell and cytokine preparation having treatment effects on premature ovarian failures and perimenopausal syndromes, and preparing method for preparation Download PDFInfo
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Abstract
The invention belongs to the field of biological medicines, and particularly discloses a mesenchymal stem cell and cytokine preparation having treatment effects on premature ovarian failures and perimenopausal syndromes, and a preparing method for the preparation. The preparation is composed of umbilical cord or placenta mesenchymal stem cells and cytokines, as well as hyaluronate which is used for bearing the umbilical cord or placenta mesenchymal stem cells and cytokines and has the slow-release and anti-inflammatory effects; and the preparation is injected into a part of the endometrium and ovary in a targeted manner to be slowly released and absorbed. The curative effect of repairing the ovary and endometrium can be achieved, functional ovulation can be promoted, the premature ovarian failures can be treated, and the various perimenopausal syndromes can be remitted. Compared with an existing intravenous re-transfusion and intervention path, untoward effects of intravenous transfusion are avoided through the preparation and a using method of the preparation, abdomen and local wounds caused by interventional treatment are avoided, and side effects and hurts caused by treatment to a patient can be effectively prevented, so that the preparation is safer and more effective, is high in practicability and easy to popularize, and has the huge application value.
Description
Technical field
The present invention relates to biomedicine technical field, relate to the Transformation Application of mescenchymal stem cell in regenerative medicine field, be specifically related to the mescenchymal stem cell and cytokine and preparation method thereof thereof with premature ovarian failure and perimenopausal syndrome therapeutical effect.
Background technology
Premature ovarian failure, refer to the women of built Regulations of Error Equation menstruation, before 40 years old, persistence amenorrhea and sexual organ's atrophy is there is due to ovarian failure, often there are the rising of Gonadotropin Level and estrogenic decline, before 40 years old, the sickness rate of premature ovarian failure is about 1% ~ 3%, and the sickness rate in 30 years old is about 0.1%.Clinical manifestation is with symptom before and after the menopause such as hectic fever hyperhidrosis in various degree, insomnia and dreamful sleep, irritable, cardiopalmus, tired, nervous, vaginal dryness, sexual disorder, sterility and infertility, amenorrhea in advance, make patient's geromorphism, if can not get adjusting timely and treating, the appearance of osteoporosis, cardiovascular and cerebrovascular disease chronic diseases can be caused, directly affect female life quality, have a strong impact on the normal work of women, bring considerable distress to its physical and mental health and love life simultaneously.
The underlying cause that perimenopausal syndrome occurs is the ovarian function failure caused due to physiological or pathologic or operation.Ovarian function is once exhaustion or cut and destruction, and the estrogen of ovarian secretion will reduce.Women's whole body has multiple estrogen receptor, is distributed in tissue and organ that almost women's whole body is all, accepts estrogenic control and domination, once estrogen decrease, will cause the degeneration change of Organ and tissue, occur a series of symptom.As can be seen here, premature ovarian failure can cause perimenopausal syndrome, and treatment premature ovarian failure can be conducive to treating perimenopausal syndrome simultaneously.Premature ovarian failure, its cause of disease it be unclear that so far, so become focus and the difficult point of domestic and international reproductive development circle to the research of this disease.At present, research both at home and abroad think more premature ovarian failure and hereditary, immune, metabolism and the factor such as iatrogenic relevant.Existing research data display chromosome mutation, FSH (FSH) interstitialcellstimulating hormone (ICSH) (LH) and receptor variant thereof, Developmental and Metabolic Disorder, drug effect, chemicotherapy damages, viral infection, immunity and other high risk factors such as factor such as smoking, depression may be its reasons, these factors or make the congenital ovum decreased number of ovary, or its follicular atresia is accelerated, or directly destroyed, make follicle premature depletion, the treatment modern medicine for premature ovarian failure generally adopts hormone replacement therapy.But premature ovarian failure treatment is more difficult, and doctor trained in Western medicine adopts Hormone Replacement Therapy clinically, needs to use estrogen and progestogen under the guidance of doctor; Time research shows this estrogen and progestogen therapy, have certain effect, can symptom be improved, but unsatisfactory to the recovery of ovarian function, and after drug withdrawal simultaneously, symptom reappears, ovary is made to be in dependent status, have and cause the worse possibility of ovarian function, that is Hormone Replacement Therapy only serves the effect taken stopgap measures, and this method side effect is large, life-time service exogenous hormones has the danger causing endometrium canceration, and increases the sickness rate of coronary heart disease and cholecystopathy.Therefore, the effect of this hormone therapy is undesirable, there is potential safety hazard simultaneously.
In recent years, along with the development of gene therapy and stem cells technology, stem cells technology is adopted to replace the therapy of hormone to be more and more concerned; At present, have and adopt venous re-transfusion mescenchymal stem cell/cytokine, or adopt the method having wound to get involved Laparoscopic Ovarian in-situ injection mescenchymal stem cell/cytokine through abdominal part to treat premature ovarian failure, obtain certain curative effect, but this curative effect is not very desirable, effective percentage is low, is in particular in:
First, venous re-transfusion mescenchymal stem cell easily adheres to agglomerating, and filtered input easily causes pulmonary vein thromboembolism, shows as rapid breathing, cyanosis, restless and convulsions etc., also has the case causing cardiovascular and cerebrovascular vessel stroke hemiplegin;
Secondly, although mescenchymal stem cell is reduced immunogenicity, do not express or low expression II class antigen, but repeatedly vein input mescenchymal stem cell, still has certain proportion to produce leucocyte antibody, to the amount of input cell/time to be directly proportional, antigen antibody reaction can cause the reactions such as low grade fever, tired, respiratory distress, vein input cell or the factor, the amount of going back to the nest is limited, also to a certain degree have impact on curative effect.
Interventional therapy generally will at abdominal part opening, and under peritoneoscope, interposing catheter is to blood vessel or pathological changes local, and disposable interventional therapy is limited, repeatedly gets involved and repeatedly brings Iatrogenic injury to patient, can not accept, not easily popularize.
Therefore, up to the present, also do not have definite effective method can recover the function of ovary, so the premature ovarian failure cause of disease is complicated, treatment difficulty is comparatively large, modern medicine not yet makes a breakthrough progress so far.
Summary of the invention
An object of the present invention is to provide a kind of mescenchymal stem cell and the cytokine thereof with premature ovarian failure and perimenopausal syndrome therapeutical effect, solve current premature ovarian failure and perimenopausal syndrome medicine for treatment thing absorbance is low, offer limited effectiveness and use time the wound that causes large problem.
Another object of the present invention is to provide above-mentionedly has the mescenchymal stem cell of premature ovarian failure and perimenopausal syndrome therapeutical effect and the preparation method of cytokine thereof.
To achieve these goals, the technical solution used in the present invention is:
There is mescenchymal stem cell and the cytokine thereof of premature ovarian failure and perimenopausal syndrome therapeutical effect, the suspension be made up of umbilical cord or placenta mesenchyma stem cell and cytokine thereof and the hyaluronate for carrying described umbilical cord or placenta mesenchyma stem cell and cytokine thereof; This hyaluronate non-immunogenicity, is extensively present in human connective tissue, has certain antiinflammatory action and medicament slow release effect, no cytotoxicity;
The volume ratio of described umbilical cord or placenta mesenchyma stem cell and cytokine and hyaluronate is 1:1 ~ 3;
The final concentration of described umbilical cord or placenta mesenchyma stem cell is 0.5 × 10
7~ 5 × 10
7individual/ml, the final concentration of described hyaluronate is 0.01 ~ 0.1%.
In technique scheme, the molecular weight of described hyaluronate is 30 ~ 3,000,000 dalton.
In technique scheme, the allosome umbilical cord within Secondary Culture 8 generation that described umbilical cord or placenta mesenchyma stem cell and cytokine thereof are people or placenta mesenchyma stem cell and cytokine thereof; More preferably, described mescenchymal stem cell and cytokine thereof derive from the Placenta Hominis or neonatal umbilical cord that detect qualified healthy puerpera, described Placenta Hominis or umbilical cord are cultivated the primary mescenchymal stem cell of acquisition through tissue culture method, within more primary mesenchymal stem cell serum-free being cultured to for 8 generations, collect mescenchymal stem cell and cytokine thereof, frozen stand-by; Wherein, adopting Placenta Hominis or umbilical cord as the source of mescenchymal stem cell and cytokine thereof, is that the ability of placenta secretion cytokine is stronger because the multiplication capacity of umbilical cord is stronger.
Above-mentioned mescenchymal stem cell and the cytokine thereof with premature ovarian failure and perimenopausal syndrome therapeutical effect, comprising:
Prepare umbilical cord or placenta mesenchyma stem cell and cytokine thereof;
Described umbilical cord or placenta mesenchyma stem cell and cytokine thereof and hyaluronate are mixed according to described ratio, obtained; The final concentration of described umbilical cord or placenta mesenchyma stem cell is 0.5 × 10
7~ 5 × 10
7individual/ml, the final concentration of described hyaluronate is 0.01 ~ 0.1%.
In technique scheme, the preparation method of described umbilical cord or placenta mesenchyma stem cell is:
The preparation of mescenchymal stem cell in umbilical cord source: get that aspiration, health are qualified, the cold preservation neonatal umbilical cord in 24 ~ 72 hours puerperal, after soaking disinfection, cut into the segment of 3 ~ 4cm, the blood vessel (radicular vein two radicular arteries) in every section of umbilical cord is removed with mosquito forceps, umbilical cord tissue block is separated with organizing surface, after soaking and washing, umbilical cord tissue block is decocted into 3 ~ 4mm
3bulk, soaking and washing, the centrifugal 3min of 2000rpm, abandons supernatant, is inoculated in T175 culture bottle by the umbilical cord tissue obtained by the mass concentration of 0.5g/ bottle, then in culture bottle, add DMEM culture medium and 10% hyclone, shakes up, in 37 DEG C, 5%CO
2cultivate in incubator, be cultured to the 5th day and the 8th day time change culture fluid respectively, after 2 weeks, when cell fusion degree reaches 40 ~ 80%, add pancreatin had digestive transfer culture first, use serum-free medium instead, collect 3rd ~ 8 generation mescenchymal stem cell, frozen for subsequent use.
The preparation of mescenchymal stem cell in Placenta Hominis source: get that aspiration, health are qualified, the cold preservation Placenta Hominis in 24 ~ 72 hours puerperal, after covering the amnion tissue on placental villi plate surface with mosquito forceps stripping, the enough Chorionic villi of placenta piece of tissue of clip from Placenta Hominis are cut again with meticulous, after cleaning, vascular tissue in above-mentioned Chorionic villi of placenta piece of tissue and Trophoblast tissue is rejected with tissue clamps, again cleaning after, with curved narrow meticulous cut by reject after Chorionic villi of placenta piece of tissue decoct into 3 ~ 5mm
3the piece of tissue of size, the centrifugal 5min of 2000rpm, abandons supernatant, is inoculated in T175 culture bottle by the placental villi membrane tissue obtained by the mass concentration of 1g/ bottle, then in culture bottle, add DMEM culture medium and 10% hyclone, shakes up, in 37 DEG C, 5%CO
2cultivate in incubator, after 5 days, change liquid first, after 2 weeks, go down to posterity when cell fusion degree reaches 40% ~ 80%, use serum-free medium instead, collection 3 ~ 8 generation mescenchymal stem cell, frozen for subsequent use.
In technique scheme, the preparation method of the cytokine of described umbilical cord or placenta mesenchyma stem cell is:
After umbilical cord or placenta mesenchyma stem cell were reached for 3rd ~ 8 generations, add trypsinization and again go down to posterity, when cell fusion degree arrives 60% ~ 70%, adopt 1% ~ 10%O
2hypoxia is cultivated, or changes culture fluid and remove hyclone hunger and cultivate, 72 h before harvest culture medium supernatants, limit is outer cross and filter small-molecule substance after, frozen stand-by.
In technique scheme, described mescenchymal stem cell and cytokine thereof be for local targeting injection mescenchymal stem cell and cytokine; In the inventive solutions, the using method of this mescenchymal stem cell and cytokine thereof is local targeting injection;
In technique scheme, more preferred targeting injection site is mesodesma, mescenchymal stem cell and cytokine transvaginal lateral fornix thereof enter mesodesma, and said preparation can absorb through slow release in mesodesma again and arrive ovary and endometrium is played effectiveness.
In technique scheme, targeting injection site, described local is not limited to mesodesma, according to individual need, can also be expelled to ovary, vagina or other genitals and adnexa thereof by targeting.
The work process of invention formulation is: after the mode of being injected by local targeting injects umbilical cord or placenta mesenchyma stem cell and cytokine thereof, said preparation can carry out slow release in targeting injection site, then plays effect by local blood circulation absorption arrival ovary and uterus position; Mescenchymal stem cell can secrete hundreds of cytokine, comprise vascular endothelial cell growth factor, transforming growth factor, fibroblast growth factor, epithelial cell growth factor, insulin-like growth factor, platelet derived growth factor, chemotactic factor, interleukin and immunoregulatory factor etc., they all play a part powerful to the endogenous neurogenesis of tissue; Hyaluronate is according to the characteristic of itself, and it is to cytotoxic in given concentration range, and it is as the material extensively existed in human connective tissue, and itself also has certain antiinflammatory action.Therefore, can find out, said preparation and use said preparation to there is no wound for the treatment of premature ovarian failure and perimenopausal syndrome, also not easily produce antibody, adopt hyaluronate as slow-released carrier, under the slow releasing function of this carrier, more lasting to the absorption of said preparation, better efficacy.
Preparation of the present invention has following beneficial effect: the present invention uses the cytokine of umbilical cord within 8 generations or placenta mesenchyma stem cell and secretion thereof, match with a certain proportion of hyaluronate and make suspension, again this suspension is expelled to focus region by the mode of local targeting injection, absorbed by the mode of local sustained release, the curative effect of effectively repairing utero-ovarian inner membrance can be reached, promotion functions is ovulated, and effectively treats premature ovarian failure, alleviates the various symptoms of climacteric; With existing venous re-transfusion and get involved compared with path, said preparation and using method thereof avoid the untoward reaction of venoclysis, as cell embolism and immunoreation etc., avoiding abdominal part and local wound that interventional therapy brings, can effectively preventing patient because treating the side effect and damage that bring, therefore, said preparation is safer and more effective, practical, be easy to promote, there is huge using value.
Detailed description of the invention
For making the object of the application, technical scheme and advantage clearly, below in conjunction with specific embodiment, the application is described in further detail.
embodiment 1
The mescenchymal stem cell with premature ovarian failure and perimenopausal syndrome therapeutical effect of the present embodiment and cytokine thereof are the suspensions be mixed according to the ratio of 1:1 by placenta mesenchyma stem cell and cytokine thereof and hyaluronate; In this suspension, the final concentration of placenta mesenchyma stem cell is 1 × 10
7individual/ml, the final concentration of hyaluronate is mass fraction is 0.01%, and the molecular weight of hyaluronate is 1,000,000 dalton.
Wherein, above-mentioned placenta mesenchyma stem cell is prepared by following methods:
Get aspiration, the cold preservation Placenta Hominis in healthy qualified, 24 ~ 72 hours puerperal, after peeling off the amnion tissue on Placenta Hominis with mosquito forceps, the enough Chorionic villi of placenta piece of tissue of clip from Placenta Hominis are cut again with meticulous, after cleaning, vascular tissue in above-mentioned Chorionic villi of placenta piece of tissue and Trophoblast tissue is rejected with tissue clamps, again cleaning after, with curved narrow meticulous cut by reject after Chorionic villi of placenta piece of tissue shred into 3 ~ 5mm
3piece of tissue, the centrifugal 5min of 2000rpm, abandons supernatant, is inoculated in T175 culture bottle by the placental villi membrane tissue obtained by the mass concentration of 1g/ bottle, then in above-mentioned culture bottle, add DMEM culture medium and 10% hyclone, in 37 DEG C, 5%CO
2cultivate in incubator, after 5 days, change liquid first, after 2 weeks, go down to posterity when cell fusion degree reaches 40% ~ 80%, by 0.25% pancreatin had digestive transfer culture first, use serum-free medium instead, collection 3 ~ 8 generation mescenchymal stem cell, frozen for subsequent use.
The cytokine of above-mentioned placenta mesenchyma stem cell is prepared by following methods:
After above-mentioned placenta mesenchyma stem cell being reached the 3rd generation, add trypsinization and again go down to posterity, when cell fusion degree arrives 60% ~ 70%, adopt 5%O
2hypoxia is cultivated, 72 h before harvest culture medium supernatants, and limit is outer to be filtered, after removing small-molecule substance, and degerming subpackage, frozen stand-by.
embodiment 2
The mescenchymal stem cell with premature ovarian failure and perimenopausal syndrome therapeutical effect of the present embodiment and cytokine thereof are the suspensions be mixed according to the ratio of 1:3 by umbilical cord mesenchymal stem cells and cytokine thereof and hyaluronate; In this suspension, the final concentration of umbilical cord mesenchymal stem cells is 5 × 10
7individual/ml, the final concentration of hyaluronate is mass fraction is 0.02%, and the molecular weight of hyaluronate is 1,500,000 dalton.
Wherein, above-mentioned umbilical cord mesenchymal stem cells is prepared by following methods:
Get aspiration, the cold preservation neonatal umbilical cord in healthy qualified, 24 ~ 72 hours puerperal, after soaking disinfection, be trimmed to the segment that length is 3 ~ 4cm, the blood vessel (radicular vein two radicular arteries) in every section of umbilical cord is removed with mosquito forceps, umbilical cord tissue block is separated with organizing surface, after soaking and washing, umbilical cord tissue block is decocted into 3 ~ 4mm
3bulk, the centrifugal 3min of 2000rpm/min, abandons supernatant, is inoculated in T175 culture bottle by the umbilical cord tissue obtained by the mass concentration of 0.5g/ bottle, then adds DMEM culture medium and 10% hyclone in above-mentioned culture bottle, shakes up, in 37 DEG C, 5%CO
2cultivate in incubator, be cultured to the 5th day and the 8th day time change culture fluid respectively, after 2 weeks, add trypsinization and go down to posterity, when cell fusion degree reaches 40 ~ 80%, use serum-free medium instead, collect the 3rd ~ 8th generation mescenchymal stem cell, frozen for subsequent use.
The cytokine of above-mentioned umbilical cord mesenchymal stem cells is prepared by following methods:
After above-mentioned umbilical cord mesenchymal stem cells being reached the 5th generation, add trypsinization and again go down to posterity, when cell fusion degree arrives 60% ~ 70%, adopt 3%O
2hypoxia is cultivated, and after 72 hours, collects culture medium supernatant, after limit outer filtration removing small-molecule substance, and degerming subpackage, frozen stand-by.
Detect the content of the cytokine of the above-mentioned umbilical cord mesenchymal stem cells prepared, testing result is as follows:
The testing result of table 1 many kinds of somatomedin
Note: VEGF: vascular endothelial cell growth factor; TGF: transforming growth factor; EGF: epithelial cell growth factor;
PDGF: platelet derived growth factor.
By above-mentioned detection, can find out, under the condition that umbilical cord mesenchymal stem cells is cultivated at hypoxia, the concentration of TGF and EGF cytokine secretion significantly increases.
embodiment 3
With the mescenchymal stem cell cytokine treatment perimenopausal syndrome that embodiment 1 is obtained
Patient: Lee * *, female, 49 years old, symptom: vaginal dryness, lax, libido obviously decline, poor, menolipsis 1 year of sleeping, and suffering from " colpitis mycotica ", recurrent exerbation.
patient features:1, female middle-aged, G3P2(are along 2); 2, menstruation 9 months is stopped; 3, want to improve the symptoms such as vaginal dryness, lax, libido is poor.
gynaecological examination situation:. pudendum is dry and astringent, lax, both sides nympha pigmentation; Slack vagina about 3 finger, vagina parcel power and suction poor; Retroposition of uterus, normal size, activity, partially firmly tenderness, bilateral adnexa are not touched obviously different.
auxiliary examination:leucorrhea is chemically examined: cleannes II; HBsAg:(-), HBeAg:(-), HIV:(-), HCV:(-), TP:(-), B ultrasonic, Electrocardioscopy are without exception.
diagnosis: 1, perimenopausal syndrome; 2, female reproductive function decline.
therapeutic scheme:adopt the obtained mescenchymal stem cell cytokine of embodiment 1 to treat a course for the treatment of, targeting injects 3 times, 6 months observation of curative effect phases.
Concrete grammar: cytokine targeting obtained for embodiment 1 is injected to mesodesma position, each 6ml ~ 10ml, 1 month, interval, totally 3 times; Said preparation absorbs through slow release in local and arrives the performance such as ovary and uterus adnexa effect.
observation of curative effect:effect after first time treatment: vaginal dryness, lax, libido declines, sleep difference and the mental status are obviously improved, menopause 1 year is in treatment latter one month compound tides, and through amount and color ditto, normally, colpitis mycotica does not recur again.
Effect after second time treatment: vaginal dryness, lax, libido decline symptom are improved obviously, and sleep difference and the mental status are improved completely, and B ultrasonic finds: endometrium 5CM(is double-deck), evenly, bilateral attachment area does not find agglomerate echo to essence echo.
Effect after third time treatment: vaginal dryness, lax, libido decline symptom are obviously improved, and sleep difference and the mental status are improved completely; Treat after 6 months, vaginal dryness, lax continuative improvement, readme libido decline sleep waits Symptoms last to improve.
embodiment 4
The general pharmacology test of the mescenchymal stem cell that embodiment 2 is obtained and cytokine thereof
1, acute toxicity test
The mescenchymal stem cell that embodiment 2 is obtained and cytokine thereof carry out the acute toxicity test of single intravenous administration to mice
Object: dosage ~ reaction relation and the fouling characteristics of illustrating subject cell acute toxicity; Obtain subject cell to the half lethal dose of test mice and maximum tolerated dose, to assess the safety of subject cell.
Conclusion: the mescenchymal stem cell that embodiment 2 is obtained and cytokine thereof are when carrying out acute toxicity test, when injected dose is greater than maximum tolerated dose, there is rapid breathing in animal, palpitate quickly, pant or the phenomenon such as cyanosis, except above-mentioned symptom, without other side effect; By test, determine that the LD50 of mescenchymal stem cell and cytokine thereof is 1.632 × 10
8individual/kg, MTD is 1.25 × 10
8individual/kg.
2, long term toxicity test
Mescenchymal stem cell and the cytokine test thereof that continuous several times repeats intravenous injection various dose is carried out to rat, observe the toxic reaction of animal subject, and subject cell long term toxicity response situation, finally determining nontoxic crude protein, providing reference for drafting people's safe dose.
, in whole process of the test, there is not the phenomenon that rat midway is dead in conclusion: in long term toxicity test.
3, tests for tumorigenicity
1. material
Adopt the mice in 1 ~ 2 week age;
Medicine: a, mescenchymal stem cell and cytokine thereof, wherein containing stem cell 3 × 10
7individual/ml, hyaluronate sodium 0.04%; B, normal saline; C, Hela cell suspension, containing Hela cell 3 × 10
7individual/ml.
2. method
Get 60 mices and be divided into 2 groups at random, often organize 20; Once, each metering is 1ul/kg to three group tail intravenously administrables every day; The injectable drug of experimental group is mescenchymal stem cell and cytokine thereof, blank group injecting normal saline, matched group injection Hela groups of cells; Continuous injection 90 days.
3. experimental result
There is not tumor in injection in 1 ~ 90 day in experimental group and blank group, matched group obviously occurs tumor in 4 days afterwards in injection, in injection after 90 days, occurs larger tumor.This description of test mescenchymal stem cell of the present invention suspension is without potential oncogenicity.
Can be found out the pharmacology test that mescenchymal stem cell and cytokine thereof carry out by above-mentioned, in use, safety is good, has no side effect, without oncogenicity for said preparation.
embodiment 5
The effectiveness experiment of the animal of the mescenchymal stem cell that embodiment 1 is obtained and cytokine thereof
One, the foundation of premature ovarian failure mouse model:
1.1 chemical induction premature ovarian failure models:
Get adult female mice 60, be divided into matched group and experimental group at random, often organize each 30.Experimental group: intraperitoneal injection of cyclophosphamide is 50 mg/kg first; Matched group: lumbar injection same volume normal saline operation repetitive; The change of the ovarian histology morphology and function of two groups of mices is observed in continuous injection 15 days (10mg latter 14 day every day) afterwards.
Two, the evaluation index of premature ovarian failure mouse model:
2.1 oestrous cycles observed:
Vaginal exfoliated and the Ovulation prediction of getting matched group and premature ovarian failure model group mice morning every day make smear, put basis of microscopic observation, determine the oestrous cycle of matched group and model group mice respectively by Ovulation prediction crystallization process, vaginal exfoliated form inspection technique or vaginal prolapse keratinocyte counting method.
2.2 serum estrogen levels detect:
Adopt the level change of the serum estradiol (E2) of enzyme-linked immunosorbent assay (ELISA) method mensuration matched group and premature ovarian failure model mice, follicle stimulating hormone (FSH), luteotropic hormone (LH).
2.3 folliculus ovarii quantity detect:
Mouse ovarian formaldehyde is fixed, paraffin embedding, and after conventional section, HE dyeing, change, and comparative control group and the non-latching Follicle number of premature ovarian failure model group mouse ovarian evaluates the function of ovary in the tectology of light Microscopic observation ovary.
Three, mescenchymal stem cell sends mode:
Ovary gap in-situ injection:
Four, result
Normal group: visible normal oestrous cycle;
Model control group: Mouse Oestrous Cycle is disorderly, serum FSH concentration raises, and E2 concentration reduces, and Follicle number reduces, stroma of ovary fibrosis;
Transplant matched group: about 40% ~ 50% mice recovers the oestrous cycle in 15 days ~ 30 days after mesenchymal stem cell transplantation, E2 level is higher than model group, and FSH concentration reduces; 40 days ~ 60 days, Follicles number increased than model group.
Five, conclusion
The mouse ovarian senilism of ovary gap in-situ injection to chemical induction has obvious curative effect.
Claims (9)
1. there is mescenchymal stem cell and the cytokine thereof of premature ovarian failure and perimenopausal syndrome therapeutical effect, by umbilical cord or placenta mesenchyma stem cell and cytokine thereof with for carrying described umbilical cord or placenta mesenchyma stem cell and cytokine thereof and the hyaluronate with slow release and antiinflammatory action forms;
The volume ratio of described umbilical cord or placenta mesenchyma stem cell and cytokine and hyaluronate is 1:1 ~ 3;
The final concentration of described umbilical cord or placenta mesenchyma stem cell is 0.5 × 10
7~ 5 × 10
7individual/ml, the final concentration of described hyaluronate is 0.01 ~ 0.1%.
2. mescenchymal stem cell according to claim 1 and cytokine thereof, is characterized in that, the molecular weight of described hyaluronate is 30 ~ 3,000,000 dalton.
3. mescenchymal stem cell according to claim 1 and cytokine thereof, it is characterized in that, the allosome umbilical cord within Secondary Culture 8 generation that described umbilical cord or placenta mesenchyma stem cell and cytokine thereof are people or placenta mesenchyma stem cell and cytokine thereof.
4. the preparation method of mescenchymal stem cell according to claim 1 and cytokine thereof, is characterized in that, comprising:
Prepare umbilical cord or placenta mesenchyma stem cell and cytokine thereof;
Described umbilical cord or placenta mesenchyma stem cell and cytokine thereof and hyaluronate are mixed according to described ratio, obtained; The final concentration of described umbilical cord or placenta mesenchyma stem cell is 0.5 × 10
7~ 5 × 10
7individual/ml, the final concentration of described hyaluronate is 0.01 ~ 0.1%.
5. the preparation method of mescenchymal stem cell according to claim 4 and cytokine thereof, is characterized in that, the preparation method of described umbilical cord mesenchymal stem cells is:
Get the cold preservation neonatal umbilical cord in healthy qualified, 24 ~ 72 hours puerperal, after soaking disinfection, be trimmed to the segment that length is 3 ~ 4cm, remove the blood vessel in described every section of umbilical cord, umbilical cord tissue block is separated with organizing surface; Then described umbilical cord tissue block is trimmed to 3 ~ 4mm
3bulk, the centrifugal 3min of 2000rpm/min, abandons supernatant, is inoculated in T175 culture bottle by the umbilical cord tissue obtained by the mass concentration of 0.5g/ bottle, adds DMEM culture medium and 10% hyclone, shake up in described culture bottle, in 37 DEG C, 5%CO
2cultivate in incubator, be cultured to the 5th day and the 8th day time change culture fluid respectively, after 2 weeks, when cell fusion degree reaches 40 ~ 80%, by 0.25% pancreatin had digestive transfer culture first, use serum-free medium instead, collect 3rd ~ 8 generation mescenchymal stem cell, frozen for subsequent use.
6. the preparation method of mescenchymal stem cell according to claim 4 and cytokine thereof, is characterized in that, the preparation method of described placenta mesenchyma stem cell is:
Get the cold preservation Placenta Hominis in healthy qualified, 24 ~ 72 hours puerperal, after peeling off the amnion tissue on described Placenta Hominis, clip is positioned at the Chorionic villi of placenta piece of tissue on Placenta Hominis, rejects the vascular tissue in described Chorionic villi of placenta piece of tissue and Trophoblast tissue, cleaning, is then cut into 3 ~ 5mm
3piece of tissue, the centrifugal 5min of 2000rpm, abandons supernatant, is inoculated in T175 culture bottle by the placental villi membrane tissue obtained by the mass concentration of 1g/ bottle, adds DMEM culture medium and 10% hyclone in described culture bottle, in 37 DEG C, 5%CO
2cultivate in incubator, after 5 days, change culture fluid first, after 2 weeks, go down to posterity when cell fusion degree reaches 40% ~ 80%, use serum-free medium instead, collection 3rd ~ 8 generation mescenchymal stem cell, frozen for subsequent use.
7. the preparation method of mescenchymal stem cell according to claim 4 and cytokine thereof, is characterized in that, the preparation method of the cytokine of described umbilical cord or placenta mesenchyma stem cell is:
After obtained umbilical cord or placenta mesenchyma stem cell were reached for 3rd ~ 8 generations, add trypsinization and again go down to posterity, when cell fusion degree reaches 60 ~ 70%, adopt 1% ~ 10%O
2hypoxia cultivation, or change culture fluid, remove hyclone hunger and cultivate, 72 h before harvest culture medium supernatants, cross after filtering small-molecule substance outside limit, frozen stand-by.
8. mesenchymal cell and the cytokine thereof with premature ovarian failure and perimenopausal syndrome therapeutical effect according to claim 1, it is characterized in that, described mescenchymal stem cell and cytokine thereof be for local targeting injection mescenchymal stem cell and cytokine.
9. mesenchymal cell and the cytokine thereof with premature ovarian failure and perimenopausal syndrome therapeutical effect according to claim 8, is characterized in that, the position of described local targeting injection is mesodesma, ovary vagina genitals and adnexa position.
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