CN109893543A - Treat the Endometrial stem cell of endometrial impairment and premature ovarian failure and combination formulations, preparation method and the medication of excretion body - Google Patents
Treat the Endometrial stem cell of endometrial impairment and premature ovarian failure and combination formulations, preparation method and the medication of excretion body Download PDFInfo
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Abstract
The invention discloses a kind of Endometrial stem cell for treating endometrial impairment and premature ovarian failure and the combination formulations and preparation method and medication of excretion body.Endometrial stem cell excretion body concentrate is obtained including Endometrial stem cell and by hungry culture.Method by cultivating Endometrial stem cell starvation promotes the secretion of its bioactive substance.The bioactive substance that Endometrial stem cell secretion is recycled in the form of excretion body, it is more easy to substantially increase recovery efficiency, way of recycling.By the method for the biomembrane gradient filtration of different pore size, the excretion body of Endometrial stem cell secretion is collected and be concentrated, the process of excretion body recycling is simplified, improves the purity of excretion body.
Description
Technical field
The present invention relates to Endometrial stem cell and its combination formulations of excretion body, especially a kind of to treat endometrium damage
Combination formulations, preparation method and the medication of the Endometrial stem cell and excretion body of wound and premature ovarian failure.
Background technique
Endometrial impairment is mainly the damage of inner membrance basal layer, and main cause is related with gestational period dilatation and curettage, and normal
Endometrium is compared, and Uterus during Pregnancy inner membrance basal layer is loose, it is easier to be damaged.Based on China's national situation and induced abortion
The status that rate gradually increases, endometrium is impaired, and a situation arises can not be ignored.Basal layer is impaired, may cause son
Endometrial stem cells are impaired or lack；At the same time, stem cell can be destroyed by damaging the infection of inner membrance part and aseptic inflammation
Alcove microenvironment causes epithelium and interstitial cell Regeneration and Repair that obstacle occurs, and vascularization is obstructed, and dense fibrous tissue is formed,
It eventually leads to uterine cavity and only covers a small amount of inner membrance, or even without inner membrance, body of gland atrophy, uterine cavity loses normal morphology and function.
Endometrial stem cell belongs to adult stem cell, has a variety of function such as Multidirectional Differentiation, tissue repair, immunological regulation
Can, there is great advantage in clinical application.Studies have shown that Endometrial stem cell can treat and alleviate to a certain extent
Endometrial impairment.But direct venous re-transfusion umbilical cord Endometrial stem cell is since cell is difficult to reach uterus position, so
Therapeutic effect is limited.
The study found that stem cell exercise biological function a most important approach be by secrete a large amount of bioactivity because
Son, and then adjust the function of pathological tissues, promote revascularization, accelerate regeneration and restoration.And a large amount of bioactie agent
Secretory pathway mainly pass through excretion body, a kind of biomembrane includes, and diameter is in 40-200nm not equal excretion vesicles.
Summary of the invention
The technical problem to be solved by the invention is to provide a kind of intrauterines for treating endometrial impairment and premature ovarian failure
The combination formulations of film stem cell and excretion body, preparation method and medication cultivate Endometrial stem cell by starvation and go forward side by side
The method of row enrichment concentration, obtains the biologically active Endometrial stem cell excretion body of high concentration, by excretion body and son
Endometrial stem cells, which are combined, prepares preparation by being injected directly into inside uterus for treating endometrial impairment.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention is that: one kind is for treating endometrial impairment
With the preparation method of the combination formulations of the Endometrial stem cell and its excretion body of premature ovarian failure, comprising the following steps:
(1) culture of people's Endometrial stem cell: selection growth conditions are good, and microorganism detection is negative, P2-
The source of people Endometrial stem cell of P8, according to 3-5*104/cm2Initial density be inoculated in Tissue Culture Flask or Tissue Culture Dish
In, culture medium is the cultivating system of the fetal calf serum containing volume by volume concentration 5-15% in basic culture medium；
(2) after inoculated and cultured 2-4 days, cell confluency reaches 80-95%, and cell growth state is good, inhales and abandons all trainings
Supernatant is supported, and with medical Multiple electrolytes injection cleaning cell 2-4 times, appropriate compound electrolyte solution is then added, makes molten
Liquid height reaches 2-3mm, is put into carbon dioxide incubator hungry culture；
(3) after hungry culture 18-30h, whole culture supernatants are recycled, with the aperture 0.22um membrane filtration, filtered training
It supports supernatant and is centrifuged 10-20min under the conditions of the centrifugal force of 3000-5000g, again recycling centrifugation supernatant；
(4) 30-60min is centrifuged to the supernatant of recycling under the conditions of the centrifugal force of 200000-500000g, after centrifugation gently
Centrifuge tube is taken out, the liquid for abandoning top 70%-90% volume is inhaled with pipette, by the liquid of lower part 10%-30% volume and is sunk
It forms sediment after mixing as the excretion body solvent for preparing endometrium treatment preparation, is saved under the conditions of 2-8 DEG C；
(5) culture of people's Endometrial stem cell: selection growth conditions are good, and microorganism detection is negative, P2-
The source of people Endometrial stem cell of P8, according to 3-5*104/cm2Initial density be inoculated in Tissue Culture Flask or Tissue Culture Dish
In, culture medium is the cultivating system of the fetal calf serum containing volume by volume concentration 5-15% in basic culture medium；
(6) after inoculated and cultured 2-4 days, cell confluency reaches 80-95%, and cell growth state is good, and recycling is all thin
Born of the same parents, and with compound electrolyte solution cleaning cell 3-4 times, by the cell finally obtained according to 0.5-2*106The concentration of/ml is resuspended
In the excretion body solvent that the step of rewarming is to 18-24 DEG C (4) obtains, it is prepared into combination formulations.
The Endometrial stem cell and excretion body for the treatment of endometrial impairment and premature ovarian failure that above-mentioned preparation method obtains
Combination formulations.
The combination formulations of the Endometrial stem cell and excretion body of above-mentioned treatment endometrial impairment and premature ovarian failure, pass through
It is directly injected into inside uterus, the medication of venoclysis or Local Multipoint injection.
The beneficial effects of the present invention are: Endometrial stem cell can secrete a variety of a large amount of excretion bodies during the cultivation process,
The excretion body of human umbilical cord mesenchymal stem cells secretion can promote the life of skin histology differentiation, angiogenesis and granulation tissue
It is long, local organization microenvironment is adjusted, promotes angiogenesis, improves local blood supply, also, Endometrial stem cell itself has
The effect of endometrial impairment and premature ovarian failure, excretion body solvent can provide optimal existence ring for Endometrial stem cell
Border can maintain the activity and function of cell to the maximum extent.The present invention using hungry Endometrial stem cell method, compared with
People's Endometrial stem cell excretion body extract can be obtained in short time, is concentrated by ultracentrifugal method highly concentrated to obtain
People's Endometrial stem cell excretion body of degree.Endometrial stem cell is resuspended in the Endometrial stem cell excretion body of concentration
In, substantially increase therapeutic effect.
Specific embodiment
Invention is further described in detail With reference to embodiment:
The joint of of the invention a kind of Endometrial stem cell for treating endometrial impairment and premature ovarian failure and excretion body
The preparation method of preparation, comprising the following steps:
(1) culture of people's Endometrial stem cell: selection growth conditions are good, and microorganism detection is negative, P2-
The source of people Endometrial stem cell of P8, according to 3-5*104/cm2Initial density be inoculated in Tissue Culture Flask or Tissue Culture Dish
In, culture medium is the cultivating system of the fetal calf serum containing volume by volume concentration 5-15% in basic culture medium；
(2) after inoculated and cultured 2-4 days, cell confluency reaches 80-95%, and cell growth state is good, inhales and abandons all trainings
Supernatant is supported, and with medical Multiple electrolytes injection cleaning cell 2-4 times, appropriate compound electrolyte solution is then added, makes molten
Liquid height reaches 2-3mm, is put into carbon dioxide incubator hungry culture；
(3) after hungry culture 18-30h, whole culture supernatants are recycled, with the aperture 0.22um membrane filtration, filtered training
It supports supernatant and is centrifuged 10-20min under the conditions of the centrifugal force of 3000-5000g, again recycling centrifugation supernatant；
(4) 30-60min is centrifuged to the supernatant of recycling under the conditions of the centrifugal force of 200000-500000g, after centrifugation gently
Centrifuge tube is taken out, the liquid for abandoning top 70%-90% volume is inhaled with pipette, by the liquid of lower part 10%-30% volume and is sunk
It forms sediment after mixing as the excretion body solvent for preparing endometrium treatment preparation, is saved under the conditions of 2-8 DEG C；
(5) culture of people's Endometrial stem cell: selection growth conditions are good, and microorganism detection is negative, P2-
The source of people Endometrial stem cell of P8, according to 3-5*104/cm2Initial density be inoculated in Tissue Culture Flask or Tissue Culture Dish
In, culture medium is the cultivating system of the fetal calf serum containing volume by volume concentration 5-15% in basic culture medium；
(6) after inoculated and cultured 2-4 days, cell confluency reaches 80-95%, and cell growth state is good, and recycling is all thin
Born of the same parents, and with compound electrolyte solution cleaning cell 3-4 times, by the cell finally obtained according to 0.5-2*106The concentration of/ml is resuspended
In the excretion body solvent that the step of rewarming is to 18-24 DEG C (4) obtains, it is prepared into combination formulations.
The Endometrial stem cell and excretion body for the treatment of endometrial impairment and premature ovarian failure that above-mentioned preparation method obtains
Combination formulations.
The combination formulations of the Endometrial stem cell and excretion body of above-mentioned treatment endometrial impairment and premature ovarian failure, pass through
It is directly injected into inside uterus, the medication of venoclysis or Local Multipoint injection.
In invention formulation include Endometrial stem cell and Endometrial stem cell excretion is obtained by hungry culture
Body concentrate.Method by cultivating Endometrial stem cell starvation promotes the secretion of its bioactive substance.With excretion body
Form recycling Endometrial stem cell secretion bioactive substance, it is more simple to substantially increase recovery efficiency, way of recycling
Just.By the method for the biomembrane gradient filtration of different pore size, the excretion body of Endometrial stem cell secretion, letter are collected and are concentrated
The process for having changed the recycling of excretion body, improves the purity of excretion body.
Endometrial stem cell can secrete a variety of a large amount of excretion bodies during the cultivation process in the present invention, and people's endometrium is dry
The excretion body of cell secretion can promote the growth of skin histology differentiation, angiogenesis and granulation tissue, and part group is adjusted
Microenvironment is knitted, angiogenesis is promoted, improves local blood supply, so that reaching improves tissue blood supply, promotes injury tissue regeneration, treatment
The effect of endometrial impairment.Also, Endometrial stem cell itself has the effect for the treatment of endometrial impairment, and excretion body is molten
Agent can provide optimal living environment for Endometrial stem cell, can maintain the activity and function of cell to the maximum extent.
The present invention can obtain the extraction of people's endometrium excretion body using the method for hungry Endometrial stem cell within a short period of time
Object is concentrated to obtain people's Endometrial stem cell excretion body of high concentration by ultracentrifugal method.
Case study on implementation 1:
Patient Zhang, 34 years old, endometrium thickness was lower than 8mm after induced abortion dilatation and curettage, and clinical diagnosis is that endometrium is thin.
The preparation of combination formulations:
(1) culture of people's Endometrial stem cell: selection growth conditions are good, and microorganism detection is negative, P4
Source of people umbilical cord mesenchymal stem cells, according to 3*104/cm2Initial density be inoculated in 10 bottles of T-175 Tissue Culture Flasks, cultivate
Base is the cultivating system of the fetal calf serum containing volume by volume concentration 10% in basic culture medium；
(2) after inoculated and cultured 4 days, cell confluency reaches 90%, and cell growth state is good, inhales and abandons in all cultures
Clearly, and with medical Multiple electrolytes injection cleaning cell 3 times, 50ml compound electrolyte solution is then added, is put into titanium dioxide
Hungry culture in carbon incubator；
(3) after hungry culture for 24 hours, whole culture supernatants are recycled, with the aperture 0.22um membrane filtration, in filtered culture
It is centrifuged 15min under the conditions of the centrifugal force of 5000g clearly, again recycling centrifugation supernatant；
(4) 60min is centrifuged to the supernatant of recycling under the conditions of the centrifugal force of 400000g, centrifuge tube is gently taken out after centrifugation,
The liquid for abandoning top 80% is inhaled with pipette, and the liquid of lower part 20% and precipitating are completely recovered as preparation after mixing
The excretion body solvent of Endometrium injury in treating preparation, saves under the conditions of 4 DEG C；
(5) culture of people's Endometrial stem cell: selection growth conditions are good, and microorganism detection is negative, P6
Source of people Endometrial stem cell, according to 3*104/cm2Initial density be inoculated in Tissue Culture Flask or Tissue Culture Dish, train
Support the cultivating system that base is the fetal calf serum containing volume by volume concentration 10% in basic culture medium；
(6) after inoculated and cultured 4 days, cell confluency reaches 90%, and cell growth state is good, recycles whole cells, and
With compound electrolyte solution cleaning cell 3 times, by the cell finally obtained according to 1*106The concentration of/ml is resuspended in rewarming
In the excretion body solvent that the step of to 22 DEG C (4) obtains, it is prepared into combination formulations.
Patient receives seance, injects 0.5ml combination formulations inside disposable uterus.Treatment end the latter monthly test
It surveys (after menstruation 10 days), endometrium thickness reaches 8.4mm, and endometrial impairment symptom is alleviated.
Case study on implementation 2:
Patient Xu certain, 39 years old, long term service pressure caused endocrine numerous imbalances, and endometrium thickness is lower than 8mm, clinical
It is thin to be diagnosed as endometrium.
The preparation of combination formulations:
(1) culture of people's Endometrial stem cell: selection growth conditions are good, and microorganism detection is negative, P6
Source of people Endometrial stem cell, according to 3*104/cm2Initial density be inoculated in 20 bottles of T-175 Tissue Culture Flasks, culture medium
For the cultivating system of the fetal calf serum containing volume by volume concentration 5% in basic culture medium；
(2) after inoculated and cultured 4 days, cell confluency reaches 85%, and cell growth state is good, inhales and abandons in all cultures
Clearly, and with medical Multiple electrolytes injection cleaning cell 3 times, 40ml compound electrolyte solution is then added, is put into titanium dioxide
Hungry culture in carbon incubator；
(3) after hungry culture for 24 hours, whole culture supernatants are recycled, with the aperture 0.22um membrane filtration, in filtered culture
It is centrifuged 15min under the conditions of the centrifugal force of 4000g clearly, again recycling centrifugation supernatant；
(4) 60min is centrifuged to the supernatant of recycling under the conditions of the centrifugal force of 300000g, centrifuge tube is gently taken out after centrifugation,
The liquid for abandoning top 90% is inhaled with pipette, and the liquid of lower part 10% and precipitating are used as after mixing and prepare endometrium damage
The excretion body solvent of wound treatment preparation, saves under the conditions of 4 DEG C；
(5) culture of people's Endometrial stem cell: selection growth conditions are good, and microorganism detection is negative, P5
Source of people Endometrial stem cell, according to 4*104/cm2Initial density be inoculated in Tissue Culture Flask or Tissue Culture Dish, train
Support the cultivating system that base is the fetal calf serum containing volume by volume concentration 10% in basic culture medium；
(6) after inoculated and cultured 3 days, cell confluency reaches 85%, and cell growth state is good, recycles whole cells, and
With compound electrolyte solution cleaning cell 3 times, by the cell finally obtained according to 1.5*106The concentration of/ml, which is resuspended in, has answered
In the excretion body solvent that the step of temperature is to 24 DEG C (4) obtain, it is prepared into combination formulations.
Patient receives two treatments, is spaced one month, and each endometrium injects 0.5ml combination formulations.Treatment is all
Terminate the latter moon detection (after menstruation 10 days), endometrium thickness reaches 10mm, and endometrial impairment symptom is alleviated.
Case study on implementation 3:
Patient Zhang, 40 years old, endometrium thickness was lower than 8mm, and clinical diagnosis is that endometrium is thin.
The preparation of combination formulations:
(1) culture of people's Endometrial stem cell: selection growth conditions are good, and microorganism detection is negative, P3
Source of people Endometrial stem cell, according to 3.5*104/cm2Initial density be inoculated in 10 bottles of T-175 Tissue Culture Flasks, cultivate
Base is the cultivating system of the fetal calf serum containing volume by volume concentration 10% in basic culture medium；
(2) after inoculated and cultured 3 days, cell confluency reaches 90%, and cell growth state is good, inhales and abandons in all cultures
Clearly, and with medical Multiple electrolytes injection cleaning cell 4 times, 40ml compound electrolyte solution is then added, is put into titanium dioxide
Hungry culture in carbon incubator；
(3) after hungry culture 20h, whole culture supernatants are recycled, with the aperture 0.22um membrane filtration, in filtered culture
It is centrifuged 13min under the conditions of the centrifugal force of 4000g clearly, again recycling centrifugation supernatant；
(4) 60min is centrifuged to the supernatant of recycling under the conditions of the centrifugal force of 200000g, centrifuge tube is gently taken out after centrifugation,
The liquid for abandoning top 70% is inhaled with pipette, and the liquid of lower part 30% and precipitating are completely recovered as preparation after mixing
The excretion body solvent of Endometrium injury in treating preparation, saves under the conditions of 4 DEG C；
(5) culture of people's Endometrial stem cell: selection growth conditions are good, and microorganism detection is negative, P4
Source of people Endometrial stem cell, according to 3*104/cm2Initial density be inoculated in Tissue Culture Flask or Tissue Culture Dish, train
Support the cultivating system that base is the fetal calf serum containing volume by volume concentration 10% in basic culture medium；
(6) after inoculated and cultured 4 days, cell confluency reaches 90%, and cell growth state is good, recycles whole cells, and
With compound electrolyte solution cleaning cell 3 times, by the cell finally obtained according to 1*106The concentration of/ml is resuspended in rewarming
In the excretion body solvent that the step of to 20 DEG C (4) obtains, it is prepared into combination formulations.
Patient receives secondary treatment three times, is spaced one month, and each endometrium injects 0.5ml combination formulations.Treatment is complete
Portion terminates the latter moon detection (after menstruation 12 days), and endometrium thickness reaches 9.4mm, and endometrial impairment symptom is delayed
Solution.
Case study on implementation 4:
Patient Zhang, 35 years old, amenorrhoea 6 months or more, (interval 2 months) blood FSH detection was all larger than 40mIU/ml twice, faced
Bed is diagnosed as premature ovarian failure.
The preparation of combination formulations: with embodiment 1
Patient receives seance, and disposable vein is transfused 40ml combination formulations.After one week treatment starts statistics treatment
Effect, continuously (interval 1 month) detection blood FSH is detected as 33mIU/ml, 28mIU/ml, 25mIU/ml three times.Patient continuous three
Normal ovulation in a month.
Case study on implementation 5:
Patient Xu certain, 37 years old, amenorrhoea 6 months or more, (interval 2 months) blood FSH detection twice was all larger than 40mIU/ml, faced
Bed is diagnosed as premature ovarian failure.
The preparation of combination formulations: with embodiment 2
Patient receives two treatments, is spaced one month, each venoclysis 20ml combination formulations.Two treatments terminate one
Start to count therapeutic effect after week, continuously three times (interval 1 month) detection blood FSH be detected as 23mIU/ml, 24mIU/ml,
27mIU/ml.Normal ovulation in patient continuous three months.
Case study on implementation 6:
Patient Ma, 35 years old, amenorrhoea 6 months or more, (interval 2 months) blood FSH detection was all larger than 40mIU/ml twice, faced
Bed is diagnosed as premature ovarian failure.
Patient receives an ovary Local Multipoint injection treatment, point 3 points of disposable ovary locally injecting 2ml joint systems
Agent.After one week treatment starts to count therapeutic effect, continuously three times (interval 1 month) detection blood FSH be detected as 22mIU/ml,
25mIU/ml,20mIU/ml.Normal ovulation in patient continuous three months.
In conclusion the contents of the present invention are not limited in the above embodiments, the knowledgeable people in same area can
Can propose other embodiments easily within technological guidance's thought of the invention, but this embodiment is included in this hair
Within the scope of bright.
Claims (3)
1. a kind of preparation of the combination formulations of Endometrial stem cell for treating endometrial impairment and premature ovarian failure and excretion body
Method, which comprises the following steps:
(1) culture of people's Endometrial stem cell: selection growth conditions are good, and microorganism detection is feminine gender, P2-P8's
Source of people Endometrial stem cell, according to 3-5*104/cm2Initial density be inoculated in Tissue Culture Flask or Tissue Culture Dish, train
Support the cultivating system that base is the fetal calf serum containing volume by volume concentration 5-15% in basic culture medium；
(2) after inoculated and cultured 2-4 days, cell confluency reaches 80-95%, and cell growth state is good, inhales and abandons in all cultures
Clearly, and with medical Multiple electrolytes injection cleaning cell 2-4 times, appropriate compound electrolyte solution is then added, makes solution height
Degree reaches 2-3mm, is put into carbon dioxide incubator hungry culture；
(3) after hungry culture 18-30h, whole culture supernatants are recycled, with the aperture 0.22um membrane filtration, in filtered culture
It is centrifuged 10-20min under the conditions of the centrifugal force of 3000-5000g clearly, again recycling centrifugation supernatant；
(4) 30-60min is centrifuged to the supernatant of recycling under the conditions of the centrifugal force of 200000-500000g, is gently taken out after centrifugation
Centrifuge tube inhales the liquid for abandoning top 70%-90% volume with pipette, and the liquid of lower part 10%-30% volume and precipitating is mixed
As the excretion body solvent for preparing endometrium treatment preparation after closing uniformly, saved under the conditions of 2-8 DEG C；
(5) culture of people's Endometrial stem cell: selection growth conditions are good, and microorganism detection is feminine gender, P2-P8's
Source of people Endometrial stem cell, according to 3-5*104/cm2Initial density be inoculated in Tissue Culture Flask or Tissue Culture Dish, train
Support the cultivating system that base is the fetal calf serum containing volume by volume concentration 5-15% in basic culture medium；
(6) after inoculated and cultured 2-4 days, cell confluency reaches 80-95%, and cell growth state is good, recycles whole cells, and
With compound electrolyte solution cleaning cell 3-4 times, by the cell finally obtained according to 0.5-2*106The concentration of/ml is resuspended in
In the excretion body solvent that the step of through rewarming to 18-24 DEG C (4) obtains, it is prepared into combination formulations.
2. the Endometrial stem cell for the treatment of endometrial impairment and premature ovarian failure that preparation method as described in claim 1 obtains
And the combination formulations of excretion body.
3. the joint of the Endometrial stem cell and excretion body for the treatment of endometrial impairment and premature ovarian failure as claimed in claim 2
Preparation, by being directly injected into inside uterus, venoclysis or Local Multipoint injection medication.
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| CN113244272A (en) * | 2021-06-18 | 2021-08-13 | 陕西中鸿科瑞再生医学研究院有限公司 | Composition for improving premature ovarian failure and preparation method and application thereof |
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| CN110585118A (en) * | 2019-09-30 | 2019-12-20 | 广东华夏健康生命科学有限公司 | Temperature-sensitive gel preparation of endometrial stem cells and preparation method and application thereof |
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| CN113244272B (en) * | 2021-06-18 | 2024-05-17 | 陕西中鸿科瑞再生医学研究院有限公司 | Composition for improving premature ovarian failure and preparation method and application thereof |
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