CN109394787A - Mescenchymal stem cell, its gel and composition are repairing the application in uterine cavity scar and adhesion - Google Patents

Mescenchymal stem cell, its gel and composition are repairing the application in uterine cavity scar and adhesion Download PDF

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CN109394787A
CN109394787A CN201811309118.7A CN201811309118A CN109394787A CN 109394787 A CN109394787 A CN 109394787A CN 201811309118 A CN201811309118 A CN 201811309118A CN 109394787 A CN109394787 A CN 109394787A
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stem cell
mescenchymal stem
culture medium
concentration
cell
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刘世红
董明珠
潘新
卢承前
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CENTURY BIOSTRENGTH BEIJING Pty Ltd.
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Beijing Century Biotechnology Co Ltd
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Abstract

The present invention provides mescenchymal stem cell, its gel and compositions to repair the application in uterine cavity scar and adhesion, test confirms that mescenchymal stem cell, mescenchymal stem cell gel or the composition containing mescenchymal stem cell have significant therapeutic effect to women uterine cavity scar and adhesion.

Description

Mescenchymal stem cell, its gel and composition are in repairing uterine cavity scar and adhesion Using
Technical field
The present invention relates to stem cell applied technical field, in particular to mescenchymal stem cell, its gel and composition is being repaired Application in multiple uterine cavity scar and adhesion.
Background technique
Asherman's syndrom is a kind of common gynaecology's secondary disease, mainly leads to uterus by endometrium wound or infection Or uterine neck partially or completely adhesion, so as to cause hypomenorrhea or amenorrhoea, periodic abdominalgia, infertile or recurrent abortion, serious shadow The fecundity of Women of Childbearing Age patient is rung, it is reported that since Asherman's syndrom leads to that infertile probability occurs to be 43%, Asherman's syndrom probability occurs in Sterility patient and is up to 13%, clinical research shows after carrying out separating operation to Asherman's syndrom Recurrence rate is higher, and up to 44.9%；Uterine cavity scar refers to the scar formed in uterine cavity, it is more difficult to heal, therefore, finding one kind has Effect improves Asherman's syndrom and the method for uterine cavity scar is very necessary.
The clinical research of mescenchymal stem cell is carried out in many countries, the study found that in addition to being used to promote to restore to make Blood inhibits other than leukaemia and refractory anemia etc. jointly with candidate stem cell, it may also be used for cardiovascular and cerebrovascular disease, cirrhosis, Bone and the autoimmune disease such as muscle degenerative disease, brain and neurologic defict, senile dementia and lupus erythematosus and chorionitis Disease, but the prior art rarely has purposes of the open mescenchymal stem cell on treatment women uterine cavity scar and adhesion.
Summary of the invention
The present invention provides mescenchymal stem cell, its preparation or compositions containing mescenchymal stem cell to treat female in preparation Application in property uterine cavity scar and the drug of adhesion.
Further to improve, mescenchymal stem cell is Amniotic Fluid-derived Mesenchymal Stem Cells.
Further to improve, Amniotic Fluid-derived Mesenchymal Stem Cells cultivate acquisition by the following method:
1) in vitro amniotic fluid tissue is placed in centrifuge tube, 6-8min is centrifuged under the conditions of 800-1000rpm, remove supernatant Liquid, the WPM culture medium that 1/2 supernatant volume is added are resuspended, and are centrifuged 10-14min, and the first culture medium is added and is resuspended, obtains slender Born of the same parents' suspension；
2) six orifice plates are added in the gelatin of 0.2% concentration, every hole adds 1mL, places 30min under room temperature；
3) the second culture medium is added in the single cell suspension obtained to step 1), adjustment cell-seeding-density is 4-6 × 106 A/mL is inoculated in six orifice plates of step 2), and inoculum concentration is the hole 2mL/, is mixed, in 37 ± 0.5 DEG C, carbon dioxide volume fraction Cell culture is carried out under conditions of 5 ± 0.2% to get Amniotic Fluid-derived Mesenchymal Stem Cells.
Wherein, WPM culture medium includes: the K of 900mg/L2SO4, 370mg/L MgSO4·7H2O, the NH of 170mg/L4NO3、 22.5mg/L MnSO4·4H2O, the ZnSO of 8.6mg/L4·7H2O, the H of 6.2mg/L3BO3With the CuSO of 0.25mg/L4· 5H2O, the Na of 0.25mg/L2MoO4·2H2O, the Ca (NO of 556mg/L3)2·4H2O, the FeSO of 27.8mg/L4·7H2O、 37.3mg/L Na2The inositol of EDTA, 100mg/L, the glycine of 2mg/L, 1mg/L VB1, 0.5mg/L VB6、0.5mg/L VB5。
Further to improve, the first culture medium further include concentration is 20-40 μ g/L based on BME cell culture medium The panax japonicus majoris saponin(e of platelet derived growth factor, the Astragaloside IV of 10-20 μ g/L and 20-40 μ g/L.
Wherein, platelet derived growth factor is the PDGF I containing 7% sugar, and BME cell culture medium is purchased from friend's Kang Jiye biology section Skill Co., Ltd, article No. CM0801-CM0802.
Further to improve, the second culture medium further include concentration is 4-6 μ g/L based on IMDM culture medium Exendin-4, the putrescine of 12-16 μ g/L, the cholera toxin of 5-9 μ g/L and 20-30 μ g/L parathyroid hormone.
Wherein, IMDM culture medium is purchased from Shanghai Hui Ying Biotechnology Co., Ltd, article No. SH30228.01；The present invention is logical The mescenchymal stem cell that above method is separately cultured acquisition is crossed, mescenchymal stem cell treatment women uterine cavity can be further improved The effect of scar and adhesion.
Further to improve, the composition containing mescenchymal stem cell includes that concentration is 2-4 × 107The mesenchyma of a/mL Stem cell and concentration are the Chinese medical extract of 20-30ng/mL, the Chinese medical extract by each ingredient preparation of following parts by weight and At:
Further to improve, Chinese medical extract is prepared by each ingredient of following parts by weight:
Wherein, concentration is 2-4 × 107The mescenchymal stem cell and concentration of a/mL is the Chinese medical extract of 20-30ng/mL What is referred to is the concentration of mescenchymal stem cell and Chinese medical extract in physiological saline respectively；The present invention passes through in mescenchymal stem cell On the basis of add Chinese medical extract, mescenchymal stem cell can be improved significantly, the treatment of women uterine cavity scar and adhesion is made With.
Further to improve, Chinese medical extract is prepared by the following method:
Caulis lonicerae, myrobalan, rhizoma anemarrhenae, lepidium seed, carpet bugle and the aleppo avens of formula ratio are taken, drying crushes, 80 meshes are crossed, With water ultrasonic extraction 3 times of 100 DEG C, 60-80min, second of extraction 40-50min are extracted for the first time, and third time extracts 30- 40min, supersonic frequency 40-80KHZ, caulis lonicerae, myrobalan, rhizoma anemarrhenae, lepidium seed, the total weight of carpet bugle and aleppo avens and water Solid-to-liquid ratio is 1:30-40, merges each secondary filtrate, is concentrated into the 1/5-1/4 of original volume, is placed in 60-70 DEG C of baking oven dry To powder to get Chinese medical extract.
Wherein, solid-to-liquid ratio refers to the ratio of the weight g of something and the volume mL of liquid.
The Chinese medical extract prepared by method made above can be improved the content of effective component, and removal is to treatment female The property ingredient of uterine cavity scar and adhesion without therapeutic effect, has the function that Synergy and attenuation.
Further to improve, preparation is gelling agent, and the gelling agent is mainly prepared by mescenchymal stem cell and pharmaceutic adjuvant It forms, the pharmaceutic adjuvant includes that parts by weight are 80-100 parts of Sodium Polyacrylate, 120-130 parts of propylene glycol and 40-60 The chitosan of part, volumetric concentration of the mescenchymal stem cell in Sodium Polyacrylate and propylene glycol are 1-2 × 106A/mL.
Wherein, volumetric concentration=mescenchymal stem cell of the mescenchymal stem cell in Sodium Polyacrylate and propylene glycol Volume+propylene glycol volume of number/Sodium Polyacrylate；The present invention provides mescenchymal stem cell, its preparation or contain mesenchyma Application of the composition of stem cell in preparation treatment women uterine cavity scar and the drug of adhesion, test confirm that mesenchyma is dry thin Born of the same parents, mescenchymal stem cell gelling agent or the composition containing mescenchymal stem cell have women uterine cavity scar and adhesion significant Therapeutic effect.
Specific embodiment
Embodiment 1
A kind of mescenchymal stem cell, the mescenchymal stem cell cultivate acquisition by the following method:
1) in vitro amniotic fluid tissue is placed in centrifuge tube, 6min is centrifuged under the conditions of 800rpm, remove supernatant, be added The WPM culture medium of 1/2 supernatant volume is resuspended, and is centrifuged 10min, and the first culture medium is added and is resuspended, obtains single cell suspension；
2) six orifice plates are added in the gelatin of 0.2% concentration, every hole adds 1mL, places 30min under room temperature；
3) the second culture medium is added in the single cell suspension obtained to step 1), adjustment cell-seeding-density is 4 × 106 A/mL is inoculated in six orifice plates of step 2), and inoculum concentration is the hole 2mL/, is mixed, in 37 ± 0.5 DEG C, carbon dioxide volume fraction Cell culture is carried out under conditions of 5 ± 0.2% to get Amniotic Fluid-derived Mesenchymal Stem Cells；
Wherein, the first culture medium is based on BME cell culture medium, further include concentration be 20 μ g/L it is platelet-derived because The panax japonicus majoris saponin(e of son, the Astragaloside IV of 10 μ g/L and 20 μ g/L；
Wherein, the second culture medium further includes exendin-4, the 12 μ g/L that concentration is 4 μ g/L based on IMDM culture medium Putrescine, the cholera toxin of 5 μ g/L and the parathyroid hormone of 20 μ g/L.
Embodiment 2
A kind of mescenchymal stem cell, the mescenchymal stem cell cultivate acquisition by the following method:
1) in vitro amniotic fluid tissue is placed in centrifuge tube, 7min is centrifuged under the conditions of 9000rpm, removed supernatant, add The WPM culture medium for entering 1/2 supernatant volume is resuspended, and is centrifuged 12min, and the first culture medium is added and is resuspended, obtains single cell suspension；
2) six orifice plates are added in the gelatin of 0.2% concentration, every hole adds 1mL, places 30min under room temperature；
3) the second culture medium is added in the single cell suspension obtained to step 1), adjustment cell-seeding-density is 5 × 106 A/mL is inoculated in six orifice plates of step 2), and inoculum concentration is the hole 2mL/, is mixed, in 37 ± 0.5 DEG C, carbon dioxide volume fraction Cell culture is carried out under conditions of 5 ± 0.2% to get Amniotic Fluid-derived Mesenchymal Stem Cells；
Wherein, the first culture medium is based on BME cell culture medium, further include concentration be 30 μ g/L it is platelet-derived because The panax japonicus majoris saponin(e of son, the Astragaloside IV of 15 μ g/L and 30 μ g/L；
Wherein, the second culture medium further includes exendin-4, the 14 μ g/L that concentration is 5 μ g/L based on IMDM culture medium Putrescine, the cholera toxin of 7 μ g/L and the parathyroid hormone of 25 μ g/L.
Embodiment 3
A kind of mescenchymal stem cell, the mescenchymal stem cell cultivate acquisition by the following method:
1) in vitro amniotic fluid tissue is placed in centrifuge tube, 8min is centrifuged under the conditions of 1000rpm, removed supernatant, add The WPM culture medium for entering 1/2 supernatant volume is resuspended, and is centrifuged 14min, and the first culture medium is added and is resuspended, obtains single cell suspension；
2) six orifice plates are added in the gelatin of 0.2% concentration, every hole adds 1mL, places 30min under room temperature；
3) the second culture medium is added in the single cell suspension obtained to step 1), adjustment cell-seeding-density is 6 × 106 A/mL is inoculated in six orifice plates of step 2), and inoculum concentration is the hole 2mL/, is mixed, in 37 ± 0.5 DEG C, carbon dioxide volume fraction Cell culture is carried out under conditions of 5 ± 0.2% to get Amniotic Fluid-derived Mesenchymal Stem Cells；
Wherein, the first culture medium is based on BME cell culture medium, further include concentration be 40 μ g/L it is platelet-derived because The panax japonicus majoris saponin(e of son, the Astragaloside IV of 20 μ g/L and 40 μ g/L；
Wherein, the second culture medium further includes exendin-4, the 16 μ g/L that concentration is 6 μ g/L based on IMDM culture medium Putrescine, the cholera toxin of 9 μ g/L and the parathyroid hormone of 30 μ g/L.
Embodiment 4
A kind of composition containing mescenchymal stem cell, should composition containing mescenchymal stem cell include concentration be 2 × 107The mescenchymal stem cell and concentration of a/mL is the Chinese medical extract of 20ng/mL, and the Chinese medical extract is by following parts by weight Each ingredient be prepared:
Wherein, mescenchymal stem cell is separately cultured acquisition using the method for embodiment 2.
Embodiment 5
A kind of composition containing mescenchymal stem cell, should composition containing mescenchymal stem cell include concentration be 3 × 107The mescenchymal stem cell and concentration of a/mL is the Chinese medical extract of 25ng/mL, and the Chinese medical extract is by following parts by weight Each ingredient be prepared:
Wherein, mescenchymal stem cell is separately cultured acquisition using the method for embodiment 2；
The Chinese medical extract is prepared by the following method:
Caulis lonicerae, myrobalan, rhizoma anemarrhenae, lepidium seed, carpet bugle and the aleppo avens of formula ratio are taken, drying crushes, 80 meshes are crossed, With water ultrasonic extraction 3 times of 100 DEG C, 60min, second of extraction 40min are extracted for the first time, and third time extracts 30min, supersonic frequency Rate is 40KHZ, and caulis lonicerae, myrobalan, rhizoma anemarrhenae, lepidium seed, carpet bugle and the total weight of aleppo avens and the solid-to-liquid ratio of water are 1:30, is closed And each secondary filtrate, it is concentrated into the 1/5 of original volume, is placed in 60 DEG C of baking oven and dries to powder to get Chinese medical extract；
Wherein, mescenchymal stem cell is separately cultured acquisition using the method for embodiment 2.
Embodiment 6
A kind of composition containing mescenchymal stem cell, should composition containing mescenchymal stem cell include concentration be 4 × 107The mescenchymal stem cell and concentration of a/mL is the Chinese medical extract of 30ng/mL, and the Chinese medical extract is by following parts by weight Each ingredient be prepared:
Wherein, mescenchymal stem cell is separately cultured acquisition using the method for embodiment 2；
The Chinese medical extract is prepared by the following method:
Caulis lonicerae, myrobalan, rhizoma anemarrhenae, lepidium seed, carpet bugle and the aleppo avens of formula ratio are taken, drying crushes, 80 meshes are crossed, With water ultrasonic extraction 3 times of 100 DEG C, 80min, second of extraction 50min are extracted for the first time, and third time extracts 40min, supersonic frequency Rate is 80KHZ, and caulis lonicerae, myrobalan, rhizoma anemarrhenae, lepidium seed, carpet bugle and the total weight of aleppo avens and the solid-to-liquid ratio of water are 1:40, is closed And each secondary filtrate, it is concentrated into the 1/4 of original volume, is placed in 70 DEG C of baking oven and dries to powder to get Chinese medical extract；
Wherein, mescenchymal stem cell is separately cultured acquisition using the method for embodiment 2.
Embodiment 7
A kind of gelling agent, the gelling agent are prepared by mescenchymal stem cell and pharmaceutic adjuvant, the pharmaceutic adjuvant packet Including parts by weight is 80 parts of Sodium Polyacrylate, 120 parts of propylene glycol and 40 parts of chitosan, and the mescenchymal stem cell is poly- Volumetric concentration in sodium acrylate and propylene glycol is 1 × 106A/mL；
Wherein, mescenchymal stem cell is separately cultured acquisition using the method for embodiment 2.
Embodiment 8
A kind of gelling agent, the gelling agent are prepared by mescenchymal stem cell and pharmaceutic adjuvant, the pharmaceutic adjuvant packet Including parts by weight is 90 parts of Sodium Polyacrylate, 125 parts of propylene glycol and 50 parts of chitosan, and the mescenchymal stem cell is poly- Volumetric concentration in sodium acrylate and propylene glycol is 1.5 × 106A/mL；
Wherein, mescenchymal stem cell is separately cultured acquisition using the method for embodiment 2.
Embodiment 9
A kind of gelling agent, the gelling agent are prepared by mescenchymal stem cell and pharmaceutic adjuvant, the pharmaceutic adjuvant packet Including parts by weight is that 100 parts of Sodium Polyacrylate, 130 parts of propylene glycol and 60 parts of chitosan, the mescenchymal stem cell exist Volumetric concentration in Sodium Polyacrylate and propylene glycol is 2 × 106A/mL；
Wherein, mescenchymal stem cell is separately cultured acquisition using the method for embodiment 2.
Reference examples 1
A kind of mescenchymal stem cell, the mescenchymal stem cell cultivate acquisition by the following method:
1) in vitro amniotic fluid tissue is placed in centrifuge tube, 7min is centrifuged under the conditions of 9000rpm, removed supernatant, add α-MEM the culture medium for entering 1/2 supernatant volume is resuspended, and is centrifuged 12min, and the first culture medium is added and is resuspended, obtains single cell suspension；
2) six orifice plates are added in the gelatin of 0.2% concentration, every hole adds 1mL, places 30min under room temperature；
3) the second culture medium is added in the single cell suspension obtained to step 1), adjustment cell-seeding-density is 5 × 106 A/mL is inoculated in six orifice plates of step 2), and inoculum concentration is the hole 2mL/, is mixed, in 37 ± 0.5 DEG C, carbon dioxide volume fraction Cell culture is carried out under conditions of 5 ± 0.2% to get Amniotic Fluid-derived Mesenchymal Stem Cells；
Wherein, the first culture medium is based on BME cell culture medium, further include concentration be 30 μ g/L it is platelet-derived because The panax japonicus majoris saponin(e of son, the Astragaloside IV of 15 μ g/L and 30 μ g/L；
Wherein, the second culture medium further includes exendin-4, the 14 μ g/L that concentration is 5 μ g/L based on IMDM culture medium Putrescine, the cholera toxin of 7 μ g/L and the parathyroid hormone of 25 μ g/L.
Reference examples 2
A kind of mescenchymal stem cell, the mescenchymal stem cell cultivate acquisition by the following method:
1) in vitro amniotic fluid tissue is placed in centrifuge tube, 7min is centrifuged under the conditions of 9000rpm, removed supernatant, add The WPM culture medium for entering 1/2 supernatant volume is resuspended, and is centrifuged 12min, and the first culture medium is added and is resuspended, obtains single cell suspension；
2) six orifice plates are added in the gelatin of 0.2% concentration, every hole adds 1mL, places 30min under room temperature；
3) the second culture medium is added in the single cell suspension obtained to step 1), adjustment cell-seeding-density is 5 × 106 A/mL is inoculated in six orifice plates of step 2), and inoculum concentration is the hole 2mL/, is mixed, in 37 ± 0.5 DEG C, carbon dioxide volume fraction Cell culture is carried out under conditions of 5 ± 0.2% to get Amniotic Fluid-derived Mesenchymal Stem Cells；
Wherein, the first culture medium is based on BME cell culture medium, further include concentration be 30 μ g/L it is platelet-derived because The ginseng sapoglycoside Rg 3 of son, the Radix Glycyrrhizae first glycosides of 15 μ g/L and 30 μ g/L；
Wherein, the second culture medium further includes exendin-4, the 14 μ g/L that concentration is 5 μ g/L based on IMDM culture medium Putrescine, the cholera toxin of 7 μ g/L and the parathyroid hormone of 25 μ g/L.
Reference examples 3
A kind of mescenchymal stem cell, the mescenchymal stem cell cultivate acquisition by the following method:
1) in vitro amniotic fluid tissue is placed in centrifuge tube, 7min is centrifuged under the conditions of 9000rpm, removed supernatant, add The WPM culture medium for entering 1/2 supernatant volume is resuspended, and is centrifuged 12min, and the first culture medium is added and is resuspended, obtains single cell suspension；
2) six orifice plates are added in the gelatin of 0.2% concentration, every hole adds 1mL, places 30min under room temperature；
3) the second culture medium is added in the single cell suspension obtained to step 1), adjustment cell-seeding-density is 5 × 106 A/mL is inoculated in six orifice plates of step 2), and inoculum concentration is the hole 2mL/, is mixed, in 37 ± 0.5 DEG C, carbon dioxide volume fraction Cell culture is carried out under conditions of 5 ± 0.2% to get Amniotic Fluid-derived Mesenchymal Stem Cells；
Wherein, the first culture medium is BME cell culture medium；
Wherein, the second culture medium further includes exendin-4, the 14 μ g/L that concentration is 5 μ g/L based on IMDM culture medium Putrescine, the cholera toxin of 7 μ g/L and the parathyroid hormone of 25 μ g/L.
Reference examples 4
A kind of mescenchymal stem cell, the mescenchymal stem cell cultivate acquisition by the following method:
1) in vitro amniotic fluid tissue is placed in centrifuge tube, 7min is centrifuged under the conditions of 9000rpm, removed supernatant, add The WPM culture medium for entering 1/2 supernatant volume is resuspended, and is centrifuged 12min, and the first culture medium is added and is resuspended, obtains single cell suspension；
2) six orifice plates are added in the gelatin of 0.2% concentration, every hole adds 1mL, places 30min under room temperature；
3) the second culture medium is added in the single cell suspension obtained to step 1), adjustment cell-seeding-density is 5 × 106 A/mL is inoculated in six orifice plates of step 2), and inoculum concentration is the hole 2mL/, is mixed, in 37 ± 0.5 DEG C, carbon dioxide volume fraction Cell culture is carried out under conditions of 5 ± 0.2% to get Amniotic Fluid-derived Mesenchymal Stem Cells；
Wherein, the first culture medium is based on BME cell culture medium, further include concentration be 30 μ g/L it is platelet-derived because The panax japonicus majoris saponin(e of son, the Astragaloside IV of 15 μ g/L and 30 μ g/L；
Wherein, the second culture medium further includes exendin-4, the 14 μ g/L that concentration is 5 μ g/L based on IMDM culture medium Putrescine, the podophyllotoxin of 7 μ g/L and the thyroid hormone of 25 μ g/L.
Reference examples 5
A kind of mescenchymal stem cell, the mescenchymal stem cell cultivate acquisition by the following method:
1) in vitro amniotic fluid tissue is placed in centrifuge tube, 7min is centrifuged under the conditions of 9000rpm, removed supernatant, add The WPM culture medium for entering 1/2 supernatant volume is resuspended, and is centrifuged 12min, and the first culture medium is added and is resuspended, obtains single cell suspension；
2) six orifice plates are added in the gelatin of 0.2% concentration, every hole adds 1mL, places 30min under room temperature；
3) the second culture medium is added in the single cell suspension obtained to step 1), adjustment cell-seeding-density is 5 × 106 A/mL is inoculated in six orifice plates of step 2), and inoculum concentration is the hole 2mL/, is mixed, in 37 ± 0.5 DEG C, carbon dioxide volume fraction Cell culture is carried out under conditions of 5 ± 0.2% to get Amniotic Fluid-derived Mesenchymal Stem Cells；
Wherein, the first culture medium is based on BME cell culture medium, further include concentration be 30 μ g/L it is platelet-derived because The panax japonicus majoris saponin(e of son, the Astragaloside IV of 15 μ g/L and 30 μ g/L；
Wherein, the second culture medium is IMDM culture medium.
Reference examples 6
A kind of composition containing mescenchymal stem cell, should composition containing mescenchymal stem cell include concentration be 3 × 107The mescenchymal stem cell and concentration of a/mL is the Chinese medical extract of 25ng/mL, and the Chinese medical extract is by following parts by weight Each ingredient be prepared:
Wherein, Chinese medical extract is prepared using the method for embodiment 5；
Wherein, mescenchymal stem cell is separately cultured acquisition using the method for embodiment 2.
Reference examples 7
A kind of composition containing mescenchymal stem cell, should composition containing mescenchymal stem cell include concentration be 3 × 107The mescenchymal stem cell and concentration of a/mL is the Chinese medical extract of 25ng/mL, and the Chinese medical extract is by following parts by weight Each ingredient be prepared:
Wherein, Chinese medical extract is prepared using the method for embodiment 5；
Wherein, mescenchymal stem cell is separately cultured acquisition using the method for embodiment 2.
Reference examples 8
A kind of composition containing mescenchymal stem cell, should composition containing mescenchymal stem cell include concentration be 3 × 107The mescenchymal stem cell and concentration of a/mL is the Chinese medical extract of 25ng/mL, and the Chinese medical extract is by following parts by weight Each ingredient be prepared:
Wherein, Chinese medical extract is prepared using the method for embodiment 5；
Wherein, mescenchymal stem cell is separately cultured acquisition using the method for embodiment 2.
Therapeutic effect of the test example 1 to Asherman's syndrom
1. Asherman's syndrom modeling and medication:
SPF grades of SD female rats 210 are taken, is divided between blank group, model group, test 1-5 group, control 1-8 group, amniotic fluid and filling Matter stem cell group, mesenchymal stem cell group, fat mesenchymal stem cell group, umbilical cord mesenchymal stem cells group, traditional Chinese medicine extraction Object group and positive controls, every group 10, each group is with following methods modeling in addition to blank group: one day before surgery evening fasting is not Prohibit water 12h, after 3% yellow Jackets 30mg/kg anesthesia, in median incision after lower abdomen routine disinfection, 1mL is used in exposure Y type uterus Syringe divides inserting needle at basin to enter in uterine cavity in uterus, slowly injects 25% Hydroxybenzene mucilage 0.06mL towards ovary direction, modeling is complete Abdomen is closed at rear layering；Start to be administered after modeling success, 1 group of the test mescenchymal stem cell using the embodiment of the present invention 2, test 2 Mescenchymal stem cell of the group using the embodiment of the present invention 3,3 groups of the test compositions using the embodiment of the present invention 5,4 groups of test example Using the composition of the embodiment of the present invention 6, implement 5 groups of gelling agents for using the embodiment of the present invention 7,1 group of control uses reference examples 1 Mescenchymal stem cell, 2 groups of the control mescenchymal stem cells for using reference examples 2,3 groups of control are dry using the mesenchyma of reference examples 3 Cell, 4 groups of the control mescenchymal stem cells using reference examples 4,5 groups of control use the mescenchymal stem cells of reference examples 5, control 6 Group uses the mescenchymal stem cell of reference examples 6, and 7 groups of the control mescenchymal stem cells using reference examples 7 compare 8 groups using control The mescenchymal stem cell of example 8, Amniotic Fluid-derived Mesenchymal Stem Cells group use the Amniotic Fluid-derived Mesenchymal Stem Cells bought in the market, fill between marrow Matter stem cell group uses the mesenchymal stem cell bought in the market, the use of fat mesenchymal stem cell group to buy in the market Fat mesenchymal stem cell, umbilical cord mesenchymal stem cells group use the umbilical cord mesenchymal stem cells bought in the market, traditional Chinese medicine extraction Object group uses biochemical ball using the Chinese medical extract in embodiment 5, positive controls；It tests between 1-5 group, control 1-8 group, amniotic fluid Mesenchymal stem cells group, mesenchymal stem cell group, fat mesenchymal stem cell group, umbilical cord mesenchymal stem cells group give medicament Amount is 0.5mL/ pcs/day, and Chinese medical extract group and positive controls are with 0.05g/ pcs/day of Rapid Dose Calculation, blank group and model Group gives isometric physiological saline, is administered 18 days altogether.
2. the content assaying method and result of each index
Previous evening of drawing materials is deprived of food but not water, the 19th day abdominal aortic blood under anaesthesia after the last administration, the measurement of Elisa method The content of IL-2, IL-10 in serum, blood rheological instrument measure the content that proteinogen is limited in blood plasma, and Masson dyeing uses Imagepro Plus quantitative analysis rat uterus degree of fibrosis, each group of data are taken from the average value of 10 Duplicate Samples, as a result It is shown in Table 1.
Therapeutic effect of 1 each group of table to female rats Asherman's syndrom
3. conclusion
As shown in Table 1, the mescenchymal stem cell and contain mescenchymal stem cell that the embodiment of the present invention 2,3,5,6,7 provides Composition and mescenchymal stem cell gel can be used in treating women Asherman's syndrom, test the mescenchymal stem cell of 2-3 group The therapeutic effect of female rats Asherman's syndrom is significantly higher than and compares 1-5 group and mescenchymal stem cell group, illustrates that the present invention mentions The mescenchymal stem cell that the isolated culture method of the mescenchymal stem cell of confession obtains is more preferable to the therapeutic effect of female rats adhesion, When changing the individual components in culture medium, the therapeutic effect to female rats Asherman's syndrom will be reduced significantly, is filled between amniotic fluid It is dry thin that matter stem cell group is significantly higher than mesenchymal stem cell group, fat mesenchymal to the therapeutic effect of women Asherman's syndrom Born of the same parents' group and umbilical cord mesenchymal stem cells group, it was demonstrated that Amniotic Fluid-derived Mesenchymal Stem Cells treat the effect of women Asherman's syndrom than filling between marrow Matter stem cell, fat mesenchymal stem cell and umbilical cord mesenchymal stem cells are more preferable；The mescenchymal stem cell of 3-4 group is tested to female The therapeutic effect of rat Asherman's syndrom, which is significantly higher than, compares 6-8 group, Chinese medical extract group and positive controls, illustrates the present invention There is provided the composition containing mescenchymal stem cell to Asherman's syndrom have good therapeutic effect, when it is provided by the invention containing It, should the group containing mescenchymal stem cell after ingredient in the composition of mescenchymal stem cell reduces or is replaced by other ingredient Closing object significantly reduces the therapeutic effect of Asherman's syndrom, and the Chinese medical extract being used alone in the present invention is viscous to female rats uterine cavity Even almost without therapeutic effect, exclusive use mescenchymal stem cell is also not ideal enough to the therapeutic effect of female rats adhesion, only Have by mescenchymal stem cell in conjunction with Chinese medical extract, act synergistically, the treatment that could improve Asherman's syndrom significantly is made With.
Therapeutic effect of the test example 2 to uterine cavity scar
1. the modeling of uterine cavity scar and medication:
Rat 200 are chosen, blank group, model group, test 1-5 group, control 1-8 group, Amniotic Fluid-derived Mesenchymal Stem Cells are divided into Group, mesenchymal stem cell group, fat mesenchymal stem cell group, umbilical cord mesenchymal stem cells group and Chinese medical extract group, often Group 10, each group is with following methods modeling in addition to blank group: rat anesthesia, and lower abdomen center longitudinal incision is gone to enter abdominal cavity, In uterus terminal mesometrium opposite side, excision is about 1.5 centimetres, wide about 0.5 centimetre of holostrome uterine wall tissue；After modeling success Start to be administered, 1 group of the test mescenchymal stem cell using the embodiment of the present invention 2,2 groups of test is using between the embodiment of the present invention 3 Mesenchymal stem cells, 3 groups of the test compositions using the embodiment of the present invention 5,4 groups of the test example combinations using the embodiment of the present invention 6 Object, implements 5 groups of gelling agents for using the embodiment of the present invention 7, and 1 group of the control mescenchymal stem cell using reference examples 1 compares 2 groups Using the mescenchymal stem cell of reference examples 2,3 groups of the control mescenchymal stem cells using reference examples 3,4 groups of control uses reference examples 4 Mescenchymal stem cell, 5 groups of the control mescenchymal stem cells for using reference examples 5,6 groups of control are dry using the mesenchyma of reference examples 6 Cell, 7 groups of the control mescenchymal stem cells for using reference examples 7,8 groups of the control mescenchymal stem cells for using reference examples 8, between amniotic fluid Mesenchymal stem cells group is used and is bought in the market using the Amniotic Fluid-derived Mesenchymal Stem Cells bought in the market, mesenchymal stem cell group Mesenchymal stem cell, fat mesenchymal stem cell group is using the fat mesenchymal stem cell bought in the market, between umbilical cord Mesenchymal stem cells group uses the umbilical cord mesenchymal stem cells bought in the market, Chinese medical extract group using the Chinese medicine in embodiment 5 Extract；It is dry to test 1-5 group, control 1-8 group, Amniotic Fluid-derived Mesenchymal Stem Cells group, mesenchymal stem cell group, fat mesenchymal Groups of cells, umbilical cord mesenchymal stem cells group dosage be 0.5mL/ pcs/day, Chinese medical extract group is with 0.05g/ pcs/day Rapid Dose Calculation, blank group and model group give isometric physiological saline, are administered 30 days altogether.
2. the content assaying method and result of each index
Previous evening of drawing materials is deprived of food but not water, and after the last administration by each group rat anesthesia, takes out rat uterus, measures intrauterine Thickness at film modeling, the results are shown in Table 2.
Heights Experiment result at 2 each group rat endometrium modeling of table
3. conclusion
As shown in Table 2, the mescenchymal stem cell and contain mescenchymal stem cell that the embodiment of the present invention 2,3,5,6,7 provides Composition and mescenchymal stem cell gel can be used in treating uterine cavity scar, test the mescenchymal stem cell of 2-3 group to female Property rat uterine cavity scar therapeutic effect be significantly higher than and compare 1-5 group and mescenchymal stem cell group, illustrate provided by the invention The mescenchymal stem cell that the isolated culture method of mescenchymal stem cell obtains is more preferable to the therapeutic effect of female rats scar, when changing When becoming the individual components in culture medium, the therapeutic effect to female rats uterine cavity scar will be reduced significantly, and amniotic fluid mesenchyma is dry Groups of cells is significantly higher than mesenchymal stem cell group, fat mesenchymal stem cell group to the therapeutic effect of women uterine cavity scar With umbilical cord mesenchymal stem cells group, it was demonstrated that the effect that Amniotic Fluid-derived Mesenchymal Stem Cells treat women uterine cavity scar is more dry than medulla mesenchyma Cell, fat mesenchymal stem cell and umbilical cord mesenchymal stem cells are more preferable；The mescenchymal stem cell of 3-4 group is tested to female rats The therapeutic effect of uterine cavity scar, which is significantly higher than, compares 6-8 group and Chinese medical extract group, illustrates provided by the invention containing filling The composition of matter stem cell has good therapeutic effect to uterine cavity scar, when provided by the invention containing mescenchymal stem cell After ingredient in composition reduces or is replaced by other ingredient, the composition containing mescenchymal stem cell is somebody's turn to do to uterine cavity scar Therapeutic effect significantly reduce, be used alone the present invention in Chinese medical extract to female rats uterine cavity scar almost without treatment Effect, exclusive use mescenchymal stem cell is also not ideal enough to the therapeutic effect of female rats scar, only that mesenchyma is dry thin Born of the same parents act synergistically in conjunction with Chinese medical extract, could improve the therapeutic effect to uterine cavity scar significantly.

Claims (9)

1. a kind of mescenchymal stem cell, its preparation or the composition containing mescenchymal stem cell treat women uterine cavity scar in preparation And the application in the drug of adhesion.
2. application as described in claim 1, which is characterized in that the mescenchymal stem cell is Amniotic Fluid-derived Mesenchymal Stem Cells.
3. application as claimed in claim 2, which is characterized in that the Amniotic Fluid-derived Mesenchymal Stem Cells are cultivated obtain by the following method :
1) in vitro amniotic fluid tissue is placed in centrifuge tube, 6-8min is centrifuged under the conditions of 800-1000rpm, remove supernatant, The WPM culture medium that 1/2 supernatant volume is added is resuspended, and is centrifuged 10-14min, and the first culture medium is added and is resuspended, and obtains unicellular outstanding Liquid；
2) six orifice plates are added in the gelatin of 0.2% concentration, every hole adds 1mL, places 30min under room temperature；
3) the second culture medium is added in the single cell suspension obtained to step 1), adjustment cell-seeding-density is 4-6 × 106A/ ML is inoculated in six orifice plates of step 2), inoculum concentration be the hole 2mL/, mix, in 37 ± 0.5 DEG C, carbon dioxide volume fraction 5 ± Cell culture is carried out under conditions of 0.2% to get Amniotic Fluid-derived Mesenchymal Stem Cells.
4. application as claimed in claim 3, which is characterized in that first culture medium is based on BME cell culture medium, also Platelet derived growth factor, the Astragaloside IV of 10-20 μ g/L and the panax japonicus majoris soap of 20-40 μ g/L for being 20-40 μ g/L including concentration Glycosides.
5. application as claimed in claim 3, which is characterized in that second culture medium is also wrapped based on MSCs culture medium Include the first shape that concentration is the exendin-4 of 4-6 μ g/L, the putrescine of 12-16 μ g/L, the cholera toxin of 5-9 μ g/L and 20-30 μ g/L Other glandular hormone.
6. application as described in claim 1, which is characterized in that the composition containing mescenchymal stem cell includes that concentration is 2-4×107The mescenchymal stem cell and concentration of a/mL be 20-30ng/mL Chinese medical extract, the Chinese medical extract by with Each ingredient of lower parts by weight is prepared:
7. application as claimed in claim 6, which is characterized in that the Chinese medical extract is prepared by each ingredient of following parts by weight It forms:
8. application as claimed in claim 6, which is characterized in that the Chinese medical extract is prepared by the following method:
Caulis lonicerae, myrobalan, rhizoma anemarrhenae, lepidium seed, carpet bugle and the aleppo avens of formula ratio are taken, drying crushes, and 80 meshes is crossed, with 100 DEG C water ultrasonic extraction 3 times, extract 60-80min for the first time, second extracts 40-50min, and third time extracts 30-40min, surpasses Acoustic frequency is 40-80KHZ, and caulis lonicerae, myrobalan, rhizoma anemarrhenae, lepidium seed, carpet bugle and the total weight of aleppo avens and the solid-to-liquid ratio of water are 1:30-40 merges each secondary filtrate, is concentrated into the 1/5-1/4 of original volume, is placed in 60-70 DEG C of baking oven and dries to powder, i.e., Obtain Chinese medical extract.
9. application as described in claim 1, which is characterized in that the preparation is gelling agent, and the gelling agent is mainly filled by Matter stem cell and pharmaceutic adjuvant are prepared, the pharmaceutic adjuvant include parts by weight be 80-100 parts Sodium Polyacrylate, The chitosan of 120-130 parts of propylene glycol and 40-60 part, body of the mescenchymal stem cell in Sodium Polyacrylate and propylene glycol Product concentration is 1-2 × 106A/mL.
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