CN106913584A - A kind of Endometrial stem cell preparation and its application - Google Patents
A kind of Endometrial stem cell preparation and its application Download PDFInfo
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Abstract
The application belongs to regenerative medicine and biology techniques field, and in particular to a kind of Endometrial stem cell preparation and its application.Endometrial stem cell preparation provided by the present invention is included：Endometrial stem cell, quinquenoside L1 and American ginseng saponin R1, from Endometrial stem cell as source of human stem cell, convenient material drawing, non-invasive, without ethics limitation；Endometrial stem cell and American ginseng saponin play synergy, effectively inhibit premature ovarian failure, greatly shorten the tissue repair time.Present invention also offers application of the above-mentioned Endometrial stem cell preparation in treatment premature ovarian failure medicine is prepared.
Description
Technical field
The invention belongs to regenerative medicine and biology techniques field, and in particular to a kind of Endometrial stem cell preparation and its
Using.
Background technology
Premature ovarian failure (premature ovarian failure, POF) refers to the female of oneself formation rule of menstruation before 40 years old
Property, occur the symptoms such as irregular menstruation, amenorrhoea, sexual organ atrophy due to ovarian failure, and it is often sharp along with sexual gland is promoted
The rising of plain level and the decline of estrogen level.Premature ovarian failure is the common disease and frequently-occurring disease in gynemetrics's clinical position,
Through the main cause of disease as current woman's acyesis.At present, ovum is mainly recovered by methods such as hormone therapy and immunization therapies
Nest function, but the above method can only temporarily relief of symptoms, it is impossible to the Regeneration and Repair of self-inflicted injury ovary tissue is effectively facilitated,
Cannot radical curing of disease.Treated by injection of bone marrow mescenchymal stem cell alternative medicine and operative treatment, be clinical practice in recent years
In be used for treating the new tool of premature ovarian failure.But mesenchymal stem cells MSCs must could be obtained by bone marrow aspiration, not only
Sampling is difficult, and easily causes certain somatic damage to patient, hinders the wish that healthy population contributes marrow.In addition,
The content of mesenchymal stem cells MSCs is very rare in marrow, and multiplication capacity is relatively low.
The content of the invention
In view of this, it is an object of the invention to provide a kind of Endometrial stem cell preparation, it is applied to prepare treatment ovary
The medicine of early ageing, source of human stem cell is convenient, non-invasive.
Concrete technical scheme of the invention is as follows：
The invention provides a kind of Endometrial stem cell preparation, comprising：Endometrial stem cell, quinquenoside L1 and
American ginseng saponin R1.
Endometrial stem cell (endometrial stem cells, EnSCs) is a kind of adult stem cell, with self
The potential of renewal, Multidirectional Differentiation and hyperproliferation, carcinogenic risk is low, few by ethics dispute.Endometrial stem cell have with
BMMSCs similar biological characteristics, expresses the special surface mark of the mescenchymal stem cells such as CD29, CD44, CD73 and CD90
Will, the endothelial cells such as CD14, CD34, CD45, CD133 and Stro-1, hemopoietic stem cell surface mark are not expressed, do not express MHC-
I, expresses MHC-II on a small quantity, does not express or few expression of HLA-DR, illustrates that EnSCs has compared with low immunogenicity.EnSCs can be from warp
Noninvasive acquisition in blood, growth rate is fast, and passage stabilization, immunogenicity is low, with stronger paracrine ability.
Preferably, the concentration of the Endometrial stem cell is 1.0 × 106~5.0 × 106Individual/mL.
American ginseng total saponins Central nervous system, cardiovascular system, digestive system, immune system, internal system and
Genitourinary has extensive effect, with resisting myocardial ischemia and protect cardiac muscle, anti-arrhythmia, suppression aging apoptosis and resist
Inflammation effect.
Preferably, the concentration of the quinquenoside L1 be 0.1~5mg/mL, more preferably 0.5~3mg/L, most preferably
It is 1mg/mL.
Preferably, the concentration of the American ginseng saponin R1 be 0.1~5mg/mL, more preferably 0.5~3mg/L, most preferably
It is 1mg/mL.
Preferably, the Endometrial stem cell preparation is also included：Physiological saline.
Present invention also offers a kind of preparation method of above-mentioned Endometrial stem cell preparation, by Endometrial stem cell,
The mixing of quinquenoside L1, American ginseng saponin R1 and physiological saline, obtains the Endometrial stem cell preparation.
The endometrium obtained present invention also offers above-mentioned Endometrial stem cell preparation or above-mentioned preparation method is dry thin
Application of born of the same parents' preparation in treatment premature ovarian failure medicine is prepared.
Preferably, the Endometrial stem cell preparation is ejection preparation.
Compared with prior art, Endometrial stem cell preparation of the invention has advantages below：
1) from Endometrial stem cell as source of human stem cell, convenient material drawing, non-invasive is limited the present invention without ethics
System；And the Endometrial stem cell preparation immunogenicity is low, immunological rejection can be avoided, it is safe and reliable；
2) Endometrial stem cell preparation of the present invention also includes quinquenoside L1 and American ginseng saponin R1, American ginseng saponin
The anti-adversity ability of senile cell can directly be strengthened, Endometrial stem cell repairs premature ovarian failure tissue, west by paracrine action
Radix Panacis Quinquefolii saponin and Endometrial stem cell act synergistically, and effectively inhibit premature ovarian failure, greatly shorten the tissue repair time；
3) invention formulation is from physiological saline as solvent, and its osmotic pressure is consistent with the osmotic pressure of human body, can avoid damaging
Hinder ovary tissue, its bioactivity stabilization under Cord blood；
4) Endometrial stem cell preparation of the present invention is ejection preparation, can avoid blood flow and cause the loss of preparation；
5) preparation process of invention formulation is simple, takes short, achievable large-scale production.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
The accompanying drawing to be used needed for having technology description is briefly described, it should be apparent that, drawings in the following description are only this
Inventive embodiment, for those of ordinary skill in the art, on the premise of not paying creative work, can also basis
The accompanying drawing of offer obtains other accompanying drawings.
Fig. 1 is the ovarian follicle quantity result of each group ovary tissue section.
Specific embodiment
Stem cell is substituted by from Endometrial stem cell, while optimize the constituent in preparation, the present invention
There is provided a kind of Endometrial stem cell preparation, it is applied to prepare the medicine for the treatment of premature ovarian failure, stem cell survival is stable, next
Source is convenient, non-invasive, and the preparation time of preparation is short, efficiency high.
The present invention is not particularly limited to the source of Endometrial stem cell, quinquenoside L1 and American ginseng saponin R1,
It is using conventional Endometrial stem cell well-known to those skilled in the art, quinquenoside L1 and American ginseng saponin R1
Can, or Endometrial stem cell, the American Ginseng soap for obtaining are prepared using routine techniques well-known to those skilled in the art
Glycosides L1 and American ginseng saponin R1.
Technical scheme is clearly and completely described below in conjunction with the specific embodiment of the invention, it is clear that
Described embodiment is a part of embodiment of the invention, rather than whole embodiments.Those skilled in the art should manage
Solution, modifies to specific embodiment of the invention or some technical characteristics is replaced on an equal basis, without deviating from the present invention
The spirit of technical scheme, all should cover in the scope of protection of the invention.
In following examples, the quinquenoside L1 and American ginseng saponin R1 for being used are from the intelligent section plant exploitation in Shaanxi
Co., Ltd.
The preparation of the Endometrial stem cell of embodiment 1
1st, after agreeing to through consulting, according to menses collection suit (Hangzhou Yi Wensai, S-Evans biosciences) specification
Collect healthy women through blood sample；Then these samples are transferred to the phosphorus containing dual anti-(penicillin/streptomycin) and heparin
In phthalate buffer, preserved under the conditions of 4 DEG C, sample is sent to laboratory centrifugal within 24h~48h；Then take supernatant and
Quick detection whether there is germ contamination under aseptic condition, if feminine gender then continues to be separately cultured and identify, table 1 is endometrium
The streaming qualification result of stem cell.Wherein, Endometrial stem cell be separately cultured and qualification process refer to following documents：
Zhou Hua, Song Lina, quick people's Endometrial stem cell in-vitro separation, culture and identification China traditional Chinese medicine academic periodical
[J].2014.32(8):1907-1908。
Table 1
| CD73 | CD90 | CD105 | CD11b | CD19 | CD34 | CD45 | HLA-DR | |
| Expression quantity | 98.4% | 97.5% | 99.3% | 1.8% | 1.2% | 1.0% | 0.4% | 1.4% |
Embodiment 2
(1) it is formulated：
Endometrial stem cell 2.5 × 106Individual/mL；
Quinquenoside L1 1mg/mL；
American ginseng saponin R1 1mg/mL；
Physiological saline is supplied.
(2) prepare：
1) quinquenoside L1 and American ginseng saponin R1 are dissolved using physiological saline, is obtained American ginseng saponin molten
Liquid；Wherein, the concentration of quinquenoside L1 and American ginseng saponin R1 is 1mg/mL；
2) using step 1) Endometrial stem cell that obtains in the resuspended embodiment 1 of American ginseng saponin solution that obtains, obtain
To the Endometrial stem cell preparation, the wherein concentration of Endometrial stem cell is 2.5 × 106Individual/mL；
3) step 2) the Endometrial stem cell preparation that obtains dispensed into syringe, is then put into aseptic sylphon
In, the sylphon for turning to have preparation is transferred in the storage box for being placed with ice bag, preserved at 0~4 DEG C.
Embodiment 3
Endometrial stem cell 1 × 106Individual/mL；
Quinquenoside L1 0.3mg/mL；
American ginseng saponin R1 0.3mg/mL；
Physiological saline is supplied.
Embodiment 4
Endometrial stem cell 5 × 106Individual/mL；
Quinquenoside L1 3mg/mL；
American ginseng saponin R1 3mg/mL；
Physiological saline is supplied.
Embodiment 5
1st, 2-3 monthly ages for being provided from experimental animal portion of Traditional Chinese Medicine University Of Guangzhou, 200 ± 20g of body weight, with normally emotionally
The wistar female rats in cycle 120, adaptability raise 1 week after, be randomly divided into 4 groups, be followed successively by control group 1, control group 2,
Control group 3 and experimental group, every group each 30.
Build rat ovary morning failure model：Disposable celiac injects cyclophosphamide solution (120mg/kg) and busulfan solution
(12mg/kg) each 0.1mL, daily 8：00 beats easily vaginal fluid smear, then pap staining with cotton swab, observes vaginal exfoliated
Change, when the phase between continuous 10d observes Constant Estrus, is considered as premature ovarian failure and models successfully.Wherein build rat ovary early ageing mould
The detailed process of type refers to following document：
Yingying Zhang, Yin Huiqun, Ni Feng Bone Marrow Mesenchymal Stem Cells Transplantations should in premature ovarian failure mouse ovarian reconstruction
With Agricultural University Of Anhui journal, 2017,44 (1):1-6.
2nd, after modeling success, every experimental rat intraperitoneal injection sodium phenobarbital 60mg/kg, by rat back after 15min
It is fixed on animal operating table upwards, skin of sterilizing holds ophthalmic tweezers and clutches lower abdomen median line skin, and small horizontal stroke is made with clipper for surgical use
Otch, scissors blunt separation fully exposes surgical field of view, cuts manadesma, and fat pad, ovary are hauled out with tweezers.Fixed with tweezers again
Firmly, be injected separately into preparation described in table 2 in rat ovary tissue with micro syringe then by ovary, and injection dosage is 20 μ L/.
Table 2
| Group | Component |
| Control group 1 | Physiological saline |
| Control group 2 | |
| Control group 3 | |
| Experimental group | Constituent and its proportioning are as described in Example 2 |
3rd, the important gene for participating in apoptosis regulation has many kinds, wherein Bcl-2 and Bax gene pairs muroid ovarian granulosa
Apoptosis plays a decisive role.Wherein, Bax increases promotion Apoptosis, and Bcl-2 increases suppression Apoptosis.
(1) expression of method test experience rat ovary Bax, Bcl-2 albumen of immunohistochemical staining is used,
Its detailed process is：The ovary tissue serial section of 6 μ m-thicks is taken, using SABC method row immunohistochemical stainings, ovary tissue section
Air drying；The 30%H of 20mL2O2With the mixing of 200mL distilled water, it is subsequently adding in ovary tissue section, room temperature 10min is going out
Endogenous peroxydase living, then with distilling water washing 3 times, each 5min；After carrying out the multiple antigen of hot repair with micro-wave oven, with 5%
BSA confining liquids room temperature close 30min, then respectively be added dropwise Bcl-2 and Bax primary antibodies mixed liquor (volume ratio is 1：200) at 4 DEG C
Overnight；On next day, biotinylated goat anti-rabbit IgG effect 10min are added dropwise at room temperature, then SABC effect 30min are added dropwise, then DAB
3~10min of colour developing, haematoxylin is redyed, and running water returns blue 15min；Conventional gradients dehydration of alcohol, dimethylbenzene are transparent, neutral gum
Mounting.
Table 3 is the expression of results of each group experimental rat ovary Bax, Bcl-2 albumen, as shown in table 3, the He of control group 2
Bax, Bcl-2 protein expression of control group 3 compared with control group 1, P<0.05, with statistical significance, show medulla mesenchyma
Stem cell and Endometrial stem cell can suppress premature ovarian failure；Control group 2 is close with the result of control group 3, between showing marrow
The effect of the reparation premature ovarian failure of mesenchymal stem cells and Endometrial stem cell is close, Endometrial stem cell can effectively replace bone
Bone marrow-drived mesenchymal stem is used as the seed cell for treating premature ovarian failure.Experimental group compared with control group 1, P<0.01, have
Notable statistical significance, shows that the effect of experimental group suppression premature ovarian failure is obvious；Compared with control group 2 and control group 3, experimental group
The effect for suppressing premature ovarian failure is more excellent.
The expression of each group ovary Bax, Bcl-2 albumen of table 3
| Group | Experiment mice number | Bax | Bcl-2 |
| Control group 1 | 10 | 0.3358±0.0072 | 0.2619±0.0224 |
| Control group 2 | 10 | ||
| Control group 3 | 10 | ||
| Experimental group | 10 |
* represent, compared with control group, P<0.05, with statistical significance；
# is represented, compared with control group, P<0.01, tool is statistically significant.
(2) using the expression of Western blot test experience rat ovary Bax, Bcl-2 albumen, its detailed process
For：Every group takes 10 rat cervical dislocations and puts to death, and peels off and takes out bilateral ovaries, is shredded；0.5mL lysates are added,
30min is homogenized on ice, and 12000g/min centrifugations 30min, takes supernatant at 4 DEG C, and protein quantification is carried out using BCA methods, calculates sample
Protein concentration；According to volume ratio 5:1 ratio adds sample-loading buffer, and 5min, cooled on ice, after being denatured are boiled in water-bath
Albumen；By the albumen equivalent loading after denaturation, SDS- polyacrylamide gel electrophoresises are then carried out；Using wet method transferring film by gel
In protein delivery to pvdf membrane on, transferring film condition is：60v transferring films 2h；Pvdf membrane (is prepared) with 5% skimmed milk power with TBST
Room temperature closes 1h, is separately added into Bax (1：200)、Bcl-2(1：200) with β-actin (1：200), 4 DEG C of overnight incubations, TBST is washed
Film 10min × 3 time；By pvdf membrane and the secondary antibody (1 of horseradish peroxidase (HRP) mark：5000) 1h is incubated at room temperature,
TBST washes film 10min*3 times；Pvdf membrane is placed on preservative film, A, B luminescent solution press 1：1 ratio mixes, and A, B mixed liquor is added dropwise,
Imaging system is automatically analyzed through gel to be imaged, IMAQ and analysis.
Table 4 is each group rat ovary Bax and Bcl-2 protein expression result, as shown in the result of table 4, control group 2 and control group
3 Bax, Bcl-2 protein expression compared with control group 1, P<0.05, with statistical significance, show mesenchymal stem cells MSCs
Can suppress premature ovarian failure with Endometrial stem cell；Control group 2 is close with the result of control group 3, shows that medulla mesenchyma is done
The effect of the reparation premature ovarian failure of cell and Endometrial stem cell is close, is filled between Endometrial stem cell can effectively replace marrow
Matter stem cell is used as the seed cell for treating premature ovarian failure.Experimental group compared with control group 1, P<0.01, with significantly system
Meter learns meaning, shows that the effect of experimental group suppression premature ovarian failure is obvious；Compared with control group 2 and control group 3, experimental group suppresses ovum
The effect of nest early ageing is more excellent.
The comparing result of each group rat ovary Bax and the Bcl-2 albumen of table 4 and β-actin
| Group | Experiment mice number | Bax | Bcl-2 |
| Control group 1 | 10 | 1.34±0.065 | 0.64±0.055 |
| Control group 2 | 10 | ||
| Control group 3 | 10 | ||
| Experimental group | 10 |
* represent, compared with control group, P<0.05, with statistical significance；
# is represented, compared with control group, P<0.01, tool is statistically significant.
4th, HE dyeing observation ovary tissue section
Every group takes remaining 10 rat cervical dislocations and puts to death, breaks end, peels off and take out bilateral ovaries, with 4% poly first
Aldehyde uses FFPE, then serial section after fixing 24h, and one, 5 μm of slice thick are taken every 20 μm；Take 20 sections, conventional H E dyes
After color under 400 times of light microscopics, the ovarian follicle situation in each stage in four groups of sections of sample of observation, while observing the follicle number in ovary
Amount.Fig. 1 is the ovarian follicle quantity result of each group ovary tissue section.
Claims (8)
1. a kind of Endometrial stem cell preparation, comprising：Endometrial stem cell, quinquenoside L1 and American ginseng saponin R1.
2. Endometrial stem cell preparation according to claim 1, it is characterised in that the Endometrial stem cell it is dense
Spend is 1.0 × 106~5.0 × 106Individual/mL.
3. Endometrial stem cell preparation according to claim 1, it is characterised in that the concentration of the quinquenoside L1
It is 0.1~5mg/mL.
4. Endometrial stem cell preparation according to claim 1, it is characterised in that the concentration of the American ginseng saponin R1
It is 0.1~5mg/mL.
5. Endometrial stem cell preparation according to claim 1, it is characterised in that also include：Physiological saline.
6. the preparation method of the Endometrial stem cell preparation described in a kind of claim 5, it is characterised in that do endometrium
The mixing of cell, quinquenoside L1, American ginseng saponin R1 and physiological saline, obtains the Endometrial stem cell preparation.
7. the Endometrial stem cell preparation as described in claim 1 to 5 any one or preparation method as described in claim 6 are obtained
To Endometrial stem cell preparation prepare treatment premature ovarian failure medicine in application.
8. application according to claim 7, it is characterised in that the Endometrial stem cell preparation is ejection preparation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108815190A (en) * | 2018-09-27 | 2018-11-16 | 天津欣普赛尔生物医药科技有限公司 | It is a kind of for treating the preparation and preparation method of premature ovarian failure |
| CN108998411A (en) * | 2018-08-15 | 2018-12-14 | 山西医科大学 | The isolated culture method of people's endometrial tissue derived mesenchymal stem cell |
| CN110613737A (en) * | 2019-09-27 | 2019-12-27 | 陕西中鸿科瑞再生医学研究院有限公司 | Preparation method and application of endometrium stem cell preparation |
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| CN105055450A (en) * | 2015-02-13 | 2015-11-18 | 中国福利会国际和平妇幼保健院 | Applications of human endometrium stem cells in preparation of drugs for premature ovarian failure treatment |
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| CN108998411A (en) * | 2018-08-15 | 2018-12-14 | 山西医科大学 | The isolated culture method of people's endometrial tissue derived mesenchymal stem cell |
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| CN110613737A (en) * | 2019-09-27 | 2019-12-27 | 陕西中鸿科瑞再生医学研究院有限公司 | Preparation method and application of endometrium stem cell preparation |
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Application publication date: 20170704 |