CN101129405B - Biological formulation product for treating habitual abortion - Google Patents
Biological formulation product for treating habitual abortion Download PDFInfo
- Publication number
- CN101129405B CN101129405B CN2007100624876A CN200710062487A CN101129405B CN 101129405 B CN101129405 B CN 101129405B CN 2007100624876 A CN2007100624876 A CN 2007100624876A CN 200710062487 A CN200710062487 A CN 200710062487A CN 101129405 B CN101129405 B CN 101129405B
- Authority
- CN
- China
- Prior art keywords
- cell
- habitual abortion
- culture
- treatment
- exosomes
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to a biological agent for treating habitual abortion in the medicine technological field, which comprises the following steps: extracting peripheral blood of healthy adult men; diluting the blood to 1. 5 times through glass fibers from the ball and Hanks liquid to separate lymphocyte to collect single core cells; washing by Hanks liquid to culture and flow and count with absolute culture liquid of RPMI-1640; adjusting the cell concentration to 2*10<6>/ml to add phasecolosaxin to culture at 37 DEG C for 72hours; collecting supernatant to produce T cells through centrifuging of centrifugal ultrafiltration and cane sugar density gradient centrifugation; filtering by sterile filter membrane to be split charging to produce completed product at 80 DEG C below zero. The invention evokes immune tolerance of body effectively and overcomes the more faults of evoking immune tolerance through cell adoptive immunotherapy to make easier clinical application, which can be produced in a large industrialization scale to get more stabilizing mass control and higher controllability for treating the habitual abortion with good curative effect.
Description
Technical field
The present invention relates to a kind of acellular composition that utilizes and replace the immunology preparation that the conventional cell composition is made treatment unknown cause habitual abortion, belong to medical technical field.
Background technology
Habitual abortion is meant and recurs three times or above Aborter that sickness rate accounts for 1% of total pregnant number, accounts for 15% of spontaneous abortion number, is the able-bodied obstetrics of a kind of serious harm anemia of pregnant woman disease.Clinical once had case to recur the Aborter 7 times, and patient is very painful.This kind disease cause of disease is very complicated, comprise inherited genetic factors, anatomic abnormalities, cryptorrhea, infection or the like, but, get rid of above-mentioned clinical disease because of after, still have 59.42% habitual abortion patients, be called unknown cause habitual abortion URSA (Unexplanted recurrent spontaneous abortion), it is relevant with the maternal immunity factor, has become the focus that present people pay close attention to.Clinically URSA patient is divided into autoimmune type and alloimmunity type two big classes at present, wherein both at home and abroad at alloimmunity type URSA patient, the conventional employing " immunocyte adopt therapy " treated, be intended to induce the immunologic tolerance of double allogeneic fetal antigen of parent of becoming pregnant, help the anemia of pregnant woman to tide over a critical period.
Though the curative effect of the method is more sure, reports that both at home and abroad effective percentage reaches 70%-95%, but still have the following disadvantages, thereby limited its clinical practice: (1) treatment is bigger with the preparation difficulty of immunocyte; (2) interindividual variation is bigger, the stability that affects the treatment; (3) the fresh separated poor stability of immunocyte, therefore, using the technical scheme of immunocyte still is not the ideal scheme of people.
There is data to show, the curative effect no difference of science of statistics that adopts husband or irrelevant male individual lymphocyte to adopt and treat, and the effect of performance tolerance induction is the T cell.This explanation is adopted male's Allogeneic T cell to adopt and is transferred all and may play a role by some the common antigen composition on the different male individual T cells, induces the immunologic tolerance of double allogeneic fetal antigen of parent; And the T cell is secreted a kind of acellular composition film microcapsule structure that is called exosomes that discharges in the growing multiplication process, concentrated the antigenic component of a whole set of T cell self, and can overcome many shortcomings that cell component is treated, might open up the road of a new treatment immune disease.The laboratory that adopts exosome to carry out clinical practice is at present explored, is confined to be applied to oncotherapy as the Biotherapeutics carrier more, and be purpose with the anti-tumor immunological effect that promotes body.But relevant its is used for the treatment of habitual abortion as immunoloigcal tolerance-inducting agent, then do not see bibliographical information as yet, said nothing of the preparation that entered practical treatment.
Summary of the invention
The present invention is used for the deficiency that overcomes prior art thereby a kind of biology preparation of being convenient to the treatment habitual abortion of clinical standard use is provided.
Problem of the present invention realizes with following technical proposals:
A kind of biology preparation for the treatment of habitual abortion, it is a raw material with male's peripheral blood, and is undertaken by following step:
A. cultivate male's periphery blood T cell:
Extract the peripheral blood 50ml of NAM; 1.5 times of dilutions of glass bead defiber Hanks liquid, isolated lymphocytes is collected mononuclearcell; Hanks liquid cleans,, counting resuspended with the RPMI-1640 complete culture solution, and adjust cell concentration to 2 * 10
6/ ml adds phytohaemagglutinin (PHA), and making final concentration is 100 μ g/ml; Cultivate 72h for 37 ℃; Collect the supernatant of culture fluid, 4 ℃ of preservations are standby;
B. prepare T cell exosomes:
Get above-mentioned periphery blood T cell culture supernatant, remove cell and cell debris with the centrifugal 10min of 1500g; After 5 times of the molecular weight 100 000 ultrafiltration pipe filtering and concentrating; With 30mg/ml sucrose/D
2O is the density pad, with 100 000g hypervelocity low-temperature centrifugation 75min, collects the cushion pad of bottom; The PBS dilution, 100 000g hypervelocity low-temperature centrifugation 75min obtains exosomes; 0.22 the aseptic membrane filtration packing of μ m ,-80 ℃ of preservations are standby.
The biology preparation of above-mentioned treatment habitual abortion, described In vitro culture should use the culture fluid of the fresh PHA of containing instead, in external repetitious stimulation, cultivation, all observe conversion ratio down greater than 75% to mirror.
A kind of purposes for the treatment of the biology preparation of habitual abortion can be used for preparing the derivant of immunologic tolerance after the medicament of treatment habitual abortion, autoimmune disease or the organ transplantation.
The invention provides a kind of acellular immunobiology preparation, it can induce the immunologic tolerance of body effectively, has overcome adopt many deficiencies of therapy inducing immune tolerance of immunocyte, makes clinical practice more simple; This preparation can carry out heavy industrialization preparation according to the GMP standard, makes that quality control is more stable, controllability is stronger.Be applied to treat habitual abortion, obtain good curative effect.Biology preparation provided by the invention, its mechanism also can be used for inducing of immunologic tolerance after other autoimmune diseasees, the organ transplantation, or the like.
The specific embodiment
The present invention is divided into two big steps, and the one, the stimulated in vitro of NAM's periphery blood T cell and cultivation, the 2nd, prepare T cell exosomes with centrifugal ultrafiltration and sucrose density gradient centrifugation.
At first extract the peripheral blood 50ml of NAM, the glass bead defiber is after 1.5 times of dilutions, with lymphocyte separation medium separated and collected mononuclearcell; After Hanks (cell cleanout fluid) liquid thoroughly cleans twice, RPMI-1640 (cell culture medium) complete culture solution re-suspended cell, counting is also adjusted cell concentration to 2 * 10
6/ ml, it is 100 μ g/ml that adding phytohaemagglutinin (PHA) makes final concentration; Mirror is observed T cell transformation situation down behind 37 ℃ of cultivation 72h; Use the external repetitious stimulation of culture fluid, the cultivation of the fresh PHA of containing instead.The culture supernatant of collecting can be standby in 4 ℃ of preservations.
Prepare biological preparation then, the T cell (2 * 10 of the stimulated in vitro of learning from else's experience, cultivation
7Individual) culture supernatant, 1500g 10min removes cell and cell debris; Behind 5 times of the molecular weight 100 000 MWCO Centriplus ultrafiltration pipe ultrafiltration and concentration, with 30mg/ml sucrose/D
2O is the density pad, and 100 000g hypervelocity low-temperature centrifugation 75min collects the cushion pad of bottom; PBS (phosphate buffer) dilution, 100 000g hypervelocity low-temperature centrifugation 75min obtains exosomes, the aseptic membrane filtration packing of 0.22 μ m ,-80 ℃ of preservations get product.
The present invention makes the T cell can secrete the film microcapsule structure that discharges a kind of exosomes of being called (allochthon) in the growing multiplication process, compare with the T cell, T cell exosomes is as a kind of acellular composition, have following characteristics: 1. the antigenic component that has concentrated a whole set of T cell self, contain the albumen relevant with self cell specific function (MHC-II quasi-molecule, TCR, integration element etc.), bootable exosomes arrives target cell; 2. limitans is stable, in case obtain to get final product long-time chilled storage, has saved the inconvenient and difficult of fresh separated and long-term cultured; 3. separation and purification are easy, and its operability and controllability are stronger; 4. acellular composition, volume is little, easy-clear, no fertility, a little less than the antigenicity, it is very little to be applied to the human body side effect, stability and high efficiency.
It is quantitative with ultraviolet spectrophotometer to separate the exosomes that obtains, and adopts immuno-electron microscope, Western trace to detect its entrained characteristic molecule (HLA-I, HLA-II, ICAM-1 etc.), as quality control standard.
Product of the present invention is treated URSA patient with the hypodermic vaccination ways of multiple spot, and in each 3 weeks at interval, the back after treatment of becoming pregnant was miscarried month to surpassing in the past.
Clinical efficacy is passed judgment on by following standard:
To miscarry once more greater than miscarried month effectively in the past as treatment; With the term labor normal newborn as treating successfully; With miscarry once more less than the month of in the past miscarrying as treatment failure, can compare with the routine immunization cell therapy of adopting.Consequently pregnant success rate is about 90.0%, and 84.80% pregnant success rate of the transfusion cell method of adopting has remarkable rising.
The present invention also obtains good result in the zoopery that is used for the treatment of URSA, its animal experiment is as follows:
Set up animal model.
Laboratory animal be 8～10 ages in week cleaning level CBA/J (female ♀), DBA/2 (male
) and BALB/c (male
) inbred mouse.With ♀ CBA/J *
DBA/2 is URSA experimental group (bibliographical information embryo Loss Rate is up to 25%～45%), ♀ CBA/J *
BALB/c is normal control group (bibliographical information embryo Loss Rate only is 5%～8%).The male and female mice was by copulation in 2: 1, and from living together certainly, every day, 8:00 and 15:00 observed once cloudy bolt respectively, as saw cloudy bolt, promptly was defined as gestation the 1st day, in pregnant the 12nd～14 day mice was put to death, and observed the fetal development situation.
The preparation of paternal periphery mononuclearcell (PBMC) exosomes.
Get male DBA/2 mice, pluck the eyeball sacrificed by exsanguination, spleen is got in aseptic dissection, and grinding, 200 order copper mesh filter and obtain the individual cells suspension; After the centrifugal abandoning supernatant, add distilled water 1.5mL, equivalent 1.8%NaCl successively, washing back counting, culture fluid is mixed with 2.5 * 10
6/ ml, it is 10 μ g/ml that adding concanavalin A, Con A (ConA) makes final concentration; Mirror is observed T cell transformation situation down behind 37 ℃ of cultivation 72h.
URSA animal model mice is divided at random do not have the treatment matched group, immunocyte is adopted treatment group, exosomes treatment group:
To there not being the treatment matched group: after determining pregnancy, do not carry out any treatment;
To the immunocyte treatment group of adopting: after determining pregnancy, get paternal PBMC and carry out injection for curing;
To exosomes treatment group: after determining pregnancy, the exsomes of paternal PBMC after ConA stimulates that extracts is carried out injection for curing;
The criterion that influences embryo's absorption to the pregnant mouse embryo absorbance is that embryo's volume obviously dwindles, and fetoplacental unit has obviously hemorrhage or bad the dead.Placenta Hominis absorbance=absorption embryo number/(absorbing embryo number+survival embryo number)
Its result is:
Embryo's absorbance that the URSA animal model does not have the treatment matched group is 28.21% ± 2.56%, is significantly higher than normal control group 5.26% ± 0.69%.After traditional immunocyte was adopted treatment, embryo's absorbance 11.40% ± 1.84% significantly reduced.Embryo's absorbance of exosomes treatment group is 9.76% ± 1.27%, and all significantly being lower than miscarriage does not have treatment matched group and the traditional immunocyte treatment group of adopting.Thereby illustrate and adopt preparation can obtain better effect, details see the following form:
Different therapeutic modalities of subordinate list or preparation are to the comparison of mice URSA therapeutic effect (x ± s)
Compare with the normal control group,
*: P＜0.05,
*: P＜0.001,
* *: P＜0.0005
Do not compare with there being the treatment matched group,
#: P＜0.05,
##: P＜0.001
Compare with the immunocyte treatment group of adopting,
$: P＜0.05.
Claims (2)
1. a biology preparation for the treatment of habitual abortion is characterized in that, it is a raw material with male's peripheral blood, and is undertaken by following step:
A. cultivate male's periphery blood T cell:
With the peripheral blood 50ml of NAM 1.5 times of dilutions of glass bead defiber Hanks liquid, isolated lymphocytes is collected mononuclearcell; Hanks liquid cleans,, counting resuspended with the RPMI-1640 complete culture solution, and adjust cell concentration to 2 * 10
6/ ml adds phytohaemagglutinin (PHA), and making final concentration is 100 μ g/ml; Cultivate 72h for 37 ℃; Collect the supernatant of culture fluid, 4 ℃ of preservations are standby;
B. prepare T cell exosomes:
Get above-mentioned periphery blood T cell culture supernatant, remove cell and cell debris with the centrifugal 10min of 1500g; After 5 times of the molecular weight 100000 ultrafiltration pipe filtering and concentrating; With 30mg/ml sucrose/D
2O is the density pad, with 100000g hypervelocity low-temperature centrifugation 75min, collects the cushion pad of bottom; The PBS dilution, 100000g hypervelocity low-temperature centrifugation 75min obtains exosomes; 0.22 the aseptic membrane filtration packing of μ m ,-80 ℃ of preservations are standby.
2. the biology preparation of treatment habitual abortion according to claim 1, it is characterized in that, in described a. step, after adopting 37 ℃ to cultivate 72h, use culture fluid, repetitious stimulation, the cultivation of the fresh PHA of containing instead, to mirror, observe T cell transformation rate greater than 75%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100624876A CN101129405B (en) | 2007-07-31 | 2007-07-31 | Biological formulation product for treating habitual abortion |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100624876A CN101129405B (en) | 2007-07-31 | 2007-07-31 | Biological formulation product for treating habitual abortion |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101129405A CN101129405A (en) | 2008-02-27 |
| CN101129405B true CN101129405B (en) | 2011-05-11 |
Family
ID=39126915
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2007100624876A Expired - Fee Related CN101129405B (en) | 2007-07-31 | 2007-07-31 | Biological formulation product for treating habitual abortion |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101129405B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2556825C1 (en) * | 2014-09-15 | 2015-07-20 | Федеральное государственное бюджетное учреждение науки Институт химической биологии и фундаментальной медицины Сибирского отделения Российской академии наук (ИХБФМ СО РАН) | Method of extraction of exosomes from blood |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111254111A (en) * | 2020-02-29 | 2020-06-09 | 贵州医科大学 | Cell for targeted therapy of habitual abortion and immune infertility and preparation method thereof |
| CN113082053B (en) * | 2021-04-06 | 2022-05-24 | 武汉济源高科技有限公司 | Kit for treating habitual abortion caused by reduction or deletion of blocking antibody |
| CN114591908B (en) * | 2022-04-18 | 2023-11-21 | 上海交通大学医学院附属新华医院 | Stimulating factor, culture medium and method for inducing Tregs to obtain embryo antigen specificity iTregs and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1265038A (en) * | 1997-07-16 | 2000-08-30 | 法国国家卫生及研究医学协会 | Cellular vesicle called 'exosome', prepn. and use thereof in immune stimulation |
| CN1275618A (en) * | 2000-06-21 | 2000-12-06 | 张国庆 | Method for cultivating T-lymphocytes |
| CN1566331A (en) * | 2003-06-27 | 2005-01-19 | 北京天润善达生物科技有限责任公司 | Serum-free culture medium for lymphocyte |
-
2007
- 2007-07-31 CN CN2007100624876A patent/CN101129405B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1265038A (en) * | 1997-07-16 | 2000-08-30 | 法国国家卫生及研究医学协会 | Cellular vesicle called 'exosome', prepn. and use thereof in immune stimulation |
| CN1275618A (en) * | 2000-06-21 | 2000-12-06 | 张国庆 | Method for cultivating T-lymphocytes |
| CN1566331A (en) * | 2003-06-27 | 2005-01-19 | 北京天润善达生物科技有限责任公司 | Serum-free culture medium for lymphocyte |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2556825C1 (en) * | 2014-09-15 | 2015-07-20 | Федеральное государственное бюджетное учреждение науки Институт химической биологии и фундаментальной медицины Сибирского отделения Российской академии наук (ИХБФМ СО РАН) | Method of extraction of exosomes from blood |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101129405A (en) | 2008-02-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102586182A (en) | Preparation and application of human-stem-cell-secreted bioactive factor and lysis solution | |
| CN101310751B (en) | Detection method of medicine composition for replenishing qi and blood | |
| CN103655257A (en) | Chinese medicine hair nourishing shampoo and preparation method thereof | |
| EP3031463A1 (en) | Method of preparing injection solution | |
| CN101129405B (en) | Biological formulation product for treating habitual abortion | |
| CN103405577B (en) | The Pharmaceutical composition of a kind of enhancing immunity, fatigue alleviating | |
| CN1785055A (en) | Health-care food with function for improving immunity and its prepn. method | |
| CN107098984A (en) | Root of bidentate achyranthes Thick many candies, polysaccharide component and homogeneous polysaccharide, Its Preparation Method And Use | |
| CN110025769A (en) | The combination of stem cell and cell factor and its in the purposes for improving sperm motility | |
| CN109731002A (en) | Hematopoietic stem cell mobilization agent and its application | |
| CN102755512A (en) | Traditional Chinese medicine extract capable of improving CIK cell proliferation rate as well as preparation method and application of same | |
| CN101322810A (en) | Chinese traditional medicine composition for treating diabetes respiratory tract infection | |
| CN100497370C (en) | Bursopoietin extracting method and its use in disease treating and immune | |
| CN1663596A (en) | Suppository for treating chronic cervicitis | |
| CN105920044B (en) | Hydra stem cell preparation as well as preparation method and application thereof | |
| CN100381164C (en) | Medicine for treating radiation diseases | |
| CN102319322A (en) | Radioprotective medicament | |
| CN102406172B (en) | Health-care food for enhancing immunity and preparation method thereof | |
| CN105663684A (en) | Medicine for treating aplastic anemia | |
| CN1047527C (en) | Chinese herb medicine instant granule for preventing and curing cardiovascular and cerebrovascular diseases | |
| JP6495482B2 (en) | Composite material, preparation method and apparatus for preparing particle energy multifunctional active water | |
| CN103845524B (en) | It is a kind of to treat the Chinese medicine composition and preparation method and purposes that kidney essence deficiency is demonstrate,proved after tumor radiotherapy, chemotherapy | |
| TW201819623A (en) | Composition and method for decreasing bilirubin level | |
| EP0506773A1 (en) | A stimulator of vascular endothelial cells and use thereof. | |
| TW201829769A (en) | Use of somatic stem cells for decreasing il-6 level |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20190603 Address after: 050081 No. 450 Zhongshan West Road, Shijiazhuang City, Hebei Province Co-patentee after: Cui Cheng Patentee after: NONCOMMISSIONED OFFICER ACADEMY OF ARMY MEDICAL University Address before: 050081 Educational Services Department, Training Department, Bethune Military Medical College, 450 Zhongshan West Road, Shijiazhuang City, Hebei Province Patentee before: Cui Cheng |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200703 Address after: 050081 No. 450 West Zhongshan Road, Hebei, Shijiazhuang Patentee after: NONCOMMISSIONED OFFICER ACADEMY OF ARMY MEDICAL University Address before: 050081 No. 450 West Zhongshan Road, Hebei, Shijiazhuang Co-patentee before: Cui Cheng Patentee before: NONCOMMISSIONED OFFICER ACADEMY OF ARMY MEDICAL University |
|
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20110511 |