CN103385899B - The purposes of Chinese medicine compound pharmaceutical composition - Google Patents
The purposes of Chinese medicine compound pharmaceutical composition Download PDFInfo
- Publication number
- CN103385899B CN103385899B CN201310313637.1A CN201310313637A CN103385899B CN 103385899 B CN103385899 B CN 103385899B CN 201310313637 A CN201310313637 A CN 201310313637A CN 103385899 B CN103385899 B CN 103385899B
- Authority
- CN
- China
- Prior art keywords
- parts
- umbilical cord
- ovum
- stem cell
- crude drug
- 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.)
- Active
Links
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses crude drug containing following weight proportioning preparing Abortion embryo umbilical cord perivascular stem cell directional is divided into purposes in the induced liquid of class ovum: Radix Cyathulae 1-16 part, Colla Plastri Testudinis 1-16 part.The invention also discloses induced liquid and method of inducing differentiation that Abortion embryo umbilical cord perivascular stem cell directional is divided into class ovum.Induced liquid of the present invention can induce Abortion embryo umbilical cord perivascular differentiation of stem cells to be stack of eggs cell, and potential applicability in clinical practice is good.
Description
Technical field
The present invention relates to a kind of novelty teabag of Chinese medicine compound pharmaceutical composition, particularly, is be divided into purposes in the induced liquid of class ovum preparing Abortion embryo umbilical cord perivascular stem cell directional.
Background technology
In recent years, because the factors such as environmental pollution is serious, operating pressure increasing, dietary habit change, reproductive system disease cause adverse effect to normally becoming pregnant, Sterility patient quantity gains momentum, and sickness rate is about 12.5% ~ 15%, and has and increase trend year by year.Therefore World Health Organization (WHO) (WHO) announces three large principal diseases infertility and cardiovascular diseases, tumor being listed as nowadays influence human lives and health.Since global the first test-tube baby in 1978 is born, the introducing of the auxiliary procreation technology (ART) being representative with IVF-ET cycle (IVF-ET) provides larger possibility to the successful treatment of infertility.The correlational study of gravidity assisting has the meaning of reality for the practical problem solving sterility and infertility patient.
External fertilization (IVF) refers to that mammiferous sperm and ovum complete the technology of fertilization process in vitro in manually operated environment.At present, external fertilization comprises induced ovulation, follicle detects and get ovum, preferred and process and the external fertilization step of sperm.The induced ovulation bringing out multiple follicular development is the important foundation of infertile treatment, this link early time use ovulation free period, but generally only have a follicle maturity each free period, there is provided ovum few, step difficulty after causing implementing, and pregnancy rate is low, now replace by super ovulation techniques.Super ovulation techniques refers to that application mankind promoting sexual gland hormone promotes a kind of technology of more follicle maturitys ovulation.But grow because this process is multiple simultaneously, estradiol increases, lutropin (LH) peak of feedback is caused to occur, cause ovum precocious, of poor quality, pregnancy rate is low, and excessively uses the complication caused because of medicine, as abnormal in ovarian hyperstimulation syndrome (OHSS), multiple pregnancy, birth defect and table genetic modification etc.
Kidney-nourishing tcm drug gets involved auxiliary procreation technology, has regulating menstruation, gravidity assisting, urgees follicular development and ovulation, raising oocyte quality and improves ovum fertilization rate, promotes to be confirmed the effect of early embryonic development.The above-mentioned effect of kidney-nourishing tcm drug is relevant with the factor such as hormone, cytokine.Link mainly Ovarian hyperstimulation and embryo transfer comparatively is closely contacted with kidney-nourishing tcm drug at present in auxiliary procreation technology.Research main with oocyte quality, external fertilization and fetal development, endometrium receptivity for point of penetration.
Bolus as a Kidney-Yin-Tonic and Yougui Wan, the kidney-Yang-Reinforcing Bolus come from " new eight gusts, the side of Jing Yue's complete work " of Ming Dynasty temperature compensation famous expert Zhang Jingyue, are the classics side of benefiting kidney-YIN, kidney yang.There are some researches show that Bolus as a Kidney-Yin-Tonic can promote embryonic stem cell proliferation, maintain its undifferentiated state, retardance embryonic stem cell apoptosis (Hu Bing, peace Fructus Mume, the Chinese TCM basis medical journal such as Shi Yunfeng the 13rd volume the 5th phase 365-367 in 2007).Also bibliographical information Serum Containing Zuogui Pill Substances is had at mescenchymal stem cell (mesenchymalstemcells, MSCs) can promote that MSCs breeds in chondrocyte directed differentiation process, and improve II Collagen Type VI and proteoglycan gene expression (Bolus as a Kidney-Yin-Tonic is on Chinese combination of Chinese and Western medicine magazine December the 31st volume the 12nd phase 1662-1668 in 2011 such as Xu Lingxiao Wang Fang Guo Dun is bright of affecting of II Collagen Type VI in mesenchymal stem cells into chondrocytes atomization and proteoglycan gene expression).Bolus as a Kidney-Yin-Tonic Aqueous extracts can improve within the scope of finite concentration rat bone marrow mesenchymal stem cells in-vitro multiplication ability (Bolus as a Kidney-Yin-Tonic SD rat bone marrow mesenchymal stem cells is bred affect Tan Feng, the 145-148 in July, 2011 Chinese experimental pharmacology of Chinese medical formulae magazine the 17th volume the 13rd phase such as Fan Qiaoling, Wang Mingyan).To sum up, compound Chinese medicine for reinforcing kidney Bolus as a Kidney-Yin-Tonic can promote the propagation of embryonic stem cell and mescenchymal stem cell, and affects the hepatocellular collagen of mesenchyme, proteoglycan gene expression, has no the report affecting differentiation of stem cells about it.
Summary of the invention
Technical scheme of the present invention there is provided a kind of novelty teabag of Chinese medicine compound pharmaceutical composition.Particularly, be purposes in class ovum for the preparation of induction Abortion embryo umbilical cord perivascular differentiation of stem cells.
Abortion embryo umbilical cord perivascular stem cell, FirstTrimester-derivedPerivascularCells, is called for short FTM-PVCs, derives from Abortion embryo umbilical cord, there is the ability of very strong self duplication, polytype cell can be induced to differentiate into.
Class ovum, refers to have ovum spline structure, expresses the cell of ovum mark.
The crude drug that the present invention contains following weight proportioning is divided into purposes in the induced liquid of class ovum preparing Abortion embryo umbilical cord perivascular stem cell directional: Radix Cyathulae 1-16 part, Colla Plastri Testudinis 1-16 part.
Preferably, the weight proportion of described crude drug is: Radix Cyathulae 9 parts, Colla Plastri Testudinis 12 parts.
Wherein, also containing Radix Rehmanniae Preparata 18-30 part, Fructus Corni 9-12 part, Rhizoma Dioscoreae 8-16 part, Fructus Lycii 9-12 part, Semen Cuscutae 8-16 part, Colla cornus cervi 8-16 part in described crude drug.
Preferably, Radix Rehmanniae Preparata is 24 parts, Fructus Corni is 9-12 part, Rhizoma Dioscoreae is 12 parts, Fructus Lycii is 9-12 part, Semen Cuscutae is 12 parts, Colla cornus cervi is 12 parts.
Further preferably, Radix Rehmanniae Preparata is 24 parts, Fructus Corni is 12 parts, Rhizoma Dioscoreae is 12 parts, Fructus Lycii is 12 parts, Semen Cuscutae is 12 parts, Colla cornus cervi is 12 parts.
Abortion embryo umbilical cord perivascular stem cell directional of the present invention is divided into the induced liquid of class ovum, and it is culture fluid based on MEME culture fluid, is added with the Contained Serum of 50IU/ml penicillin, 50mg/ml streptomycin and 2 ~ 20% (v/v); Described Contained Serum is prepared as follows:
(1) get crude drug according to aforementioned proportioning, preparation becomes micropowder, and be applied to Mus with the dosage of 5.0g/kg, method of application is oral administration;
(2) after administration 30min, 60min, 90min, eyeground vein clump gets blood, leaves standstill respectively, centrifugal supernatant, and mixing, is Contained Serum.
The concentration of described penicillin is 50IU/ml, the concentration of described streptomycin is 50mg/ml, and the concentration of described Contained Serum is 5 ~ 10% (v/v).
In step (2), time of repose is 2h, and centrifugal rotational speed is 3000rpm, and centrifugation time is 10min.
The present invention induces Abortion embryo umbilical cord perivascular stem cell directional to be divided into the method for class ovum, comprises the steps:
A, get Abortion embryo umbilical cord perivascular stem cell;
B, be placed in aforesaid induced liquid and cultivate, incubation time is not less than 5 days, obtains class oocyte.
In step b, incubation time is 5 ~ 24 days.
Compound Chinese medicine for reinforcing kidney Bolus as a Kidney-Yin-Tonic of the present invention and the side's of tearing open component thereof can promote that Abortion embryo umbilical cord perivascular stem cell (FTM-PVCs) is divided into class ovum, significant, are the developing of test-tube baby field new approaches, new way.
Obviously, according to foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, not departing under the present invention's above-mentioned basic fundamental thought prerequisite, the amendment of other various ways, replacement or change can also be made.
The detailed description of the invention of form by the following examples, is described in further detail foregoing of the present invention again.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following example.All technology realized based on foregoing of the present invention all belong to scope of the present invention.
Accompanying drawing explanation
Fig. 1 can induce people FTM-PVCs to occur class ovarian cumulus-ovum structure, amplification 10 × 10 containing No. 3 medicine Mus serum.A. 2%, 5%, 10%, 20% is cultivated after 5 days containing No. 3 medicine Mus serum group morphocytologyes changes; B. 2%, 5%, 10%, 20% is cultivated after 17 days containing No. 3 medicine Mus serum group morphocytologyes changes;
Fig. 2 can induce people FTM-PVCs faint expression ovum mark, amplification 20 × 10 containing No. 3 medicine Mus serum.A.Positivecontrol group; B.5% ovum mark is expressed containing No. 3 medicine Mus serum group; C.10% ovum mark is expressed containing No. 3 medicine Mus serum group; D.5% blank Mus serum group does not express ovum mark;
Fig. 3 can induce people FTM-PVCs to occur class ovum spline structure, amplification 10 × 10 containing No. 5 medicine Mus serum.A. 5% is cultivated after 5 days containing No. 5 medicine Mus serum group cell aggregationes and with border is obvious around; B. to cultivate after 17 days 5% agglomerating containing No. 5 medicine Mus serum group cell aggregationes, edge has network structure to occur;
Figure 45 % can induce people FTM-PVCs to express ovum mark, amplification 20 × 10 containing No. 5 medicine Mus serum.A.control group; B.5% ovum mark is expressed containing No. 5 medicine Mus serum group; C.5% blank Mus serum group does not express ovum mark.
Detailed description of the invention
The preparation of embodiment 1 medicine of the present invention
Get Radix Cyathulae 9g, Colla Plastri Testudinis 12g, be ground into fine powder, mixing of sieving, dry sealing and preserve, be watered and take, or every 100g powder adds refined honey 10g and appropriate water.
The preparation of embodiment 2 medicine of the present invention
Get Radix Cyathulae 1g, Colla Plastri Testudinis 1g, prepare by embodiment 1 method.
The preparation of embodiment 3 medicine of the present invention
Get Radix Cyathulae 16g, Colla Plastri Testudinis 16g, prepare by embodiment 1 method.
The preparation (Bolus as a Kidney-Yin-Tonic) of embodiment 4 pharmaceutical composition 1 of the present invention
Take raw material: Radix Rehmanniae Preparata 200g, Semen Cuscutae 100g, Radix Achyranthis Bidentatae 75g, Colla Plastri Testudinis 100g, Colla cornus cervi 100g, Rhizoma Dioscoreae 100g, Fructus Corni 100g, Fructus Lycii 100g;
Preparation method: above eight tastes, except Colla cornus cervi, Colla Plastri Testudinis, the Six-elements such as all the other Radix Rehmanniae Preparata are ground into fine powder, mixing of sieving.Get Colla cornus cervi, Colla Plastri Testudinis molten, mix with above-mentioned fine powder.Every 100g powder adds refined honey 10g and appropriate water, general ball, dry, to obtain final product; Or do not add honey, be only powder powder.
The preparation of embodiment 5 pharmaceutical composition 1 of the present invention
Take raw material: Radix Rehmanniae Preparata 18g, Semen Cuscutae 8g, Radix Achyranthis Bidentatae 1g, Colla Plastri Testudinis 1g, Colla cornus cervi 8g, Rhizoma Dioscoreae 8g, Fructus Corni 9g, Fructus Lycii 9g;
Prepare according to the method for embodiment 4.
The preparation of embodiment 6 pharmaceutical composition 1 of the present invention
Take raw material: Radix Rehmanniae Preparata 30g, Semen Cuscutae 12g, Radix Achyranthis Bidentatae 16g, Colla Plastri Testudinis 16g, Colla cornus cervi 16g, Rhizoma Dioscoreae 16g, Fructus Corni 12g, Fructus Lycii 16g;
Prepare according to the method for embodiment 4.
Above-mentioned raw materials can use crude drug in whole, also can use the processed product of crude drug in whole.
Below by effect experiment and clinical trial certificate beneficial effect of the present invention.
Explanation of nouns:
FTM-PVCs:FirstTrimester-derivedPerivascularCells Abortion embryo umbilical cord perivascular stem cell
FSH:follicle-stimulatinghormone follicle stimulating hormone
LH:LuteinizingHormone lutropin
E2:Estradiol estradiol
FF:Follicularfluid follicular fluid
GDF-9:Growthdifferentiationfacter9 growth differentiation factor 9
GDF-9B:Growthdifferentiationfacter9B growth differentiation factor 9 B
SCP1:SynaptonemalComplexProtein1 synaptonemal complex protein 1
SCP2:SynaptonemalComplexProtein2 synaptonemal complex protein 2
SCP3:SynaptonemalComplexProtein3 synaptonemal complex protein 3
ZP-1:zonapellucidaglycoprotein1 Zona Pellucida Glycoprotein 1
ZP-2:zonapellucidaglycoprotein2 Zona Pellucida Glycoprotein 2
ZP-3:zonapellucidaglycoprotein3 Zona Pellucida Glycoprotein 3
Experimental example 1 induced liquid induction of the present invention FTM-PVCs is divided into class ovum
1, experiment material
1.1 experimental apparatus
Abortion embryo umbilical cord perivascular stem cell, i.e. gestation period 1 umbilical cord tissue cell (Humanfirsttrimesterumbilicalcordcells), purchased from CReATe unbilical blood bank.
Ultrasonic cleaner (Ultrasoniccleaner): Kunshan Ultrasonic Instruments Co., Ltd., KQ5200B;
Electronic balance (Electronicbalance): Mettler Toledo Inc. of Switzerland, AB265-5;
Water purification machine (Ultra-PureWaterProcessor): the general company of middle national best, UPR-IV-10T;
Ultrapure water system (Ultra-purewatersystem): ELGA company of Britain, UHQ2;
CO2 cell culture incubator (CO2CellCultureIncubator): ThermoScientific company of the U.S., 3111;
Double one side superclean bench (Double-personSingle-faceSuper-cleanBench): Chinese Suzhou purification company, SW-CJ-2FD;
Long ultraviolet light/the visible spectrophotometer (FullRangeWavelengthUV/VisSpectrophotometer) of all-wave: Beckmancoulter company of the U.S., model: DU730;
Regular-PCR instrument (GeneralPCRSystem): MJResearch company of the U.S., PTC-200;
CO2 concentration detector (CO2ConcentrationDetector): German Labotect company, InControl1050;
Just putting fluorescence microscope (UprightFluorescenceMicroscope): German Leica company, DM4000B;
Inverted microscope (InvertedMicroscope): German Leica company, DMI3000B;
Inverted microscope (InvertedMicroscope): Japanese Olympus company, CK-40;
Artificial insemination (IVF) work station (IVFWorkingStation): Labogene company of Denmark, F-1800;
-80 DEG C of ultra cold storage freezers (-80 DEG C of Ultra-lowTemperatureFreezer): Japanese Sanyo company, MDF-U73C;
-40 DEG C of cryogenic refrigerators (-40 DEG C of LowTemperatureFreezer): Japanese Sanyo company, MDF-U5412;
4 DEG C of refrigerator-freezers (4 DEG C of Refrigerator): Japanese Sanyo company, MPR-1411R;
Large-scale tabletop refrigerated centrifuge (Large-sizedDesktopCentrifugewithRefrigerator): ThermoScientific company of the U.S., SorvallBiofugeStratos;
Tabletop refrigerated centrifuge (DesktopCentrifugewithRefrigerator): U.S. ThermoScientific, LegendMicro17R;
Desk centrifuge (DesktopCentrifuge): ThermoScientific company of the U.S., Micromax;
Stereomicroscope (StereoMicroscope): Japanese Olympus company, SZX10;
Electrophoresis system (ElectrophoresisSystem): Bio-Rad company of the U.S., MP4;
Wash trigger (MicroplateWasher): Bio-Tek company of the U.S., ELx-50;
Cultivate shaking table (Culturingshakingincubator): Chinese Shanghai Zhi Cheng company, ZHWY-111B;
Large Copacity liquid nitrogen container (LargeVolumeLiquidNitrogenCanister): Chinese Chengdu Jinfeng company, YDS-47-127;
Large Copacity liquid nitrogen container (Largevolumeliquidnitrogencanister): MVE company of the U.S., XC47/11-10;
High-pressure sterilizing pot (Autoclaver): Japanese SANYO company, MLS-3020;
The long microplate reader of all-wave (FullWavelengthMicroplateReader): ThermoScientific company of the U.S., Multiskan;
Visible/ultraviolet gel imaging system (Vis/UVGel-ImagingSystem): Bio-Rad company of the U.S., XR;
Accurate pH meter (pHMeter): German Sartorius company, PP-15;
Constant temperature roaster: the upper grand experimental facilities company limited of Nereid, DHG-9053A;
Thermostat water bath: Chengdu, Sichuan Sai Kelong plant equipment company limited, esun-0048;
Constant temperature blender with magnetic force: Mei Ying Pu, Shanghai instrument and meter Manufacturing Co., Ltd, H01-1A;
The manual single track adjustable pipette of 0.1-2.5ul: eppendorf;
The manual single track adjustable pipette of 0.5-10ul: eppendorf;
The manual single track adjustable pipette of 10-100ul: eppendorf;
The manual single track adjustable pipette of 20-200ul: eppendorf;
The manual single track adjustable pipette of 100-1000ul: eppendorf;
The manual single track adjustable pipette of 50-5000ul: eppendorf;
The manual single track adjustable pipette of 100-10000ul: eppendorf;
30-300ul8 road adjustable pipette: eppendorf;
10-100ul8 road adjustable pipette: eppendorf.
1.2 experiment material
Physiological saline solution: Kelun Pharm Ind Co., Ltd., Sichuan
I-type collagen enzyme: Sigma-Aldrich, St.Louis, USA
Hyclone (FetalBovineSerum, FBS): Gibco-BRL, MD, USA
MEME culture medium (MinimumEssentialMediumEagle): AlphaModification, Sigma-Aldrich, M4526
EB culture medium (SCM018EmbryoidBodyFormationMedium): millipore, MD, USA
PBS: Chengdu Harry is biological
Antibiotic (Penicillin-Streptomycin): Gibco-BRL, MD, USA
0.05% trypsin-0.01%EDTA:Gibco-BRL, MD, USA
Low adhesion culture plate: STEMCELLTechnologies, BC, Canada
Six porocyte culture plates (6wellcellcultureplate), 12 porocyte culture plate (12wellcellcultureplate): Labserv
Culture dish (60mm, 100mm): Corningcostar
Vent cap 75cm2,25cm2 culture bottle: Corningcostar, NUNC
15ml conical centrifuge tube: Corningcostar
50ml conical centrifuge tube: Corningcostar
The disposable cryopreservation tube of 2ml: Corningcostar, NUNC
Coverslip: 24 × 24mm, Citotest Labware Manufacturing Co., Ltd.
Disposable graduated plastic's suction pipe 2ml:Corningcostar, Axygen
Disposable graduated plastic's suction pipe 5ml, 10ml, 25ml:Corningcostar, Axygen
Disposable graduated plastic's suction pipe 50ml:KIMBLE
Octal, four holes, two porocyte cell (8wellglassslide, 4wellglassslide, 2wellglassslide): Lab-Tek
The packed suction nozzle of 10ul: Axygen
The packed suction nozzle of 200ul: Axygen
The aseptic box-packed suction nozzle of 200ul: Axygen
The aseptic box-packed suction nozzle of 10ul: Axygen
The packed suction nozzle of 1000ul: Axygen
The aseptic box-packed suction nozzle of 1000ul: Axygen
0.5ml centrifuge tube: Axygen
1.5ml centrifuge tube: Axygen
0.5ml light-wall pipe [flat cover]: Axygen
Primary antibodie: rabbitanti-OCT-4 (1:200, Abcam), rabbitanti-Nanog (1:100, SantaCruzBiotechnology), mouseanti-Fragilis/IFITM3 (1:100, SantaCruzBiotechnology), rabbitanti-SCP1 (1:200, CellSignalingTechnology), rabbitanti-VASA (1:100, SantaCruzBiotechnology), rabbitanti-DAZL (1:100, SantaCruzBiotechnology), rabbitanti-STELLAR (1:200, Abcam), rabbitanti-SCP3 (1:200, CellSignalingTechnology), rabbitanti-GDF-9 (1:100, Abcam), rabbitanti-GDF-9B (1:200, SantaCruzBiotechnology), rabbitanti-ZP1 (1:200, SantaCruzBiotechnology), rabbitanti-ZP2 (1:100, Sigma), rabbitanti-ZP3 (1:200, SantaCruzBiotechnology)
Two resist: goat antirabbit (HRP, the green skies), goat anti-mouse (HRP, the green skies), blue DyLight anti-rabbit (SantaCruzBiotechnology), green FITC anti-rabbit (SantaCruzBiotechnology)
Hoechst33258: the green skies
Paraformaldehyde: Ming Rui bio tech ltd, Shanghai
TxitonX-100：Amresco
H2O2: Chengdu Ke Long chemical reagent factory
HRP-DAB substrate colour reagent box: the green skies
Haematoxylin: the green skies
DMSO analytical pure: the special chemical drugs company limited in Rui Jin, Tianjin
Plug-in type Tissue Culture Dish: millipore, 0.4 μm
0.22 μm of filter membrane: millipore
Injection high-purity menotrophin (HMG): FerringGmbH
Estradiol (E2, Estradiol): CaymanChemicalCompany
Folic acid (Folicacid): Sigma
Dehydrated alcohol: Chengdu Ke Long chemical reagent factory
Isopropyl alcohol: Chengdu Ke Long chemical reagent factory
Chloroform: Chengdu Ke Long chemical reagent factory
Anti-fluorescent quenching mounting liquid: the green skies
Primer: Shanghai Jierui Biology Engineering Co., Ltd, prompt base (Shanghai) trade Co., Ltd in the English Weihe River
reagents：Macherey-Nagel
People's estradiol (E2) enzyme linked immunological kit: SanAntonio, TX, USA
Mineral oil (oilfortissueculture): InVitroFertilization, Inc, Trumbull
HTF(Quinns’sAdvantageFertilization):In-VitroFertilization，SAGE
Human albumin succedaneum (Quinns ' sAdvantageTMSpsSerumProteinSubstitute): In-VitroFertilization, SAGE, USA
Six orifice plate Dual culture cell (6wellmillicellHangingCellCultureInserts): Millipore
5ml, 10ml disposable syringe: two Columba livia
CDNA test kit (RevertAidTMHMinusFirstStrandcDNASynthesisKit): FermentasLifeSciences
One step RT-PCR test kit: FermentasLifeSciences
Agarose gel: BiowestAgarose
Goldview: match Parkson
reagents：Macherey-Nagel
TAE buffer: Tris, Na2EDTA.2H2O, glacial acetic acid
Eye scissors, ophthalmology are taken the photograph, operating scissors, aseptic cotton balls, 75% ethanol etc.
Bolus as a Kidney-Yin-Tonic compound recipe, Yougui Wan, the kidney-Yang-Reinforcing Bolus compound recipe, left Yougui Wan, the kidney-Yang-Reinforcing Bolus have Chinese medicine, the exclusive Chinese medicine micropowder of Bolus as a Kidney-Yin-Tonic: greenery pharmacy group.
2, experimental technique
The 2.1 all kinds of preparations containing kidney-nourishing tcm drug Mus serum
SD rat, cleaning grade, 6 ~ 8 week age, body weight 220 ~ 250g, ♀ ♂ dual-purpose, each 15, credit number: SCXK (river) 2004-15; Institute of lab animals of Sichuan Academy of Medical Sciences provides.Animal feeding is in cleaning grade animal housing, and temperature 22 ± 2 DEG C, humidity 40 ~ 70%, 10h/14h light and shade alternately, once, feed every 3min automatic air-exchanging, and freely drink water, ♀ ♂ separately raises in rustless steel rearging cage by conventional full-valence pellet feed.
According to previous experiments result, at random 30 rats are divided into 3 groups when body weight is homogeneous: be respectively blank group, No. 3 medicine groups, No. 5 medicine groups, often organize 10, male and female half and half.Blank group rats gavaged distilled water, Bolus as a Kidney-Yin-Tonic prescription Chinese medicine mixing micropowder (is prepared according to the method for embodiment 4, be called for short No. 3 medicines, containing Radix Rehmanniae Preparata, Rhizoma Dioscoreae, Fructus Corni, Fructus Lycii, Semen Cuscutae, Colla cornus cervi, Radix Cyathulae, Colla Plastri Testudinis), Bolus as a Kidney-Yin-Tonic prescription exclusive Chinese medicine mixing micropowder (prepared according to the method for embodiment 1, be called for short No. 5 medicines, containing Radix Cyathulae, Colla Plastri Testudinis), dosage is 5.0g/kg, and administration capacity is 20ml/kg.Get blood respectively at 30min, 60min, 90min eyeground vein clump after administration, blood left standstill after 2 hours, and the centrifugal 10min of 3000rpm, is separated each group of variant time period serum, by for subsequent use for the serum mixing often organizing three time periods.
Immersion method, L-L extraction, solid phase extraction, precipitation of protein is compared through trial test, choose precipitation of protein and carry out blood serum sample pre-treatment, namely rat blood serum 150 μ l is got, add perchloric acid-methanol solution (15 μ l50% perchloric acid+5 μ l methanol), vortex oscillation 2min, then the centrifugal 10min of 20000rpm, gets supernatant and does efficient liquid phase chromatographic analysis as test liquid, by analysis result, proportionately swarming prepares pastille rat blood serum.All separating obtained serum all uses the filtering with microporous membrane of 0.22 μm degerming, and-20 DEG C of preservations are stand-by.
2.2 variable concentrations break up containing No. 3, No. 5 medicine Mus serum inducing in vitro human FTM-PVCs
According to above-mentioned experimental result, regulation experiment scheme: people FTM-PVCs is inoculated in 8 porocyte cells, inoculum density is 2 ~ 3 × 10
3cells/cm2, MEME culture fluid is containing 10%FBS, 50IU/ml penicillin, 50mg/ml streptomycin.
After cell attachment, with as in following table 1 culture fluid cultivate, every 3 days replacing culture fluid once, by each group of culture fluid at every turn changed with aseptic 1.5ml disposable plastic centrifuge tube be stored in-20 DEG C to be measured, Microscopic observation is often organized morphocytology and is changed and take pictures.After each group of people FTM-PVCs cultivates 24 days at different conditions, the expression of immuno-fluorescence assay ovum mark GDF-9.
Table 1 divides into groups and culture fluid composition
The each group of cell culture fluid that different time sections is collected is centrifugal with 3000 × g, detects the content of E2 in culture fluid with enzyme linked immunological kit.In strict accordance with enzyme linked immunological kit description operation, in microplate reader 450nm absorbance, read OD value, make standard curve according to the concentration of standard substance and OD value, calculate
3, experimental result
The result of No. 3.13 medicine induction people FTM-PVCs
3.1.1 morphological change
As shown in Table 2 and Figure 1:
There is morphological change in table 23 medicine Mus Serum-induced FTM-PVCs
Can find out, No. 3 medicines of variable concentrations, for cultivating FTM-PVCs, all there will be ovum spline structure after 5 days---and, there is granular cell spline structure in 17 days in agglomerating gathering, wherein, when concentration is 20%, phenomenon is the most obvious.
3.1.2 the expression of ovum mark GDF-9
As shown in Table 3 and Figure 2:
Table 33 medicine Mus Serum-induced FTM-PVCs expresses GDF-9
| Group | The expression of GDF-9 | Corresponding picture |
| Blank Mus serum group (5%) | Do not express | Fig. 2 D (5%blank) |
| Positive controls | Express | Fig. 2 A (positive control) |
| No. 3 medicine groups (5%) | Express | Fig. 2 B (5%3#) |
| No. 3 medicine groups (10%) | Express | Fig. 2 C (10%3#) |
Can find out, when cultivating FTM-PVCs, variable concentrations of the present invention No. 3 medicines are identical with positive controls, and cellular expression ovum mark GDF-9, blank Mus serum group then can not express GDF-9.
The result of No. 3.25 medicine induction people FTM-PVCs
3.2.1 morphological change
As shown in table 4 and Fig. 3:
There is morphological change in table 45 medicine Mus Serum-induced FTM-PVCs
Can find out, No. 5 medicines of variable concentrations were cultivated after FTM-PVCs5 days, and cell all there will be ovum spline structure---and, there is network structure in 17 days in agglomerating gathering, wherein, when concentration is 5%, phenomenon is the most obvious.
3.2.2 the expression of ovum mark GDF-9
As shown in table 5 and Fig. 4:
Table 55 medicine Mus Serum-induced FTM-PVCs expresses GDF-9
| Group | The expression of GDF-9 | Corresponding picture |
| Blank Mus serum group (5%) | Do not express | Fig. 4 C (5%blank) |
| Negative control group | Do not express | Fig. 4 A (control) |
| No. 5 medicine groups (5%) | Express | Fig. 4 B (5%5#) |
Can find out, No. 5 medicines are for cultivating FTM-PVCs, and cellular expression ovum mark GDF-9, blank Mus serum group and negative control group then do not express GDF-9.
To sum up, the present invention's No. 3 medicines and No. 5 medicine inducing culture people are after FTM-PVCs3 days, all there is class ovum spline structure in cell, also express ovum mark GDF-9 simultaneously, illustrate that induced liquid of the present invention can induce FTM-PVCs to be divided into class ovum, wherein, the crude drug that the induced liquid containing No. 5 medicines uses is less, and cost is lower.
Medicine of the present invention can induce FTM-PVCs to be divided into class ovum, and can provide raw material for external fertilization technology, overcome the many disadvantages of super ovulation techniques, application prospect is very excellent.
Claims (8)
1. the crude drug of following weight proportioning is divided into purposes in the induced liquid of class ovum preparing Abortion embryo umbilical cord perivascular stem cell directional: Radix Cyathulae 9 parts, Colla Plastri Testudinis 12 parts.
2. the crude drug of following weight proportioning is divided into purposes in the induced liquid of class ovum preparing Abortion embryo umbilical cord perivascular stem cell directional: Radix Cyathulae 9 parts, Colla Plastri Testudinis 12 parts of Radix Rehmanniae Preparata 18-30 parts, Fructus Corni 9-12 part, Rhizoma Dioscoreae 8-16 part, Fructus Lycii 9-12 part, Semen Cuscutae 8-16 part, Colla cornus cervi 8-16 part.
3. purposes according to claim 2, is characterized in that: in described crude drug, and Radix Rehmanniae Preparata is 24 parts, Fructus Corni is 9-12 part, Rhizoma Dioscoreae is 12 parts, Fructus Lycii is 9-12 part, Semen Cuscutae is 12 parts, Colla cornus cervi is 12 parts.
4. purposes according to claim 3, is characterized in that: in described crude drug, and Radix Rehmanniae Preparata is 24 parts, Fructus Corni is 12 parts, Rhizoma Dioscoreae is 12 parts, Fructus Lycii is 12 parts, Semen Cuscutae is 12 parts, Colla cornus cervi is 12 parts.
5. induce Abortion embryo umbilical cord perivascular stem cell directional to be divided into a method for class ovum, it is characterized in that: comprise the steps:
A, get Abortion embryo umbilical cord perivascular stem cell;
B, be placed in induced liquid and cultivate, incubation time is not less than 5 days, obtains class oocyte;
Described induced liquid is culture fluid based on MEME culture fluid, is added with the Contained Serum of 40 ~ 60IU/ml penicillin, 40 ~ 60mg/ml streptomycin and 2 ~ 20%v/v; Described Contained Serum is prepared as follows:
(1) get crude drug according to proportioning described in Claims 1 to 4 any one, preparation becomes micropowder, and be applied to Mus with the dosage of 5.0g/kg, method of application is oral administration;
(2) after administration 30min, 60min, 90min, eyeground vein clump gets blood, leaves standstill respectively, centrifugal supernatant, and mixing, is Contained Serum.
6. method according to claim 5, is characterized in that: in step b, and incubation time is 5 ~ 24 days.
7. method according to claim 5, is characterized in that: the concentration of described penicillin is 50IU/ml, the concentration of described streptomycin is 50mg/ml, and the concentration of described Contained Serum is 5 ~ 10%v/v.
8. method according to claim 5, is characterized in that: in step (2), and the time left standstill is 2h, and centrifugal rotational speed is 3000rpm, and centrifugation time is 10min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310313637.1A CN103385899B (en) | 2013-07-24 | 2013-07-24 | The purposes of Chinese medicine compound pharmaceutical composition |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310313637.1A CN103385899B (en) | 2013-07-24 | 2013-07-24 | The purposes of Chinese medicine compound pharmaceutical composition |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103385899A CN103385899A (en) | 2013-11-13 |
| CN103385899B true CN103385899B (en) | 2016-03-16 |
Family
ID=49530434
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310313637.1A Active CN103385899B (en) | 2013-07-24 | 2013-07-24 | The purposes of Chinese medicine compound pharmaceutical composition |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103385899B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105343414B (en) * | 2014-08-22 | 2019-06-11 | 成都中医药大学 | The new application of Chinese medicine compound pharmaceutical composition |
| CN107034175B (en) * | 2016-02-03 | 2021-07-02 | 成都中医药大学 | New application of wolfberry fruit |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101011553A (en) * | 2006-04-18 | 2007-08-08 | 李运乃 | Chinese traditional medicine preparation for treating female barrenness |
-
2013
- 2013-07-24 CN CN201310313637.1A patent/CN103385899B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101011553A (en) * | 2006-04-18 | 2007-08-08 | 李运乃 | Chinese traditional medicine preparation for treating female barrenness |
Non-Patent Citations (1)
| Title |
|---|
| 促排卵的中医研究进展;尹巧芝等;《中国中西医结合杂志》;20020228;第88卷(第8期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103385899A (en) | 2013-11-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103446382B (en) | The purposes of Chinese medicine compound pharmaceutical composition | |
| CN106701672B (en) | Human adipose-derived mesenchymal stem cell factor and preparation method and application thereof | |
| US10251824B2 (en) | Method for inducing pluripotent stem cells and pluripotent stem cells prepared by said method | |
| CN105132369B (en) | A kind of derivant and inducing culture that mescenchymal stem cell is converted into testosterone secretion cell | |
| CN102465148A (en) | Human-derived stem cell bioactive substance, its preparation and application | |
| CN111500540A (en) | Breast cancer organoid culture kit | |
| CN105039245A (en) | Method for promoting in-vitro maturing of human immature oocyte by utilizing 3D printing technology | |
| CN109988746A (en) | A kind of mescenchymal stem cell adipogenic induction differentiation method | |
| CN109430252A (en) | A kind of stem cell cryopreserving liquid and preparation method thereof | |
| CN109943533A (en) | A kind of method preparing fat stem cell excretion body, fat stem cell excretion body and its application | |
| CN103385899B (en) | The purposes of Chinese medicine compound pharmaceutical composition | |
| CN105018418B (en) | Human oocytes In-vitro maturation liquid containing Endothelin-1 and application | |
| CN103184190A (en) | Inducing agent and culture medium for transformation of adipose-derived stem cell into testosterone cell | |
| CN105505875A (en) | Culture medium and culture method for efficiently inducing stem cell directional differentiation | |
| CN109260228A (en) | A kind of compound for treating tumour | |
| CN112022898B (en) | Flavanone compound for preventing and treating lipid metabolism disorder | |
| CN104152404B (en) | Embryo heat resistance improving culture solution and using method thereof | |
| CN104762253B (en) | The new application of Chinese medicine compound pharmaceutical composition | |
| CN106566802A (en) | Preparation method for preparing culture kit of umbilical cord blood mesenchemal stem cells | |
| CN105412059A (en) | New application of ethyl gallate to osteosarcoma treatment | |
| CN105343414B (en) | The new application of Chinese medicine compound pharmaceutical composition | |
| CN110038021A (en) | Benzopyrans compounds are preparing the application and combinations thereof in regulating lipid metabolism product | |
| CN110279728A (en) | Herba Visci extract improves the purposes in gonad granulocyte activity tcm product in preparation | |
| CN108888665A (en) | E Seye and its extract are preparing the purposes in blood lipid-lowering medicine | |
| CN108888674A (en) | Oldenlandia diffusa is proliferated in anti-human brain glioblastoma cell, promotees apoptosis, the application in anti-migration, anti-invasion |
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 |