CN1043350C - Method for preparation of cardiac muscle cell growth stimulus peptide - Google Patents
Method for preparation of cardiac muscle cell growth stimulus peptide Download PDFInfo
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- CN1043350C CN1043350C CN94102798A CN94102798A CN1043350C CN 1043350 C CN1043350 C CN 1043350C CN 94102798 A CN94102798 A CN 94102798A CN 94102798 A CN94102798 A CN 94102798A CN 1043350 C CN1043350 C CN 1043350C
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- cmgsp
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- peptide
- cardiac muscle
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Abstract
The present invention relates to a preparation method of peptide (CMGSP) for simulating the growth of cardiac muscle cells and a freeze-drying curve thereof. After hearts selected from healthy young mammals are mashed by any mechanical mode, frozen deeply, heated, frozen deeply and centrifuged, supernatant liquid enters trapping columns with negative pressure to be sterilized, subpackaged, dried by freezing and packaged; thereby, the peptide for simulating the growth of cardiac muscle cells is prepared. The method of keeping bioactivity comprises the steps that CMGSP liquid is added to mannitol and is subpackaged in 5 ml of medicinal ampoule which is easy to dissolve for quick freezing, temperature rise, temperature enhancement and temperature maintenance; under the light shielding condition at-20 DEG C to 30 DEG C, the bioactivity of CMGSP can be stably kept for three years, and the bioactivity of CMGSP is equal to that of peptide liquid for simulating the growth of cardiac muscle cells.
Description
What the present invention relates to is CMGSP (CMGSP) technical field.
It is synthetic synthetic with albumen generally to have the myocardial cell of stimulation DNA, promotes the cytokine of its division growth, is to extract from the cardiac muscle of spontaneous hypertensive rat or experimental hypertension animal.According to the document introduction: still there are people such as people, Subha S such as NaganoM to carry out this research.Its common feature is that pathologic hypertension animal cardiac muscle is all used in the extraction of heart somatomedin (CGF).
The object of the present invention is to provide a kind of cardiac muscle of the young mammals from health, prepare a kind of method with higher bioactive CMGSP, and adopt special lyophilize curve (method), with the CMGSP freeze-drying, but make its longer-term keep active.
Purpose of the present invention can reach by following technical measures, choose healthy young mammals heart, adopt any mechanical system, it is smashed to pieces-20 ℃ and freeze-thawly separate post-heating 60-100 ℃ deeply, 15 minutes-return to after the room temperature again-20 ℃ dark freeze-thaw separating after, centrifugal 3000rpm, 30 minutes-supernatant liquor advance negative pressure and hold back post-degerming-packing-freeze-drying-packing
Purpose of the present invention can also reach by following technical measures: holds back post system and adopts the Hollow Fiber Ultrafiltration post, and its molecular weight cut-off≤20000Da, negative pressure is 0.5 * 10
4Pa, ultrafiltration column combination mode can be single post or the multicolumn series connection is used; The CMGSP (CMGSP) of preparation is active to keep lyophilize to be: add the 3-8% sweet dew, after-40 ℃ of quick-frozens are kept 2 hours 30 minutes, in half an hour, be warming up to 0 ℃, continue 3 hours, in 1 hour, be warming up to 37-38 ℃ then, kept 20 hours.
Freezing-thawing method can repeat 1 to for several times.
(CMGSP) of the present invention's preparation has higher biological activity, performance biological activity method: get SD mouse embryo (pregnant 12-16 days), aseptic isolating cardiac, trypsinase separating myocardium cell, after DMEM substratum washing 3 times, with DME/F12 substratum preparation 2-5 * 10 that contain 10% calf serum
5Individual cell/ml puts in 96 well culture plates, every hole 0.15ml, 37 ℃, 5%CO
2Hatched under the condition 24 hours.After changing liquid with serum-free DME/F12 substratum, experimental port adds the CMGSP in 5-40 μ g/ hole, control wells only adds the DME/F12 substratum, continue to cultivate 48 hours, every hole adds 5 μ IMTT (1.5mg/ml, the preparation of DME substratum), continues to cultivate 4-6 hour, every hole adds dimethyl sulfoxide (DMSO) 100 μ l termination reactions puts and surveys the OD value under the microplate reader 570nm condition, with experimental port/control wells＞1.7 times for activity is arranged.Measurement result is as follows:
CMGSP is to the influence of Culture Center myocyte mitochondrial dehydrogenase vigor
N OD multiple P value
Control group 8 0.108 ± 0.01
Experimental group (μ g)
5 4 0.143±0.02 1.3 ＞0.05
10 4 0.233±0.03 2.2 ＜0.01
20 4 0.243±0.02 2.3 ＜0.01
40 4 0.195±0.04 1.8 ＜0.05
The pharmacological action of the CMGSP of the present invention's preparation is obvious, and it is few that the myocardial cell encircles doomed dead amount, and the lymphocytic infiltration degree is light, myocardial cell's clear in structure.
Mammal childhood of the health of the CMGSP (CMGSP) of the present invention's preparation is selected from pig, ox, horse, sheep, dog, rabbit etc.
The CMGSP (CMGSP) of the present invention's preparation can be made into oral medicine and intramuscular injection, or other medicines compatibility vein splashes into administration.
The invention process: 1. CMGSP (CMGSP) liquid is added that 3-8% sweet dew packing 5ml is medicinal easily newly to analyse in the ampoule, every bottle of 5ml liquid,-40 ℃ of quick-frozens were kept 2.5 hours, improve temperature to 0 ℃ in half an hour, continue 3 hours, in 1 hour, improve temperature then to 37-38 ℃, kept 20 hours.
Freeze-drying is the medicinal ampoule of easily analysing of 5ml, also is suitable for medicinal pipe bottle of other 5-10ml or mould bottle.
Claims (3)
1. the preparation method of a CMGSP is characterized in that described preparation method comprises:
Choose healthy young mammals heart, do not comprise human heart;
Smash-20 ℃ to pieces with any mechanical system and freeze-thawly separate post-heating 60-100 ℃ deeply, 15 minutes-return to after the room temperature again-20 ℃ dark freeze-thaw separating after, centrifugal 3000rpm, 30 minutes-supernatant liquor advance negative pressure and hold back post-degerming-packing-freeze-drying-packing;
Wherein, the molecular weight≤20000Da that holds back.
2. CMGSP preparation method according to claim 1 is characterized in that holding back post system and adopts the Hollow Fiber Ultrafiltration post, its molecular weight cut-off≤20000Da, and negative pressure is 0.5 * 10
4Pa, ultrafiltration column combination mode can be single post or the multicolumn series connection is used.
3. the CMGSP of preparation according to claim 1 activity keeps the lyophilize curve, it is characterized in that adding the 3-8% sweet dew, after-40 ℃ of quick-frozens are kept 2 hours 30 minutes, in half an hour, be warming up to 0 ℃, continue 3 hours, in 1 hour, be warming up to 37-38 ℃ then, kept 20 hours.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN94102798A CN1043350C (en) | 1994-03-15 | 1994-03-15 | Method for preparation of cardiac muscle cell growth stimulus peptide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN94102798A CN1043350C (en) | 1994-03-15 | 1994-03-15 | Method for preparation of cardiac muscle cell growth stimulus peptide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1108663A CN1108663A (en) | 1995-09-20 |
| CN1043350C true CN1043350C (en) | 1999-05-12 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN94102798A Expired - Fee Related CN1043350C (en) | 1994-03-15 | 1994-03-15 | Method for preparation of cardiac muscle cell growth stimulus peptide |
Country Status (1)
| Country | Link |
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| CN (1) | CN1043350C (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1300171C (en) * | 2003-06-04 | 2007-02-14 | 大连珍奥药业有限公司 | Preparation of myocardium peptide |
| WO2004108751A1 (en) * | 2003-06-04 | 2004-12-16 | Dalian Zhen-Ao Pharmaceutical Co., Ltd. | A cardio myopeptidin, the production and the use thereof |
| CN101265292B (en) * | 2007-03-16 | 2011-09-14 | 大连珍奥药业有限公司 | Polypeptides substances, preparing method and use thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1989004325A1 (en) * | 1987-11-06 | 1989-05-18 | Ferring Arzneimittel Gmbh | Neutrophil-activating polypeptide, process for its manufacture and its use as a drug and diagnosticum |
| CN1044468A (en) * | 1989-01-28 | 1990-08-08 | 杭州市第三人民医院 | Cell growth stimulant and manufacture method thereof |
-
1994
- 1994-03-15 CN CN94102798A patent/CN1043350C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1989004325A1 (en) * | 1987-11-06 | 1989-05-18 | Ferring Arzneimittel Gmbh | Neutrophil-activating polypeptide, process for its manufacture and its use as a drug and diagnosticum |
| CN1044468A (en) * | 1989-01-28 | 1990-08-08 | 杭州市第三人民医院 | Cell growth stimulant and manufacture method thereof |
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| Publication number | Publication date |
|---|---|
| CN1108663A (en) | 1995-09-20 |
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