CN1035040C - Preparation method of thymus protein of sucking pig - Google Patents
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- CN1035040C CN1035040C CN92105643A CN92105643A CN1035040C CN 1035040 C CN1035040 C CN 1035040C CN 92105643 A CN92105643 A CN 92105643A CN 92105643 A CN92105643 A CN 92105643A CN 1035040 C CN1035040 C CN 1035040C
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Abstract
The invention relates to animal thymus protein with cell proliferation promoting effect, in particular to piglet thymus protein with cell proliferation promoting effect, and a preparation method and application thereof. The present invention mainly utilizes the method including centrifugal separation and dialysis to prepare a group of middle molecular weight thymus proteins of suckling pigs which have the capability of promoting cell proliferation and can obviously promote the growth of endothelial cells and fibroblasts from the thymus of suckling pigs. The invention also aims to utilize the cell proliferation promoting capacity of the thymus protein of the medium molecular weight suckling pig as one of the main components of the chemical product, and can effectively promote the proliferation and metabolism of epidermal cells of a human body, thereby achieving the purposes of beauty treatment and the like.
Description
What the present invention relates to is to have the animal thymus albumen that promotes cell propagation effect, especially has the thymus histone from baby pig that promotes cell propagation effect, and their manufacture method and purposes.
Thymus is a secreting hormone and the immune organ that produces the various kinds of cell factor, immunoreactive quality and quantity by gang's polypeptide and albumen mass-energy regulating and controlling that thymus produces.In recent years, external research to thymic extract draws attention.Be subjected to thymus active matter mass-energy to influence the inspiration of cornea and the research of lymph node epithelial cell proliferation, we extract molecular weight water soluble protein in one group from suckling pig thymus, be called for short thymus protein, and it is urged the research of cell-proliferation activity.
1972, people such as Goldstein successfully are separated to one group from calf thymus can regulate immune polypeptide, called after thymosin F5, and biochemical analysis shows, thymus F5 has active single peptide class, be the small-molecular peptides constituents, molecular weight is all less than 15000 dalton, but in the Goldstein reported method, it is centrifugal to need ultrahigh speed, processes such as acetone precipitation, complex process is difficult to promote the use of.
1975, people such as Hopper adopted hyperfiltration technique for the first time in the preparation of calf thymus peptide, by molecular cut off, prepare preparation less than 15000 daltonian thymosin components, have simplified method of purification.
In these several years, many people have bioactive single peptide class to do a large amount of work to the further separation of calf thymus peptide, isolating molecular weight such as following table with immune active ingredient from the thymosin tissue:
| Goods | Molecular weight (dalton) | The researcher age |
| HTH F THF thymopoietin TFX α α 7 β | ??1800-2500 ??＜15000 ????3200 ????5560 ????4200 ????3108 ????2500 ????4982 | ?Comsa????????????1973 ?Goldstein????????1975 ?Traainin?????????1975 ?Schlessinger?????1975 ?Aleksandrowiz????1976 ?Goldstein????????1977 ?Ahmed????????????1979 ?Low??????????????1981 |
It can be seen from the above, disclosed at present result of study, mainly concentrate on the micromolecule thymosin, molecular weight is less than 15000 dalton, the scholar who also has thinks that the composition of biologically active all is molecular weight smaller polypeptides below 6000 dalton in the thymus, is not difficult to find out, the thymosin that is extracted at present mainly is a small-molecular weight, and the process conditions complexity.Sometimes also need by Sep-hader-150 post layer purification, also the someone adopts the normal saline lixiviate, freeze thawing after the dialysis form.
Main purpose of the present invention is to utilize to comprise centrifugalize, and the method for dialysis is produced the thymus histone from baby pig of molecular weight in a group from suckling pig thymus.
Another object of the present invention provides a kind of simple method, thereby can effectively can satisfy the thymus histone from baby pig of this middle molecular weight of producing of actual needs.
A further object of the present invention is to utilize the thymus histone from baby pig of above-mentioned middle molecular weight, and utilize this in the biological activity that thymus histone from baby pig had of molecular weight, that is: have the ability that promotes cell proliferation, can obviously promote endotheliocyte, the growth of fibroblast.
A further object of the invention, it is the ability of utilizing the promotion cell proliferation that thymus protein had of this middle molecular weight piglets, as one of main component of changing the shape product, can effectively promote the propagation and the metabolism of human epidermal cell, thereby reach beauty treatment or similar purpose.
Specifically describe the preparation technology of the thymus histone from baby pig of middle molecular weight of the present invention below.
At first, select the thymus of the healthy piglets in 40-60 days pig ages for use, pull out the fatty tissue on the thymus, thymus is smashed to pieces at a high speed, be noted that and should in the short as far as possible time, finish these work, during this period, homogenate belongs to low-temperature and high-speed smashs to pieces, and the homogenate after smashing to pieces is heated, and temperature remains between 80-90 ℃, time is 10-30 minute, then, precipitation is gone in centrifugalize after the centrifugalize, stay supernatant, dialyse, described dialysis can be adopted dialyser, dialyzer, it also can be other dialysis apparatus that can reach this purpose, the molecular cut off of dialyzer or dialyser is 50,000, and then is that 30,000 dialysis apparatus carries out the dialysis second time with molecular cut off, and the liquid of secondary dialysis is carried out dense volume, pass through the processing of conventional means again, as: degerming, packing, lyophilizing has just obtained molecular weight molecule thymus protein in 3-5 ten thousand is daltonian at last.
Above-mentioned example is to be used for illustrating technology of the present invention, rather than is used for limiting the present invention, and similarly method is all within category of the present invention.
Fig. 1 is the propagation situation of the thymus histone from baby pig (TP) of variable concentrations to endotheliocyte (EC).
Fig. 2 is the propagation situation of the thymus histone from baby pig (TP) of variable concentrations to mice 3T3 fibroblast (FC).
Fig. 3 is thymus histone from baby pig (TP), become porcine thymus albumen (TP), injection liquid of thymic peptide alpha 1 (TF) hepatocyte growth factor injection (HCF) is to the multiplication effect of endotheliocyte.
Fig. 4 is thymus histone from baby pig (TP), becomes porcine thymus albumen (TP), injection liquid of thymic peptide alpha 1 (TF), hepatocyte growth factor injection (HCF) to the fibroblasts proliferation effect.
1 is thymus histone from baby pig (TP), the 2nd among Fig. 3, Fig. 4, becomes porcine thymus albumen (TP), the 3rd, injection liquid of thymic peptide alpha 1 (TF), the 4th, hepatocyte growth factor injection (HGF).
The middle molecule thymus protein of method for preparing is carried out each time test, and particular content is as follows.
Sample comprises: the thymus histone from baby pig of molecular weight (TP) among the present invention, molecular weight 3-5 ten thousand.Injection liquid of thymic peptide alpha 1 (TF) is provided by the institute of biological products, Guangdong Province.Hepatocyte growth-promoting factor injection is provided by Yangjiang, Guangdong Province pharmaceutical factory.The preparation of endotheliocyte (EC): get the umbilical cord in 3 hours behind the healthy parturient childbirth, wash umbilical vein repeatedly to there not being bloodstain with D-Hanks ' liquid, pour into 0.25% trypsin 5ML, put 37 ℃ of water-baths interior 10 minutes, collect Digestive system, the RPMI-1640 flushing umbilical vein that contains 20% calf serum with 5ml, collect flushing liquor, in Digestive system and flushing liquor consolidated revenue centrifuge tube, 1000rpm, centrifugal 10 minutes, remove supernatant.The adding culture fluid of endothelial cell (include 20% calf serum, the 200u/ml penicillin, the 0.2ug/ml streptomycin, 200ug/mlECCS) the dilution sedimentation cell in the glass culture bottle, is put 50%CO with cell inoculation
2, cultivate in 37 ℃ of cell culture incubators, changed liquid once in every 2-3 days, form the cultivation of promptly going down to posterity until cell monolayer.
Go down to posterity when cultivating, use D-Hanks ' liquid fine laundering cell monolayer 2-3 time.At room temperature digested 1-2 minute with 0.06% trypsin, abandon Digestive system, add culture fluid of endothelial cell, and, make cell suspension with suction pipe piping and druming 20-30 time.With 1: the 2-3 ratio goes down to posterity.Be used to test for 2-3 for cell, through the antibody test of V factor related antigen (VR.Ag), confirm as the vascular endothelial cell rear and measure as biological effect and use cell.
Mice 3T3 fibroblast (FC) provides (culture medium is that 1640 culture medium add 20% calf serum) by Zhujiang Hospital, No.1 Military Medical Univ..
Thymus histone from baby pig biological effect of the present invention is measured: get 3 of 24 hole plastics Tissue Culture Plates at every turn, (area 2cm) adds 2 * 10 cells in each culture hole, each 72 hole of endotheliocyte or fibroblast, (thymus histone from baby pig, the porcine thymus albumen of growing up, injection liquid of thymic peptide alpha 1 (TF), hepatocyte growth factor injection (HCF) add parallel detection in 3 porocytes respectively by variable concentrations (0.1,0.2,0.3,0.4,0.5mg/ml) with each test sample.Every duplicate samples is set up 3 hole blanks (doing contrast with saline).Above-mentioned cell is put 5%CO
2, leave standstill in 37 ℃ of cell culture incubators and cultivated 72 hours, harvesting, viable count in every hole is counted in trypan blue dyeing.Method of counting is: put the every hole of microscopically and count 5 cell number under the high power field at random, get its average, be converted into every square centimeter of cell number of turning out by following equation.
ECs cm=cell counting average/0.19625 * 100
[ECs endotheliocyte number, 0.19625 is every high power lens visual field (X400) area, the mm of unit], data are learned processing by statistics.
The thymus histone from baby pig of variable concentrations (TP) is seen Fig. 1 to endotheliocyte (EC), mice 3T3 fibroblast (FC) propagation situation. Fig. 2.
Fig. 1, Fig. 2 prompting, thymus histone from baby pig of the present invention (TP) all has significantly short propagation biological effect to endotheliocyte (EC), mice 3T3 fibroblast (FC), with matched group significant difference (P＜0.01) is arranged relatively, and multiplication effect relevant with the optium concentration of thymus histone from baby pig (TP) (0.2mg-0.4mg/ml).
Various different test sample compare the influence of endotheliocyte (EC), mice 3T3 fibroblast (FC) multiplication effect, the results are shown in Figure 3, Fig. 4.
Fig. 3, Fig. 4 prompting: 1. thymus histone from baby pig of the present invention (TP) has the effect of obvious promotion endotheliocyte (EC), mice 3T3 fibroblast (FC) propagation.2. adult porcine thymus albumen, injection liquid of thymic peptide alpha 1 (TF), three kinds of test samples of hepatocyte growth-promoting factor injection (HCF) all do not have obviously short endotheliocyte (EC), mice 3T3 fibroblast (FC) proliferation activity, compare no significant difference (P＞0.05) with matched group.3. hepatocyte growth-promoting factor injection (HCF) obviously has low-level stimulation phenomenon to endotheliocyte (EC), but compare with thymus histone from baby pig (TP) and to still have significant difference, relatively do not have clear meaning with adult porcine thymus albumen, injection liquid of thymic peptide alpha 1 (TF) result.Illustrate and really exist one group of active component that can improve endotheliocyte (EC), mice 3T3 fibroblast (FC) propagation in the thymus histone from baby pig (TP).
At first study the acetone extract of calf thymus from Goldstein in 1966 etc., since further dividing the Fr5 obtain in 1975 with ammonium sulfate, Most scholars all concentrates on the physicochemical property of micromolecule Fr5, biological activity, the research of the mechanism of action and development and use aspect.Less to molecule in the thymus and the research of high molecular weight protein class.The middle molecular weight thymus histone from baby pig (TP) that the present invention extracts has 6 component peaks, 6 electrophoresis bands through efficient liquid phase chromatographic analysis, and UV scanning has a main absworption peak in wavelength 280nm, still belong to one group of middle molecule protein class mixture.Molecular weight thymus histone from baby pig (TP) simple and convenient extraction in of the present invention, stable in properties, cost is low, through using endotheliocyte (EC), mice 3T3 fibroblast (FC) breeds the culture studies that goes down to posterity continuously and finds, exist one group in the thymus histone from baby pig (TP) and can obviously promote endotheliocyte (EC), the active component of mice 3T3 fibroblast (FC) multiplication effect, and the relevant phenomenon of optium concentration of multiplication effect and thymus histone from baby pig (TP) appears.Whether exist one group and endothelial cell growth factor (ECGF) (ECF) among the piglets TP, fibroblast growth factor (FCF) identical or similar or the component material that function is identical with structure is different, do not understand yet at present, but extract the source of material from thymus histone from baby pig (TP), extraction process and molecular weight of material interpretation of result are inferred, molecular weight thymus histone from baby pig (TP) and micromolecule (the molecular weight 1.8 ten thousand) polypeptides matter of reporting both at home and abroad at present in of the present invention, endothelial cell growth factor (ECGF) (ECF), fibroblast growth factor (FCF), injection liquid of thymic peptide alpha 1 (TF), hepatocyte growth-promoting factor injection (HCF) differs greatly, and Fig. 3 of the present invention, Fig. 4 result also shows, thymus histone from baby pig (TP) and adult porcine thymus albumen (TP), injection liquid of thymic peptide alpha 1 (TF), the action effect difference of hepatocyte growth-promoting factor injection (HCF).
We are according to Komarov FI thymic extract and stomach, the relevant report of duodenal ulcer, adopt middle molecular weight thymus histone from baby pig (TP) external curing oral ulcer (1 * 1 of the present invention, 2cm) the skin infection erosion for a long time not the wound healing mouth (patient of 4 * 5cm, thymus histone from baby pig (TP) shows good infection and obviously promotes the granulation tissue growth result.Research shows that tentatively thymus histone from baby pig of the present invention (TP) promotes the result of wound healing to match at the short endotheliocyte (EC) of laboratory, mice 3T3 fibroblast (FC) multiplication effect and clinical external curing.Thymus histone from baby pig of the present invention (TP) is a kind of middle molecule protein that does not also come into one's own, and the active substance of obvious promotion endotheliocyte (EC), mice 3T3 fibroblast (FC) propagation is arranged.As for the physicochemical property of thymus protein itself how? difference between suckling pig thymus and the adult porcine thymus, thymus histone from baby pig (TP) has any internal relation still to need further comprehensively further investigation exploration with the various cytokines of reporting at present both at home and abroad, we think: the experimentation of carrying out suckling pig thymus middle molecule protein class has new bright prospects, and further Application and Development suckling pig thymus is had crucial value.
Molecule thymus histone from baby pig Preliminary Clinical had obtained good result in treatment oral ulcer and skin injury during we adopted.With molecule thymus histone from baby pig in the water for injection dilution is the aqueous solution of 2mg/ml to concentration.10 routine oral ulcer patients use this liquid and smear the ulcer surface, and 2-3 treatment 1-3 day of every day, (2 example treatments are after 1 day for 10 routine patients, 6 examples are after 2 days, 2 examples are after 3 days) all healings fully of ulcer skin ulcer face, there is no and infect phenomenon and take place, evident in efficacy than concurrent control group (treat 4-5 days then heal).Other has 3 routine skin injury patients in order to the local external application skin of the wetted gauze of this product aqueous solution skin ulcer face, change dressings every day once, after application 4-5 day, find that wherein the rotten to the corn wound of 2 example infection has newborn granulation tissue and epidermis to generate, skin ulcer face inflammatory exudation and red and swollen obviously elimination continuously.The results suggest thymus protein has good infection and obviously promotes the effect of granulation tissue growth, and this and thymus protein laboratory promote the multiplication effect of endotheliocyte (EC), mice 3T3 fibroblast (FC) to match.
Fig. 3,4 square frame empty are sample, shade is contrast.
Claims (1)
1. the preparation technology of a thymus histone from baby pig, comprise select materials, smash to pieces, centrifugalize, dialysis, dense volume, degerming, packing lyophilizing, it is characterized in that selecting for use the healthy piglets in 40-60 days pig ages; Dial the fatty tissue of removing on the thymus; Under cryogenic conditions, thymus is smashed to pieces with the short as far as possible time; The homogenate of smashing to pieces is incubated 10-30 minute between the 80-90 degree; Centrifugalize; Being that 50,000 dialysis apparatus is dialysed with supernatant with molecular cut off then, is that 30,000 dialysis apparatus carries out the dialysis second time with molecular cut off again, is that the dialysis solution of 3-5 ten thousand concentrates with the molecular weight of secondary dialysis; The thymus protein of gained has 6 component peaks under the condition of efficient liquid phase chromatographic analysis at 261nm, 6 electrophoresis bands, and UV scanning has a main absworption peak under the condition of wavelength 280nm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN92105643A CN1035040C (en) | 1992-07-18 | 1992-07-18 | Preparation method of thymus protein of sucking pig |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN92105643A CN1035040C (en) | 1992-07-18 | 1992-07-18 | Preparation method of thymus protein of sucking pig |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1081445A CN1081445A (en) | 1994-02-02 |
| CN1035040C true CN1035040C (en) | 1997-06-04 |
Family
ID=4941506
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN92105643A Expired - Lifetime CN1035040C (en) | 1992-07-18 | 1992-07-18 | Preparation method of thymus protein of sucking pig |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1035040C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1035184C (en) * | 1993-11-22 | 1997-06-18 | 中国人民解放军第458医院 | Water-soluble medium molecular weight extractant from animal thymus gland |
| CN103690929A (en) * | 2013-12-10 | 2014-04-02 | 吉林修正药业新药开发有限公司 | Enema for treating ulcerative colitis and preparation method thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1009725B (en) * | 1986-08-21 | 1990-09-26 | 株式会社细川洋行 | beverage container |
-
1992
- 1992-07-18 CN CN92105643A patent/CN1035040C/en not_active Expired - Lifetime
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1009725B (en) * | 1986-08-21 | 1990-09-26 | 株式会社细川洋行 | beverage container |
Non-Patent Citations (2)
| Title |
|---|
| CN,VOL91,62624U 1979.1.1 Properties of TFX-A product obtained from calf thymus * |
| 广东医学，13(6),92 1992.11.15 陈光明，杨富强等乳猪胸腺蛋白促细胞增殖效应的研究 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1081445A (en) | 1994-02-02 |
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