CN1042833C

CN1042833C - Process for preparing growth factor of human nerve by embryonic down or decidual

Info

Publication number: CN1042833C
Application number: CN94111364A
Authority: CN
Inventors: 李法卿; 谭维国
Original assignee: Pla Nanjing Military Area Logistics Department Military Medicine Research Institute
Current assignee: GUIZHOU FACTORR BIOTECHNOLOGY CO.,LTD.; MILITARY MEDICAL INST NANJING MILITARY AREA COMMAND OF CHINESE PLA
Priority date: 1994-06-20
Filing date: 1994-06-20
Publication date: 1999-04-07
Anticipated expiration: 2014-06-20
Also published as: CN1114322A

Abstract

The present invention relates to a method for preparing the growth factors of human nerves by villus and decidua. The embryo villus and the decidua of a human body are used as raw materials; hybrid protein is removed by the steps of homogenate, freeze thawing, enzymolysis, centrifugal separation, ultrafiltration, dialysis, depolymerization, secondary ion exchange chromatography separation, primary molecular separation in a sieve adsorption mode, etc.; the growth factors of human nerves, which satisfy the requirements of the human body, is obtained after purifying. The method has the advantages of wide raw material sources, simple preparation process and low cost. The prepared growth factors of human nerves have the advantages of no heterologous protein and no poison and side effect, have high safety to the human body, promote the growth activity of ganglion nerve fibers, and have certain curative effects on senile dementia, encephalodysplasia, cerebrovascular diseases, etc. in clinic.

Description

The method of fine hair, decidual growth factor of human nerve

The present invention relates to a kind of manufacture method of nerve growth factor, particularly use human embryo's fine hair, the method for decidual growth factor of human nerve.

Nerve growth factor (NGF) is peripheral sensory and the sympathetic nerve differentiation and the essential a kind of protein of surviving, at first found in 1948 by Bucker, from the tissue of male mice submaxillary gland and some poisonous snake extract achieving success by Levi-Montalcini and Cohen the seventies, and won Nobel Prize in medicine in 1986.Nerve growth factor can promote neural growth, normal neurocyte had trophism, injured nerve is regained the function, in recent years, many scholars think that being expected to use NGF treats senile dementia, Parkinson, diseases such as sick amyotrophic lateral sclerosis, more external companies are also developing NGF and are being used for this respect treatment of diseases, and have obtained certain curative effect.Yet complicated from mouse submandibular gland and poisonous snake extraction NGF technology, it must just can be applicable to human body through a series of processing, and the few extraction cost height of raw material sources far can not satisfy social needs.

The purpose of this invention is to provide and directly extract the method for going into neural somatomedin that meets the human body needs a kind of fine hair, the decidua,, reduce cost, improve result of treatment to simplify technology from the human embryo.

The present invention is a raw material with people's embryo villi, the decidua of Gestation period miscarriage, makes through the following step:

(1) with homogenate behind fine hair, the decidua dehematize;

(2) material after the homogenate is freezed at low temperatures, under＜38 ℃ room temperature, melt subsequently, so repeat freeze thawing and break fully to cell walls for several times;

(3), the material with step (2) adds C-6885 collagenase enzymolysis;

(4), solid impurity is removed in the centrifugation of step (3) gained material, the collection supernatant liquor;

(5), the supernatant liquor of step (4) is removed the foreign protein of molecular weight＞30000 with 30000 ultra-filtration membrane ultrafiltration;

(6), step (5) gained material is removed the foreign protein of molecular weight＜10000 with 10000 ultra-filtration membrane ultrafiltration, molecular weight be the material concentrated solution of 10000-30000;

(7), with step (6) gained material with the dialysis of the phosphate buffered saline buffer of pH=6.8-7.2, make material pH value be adjusted into 6.8-7.2;

(8), step (7) dialysed to the material of pH=6.8-7.2 moves into the ion-exchange cellulose chromatography column, the damping fluid flushing pillar with pH=6.8-7.2 is received in the material that washes in several containers successively;

(9) merging collection step (8) medium wavelength is 280nm, the material of absorption value＞0.5;

(10) add the acetate buffer of pH=4 in the collected material of step (9), general-NGF dimer depolymerizes to monomer, and wherein the acetate buffer add-on is 0.1-0.13 a times of volume of material;

(11) material after the depolymerization is proofreaied and correct to pH=3.8-4.2 with acetate buffer, people's ion-exchange cellulose chromatography column, NGF is adsorbed, and washes post by follow procedure:

A, contain the foreign protein that the acetate buffer wash-out of NaCl is not adsorbed with pH=3.8-4.2;

Tris-HCl damping fluid flush away another part foreign protein of b, usefulness pH=8.5-9;

C, with the Tris-HCl damping fluid flushing pillar that contains NaCl of pH=8.5-9, NGF is collected the NGF elutriant by wash-out;

(12) the NGF elutriant that step (11) is collected is crossed molecular sieve column, washes post with the Tris-HCl damping fluid that contains NaCl of pH=8.5-9, the collection elutriant, and filtration sterilization promptly obtains the growth factor of human nerve HuNGF that conforms to the human nerve somatomedin.

Among the present invention, the used acetate buffer concentration of step (10) is 0.4-0.6mol/L; Acetate buffer concentration used among step (11)-a is 0.04-0.06mol/L, contains NaCl0.3-0.4mol/L, and used Tris-HCl buffer concentration is 0.04-0.06mol/L among step (11)-b; Tris-HCl buffer concentration used among step (11)-c is 0.04-0.06mol/L, contains NaCl0.4-0.6mol/L.Used Tris-HCl buffer concentration is 0.04-0.06mol/L in the step (12), contains NaCl0.15-0.3mol/L.

The present invention removes molecular weight more than 30000 and 10000 following most of foreign proteins by step (5) and (6) earlier, separates the foreign protein of removing more than 95% by step (8) with (11) secondary ions displacement chromatography post again, makes concentrated 50-100 times of NGF.The latter is the basic protein of iso-electric point about pH9.5 according to NGF, in acidic buffer easily by cellulose adsorption, in ealkaline buffer owing to be not adsorbed or easy eluted principle near iso-electric point, in step (8) chromatography column, the part foreign protein is adsorbed earlier with the damping fluid about pH7, and with the NGF wash-out, be that about 4 damping fluid is washed post with pH in step (11) chromatography column again, NGF is adsorbed, and another part foreign protein is removed by wash-out.The foreign protein close with NGF to a small amount of iso-electric point adopts the isolating method Ex-all of molecular sieve adsorption of step (12).In purge process of the present invention, along with foreign protein content reduces gradually, the active phenomenal growth of NGF extract.Step (10) makes dimeric two polypeptide chains of NGF depolymerize to monomer, thereby improves molecular activity, is easy to pass through hemato encephalic barrier.

The growth factor of human nerve that the present invention makes can be made the injection of 1mg/ml with the stroke-physiological saline solution dilution, for clinical use.

Raw material fine hair, decidua that the present invention makes growth factor of human nerve mainly are taken at the artificial abortion aspirate, and domestic raw material is easy to get.Because raw material sources are in human body, the nerve growth factor of extraction does not have different in nature albumen, conforms to the nerve growth factor of needed by human body fully, can be directly used in human body, has no side effect.After measured, it is that molecular weight is about 14000 polypeptide, and activity test shows that the chicken dorsal root ganglion is in the nutrient solution of the HuNGF of the present invention that contains 5-10ng/ml, and the Sensory neurone projection is not only long but also close, has the neuroganglion of promotion nerve fiber growth activity.Clinical application proves that the growth factor of human nerve that the present invention makes is safe in utilization, has no side effect, and to the treatment senile dementia, cerebral dysplasia and cerebrovascular disease etc. has certain curative effect.Preparation technology of the present invention is simple, and cost reduces greatly, helps applying, and satisfies social needs.

Embodiment one: preparation process

Take from 40 days-3 month stream of peoples' of about 600 people's pregnancy periods fine hair, decidua, homogenate behind dehematize, 21800 milliliters of homogenate meters, be divided in the 500ml Sterile Saline bottle, through-25 ℃～37 ℃ freeze thawing 6 times, add the C-6885 enzyme, 37 ℃ of following enzymolysis 6 hours, thereafter through 25000g centrifugal 1 hour, collect about 20000 milliliters of supernatant liquor, with molecular weight 30,000 filter membranes, with the ultrafiltration of crossing current ultra-fine filter, collecting filtrate is 18500 milliliters, be 10,000 filter membrane ultrafiltration with molecular weight again, collect 2100 milliliters of concentrated solutions, thereafter use 0.02mol/L, the phosphate buffered saline buffer dialysis of pH6.8 6 hours adds the CM32I chromatography column, collects wavelength 280nm, absorption value is greater than 0.5 not 780 milliliters of protein ingredients of absorption, add 87 milliliters of 0.5mol/L, the acetate buffer of pH4.0, effect is 5 minutes behind the mixing, join the CM32I chromatography column, with containing 0.4mol/L NaCl, concentration is 0.05mol/L, the foreign protein that the acetate buffer wash-out of pH4.0 does not adsorb earlier, use 0.05mol/L again, more remaining foreign proteins of Tris-HCl damping fluid flush away of pH9.0 are used pH9.0 at last, and the Tris-HCl of 0.05mol/L adds 0.5m/L NaCl and washes post, get 480 milliliters of NGF elutriants, join the G100 molecular sieve column again, wash post with the pH9.0Tris-HCl damping fluid of the 0.05mol/L that contains 0.2mol/L NaCl concentration, collection contains 610 milliliters of NGF components.Record the 1mg/ml injection for 3600 milliliters with the rare one-tenth of Injectable sterile physiological saline.

Embodiment two: the test of pesticide effectiveness

Be male rat with wistar earlier; by the three-dimensional location of brain; the micro-Ibotenic acid of substantia innominata zone injection at the mouse brain; cause central cholinergic system to be corrupted to dull-witted animal model; and inject HuNGF of the present invention by the ventricles of the brain it is treated; by the painted method of groupization; HuNGF treatment group substantia innominata district's acetylcholinesterase (Ache) positive cell increases than control group; frontal lobe, top Ache positive fiber density show that also obviously more than control group infringement has many-sided provide protection to HuNGF to central cholinergic system.

Claims

A kind of fine hair from artificial abortion, decidua prepare the method for growth factor of human nerve, and this method is a raw material with people's embryo villi, the decidua of artificial abortion, comprises following preparation process:

(1). with people's embryo villi, the homogenate of decidua dehematize of artificial abortion;
(2). the material after the homogenate is freezed under-25-0 ℃, at room temperature melt subsequently, repeat freeze thawing to cell walls and break fully;
(3). the material of step (2) is added C-6885 collagenase enzymolysis;
(4). with step (3) gained material centrifugal a hour, separate and remove solid impurity, collect supernatant liquor through 25000g;
(5). the supernatant liquor of step (4) is removed the foreign protein of molecular weight＞30000 with 30000 ultra-filtration membrane ultrafiltration;
(6). step (5) gained material is removed the foreign protein of molecular weight＜10000 with 10000 ultra-filtration membrane ultrafiltration;
(7). with step (6) gained material 0.02mol/L, the dialysis of the phosphate buffered saline buffer of pH=6.8-7.2;
(8). the material after step (7) dialysis is moved into the ion-exchange cellulose chromatography column, wash pillar, the material that washes is received in several containers successively with the damping fluid of pH=6.8-7.2;
(9). merging collection step (8) medium wavelength is 280nm, the material of absorption value＞0.5;
(10). adding concentration in the collected material of step (9) is the acetate buffer mixing of 0.4-0.6mol/L pH=4, and β-NGF dimer is depolymerized to monomer, and the acetate buffer add-on is 0.1-0.13 a times of volume of material;
(11). the material after the depolymerization is proofreaied and correct pH=3.8-4.2 with acetate buffer, go into the ion-exchange cellulose chromatography column, wash post by follow procedure:

A. be 0.04-0.06mol/L with concentration, the foreign protein that the acetate buffer wash-out that contains NaCl0.3-0.4mol/L of pH=3.8-4.2 is not adsorbed;

B. be 0.04-0.06mol/L with concentration, Tris-HCl damping fluid flush away another part foreign protein of pH=8.5-9;

C. be 0.04-0.06mol/L with concentration, contain NaCl0.4-0.6mol/L, the Tris-HCl damping fluid of pH=8.5-9 is washed post, collects elutriant;
(12). the NGF elutriant that step (11) is collected is crossed molecular sieve column, is 0.04-0.06mol/L with concentration, and the Tris-HCl damping fluid that contains NaCl 0.15-0.3mol/L of pH=8.5-9 is washed post, collects elutriant, obtains growth factor of human nerve HuNGF.