JPS61229894A - Growth factor existing in human placenta and separation thereof - Google Patents

Abstract

PURPOSE:To obtain the titled growth factor having specific molecular weight and isoelectric point, by partially purifying the salt extraction of human placenta with a weakly acidic cation exchange material and treating the fraction containing the growth factor with heparin-immobilized Sepharose. CONSTITUTION:A term human placenta is disintegrated in a solution of a salt (e.g. NaCl) having an electrical conductivity of about <=1.5mS and centrifuged to obtain an extracted liquid, which is salted out with ammonium sulfate. The obtained precipitate is dialyzed with a 0.1M Na phosphate buffer solution of 6.0pH and made to contact with a weakly acidic ion exchange material to effect the adsorption of the growth factor. A fraction containing the growth factor is eluted with a 0.1M Na phosphate buffer solution containing 0.6M NaCl and having 6.0pH and subjected to the chromatography using a heparin- immobilized Sepharose to separate the objective growth factor having a molecular weight of about 16,000-18,000, an isoelectric point of 9.5-10.5 and strong affinity to the heparin-immobilized Sepharose.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to growth factors contained in human placenta and methods for their isolation.

Conventional technology Cell culture using various animal cells and plant cells is rapidly becoming popular as technology advances. It has attracted considerable attention in recent years as an extremely useful tool. Examples include fused cells of human lymphocytes and human myeloma cells that continuously produce antibodies and lymphokines, or fibroblasts that produce interferon.

In many cases, the addition of serum such as fetal bovine serum or neonatal bovine serum to the culture medium is essential for culturing these cells.

Problems to be Solved by the Invention However, there are several drawbacks to using serum for culture. First, the drastic variation between serum lofts affects cell proliferation, so it is necessary to always use a tonic before use, and it is also necessary to ensure a stock of usable lofts. A second drawback is that when trying to release useful substances from cultured cells into a medium and then purifying the target substance from the medium, substances derived from added serum always become contaminants, and purification is often difficult. cause it to become difficult. In particular, when administering purified substances to humans as pharmaceuticals, extreme care must be taken, as even minute amounts of substances derived from non-human serum can cause life-threatening symptoms. need to pay.

In order to improve these drawbacks, there is an urgent need to develop a serum-free medium consisting only of known substances.

Development of this serum-free medium is thought to require various growth factors, and so far, epidermal growth factor (Epidermal growth factor), which is shown in US Pat. No. 3,948,875, is considered necessary.
Growth Factor; EGF),
Platelet Derived Growth Factor (Platelet Derived Growth Factor) shown in U.S. Pat. No. 4,479,896
wth Factor; PDGF) etc. have been discovered and isolated. However, among these growth factors, the only human-derived growth factors include epithelial cell growth factor derived from human urine and platelet-derived growth factor.

Means to Solve the Problem Obtaining human-derived growth factors means that it becomes possible to prepare a culture medium using only human-derived substances and substances that do not have antigenicity to humans. This is where the isolation of human-derived growth factors has great significance. As shown in JP-A-58-116677, human placental extracts are useful for culturing at least some types of cells. As a result of extensive research in search of human-derived growth factors, the present inventors discovered that term human placenta has the activity of promoting the proliferation of at least endothelial cells and fibroblasts, and isolated this. We were able to.

Human term placenta was crushed with a saline solution, and the extract obtained by centrifugation was treated with a weakly acidic cation exchanger to obtain a crude fraction containing growth factors, which was further immobilized with heparin.
- Isolate the desired growth factor by treatment with water.

A feature of the present invention is the isolation of growth factors from human placental extracts. In other words, human placenta extract can be used as it is to culture certain types of cells, such as human IJ lymphocytes, but fibroblasts and other cells contain strong growth inhibitory factors in addition to growth factors. Because it is included, it cannot be used as is.

As mentioned above, human term placenta was soaked in a saline solution (sodium chloride, ammonium sulfate, etc.) with an electrical conductivity of approximately 1.5 m.
8 or less) to obtain an extract by centrifugation or the like, and this extract is salted out with ammonium sulfate at 30% saturation to 75% saturation.

The resulting precipitate was reduced to 0.1 M! After thorough dialysis against sodium sodium chloride buffer pH 6.0, contact with a weakly acidic cation exchanger (e.g. CM-Cef1Dex C-50, CM-52, etc.) equilibrated with the same buffer. After allowing growth factors to be adsorbed, the exchanger was washed with the buffer used for equilibration and 0.1M sodium phosphate, pH 6.0, containing 0.15M sodium chloride, and then washed with 0.1M sodium phosphate, pH 6.0, containing 0.6M sodium chloride. 0.1M sodium phosphate buffer, l) H6,0
Elute the fraction containing growth factors. The obtained growth factor-containing fraction was diluted with 0.01 M containing 0.6 M sodium chloride.
Sodium phosphate buffer, 1) The growth factors were adsorbed by contacting with heparin-immobilized Sepharose equilibrated with H7.0, and the same resin was added to the buffer solution used for equilibration and 01M phosphate containing 1.0M sodium chloride. sodium acid, pH
After washing with 7.0, growth factors are isolated using buffers with increased concentrations of sodium chloride.

Even if one or both of the salting out with ammonium sulfate and subsequent dialysis steps are omitted in the isolation step, there will be no essential difference in the obtained growth factor. However, treatment with a weakly acidic cation exchanger and treatment with a heparin-immobilized Ceph 10-seed are indispensable.

The molecular weight of the growth factor thus obtained was determined by 5D8-polyacrylamide electrophoresis, and the molecular weight was approximately 16°000 to 18°.
,000, and an isoelectric point of 9.5 to 10.5 by isoelectric focusing using ampholine. Fetal and splenic endothelial cysts with growth factor activity
ne Heart Endothelial Ce1l
.

In the measurement of cell proliferation promoting activity using ATCC%CRL-1395) and the measurement of 3H-thymidine uptake activity using mouse 3T3 cells, a promoting effect was shown even at several tens of pg/tnl.

Effects of the Invention Comparing the growth factor activities, the effective concentration of the above-mentioned epidermal growth factor is several tens of ng/d, and the effective concentration of platelet-derived growth factor, which is said to have strong growth factor activity, is several ng/d.
/td, the growth factor contained in the human placenta of the present invention exhibits a promoting effect even at tens of pg/IAI and has extremely strong activity, and the scope of its application will be quite wide in the future. Seem.

Examples Example 1 Four term human placentas were cut into small pieces, 8 tons of 0.15M ammonium sulfate aqueous solution was added thereto, and the pieces were crushed using a homogenizer. After adjusting the pH to 4.5 by adding phosphoric acid, the mixture was centrifuged to obtain a supernatant. Add sodium hydroxide to this supernatant to adjust the pH.
After returning to about 7, add ammonium sulfate to 30%
The mixture was saturated, centrifuged, and the precipitate was discarded. Ammonium sulfate was further added to the supernatant to achieve 75% saturation, followed by centrifugation and the supernatant was discarded. The obtained precipitate was dissolved in water and diluted with 0.1M
Extensive dialysis was performed against sodium phosphate buffer, pH 6.0. After centrifuging to remove the insoluble matter generated after dialysis, a nine column (column size 3.6 x After adsorption of the growth factors through 15 crn) K, the column was washed with the buffer used for equilibration and 0.1 M sodium phosphate buffer, pH 6.0, containing 0.15 M sodium chloride. 6M
001M Sodium Phosphate with Sodium Chloride, p
The growth factor fraction was eluted with H6,0. The obtained two parts are 0
．． A column (column size 1-2
The same buffer was used for equilibration and 0.01 M sodium phosphate buffer containing 1.0 M sodium chloride, pH 7.0.
After washing with 0.01 M sodium phosphate buffer,
Growth factors were eluted using a linear concentration gradient elution method of sodium chloride from 1.0 M to 2.0 M based on pH 7.0. The growth factor was eluted at a sodium chloride concentration of around 1.5M.

In the growth factor obtained by this method, only a single protein band was detected at a molecular weight of about 17,000 by 5DS-polyacrylamide gel electrophoresis. Growth factor activity was determined in bovine fetal cardiac endothelial cells (Feta).
l Bovine Heart Endothelia
The results shown in Table 1 were obtained when the cell proliferation promoting activity was measured using CRL-1395, ATCC, CRL-1395).

(Left below) Table 1 0,122 0,325 1,039 3,955 3Q 110 Example 2 Purification was carried out according to Example 1, and the final step of salt concentration gradient elution was carried out using 2.0 M sodium chloride. Growth factors were eluted instead of using a stepwise elution method. The nine fractions obtained were divided into 8D
When analyzed by S-polyacrylamide gel electrophoresis, the same results as in Example 1 were obtained, except that a few bands of extremely small amounts of inclusions were observed. The measurement results of cell proliferation promoting activity were also the same as in Example 1.

Example 3 After obtaining a 30% to 75% saturated ammonium sulfate precipitate according to Example 1, the precipitate was dissolved in water and the electrical conductivity was adjusted to 1.5.
m After adjusting S K, CM-52 equilibrated with 0.1 M + 7 phosphate buffer, l) H6,Q, i
sOx/ was added and stirred at 5°C for 1 hour. Thereafter, after filtration on a glass filter, the same resin was washed with the buffer used for equilibration, packed into a column with a diameter of 3.6 (7), and further treated with 0.1M phosphoric acid containing 0.15M sodium chloride. Washed with sodium buffer, pH 6.0. 0.6
Both fractions containing growth factors were eluted with 0.1 M sodium phosphate containing IJ (M chloride), pH 6.0, and the obtained fractions were treated with heparin-immobilized Cef10-scalar using the method shown in Example 2. Growth factors were isolated using mucochromatography.

The same results as in Example 2 were obtained in both analysis by 5DR-polyacrylamide gel electrophoresis and measurement of cell proliferation promoting activity.

Example 4 Four full-term human placentas were cut into small pieces, 8 tons of 0.05M sodium chloride was added, and the pieces were crushed using a homogenizer.

An extraction supernatant was obtained in the same manner as in Example 1, sodium hydroxide was added to this supernatant to make p)(6.0, and then 0.1M
Sodium phosphate, I) CM- equilibrated with H6,0
52,159 mJ was added and stirred at 5°C for 1 hour. After washing according to the method shown in Example 3, the column was filled, and further washing and elution were performed. The obtained growth factor-containing fraction was subjected to heparin-immobilized Sepharose column chromatography in the manner described in Example 1 to isolate growth factors.

The same results as in Example 1 were obtained in both analysis by 8DS-polyacrylamide gel electrophoresis and measurement of cell proliferation promoting activity.

Claims (2)

[Claims] (1) Growth factors contained in human placenta and having the following properties: (A) Molecular weight approximately 16,000-18,000 (B) Isoelectric point 9.5-10.5 (C) Heparin-immobilized Sepharose have a strong affinity for (2) Molecular weight contained in human placenta: approximately 16,000 to 1
8,000, an isoelectric point of 9.5 to 10.5, a method for isolating a growth factor comprising the following steps in the process of isolating a growth factor having an affinity for heparin-immobilized sepharose, (a) Partially purified from human placenta extract using a weakly acidic cation exchanger. (b) Growth factors are isolated from the growth factor-containing fraction obtained in (a) using heparin-immobilized Sepharose.