JPH0761278B2 - Method for measuring cartilage growth promoting activity of growth factors - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for measuring the activity of growth factors. More specifically, the present invention relates to a method for measuring a cartilage growth promoting activity of a growth factor using an organ culture method for embryonic cartilage such as chicken.

Many growth factors have been discovered in recent years, and due to the progress of genetic engineering, it becomes possible to mass-produce these growth factors, and the application to humans and other mammals, that is, to humans and others. Application as a remedy for diseases such as mammals is considered. However, when it is marketed as a therapeutic drug or the like, it is essential to test the biological activity of the drug, and in that case, the present invention can be a useful means.

[Conventional technology]

Growth hormone (GH) and somatomedins are known as growth factors that act on cartilage. Somatomedins include somatomedin A, somatomedin C, insulin-like growth factors I and II, and multiplex stimulating activity ( MSA), cartilage-derived factor (CD
F) and the like are known, and the method for measuring the biological activity thereof is as follows. In the case of growth hormone, GH is intraperitoneally administered to a hypophysectomized rat on the 26th to 28th day after birth for 4 days and tibia cartilage The tibial test, which measures the increase in epiphyseal cartilage width, is commonly used [FS Greenspan et al. Endocrinology, 45 , 455 (194
9)]. For somatomedins, the method of Salmon and Daughaday et al., Which measures the amount of radiolabeled inorganic sulfate ³⁵ SO ₄ incorporated into the assisted cartilage taken out after administration of somatomedins to hypophysectomized rats (J. Clin.Endocrin
ol.Metab., 19 , 743, 1959) and a method using a pelvic cartilage fragment of an 11-day-old chicken embryo by the method of Hall (Acta End.
ocrinol. (kbh), 63, 338,1970) it is known. In addition, in recent years somatomedins were added to the cell culture system of growth cartilage of young rat or rabbit auxiliary cartilage, and its cell growth activity was determined by the uptake of tritium-labeled thymidine as an index,
Also known is a method of measuring the cartilage matrix synthesis promoting activity using the incorporation of labeled inorganic sulfate ³⁵ SO ₄ as an index. (Tissue culture
8 , 46, 1982), (Tissue culture applied research method, pages 116-128, 1
985, Soft Science) [Problems to be solved by the invention] As described above, in the conventional method for measuring growth-promoting activity of growth factors on cartilage, when a pituitary extirpated animal is used, an experimental animal used for measurement It is necessary to give a special treatment to the pituitary gland. It requires considerable empirical skill to acquire the technique. In addition, such treated animals are very expensive to purchase and impose a significant financial burden on animal care facilities and management. Furthermore, the sensitivity of the measurement is not so high, and it is necessary to use a very large number of animals in consideration of the individual difference of animals and the procedural error of surgery, and a considerable labor is required for quality control. In addition, radioactive material control is also required for measuring the uptake of radioactively labeled inorganic sulfuric acid, and the operation is complicated. Even when the pelvic cartilage of chicken embryo is used, the use of a labeling compound does not change the need for radioactive material management as in the case of using a hypophysectomized animal. Further, in the method using cell culture, a long time is required from the collection of tissue for separating the cultured cells to the cell separation and culture, and cell separation operations such as tissue fragmentation and enzyme treatment with collagenase are extremely complicated. Is. In addition, since a juvenile animal is used, a large number of animals are required to obtain the cell amount necessary for measuring a large number of specimens, and a long culture time is required to obtain a measurable state. In addition, before or during measurement, it is necessary to add serum components such as fetal bovine serum to the culture conditions, and these biological components may contain the growth factor to be measured or other unknown growth factors. Therefore, it is doubtful whether or not the growth factor, which is the sample, can be specifically measured in the measurement. The use of tritium-labeled thymidine or labeled inorganic sulfuric acid is the same as in the above case.

[Means for solving problems]

In order to solve these problems of the prior art, that is, the present invention relates to a method for measuring the cartilage growth promoting activity of growth factors, 1. The operation is technically simple, 2. The economical burden is small, and 3. As a result of various studies to provide products that satisfy the requirements of sensitivity, accuracy and reproducibility, 4. Radioactive substance control is not required, 5. Serum component addition is not required for culture conditions, etc. I got new knowledge of.

That is, by adding a growth factor to an organ culture system of an embryonic bone primordia consisting mostly of cartilage and culturing in a culture medium containing no serum, the size of the cultured embryonic bone primordia, It was found that the dry weight, the total protein amount, and the hexosamine amount significantly increased in a concentration-dependent manner as compared with those of the tissue cultured without addition.

The present invention has been completed based on such new findings, and in measuring the growth-promoting activity of growth factors on cartilage, after the growth factors were added to the organ culture system of embryonic bone primordium, the cells were cultured, We propose a method for measuring the cartilage growth promoting activity of growth factors, which comprises measuring the weight of the tissue.

The growth factor to be measured in the present invention is an animal, particularly a mammal (human, cow, pig, horse, mouse, rat, etc.).
There is no particular limitation as long as it has a growth promoting action on cartilage, and the origin of the animal is not particularly limited. For example, human origin includes growth hormone (G
H) and somatomedins are exemplified, and somatomedins include somatomedin A, somatomedin C, insulin-like growth factors I and II, and the like. In addition, other animal origins include cartilage-derived factor (CDF), which is a cartilage growth factor derived from fetal bovine cartilage, such as multiplication stimulation activity (MSA) produced in rat hepatocyte culture medium. It is illustrated.

The embryonic bone primordium used in the present invention refers to a part that will become bone or cartilage of an animal in the embryo state in the future, and is generally at a time when it can react with a growth factor intended to be measured, and is cultured. In the liquid, there is used one that is grown up to a size such that the culture liquid can be absorbed into the inside and that is mostly composed of cartilage tissue. The embryonic bone primordia may be appropriately selected depending on the type of growth factor used as a control for measurement, and is preferably embryonic bone primordia of birds, particularly preferably chicken, quail, turkey, etc. Base, tibia primordia) is used.
The embryonic development period is, for example, 6 to 6 in birds.
Those that occur on the 12th day are preferable, and those that occur on the 14th to 17th day are preferable in mice, rats and the like.

In the assay method of the present invention, by comparing the weight of the embryonic bone primordium cultured in the presence of growth factor and the embryonic bone primordium cultured in the absence of growth factor, the growth It measures the activity of a factor.

In the measurement, usually, in a culture plate of multiple wells, culture was performed for wells to which various concentrations of growth factors were added and wells to which growth factors were not added, and to compare the increase in cartilage tissue of embryonic bone primordium in each. To measure the activity of growth factors.

Prior to culturing, the embryonic bone primordium is, for example, a buffer solution (preferably pH 7 to 8, preferably calcium-free phosphate buffered saline) or the like.
In particular, it is preferable to keep the viability of the separated tissue by immersing the tissue in pH 7.4).

Culture conditions for organ culture of embryonic bone primordium are as follows, for example.

Examples of the medium include Eagle's MEM culture medium (MEM, Dulbecco's modified MEM, α-MEM, etc.) and Ham's F-10 and F-1.
2, CMRL1066, BGJb, BGJb-HW2 etc. are used. Cultivation is preferably performed in the presence of carbon dioxide (0 to 10%, preferably 5%).

The culture time is 48 hours to 6 days, preferably about 3 days,
The culture temperature is 36 to 38 ° C, preferably about 37 ° C.

As a pre-culture before the culture, the same medium as above is used for 20 hours to 2 days, preferably about 24 hours, at a culture temperature of 36.
~ 38 ℃, preferably by culturing under the conditions of about 37 ℃,
It is preferable to acclimate the cartilage tissue of the embryonic bone primordium to a culture state.

〔Example〕

Aseptically remove the embryos from the hatched eggs of the normally developed white leghorn species that had been incubated for 9 days, separated the left and right femoral primordia with tweezers, and rolled the separated femurs to attach them. The soft tissues such as muscles were carefully and almost completely removed, and the cells were allowed to stand in a small amount of calcium-free phosphate buffered saline (pH 7.4) until the culture was started.
Then, 0.5 ml of synthetic culture medium α-MEM culture medium (GIBCO)
24-well plastic culture plate (Falc
on 3047), one femur was placed per well, and precultured for 1 day in a carbon dioxide incubator at 37 ° C., 5% CO ₂ . The culture medium containing insulin-like growth factor-1 (IGF-1), which is a cartilage growth factor, at various concentrations was used for one of a pair of left and right femoral discs separated from the same embryo that had been precultured the next day. Using the other one as a control, a 6-well plastic culture plate (Falcon 3
046) transfer one femur per well for 3 days, 3
The cells were cultured in a carbon dioxide incubator at 7 ° C and 5% CO ₂ . (In addition, the culture was carried out in a pair culture with the number of one group being 5) IGF-1
Preparation of the culture solution containing, was dissolved in 0.1M acetic acid aqueous solution of the freeze-dried preparation of IGF-1 wholly synthesized by the solid-phase synthesis method, and then diluted with α-MEM culture solution to a concentration of 0.3 nM to 100 nM. Used. At this time, the final concentration of acetic acid was 0.1 mM or less, and the amount of culture solution was 2 ml / well. After culturing for 3 days, each of the femurs was washed with phosphate buffered saline, the water adhering to the filter paper was removed, and the femur was transferred to a glass test tube containing 2 ml of acetone and immersed for about 30 minutes for dehydration. . Then, the acetone was discarded, the mixture was transferred into a desiccator containing dried silica gel, suctioned under negative pressure for several minutes, the desiccator was tightly stoppered under negative pressure and left for 1 day to be completely dried. The weight of each dried femur was measured by a micro-chemical balance (Mettler AE163). The IGF-1 activity was based on the dry weight ratio of the added group and the non-added group as an index. In this case, it has been confirmed that the pair of left and right femurs have exactly the same weight before culturing and almost the same weight after culturing under the same conditions.

The results are shown in Fig. 1. When IGF-1 was added to the culture medium at a concentration of 1.0 nM to 100 nM, the weight of the cultured cartilage was increased in a concentration-dependent manner, up to about 2.3 times that of the control. It was confirmed that the IGF-1 was able to fully evaluate the cartilage growth promoting activity.

[Action / effect]

According to the present invention, by adding a growth factor to an organ culture system of embryonic bone primordium and culturing in a culture medium containing no serum, the size, dry weight and total amount of the cultured embryonic bone primordia are calculated. Since the amount of protein significantly increases in a concentration-dependent manner as compared with those of tissues cultured without addition, this can be used to measure the growth promoting activity of growth factors on cartilage. Thus, by measuring the growth-promoting activity, 1. operation is technically easy, 2. economical burden is small, 3. high sensitivity, accuracy and reproducibility are obtained 4. radioactive material control is not required, 5 The effect that the addition of serum components is not necessary for the culture conditions can be obtained.

[Brief description of drawings]

FIG. 1 shows the cartilage growth promoting activity of IGF-1. The vertical axis is IGF
The dry weight ratio of the -1 addition group and the non-addition group is shown, and the horizontal axis shows the concentration (nM) of IGF-1 added to the culture solution.

Claims (1)

[Claims] 1. When measuring the growth-promoting activity of a growth factor on cartilage, growth factor is added to an organ culture system of embryonic bone primordium, and the weight of the cultured tissue is measured. Method for measuring the cartilage growth promoting activity of growth factors.