Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using tissue cell culture to make a protein or polypeptide
Patent
1991-03-20
1993-11-02
Robinson, Douglas W.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using tissue cell culture to make a protein or polypeptide
514 2, 514 21, 530350, 530351, 530399, C12P 2102, A01N 3718, A61K 3700, C07K 300
Patent
active
052583244
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to a novel megakaryocyte colony stimulating factor comprising or composed of such a protein which has been isolated from the supernatant of the culture as obtained by cultivation or culturing of a cell strain of a human large cell lung cancer, and which exhibits an activity of forming a megakaryocyte colony from human or murine bone marrow cells when the factor is applied to the bone marrow cells in vitro, and also exhibits an activity of proliferating megakaryocytes in spleen and an activity of increasing the number of platelets in peripheral blood in vivo. This invention also relates to its preparation process.
BACKGROUND ART
Platelets play important roles for promotion of thrombus formation and blood coagulation which both take place in such course of hemostasis that the bleeding as caused by a vascular puncture can stop naturally. In human beings, these platelets are released into the blood stream from the megakaryocytes which are present in the bone marrow and which have been differentiated from myeloid stem cells via the megakaryocyte progenitor cells.
Megakaryocyte colony stimulating factor (abbreviated as "Meg-CSF") is present in the blood of healthy human beings or mammals, and is a physiologically active substance which is said to act on the myeloid stem cells and/or megakaryocyte progenitor cells to promote the differentiation of these cells into megakaryocytes and proliferation of such megakaryocytes. The activities of Meg-CSF are measured in terms of the activity of forming the megakaryocyte colony from human or murine bone marrow cells in vitro. At present, it is recognized that the activities of the megakaryocyte colony stimulating factor are found in the urine from patients with aplastic anemia [Kawakita, M. et al., "Blood", 61, 556 (1983)], the plasma from patients with amegakaryocytic thrombocytopenic purpurea [Hoffman, R. et al., "J. Clin. Invest.", 75, 1174 (1985)] and the supernatant recovered from the culture of human peripheral lymophocytes stimulated by phytohemagglutinin (PHA) [Messner, H. A., et al., "J. Cell Physiol. Suppl.", 1, 45]. When intentions are made to recover and purify the megakaryocyte colony stimulating factor and then to formulate it into medicinal drugs, it is however difficult to obtain the urine and plasma etc. of such patients in the form of a uniform raw material in a large quantity as the starting material to be used for the preparation of Meg-CSF, because they are all biological materials, exhibit individual changes and carry the potential danger of infection by virus or bacteria.
On the other hand, there have been reported a megakaryocyte stimulatory factor (MSF) which has been isolated from the supernatants of the cultures of human embryonic kidney cells or from thrombocytopenic patient's plasma and which has a molecular weight of 15,000 daltons on SDS-PAGE, an isoelectric point of 5.1 and the activity of promoting the protein-synthesis in the megakaryocyte cells, and also process for preparation of MSF (see European Patent Publication No. 0 260 918 A2 or Japanese Patent Application first publication "Kokai" No. 239298/88). This known MSF can specifically act on megakaryocytes but cannot show the activities of Meg-CSF.
An object of the present invention is to find out a stable material source available for the preparation of the megakaryocyte colony stimulating factor (Meg-CSF) and also to produce the megakaryocyte colony stimulating factor from that material source. Another object of the present invention is to purify the thus-obtained Meg-CSF substance to a purity such that it can be used as a medicinal drug.
We, the present inventors, have thus investigated into a variety of animal cell strains which were available in a large quantity as the uniform starting materials. As a result, the activities of the megakaryocyte colony stimulating factor (Meg-CSF) have now been discovered to be developed in the supernatants of the culture (hereinafter called simply "the supernatant") which are obtained by culturing a human
REFERENCES:
patent: 4965195 (1990-10-01), Naren et al.
Burgess et al., Biochem J. 185, 1980, pp. 301-314.
R. Hoffman et al., Journal of Clinical Investigation, vol. 75, No. 4, "Purification and Partical Characterization of a Megakaryocyte Colony-Stimulating Factor from Human Plasma," pp. 1174-1182 (1985).
D. Geissler et al., Journal of Immunology, vol. 137, No. 8, "The Influence of T-Lymphocyte Subsets and Humoral Factors on Colony Formation by Human Bone Marrow and Blood Megakaryocyte Progenitor Cells In-Vitro," pp. 2508-2513 (1986).
Hamaguchi Hiroyuki
Kuriya Shinichiro
Makabe Osamu
Matsunaga Keita
Nagaoka Kozo
Lankford L. Blaine
Meiji Seika Kaisha Ltd.
Robinson Douglas W.
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