CN1148342A - Lymphoblastoid natural interferon alpha production - Google Patents
Lymphoblastoid natural interferon alpha production Download PDFInfo
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- CN1148342A CN1148342A CN96190195.0A CN96190195A CN1148342A CN 1148342 A CN1148342 A CN 1148342A CN 96190195 A CN96190195 A CN 96190195A CN 1148342 A CN1148342 A CN 1148342A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Abstract
An extract of human fetal thymus which, when added to lymphoblastoid cell cultures, enables the cells to remain in a differentiated state and allows the cells to remain responsive to stimuli enabling the lymphoblastoid cells to produce interferon alpha.
Description
Background of invention
Interferon all is found in many vertebratess, and it is the main method of the non-specific opposing viral infection of body seemingly.Interferon generally includes one group of vertebrate glycoprotein and protein, and known have various biological activitys, for example antiviral, antiproliferative and an immunoregulatory activity.Interferon is the polypeptide that contains about 150 amino acid residues, and it is tried out in treatment hepatitis, herpes simplex, flu even tumor, has all obtained experimental effect.Yet use interferon to exist a problem: expense is huge, and this is owing to use traditional method to be difficult to obtain a large amount of human interferons.
The name of interferon, usually according to the zooblast kind (as people, Mus, cattle etc.) and the cell type (as leukocyte, Lymphoblastoid, fibroblast) that produce this material, the type of the inducer that also produces according to inducing interferon is named (as viral, immunity) once in a while.Some researcheres are not strictly divided into I type and II type according to inducing mode with interferon, and the former comprises that virus and nucleic acid induces the interferon of generation, and the latter comprises that antigen and mitogen induce generation to be called as the material of lymphokine.Recently, proposed a kind of systematic nomenclature, this method is carried out typing according to antigenic specificity to interferon, and according to this newer categorizing system, α, β, IFN-be respectively corresponding to original leukocyte, fibroblast and II type (immunity) interferon.α and interferon-are generally antacid, promptly original alleged I type interferon, and it is unsettled that IFN-is generally acid, promptly original alleged II type interferon.
With the interferon therapy mankind and Animal diseases, this is that a lot of test chambers of global industry and educational institution are being made great efforts the content studied.In some research activitiess the earliest, interferon shows ntiviral characteristic, and interferon the most successful application on clinical treatment so far is to be used for the treatment of and virus-associated diseases.Recently, it is found that exogenous interferon can make some metastatic diseases disappear or alleviates.About the research and the progress report of the activity of determining interferon and potential therapeutic use extremely many in the literature, the great majority report relates to the external activity of interferon, or the in vivo test result behind intestinal external administration, especially intramuscular or the intradermal administration, local use and nasal cavity use some successful reports.
Yet interferon seldom adopts intravenous administration.This is because semifinished product, even all exist impurity in high-purity separator, these impurity have negative interaction widely, although the appearance of recombinant DNA technology makes the production of pure interferon become possibility, carry out with this pure composition that intravenous administration neither be free from side effects and shortcoming.
The interferon of supply exists a lot of problems now.At present, the natural interferon alpha of commercially available all brands and form all derives from leukocyte alive (leukocyte) on the market, the supply of this cell must rely on the blood donor, and therefore, the production of the natural interferon alpha of any specific manufacturer all is subjected to the restriction of blood donor's quantity that it had.And from leukocytic natural interferon alpha, except its expense costliness, whether its blood donor's health and hereditary situation be suitable makes increasing user and family members thereof uneasy day by day to this.
One of purpose of the present invention provides a kind of complex, and it can be used for keeping the cell strain that breaks up, particularly, is the Lymphoblastoid strain that can produce the natural human interferon-ALPHA.Summary of the invention
The present invention relates to a kind of new complex, and use it to keep the method for replying the Lymphoblastoid strain of differentiation, particularly, this new complex is people's tire thymus extract.
In human body, the T lymphocyte is the source of interferon-ALPHA.Lymphoblastoid is T lymphocytic " cell childhood ", and it is one of multiple leukocyte that exists, and Lymphoblastoid is given the characteristic that the T-lymphocyte produces interferon-ALPHA.
Usually, the method that the production of interferon is all invented at first based on the Cantell of Finland, its method are exactly to stimulate leukocyte with different non-pathogenic Sendai virus basically, make leukocyte produce the interferon composition.
Researcheres are being told about a kind of viewpoint always: under aseptic laboratory condition, suppress auxostatic culture medium culturing Lymphoblastoid culture with ideal growth, utilization Cantell method produces interferon-ALPHA to stimulate these cells, and needn't extract interferon-ALPHA in the hemocyte by the blood donor.
The huge superiority of this production technology is conspicuous.At first, production cost can descend significantly; Secondly, this production is unrestricted, promptly only is subjected to the space that had and the restriction of equipment; The 3rd, production can preferably be predicted, this means under standard, ideal laboratory condition, and the probability that will decontaminate with being sure about, change coefficient also becomes insignificant factor on the statistics; The 4th, this production will not rely on the blood donor fully, initial starting culture cell will be from the careful blood donor's who screens of a collection of warp lymphocytoid cell tissue, in addition, need not the blood donor and can produce natural interferon alpha, the life expectancy that promptly is implanted into this cell culture in the artificial ideal culture medium is quite long, and predictable interval is changed easily.
Yet, above-mentioned technology exists a problem always, because after cell is implanted in the ideal artificial environment, its functional requirement forfeiture, in other words, after cell moves into an optimal medium that continues, it is no function purpose, the forfeiture of this function finally can influence its form, thereby, after generation, this cells in culture will be lost their differentiation, changes one of four kinds of initial cell forms into through four to five mitosiss, they will can not show as the specialized cell of differentiation, and say and all become the free migration cell do not have real purpose and live from every side, all are devoted to produce in the research of natural interferon alpha with the Lymphoblastoid culture and all ran into this problem.After some generations, Lymphoblastoid begins to lose its differentiation state.In case its metamorphosis is finished, they are reactionless to any stimulating factor.All attempt to stop the effort of the forfeiture of cell differentiation state all to fail, and people attempted several different methods, to the periodically-varied of culture medium, all do not have effect from enzyme or chemical substance.
Method provided by the invention is: suppress culture medium to the growth that contains the Lymphoblastoid culture, the mixture that adds natural materials, to keep the differentiation state of cell, particularly, this material is the extract of people's tire thymus, end user's fetus cells and do not use adult's cell of differentiation is in order to keep more active mitosis speed, in fact also just to produce interferon more economically.By using this technology, Lymphoblastoid is not lost its differentiation state, and to the stimulation hold acknowledge in the Cantell method, thereby, these cultures can be produced endless natural interferon alpha really, and need not the blood donor fully, and this production can definitely be predicted.Its output only is subjected to the restriction of getable space and technical equipment.Description of Preferred Embodiments
For the systematic naming method method that designs a kind of interferon has been held an international conference, meeting accepted " interferon " to give a definition: as interferon, must be a kind of by involving the metabolic process of RNA and proteinic synthetic cell, at least in homologous cell, show the protein of nonspecific antiviral activity, " interferon " mentioned among the present invention will be considered to meet above-mentioned definition, it can be a kind of protein, comprises glycoprotein, no matter and which kind of cell it derives from.
The interferon that produces can be from human or animal's cell culture, and the interferon in the preferred embodiment is α or interferon-.According to the present invention, contain the aminoacid sequence of modification, but have the active protein of similar natural interferon, also be considered to useful.Among the present invention, the interferon that is selected to clinical generation is human leukocyte interferon and Lymphoblastoid interferon, but they are mass production by the following method: adopt the Lymphoblastoid culture, produce by Cantell method inducing interferon, subsequently by separating interferon in the culture medium.
Tire thymus extract is added Lymphoblastoid culture, the blood donor after the clinical screening that it is careful that thymus is hung oneself.Thymocyte cell carries out extracting quick-freezing then according to surgery sterile working rules, grind into oo level ice powder with the special device for grinding that can under utmost point low temperature, operate, ice powder is spread into out the thin layer of a millimeters thick in a big bottom utensil, room temperature is thawed under vacuum condition, after thawing, to destroy their film, lymphoblast sample (thymus) cell of exposure carries out super centrifugal the dehydration cellular exposure, to separate DNA contained in its nucleus in acid flow.The material that obtains is added to grow and suppresses to contain growth Lymphoblastoid culture thereon in this culture medium in the culture medium with the dilution of sterile physiological saline solution like this.
During the adult lymphoid blastoid cell culture growth in vitro, with the lasting people's tire thymus extract that adds of the amount of 2 milliliters of per 100 liters of cultures addings in per 48 hours, doing like this is in order to prevent cell culture forfeiture differentiation state.This cell can be successfully used to Cantell interferon separation process subsequently, as long as above-mentioned fetal thymus extract constantly adds, cell promptly can be not dead, the sustainable mitosis that carries out.
These cells can be used for producing interferon subsequently.The human can be by following method preparation, the promptly common alleged Cantell method of this method, and its process starts from a large amount of human leukocyte, and the yellowish chromatograph of these a large amount of cells is collected in the centrifuge tube, dilutes with 0.83% ammonium chloride.Gained mixture insulation 15 minutes is intermittently shaken, and 2000rpm is centrifugal 20 minutes subsequently, and supernatant discards, and cell precipitation is resuspended with the aseptic medicine level Radix Acaciae senegalis of minimum.The gained mixture dilutes with ammonium chloride, centrifugal back supernatant discarded, remaining cell precipitation is resuspended with the tissue culture medium (TCM) of minimum, and resuspended substrate can adopt from the MEM of KC Biological (MEM), and cell concentration is determined with the Coulter enumerator.
Interferon-induced can in glass or plastic bottle, carrying out.Inducing culture comprises MEM, 75mM Hepes (from Calbiochem), and 75mM Tricine (from Sigma Chemical co.), people γ serum (18mg/ml) and gentamycin sulfate are (from M.A.Bioproducts; 50mcg/ml).With cell add induce in the container to final concentration be ten thousand cell induction containers of every milliliter of 500-1000 in 37 ℃ water-soluble in insulation, the adding alpha-interferon is as primer.
After two hours, the Sendai virus adding is induced in the mixture, leukocyte produces interferon-alpha in supernatant.Be incubated after 12-18 hour, induce mixture centrifugal, cell discards, and supernatant is carried out purification.
The interferon semifinished product is chilled in ice is molten and is not higher than 10 ℃, and adding 5 molar sulphur potassium cyanates to final concentration is 0.5M, and agitation 15 minutes adds hydrochloric acid then and makes its PH reduce to 3.3, mixture centrifugal 30 minutes in 2800rpm, and supernatant discards.
Precipitation is resuspended in 95% ethanol subsequently, stirred 15 minutes, and suspension centrifugal 20 minutes in 2800rpm, precipitation discards.The gained mixture stirred 10 minutes, and in 280rpm centrifugal 20 minutes then, precipitation discarded, and supernatant PH transfers to 8 with sodium hydroxide.Agitation 10 minutes, 2800rpm is centrifugal 20 minutes subsequently, and supernatant discards, and precipitation is resuspended in order to the 0.5M potassium thiocyanate of 0.1M sodium phosphate buffer preparation, and suspension is in 4 ℃ of stirrings.
With rear suspension liquid in 2800rpm centrifugal 20 minutes, precipitation discarded, and supernatant PH transfers to 5.3 with hydrochloric acid, stirred centrifugally after 10 minutes, and supernatant PH transfers to 2.8 with hydrochloric acid, stirs 20 minutes again.Mixture is centrifugal in 2800rpm, and the gained precipitation is the human of purification.
Precipitation is resuspended in order to the 0.5M potassium thiocyanate of 0.1M kaliumphosphate buffer preparation, and PH is about 8.Under 4 ℃ PBS is dialysed then, PBS changes twice, and the gained mixture is centrifugal, and precipitation discards, the purification interferon-alpha 0.2 micron membrane filtration degerming in footpath that stays.
Certainly, also have additive method to can be used to produce interferon.For example United States Patent (USP) discloses the method for producing human gamma-interferon No. 4376821 and No. 4460685.United States Patent (USP) discloses the method for producing the Lymphoblastoid interferon No. 4276282.A kind of method of producing cattle fibroblast (β) interferon is disclosed in inventor's No. the 4462985th, United States Patent (USP), the interferon goods can be purchased to Hoffmann-LaRoche.Burroughs-Wellcome and Schering-Plough, and tire thymocyte cell extract also can be used for these methods.
Claims (14)
1. Lymphoblastoid culture comprises:
Be in the Lymphoblastoid of differentiation state, can reply interferon and produce stimulus object generation interferon; With a kind of complex that prolongs the differentiation state of said Lymphoblastoid, wherein this complex is a kind of extract of mammal fetal thymus.
2. the cell culture of claim 1, wherein mammal fetal thymus extract is a human fetal thymus extract.
3. the cell culture of claim 1, wherein stimulus object is a kind of non-Causative virus.
4. the cell culture of claim 3, wherein non-Causative virus is a Sendai virus.
5. the cell culture of claim 1, wherein interferon is an interferon-ALPHA.
6. the cell culture of claim 5, wherein to produce stimulus object be that a kind of interferon-ALPHA produces stimulus object to interferon.
7. method of replying the Lymphoblastoid strain that keeps producing the differentiation of interferon comprises:
Add mammal fetal thymus extract to Lymphoblastoid;
Introduce interferon to Lymphoblastoid and produce stimulus object.
8. the method for claim 7, wherein mammal fetal thymus extract is a human fetal thymus extract.
9. the method for claim 7, wherein to produce stimulus object be a kind of non-Causative virus to interferon.
10. the method for claim 8, wherein non-Causative virus is a kind of Sendai virus.
11. the method for claim 7, wherein interferon generation stimulus object is that interferon-ALPHA produces stimulus object.
12. one kind keeps the method for replying the Lymphoblastoid strain of breaking up, comprising:
In Lymphoblastoid, introduce human fetal thymus extract.
13. a Lymphoblastoid culture comprises:
Be in the Lymphoblastoid of differentiation state, keep its differentiation state because of introducing mammal fetal thymus extract.
14. the cell culture of claim 13, wherein mammal fetal thymus extract is a human fetal thymus extract.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US40466795A | 1995-03-15 | 1995-03-15 | |
| US08/404,667 | 1995-03-15 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1148342A true CN1148342A (en) | 1997-04-23 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN96190195.0A Pending CN1148342A (en) | 1995-03-15 | 1996-03-15 | Lymphoblastoid natural interferon alpha production |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0814830A4 (en) |
| CN (1) | CN1148342A (en) |
| AU (1) | AU5254596A (en) |
| WO (1) | WO1996028184A1 (en) |
-
1996
- 1996-03-15 CN CN96190195.0A patent/CN1148342A/en active Pending
- 1996-03-15 AU AU52545/96A patent/AU5254596A/en not_active Abandoned
- 1996-03-15 EP EP96908839A patent/EP0814830A4/en not_active Withdrawn
- 1996-03-15 WO PCT/US1996/003613 patent/WO1996028184A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| WO1996028184A1 (en) | 1996-09-19 |
| EP0814830A4 (en) | 2001-12-12 |
| EP0814830A1 (en) | 1998-01-07 |
| AU5254596A (en) | 1996-10-02 |
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| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |