CN1754574A - Use of immune cell for treating blood coloboma symptom and arrest of bone marrow - Google Patents

Abstract

The invention provides a method of treatment which comprises, activating immunocyte externally, and supplying these activated cells to patients suffering from blood deficiency disease, wherein co-culture is carried out between cytokine and calcium ion carriers with auto- or iso- hemocytes externally, the cultured hemocytes show activated state, and the activated immunocytes have therapeutic actions.

Description

Use immunocyte treatment damaged disease of blood and bone marrow depression

Technical field

The present invention is a Therapeutic Method, treat anemia that the damaged disease of multiple blood comprises that primary aplastic anemia, idiopathic thrombocytopenic purpura, medicine or toxin cause, and the blood that causes of chemotherapy of tumors radiotherapy damaged.Especially specifically, the present invention uses external activated immunocyte and treats above-mentioned disease.Moreover, the present invention is relevant with activated method of cell in vitro and corresponding cell culture processes.

Background technology

Anemia be a kind of be the disease of feature with the hemopoietic shortage.Patient has ANOMALOUS VARIATIONS in various degree when generating various hemocyte.Though in most of the cases its cause of disease is not also known,, radioactive substance, benzene and derivative compound thereof, virus (as hepatitis virus), the toxin that environment produces and a large amount of prescriptions and over-the-counter drug etc. are suspect to be energy damaged bone marrow, cause the apoptosis of bone marrow stem cell.No matter be that what reason causes, patient demonstrates similar clinical symptoms and pathogenic process.Exsanguine morbidity does not have too big difference mainly adolescence and old people on the sex.Have 2 to 6 people morbidity every year among worldwide every million people, its sickness rate is higher than the Europe and the U.S. in the Asia.Some exsanguine pathogenic factors among research, as: geneogenous, pregnancy cause, virus causes, drug-induced and chemical factor causes.

What discussion was maximum in aplastic anemia disease is that medicine and chemical substance cause.Some materials such as antibiotics, benzene, the ionizing radiation material, antiepileptics etc. can both cause the serious aplastic anemia in various degree of people.Other material comprises insecticide, anticoagulant and antimicrobial drug etc., and they cause damage to blood system, and this damage is depended on patient to its sensitivity, and often very little dosage just can cause very big injury, with dosage indifference.In these Drug therapy processes of application, promptly after the drug withdrawal, anemia also can take place.Compare with primary aplastic anemia, said medicine or toxin demonstrate similar clinical symptoms and the geographical statistics feature of population, similar prognosis and the more or less similar reaction to treating.

The aplastic anemia disease that is caused by benzolism, when patient stops with after benzene contacts, patient's symptom just can transfer to and relaxes even disappear.But the patient who is in a bad way more back is bad, needs constantly blood transfusion.Also do not have fool proof effective Therapeutic Method up to now, bone marrow transplantation is present only cure method.

The patient that symptom is not too serious normally treats with the as far as possible little therapy of harm.The basic theories of this therapy is that a factor of bringing out is removed, and makes the recovery from illness that he can be spontaneous.Suffer from serious exsanguine young patient, bone marrow transplantation is an alternative treatment method, and he must be complementary with the damage person's of offering HLA.The effective percentage of bone marrow transplantation is near 80%.When donor and receiver have two or more histocompatibility antigens not match, the chance of survival will reduce to 10% to 20%.Related complication after the transplanting comprises graft-rejection, and acute or chronic graft versus host disease infects and the various damages in various degree of organ.The risk that develops malignant tumor at a specified future date increases after accepting bone marrow transplantation.

In drug-induced cytopenia, bone marrow depression is the modal side effect of cancer patient chemoradiotherapy.Behind the chemoradiotherapy of standard dose, use somatomedin such as Filgrastim (G-CSF), erythropoietin and interleukin 11 can reduce granulocytopenia, anemia and hemorrhage generation respectively.Because high-dose chemotherapy has reasonable therapeutic effect to tumor, is advocated widely and uses recently.Therefore, the serious and secular bone marrow depression that causes of high-dose chemotherapy increasing gradually.Serious and secular bone marrow depression treats and uses hematometachysis usually with somatomedin, adjust that chemotherapeutics is mensuration to be controlled.But, the general difficult healing of serious and secular bone marrow depression with thrombocytopenia, interleukin 11, it is the medicine that unique now approval is used for the clinical treatment thrombocytopenia, hematoblastic regeneration there is certain effect, but undesirable to serious or chronic thrombocytopenia effect.Thrombopoietin (TPO) is confirmed to be and can promotes megalokaryocyte and hematoblastic existence and the ripe a kind of somatomedin that discharges in the carninomatosis people, but clinical trial in early days shows that thrombopoietin has antigenicity and causes production of antibodies in some patients, consequently sb.'s illness took a turn for the worse, can jeopardize patient's life when serious.It is also uncertain whether thrombopoietin can prevent and treat serious thrombocytopenia.

At blood that tumor chemoradiotherapy causes in the damaged and bone marrow depression, though above-mentioned somatomedin is effective medicine, but each somatomedin only works to the some cell line of hemocyte, for example the Filgrastim can only stimulate leukocyte, erythropoietin is to erythrocyte, and interleukin 11 is to platelet.So far also there are not a kind of medicine or the method can the polyclonal growth of effective stimulus.Moreover, all growths

The factor and method all do not have ideal treatment for serious with chronic bone marrow depression, especially to being that the healing of aleucia remains one problem to be solved is arranged.

Summary of the invention

The present invention goes to treat anemia, former and the secondary aplastic anemia that damaged disease of multiple blood such as medicine or toxin cause, the bone marrow depression that the radiotherapy of idiopathic thrombocytopenic purpura and tumorization causes with external activated hemocyte.Wherein content of the present invention is when the damaged disease of treatment blood the immunocyte of the effective dose of some to be injected in the patient body.These immunocytes need elder generation at In vitro culture, contain cytokine and Calcium ionophore in the culture fluid.These cytokines and Calcium ionophore are effectively working in the concentration.Cytokine comprises interleukin II (IL-2), granulocyte macrophage colony stimulating factor (GM-CSF).Optionally Calcium ionophore then comprises calcium ion carrier A 23187.

Another content of the present invention has provided the process of the damaged disease of treatment blood, and this process comprises with activated immunocyte behind the In vitro culture gives patient's medication, and these cells must be the immunocytes with effective dose.This immunocyte can derive from patient's same somatic cell, or patient's variant cell, or the cell that the acceptable donor provides on the immunology.This immunocyte is cultivated then in cytokine that contains valid density and ionophoric culture fluid.

Another content of the present invention has provided a kind of method of cultivating immunocyte, and this method is included in the culture fluid that contains cytokine and Calcium ionophore and cultivates hemocyte.For example, hemocyte will be cultivated in the cytokine of valid density and ion concentration, hemocyte is cultivated containing mammalian blood serum or do not contain in the culture fluid of serum, is cultivating 2 hours to 200 hours or longer time, and cultivation temperature is between 30 ℃ to 42 ℃.Cytokine comprises interleukin II and granulocyte-macrophage colony stimutaing factor.Calcium ionophore comprises calcium ion carrier A 23187.

Description of drawings

Fig. 1 is the platelet variation diagram of a trouble idiopathic thrombocytopenic purpura child in cell therapy treatment overall process.

Fig. 2 shows that repeatedly the cellular immunization treatment can significantly improve because the survival rate of the serious myelosuppressive BALB/c mouse that chemoradiotherapy causes.

Fig. 3 shows that a cellular immunization treatment can significantly improve because the survival rate of the serious myelosuppressive BALB/c mouse that chemoradiotherapy causes.

Fig. 4 shows the comparison of the serious myelosuppressive BALB/c mouse that adherent and non-adherent cell causes at the immunization therapy chemoradiotherapy.

Fig. 5 shows the recovery of the peripheral blood hemogram of serious myelosuppressive BALB/c mouse after accepting the cellular immunization treatment that chemoradiotherapy causes.

Serious bone marrow depression followed the platelet of the cancer patient of thrombocytopenia to change after Fig. 6 showed the activated hemocyte chemoradiotherapy of In vitro culture.

Fig. 7 shows that serious bone marrow depression behind the activated hemocyte chemoradiotherapy of In vitro culture follows the leukocytic variation of the cancer patient of thrombocytopenia.

The specific embodiment

The present invention comprises a Therapeutic Method on the immunology, it be the activated cell of effective dose on the immunology of In vitro culture is given suffer from the damaged disease of blood such as anemia, aplastic anemia, thrombocytopenic purpura and chemoradiotherapy cause damaged disease of blood and myelosuppressive patient.This term " effective dose on the immunology " then is when the damaged patient of treatment blood, the immunocyte that is activated of sufficient amount can increase the quantity of patient's hemocyte, that is to say to increase any blood constituent that is lower than normal concentration in the patient blood, as erythrocyte, leukocyte, cell that platelet or other bone marrow are made and blood constituent.The immunocyte of cultivating or obtain from patient, or on immunology, can obtain the received donor.The step of In vitro culture immune cell activated is from patient or can obtains blood sample (adopting spissated leukocyte composition as 10 to 2000 milliliters of whole bloods or machine) the received donor, from blood sample, separate mononuclearcell, more isolating mononuclearcell is cultivated." on the immunology can received donor " be meant that the immunocyte that the donor contributes can not produce medically unacceptable rejection (as the haemolysis anemia, cardiac dysfunction, renal function are not congruent) on one's body the receiver.The separation of mononuclearcell separates with centrifuging.Isolating hemocyte will under the aseptic condition, containing in the culture fluid of one or more cytokine (as cell stimulating factor) and Calcium ionophore and cultivating.The mononuclearcell of separating will be cultivated under following environment, for example, cultivate above one hour, be specifically in 4 to 200 hours, between 20 hours to 80 hours, between 30 hours to 60 hours, temperature is between 30 ℃ to 42 ℃, be between 32 ℃ to 40 ℃ or 37 ℃ to 38 ℃ specifically, perhaps any temperature that is included in this scope.Professional person with general knowledge and technical ability can recognize that technically the time of other scope and temperature also can reach same effect, should be included among the present invention.

The disease that blood is damaged is treatable in method described above.In general, the damaged disease of blood comprises the reduction of blood constituent concentration, these compositions or be from bone marrow, directly to produce, or be the special cells division that produces from bone marrow.The blood deficiencies disease comprises, anemia for example, the bone marrow depression that former and secondary aplastic anemia, thrombocytopenic purpura and chemicotherapy cause.Anemia is generally thought lacking of a certain composition of blood to be to refer in particular to erythrocytic minimizing in some documents.Aplastic anemia is lacking of peripheral blood all the components.Thrombocytopenic purpura such as former 's thrombocytopenic purpura mainly is lacking of platelet counts.Below by a special example, describe in detail and how to treat damaged disease of these blood and bone marrow depression.

Mononuclearcell then is cleaned (twice aseptic normal saline handled) after cultivating.In treatment, give patient's medication the mononuclearcell of activated effective dose.Wherein a kind of effective administering mode is to give the venous patient administration activated mononuclearcell.Though activated mononuclearcell can disposable administration, also can the component multiple dosing.For example, activated mononuclearcell gives patient weekly, around continuing.Also can use At All Other Times at interval, for example, 10 days, 14 days, 21 days, etc.Certainly, during therapeutic process, can change blanking time.For example, initial dosage can be once a day, and two days once, once in a week or whenever biweekly.The cell quantity of each treatment can be 2 * 10 ⁵To 1 * 10 ⁹Between, and concrete dosage depends on patient's age and health.That treats total time (cell administration time) depends on the effectiveness of cell and patient's reaction.Patient's reaction can be observed, and for example weighs with the improvement that returns to the total health status of normal plasma cell quantity and patient.Obviously, can be from the multiple extraction on one's body hemocyte of patient, and using in the treatment continuously.Moreover the cell that obtains from the donor can use during initial and whole successive treatment.With the activated mononuclearcell administration that activated mononuclearcell of donor and patient obtain on one's body, this two step can carry out in turn.

The definition of now serious aplastic anemia is to be outstanding feature with the pancytopenia, and following at least two characteristics are arranged: 1) granulocyte is less than 500/ milliliter; 2) platelet is less than 20,000/ milliliters; 3) effectively reticulocyte counts is less than 1%, medullary cell hypoplasia, and hematopoietic cell disappears.Aplastic anemia slight or moderate is the minimizing of medullary cell, can have the hemocyte of two cell lines to descend, but the order of severity is less than the scope of severe.The episode process of disease may be slower, initial uncomfortable fatigue and the health weakness gradually that shows as gradually, and these all are because anemia, hemorrhage under the simultaneous certain situation.The hemorrhage of the hemorrhage and health internal layer mucosa of skin then is because thrombocytopenia.Although serious granulocytopenia is arranged, infecting might not be universal phenomenon.Physical examination has shown that pale and skin have congestion and petechia.The aplastic amenia people does not demonstrate lymphadenopathy and splenomegaly.Sometimes do not have during fever.The content analysis of peripheral blood is pancytopenia normally.

If very low of normal skein cell approaches zero even, then show the shortage hemopoietic function.Can prolong the setting time of blood.Irony and transferrins in patient's immune serum all increase, so that transferrins rises to saturation.Irony in the blood plasma is relevant with erythrocytic generation.The bone marrow of extracting out is exsiccant.The bone marrow that cut sections for microscopic examination show the minimizing of cell and lose regeneration capacity is replaced by excess fat.Because initial bone marrow biopsy cut sections for microscopic examination show a large amount of unusual cells is arranged, therefore be necessary to do more tissue slice and diagnose accurately.Serious bone marrow depression can be observed from all hematopoietic cells, and these cells comprise medullary cell, red blood cell, multi-functional cell line and megalokaryocyte.These diagnosis are reflected in three kinds of typical blood cell lines usually: anemia, sexual cell reduces disease, thrombocytopenia.X ray is used for diagnosing bone marrow injury and tumor infiltration.Above-mentioned diagnosis is to get rid of one of method of other cause of disease, adds other experimental analysis, just can make a definite diagnosis aplastic anemia.

The most basic cause of disease of aplastic anemia is that cell loses hemopoietic function.The pathogenesis of aplastic anemia has: 1) lack or do not exist hematopoietic stem cell; 2) bone marrow microenvironment is unusual; 3) cell is adjusted mechanism unusually; 4) immunocyte suppresses hemoposieis.

The pathophysiology of aplastic anemia current (Young et al., The pathophysiology of acquiredaplastic anemia, N.Engl.J.Med.1997 also not fully aware of; 336:1365-1372 and Young et al., The treatment of severeacquired aplastic anemia, Blood.1995; 85 (12): 3367-3377), it is immune diseases that some evidence proof aplastic anemia are arranged.The method and the medicine of inhibition such as bone marrow transplantation and Antilymphocyte Globulin (ATG) and ciclosporin immunity are used for the treatment of (Rosenfeld etc., Intensive immunosuppression with antithymocyte globulin and cyclosporine astreatment for severe aplastic anemia, Blood 1995; 85 (11): 3058-3065 and Halperin etc., Severeacquired aplastic anemia in children:11-year experience with bone marrow transplantation andimmunosuppressive therapy, Am.J.Pediatr.Hematol.Oncol.1989; 11 (3): 304-309).Immunosuppressive therapy is not all effective to all patients, and serious adverse is arranged.In addition, the hematopoietic stimulation factor such as granulocyte colony-stimulating factor have certain short run effect (Kojima etc., Treatment of aplastic anemia in children with recombinant humangranulocyte-colony stimulating factor, Blood 1991; 77 (5): 937-941; Sonoda etc., Multilineageresponse in aplastic anemia patients following long-term administration of filgrastim (recombinant human granulocyte colony stimulating factor), Stem Cells 1993; 11:543-554), granulocyte macrophage colony-stimulating factor; Champlin etc., Treatment of refactory aplasticanemia with recombinant human granulocyte-macrophage-colony-stimulating factor, Blood 1989; 73 (3): 694-699 and Guinan etc., A phase I/II trial of recombinant granulocyte-macrophagecolony-stimulating factor for children with aplastic anemia, Blood 1990; 76 (6): 1077-1082); Ganser etc., Effect of recombinant human interleukin-3 in patients with normal hematopoiesis andin patients with bone marrow failure, Blood 1990; 76 (4): 666-676 and Nimer etc., A phase I/II studyof interluekin-3 in patients with aplastic anemia and myelodysplasia, Exp.Hematol.1994; 22:875-880).

Immunosuppressive therapy is effective to some patient, and many aplastic amenia people's immune molecule is in improper level.For example: interleukin-1, generally by macrophage, natural killer cell, bone-marrow-derived lymphocyte, endotheliocyte produces.Interleukin-1 discharges multiple stimulating factor and proerythroblast by stimulating bone marrow interstital, plays an important role aspect regulating these two in immunne response and hemopoietic, stimulates early stage mother cell and stem cell to recover the influence that bone marrow depression brings.The immunologic derangement that aplastic anemia disease occurs comprises the reduction of natural killer cell activity, suppresses that T lymphocyte number purpose increases and interleukin II and gamma interferon expression unusual.

Natural killer cell is huge granular lymphocyte, and it can dissolve tumor cell and by the cell of viral infection.Natural killer cell can generate γ-interleukin, interleukin II and the formation that stimulates colony.But the formation that these cells can stop red system in the bone marrow and grain assembly to fall under certain conditions.For example, when not adding adventitious agents in the culture fluid, natural killer cell will produce cytokine and hematopoiesis support effect.But manual change's condition of culture can be induced the natural killer cell bone marrow hematogenesis that disinthibites.After the state of an illness recovery from illness of Patients with Aplastic Anemia, the function of natural killer cell is also got back to normally.

Gamma interferon is generated by activated lymphocyte, and it can suppress hemoposieis.Though the patient of aplastic anemia has generated excessive γ-interleukin, the reduction of gamma interferon level has suppressed immunity.Interferon is the potent inhibitor that forms colony in the hemoposieis, and it suppresses bone marrow hematogenesis by acting directly on hemocytoblast or indirect action at other immunocyte.

Tumor necrosis factor-alpha is another cytokine, and it is many especially in aplastic anemia disease.Its function is to suppress normal blood precursor growth.The tumor necrosis factor-alpha of high concentration then with platelet, hemoglobin and leukocytic minimizing are associated.Tumor necrosis factor-alpha and gamma interferon can strengthen its bone marrow hematogenesis inhibitory action mutually.

Tumor necrosis factor-alpha and gamma interferon that the patient of aplastic anemia generates are excessive, and t helper cell and inhibition cells ratio are inverted in the blood, have excessive inhibition cell in bone marrow.These suppress cell and suppress bone marrow by producing cytokine.Bone marrow contains very a high proportion of T killer cell than peripheral blood.The disorder of immunologic function and clinical dependency can prove by the immunosuppressant therapy of success, and the activated lymphocyte quantity in patient's bone marrow of recovery from illness is less than when ill.

The pathogenesis of the aplastic anemia that acquired aplastic anemia and benzolism cause is all illustrated completely.But this aplastic anemia of two types all has very high similarity on Pathophysiology and clinical symptoms.Mechanism at aplastic anemia has two kinds of hypothesis at present: coup injury and immune-mediated damage.These two kinds of hypothesis have all obtained the support of experimentation data and clinical research.Have Cytotoxic chemotherapy and radiation temporary transient to bone marrow caused with reversible damage may be the reason of medullary cell coup injury.It is very difficult curing immune-mediated bone marrow injury.As if the aplastic anemia that benzolism causes all be related with these two kinds of pathogenesis.Discover that benzene can suppress many biochemical reactions of medullary cell, prove that this has produced direct infringement to medullary cell.In addition, benzene can damage the macrophage in the bone marrow matrix, has therefore caused the deficiency of the interleukin-1 of generation.(Niculescu etc., Inhibition of theconversion of pre-interleukins-1[alpha] and 1[beta] to mature cytokines by p-benzoquinone, ametabolite of benzene, Chemico-Biological Interactions; 1995; 98:211-222; Kalf etc., p-benzoquinone, a reactive metabolite ofbenzene, prevents the processing ofpre-interleukins-1[alpha] and-1[beta] to active cytokines by inhibition of the processingenzymes, calpain, and interluekin-1[beta] converting enzyme, Environmental Health Perspectives; 1996; 104 (suppl.6): 1251-1256) interleukin-1 has important effect (Bagby to growth and the differentiation of stem cell, G.C., Production of multi lineage growth factors by hematopoietic stromal cells:anintercellularregulatory network involving mononuclear phagocytes and interleukin-1, BloodCells 1987; 13:147-159 and Fibbe etc., Human fibroblasts produce granulocyte-CSF, macrophage-CSF andgranulocyte-macrophage-CSF following stimulation by interleukin-1 andpoly (rl) .poly (rC), Blood1988; 72 (3): 860-866).Yet Patients with Aplastic Anemia comprises the permanently effective reaction that interleukin-1 is treated to hemopoietic growth factor, does not appear in the newspapers yet so far.

The most basic nutrient substance of the external active cell of culture fluid is suitable culture fluid.These culture fluid are generally by normal saline, aminoacid, and vitamin and other chemical compounds are formed, and they can directly be utilized by cell.In so many culture fluid, RPMI1640 is a kind of proper culture fluid.Certainly, other culture fluid are as serum-free medium AIM-V, for cultivating and to keep hemocyte suitable equally.Culture fluid can replenish mammalian immune serum, as the level of embryo's ICS at 0.1-50%, and in the level of 1-40%, perhaps 5% and 15% level.

Cytokine is when cultivating, and one or more cytokines can be used for activating hemocyte.Cytokine is a kind of small protein (molecular size is generally 5-20kD), and these cytokines are discharged by cell, and cell between communication produce some special effects.Cytokine generally includes as interleukin, lymphokine and signal transduction molecule, for example tumor necrosis factor (TNF) and interferon.Though the n cell factor can be used, used more widely by the excretory recombinant cytokine of the express cell of having set up.In addition, carry out that molecular modification is crossed but have the recombinant cytokine of same biological function also extensively to be used with the n cell factor.Typical cytokine used in the present invention comprises:

A. interleukin.Just can the generation effect when polypeptide of one group of natural generation can act on dissimilar cells and trace.The secretion constantly in participating in antigen and non-antigenic irritant reaction of lymphocyte, mononuclear cell and other various cells produces these protein.Up to the present these interleukin have found 16 kinds, and they are by being used for regulating inflammation and immunoreation to immunocyte and the growth of other cells, motion, differentiation.Usually the concentration of interleukin is 10 to 50,000IU/ml, and perhaps 100 to 5,000IU/ml, or 100 to 1,000IU/ml.Other concentration are perhaps effective.

I. interleukin-1 (IL-1).IL-1 is a kind of soluble protein (molecular size is a 17kD:152 aminoacid), by mononuclear cell, macrophage and some attached emiocytosis, these cells have participated in the activation of T lymphocyte and bone-marrow-derived lymphocyte, and add the reaction of strong antigen and mitogen.The biological action of IL-1 comprises the replacement macrophage, and activated T cell influences the cell of other a lot of types simultaneously.The gene that has two IL-1 at least is to be identified, and they are α and β.Mononuclear cell and macrophage discharge IL-1 in early days at immune system response.It can evoke the propagation and proteinic the synthesizing of T cell.Another effect that IL-1 produces is to cause heating.

Ii. interleukin II (IL-2).IL-2 is a kind of hormonal material that is similar to, by discharged by activated T lymphocyte.IL-2 is not having to cause lymphocytic activation of other T and differentiation under the antigenic situation.Swash the CD8 cytotoxic T lymphocyte by Th1 cd4 cell secretion IL-2 deburring.IL-2 also can increase the propagation and the maturation of cd4 cell self.

Iii. interleukin 3 (IL-3).IL-3 is the product of T cell under the mitosis primary stimuli.IL-3 is bone marrow stem cell and mastocyte colony stimulating factor.IL-3 is considered to a kind of of the hematopoietic stimulation factor.

Iv. interleukin 4 (IL-4).IL-4 is a kind of soluble cell factor, is generated by activated T lymphocyte, and the T lymphocyte stimulates production of antibodies by propagation and the differentiation that promotes the B cell.IL-4 can induce Fc receptor expression on Type II major histocompatibility complex and the B cell.IL-4 also acts on the T lymphocyte, and mast cell line and other hematopoietic cells system comprise granulocyte, megalokaryocyte, erythrocyte mother cell and macrophage.

V. interleukin 5 (IL-5).IL-5 is a kind of differentiation and activation that promotes the eosinophilic granulocyte in hemoposieis.It can also excite the B cell finally to be divided into IgSC.

Vi. interleukin-6 (IL-6).IL-6 is the growth and the differentiation of stimulating human B cell, also is the somatomedin of hybridoma and plasmocytoma simultaneously.It comprises the T cell by many different cells, mononuclear cell, and fibroblast generates.IL-6 is the strand cytokine, and molecular size is 25KD, is described as pre-B cell growth factor at first, at present known to, it comprises that to other various kinds of cell types the T cell has stimulation.

Vii. interleukin 7 (IL-7).IL-7 is a kind of hemopoietic growth factor, can promote the growth of B precursor, simultaneously with the collaborative activation that promotes mature T cells of interleukin II.IL-7 is produced by marrow stromal cell.

Viii. interleukin 8 (IL-8).IL-8 is a kind of cytokine, and it can activate neutrophilic granulocyte and attract neutrophilic granulocyte and the T lymphocyte.IL-8 is discharged by some cells and comprises mononuclear cell, macrophage, T lymphocyte, fibrocyte, the keratinocyte that endotheliocyte and some inflammatory stimulus produce.IL-8 is one of member of β-thrombopoietin family, and it structurally is associated with platelet factor 4.

Ix. interleukin 9 (IL-9).IL-9 is a kind ofly particularly produced by the mitosis primary stimuli by the T cell.IL-9 activates the propagation of erythrocyte mother cell (BFUE), is considered to the moderator of hemoposieis.IL-9 and erythropoietin one work.The IL-9 receptor belongs to the receptor of hemopoietic family.IL-9 shows as propagation and the muroid helper T cell clone who improves human mast cell and original macronucleus blood cell.IL-9 is a kind of glycoprotein, obtains from the T cell, is positioned on human No. 5 chromosomes.

X. interleukin 10 (IL-10).IL-10 is a kind of by the Th2 helper T cell, the cytokine that the activated mononuclear cell of some B cells and bacteria lipopolysaccharide (LPS) produces.It participates in the propagation of mastocyte.

Xi. interleukin 11 (IL-11).IL-11 is a kind of polyphenic cytokine, and initial separation is in the marrow stromal cell of primate, and it has the antibody response of regulating antigenic specificity, strengthens megalokaryocyte, regulates the ability of bone marrow fat composition.IL-11 stimulates maturation, the generation of the giant cell of bone marrow and the multiple granulocytic differentiation of T cell dependency B cell.

Xii. interleukin 12 (IL-12).IL-12 is that a kind of molecular size is the cytokine of 75kD, is formed by disulfide-bonded by 40kD and 35kD submolecule, and initial Function Identification comes from it and micro-interleukin II co-induction killer cell.IL-12 is discharged in infecting reaction by macrophage, and it can improve the vigor of cellular immunization.Particularly, IL-12 caused the Th1 cd4 cell maturation, antigenic specificity cytotoxic T lymphocyte reaction and increase the vigor of natural killer cell.Therefore, IL-12 is the starting material of cellular immunization.It can improve the kill capability of natural killer cell, the generation of inducing interferon, the propagation of promotion activating T cell and natural killer cell.It is secreted by human B lymphoblast.(NC 37)。

Xiii. Interleukin-13 (IL-3).IL-13 is the cytokine that is produced by the T lymphocyte, and it can promote the hypotype conversion and the immature bone-marrow-derived lymphocyte of breeding immunoglobulin to produce immunoglobulin.IL-13 is produced by activated T cells, can suppress mononuclear cell and produce IL-6, and suppress the generation of other inflammatory factors, tumor necrosis factor (TNF) for example, IL-1 and IL-8.IL-13 activates the B cell.It is the same with the gene of IL-4 to be positioned on No. 5 chromosomes.

Xiv. interleukin-1 4 (IL-14).IL-14 is a kind of energy inducing B cell proliferation, suppresses the cytokine that immunoglobulin secretion and selectivity increase the B cell quantity.

Xv. Interleukin-15 (IL-15).IL-15 is a kind of T of stimulation cell proliferation and with IL-12 the cytokine of identical biologic activity is arranged.IL-15 is the propagation and the differentiation of energy inducing B cell equally.

Xvi. interleukin-1 6 (IL-16).IL-16 is a kind of cytokine that is produced by activated T cells, and it can stimulate CD-4 positive lymphocyte and monocytic transfer.

B. lymphokine.Lymphokine is that leukocyte produces, and acts on other cells, interleukin for example, alpha-interferon, lymphotoxin (tumor necrosis factor-alpha), granulocyte, mononuclear cell colony stimulating factor (GM-CSF).

I. interferon (IFN) is a human glycoprotein family, breeds by the inhibition intracellular virus usually and resists viral infection.Interferon can work with identical concentration with interleukin.The valid density of interferon is for activating the concentration of hemocyte arbitrarily in process of the present invention.Alpha-interferon is secreted by leukocyte, and gamma interferon is then secreted by fibroblast behind infective virus.

1. gamma interferon is to be produced by the t cell responses of antigenic specificity mitosis primary stimuli.

2. alpha-interferon divides different hypotypes to be produced by the leukocyte that viral infection or double-stranded RNA stimulate.IFN-α-2A and 2B are the protein that produces by recombinant DNA technology, and are applied on the antineoplastic agent.Alpha-interferon belongs to I type interferon, produces when running into virus, double-stranded RNA or bacterial product by peripheral blood, leukocyte or lymphoblast.It is again the excretory main type of leukocyte of the cultivation of viral infection simultaneously.In addition, it has very strong antiviral activity and can activate natural killer cell.

3. alpha-interferon-2a is an I type interferon, is made up of 165 amino acid residues, and wherein lysine is in the position 23.This protein is produced by recombinant DNA technology, is similar to by the excretory natural interferon of leukocyte.It uses widely and is antiviral or antineoplastic agent.

4. alpha-interferon-2b is an I type interferon, and it is made up of 165 amino acid residues, and wherein arginine is on position 23.This protein is produced by recombinant DNA technology, is similar to by the excretory natural interferon of leukocyte.It uses widely and is antiviral or antineoplastic agent.

5. beta-interferon is a kind of interferon that is produced by fibroblast, and is the same as with alpha-interferon identical stimulation generation.It is a member that belongs in the type i interferon family, is produced by fibroblast after live virus or inactivation of viruses or double-stranded RNA stimulation.It is the immunoregulatory cytokine of the anticancer disease of a kind of antiviral.

6. interferon-beta 2 (interleukin-6) is a kind of somatomedin of growth and differentiation of the B of stimulation cell, also is the somatomedin of hybridoma and plasmocytoma simultaneously.It comprises the T cell by many different cells, mononuclear cell, and the former blast cell of fiber generates.INF-b2 is the cytokine of strand, and molecular size is 25KD, is described as the somatomedin of B cell precursor at first, at present known to, its biological action is to stimulate other cells to comprise the propagation of T cell, irritation cell mitogen simultaneously.INF-β 2 induces the generation of acute protein and acts on mouse bone marrow cells as colony stimulating factor.

7. gamma interferon is produced under specific antigen and mitogenesis primary stimuli by the T cell.

Ii. tumor necrosis factor (TNF) is a kind of tumor-inhibiting factor that produces behind animal bacterial injection lipopolysaccharide.Tumor necrosis factor (TNF) can be in vivo and external kill tumor cell, causes to be planted in the intravital neoplasm necrosis of mice, and suppress experimental tumor and shift.The protein that human α-TNF is made up of 157 aminoacid can extensively promote inflammatory reaction.Tumor necrosis factor exists with identical concentration with interleukin.In addition, tumor necrosis factor can exist with a kind of valid density.Any valid density is any concentration that hemocyte can be activated in the therapeutic process of the present invention.

C. cell stimulating factor.In the present invention, all might be in the content that comprises of the present invention by the activated hemocyte of one or more cell stimulating factor to anemia generation effect.The cell stimulating factor of indication of the present invention comprises picture granulocyte colony-stimulating factor, M-CSF class material.The valid density of cell stimulating factor should be 10 to 50,000IU/ milliliter, or 10 to 10, the 000IU/ milliliter, or 10 to the 1000IU/ milliliter.In addition, the valid density of other stimulating factors may be present in outside the above-mentioned scope.The valid density of any stimulating factor can be defined as any concentration that hemocyte can be activated in the therapeutic process of the present invention.

1. granulocyte colony-stimulating factor (G-CSF): G-CSF is by the synthetic glycoprotein of various kinds of cell, and it works in the growth of hematopoietic stem cell and differentiation.Moreover, these factors can also stimulate the final function of stem cell.

2. granulocyte-macrophage colony stimutaing factor (GM-CSF): GM-CSF is that molecular size is the acidic protein sugar substance of 23kD, is linked to each other by disulfide bond.The inflammatory factor induction of immunity reacts GM-CSF and GM-CSF produces when the Interstitial cell at bone marrow and periphery inflammatory position discharges.GM-CSF stimulates the neutrophilic granulocyte in the medullary cell, macrophage and mangcorn cell, and the formation of macrophage colony, and can stimulate forming of the eosinophilic granulocyte colony next by the fetal liver precursor.

3. M-CSF (M-CSF): M-CSF is by the synthetic cytokine of Interstitial cell, and it can stimulate the differentiation of stem cells that has multiple potentiality in the bone marrow to become mononuclear cell (mononuclear phagocyte).This material can stimulate differentiation, propagation and the survival of the hematopoietic cell of mononuclear phagocyte system.It is that a kind of molecular size is the dimer by disulfide-bonded of 70kD, and combining with a kind of receptor of high-affinity of single kind connects, and this receptor is identical with the product of c-fms oncogene.

D. Calcium ionophore. Calcium ionophore is a kind of specific cation material (as polypeptrates), and it can be free by lipid bilayer and solubility lipid.Two kinds of ionophores are arranged: carrier and ion channel form thing.Carrier as valinomycins, forms a structure as the picture cage shape around special ion, can carry out freely spreading at hydrophobicity bilayer hydrophobic region.Ion channel forms thing, as gram positive bacteria, forms the pole-face of successive liquid in double-deck molecular film, allows ions diffusion to pass through.In addition, except above-mentioned described carrier, the ionophore that the present invention is fit to comprises calcium ion carrier A 23187 (A-23187), ionomycin, geldanamycin, monensin (sodium salt), nystatin, polymyxin-B sulfur salt and rapamycin.As calcium ion carrier A 23187, these carriers can be reacted to the variation of PH gradient, to assemble the calcium cation.Calcium ion carrier A 23187 has an acid carboxyl group, and it can carry out proton exchange with other cationes in whole biomembrane.When the molecule of two A23187 carries a plurality of carboxyl anions, therefore, can carry out the ion exchange of proton or other relevant unit's materials, after ion exchange is finished, then get back to the other end of film.Ionophoric valid density is 1 to 10, and the 000ng/ milliliter arrives the 1000ng/ milliliter 1, arrives the 500ng/ milliliter 10.In other words, ionophore will be present in the valid density.Ionophoric valid density is the activated any concentration of hemocyte, but is not excessive concentration.In view of above-mentioned, the material of excessive concentration can not produce the said effective therapeutic effect of the present invention.

Usage, pack and transport and significantly to improve patient's clinical indices after activated immunocyte is given patient injection, for example, can cause leukocyte in the activated immunocyte administration of treatment of the present invention, erythrocyte, the increase of platelet counts and the raising of hemoglobin level.Generally come, as previously mentioned, the process of treatment can make blood return to normal level continuously.In concrete condition, through after this treatment, patient's quantity of leucocyte, erythrocyte number, hemoglobin level can be increased to 20% at least, and perhaps 35%, even 50%.Similarly, hematoblastic quantity can increase at least 25%, and perhaps 50%, even can reach 100%.The effectiveness that people with Professional knowledge can judge treatment according to the improvement amplitude and the scope of other blood parameters.

As for the material that is used for active cell, can be one or more cytokines, and one or more Calcium ionophore, mix with suitable cell culture fluid or distribute according to a certain percentage and use.The another kind of selection be, one or more activated materials can separate with cell culture fluid to be packed or packed and transported in proportion.Same, an ideal culture fluid for preparing comprises the activated material of one or more cytokines and one or more Calcium ionophores, can packaging togetherly transport, and these materials can be placed in one or more containers.Any scheme no matter, no matter the culture medium for preparing is according to any pro rate, and cultured cells is activated under described condition.In addition, no matter be any traffic program, test kit should comprise description and cell culture rule of operation.

Treatment patient's cell culture can be finished in specific laboratory, and patient can be in the same localities or receive treatment in the different location.In either case, activated cell should be injected to patient at short notice to guarantee cell activity.Another way, cell is preserved under the condition that can keep cytoactive.For example, cell can be kept under the liquid nitrogen.Before the treatment patient, prepare with known way pair cell.For example, when giving patient's medication, cell can be suspended in gives patient injection in the normal saline, and other known available injection also can use with cell.

The treatment case for example

Give an example 1

The treatment of aplastic anemia

I. patient

Eight patients have 1 to the 6 year reason contact benzene owing to occupation through finding out, agree through me, carry out treatment of the present invention.1 male 7 woman are arranged among eight patients, and the age is between 24 to 41 years old.All physical weakness has appearred in patient, feels dizzy symptoms such as faintness and heart rate quickening.In these patients, wherein there are 4 people to be hospitalized for treatment and are because they have acute bleeding, need regular blood transfusion after being admitted to hospital.Other 4 people then are chronic symptons, carried out 4 months respectively, and 6 months, the standard care that waited in 15 months.All patients pass through bone marrow biopsy and bone marrow depression has been made a definite diagnosis in examination of bone marrow smear.Deleterious benzene is present in these patients'blood and the bone marrow.

II. the purification of periphery monokaryon hemocyte and cell culture

Get patient 40-50 milliliter venous blood and isolate blood mononuclearcell (PBMC) by the Ficoll-Hypaque density.Mononuclearcell of separating and RPMI1640 culture fluid were cultivated 48 hours under aseptic condition.Contain 10% embryo's Ox blood serum in the culture fluid, 500IU/ milliliter interleukin II (IL-2) (Chiron, Emeryville, California, USA), 200IU/ milliliter granulocyte-macrophage colony stimutaing factor (GM-CSF) (Immunex, Seattle, California, USA), 100ng/ milli A23187 Calcium ionophore (Sigma, U.S. Saint Louis city), cell culture concentration is 2 * 10 ⁶/ milliliter.After cultivation finishes, attached cell is scraped gently from culture bottle bottom, and with the together centrifugal collection of non-adherent cell.Cell after centrifugal forms agglomerate in the bottom.Different patients obtains the cell of varying number according to state of an illness weight.The cell of collecting wash at least twice in normal saline, the cell that washes is dissolved in earlier in 5～10 ml physiological salines to be counted, and then is diluted in 50 ml physiological salines and inputs to patient by vein.

Therapeutic Method

Wherein a patient is owing to be in a bad way, and cytometry is extremely low and with hemorrhage and infect, and this patient has been used the activated hemocyte of allosome in treatment of first three time.Patient to other uses from the activated cell of body.Around this treatment is treated once in a week at least continuously.The cell quantity of administration depends on the actual cell quantity that obtains from patient.

III. result

Hematological parameter

Hematological parameter, quantity of leucocyte, erythrocyte number, hemoglobin level and platelet counts are all being monitored and are being listed in the table 1 before and after to each patient.The data that obtain from these patients show that this treatment is effective at the quantitative aspects that increases peripheral blood cells.Six patients have in listed index to increase and improves, two other then platelet counts take a favorable turn.Most patient hemocyte quantity after two treatments begins to improve, and continues to improve by further treatment patient.There is among back eight patients 7 hematologic parameter to arrive normal level or near normal level by four treatments.Though patient's improvement degree is different on the whole, for example, some patient has the erythrocyte of little spoke degree to improve the platelet counts that big spoke degree is arranged to be increased.Notice that in treatment all patients' platelet all has increase in various degree.

Patient HC has the acute symptom even more serious than other patients.In addition, patient HC also has with bleeding.Because the venous blood cell quantity that obtains from patient HC is limited, so the allosome hemocyte is used to treat this patient.After cell was treated through three times, the state of an illness of patient HC began to take a turn for the better.Patient is used to subsequently treatment at patient's autogenous cell after this.After six treatments, though the patients'blood mathematic(al) parameter does not also reach normally, patient's the state of an illness and blood parameters continue to take a turn for the better.

The toxicity of this treatment is on the whole from light to moderate.5 patients do not show discomfort, and shivering appears after having inculcated cell in 3 patients, generate heat to 37 ℃ to 39 ℃ headache, nausea and vomiting, reactions such as appetite depression.However, these symptoms are temporary transient, most lasting one to two day.If patient has not in good time, with medicine controlling symptoms such as aspirin class and promethazines.

Bone marrow hematogenesis

Before treatment and among all patients of fortnight of treatment back, all to carry out bone marrow biopsy.Before treatment, from three patient's bone marrow biopsies the most serious, find out the existing grievous injury before treatment of patient's bone marrow, the cavityization.After the treatment, myeloid tissue is obviously improved.Hemopoietic is active, and nucleated cell significantly increases.However, patient HC peripheral blood counting improves the improvement of postpone in bone marrow biopsy.

Before the aplastic amenia human therapy that table 1. benzolism causes and treatment back hemogram parameter

Patient	Age/gender	The treatment number of times	Disease type	After treating before the treatment
												Leukocyte (* 10 3/mL)	Erythrocyte (* 10 6/mL)	Hemoglobin (g/dl)	Platelet (* 10 3/mm 3)	Leukocyte (* 10 3/mL)	Erythrocyte (* 10 6mL)	Hemoglobin (g/dl)	Platelet (* 10 3/mm 3)
				HC TM TB LC YX JX ZL SC	32/ woman, 29/ woman, 33/ woman, 25/ man, 25/ woman, 29/ woman 41/ women 29/ woman	6 4 4 4 4 5 4 4	Acute chronic acute chronic acute chronic	2.7+/-0.2 3.6+/-0.3 3.2+/-0.2 1.4+/-0.2 2.5+/-0.3 2.7+/-0.7 3.1+/-0.6 2.4+/-0.3	1.2+/-0.1 3.8+/-0.5 2.3+/-0.3 1.6+/-0.3 2+/-0.3 3.+/-0.2 4.0+/-0.6 2.+/-0.1	40+/-0.3 11.1+/-0.4 8.4+/-0.4 5.7+/-0.3 8.2+/-0.4 8.1+/-3.4 12.6+/-1.9 10.0+/-3.5	28+/-3 99+/-11 47+/-5 16+/-7 22+/-7 4.5+/-18 154+/-35 54+/-4									3.8+/-0.25 6.7+/-0.3 4.7+/-0.3 5.7+/-0.2 3.5+/-0.3 4.5+/-0.7 3.7+/-0.1 2.8+/-0.2	2.0+/-0.2 3.9+/-0.4 3.2+/-0.3 4.8+/-0.3 2.8+/-0.2 3.6+/-0.1 4.3+/-0.02 2.9+/-0.2	7.0+/-0.3 12.7+/-0.5 11.5+/-0.7 13.5+/-0.7 9.8+/-0.5 12.0+/-0.4 13.2+/-1.3 11.0+/-0.3	43+/-3 182+/-1 107+/-1 135+/-1 100+/-1 126+/-8 187+/-2 71+/-8

Blood transfusion

Before the treatment, there are 4 patient symptoms more serious among eight patients, are attended by hemorrhage.These four patients will import whole blood or platelet to them termly before treatment and during the treatment.After four treatments, the neither one patient has hemorrhage, and whole blood and hematoblastic input have also stopped.

Persistent period

The effect that produces with cell therapy is not temporary transient.After treatment stopped, all patients continued to take a favorable turn and hematological parameter is stable.Some aegs' hemogram is in some fluctuation of menstrual phase, but the recurrence of neither one patient's disease.Patient LC shows as stable situation by treating existing two wheat harvesting periods in back.

Give an example 2

The treatment of aleucia

As described herein is a treatment of suffering from congenital thrombocytopenic purpura patient of 1 years old 05 months.This patient was made a definite diagnosis in treatment in preceding 9 months.It is to treat with simple corticosteroid hormone and immunoglobulin'intravenous that this patient begins.Though this treatment is effective to conditions of patients, the place one's entire reliance upon corticosteroid hormone of high concentration of this patient.In order to keep hematoblastic quantity, the hormone dosage that the patient took is also in continuous increase.In case reduce or without hormone, patient's platelet counts can sharply descend.Therefore, patient has accepted the therapy of above-mentioned said cellular immunization treatment.The method of this treatment and process and example 1 described basically identical, the amount for taking blood that different is each time is 20 milliliters to 30 milliliters.The treatment of patient's jede Woche once continued for 9 week.External activated immunocyte is respectively at the 1st day, and the 8th day, the 15th day, the 22nd day, the 29th day, the 35th day, the 42nd day, the 49th day, administration in the 56th day.With the cell therapy while, corticosteroid hormone and other medicine are stopped using.Patient's platelet levels is little by little improved during the treatment, as shown in Figure 2.Patient had pulmonary infection and with the minimizing of platelet counts at the 49th day.After the recovery from illness, hematoblastic quantity has been got back to normal level (Fig. 1) again to patient from infect.

Give an example 3

The mice chemicotherapy causes myelosuppressive treatment

Experiment mice

Use the female Mus of BALB/c in 10 to 12 week, about body weight 20g (Zhongshan University's Experimental Animal Center).The animal grouping, ten every group.Bone marrow depression adds what the whole body radiotherapy of single dose 250cGy was produced by 1.5 milligrams of carboplatins of every mouse peritoneal injection.The dosage of used carboplatin and radiotherapy is that test obtains through test of many times in advance, and mortality of mice reaches 80%～100% under this dosage.

Cell culture

Mouse cell

Obtain spleen from normal BALB/c mouse, with distilled water with erythrocyte splitting.Remaining cell culture is containing 10% calf serum, and mice GM-CSF is in the RPMI1640 culture medium of mice IL-2 and calcium ion carrier A 23187.Cultivated 48 hours.

Human blood cell's cultivation

With the leukocyte that normal person's donor provides, it gets blood, separates, and cultivates as with 1 identical for example.

Research grouping and treatment

Suffering from myelosuppressive mice for every group ten treats with external activated splenocyte before and after chemotherapy and radiation.Every mice the 48th hour, the 96th hour, carried out dosage and is respectively 1 * 10 at the 3rd hour on the 144th hour behind chemotherapy and radiation ⁴, 1 * 10 ⁵, 1 * 10 ⁶Tail vein injection, matched group is not for to accept the mice of any cell therapy and only to accept fresh uncultivated splenocyte.One group of mice is induced in bone marrow depression and accepted 1 * 10 in preceding 24 hours ⁷Activated mouse cell.

The analysis of cytokine

The described in the above GM-CSF of human peripheral mononuclear cell from the donor of health obtains cultivated 2 days in IL-2 and the Calcium ionophore.Cultured cells is collected handy PBS washed twice.These cells then continue to cultivate 3 days in not containing the fresh culture fluid of cytokine and Calcium ionophore.Centrifugal, collect supernatant for cytokine analysis.

The analysis of peripheral blood

The observation that recovers behind chemotherapy and radiation hemogram weekly changes, and collects peripheral blood from the mouse vein tip.Mice complete blood cell quantitative measurement is finished by Thechnicon H-1E (Technicon Instruments Corp, Tarrytown, New York, United States) automatic counter for counting.

Stem cell mobilization

Every vein of normal mouse injects activated people's immunocyte 1 * 10 ⁷, injected continuously 3 days.The 5th day, get blood from vena ophthalmica, destroy erythrocyte with distilled water, remaining cell in ice bath with monoclonal antibody (the BD BiosciencesPharmingen that has fluorescently-labeled anti-mice CD34, San Diego city, the U.S.) in conjunction with 20 minutes, wash 2 times with normal saline then.The cell BD FACSCalibur that handles ^TMFlow cytometry analysis.The scope that door is set comprises all single karyolymph cells, and each sample is got 30,000 cells at every turn and analyzed.

III. result

Short-term and secular survival rate

Mice at 3 hours, 48 hours, 96 hours, was accepted the treatment of activated splenocyte in 144 hours behind chemotherapy and radiation.Control mice is accepted the fresh external activated splenocyte that do not carry out.As shown in Figure 2, this therapy makes the survival rate of mice be increased to 80-100% (Fig. 2) from 0-20%.In treatment, 1 * 10 of mice acceptance ⁵Activated splenocyte and 1 * 10 ⁷Activated splenocyte does not have very big difference on survival rate, show to be dose therapeutically effective in this scope.Any dose therapeutically effective can be defined as any concentration that splenocyte is activated in the therapeutic process of the present invention.Saved most of or whole mices with 4 treatments of external activated splenocyte, otherwise they just die from serious bone marrow depression.Whether we also explore seance can improve the survival rate of suffering from myelosuppressive mice.This result shows at chemotherapy and radiation after 3 hours that one-time treatment is given every mice 2 * 10 ⁷Activated splenocyte can significantly improve the survival rate (Fig. 3) of suffering from the bone marrow depression mice.

Above-mentionedly experimental results show that external activated mouse cell of the same race can significantly improve the survival rate of suffering from serious bone marrow depression mice, whether we have tested heterogenous cell simultaneously same effect.Suffered from serious myelosuppressive mice respectively at 3 hours, 48 hours, 96 hours and 144 hours outer activated hemocytees of injection human body are the same with the effect of allogenic cell treatment, the big spoke degree of its survival rate of mice through treatment improves, and brings up to 80-100% from 0-20%.

In order to study that a kind of cell subsets this treatment is had better therapeutic, the characteristic that we utilize cell itself to cultivate is divided into adherent cell and non-adherent cell, gives respectively and suffers from the disposable injection of serious myelosuppressive mice.The result shows that the effect of attached cell is better than non-adherent cell (Fig. 4).

In addition, experimental result can not improve the survival rate of mice for the external activated cell of injected in mice before being presented at chemoradiotherapy, so the bone marrow depression that this Therapeutic Method causes chemoradiotherapy does not have too big preventive effect.

Handle normal mouse with external activated people's mononuclearcell, express the ratio of stem cell in peripheral blood of CD34 after 3 administrations with flow cytometry analysis, find that the 0.20-0.4% of the ratio of CD34 stem cell in whole blood before the administration is increased to 0.5-0.6%, though the amplitude that increases is not very big, but uniform.

Give an example 4

The myelosuppressive treatment of the severe chronic that chemoradiotherapy causes

I. patient

Patient's selection.The adult suffers from hematologic cancers and solid tumor and be selected in the middle of the current research with the patient who extensively shifts in late period.Before beginning one's study, patient need accept a large amount of secular chemotherapy and radiations.≤ 500/ milliliter of its peripheral blood leucocyte counting, hemochrome concentration≤6.5mg/ milliliter, and platelet counts≤20,000/ milliliter.What patient need accept to continue four weeks contains granulocyte colony-stimulating factor (G-CSF), the therapeutic alliance of erythropoietin and interleukin-11 (IL-11); Though other blood cell line of these patients makes moderate progress after accepting somatomedin, platelet count continues to be less than or equal to 20,000/ milliliters.Patient need rely on the input platelet to prevent hemorrhage; Patient's major part of the trouble solid tumor of receiving treatment need be accepted radiotherapy with alleviating pain with having an intense pain that bone marrow shifts and causes thus.Because these patient's platelet counts wretched insufficiencies, this type of radiotherapy can cause bleeding even threat to life.Patient need satisfy the qualification that following standard just can have reception test research under study for action: the performance state mark A2 of eastern united states tumor cooperative association (ECOG); There are not great heart disease and disease of metabolism clinically.Normal hepatic and renal function (the total level of bilirubin is 52.0 mg/ml, the nitrogen in the blood urea (BUN) 530 mg/ml, immune serum creatinine levels 52.0 mg/ml); The rate that pumps (pumping rate [RNEF] 〉=50%) of normal left ventricle.

II. the purification of the single nuclear blood cell of periphery and cell culture

With the leukocyte that normal person's donor provides, it gets blood, separates, and cultivates as with 3 identical for example.

Therapeutic Method

12 qualified patients are selected into.Patient was at the 1st day, and the 2nd day, injected 5 * 10 the 3rd day every day ⁷The activated hemocyte of dosage was then had a rest at 4 to 7 days.Carry out the circulation treatment of treatment in 3 days like this and 4 days rest each week, lasting 4 week.Increase above 40,000/ milliliters if find platelet counts in treatment, then treatment stops.Platelet counts was monitored in each week, monitored at least 4 times.It is hemorrhage to prevent then to need to import platelet during platelet counts≤20,000/ milliliter.Thisly observed by all research worker in this research for the strategy that prevents the hemorrhage platelet transfusion of carrying out.If find to surpass 40,000/ milliliters at the last patient's platelet counts, then enumeration of thrombocytes repeated after 3 days once to confirm the improvement of platelet count.The use that does not allow any somatomedin in whole therapeutic process is to reduce the interference to this treatment curative effect.Patient accepts promethazine usually to reduce side effect before cell injects.

Patient characteristic

Patient's number age intermediate range sex masculinity femininity ECOG mark diagnosing non-small cell lung cancer non_hodgkin lymphoma colon cancer Preceding acute lymphoblastic leukemia Zhi treats lasting time granulocyte and leukopenia and the anemia of number intermediate range chemotherapy of lumbar vertebrae backbone pelvis radiotherapy chemotherapy scheme

12 55(20-74) 6 6 1(0-2) 4 3 3 2 6 3(1-5) 24(2-52) 6

The analysis of cytokine

Cell begins to cultivate two days later, do not have adherent cell to be collected and with the normal saline washed twice adherent cell in culture bottle equally with normal saline washed twice lightly.Washed do not have adherent cell to be put back in the identical culture bottle, upgrades culture fluid continuous culture 3 days again, and new culture medium does not contain any cytokine but contains Calcium ionophore.After supernatant was collected, (Luminex, Austin TX) carried out cytokine analysis to concentrate in together usefulness Luminex  xMAP  system.

III. result

Hematoblastic recovery

External activated immunocyte can strengthen hematoblastic recovery capability for patient's administration of suffering from long-term thrombocytopenia.Among 12 patients that receive treatment, have through after the treatment of different number of times, 10 patients' platelet counts increases and has reached 40,000/ milliliters, and two other patient reactionless (Fig. 5).

Leukocytic recovery

Its leukocytic recovery of patient that all 6 leukocyte are low is consistent with hematoblastic recovery, and the speed of recovering faster and better (as shown in Figure 7).

The secretion of cytokine

External activated people's mononuclearcell secretion various kinds of cell factor in 19 kinds of cytokines that detect, has detected 15 kinds (its secretion level is significantly higher than contrast), comprises IL-1, IL-8, GM-CSF, G-CSF.

Table 2. human blood cell activates the excretory cytokine in back at In vitro culture

Cytokine	Concentration (pg/mL)
			The culture fluid contrast	Cells and supernatant
	IL-1b IL-2 IL-4 IL-5 IL-6 IL-7 IL-8 IL-10 IL-12(p40) IL-13 IL-17 IL-18 IFN-gamma TNF-a G-CSF GM-CSF MCP-1 MIP-1b Eotaxin	0 0 0 0.14 0 0 0 3.54 0 0.13 0 0 0 0 0 19.7 0 0 0			2777 24342 164.87 141 334881 2 ＞200000 415 6 1167 50 9 634 461 178906 198833 215 3202 8

Clinical tolerance

External activated immunotherapy has good toleration to patient.Do not find to have skin allergy, unusual hydrops in the body, phenomenons such as thrombosis and organ toxicity.Modal side effect is to shiver and generate heat between 37 ℃ to 39 ℃, and headache is felt sick, vomiting, and degradation under the appetite, they are temporary transient similar to typical infusion reaction, major part is all alleviated fully in inject at cell 24 hours.Patient these symptoms occurred and has just come symptomatic treatment with corresponding routine treatment and medicine.Promethazine hydrochloride usually is used to reduce side effect.The neither one patient is owing to this research is withdrawed from side effect.

Discuss

The result of this research shows with activated hemocyte has good effect to patient's administration of suffering from aplastic anemia.Its bone marrow of some patient improves approaching normal, but these patient's peripheral blood parameters also do not reach normal fully.About this point, we find, the asynchronism(-nization) step that the quantity of histology's bone marrow and peripheral blood cells is improved.Although as follows, the parameter of patient's peripheral blood continues to improve.These patients need the quantity of its peripheral blood cells of time of several weeks or some months just can reach normal sometimes.

The data that obtain in analysis and research can see that in the blood constituent of different parts, patients undergo is treated, and the increase of platelet counts is very tangible.Other blood constituent also is improved.The improvement speed of this platelet counts is far away faster than other standard care.

Aplastic anemia is a kind of more refractory disease, and current immunosuppressive therapy is not very effective, and having only bone marrow transplantation is known cure method.Although bone marrow transplantation has obtained success, this therapy can produce severe complications, as tumor, and (Socie et al., Malignant tumors occurring after treatment of aplastic anemia, N.Eng.J.Med.1993; 329 (16): 1152-1157) and graft versus host disease (Ferrara et al., Graft-versus-host disease, N.Engl.J.Med.1991 (324); 324:667-674). in addition, many patients can not carry out bone marrow transplantation because the treatment expense or can not find the blood donor who is complementary.Now, need a kind of simple effectively and have the Therapeutic Method of low side effect to treat the aplastic anemia disease.The result of the present invention research shows that aplastic anemia can be treated effectively and can be reduced side effect.Believe that this immunotherapy based on cell is equally applicable to other anemias and bone marrow depression disease.These bone marrow depression diseases comprise that the AIDS patient accepts the anemia that causes after the HAART, the bone marrow depression that the cancer patient radiation and chemotherapy causes, congenital aplastic anemia and idiopathic thrombocytopenic purpura.

Though the present invention can not explain with a clear and definite mechanism of action, has this following possible mechanism of action at present.First is that this therapy allows activated cell secrete the multiple effective factor (some is not also for known to us) simultaneously.These factors are done the time spent, can produce the drug effect effect of associating.The second, external activated hemocyte is secreted some, and effect plays a crucial role to bone marrow hematogenesis, but existing also not known Hemopoietic factor.The all or part of curative effect to therapy of the present invention of these lost Hemopoietic factors works.The 3rd factor is that immunocyte can arrive bone marrow and the liftoff secrete cytokines of low coverage is given the hematopoietic stem cell mother cell relevant with other.Also have, activated immunocyte can improve the hematopoieticmicroenviron-ment in the bone marrow.The 4th is that cell is in contact with one another to treating effective factor, and its result has promoted the differentiation and the growth of hematopoietic stem cell and other hemocytoblast.The 5th factor is activated immunocyte, even quantity seldom also can stop the adverse immune response that destroys normal hematopoiesis mechanism.In above-mentioned four kinds of mechanism, cytokine must play a part crucial.Because clear people's the immunocyte that shows has very strong effect to mice in the experiment of mice, it is the general knowledge of generally acknowledging that Humanized cell factor can work to the Mus cell.

In the present invention, activated hemocyte has produced for patient's medication and is much better than the result of study of most somatomedin in the past.Reports such as Young find can to help aplastic anemia to patient with granulocyte colony-stimulating factor and granulocyte macrophage colony stimulating factor, and others improves neutrophilic granulocyte quantity, but only only limit to neutrophilic granulocyte (The treatment ofsevere acquired aplastic anemia, Blood.1995; 85 (12): 3367-3377).The research report of sole exception shows that granulocyte colony-stimulating factor stimulates the increase of granulocyte and platelet counts simultaneously.The effect of the individually dosed generation of interleukin 3 is undesirable, but the effect that then produces with the granulocyte-macrophage colony stimutaing factor administering drug combinations is poorer.Similarly discover (Liu etc., Cellular interactions in hemopoiesis, Blood Cells 1987; 13:101-110 and Ettinghausen etc., Hematologic effects if immunotherapy with lymphokine-activated killer cellsand recombinant interleukin-2 in cancer patients, Blood 1987; 69 (6): 1654-1660), the cancer patient who is subjected to chemotherapy to cause anemia can increase the weight of patient's severity of anemia when accepting activated tumor-killing lymphocyte of interleukin II (LAK) and interleukin-2 administering drug combinations.

The present invention relates to other required when producing product key element simultaneously, includes the container of one or more somatomedin and Calcium ionophore, proves absolutely among the present invention of these key elements.In the invention of setting forth current treatment, the material that comprises in the container is to be used for cultivating and activating hemocyte.The suitable culture fluid of cultivating hemocyte also can be included in wherein.

The cytokine and the chemokine that are produced by external activated immunocyte show some features, γ-IFN, IL-12, these four kinds of cytokines of IL-4 and IL-10 are being represented t helper cell Th1 and the secreted main cytokine of Th2, preceding two kinds by Th1 secretion then two kinds secrete by Th2.But these cytokines are limited in the effect that promotes bematogenesis, on the contrary, other cytokine such as IL-6, IL-8, G-CSF, GM-CSF but has very strong stimulation to hemopoietic function of bone marrow.Institute of the present invention cultured cells secreting high levels bone marrow hematogenesis is had the cytokine of facilitation, comprise above-mentioned four kinds.

The IL-2 that uses in cell culture can control the T cell and make t cell activation.By the IL-2 activated T cells than by phytohaemagglutinin (PHA) or concanavalin A, Con A (ConA) activated T cells in the external ability that longer vitality and stronger generation cytokine are arranged.It is cell that GM-CSF is used to activate grain, comprises mononuclear cell, dendritic cell, and neutrophilic granulocyte, to have a liking for the acid grain thin, and basophil is hugely bitten Fine born of the same parents etc.The purpose of using Calcium ionophore is the stimulation that strengthens IL-2 and GM-CSF pair cell, because immunocyte needs two or more stimulus signal fully to be activated usually, reaches the cytokine of secretion maximum after the complete state of activation.

US Patent No 5,643,786 (separation methods of Cohen etc. " Method for Isolating Dendritic Cells " dendritic cell) are used IL-2, GM-CSF, IL-12 and calcium ion carrier A 23187 are cultivated and the isolated dendritic cell, these dendritic cell be used as vaccine after antigen combines, immune host with improve the host at antigenic immunoreation.It is in order to reduce the expression of CD14 on the mononuclear cell surface that calcium ion carrier A 23187 is used in this patent, because CD14 is monocytic peculiar molecule, dendritic cell should not expressed this molecule.Calcium ion carrier A 23187 has the effect of very strong reduction CD14, and this patent does not relate to damaged disease of blood and bone marrow depression fully.Most document is mentioned and cultivated the ripe and effective optimum time of dendritic cell is 6 to 8 days, and dendritic cell just can reach best maturity state like this, becomes the antigen-presenting cell that function is arranged.The used cell culture time of treatment of the present invention is 2 days, has probably reached the peak of active cell factor growth.In addition, this therapy is very practical and operation easily.

Absorbingly notice that this current treatment is to be different from G-CSF to mobilize lymphocytic treatment in stem cell and the storage pool.G-CSF has cell in the very strong mobilization storage pool to the ability of peripheral blood, but the effect that stimulates stem cell growth a little less than.Method of the present invention can promote the growth of stem cell in bone marrow in bone marrow.The effect of growth factor for treating congenital anemia more is the cell that stimulates differentiation, mobilize effect strong, but they lacks the lasting effect of bone marrow hematogenesis effect.On the contrary, Therapeutic Method of the present invention more produces effect to serious and chronic bone marrow depression, is more suitable in long-term treatment.In the serious and chronic bone marrow depression that causes by chemotherapy and radiation, whether mutual the replenishing of somatomedin and current this therapy, uniting these two kinds of therapies of utilization when the different phase of treatment disease, whether can to produce better effect be that we will study and understand from now on.

In this research of the present invention, external immune cell activated therapy has strengthened significantly because the thrombocytopenic recovery that chemotherapy and radiation causes.As a result, great majority have its platelet counts of patient of long-term thrombocytopenia to obtain recovery to a certain degree, make these patients have enough platelet to accept further chemotherapy and radiation.Because this therapy has been used single dosage under study for action, and the valid density of any stimulating factor of its ideal biological dose can be defined as any concentration that hemocyte can be activated in the therapeutic process of the present invention.Many PDGFs are found the generation that can induce anti-human body factor antibody of the same race in clinical application, these antibody can cause serious even life-threatening toxicity, thereby its utilization prospect clinically allows of no optimist.Interleukin 11 is unique a kind of approved thrombopoietic stimulating factor.Selected patient had accepted the treatment of IL-11 in our research, but without any improvement.Inoperative to existing medicine of these patients and treatment, and treatment of the present invention has significant curative effect.

Claims (27)

1, method is used for the treatment of and suffers from leiphemia disease and myelosuppressive patient, and this method is to give patient's medication to increase blood constituent concentration with the immunocyte that external activation is cultivated;

2, the patient in the claim 1 is human;

3, the leiphemia disease in the claim 1 is relevant with cancer condition with bone marrow depression;

4, the leiphemia disease in the claim 1 comprises granulocytopenia, leukopenia, thrombocytopenia and the combination in any between them;

5, the leiphemia disease in the claim 1 comprises the severe chronic thrombocytopenia;

6, leiphemia disease and the bone marrow depression in the claim 1 caused by chemoradiotherapy;

7, the leiphemia disease in the claim 1 is relevant with former and secondary aplastic anemia with bone marrow depression;

8, the leiphemia disease in the claim 1 is relevant with idiopathic thrombocytopenic purpura;

9, patient accepts the hemocyte of dose therapeutically effective in the claim 1;

10, the immunocyte in the claim 1 is cultivated at the culture fluid that contains cytokine and Calcium ionophore;

11, the cultivation of the immunocyte in the claim 1 is a calcium ion carrier A 23187 containing cytokine and Calcium ionophore;

12, the immunocyte in the claim 1 is cultivated in containing the culture fluid of interleukin II;

13, the immunocyte in the claim 1 is cultivated in containing the culture fluid of granulocyte-macrophage colony stimutaing factor;

14, the immunocyte in the claim 1 was cultivated 2 days or was less than 2 days;

15, the immunocyte in the claim 1 is patient's same somatic cell or a variant cell;

16, the immunocyte in the claim 1 is separated from serum by centrifugation;

17, the immunocyte in the claim 1 is the peripheral blood mononuclear hemocyte;

18, the immunocyte in the claim 1 is cultivated in mammiferous serum or in the culture fluid of serum-free;

19, the immunocyte in the claim 1 is can obtain the received donor on immunology;

20, cultured cells is to pass through intravenous administration in the claim 1;

21, the cell intravenously administrable with In vitro culture in the claim 1 comprises multiple dosing;

22, the immunocyte of the In vitro culture in the claim 1 single nuclear blood cell of peripheral blood of separation and purification;

23, the immunocyte in the claim 1 is cultivated and is being contained an interleukin, in the culture fluid of a cell stimulating factor and a Calcium ionophore;

24, the immunocyte in the claim 1 comprises 3 types cell at least;

25, the single nuclear blood cell of separation and purification is adopted granulocyte composition blood system from purification by venous blood or machine in the claim 1, and produces required immunocyte through In vitro culture;

26, the single nuclear blood cell of separation and purification is separated into attached cell and non-adherent cell in the claim 1 in incubation, and attached cell and non-adherent cell are used to separately cultivate and the immunocyte of the required In vitro culture of output;

27, the immunocyte of In vitro culture causes polyclonal bone marrow hematogenesis in the claim 1.

Images