CN1114443C - Medicine for induction-differential therapy and its application - Google Patents
Medicine for induction-differential therapy and its application Download PDFInfo
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- CN1114443C CN1114443C CN99119912A CN99119912A CN1114443C CN 1114443 C CN1114443 C CN 1114443C CN 99119912 A CN99119912 A CN 99119912A CN 99119912 A CN99119912 A CN 99119912A CN 1114443 C CN1114443 C CN 1114443C
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Abstract
The present invention relates to a medicine for induction and differentiation treatment, which contains astragalus root, etc. Beta-globinmRNA is promoted to be translated into globin and synthesized into haemoglobin by inducing beta-globin genes in tumor cells from not being expressed to being expressed, and the tumor cells are induced to tend terminal differentiation to become normal or nearly normal cells, so that the goal of resisting tumors is achieved. The medicine of the present invention is obviously superior to an induction and differentiation medicine hydroxyurea which is internationally accepted at present, and has no toxic and side effect. The present invention can be used for treating hemopoietic system tumours, beta-mediterranean anemia, sickle cell anaemia, etc., and shows wide application prospects.
Description
The present invention relates to treat the medicine of tumor, especially about a kind of medicine of induction-differential therapy hematopoietic system cancer.
In recent years, the sickness rate of hematopoietic system cancer (as leukemia etc.) rises year by year, accounted for the tumor incidence first place at present in adolescent and child, and its mortality rate also rises to former positions of this age group gradually.But, wait the problem that solves in addition in a large number around the treatment of hematopoietic system cancer, lack gratifying treatment means (Ai Huisheng etc., " modern leukemia ", the People's Medical Officer Press, Beijing, 1997, P.171～178).For leukemic treatment, mainly contain three kinds of forms at present: chemotherapy is the leukemic primitive form of treatment, especially for acute leukemia, but, because chemical sproof existence may cause the failure of chemotherapy, simultaneously, follow the serious toxic and side effects of chemotherapy also to make the patient be difficult to finish chemotherapy.Immunization therapy comes also to be applied to gradually clinical at recent two decades, but because its side effect is more outstanding, also seldom succeeds.Bone marrow transplantation, operative treatment and radiotherapy etc. are used very limited so far in leukemia treating, are only applicable to small number of patients as bone marrow transplantation; Operative treatment only limits to before treatment or the emergency case surgery that occurs in the treatment; Radiotherapy is applicable to that symptom control before appears in central nervous system leukemia.Though external in recent years gene therapy of rising has obtained some challenging result, but still be in the starting stage, and be mainly used in the treatment single gene inheritance disease, the leukemic practical application of distance treatment also has very long stretch, exists a lot of difficulties (Lu Daopei, " leukemia treating ", Science Press, Beijing, 1992, P.23～39; Evans WE, et al.Leuk.Res., 1997,21:435～437; Balis FM, et al.J.Clin.Oncol., 1985,3:485～489).
Leukemic induction-differential therapy is being proved to be an effective treatment approach recently, and be confirmed in the inductive treatment of acute promyelocytic leukemia (M3) (Chen GQ, etal.Leukemia, 1996,10:825～828).As the medicine hydroxyurea (Hydroxyurea) of the inducing leukemia cell differentiation of generally acknowledging in the world at present etc., but the hydroxyurea toxic and side effects is bigger.Because the key of induction-differential therapy is the discovery of new differentiating inducer and illustrating of the mechanism of action thereof, therefore, medicine scholar both domestic and external and pharmacologist just are being devoted to set up model, and screen a large amount of chemical compounds.Come out though existing multiple chemical compound is screened, nearly 1000 kinds of chemical compounds have promptly been screened as Shanghai Pharmaceutical Inst., Chinese Academy of Sciences only, and find that some chemical compounds have stronger differentiation-inducing action, but, these chemical compounds also have outstanding toxic and side effects to body, as intensive tissue irritation, bone marrow depression, immune function infringement, gastrointestinal reaction, heart Toxicity of Kidney, dermal toxicity and neurotoxicity or the like (Ai Huisheng etc., " modern leukemia ", the People's Medical Officer Press, Beijing, 1997, P.171～178; Gunz FW, et al. " Leukemia ", Grune ﹠amp; Stratton, Inc.1983, P.137～213).
Chinese herbal medicine is bright bright jewel in motherland's traditional medicine treasure-house.Use tumors such as Chinese medicine method treatment pulmonary carcinoma, gastric cancer, hepatocarcinoma to obtain good efficacy clinically, Chinese medicine has the enhancing human body immunity function, alleviate clinical symptoms, improve life quality, alleviate characteristics (Cheng Xiaodong etc. such as chemotherapy radiotherapy toxic and side effects.China's combination of Chinese and Western medicine magazine, 1997,17 (2): 88～90; Cheng XD, et al.Chinese Journal of Immunology, 1995,11 (Suppl.): 218～220).Though the clinical case of late malignant tumours such as useful treatment by Chinese herbs pulmonary carcinoma, hepatocarcinoma early in the traditional medicine,, with herb induction tumor cell trend eventually the end differentiation with the treatment hematopoietic system cancer, so far from not having report and discussing.Therefore, the medicine of induction-differential therapy and the research of application thereof are a job highly significant.
The purpose of this invention is to provide the medicine of induction-differential therapies such as a kind of Radix Astragali, be used for the treatment of hematopoietic system cancer, evident in efficacy and have no side effect.
The invention provides the medicine of induction-differential therapies such as a kind of Radix Astragali.Behind the herb induction K562 cells such as the Radix Astragali; can make never expression of beta-globin in the K562 cell; promote that β-globin mRNA translates into globin and synthetic hemoglobin; induce the end differentiation eventually of K562 cell trend; its grade malignancy is reduced; make it " turning over a new leaf ", become normal or approaching normal cell, thereby reach the purpose of treatment tumor.
The medicine of induction-differential therapy of the present invention can distinctly adopt following four kinds of prescriptions:
Prescription A is the Radix Astragali;
Prescription B comprises the Radix Astragali and Radix Angelicae Sinensis, and both spice part by weight Radixs Astragali are 0.1-99.9%: Radix Angelicae Sinensis 99.9-0.1%;
Prescription C comprises the Radix Astragali and Fructus Jujubae, and both spice part by weight are Radix Astragali 0.1-99.9%: Fructus Jujubae 99.9-0.1%; Or
Prescription D comprises Radix Angelicae Sinensis, Rhizoma Chuanxiong, Radix Rehmanniae Preparata and the Radix Paeoniae Alba, and its spice part by weight is Radix Angelicae Sinensis 0.1-99.7%, Rhizoma Chuanxiong 99.7-0.1, Radix Rehmanniae Preparata 99.7-0.1%, Radix Paeoniae Alba 99.7-0.1%.
The preparation method of induction-differential therapy medicine of the present invention: earlier the spice of each prescription is used water rinse respectively, after in vial, adding the tri-distilled water decoction, remove slag, filter, concentrate, operate (Du Keli etc. routinely, " simple and clear pharmaceutics " People's Health Publisher, Beijing 1994) make various dosage forms such as decoct, pill, electuary, tablet, capsule, oral liquid or injection etc.The medicine of induction-differential therapy of the present invention is Chinese herbal medicine, is natural plants, and human body is not had any toxicity.
The present invention adopts human erythroleukemia cell's strain (K562 cell) as external model, carries out the in vitro tests of Chinese medicines such as the Radix Astragali to hematopoietic system cancer cell induction differentiation.
1) cells in vitro goes down to posterity and cultivates and inducing culture:
The K562 cell uses RPMI-1640 (Gibco product) culture fluid at 37 ℃, CO
2Cultivate in the incubator,, in cell training liquid, add each prescription (A, B, C, D) medicinal liquid of Chinese medicine respectively for inductive K562 cell, in the experiment to induce differentiation medicament hydroxyurea (Hydroxyurea, Hu) in contrast, collected the K562 cell in back 7 days, be used for the extracting of cell total rna in inducing.
2) extracting of total RNA in the cell:
Total RNA in the extracting cell is with the extracting of guanidinium isothiocyanate one-step method.
3) RT-PCR detect α in the cell-, β-and γ-globin gene expression:
Get cell total rna and carry out the pcr amplification test, the concentration of various compositions is the same with conventional PCR reaction in the PCR reaction.
4) phenylhydrazine of cell dyeing:
Collect an amount of cell, after the phenylhydrazine dyeing liquor dyeing of the new preparation of adding, counting blue-stained cell is represented positive rate with the percentage ratio that blue transfect cell number accounts for total cell number.
Each prescription (A, B, C, D) of Chinese medicine of the present invention is as follows to the in vitro tests result of K562 cell induction differentiation:
1) each prescription (A, B, C, D) of Chinese medicine is to the influence of beta-globin gene expression in the K562 cell
Use in the experiment without inductive K562 cell as negative control, with the inductive K562 cell of hydroxyurea as positive control.The result shows: the K562 cell can not be expressed as model year beta-globin gene when inducing, and cell can not reach end differentiation eventually; After inducing the K562 cell with 200 μ mol/L hydroxyureas, can detect beta-globin gene transcription product, promote the K562 cell to end differentiation eventually; Induce the K562 cell after 7 days respectively with Chinese medicine preparation A, B, C, D medicinal liquid, can detect beta-globin gene transcription product too, as shown in Figure 1.This result shows that Chinese medicine preparation A, B, C, D can induce never expression of beta-globin gene in the K562 cell, promotes the end differentiation eventually of K562 cell trend.
2) each prescription (A, B, C, D) of Chinese medicine is to the influence of α-Zhu Danbai gene expression in the K562 cell
The K562 cell can express alpha-globin gene when inducing; Induce the K562 cell after 7 days respectively with hydroxyurea and Chinese medicine preparation A, B, C, D medicinal liquid, still can detect α-Zhu Danbai gene transcription product, do not see significant change, as shown in Figure 2.This result shows that Chinese medicine preparation A, B, C, D do not have a significant effect to α-Zhu Danbai expression of gene in the K562 cell.
3) each prescription (A, B, C, D) of Chinese medicine is to the influence of γ-globin gene expression in the K562 cell
The K562 cell can be expressed fetal type γ-globin gene when inducing; Induce the K562 cell after 7 days respectively with hydroxyurea and Chinese medicine preparation A, B, C, D medicinal liquid, still can detect the transcription product of fetal type γ-globin gene, do not see significant change, as shown in Figure 3.This result shows that Chinese medicine preparation A, B, C, D do not have a significant effect to the expression of fetal type γ-globin gene in the K562 cell.
4) each prescription (A, B, C, D) of Chinese medicine is to the synthetic influence of hemoglobin in the K562 cell
Cell shows the conventional K562 cell of cultivating before inducing after phenylhydrazine dyeing, the blue transfect cell number that contains hemoglobin only accounts for 1% of total cellular score; After inducing with hydroxyurea, the blue transfect cell number of phenylhydrazine obviously increases, and reaches 13%; After inducing respectively with Chinese medicine preparation A, B, C, D, the increase of the blue transfect cell number of phenylhydrazine is more remarkable, reaches 30%, 31%, 33%, 28% respectively, as shown in Figure 4.This result shows: Chinese medicine preparation A, B, C, D not only can induce the K562 cell to make beta-globin gene expression, and can promote β-globin mRNA to translate into globin and synthetic hemoglobin, and its effect significantly is better than the chemical inducer hydroxyurea.
Above-mentioned in vitro tests result shows: respectively fill a prescription A, B, C, D of Chinese medicine induces the K562 cell after 7 days, can make the never expression of adult beta-globin gene, and the expression of α-Zhu Danbai gene and γ-globin gene is not obviously influenced, be similar to the effect of inducing the differentiation agent hydroxyurea, show that respectively fill a prescription A, B, C, D of Chinese medicine activated K562 cell beta-globin gene transcription mechanism.Simultaneously, the K562 cell is under routine is cultivated, contain the blue transfect cell number few (＜1%) of hemoglobin, though illustrate the K562 cell have transcribe ε-, γ-and the α-Zhu Danbai gene pairs mRNA ability of answering, still can not translate into globin and synthetic hemoglobin.Respectively fill a prescription after A, B, C, D induce through Chinese medicine, the blue transfect cell digital display work that contains hemoglobin increases, and the K562 cell be described after inducing, synthetic hemoglobin effectively, and also it acts on and obviously is better than hydroxyurea.Show to exist the mechanism that suppresses the globin gene translation in the K562 cell, Chinese medicine is respectively filled a prescription after A, B, C, D induce, and this inhibition mechanism is reversed.Respectively fill a prescription A, B, C, D of experimental result proof Chinese medicine not only can induce human erythroleukemia cell's strain (K562 cell) beta-globin expression of gene, and, promote the end differentiation eventually of tumor cell trend to β-globin mRNA translates into globin and synthetic hemoglobin has facilitation.
According to motherland's theory of medicine " in healthy energy deposits; heresy can not be done; the institute of heresy gathers; its gas must be empty ", the generation development of positive QI-insufficiency and tumor is closely related, be the main pathomechanism of tumor body, setting upright therapy is the common method of tcm syndrome differentiation and treatment tumor, reaches the purpose of driving away pathogenic factor, treatment disease by reinforcing body resistance.But until now, the present invention treats hematopoietic system cancer with the method for herb induction tumor cell differentiation first, and from gene level research Chinese medicine antineoplastic action.Motherland's theory of medicine is thought: " qi promoting blood production ", " tangible blood; can not be spontaneous; be born in invisible gas ", Chinese medicine preparation A, B, C, D all according to QI invigorating, theoretical prescription nourishes blood, the gas of big spleen reinforcing lung as a means of the source of hemopoietic, can make Yin grows while yang is generating, the prosperous blood of gas is given birth to, and reaches the purpose of strengthening vital QI to eliminate pathogenic factors, treatment disease.The present invention not only provides experimental basis for the theory of the traditional Chinese medical science " qi-supplementing, blood-engendering " theory and strengthening vital QI to eliminate pathogenic factors treatment tumor, and provide a medicine that has no side effect evident in efficacy for significant anaemia such as clinical treatment hematopoietic system cancer and β-thalassemia, sickle anemias, also can be used as rehabilitation of patients health nutrient health product such as hematopoietic system cancer, β-thalassemia and sickle anemias, show wide application prospect, had huge social and economic benefit.
During clinical trial of the present invention is being carried out smoothly.
Advantage of the present invention: the present invention at home and abroad proposes first with the end differentiation eventually of herb induction hematopoietic system cancer cells such as Radix Astragali trend; the tumor cell grade malignancy is reduced; make it " turning over a new leaf ", become normal or approaching normal cell, thereby reach the purpose of treatment tumor.Medicine provided by the invention is different with common antineoplastic chemotherapy medicine, but the division and the propagation of common chemotherapeutics killing tumor cell, inhibition tumor cell, but organism normal cell also there is obvious impairment, there is serious toxic and side effects, such as intensive tissue irritation, bone marrow depression, immune function infringement, gastrointestinal reaction, heart Toxicity of Kidney, dermal toxicity and neurotoxicity or the like.And medicine advantage provided by the invention both had been inducing tumor cell trend end differentiation eventually effectively, simultaneously again organism normal cell there is not any damaging action, and can be by adjusting the balance of each system's physiological function of body, as regulate neuroendocrine-immune network, regulate cardiopulmonary hepatic and renal function, compensate qi and blood etc., to improve clinical symptoms, improve life quality.
Description of drawings:
Fig. 1 is the figure as a result that Chinese medicine preparation A, B, C, D induce beta-globin gene expression in the K562 cell
Wherein 1: the molecular weight sign; 2: not inductive K562 cell; 3: the inductive K562 cell of hydroxyurea; 4: the inductive K562 cell of Chinese medicine preparation A; 5: the inductive K562 cell of Chinese medicine preparation B; 6: the inductive K562 cell of Chinese medicine preparation C; 7: the inductive K562 cell of Chinese medicine preparation D.
Fig. 2 is the figure as a result that Chinese medicine preparation A, B, C, D induce α-Zhu Danbai gene expression in the K562 cell
Wherein 1: the molecular weight sign; 2: not inductive K562 cell; 3: the inductive K562 cell of hydroxyurea; 4: the inductive K562 cell of Chinese medicine preparation A; 5: the inductive K562 cell of Chinese medicine preparation B; 6: the inductive K562 cell of Chinese medicine preparation C; 7: the inductive K562 cell of Chinese medicine preparation D.
Fig. 3 is the figure as a result that Chinese medicine preparation A, B, C, D induce γ-globin gene expression in the K562 cell
Wherein 1: the molecular weight sign; 2: not inductive K562 cell; 3: the inductive K562 cell of hydroxyurea; 4: the inductive K562 cell of Chinese medicine preparation A; 5: the inductive K562 cell of Chinese medicine preparation B; 6: the inductive K562 cell of Chinese medicine preparation C; 7: the inductive K562 cell of Chinese medicine preparation D.
Fig. 4 is the figure as a result that Chinese medicine preparation A, B, C, D induce K562 cell synthetic hemoglobin
Wherein 1: not inductive K562 cell; 2: the inductive K562 cell of hydroxyurea; 3: the inductive K562 cell of Chinese medicine preparation A; 4: the inductive K562 cell of Chinese medicine preparation B; 5: the inductive K562 cell of Chinese medicine preparation C; 6: the inductive K562 cell of Chinese medicine preparation D.
The present invention is further elaborated by following examples, but do not limit the scope of the invention.
Embodiment 4 Radixs Astragali 60 grams, Fructus Jujubae 30 grams add water 400ml and decoct 2 times, and each 1 hour, decocting liquid merged filtration, is condensed into cream, drying, granulate, tabletting, every contains crude drug 20 grams, promptly gets tablet.
1) cells in vitro goes down to posterity and cultivates and inducing culture
The K562 cell uses RPMI-1640 (Gibco product) culture fluid at 37 ℃, CO
2Cultivate in the incubator, contain 10% newborn calf serum, 100 μ g/ml streptomycins, 100u/ml penicillin in the culture fluid.For inductive K562 cell, in cell training liquid, add each prescription (A, B, C, D) medicinal liquid of Chinese medicine respectively, the medicine final concentration is 7mg/ml; (Hydroxyurea, Hu) in contrast, the hydroxyurea final concentration is 200 μ mol/L to induce the differentiation medicament hydroxyurea in the experiment.Collect the K562 cell in back 7 days in inducing, be used for the extracting of cell total rna.
2) extracting of total RNA in the cell
Total RNA guanidinium isothiocyanate one-step method extracting in the extracting cell.
3) RT-PCR detect α in the cell-, β-and γ-globin gene expression
Get 1-1.5 μ g cell total rna, the concentration of various compositions is the same with Standard PC R reaction in the PCR reaction.The reaction cumulative volume is 50 μ l.
4) phenylhydrazine of cell dyeing
Collect an amount of cell, press 1/5 of cell volume, add phenylhydrazine dyeing liquor (1.25ml phenylhydrazine solution, the 0.1ml H of new preparation
2O
2), behind the dyeing 5min, counting blue-stained cell is represented positive rate with the percentage ratio that blue transfect cell number accounts for total cell number.
Experimental result
As shown in Figure 1.Show: Chinese medicine preparation A, B, C, D can induce never expression of beta-globin gene in the K562 cell, promote the end differentiation eventually of K562 cell trend.
As shown in Figure 2.Show: Chinese medicine preparation A, B, C, D do not have a significant effect to α-Zhu Danbai expression of gene in the K562 cell.
As shown in Figure 3.Show: Chinese medicine preparation A, B, C, D do not have a significant effect to the expression of fetal type γ-globin gene in the K562 cell.
Cell is through showing after the phenylhydrazine dyeing: before the conventional K562 cell of cultivating was being induced, the blue transfect cell number that contains hemoglobin only accounted for 1% of total cellular score; After inducing with hydroxyurea, the blue transfect cell number of phenylhydrazine obviously increases, and reaches 13%; After inducing respectively with Chinese medicine preparation A, B, C, D, the increase of the blue transfect cell number of phenylhydrazine is more remarkable, reaches 30%, 31%, 33%, 28% respectively, as shown in Figure 4.This experimental result shows that Chinese medicine preparation A, B, C, D not only can induce the K562 cell to make beta-globin gene expression, and can promote β-globin mRNA to translate into globin and synthetic hemoglobin, and its effect significantly is better than the chemical inducer hydroxyurea.
Claims (3)
1. pharmaceutical composition for the treatment of hematopoietic system cancer, β-thalassemia and reaping hook anemia, it is 5: 1 the Radix Astragali and when being grouped into by weight ratio.
2. pharmaceutical composition for the treatment of hematopoietic system cancer, β-thalassemia and reaping hook anemia, it is that 2: 1 the Radix Astragali and Fructus Jujubae formed by weight ratio.
3. by claim 1 or 2 described pharmaceutical compositions, the dosage form that it is characterized in that described medicine can be decoct, pill, electuary, tablet, capsule, oral liquid or injection.
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| ES2407090T3 (en) | 2002-12-04 | 2013-06-11 | Virginia Commonwealth University | Use of heterocyclic derivatives of carbaldehyde against sickle cell anemia |
| CN101991637B (en) * | 2004-03-09 | 2013-02-06 | 中国科学院上海生命科学研究院 | Medicament for inducing apoptosis and application thereof |
| CN100567490C (en) * | 2004-11-26 | 2009-12-09 | 上海交通大学附属儿童医院 | RNA interference fragment and application thereof |
| CN114073729A (en) * | 2020-07-31 | 2022-02-22 | 株洲千金药业股份有限公司 | Traditional Chinese medicine composition for preventing and treating thalassemia |
| CN113413408A (en) * | 2021-07-16 | 2021-09-21 | 安徽康信制药股份有限公司 | A Chinese medicinal capsule with anti-tumor effect, and its preparation method |
| CN115837042A (en) * | 2022-11-18 | 2023-03-24 | 重庆市中医院 | Application of angelica sinensis blood-enriching decoction in preparation of medicine for enhancing curative effect of immune checkpoint inhibitor |
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| CN1127652A (en) * | 1995-08-28 | 1996-07-31 | 陈尔东 | Oral liquid for restoration of cancer |
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| CN1127652A (en) * | 1995-08-28 | 1996-07-31 | 陈尔东 | Oral liquid for restoration of cancer |
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| 《中药大辞典》第1版 1977-10-31 江苏新医学院编上海科学技术出版社 * |
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