CN1204142C - Antigene V-erb B oligonucleotide and its use - Google Patents

Antigene V-erb B oligonucleotide and its use Download PDF

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CN1204142C
CN1204142C CN 00109023 CN00109023A CN1204142C CN 1204142 C CN1204142 C CN 1204142C CN 00109023 CN00109023 CN 00109023 CN 00109023 A CN00109023 A CN 00109023A CN 1204142 C CN1204142 C CN 1204142C
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oligonucleotide
gene
antigene
mds
erbb
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CN1326938A (en )
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冯宝章
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冯宝章
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Abstract

本发明涉及反基因V-erbB寡核苷酸,其多肽产物,包含其的药物组合物及其在诊断和治疗骨髓增生异常综合征、白血病和其他多种肿瘤中的应用。 The present invention relates to a V-erbB antigene oligonucleotide, polypeptide products thereof, pharmaceutical compositions containing them and their diagnosis and treatment of myelodysplastic syndrome, leukemia and other tumors in a variety of applications.

Description

反基因V-erbB寡核苷酸及其应用 V-erbB antigene oligonucleotides and their Applications

本发明涉及反基因V-erbB寡核苷酸,其多肽产物,包含其的药物组合物及其在诊断和治疗骨髓增生异常综合征、白血病和其他多种肿瘤中的应用。 The present invention relates to a V-erbB antigene oligonucleotide, polypeptide products thereof, pharmaceutical compositions containing them and their diagnosis and treatment of myelodysplastic syndrome, leukemia and other tumors in a variety of applications.

基因治疗是近十多年来发展起来的疾病治疗新技术,它是通过纠正致病基因的异常或用新的基因进行替换的办法达到治疗疾病的目的。 Gene therapy is developed in the last decade of the new disease treatment technology, it is through abnormal or replace it with a new way to correct genetic disease genes to achieve the purpose of treating disease. 鉴于目前肿瘤治疗手段的局限性和不彻底性,乃至多数肿瘤患者治疗后若干年便复发和死亡,人们对肿瘤的基因治疗抱有很高的期望。 Given the limitations of current methods of cancer treatment and lack of thoroughness, several years after recurrence and death and even then the majority of cancer patients, people on cancer gene therapy have high expectations. 然而现有技术中用病毒载体将正常基因导入体内的方法,虽有某些疗效,但面临一系列难题。 However, using the prior art viral vectors normal method of gene transfer in vivo, although some effect, but faces a series of problems. 而用反义寡核苷酸硫代磷酸酯作基因治疗则避免了因使用病毒载体而带来的一系列问题。 And with phosphorothioate antisense oligonucleotides for gene therapy is to avoid a series of problems arising from the use of viral vectors brought. 硫代磷酸所带来的某些毒性是人体可接受和能克服的,因而十分安全。 Some phosphorothioate toxicity is caused by the human body and overcome acceptable, and therefore very safe. 然而,由于反义寡核苷酸是针对癌细胞靶基因的mRNA而设计的,而癌细胞能源源不断地从细胞核内产生mRNA,因而不可能有巩固的疗效。 However, due to the antisense oligonucleotide against mRNA target cancer gene designed, but cancer cells can continuously produce mRNA from the nucleus, and therefore can not have the effect of consolidation. 而在此基础上发展起来的反基因寡核苷酸则针对癌基因的特定的突变位置而设计,使之进入细胞后能在细胞核内形成巩固的DNA三链结构而抑制该基因的表达和扩增,从而为肿瘤基因治疗带来新的希望。 In this developed on the basis of the antigene oligonucleotides specific position of the mutation designed oncogenes, so that the DNA triplex into the cell structure can be formed after consolidation in the nucleus and inhibits expression of the gene and expansion increase, bringing new hope to cancer gene therapy.

骨髓增生异常综合征(myelodysplastic syndrome,MDS)俗称白血病前期(preleukemia),是一组恶性的骨髓造血干细胞病,多数病人骨髓增生过度但分化成熟障碍,因而其外周血中有形成分减少,出现贫血、出血、血细胞减少及头晕、乏力等症状。 MDS (myelodysplastic syndrome, MDS), commonly known as pre-leukemia (preleukemia), is a group of malignant bone marrow hematopoietic stem cell disease, the majority of patients, but bone marrow differentiation and maturation hyperproliferative disorder, thereby reducing peripheral blood formed elements, anemia , bleeding, and cytopenia dizziness, fatigue and other symptoms. 多数病人诊断后若干月或若干年发展为急性白血病,且无有效治疗方法。 The majority of patients after diagnosis of several months or several years the development of acute leukemia, and no effective treatment.

已知V-erbB和V-erbA为鸡原始红细胞增多症(AvianErythroblastosis)病毒癌基因。 Known V-erbB and V-erbA raw chicken polycythemia (AvianErythroblastosis) viral oncogene. 前者同源于表皮生长因子受体(EGFR)基因,具有转化红系祖细胞的功能;后者具有阻断上述红系祖细胞分化成熟的作用。 The former homologous to the epidermal growth factor receptor (EGFR) gene, having a conversion function of erythroid progenitor cells; the latter having the above-described blocking erythroid maturation of progenitor cells. 二者协同作用导致鸡原始红细胞增多症或称红白血病。 Both lead to synergies chicken original polycythemia also known as red leukemia.

骨髓增生异常综合征(MDS)具有五个不同亚型,其中难治性贫血(Refractory Anemia,RA)是其基本型。 Myelodysplastic syndrome (MDS) has five different subtypes, wherein the refractory anemia (Refractory Anemia, RA) is the basic type. 它可以转化为原始粒细胞增多RA(即RAEB)和转化中的RAEB(即RAEBT)和急性白血病(Acute Leukemia,AL)。 It can be transformed into an increase in myeloblasts RA (i.e., RAEB) and RAEB in transformation (i.e. RAEBT) and acute leukemia (Acute Leukemia, AL). RAEBT患者骨髓中原始和早幼红细胞增多时还可以再诊断为红白血病而与上述鸡红白血病相同。 RAEBT increased bone marrow of patients and early primitive erythroblast diagnosis can have again the same as described above chicken leukemia erythroleukemia. RA患者骨髓单一红系统过度增生时亦可诊断为红血病。 RA patients with bone marrow red single system can also be diagnosed with leukemia when red hyperplasia. 而红血病可以发展为红白血病。 The Red leukemia can progress to leukemia. 说明红血病—红白血病与MDS-RA和RAEB、RAEBT等并无本质区别。 Description Red leukemia - erythroleukemia no essential difference with MDS-RA and RAEB, RAEBT like. 提示V-erbB和V-erbA之血细胞副本C-erbB和C-erbA异常可能在人MDS、白血病发病中起重要作用。 Tip V-erbB and V-erbA copy of C-erbB blood cells and C-erbA abnormalities may play an important role in human MDS, leukemia.

以往的研究业已证明,人外周血淋巴细胞染色体脆性部位(Fragile Site,FS)高表达提示肿瘤易感性。 Previous studies have demonstrated that human peripheral blood lymphocyte chromosome fragile site (Fragile Site, FS) with high expression of cancer susceptibility. 肿瘤患者FS往往与肿瘤细胞染色体断点以及癌基因位置相同或相近。 FS tend to the patient's tumor cells and tumor chromosomal breakpoints and the same or similar position oncogene. 本发明人发现一例白血病前期患者外周淋巴细胞表达Fra(7)(P11)和Fra(3)(P14)等。 The present inventors have found that one case of leukemia patients with pre outer peripheral lymphocytes express Fra (7) (P11) and Fra (3) (P14) and the like. Fra(7)(P11)与C-erbB相毗邻,患者骨髓红系病态造血(Myelodysplasia)明显,因而高度怀疑C-erbB受累。 Fra (7) (P11) and C-erbB adjacent to, bone marrow erythroid dysplasia (myelodysplasia) significantly, thus C-erbB highly suspected involvement. 于是,本发明人选用V-erbB和V-erbA作探针进行Southetn印迹杂交,证明MDS与C-erbB重排/扩增和C-erbA缺失/失活相关,杂交结果有诊断和治疗意义。 Accordingly, the present invention is selected V-erbB and V-erbA Southetn blot hybridization as a probe, demonstrated MDS and C-erbB rearrangement / C-erbA amplification and deletion / inactivation of, hybridization diagnostic and therapeutic implications. 在此基础上,本发明人应用V-erbB PCR和正基因寡核苷酸原位杂交方法,进而确定C-erbB重排位置,其PCR产物和原位杂交结果均有诊断和发病学意义。 On this basis, the present invention is applied and a positive V-erbB PCR gene oligonucleotide in situ hybridization, and to determine the position of C-erbB rearrangement which results in situ hybridization and PCR products have diagnostic and pathogenic significance. 由此完成本发明。 Thereby completing the present invention.

因此,本发明的第一个目的在于提供应用反基因技术原理用于治疗MDS、白血病和其他多种肿瘤的反基因寡核苷酸及其较短的副本。 Accordingly, a first object of the present invention to provide a technical principles apply inverse gene therapy for the MDS, leukemia and various other anti-tumor genes oligonucleotides shorter and copies. 所述的反基因寡核苷酸是针对V-erbB DNA序列中可疑的重排位置即原位杂交中所用正基因寡核苷酸而设计合成的反基因寡核苷酸。 The anti-gene oligonucleotides for the V-erbB DNA sequence suspected rearrangement position i.e. designed as in situ hybridization antigene oligonucleotides synthesized oligonucleotides with positive gene.

本发明的第二个目的在于提供一种药物组合物,其包括本发明的寡核苷酸及一种药物学可接受的载体,用于治疗MDS、白血病和其他多种肿瘤。 A second object of the present invention is to provide a pharmaceutical composition which comprises an oligonucleotide of the present invention and a pharmaceutically acceptable carrier, for the treatment of the MDS, leukemia and various other cancers.

本发明的第三个目的在于提供一种反基因寡核苷酸组合(配方),其包括本发明的寡核苷酸和与癌症发病相关基因的反基因寡核苷酸。 A third object of the present invention is to provide an anti-gene oligonucleotide composition (formulation) which comprises an oligonucleotide of the present invention and an anti-cancer related genes gene oligonucleotide.

本发明的第四个目的在于提供与所述反基因寡核苷酸相对应的正基因寡核苷酸,其能用于诊断MDS、白血病和其他多种肿瘤。 A fourth object of the present invention is to provide the antigene oligonucleotides corresponding to n-gene oligonucleotides, which can be used to diagnose the MDS, leukemia and various other cancers.

本发明的第五个目的在于由本发明的反基因或正基因寡核苷酸转录得到的mRNA,及所述mRNA表达的多肽,包含该多肽的疫苗。 A fifth object of the present invention is expressed polypeptide mRNA, the mRNA, and obtained by antigene oligonucleotide transcripts or gene of the present invention n, the vaccine comprising the polypeptide.

本发明的第六个目的在于提供一种诊断试剂盒,它含有本发明的第四个目的中所述的寡核苷酸。 A sixth object of the present invention to provide a diagnostic kit comprising an oligonucleotide fourth object of the present invention.

具体地说,首先本发明涉及反基因V-erbB寡核苷酸,其能用于治疗MDS、白血病和其他多种肿瘤。 Specifically, first, the present invention relates to a V-erbB antigene oligonucleotides, which can be used to treat the MDS, leukemia and various other cancers. 所述的寡核苷酸具有如下核苷酸序列:5′ATGGCAGAGCTGGCAAAC(SEQ ID NO:1)5′AATTCTCAGGTGGGCCTG(SEQ ID NO:2)本发明的反基因V-erbB寡核苷酸是朝着C-erbB的重排部位而设计合成的,因其与正基因C-erbB重排部位碱基互补而能靶向地结合并抑制正基因C-erbB的异常表达或转录,或者同正基因C-erbB mRNA结合而阻断其翻译为致癌性蛋白。 The oligonucleotide having the following nucleotide sequence: 5'ATGGCAGAGCTGGCAAAC (SEQ ID NO: 1) 5'AATTCTCAGGTGGGCCTG (SEQ ID NO: 2) Anti-V-erbB gene oligonucleotides of the present invention towards C -erbB rearrangement of parts designed synthesized, because the positive C-erbB gene rearrangement nucleotide site complementary to and capable of targeting gene binds to and inhibits the positive C-erbB abnormal expression or transcription or gene Far C- erbB mRNA which block binding oncogenic protein translation. 因此,运用本领域人员公知的反基因技术,本发明的寡核苷酸可用于治疗C-erbB重排/扩增和C-erbA缺失/失活相关的MDS、白血病及其他多种肿瘤。 Thus, those skilled in the use of known anti-gene technology, oligonucleotides of the invention are useful for the treatment of C-erbB rearrangement / C-erbA amplification and deletion / inactivation of the MDS to, leukemia and various other cancers. 在本发明的一个优选实施方案中,本发明的寡核苷酸经硫代磷酸修饰,这种修饰后的产物具有抑制核酸酶的作用并使其达到细胞内的靶位置。 In a preferred embodiment of the invention, the oligonucleotides of the invention via phosphorothioate modified product of such modification has the effect of inhibiting nuclease and allowed to reach a target location within the cell.

因此,本发明的另一方面涉及包含SEQ ID NO:1和/或SEQ IDNO:2的寡核苷酸及药物学可接受的载体的药物组合物,所述的药物组合物可给予MDS、白血病及其他多种肿瘤患者,药物组合物中包含的载体可以是本领域熟知的载体或赋形剂。 Accordingly, another aspect of the present invention relates to a SEQ ID NO: 1 and / or SEQ IDNO: oligonucleotide acceptable carrier and a pharmaceutically 2 a pharmaceutical composition, said pharmaceutical composition can be administered to the MDS, leukemia and a variety of other patients, a pharmaceutical carrier composition comprising a tumor may be known in the art carriers or excipients. 一种反基因寡核苷酸组合(配方),其包括本发明的寡核苷酸和与癌症发病相关基因的反基因寡核苷酸。 Gene an oligonucleotide composition (formulation) of trans, comprising the oligonucleotides of the invention and an anti-cancer related genes gene oligonucleotide.

已知骨髓中原始和早幼粒细胞比例的增加,不仅是MDS转白血病,也是慢性粒细胞白血病急性转变的集中表现,同时也是白血病原发和复发的集中表现。 Known to increase the proportion of cells in the bone marrow and promyelocytic original, not only switch MDS leukemia, acute leukemia is chronic myeloid transition concentrated expression, but also leukemias concentrated expression of primary and recurrent. 从以下实施例可知运用本发明的反基因V-erbB寡核苷酸能快速而有效地使MDS模型大鼠骨髓中原始和早幼粒细胞降低到正常水平(≤5%),从而为基因治疗MDS等恶性肿瘤疾病提供了一种途径。 Apparent from the following examples of the present invention is the use of V-erbB antigene oligonucleotides capable of quickly and effectively so that the original model of rat bone marrow MDS and promyelocytic reduced to normal levels (≤5%), such as gene therapy cancer and other diseases MDS provides a way.

根据本发明的另一方面,本发明还涉及与所述的反基因寡核苷酸相对应的正基因寡核苷酸,所述的正基因寡核苷酸经标记后例如可以用作诊断探针,与骨髓细胞样品进行原位杂交以判断个体是否患有MDS等恶性肿瘤。 According to another aspect of the present invention, the present invention also relates to the anti-gene oligonucleotides corresponding to n-gene oligonucleotide, after the positive gene labeled oligonucleotide can be used as a diagnostic probe e.g. needle, and bone marrow cell sample in situ hybridization to determine whether the subject MDS and other malignancies. 或者应用本发明人发明的引物,经V-erbBPCR方法检测患者骨髓的C-erbB基因异常与否,从而诊断MDS等恶性肿瘤。 Primer or application of the present inventors, V-erbBPCR detected by bone marrow of patients abnormal or not C-erbB gene, to diagnose MDS and other malignancies. 所述的正基因寡核苷酸可以以本领域已知的任何方法进行标记,如生物素、地高辛等。 The positive gene oligonucleotides may be labeled in any manner known in the art, such as biotin, digoxin and the like.

与病毒介导的基因治疗相比,用本发明的反基因寡核苷酸作基因治疗有许多优点。 Compared to virus-mediated gene therapy, with anti-gene oligonucleotides of the present invention has many advantages for gene therapy. 除了作用快速,操作简便外,没有因载体(如逆病毒或腺病毒)DNA或RNA的导入细胞而带来一系列难以评估的不良后果是最大的优点。 In addition to the role of fast, easy to operate, but because there is no carrier (e.g., retrovirus, or adenovirus) DNA or RNA into a cell brought difficult to assess a series of negative consequences is the greatest advantage. 本发明的寡核苷酸的作用特点是癌基因重排位置的原位修复,而非导入外源基因,因而它不同于一般的反义基因治疗。 Oligonucleotide effect characteristic of the present invention is a nucleotide cancer gene rearrangement in situ repair location, rather than the introduction of exogenous genes, so it is different from the antisense gene therapy. 后者是针对癌基因mRNA而设计合成反义寡核苷酸,而由于癌细胞能源源不断地从细胞核内产生mRNA,所以有限的反义寡核苷酸的治疗作用是有限和短暂的。 The latter is designed for cancer gene mRNA Antisense oligonucleotides, since the cancer cells can continuously produce mRNA from the nucleus, so limited antisense oligonucleotide therapeutic effects are limited and temporary.

以下将参照附图和实施例详细描述本发明。 The invention will be described in detail with reference to the accompanying drawings and embodiments.

图1显示实施例2中用本发明的反基因V-erbB寡核苷酸治疗后大鼠MDS的效果。 Figure 1 shows the effect of Example 2 in rat MDS antigene V-erbB oligonucleotide treatment by embodiments of the present invention.

图2显示实施例2中用本发明的反基因V-erbB寡核苷酸治疗后,大鼠MDS血象恢复正常。 Figure 2 shows the V-erbB antigene oligonucleotides of the present invention used in Example 2 after treatment, MDS blood return to normal rats.

图3显示实施例2中用本发明的反基因V-erbB寡核苷酸治疗过程中大鼠MDS体重增加。 Figure 3 shows the embodiment 2 antigene V-erbB MDS rat body weight gain during the treatment with the oligonucleotides of the present invention.

实施例1反基因和正基因V-erbB寡核苷酸的合成在DNA合成仪(型号:394型,来源:PE公司)上自动合成如下的寡核苷酸:5′ATGGCAGAGCTGGCAAAC(SEQ ID NO:1)5′AATTCTCAGGTGGGCCTG(SEQ ID NO:2)实施例2反基因V-erbB寡核苷酸治疗MDS的效果选择幼年天津大鼠(TR,来自天津医科大学动物室)和Wistar大鼠(WR,来自军事医学科学院营养所动物室)各10只,分为4组,每组5只。 Example 1 Synthesis of n and antigene V-erbB genes embodiment the oligonucleotide DNA synthesizer (Model: Model 394, Source: PE Corporation) automated synthesis of oligonucleotides are as follows: 5'ATGGCAGAGCTGGCAAAC (SEQ ID NO: 1 ) 5'AATTCTCAGGTGGGCCTG (SEQ ID NO: 2) Example 2 antigene V-erbB oligonucleotide treatment of MDS embodiment Tianjin selected young rats (TR, animal by Tianjin Medical University) and Wistar rats (WR, from the animal Center of military Medical Sciences nutrition) each 10, divided into four groups of five each. 按以往的方法(冯宝章等,中国实验血液学杂志,1996,4(3):309),经尾静脉注射二甲基苯蒽(DMBA)。 According to the conventional method (FENG Zhang et al., Chinese Journal of Experimental Hematology, 1996,4 (3): 309), injection DMBA (DMBA) via the tail vein. TR二组中一组为空白对照,另一组为实验组,每只动物每周注射1次,连续注射4次。 Two groups of TR was the control group, the other groups as experimental groups, each animal was injected once a week for four injections. WR二组中第一组和第二组分别注射1次和3次。 WR two groups of the first and second groups were injected once and three times. 而后观察各组动物发病情况。 Then each group of animals were observed incidence.

根据以往的工作,3次注射DMBA的大鼠有9/14发生MDS。 Based on previous work, three injections of rat DMBA occurred 9/14 MDS. 注射后2个月发病,并有3-4个月MDS期。 2 months after the onset of injection, and MDS have a 3-4 month period. 选择3次注射DMBA的Wistar大鼠作基因治疗模型。 Select three injections Wistar rats DMBA model for gene therapy.

将实施例1合成的2条反基因V-erbB寡核苷酸,经磷酸硫代修饰。 2 antigene V-erbB Example 1 Synthesis of oligo nucleotide embodiment, modified with phosphorothioate. 使用之前用生理盐水溶解,浓度为10.0OD/ml,MDS大鼠每天尾静脉注射1次,连续注射3天。 Dissolved in physiological saline prior to use, at a concentration of 10.0OD / ml, MDS rat tail vein injection of 1 day, 3 days continuous injection. 剂量为0.56mg/kg体重。 Dose of 0.56mg / kg of body weight.

在对3次给药组(5只)进行观察过程中,发现2只WR几乎同时发病并诊断为MDS-RA(即难治性贫血)。 In the administration group of 3 (5) was observed during 2 WR was found almost the same disease and a diagnosis of MDS-RA (i.e., refractory anemia). 当它们从RA发展到RAEB(原始细胞增多型RA)阶段时,将其中1只(WR1)选作基因治疗实验鼠,另一只(WR2)作为对照鼠。 When they are developed to RAEB from the RA (RA blasts type) phase, in which a (WR1 is) mice selected for gene therapy, the other (WR2 of) as the control mice. 实验鼠经尾静脉注射V-erbB反基因寡核苷酸,每天1次,连续3天为一个疗程。 Mice via the tail vein V-erbB antigene oligonucleotides, once a day, for three consecutive days as a course. 而后进行观察。 Then observed. 注射反基因寡核苷酸后1个月左右,发现对照鼠精神萎摩不振,且见尾根部生长3-4公分大小的瘤子(病理检查确认为肉瘤)。 1 month after injection antigene oligonucleotides, the control mice was found weak friction spirit wilt, root growth and see the end of 3-4 cm in size build-up (as confirmed pathological sarcoma). 骨髓检查确诊为红白血病,并死于尿路受肉瘤压迫而致的尿潴留。 Bone marrow examination diagnosed with leukemia and died by the urinary tract sarcomas of oppression caused. 而实验鼠不仅精神好,而且骨髓检查发现实验鼠的原始和早幼粒细胞从V-erbB反基因寡核苷酸治疗前的13.0%下降为3.0%。 Mice while only good spirit, mice and bone marrow examination revealed the original and promyelocytic decreased from 13.0% before the V-erbB antigene oligonucleotide treatment was 3.0%. 血象和骨髓象明显改善。 Blood and bone marrow significantly improved. 反基因治疗后3个月再作骨髓象和血象检查时发现二者均正常。 Found that both normal and then for 3 months, bone marrow and blood examination after anti-gene therapy. 详见图1、2。 See FIGS. 初步结果说明,本发明的反基因寡核苷酸对大鼠MDS有良好的疗效,而治疗前后体重检查结果说明此反基因寡核苷酸毒性不明显(图3)。 Preliminary results indicate, antigene oligonucleotides of the present invention have a good effect in rats MDS, and weight after treatment test results illustrate this obvious toxicity antigene oligonucleotides (FIG. 3). 将此只雄性实验鼠与1只未孕的雌性Wistar大鼠同窝喂养48天后,雌鼠生育一胎14只健康活泼的幼仔,进一步说明本发明的反基因寡核苷酸虽经硫代但毒性不显且安全性能好。 This female male littermates mice with a non-pregnant Wistar rats fed 48 days after the birth of a child 14 female healthy pups lively, antigene oligonucleotides further illustrate the present invention despite thio However, no significant toxicity and good safety performance.

本发明的反基因V-erbB寡核苷酸不同给药次数和方案对大鼠MDS骨髓的影响见表1。 V-erbB antigene oligonucleotides of the present invention and the number of doses of different nucleotide programs on rat marrow MDS Table 1. 从表1可见,进行治疗的16只大鼠原始和早幼粒细胞均有明显下降,其中15只大鼠的骨髓象恢复正常。 Seen from Table 1, the original 16 treated rats and promyelocytic decreased significantly, where 15 normal bone marrow of rats. 并且本发明的两种寡核苷酸单独给药或联合给药均有疗效。 Two oligonucleotides and polynucleotides of the present invention administered alone or administered in combination are effective. 而对照鼠(0-0,B-7)骨髓原始和早幼粒细胞百分比上升,提示疾病在进展。 While the control mice (0-0, B-7) and the original bone marrow promyelocytic percentage rises, indicating disease progression. 随后应用相同的剂量,1-3个疗程对8只MDS-RAEB大鼠进行口服治疗,取得与上述相同的结果。 The subsequent application of the same dose, 1-3 treatment of MDS-RAEB eight rats were treated orally, to obtain the same results described above. 局部用药治疗3例食管癌和2例宫颈癌早期病人均有效,使重度增生逆转为正常。 3 cases of local treatment of esophageal cancer and 2 cases of early cervical cancer patients are effectively the severe hyperplasia reversed to normal.

表1 16只大鼠MDS注射本发明的反基因寡核苷酸(B1和B2)后骨髓变化的观察MDS MDS 注射后 骨髓原始+早幼粒细胞% 给药方法大鼠 亚型 观察时间(月) 治疗前 治疗后(变量)WR 0-0 RAEB 3.0 10.0 21.0(-11.0) 0B-0 RAEB 3.0 13.5 3.0(10.5) B1,B2×3B-1*RAEB 2.5 15.0 无恶性细胞 B1,B2×3B-4 RAEB 2.5 11.0 6.0(5.0) B1,B2×2B-7 RAEB 2.5 11.5 16.0(-5.5) 0A-4 RAEB 3.0 17.0 12.0(5.0) B1,B2×2C-2 RAEB 3.5 9.0 5.0(4.0) B1,B2×3C-8 RAEB 3.5 8.5 4.5(4.0) B1,B2×3D-3 RAEB 3.0 13.0 6.0(7.0) B1,B2×1D-5 RAEB 3.0 9.5 2.5(7.0) B1,B2×1E-2 RAEB 3.0 7.0 4.5(2.5) B2×3E-5 RAEB 3.0 6.5 MDS MDS observed after injection of bone marrow changes after the original table 116 rats injected MDS antigene oligonucleotides of the present invention (B1 and B2) +% promyelocytic subtype of administration was observed in rats (months ) after treatment before treatment (variable) WR 0-0 RAEB 3.0 10.0 21.0 (-11.0) 0B-0 RAEB 3.0 13.5 3.0 (10.5) B1, B2 × 3B-1 * RAEB 2.5 15.0 no malignant cells B1, B2 × 3B- 4 RAEB 2.5 11.0 6.0 (5.0) B1, B2 × 2B-7 RAEB 2.5 11.5 16.0 (-5.5) 0A-4 RAEB 3.0 17.0 12.0 (5.0) B1, B2 × 2C-2 RAEB 3.5 9.0 5.0 (4.0) B1, B2 × 3C-8 RAEB 3.5 8.5 4.5 (4.0) B1, B2 × 3D-3 RAEB 3.0 13.0 6.0 (7.0) B1, B2 × 1D-5 RAEB 3.0 9.5 2.5 (7.0) B1, B2 × 1E-2 RAEB 3.0 7.0 4.5 (2.5) B2 × 3E-5 RAEB 3.0 6.5 3.0(3.5) B2×1D-4 RAEB 2.5 6.5 2.5(4.0) B1,B2×1D-1 RAEB 2.5 9.5 2.0(7.5) B1×3D-2 RAEB 2.5 11.5 3.0(8.5) B1×3F-5 RA 2.5 5.0 1.0(4.0)**B1,B2×1F-1 RAEB 2.5 7.5 1.5(6.0) B1,B2×1TR G-2 RAEB 1.0 10.0 5.0(5.0) B1,B2×3*该鼠观察期间死于炎症**该鼠在尾部发生肉瘤,转移至骨髓如上所述,我们建立的大鼠MDS模型同时也是肉瘤等其他肿瘤模型。 3.0 (3.5) B2 × 1D-4 RAEB 2.5 6.5 2.5 (4.0) B1, B2 × 1D-1 RAEB 2.5 9.5 2.0 (7.5) B1 × 3D-2 RAEB 2.5 11.5 3.0 (8.5) B1 × 3F-5 RA 2.5 5.0 1.0 (4.0) ** B1, B2 × 1F-1 RAEB 2.5 7.5 1.5 (6.0) B1, B2 × 1TR G-2 RAEB 1.0 10.0 5.0 (5.0) B1, B2 × 3 * mice died during this observation inflammation ** the murine sarcoma occurs in the tail, transferred to the bone marrow as described above, MDS rat model we have established are also other sarcoma tumor model. 本发明的V-erbB反基因寡核苷酸对MDS有良好疗效,对其他肿瘤看来也有疗效。 V-erbB invention antigene oligonucleotides have a good effect on the MDS, on the other tumors also appears to have effect. 鉴于脑胶质瘤有C-erbB基因扩增,应用本发明的V-erbB反基因寡核苷酸治疗其早期病变可能也有相似的疗效。 Given glioma have C-erbB gene amplification, V-erbB present invention is applied antigene oligonucleotide therapy early lesions which may also have a similar effect. 常见恶性肿瘤如食管癌、胃癌、肺癌和宫颈癌等的早期病变看来也有疗效,因为它们均有与MDS相同的C-erbB重排/扩增。 Common malignancies such as esophageal, stomach, lung and cervical cancer such early lesions appear also effective, as they have the same C-erbB MDS rearrangement / amplification.

实施例3用V-erbB正基因寡核苷酸诊断MDS的效果1.原位杂交法检测:如实施例1所述合成对应于SEQ ID NO:1的V-erbB正基因寡核苷酸,并用地高辛(DIG)标记该寡核苷酸,采用该标记的寡核苷酸作为探针,经原位杂交方法检测了33例MDS和19例对照及3例可疑MDS的C-erbB基因的扩增情况,结果显示全部33例MDS的原位杂交均呈阳性,19例对照组中有1例呈阳性,3例可疑MDS均为阳性。 3 Effect nucleotide diagnosis of MDS with n-V-erbB gene situ hybridization oligonucleotide 1. Example: Synthesis as described in example 1 corresponding to SEQ ID NO: V-erbB 1 gene positive oligonucleotide, and digoxigenin (DIG) labeled the oligonucleotide, using the labeled oligonucleotide as a probe, in situ hybridization detection of C-erbB gene MDS 33 cases and 19 controls and three cases of suspected MDS amplification, the in situ hybridization results show that all 33 cases were positive MDS, the control group, 19 cases were positive in 1 case 3 cases of suspected MDS were positive.

2.PCR方法A.按常规方法提取骨髓细胞模板DNA。 Methods A. 2.PCR template DNA extraction of bone marrow cells by conventional methods.

B.以5'CACTGTGTGAAGGCCTGC(P1)和5'CTTATAAATAGTGCCAAAAGC(P2)为引物,97℃7分钟进行模板变性,然后进行32个循环,每个循环中变性条件为94℃0.5分钟,退火条件为52℃1分钟和延伸条件为72℃2分钟。 B. In a 5'CACTGTGTGAAGGCCTGC (P1) and 5'CTTATAAATAGTGCCAAAAGC (P2) primers, 97 ℃ 7 min template denaturation, followed by 32 cycles, each cycle of denaturing conditions 94 ℃ 0.5 min annealing conditions of 52 ℃ 1 minute and extension conditions of 72 ℃ 2 minutes. 最后一次循环后72℃延伸5分钟。 After the last cycle 72 ℃ for 5 minutes. PCR产物在1.8%琼脂糖凝胶上电泳检测。 PCR products were electrophoresed on a 1.8% agarose detected gel. 根据电泳带型作基因诊断。 According electrophoresis pattern for genetic diagnosis.

依照上述方法检测了23例MDS和25例对照及3例可疑MDS的C-erbB基因的重排情况,结果23例MDS中有18例PCR结果呈阳性,25例对照中有1例为阳性,3例可疑MDS中有2例为阳性。 Detecting according to the above method for the rearrangement C-erbB genes in 23 cases of MDS and 25 controls and three cases of suspected MDS, the results of 23 cases of MDS 18 cases PCR results were positive, 25 cases in control in 1 case was positive, 3 cases of suspected MDS in 2 cases were positive.

Claims (7)

  1. 1.一种反基因寡核苷酸,具有如下核苷酸序列:5′ATGGCAGAGCTGGCAAAC;或5′AATTCTCAGGTGGGCCTG。 An anti-gene oligonucleotide having the following nucleotide sequence: 5'ATGGCAGAGCTGGCAAAC; or 5'AATTCTCAGGTGGGCCTG.
  2. 2.由权利要求1所述的反基因寡核苷酸转录得到的mRNA。 Antigene oligonucleotide transcripts obtained from the mRNA 2. claimed in claim 1.
  3. 3.一种与权利要求1所述的反基因寡核苷酸互补的正基因寡核苷酸。 N antigene oligonucleotides gene oligonucleotide according to claim 1 and 3. A polynucleotide complementary nucleotide.
  4. 4.一种药物组合物,它含有权利要求1所述的反基因寡核苷酸以及药物学上可以接受的载体。 4. A pharmaceutical composition, comprising the claims acceptable carrier antigene oligonucleotides and pharmaceutically 1.
  5. 5.一种诊断试剂盒,它含有权利要求3中所述的寡核苷酸。 A diagnostic kit comprising the oligonucleotide as claimed in claim 3.
  6. 6.权利要求1中所述的反基因寡核苷酸在制备治疗骨髓异常增生综合征、白血病和其他多种肿瘤药物中的应用。 Application of leukemia and other drugs in a variety of tumors in the antigene oligonucleotide according to claim 1 for the preparation of nucleotide treating myelodysplastic syndromes.
  7. 7.权利要求3中所述的寡聚核苷酸在制备用于诊断骨髓异常增生综合征基因诊断试剂中的应用。 Said oligomer according to claim 3 in the preparation of nucleotide gene diagnostic agent for the diagnostic applications in myelodysplastic syndromes.
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