CN110592211A - 基因foxd4在制备急性髓系白血病诊断试剂盒的应用及试剂盒 - Google Patents
基因foxd4在制备急性髓系白血病诊断试剂盒的应用及试剂盒 Download PDFInfo
- Publication number
- CN110592211A CN110592211A CN201910751243.1A CN201910751243A CN110592211A CN 110592211 A CN110592211 A CN 110592211A CN 201910751243 A CN201910751243 A CN 201910751243A CN 110592211 A CN110592211 A CN 110592211A
- Authority
- CN
- China
- Prior art keywords
- gene
- foxd4
- diagnostic kit
- kit
- myeloid leukemia
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 101001029302 Homo sapiens Forkhead box protein D4 Proteins 0.000 title claims abstract description 48
- 208000031261 Acute myeloid leukaemia Diseases 0.000 title claims abstract description 35
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 title claims abstract description 34
- 238000009007 Diagnostic Kit Methods 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims description 9
- 102100037043 Forkhead box protein D4 Human genes 0.000 claims abstract description 23
- 238000003752 polymerase chain reaction Methods 0.000 claims abstract description 12
- 108700039887 Essential Genes Proteins 0.000 claims abstract description 11
- 108010043121 Green Fluorescent Proteins Proteins 0.000 claims abstract description 4
- 239000007853 buffer solution Substances 0.000 claims abstract description 4
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 11
- 101001025074 Homo sapiens Forkhead box protein D4-like 1 Proteins 0.000 claims description 10
- 238000010839 reverse transcription Methods 0.000 claims description 10
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 claims description 8
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 claims description 8
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 6
- 101150114335 FOXD4L1 gene Proteins 0.000 claims description 5
- 102100038038 Forkhead box protein D4-like 1 Human genes 0.000 claims description 5
- 239000000243 solution Substances 0.000 claims description 4
- 102100034343 Integrase Human genes 0.000 claims description 3
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 3
- 239000003161 ribonuclease inhibitor Substances 0.000 claims description 3
- 239000012295 chemical reaction liquid Substances 0.000 claims description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims 1
- 210000005259 peripheral blood Anatomy 0.000 abstract description 11
- 239000011886 peripheral blood Substances 0.000 abstract description 11
- 238000003745 diagnosis Methods 0.000 abstract description 8
- 206010028980 Neoplasm Diseases 0.000 abstract description 7
- 201000011510 cancer Diseases 0.000 abstract description 6
- 238000001514 detection method Methods 0.000 abstract description 6
- 238000013518 transcription Methods 0.000 abstract description 4
- 230000035897 transcription Effects 0.000 abstract description 4
- 238000013399 early diagnosis Methods 0.000 abstract description 3
- 230000014509 gene expression Effects 0.000 description 18
- 102000004315 Forkhead Transcription Factors Human genes 0.000 description 10
- 108090000852 Forkhead Transcription Factors Proteins 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 9
- 208000025113 myeloid leukemia Diseases 0.000 description 8
- 230000019491 signal transduction Effects 0.000 description 7
- 210000000130 stem cell Anatomy 0.000 description 7
- 238000011529 RT qPCR Methods 0.000 description 6
- 238000010171 animal model Methods 0.000 description 4
- 210000001185 bone marrow Anatomy 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000003753 real-time PCR Methods 0.000 description 4
- 101100289995 Caenorhabditis elegans mac-1 gene Proteins 0.000 description 3
- 102100038024 Forkhead box protein D4-like 4 Human genes 0.000 description 3
- 102100038037 Forkhead box protein D4-like 5 Human genes 0.000 description 3
- 101001025072 Homo sapiens Forkhead box protein D4-like 4 Proteins 0.000 description 3
- 101001025075 Homo sapiens Forkhead box protein D4-like 5 Proteins 0.000 description 3
- 241000699660 Mus musculus Species 0.000 description 3
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 3
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 210000003714 granulocyte Anatomy 0.000 description 3
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 3
- 102000058051 human FOXD4L1 Human genes 0.000 description 3
- 208000032839 leukemia Diseases 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 238000011830 transgenic mouse model Methods 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 238000002247 constant time method Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000003211 malignant effect Effects 0.000 description 2
- 238000011880 melting curve analysis Methods 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical group C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 1
- 102000038594 Cdh1/Fizzy-related Human genes 0.000 description 1
- 108091007854 Cdh1/Fizzy-related Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 102100038025 Forkhead box protein D4-like 3 Human genes 0.000 description 1
- 102100038022 Forkhead box protein D4-like 6 Human genes 0.000 description 1
- 101150057663 Foxa2 gene Proteins 0.000 description 1
- 101150019145 Foxd4 gene Proteins 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 1
- 101001025071 Homo sapiens Forkhead box protein D4-like 3 Proteins 0.000 description 1
- 101001025065 Homo sapiens Forkhead box protein D4-like 6 Proteins 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- 101000633045 Homo sapiens Zinc finger protein SNAI3 Proteins 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 101100125257 Mus musculus Hoxa3 gene Proteins 0.000 description 1
- 208000033761 Myelogenous Chronic BCR-ABL Positive Leukemia Diseases 0.000 description 1
- 208000014767 Myeloproliferative disease Diseases 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 208000015634 Rectal Neoplasms Diseases 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- -1 TGF-beta small molecule Chemical class 0.000 description 1
- 102000013530 TOR Serine-Threonine Kinases Human genes 0.000 description 1
- 108010065917 TOR Serine-Threonine Kinases Proteins 0.000 description 1
- 102100029573 Zinc finger protein SNAI3 Human genes 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 238000010322 bone marrow transplantation Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000005757 colony formation Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000003831 deregulation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000008482 dysregulation Effects 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 230000007705 epithelial mesenchymal transition Effects 0.000 description 1
- 230000005713 exacerbation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 206010038038 rectal cancer Diseases 0.000 description 1
- 201000001275 rectum cancer Diseases 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000003393 splenic effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 230000005740 tumor formation Effects 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Hospice & Palliative Care (AREA)
- Biophysics (AREA)
- Oncology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
基因FOXD4在制备急性髓系白血病诊断试剂盒的应用及试剂盒，诊断试剂盒由SYBR Green聚合酶链式反应体系，用于扩增FOXD4基因和管家基因的引物对。SYBR Green聚合酶链式反应体系包括PCR缓冲液，dNTPs，SYBR Green荧光染料。本发明以FOXD4作为检测诊断的靶点应用于临床制备的诊断试剂盒，可以对急性髓系白血病患者的外周血或骨髓有核细胞进行快速诊断和预测；检测样本中目标基因FOXD4的转录水平，可以判断受试者是否患有急性髓系白血病，为癌症的早期诊断和治疗提供依据。
Description
技术领域
本发明属于生物医药技术和血液学白血病领域，涉及核酸的检测试剂盒及其在急性髓系白血病体外诊断中的应用。
背景技术
急性髓系白血病(Acute Myeloid Leukemia，AML)是造血干细胞恶性变化所产生的高死亡率疾病之一，在全世界癌症病例中恶性程度较高。在治疗过程中，一般是放射治疗、化学药物治疗、骨髓移植等，但临床成功率、移植配型和预后方面都有一定的问题。白血病的早期诊断在疾病的治疗上十分重要，因此寻找和确定有效的生物标志甚至作为治疗靶点是迫切需要解决的问题。分子诊断技术对判断AML具有独特的客观优势，而基于Real-time PCR技术的体外检测系统使对疾病的诊断更加便利和精准。
Forkhead蛋白是一类在进化上高度保守的重要转录因子家族。Forkhead蛋白的典型结构特征是含有一段由100个氨基酸组成的进化上高度保守的Forkhead(或称为Wingedhelix)DNA结合结构域。Forkhead转录因子的主要功能包括调控与胚胎发育，细胞生长，增殖，分化，干细胞干性保持，语言形成以及长寿相关基因表达。Forkhead基因突变或调控异常导致发育缺陷以及肿瘤形成。Forkhead结构域在真核生物中十分保守，存在于从酵母到人类中，在人类基因组含有超过40种Forkhead蛋白。除了可以直接结合DNA之外，Forkhead转录因子还可以与多种信号通路中的激酶或成员有直接的相互作用，从而在信号转导与转录调控之间发挥承上启下的桥梁作用。
近期研究证明，FOXD4高表达在造血细胞系统的细胞系中，并被称为髓系因子myeloid factorα(Freyaldenhoven，B.S.，C.Fried，and K.Wielckens，FOXD4a andFOXD4b，two new winged helix transcription factors，are expressed in humanleukemia cell lines.Gene，2002.294(1-2)：p.131-140.)。在骨髓增殖性疾病病人(myeloproliferative neoplasm，MPN)的循环CD34+细胞和粒细胞中发现FOXD4表达失调控(Cokic，V.P.，et al.，Microarray and Proteomic Analyses of MyeloproliferativeNeoplasms with a Highlight on the mTOR Signaling Pathway.PLoS One，2015.10(8)：p.e0135463.)。并且在直肠癌病人中，最近研究表明FOXD4上调，并增强直肠癌的恶性能力和上皮-间质转化能力(Chen，C.，et al.，FOXD4 induces tumor progression incolorectal cancer by regulation of the SNAI3/CDH1 axis.Cancer Biol Ther，2018：p.1-7.)。但是目前尚无研究报道FOXD4基因家族成员在急性髓系白血病AML临床病人骨髓和外周血有核细胞中的表达情况。
并且，本发明研究者设计使用AML动物模型KrasG12D/+；CD2Cre转基因小鼠，使癌基因KrasG12D特异表达在造血细胞谱系。我们发现此小鼠进展为AML，粒系/单核祖细胞(granulocyte-macrophage progenitors，GMP)增多，形成白血病干细胞。表达谱芯片分析发现GMP细胞的TGF-β信号通路增强。之前研究说明Forkhead转录因子家族是TGF-β信号通路的调控转录因子，我们发现在AML小鼠中，其下游的调控转录因子FOXD4高表达。并且我们使用免疫荧光，集落形成实验和TGF-β小分子抑制剂证实KrasG12D的粒系/单核祖细胞的TGF-β-FOXD4信号通路活化。
人源FOXD4基因家族有FOXD4，FOXD4L1，FOXD4L3，FOXD4L4，FOXD4L5和FOXD4L6，一共6个同源基因成员。我们进行核苷酸同源比对，找到这6个同源基因成员的共同保守位点，设计引物为人FOXD4基因家族的引物对。鼠源FOXD4基因只有一个，其核苷酸序列与人源FOXD4L1具有很高同源性，因而在人的6个同源基因中，我们关注人源FOXD4L1并设计人FOXD4L1特异性引物。本发明根据AML小鼠动物模型的实验结果，在此探讨临床AML病人数据分析证明FOXD4通路有相关性，从而为研究AML的发病机制奠定基础。
发明内容
我们经过研究发现，Forkhead转录因子家族成员FOXD4在临床急性髓系白血病患者的骨髓和外周血单个核细胞中特异性表达，而在正常骨髓和外周血中不表达。因此，本发明的目的是提供基因FOXD4在制备急性髓系白血病诊断试剂盒的应用，以及提供一种诊断试剂盒，利用对基因FOXD4的检测用于急性髓系白血病的诊断。
为达上述目的，本发明的一个实施例中提供了基因FOXD4在制备急性髓系白血病诊断试剂盒的应用，诊断试剂盒包括SYBR Green聚合酶链式反应体系、用于扩增FOXD4、FOXD4L1基因和管家基因的引物对；SYBR Green聚合酶链式反应体系包括PCR缓冲液、dNTPs、SYBR Green荧光染料。
优选的，所述诊断试剂盒中，扩增FOXD4基因家族的引物对的序列如下：
正向引物序列为5，-CTCAGAGTTTGGCACCGAGT-3，；
反向引物序列为5，-AGTCGTTCAGCGAGAGGTTG-3，。
优选的，所述诊断试剂盒中，扩增FOXD4L1基因的引物对的序列如下：
正向引物序列为5，-CTAGCGTTCCTGCTTCTTGC-3，；
反向引物序列为5，-GTAGGTAGCGAGGAGGATGC-3，。
优选的，管家基因是GAPDH，扩增管家基因GAPDH的引物对的序列如下：
正向引物序列为5，-GAAGGTGAAGGTCGGAGTC-3，；
反向引物序列为5，-GAAGATGGTGATGGGATTTC-3，。
所述诊断试剂盒中包括逆转录体系，即包括Oligo dT、逆转录反应液、M-MLV逆转录酶、RNA酶抑制剂、dNTPs。所述逆转录反应液包括250mM pH8.3Tris-HCl、375mM KCl、15mMMgCl2、50mM DTT。
本发明具有以下优点：
本发明以FOXD4作为检测诊断的靶点应用于临床制备得到诊断试剂盒，可以对急性髓系白血病进行快速诊断和预测；能够通过检测FOXD4基因的转录水平判断受试者是否患有癌症，为疾病的早期诊断和治疗提供依据。
附图说明
图1为AML动物模型的KrasG12D/+；CD2Cre转基因小鼠中，骨髓中粒系/单核祖细胞及脾脏的粒细胞上调TGF-βFOXD4信号通路基因。(A，B)定量Real Time-PCR测定候选基因的转录水平差异。RNA从对照和KrasG12D/+；CD2Cre+的骨髓中粒系/单核祖细胞(A)或脾脏中Gr1+Mac1+的髓系白血病细胞(B)中提取。相对表达量使用相对GAPDH的表达倍数来表示。
图2为FOXD4在临床急性髓系白血病病人外周血中基因表达水平。Real Time-PCR检测临床髓系白血病病人外周血的FOXD4基因家族成员FOXD4(A)和FOXD4L1(B)的表达。FOXD4L4和FOXD4L5没有明显差异。
图3为FOXD4在Oncomine数据库中，髓系白血病病人中的表达情况。
具体实施方式
基因FOXD4在制备检测急性髓系白血病诊断试剂盒中的应用及试剂盒，诊断试剂盒包括SYBR Green聚合酶链式反应体系、用于扩增FOXD4、FOXD4L1和管家基因的引物对；SYBR Green聚合酶链式反应体系包括PCR缓冲液、dNTPs、SYBR Green荧光染料。
诊断试剂盒中，扩增FOXD4基因家族的引物对的序列如下：
正向引物序列为5，-CTCAGAGTTTGGCACCGAGT-3，；
反向引物序列为5，-AGTCGTTCAGCGAGAGGTTG-3，。
诊断试剂盒中，扩增FOXD4L1基因的引物对的序列如下：
正向引物序列为5，-CTAGCGTTCCTGCTTCTTGC-3，；
反向引物序列为5，-GTAGGTAGCGAGGAGGATGC-3，。
管家基因是GAPDH，扩增管家基因GAPDH的引物对的序列如下：
正向引物序列为5，-GAAGGTGAAGGTCGGAGTC-3，；
反向引物序列为5，-GAAGATGGTGATGGGATTTC-3，。
诊断试剂盒中包括逆转录体系，即包括Oligo dT、逆转录反应液、M-MLV逆转录酶、RNA酶抑制剂、dNTPs。所述逆转录反应液包括250mM pH8.3Tris-HCl、375mM KCl、15mMMgCl2、50mM DTT。
本发明提供了一种所述诊断试剂盒的使用方法，包括以下步骤：
(1)使用Ficoll分离液从患者骨髓或外周血样本中分离出有核细胞。
(2)使用Trizol或血液RNA提取试剂盒提取样本总RNA，溶解在20-30μl DEPC水中，-20℃(最好-80℃)保存，检测RNA浓度。
(3)将样本总RNA逆转录为cDNA。所述RNA逆转录为cDNA的反应体系为：RNA(2-5μg)，Oligo dT(1μl)，2×逆转录反应液(10μl)，Enzyme Mix(1μl)，以及使体系达到20μl的水。PCR程序设置为①25℃，10min；②42℃，30min；③4℃，forever。
(4)使用PCR扩增仪对FOXD4和FOXD4L1基因和管家基因进行扩增；扩增完毕后进行定量检测。以cDNA为模板的实时定量聚合酶链式反应：cDNA模版1μl，引物对1μl，SYBRGreen Master Mix 10μl，ddH2O 8μl，总体系为20ul。
设置扩增程序：①94℃，5min ②94℃，30sec ③57℃，30sec ④72℃，1min ⑤72℃，10min ⑥4℃，forever。其中②-④，循环39-40次。以SYBR Green作为荧光标记物，通过融解曲线分析和电泳确定，ΔΔCT法进行相对定量。
(5)若FOXD4或FOXD4L1基因表达提高，则说明诊断对象为急性髓系白血病患者。
实施例1
我们首先验证在AML动物模型的KrasG12D/+；CD2Cre转基因小鼠中，骨髓中粒系/单核祖细胞及脾脏的髓系白血病细胞上调FOXD4信号通路基因(图1)。(A，B)定量qPCR测定候选基因的转录水平差异。RNA从对照和KrasG12D/+；CD2Cre+的骨髓中粒系/单核祖细胞(A)或脾脏中Gr1+Mac1+的髓系白血病细胞(B)中提取。相对表达量使用相对GAPDH的表达倍数来表示。
结果是qPCR验证了AML小鼠的粒细胞系TGF-β通路的基因表达升高(图1A，B)。qPCR结果证明Tgfβr1，Tgfβ1，Foxd4在KrasG12D/+；CD2Cre+的粒系/单核祖细胞和Gr1+Mac1+的髓系白血病细胞中高表达(图1A，B)，而Hoxa3，foxa2，和foxa3表达差异较小。
实施例2
评价FOXD4在临床急性髓系白血病病人外周血中基因表达水平。
实验材料：收集8例急性髓系白血病病人外周血样本，同时收集3个正常人外周血样本，采用荧光实时定量PCR进行经典的分子生物学实验验证(qRT-PCR)。
实验结果：通过融解曲线分析和电泳确定目的条带，ΔΔCT法进行相对定量，结果如图2所示。与正常外周血样本相比，q-PCR检测3例临床髓系白血病病人外周血的FOXD4基因家族成员FOXD4(A)和FOXD4L1(B)的表达上调。FOXD4L4和FOXD4L5没有明显差异。
实施例3
FOXD4在Oncomine数据库中，髓系白血病病人中的表达情况。
我们在Oncomine数据库中找到FOXD4基因表达与急性髓系白血病以及慢性髓系白血病的恶化期呈正相关(图3)。
Claims (6)
1.基因FOXD4在制备急性髓系白血病诊断试剂盒的应用及试剂盒，所述的基因FOXD4序列为NCBI Gene ID：2298所示序列，并且所述的基因FOXD4L1序列为NCBI Gene ID：200350所示序列。
2.根据权利要求1所述，其诊断试剂盒包括SYBR Green聚合酶链式反应体系、用于扩增FOXD4、FOXD4L1和管家基因的引物对；所述SYBR Green聚合酶链式反应体系包括PCR缓冲液、dNTPs、SYBR Green荧光染料。
3.根据权利要求1所述的基因FOXD4在制备急性髓系白血病诊断试剂盒的应用，其特征在于：所述诊断试剂盒中，所述扩增FOXD4基因家族的引物对的序列如下：
正向引物序列为5，-CTCAGAGTTTGGCACCGAGT-3，；
反向引物序列为5，-AGTCGTTCAGCGAGAGGTTG-3，。
4.根据权利要求1所述的基因FOXD4在制备急性髓系白血病诊断试剂盒的应用，其特征在于：所述诊断试剂盒中，所述扩增FOXD4L1基因的引物对的序列如下：
正向引物序列为5，-CTAGCGTTCCTGCTTCTTGC-3，；
反向引物序列为5，-GTAGGTAGCGAGGAGGATGC-3，。
5.根据权利要求1所述的基因FOXD4在制备急性髓系白血病诊断试剂盒的应用，其特征在于：所述管家基因是GAPDH，扩增管家基因GAPDH的引物对的序列如下：
正向引物序列为5，-GAAGGTGAAGGTCGGAGTC-3，；
反向引物序列为5，-GAAGATGGTGATGGGATTTC-3，。
6.如权利要求1所述的基因FOXD4在制备急性髓系白血病诊断试剂盒的应用，其特征在于：所述诊断试剂盒中包括逆转录体系，即包括Oligo dT、逆转录反应液、M-MLV逆转录酶、RNA酶抑制剂、dNTPs。所述逆转录反应液包括250mM pH8.3 Tris-HCl、375mM KCl、15mMMgCl2、50mM DTT。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910751243.1A CN110592211A (zh) | 2019-08-15 | 2019-08-15 | 基因foxd4在制备急性髓系白血病诊断试剂盒的应用及试剂盒 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910751243.1A CN110592211A (zh) | 2019-08-15 | 2019-08-15 | 基因foxd4在制备急性髓系白血病诊断试剂盒的应用及试剂盒 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110592211A true CN110592211A (zh) | 2019-12-20 |
Family
ID=68854232
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910751243.1A Pending CN110592211A (zh) | 2019-08-15 | 2019-08-15 | 基因foxd4在制备急性髓系白血病诊断试剂盒的应用及试剂盒 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110592211A (zh) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080241110A1 (en) * | 2005-11-29 | 2008-10-02 | Nevada Cancer Institute | Targeting of sall4 for the treatment and diagnosis of proliferative disorders associated with myelodysplastic syndrome (MDS) |
| WO2012031008A2 (en) * | 2010-08-31 | 2012-03-08 | The General Hospital Corporation | Cancer-related biological materials in microvesicles |
-
2019
- 2019-08-15 CN CN201910751243.1A patent/CN110592211A/zh active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080241110A1 (en) * | 2005-11-29 | 2008-10-02 | Nevada Cancer Institute | Targeting of sall4 for the treatment and diagnosis of proliferative disorders associated with myelodysplastic syndrome (MDS) |
| WO2012031008A2 (en) * | 2010-08-31 | 2012-03-08 | The General Hospital Corporation | Cancer-related biological materials in microvesicles |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cheng et al. | Molecular testing for BRAF mutations to inform melanoma treatment decisions: a move toward precision medicine | |
| Stewart et al. | Circulating cell-free DNA for non-invasive cancer management | |
| CN106715723B (zh) | 测定样品中pik3ca突变状态的方法 | |
| Saudy et al. | BMI1 gene expression in myeloid leukemias and its impact on prognosis | |
| CA2945531C (en) | Mirna expression signature in the classification of thyroid tumors | |
| CN109182521B (zh) | circRNA作为甲状腺乳头状癌标志物的应用 | |
| AU2017204894A1 (en) | Prediction of treatment response to JAK/STAT inhibitor | |
| CN102676663A (zh) | 用于检测BRCA1mRNA的核酸检测试剂盒 | |
| JP2022189882A (ja) | ｍｉｎｏｒ ＢＣＲ－ＡＢＬ１遺伝子を検出する方法 | |
| JP6453781B2 (ja) | ヒトｐｉ３ｋｃａ（ｐｉｋ３ｃａ）遺伝子変異検出のための方法及び組成物 | |
| CN109735622A (zh) | 与结直肠癌相关的lncRNA及其应用 | |
| CN106555004A (zh) | 缺血性脑卒中的lncRNA标志物 | |
| CN102912018B (zh) | 一种检测WT1基因mRNA表达量的试剂盒 | |
| CN106048073B (zh) | 一种卵巢癌辅助诊断或者疗效预测用的生物标记物及其应用 | |
| JP6827067B2 (ja) | ループス腎炎の検出またはそのリスクを予測する方法およびその応用 | |
| JP2011211955A (ja) | 肝ガン患者予後予測用組成物及び方法 | |
| CN107988370B (zh) | 一种circRNA基因在制备诊断慢性粒细胞性白血病试剂中的应用 | |
| CN102827935B (zh) | 定量检测FIP1L1-PDGFRA融合基因mRNA水平的试剂盒 | |
| CN102443638A (zh) | 一种用于血清/血浆miRNA检测的内参及其应用 | |
| CN110592211A (zh) | 基因foxd4在制备急性髓系白血病诊断试剂盒的应用及试剂盒 | |
| WO2010076887A1 (en) | Predictive biomarkers useful for cancer therapy mediated by a wee1 inhibitor | |
| CN103589786B (zh) | 检测RRM1 mRNA相对表达量的方法、试剂盒及引物和探针 | |
| CN103725787B (zh) | 一种白血病融合基因荧光pcr检测试剂盒 | |
| EP2652144A2 (en) | Plasma cell disorders | |
| CN110551817A (zh) | 检测人wt1融合基因的试剂盒及其使用方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20191220 |