CN112213492B - Clic4在制备放射治疗鼻咽癌制剂中的应用 - Google Patents
Clic4在制备放射治疗鼻咽癌制剂中的应用 Download PDFInfo
- Publication number
- CN112213492B CN112213492B CN201910615916.0A CN201910615916A CN112213492B CN 112213492 B CN112213492 B CN 112213492B CN 201910615916 A CN201910615916 A CN 201910615916A CN 112213492 B CN112213492 B CN 112213492B
- Authority
- CN
- China
- Prior art keywords
- radiation
- clic4
- nasopharyngeal carcinoma
- preparation
- cells
- 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.)
- Active
Links
- 101000906636 Homo sapiens Chloride intracellular channel protein 4 Proteins 0.000 title claims abstract description 92
- 102100023508 Chloride intracellular channel protein 4 Human genes 0.000 title claims abstract description 90
- 230000005855 radiation Effects 0.000 title claims abstract description 87
- 208000002454 Nasopharyngeal Carcinoma Diseases 0.000 title claims abstract description 80
- 206010061306 Nasopharyngeal cancer Diseases 0.000 title claims abstract description 76
- 201000011216 nasopharynx carcinoma Diseases 0.000 title claims abstract description 76
- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 230000035945 sensitivity Effects 0.000 claims abstract description 20
- 239000012474 protein marker Substances 0.000 claims abstract description 14
- 239000003814 drug Substances 0.000 claims abstract description 13
- 238000001959 radiotherapy Methods 0.000 claims abstract description 12
- 229940079593 drug Drugs 0.000 claims abstract description 11
- 238000012216 screening Methods 0.000 claims abstract description 9
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 6
- 210000004881 tumor cell Anatomy 0.000 claims abstract description 5
- 238000004393 prognosis Methods 0.000 claims abstract description 3
- 206010028980 Neoplasm Diseases 0.000 claims description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 206010070834 Sensitisation Diseases 0.000 claims description 4
- 239000003596 drug target Substances 0.000 claims description 4
- 230000008313 sensitization Effects 0.000 claims description 4
- 238000009472 formulation Methods 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 97
- 238000002474 experimental method Methods 0.000 abstract description 19
- 230000004083 survival effect Effects 0.000 abstract description 7
- 230000015572 biosynthetic process Effects 0.000 abstract description 6
- 230000014509 gene expression Effects 0.000 description 32
- 108090000623 proteins and genes Proteins 0.000 description 26
- 102000004169 proteins and genes Human genes 0.000 description 19
- 239000000523 sample Substances 0.000 description 17
- 101150080924 CNE1 gene Proteins 0.000 description 11
- 238000011529 RT qPCR Methods 0.000 description 9
- 238000005516 engineering process Methods 0.000 description 9
- 108091027967 Small hairpin RNA Proteins 0.000 description 8
- 239000004055 small Interfering RNA Substances 0.000 description 8
- 238000001262 western blot Methods 0.000 description 8
- 239000007850 fluorescent dye Substances 0.000 description 7
- 230000003834 intracellular effect Effects 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 238000001514 detection method Methods 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 230000001105 regulatory effect Effects 0.000 description 6
- 201000011510 cancer Diseases 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 102000007469 Actins Human genes 0.000 description 4
- 108010085238 Actins Proteins 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 238000001819 mass spectrum Methods 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 238000000692 Student's t-test Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000003766 bioinformatics method Methods 0.000 description 2
- 230000004709 cell invasion Effects 0.000 description 2
- 230000005757 colony formation Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 230000037353 metabolic pathway Effects 0.000 description 2
- 239000013610 patient sample Substances 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- GPNYJXANBXUOLW-UHFFFAOYSA-N 2-(2,3-dihydro-1h-inden-1-yloxy)acetic acid Chemical compound C1=CC=C2C(OCC(=O)O)CCC2=C1 GPNYJXANBXUOLW-UHFFFAOYSA-N 0.000 description 1
- 108091006146 Channels Proteins 0.000 description 1
- 102000034573 Channels Human genes 0.000 description 1
- 108010062745 Chloride Channels Proteins 0.000 description 1
- 102000011045 Chloride Channels Human genes 0.000 description 1
- 206010073306 Exposure to radiation Diseases 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 102000009195 Intracellular chloride channels Human genes 0.000 description 1
- 108050000080 Intracellular chloride channels Proteins 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 108010026552 Proteome Proteins 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 231100000991 Reactive Oxygen Species (ROS) Photosafety Assay Toxicity 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000003471 anti-radiation Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229940125400 channel inhibitor Drugs 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 210000001989 nasopharynx Anatomy 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 229940121649 protein inhibitor Drugs 0.000 description 1
- 239000012268 protein inhibitor Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- 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
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明属于生物医学技术领域,涉及CLIC4作为蛋白标志物在制备预测鼻咽癌光子辐射耐受制剂中应用;本发明运用细胞存活克隆形成实验检测辐射耐受细胞与敏感细胞在细胞不同光子剂量下的存活能力差异,结果显示,相同剂量下,鼻咽癌辐射耐受细胞的克隆形成能力显著强于辐射敏感细胞,即鼻咽癌辐射耐受细胞辐射敏感性低。所述的CLIC4可作为蛋白标志物制备预测鼻咽癌光子辐射耐受制剂,以及制备判断鼻咽癌放疗预后情况制品和制备抑制鼻咽癌细胞辐射耐受药物和用于筛选增加肿瘤细胞辐射敏感性的潜在药物。
Description
技术领域
本发明涉及生物医学技术领域,涉及CLIC4作为蛋白标志物的新用途,具体涉及CLIC4作为蛋白标志物在制备预测鼻咽癌光子辐射耐受制剂中应用;尤其涉及CLIC4的表达用于制备在鼻咽癌光子辐射敏感性差异以及其在鼻咽癌放疗效果预测和治疗制品中的应用。
背景技术
现有技术公开了鼻咽癌是发生在鼻咽侧壁和顶部的恶性肿瘤,发病率在耳鼻咽喉恶性肿瘤中居于第一位,临床研究显示,鼻咽癌主要好发于东南亚,特别是中国华南地区。实践显示,放射治疗是治疗鼻咽癌的首选方法之一。随着科学技术的进步,立体定向放疗技术的应用越来越广泛,据统计,早期鼻咽癌的放疗后5年生存率达到了90%多,晚期生存率也达到了70%多,但遗憾的是,放疗耐受会导致治疗效果差和早期复发,因此,寻找鼻咽癌产生光子耐受的基因/蛋白对今后鼻咽癌治疗的发展具有非常重要的实践指导意义。
近年来,随着经济的发展和科技的进步,TMT(Tandem Mass Tag)差异蛋白质组学定量分析技术应用越来越广泛。本发明的研究退队通过将TMT标记、高效液相色谱分级技术以及基于质谱的定量蛋白质组学技术等一系列前沿技术的有机结合,对有关样本进行定量蛋白组的研究,之后对包含定量信息的蛋白质进行了系统的生物信息学分析,包括蛋白注释、功能分类、功能富集及基于功能富集的聚类分析,并综合以上信息,结合临床基因芯片筛选出可能的辐射敏感性差异相关的分子标志物,将为今后鼻咽癌预后相关标志物的研究提供理论基础和科学依据。
本发明通过质谱结果筛选出在鼻咽癌辐射耐受细胞株与辐射敏感细胞株中差异表达的基因,显示CLIC4在鼻咽癌辐射耐受株细胞中的表达明显高于鼻咽癌辐射敏感株细胞中的表达,检索Pubmed及相关数据库发现,CLIC4在鼻咽癌辐射敏感性中的功能未见报道。
基于现有技术的基础与现状,本申请的发明人拟提供CLIC4作为蛋白标志物的新用途,具体涉及CLIC4作为蛋白标志物在制备预测鼻咽癌光子辐射耐受制剂中应用。
发明内容
本发明的目的是基于现有技术的基础与现状,提供CLIC4作为蛋白标志物的新用途,具体涉及CLIC4作为蛋白标志物在制备预测鼻咽癌光子辐射耐受制剂中应用。
本发明通过质谱结果筛选出在鼻咽癌辐射耐受细胞株与辐射敏感细胞株中差异表达的基因CLIC4,结果显示CLIC4在鼻咽癌辐射耐受株细胞中的表达明显高于鼻咽癌辐射敏感株细胞中的表达。
更具体的,本发明取待测样本,然后分析比较待测样本与正常对照中CLIC4的表达量,CLIC4的表达量越高,鼻咽癌细胞的光子辐射敏感性越差,耐受性越强。
本发明中,所述的待测样本可以是细胞样本,组织样本,或者细胞、组织的蛋白抽提物;正常对照可以是正常人的样本,较好的是取自患者本人肿瘤组织周边的组织或者细胞样本;检测时,通常采用相关抗体等检测样本中CLIC4含量;实验结果用三次独立实验的平均值和标准差表示,用双侧t检验来比较差异;p<0.01认为具有统计学差异;所有数据采用SPSS V13.0软件或者具备类似统计功能的软件分析。
本发明的CLIC4是细胞内氯离子通道蛋白,其序列及相关信息可以参考Genbank数据库。
本发明进行了实验,其中,先对鼻咽癌细胞系(即鼻咽癌辐射敏感株CNE-1,CNE-2)进行γ射线照射,分割照射累计剂量至60Gy,建立辐射耐受株(CNE-1R,CNE-2R);接着利用TMT技术,分析鼻咽癌辐射耐受株与辐射敏感株中的差异蛋白,结果显示CLIC4在鼻咽癌辐射耐受株中的表达高于辐射敏感株;其次,结合临床基因芯片,验证CLIC4在鼻咽癌放疗抵抗样本组中的表达显著高于放疗敏感样本组;
本发明中,在细胞水平,证实CLIC4在鼻咽癌辐射耐受株中的表达高于辐射敏感株,结果表明,本发明的CLIC4可作为蛋白标志物用于制备预测鼻咽癌光子辐射敏感性的制剂。
本发明中,运用细胞存活克隆形成实验检测辐射耐受细胞与敏感细胞在细胞不同光子剂量下的存活能力差异,结果显示,相同剂量下,鼻咽癌辐射耐受细胞的克隆形成能力显著强于辐射敏感细胞,即鼻咽癌辐射耐受细胞辐射敏感性低。
本发明中,运用qRT-PCR和western blot技术对鼻咽癌辐射耐受细胞与敏感细胞中CLIC4的表达进行检测,结果显示,鼻咽癌辐射耐受细胞中的CLIC4的mRNA和蛋白水平均明显高于辐射敏感细胞。
本发明中,运用shRNA显著下调鼻咽癌辐射耐受细胞和辐射敏感细胞中的CLIC4,结果显示,CLIC4下调后细胞株的辐射敏感性均提高。
本发明还提供了CLIC4在制备抑制鼻咽癌细胞辐射耐受制品中的应用,所述的CLIC4可以作为筛选鼻咽癌细胞辐射耐受药物的药靶;降低CLIC4表达量的物质可作为抑制肿瘤细胞侵袭药物的候选药物。本发明同时提供了IAA94(Indanyloxyacetic acid 94,R(+)-IAA-94)是一类潜在的氯离子通道蛋白抑制剂,可以抑制CLIC4及其同家族氯离子蛋白的表达,进一步可以制备作为临床鼻咽癌放疗时增加肿瘤细胞辐射敏感性的潜在药物。
本发明运用了ROS荧光探针检测技术对CLIC4表达发生改变时细胞内ROS的改变进行检测,结果显示,CLIC4高表达细胞在受到辐射后ROS的水平上升较弱,反之,敲低细胞内CLIC4后,细胞在受到辐射后ROS的水平明显上调,表明CLIC4通过抑制细胞内ROS水平促进细胞辐射耐受,进一步,本发明的CLIC4可以制备作为筛选鼻咽癌辐射增敏的药物的药靶。
本发明提供CLIC4作为蛋白标志物的新用途,尤其CLIC4作为蛋白标志物在制备预测鼻咽癌光子辐射耐受制剂中应用。本发明通过质谱结果筛选出在鼻咽癌辐射耐受细胞株与辐射敏感细胞株中差异表达的基因CLIC4,经试验,结果显示CLIC4在鼻咽癌辐射耐受株细胞中的表达明显高于鼻咽癌辐射敏感株细胞中的表达。进一步,所述的CLIC4可作为蛋白标志物用于制备预测鼻咽癌光子辐射敏感性的制剂,以及采用降低CLIC4表达量的物质用于制备抑制肿瘤细胞侵袭药物的候选药物,和,所述的CLIC4可以制备作为筛选鼻咽癌辐射增敏的药物的药靶。
本发明的实施例中以鼻咽癌细胞为模型,但是,本发明也可以应用于其他一些肿瘤,。因此CLIC4的特性及其与胞内ROS代谢通路作用的分子机制可以为肿瘤辐射抵抗的基础研究和辐射增敏药物的研发提供新的思路。
附图说明
图1显示克隆形成实验说明gamma射线0、2、4、6Gy照射下,鼻咽癌辐射耐受株(CNE-1R、CNE-2R)与辐射敏感株(CNE-1、CNE-2)的剂量存活曲线。
图2显示qRT-PCR结果中的CLIC4在CNE-1R、CNE-2R和CNE-1、CNE-2中的相对表达。
图3显示蛋白印迹实验结果中的CLIC4在CNE-1R、CNE-2R和CNE-1、CNE-2中的表达。
图4显示qRT-PCR结果中的CNE-1R、CNE-2R和CNE-1、CNE-2细胞受到光子照射(4Gy)后,CLIC4的相对表达情况。
图5显示shRNA干扰后,qRT-PCR结果中的CLIC4在CNE-1R、CNE-2R中的表达。
图6显示shRNA干扰后,蛋白印迹实验结果中的CLIC4在CNE-1R、CNE-2R、CNE-1、CNE-2中的表达及细胞受到辐射后CLIC4变化情况。
图7显示shRNA病毒干扰后,克隆形成实验说明gamma射线0、2、4、6Gy照射下,CNE-1R、CNE-2R、CNE-1、CNE-2的阴性对照组(Negative control组)、CLIC4敲低组(shRNA组)剂量存活曲线。
图8显示ROS荧光探针检测实验中CNE-1R、CNE-2R和CNE-1、CNE-2受到辐射(4Gy)后不同时间ROS的变化情况。
图9显示shRNA病毒干扰后,ROS荧光探针检测实验中CNE-1R、CNE-2R、CNE-1、CNE-2的阴性对照组(Negative control组)、CLIC4敲低组(shRNA组)受到辐射(4Gy)后不同时间ROS的变化情况。
具体实施方式
下面结合附图对本发明的具体的实施方案进行详述。除非特别说明,本发明中所涉及的技术手段均为本领域内的技术人员所公知的方法。此外,实施方案应理解为说明性的,而非限制本发明的范围,本发明的实质和范围仅由权利要求书所限定。对于本领域内的技术人员而言,在不违背本发明的实质和范围的前提下,对本发明涉及的试剂进行成分和用量的各种改变或改动也属于本发明的保护范围。本发明所涉及细胞及试剂均在市场上有销售。
实施例1
1)方法
1.1细胞系
人鼻咽癌细胞株(NPC)CNE1、CNE2细胞由复旦大学附属肿瘤医院惠赠。
1.2主要试剂
胎牛血清购于美国Gibco公司;RPMI1640培养基购于美国Gibco公司;FastQuantcDNA第一链合成试剂盒、荧光定量检测试剂盒(SuperReal PreMix(SYBR Green))购于北京天根生物技术有限公司;qRT-PCR的引物由上海赛百盛基因技术有限公司合成,纯化方式为PAGE;CLIC4的siRNA购于广州市锐博生物科技有限公司;β-actin、CLIC4抗体购于Abclonal武汉爱博泰克生物科技有限公司;山羊抗兔IgG(H+L)、山羊抗鼠IgG(H+L)(荧光二抗)购于江苏碧云天生物科技有限公司;预染三色蛋白Marker购于上海翊圣生物技术有限公司;ROS荧光探针DCFH-DA购于江苏碧云天生物科技有限公司。
1.3细胞培养
人鼻咽癌细胞株CNE1、CNE2细胞的培养条件为含有10%胎牛血清的RPMI1640培养基,37℃下在含有5%CO2的培养箱中培养。
1.4建立辐射耐受株
Cs-137γ源(Cs-137,0.73Gy/min)分割照射,分割照射2,2,4,4,4,4,6,6,6,6,8,8Gy累积剂量60Gy。
1.5细胞集落形成实验
每种细胞设置对照组和辐射组(2、4、6Gy)。将处于对数生长期的对照组和辐射组的细胞,胰蛋白酶消化,稀释细胞至适当浓度,加入2ml培养液的6孔培养板中,每组处理均设置3个平行样,放置5%CO2、37℃恒温细胞培养箱中继续培养10~14天。培养结束后将细胞用4%多聚甲醛固定,结晶紫染液染色,计数细胞集落数目。
1.6细胞转染
用CLIC4的shRNA病毒转染CNE1、CNE2、CNE1R、CNE2R细胞,获得稳定细胞株。其干扰序列如表3。
表3
1.7 qRT-PCR应用
qRT-PCR检测转染细胞中CLIC4的表达,转染后48h收集各组细胞,利用柱式离心法提取细胞中总RNA,用Nano Vue核酸蛋白检测仪定量检测。RNA样品用逆转录反应合成cDNA后,用SYBR Green PCR试剂盒检测样本中CLIC4含量,其引物序列如表4。反应条件为95℃预变性30s,95℃、5s,55℃、30s;72℃、1min,40个循环,反应结束后得到各个样本CLIC4和内参β-actin的基本循环数(Ct值)。根据公式计算目的基因相对含量,即为CLIC4相对含量,实验重复三次。
表4
1.8 Western blot实验
Western blot检测转染细胞中相关蛋白的表达,收集不同条件处理后的鼻咽癌细胞(NPC、NPC-R),利用RIPA裂解液(强)提取细胞中总蛋白并进行BCA蛋白定量,然后加入0.25倍体积的5×loading buffer,混匀,煮沸法进行蛋白变性。SDS-PAGE电泳条件:根据蛋白相对分子量的大小配制适宜浓度的分离胶和5%的浓缩胶,每孔加入等量经过变形处理的蛋白样本,样品两侧的孔内加入预染三色蛋白Marker;打开电源,将电压设置到80V恒压,当待测样本的蛋白跑至分离胶时调换至120V,待Marker最下面蓝色条带距离胶底1cm左右,终止电泳。转膜条件:冰水浴,300mA,1~2h(具体时间根据目的蛋白相对分子量大小进行调整)。封闭条件:放入1×TBST配制的封闭液(5%脱脂奶粉),放置水平摇床上,室温下缓慢晃动2h。一抗孵育条件:按照说明书(1:1000)用一抗稀释液配制相应的一抗孵育液,4℃孵育过夜。二抗孵育:1×TBST清洗三次;按照说明书(1:3000~10000)用1×TBST配制所需体积二抗孵育液,浸没目的条带,放置垂直摇床上,室温下上下缓慢晃动2h。显影:1×TBST清洗三次,按照说明书配制化学发光底物(A液:B液=1:1),使其均匀覆盖目的条带膜表面,启动化学发光成像系统,进行显影成像,最终使用Quantity One软件分析所有检测目标条带的灰度值。
1.9细胞内ROS检测实验
细胞接种于96孔板中,完全贴壁后进行4Gy gamma照射。照射后弃去培养基,加入稀释后的DCFH-DA探针溶液,终浓度为3μM。37℃培养箱内孵育30min后,用PBS洗涤细胞,充分去除未进入细胞内的荧光探针。再次加入无血清培养基孵育1.5小时。孵育结束后,采用多功能荧光酶标仪进项细胞内ROS含量测定,读取平均荧光强度。
2)统计学处理
采用SPSS 20.0统计学软件进行分析,论文中的实验结果均为3~5次重复实验的平均值,Student’s-t检验进行统计分析,P<0.05则认为差异具有统计学意义。
3)
构建鼻咽癌CNE1、CNE2细胞株辐射耐受株后,使用γ源(Cs-137,0.73Gy/min)验证NPC-R辐射敏感性(如图1所示);由剂量-存活曲线可以看出,CNE1R和CNE2R和其亲本细胞CNE1、CNE2相比,出现明显的辐射抗性。以辐射敏感性较低的细胞株与辐射敏感性较高的细胞株做比对:CNE1R/CNE1,CNE2R/CNE2,对鼻咽癌细胞株TMT检测结果进行生物信息学分析,筛选表达差异在1.5倍以上的蛋白,确定研究目标CLIC4(如表1所示);
表1显示TMT结果中的CLIC4在CNE-1R、CNE-2R和CNE-1、CNE-2中的表达
利用鼻咽癌临床样本(鼻咽癌放疗敏感、放疗抵抗病人病灶活检组织)的基因表达谱芯片检测结果,查询CLIC4的表达情况,确定CLIC4在放疗抵抗病人中高表达,与鼻咽癌细胞株TMT结果一致(如表2所示);
表2显示临床基因芯片中的CLIC4在鼻咽癌放疗抵抗组病人样本和鼻咽癌放疗敏感组病人样本中的表达
实验结果表明,CLIC4可以作为鼻咽癌辐射耐受的标志。
实施例2
利用qRT-PCR、Western blot实验检测鼻咽癌细胞株CNE1、CNE2及其辐射耐受株CNE1R、CNE2R中CLIC4表达情况,及经过辐射后CLIC4在细胞株中随时间的变化情况。qRT-PCR、Western blot实验结果显示CNE1R、CNE2R的CLIC4的转录水平与蛋白水平均高于CNE1、CNE2,与TMT和表达谱芯片结果一致(如图2-3所示),受到4Gy光子照射后,相比于CNE1照后升高的水平,CNE1R的CLIC4持续低表达,CNE2在1小时左右表达明显降低,CNE2R在4小时内表达逐渐降低,最终均维持较低水平(如图4所示);
实验结果表明,CLIC4的高表达水平与鼻咽癌细胞辐射耐受相关。
实施例3
利用sh-CLIC4病毒对鼻咽癌细胞进行CLIC4敲低实验,利用qRT-PCR、Westernblot实验验证细胞株内CLIC4的敲低水平,获得稳定的CLIC4低表达细胞株(如图5-6所示),并检测细胞受照后不同时间点的CLIC4蛋白表达含量,实验结果与mRNA水平基本保持一致,获得稳定CLIC4低表达细胞株后,通过细胞集落形成实验,检测癌细胞光子放射敏感性的变化(如图7所示),实验结果显示,CLIC4表达下调后,癌细胞的光子辐射敏感性明显升高;
表明CLIC4在调控细胞辐射敏感性上能够发挥稳定作用。
实施例4
利用ROS荧光探针实验,通过对鼻咽癌细胞株及CLIC4敲低细胞株受辐射后胞内ROS含量的测定,进一步分析CLIC4对辐射敏感性的影响;。ROS荧光探针实验显示,高表达CLIC4的CNE1R、CNE2R细胞在受到辐射后,ROS水平明显低于CNE1、CNE2细胞(如图8所示);同时,经过CLIC4敲低后,CNE1 sh-CLIC4、CNE1R sh-CLIC4、CNE2sh-CLIC4、CNE2R sh-CLIC4细胞在受到辐射后,ROS水平明显高于sh-NC细胞(如图9所示);
上述实验进一步证实了CLIC4通过调控细胞受辐射后胞内ROS含量,降低癌细胞的辐射敏感性。
综上所述,所述的CLIC4及其调控的ROS代谢通路,不仅可以为鼻咽癌的基础研究提供新的思路,还可以为临床鼻咽癌抗辐射耐受药物的研究和筛选提供参考;例如,氯离子通道抑制剂IAA94可以明显抑制ROS的生成,能够明显抑制鼻咽癌细胞的放疗耐受;CLIC4可以用于筛选抑制肿瘤辐射耐受的药物,尤其是抑制鼻咽癌辐射耐受的药物。
序列表
<110> 复旦大学
<120> CLIC4在制备放射治疗鼻咽癌制剂中的应用
<130> 20190709
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 53
<212> DNA
<213> siCLIC4-1
<400> 1
ccgggatggc aatgaaatga cattactcga gtaatgtcat ttcattgcca tct 53
<210> 2
<211> 58
<212> DNA
<213> si CLIC4-2
<400> 2
ccggtatgcc ctcccaagta cttaactcga gttaagtact tgggagggca tatttttg 58
<210> 3
<211> 52
<212> DNA
<213> si CLIC4-3
<400> 3
ccgggcatat agtgatgtag ccaaactcga gtttggctac atcactatat gc 52
<210> 4
<211> 21
<212> DNA
<213> CLIC4 F
<400> 4
tgaaagcata ggaaactgcc c 21
<210> 5
<211> 22
<212> DNA
<213> CLIC4 R
<400> 5
ggtcaacagt cgtcacacta aa 22
<210> 6
<211> 20
<212> DNA
<213> β-actin F
<400> 6
tgacgtggac atccgcaaag 20
<210> 7
<211> 20
<212> DNA
<213> β-actin R
<400> 7
ctggaaggtg gacagcgagg 20
Claims (4)
1.CLIC4作为蛋白标志物在制备预测鼻咽癌光子辐射耐受制剂中的用途。
2.CLIC4在制备判断鼻咽癌放疗预后情况制品中的应用。
3.CLIC4 在制备用于筛选抑制肿瘤辐射耐受的药物的试剂中的应用, 所述的肿瘤是鼻咽癌。
4.CLIC4 在制备用于筛选增加肿瘤细胞辐射敏感性的潜在药物的试剂中的应用,所述CLIC4作为筛选鼻咽癌辐射增敏的药物的药靶。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910615916.0A CN112213492B (zh) | 2019-07-09 | 2019-07-09 | Clic4在制备放射治疗鼻咽癌制剂中的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910615916.0A CN112213492B (zh) | 2019-07-09 | 2019-07-09 | Clic4在制备放射治疗鼻咽癌制剂中的应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112213492A CN112213492A (zh) | 2021-01-12 |
CN112213492B true CN112213492B (zh) | 2024-04-30 |
Family
ID=74048392
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910615916.0A Active CN112213492B (zh) | 2019-07-09 | 2019-07-09 | Clic4在制备放射治疗鼻咽癌制剂中的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112213492B (zh) |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1854313A (zh) * | 2002-09-30 | 2006-11-01 | 肿瘤疗法科学股份有限公司 | 非小细胞肺癌的诊断方法 |
CN101611154A (zh) * | 2006-12-04 | 2009-12-23 | 艾博特公司 | 用于癌症治疗的成对诊断测定 |
KR20140094767A (ko) * | 2013-01-22 | 2014-07-31 | 한국원자력의학원 | Wdfy3를 측정하는 제제를 포함하는 방사선 저항성 또는 민감성 진단용 조성물 및 이의 용도 |
CN104894229A (zh) * | 2014-03-04 | 2015-09-09 | 中南大学 | 己糖激酶2作为鼻咽癌放疗预后预测的生物标志物 |
CN106636402A (zh) * | 2016-12-22 | 2017-05-10 | 中南大学湘雅医院 | 一种判断鼻咽癌放疗敏感性的分子标志物及应用 |
WO2019103456A2 (ko) * | 2017-11-22 | 2019-05-31 | 울산대학교 산학협력단 | Pmvk를 유효성분으로 포함하는 방사선 저항성 암 진단용 또는 방사선 치료 예후 예측용 바이오마커 조성물 |
KR20200137253A (ko) * | 2019-05-29 | 2020-12-09 | 서울대학교산학협력단 | Fes 저해제를 포함하는 방사선 민감도 증진용 조성물 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008082712A2 (en) * | 2006-08-25 | 2008-07-10 | The Trustees Of Columbia University In The City Of New York | Systems and methods for biodosimetry with biochip using gene expression signatures |
-
2019
- 2019-07-09 CN CN201910615916.0A patent/CN112213492B/zh active Active
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1854313A (zh) * | 2002-09-30 | 2006-11-01 | 肿瘤疗法科学股份有限公司 | 非小细胞肺癌的诊断方法 |
CN101611154A (zh) * | 2006-12-04 | 2009-12-23 | 艾博特公司 | 用于癌症治疗的成对诊断测定 |
KR20140094767A (ko) * | 2013-01-22 | 2014-07-31 | 한국원자력의학원 | Wdfy3를 측정하는 제제를 포함하는 방사선 저항성 또는 민감성 진단용 조성물 및 이의 용도 |
CN104894229A (zh) * | 2014-03-04 | 2015-09-09 | 中南大学 | 己糖激酶2作为鼻咽癌放疗预后预测的生物标志物 |
CN106636402A (zh) * | 2016-12-22 | 2017-05-10 | 中南大学湘雅医院 | 一种判断鼻咽癌放疗敏感性的分子标志物及应用 |
WO2019103456A2 (ko) * | 2017-11-22 | 2019-05-31 | 울산대학교 산학협력단 | Pmvk를 유효성분으로 포함하는 방사선 저항성 암 진단용 또는 방사선 치료 예후 예측용 바이오마커 조성물 |
KR20200137253A (ko) * | 2019-05-29 | 2020-12-09 | 서울대학교산학협력단 | Fes 저해제를 포함하는 방사선 민감도 증진용 조성물 |
Also Published As
Publication number | Publication date |
---|---|
CN112213492A (zh) | 2021-01-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6612280B2 (ja) | エリブリンに対する応答を予測するための方法及び組成物 | |
CN106978480A (zh) | 用于癌症的分子诊断试验 | |
Cui et al. | Novel lncRNA PSMG3‑AS1 functions as a miR‑143‑3p sponge to increase the proliferation and migration of breast cancer cells | |
KR101760464B1 (ko) | 교모세포종의 진단 또는 예후 예측용 바이오 마커 및 그 용도 | |
Niu et al. | Knockdown of SMAD3 inhibits the growth and enhances the radiosensitivity of lung adenocarcinoma via p21 in vitro and in vivo | |
Shi et al. | Transgelin-2 contributes to proliferation and progression of hepatocellular carcinoma via regulating Annexin A2 | |
Yang et al. | Far upstream element-binding protein 1 (FUBP1) is a potential c-Myc regulator in esophageal squamous cell carcinoma (ESCC) and its expression promotes ESCC progression | |
Liu et al. | E2F4 promotes the proliferation of hepatocellular carcinoma cells through upregulation of CDCA3 | |
Liang et al. | Linc00467 promotes invasion and inhibits apoptosis of head and neck squamous cell carcinoma by regulating miR-1285-3p/TFAP2A | |
Liu et al. | Correlation between chemotherapy resistance in osteosarcoma patients and PAK5 and Ezrin gene expression | |
Rezaei et al. | Identification of antibody reactive proteins in pancreatic cancer using 2D immunoblotting and mass spectrometry | |
CN108977547B (zh) | Tspan1在乳腺癌转移的诊断、预后及治疗中的应用 | |
CN106047875A (zh) | lncRNA分子及其在鼻咽癌辅助诊断中的应用 | |
Zhang et al. | Identification of stemness index-related long noncoding RNA SNHG12 in human bladder cancer based on WGCNA | |
CN112213492B (zh) | Clic4在制备放射治疗鼻咽癌制剂中的应用 | |
CN110714076B (zh) | Arl14作为肿瘤标志物在制备预测肺腺癌预后及靶点药物中的用途 | |
Huang et al. | LncRNA linc01194 promotes the progress of endometrial carcinoma by up-regulating SOX2 through binding to IGF2BP1 | |
CN114427003B (zh) | Nhp2在预测癌症放疗敏感性和预后中的应用 | |
Lv et al. | Application of high-throughput gene sequencing in lymphoma | |
Chen et al. | Identification of metastasis-associated genes in cutaneous squamous cell carcinoma based on bioinformatics analysis and experimental validation | |
Wu et al. | Association between expression of nuclear receptor co‑activator 5 protein and prognosis in postoperative patients with osteosarcoma | |
CN115725735A (zh) | Fkbp5基因在作为鼻咽癌光子辐射耐受的分子标志物中的应用 | |
CN110184336B (zh) | 叶酸受体folr2及其编码基因的应用 | |
CN114703287B (zh) | CXorf56基因在治疗三阴性乳腺癌中的应用 | |
Li et al. | ERBB2-PTGS2 axis promotes intervertebral disc degeneration by regulating senescence of nucleus pulposus cells |
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 | ||
GR01 | Patent grant | ||
GR01 | Patent grant |