CN107233573A - miRNA‑324‑5p及其激活物在制备炎症性肠病及炎症性肠病相关结直肠癌临床标志物及治疗药物中的应用 - Google Patents
miRNA‑324‑5p及其激活物在制备炎症性肠病及炎症性肠病相关结直肠癌临床标志物及治疗药物中的应用 Download PDFInfo
- Publication number
- CN107233573A CN107233573A CN201610821172.4A CN201610821172A CN107233573A CN 107233573 A CN107233573 A CN 107233573A CN 201610821172 A CN201610821172 A CN 201610821172A CN 107233573 A CN107233573 A CN 107233573A
- Authority
- CN
- China
- Prior art keywords
- ibd
- mouse
- dicer
- mirna
- colorectal cancer
- 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.)
- Granted
Links
- 208000001333 Colorectal Neoplasms Diseases 0.000 title claims abstract description 33
- 206010009944 Colon cancer Diseases 0.000 title claims abstract description 32
- 239000012190 activator Substances 0.000 title claims abstract description 25
- 239000003814 drug Substances 0.000 title claims abstract description 13
- 108091070501 miRNA Proteins 0.000 title abstract description 20
- 239000002679 microRNA Substances 0.000 title abstract description 17
- 239000003550 marker Substances 0.000 title description 3
- 208000022559 Inflammatory bowel disease Diseases 0.000 claims abstract description 54
- 208000002551 irritable bowel syndrome Diseases 0.000 claims abstract description 43
- 238000001514 detection method Methods 0.000 claims abstract description 6
- 230000002757 inflammatory effect Effects 0.000 claims description 8
- 238000009472 formulation Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 239000000825 pharmaceutical preparation Substances 0.000 claims description 4
- 239000002775 capsule Substances 0.000 claims description 2
- 239000008187 granular material Substances 0.000 claims description 2
- 239000006187 pill Substances 0.000 claims description 2
- 239000003826 tablet Substances 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 108091046551 miR-324 stem-loop Proteins 0.000 abstract description 25
- 101150083707 dicer1 gene Proteins 0.000 abstract description 16
- 238000002474 experimental method Methods 0.000 abstract description 9
- 210000002490 intestinal epithelial cell Anatomy 0.000 abstract description 8
- 206010009887 colitis Diseases 0.000 abstract description 7
- 208000024891 symptom Diseases 0.000 abstract description 4
- 230000002269 spontaneous effect Effects 0.000 abstract description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 2
- TVZRAEYQIKYCPH-UHFFFAOYSA-N 3-(trimethylsilyl)propane-1-sulfonic acid Chemical compound C[Si](C)(C)CCCS(O)(=O)=O TVZRAEYQIKYCPH-UHFFFAOYSA-N 0.000 abstract 1
- 230000001154 acute effect Effects 0.000 abstract 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 52
- 102100023387 Endoribonuclease Dicer Human genes 0.000 description 36
- 101000907904 Homo sapiens Endoribonuclease Dicer Proteins 0.000 description 22
- BLFLLBZGZJTVJG-UHFFFAOYSA-N benzocaine Chemical compound CCOC(=O)C1=CC=C(N)C=C1 BLFLLBZGZJTVJG-UHFFFAOYSA-N 0.000 description 21
- 210000004027 cell Anatomy 0.000 description 15
- 238000011813 knockout mouse model Methods 0.000 description 11
- 210000001072 colon Anatomy 0.000 description 10
- 241000699670 Mus sp. Species 0.000 description 8
- 230000001939 inductive effect Effects 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 206010028980 Neoplasm Diseases 0.000 description 6
- 238000010171 animal model Methods 0.000 description 6
- 238000010586 diagram Methods 0.000 description 6
- 238000003209 gene knockout Methods 0.000 description 6
- 230000000968 intestinal effect Effects 0.000 description 6
- 108020004999 messenger RNA Proteins 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 206010061218 Inflammation Diseases 0.000 description 5
- 208000005016 Intestinal Neoplasms Diseases 0.000 description 5
- 201000010989 colorectal carcinoma Diseases 0.000 description 5
- 239000003651 drinking water Substances 0.000 description 5
- 235000020188 drinking water Nutrition 0.000 description 5
- 230000006698 induction Effects 0.000 description 5
- 230000004054 inflammatory process Effects 0.000 description 5
- 238000003757 reverse transcription PCR Methods 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 208000029742 colonic neoplasm Diseases 0.000 description 4
- 230000001276 controlling effect Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 208000037902 enteropathy Diseases 0.000 description 4
- 201000002313 intestinal cancer Diseases 0.000 description 4
- 208000028774 intestinal disease Diseases 0.000 description 4
- 210000004347 intestinal mucosa Anatomy 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 206010068676 Pneumoretroperitoneum Diseases 0.000 description 3
- 208000005727 Retropneumoperitoneum Diseases 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 230000008595 infiltration Effects 0.000 description 3
- 238000001764 infiltration Methods 0.000 description 3
- 210000004969 inflammatory cell Anatomy 0.000 description 3
- 239000007928 intraperitoneal injection Substances 0.000 description 3
- 238000010172 mouse model Methods 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 208000011231 Crohn disease Diseases 0.000 description 2
- 108700011259 MicroRNAs Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 244000144987 brood Species 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 101150042537 dld1 gene Proteins 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 210000000981 epithelium Anatomy 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 210000004209 hair Anatomy 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 206010009900 Colitis ulcerative Diseases 0.000 description 1
- 108010051219 Cre recombinase Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 201000006704 Ulcerative Colitis Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000009920 chelation Effects 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 238000005034 decoration Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 210000001842 enterocyte Anatomy 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 210000005027 intestinal barrier Anatomy 0.000 description 1
- 230000004673 intestinal mucosal barrier function Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000004682 mucosal barrier function Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 230000001124 posttranscriptional effect Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000003716 rejuvenation Effects 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 235000011083 sodium citrates Nutrition 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 210000004876 tela submucosa Anatomy 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000001256 tonic effect Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical class [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 108090000195 villin Proteins 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Epidemiology (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Molecular Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明公开了miRNA‑324‑5p激活物在制备炎症性肠病及炎症性肠病相关结直肠癌疾病临床检测标志物和在制备治疗炎症性肠病及炎症性肠病相关结直肠癌药物中的应用。本发明通过大量实验筛选，发现伴随着肠粘膜上皮细胞DICER基因的敲除，小鼠自发产生严重的炎症性肠病；同时miRNA‑324‑5p作为变化最显著的miRNAs之一，能够作为炎症性肠病的临床检测标志物。更特别的，在DSS诱导的小鼠结肠炎模型以模拟人炎症性肠病的模型中，通过腹腔注射miR‑324‑5p激活物能显著缓解小鼠急性结肠炎的症状，表明miR‑324‑5p激活物在制备用于治疗炎症性肠病及炎症性肠病相关结直肠癌患者中的用途。
Description
技术领域
本发明涉及一种微小RNA分子的新用途,尤其涉及miRNA-324-5p的新用途，属于生物医学技术领域。
背景技术
炎症性肠病(IBD)是一类病因不明的肠道慢性非特异性炎症，包括溃疡性结肠炎(UC)和克罗恩病(CD)。随着研究的深入，人们逐渐意识到IBD是导致结直肠癌的一个危险因素，并且将结直肠癌年轻化。近5年来，我国IBD患者数目是20世纪90年代同期的8倍，亟需得到广泛关注。微小RNA(microRNA)是一类在转录后水平调控基因表达的单链RNA，长度约为22bp。miRNA通过介导mRNA的降解和抑制mRNA转录这两种方式来调控基因的表达，研究表明miRNA参与多种类型肿瘤的发生及发展过程，越来越多的研究证明miRNA在结直肠肿瘤的发生发展中有重要的调控作用。DICER是miRNA合成过程中重要的酶，目前科学家已经利用体内及体外DICER基因敲除的模型作为研究miRNA在结直肠肿瘤发生发展中作用的有效手段。申请人利用小鼠肠粘膜上皮细胞Dicer基因敲除作为工具，发现纯合子(Dicerloxp/loxp&VillinCre)小鼠和杂合子(Dicerloxp/+&VillinCre)小鼠肠道均有一定程度的自发炎症，且纯合敲除小鼠要重于杂合敲除小鼠；进一步的利用AOM合并DSS诱导结直肠癌的模型发现，Dicer缺陷的小鼠易患更严重的肠癌。这一结果提示DICER及其下游miRNA在炎症性肠病及炎症性肠病相关结直肠癌发生中具有重要的调控作用。目前还没有进一步研究阐释DICER及其下游某一特异的miRNA参与这个病理过程，关于miR-324-5p激活物作为一种抑制炎症性肠病及炎症性肠病相关结直肠癌药物的用途和作为炎症性肠病及炎症性肠病相关结直肠癌诊断标志物的用途目前也尚未见报道。
发明内容
发明目的：本发明所要解决的技术问题是克服现有技术中治疗炎症性肠病及炎症性肠病相关结直肠癌药物的不足及临床上炎症性肠病诊断标志物的局限，通过对DICER及其下游miRNA的深入研究，在miRNA-324-5p原有研究的基础上，通过大量实验筛选，开发出miRNA-324-5p新用途。
技术方案：为了实现以上目的，本发明采取的技术方案为：
本发明提供miRNA-34-5p在制备炎症性肠病临床检测标志物中的应用。
本发明另一个目的是提供miRNA-324-5p激活物在制备治疗炎症性肠病药物中的应用。
本发明另一个目的是提供miRNA-324-5p激活物在制备治疗炎症性肠病相关结直肠癌中的应用。
一种具有治疗炎症性肠病及炎症性肠病相关结直肠癌的药物制剂，该药物制剂为含有miRNA-324-5p化合物的口服制剂或注射剂。
作为优选方案，以上所述的具有治疗炎症性肠病及炎症性肠病相关结直肠癌的药物制剂，其特征在于，所述的口服制剂为肠溶性片剂、胶囊剂、丸剂或颗粒剂。
miR-324-5p的核苷酸序列为cgcauccccuagggcauuggugu。其序列如NO1。
本发明所述的miR-324-5p激活物为经过化学修饰过的miR-324-5p(cgcauccccuagggcauuggugu)序列，其序列如NO2。实验中通过腹腔注射给药的方式将miR-324-5p激活物(AgomiR-324-5p)注入小鼠体内。
本发明首先采用基因芯片的方法对3对(RKO、DLD1、HCT116)DICER基因敲除(DICER-/-)的结直肠癌细胞系及其对照细胞系(DICER-WT)进行筛选，实验结果表明miR-324-5p水平在DCIER敲除的细胞中下降最为明显；对野生型小鼠和肠粘膜上皮细胞Dicer基因敲除小鼠的肠粘膜组织进行RT-PCR实验，实验结果表明随着小鼠自发IBD症状的加剧，miR-324-5p的水平也显著下降。另外，用DICER基因敲除小鼠(Dicerloxp/+&VillinCre)和其同窝野生型小鼠(Dicerloxp/+)作为工具，AOM合并DSS诱导小鼠结直肠癌的动物模型中，Dicerloxp/+&VillinCre小鼠更易患严重的结直肠癌。以上结果都表明miRNA-324-5p水平下调和IBD及IBD相关结直肠癌的发生直接相关，因此通过miRNA-324-5p激活物过表达可抑制IBD的发生及缓解其发展，也能够成为治疗IBD相关结直肠癌的有效药物。
本发明应用DSS诱导的小鼠结肠炎动物模型模拟人炎症性肠病模型，用野生型小鼠(Dicerloxp/+)和DICER基因敲除小鼠(Dicerloxp/+&VillinCre)作为工具；通过腹腔注射miR-324-5p激活物的方式提高局部miR-324-5p的水平。实验结果表明miR-324-5p过表达后，DSS诱导的Dicerloxp/+&VillinCre小鼠肠粘膜损伤显著下降，肠粘膜屏障完整性升高，炎性细胞浸润显著减少，炎症性肠病的症状得到显著缓解，表明miR-324-5p激活物可提高肠粘膜完整性，发挥很好的抑制IBD的功效，因此对炎症性肠病相关结直肠癌的预防及治疗都有一定的作用。
有益效果：本发明通过大量实验研究筛选，实验结果表明miRNA-324-5p在炎症性肠病及炎症性肠病相关结直肠癌发生发展中的重要作用。miR-324-5p下降伴随着肠粘膜屏障损伤以及粘膜上皮细胞大量脱落，炎性细胞浸润显著增加，肠道炎症显著加剧；在持续的炎症刺激下，患者易患结直肠肿瘤。因此，通过miRNA-324-5p激活物来提高局部的miR-324-5p水平，能显著缓解肠道炎症，进而预防与治疗IBD及IBD相关结直肠肿瘤。因此miR-324-5p激活物可作为制备防治炎症性肠病及炎症肠病相关结直肠癌的药物，同时miR-324-5p也可作为炎症性肠病及的临床检测标志物。
附图说明
图1是DICER在3对结直肠癌DICER敲除细胞系中mRNA水平的柱状图。
图2是miRNA-324-5p在3对结直肠癌DICER敲除细胞系中mRNA水平的柱状图。
图3是Dicer基因敲除小鼠肠粘膜上皮细胞中Dicer基因mRNA水平的柱状图。
图4是miR-324-5p在Dicer基因敲除小鼠肠粘膜上皮细胞中mRNA水平的柱状图。
图5是野生型小鼠(Dicerloxp/+)和肠粘膜上皮细胞Dicer敲除小鼠(Dicerloxp/+&VillinCre)在AOM合并DSS诱导小鼠结直肠癌模型的模式图以及小鼠处死后肠肿瘤的照片。
图6是图5中所述动物模型中肿瘤最大径总和的数据统计图(n＝8)。
图7是图5中所述动物模型中结肠的重量/长度的比值的统计图(n＝8)。
图8是图5中所述动物模型中每只小鼠结肠肿瘤数目的统计图(n＝8)
图9是Dicerloxp/+和Dicerloxp/+&VillinCre小鼠用DSS诱导小鼠结肠炎后腹腔注射miR-324-5p激活物的小鼠动物模型处死后的结肠照片图。
图10是Dicerloxp/+和Dicerloxp/+&VillinCre小鼠用DSS诱导小鼠结肠炎后腹腔注射miR-324-5p激活物的小鼠动物模型中将小鼠结肠称重及测量长度，计算结肠重量与长度比值的柱状图。
图11是Dicerloxp/+和Dicerloxp/+&VillinCre小鼠用DSS诱导小鼠结肠炎后腹腔注射miR-324-5p激活物的小鼠动物模型，小鼠结肠组织HE染色的结果图。
具体实施方式
下面结合具体实施例，进一步阐明本发明，应理解这些实施例仅用于说明本发明而不用于限制本发明的范围，在阅读了本发明之后，本领域技术人员对本发明的各种等价形式的修改均落于本申请所附权利要求所限定的范围。
实施例1 miR-324-5p作为炎症性肠病诊断标志物的实验
1.体外结直肠肿瘤细胞的培养：
3种野生型结直肠癌细胞(RKO、DLD1、HCT116)及其对应的DICER基因敲除的细胞(DICER-/-)培养在含10％FBS的1640培养基中，置于37℃、5％CO2培养箱中生长，至对数生长期用于试验；细胞生长至融合度大于80％时，进行传代：PBS缓冲液洗2遍，胰酶体积约覆盖细胞表面，37℃培养箱作用1min，显微镜明场下观察，可见细胞收缩边缘清晰时，弃去消化液，加入完全培养基终止消化，用移液器吹打瓶壁上的细胞制成单细胞悬液，接种到新的培养皿内培养。
2.小鼠的培育及观察
小鼠饲养条件保持在温度22-28℃，相对湿度40％-60％，噪音<60dB，日夜温差为2℃，昼夜交替各为12h。饮用水为高压灭菌纯净水，每周换水3次，鼠笼及垫料每周更换。应用含有LoxP序列的Dicerloxp/loxp小鼠与含有肠粘膜上皮特定启动子Villin驱动的Cre重组酶小鼠进行数代的杂交。每天对比观察野生型小鼠(Dicerloxp/+)、Dicer杂合缺失型小鼠(Dicerloxp/+&VillinCre)和Dicer纯和缺失型小鼠(Dicerloxp/loxp&VillinCre)的进食、饮水及活动等日常状态，定期记录小鼠体重、生存时间。
3.RNA提取
细胞的RNA直接收取细胞后按照Trizol法抽提总RNA。小鼠原代肠粘膜上皮细胞的分离需要将小鼠结肠取出后置于冰PBS中清洗数次，将肠剪成长度为2-3mm长度的片段后放入螯合缓冲液(27mM柠檬酸钠、5mM Na2PO4、96mM NaCl、8mM KH2PO4、1.5mM KCl、0.5mM DTT、55mM山梨醇、44mM蔗糖、6mM EDTA、5mM EGTA、pH7.3)中4℃剧烈摇晃45min；用100μm细胞过滤器去除组织碎片，细胞悬液1500rpm离心后得到肠上皮细胞；然后按照Trizol法提取总RNA。测定浓度后取1μg总RNA进行反转录，得到的cDNA产物稀释10倍用于RT-PCR的模板。
4.采用RT-PCR进行miRNA定量分析
miRNA的定量分析采用特异性的茎环结构反转录引物进行反转录后再进行定量PCR检测。购买锐博生物公司(中国、广州)的特异miR-324-5p颈环引物试剂盒进行实验(Bulge-loopTM miRNA qRT-PCRPrimer Sets)。
如图1和图2所示，采用RT-PCR的miRNA定量分析结果表明，miRNA-324-5p在DICER基因敲除的细胞中显著降低；如图3和图4所示，取野生小鼠、Dicer杂合敲除小鼠和Dicer纯合敲除小鼠肠粘膜细胞进行miRNA的RT-PCR实验，结果显示随着Dicer基因敲除程度的增加，miR-324-5p的水平显著降低。以上结果表明随着小鼠肠IBD的加剧，miR-324-5p作为DICER下游特异的miRNA会显著下降，因此miR-324-5p可作为炎症性肠病临床检测标志物。
5.AOM合并DSS诱导小鼠结肠癌的动物模型
随机选取6-8周Dicerloxp/+&VillinCre小鼠及同窝Dicerloxp/+小鼠，按注射浓度为10mg/kg氧化偶氮甲烷(AOM)于第1、22、53天分三次腹腔注射于小鼠体内，同时在第1、4、7周自由饮用含2％葡聚糖硫酸钠(DSS)的蒸馏水，第10周处死小鼠。如图5所示，Dicerloxp/+&VillinCre小鼠结肠上肉眼所见肿瘤显著多于Dicerloxp/+小鼠；图6对肿瘤最大径的总和进行统计，表明Dicerloxp/+&VillinCre小鼠上肿瘤显著大于Dicerloxp/+小鼠；图7对两组小鼠结肠重量/长度的比值进行统计(侧面反映肿瘤重量)，结果显示Dicerloxp/+&VillinCre小鼠显著高于Dicerloxp/+小鼠；图8对两组小鼠上肿瘤的数目进行统计，发现Dicerloxp/+&VillinCre小鼠显著多于Dicerloxp/+小鼠。以上结果表明Dicer缺失的小鼠易患更严重的结肠癌，而miR-324-5p作为DICER基因下游特异的miRNA，其下降也会导致IBD相关肠癌的发生。
实施例2 miRNA-324-5p激活物治疗炎症性肠病的动物实验
1.实验分组：
选用6-8周、雄性野生型小鼠(Dicerloxp/+)和Dicer杂合敲除小鼠(Dicerloxp/+&VillinCre)各自随机分为三组：对照饮水组(水)，DSS诱导组(DSS)，DSS诱导同时miR-324-5p激活物腹腔注射组(DSS+AgomiR-324-5p)。
2.实验方法：
将分子量为DSS(MW：36000-50000)溶于饮用水，配成3％DSS溶液。同窝野生型小鼠(Dicerloxp/+)和Dicer杂合敲除小鼠(Dicerloxp/+&VillinCre)小鼠分别自由饮用蒸馏水和含3％DSS的蒸馏水。在第2天时进行腹腔注射浓度为10nM的AgomiR-324-5p或其对照200μl/只，每天观察小鼠进食、饮水及活动等日常状态，并记录小鼠体重。第7天处死小鼠观察。
3.实验结果
DSS诱导结肠炎后，miRNA-324-5p激活物干预治疗组小鼠体重比未干预的Dicerloxp/+&Villin-Cre小鼠体重显著回升，具有明显差异(P＜0.05)。
如图9为小鼠处死后结肠的拍照图，结果显示DSS诱导下Dicerloxp/+小鼠和Dicerloxp/+&VillinCre小鼠的结肠长度都显著减少；当腹腔注射AgomiR-324-5p后，Dicerloxp /+&VillinCre小鼠的结肠长度出现回升。
如图10为小鼠结肠重量与长度(g/cm)比值的统计柱状图，结果显示AgomiR-324-5p干预后能够显著降低这个比值，表明干预后的炎症性肠病症状得到明显减轻，具有明显差异(P＜0.05)。
如图11为小鼠结肠组织石蜡切片的HE染色结果，结果显示DSS诱导下Dicerloxp/+&VillinCre小鼠肠粘膜上皮细胞大量脱落，炎性细胞浸润加剧，粘膜上皮失去原有组织形态，粘膜屏障消失；当用miRNA-324-5p激活物AgomiR-324-5p干预后，粘膜上皮细胞形态得到很好的恢复，炎症程度减轻。
并且实验过程中，未见有明显不良反应，有望开发成新一代安全有效，防治炎症性肠病及炎症性肠病相关结肠癌的新药。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理和构思的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (5)
1.miRNA-324-5p在制备炎症性肠病临床检测标志物中的应用。
2.miRNA-324-5p激活物在制备治疗炎症性肠病药物中的应用。
3.miRNA-324-5p激活物在制备治疗炎症性肠病相关结直肠癌药物中的应用。
4.一种具有治疗炎症性肠病及炎症性肠病相关结直肠癌的药物制剂，其特征在于，该药物制剂为含有miRNA-324-5p激活物的口服制剂、注射剂或外用制剂。
5.根据权利要求4所述的具有治疗炎症性肠病及炎症性肠病相关结直肠癌的药物制剂，其特征在于，所述的口服制剂为肠溶性片剂、胶囊剂、丸剂或颗粒剂。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610821172.4A CN107233573B (zh) | 2016-09-13 | 2016-09-13 | miRNA-324-5p及其激活物在制备炎症性肠病及炎症性肠病相关结直肠癌临床标志物及治疗药物中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610821172.4A CN107233573B (zh) | 2016-09-13 | 2016-09-13 | miRNA-324-5p及其激活物在制备炎症性肠病及炎症性肠病相关结直肠癌临床标志物及治疗药物中的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107233573A true CN107233573A (zh) | 2017-10-10 |
| CN107233573B CN107233573B (zh) | 2020-06-26 |
Family
ID=59982960
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610821172.4A Expired - Fee Related CN107233573B (zh) | 2016-09-13 | 2016-09-13 | miRNA-324-5p及其激活物在制备炎症性肠病及炎症性肠病相关结直肠癌临床标志物及治疗药物中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107233573B (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111686106A (zh) * | 2020-04-05 | 2020-09-22 | 温州医科大学附属第一医院 | 提高Dicer表达的化合物在制备药物中的应用 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012078558A2 (en) * | 2010-12-06 | 2012-06-14 | Rush University Medical Center | microRNAs AS NEW THERAPEUTIC TARGETS FOR THE PREVENTION AND/OR TREATMENT OF RETINOPATHY |
| CN103025890A (zh) * | 2010-04-06 | 2013-04-03 | 卡里斯生命科学卢森堡控股 | 疾病的循环生物标志物 |
-
2016
- 2016-09-13 CN CN201610821172.4A patent/CN107233573B/zh not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103025890A (zh) * | 2010-04-06 | 2013-04-03 | 卡里斯生命科学卢森堡控股 | 疾病的循环生物标志物 |
| WO2012078558A2 (en) * | 2010-12-06 | 2012-06-14 | Rush University Medical Center | microRNAs AS NEW THERAPEUTIC TARGETS FOR THE PREVENTION AND/OR TREATMENT OF RETINOPATHY |
Non-Patent Citations (1)
| Title |
|---|
| 胡菊香等: "非编码RNA与炎症性肠病关系的研究进展", 《新医学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111686106A (zh) * | 2020-04-05 | 2020-09-22 | 温州医科大学附属第一医院 | 提高Dicer表达的化合物在制备药物中的应用 |
| CN111686106B (zh) * | 2020-04-05 | 2022-07-15 | 温州医科大学附属第一医院 | 提高Dicer表达的化合物在制备药物中的应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107233573B (zh) | 2020-06-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Pathania et al. | H3. 3K27M cooperates with Trp53 loss and PDGFRA gain in mouse embryonic neural progenitor cells to induce invasive high-grade gliomas | |
| Dorshorst et al. | A complex genomic rearrangement involving the endothelin 3 locus causes dermal hyperpigmentation in the chicken | |
| Alam et al. | Population genetic structure of Catla catla (Hamilton) revealed by microsatellite DNA markers | |
| Khan et al. | Pre-mRNA splicing: role of epigenetics and implications in disease | |
| Fastman et al. | Erasing the epigenetic memory and beginning to switch—the onset of antigenic switching of var genes in Plasmodium falciparum | |
| Li et al. | Role of RHOT1 on migration and proliferation of pancreatic cancer | |
| CN107208151A (zh) | 使用基因组特异性探针的循环肿瘤和肿瘤干细胞检测 | |
| Scheid et al. | The second genome: Effects of the mitochondrial genome on cancer progression | |
| EP3397051B1 (en) | Fly avatars for cancer and uses thereof | |
| CN104046644A (zh) | 人源化小鼠模型的构建方法和应用 | |
| CN102747096B (zh) | 基因搜寻载体、随机基因突变调控方法及用途 | |
| CN107233573A (zh) | miRNA‑324‑5p及其激活物在制备炎症性肠病及炎症性肠病相关结直肠癌临床标志物及治疗药物中的应用 | |
| JP2010530524A5 (zh) | ||
| CN104174012B (zh) | Otud3蛋白在制备抑制肿瘤生长的产品中的应用 | |
| CN102399870A (zh) | 一种确定食道癌病人生存预后的试剂 | |
| CN105441552A (zh) | 鸡B亚群禽白血病抗性分子标记tvb3731-3732insA及其分子诊断方法 | |
| Shibata et al. | Wild polyps of the blooming jellyfish Aurelia limbata (Brandt, 1838)(Cnidaria: Scyphozoa) found on deep-sea debris off Sanriku, Japan | |
| Lesser et al. | Effects of thermal stress and ocean acidification on the expression of the retrotransposon steamer in the softshell Mya arenaria | |
| CN104593423A (zh) | 一种抗肿瘤化合物筛选模型的建立方法及其应用 | |
| CN106119341A (zh) | miR‑20a抑制剂的用途、药物组合物和试剂盒 | |
| CN105969745A (zh) | 鱼类低氧耐受基因及其用途 | |
| CN105671195B (zh) | miR-520c核苷酸的用途、药物组合物和试剂盒 | |
| CN106222169A (zh) | 长链非编码rna apoc1p1-3基因及其用途 | |
| CN105506088A (zh) | 鸡B亚群禽白血病抗性分子标记tvb3667-3668insAG及其分子诊断方法 | |
| JP3861170B2 (ja) | リンホシスチス病抵抗性のヒラメの識別方法 |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200626 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |