论文部分内容阅读
目的 :探索妊娠期糖尿病(gestational diabetes mellitus,GDM)与正常妊娠胎盘组织微小RNA(microRNA,miRNA)的差异表达,为深入研究GDM发病机制提供基础。方法:选取于2013年1月—2014年1月在武警广东省总队医院、南方医科大学珠江医院就诊并分娩的GDM孕妇20例作为观察组(GDM组),均为粤籍汉族并且相互之间无亲缘关系,随机选取同期无妊娠合并症的正常广东汉族孕妇20例作为对照组。胎儿分娩后收集胎盘组织,用TRIzol法进行总RNA提取,经过质量和纯度分析鉴定合格后分别选取5个样本混合成GDM和对照组,采用逆转录法构建小RNA文库,使用第二代高通量测序采用边合成边测序的方法对miRNA进行测序并分析结果。结果:从胎盘组织提取到的总RNA纯度较高,D(260 nm)/D(280 nm)>1.90,并成功构建小RNA的c DNA文库。采用高通量测序得到长度为10~35 nt的小RNA片段,其中文库数量最多的小RNA长度21~24 nt,miRNA量占所有小RNA总量的30%以上,最终获得洁净的片段超过10 000 000。将得到的片段与Genebank及Rfram数据库比对,去除其他小RNA筛选出miRNA达7 000多种。通过样本两两比较分析,与对照组相比,发现GDM组有138种已知的miRNA表达明显上调,包括hsa-miR-548au-5p、hsa-miR-95a-5p、hsa-miR-373-5p、hsa-miR-216-5p等(log2-ratio>1,P<0.01);16种已知miRNAs明显表达下调,如hsa-miR-5699-5p、hsa-miR-4286等(log2-ratio<-1,P<0.01)。Mireap软件预测出27种新miRNA,其中有9种在GDM表达升高,6种表达降低。结论:GDM的胎盘组织miRNAs与对照组存在表达差异,胎盘组织miRNA可能在GDM发病过程中起一定的调节作用。
Objective: To explore the differential expression of gestational diabetes mellitus (gestational diabetes mellitus, gestational diabetes mellitus, and normal pregnancy placenta tissue microRNA (miRNA, to provide the basis for further study of the pathogenesis of GDM. Methods: From January 2013 to January 2014, 20 pregnant women with GDM who visited and delivered at Guangdong Provincial Corps Hospital of PLA Armed Police Force and Zhujiang Hospital of Southern Medical University were selected as the observation group (GDM group) In unrelated cases, 20 normal Han women of Guangdong in the same period with no pregnancy complications were selected as the control group. The placenta tissues were collected after fetus delivery, and the total RNA was extracted by TRIzol method. After passing the quality and purity analysis, 5 samples were selected and mixed into GDM and control groups respectively. The small RNA library was constructed by reverse transcription, Sequencing The miRNAs were sequenced using sequencing-by-synthesis sequencing and the results were analyzed. Results: The purity of total RNA extracted from placenta was higher than that of the control (D (260 nm) / D (280 nm)> 1.90). A small RNA fragment of 10 to 35 nt in length was obtained by high-throughput sequencing. The length of the small RNA in the library was 21-24 nt, the miRNA accounted for more than 30% of the total RNA and the final fragment was over 10 000 000. The obtained fragments were compared with Genebank and Rfram databases, and more than 7,000 miRNAs were screened out by removing other small RNAs. Compared with the control group, there were 138 known miRNAs in the GDM group were significantly up-regulated, including hsa-miR-548au-5p, hsa-miR-95a-5p, hsa-miR-373- (Such as hsa-miR-5699-5p, hsa-miR-4286 and so on) (log2-ratio> 1, P <0.01) <-1, P <0.01). Mireap software predicted 27 new miRNAs, of which 9 were elevated in GDM and 6 were decreased. Conclusion: The expression of miRNAs in placental tissue of GDM is different from that in control group. The placental miRNA may play a regulatory role in the pathogenesis of GDM.