CAJ | 학술논문

目的观察microRNA (miR)-379在正常小鼠胚胎心脏发育过程中表达水平的变化,结合其在P19细胞向心肌细胞分化过程中的表达情况,探讨其在心脏发育中可能存在的作用.方法取孕第8.5、11.5、14.5、18.5天小鼠胚胎的心脏组织,采用实时荧光定量PCR (RT-PCR)技术检测基因miR-379的表达变化.应用10 mL/L二甲基亚砜诱导P19细胞向心肌细胞方向分化,细菌培养皿中悬浮诱导至第4天,形成细胞聚集体后转移至6孑板培养,采用RT-PCR技术分别检测诱导第0、4、6、10天P19细胞中miR-379基因的表达水平.结果 miR-379在小鼠胚胎心脏发育过程中表达量逐渐减低,各时间点之间比较差异有统计学意义(F=21.13,P＜0.05).另一方面,在P19细胞诱导分化过程中心肌标志物肌钙蛋白T呈递增趋势,证明P19细胞被成功诱导为心肌样细胞.在P19细胞向心肌细胞分化的第0天表达量较低,第4天表达量最高,随后表达量又逐渐减低.结论 miR-379参与心脏发育过程,且可能与先天性心脏病的发生发展有关,但是具体机制还需要进一步的研究.
목적 관찰microRNA (miR)-379재정상소서배태심장발육과정중표체수평적변화,결합기재P19세포향심기세포분화과정중적표체정황,탐토기재심장발육중가능존재적작용.방법 취잉제8.5、11.5、14.5、18.5천소서배태적심장조직,채용실시형광정량PCR (RT-PCR)기술검측기인miR-379적표체변화.응용10 mL/L이갑기아풍유도P19세포향심기세포방향분화,세균배양명중현부유도지제4천,형성세포취집체후전이지6혈판배양,채용RT-PCR기술분별검측유도제0、4、6、10천P19세포중miR-379기인적표체수평.결과 miR-379재소서배태심장발육과정중표체량축점감저,각시간점지간비교차이유통계학의의(F=21.13,P＜0.05).령일방면,재P19세포유도분화과정중심기표지물기개단백T정체증추세,증명P19세포피성공유도위심기양세포.재P19세포향심기세포분화적제0천표체량교저,제4천표체량최고,수후표체량우축점감저.결론 miR-379삼여심장발육과정,차가능여선천성심장병적발생발전유관,단시구체궤제환수요진일보적연구.
Objective To observe the expression changes in microRNA (miR)-379 in the developmental process of the mouse heart and during the differentiation of P19 cells into cardiac myocytes,and to explore the possible relationship between miR-379 and the differentiation of cardiacmyocytes.Methods Heart tissues were collected from fetal mice in pregnant ones at their gestational age (8.5,11.5,14.5 and 18.5 days) respectively.Heart tissue sections of the fatal mice were obtained to observe the heart development process.Then total RNA was isolated from heart tissues by using the TRIzol method.Complementary DNA was synthesized from 1 μg total RNA by using a Reverse Transcriptase Kit.Finally,real-time PCR (RT-PCR) was employed to detect the expression of miR-379.At the same time,P19 cells were cultured with 10 mL/L Dimethyl sulfoxide in suspension for 4 days to form cell aggregation,and these aggregations were transferred into 6-wells plate for culturing by adherence.Beating cells were detected with microscopy on the 10th day after induction.Afterwards,total RNA was extracted from cultured P19 cells at different time points.Reverse transcription was executed to get DNA.At last,RT-PCR was used to explore the expression of miR-379 on 0,4,6,10 days after aggregation.Results The expression level of miR-379 was down-regulated gradually in the developing heart (at gestational age of 8.5,11.5,14.5,16.5 days,respectively),and there were significant differences on the different days (F =21.13,P ＜ 0.05).On the other hand,myocardial markers of troponin T represented an increasing trend during the process of P19 cells induction,which demonstrated that P19 cells were successfully induced into cardiomyocyte-like cells.Meanwhile,miR-379 showed a low expression on day 0 of P19 cells aggregation.On day 4,miR-379 demonstrated a higher level.Afterwards,miR-379 proved to be down-regulated gradually.Conclusions miR-379 plays a role in the process of the heart development,but the specific mechanisms need further research.