中华临床医师杂志（电子版）
中華臨床醫師雜誌（電子版）
중화림상의사잡지（전자판）
CHINESE JOURNAL OF CLINICIANS(ELECTRONIC VERSION)
2014年
21期
3850-3856
,共7页
虞勇%虞莹%王兴冈%邹云增%陈瑞珍
虞勇%虞瑩%王興岡%鄒雲增%陳瑞珍
우용%우형%왕흥강%추운증%진서진
柯萨奇B3病毒%心脏微血管内皮细胞%miRNA21
柯薩奇B3病毒%心髒微血管內皮細胞%miRNA21
가살기B3병독%심장미혈관내피세포%miRNA21
Coxsackie B3 virus%Cardiac microvascular endothelial cells%miRNA21
目的：本研究通过体外培养心脏微血管内皮细胞（CMVECs），经柯萨奇 B3病毒（CVB3）感染后，利用miRNA寡核苷酸基因芯片技术筛选差异表达的miRNAs，进一步探讨miRNA在 CVB3诱导 CMVECs 凋亡中的潜在作用机制。方法原代分离培养大鼠 CMVECs 细胞，以100TCID50CVB3病毒感染48 h 后检测Caspase-3活性和细胞凋亡；提取CMVECs细胞RNA，用Agilent大鼠miRNA寡核苷酸基因芯片进行检测，并通过TargetScan、miranda、mirbase和mirdb数据库选取出差异表达明显并与心血管疾病相关的miRNA，经qPCR对其进行验证，通过生物信息学分析预测其调控靶基因；合成miRNA21 mimics转染CMVECs细胞，同时合成miRNA21 inhibitor，与 CVB3共转染 CMVECs 细胞，检测各组细胞 Caspase-3活性变化和细胞凋亡。结果 CVB3感染CMVECs细胞48 h后与正常对照组比较Caspase-3活性显著上调（P＜0.01），细胞凋亡明显增加；通过基因芯片检测及生物信息学分析后发现，与心血管系统疾病密切相关的miRNA为miRNA21，经qPCR验证结果一致，预测其靶基因为PDCD4；miRNA21mimics转染CMVECs细胞后与正常对照组相比Caspase-3活性显著上调，细胞凋亡增加（P＜0.05）；而miRNA21 inhibitor与CVB3共转染 CMVECs 细胞后与 CVB3感染组相比 Caspase-3活性显著下调，细胞凋亡明显减少（P＜0.05）。结论 miRNA21在CVB3感染的CMVECs细胞中的表达有显著变化，并与CMVECs细胞凋亡密切相关，提示miRNA21在CVB3诱导的病毒性心肌炎发病过程中可能起着重要作用。
目的：本研究通過體外培養心髒微血管內皮細胞（CMVECs），經柯薩奇 B3病毒（CVB3）感染後，利用miRNA寡覈苷痠基因芯片技術篩選差異錶達的miRNAs，進一步探討miRNA在 CVB3誘導 CMVECs 凋亡中的潛在作用機製。方法原代分離培養大鼠 CMVECs 細胞，以100TCID50CVB3病毒感染48 h 後檢測Caspase-3活性和細胞凋亡；提取CMVECs細胞RNA，用Agilent大鼠miRNA寡覈苷痠基因芯片進行檢測，併通過TargetScan、miranda、mirbase和mirdb數據庫選取齣差異錶達明顯併與心血管疾病相關的miRNA，經qPCR對其進行驗證，通過生物信息學分析預測其調控靶基因；閤成miRNA21 mimics轉染CMVECs細胞，同時閤成miRNA21 inhibitor，與 CVB3共轉染 CMVECs 細胞，檢測各組細胞 Caspase-3活性變化和細胞凋亡。結果 CVB3感染CMVECs細胞48 h後與正常對照組比較Caspase-3活性顯著上調（P＜0.01），細胞凋亡明顯增加；通過基因芯片檢測及生物信息學分析後髮現，與心血管繫統疾病密切相關的miRNA為miRNA21，經qPCR驗證結果一緻，預測其靶基因為PDCD4；miRNA21mimics轉染CMVECs細胞後與正常對照組相比Caspase-3活性顯著上調，細胞凋亡增加（P＜0.05）；而miRNA21 inhibitor與CVB3共轉染 CMVECs 細胞後與 CVB3感染組相比 Caspase-3活性顯著下調，細胞凋亡明顯減少（P＜0.05）。結論 miRNA21在CVB3感染的CMVECs細胞中的錶達有顯著變化，併與CMVECs細胞凋亡密切相關，提示miRNA21在CVB3誘導的病毒性心肌炎髮病過程中可能起著重要作用。
목적：본연구통과체외배양심장미혈관내피세포（CMVECs），경가살기 B3병독（CVB3）감염후，이용miRNA과핵감산기인심편기술사선차이표체적miRNAs，진일보탐토miRNA재 CVB3유도 CMVECs 조망중적잠재작용궤제。방법원대분리배양대서 CMVECs 세포，이100TCID50CVB3병독감염48 h 후검측Caspase-3활성화세포조망；제취CMVECs세포RNA，용Agilent대서miRNA과핵감산기인심편진행검측，병통과TargetScan、miranda、mirbase화mirdb수거고선취출차이표체명현병여심혈관질병상관적miRNA，경qPCR대기진행험증，통과생물신식학분석예측기조공파기인；합성miRNA21 mimics전염CMVECs세포，동시합성miRNA21 inhibitor，여 CVB3공전염 CMVECs 세포，검측각조세포 Caspase-3활성변화화세포조망。결과 CVB3감염CMVECs세포48 h후여정상대조조비교Caspase-3활성현저상조（P＜0.01），세포조망명현증가；통과기인심편검측급생물신식학분석후발현，여심혈관계통질병밀절상관적miRNA위miRNA21，경qPCR험증결과일치，예측기파기인위PDCD4；miRNA21mimics전염CMVECs세포후여정상대조조상비Caspase-3활성현저상조，세포조망증가（P＜0.05）；이miRNA21 inhibitor여CVB3공전염 CMVECs 세포후여 CVB3감염조상비 Caspase-3활성현저하조，세포조망명현감소（P＜0.05）。결론 miRNA21재CVB3감염적CMVECs세포중적표체유현저변화，병여CMVECs세포조망밀절상관，제시miRNA21재CVB3유도적병독성심기염발병과정중가능기착중요작용。
Objective In the study, we cultured cardiac microvascular endothelial cells (CMVECs) in vitro and infected CMVECs with CVB3 to scan miRNA by miRNAs oligonucleotide microarray technology. Moreover, we explored the effects of miRNA on CVB3-induced CMVECs apoptosis. Methods We cultured primary rat CMVECs and assessed activity of caspase-3 in CMVECs exposure to CVB3 with 48 hours. RNA was isolated and scanned by miRNAs oligonucleotide microarray. We choosed the miRNA which was expressed significantly different and closely related with cardiovascular diseases, and furtherly validated by qPCR. Additionally, we respectively used miRNA mimics and miRNA inhibitors to transfect CMVEC and detect caspases-3 activity. Results CVB3 significantly up-regulated caspase-3 activity in 48 hours, as compared with control (P<0.05). miRNA21 was increased by CVB3 and displayed close relation with cardiovascular diseases. Similarly, qPCR also showed miRNA21 levels were significantly raised. The activity of caspase-3 was elevated in miRNA21 mimics+CVB3 group, which was decreased in miRNA21 inhibitors+CVB3 group. Conclusion Our investigations showed that the expression of miRNA21 was markedly increased by CVB3 and miRNA21 regulated CMVECs apoptosis,suggesting miRNA21 had vital roles in CVB3-induced viral myocarditis.
