中国修复重建外科杂志

中国修复重建外科杂志

TBX3、TBX18 在人源诱导多能干细胞向窦房结样细胞富集分化中的作用

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目的 探索过表达 TBX3、TBX18 诱导人源诱导多能干细胞(human induced pluripotent stem cells,HiPS)向窦房结样细胞富集分化的作用。方法 取 HiPS,采用实时荧光定量 PCR(real-time fluorescence quantitative PCR,qRT-PCR)检测其干性标记物 OCT3/4、SOX2、NANOG 表达,并与人胚胎干细胞(human embryonic stem cells,hESCs)比较;免疫荧光染色观察 HiPS 干性标记物 OCT3/4、NANOG、SSEA4 和 TRA-1-60 表达。将 HiPS 向心肌细胞定向分化,qRT-PCR 检测心肌前体细胞特异性基因 ISL1、NKX2-5(NK2 homeobox 5)以及心肌细胞特异标记物 ACTN1、TNNT2 表达,以成人心肌细胞(human adult cardiomyocytes,hACM)作为阳性对照;免疫荧光染色观察心肌前体细胞特异核转录因子 NKX2-5,心肌细胞特异性收缩蛋白肌球蛋白(cardiac troponin,cTnT)、α-辅肌动蛋白(α-actinin),以及心房肌球蛋白轻链(myosin light chain 2A,MLC-2A)和心室肌球蛋白轻链(myosin light chain 2V,MLC-2V)表达;流式细胞术检测 α-actinin 阳性率。在 HiPS 向心肌细胞定向分化第 3 天(中胚层阶段),以慢病毒方式过表达窦房结相关基因 TBX3、TBX18,继续培养至 21 d,采用 qRT-PCR 检测窦房结细胞特异性标记物 TBX3、TBX18、SHOX2、NKX2-5、HCN4、HCN1 相对表达量,以增强绿色荧光蛋白空白病毒为对照。结果 OCT3/4、SOX2、NANOG 在 HiPS 和 ESCs 中均高表达,各基因相对表达量比较差异无统计学意义(P>0.05);OCT3/4 和 NANOG 在 HiPS 中特异分布于细胞核中,SSEA4 和 TRA-1-60 分布于细胞膜上。HiPS 向心肌细胞分化 5、7、21、28 d 时 ISL1 基因以及分化 7、21、28 d 时 NKX2-5 基因相对表达量均显著高于 hACM(P<0.05),分化 3、5、7、21 d ACTN1 和 TNNT2 基因相对表达量均显著低于 hACM(P<0.05)。NKX2-5 在绝大部分细胞核中表达;cTnT 和 α-actinin,MLC-2A 和 MLC-2V 信号定位于细胞质中,并初步呈现出肌小结纹理样结构。流式细胞术检测示 HiPS 向心肌细胞诱导分化成功。过表达 TBX3 组中,TBX18、SHOX2、HCN4、HCN1 较对照组表达均有上调,且 SHOX2 基因相对表达量差异有统计学意义(P<0.05);NKX2-5 基因相对表达量虽低于对照组,但差异无统计学意义(P>0.05)。过表达 TBX18 组中,各基因相对表达量与对照组比较差异均无统计学意义(P>0.05)。结论 HiPS 和 hESCs 的多能性相近,建立了稳定的 HiPS 干性维持及培养方法;成功建立 HiPS 向心肌细胞高效分化技术平台;尽管 TBX3、TBX18 在 HiPS 向窦房结样细胞富集分化中的促进作用不显著,但 TBX3 呈现出一定促进趋势,未来可进一步探索。

Objective To explore the role of over-expression of TBX3 and TBX18 in inducing human induced pluripotent stem cells (HiPS) to enrich and differentiate into sinoatrial node-like cells. Methods The expression of stemness markers OCT3/4, SOX2, and NANOG in HiPS was detected by real-time fluorescence quantitative PCR (qRT- PCR), and compared with human embryonic stem cells (hESCs). Immunofluorescence staining was used to observe the expression of HiPS stemness markers OCT3/4, NANOG, SSEA4, and TRA-1-60. The HiPS were directional differentiated into cardiomyocytes, the expression of ISL1, NK2 homeobox 5 (NKX2-5), ACTN1, and TNNT2 were detected by qRT-PCR, human adult cardiomyocytes (hACM) were used as positive control. Immunofluorescence staining was used to observe the expression of NKX2-5, cardiac troponin (cTnT), α-actinin, atria myosin light chain 2A (MLC-2A), and ventricular myosin light chain 2V (MLC-2V). The positive rate of α-actinin was detected by flow cytometry. On the 3rd day after HiPS differentiated into cardiomyocytes (mesodermal stage), lentiviral over-expression of sinoatrial node-related genes TBX3 and TBX18 was carried out for 21 days. The relative expression of specific markers TBX3, TBX18, SHOX2, NKX2-5, HCN4, and HCN1 in sinoatrial node cells was detected by qRT-PCR, and compared with enhanced green fluorescent protein blank virus. Results OCT3/4, SOX2, and NANOG were highly expressed in HiPS and ESCs, and there was no significant difference in the relative expression of each gene (P>0.05); OCT3/4 and NANOG were specifically distributed in the nucleus of HiPS, while SSEA4 and TRA-1-60 were distributed in the cell membrane. The relative expression of ISL1 gene at 5, 7, 21, and 28 days and NKX2-5 gene at 7, 21, and 28 days of HiPS differentiation into cardiomyocytes was significantly higher than that of hACM (P<0.05), and the relative expression of ACTN1 and TNNT2 genes at 3, 5, 7, and 21 days of HiPS differentiation into cardiomyocytes was significantly lower than that of hACM (P<0.05). NKX2-5 was expressed in most of the nuclei, cTnT and α-actinin, MLC-2A and MLC-2V signals were localized in the cytoplasm, presenting a texture-like structure of muscle nodules. Flow cytometry showed that HiPS was successfully induced to differentiate into cardiomyocytes. The expression of TBX18, SHOX2, HCN4, and HCN1 in the over-expression TBX3 group was up-regulated when compared with the control group, and the relative expression of SHOX2 gene had significant difference (P<0.05); the relative expression of NKX2-5 gene was lower than that in the control group, but there was no significant difference (P>0.05). There was no significant difference in the relative expression of each gene between the over-expressed TBX18 group and the control group (P>0.05). Conclusion HiPS and hESCs have similar pluripotency, and have established a stable method for maintaining and culturing the stemness of HiPS. A technological platform for the efficient differentiation of HiPS into cardiomyocytes has been successfully established. Although TBX3 and TBX18 do not play a significant role in promoting the enrichment and differentiation of HiPS into sinoatrial node-like cells, TBX3 shows a certain promoting trend, which can be further explored in the future.

关键词: 人诱导多能干细胞; TBX3; TBX18; 窦房结样细胞

Key words: Human derived induced pluripotent stem cells; TBX3; TBX18; sinoatrial node-like cells

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