Sivasubramaniyan Kavitha, Atluri Rajesh Reddy, Sarda Kanchan, Arvind Milan, Balaji Vishnu, Deb Kaushik Dilip
Manipal University, Embryonic Stem Cell and Developmental Biology Program, Manipal Institute of Regenerative Medicine, #10 Service Road, Domlur Layout, Bangalore 560071, India.
Regen Med. 2008 Jan;3(1):23-31. doi: 10.2217/17460751.3.1.23.
Mechanisms underpinning Gram-negative bacterial vaginosis-induced birth anomalies are obscure. Ethical issues limit such studies on peri-implantation-stage human embryos. Here we have used embryoid bodies (EBs) as an in vitro model to examine the effect of Gram-negative bacterial endotoxins/lipopolysaccharides (LPS) on the faithful induction of germ lineages during embryogenesis. The role of LPS-inducible cytokine and pluripotency-related DNA-binding protein HMGB1 was also studied in these EBs.
EBs derived from the human embryonic stem cell line HUES9 were exposed to 12.5 pg/ml of LPS for 48 h. The expression profile of the ectoderm, endoderm, mesoderm and trophectoderm lineage markers, such as beta III-tubulin, GATA4, BMP2, Brachury and beta-hCG, were studied, by RT-PCR and immunofluorescence. Inhibition of mesoderm induction was confirmed by RT-PCR analysis for hANP, cTnT, ABCG2, GATA2, BMP4 and HAND1. Osteoblast differentiation was induced in the EBs, and confirmed by von Kosa and Alizarin red staining. A comet assay was also carried out to assess the degree of apoptosis in these EBs.
We found that the LPS-treated EBs were selectively silenced for mesoderm markers and failed to differentiate into functional osteoblasts. HMGB1 expression was absent in the normal EBs and was found to be localized in the cytoplasm of the LPS-treated EBs. Overall, our data indicate that endotoxin-induced HMGB1 expression in the peri-implantation-stage embryos can bring about severe birth defects of, for example, the bone and heart. This study also indicates that HMGB1 could be involved in maintenance of pluripotency in the human embryonic stem cells by impeding their differentiation.
革兰氏阴性菌性阴道炎导致出生异常的潜在机制尚不清楚。伦理问题限制了对植入前期人类胚胎进行此类研究。在此，我们使用类胚体（EBs）作为体外模型，以研究革兰氏阴性菌内毒素/脂多糖（LPS）对胚胎发生过程中生殖系忠实诱导的影响。还在这些EBs中研究了LPS诱导的细胞因子和多能性相关DNA结合蛋白HMGB1的作用。
将源自人类胚胎干细胞系HUES9的EBs暴露于12.5 pg/ml的LPS中48小时。通过逆转录聚合酶链反应（RT-PCR）和免疫荧光研究外胚层、内胚层、中胚层和滋养外胚层谱系标志物的表达谱，如βIII微管蛋白、GATA4、骨形态发生蛋白2（BMP2）、短尾相关蛋白（Brachury）和β人绒毛膜促性腺激素（β-hCG）。通过对心房钠尿肽（hANP）、心肌肌钙蛋白T（cTnT）、ATP结合盒转运体G2（ABCG2）、GATA结合蛋白2（GATA2）、BMP4和心脏和神经嵴衍生蛋白1（HAND1）进行RT-PCR分析，证实中胚层诱导受到抑制。在EBs中诱导成骨细胞分化，并通过冯科萨染色和茜素红染色进行确认。还进行了彗星试验以评估这些EBs中的凋亡程度。
我们发现，经LPS处理的EBs中中胚层标志物被选择性沉默，并且未能分化为功能性成骨细胞。正常EBs中不存在HMGB1表达，而在经LPS处理的EBs的细胞质中发现有HMGB1表达。总体而言，我们的数据表明，植入前期胚胎中内毒素诱导的HMGB1表达可导致严重的出生缺陷，例如骨骼和心脏方面的缺陷。这项研究还表明，HMGB1可能通过阻碍人类胚胎干细胞的分化而参与维持其多能性。
