Tseng C E, Miranda E, Di Donato F, Boutjdir M, Rashbaum W, Chan E K, Buyon J P
Department of Medicine, New York University School of Medicine, Hospital for Joint Diseases, New York 10003, USA.
Pediatr Res. 1999 Feb;45(2):260-9. doi: 10.1203/00006450-199902000-00018.
Irreversible congenital heart block (CHB) and the transient rash of neonatal lupus are strongly associated with maternal antibodies to SSA/Ro and SSB/La proteins; however, the precise mechanism by which these antibodies mediate organ-specific injury is not yet defined. Culturing of keratinocytes has provided critical insights. Accordingly, successful culturing of human fetal cardiac myocytes at high yield would constitute a powerful tool to directly examine conditions that promote expression of the target autoantigens. To accomplish this aim, fetal cardiac myocytes from 18- to 22-wk abortuses were established in culture using a novel technique in which cells were isolated after perfusion of the aorta with collagenase in a Langendorff apparatus. After preplating to decrease fibroblast contamination, cardiocytes were grown in flasks and slide chambers. Staining with monoclonal anti-sarcomeric alpha-actinin revealed the expected striations typical of cardiac myocytes in 70-90% of the cells after 4 d in culture. Furthermore, the cells were observed to beat at rates varying between 25-75 beats per minute (bpm) after the addition of 1.8 mM CaCl2. An average yield of 45-60 x 10(6) cells was obtained from a 3- to 5-g heart. Cellular localization of SSA/Ro and SSB/La by indirect immunofluorescence and demonstration of mRNA expression by reverse transcriptase polymerase chain reaction supports the feasibility of cultured cardiac myocytes for the study of congenital heart block. In contrast to the increased expression of SSA/Ro reported for keratinocytes, incubation of cultured human cardiac myocytes with either 17beta-estradiol or progesterone did not alter mRNA expression or cellular localization of 48 kD SSB/La, 52 kD SSA/Ro, or 60 kD SSA/Ro. In summary, we describe a novel method to successfully culture human fetal cardiac myocytes that should provide a valuable resource for investigation of the molecular mechanism(s) contributing to the development of congenital heart block. Differential constitutive and estradiol-induced expression of 52 and 60 kD SSA/Ro in human cardiac myocytes compared with keratinocytes may be a factor contributing to the marked discordance of clinically detectable injury in these two target tissues.
不可逆性先天性心脏传导阻滞（CHB）和新生儿狼疮的短暂皮疹与母体抗SSA/Ro和SSB/La蛋白抗体密切相关；然而，这些抗体介导器官特异性损伤的确切机制尚未明确。角质形成细胞的培养提供了重要的见解。因此，高产率地成功培养人胎儿心肌细胞将成为直接检测促进靶自身抗原表达条件的有力工具。为实现这一目标，使用一种新技术对18至22周流产胎儿的心肌细胞进行培养，该技术是在Langendorff装置中用胶原酶灌注主动脉后分离细胞。在预先铺板以减少成纤维细胞污染后，心肌细胞在培养瓶和载玻片培养室中生长。培养4天后，用单克隆抗肌节α-肌动蛋白染色显示，70%-90%的细胞呈现出心肌细胞典型的预期条纹。此外，添加1.8 mM氯化钙后，观察到细胞以每分钟25-75次的速率跳动。从3至5克的心脏中平均可获得45-60×10⁶个细胞。通过间接免疫荧光法对SSA/Ro和SSB/La进行细胞定位，并通过逆转录聚合酶链反应证明mRNA表达，支持了培养的心肌细胞用于先天性心脏传导阻滞研究的可行性。与角质形成细胞中报道的SSA/Ro表达增加相反，用17β-雌二醇或孕酮孵育培养的人心肌细胞，并未改变48 kD SSB/La、52 kD SSA/Ro或60 kD SSA/Ro的mRNA表达或细胞定位。总之，我们描述了一种成功培养人胎儿心肌细胞的新方法，该方法应为研究导致先天性心脏传导阻滞发生的分子机制提供有价值的资源。与角质形成细胞相比，人心肌细胞中52 kD和60 kD SSA/Ro的差异组成性表达和雌二醇诱导表达可能是导致这两个靶组织中临床可检测损伤明显不一致的一个因素。
