E1A激活基因阻遏子在不同表型血管平滑肌细胞中的表达

Expression of cellular repressor of E1A-sti mulated genes in vascular smooth muscle cells of different phenotypes

目的 探讨血管平滑肌细胞表型转换、分化过程中E1A激活基因阻遏子(cellularrepressorofE1A stimulatedgenes, CREG)的表达变化及其相关机制。方法 将PCR扩增的CREG片段插入pGEX -4T -1原核表达载体,纯化的CREG融合蛋白免疫家兔以制备多克隆抗体。以人胸廓内动脉平滑肌细胞(humaninternalthoracicarterysmoothmusclecells,HITASY)为模型, [3H]标记脱氧胸苷掺入法测定去血清培养HITASY细胞DNA合成变化。以Western印迹观察HITASY细胞在去血清培养过程中SMα肌动蛋白、肌钙结合蛋白(calponin)的表达,RT- PCR和Western印迹分析去血清培养诱导CREGmRNA和蛋白表达变化;免疫组化法观察CREG在细胞内的表达及定位。结果 构建载体并成功得到抗CREG多克隆抗体。去血清培养可使HITASY细胞DNA合成明显降低。随去血清培养时间延长,除SMα肌动蛋白和肌钙结合蛋白表达逐渐上调之外,CREGmRNA转录活性和蛋白翻译合成亦上调。免疫组化显示去血清培养后,CREG蛋白在细胞内表达并主要位于细胞核周。结论 去血清能促使HITASY细胞由合成表型向收缩表型转换,同时伴CREGmRNA和蛋白表达上调。

Objectives The phenotypic modulat io n of vascular smooth muscle cells (VSMC) plays a central role in the pathogenesi s of arteriosclerosis. The purpose of this study was to investigate the expressi on of the cellular repressor of E1A-activated genes (CREG) at the transcription al and protein level in human internal thoracic artery smooth muscle cells(HITAS Y),which express different patterns of differentiation markers after serum with drawal.Methods After cloning and recombining the CREG vector, th e antiserum against the CREG protein was produced from the rabbits immunized by the purification CREG protein. The specificity of purified polyclonal antibody w as detected by Western blot assay. The DNA synthesis of HITASY cultured in serum -free and serum-supplemented medium was measured by the [3H]-thymidine in corporation. Western blot analysis detected the expression of smooth muscle-spe cific markers (smooth muscle α-actin, calponin). The localization of CREG in c ells was examined with immunohistochemistry staining and expression of CREG mRNA and protein were analyzed by RT-PCR and Western blot in HITASY after serum wit hdrawal.Results The high specificity of polyclonal antibody again st CREG obtained from rabbits was confirmed by Western blot assay. In response to serum withdrawal, cultured HITASY cells exhibited phenotypic conversion from synthetic into contractile one as evidenced by the data of [3H]-thymidine i ncorporation and Western blot. The DNA synthesis of HITASY precipitously dropped to background levels after serum withdrawal and nearly restored after reintrodu ction of serum to culture medium 2 days later. Western blot revealed a reversibl e upregulation of smooth muscle α-actin and calponin in HITASY after serum dep rivation. Moreover, serum withdrawal also induced a prominent increase of CREG m RNA and protein expression which reached a peak on 3 days and decreased graduall y on 5~7 days after serum withdrawal. Immunohistochemistry stain indicated the C REG protein mainly localizes in a perinuclear pattern in HITASY cells. Conclusions Those data provide evidence that the coordina ted changes in CREG gene and protein expression as well as smooth muscle-specif ic markers may take place in connection with the process of phenotypic modulatio n of VSMC in vitro.