中文摘要：

钙网蛋白（calreticulin，CRT）是内质网（endoplasmic reticulum，ER）／肌浆网（sarcoplasmic reticulum，SR）中主要的Ca^2＋结合分子伴侣，参与调节细胞Ca^2＋稳态和协助蛋白质折叠，在内源性保护现象——缺血后处理（ischemic postconditioning，I-postC）过程中表达上调，但其发挥作用的分子机制尚未完全阐明。本工作在原代培养Sprague-Dawley（SD）乳鼠心肌细胞低氧／复氧（hypoxia／reoxygenation，H／R）模型上，采用反义寡核苷酸（antisense oligodeoxynucleotides，AS-ODNs）抑制CRT表达，观察低氧后处理（hypoxic postconditioning，H-postC）过程中CRT下游分子钙调神经磷酸酶（calcineurin，CaN）活性以及CaN、核转录因子κB（nuclear factorkappa B，NFκB）、凋亡相关分子Bcl-2、Bax、C／EBP同源蛋白（C／EBP homologous protein，CHOP）等蛋白表达变化，及其与细胞保护的关系。心肌细胞随机分为6组（n=4）：对照组、低氧／复氧（H／R）组、低氧后处理（H-postC）组、AS-ODNs抑制CRT表达（AS）组、AS＋H／R组和AS＋H-postC组，采用台盼蓝排斥实验、培养基乳酸脱氢酶（1actate dehydrogenase，LDH）活性测定及流式细胞术检测细胞损伤；经Fluo-3／AM染色，采用激光共聚焦显微镜测定细胞浆游离Ca^2＋浓度；采用对硝基磷酸酚（p-nitrophenyl phosphate，PNPP）底物发色法测定CaN活性；Western blot检测相关蛋白表达。结果显示：（1）H-postC可减轻H／R诱导的心肌细胞损伤，与H／R组比较，细胞存活率升高17．1％，凋亡率和LDH漏出分别降低6．67％和27．9％（P均〈0．05）；（2）H-postC轻度上调CRT，AS-ODNs抑制CRT表达后，部分消除后处理的心肌保护作用，与H-postC组相比，AS＋H-postC组细胞存活率降低8．98％、凋亡率和LDH漏出分别升高1．74％和13．6％（P均〈0．05），而胞浆游离Ca^2＋浓度、CaN活性、CaN及NFKB表达均未发生明显变化（P〉0?

英文摘要：

Calreticulin （CRT） is an essential Ca^2＋-binding chaperone existing in endoplasmic reticulum （ER） or sarcoplasmic reticulum （SR）, and is involved in intracellular Ca^2＋ homeostasis and protein folding. Ischemic postconditioning （I-postC）, a newly discovered endogenous protective phenomenon, induces CRT up-regulation. The present study aimed to investigate the cardioprotective mechanism of CRT up-regulation induced by hypoxic postconditioning （H-postC）. Primary cultured neonatal rat cardiomyocytes were exposed to 2 h of hypoxia followed by 24 h of reoxygenation. Postconditioning was carried out by two cycles of 10 min of reoxygenation and 20 min of rehypoxia after 2 h of hypoxia. Antisense oligodeoxynucleotides （AS-ODNs） were used to inhibit CRT expression 36 h before hypoxia. Cardiomyocytes were randomly divided into 6 groups as follows （n=4）： control, hypoxia/reoxygenation （H/R）, H-postC, AS, AS ＋ H/R, and AS ＋ H-postC. Morphological studies, lactate dehydrogenase （LDH） activity assay in culture medium, and flow cytometry were used to detect cardiomyocyte necrosis and apoptosis. Intracellular Ca^2＋ concentration was detected by fluorescent Fluo-3/AM staining through laser confocal microscope, and p-nitrophenyl phosphate （PNPP） was used as substrate to measure calcineurin （CAN） activity. The expression of CRT, CaN, nuclear factor kappa B （NFκB） and apoptosis-related proteins, such as Bcl-2, Bax and C/EBP homologous protein （CHOP） were detected by Western blot. The results were as follows. （1） H-postC protected neonatal cardiomyocytes from H/R injury. Compared with H/R group, cell survival rate increased by 17.1%, apoptotic rate and LDH leakage decreased by 6.67% and 27.9% in H-postC group, respectively （P〈0.05）. （2） H-postC induced mild up-regulation of CRT expression. Inhibition of CRT by AS-ODNs attenuated the cardioprotection of H-postC partly. Compared with H-postC group, cell survival rate decreased by 8.98%, and apoptotic rate an