Clinical implications of forkhead box M1, cyclooxygenase-2, and glucose-regulated protein 78 in breast invasive ductal carcinoma

BACKGROUND Breast infiltrating ductal carcinoma(BIDC)represents the largest heterotypic tumor group,and an in-depth understanding of the pathogenesis of BIDC is key to improving its prognosis.AIM To analyze the expression profiles and clinical implications of forkhead box M1(FOXM1),cyclooxygenase-2(COX-2),and glucose-regulated protein 78(GRP78)in BIDC.METHODS A total of 65 BIDC patients and 70 healthy controls who presented to our hospital between August 2019 and May 2021 were selected for analysis.The peripheral blood FOXM1,COX-2,and GRP78 levels in both groups were measured and the association between their expression profiles in BIDC was examined.Additionally,we investigated the diagnostic value of FOXM1,COX-2,and GRP78 in patients with BIDC and their correlations with clinicopathological features.Furthermore,BIDC patients were followed for 1 year to identify factors influencing patient prognosis.RESULTS The levels of FOXM1,COX-2,and GRP78 were significantly higher in BIDC patients compared to healthy controls(P<0.05),and a positive correlation was observed among them(P<0.05).Receiver operating characteristic analysis demonstrated that FOXM1,COX-2,and GRP78 had excellent diagnostic value in predicting the occurrence of BIDC(P<0.05).Subsequently,we found significant differences in FOXM1,COX-2,and GRP78 levels among patients with different histological grades and metastasis statuses(with vs without)(P<0.05).Cox analysis revealed that FOXM1,COX-2,GRP78,increased histological grade,and the presence of tumor metastasis were independent risk factors for prognostic death in BIDC(P<0.001).CONCLUSION FOXM1,COX-2,and GRP78 exhibit abnormally high expression in BIDC,promoting malignant tumor development and closely correlating with prognosis.These findings hold significant research implications for the future diagnosis and treatment of BIDC.

Prostaglandin E1 protects hepatocytes against endoplasmic reticulum stress-induced apoptosis via protein kinase A-dependent induction of glucose-regulated protein 78 expression

AIM To investigate the protective effect of prostaglandin E1(PGE1) against endoplasmic reticulum(ER) stressinduced hepatocyte apoptosis, and to explore its underlying mechanisms.METHODS Thapsigargin(TG) was used to induce ER stress in the human hepatic cell line L02 and hepatocarcinomaderived cell line Hep G2. To evaluate the effects of PGE1 on TG-induced apoptosis, PGE1 was used an hour prior to TG treatment. Activation of unfolded protein response signaling pathways were detected by western blotting and quantitative real-time RTPCR. Apoptotic index and cell viability of L02 cells and Hep G2 cells were determined with flow cytometry and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium] assay. RESULTS Pretreatment with 1 μmol/L PGE1 protected against TG-induced apoptosis in both L02 cells and Hep G2 cells. PGE1 enhanced the TG-induced expression of C/EBP homologous protein(CHOP), glucose-regulated protein(GRP) 78 and spliced X box-binding protein 1 at 6 h. However, it attenuated their expressions after 24 h. PGE1 alone induced protein and m RNA expressions of GRP78; PGE1 also induced protein expression of DNA damage-inducible gene 34 and inhibited the expressions of phospho-PKR-like ER kinase, phosphoeukaryotic initiation factor 2α and CHOP. Treatment with protein kinase A(PKA)-inhibitor H89 or KT5720 blocked PGE1-induced up-regulation of GRP78. Further, the cytoprotective effect of PGE1 on hepatocytes was not observed after blockade of GRP78 expression by H89 or small interfering RNA specifically targeted against human GRP78.CONCLUSION Our study demonstrates that PGE1 protects against ER stress-induced hepatocyte apoptosis via PKA pathwaydependent induction of GRP78 expression.

Role of Glucose-regulated Protein 78 in Early Brain Injury after Experimental Subarachnoid Hemorrhage in Rats

Early brain injury（EBI） plays a key role in the pathogenesis of subarachnoid hemorrhage（SAH）. This study investigated the role of glucose-regulated protein 78（GRP78） in EBI after SAH. Male Sprague-Dawley rats（n=108） weighing 260±40 g were divided into control, sham-operated, and operated groups. Blood was injected into the prechiasmatic cistern of rats in the operated group. Neurological scores, ultrastructures of neurons, apoptosis, and GRP78 expression in the hippocampus were examined using Garcia scoring system, transmission electron microscopy, terminal deoxynucleotidyl transferase-mediated d UTP nick-end labelling, and Western blotting at 1, 6, 12, 24, 48, and 72 h after SAH, respectively. The results showed that neurological scores were significantly decreased in the operated group as compared with those in control and sham-operated groups at 12, 24, 48, and 72 h. Metachromatin, chromatin pyknosis at the edge, endoplasmic reticulum swelling, and invagination of nuclear membrane were observed at 24 h in the operated group, indicating the early morphological changes of apoptosis. The number of apoptotic cells was significantly increased in the operated group as compared with that in control and sham-operated groups at 6, 12, 24, 48, and 72 h. The GRP78 protein expression levels in the operated group were significantly elevated at all time points and reached the peak at 12 h. GRP78 expression was positively associated with apoptosis cells and negatively with neurological scores. In conclusion, EBI was demonstrated to occur after SAH and GRP78 was involved in the development of EBI after SAH.

Effects of serum containing natural cerebrolysin on glucose-regulated protein 78 and CCAAT enhancer-binding protein homologous protein expression in neuronal PC12 cells following tunicamycin-induced endoplasmic reticulum stress

BACKGROUND： Glucose-regulated protein 78 （GRP78）, a marker of endoplasmic reticulum stress, can prolong cell survival. Alternatively, CCAAT enhancer-binding protein homologous protein （CHOP）, a transcription factor specific for endoplasmic reticulum stress, can cause cell cycle arrest and cell apoptosis. OBJECTIVE： To study the protective effects of serum containing natural cerebrolysin on endoplasmic reticulum stress in tunicamycin-induced neuronal PC12 cells, and analyze the influence on GRP78 and CHOP expressions. DESIGN, TIME AND SETTING： A parallel controlled study was performed at the Institute of Integrated Western and Traditional Chinese Medicine, Shenzhen Hospital, Southern Medical University, between March 2006 and August 2008. MATERIALS： Adult Sprague-Dawley rats were perfused with natural Cerebrolysin aqueous extract （0.185 g/kg/d） to produce serum containing natural Cerebrolysin. Physiological saline was used to produce blank serum. PC12 cell line was provided by Shanghai Institute of Cell Biology, Chinese Academy of Science. Tunicamycin was provided by Sigma （St. Louis, USA）, and natural Cerebrolysin, containing ginseng, rhizoma gastrodiae, and gingko leaf （1：2：2）, by Shengzhen Institute of Integrated Western and Traditional Chinese Medicine. METHODS： PC12 cells were treated with DMEM culture media containing 10% blank serum （normal control group）, tunicamycin （1 μg/mL; model group）, and 5%, 10%, and 15% serum containing natural cerebrolysin and tunicamycin （1 μ g/mL; low-, moderate-, and high-dose serum containing natural cerebrotysin groups）, for 2 hours. MAIN OUTCOME MEASURES： PC12 cells were treated with tunicamycin for 48 hours after which apoptosis was measured using the TUNEL method to calculate apoptotic index. GRP78 expression was detected using immunocytochemistry. After 24 hours of treatment with tunicamycin, GRP78 and CHOP mRNA expressions were measured using RT-PCR. RESULTS： The apoptotic index and CHOP mRNA expression were in the model group and three cerebrolysin groups were significantly increased when compared to the normal control group （P 〈 0.05）. In contrast, GRP78 mRNA and protein expressions were significantly decreased （P 〈 0.05）. CONCLUSION： Serum containing natural cerebrolysin significantly reduced apoptosis in neuronal PC12 cells following tunicamycin-induced endoplasmic reticulum stress. These results may be related to an up-regulation of GRP78 expression and down-regulation of CHOP expression, both of which displayed dose-dependent effects.

Glucose-regulated protein 78 inhibits scavenger receptor A-mediated internalization of acetylated low density lipoprotein

Class A scavenger receptor(SR-A) plays an important role in foam cell formation.However, the mechanism underlying the internalization of the receptor-ligand complexes remains unclear.The aim of the present study was to investigate the molecular mechanism to regulate SR-A-mediated intracellular lipid accumulation in macrophages A pull-clown assay was performed and glucoseregulated protein 78(GRP78) was identified to bind with the cytoplasmic domain of SR-A(CSR-A).Immunoprecipitation and artificially expressed protein binding assay demonstrated the direct specific binding of GRP78 with SR-A in cells.Indirect immunofluorescence assay and western blot analysis showed their co-localization in membrane and cytoplasm.Over-expression of GRP78 specifically inhibited SR-A-mediated uptake of fluorescent acetylated low-density lipoprotein, a specific ligand for SR-A, without altering cellular SR-A expression and binding ability, and significantly inhibited cholesterol ester accumulation in cells, which can be partly attributed to the suppression of c-Jun-NH2-terminal kinase signaling pathway.These results suggest that GRP78 may act as an inhibitor of SR-A-mediated internalization of modified low-density lipoprotein into macrophages(C) 2009 Elsevier Inc.All rights reserved.

绝经后骨质疏松症患者外周血中GRP78和ATF4的表达水平及临床意义

目的探究绝经后骨质疏松症(postmenopausal osteoporosis,PMO)患者外周血中葡萄糖调节蛋白78(glucoseregulated protein 78,GRP78)和激活转录因子4(activating transcription factor 4,ATF4)的水平及临床意义。方法选取2018年12月—2021年5月石家庄市第三医院创伤骨科收治的96例PMO患者为PMO组,另选取同期96名体检健康的绝经女性为正常组。比较PM O组和正常组的一般资料;采用酶联免疫吸附法测定血清GR P78、ATF4水平;利用电化学发光免疫分析仪测定血清β-胶原特殊序列(β-Crosslaps)、Ⅰ型前胶原氨基端前肽(procollagenⅠtype N-terminal propeptide,PINP)水平;利用双能X线骨密度仪检测腰椎骨密度(bone mineral density,BMD);采用Pearson法分析PMO患者血清GRP78、ATF4水平与β-Crosslaps、PIN P、BM D的相关性及PMO患者血清GR P78表达水平与ATF4的相关性;用Logistic回归分析PMO发生的影响因素。结果PMO组患者腰臀比、BMD低于正常组(t=9.76、15.47,P均<0.001);血清GRP78、ATF4、β-Crosslaps、PINP水平高于正常组(t=18.32、16.81、8.15、14.84,P均<0.001);PMO患者血清GRP78、ATF4水平与β-Crosslaps、PINP均呈正相关(r=0.56、0.48、0.48、0.52,P<0.001、<0.001、<0.001、=0.006),与BMD呈负相关(r=-0.54、-0.44,P均<0.001);PMO患者血清GRP78水平与ATF4呈正相关(r=0.595,P<0.001);PINP、GRP78、β-Crosslaps、ATF4是影响PMO发生的危险因素(OR=2.518、2.672、2.271、2.336,P均<0.001),BMD是影响PMO发生的保护因素(OR=0.583,P<0.001)。结论PMO患者血清GRP78、ATF4水平较高,均与β-Crosslaps、PINP、BMD相关,GRP78、ATF4可能是诊治PMO的潜在靶标,测定血清GRP78、ATF4水平有助于临床防治PMO。

血清ADAM15、GRP78联合CT肺动脉造影对急性肺栓塞的诊断价值

目的探究血清整合素金属蛋白酶15(ADAM15)、葡萄糖调节蛋白78(GRP78)联合CT肺动脉造影(CTPA)对急性肺栓塞(APE)的诊断价值。方法选取本院2021年6月至2023年6月收治的86例疑似APE患者,患者均行CTPA检查;根据病情严重程度将APE患者分为中高危组和低危组;酶联免疫吸附法检测ADAM15、GRP78水平;Pearson相关性分析血清ADAM15、GRP78与CTPA指标的相关性;中高危APE的影响因素采用多因素Logistic回归分析;绘制ROC曲线分析血清ADAM15、GRP78对中高危APE的诊断价值。结果86例患者经过CTPA检测出栓子702个,86例患者在不同肺动脉部位表现出充盈缺损等,病变部位主要位于双肺29例,左肺30例,右肺27例。4种栓塞类型42例中心型,98例偏心型,26例附壁血栓型,30例完全堵塞型。中高危组RVD/LVD、RV-LD/LV-LD、Qanadli栓塞指数显著高于低危组(P<0.05)。中高危组血清ADAM15、GRP78水平显著高于低危组(P<0.05)。根据Pearson相关性分析得知,血清ADAM15与GRP78呈正相关(P<0.05),二者均与RVD/LVD、RVLD/LV-LD、Qanadli栓塞指数呈正相关(P<0.05)。多因素Logistic回归分析得知ADAM15、GRP78、RVD/LVD、RV-LD/LV-LD、Qanadli栓塞指数为影响中高危APE患者的危险因素(P<0.05)。根据ROC曲线得知,血清ADAM15、GRP78、RVD/LVD、RVLD/LV-LD和Qanadli栓塞指数五者联合诊断中高危APE的AUC为0.990,五者联合优于各自单独诊断(Z_(联合vs ADAM15)=2.691、Z_(联合vs GRP78)=2.578、Z_(联合vs RVD/LVD)=2.710、Z_(联合vs RV-LD/LV-LD)=2.714、Z联合vs Qanadli栓塞指数=2.698,P均<0.05)。结论血清ADAM15、GRP78在APE患者中显著升高,二者联合CTPA可提高对APE的诊断价值。

Correlation between clinicopathology and expression of heat shock protein 70 and glucose-regulated protein 94 in human colonic adenocarcinoma

AIM: To investigate the correlation between clinicopathology and expression of heat shock protein 70 (HSP70) and glucose-regulated protein 94 (grp94) in human colonic carcinoma. METHODS: The expression of HSP70 and grp94 was studied in 80 human colonic cancers with or without metastasis as well as in their adjacent mucous membrane by way of immunohistochemistry and pathology photograph analysis. RESULTS: The expression of HSP70 and grp94 was significantly higher in cancer than that in adjacent mucous membrane (92.5%, 85.0% vs 56.3%, 42.5%, P<0.01). HSP70 and grp94 expressed higher in moderately- and poorly-differentiated colonic cancers than that in their adjacent tissues (93.7%, 87.5%; 100%, 90% vs56.3%, 42.5%;P<0.01). Dukes C and D stages of colonic cancers showed higher positive rates than Dukes A and B stage groups (97.1%, 91.2%; 100%, 90.9%; vs 80%, 70%; 78.6%, 71.4%; P<0.05). There were definite differences in HSP70 and grp94 expression between metastasis groups and non-metastasis groups (100% vs 75%, 100% CONCLUSION: The HSP70 and grp94 expression rates in colonic cancer groups are significantly higher than that in their adjacent mucous membrane. The HSP70 and grp94 expression in poorly-differentiated colonic cancers with metastasis is significantly higher than well-differentiated cancers without metastasis. The overexpression of HSP70 and grp94 can be used as diagnostic or prognostic markers for colonic cancer.

Expression and significance of heat shock protein 70 and glucose-regulated protein 94 in human esophageal carcinoma

AIM: To investigate the expression and significance of heat shock protein 70 (HSP70) and glucose-regulated protein 94 (grp94) in human esophageai carcinoma and adjacent normal tissues. METHODS: The expression of HSP70 and grp94 in 78 human esophageai cancer and adjacent normal tissues was studied by immunohistochemistry and pathology photograph analysis. RESULTS: Both esophageai cancer and adjacent normal tissues could express HSP70 and grp94. Of the 78 cases of esophageai carcinoma, 95.0%(72/78) showed positive HSP70, mainly stained in nuclei, while grp94 was mainly stained in cell plasma, and the positive rate was 71.8% (56/78).There was a significant difference in the expression of HSP70 and grp94 between esophageai cancer and adjacent normal tissues (P<0.01). Compared with adjacent normal tissues, there was a significant difference between differential types and HSP70 expression (P<0.01). CONCLUSION: HSP70 and grp94 express differently in cell plasma and nuclei. The expression intensity of HSP70 is related to the differentiation of esophageai carcinoma.

Co-expression of heat shock protein 70 and glucose-regulated protein 94 in human gastric carcinoma cell line BGC-823

AIM:To investigate the co-expression and significance of heat shock protein 70 (HSP70) and glucose-regulated protein 94 (grp94) in human gastric carcinoma cell line BGC-823. METHODS: The expression and localization of HSP70 and grp94 in human gastric carcinoma cell line BGC-823 were determined by immunocytochemistry and indirect immunofluorescence cytochemical staining. Flow cytometry was used to analyze the correlation between expression of HSP70, grp94 and cell cycle in BGC-823 cell line. RESULTS: Gastric cancer cell line BGC-823 expressed high level of HSP70 and grp94. The positive rate of HSP70 and grp94 was 84.9±4.94% and 79.6±5.16%, respectively. Both of them were stained in cell plasma. There was a significant difference compared with control group (1.9±0.94%, P<0.01). During the cell cycle, HSP70 and grp94 were continuously expressed in BGC-823. CONCLUSION: HSP70 and grp94 are highly expressed in human gastric carcinoma BGC-823 cells through the whole cell cycle. There is no relationship between expression of HSP70, grp94 and cell cycle.

流体剪应力对人脐静脉内皮细胞葡萄糖调节蛋白78和C/EBP同源蛋白表达的影响

目的探索流体剪应力对人脐静脉内皮细胞(HUVECs)葡萄糖调节蛋白78(GRP78)和C/EBP同源蛋白(CHOP)表达的影响。方法以HUVECs作为实验细胞,设计并构建流体动力学模拟实验系统,控制流体动力学模拟实验系统中灌流液的流速,以实现对实验细胞施加不同的流体剪应力。按实验细胞在实验系统中所承受的不同流体剪应力,将实验细胞分为低剪应力组(A组;0.4 Pa)、中剪应力组(B组;0.8 Pa)和高剪应力组(C组;1.2 Pa)。每组HUVECs包含3个细胞玻片,每个玻片经实验系统灌流液反复循环流经12 h。采用蛋白质印迹法对各组细胞中GRP78和CHOP蛋白水平进行检测,采用实时荧光定量逆转录聚合酶链反应技术测定各组细胞GRP78和CHOP蛋白及其信使RNA(mRNA)相对水平。应用GraphPad Prism 8.0软件对数据进行统计学分析。结果(1)A、B、C组HUVECs中GRP78蛋白相对表达水平分别为1.33±0.46、0.93±0.34、0.64±0.30;多组间比较差异有统计学意义(F=36.17,P<0.05)。A组GRP78蛋白相对表达水平高于B组、C组(均P<0.01),B组GRP78蛋白相对表达水平高于C组(P=0.0013)。3组HUVECs中CHOP蛋白相对表达水平分别为:A组1.29±0.38,B组0.90±0.34,C组0.59±0.29;多组间比较差异有统计学意义(F=41.27,P<0.05)。A组CHOP蛋白相对表达水平高于B组、C组(均P<0.01),B组CHOP蛋白相对表达水平高于C组(P=0.0004)。(2)A、B、C组HUVECs中GRP78 mRNA相对表达水平分别为18.3±3.4、11.3±1.8、5.4±2.2;多组间比较差异有统计学意义(F=189.20,P<0.05)。A组GRP78 mRNA相对表达水平高于B组、C组(均P<0.01),B组GRP78 mRNA相对表达水平高于C组(P<0.01)。3组HUVECs中CHOP mRNA相对表达水平分别为:A组20.4±3.8,B组14.2±2.1,C组7.8±1.3;多组间比较差异有统计学意义(F=171.80,P<0.05)。A组CHOP mRNA相对表达水平高于B组、C组(均P<0.01),B组CHOP mRNA相对表达水平高于C组(P<0.01)。结论低流体剪应力可能增加HUVECs中GRP78、CHOP的蛋白及其mRNA表达水平。

天香丹通过GRP78/NLRP3信号通路调控内质网应激抗动脉粥样硬化的作用机制研究

目的:基于葡萄糖调节蛋白78(GRP78)/NOD样受体蛋白3(NLRP3)信号通路探讨天香丹通过调控内质网应激抗动脉粥样硬化的作用机制。方法:将雄性载脂蛋白E基因敲除(ApoE^(-/-))小鼠随机分为模型组、阿托伐他汀组、天香丹组,每组11只,给予高脂饲料诱导动脉粥样硬化模型;另取10只雄性C57BL/6J小鼠作为正常组。于造模10周后给予相应药物治疗,连续干预12周。采用苏木精-伊红(HE)染色观法察胸主动脉病理特点;酶联免疫吸附试验(ELISA)检测血清白细胞介素1β(IL-1β)、白细胞介素18(IL-18)、活性氧(ROS)含量;免疫组织化学法(IHC-P)检测胸主动脉中GRP78和NLRP3蛋白阳性表达水平。结果:各实验组存活6只。与模型组比较,阿托伐他汀组和天香丹组IL-1β、IL-18、ROS表达含量均降低(P<0.05或P<0.01);GRP78、NLRP3蛋白阳性表达均明显减少(P<0.05或P<0.01)。结论:天香丹能有效改善动脉粥样硬化性心血管疾病小鼠炎症反应,其作用机制可能与调控内质网应激GRP78/NLRP3信号通路抑制炎症小体活化、减轻动脉粥样硬化炎症损伤有关。

血清葡萄糖调节蛋白78在多发性骨髓瘤病人中的表达及其预后意义

目的探讨葡萄糖调节蛋白78(GRP78)在初诊多发性骨髓瘤(MM)病人血清中的表达,及其对病人治疗反应及预后的预测价值。方法选择2017年1月至2020年12月大竹县人民医院收治的78例MM病人为MM组,同期该院门诊就诊的62例健康成人作为对照组。收集所有参与者的外周静脉血样本,使用酶联免疫吸附测定(ELISA)法检测血清中GRP78的表达,分析GRP78表达与临床特征及治疗反应的关系。通过受试者操作特征曲线(ROC曲线)确定血清GRP78预测MM病人预后的最佳临界值,采用Kaplan-Meier曲线与Cox比例风险模型评估血清GRP78表达与MM病人预后的关系。结果MM病人的血清GRP78表达水平显著高于对照组(Z=4.12,,P<0.001),中位表达量分别为2.49(1.23,5.13)mg/L和1.29(0.75,2.14)mg/L。与多发性骨髓瘤国际预后分期(ISS)Ⅰ、Ⅱ期病人2.05(1.09,4.40)mg/L相比,血清GRP78表达在ISSⅢ期病人2.99(1.66,5.63)mg/L显著升高(Z=2.18,P=0.030)。然而,血清GRP78表达与接受硼替佐米诱导方案的MM病人的治疗反应无关。ROC曲线显示血清GRP78预测总体生存(OS)的最佳临界值为1.68 mg/L,曲线下面积(AUC)为0.70,95%CI:(0.58,0.81),灵敏度与特异度分别为78.7%和58.4%。Kaplan-Meier曲线表明GRP78高表达(≤1.68 mg/L,n=50)组生存期明显短于低表达组(>1.68 mg/L,n=28),差异有统计学意义(χ^(2)=10.99,P=0.001)。单、多因素Cox分析揭示ISS分期[HR=2.04,95%CI:(1.11,3.76),P=0.022]、治疗反应[HR=2.42,95%CI:(1.28,4.58),P=0.007]和血清GRP78表达[HR=2.96,95%CI:(1.43,6.12),P=0.003]是MM病人的独立预后因素。结论GRP78在MM病人血清中呈现异常高表达,且与ISS分期及不良预后有关,或许是MM诊断与预后风险分层的重要标志物。

过表达葡萄糖调节蛋白78人乳腺癌细胞系HS578T、MDA-MB-231的构建

目的构建过表达内质网应激标志蛋白葡萄糖调节蛋白78(Glucose-regulated protein 78,GRP78)的人乳腺癌细胞系HS578T、MDA-MB-231,为探讨GRP78在乳腺癌发生发展中的作用机制提供基础。方法采用分子克隆技术构建表达GRP78的慢病毒载体pCDH-CMV-GRP78-HA-EF1-Puro质粒,采用PCR法扩增质粒,后使用EcoRⅠ、NotⅠ限制性内切酶切割质粒,对酶切产物进行测序鉴定,成功构建pCDH-CMV-GRP78-HA-EF1-Puro质粒。取对数生长期293FT细胞系(克隆分离株),在培养皿中加入pCDH-CMV-GRP78-HA-EF1-Puro混合液,培养48 h收集含GRP78慢病毒颗粒的上清液,保存备用。另取对数生长期人乳腺癌细胞系HS578T及MDA-MB-231,置于含GRP78慢病毒颗粒上清液的培养基中培养,培养48 h时在培养基中加入1∶500稀释嘌呤霉素,筛选GRP78过表达的HS578T、MDA-MB-231细胞系。加入嘌呤霉素培养48 h采用Western Blotting法检测HS578T、MDA-MB-231细胞GRP78。采用激光共聚焦显微镜对HS578T、MDA-MB-231细胞中GRP78蛋白进行定位。结果构建表达GRP78的慢病毒载体pCDH-CMV-GRP78-HA-EF1-Puro质粒。GRP78过表达的HS578T、MDA-MB-231细胞系在78 kD左右可见明显GPR78蛋白电泳条带。HS578T细胞系中存在GRP78蛋白表达,主要分布在细胞质中。结论成功构建GRP78过表达的MDA-MB-231、HS578T细胞系,GRP78主要表达于细胞质中。

GRP78-c-Src信号通路介导周期性牵张力作用下牙周膜成纤维细胞成骨分化的机制研究

目的探讨在周期性牵张力作用下葡萄糖调节蛋白78(GRP78)对牙周膜成纤维细胞成骨分化的影响,并阐述其机制。方法应用FlexCell 5000细胞应力装置模拟牙齿正畸受力环境;应用流式细胞术细胞分选技术获得牙周膜成纤维细胞GRP78高表达细胞和GRP78低表达细胞;采用基因转染技术敲减GRP78、c-Src的表达以及过表达c-Src;蛋白质印迹实验检测成骨转录因子Runt相关基因2(RUNX2)、锌指结构转录因子(Osterix)以及成骨标志蛋白骨钙蛋白(OCN)、骨桥蛋白(OPN)的表达;免疫共沉淀实验检测GRP78与c-Src激酶的相互作用;茜素红染色实验检测细胞矿化结节的形成。结果GRP78在牙周膜成纤维细胞呈异质性表达,流式分选实验获得GRP78高表达和GRP78低表达细胞。周期性牵张力处理后,与GRP78低表达细胞相比,GRP78高表达细胞成骨分化能力及c-Src激酶磷酸化水平均升高,差异具有统计学意义(P<0.05);GRP78与c-Src激酶存在相互作用;与对照组相比,过表达c-Src组细胞成骨分化能力升高(P<0.05),sic-Src组细胞成骨分化能力降低(P<0.05)。结论GRP78通过与c-Src激酶相互作用并上调其表达,进而促进周期性牵张力诱导的牙周膜成纤维细胞成骨分化。

溃疡性结肠炎患儿血清sB7-H3、GRP78水平及与病情、预后的关系

目的探究溃疡性结肠炎(UC)患儿血清可溶性B7同源体3(sB7-H3)、葡萄糖调节蛋白78(GRP78)水平及与病情、预后的关系。方法选取2019年8月至2020年8月在本院诊治的UC患儿78例作为研究组,选取同时期在本院进行健康体检的儿童75例作为对照组。根据研究组病情严重情况分为轻度组29例,中度组32例,重度组17例。对研究组进行3年随访,根据预后情况分为预后不良组22例,预后良好组56例。采用酶联免疫吸附法测定血清sB7-H3、GRP78及炎症因子干扰素-r(IFN-r)、白细胞介素-1β(IL-1β)、白细胞介素-33(IL-33)的水平;根据血压测量仪检测舒张压和收缩压水平。Pearson法分析血清sB7-H3和GRP78分别与炎症因子的相关性;Logistic回归分析影响UC患儿预后发生不良的因素;受试者工作特征(ROC)曲线分析血清sB7-H3和GRP78水平预测UC患儿预后发生不良的临床价值。结果研究组血清sB7-H3、GRP78、IFN-r、IL-1β、IL-33水平显著高于对照组(P<0.05)。随着UC患儿病情越严重,血清sB7-H3、GRP78、IFN-r、IL-1β、IL-33水平越高(P<0.05)。血清sB7-H3和GRP78分别与IFN-r、IL-1β、IL-33呈正相关(P<0.05)。不良组血清sB7-H3、GRP78、IFN-r、IL-1β、IL-33水平显著高于良好组(P<0.05)。血清sB7-H3、GRP78、IL-33是影响UC患儿预后发生不良的危险因素(P<0.05)。血清sB7-H3、GRP78及二者联合检测UC患儿预后发生不良的曲线下面积(AUC)为0.874、0.884、0.969,二者联合检测明显高于单独检测(Z二者联合-sB7-H3=1.878,Z二者联合-GRP78=1.931,P<0.05)。结论UC患儿血清sB7-H3、GRP78水平升高,其随病情越严重水平越高,二者联合可较好预测UC患儿预后发生不良的情况。

胸部CT影像特征联合血清GRP78和CST1对良恶性肺结节的诊断价值

目的 探讨葡萄糖调节蛋白78(GRP78)、半胱氨酸蛋白酶抑制剂1(CST1)在良恶性肺结节患者血清中的表达,以及联合胸部CT影像特征在诊断良恶性肺结节中的价值。方法 收集98例2020年1月至2021年12月在本院治疗的肺结节患者作为研究对象,根据病理学结果,将患者分为良性组(n=45)和恶性组(n=53)。比较两组血清GRP78、CST1表达水平和胸部CT影像特征;ROC曲线分析血清GRP78、CST1对恶性肺结节的诊断效能;四表格法分析胸部CT影像特征联合血清GRP78、CST1对恶性肺结节的诊断效能。结果 恶性组血清GRP78、CST1的表达水平高于良性组(P<0.05)。良性组和恶性组在分叶征、毛刺征、血管集束征、胸膜凹陷征之间存在显著差异(P<0.05)。胸部CT影像特征联合血清GRP78和CST1诊断恶性肺结节的准确率为92.86%,敏感度为96.23%,特异度为88.89%。其中三项联合的准确率高于胸部CT影像、血清CST1单独诊断(P<0.05),敏感度高于血清GRP78和CST1单独诊断(P<0.05)。结论 恶性肺结节患者血清GRP78、CST1表达水平显著高于良性患者;胸部CT影像特征联合血清GRP78和CST1在良恶性肺结节的诊断中具有较高的应用价值。

急性冠脉综合征患者血清GRP78、suPAR水平与心血管预后的关系及预测价值研究

目的探讨急性冠脉综合征(ACS)患者血清葡萄糖调节蛋白78(GRP78)、可溶性尿激酶型纤溶酶原激活物受体(suPAR)水平与心血管预后的相关性及预测价值研究。方法回顾性纳入2021年11月至2022年3月江汉大学附属武汉市第六医院收治的ACS患者179例作为ACS组,并选取冠脉造影检查正常的受检者80例作为对照组,均行血清GRP78、suPAR水平的测定并比较。按随访1年是否发生主要不良心血管事件(MACE)将患者分为预后不良组(发生MACE,n=31)与预后良好组(未发生MACE,n=148)。比较不同预后ACS患者的一般资料[性别、年龄、吸烟史、饮酒史、冠心病家族史、体重指数、既往病史、疾病诊断、收缩压、舒张压和左室射血分数(LVEF)等]和实验室指标[空腹血糖、总胆固醇、甘油三酯、低密度脂蛋白胆固醇(LDL-C)、血肌酐、尿素氮和脑利钠肽、GRP78、suPAR水平]差异,并采用多因素Logistic回归分析影响ACS患者预后的因素,采用受试者操作特征(ROC)曲线评价GRP78、suPAR对ACS患者预后的预测能力。结果ACS组血清GRP78、suPAR水平分别为(1.42±0.43)、(2.59±0.65)ng/mL,均高于对照组[(1.01±0.35)、(1.54±0.43)ng/mL],差异均有统计学意义(P<0.05)。预后不良组的LVEF为(55.47±7.58)%,低于预后良好组[(59.44±10.25)%],血清脑利钠肽、GRP78及suPAR水平分别为(156.25±29.27)pg/mL、(1.72±0.51)ng/mL、(3.08±0.64)ng/mL,均高于预后良好组[(142.54±26.37)pg/mL、(1.36±0.39)ng/mL、(2.49±0.57)ng/mL],差异均有统计学意义(P<0.05);两组其余各项一般资料和实验室指标比较,差异均无统计学意义(P>0.05)。Logistic回归分析得出,较高GRP78水平和较高suPAR水平是ACS患者预后不良的危险因素(OR=1.669,95%CI:1.186~2.347;OR=1.517,95%CI:1.133~2.032;P<0.05),LVEF是其保护因素(OR=0.674,95%CI:0.507~0.898;P<0.05)。ROC曲线分析得出,GRP78、suPAR预测ACS患者不良预后的曲线下面积(AUC)分别为0.708、0.747,二者联合的AUC达到0.802。结论ACS患者血清GRP78、suPAR水平与心血管预后密切相关,二者均对不良预后有一定预测价值,且联合应用的预测效能更高。

Transformation of Arabidopsis by Rice OsWRKY78::GFP Fusion Gene and Subcellular Localization of OsWRKY78 Protein

[Objective] The study was to understand the subcellular localization of OsWRKY78 protein in plants. [Method] Primers specific for OsWRKY78 gene were designed according to the OsWRKY78 full length sequence in Genbank. The gene was cloned by RT-PCR method. The gene was then recombined into a plasmid expression vector carrying green fluorescent protein (GFP) gene, pBinGFP. The recombinant was confirmed by PCR and enzyme digestion. The recombinant plasmid pBinGFP-OsWRKY was transformed into Arabidopsis through Agrobacterium tumefaciens strain GV3101 and transgenic plants were obtained. [Result] Measured by fluorescence microscopy, the expression of OsWRKY78 and GFP fusion protein in root tip cells was localized in the nucleus. [Conclusion] This study laid the foundation for further investigating the function of OsWRKY78 gene and its role in related signal transduction and provided theoretical basis for exploring the relation between OsWRKY78 gene and brown planthoppers.

急性心肌梗死患者血清GRP78,HSP47,miR-126表达与心功能的相关性

目的 探讨急性心肌梗死患者血清葡萄糖调节蛋白78(GRP78)、热休克蛋白47(HSP47)、微小RNA-126(miR-126)表达与心功能的相关性。方法 选取医院2021年6月至2022年6月收治的急性心肌梗死患者80例为观察组,另选取同期健康体检者50例为对照组。采用酶联免疫吸附试验检测血清GRP78和HSP47水平及心肌肌钙蛋白I(cTnI)、肌酸激酶MB型同工酶(CK-MB)水平,采用实时荧光定量聚合酶链式反应(qRT-PCR)检测miR-126表达水平,采用超声心动图测定左心室射血分数(LVEF)。采用Pearson相关系数法进行相关性分析,根据全球急性冠状动脉事件注册研究(GRACE)评分将观察组患者分为高危组(12例)、中危组(31例)、低危组(37例),建立受试者操作特征(ROC)曲线,评价GRP78,HSP47,miR-126鉴别高危险分层的诊断价值。结果 观察组患者血清cTnI和CK-MB水平均显著高于对照组(P <0.05),LVEF显著低于对照组(P <0.05)。观察组患者血清GRP78和HSP47水平均显著高于对照组(P <0.05),且低危组、中危组、高危组呈持续上升趋势(P <0.05);miR-126相对表达显著低于对照组(P <0.05),且低危组、中危组、高危组呈持续下降趋势(P <0.05)。GRP78,HSP47与cTnI,CK-MB均呈正相关(P <0.05),与LVEF均呈负相关(P <0.05);miR-126与cTnI,CK-MB均呈负相关(P <0.05),与LVEF呈正相关(P <0.05)。GRP78诊断急性心肌梗死灵敏度为50.00%,特异度为98.50%;HSP47灵敏度为91.70%,特异度为64.70%;miR-126灵敏度为75.00%,特异度为89.70%;GRP78,HSP47,miR-126 3项联合预测的曲线下面积为0.928(P <0.05)。结论 急性心肌梗死患者血清GRP78和HSP47存在异常高表达,miR-126存在异常低表达,且3项指标与心功能有一定相关性,单独或联合分析时对临床鉴别患者的风险层级均有参考价值。