Gp78 regulates PMP22 and causes ER stress and autophagy in EV71-VP1-overexpressing mouse Schwann cells

Background:During Enterovirus type 71(EV71)infection,the structural viral protein 1(VP1)activates endoplasmic reticulum(ER)stress associated with peripheral myelin protein 22(PMP22)accumulation and induces autophagy.However,the specific mechanism behind this process remains elusive.Methods:In this research,we used the VP1-overexpressing mouse Schwann cells(SCs)models co-transfected with a PMP22 silencing or Autocrine motility factor receptor(AMFR/gp78)overexpressing vector to explore the regulation of gp78 on PMP22 and its relationship with autophagy and apoptosis.Results:The activity of gp78 could be influenced by EV71-VP1,leading to a decrease in the ubiquitination and degradation of PMP22,resulting in PMP22 accumulation in ER.In VP1-overexpressing mouse SCs,all three ER stress sensors,including pancreatic endoplasmic reticulum kinase(PERK),activating transcription factor 6(ATF6)and inositol-requiring enzyme 1(IRE1)and the related downstream signals(C/EBP-homologous protein(CHOP)and Caspase 12)were activated,as well as the ER-resident chaperone Glucose-regulated protein 78(GRP78).In addition,VP1 upregulated the autophagy marker Microtubule-associated protein 1 light chain 3 beta(LC3B),while PMP22 silencing or gp78 overexpression reversed the phenomenon.Meanwhile,PMP22 silencing or gp78 overexpression increased proliferation of EV71-VP1-transfected mouse SCs.Conclusion:Gp78 could regulate PMP22 accumulation through ubiquitination degradation and cause ER stress and autophagy in EV71-VP1-overexpressing mouse SCs.Therefore,the gp78/PMP22/ER stress axis might emerge as a promising therapeutic target for myelin and neuronal damage induced by EV71 infection.

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.

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。

Constitutive Overexpression of Bcl-2, Survivin and ER Stress Chaperone GRP-78 Confers Intrinsic Radioresistance in Human Hepatocellular Carcinoma Cells: Insight into the Mechanistic Pathways Involved

We present evidence here that abundantly expressed b-catenin-triggered NF-kB-dependent upregulation of inducible nitric oxide synthase(iNOS) found in hepatoma Mahlavu cells (RT-resistant variant designated as RR-Mal), but not in Hep 3B cells (RT-sensitive variant designated as RS-3B) is a key element contribrting to the radioresisitance through the activation of two prominent radioprotective pathways. First, high iNOS expression found in RR-Mal, but not in RS-3B cells was found to perturb calcium homeostasis that triggered ER stress response leading to the overproduction of ER chaperone GRP-78 via robust generation of cleaved ATF-6a (50 kDa) subunits and their nuclear translocation. Meanwhile, both abundantly expressed NF-κB and COX-2 found in RR-Mal cells could also provoke an increased production of PGE2 resulting in robust production of Bcl-2. Interestingly, when RR-Mal cells were treated with PDTC (a NF-κB inhibitor) or celecoxib (a COX-2 inhibitor), a concentration-dependent downregulation of Bcl-2 could be demonstrated implying that Bcl-2 overexpression was indeed mediated through NF-κB/Cox-2/PGE2 pathway. Importantly, we also unveiled that siRNA-mediated silencing of survivin in RR-Mal cells could result in a concomitant downregulation of GRP-78 due to a severe inhibition of ATF-6a (50 kDa) expression. Taken together, our data demonstrate that constitutively overexpressed b-catenin/NF-κB/iNOS and NF-κB/COX-2/PGE2 pathways that overproducing GRP-78, survivin and Bcl-2 expressions are responsible for radioresistance acquisition in RR-Mal cells. Thus, both pathways could be served as potential targets for overcoming radioresistance.

双氢青蒿素诱导外周T细胞淋巴瘤Hut-78细胞凋亡及其可能的机制

目的：考察双氢青蒿素（dihydroartemisinin,DHA）诱导的外周T细胞淋巴瘤Hut-78细胞凋亡,并进一步研究其作用机制。方法：CCK-8法检测1～30μg/ml DHA对Hut-78细胞增殖的影响,运用Hochest 33258染色技术在共聚集显微镜下观察DHA对Hut-78细胞核形态的影响,流式细胞术检测DHA对Hut-78细胞凋亡的影响,10 mmol/L的活性氧（reactive oxygen species,ROS）清除剂N-乙酰半胱氨酸（NAC）预处理细胞1 h,观察ROS在DHA引起的细胞线粒体膜电位变化中的作用,Western blotting检测在Hut-78细胞凋亡过程中ROS对DHA引起的细胞色素C释放的影响。结果：DHA呈浓度依赖性抑制Hut-78细胞的增殖,并引起细胞核固缩,形成凋亡小体。5、10、20μg/ml DHA引起的细胞凋亡率分别为（25.1±2.8）%、（43.6±3.1）%、（68.9±2.6）%,与对照组相比差异有统计学意义（均P〈0.01）。20μg/ml DHA处理导致Hut-78细胞线粒体膜电位下降（59.4±2.6）%,而经ROS抑制剂NAC预处理之后,线粒体膜电位下降（38.4±2.1）%。DHA能够引起线粒体中细胞色素C的释放,而NAC的预处理能够明显地抑制这一过程,统计结果进一步证明这一点。结论：DHA能够抑制外周T细胞淋巴瘤Hut-78细胞的增殖并诱导细胞凋亡,其作用机制可能与DHA促进ROS依赖的细胞色素C释放有关。

人皮肤T细胞淋巴瘤系Hut-78细胞FHIT基因缺失的研究

目的 探讨抑癌基因FHIT在Hut 78细胞中的缺失情况。方法 用巢式RT PCR方法研究FHIT的缺失情况。结果 Hut 78细胞中FHIT基因mRNA比正常缩短 ,外显子 5丢失。结论 FHIT基因外显子 5的缺失可能是皮肤T细胞淋巴瘤FHIT基因失活的主要方式 。

抗肝癌单链双功能抗体在Hut-78细胞系中的表达及体外杀伤活性

目的 用携带分泌型抗肝癌单链双功能抗体基因(sFv TNF α)的重组逆转录病毒感染T细胞淋巴瘤Hut 78细胞 ,使其表达并分泌针对人肝癌细胞的sFv TNF α融合蛋白 ,观察转导的T细胞对体外培养肝癌细胞的杀伤作用 .方法 用感染性重组病毒产生细胞C2 2 (PA317/PST)产生的病毒上清转导人T细胞淋巴瘤Hut 78细胞 ,采用PCR、RT PCR、细胞原位杂交及免疫组织化学染色等方法 ,对转导的Hut 78细胞进行DNA、mRNA及蛋白水平的分析 .转导的Hut 78细胞分别与SMMC 772 1、HHCC共培养 ,MTT法检测Hut/PST细胞表达产物对两种肝癌细胞的杀伤作用 .结果 PCR、RT PCR结果显示转导Hut细胞中扩增出外源目的基因对应的电泳条带 .neo基因探针原位杂交显示转导细胞质内有较强的紫蓝色阳性反应信号 ,其阳性率约为 95 % .鼠抗人TNF α多克隆抗体免疫组织化学染色 ,转导细胞质内有明确的棕黄色阳性反应信号 .MTT法检测结果 ,分泌型抗肝癌单链双功能抗体对体外培养的两种肝癌细胞的杀伤率分别为 15 .4 %和2 6 .5 % .结论 分泌型抗肝癌单链双功能抗体基因可以在T细胞中整合并稳定表达 ,其分泌的表达产物对两种肝癌细胞均具有一定的亲合活性 ,并且具有一定的体外杀伤作用 .

吉西他滨联合培美曲塞不同序贯用药时序对HUT-78细胞增殖及凋亡的影响

目的观察吉西他滨与培美曲塞单药及不同时序联合用药方案对人皮肤T细胞淋巴瘤HUT-78细胞增殖及凋亡的影响。方法采用体外培养技术,应用不同浓度的吉西他滨及培美曲塞处理HUT 78细胞,CCK-8法确定两药的IC50值,后应用CCK-8法及Annexin V-FITC法检测两药不同顺序联合应用对HUT-78细胞增殖及凋亡的影响。结果吉西他滨及培美曲塞单药对HUT-78细胞的增殖的抑制具有时间依赖性及浓度依赖性,吉西他滨的IC50值为(7.50±0.71)×10-3μg/ml,培美曲塞的IC50值为(1.72±0.52)μg/ml。不同顺序药物联合应用效果不同,先用培美曲塞后用吉西他滨及同时应用具有协同作用,抑制率及凋亡率均较各单药组高,而先用吉西他滨后用培美曲塞则无协同作用。结论吉西他滨与培美曲塞单药以及不同时序联合应用均可抑制HUT-78的增殖促进凋亡,作用大小与应用时序有关,先用培美曲塞再用吉西他滨的作用最强。

菝葜提取物对Hut-78细胞Bcl-2基因转录水平的影响

目的探讨菝葜提取物对Hut-78细胞Bcl-2基因转录水平的影响。方法利用半定量逆转录-聚合酶链反应(RT-PCR)测定给药后各组的吸光度值,计算各组的Bcl-2/GAPDH值,从而分析Bcl-2基因转录水平的变化。结果菝葜提取物对Bcl-2基因的转录水平具有下调作用,药物浓度在8μg·mL-1时的效果最为明显。结论该菝葜提取物可降低Hut-78细胞的Bcl-2基因的转录。

Cell-free derivatives from mesenchymal stem cells are effective in wound therapy

AIM:To compare the efficacy of cell-free derivatives from Bone marrow derived human mesenchymal stem cells(hMSCs) in wound therapy.METHODS:hMSCs have been shown to play an important role in wound therapy.The present study sought to compare efficacy of hMSCs and cell-free derivatives of hMSCs,which may be clinically more relevant as they are easier to prepare,formulate and transport.hMSCs were isolated from human bone marrow and cultured.Multi lineage differentiation of hMSCs was performed to confirm their identity.The ability of hMSCs to migrate was evaluated using in vitro and in vivo migration assays.Cell lysates and conditioned medium concentrate was prepared from hMSCs(see Methods for details).Wounds were induced in mice and wound areas were measure before and after cell and cell-free derivative treatment.RNA and proteins were extracted from the skin and cytokine levels were measured.RESULTS:Co-culture of hMSCs with keratinocytes resulted in increased expression of CXCL-12(SDF1) and ENA78(CXCL-5) in the conditioned media indicating that the hMSCs can respond to signals from keratinocytes.Accelerated wound closure was observed when hMSCs were injected near the site of excisional wounds in athymic as well as NOD/SCID mice.Interestingly,cell-free lysates prepared from hMSCs were also effective in inducing accelerated wound closure and increased expression of SDF1 and CXCL-5 at the wound bed.Additionally,concentrated media from hMSCs as well as an emulsion containing lysates prepared from hMSCs was also found to be more effective in rapid re-epithelialization than fibroblasts or vehicle-alone control.Use of cell-free derivatives may help replace expensive wound care approaches including use of growth factors,epidermal/dermal substitutes,synthetic membranes,cytokines,and matrix components,and most importantly avoid transmission of pathogens from human and animal products.CONCLUSION:These results encourage development of derivatives of hMSCs for wound care and re-epithelialization applications.

Valproate reduces retinal ganglion cell apoptosis in rats after optic nerve crush

The retinal ganglion cells of the optic nerve have a limited capacity for self-repair after injury.Valproate is a histone deacetylase inhibitor and multitarget drug,which has been demonstrated to protect retinal neurons.In this study,we established rat models of optic nerve-crush injury and injected valproate into the vitreous cavity immediately after modeling.We evaluated changes in the ultrastructure morphology of the endoplasmic reticulum of retinal ganglion cells over time via transmission electron microscope.Immunohistochemistry and western blot assay revealed that valproate upregulated the expression of the endoplasmic reticulum stress marker glucose-regulated protein 78 and downregulated the expression of transcription factor C/EBP homologous protein,phosphorylated eukaryotic translation initiation factor 2α,and caspase-12 in the endoplasmic reticulum of retinal ganglion cells.These findings suggest that valproate reduces apoptosis of retinal ganglion cells in the rat after optic nerve-crush injury by attenuating phosphorylated eukaryotic translation initiation factor 2α-C/EBP homologous protein signaling and caspase-12 activation during endoplasmic reticulum stress.These findings represent a newly discovered mechanism that regulates how valproate protects neurons.

Establishment of HIV-1 model cell line GHOST（3） with stable DRiP78 and NHERF1 knockdown

Chemokine receptors CXCR4 and CCR5 are indispensable co-receptors for HIV-1 entry into host cells. In our previous study, we identified that dopamine receptor-interacting protein 78（DRi P78） and Na＋-H＋ exchanger regulatory factor 1（NHERF1） are the CXCR4 and CCR5 homo- or hetero-dimerinteracting proteins. DRi P78 and NHERF1 are able to influence the co-receptor internalization and intracellular trafficking. Over-expression of NHERF1 affects the ligands or HIV-1 gp120-induced CCR5 internalization and HIV-1 production. It is reasonable to speculate that DRi P78 and NHERF1, as well as the signaling pathways involved in viral replication, would probably affect HIV-1 replication through regulating the co-receptors. In this present study, we designed two short hairpin RNAs（sh RNAs） targeting the DRi P78 and NHERF1, respectively, and constructed the p Lenti6/BLOCK-i T-DEST lentiviral plasmids expressing DRi P78 or NHERF1 sh RNA. The packaged lentiviruses were used to transduce the widely-applied HIV-1 model cell line GHOST（3）. Then, cells with stable knockdown were established through selecting transduced cells with Blasticidin. This study, for the first time, reported the establishment of the GHOST（3） with DRi P78 and NHERF1 knockdown, which is the first stable cell line with HIV-1 co-receptor-interacting molecular defects.

GRP78 expression beyond cellular stress:A biomarker for tumor manipulation

Physiological stress takes place in the endoplasmicreticulum （ER） of cells where activation and up-regulationof genes and proteins are primarily induced to enhancepro-survival mechanisms such as the unfolded protein response （UPR）. A dominant protein in the UPR response is the heat shock GRP78 protein. Although GRP78 is primarily located in the ER, under certain conditions it is transported to the cell surface, where it acts as a receptor inducing pathways of cell signaling such as proliferation or apoptosis. In the prolonged chronic stress transportation of the GRP78 from the ER to the cell membrane is a major event where in addition to the presentation of the GRP78 as a receptor to various ligands, it also marks the cells that will proceed to apoptotic pathways. In the normal cell that under stress acquires cell surface GRP78 and in the tumor cell that already presents cell surface GRP78, cell surface GRP78 is an apoptotic fag. The internalization of GRP78 from the cell surface in normal cells by ligands such as peptides will enhance cell survival and alleviate cardiovascular ischemic diseases. The absence of cell surface GRP78 in the tumor cells portends proliferative and metastatic tumors. Pharmacological induction of cell surface GRP78 will induce the process of apoptosis and might be used as a therapeutic modality for cancer treatment.

低浓度氟化钠对人牙髓细胞的成骨/成牙本质分化的影响

目的探讨低浓度氟化钠对人牙髓细胞(human dental pulp cells,hDPCs)成骨/成牙本质分化的影响。方法本研究已通过单位伦理委员会审查批准。原代培养hDPCs,采用MTT法检测不同浓度氟化钠对hDPCs增殖的影响;选取合适浓度的氟化钠加入成骨/成牙本质分化诱导培养液中,对hDPCs进行体外诱导,通过茜素红染色检测hDPCs成骨/成牙本质分化能力的变化,RT⁃qPCR检测分化相关基因的mRNA表达;同时通过RT⁃qPCR和Western blot检测hDPCs成骨/成牙本质分化过程中内质网应激相关基因的表达。结果低浓度氟化钠(0.1 mmol/L)在体外可刺激hDPCs增殖,高浓度氟化钠(5~10 mmol/L)可抑制hDPCs增殖(P<0.05)。选取0.1 mmol/L氟化钠体外混合成骨/成牙本质分化诱导培养后hDPCs的茜素红染色增加,成骨/成牙本质分化相关基因牙本质涎磷蛋白(dentin sialophosphoprotein,DSPP)、骨涎蛋白(bone sialoprotein,BSP)和骨钙蛋白(osteocalcin,OCN)mRNA表达水平升高(P<0.05)。同时在此过程中RT⁃qPCR检测出mRNA水平hDPCs内质网应激相关基因:剪切x盒结合蛋白1(splicing x⁃box binding protein⁃1,sXBP1)、葡萄糖调节蛋白78(glucose⁃regulated protein 78,GRP78)以及活化转录因子4(activating transcription factor 4,ATF4)表达升高(P<0.05);Western blot检测出氟化钠混合成骨/成牙本质分化培养后细胞磷酸化真核起始因子⁃2α(phosphorylated eukary⁃otic initiation factor⁃2α,p⁃eIF2α)、磷酸化蛋白激酶样内质网激酶(phosphorylated the RNA⁃activated protein kinase⁃like ER⁃resident kinase,p⁃PERK)和ATF4蛋白表达增加(P<0.05)。结论低剂量氟化钠促进人牙髓细胞的成骨/成牙本质分化并伴有内质网应激水平的升高。

PI3K/Akt调控内质网应激对GRP78的诱导

目的:研究内质网应激条件下PI3K/Akt信号通路对HEK293细胞中葡萄糖调节蛋白78(GRP78)表达水平的调控作用。方法:采用PI3K抑制剂LY294002、Akt1失活型突变载体Akt1(K179M)及Akt siRNAs阻断内质网应激介导的Akt活化,采用Akt激活型突变载体Myr-Akt过度激活内质网应激介导的Akt活化,并利用RT-PCR和Western blotting技术分析内质网应激条件下PI3K/Akt信号途径对HEK293细胞中GRP78表达水平的调控作用。结果:LY294002、Akt1(K179M)及Akt1 siRNA均明显抑制了内质网应激对GRP78的诱导。Myr-Akt1明显促进内质网应激对GRP78的诱导。Myr-Akt2/3及Akt2/3 siRNA对GRP78的诱导均无影响。PI3K/Akt信号通路阻断或过度激活对GRP78 mRNA水平的诱导无影响,但是对GRP78的降解有显著影响。结论:HEK293细胞中,PI3K/Akt通过蛋白稳定性调节促进内质网应激对GRP78的诱导。

钙离子调控KLK4表达对成釉细胞生长的影响

目的探讨钙离子对成釉细胞中激肽释放酶4(kallikrein-4,KLK4)表达及细胞生长的影响,为钙离子促进牙釉质正常矿化提供实验依据。方法采用不同浓度CaCl_(2)(0、2.0、2.5、3.0、3.5 mmol/L)处理成釉细胞株ALC(ameloblast-lineage cell)24 h、48 h,q RT-PCR和Western blot检测KLK4 mRNA和蛋白表达水平;CCK-8检测细胞相对活力;流式细胞术、Hoechst 33342染色检测钙离子对细胞周期和细胞凋亡的影响;Western blot检测葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)的蛋白表达水平。结果与对照组(0 mmol/L CaCl_(2))相比,2.5、3.0、3.5 mmol/L CaCl_(2)处理细胞24 h后,KLK4 mRNA表达上升(P<0.05),2.0、2.5、3.0、3.5 mmol/L CaCl_(2)处理细胞24 h后,KLK4蛋白表达上升(P<0.05);3.0、3.5 mmol/L CaCl_(2)处理细胞48 h后,KLK4 mRNA和蛋白表达上升(P<0.05)。与对照组相比,2.0、2.5、3.0 mmol/L CaCl_(2)处理ALC细胞24 h、48 h后,细胞活力增加(P<0.05),其中2.5 mmol/L CaCl_(2)组中细胞活力最高。Hoechst 33342染色结果显示,3.0、3.5 mmol/L CaCl_(2)促使ALC细胞发生凋亡。流式细胞仪检测结果显示,与0、2.0、2.5、3.0 mmol/L CaCl_(2)组相比,3.5 mmol/L CaCl_(2)处理ALC细胞24 h后,G2/M期细胞比例增加,细胞凋亡率上升(P<0.05)。3.0、3.5 mmol/L CaCl_(2)处理细胞24 h后,与对照组相比,GRP78蛋白表达下降(P<0.05);2.5 mmol/L CaCl_(2)处理细胞48 h后,与对照组相比,GRP78蛋白表达下降(P<0.05)。结论钙离子促进ALC细胞中KLK4表达上升、细胞活力增加,但较高浓度的钙离子可使ALC细胞的G2/M期阻滞,诱发ALC细胞凋亡,降低凋亡相关蛋白GRP78的表达。

非小细胞肺癌体外化疗药物敏感性与GRP78表达的相关性

背景和目的糖调节蛋白78(GRP78)在乳腺癌细胞中表达增高并与其对化疗药物的耐药性有关,而非小细胞肺癌(NSCLC)中GRP78的表达与其对化疗药物的耐药性是否相关,研究较少。本研究拟探讨NSCLC对8种化疗药物的体外敏感性与GRP78表达的相关性。方法采用四噻唑蓝(MTT)法对52例手术切除的新鲜NSCLC组织进行8种化疗药物体外敏感性检测;采用免疫组织化学方法检测其GRP78的表达水平。采用Spearman相关分析对NSCLC的耐药性与GRP78表达的相关性进行分析。结果52例NSCLC对紫杉醇(PTX)、阿霉素(ADM)、卡铂(CBP)、拓扑替康(TPT)、长春瑞滨(NVB)、长春新碱(VCR)、顺铂(DDP)和鬼臼乙叉苷(VP-16)8种化疗药物的耐药率分别为42.31%、57.69%、63.46%、65.38%、67.31%、73.08%、78.85%、90.38%,其中14例表现为完全耐药。GRP78的表达水平随着肺癌恶性程度的提高而增加,且其高表达与肺癌细胞对VP-16、ADM、VCR和TPT的耐药具有相关性(P<0.05)。结论MTT法体外化疗药物敏感性实验对NSCLC的治疗具有一定的指导作用,GRP78对判定NSCLC的恶性程度及其耐药性具有一定的参考意义。

葡萄糖调节蛋白78在子痫前期胎盘滋养细胞中的表达及意义

目的:探讨葡萄糖调节蛋白78(GRP78)在子痫前期孕妇胎盘组织中表达及其与子痫前期发病机制的关系。方法:选择2009年1～12月在青岛市市立医院及青岛市第八人民医院住院的子痫前期孕妇120例,其中早发型轻度子痫前期孕妇30例(早发轻度组),晚发型轻度子痫前期孕妇30例(晚发轻度组),早发型重度子痫前期孕妇30例(早发重度组),晚发型重度子痫前期孕妇30例(晚发重度组);另选同期健康孕妇30例为健康孕妇组。采用半定量逆转录-聚合酶链反应(RT-PCR)检测胎盘组织中GRP78 mRNA相对表达量;采用免疫组织化学方法、Western-Blotting检测胎盘组织GRP78蛋白的表达。结果:①子痫前期各组胎盘组织中GRP78 mRNA表达水平均高于健康孕妇组,差异有统计学意义(P<0.05);但子痫前期各组间分别比较,差异无统计学意义(P>0.05)。②子痫前期各组和健康孕妇组胎盘组织中都有GRP78蛋白表达,主要表达于细胞质,呈棕黄色染色。Western-Blotting检测结果显示,子痫前期各组胎盘组织中GRP78蛋白表达水平均高于健康孕妇组,差异有统计学意义(P<0.05);但子痫前期各组间分别比较,差异无统计学意义(P>0.05)。结论:GRP78在子痫前期胎盘组织中表达升高,GRP78的表达水平变化可能与子痫前期发病有关。

葡萄糖调节蛋白78和基质金属蛋白酶在口腔鳞状细胞癌中的表达

目的探讨葡萄糖调节蛋白78(GRP78)和基质金属蛋白酶(MMPs)在口腔鳞状细胞癌组织中的表达及意义,阐明GRP78在口腔鳞状细胞癌发生、发展中的作用。方法采用免疫组织化学方法检测26例口腔鳞状细胞癌组织和10例正常口腔组织中GRP78蛋白的表达;采用逆转录PCR检测正常口腔组织和口腔鳞状细胞癌组织中GRP78 m RNA的表达;采用免疫组织化学方法检测26例口腔鳞状细胞癌组织中MMP-2和MMP-9蛋白的表达。结果 GRP78蛋白在口腔鳞状细胞癌组织中的阳性表达率为100%(26/26),在正常口腔组织中的阳性表达率为20%(2/10),两者比较差异有统计学意义(P<0.01);与高分化和中分化口腔鳞状细胞癌组织比较,低分化口腔鳞状细胞癌组织中的GRP78蛋白阳性表达强度明显增高(P<0.05)。与正常口腔组织比较,GRP78 m RNA表达水平在口腔鳞状细胞癌组织中的表达增高(P<0.05)。MMP-2和MMP-9蛋白在口腔鳞状细胞癌组织中的阳性表达率均为100%(26/26)。结论 GRP78和MMPs在口腔鳞状细胞癌组织中表达明显升高,提示口腔鳞状细胞癌中GRP78蛋白的表达可能与肿瘤的侵袭和转移相关。

人食管鳞状细胞癌组织中GRP78的表达及其与肿瘤生物学行为的关系

目的:探讨葡萄糖调节蛋白78(glucose-regulated proteins78,GRP78)在食管鳞状细胞癌组织及正常鳞状上皮组织中的表达情况,并分析其与临床病理特征的关系。方法:取自2007年10月至2008年11月在山东大学附属济南中心医院胸外科手术切除的新鲜食管鳞状细胞癌标本59例及距癌组织5cm以上的手术远端切缘的正常食管鳞状上皮组织20例,RT-PCR检测GRP78mRNA的表达,应用Western blotting检测GRP78蛋白的表达,并从mRNA和蛋白水平分析GRP78的表达与患者性别、肿瘤长度、浸润深度、分化程度、病理分期及淋巴转移等临床病理特征之间的关系。结果:食管鳞状细胞癌组织中GRP78的表达在mRNA和蛋白水平均明显高于食管正常鳞状上皮组织(均P<0.01)。GRP78在食管鳞状细胞癌组织中的高表达与食管鳞状细胞癌的浸润深度(P<0.05或P<0.01)、分化程度(P<0.05或P<0.01)、病理分期(pTMN,P<0.01)及淋巴转移密切相关(P<0.01),而与患者性别及肿瘤长径无关(P>0.05)。结论:GRP78参与了人类食管鳞状细胞癌的发生、发展,表达水平随着食管鳞状细胞癌组织恶性程度的增高而增高,其可作为衡量食管鳞状细胞癌恶性程度的一种有潜在价值的分子标志物。