辛伐他汀诱导人红白血病K562细胞凋亡的实验研究

目的:探讨辛伐他汀诱导K562细胞凋亡及其机制。方法:取浓度为0(阴性对照组)、10、20、30μmol·L-1的辛伐他汀作用K562细胞72h后,双染色法检测细胞凋亡率;DNALadder实验检测细胞凋亡条带;用线粒体分离试剂分离出胞浆蛋白、微粒体蛋白、线粒体蛋白;逆转录-聚合酶链反应法检测GRP78、caspase-9、caspase-3、GADD153mRNA表达;Western blot法检测GRP78、caspase-12、caspase-9、caspase-3、GADD153、细胞色素C蛋白水平。结果:与阴性对照组比较,10、20、30μmol·L-1辛伐他汀作用K562细胞72h后出现典型的凋亡条带,凋亡率分别为12.41%、19.08%、23.41%(P<0.01),GRP78、caspase-9、caspase-3和GADD153 mRNA表达上调(P<0.05),caspase-12、caspase-9、caspase-3蛋白水平下降,caspase-12定位于内质网,细胞色素C从线粒体释放,GRP78、GADD153蛋白表达上调。结论:辛伐他汀可通过内质网和线粒体途径诱导K562细胞凋亡。     

辣椒素经内质网应激反应途径诱导K562/ADM耐药细胞凋亡

目的：研究辣椒素（capsaicin）是否经内质网途径诱导耐药性白血病K562/ADM细胞凋亡。方法：以耐药性白血病K562/ADM细胞为靶细胞,采用MTT比色法测定细胞增殖活性;细胞形态学和AnnexinV/PI双染色法检测细胞凋亡,电镜观察凋亡细胞内质网形态结构变化;实时定量RT-PCR检测GRP78mRNA的表达;Western blot法检测GRP78蛋白的表达。结果：不同浓度的辣椒素显著抑制K562/ADM细胞的增殖活性,20、50μmol/L辣椒素诱导后K562/ADM细胞出现典型的凋亡形态学改变,细胞凋亡率明显增高,分别为39.67%和41.78%。辣椒素诱导凋亡过程中,K562/ADM细胞出现内质网明显扩张和脱颗粒现象,GRP78mRNA的表达增高,GRP78蛋白的表达量分别增高1.5和2.2倍,随时间延长有所降低。结论：辣椒素可能通过内质网应激反应性途径诱导K562/ADM细胞发生凋亡。     

辛伐他汀诱导K562细胞凋亡时caspase-3，8，9蛋白活性与mRNA的动态变化

目的:研究辛伐他汀诱导K562细胞凋亡过程中caspase-3,8,9活性与mRNA的动态变化,探讨其凋亡通路。方法:20μmol.L-1辛伐他汀处理K562细胞,48 h观察细胞形态学;24,48和72 h流式细胞技术检测细胞凋亡率;比色法测定caspase-3,8和9活性;半定量RT-PCR检测caspase-3,8,9的mRNA变化。结果:20μmol.L-1辛伐他汀作用K562细胞48 h后细胞出现核固缩、核碎裂和凋亡小体等形态学改变;24,48和72 h辛伐他汀处理组细胞凋亡率较对照组的增加值分别为(6.10±0.35)%,(14.15±0.42)%和(30.70±0.65)%,差异均具有显著性(P<0.01);不同时间辛伐他汀处理组的caspase-3,8,9活性与mRNA表达量均比对照组明显升高(P<0.01)。结论:caspase-3,8,9蛋白活性与mRNA上调是辛伐他汀诱导K562细胞凋亡的重要原因和机制。     

二氢青蒿素下调粒系白血病细胞转铁蛋白受体表达

通过建立常铁HL60和K562细胞以及富铁K562细胞体外模型，研究二氢青蒿素对粒系白血病细胞转铁蛋白受体(transferrin receptor，TfR)的调控作用。采用流式细胞术检测二氢青蒿素对粒系白血病细胞TfR密度的调控作用，Western blotting和RT-PCR法检测二氢青蒿素对粒系白血病细胞TfR表达的调控作用，原子吸收分光光度法检测二氢青蒿素对常铁和富铁K562细胞铁含量的影响，以及MTT法和台盼蓝拒染法分析二氢青蒿素对粒系白血病细胞增殖的作用。结果显示，二氢青蒿素能显著降低常铁HL60和K562细胞TfR的密度和下调TfR蛋白的表达，且呈浓度和时间依赖性，并能有效地抑制细胞增殖，IC₅₀值分别为1.74和11.33 μmol·L^-1。二氢青蒿素对富铁K562细胞的TfR蛋白和mRNA表达能进一步增强下调作用，与常铁培养组比较，10 μmol·L^-1二氢青蒿素对富铁K562细胞TfR蛋白和TfR mRNA表达量分别下调了28.1%(P<0.01)和26.2%(P<0.05)，并能显著下降富铁K562细胞铁的含量(P<0.05)，更有效地抑制富铁K562细胞增殖。由此可见，二氢青蒿素能下调粒系白血病细胞TfR密度以及TfR蛋白和mRNA的表达，有效抑制常铁HL60和K562细胞的增殖，对富铁K562细胞增殖的抑制作用能进一步增强。     

毒胡萝卜素诱导大鼠皮层神经元内质网应激及丹红注射液的干预作用

目的：探讨毒胡萝卜紊诱导大鼠皮层神经元内质网应激凋亡的机制及丹红注射液的干预作用。方法：体外培养SD乳鼠皮层神经元，免疫组织化学、免疫荧光染色鉴定神经元纯度。流式细胞术Annexin V、PI双标检测凋亡率及活性caspase-3、caspase-8、caspase-7、caspase-9表达，Western Noting免疫印迹分析caspase-12、GRP78、Bcl-2、细胞色素C蛋白表达，Fura-2／AM法荧光分光光度计检测细胞内钙浓度（[Ca^2＋]i）。结果：SD乳鼠皮层神经元可纯化体外培养。2μmol／L毒胡萝卜素作用神经元24、48h细胞凋亡率分别是17.88％、21.38％，丹红治疗组分别是6．30％、6．11％，两组比较差异有统计学意义（P〈0．05）。毒胡萝卜素诱导神经元GRP78表达上调，剪切活化caspase-3、caspase-8、caspase-9、caspase-12，使细胞色素C表达增加，Bcl-2表达减少。丹红注射液促进细胞Bcl-2表达，抑制细胞色素C释放，减少活化的caspase-3、caspase-8、caspase-9含量，稳定游离钙浓度。结论：毒胡萝卜素诱导神经元内质网应激反应性凋亡。丹红注射液能抑制体外培养神经元内质网应激所致凋亡。     

青蒿琥酯对人白血病K562，K562 /ADM细胞凋亡的作用及对NF-κB p65表达的影响

摘 要 目的:观察青蒿琥酯对人白血病K562、K562 /ADM细胞凋亡以及对p65表达的影响。方法: 采用流式细胞仪检测青蒿琥酯在不同浓度和不同时间段对K562、K562 /ADM细胞凋亡的影响,采用Western blot法检测15 μmol·L^-1青蒿琥酯在不同时间对K562细胞NF-κB p65表达的影响。结果 青蒿琥酯对K562细胞凋亡影响不明显,但对阿霉素耐药K562 /ADM细胞影响较大,青蒿琥浓度为7.5 μmol·L^-1和15 μmol·L^-1时,K562 /ADM细胞的调亡率明显高于K562细胞（P<0.01）,15 μmol·L^-1青蒿琥酯作用后4 h后,K562 /ADM细胞的调亡率明显增加（P<0.01）;经15 μmol·L^-1青蒿琥酯作用后, p65表达随时间的增加而明显降低（P<0.01）。结论 青蒿琥酯通过下调P65的表达而诱导白血病细胞凋亡。     

ASPP2调控GRP78在L-NAME诱导胎盘滋养细胞凋亡中的作用

目的探讨p53凋亡刺激蛋白2(apoptosis stimulating protein 2 of p53,ASPP2)调控葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)在N-硝基-L-精氨酸甲酯(L-nitroarginine methyl ester,L-NAME)诱导胎盘滋养细胞凋亡中的作用,为临床研究妊娠期高血压疾病提供理论依据。方法体外培养HTR-8/SVneo人胎盘滋养细胞,分为对照组(0μmol·L~(-1)L-NAME)和L-NAME组(100μmol·L~(-1)LNAME),干预48 h后,分别采用Annexin V-FITC/PI双标记流式细胞术和吖啶橙/溴化乙锭染色,检测胎盘滋养细胞凋亡水平的变化;运用Western blot检测caspase-12、GRP78和ASPP2的蛋白变化; ASPP2干扰腺病毒感染人胎盘滋养细胞后,采用实时荧光定量PCR(qRT-PCR)检测ASPP2的mRNA表达水平,Western blot检测GRP78的蛋白表达水平;给予100μmol·L~(-1) L-NAME干预48 h后,Western blot和免疫荧光分别检测caspase-12和GRP78的蛋白表达。结果与对照组相比,L-NAME组胎盘滋养细胞凋亡水平明显增加(P <0. 05);吖啶橙染色显示,与对照组相比,L-NAME组细胞多数呈现鲜亮的橙色,晚期凋亡细胞数明显增加;同时caspase-12、GRP78和ASPP2蛋白的表达明显增加(P <0. 05,P <0. 01);干扰ASPP2后,caspase-12和GRP78蛋白表达明显降低(P <0. 05)。结论下调ASPP2可降低GRP78的表达,进而抑制L-NAME诱导的胎盘滋养细胞凋亡。     

λ-卡拉胶寡糖对人脐静脉内皮细胞的增殖抑制作用及凋亡相关基因表达

为了探讨λ-卡拉胶寡糖(λ-carrageenan oligosaccharides， λ-CO)在体外抑制人脐静脉内皮细胞(HUVEC)增殖作用及其分子机制， 采用MTT法检测λ-CO对HUVEC存活率的影响， 通过流式细胞技术检测λ-CO对HUVEC增殖周期的影响， 测定细胞凋亡率， 检测用药前后活性半胱氨酸蛋白酶-3(caspase-3)的变化。同时采用半定量RT-PCR方法检测凋亡相关基因mRNA的表达情况。MTT检测结果显示，λ-CO在高浓度(1 mg·mL^-1)能明显抑制细胞增殖。流式细胞仪检测到λ-CO作用HUVEC后， 出现早期凋亡细胞， 且凋亡呈浓度和时间依赖性； 对细胞增殖周期的检测结果显示， 与对照组相比，λ-CO作用后G₁期细胞比例减少， S期比例增加， 并出现凋亡峰； 活化的caspase-3比例亦随用药浓度的加大而增加。RT-PCR检测发现0.3 mg·mL^-1λ-CO作用人脐静脉内皮细胞12、 24和36 h后，细胞TNFα、 p53、 caspase-8、 caspase-3的mRNA表达上调。表明λ-CO能剂量依赖性地诱导HUVEC细胞凋亡， 并引起HUVEC的S期阻滞， 其机制可能与促进TNFα、 p53、 caspase-8、 caspase-3等基因的转录， 使细胞内活化的caspase-3水平增加有关。     

冬凌草甲素对K562细胞端粒酶活性调控及细胞周期的影响

目的研究冬凌草甲素(ORI)对K562细胞端粒酶活性及其细胞周期的影响。方法免疫细胞化学法测定K562细胞中hTERT和C-myc蛋白的表达；TRAP-PCR-ELISA法检测了端粒酶活性变化；流式细胞仪测定细胞周期各时相百分比。结果用3.43 μmol·L^-1 ORI作用于Ｋ562细胞48 h后，hTERT和C-myc蛋白的表达降低；在一定的浓度范围内，ORI可下调K562细胞端粒酶活性。同时细胞周期各时相分布发生变化，G₀/G₁期或G₂/M期细胞增多，S期细胞减少。结论ORI可下调K562细胞的端粒酶活性，其机制可能与其细胞周期阻滞作用及抑制hTERT和C-myc蛋白的表达有关。     

α-维尼非林经线粒体凋亡途径诱导K562细胞凋亡

目的旨在研究α-维尼非林(α-viniferin)对人慢性髓系白血病细胞K562的作用与相关机制。方法 MTT法评价α-viniferin对K562细胞的细胞毒活性。采用细胞形态学和生物化学方法检测细胞凋亡。通过化学荧光法对细胞内线粒体膜电位、caspase-9、caspase-3活性分析。通过半定量RT-PCR表达分析来确定Bcl-2家族相关基因在α-viniferin诱导K562细胞凋亡中的作用。结果α-viniferin能抑制K562细胞增殖,呈剂量和时间依赖性,IC50为13.61 mg·L-1。α-viniferin引起K562细胞出现死亡并伴随有染色质聚集、核破碎、凋亡小体等典型的凋亡形态学特征;此外还伴随有线粒体膜电位显著降低,caspase-9、caspase-3活性升高等现象;α-viniferin(2~32 mg·L-1)引起K562细胞caspase-3 mRNA表达持续升高,Bax、Bad、Bim、Bid促凋亡基因mRNA表达增加,而Bcl-2、Bcl-xL抗凋亡基因mRNA表达持续下降。结论α-viniferin通过线粒体途径诱导K562细胞凋亡。     

Calcium plays a key role in paraoxon-induced apoptosis in EL4 cells by regulating both endoplasmic reticulum- and mitochondria-associated pathways

Context and objective: Paraoxon (POX) is one of the most toxic organophosphorus pesticides, but its toxic mechanisms associated with apoptosis remain unclear. The aim of this study was to investigate calcium-associated mechanisms in POX-induced apoptosis in EL4 cells.

Materials and methods: EL4 cells were exposed to POX for 0-16?h. EGTA was used to chelate Ca^2+? in extracellular medium, and heparin and procaine were used to inhibit Ca^2+?efflux from the endoplasmic reticulum (ER). Z-ATAD-FMK was used to inhibit caspase-12 activity. The apoptotic rate assay, western blotting and immunocytochemistry (ICC) were used to reveal the mechanisms of POX-induced apoptosis.

Results and discussion: POX significantly increased the expression and activation of caspase-12 and caspase-3, enhanced expression of calpain 1 and calpain 2, and induced the release of cyt c, but did not change the expression of Grp 78. Inhibiting caspase-12 activity alleviated POX-induced upregulation of calpain 1 and caspase-3, promoted POX-induced upregulation of calpain 2, and reduced POX-induced cyt c release, suggesting that there was a cross-talk between the ER-associated pathway and mitochondria-associated apoptotic signals. Attenuating intracellular calcium concentration with EGTA, heparin or procaine decreased POX-induced upregulation of calpain 1, calpain 2, caspase-12 and caspase-3, and reduced POX-induced cyt c release. After pretreatment with EGTA or procaine, POX significantly promoted expression of Grp 78.

Conclusions: Calcium played a key role in POX-induced apoptosis in EL4 cells by regulating both ER- and mitochondria-associated pathways. The cross-talk of ER- and mitochondria-associated pathways was accomplished through calcium signal.     

EGCG对三阴乳腺癌细胞MDA-MB-231的增殖抑制作用及机制

目的探讨表没食子儿茶素没食子酸酯[(-)-epigallo-catechin-3-gallate,EGCG]对三阴乳腺癌细胞MDA-MB-231增殖和凋亡的影响及其可能作用机制。方法通过CCK-8实验观察不同浓度EGCG(10、20、40、80、160 mg.L-1)对MDA-MB-231细胞增殖的影响;Hoechst33258染色法观察EGCG对MDA-MB-231细胞凋亡的影响;JC-1法测定细胞线粒体膜电位;caspase-3活性检测试剂盒检测caspase-3活性的变化;Western blot法检测葡萄糖调节蛋白78(glucose reg-ulatd protein 78,GRP78)和caspase-3蛋白表达变化。结果EGCG能明显抑制MDA-MB-231细胞增殖,且随EGCG浓度的增加和作用时间的延长而增强,EGCG作用MDA-MB-231细胞12、24、48 h的IC50分别为69.1、40.4、29.4 mg.L-1。EGCG作用MDA-MB-231细胞24 h后,细胞出现体积变小、染色质聚集、细胞核边缘化等典型的凋亡形态学改变,且随EGCG浓度的增加,MDA-MB-231细胞凋亡率逐渐增加,线粒体膜电位明显降低,caspase-3活性明显增强,West-ern blot结果显示EGCG可抑制GRP78蛋白表达,增强活性caspase-3蛋白表达。结论 EGCG能够促进MDA-MB-231凋亡,其机制可能与内质网应激(Endoplasmic reticulumstress,ERS)引起的caspase-3激活有关。     

Capsaicin-induced apoptosis is regulated by endoplasmic reticulum stress- and calpain-mediated mitochondrial cell death pathways

Capsaicin, a pungent compound found in hot chili peppers, induces apoptotic cell death in various cell lines, however, the precise apoptosis signaling pathway is unknown. Here, we investigated capsaicin-induced apoptotic signaling in the human breast cell line MCF10A and found that it involves both endoplasmic reticulum (ER) stress and calpain activation. Capsaicin inhibited growth in a dose-dependent manner and induced apoptotic nuclear changes in MCF10A cells. Capsaicin also induced degradation of tumor suppressor p53; this effect was enhanced by the ER stressor tunicamycin. The proteasome inhibitor MG132 completely blocked capsaicin-induced p53 degradation and enhanced apoptotic cell death. Capsaicin treatment triggered ER stress by increasing levels of IRE1, GADD153/Chop, GRP78/Bip, and activated caspase-4. It led to an increase in cytosolic Ca²⁺, calpain activation, loss of the mitochondrial transmembrane potential, release of mitochondrial cytochrome c, and caspase-9 and -7 activation. Furthermore, capsaicin-induced the mitochondrial apoptotic pathway through calpain-mediated Bid translocation to the mitochondria and nuclear translocation of apoptosis-inducing factor (AIF). Capsaicin-induced caspase-9, Bid cleavage, and AIF translocation were blocked by calpeptin, and BAPTA and calpeptin attenuated calpain activation and Bid cleavage. Thus, both ER stress- and mitochondria-mediated death pathways are involved in capsaicin-induced apoptosis.     

Endoplasmic reticulum may not be involved in the lead‐induced apoptosis in PC 12 cells in vitro

Recent researches indicated that mitochondrial pathway might play an important role in lead‐induced apoptosis. Our previous study also found that lead could induce apoptosis in PC 12 cells, and mitochondrial pathway events were involved in this process. As lead can disturb Ca²⁺ homeostasis, the present study was undertaken to determine whether lead can activate key cellular events in the endoplasmic reticulum (ER) pathway, including the expressions of C/EBP homology protein (CHOP) and glucose‐regulated protein 78 (GRP78), and the activation of caspase‐12 and calpain. The results showed that lead could increase the expression of GRP78, while the expressions of CHOP and procaspase‐12 remained unchanged. Moreover, the caspase‐12 and calpain were not activated, and the ultrastructure of endoplasmic reticulum was not altered. Therefore, it suggests that lead may induce apoptosis in PC 12 cells through mitochondrial pathway, but not through the endoplasmic reticulum pathway. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2010.     

新藤黄酸通过内质网应激诱导人鼻咽癌 CNE-2Z细胞凋亡的研究

目的 探讨新藤黄酸(gambogenic acid,GNA)对人鼻咽癌CNE-2Z细胞的影响和相关分子机制.方法 采用倒置显微镜观察药物处理组和对照组的CNE-2Z细胞形态的变化;MTT法检测药物处理组的CNE-2Z细胞增殖的抑制作用;DAPI染色和AV/PI 双染实验观察细胞凋亡情况;蛋白质印迹法检测GRP78、CHOP和ATF4蛋白的表达.结果 倒置显微镜和荧光显微镜观察,发现与对照组相比,GNA作用CNE-2Z细胞后,体积缩小变圆,细胞核发生典型核染色质固缩等细胞凋亡形态学的变化.GNA可以抑制人鼻咽CNE-2Z细胞的增殖,并有时间和浓度依赖性.AV/PI染色后早凋细胞膜呈苹果绿色,晚凋或坏死细胞质呈不同程度的黄-红色.蛋白质印迹法检测表明GRP78蛋白表达总体呈现浓度依赖性下调,上调了CHOP蛋白和ATF4蛋白.结论 GNA可以影响人鼻咽癌CNE-2Z细胞的增殖,引发细胞凋亡,这可能与内质网应激相关蛋白(GRP78、CHOP和ATF4蛋白)有关.     

辛伐他汀通过线粒体途径诱导K562细胞凋亡

目的观察辛伐他汀诱导K562细胞凋亡不同时间的膜电位(Δψm),caspase-3、9和细胞色素C的改变,以推测其凋亡通路。方法采用浓度为20μmol.L-1的辛伐他汀处理K562细胞24、48、72 h,采用流式细胞技术检测细胞凋亡率和线粒体膜电位,分光光度法检测caspase-3、9蛋白活性,免疫组织化学法检测细胞色素C蛋白。结果浓度为20μmol.L-1辛伐他汀作用K562细胞24、48、72 h后,凋亡率分别为(6.1±0.35)%、(14.15±0.42)%(、30.70±0.65)%,随着凋亡率增加线粒体膜电位降低分别为(39.6±4.80)%,(24.4±2.45)%,(6.0±1.62)%;caspase-3、9蛋白活性与对照组相比上调,细胞浆内细胞色素C升高。结论辛伐他汀诱导K562细胞凋亡时线粒体膜电位下降,caspase-3、9活性增高和细胞色素C释放,推测辛伐他汀诱导K562细胞的凋亡可能经过线粒体凋亡途径。     

Indomethacin induces apoptosis in 786-O renal cell carcinoma cells by activating mitogen-activated protein kinases and AKT

Studies on chemoprevention of cancer are generating increasing interest. The anti-neoplastic effect of nonsteroidal anti-inflammatory drugs (NSAIDs) involves cyclooxygenase (COX)-dependent and COX-independent mechanisms. Evidence suggests that mitogen-activated protein kinases (MAPKs) may mediate apoptotic signaling induced by anti-neoplastic agents. While many reports have revealed the existence of MAPK activation in apoptosis induced by various stimuli, the signaling transduction pathways used by NSAIDs to trigger apoptosis in human renal cell carcinoma (RCC) remain largely unknown. Treatment of RCC 786-O cells with indomethacin resulted in growth regression and apoptosis. Caspase-dependent apoptosis was evidenced by the detection of enzymatic activities of caspase-3, caspase-6, and caspase-9 and suppression of toxicity using a caspase inhibitor. Indomethacin treatment was associated with increased expression of glucose-regulated protein 78 (GRP78) and C/EBP homologus protein (CHOP) and activation of ATF-6, characteristics of endoplasmic reticulum stress. In addition, the concomitant induction of peroxisome proliferator-activated receptor (PPAR), especially PPAR-beta, was apparent in treated cells. Western blotting revealed the activation of extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun N-terminal kinase (JNK) with indomethacin treatment. Selective inhibitors of ERK, p38 MAPK, and JNK suppressed the induction of GRP78, CHOP, and PPAR-beta, attenuated indomethacin-induced cytotoxicity and reduced increased caspase activity. LY294002, a phosphoinositide-3 kinase (PI3K)/AKT inhibitor, and Trolox, an antioxidant, suppressed indomethacin-induced cytotoxicity and caspase activation. Furthermore, Trolox attenuated indomethacin-induced increased phosphorylation in ERK, p38 MAPK, JNK, and AKT. In conclusion, our findings establish a mechanistic link between the oxidative stress, PI3K/AKT pathway, MAPK pathway and indomethacin-induced cellular alterations and apoptosis in 786-O cells.     

环氧合酶-2抑制剂对K562白血病细胞增殖及凋亡的影响

目的:体外观察非甾体药物选择性环氧合酶-2(COX-2)抑制剂NS-398对白血病细胞株K562细胞增殖及凋亡影响。方法:采用噻唑蓝法观察NS-398对K562细胞增殖的影响,流式细胞仪检测细胞周期的改变及凋亡百分率的变化,DNA梯状电泳检测凋亡的发生。Western印迹法检测K562细胞半胱氨酸天冬氨酸特异性蛋白酶Caspa-se-3和COX-2的表达。结果:NS-398呈剂量依赖性的方式抑制K562细胞增殖,并诱导其凋亡。并呈浓度依赖性改变细胞周期的分布,一方面增高Go/G1期细胞比例,另一方面降低S期和G2/M期细胞比例,与对照组相比差异具显著性(P<0.05)。NS-398干预的K562细胞Caspase-3的蛋白表达均显著升高,而COX-2的蛋白表达降低,并呈浓度依赖性。结论:体外NS-398能有效的发挥抗K562细胞白血病效应,对白血病细胞增殖有抑制作用并诱导其凋亡,这可能与抑制COX-2表达及上调Caspase有关。