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[摘要]目的探讨70 kDa热休克蛋白8（HSPA8）在人腰椎间盘髓核组织中的表达及其与人椎间盘退变程度的关系。方法收集人腰椎间盘髓核组织50例，其中15例为正常对照组，35例为退变腰椎间盘髓核组织（实验组，根据术前MRI分为突出组12例，脱出组13例，游离组10例）。分别应用Western`-blot、苏木精`-伊红（HE）染色、免疫组织化学方法检测各组髓核组织中HSPA8的表达。结果3种方法检测结果均显示，对照组、突出组、脱出组、游离组腰椎间盘髓核组织中HSPA8表达依次降低，差异有显著性（F=153.52～1 662.43，P<0.001）。结论HSPA8在人椎间盘髓核组织中稳定表达，并且与椎间盘退变的程度相关。
[关键词]HSC70热休克蛋白质类;椎间盘退行性变;印迹法，蛋白质;苏木精;免疫组织化学
EXPRESSION OF HSPA8 IN THE NUCLEUS PULPOSUS TISSUE OF LUMBAR INTERVERTEBRAL DISCS AND ITS EFFECT ON DEGENERATION OF INTERVERTEBRAL DISCS WANG Huacong， CHEN Bohua， JIN Canghai， XIANG Hongfei， LIU Yong（Department of Spinal Surgery， The Afflicated Hospital of Qingdao University， Qingdao 266100， China）
[ABSTRACT]ObjectiveTo investigate the expression of the 70 kDa heat shock protein member 8 （HSPA8） in the nucleus pulposus tissue of human lumbar intervertebral discs （LIDs） and its correlation with the degree of degeneration of human intervertebral discs. MethodsThe nucleus pulposus tissue of human LIDs was collected from 50 cases， including 15 cases as control group and 35 cases with degenerated LIDs as experimental group. According to preoperative MRI results， the experimental group was subdivided into protrusion group （n=12）， prolapse group （n=13）， and sequestration group （n=10）. The expression of HSPA8 in the nucleus pulposus tissue of each group was determined by Western blot， hematoxylin`-eosin staining， and immunohistochemistry. ResultsAll the results obtained from the three determination methods showed decreased expression of HSPA8 in the nucleus pulposus tissue of each group， with significant differences observed between groups （highest expression seen with the control group， successively followed by the protrusion group， the prolapse group， and the sequestration group） （F=153.52-1 662.43， all P<0.001）. ConclusionHSPA8 is expressed stably in the nucleus pulposus tissue of human intervertebral discs， and its expression is correlated with the degree of degeneration of intervertebral discs.
[KEY WORDS]HSC70 heat`-shock proteins; intervertebral disc degeneration; blotting， Western; hematoxylin; immunohistochemistry
椎間盘退变是骨科常见的疾病，据统计，全球有70%～90%的人会受到其困扰[1`-3]，腰腿痛是导致人类生活质量下降、劳动力丧失的最常见原因[4`-6]。腰椎管狭窄症、腰椎间盘突出症是导致腰腿痛最常见的疾病[7]。腰椎间盘退变与遗传和环境因素有关，其特征之一就是椎间盘髓核蛋白多糖和水分含量丧失[8`-10]。热休克蛋白是一种能够在细胞核和细胞质之间穿梭的多功能蛋白[11]，参与多种细胞功能的调控，是分子伴侣系统重要的一部分[12`-13]。70 kDa热休克蛋白8（HSPA8）是HSC70家族中高度保守的成员，具有多效性，对细胞的存活起着至关重要的作用，在应激反应中发挥着不可或缺的作用[14`-16]。LOEFFLER等[17]研究显示，脑脊液中的HSPA8随着年龄的增长而降低，并且与氧化应激有关。YE等[18]首次报道HSPA8在退变椎间盘纤维环细胞中表达量降低。但是，HSPA8在人腰椎间盘髓核组织是否表达及其与椎间盘退变程度的关系尚不清楚。本文研究联合应用Western`-blot、苏木精`-伊红（HE）染色、免疫组织化学方法，对人腰椎间盘髓核组织中HSPA8表达及其与腰椎间盘退变程度的关系进行研究，为腰腿痛病人的治疗提供理论依据。 [14]HUNT C， MORIMOTO R I. Conserved features of eukaryotic hsp70 genes revealed by comparison with the nucleotide sequence of human hsp70[J]. Proceedings of the National Academy of Sciences of the United States of America， 1985，82（19）：6455`-6459.
[15]LIU T， DANIELS C K， CAO S. Comprehensive review on the HSC70 functions， interactions with related molecules and involvement in clinical diseases and therapeutic potential[J]. Pharmacology &Therapeutics， 2012，136（3）：354`-374.
[16]KITYK R， KOPP J， SINNING I， et al. Structure and dyna`-mics of the ATP`-bound open conformation of Hsp70 chaperones[J]. Molecular Cell， 2012，48（6）：863`-874.
[17]LOEFFLER D A， KIAVER A C， COFFEY M P， et al. Age`-related decrease in heat shock 70 kDa protein 8 in cerebrospinal fluid Is associated with increased oxidative stress[J]. Front Aging Neurosci， 2016，8：178.
[18]YE Dongping， LIANG Weiguo， DAI Libing， et al. Comparative and quantitative proteomic analysis of normal and degene`-rated human annulus fibrosus cells[J]. Clinical and Experimental Pharmacology &Physiology， 2015，42（5）：530`-536.
[19]SPENGLER D M. Lumbar discectomy. Results with limited disc excision and selective foraminotomy[J]. Spine， 1982，7（6）：604`-607.
[20]VADALA G， RUSSO F， Di MARTINO A， et al. Intervertebral disc regeneration： from the degenerative cascade to mole`-cular therapy and tissue engineering[J]. Journal of Tissue Engineering and Regenerative Medicine， 2015，9（6）：679`-690.
[21]GORNET M G， PEACOCK J， CIAUDE J， et al. Magnetic resonance spectroscopy （MRS） can identify painful lumbar discs and may facilitate improved clinical outcomes of lumbar surgeries for discogenic pain[J]. Eur Spine J， 2019. doi：10.1007/s00586`-018`-05873`-3.
[22]ZEHRA U， CHEUNG J P Y， BOW C， et al. Multidimensio`-nal vertebral endplate defects are associated with disc degeneration， Modic changes， facet joint abnormalities and pain[J]. J Orthop Res， 2018. doi：10.1002/jor.24195.
[23]STRICHERR F， MACRI C， RUFF M， et al. HSPA8/HSC70 chaperone protein： structure， function， and chemical targeting[J]. Autophagy， 2013，9（12）：1937`-1954.
[24]GOLDFA R B， KASHLAN O B， WATKINS J N， et al. Diffe`-rential effects of Hsc70 and Hsp70 on the intracellular trafficking and functional expression of epithelial sodium channels[J]. Proceedings of the National Academy of Sciences of the United States of America， 2006，103（15）：5817`-5822. [25]DEANE E E， WOO N Y. Impact of heavy metals and organochlorines on Hsp70 and Hsc70 gene expression in black sea bream fibroblasts[J]. Aquatic Toxicology （Amsterdam， Ne`-therlands）， 2006，79（1）：9`-15.
[26]GERACI F， PINSINO A， TURTURICI G， et al. Nickel， lead， and cadmium induce differential cellular responses in sea urchin embryos by activating the synthesis of different HSP70s[J]. Biochemical and Biophysical Research Communications， 2004，322（3）：873`-877.
[27]MAKINO H， SEKI S， YAHARA Y， et al. A selective inhibition of c`-Fos/activator protein`-1 as a potential therapeutic target for intervertebral disc degeneration and associated pain[J]. Scientific Reports， 2017，7（1）：16983.
[28]姜棚菲，馬张稳，张民泽，等. 腰椎间盘退行性改变患者ADAMTS`-7表达及其机制研究[J]. 实用医院临床杂志， 2017，14（6）：101`-104.
[29]NIKOLAOU G， ZIBIS A H， FYIIOS A H， et al. Detection of O`-Linked`-N`-Acetylglucosamine modification and its associated enzymes in human degenerated intervertebral discs[J]. Asian Spine J， 2017，11（6）：863`-869.
[30]JOHNSON Z I， GOGATE S S， DAY R， et al. Aquaporin 1 and 5 expression decreases during human intervertebral disc degeneration： novel HIF`-1`-mediated regulation of aquaporins in NP cells[J]. Oncotarget， 2015，6（14）：11945`-11958.
[31]WANG Jingjie， LIU Xiaoyan， SUN Bing， et al. Upregulated miR`-154 promotes ECM degradation in intervertebral disc degeneration[J]. J Cell Biochem， 2019. doi：10.1002/jcb.2847.
[关键词]HSC70热休克蛋白质类;椎间盘退行性变;印迹法，蛋白质;苏木精;免疫组织化学
EXPRESSION OF HSPA8 IN THE NUCLEUS PULPOSUS TISSUE OF LUMBAR INTERVERTEBRAL DISCS AND ITS EFFECT ON DEGENERATION OF INTERVERTEBRAL DISCS WANG Huacong， CHEN Bohua， JIN Canghai， XIANG Hongfei， LIU Yong（Department of Spinal Surgery， The Afflicated Hospital of Qingdao University， Qingdao 266100， China）
[ABSTRACT]ObjectiveTo investigate the expression of the 70 kDa heat shock protein member 8 （HSPA8） in the nucleus pulposus tissue of human lumbar intervertebral discs （LIDs） and its correlation with the degree of degeneration of human intervertebral discs. MethodsThe nucleus pulposus tissue of human LIDs was collected from 50 cases， including 15 cases as control group and 35 cases with degenerated LIDs as experimental group. According to preoperative MRI results， the experimental group was subdivided into protrusion group （n=12）， prolapse group （n=13）， and sequestration group （n=10）. The expression of HSPA8 in the nucleus pulposus tissue of each group was determined by Western blot， hematoxylin`-eosin staining， and immunohistochemistry. ResultsAll the results obtained from the three determination methods showed decreased expression of HSPA8 in the nucleus pulposus tissue of each group， with significant differences observed between groups （highest expression seen with the control group， successively followed by the protrusion group， the prolapse group， and the sequestration group） （F=153.52-1 662.43， all P<0.001）. ConclusionHSPA8 is expressed stably in the nucleus pulposus tissue of human intervertebral discs， and its expression is correlated with the degree of degeneration of intervertebral discs.
[KEY WORDS]HSC70 heat`-shock proteins; intervertebral disc degeneration; blotting， Western; hematoxylin; immunohistochemistry
椎間盘退变是骨科常见的疾病，据统计，全球有70%～90%的人会受到其困扰[1`-3]，腰腿痛是导致人类生活质量下降、劳动力丧失的最常见原因[4`-6]。腰椎管狭窄症、腰椎间盘突出症是导致腰腿痛最常见的疾病[7]。腰椎间盘退变与遗传和环境因素有关，其特征之一就是椎间盘髓核蛋白多糖和水分含量丧失[8`-10]。热休克蛋白是一种能够在细胞核和细胞质之间穿梭的多功能蛋白[11]，参与多种细胞功能的调控，是分子伴侣系统重要的一部分[12`-13]。70 kDa热休克蛋白8（HSPA8）是HSC70家族中高度保守的成员，具有多效性，对细胞的存活起着至关重要的作用，在应激反应中发挥着不可或缺的作用[14`-16]。LOEFFLER等[17]研究显示，脑脊液中的HSPA8随着年龄的增长而降低，并且与氧化应激有关。YE等[18]首次报道HSPA8在退变椎间盘纤维环细胞中表达量降低。但是，HSPA8在人腰椎间盘髓核组织是否表达及其与椎间盘退变程度的关系尚不清楚。本文研究联合应用Western`-blot、苏木精`-伊红（HE）染色、免疫组织化学方法，对人腰椎间盘髓核组织中HSPA8表达及其与腰椎间盘退变程度的关系进行研究，为腰腿痛病人的治疗提供理论依据。 [14]HUNT C， MORIMOTO R I. Conserved features of eukaryotic hsp70 genes revealed by comparison with the nucleotide sequence of human hsp70[J]. Proceedings of the National Academy of Sciences of the United States of America， 1985，82（19）：6455`-6459.
[15]LIU T， DANIELS C K， CAO S. Comprehensive review on the HSC70 functions， interactions with related molecules and involvement in clinical diseases and therapeutic potential[J]. Pharmacology &Therapeutics， 2012，136（3）：354`-374.
[16]KITYK R， KOPP J， SINNING I， et al. Structure and dyna`-mics of the ATP`-bound open conformation of Hsp70 chaperones[J]. Molecular Cell， 2012，48（6）：863`-874.
[17]LOEFFLER D A， KIAVER A C， COFFEY M P， et al. Age`-related decrease in heat shock 70 kDa protein 8 in cerebrospinal fluid Is associated with increased oxidative stress[J]. Front Aging Neurosci， 2016，8：178.
[18]YE Dongping， LIANG Weiguo， DAI Libing， et al. Comparative and quantitative proteomic analysis of normal and degene`-rated human annulus fibrosus cells[J]. Clinical and Experimental Pharmacology &Physiology， 2015，42（5）：530`-536.
[19]SPENGLER D M. Lumbar discectomy. Results with limited disc excision and selective foraminotomy[J]. Spine， 1982，7（6）：604`-607.
[20]VADALA G， RUSSO F， Di MARTINO A， et al. Intervertebral disc regeneration： from the degenerative cascade to mole`-cular therapy and tissue engineering[J]. Journal of Tissue Engineering and Regenerative Medicine， 2015，9（6）：679`-690.
[21]GORNET M G， PEACOCK J， CIAUDE J， et al. Magnetic resonance spectroscopy （MRS） can identify painful lumbar discs and may facilitate improved clinical outcomes of lumbar surgeries for discogenic pain[J]. Eur Spine J， 2019. doi：10.1007/s00586`-018`-05873`-3.
[22]ZEHRA U， CHEUNG J P Y， BOW C， et al. Multidimensio`-nal vertebral endplate defects are associated with disc degeneration， Modic changes， facet joint abnormalities and pain[J]. J Orthop Res， 2018. doi：10.1002/jor.24195.
[23]STRICHERR F， MACRI C， RUFF M， et al. HSPA8/HSC70 chaperone protein： structure， function， and chemical targeting[J]. Autophagy， 2013，9（12）：1937`-1954.
[24]GOLDFA R B， KASHLAN O B， WATKINS J N， et al. Diffe`-rential effects of Hsc70 and Hsp70 on the intracellular trafficking and functional expression of epithelial sodium channels[J]. Proceedings of the National Academy of Sciences of the United States of America， 2006，103（15）：5817`-5822. [25]DEANE E E， WOO N Y. Impact of heavy metals and organochlorines on Hsp70 and Hsc70 gene expression in black sea bream fibroblasts[J]. Aquatic Toxicology （Amsterdam， Ne`-therlands）， 2006，79（1）：9`-15.
[26]GERACI F， PINSINO A， TURTURICI G， et al. Nickel， lead， and cadmium induce differential cellular responses in sea urchin embryos by activating the synthesis of different HSP70s[J]. Biochemical and Biophysical Research Communications， 2004，322（3）：873`-877.
[27]MAKINO H， SEKI S， YAHARA Y， et al. A selective inhibition of c`-Fos/activator protein`-1 as a potential therapeutic target for intervertebral disc degeneration and associated pain[J]. Scientific Reports， 2017，7（1）：16983.
[28]姜棚菲，馬张稳，张民泽，等. 腰椎间盘退行性改变患者ADAMTS`-7表达及其机制研究[J]. 实用医院临床杂志， 2017，14（6）：101`-104.
[29]NIKOLAOU G， ZIBIS A H， FYIIOS A H， et al. Detection of O`-Linked`-N`-Acetylglucosamine modification and its associated enzymes in human degenerated intervertebral discs[J]. Asian Spine J， 2017，11（6）：863`-869.
[30]JOHNSON Z I， GOGATE S S， DAY R， et al. Aquaporin 1 and 5 expression decreases during human intervertebral disc degeneration： novel HIF`-1`-mediated regulation of aquaporins in NP cells[J]. Oncotarget， 2015，6（14）：11945`-11958.
[31]WANG Jingjie， LIU Xiaoyan， SUN Bing， et al. Upregulated miR`-154 promotes ECM degradation in intervertebral disc degeneration[J]. J Cell Biochem， 2019. doi：10.1002/jcb.2847.