酸敏感离子通道1a偶联内质网应激在人髓核细胞退变中的作用机制初探

[目的]模拟人椎间盘髓核(nucleus pulposus,NP)酸性环境,探讨酸敏感离子通道1a(acid-sensing ion channel 1a,ASIC1a)的活化与内质网应激的关系。[方法]体外单层培养人正常髓核细胞(nucleus pulposus cells,NPCs)系,不同p H值培养不同时间,模拟椎间盘酸性微环境,建立酸诱导的退变髓核细胞模型。CCK-8检测细胞增殖能力。Western blot、q PCR检测内质网应激。Western blot检测ASIC1a的表达。Fura-2/AM荧光探针检测ASIC1a活化介导的Ca2+内流。流式细胞术检测ASIC1a活化后细胞凋亡率。Pc TX1(ASIC1a特异性阻断剂)阻断ASIC1a后,观察细胞凋亡及内质网应激指标变化情况。[结果]酸诱导髓核细胞凋亡,酸激活ASIC1a及内质网应激,Pc TX1能降低促凋亡的内质网应激通路和髓核细胞凋亡率(P0.05),而未折叠蛋白反应相关指标无明显变化(P0.05)。[结论]ASIC1a能够调控内质网应激中的促凋亡通路,阻断ASIC1能够保护酸诱导的髓核细胞凋亡。     

大鼠椎间盘软骨终板细胞凋亡体外模型的建立

[目的]建立大鼠椎间盘软骨终板细胞凋亡体外模型.[方法]为了模拟椎间盘内部低营养低代谢环境,采用低胎牛血清培养法培养椎间盘软骨终板细胞分别含0％,1％,3％,5％,8％,10％胎牛血清,设置不同浓度梯度筛选最佳浓度,检测凋亡率、凋亡蛋白表达及caspase酶活性.[结果]低胎牛血清培养组细胞发生形态改变,DAPI染色阳性细胞增多;流式细胞仪检测凋亡率随着FBS浓度降低而升高,1％为最有效诱导凋亡浓度;Western blot示FAS、caspase-3、PARP、细胞色素C表达在1％ FBS组明显高于10％时,同时caspase-3/8/9酶活性增高.[结论]低胎牛血清培养法能诱导体外培养的软骨终板细胞发生凋亡,最终会引起细胞功能丧失和椎间盘退变,caspase家族可能参与了这一过程.     

人退变椎间盘髓核细胞体外平面培养的形态学及活性观察

[目的]观察平面培养体系内人退变椎间盘髓核细胞的形态及活性变化.[方法]收集20例人退变椎间盘髓核,分离髓核细胞行平面培养,倒置相差显微镜和HE染色观察髓核细胞的生长过程与形态变化,流式细胞仪量化细胞周期分布和凋亡率,甲苯胺蓝染色和免疫细胞化学染色髓核细胞聚集蛋白聚糖和Ⅱ型胶原的表达,观察平面培养传代对髓核细胞活性和基质合成能力的影响.[结果]原代髓核细胞呈类圆形或多角形,平均7d贴壁,31 d融合至95％,P1代髓核细胞呈长梭形或多角形,平均12h贴壁,6.6d融合至95％,两代细胞增殖能力的差异有统计学意义(P＜0.01).原代与P1代髓核细胞的细胞浆阳性染色聚集蛋白聚糖和Ⅱ型胶原,生长融合至95％后约90％的细胞分布于G1期,约16％的细胞凋亡,两代细胞的细胞活性和基质合成能力无统计学差异(P＞0.05).[结论]人退变椎间盘髓核细胞体外平面培养将经历显著的形态学变化.传一代后髓核细胞增殖能力提高,但能维持细胞活性以及蛋白聚糖和Ⅱ型胶原的合成能力.     

白藜芦醇对硝普钠诱导的软骨细胞凋亡bax及bcl-2表达的影响

[目的]探讨白藜芦醇对硝普钠诱导的软骨细胞凋亡及凋亡调控基因bax及bcl-2表达的影响。[方法]常规培养关节软骨细胞后,按不同浓度白藜芦醇(分别为25μmol/L、50μmol/L、100μmol/L)处理后分组并用硝普钠(SNP)(2.5 mmol/L)诱导细胞凋亡。采用MTT法检测各组软骨细胞活性、DAPI染色和流式细胞术检测各组细胞凋亡,采用western-blot技术检测各组细胞sirt1、bax、bcl-2的表达,RT-PCR法检测各组细胞sirt1mRNA的表达。[结果]随着白藜芦醇浓度的升高,MTT法结果显示处理后细胞活性依次增高;DAPI染色、流式细胞术检测显示处理后细胞凋亡相对较少;western-blot检测显示随着白藜芦醇浓度的升高,处理后细胞sirt1、bcl-2依次增高,而bax逐渐降低;RT-PCR法检测显示白藜芦醇处理后细胞sirt1 mRNA表达随浓度逐渐增高。[结论]白藜芦醇激活sirt1,使bcl-2/bax表达增高,是白藜芦醇抑制软骨细胞凋亡、预防骨关节炎的机制之一。     

羧甲基壳聚糖对体外培养大鼠椎间盘髓核细胞增殖、细胞周期及细胞外基质分泌的影响

目的 观察羧甲基壳聚糖(CMCS)对体外培养大鼠椎间盘髓核细胞细胞周期进程、细胞周期蛋白表达及分泌细胞外基质的影响.方法 使用酶消化法提取大鼠椎间盘髓核细胞并进行体外培养,Ⅱ型胶原免疫组织化学鉴定髓核细胞.将髓核细胞分为:对照组、不同浓度CMCS处理组(100、200、500 mg/L),作用24 h后通过碘化丙锭(PI)染色流式细胞技术检测细胞周期进程.通过荧光定量聚合酶链反应(FQ-PCR)及Western blot法检测不同浓度(100、200、500 mg/L) CMCS对髓核细胞内增殖相关蛋白增殖细胞核抗原(PCNA)及细胞周期蛋白(Cyclin)D表达的影响,并通过FQ-PCR法检测不同浓度(100、200、500 mg/L) CMCS对髓核细胞分泌细胞外基质Ⅱ型胶原的影响.结果 培养的细胞经Ⅱ型胶原免疫组织化学染色鉴定显示其表达阳性,表明为髓核细胞;通过FQ-PCR及Western blot检测增殖相关蛋白PCNA可见通过100、200、500 mg/L CMCS作用后髓核细胞表达PCNA与对照组比较分别增加12.3％、18.2％、54.2％与25.1％、52.7％、66.9％ (P ＜0.05);经流式细胞仪检测发现经100、200、500 mg/L CMCS处理的髓核细胞处于S期细胞比例是对照组的1.58、2.34与2.61倍(P＜0.05);Cyclin D表达分别是对照组的1.26、2.15、2.64倍与1.53、1.69、1.83倍(P ＜0.05);FQ-PCR及Western blot检测结果表明100、200、500 mg/L CMCS分别促进髓核细胞分泌Ⅱ型胶原表达是对照组的1.67、2.24、2.79倍与1.48、1.65、1.77倍(P＜0.05).结论 CMCS对体外培养髓核细胞增殖相关蛋白PCNA表达具有促进作用,可促进细胞周期进程及细胞周期蛋白表达,对髓核细胞分泌细胞外基质具有促进作用.     

双环铂对激素抵抗型前列腺癌细胞PC3的凋亡诱导作用

目的采用体外实验观察双环铂对激素抵抗型前列腺癌细胞PC3的凋亡诱导作用,并初步探讨其凋亡机制。方法 CCK-8法观察双环铂对PC3细胞增殖的抑制,Hoechst染色荧光显微镜下观察凋亡细胞,Annexin-V-FITC/PI染色流式细胞仪检测细胞凋亡率,PI单染流式细胞仪检测细胞周期变化。结果双环铂可抑制PC3细胞的增殖,48hIC50为（195±19.0）×10-6 mol/L.;PC3细胞在经双环铂处理后,漂浮细胞呈明显的凋亡细胞形态,经Hoechst染色荧光显微镜下可见凋亡小体等典型的细胞核染色质形态学改变;双环铂诱导的PC3细胞凋亡呈剂量依赖性及时间依赖性。低浓度的双环铂可引起PC3细胞G2期阻滞,随药物浓度的升高sub-G1期细胞比例明显增加。结论体外实验显示双环铂可诱导PC3细胞的凋亡,双环铂对细胞周期的影响可视为其抗肿瘤活性的机制之一。     

pH值对人退变椎间盘髓核细胞凋亡的影响

[目的]观察不同pH值对体外培养人退变椎间盘髓核细胞凋亡的影响,为椎间盘退变的预防与治疗提供理论依据。[方法]对体外培养的人退变椎间盘髓核细胞利用甲苯胺蓝和番红O染色进行鉴定,并对P3代细胞分别用普通培养基(pH值7.4)、酸性培养基(pH值6.8)和酸性培养基(pH值6.8)+Cucl_2(酸质子受体抑制剂)培养24 h,用流式细胞仪测定细胞凋亡,计算细胞凋亡率。[结果]细胞染色结果均为阳性,证明实验中所培养的细胞为髓核类软骨细胞。普通培养基组的凋亡率为(8.86±0.20)%,酸性培养基组的凋亡率为(11.23±0.77)%,酸性培养基+Cucl_2组的凋亡率为(9.60±0.49)%。与其他两组相比,酸性培养基组的凋亡率明显增加(P0.05)。[结论]在低pH值的环境下,人退变椎间盘髓核细胞的凋亡率增加。     

羧甲基壳聚糖通过抑制线粒体途径保护氧化损伤诱导雪旺细胞凋亡

[目的]探讨线粒体途径在羧甲基壳聚糖(carboxymethylated chitosan,CMCS)保护过氧化氢(H_2O_2)诱导雪旺细胞(Schwann cells,SCs)凋亡中的作用及机制。[方法]体外培养SCs,S-100免疫荧光染色鉴定。将SCs分为空白对照组、H_2O_2诱导组、H_2O_2加CMCS处理组。流式细胞仪检测细胞凋亡比率,罗丹明(Rhodamine123)荧光染色检测细胞线粒体膜电位水平,Western blot检测SCs内细胞色素C(Cytochrome C)表达水平。[结果]本实验培养细胞经S-100荧光染色鉴定阳性率达95%以上,H_2O_2诱导SCs凋亡,降低线粒体膜电位,增加细胞色素C释放;而在加入CMCS后SCs凋亡比例降低,线粒体膜电位增加,细胞色素C释放减少。[结论]CMCS通过抑制线粒体细胞凋亡途径保护H_2O_2诱导SCs凋亡。     

siRNA转染对大鼠髓核细胞TRAIL表达和细胞行为的影响

[目的]探讨siRNA转染对大鼠髓核细胞TRAIL表达和细胞行为的影响。[方法]采用弯曲鼠尾法建立椎间盘退变模型。处死动物。取退变椎间盘组织分离培养髓核细胞。P1髓核细胞分为4组,分别为空白对照组、TRAIL-siRNA转染组(siTRAIL组)、TNF-α处理组(TNF-α组)和TNF-α处理+TRAIL-siRNA转染组(TNF-α+siTRAIL组)。采用MTT法检测P1髓核细胞增殖,流式细胞仪检测细胞凋亡,Western blot法检测髓核细胞Caspase-3的活性和表达。[结果]相较于空白对照细胞,TRAIL siRNA转染对细胞增殖、凋亡、Caspase-3的表达无明显影响(P0.05);TNF-α处理引发髓核细胞增殖显著下降(P0.05),而凋亡率显著升高(P0.05),Caspase-3的表达显著升高(P0.05)。TNF-α处理后再用TRAIL-siRNA转染可显著逆转TNF-α对细胞增殖、凋亡和Caspase-3表达的影响(P0.05)。[结论] TRAIL siRNA转染可沉默TRAIL表达,从而逆转TNF-α诱导大鼠髓核细胞增殖抑制、细胞凋亡和Caspase-3表达。     

高浓度抗生素溶液对椎间盘细胞影响的研究

[目的]检测高浓度的头孢唑林钠及克林霉素对兔腰椎间盘髓核细胞活力及增殖能力的影响.[方法]通过在体外分离、培养新西兰兔髓核细胞,体外扩增培养至第1代,采用CCK-8及RT-PCR等方法,检测4个浓度(0 mg/ml、0.25 mg/ml、0.5 mg/ml、1 mg/ml)的头孢唑啉钠及克林霉素对于兔腰椎间盘髓核细胞活力及增殖能力的影响.[结果]当头孢唑啉钠浓度超过0.5mg/ml、克林霉素浓度超过0.25 mg/ml时,髓核细胞Ⅱ型胶原及蛋白多糖的表达下降,浓度在1.0 mg/ml时达到最低.头孢唑啉钠浓度超过0.5 mg/ml、克林霉素浓度超过0.25 mg/ml时,明显影响细胞活力,小于以上浓度时细胞活性无明显影响.[结论]高浓度的头孢唑林钠及克林霉素抗生素对于体外培养的兔髓核细胞的活力、增殖能力具有抑制作用.     

Activation of rat nucleus pulposus cells by coculture with whole bone marrow cells collected by the perfusion method

Cell proliferation and matrix synthesis were compared for rat nucleus pulposus cells cocultured with mesenchymal stem cells (MSCs) or fresh whole bone marrow cells (BMCs), harvested by the perfusion or aspiration methods. Nucleus pulposus cells were isolated from tail intervertebral discs of F344/slc rats, and BMCs were obtained from femora. Proteoglycan synthesis, DNA synthesis, and aggrecan mRNA expression were measured. The level of transforming growth factor‐β in supernatants from the culture system was also measured. Cell number, aggrecan mRNA expression, and uptake of [³⁵S]‐sulfate and [³H]‐thymidine by nucleus pulposus cells cocultured with fresh whole BMCs all increased significantly compared with nucleus pulposus cells cocultured with MSCs. TGF‐β secreted by nucleus pulposus cells cocultured with fresh whole BMCs also significantly increased when compared with cocultures with MSCs. The perfusion method was superior to the aspiration method for preventing contamination of BMCs with peripheral red blood cells and lymphocytes, which may cause an autoimmune response in the disc. In conclusion, we suggest that fresh whole BMCs harvested by the perfusion method are more effective for increasing the proliferative and matrix synthesis capacity of nucleus pulposus cells. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:222–228, 2009     

Nucleus pulposus inhibits the axonal outgrowth of cultured dorsal root ganglion cells

Although it is well established that nucleus pulposus cells may induce structural and functional changes in adjacent nerve roots when placed epidurally, it is not known whether this is due to direct neurotoxic effects or whether the nerve roots are affected indirectly by reduction of nutrition and inflammatory/immunologic mechanisms. In the present study we assessed the effects of various tissues on cultured dorsal root ganglions from newborn rats. Nucleus pulposus was found to have a toxic effect on the axons by blocking axonal outgrowth, but no similar effects on the nerve cell bodies (extra-ganglionic nerve cell density, nerve cell arborisation) were found as compared to the series with only culture medium. Sterile water for 1 or 24 h (positive controls) induced significant effects by all four criteria, whereas medium without nerve growth factor, fat and frozen nucleus pulposus had no statistically significant effects. The study thus showed that there are direct axonotoxic effects induced by the nucleus pulposus, and since frozen nucleus pulposus did not have any effects, it may be assumed that the mechanisms are related to substances produced by the nucleus pulposus cells. The presented model allows for future studies on the neurotoxic properties of nucleus pulposus cell-derived candidate substances. Received: 3 April 1999/Revised: 16 October 1999/Accepted: 8 November 1999     

高渗应激下ERK信号转导通路在兔髓核细胞凋亡中的作用

[目的]观察高渗透压对体外培养的兔髓核细胞凋亡的影响及细胞外信号调节激酶(ERK1/2)信号转导通路在此过程中的作用.[方法]用不同的渗透压梯度以及ERK1/2特异性抑制剂PD 98 059(50 μmol/L)分不同作用时间处理髓核细胞;流式细胞仪检测髓核细胞在不同处理组的凋亡情况,蛋白质免疫印迹技术检测各组ERK1/2和p- ERK1/2蛋白的表达情况;免疫荧光标记技术检测p- ERK1/2蛋白在髓核细胞内的分布情况.[结果]高渗透压(600 mOsm)培养基中兔髓核细胞凋亡显著增加,与对照组相比差异有统计学意义(P=0.013),并呈时间依赖性;PD98059预处理组与相应时间段同渗透压组相比,细胞凋亡进一步显著地增加(P=0.035);400 mOsm组及其抑制组在各时间段细胞凋亡均不明显;600 mOsm高渗透压显著激活p- ERK1/2的表达,并且在3h组表达水平最高,与对照组比较差异显著(P--0.027),而PD98059几乎阻断p- ERK1/2的表达;免疫荧光标记检测到p- ERK1/2在600 mOsm高渗组的细胞胞浆和胞核中均有分布.[结论]高渗透压(600 mOsm)应激下兔髓核细胞凋亡显著增加,而且激活的ERK1/2信号转导通路具有抵抗髓核细胞凋亡的作用;并且髓核细胞能够适应轻度增加的渗透压(400mOsm)环境.     

Cell culture of the intervertebral disc of rats: factors influencing culture, proteoglycan, collagen, and deoxyribonucleic acid synthesis.

To establish cell culture of the nucleus pulposus and anulus fibrosus of rat intervertebral disc, the effects of culture conditions on the growth of cells and the synthesis of DNA, proteoglycan, and collagen were studied. For cell culture of the nucleus pulposus, the use of 3-week-old rats and a medium adjusted to pH 7.0 was optimal. There was almost no difference in growth between cells in Ham's F12 medium and those in Dulbecco's Modified Eagle Medium. In cells isolated from the anulus fibrosus, a medium adjusted to pH 7.0-7.6 was preferable, but irrespective of rat age. Culture cells of the nucleus pulposus were composed of large cells with vacuoles and small polygonal cells. These cells had a slight growth activity and a fair capability of proteoglycan and collagen synthesis. Culture cells of the anulus fibrosus were composed of polygonal and spindle-shape cells, and the growth was more vigorous with the potentials for proteoglycan and collagen synthesis than the nucleus cells.     

Low‐intensity pulsed ultrasound stimulation enhances TIMP‐1 in nucleus pulposus cells and MCP‐1 in macrophages in the rat

Recent studies have reported that low‐intensity pulsed ultrasound (LIPUS) stimulates cell proliferation and proteoglycan production in rabbit intervertebral disc cells, and moreover promotes the secretion of MCP‐1 (monocyte chemotaxis protein‐1) from macrophages in a disc organ culture model. These findings suggest the possible application of LIPUS for biological repair of disc degeneration and herniation. Although the mechanisms involved are not well understood, several cytokine pathways may play a role. Therefore, in order to evaluate the effect of LIPUS stimulation on cytokine production by nucleus pulposus cells and macrophages, in vitro culture studies were designed. Nucleus pulposus cells and macrophages were collected from Sprague‐Dawley rats, cultured separately in a monolayer, and stimulated with LIPUS for 7 days. After culture, the culture medium and the cells were analyzed by cytokine array, RT‐PCR, and ELISA. Cytokine array showed that LIPUS stimulation significantly upregulated TIMP‐1 (tissue inhibitor of metalloproteinase‐1) in the nucleus pulposus and MCP‐1 in macrophages in comparison with the control. This was confirmed at the gene level by RT‐PCR in nucleus pulposus cells and macrophages after stimulation with LIPUS. Quantitative evaluation of these proteins by ELISA showed higher levels in nucleus pulposus cells and macrophages stimulated by LIPUS than in controls. These results showed that LIPUS stimulation significantly activated TIMP‐1 and MCP‐1 in nucleus pulposus cells and macrophages at both the protein and gene levels, suggesting that LIPUS may be a promising supplemental treatment for intervertebral disc herniation. © 2008 Orthopaedic Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:865–871, 2008     

仿生髓核组织工程材料支架对体外培养髓核细胞合成代谢的影响

目的：观察自行设计构建的仿生髓核组织工程材料——CⅡ／HyA-CS（CHCS）支架对体外培养髓核细胞合成代谢的影响。方法：将体外培养的传1代髓核细胞接种于培养瓶内，置入CHCS支架，体外培养10d后，分别测定^3H-脯氨酸掺入量、培养液中糖胺聚糖（GAG）含量、髓核细胞可凝集蛋白多糖（Aggrecan）、核多糖（Decorin）、二聚糖（Biglycan）的mRNA表达及Aggrecan的蛋白表达等。结果：实验组与对照组的^3H-脯氨酸掺入量、培养液中GAG含量、髓核细胞Aggrecan、Decorin、Biglycan mRNA表达、Aggrecan蛋白表达等均无统计学差异。结论：CHCS支架对体外培养髓核细胞的合成代谢无明显抑制作用。     

兔腰椎间盘髓核细胞的培养及形态观察

目的：通过对兔腰椎间盘髓核细胞的培养,观察细胞内的演变,探讨髓核细胞的生物学行为及影响因素。方法：取兔腰椎间盘髓核细胞,在加10％灭活胎牛血清的F12－DMEM液中培养,建立体外髓核细胞培养模型。通过光镜、电镜观察,同时进行细胞活力测定。结果：（1）原代细胞生物学性状最接近体内细胞,传代后细胞呈现衰老现象。（2） 活力测定提示随着培养时间的延长及传代,活力逐渐降低。（3）髓核细胞内细胞器的变化与其生物学活性变化相一致。结论：兔腰椎髓核细胞的体外培养成功为人腰椎髓核细胞的培养奠定了基础。     

Prolonged down regulation of specific gene expression in nucleus pulposus cell mediated by RNA interference in vitro.

To investigate the efficacies and the longevity of RNA interference in nucleus pulposus cells from rat and human, two reporter luciferase plasmids (Firefly and Renilla) were used. These plasmids were cotransfected with siRNA targeting Firefly luciferase to the nucleus pulposus cells extracted from Sprague Dawley rats and scoliosis patients. The inhibitory effects were evaluated by dual luciferase assay for 3 weeks. Proliferation activity of fibroblast-like cells extracted from the subcutaneous tissue of Sprague Dawley rats and the nucleus pulposus cells were measured by proliferation assay (WST-8 assay) every 2 days after plating. The expression of Firefly luciferase was drastically inhibited both in rats (94.7%) and in humans (93.7%). The inhibitory effects were maintained for 2 weeks and had disappeared completely by 3 weeks. The proliferation activity of nucleus pulposus cells was significantly lower than fibroblast-like cells. We have shown, for the first time, siRNA-mediated gene silencing in rat and human disc cells for a relatively sustained period, probably due to the stability of the nucleus pulposus cells in terms of cell proliferation. The demonstration of this study may allow further exploration of the use of siRNA for scientific research and the treatment of disc degenerative diseases.     

Fas ligand plays an important role for the production of pro‐inflammatory cytokines in intervertebral disc nucleus pulposus cells

It is suggested that pro‐inflammatory cytokines, which are produced by interaction of the intervertebral nucleus pulposus cells and macrophages, may be linked to the cause of pain of the intervertebral disc herniation. This study carries out the in vitro experiments to examine the mechanism, with the use of the co‐culture of an immortalized cell line of nucleus pulposus of the human intervertebral disc and the macrophage cell line. As a result, it is found that the production of pro‐inflammatory cytokines is significantly larger at the co‐culture group than at the independent culture group. Also, at the co‐culture group of macrophages and intervertebral nucleus pulposus cells with over‐expression of fas ligand (FasL), the production of pro‐inflammatory cytokines is found to be far larger. Furthermore, it is found that these pro‐inflammatory cytokines are produced mainly by the intervertebral nucleus pulposus cells with over‐expression of FasL, and that the expression of a disintegrin and metalloproteinase (ADAM) 10, which controls the expression of FasL and activates reverse signaling inside cells, also increases. From these findings, it is suggested that FasL and ADAM10 play an important role in the production of pro‐inflammatory cytokines coming from interaction of the intervertebral nucleus pulposus cells and macrophages. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 608–615, 2013