髓核内注射过氧化还原酶Ⅱ对兔椎间盘退变过程的影响

目的探讨髓核内注射人重组过氧化还原酶（Peroxiredoxin,Prx）对兔椎间盘退变过程的影响。方法通过纤维环穿刺法建立新西兰大白兔椎间盘退变模型（L3～4～L5～6）,24只兔随机分为高浓度组、低浓度组和对照组,每组各8只。4周后MRI显示造模成功,然后行L3～4～L5～6椎间盘内注射,高浓度组和低浓度组分别注射1 mg/mL、10μg/mL Prx蛋白,对照组注射0.1%磷酸盐缓冲液,注射量均为25μL。在注射后2、4、12、16周每组分别随机取2只兔行脊柱MRI检查,采用硫酸咔唑法检测髓核蛋白多糖的含量,免疫组化检测型胶原含量,并做病理切片HE染色观察组织及细胞的改变情况。结果蛋白注射后三组椎间盘均发生不同程度退变,注射后16周,高浓度组髓核内水分的含量明显低于低浓度组和对照组（P〈0.05）;髓核内蛋白多糖含量高浓度组显著低于低浓度组和对照组（P〈0.05）;细胞外基质型胶原灰度值高浓度组显著低于低浓度组和对照组（P〈0.05）,而低浓度组和对照组比较差异没有统计学意义（P〉0.05）。结论高浓度（1 mg/mL）Prx在椎间盘退变过程中起促进退变作用。     

直立体位下无创轴向加载建立兔椎间盘退变动物模型

目的:通过直立体位下无创性轴向加载的方式,建立一种新型兔腰椎间盘早期退变的动物模型。方法:24只4月龄雄性新西兰大白兔随机分为实验组及对照组。将实验组动物置于特制筒内使之保持直立体位,并自颈部施以600g轴向载荷,每日6h;对照组动物不接受任何处理而常规饲养。在实验开始前及实验开始后4周、8周、12周时对全部动物行X线及MRI检查,观察实验组动物在筒内时腰椎的骨性结构,通过椎间盘高度指数(disc height index,DHI)测量,比较两组动物侧卧位时L2/3、L4/5和L6/7节段椎间隙高度改变;通过髓核相对灰度值测量比较两组动物髓核含水量变化。14周后全部动物处死,取L5/6节段胶冻状髓核组织,应用rtPCR检测Ⅰ型胶原、Ⅱ型胶原及蛋白多糖表达水平;取L6/7节段椎间盘连同上、下各1mm软骨下骨,应用HE及天狼星红染色观察各组动物椎间盘组织结构改变。结果:2只实验组动物在实验过程中死亡,其对应的实验数据从结果中剔除。腰椎X线片显示,在直立体位负重条件下,实验组兔腰椎形成明显后凸,较侧卧位下腰椎间隙明显变窄,在L2/3节段,直立体位下椎间隙高度为侧卧位时的75.1%(P0.05);在L4/5节段为54.8%(P0.05);在L6/7节段为47.9%(P0.05)。DHI测量显示两组动物腰椎各节段DHI在任何时间点均无显著差异(P0.05)。MRI结果显示,在L2/3节段,实验组与对照组髓核灰度值在任何时间点均无统计学差异(P0.05),在L4/5节段,实验组与对照组髓核相对灰度值在12周时开始具有显著差异(P0.05);在L6/7节段,两组间在8周时即开始有显著性差异(P0.05)。rt-PCR结果显示,实验组Ⅰ型胶原m RNA表达明显高于对照组(3.57倍,P0.05);而Ⅱ型胶原及蛋白多糖表达明显低于对照组(分别为0.35倍和0.43倍,P0.05)。病理学检查显示对照组与实验组椎间盘组织结构存在显著差异,对照组髓核组织与纤维环间边界清晰,髓核内富含均匀分布的髓核细胞;实验组内层纤维环明显增生,而髓核区域相应减小。结论:直立体位下无创性轴向加载方式可显著加速兔腰椎间盘的退变进程,其在发病机理上与人类椎间盘退变更加相似,该腰椎间盘退变动物模型操作简单、可重复性强。     

腺病毒介导hTGF-β1基因体内转染兔腰椎间盘髓核细胞对蛋白多糖含量的影响

目的观察构建腺病毒载体介导外源性人转化生长因子(humantransforminggrowthfac-tor,hTGF-β1)基因(Ad/CMV-hTGF-β1)转染到兔椎间盘髓核细胞后,髓核组织中蛋白多糖含量的变化。方法(1)纯种成年新西兰大白兔35只,其中25只对腰椎间盘髓核组织注射Ad/CMV-hTGF-β1,每只注射2个椎间盘作为实验组,注射量为每个腰椎间盘20μl(6×106pfu);同时每只取2个未做注射的腰椎间盘作为自身空白对照组。其余10只兔每只注射2个腰椎间盘各20μl磷酸盐缓冲液(PBS)作为实验对照组。全组共120个椎间盘。(2)手术后1～12周的不同时间段分别取出各组椎间盘组织,采用间苯三酚分光光度法测定髓核组织中蛋白多糖的含量,所得数据经SPSS10.0统计学软件进行统计学处理。结果(1)术后1周,实验组与自身空白对照组蛋白多糖测定值经配对t检验,t=3.968,P<0.05。(2)术后2周,三组结果经方差分析显示,F=17.871,P<0.01;各组间再行两两q检验,实验组与自身空白对照组间q=7.686,P<0.01;实验组与实验对照组间q=6.894,P<0.01;实验对照组与自身空白对照组间q=0.792, P>0.05。实验组与自身空白对照组间配对t检验,t=5.276,P<0.01。(3)术后4周,实验组与自身空白对照组经配对t检验,t=8.352,P<0.01。(4)术后8周,实验组与自身空白对照组经配对t检验,t=7.086,     

重组腺相关病毒2介导hTGF-β_1基因体内转染兔退变髓核细胞对蛋白多糖含量的影响

目的应用重组腺相关病毒2(recombinant adeno-associated virus2,rAAV2)介导hTGF-β1基因体内转染兔退变椎间盘髓核细胞,观察基因产物的表达及其对退变髓核细胞蛋白多糖合成的生物调节作用。方法于24只成年新西兰大白兔,雌雄不限,体重1.7～2.2kg,L1、2、L2、3、L3、4、L4、5椎间盘注射25μL浓度为1mmol/L的纤维结合素片段(fi bronectin fragment,Fn-f)制备椎间盘退变模型,注射Fn-f4周时的造模椎间盘模拟早期退变的椎间盘。将24只兔随机分为3组(n=8),A、B、C组分别于造模椎间盘髓核内注射25μL rAAV2-hTGF-β1(1×1012vg/mL)、rAAV2-增强绿色荧光蛋白(enhanced green?uorescent protein,EGFP,rAAV2-EGFP)、PBS。术后1周A、C组各处死2只兔,取髓核组织采用免疫组织化学染色观察hTGF-β1的表达;术后4、8、12周每组取2只兔髓核组织,35S整合分析法检测新合成蛋白多糖的含量;术后12周处死B组2只兔,荧光显微镜观察髓核组织中EGFP的表达。结果术后1周,免疫组织化学染色示A组髓核细胞及基质中广泛存在强阳性染色颗粒,C组仅存在少量阳性颗粒。35S整合法检测示,各时间点A组35S蛋白多糖合成率均高于B、C组,比较差异有统计学意义(P0.05);B、C组间比较差异无统计学意义(P0.05)。术后12周,荧光显微镜下观察B组盘髓核组织可见大量绿色荧光表达。结论新型基因转导载体rAAV2可有效介导hTGF-β1基因体内转染兔退变髓核细胞,基因产物可持续表达超过12周,hTGF-β1可有效促进退变髓核细胞蛋白多糖的合成。     

腺病毒介导hTGT—β1基因体内转染兔腰间盘髓核细胞对蛋白多糖含量的影响

目的观察构建腺病毒载体介导外源性人转化生长因子(humantransforminggrowthfac-tor,hTGF-β1)基因(Ad/CMV-hTGF-β1)转染到兔椎间盘髓核细胞后,髓核组织中蛋白多糖含量的变化。方法(1)纯种成年新西兰大白兔35只,其中25只对腰椎间盘髓核组织注射Ad/CMV-hTGF-β1,每只注射2个椎间盘作为实验组,注射量为每个腰椎间盘20μl(6×106pfu);同时每只取2个未做注射的腰椎间盘作为自身空白对照组。其余10只兔每只注射2个腰椎间盘各20μl磷酸盐缓冲液(PBS)作为实验对照组。全组共120个椎间盘。(2)手术后1～12周的不同时间段分别取出各组椎间盘组织,采用间苯三酚分光光度法测定髓核组织中蛋白多糖的含量,所得数据经SPSS10.0统计学软件进行统计学处理。结果(1)术后1周,实验组与自身空白对照组蛋白多糖测定值经配对t检验,t=3.968,P<0.05。(2)术后2周,三组结果经方差分析显示,F=17.871,P<0.01;各组间再行两两q检验,实验组与自身空白对照组间q=7.686,P<0.01;实验组与实验对照组间q=6.894,P<0.01;实验对照组与自身空白对照组间q=0.792, P>0.05。实验组与自身空白对照组间配对t检验,t=5.276,P<0.01。(3)术后4周,实验组与自身空白对照组经配对t检验,t=8.352,P<0.01。(4)术后8周,实验组与自身空白对照组经配对t检验,t=7.086,     

自体富血小板血浆干预兔早期椎间盘退变的初步研究

目的富血小板血浆(platelet-rich plasma,PRP)具有刺激椎间盘细胞增殖、促进细胞外基质合成代谢及抑制纤维环细胞凋亡等作用。通过观察自体PRP干预兔早期椎间盘退变,明确其治疗效果,为临床应用提供理论依据。方法取健康成年新西兰大白兔45只,体重2.5～3.0 kg,雌雄不限;随机分为实验组、对照组、假手术组(n=15)。取实验组兔耳中央动脉血,采用Landesberg等方法制备PRP,同时对全血及PRP行血小板计数。实验组及对照组采用纤维环针刺法建立L4、5及L5、6椎间盘退变模型,造模2周后于L4、5及L5、6椎间隙分别注入100μL自体PRP及100μL PBS液;假手术组仅分离暴露椎间盘,不作处理。观察实验动物造模后一般情况;造模2周及干预1、2周时各组取5只实验动物行腰椎MRI、HE染色及Ⅱ型胶原免疫组织化学染色观察,腰椎MRI退变程度分级及Ⅱ型胶原阳性积分吸光度(IA)值检测。结果兔PRP中血小板计数约为外周血的4.92倍。实验动物均存活至实验完成。造模2周时,与假手术组相比,实验组和对照组椎间盘信号降低,髓核细胞减少,基质退变,Ⅱ型胶原表达降低。腰椎MRI退变程度分级及Ⅱ型胶原阳性IA值结果显示,各时间点实验组、对照组与假手术组相比差异均有统计学意义(P<0.05),干预1、2周时,实验组MRI退变程度分级显著低于对照组(P<0.05),但仍与假手术组有差异(P<0.05);干预1、2周时,实验组髓核细胞及软骨样基质较对照组增多,基质纤维化程度轻,Ⅱ型胶原表达明显强于对照组(P<0.05)。结论椎间盘内注射自体PRP可终止甚至一定程度逆转兔早期椎间盘退变,可能与PRP含有多种生长因子调控细胞功能、改善组织微环境、促进组织再生修复有关。     

兔退变椎间盘中髓核细胞凋亡与BNIP3基因表达的意义

目的髓核细胞凋亡可能与髓核组织代谢障碍产生缺氧,导致BNIP3基因表达有关。通过观察兔退变椎间盘中髓核组织的细胞密度、细胞凋亡率及BNIP3的表达,为进一步了解髓核细胞凋亡机制提供实验依据。方法健康3月龄雄性新西兰大白兔30只,体重(2.3±0.2)kg,随机分为实验组(n=20)及对照组(n=10)。实验组大白兔采用针刺L3、4、L4、5及L5、6椎间盘制备椎间盘退变模型;对照组仅暴露椎间盘后缝合。术后4、8周通过MRI检查评价椎间盘退变情况,采用组织学观察和TUNEL法检查椎间盘髓核组织中凋亡细胞,用免疫组织化学染色法检测兔椎间盘髓核细胞BNIP3的表达。结果 MRI检查示实验组术后4、8周椎间盘髓核信号强度呈逐渐降低趋势。根据Pfirrmann分级标准,实验组术后4、8周椎间盘退变分级比较,差异均有统计学意义(P0.05)。组织学观察及TUNEL检查示:对照组椎间盘髓核中细胞密度高,可见少量散在的凋亡细胞;实验组术后4、8周时椎间盘髓核组织内细胞密度逐渐降低,可见较多凋亡细胞。各时间点实验组细胞密度、TUNEL染色阳性细胞率与对照组比较,以及实验组各指标两时间点间比较,差异均有统计学意义(P0.05)。对照组椎间盘髓核组织细胞中无BNIP3表达;实验组术后4、8周椎间盘髓核组织细胞中BNIP3表达逐渐增多,BNIP3染色阳性细胞率分别为13.45%±1.16%、32.00%±1.82%,BNIP3灰度值分别为194.32±4.65、117.54±2.11,各时间点间比较差异均有统计学意义(P0.05)。结论椎间盘退变与髓核组织中细胞密度下降有关,细胞凋亡是椎间盘髓核细胞减少的原因之一,BNIP3参与了椎间盘髓核细胞凋亡。     

微囊化同种异体骨髓间充质干细胞移植预防兔椎间盘退变的实验研究

目的:探讨超顺磁性氧化铁(superparamagnetic iron oxide,SPIO)磁粒子标记的微囊化同种异体骨髓间充质干细胞(bone marrow mesenchymal stem cells,bMMSCs)移植预防椎间盘退变的可行性.方法:选用60只健康新西兰大白兔.随机平均分为A、B、C、D、E 5组,每组12只.A、B、C、D组应用髓核抽吸法制作L2/3、L3/4、L4/5椎间盘退变模型,取同种异体兔bMMSCs行体外培养,并在体外纯化扩增,用SHO标记后行微囊化,于造模手术当时植入(A组)兔手术节段椎间盘内;B组植入未微囊化bMMSCs;C组移植空微囊;D组仅制作模型,不移植;E组为正常对照组.分别于建模后2、4、6、8周时用MRI扫描对标记干细胞在椎间盘内分布进行示踪,并于相应时间点处死动物后取L2/3、L3/4、L4/5髓核组织,用间苯三酚分光光度法测定蛋白多糖含量的变化,免疫组化法测定Ⅱ型胶原含量的变化.所得数据进行统计学分析.结果:bMMSCs能被SPIO有效标记,应用MRI扫描可观察到其在体内的分布,8周时MRI图像与4周时相比,干细胞由注射部位向周边发生了迁徙.建模后2、4、6、8周各时间点.A组髓核中蛋白多糖和Ⅱ型胶原的含量均高于B组、C组和D组,差异有统计学意义(P<0.05);B组均高于C组及D组,差异有统计学意义(P<0.05);各时间点E组与B组、C组及D组相比差异有统计学意义(P<0.05);术后6、8周时A组与E组之间差异无统计学意义(P>0.05);各时间点C组和D组之间差异无统计学意义(P>0.05).结论:微囊化bMMSCs移植后能恢复兔髓核中细胞外基质含量,其效果优于单纯bMMSCs移植.     

经皮穿刺纤维环建立兔腰椎间盘退变模型

目的探讨使用自制穿刺针经皮穿刺纤维环制备兔腰椎间盘退变模型的可行性。方法将18只新西兰大白兔分为实验组、假手术组及空白对照组。实验组及假手术组使用自制穿刺针穿刺L_(3~4)、L_(4~5)和L_(5~6)椎间盘位置,实验组穿刺椎间盘深度为5 mm,假手术组钝性穿刺但不损伤椎间盘,空白对照组不作处理。术后3、6、9周每组取2只兔麻醉后行腰椎MRI检查,处死行大体观察并取椎间盘行HE染色及髓核蛋白多糖含量测定。结果术后3周开始实验组MRI信号强度、髓核蛋白多糖含量与其他两组相比明显下降(P0.05),其后呈逐渐下降趋势,大体观察及HE染色显示实验组髓核及纤维环呈逐渐退变趋势。结论自制穿刺针经皮穿刺纤维环法能成功建立兔腰椎间盘退变模型,具有操作简单、损伤小、动物存活率高等优点。     

微创针刺旋切制备兔椎间盘退变模型

目的探讨微创针刺旋切制备兔椎间盘退变(intervertebral disc degeneration,IDD)模型的可行性。方法取40只新西兰大白兔,雌雄不限,体质量(2.9±0.3)kg;随机分为对照组和实验组(n=20)。对照组不予处理;实验组采用18G穿刺针在C臂X线机引导下经皮侧后方穿刺进入L4、5、L5、6椎间盘内,旋切髓核组织以促进椎间盘的退变。术后4、8、12、16周行大体观察、MRI观察并根据Pfirrmann分级法评价椎间盘退变情况,然后处死动物取材行Masson染色和番红O染色观察。结果实验组髓核组织颜色较对照组暗,弹性降低。对照组MRI T2加权像椎间盘信号强度早期未见明显改变,后期略减弱;实验组椎间盘信号强度随时间延长呈减弱趋势。根据Pfirrmann分级法评价椎间盘退变程度,两组随时间延长椎间盘退变程度均逐渐加重(P0.05);两组间比较除术后4周差异无统计学意义(P0.05)外,其余术后各时间点实验组椎间盘退变程度较对照组严重(P0.05)。Masson染色示随时间延长,对照组纤维环出现排列不规整,但结构仍完整;实验组纤维环排列紊乱,甚至出现断裂现象。番红O染色示对照组髓核细胞未见明显减少,实验组髓核细胞明显减少。结论微创针刺旋切法可成功制备兔IDD模型。     

Modulation of the biologic activity of the rabbit intervertebral disc by gene therapy: an in vivo study of adenovirus-mediated transfer of the human transforming growth factor beta 1 encoding gene

STUDY DESIGN: In vivo studies using a rabbit model to determine the biologic effects of direct, adenovirus-mediated transfer of a therapeutic gene to the intervertebral disc. OBJECTIVES: 1) To deliver an exogenous therapeutic gene to rabbit lumbar intervertebral discs in vivo, 2) to quantify the resulting amount of gene expression, and 3) to determine the effect on the biologic activity of the discs. SUMMARY OF BACKGROUND DATA: Although growth factors such as transforming growth factor beta 1 appear to have promising therapeutic properties, there currently is no practical method for sustained delivery of exogenous growth factors to the disc for the management of certain chronic types of disease (e.g., disc degeneration). A possible solution is to modify the disc cells genetically through gene transfer such that the cells manufacture the desired growth factors endogenously on a continuous basis. METHODS: Saline, with or without virus, was injected directly into lumbar discs of 22 skeletally mature female New Zealand white rabbits. Group 1 (n = 11) received the adenovirus construct Ad/CMV-hTGF beta 1 containing the therapeutic human transforming growth factor beta 1-encoding gene. Group 2 (n = 6) received adenovirus containing the luciferase marker gene. Group 3 (n = 5) received saline only. The rabbits were killed 1 week after injection. Immunohistochemical staining for human transforming growth factor beta 1 was performed on the disc tissues of one rabbit from Group 1. Nucleus pulposus tissues from the remaining rabbits were cultured in serumless medium. Bioassays were performed to determine human transforming growth factor beta 1 production and proteoglycan synthesis. RESULTS: Discs injected with Ad/CMV-hTGF beta 1 exhibited extensive and intense positive immunostaining for transforming growth factor beta 1. The nucleus pulposus tissues from the discs injected with Ad/CMV-hTGF beta 1 exhibited a 30-fold increase in active transforming growth factor beta 1 production, and a 5-fold increase in total (active + latent) transforming growth factor beta 1 production over that from intact control discs (P < 0.05). Furthermore, these tissues exhibited a 100% increase in proteoglycan synthesis compared with intact control tissue, which was statistically significant (P < 0.05). CONCLUSIONS: The results of this study suggest that the intervertebral disc is an appropriate site for adenovirus-mediated transfer of exogenous genes and subsequent production of therapeutic growth factors. Gene therapy therefore may have useful applications for study of the basic science of the intervertebral disc and for clinical management of degenerative disc disease.     

Does long-term compressive loading on the intervertebral disc cause degeneration?

STUDY DESIGN: Coil springs were stretched and attached to produce a compressive force across the lumbar intervertebral discs of dogs for up to 53 weeks. OBJECTIVE: To test the hypothesis that compressive forces applied to the intervertebral disc for a long period of time cause disc degeneration in vivo in a dog model. SUMMARY OF BACKGROUND DATA: It is a commonly held belief that high forces applied to the intervertebral disc, and to joints in general, play a role in causing degeneration. METHODS: Coil springs were stretched and attached to produce a compressive force across the lumbar intervertebral discs (L3/L4) of 12 dogs. After up to a year, the dogs were killed, and their lumbar spines were removed and radiographed. The L3/L4 disc and the controls (T13/L1 and L4/L5) were excised and examined for visible signs of degeneration. The discs then were assessed using immunohistochemical analysis and enzyme-linked immunosorbent assay. Disc chondrocytes also were assayed for apoptosis. RESULTS: No obvious signs of degeneration in the discs (L3/L4) that had been under compression for up to a year could be observed. There was no disc bulging, anular fissures, or disc space narrowing. Some changes were observed at the microscopic level, although no thickening of the endplate was apparent. The enzyme-linked immunosorbent assay analysis provided significant data for all three regions of the disc (nucleus, inner anulus, and outer anulus). When comparing the compressed disc (L3/L4) with either of the control discs (T13/L1 and L4/L5), in the compressed disc: 1) the nucleus contained less proteoglycan and more collagen I and II; 2) the inner anulus contained less proteoglycan and collagen I; and 3) the outer anulus contained more proteoglycan and less collagen I. The collagen II differences for the inner and outer anulus were not significant. CONCLUSION: Compression applied to the lumbar intervertebral discs of dogs for up to a year does not produce degeneration in any visible form. It does produce microscopic changes and numerical changes, however, in the amounts of proteoglycan and collagen in the nucleus, inner anulus, and outer anulus. The present results add no credence to the commonly held belief that high compressive forces play a causative role in disc degeneration.     

Bone morphogenic protein-2 signaling in human disc degeneration and correlation to the Pfirrmann MRI grading system

BACKGROUND CONTEXTBack and neck pain secondary to disc degeneration is a major public health burden. There is a need for therapeutic treatments to restore intervertebral disc (IVD) composition and function.PURPOSETo quantify ALK3, BMP-2, pSMAD1/5/8 and MMP-13 expression in IVD specimens collected from patients undergoing surgery for disc degeneration, to correlate ALK3, BMP-2, pSMAD1/5/8 and MMP-13 expression in IVD specimens to the 5-level Pfirrmann MRI grading system, and to compare ALK3, BMP-2, pSMAD1/5/8 and MMP-13 expression between cervical and lumbar degenerative disc specimens.STUDY DESIGNAn immunohistochemical study assessing ALK3, BMP-2, pSMAD1/5/8, and MMP-13 expression levels in human control and degenerative IVD specimens.METHODSHuman IVD specimens were collected from surgical patients who underwent discectomy and interbody fusion at our institution between 1/2015 and 8/2017. Each patient underwent MRI prior to surgery. The degree of disc degeneration was measured according to the 5-level Pfirrmann MRI grading system. Patients were categorized into either the 1) control group (Pfirrmann grades I-II) or 2) degenerative group (Pfirrmann grades III-V). Histology slides of the collected IVD specimens were prepared and immunohistochemical staining was performed to assess ALK3, BMP-2, pSMAD1/5/8, and MMP-13 expression levels in the control and degenerative specimens. Expression levels were also correlated to the Pfirrmann criteria. Lastly, the degenerative specimens were stratified according to their vertebral level and expression levels between the degenerative lumbar and cervical discs were compared.RESULTSFifty-two patients were enrolled; however, 2 control and 2 degenerative patients were excluded due to incomplete data sets. Of the remaining 48 patients, there were 12 control and 36 degenerative specimens. Degenerative specimens had increased expression levels of BMP-2 (p=.0006) and pSMAD1/5/8 (p<.0001). Pfirrmann grade 3 (p=.0365) and grade 4 (p=.0008) discs had significantly higher BMP-2 expression as compared to grade 2 discs. Pfirrmann grade 4 discs had higher pSMAD1/5/8 expression as compared to grade 2 discs (p<.0001). There were no differences in ALK3 or MMP-13 expression between the control and degenerative discs (p>.05). Stratifying the degenerative specimens according to their vertebral level showed no significant differences in expression levels between the lumbar and cervical discs (p>.05).CONCLUSIONSBMP-2 and pSMAD1/5/8 signaling activity was significantly upregulated in the human degenerative specimens, while ALK3 and MMP-13 expression were not significantly changed. The expression levels of BMP-2 and pSMAD1/5/8 correlate positively with the degree of disc degeneration measured according to the Pfirrmann MRI grading system.CLINICAL SIGNIFICANCEBMP-SMAD signaling represents a promising therapeutic target to restore IVD composition and function in the setting of disc degeneration.     

TGF-β3修饰退变髓核细胞移植修复兔退变椎间盘的效果观察

目的探讨TGF-β3基因修饰后退变髓核细胞生物学效应以及植入兔退变椎间盘后对退变椎间盘的影响。方法将重组腺病毒载体Ad-TGF-β3与第2代退变髓核细胞按10∶1比例混合培养转染(Ad-TGF-β3组),待细胞融合后传代,MTT检测转染细胞增殖活性,Western blot检测TGF-β3蛋白含量,免疫细胞化学染色观察对数生长期转染细胞Ⅱ型胶原染色阳性率;采用病毒空载体转染髓核细胞(Adv组)和未经转染髓核细胞(空白组)作为对照。取30只新西兰兔,体重3.2～3.5 kg,雌雄不限,通过针刺L3、4、L4、5和L5、6椎间盘制备椎间盘退变模型。将实验动物按照随机数字法分为3组,转染细胞组(A组,n=12)、退变细胞组(B组,n=12)和空白对照组(C组,n=6)。A、B组将100μL浓度为1×105个/mL对应细胞悬液注射入退变椎间盘,C组同法注入等量PBS。注射后6、10、14周取A、B组各4只、C组2只实验动物处死,取L3、4、L4、5和L5、6椎间盘行组织学观察,RT-PCR检测Ⅱ型胶原和蛋白多糖mRNA表达。结果 Ad-TGF-β3转染后髓核细胞活性明显改善;转染后3、7、14 d,TGF-β3在髓核细胞内表达逐渐升高;Ad-TGF-β3组髓核细胞细胞质内见棕黄色Ⅱ型胶原阳性染色,阳性率显著高于Adv组及空白组(P<0.05)。组织学观察示,A组椎间盘退变程度较B、C组明显减轻。6、10、14周A组Ⅱ型胶原和蛋白多糖mRNA表达显著高于B、C组,差异均有统计学意义(P<0.05)。结论 TGF-β3基因修饰退变髓核细胞后可明显改善细胞生物活性,转染后髓核细胞植入兔体内可明显增加退变椎间盘的基质分泌。     

终板下椎体缺血诱导兔椎间盘退行性变模型的建立及终板中内皮素1的表达

目的通过终板下注射无水乙醇阻碍椎体-终板营养,建立一种新型兔腰椎椎间盘退行性变模型,并观察终板退行性变过程中内皮素1(ET-1)的表达情况。方法健康4月龄新西兰兔32只,随机分成4组,每组8只,选取L5,6椎体(对应L4/L5及L5/L6椎间盘)注射300μL无水乙醇,选取L4椎体(对应L3/L4椎间盘)注射磷酸盐缓冲液(PBS)作为实验对照,L7椎体(对应L6/L7椎间盘)未注入任何物质作为正常对照。其中1组造模后1个月提取软骨终板细胞,行免疫细胞化学染色检测ET-1表达;余3组分别于造模后1、3和5个月进行椎间盘X线和MRI检查,取椎间盘组织行HE染色观察形态学改变,免疫组织化学染色观察ET-1表达。结果注射无水乙醇后,随着时间进展,X线片显示椎间隙高度显著下降、椎间隙变窄、边缘骨赘增生,MRI T2WI显示椎间盘低信号;苏木精-伊红染色(HE)显示终板的生长板厚度变薄,终板结构破损,同时软骨终板细胞退化、直至消失,髓核中细胞发生转化(由空泡细胞转变为软骨样细胞,进而形成纤维软骨样细胞)造成髓核纤维化,纤维环结构排列紊乱、纤维化程度逐步加重;免疫组织化学染色显示,发生退行性变的终板组织内有ET-1表达,但随着退行性变加剧,ET-1表达强度下降;提取的退行性变软骨终板细胞(造模后1个月)也显示细胞质内ET-1强表达。结论通过注射无水乙醇阻碍椎体-终板营养途径可成功建立兔椎间盘退行性变模型,终板退行性变过程中伴随ET-1的表达。     

应用微创技术建立恒河猴腰椎间盘早期退变模型

目的 应用CT定位,经皮穿刺纤维环诱导恒河猴腰椎间盘退变,建立灵长类动物腰椎间盘早期退变模型.方法 恒河猴13只,随机分为三组:(1)造模组:在CT定位下,用20G穿刺针从左侧后方入路经皮穿刺L1,2:(n=12),L2,3、L3,4、L4,5、L5,6(n=13)椎间盘的纤维环全层至椎间盘髓核正中,共64个椎间盘.(2)穿刺对照组:15G穿刺针穿刺1只猴的L1,2椎间盘.(3)正常对照组:L6,7,L7-S1,共26个椎间盘.造模前及造模后4、8、12周对各组椎间盘行MRI检查,并行HE、Masson、番红O、免疫组织化学染色组织学观察.结果 (1)MRI:20G穿刺针穿刺的造模组椎间盘造模前及造模后4、8、12周,椎间盘信号强度按Pfirmann分级均为Ⅰ级.15G穿刺针穿刺椎间盘4周时信号降低(Pfirrmann Ⅲ级),8周时为黑色椎间盘(Pfirmann Ⅳ级).正常对照组椎间盘为Pfirmann Ⅰ级.(2)组织学:造模组椎间盘造模后4周未见改变,8周时HE染色示髓核内细胞数减少,12周时较为明显.Masson染色4周未见改变,8周时各层纤维间出现裂隙,12周时裂隙增宽.番红O染色见8、12周髓核内蛋白聚糖进行性减少.免疫组织化学结果显示4周和8周时同正常椎间盘比较差异无统计学意义(P＞0.05),12周时,Ⅱ型胶原合成减少(P＜0.05).15G穿刺对照组在8周时HE染色见髓核内细胞减少明显,Masson染色见纤维环各层间裂隙明显,呈波浪状.番红O染色示髓核内蛋白聚糖数量明显减少.免疫组织化学染色示Ⅱ型胶原合成减少.正常对照组在各时间点未见到形态学改变.结论 20G穿刺针可以诱发椎间盘缓慢进展的轻度退变.MRI平均信号强度观察椎间盘轻度退变时,不是敏感的指标,需要依靠组织学证实.     

两种骨水泥渗漏对椎间盘影响的实验研究

目的 探讨椎体成形术时骨水泥渗漏是否会引起椎间盘退变，以及椎间盘退变程度与骨水泥类型是否相关。方法 选用8只成年家犬，以每只犬L2-3、L3-4、L4-5椎间盘为实验对象，随机分为对照组、聚甲基丙烯酸甲酯（polymethylmethacrylate，PMMA）与磷酸钙骨水泥（calcium phosphate cement，CPC）3组。对照组仅行椎间盘穿刺，不注入任何物质，PMMA组及CPC组均各向椎间盘注入0．1ml骨水泥。术前及术后24周摄正、侧位X线片，计算椎间盘高度指数百分数（disc height index percentage，DHIP）。术后24周行MR检查，计算MRI指数。组织学检查参照Masuda标准对椎间盘退变程度评分并分析。结果 术后24周X线片显示对照组椎间隙无狭窄，病理学检查未见椎间盘退变。PMMA、CPC组椎间盘MRI显示：椎间隙有狭窄，R加权像髓核信号不同程度降低且不均一，其相对高信号区面积减小，髓核形态不规则，纤维环与髓核界限不清。组织学检查显示髓核细胞数量不同程度减少，空泡变小。髓核的细胞外基质不同程度压缩，纤维环断裂或扭转。3组DHIP、MRI指数、组织学评分的差异均有统计学意义（P〈0.01）。结论 PMMA、CPC注入椎间盘会导致椎间盘退变，PMMA所致椎间盘退变较CPC更为严重.     

兔退变椎间盘体内转染hTGF-β1、β3的生物学效应

目的 应用重组腺相关病毒2(recombinant adeno-associated virus,rAAV2)介导人转化生长因子β1(human transforming growth factor-β1,hTGF-β1)和β3(hTGF-β3)单基因或双基因联合体内转染退变兔髓核细胞,观察基因产物的表达及其对基质成分...