椎间盘移植的实验研究

本实验研究目的在于：（１）建立间盘移植动物模型，（２）观察椎间盘移植术后不同时期Ｘ线、组织病理学、生物化学和生物力学变化。于１５只猴体上制备自体腰椎间盘移植模型，另有３只猴作为正常生物力学对照。结果表明移植间盘高度术后１～３个月呈下降趋势，４～１２个月呈恢复上升趋势。光镜下纤维环和终板软骨结构无明显改变，髓核中有部分细胞退变，同时亦有成软骨样纤维细胞的再生。术后２～４个月蛋白多糖和含水量呈下降趋势，６～１２个月有恢复；胶原含量２～６个月增加幅度较大，６～１２个月较平缓，其中髓核较纤维环明显。生物力学结果表明术后２个月时移植间盘活动度增大，术后４～１２个月恢复至正常水平。实验结果表明移植间盘在组织病理及生物化学方面有轻度退变倾向，同时又有一定的自我修复能力，生物力学上可满足生理活动功能需要。     

同种异体椎间盘移植的实验研究

本研究是在自体椎间盘移植实验研究的基础上进一步通过Ｘ线、组织病理、生物学活性、生物化学和生物力学探索异体椎间盘移植是否可存活、功能及其归宿。１２只猴随机分为４组，移植术后３、６、９和１２个月分别处死检测。结果表明移植间盘高度术后下降，但１２个月时仍保持正常高度的６１．４％。光镜下未见明显排斥反应，终板和纤维环结构无明显改变，术后早期可见移植间盘终板软骨增生现象，髓核基质密度增大，成软骨样纤维细胞增生明显。３Ｈ－ｐｒｏｌｉｎｅ掺入较对照组明显增加。术后移植间盘的蛋白多糖和水含量降低，而胶原含量增加。生物力学动态变化表明术后早期移植间盘有失稳趋势，晚期则稳定性恢复。上述结果显示同种异体移植间盘可存活，生化代谢虽有变化但有一定的自限性，形态结构无明显改变，生物力学满足功能需要。     

颈椎间盘纤维环及髓核生化成分的分析

目的：探讨在颈椎间盘纤维及髓核退变中生化成分的变化。方法：通过正常人、单纯颈椎间盘突出症、脊髓型颈椎病３组椎间盘纤维环及髓核生成分测量,主要测定水分、胶原蛋白及蛋白多糖的含量。结果：单纯颈椎间盘突出症与正常人椎间盘内生化成分无明显变化（Ｐ〉０．０５）,而在脊髓型颈椎病人的退变间盘中的生化成较正常人有明显变化（Ｐ〈０．０５）,主要表现为前者退变间盘中水分及蛋白多糖含量明显减少,而胶原蛋白明显增加,但Ⅰ／Ⅱ型胶原比无明显变化（Ｐ〉０．０５）。结论：单纯经椎间盘突出为退变的较早期阶段为可逆期,脊髓型颈椎病为退变晚期阶段,为不可逆期;颈腰间盘退变不完全相同。     

椎间盘移植实验—组织形态学研究

观察了６只猴自体腰椎间盘移植术后不同时期间盘组织形态学变化。结果表明：移植椎间盘高度术后１～３月呈下降趋势，４～６月呈恢复上升趋势。大体形态观察，移植间盘髓核的反光性减弱，呈较粘稠状。光镜下纤维环和终板软骨结构无明显改变，髓核中有部分细胞退变，同时亦有软骨样纤维细胞再生，表明间盘组织对损伤有一定的自体修复能力。组织形态学研究结果表明，猴自体椎间盘移植是可以接受的。     

TGF-β1干预下体内兔骨髓间充质干细胞对退变椎间盘治疗的实验研究

[目的]评价在TGF-β1干预下的兔间充质干细胞移植到兔退变椎间盘后是否可诱导向髓核细胞分化并增加蛋白多糖和Ⅱ型胶原的含量。[方法]采用体外细胞培养技术，将兔骨髓间充质干细胞白骨髓血中分离、纯化和培养，成功传代、纯化后并在TGF-β1干预下植入兔退变椎间盘的模型中。分别于2、4、6、8周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型胶原的含量变化。所得数据经SPSS 11．5统计学软件进行统计学处理。[结果]原代培养及传代培养显示兔骨髓间充质干细胞具有活跃的增殖倍增能力，并且8周内蛋白多糖和Ⅱ型胶原的含量的恢复实验组对比模型组增高明显，统计学显示差异有显著性意义。[结论]将TGF-β1干预下的兔间充质干细胞移植到退变椎间盘后可以在一定时间内增加蛋白多糖和Ⅱ型胶原的含量，从而延缓椎间盘退变。     

骨髓间质干细胞移植对椎间盘基质生化成分的影响

目的：研究骨髓间质干细胞移植对椎间盘基质生化成分的影响。方法：32只新西兰大白兔,L2～L3、L3～L4、L4～L5、L5～L6被随机分为正常对照组、生理盐水对照组、细胞移植组Ⅰ、细胞移植组Ⅱ（BMP2转基因组）。在1、3、6月时间点,用Ⅰ、Ⅱ型胶原单克隆免疫组化染色分析胶原表型的改变,用实时荧光PCR分析Ⅰ、Ⅱ型胶原及蛋白多糖的表达,采用方差分析线型模型进行统计学分析。结果：术后1、3、6个月时间点,细胞移植组髓核有较多的细胞,蛋白多糖染色显示,细胞移植组髓核兰染增加,免疫组化显示Ⅰ型胶原实验各组未见明显差异,Ⅱ型胶原细胞移植组有较强的红染颗粒沉积。实时荧光PCR分析显示,在细胞移植组,蛋白多糖和Ⅱ胶原的表达增加,而Ⅰ型胶原表达则无改变。对于蛋白多糖和Ⅱ胶原的表达,与细胞移植组Ⅰ比较,细胞移植组Ⅱ表达增加。结论：骨髓间质干细胞移植能够提高髓核中蛋白多糖和Ⅱ型胶原的含量,复合BMP2转基因有“分子增强效应”,提示基于骨髓间质干细胞移植策略是治疗椎间盘退变潜在的有效方法。     

脐带华通胶间充质干细胞移植对退变椎间盘影响的实验研究

目的:探讨人华通胶间充质干细胞(Wharton′s jelly-derived mesenchymal stem cells,WJMSCs)移植对犬退变椎间盘的影响。方法:从新生儿脐带中提取WJMSCs,取增殖良好的第3代细胞,用含有绿色荧光蛋白的腺相关病毒(r AAV2-EGFP)感染标记细胞。选择20只健康成年比格犬作为实验动物,使用穿刺抽吸髓核组织法建立椎间盘退变模型(L4/5、L5/6、L6/7)。4周后将犬各节段椎间盘进行分组:L3/4为对照组(A组);L4/5为退变组(B组);L5/6为注射组(C组),注射生理盐水;L6/7为移植组(D组),移植绿色荧光蛋白标记的WJMSCs细胞悬液。造模术前、术后4、8、12、24周行腰椎X线及MRI检查。24周后处死动物取材进行冰冻切片荧光、HE染色及番红O染色等组织学检测,提取髓核组织总RNA,反转录后行Real Time PCR检测,观察蛋白多糖、Ⅱ型胶原、SOX-9及Ⅰ型胶原基因表达变化。结果:分离培养的WJMSCs贴壁生长,呈梭形形态,r AAV2-EGFP病毒感染后第3天表达绿色荧光。影像学检查结果显示各组椎间盘高度指数及相对灰度指数在造模术前、术后第4周无统计学差异,术后8、12、24周,D组椎间盘相对高度指数及相对灰度指数较B、C组高(P0.05),比A组低(P0.05)。术后24周,D组髓核组织冰冻切片内能够检测到GFP阳性的WJMSC细胞,HE染色显示D组髓核组织退变比B组和C组轻,番红O染色结果显示D组染色较B组和C组深,基因表达检测结果显示D组Ⅱ型胶原、蛋白多糖及SOX-9基因表达比B、C组高(P0.05),但比A组低(P0.05)。结论:人WJMSCs移植入犬退变椎间盘内能够存活,促进椎间盘细胞外基质Ⅱ型胶原及蛋白多糖合成,维持椎间盘高度及髓核含水量,能够有效延缓椎间盘退变进展。     

猴自体椎间盘移植的组织学研究

本实验观察了猴自体椎间盘移植术的不同时期间盘组织学变化。结果表明大体形态移植间盘髓核的含水景减少，呈较粘稠胶状，光镜下纤维环和终板软骨结构无明显变化，髓核中有部分细胞退变，同时亦有成软骨样纤维细胞再生。透射电镜发现间盘髓核中仍有脊索细胞存在，移植后不同时间组髓与纤维环中均可见部分细胞变性坏死。本实验成功地建立了椎间盘移植动物模型，为进上步深入研究提供了依据。     

颈椎后纵韧带胶原、蛋白多糖、钙含量变化的研究

[目的]通过定量测定颈椎后纵韧带总胶原、Ⅰ型胶原、Ⅱ型胶原、蛋白多糖和钙含量改变,探讨颈椎后纵韧带在颈椎病发病机理中的意义。[方法]收集脊髓型颈椎病下位颈椎后纵韧带15例作试验组,同年龄段正常下位颈椎后纵韧带10例作对照,Weossner法测定两组总胶原含量;酶联免疫吸附法测定Ⅰ型、Ⅱ型胶原含量;间苯三酚分光光度法测定蛋白多糖含量;甲基百里香酚蓝比色法测定钙含量;进行HE染色和Masson染色,对比观察后纵韧带的显微结构变化特点。[结果]试验组总胶原、Ⅰ型胶原和蛋白多糖含量低于对照组;试验组Ⅱ型胶原含量较对照组高;试验组Ⅰ型/Ⅱ型胶原比值低于对照组;试验组钙含量较对照组高,组间对比均有统计学意义(P<0.05);病理结构显示试验组弹力纤维、胶原纤维肿胀,排列紊乱。[结论]颈椎病患者颈椎后纵韧带退变,总胶原、Ⅰ型胶原、Ⅱ型胶原、蛋白多糖和钙代谢发生紊乱,颈椎后纵韧带分子水平的生物化学变化与颈椎病发生发展有密切关系。     

细胞因子对退变椎间盘成份的影响

构成椎间盘的蛋白多糖含量随年龄增长而逐渐减少 ,其涵水能力下降 ,椎间盘高度和负重能力发生改变 ,影响了椎间盘的生物力学功能。椎间盘退变也与椎间盘组织中酶系活性改变和相关细胞因子的释放有关。这种成份变化的机制迄今仍不完全清楚。可能与椎间盘的生物化学和组织学性质改变有关 ,有多种细胞因子及酶可能在椎间盘退变的病理过程中发挥调节作用。现就细胞因子对退变椎间盘成份影响的研究状况综述如下 :1　椎间盘的成份及退变时的变化椎间盘主要由胶原、蛋白多糖、非胶原蛋白、细胞成份及大部分水组成。其中 ,蛋白多糖约占髓核干重的 30…     

脊柱内固定导致邻近节段椎间盘胶原变化的实验研究

目的：设计山羊脊柱内固定的动物模型,研究脊柱内固定对固定邻近椎间盘胶原的影响。方法：采用生化法测定胶原含量、胶原类型及胶原酶的变化以及反映固定3月及6月后胶原的变化。结果：脊柱内固定后椎间中胶原含量及胶原类型都有了显著性改变,胶原酶活性无变化。结论：脊柱内固定可导致邻近节段椎间盘退变。     

冷冻保存异体椎间盘移植的实验研究

目的 观察猴冷冻保存异体椎间盘移植后的Ｘ线、组织形态学、分子生物学、生物化学少生物力学的变化,探讨移植椎间盘的长期归宿及临床应用的可能性。方法 １７只猴中的１２只随机分为０．５、１、１．５、２、６和２４个月组。移植椎间盘梯度降温至－１９６℃保存,术前复温后手术植入。结果 Ｘ线显示无脱位,２４个月极能维持正常高度的６４．９％。术后２周权在移植椎间盘终板下骨与宿主椎体骨界面区有轻度免疫排斥反应,４周时     

Swelling pressure of the lumbar intervertebral discs: influence of age, spinal level, composition, and degeneration

The fluid content of the intervertebral disc is important in determining its mechanical response and also its transport and biologic properties. Fluid content depends on the proteoglycan content of the tissue and on the relationship of the external load to the disc's swelling pressure. The influence of proteoglycan content and external load on the hydration of nuclei from 32 human lumbar discs was measured. Swelling pressure of the same specimens was measured by equilibrium dialysis. The influence of age (14-91 years) and spinal level was noted. Proteoglycan content of the discs fell with age, and for all spines tested, proteoglycan content was lowest in the L5-S1 disc; no systematic change in collagen content was found. The hydration of the discs, as received, also fell with increase in age; in each complete lumbar spine tested, the L1-L2 and the L5-S1 discs had the lowest hydration at postmortem examination. As the stress applied to the discs was increased, hydration decreased. Although a stress of 0.10-0.23 MPa maintained the disc slices at their postmortem hydration, under a stress of about 0.6-0.8 MPa, most discs lost 40-60% of their initial fluid. The relationship between change in hydration and swelling pressure was found to depend on the composition of the disc rather than on age or degree of degeneration; the relationship between equilibrium hydration and swelling pressure could be predicted satisfactorily for a disc of known collagen and proteoglycan content.     

Correlating magnetic resonance imaging with the biochemical content of the normal human intervertebral disc.

Magnetic resonance imaging was used to determine the T2 relaxation times of prepared proteoglycan solutions and of normal human intervertebral disc tissue from the annulus fibrosus (AF) and nucleus pulposus (NP). The collagen, proteoglycan, and water contents of the disc tissue samples were determined by biochemical assays after they were scanned. Correlations among 1/T2, collagen, proteoglycan, and water contents of the tissue samples and among 1/T2, water, and proteoglycan contents of the proteoglycan solutions were calculated. A moderate negative correlation between 1/T2 and water content was noted for the tissue samples, and a very high negative correlation was found between 1/T2 and water content for the proteoglycan solutions. The very high positive correlation between 1/T2 and proteoglycan content of the proteoglycan solutions is probably due to this negative correlation between 1/T2 and water content. There was no significant correlation between 1/T2 and proteoglycan content of the tissues. The moderate positive correlation between 1/T2 and collagen content is probably due to the high negative correlation between collagen content and water content. No significant correlation was found between the collagen and proteoglycan contents of the tissues. Thus it appears that the data confirm previous reports in the literature that the collagen of the disc tissue functions to control its water content.     

The influence of weight-bearing exercise on articular cartilage of meniscectomized joints. An experimental study in sheep

Unilateral medial meniscectomy was undertaken in 29 purebred adult merino sheep. Arthrotomy (without meniscectomy) was conducted in 11 animals, and six were used as nonoperated controls. All animals were followed with either three- or six-month active or passive postoperative management. Active animals traversed a total of 360 km after three months and 1040 km after six months. The passive group was housed in pens allowing limited weight-bearing exercise. After death, morphologic changes were recorded and cartilages from the medial and lateral joint regions were examined for water, collagen, and proteoglycan (as hexuronate) content. Proteoglycan aggregation and extractability under nondissociative (0.4 molar GuHCl) and dissociative (4.0 molar GuHCl) conditions were also determined. The collagen content of the medial cartilage of the passively maintained meniscectomized animals was reduced, relative to external controls. Although proteoglycan content was elevated in medial cartilage of the passive group three months postoperatively, these levels returned to the control range after six months. However, low-salt (0.4 molar GuHCl) extractability of proteoglycans still remained high. All cartilages of control and meniscectomized joints showed an elevation of proteoglycan content in the active groups three months postoperatively. The cartilages in the medial region of meniscectomized animals showed the largest increase, but these levels declined after six months. Proteoglycan aggregation and water content were still elevated relative to controls six months postoperatively. The collagen levels in the three-month or six-month actively maintained meniscectomized group were not distinguishable from control values. Morphologically, joints of the passively and actively maintained animals showed focal surface fibrillation and erosions. However, in the active group, osteophytes were common and well developed six months postoperatively. These studies indicate that, while weight-bearing exercise after meniscectomy appears to be beneficial to the quality of the cartilaginous matrix, it is also accompanied by osteophytosis and cartilage hyperplasia.     

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.     

Experimental disc degeneration due to endplate injury

The aim of this study was to create an experimental model of disc degeneration that closely mimicked human disc degeneration. In six domestic pigs, an L4 cranial endplate perforation into the nucleus pulposus was made. Three months postoperatively, compressive testing was performed on the L2-L4 motion segments, and intradiscal pressure was measured in the intervening discs. Histochemical and morphologic examinations were made on the excised degenerated and adjacent discs. A significant reduction in water content was observed in the outer anterior annulus of the degenerated disc. In the nucleus, the proteoglycan content was significantly reduced, as well as the cellularity, although not significantly. The nucleus lost its gel-like structure and was discolored, and there was delamination of annular layers. Intradiscal pressure in the nucleus was significantly lower in the degenerated disc. In conclusion, experimental degeneration of the intervertebral disc induced by endplate penetration resembled human disc degeneration, as exemplified by biochemical and structural changes.     

Hamstring腱在体内重塑与转归的组织学研究

目的探讨游离Hamstring腱作为替代腱重建前十字韧带（anterior cruciate ligament，ACL）后在体内的重塑与转归过程，初步确定其术后在体内成熟的时间。方法33例关节镜下自体同侧游离4-5股Hamstring腱重建ACL的患者在行关节镜下再视手术时，于移植腱体中下段取活检组织进行组织学观察。重建术至再视手术的平均时间为11．9个月。替代腱依据重建术至再视手术的时段分为1月～、4月～、7月～、10月～、13月～、18月～和25月～组。将替代腱与正常ACL和半腱肌腱组织进行对比。结果重建ACL的Hamstring腱随植入时间延长，组织结构呈现其胶原纤维由不规则排列逐步向同向排列，排列不规则的菱形成纤维细胞逐步向较规则排列的椭圆形类纤维细胞转变，且细胞数目逐渐减少，血管腔数目也不断减少。上述的重塑变化主要发现在7月～组以前，其后各组重塑变化过程缓慢。结论自体游离Hamstring腱重建ACL术后具有良好的早期存活、加快再血管化和重塑过程的组织特性。其术后的重塑与成熟过程与自体髌腱相似，自体Hamstring腱在体成熟的时间为7-9个月。     

腺相关病毒载体介导结缔组织生长因子对体外转染恒河猴和人腰椎间盘细胞蛋白多糖和Ⅱ型胶原影响的比较

目的比较腺相关病毒载体介导结缔组织生长因子（connective tissue growth factor,CTGF）对体外培养的恒河猴和人腰椎间盘细胞转染后蛋白多糖含量和Ⅱ型胶原的影响。方法将恒河猴及成人腰椎间盘髓核细胞进行体外培养,应用rAAV2-CTGF体外转染细胞,分别通过^35S标记蛋白多糖的方法和Ⅱ型胶原的SP-ABC免疫组化法检测腺相关病毒载体介导生长因子对椎间盘细胞蛋白多糖合成和Ⅱ型胶原的影响。结果恒河猴椎间盘细胞能进行体外培养并传代,其形态学特性与培养的成人椎间盘细胞相似,rAAV2-CTGF与对照组相比可促进恒河猴和人椎间盘髓核细胞的蛋白多糖生物合成（P〈0．01）,恒河猴和成人相比生长因子对于蛋白多糖的促进作用无明显差异（P〉0．05）。结论成功培养恒河猴及成人椎间盘细胞,腺相关病毒载体介导CTGF能显著促进髓核细胞蛋白多糖的合成,生长因子对恒河猴和人椎间盘细胞蛋白多糖含量的影响具有很大的相似性。     

Type-II collagen gene expression is transiently upregulated in experimentally induced degeneration of rabbit intervertebral disc

To clarify phenotypic alterations of intervertebral disc cells during the repair process, we cloned partial type-II collagen cDNA from rabbits and analyzed the level of expression of type-II collagen mRNA in disc degeneration. An animal model was created by surgical denucleation of rabbit intervertebral discs through, an extraperitoneal approach. Eight animals each from an experimental and a control group were killed at 2, 4, 8, or 16 weeks postoperatively, and the disc samples were used for this study. Round chondrorcyte-like cells that filled the herniated space showed intense signal of type-II collagen mRNA and significant pericellular immunostaining of type-II collagen but no clear staining of type-I collagen. Northern. blot analysis revealed that the expression of type-II collagen mRNA of the repair disc cells was transiently increased at 4 weeks postoperatively. The cells were able to change their morphology in response to mechanical stimulation by surgical denucleation and to induce a significant increase in the gene expression of type-II collagen at an early phase of disc degeneration. The present results indicate the transient enhancement of repair activity in the degenerative process of injured fibrocartilage.