成年脊髓损伤大鼠脊髓神经干细胞的体外培养及分化研究

目的 探讨大鼠脊髓损伤后脊髓神经干细胞的分离培养方法及分化情况.方法 采用Allen法制作大鼠脊髓损伤模型,利用无血清培养和单细胞克隆技术在成年脊髓损伤7 d大鼠脊髓中分离具有单细胞克隆能力的神经干细胞,并进行培养鉴定.结果 从成年脊髓损伤7 d大鼠脊髓中成功分离出神经干细胞,该细胞具有连续克隆能力,可传代培养,表达神经巢蛋白抗原.分化后的细胞表达神经元细胞、星形胶质细胞和少突胶质细胞的特异性抗原.结论 致伤7 d的成年大鼠脊髓组织体外町培养出神经十细胞,并分化为神经无细胞、星形胶质细胞和少突胶质细胞,有可能参与脊髓损伤的修复过程.     

甲基强的松龙对大鼠脊髓损伤后神经细胞凋亡的影响及其机理

目的研究甲基强的松龙(MP)对大鼠横断性脊髓损伤(SCI)后神经细胞凋亡的影响及其作用机理.方法60只成年Wistar大鼠随机分成正常对照组、脊髓损伤组和MP治疗组,每组20只,MP治疗组在横断T10脊髓组织后30min经尾静脉给予MP治疗,损伤组和对照组未予任何治疗.MP治疗组和脊髓损伤组大鼠于脊髓损伤后8h、24h、3d和1周取材,采用透射电镜、TUNEL染色观察细胞凋亡情况,采用免疫组织化学染色观察Fas、半胱氨酸蛋白酶(caspase)-8和caspase-3在SCI前后的变化情况,采用改良Tarlov评分方法观察大鼠后肢运动功能.结果SCI后24h大鼠后肢运动功能逐渐恢复,MP治疗组大鼠的后肢运动功能恢复优于损伤组,7d后尤其明显.TUNEL染色和电镜检查证实SCI后8h即有凋亡细胞出现,其中既有神经元也有胶质细胞3d凋亡细胞数达到高峰;7d仍有凋亡细胞存在,但已明显减少;各时间点治疗组凋亡细胞数量明显少于损伤组(P＜0.05).治疗组和损伤组在SCI后8h可以检测到少量的Fas和caspase-8阳性神经元及胶质细胞,Fas和caspase-8的表达在SCI后3d达到高峰,7d表达下降,各时间点治疗组的Fas和caspase-8灰度值明显大于损伤组,二者有显著性差异(P＜0.05).治疗组和损伤组caspase-3的表达在SCI后8h均逐渐增加(与对照组相比),7d达到高峰,治疗组灰度值大于损伤组,但二者无显著性差异.结论MP能抑制大鼠脊髓横断性脊髓损伤后的神经细胞凋亡,但不能推迟细胞凋亡出现的高峰时间;MP抑制细胞凋亡的途径可能通过非特异性抑制Fas和caspase-8的表达来实现.     

大剂量甲基强地松龙对大鼠脊髓损伤后iNOS表达及细胞凋亡的影响

目的：探讨大鼠脊髓损伤后应用大剂量甲基强地松龙(MP)对诱导型一氧化氮合酶(iNOS)表达及细胞凋亡的影响。方法：成年大鼠随机分为脊髓损伤后应用大剂量甲基强的松龙治疗组(A组)和应用生理盐水对照组(B组)，损伤后不同时间点(4h、8h、1d、3d、7d、14d、21d)按Tarlov标准评价大鼠双后肢神经功能恢复情况，然后处死。用HE染色观察损伤脊髓组织病理变化，用免疫组化染色检测iNOS阳性细胞，原位末端标记法(TUNEL法)标记凋亡细胞。结果：HE染色镜检发现脊髓组织病理学改变A组明显轻于B组。A、B两组均发现凋亡细胞及iNOS表达，神经细胞凋亡指数及iNOS表达均为B组＞A组(P＜0．01)。结论：大剂量甲基强的松龙能抑制大鼠脊髓损伤后iNOS表达及细胞凋亡。     

诱导型一氧化氮合酶反义核酸对脊髓损伤后神经功能的影响

目的　观察诱导型一氧化氮合酶反义核酸 (ASODN iNOS)对大鼠脊髓损伤 (SCI)后神经功能恢复的影响。方法　设计并合成ASODN iNOS ,微量注入大鼠蛛网膜下腔后制备成脊髓压迫伤动物模型 ,伤后 6h用逆转录 聚合酶链反应 (RT PCR)检测iNOSmRNA表达变化 ,2 4h后用分光光度法测定组织中一氧化氮 (NO)含量和一氧化氮合酶 (NOS)活性 ,4周后用电生理、动物行为学和病理学等指标评价神经功能的恢复情况。对照组为正常组、损伤对照组和无义核酸对照组 (NSODN)。结果　SCI后组织中存在iNOS的表达 ,应用ASODN iNOS可以抑制相应酶的表达 ,并可以降低组织中的NO含量和NOS活性 ,改善神经传导功能 ,与损伤对照相比差异有显著性 ,NSODN没有上述作用。结论　脊髓损伤后应用iNOS反义核酸可以使伤后神经功能得到改善。     

丙戊酸对大鼠急性脊髓损伤后氧化应激的影响

[目的]探讨丙戊酸(VPA)对大鼠脊髓损伤(SCI)后氧化应激的影响。[方法]72只雄性SD大鼠随机分为3组:假手术组(C组)、损伤组(SCI组)和丙戊酸保护组(VPA组)。采用改良的Allen法制作脊髓损伤动物模型。VPA组术后即刻及其后每12 h皮下注射VPA 300 mg/kg至取材;C组和SCI组在相应时间点注射等体积的生理盐水。大鼠在伤后24、48、72 h和1周先行后肢运动功能BBB评分,随后处死取材。通过石蜡切片HE染色观察脊髓组织病理变化,并用免疫组化法检测诱导型一氧化氮合酶(iNOS)的表达;通过化学比色法测定脊髓组织中丙二醛(MDA)和谷胱甘肽过氧化物酶(GSH-Px)的含量。[结果]BBB评分显示C组运动功能未受影响,VPA组的BBB评分均高于SCI组,两者相比在伤后48、72 h和1周差异有显著性(P<0.01)。HE染色示C组脊髓组织形态正常,VPA组各时间点的病理变化与SCI组相比没有明显改善。C组偶见或未见iNOS阳性表达细胞。与C组相比,SCI组和VPA组的iNOS表达均明显增加(P<0.05),在伤后72 h达高峰,但VPA组的iNOS表达在各时间点均明显低于SCI组(P<0.05)。SCI组和VPA组脊髓组织的MDA含量明显高于C组,而GSH-Px活性明显低于C组(P<0.05),VPA组和SCI组相比较,MDA含量在各时间点均明显下降,GSH-Px活性均明显升高(P<0.05)。[结论]VPA通过减轻SCI所诱导的氧化应激,从而对SCI发挥保护作用。     

大鼠脊髓损伤后不同时期移植人脐带间充质干细胞的修复效果观察

目的:观察大鼠脊髓损伤后不同时期局部移植人脐带间充质干细胞(human umbilical cord mesenchy-mal stem cells,hUCMSCs)修复脊髓损伤的效果,探讨hUCMSCs局部移植治疗脊髓损伤(spianl cord injury,SCI)的最佳移植时机。方法:雌性Wistar大鼠80只,随机均分为A、B、C、D组,以Impactor Model-Ⅱ打击器制备脊髓胸10(T10)损伤模型。A组为单纯损伤组,B组损伤后3d移植hUCMSCs,C组损伤后1w移植hUCM-SCs,D组损伤后3w移植hUCMSCs,分别于造模后24h、移植前1d及移植后8w(A组与D组损伤后相应时间点相同)每周对各组大鼠后肢功能进行行为学评分(BBB评分);移植后8周免疫组化染色观察移植细胞的存活、分化和血管再生情况,10周时用生物素葡聚糖胺(biotin dextran amine,BDA)示踪皮质脊髓束(corticospinaltract,CST)观察轴突的再生情况。结果:SCI后24h各组大鼠BBB评分无显著性差异(P0.05),D组在移植前和移植后1周与A组相应时间点的BBB评分无显著性差异(P0.05),移植后2w起各时间点评分均明显高于A组相应时间点,差异有统计学意义(P0.05);A组在SCI后9w时到达平台期,与实验结束时相比差异无统计学意义(P0.05)。移植各组移植后BBB评分逐渐上升,后一个时间点与同组前一时间点比较差异有显著性意义(P0.05),移植后6w开始C组明显高于B、D组(P0.05)。免疫组化染色A组脊髓中未见Brdu阳性细胞,损伤区血管形态欠完整,未见新生血管;损伤周围星形胶质细胞大量增生,细胞肥大、突起增多、排列不规则,形成胶质瘢痕;未见BDA标记的皮质脊髓束通过损伤区。B、C、D组脊髓中均有Brdu阳性标记的细胞存活,C、D组中细胞数量明显多于B组,但三组中均未见到移植细胞向神经元或神经胶质样细胞分化;B、D组损伤周围的星形胶质细胞较A组减少,但排列仍欠规则;C组损伤周围星形胶质细胞形态趋于正常,排列规则,且有大量星形胶质细胞通过损伤区,血管再生数目及通过损伤区的皮质脊髓束神经纤维也明显多于B、D组。结论:大鼠SCI后局部移植的hUCMSCs能长期存活,抑制胶质瘢痕形成,改善后肢运动功能;SCI后1w移植的效果优于SCI后3d和3w时移植的效果。     

损伤性氧化反应在腹主动脉瘤形成中的作用

目的探讨诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）在人腹主动脉瘤（abdominal aortic aneurysm，AAA）组织中的表达情况，了解一氧化氮（nitric oxide，NO）在人AAA形成中的作用。方法22例AAA患者及10例正常人腹主动脉组织标本。采用原位杂交及免疫荧光染色的方法探查iNOS mRNA及蛋白的表达。采用原位杂交联合免疫荧光染色的方法探查阳性细胞的性质。采用免疫荧光染色检测过氧化亚硝酸盐。结果原位杂交和免疫荧光发现iNOS在22例AAA组织中外膜均有表达。联合免疫荧光染色证实阳性细胞分别为：T、B淋巴细胞、巨噬细胞和平滑肌细胞（smooth muscle cell，SMC）。22例AAA均出现硝基酪氨酸的表达，阳性细胞为巨噬细胞和SMC。10例正常腹主动脉组织均没发现有iNOS和硝基酪氨酸的表达。结论iNOS的表达与人AAA组织的退行性变关系密切。iNOS促进过氧化亚硝酸盐的生成，导致组织和细胞的氧化损伤。     

槲皮素抑制TLR4/NF-κB通路介导的炎症反应减轻脊髓损伤

[目的] 探讨槲皮素(Quecetin,Que)对大鼠脊髓损伤保护作用及可能机制。[方法] SD大鼠60只,随机分为3组:假手术组(sham组),脊髓损伤组(SCI组),槲皮素治疗组(Que+SCI组)。采用改良的Allen's方法制备大鼠脊髓损伤模型。Que+SCI组大鼠术后每天给予20 mg/kg的槲皮素治疗,持续14 d。脊髓损伤后1、3、7和14 d采用BBB评分法评估各组大鼠后肢的运动功能。术后14 d,收集大鼠脊髓组织,HE染色法观察组织病理变化,蛋白印迹法评价槲皮素对TLR4/NF-KB信号通路的作用。炎症因子肿瘤坏死因子-α(TNF-α)和白介素-1β(IL-1β)的产生采用酶联免疫法检测。此外,通过蛋白印迹法测定凋亡相关蛋白cleaved-caspase 3、Bax和Bcl-2的表达水平。[结果] 术后1、3、7和14 d,Que+SCI组BBB评分显著高于SCI组(P0.05)。HE染色结果发现SCI组可见明显的空洞形成,空洞周围有胶质瘢痕增生,而在Que+SCI组上述病理改变显著减轻。Que+SCI组的TLR4/NF-κB信号通路蛋白、TNF-α和IL-1β,以及cleaved-caspase 3和Bax蛋白表达水平显著低于SCI组(P0.05),而Que+SCI组的Bcl-2蛋白表达水平显著高于SCI组(P0.05)。[结论] 槲皮素抑制TLR4/NF-KB信号通路介导的炎症反应和细胞凋亡从而减轻脊髓损伤。     

Ephrin-B1在大鼠脊髓损伤的表达

[目的]探讨红细胞生成素诱导肝癌细胞株配体-B1 (Ephrin-B1)在大鼠脊髓损伤的表达变化.[方法]将60只成年Wistar大鼠分正常组、假手术组、伤后3、7、14 d及28 d组.假手术组大鼠不损伤脊髓,损伤组大鼠采用Allen's氏打击法损伤T10脊髓.分别在各个时间点取材,免疫荧光和Western Blot检测Ephrin-B1蛋白表达情况.[结果]免疫荧光结果显示Ephrin-B1在正常、假手术和损伤脊髓的灰质和白质中均有表达,但在损伤脊髓组织中表达较高,脊髓组织中的星形胶质细胞表达Ephrin-B1.Western Blot显示损伤组比对照组Ephrin-B1蛋白含量显著增加(P＜0.01),从伤后3d开始持续到伤后28 d;伤后14 d蛋白含量达到高峰,明显高于对照组及伤后其他组(P＜0.01);伤后28 d蛋白含量较14 d减少(P＜0.01),与伤后7d相比无显著性差异(P＞0.05).[结论]Ephrin-B1在大鼠脊髓损伤后表达升高,伤后14d表达升高显著,表达于脊髓的星形胶质细胞.     

Intra-operative spinal sonography in spinal intramedullary tumor

Recently, operative results of intramedullary spinal cord tumors have been greatly improved since the introduction of microsurgery. It is very important to know the precise size and location of the tumor prior to the operation so that we can approach the tumor with a minimum of damage to the spinal cord. However, it is not always possible to demonstrate the precise localization of the tumor preoperatively. In this report, we emphasize that intraoperative spinal sonography is very useful in determining the extent of the tumor and differentiating solid component from cystic component of the tumor. Methods and Materials We performed intraoperative spinal sonography on ten patients with intramedullary spinal cord tumor. This series included three cases of hemangioblastoma, three cases of astrocytoma, two cases of ependymoma, one case of subependymoma, and one case of mixed glioma. Eight out of ten cases were associated with cysts. The intraoperative spinal sonographic examinations were performed after laminectomy. The linear scanning probe of 5 or 7.5 MHz transducer was used. Results 1) Solid components The acoustic pattern of the solid tumor was either hyperechoic or iso-echoic. Six cases (three hemangioblastomas, two ependymomas, and one astrocytoma) were hyperechoic. Other four cases (two astrocytomas, one subependymoma, and one mixed glioma) were iso-echoic. 2) Cystic components The cysts associated with the tumor were anechoic in six out of eight cases, which were confirmed at surgery, and multiple cysts were identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Segmental spinal instrumentation for neuromuscular spinal deformity

Seventy-six consecutive surgical cases of paralytic neuromuscular spinal deformity were retrospectively analyzed. Posterior arthrodesis with segmental spinal stabilization with Luque L-rods, sometimes preceded by anterior release, was done in all cases. The infection rate of 14.5% was observed to be markedly higher in patients with myelodysplasia. Deep placement of the rods lateral to the spine and well beneath full-thickness skin is recommended to reduce the incidence of this complication.     

Rationale for spinal fusion in lumbar spinal stenosis

In order to define the indications for spinal fusion in patients undergoing decompression for lumbar spinal stenosis, 114 patients surgically treated were reviewed. Follow-up was 24 to 108 months. Patients were grouped into four categories: 15 with lateral recess stenosis, 45 with central-mixed stenosis, 43 with stenosis following prior lumbar surgery(s), and 11 with scoliosis and spinal stenosis. Only two patients with lateral recess stenosis underwent fusion with fair results. Approximately one-third of those with central-mixed stenosis required a fusion. Results were good in 70%. In those with stenosis following prior lumbar surgeries, although not statistically significant, those who had concomitant decompression and arthrodesis had a better outcome than those in whom decompression only was done. Patients with scoliosis and stenosis had decompression for significant motor and reflex deficits and fusion over the length of their major curves. Patients having decompression for lumbar stenosis with degenerative spondylolisthesis, isolated disc resorption with degenerative facet joints, intervertebral disc disease with instability, and those with scoliosis with multidirectional instabilities benefit from concomitant spinal fusion.