一种改良方法构建大鼠脊髓损伤体外细胞模型

目的探讨大鼠脊髓神经细胞原代培养、鉴定的方法并构建大鼠脊髓损伤体外细胞模型。方法取孕15 d SD大鼠胚胎,分离脊髓组织并采用改良的胰酶消化联合机械分离法进行脊髓神经细胞的原代培养;采用免疫荧光三标染色鉴定脊髓神经细胞;应用H₂O₂干预构建大鼠脊髓损伤体外细胞模型。结果取孕15 d大鼠胚胎采用改良的胰酶消化联合机械分离法成功分离培养原代脊髓神经细胞。免疫荧光染色可见特异的NeuN及β tubulin-Ⅲ染色,符合脊髓神经细胞的特征,经鉴定脊髓神经细胞纯度为90%。应用H₂O₂干预大鼠原代脊髓神经细胞建立脊髓损伤体外细胞模型时以干预浓度700μM,时间3 h最为合适。结论采用改良的胰酶消化联合机械分离法分离培养大鼠脊髓原代神经细胞,细胞产量高、活性好,纯度可以达到90%;采用H₂O₂干预大鼠脊髓原代神经细胞以建立脊髓损伤体外细胞模型可靠性高,重复性好。     

硫酸软骨素酶ABC对大鼠急性脊髓损伤后神经中丝和胶质纤维酸性蛋白的影响

目的：探讨硫酸软骨素酶ABC（ChABC）对大鼠急性脊髓损伤后神经中丝200（NF200）和胶质纤维酸性蛋白（GFAP）的影响.方法：SD大鼠72只,雌雄不限,随机分为假手术组（A组）、损伤对照组（B组）和ChABC治疗组（C组）,每组24只.A组仅打开椎板及置管,不损伤脊髓,不给药;C组和B组均采用Allen＇s法制作大鼠T10脊髓损伤模型,分别在伤后即刻和随后每天1次连续1周蛛网膜下腔注射ChABC（6μl/次）和等量生理盐水.术后1d、1周、2周和4周每组各处死6只大鼠,B组和C组以损伤区为中心、A组在相应部位切取1cm长的脊髓组织,以HE染色观察脊髓组织形态变化,应用免疫组化方法检测脊髓组织中NF200和GFAP的变化.结果：HE染色示A组脊髓无胶质细胞增生和胶质瘢痕形成;B、C组脊髓损伤区有胶质细胞增生和胶质瘢痕,C组明显少于B组.A组术后1d、1周、2周和4周时NF200阳性细胞数及灰度值和GFAP染色阳性面积无差异,1、2、4周时B、C组脊髓损伤区NF200染色阳性细胞数及灰度值和GFAP染色阳性面积均较A组显著增加（P＜0.05或P＜0.01）,1d和1周时C组NF200染色阳性细胞数及灰度值与B组比较无显著性差异,2周和4周时C组明显高于B组（P＜0.05）;1d、1周和4周时C组GFAP染色阳性面积与B组无显著性差异,2周时C组显著小于B组（P＜0.05）.结论：ChABC能提高大鼠急性脊髓损伤后神经细胞内NF200的表达并抑制GFAP的表达,进而促进神经细胞的修复,抑制胶质细胞的增生和胶质瘢痕的形成,对脊髓损伤具有保护作用.     

牵张性脊髓损伤后神经细胞中Caspase-3表达及其作用的实验研究

目的:观察大鼠牵张性脊髓损伤后脊髓神经细胞中半胱氨酸天冬氨酶3(Caspase-3)的表达及其在神经细胞凋亡中的作用。方法:大鼠脊髓T13~L2经牵张损伤,皮层体感诱发电位监测P1-N1波幅下降至术前波幅70%后维持10m in,分别于术后6h、1、4、7、14、21d处死取材。采用流式细胞仪、原位末端脱氧核苷酸转移酶介导的生物素脱氧尿嘧啶核苷酸缺口末端标记法(TUNEL法)、免疫组织化学检测等方法观察大鼠脊髓神经细胞中Caspase-3表达变化及神经细胞凋亡情况,测定Caspase-3活性。结果:脊髓损伤后神经细胞凋亡率、TUNEL阳性细胞数、Caspase-3免疫组织化学阳性表达及Caspase-3活性测定均较空白对照组及椎板切除组显著升高(P<0.05或0.01),前三项指标改变趋势大致相同,均为术后7d达高峰,而Caspase-3活性则术后4d达高峰。结论:大鼠牵张性脊髓损伤后神经细胞中Caspase-3表达增高、Caspase-3活性增强,是检测神经细胞凋亡的早期生物学指标,对认识脊髓损伤机制具有一定的意义。     

胶质细胞源性神经营养因子对大鼠脊髓损伤后运动功能的影响

目的:研究胶质细胞源性神经营养因子（GDNF）对大鼠脊髓损伤后后肢运动功能恢复的影响。方法:采用改良Nystr(?)m法后路压迫大鼠胸段脊髓造成损伤模型,经蛛网膜下腔置管局部连续给予NGF （10μg/d）或GDNF（10μg/d）1周,对照组给予生理盐水。伤后4周3组分别观测:①伤段脊髓残存组织面积;②采用斜板试验和运动功能评分观察大鼠后肢运动功能恢复情况。结果:大鼠脊髓损伤后4～14d,GDNF治疗组后肢运动功能评分明显高于NGF组和生理盐水对照组（P<0.05）。伤后28d GDNF组伤段残存脊髓组织面积大于对照组和NGF组（P<0.01）。结论:外源性GDNF能减少脊髓损伤后伤区的坏死、萎缩并促进大鼠后肢运动功能的早期恢复。     

局部应用FK506缓释膜片对大鼠坐骨神经缝合术后脊髓神经元的影响

目的探讨大鼠坐骨神经切断缝合术后局部应用FK506缓释膜片对脊髓神经元的保护。方法建立大鼠坐骨神经切断缝合术模型。术后分为治疗组(A组):术中神经缝合后在神经缝合口周围放置FK506缓释膜片(FK506释放率为2mg·kg·d);对照组(B组):不用药物。A、B组大鼠各为25只。于术后1、3、7、14、28d5个时相点,切取L46脊髓。标本按常规固定、脱水、冰冻切片、切片厚度为5μm,每隔20张取1张切片,每个标本取10张。采用TUNEL法行细胞凋亡检测。结果A组中仅发现少量脊髓神经元凋亡,B组的凋亡细胞明显多于A组,两组差异有统计学意义(P<0.05)。结论大鼠坐骨神经切断缝合术后局部应用FK506缓释膜片对脊髓神经元有保护作用。     

活性巨噬细胞移植治疗实验动物脊髓损伤的疗效分析

目的　研究活性巨噬细胞在脊髓损伤早期移植后对星形胶质细胞和神经轴突的作用 ,明确该细胞对脊髓损伤的作用效果。方法　将 48只SD大鼠采用随机配对的方法分为 2 4组 ,每组大鼠分别行T1 0 段脊髓撞击性损伤和伤后巨噬细胞悬液髓内注射。术后 1、 4d和 1、 2、 3、 4周收集脊髓标本并冰冻切片、免疫组化染色 ,显微镜下巨噬细胞、星形胶质细胞及神经纤维计数。结果　移植组术后 4周时BBB运动功能评分无明显提高 ,镜下观察见移植组大鼠在伤后 3周内巨噬细胞数目高于对照组 ,星形胶质细胞在损伤早期的反应较轻 ,但单位截面积上的神经轴突数目少于对照组。结论　活性巨噬细胞在撞击性脊髓损伤早期移植可加重神经轴突的继发性损伤 ,影响预后     

人胚胎嗅鞘细胞移植修复大鼠脊髓全横断损伤

[目的]探索人胚胎嗅鞘细胞(hOECs)移植修复大鼠脊髓全横断损伤的可行性。[方法]分离、培养并鉴定人胚嗅鞘细胞,24只W istar大鼠,随机分为实验组和对照组,均行T10节段脊髓全横断。术后第9~10 d,实验组、对照组分别移植Hoechst33342标记的hOECs 5μl(2.5×105个细胞)、DMEM-F12培养基5μl。移植术后第1、2、4、6、8、10周进行BBB运动功能评分,取材行荧光化学(Hoechst33342)和免疫组织化学染色(P75、NF-200、Syn-aptophysin)。双盲条件下进行数据统计。[结果]移植的hOECs可以在损伤脊髓内存活10周以上,可向损伤脊髓头尾两端迁移;术后4~10周实验组的BBB运动功能评分明显高于对照组(P<0.01);P75染色实验组呈阳性反应;NF-200和Synaptophysin免疫组化染色,实验组神经纤维、突触数目和密度都较对照组高。[结论]hOECs移植对脊髓全横断损伤大鼠运动功能恢复具有促进作用。     

脊髓电刺激对糖尿病神经痛大鼠脊髓胶质细胞谷氨酸转运体-1和谷氨酸-天冬氨酸转运体的影响

目的观察脊髓电刺激对糖尿病大鼠痛性周围神经病变时脊髓胶质细胞谷氨酸转运体-1(GLT-1)和谷氨酸-天冬氨酸转运体(GLAST)表达的影响。方法选择健康雄性SD大鼠48只,2月龄,体重250~300 g,采用随机数字表法将其分为四组:对照组(C组)、糖尿病神经痛组(D组)、假刺激组(N组)和脊髓电刺激组(S组),每组12只。D、N和S组腹腔注射1%链脲佐菌素60 mg/kg制备大鼠糖尿病痛性周围神经病变模型,C组注射等容量枸橼酸-枸橼酸钠缓冲液。N组和S组于造模后19 d在硬膜外间隙置入电极,S组于造模后26~28 d行脊髓电刺激。于造模前1 d、造模后2、7、14、28 d测定机械缩足反应阈(MWT)。于造模后28 d测定痛阈后处死大鼠,取L4~5脊髓组织,采用荧光定量PCR法和Western blot法检测脊髓背角GLT-1和GLAST m RNA表达量和蛋白含量。结果与C组比较,造模后14、28 d D组、N组和S组MWT明显降低(P0.05);造模后28 d S组胶质细胞GLT-1及GLAST m RNA表达量和蛋白含量明显增加(P0.05)。与造模前1 d比较,造模后14、28d D、N、S组MWT明显降低(P0.05);与D组比较,造模后28 d S组MWT明显升高,脊髓胶质细胞GLT-1及GLAST m RNA表达量和蛋白含量明显增加(P0.05)。结论脊髓电刺激减轻糖尿病大鼠神经痛的机制可能与上调脊髓胶质细胞GLT-1及GLAST基因的表达有关。     

绿色荧光蛋白转基因大鼠骨髓间充质干细胞在急性脊髓损伤大鼠中的迁移和分化

目的观察绿色荧光蛋白转基因大鼠来源的骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)在急性脊髓损伤大鼠脊髓组织中的迁移和分化情况。方法全骨髓贴壁培养法培养BMSCs,Allen法制作脊髓损伤大鼠模型,共30只大鼠。于造模1周后进行干预,随机选取造模成功的24只大鼠并随机分为脊髓损伤组、假移植组(生理盐水注射)和细胞移植组(BMSCs注射),每组8只。于移植前、移植后7 d、14 d、21 d、28 d、35 d及42 d进行BBB(Basso,BeattieBresnahan locomotor rating scale)评分。HE染色、免疫荧光观察脊髓损伤的组织修复和BMSCs的迁移分化情况。结果从第14天始,细胞移植组大鼠的BBB评分较脊髓损伤组和假移植组大鼠高,差异具有统计学意义(P0.05)。HE染色显示细胞移植组大鼠的脊髓结构相对完整,液化和囊泡区缩小,炎性细胞减少。免疫荧光显示BMSCs聚集于脊髓损伤处,且能分化为神经元、神经胶质细胞和神经前体细胞。结论 BMSCs能向脊髓损伤部位迁移和聚集,并分化为相应的神经细胞促进脊髓功能恢复。     

B淋巴细胞瘤-2蛋白多位点磷酸化在大鼠脊髓损伤后细胞自噬中的表达研究

目的探讨大鼠脊髓损伤(spinal cord injury,SCI)后细胞自噬的变化及其与B淋巴细胞瘤-2(Bcell lymphoma-2,Bcl-2)蛋白多位点磷酸化的关系。方法取40只8周龄雄性SD大鼠,采用改良Allen法制备SCI模型;将造模成功的36只大鼠随机分为SCI组、自噬抑制剂组、自噬促进剂组,每组12只。另取12只大鼠仅切除椎板、不损伤脊髓,作为假手术组。造模结束后,自噬抑制剂组及自噬促进剂组分别于脊髓鞘内注射20μL600 nmol/L 3-甲基腺嘌呤、25 nmol/L雷帕霉素,假手术组及SCI组仅注射20μL生理盐水;每天1次,连续4周。造模后1 d及1、2、4周,采用BBB评分法评价各组大鼠后肢运动功能。末次注射后24 h处死各组大鼠并取脊髓组织,ELISA法检测脊髓组织中过氧化物酶(myeloperoxidase,MPO)活性及TNF-α、IL-1β水平;HE染色观察脊髓组织形态学变化;透射电镜观察脊髓组织中线粒体超微结构变化;免疫荧光染色检测自噬相关蛋白(Beclin1)、微管相关蛋白轻链3(microtubule-associated protein light chain 3,LC3)蛋白表达;TUNEL染色观察脊髓组织神经细胞凋亡;免疫荧光双染检测LC3/TUNEL阳性细胞表达;Western blot检测细胞Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)、Bcl-2及p-Bcl-2(Ser87)、p-Bcl-2(Ser70)蛋白表达。结果与假手术组相比,SCI组各时间点BBB评分降低,MPO活性、TNF-α、IL-1β水平升高;神经细胞周围间隙增大,细胞肿胀、出现空泡,线粒体中出现自噬小体;Beclin1及LC3蛋白阳性率、神经细胞凋亡率显著升高;LC3、TUNEL阳性细胞增多;Bax、p-Bcl-2(Ser87)、p-Bcl-2(Ser70)蛋白表达升高,Bcl-2蛋白表达降低;以上指标比较差异均有统计学意义(P0.05)。与SCI组相比,自噬抑制剂组各时间点BBB评分降低,MPO活性、TNF-α、IL-1β水平升高;线粒体中出现少量自噬囊泡;Beclin1及LC3蛋白阳性率降低,神经细胞凋亡率显著升高;LC3阳性细胞减少、TUNEL阳性细胞增多;Bax、p-Bcl-2(Ser87)、p-Bcl-2(Ser70)蛋白表达升高,Bcl-2蛋白表达降低。而自噬促进剂组结果与自噬抑制剂组相反;以上指标组间比较差异均有统计学意义(P0.05)。结论大鼠SCI后通过诱导细胞自噬可降低神经细胞凋亡,保护脊髓功能,其机制可能与抑制Bcl-2蛋白多位点磷酸化有关。     

The utilization of glucose in the brain slices during uraemia

Rat brain cortex slices were incubated in diluted sera obtained from patients with renal failure and from healthy persons. It was found that uraemic sera inhibited glucose uptake and the incorporation of¹⁴C-D-glucose into aspartate, glutamate and gamma-aminobutyrate (GABA). The concentration of these amino acids in the brain slices did not change.     

The quantitative study of the orientation of collagen in compact bone slices

We describe and validate a simple method for the study of the proportion of collagen fibers (and apatite crystals) in a bone slice parallel with the plane of section. Viewed between crossed circular polarizers, all bone areas with collagen lying more nearly in the plane of section (i.e., transverse [TS] collagen) appear bright whatever its direction in this plane; longitudinal [LS] collagen appears dark, but not as dark as the background. The degree of brightness increases with section thickness, which must therefore be standardized--we chose 100 microns plane parallel sections. We transferred the circularly polarized light [CPL] image via a CCD TV camera to an image analyzing computer. Color-coded maps of the CPL image were used to compare regions within and between sections. The new analytical procedure makes more detailed studies of the fine-structural orientation in compact bone possible, but does this have any significance? To answer this question, a bone in which the in vivo strain pattern had been clearly documented was chosen for particular study. Transverse mid-diaphyseal sections of the equine radius showed a distribution of CPL bright areas which correlated closely with previously reported strain patterns.     

马铃薯片外敷促进会阴切口愈合的效果观察

目的观察新鲜马铃薯片外敷促进会阴切口愈合的临床效果。方法将住院行会阴侧切术的产妇196例随机分为对照组与观察组各98例。观察组每天采用0.1%碘伏擦洗切口后用新鲜马铃薯片外敷切口,每日2次;对照组每天采用0.1%碘伏擦洗切口2次。结果观察组会阴切口愈合情况显著优于对照组,会阴切口处肿胀及疼痛消退时间显著短于对照组(均P<0.01)。结论使用自制马铃薯薄片贴敷会阴切口能有效加速肿胀及疼痛消退,促进会阴切口愈合。     

External application of potato slices to lateral episiotomy cut

目的 观察新鲜马铃薯片外敷促进会阴切口愈合的临床效果.方法 将住院行会阴侧切术的产妇196例随机分为对照组与观察组各98例.观察组每天采用0.1％碘伏擦洗切口后用新鲜马铃薯片外敷切口,每日2次；对照组每天采用0.1％碘伏擦洗切口2次.结果 观察组会阴切口愈合情况显著优于对照组,会阴切口处肿胀及疼痛消退时间显著短于对照组(均P＜0.01).结论 使用自制马铃薯薄片贴敷会阴切口能有效加速肿胀及疼痛消退,促进会阴切口愈合.     

Improved biochemical preservation of lung slices during cold storage

BACKGROUND: Development of lung preservation solutions typically requires whole-organ models which are animal and labor intensive. These models rely on physiologic rather than biochemical endpoints, making accurate comparison of the relative efficacy of individual solution components difficult. We hypothesized that lung slices could be used to assess preservation of biochemical function during cold storage. MATERIALS AND METHODS: Whole rat lungs were precision cut into slices with a thickness of 500 microm and preserved at 4 degrees C in the following solutions: University of Wisconsin (UW), Euro-Collins (EC), low-potassium-dextran (LPD), Kyoto (K), normal saline (NS), or a novel lung preservation solution (NPS) developed using this model. Lung biochemical function was assessed by ATP content (etamol ATP/mg wet wt) and capacity for protein synthesis (cpm/mg protein) immediately following slicing (0 h) and at 6, 12, 18, and 24 h of cold storage. Six slices were assayed at each time point for each solution. The data were analyzed using analysis of variance and are presented as means +/- SD. RESULTS: ATP content was significantly higher in the lung slices stored in NPS compared with all other solutions at each time point (P < 0.0001). Protein synthesis was significantly higher in the lung slices stored in NPS compared with all other solutions at 6, 12, and 18 h of preservation (P < 0.05). CONCLUSIONS: This lung slice model allows the rapid and efficient screening of lung preservation solutions and their components using quantifiable biochemical endpoints. Using this model, we have developed a novel solution that improves the biochemical preservation of lung slices during cold storage.     

Mechanisms of glutamate release in the rat spinal cord slices during metabolic inhibition

Glutamate toxicity is a viable hypothesis to explain the expanding tissue degeneration occurring after traumatic or ischemic spinal cord injury. One important component in this process is the acute, excessive release of glutamate. In the current communication, the glycolytic inhibitor iodoacetate was used to induce metabolic inhibition in spinal cord slices and thereby provide an in vitro model to study the mechanisms of pathological glutamate release in the spinal cord. The evoked glutamate release was not Ca2+-dependent. Exclusion of NaCl reduced the evoked release of endogenous glutamate by 56%, while excluding Na+ increased release. Glutamate release was also reduced by the PLA2 inhibitors indomethacin (40%), arachidonyltrifluoromethyl ketone (45%) and 4-bromophenacyl bromide (36%). Blocking reverse glutamate transport by preincubation with 1 mM dihydrokainic acid reduced evoked release by 41%. However, when the dihydrokainic acid and arachidonyltrifluoromethyl ketone treatments were combined, no additive effect of the two substances was seen. These findings suggest that glutamate is released by three mechanisms from the energy compromised spinal cord: (1) in response to cellular swelling, most likely by the regulatory volume decrease, (2) by PLA2-mediated breakdown of the cell membrane and diffusion of glutamate down its concentration gradient, and (3) through reversal of the glutamate transporter.