KLF7对小鼠脱细胞异体神经支架移植后运动功能的影响

目的:探讨核转录因子KLF7对脱细胞异体神经支架(ANA)移植修复坐骨神经缺损后小鼠运动轴突再生和运动功能恢复的影响。方法:制作C57BL/6小鼠坐骨神经缺损模型,用ANA进行桥接修复,在ANA注射腺相关病毒2(AA2)或腺相关病毒2-KLF-7(AAV2-KLF7,2μl,1×10~(11)病毒颗粒)。于修复术后4周,Western Blot和免疫荧光染色检测ANA内KLF7蛋白表达,NF和S100免疫荧光染色检测ANA内轴突再生和髓鞘生成,神经示踪剂荧光金(FG)逆行标记检测运动轴突再生,坐骨神经运动功能指数(SFI)和电生理检测运动功能的恢复。结果:AAV2-KLF7注射ANA后4周,ANA内KLF7蛋白表达显著增高,NF和S100表达显著增加,FG阳性标记脊髓运动神经元的数量增加,坐骨神经功能指数增加,电生理动作电位波幅增大,潜伏期缩短(P0.05)。结论:KLF7促进小鼠脱细胞异体神经支架修复坐骨神经缺损后运动轴突再生和髓鞘形成,有助于运动功能恢复。     

Hsp27联合ADSC对脱细胞神经支架修复小鼠坐骨神经缺损后caspase-3的影响

目的探讨热休克蛋白27(Hsp27)与脂肪源性干细胞(ADSC)联合应用对小鼠脱细胞同种异体神经支架移植后凋亡相关基因caspase-3表达及功能恢复的影响。方法 30只成年C57BL/6小鼠随机分为脱细胞神经支架(ANA)组、ADSC组和Hsp27+ADSC组。坐骨神经功能指数(SFI)、电生理和胫前肌湿重比方法检测神经运动功能的恢复,Western bolt检测神经支架内Hsp27和caspase-3的蛋白表达。胆碱乙酰转移酶(Ch AT)免疫荧光法检测神经移植体中运动轴突表达,并示踪PKH26标记的移植ADSC。结果与ADSC组比较,Hsp27+ADSC组神经移植体内Hsp27蛋白表达显著增高,caspase-3蛋白表达明显降低,Ch AT标记运动轴突表达增强,PKH-26标记的ADSC数量显著增高(P0.05)。与ANA组比较,ADSC组和Hsp27+ADSC组SFI增高、电生理波幅增高、神经传导速度增快、延迟期缩短、胫前肌湿重比率增高,其中Hsp27+ADSC组神经恢复作用显著优于ADSC组(P0.05)。结论Hsp27联合ADSC移植对小鼠脱细胞异体神经支架修复坐骨神经缺损后运动轴突再生和功能恢复的作用优于ADSC组,其机制可能与Hsp2抑制caspase-3凋亡信号通路,促进移植的ADSC存活有关。     

异体脂肪源干细胞移植对糖皮质激素性骨质疏松大鼠的影响

目的 基于“筋膜学假说”,探索未分化非特异性结缔组织（筋膜）支架构成的支持与储备系统中的主要细胞储备成分——脂肪源干细胞（ADSCs）移植对糖皮质激素性骨质疏松大鼠骨密度及骨生物力学的影响。方法 40只Wistar雌性大鼠随机分为空白对照组、模型组、治疗组和治疗对照组。利用糖皮质激素建立骨质疏松模型后,经尾静脉移植ADSCs 3×106个/只进行治疗。测定血清Ca、P、碱性磷酸酶（ALP）的含量,L3~L5椎体和右侧股骨骨密度以及左侧股骨最大载荷和刚度。结果 糖皮质激素注射12周后,模型组血清Ca和ALP、骨密度、最大载荷和刚度均明显低于空白对照组,而血清P明显升高 （P<0.01）。ADSCs治疗4周后,治疗组血清Ca和ALP、骨密度、最大载荷和刚度均明显升高,而血清P水平下降。 结论 移植异体ADSCs可以有效改善糖皮质激素性骨质疏松大鼠的骨密度和骨生物力学性能,为糖皮质激素性骨质疏松的治疗提供了新的治疗方法,并为“筋膜学假说”提供了部分实验依据     

异体脂肪源干细胞移植对大鼠的抗衰老作用

目的从筋膜学的角度设计观察脂肪源干细胞(ADSCs)异体移植对衰老模型大鼠体内自由基和免疫功能的影响,探索一种新的抗衰老方法。方法30只SD大鼠随机分成空白对照组(A组)、模型组(B组)和治疗组(C组)。B、C组大鼠注射D-半乳糖(D-gal)复制亚急性衰老模型。C组大鼠在造模8周后,经尾静脉输入体外扩增培养的脂肪源干细胞3×106个。治疗2周后,检测各组大鼠血清超氧化物歧化酶(SOD)、丙二醛(MDA)、一氧化氮(NO)、血清白细胞介素-2(IL-2)水平和脾脏指数。结果与空白对照组比较,模型组大鼠血清SOD、NO、IL-2水平及脾脏指数均显著性降低,血清MDA水平显著升高;移植ADSCs后,治疗组大鼠较模型组大鼠血清SOD、NO、IL-2水平及脾脏指数均有所提高,而MDA含量显著降低。结论异体移植ADSCs可有效增强大鼠机体清除自由基和抗氧化能力,提高机体免疫功能,从而延缓D-gal诱发的大鼠衰老。     

脱细胞处理的同种异体神经移植研究进展

周围神经损伤的修复是创伤外科难题之一,筛选促进神经再生理想的移植物,是解决这一难题的关键。近年来,在修复周围神经缺损研究中采用脱细胞处理的同种异体神经作为移植物,已取得促进神经再生的效果,展示了较好的应用前景。本文对脱细胞处理移植体的脱细胞处理方法,脱细胞处理后的细胞外基质所含的生物活性物质,以及这些物质的对促进神经再生的生物效应进行综述,以期为此种移植体的深入研究提供参考资料。     

大鼠脂肪源性干细胞来源的施万细胞特性分析

目的 探讨脂肪源性干细胞（ADSCs）及其分化的施万细胞（SCs）在形态、活性和标志物表达上的区别。方法 取SD大鼠腹股沟处脂肪制备ADSCs,用流式细胞术检测细胞表面标志;钙钴金属盐沉淀法检测碱性磷酸酶（ALP）,茜素红染色观察矿化结节;油红O染色观察脂滴;随机分对照组和诱导组,加入条件培养基向SCs诱导分化;MTT法检测诱导后第2、4、6和8 d各时间点的细胞活性;扫描电镜及免疫染色观察S100的表达。结果 光镜下可见ADSCs呈梭形贴壁生长;ADSCs表达抗原分化簇90(CD90)和抗原分化簇105(CD105),不表达抗原分化簇45(CD45);成骨诱导后可见ALP呈强阳性表达,茜素红染色可见橘红色矿化结节,油红O染色可见胞内红染脂滴聚集;向SCs诱导后第4 d,可见多数细胞呈纺锤形,第8 d纺锤状细胞增多;MTT检测显示第4 d诱导组的吸光度明显低于对照组,差异有统计学意义（P<0.05）;组内相比,诱导组和对照组的吸光度均随着时间的延长而逐渐增大,差异有统计学意义（P<0.05）;第6、第8 d诱导组与对照组吸光度差异无统计学意义（P>0.05）;扫描电镜...     

脱细胞异种神经桥接体联合经血源性间充质干细胞对大鼠坐骨神经缺损的影响

目的:研究脱细胞异种神经桥接体(ANX)联合经血源性间充质干细胞(MenSCs)对大鼠坐骨神经缺损的影响。方法:将SD大鼠随机分为自体移植组、单纯ANX组和ANX+MenSCs共培养组,制备SD大鼠右侧坐骨神经缺损模型,将不同组的桥接体移植到SD大鼠右侧坐骨神经缺损处,移植术后8周,用热板实验检测大鼠感觉功能的恢复,斜坡实验来检测运动功能的恢复,通过坐骨神经功能指数(SFI)检测大鼠坐骨神经功能,通过胫骨前肌湿重比率检测神经肌肉的功能,用透射电镜来观察髓鞘的恢复情况。结果:扫描电镜显示ANX组内原有的细胞及结构基本去除,完整保留了神经的基膜及外膜,ANX+MenSCs组可见圆形的细胞在基膜间稳定黏附生长;免疫荧光染色显示神经生长因子(NGF)在ANX+MenSCs共培养组中有阳性表达;移植术后8周,ANX+MenSCs组的患肢回缩时间显著短于单纯ANX组(P<0.05),爬坡坡度、SFI值以及胫骨前肌湿重比率均显著高于单纯ANX组(P<0.05),ANX+MenSCs组与自体组相比,两组的患肢回缩时间、爬坡坡度以及胫骨前肌湿重比均无显著差异(P>0.05),但自体组的SFI值显著增高(P<0.05);透射电镜显示与单纯ANX组相比,ANX+MenSCs组和自体组的髓鞘结构更完整,虽有水肿,但形状小而均匀致密。结论:ANX联合MenSCs修复SD大鼠坐骨神经缺损的效果良好,MenSCs可能成为外周神经系统疾病细胞治疗的潜在候选种子细胞。     

脱细胞异体真皮基质与人脂肪干细胞的生物相容性

背景：脱细胞异体真皮基质具有优良的生物相容性和组织细胞诱导功能。 目的：评价人脂肪干细胞与脱细胞异体真皮基质的生物相容性。 方法：取健康成年人吸脂术后的脂肪组织分离脂肪干细胞,并行原代与传代培养,传至第3代,将细胞与脱细胞异体真皮基质联合体外培养3,7 d,倒置相差显微镜和扫描电镜观察细胞在支架材料上的黏附、生长及增殖情况,并计算细胞在材料上的黏附率;XTT比色法检测细胞的生长增殖情况。 结果与结论：脂肪干细胞在支架材料上分布均匀,24 h内细胞开始伸展、黏附,二三天完全伸展变形,以梭形为主,呈网状排列;随着培养时间延长,支架上的细胞逐渐增多;人脂肪干细胞细胞与脱细胞异体真皮基质混合培养后平均黏附率为95.03%,并保持正常的生长增殖速度,表明支架对细胞具有良好的黏附性;脱细胞异体真皮基质材料与人脂肪干细胞复合后相容性良好。     

神经干细胞联合胶质细胞源性神经营养因子脑内移植对Parkinson病大鼠纹状体内多巴胺含量的影响

将原代培养的胚鼠(E14.5)腹侧中脑神经干细胞(NSCs)单独或联合胶质细胞源性神经营养因子(GDNF)移植入Parkinson病(PD)大鼠纹状体内,术后各组动物存活一段时间,观察旋转行为后处死,取纹状体组织做高效液相色谱检测多巴胺(DA)含量。探讨NSCs单独或联合GDNF脑内移植对PD大鼠纹状体内DA含量的影响。结果显示:与PD模型相比,NSCs单独或联合GDNF脑内移植均能提高PD大鼠纹状体内的DA含量(P<0.05);在术后30d和60d,DA含量在GDNF+NSCs组的增加幅度比NSCs组更加明显,动物旋转行为亦得到明显改善(P<0.05)。上述结果表明,NSCs单独或联合GDNF移植均对PD大鼠有一定的治疗作用,而NSCs联合GDNF移植的治疗效果更好。     

大鼠脑源性神经干细胞的分离培养

目的 探讨大鼠不同年龄组来源的神经干细胞体外培养和诱导分化的最佳条件。方法 分离12～15d大鼠胚胎、1～2d新生鼠的脑组织和成年大鼠脑的海马区和嗅球，在条件培养基(包含生长因子)中增殖培养，用神经干细胞的特异性单克隆抗体巢蛋白(nestin)鉴定克隆细胞；去除生长因子，降低血清浓度到2％后，诱导细胞分化，用神经细胞的特异性单克隆抗体鉴定分化细胞。结果 获得了大量可自我增殖并分化为3种神经细胞(神经元，星形胶质细胞，少突胶质细胞)的神经前体细胞。结论 在适当的分离培养条件下，可以获得大量的神经前体细胞，为神经干细胞的基础和临床研究提供细胞基础。     

Transplantation of embryonic stem cells improves nerve repair and functional recovery after severe sciatic nerve axotomy in rats

Extensive research has focused on transplantation of pluripotent stem cells for the treatment of central nervous system disorders, the therapeutic potential of stem cell therapy for injured peripheral nerves is largely unknown. We used a rat sciatic nerve transection model to test the ability of implanted embryonic stem (ES) cell-derived neural progenitor cells (ES-NPCs) in promoting repair of a severely injured peripheral nerve. Mouse ES cells were neurally induced in vitro; enhanced expression and/or secretion of growth factors were detected in differentiating ES cells. One hour after removal of a 1-cm segment of the left sciatic nerve, ES-NPCs were implanted into the gap between the nerve stumps with the surrounding epineurium as a natural conduit. The transplantation resulted in substantial axonal regrowth and nerve repair, which were not seen in culture medium controls. One to 3 months after axotomy, co-immunostaining with the mouse neural cell membrane specific antibody M2/M6 and the Schwann cell marker S100 suggested that transplanted ES-NPCs had survived and differentiated into myelinating cells. Regenerated axons were myelinated and showed a uniform connection between proximal and distal stumps. Nerve stumps had near normal diameter with longitudinally oriented, densely packed Schwann cell-like phenotype. Fluoro-Gold retrogradely labeled neurons were found in the spinal cord (T12-13) and DRG (L4-L6), suggesting reconnection of axons across the transection. Electrophysiological recordings showed functional activity recovered across the injury gap. These data suggest that transplanted neurally induced ES cells differentiate into myelin-forming cells and provide a potential therapy for severely injured peripheral nerves.     

脂肪干细胞外泌体可促进大鼠海绵体神经再生

背景:海绵体神经是调控阴茎勃起的重要神经,在盆腔手术或外伤中容易受到损伤,导致阴茎勃起功能障碍。干细胞来源外泌体对于受损海绵体神经再生的直接影响目前尚无研究报道。目的:观察脂肪干细胞外泌体对海绵体神经再生的影响。方法:体外构建大鼠盆大神经节三维培养模型,培养24 h后实验组换成含有青霉素-链霉素和脂肪干细胞来源外泌体的RPMI-1640培养基,外泌体浓度为1015 L-1,对照组仍使用含有青霉素-链霉素的RPMI-1640培养基。干预开始后第2,5,9天观察两组海绵体神经断端再生情况,并测量轴突生长长度,用以评价海绵体神经再生能力。结果与结论:①从大鼠肾周脂肪组织获得的原代脂肪干细胞贴壁生长,形态稳定,能够诱导分化为脂肪、骨和软骨;②提取自第3代脂肪干细胞的外泌体呈大小均一的囊泡结构,直径集中在85 nm附近;③三维培养体系中的海绵体神经自身具有一定的再生能力,外泌体干预后2,5,9 d实验组海绵体神经再生情况均优于对照组,轴突再生长度明显大于对照组;④结果表明,脂肪干细胞来源外泌体直接作用于海绵体神经,对其再生具有显著的促进作用。     

Chondroitinase ABC reduces time to muscle reinnervation and improves functional recovery after sciatic nerve transection in rats

Application of chondroitinase ABC (ChABC) to injured peripheral nerves improves axon regeneration, but it is not known whether functional recovery is also improved. Recordings of EMG activity [soleus (Sol) M response and H reflexes] evoked by nerve stimulation and of Sol and tibialis anterior (TA) EMG activity and hindlimb and foot kinematics during slope walking were made to determine whether ChABC treatment of the sciatic nerve at the time of transection improves functional recovery. Recovery of evoked EMG responses began as multiple small responses with a wide range of latencies that eventually coalesced into one or two more distinctive and consistent responses (the putative M response and the putative H reflex) in both groups. Both the initial evoked responses and the time course of their maturation returned sooner in the ChABC group than in the untreated (UT) group. The reinnervated Sol and TA were coactivated during treadmill locomotion during downslope, level, and upslope walking throughout the study period in both UT and ChABC-treated rats. By 10 wk after nerve transection and repair, locomotor activity in Sol, but not TA, had returned to its pretransection pattern. There was an increased reliance on central control of Sol activation across slopes for both groups as interpreted from elevated prestance Sol EMG activity that was no longer modulated with slope. Limb length and orientation during locomotion were similar to those observed prior to nerve injury during upslope walking only in the ChABC-treated rats. Thus treatment of cut nerves with ChABC leads to improvements in functional recovery.     

Partial functional recovery after complete spinal cord transection by combined chondroitinase and clenbuterol treatment

Spinal cord injury not only disrupts axonal tracts but also causes gliotic, fibrotic, and Schwannotic scarring with resulting deposition of chondroitin sulfate proteoglycans (CSPGs) which prevent axonal reconnection and recovery of locomotor function. Here, we determined whether recovery of locomotor function could be promoted after complete transection, by degrading CSPGs enzymatically within the injury site with chondroitinase ABC (chABC) together with treatment with the β₂-adrenoceptor agonist, clenbuterol, a neuroprotective agent which can promote regrowth of lower motoneurons. Partial recovery of locomotor function was observed 8–12 weeks postinjury only after combined chABC and clenbuterol treatment. The recovery of locomotor function coincided with the presence of axons caudal to the injury site arising from neurons of the reticular, vestibular, and red nuclei also only with combined chABC and clenbuterol treatment. Axons myelinated by Schwann cells were most prominent in the transection site in the combined treatment group. Clenbuterol treatment activated cAMP response element binding protein within retrogradely traced neurons which has been associated with axonal regrowth. ChABC treatment decreased scarring due to both CSPG and collagen deposition as well as the gap between intact regions of the spinal cord. ChABC also increased numbers of phagocytic cells which remove myelin debris as well as populations of astrocytes thereby aiding blood–spinal cord barrier reformation. Together the results suggest that chABC and clenbuterol can act synergistically to promote recovery of locomotor function.     

化学去细胞异体神经联合骨髓间充质干细胞修复损伤坐骨神经的生物力学评价

文题释义： 生物力学：是应用力学原理和方法对生物体中的力学问题定量研究的生物物理学分支,其研究范围从生物整体到系统、器官(包括血液、体液、脏器、骨骼等),从鸟飞、鱼游、鞭毛和纤毛运动到植物体液的输运等。生物力学的基础是能量守恒、动量定律、质量守恒定律并加上描写物性的本构方程。生物力学研究的重点是与生理学、医学有关的力学问题。依研究对象的不同可分为生物流体力学、生物固体力学和运动生物力学等。 周围神经研究热点和发展趋势：周围神经损伤的治疗已经取得了突破性的进展,但由于周围神经走行长、结构复杂、功能多样,损伤后其自身和靶器官会发生改变,而且是动态的、有时间效应的改变,因此以整体统一的理念研究周围神经损伤是今后研究重点。如何将基础研究与临床相结合,最终实现成果转化服务于患者,是研究者们的努力方向。国内外学者对自体神经损伤移植替代材料的研究从未间断过。随着组织工程和生物力学的发展,同种异体神经和人工神经已逐渐应用于临床,去细胞神经移植物也已取得了很好的临床效果。背景：以拉伸力学指标评估化学去细胞异体神经联合骨髓间充质干细胞治疗坐骨神经损伤的效果,目前鲜有报道。 目的：评估化学去细胞异体神经联合骨髓间充质干细胞移植治疗坐骨神经损伤的效果。 方法：将45只SD大鼠随机分为自体神经移植组、化学去细胞异体神经移植组、化学去细胞异体神经联合骨髓间充质干细胞移植组,每组15只,制备10 mm坐骨神经损伤模型,分别以自体神经、化学去细胞异体神经、化学去细胞异体神经联合骨髓间充质干细胞修复坐骨神经损伤,术后20周,进行坐骨神经电生理检测和坐骨神经单向拉伸实验。 结果与结论：①化学去细胞异体神经联合骨髓间充质干细胞移植组与自体神经移植组波幅值、运动传导速度值大于化学去细胞异体神经移植组(P < 0.05);②化学去细胞异体神经联合骨髓间充质干细胞移植组与自体神经移植组拉伸弹性限度应变、弹性限度应力、最大应变、最大应力大于化学去细胞异体神经移植组(P < 0.05);③结果表明,化学去细胞异体神经联合骨髓间充质干细胞移植对坐骨神经损伤具有明显的修复效果。ORCID: 0000-0002-1029-8117(吴志峰) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程