胸腺内注射异基因抗原诱导鼠异体骨移植的免疫耐受

背景：同种异体骨同其他异体组织一样具有抗原性,移植后可引起以细胞免疫为主的排斥反应,应用免疫抑制剂可抑制免疫排斥反应的发生,但对机体有一定的不良影响。 目的：探讨胸腺内注射异基因抗原在建立特异性骨移植免疫耐受中的作用。 方法：取60只Wistar大鼠随机分为3组,均制作骨缺损模型,供体异基因胸腺内注射组于同种异体骨移植前胸腺内注射受体大鼠脾细胞MHC抗原,同时另设自体骨移植组、同种异体骨移植加用免疫抑制剂组为对照,移植后观察切口情况。移植后1,2,4,6周进行X射线检查,苏木精-伊红染色,可溶性白细胞介素2受体、混合淋巴细胞培养免疫学检测。 结果与结论：大体表现、X射线及组织学检查见供体异基因胸腺内注射组、自体骨移植组表现相近,均有炎性细胞浸润及新骨形成。同种异体骨移植加用免疫抑制剂组再生血管较少,骨愈合延迟,炎性细胞浸润明显,移植骨逐渐吸收,无新骨形成。证实了异基因MHC抗原注入小鼠胸腺内,能成功诱导受体对供体鼠骨移植物的耐受。免疫学检查各项数据表明供体异基因胸腺内注射的成骨效果接近于自体骨移植组,优于同种异体骨移植加用免疫抑制剂,证实了胸腺内注射异体MHC抗原可诱导对供体骨移植物的免疫耐受。     

转化生长因子β1对坐骨神经移植后免疫耐受的影响

背景：转化生长因子β1是一种强效细胞生长增殖调节蛋白,在移植免疫的抗排斥反应、移植物血管病发展中扮演重要角色。 目的：观察经冷冻处理异体神经移植后，局部注射转化生长因子β1对移植免疫排斥反应的影响。 设计、时间及地点：随机对照动物实验，于2007-06/2008-06在哈尔滨医科大学动物实验中心完成。 材料：受体为清洁级SD大鼠60只，分为3组：自体神经移植组、异体神经移植组、转化生长因子β1质粒+异体神经移植组，每组20只。供体为40只Wistar雄性大鼠。pAdTrack-CMV-TGF-β1质粒、pAdEasy-1-Bj51833细胞由哈尔滨医科大学附属四院骨科实验室惠赠。 方法：取供体大鼠40只作双侧股后外侧纵切口,分离显露坐骨神经,切取双侧整段坐骨神经,置于无菌冷冻管中保存1周,备用。手术显微镜下将受体鼠自骨二头肌与半腱肌和半膜肌间隙剪开结缔组织，显露坐骨神经，从犁状肌孔下0.5 cm处整齐剪下长约1 cm的坐骨神经。自体神经移植组、异体神经移植组选择粗细相等、已预制冷冻的自体及异体神经移植；转化生长因子β1质粒+异体神经移植组异体神经移植后于大鼠局部肌肉及神经两断端内注射pAdTrack-CMV-TGF-β1质粒40 μg/只。 主要观察指标：术后3，6，9周各组大鼠运动神经传导速度、病理学和轴突计数检查。 结果：转化生长因子β1质粒+异体神经移植组运动神经传导速度高于新鲜异体神经移植组(P < 0.01)，与自体神经移植组比较差异无显著性意义。自体神经移植组、转化生长因子β1质粒+异体神经移植组术后9周轴突计数较新鲜异体神经移植组高(P < 0.01)。转化生长因子β1质粒+异体神经移植组光镜及电镜可见神经纤维走行正常，排列完好，神经纤维可见血管增生，髓鞘结构较好，神经纤维内见有大量再生髓鞘，许旺细胞明显增多，胞质较发达，大量粗面内质网，线粒体结构清晰，再生的轴突内微丝密集排列，与自体神经移植组接近。异体神经移植组光镜及电镜可见神经纤维数量少、排列紊乱，髓鞘轴突变性明显，大部分神经纤维脱髓鞘崩解，轴突消失，未见再生的神经纤维。 结论：局部注射转化生长因子β1质粒联合冷冻处理的冷藏异体神经移植可以协同减轻移植后产生的免疫排斥反应。     

重症肌无力患者胸腺微环境内树突状细胞的免疫组织化学变化

目的 探讨重症肌无力（MG）胸腺微环境内树突状细胞、淋巴细胞和上皮细胞的免疫组织学变化。方法 （1）25例MG病人（胸腺滤泡性增生者13例，无增生者12例）、7例先天性心脏病和1例新生儿胸腺组织，常规石蜡包埋切片，HE染色，光镜观察。（2）免疫化学标记S－100、CD1a、CD4、CD8、CD20、CD45RO、细胞角蛋白（CKPan)、上皮细胞膜抗原（EMA）和癌胚抗原（CEA），光镜观察，对其中S－100，CD1a阳性的树突状细胞半定量计数，t检验，统计分析。结果 （1）在胸腺淋巴滤泡性增生组和非增生组，胸腺单位面积内（100m^2)表达S－100阳性的树突状细胞数是23．5和47.5,CD1a为2．1和3．8，两组t检验，P＜0．05）。（2）胸腺质皮细胞和淋巴滤泡性增生的髓质分别表达CD1a、CD8和CD45RO，胸腺小体、血管周围淋巴细胞极少表达CD1a，胸腺髓质和生发中心内淋巴细胞表达CD20。（3）胸腺被膜下、皮质、胸腺小体和髓质内有表达CKPan的上皮细胞，但EMA和CEA阴性。结论 （1）MG时胸腺髓质无论有无淋巴滤泡性增生，都表达多量抑制T细胞和CD20淋巴细胞；生发中心内有多量表达CD20和少量CD8细胞。（2）MG时胸腺微环境内这些抗体的表达和树突状细胞数量的变化，将影响T、B淋巴细胞增生和淋巴滤泡形成；胸腺内树突状细胞与其基质细胞在MG的发病中起重要作用。     

同种异体肢体移植特异性免疫耐受诱导方案

背景：通过诱导受体产生免疫耐受能够解决移植领域存在的许多问题,如长期应用免疫抑制剂带来的毒副作用,慢性排斥反应等。 目的：观察联合应用亚致死量放射线照射、氟达拉滨及骨髓腔内骨髓移植对诱导大鼠同种异体肢体移植产生特异性免疫耐受的影响。 设计、时间及地点：随机同期对照动物实验,于2004-01/2008-12在哈尔滨医科大学完成。 材料：40只雄性Wistar大鼠随机分成4组：单纯移植组、氟达拉滨组、骨髓移植组、氟达拉滨＋骨髓移植组,每组各10只。40只SD大鼠为骨髓移植供体。供体DA大鼠用于皮肤移植实验。 方法：制作同种异体右后肢移植模型,单纯移植组仅进行肢体移植;氟达拉滨组在肢体移植前1 d,受体给予亚致死量60Co照射及腹腔内注射氟达拉滨50 mg/kg;骨髓移植组肢体移植当天受体接受供体骨髓腔内骨髓移植1×1012 L-1,10 μL;氟达拉滨+骨髓移植组受体联合应用亚致死量照射、注射氟达拉滨及骨髓腔内骨髓移植。 主要观察指标：观察移植物排斥反应征象及存活情况,并对氟达拉滨+骨髓移植组产生免疫耐受大鼠进行SD大鼠及DA大鼠皮肤移植及脾细胞中细胞因子mRNA检测。 结果：与其他实验组比较,氟达拉滨＋骨髓移植组移植物发生排斥反应的时间及存活时间均显著延长(P < 0.01)。氟达拉滨+骨髓移植组大鼠仅对第3方DA大鼠的皮肤呈现强烈的免疫反应,移植皮肤初为水疱、渗出,逐渐溃疡、糜烂、焦痂、变黑坏死。 与单纯移植组比较,氟达拉滨+骨髓移植组大鼠Th1型细胞因子的表达明显降低,而Th2型细胞因子的表达明显增高。 结论：氟达拉滨与骨髓移植联合方案可以诱导大鼠同种异体肢体移植产生特异性免疫耐受。     

异体神经干细胞脑移植后免疫排斥反应的研究

目的 探讨中枢神经系统异体神经干细胞移植后免疫排斥反应的问题. 方法 选取同一批次的成年克隆山羊8只,采用随机数字表法分成2组:神经干细胞移植组和对照组.采用开颅手术的方式分别将异体神经干细胞及相同剂量的生理盐水移植到山羊脑皮层下,抽血测定移植后2组动物不同时间点(移植前1周、移植当时、移植后1周、3周及3个月)血中IL-2及IL-10的水平,以了解移植后动物全身免疫排斥反应的情况;并通过免疫组化的方法 检测移植局部病理切片CD3+细胞浸润的情况,了解移植局部免疫排斥反应的情况. 结果神经干细胞移植组在移植后1周、3周及3个月时血中IL-2水平较对照组明显升高,IL-10水平较对照组明显下降,差异均有统计学意义(P＜0.05).移植局部在急性期(细胞移植后1周)和慢性期(细胞移植后3个月)两个时间点均有大量CD3+细胞浸润,与对照组相比差异均有统计学意义(P＜0.05). 结论 中枢神经系统异体神经干细胞移植后可发生急、慢性全身和局部免疫排斥反应.     

微囊包膜神经系统细胞移植

脑为人类免疫特免器官,即在自体移植时不产生免疫排斥反应。但在同种异体或异种间移植时,仍会产生免疫排斥反应。一种日趋完善的免疫隔离技术—微包囊技术可望最终解决这一难题。本文将微包囊技术原理及几种在中枢神经系统移植中研究较多的微囊包裹细胞作一介绍。     

骨髓基质细胞成年大鼠脑内移植

目的 研究骨髓基质细胞脑内移植后的分布和移行，为细胞移植治疗疾病奠定基础。方法 常规培养大鼠骨髓基质细胞，应用免疫组织学方法对细胞进行鉴定，Hoechst33258标记细胞，立体定向移植到大鼠的纹状体，经过一段时间后处死大鼠，脑组织切片，直接在荧光显微镜下检查存活的细胞。结果 细胞移植到大鼠脑内能够长时间存活，移植细胞与宿主细胞有很好的相容性，宿主脑组织的结构无破坏，移植细胞能够移行一段距离，说明脑内存在的信号诱导细胞向一定的方向迁徙。结论 骨髓基质细胞脑内移植后，能够与宿主脑组织整合在一起，无细胞过度增生和胶质瘢痕形成，这种细胞可能成为中枢神经系统自体移植的细胞来源。     

神经干细胞中枢神经系统移植中的免疫学问题

神经干细胞中枢神经系统移植作为一种细胞替代治疗方法已吸引着众多学者的关注,但神经干细胞移植的临床应用还必须面临免疫排斥等问题.中枢神经系统具有其独特的移植免疫学中的特点.大脑曾被认为是免疫豁免区域之一,然而后来的研究已经证实,中枢神经系统并不是完全意义上的免疫豁免器官.中枢神经系统内免疫反应的屏障系统,中枢神经系统抗原提呈细胞的特点以及神经干细胞自身的免疫学特性决定了大脑的相对免疫豁免地位,实际是机体调节免疫豁免和免疫反应所达到的一个平衡.     

间充质干细胞诱导移植耐受的研究进展及可能的机制

学术背景：间充质干细胞能自我更新、高度增殖,具有多向分化潜能、低免疫原性及免疫调节特性。在体内和体外均能调节同种异体免疫反应,诱导免疫耐受。在实体器官移植中发挥着越来越重要的作用,有望成为诱导移植耐受的新途径。目的：综述间充质干细胞在实体器官移植中的应用及其免疫调节机制的最新进展。检索策略：应用计算机检索Pubmed1994-01/2009-10 期间相关文章,运用MeSH主题词检索,检索词为“Mesenchymal Stem Cells,Mesenchymal Stem Cell Transplantation,Organ Transplantation,Transplantation Immunology,Immunologic Graft Enhancement, Graft vs Host Disease”,并限定文章语言种类为English。同时检索万方数据库1994-01/2009-10 期间相关文章,检索词为“间充质干细胞,器官移植,移植免疫”。对资料进行初审,并查看每篇文献后的引文。纳入标准：文章所述内容应与间充质干细胞免疫学特性、器官移植中的应用及移植免疫进展研究相关。排除标准：重复研究或Meta 分析类文章。共收集到200篇相关文献,86篇文献符合纳入标准,排除的44 篇为内容陈旧或重复文献。文献评价：文献的来源主要是关于间充质干细胞的免疫学特性、细胞移植、在实体器官移植中的应用等基础和临床相关研究。选用符合纳入标准88 篇文献中的42 篇,7 篇关于皮肤移植,6 篇关于心脏移植,5 篇关于肝脏移植,4 篇关于肾脏移植,2 篇关于胰腺移植,7 篇关于移植物抗宿主病,5篇关于MSCs在体内的迁移转化,6 篇关于MSCs在体内应用的安全性。资料综合：间充质干细胞能自我更新、高度增殖,具有多向分化潜能、低免疫原性及免疫调节特性。在体内和体外均能调节同种异体免疫反应,诱导免疫耐受。在实体器官移植（皮肤、心脏、肝脏、肾脏等移植）中的研究主要是诱导免疫耐受及组织修复。其诱导免疫耐受的机制可能与可溶性因子、调节性T细胞、耐受性树突细胞、骨髓嵌合状态、抗炎和组织修复功能有关,并受到MSCs输入方式、剂量与时间的影响。但是,MSCs诱导供体特异性免疫耐受的方案、机制及安全性仍不清楚。结论：间充质干细胞具有低免疫原性及调节免疫调节特性, 在诱导移植耐受和器官移植后组织修复中有着不可或缺的优势。但是,MSCs在体内免疫调节作用的机制及诱导移植耐受的机制;在体内的迁移、转化,定植于靶器官的过程;在活体内应用的安全性;在大动物模型中的作用还需深入系统研究。     

神经干细胞和嗅鞘细胞联合移植与神经干细胞移植治疗实验性脑出血的比较

目的对比神经干细胞(NSCs)和嗅鞘细胞(OECs)联合移植与NSCs移植治疗大鼠脑出血的疗效。方法制作大鼠脑出血模型,记录细胞移植后对照组和移植组(NSCs组、NSCsOECs组)大鼠运动功能,采用免疫组化观察移植细胞在体内存活、分化和迁徙的情况。结果NSCsOECs组和NSCs组移植NSCs分别有14.19%和2.96%分化为神经元,差异有统计学意义(χ2=154.79,P<0.01),30d时神经功能缺损评分NSCs组为1.60±0.13,NSCsOECs组为1.01±0.11,差异有统计学意义(P<0.05),OECs移植后能存活、迁徙,NSCs移植后能存活、分化为神经元、星形和少突胶质细胞,并向损伤区迁徙。结论OECsNSCs移植改善脑出血大鼠运动功能的疗效优于单纯NSCs移植。     

Receptors for nerve growth factor on rat spleen mononuclear cells

Considerable evidence is mounting to support the concept of a modulatory role for the brain and neuroendocrine system on the immune response. This neuroimmunomodulation occurs in part through the interaction of specific neurosubstances with receptors on lymphocytes and monocytes. Nerve growth factor (NGF) is a neuronotrophic factor necessary for the development and maintenance of sympathetic and embryonic sensory neurons. This trophic effect is initiated through binding of NGF at specific cell surface receptor sites on NGF-responsive cells. Several recent studies suggest that NGF may interact with cells of the immune system and may play a role in the regulation of some immunologic reactions. In this study we report on the presence of specific receptors for NGF on the surface of mononuclear cells from rat spleens. The NGF-binding sites are of the low-affinity type with Kd's in the 10(-9) M range. These receptors migrate on SDS-PAGE as two molecular species of approximately 190 and 125 kilodaltons. Our findings of receptors for NGF on lymphocytes and accessory cells support other evidence that NGF may influence immunoreactivity in vivo.     

Effect of intrathymic injection of allogene antigen on immune response to sciatic nerve transplantation in allogenic mice

BACKGROUND: The latest researches demonstrate that intrathymic injection of MHC antigen which reaches a certain dosage (2 mg, i.e., 4 × 108 cell extraction) can induce immunologic tolerance under non-antilymphocyte serum condition. OBJECTIVE: To investigate the effect of intrathymic injection of allogene antigen on survival and function of sciatic nerve in allogenic mice. DESIGN: Randomized controlled animal study. SETTING: The 4th Affiliated Hosptial of Harbin Medical University. MATERIALS: A total of 32 male donor C57BL/6(H-2b) mice of 4–8 weeks old and weighing 18–22 g and 44 female receptor Balb/c(H-2d) mice of 4–8 weeks old and weighing 18–22 g were selected from Heilongjing Veterinary Institution. The animal experiment had got confirmed consent from local ethic committee. METHODS: The experiment was carried out in the Laboratory (Provincial Key Laboratory) of the Fourth Hospital, Harbin Medical University from June 2006 to May 2007. C57BL/6(H-2b) mice were anesthetized to extract MHC (H-2b) antigen from splenic cells and sciatic nerves. Allogenous nerve transplantation group: Mice were given intrathymic injection of 100 μL saline; two weeks later, frozen sciatic nerves of donor mice were transplanted. Immunosuppressive agent group: Mice were given intrathymic injection of 100 μL saline; two weeks later, fresh sciatic nerves of donor mice were transplanted. At three days before transplantation, 10 mg/kg per day cyclosporin A was intraperitoneally injected once a day till mice were sacrificed. MHC (H-2b) antigen injection group: Mice were given intrathymic injection of MHC (H-2b) antigen from C57BL/6(H-2b) donor mice; two weeks later, fresh sciatic nerves of donor mice were transplanted. Autogenous nerve transplantation group: Mice were given intrathymic injection of 100 μL saline; two weeks later, fresh sciatic nerves were transplanted. MAIN OUTCOME MEASURES: ① Three weeks later, transplanted part of exposured sciatic nerve was used to measure the motor nerve conduction velocity. ② Transplanted nerve was stained with histochemical staining and observed light microscope and electron microscope. ③ Mice received mixed lymphocyte culture and delayed-typed hypersensitiveness to observe absorbency and measure depth of soles. RESULTS: All 76 mice were involved in the final analysis. ① Motor nerve conduction velocity: The nerve recovery in MHC (H-2b) antigen injection group was higher than that in allogenous nerve transplantation group, equal to immunosuppressive agent group and lower than autogenous nerve transplantation group. There were significant differences among them (P < 0.05). ② Histological changes of transplanted nerve: Light and electron microscopes demonstrated that there were a lot of regenerative nerve fibers in autogenous nerve transplantation group, immunosuppressive agent group and MHC (H-2b) antigen injection group, and all nerve fibers passed grafts. ③ Immunological examination: There was no significant difference in mixed lymphocyte culture among allogenous nerve transplantation group, autogenous nerve transplantation group and MHC (H-2b) antigen injection group (P < 0.05). Depth of soles in other groups was deeper than that in the MHC (H-2b) antigen injection group, and there was significant difference (P < 0.05); that was to say, delayed-typed hypersensitiveness was remarkable. CONCLUSION: The intrathymic injection of allogene MHC antigen may induce specific immune tolerance to allogenous sciatic nerve transplantation and promote nerve survival and functional recovery.     

Peripheral nerve demyelination induced by intraneural injection of experimental allergic encephalomyelitis serum.

Intraneural injection of sera from rabbits with experimental allergic encephalomyelitis, induced by sensitization with bovine brain white matter in complete Freund's adjuvant, produced focal primary demyelinative lesions in rat sciatic nerves. Demyelinating activity was removed by prior incubation of antisera with central (CNS) and peripheral nervous system (PNS) myelin but not with liver or kidney, and was heat-labile and complement-dependent. Recipient animals developed a sensorimotor disturbance of their toes and ankles on the side injected with antiserum. Twenty minutes after antiserum injection, Schwann cells showed focal cytoplasmic outpouching and their external mesaxons opened. Between 1 and 8 hours after injection vacuolation, splitting and vesiculation of myelin became increasingly prominent at Schmidt-Lanterman clefts and paranodal regions, with concomitant degenerative changes in Schwann cell cytoplasm. Polymorphonuclear cell infiltration and endoneurial edema were apparent at this time. Substantial demyelination occurred before the appearance of phagocytic cells. Between 8 hours and 3 days many nerve fibers were surrounded and attacked by invading macrophages. Axons became demyelinated progressively over several internodes by macrophage phagocytosis. Early signs of remyelination were observed by 5 days. These findings suggest that antibodies directed against antigens common to both CNS and PNS myelin can produce in vivo peripheral nerve demyelination.     

静脉注射神经生长因子基因修饰的骨髓基质干细胞对辐射诱导小鼠肝细胞凋亡的影响

背景：研究发现神经生长因子能减少神经细胞和心肌细胞的凋亡。 目的：假设神经生长因子基因修饰的骨髓基质干细胞对辐照诱导小鼠肝细胞凋亡具有保护作用，拟验证其可能性。 设计、时间及地点：随机对照动物实验，于2007-01/08在广东医学院实验动物中心实验室完成。 材料：昆明小鼠120只，随机分细胞注射组40只、单纯照射组40只和正常对照组40只。 方法：单纯照射组采用 60Coγ射线照射小鼠，吸收剂量率188.2 cGy/min，细胞注射组照射前2 d静脉给予神经生长因子基因修饰的骨髓基质干细胞5×1010 L-1;正常对照组不辐射损伤。观察小鼠30 d存活率和死亡动物存活时间；用原位末端标记法和流式细胞仪观察受照小鼠肝细胞凋亡及p53表达，再用Western blot进一步测定p53的表达水平。 主要观察指标：①受照射小鼠30 d存活率。②受照射小鼠肝细胞凋亡情况。③受照射小鼠肝细胞p53表达。 结果：细胞注射组30 d存活率高于单纯照射组。单纯照射组小鼠大量肝细胞发生凋亡，呈灶性。细胞注射组小鼠肝细胞凋亡少于单纯照射组, 凋亡细胞呈散在分布。细胞注射组肝细胞p53标记的荧光强度和细胞比例明显低于单纯照射组(P < 0.05)。细胞注射组p53带灰度值低于单纯照射组(P < 0.05)。 结论：神经生长因子基因修饰的骨髓基质干细胞能明显抑制受照小鼠肝细胞凋亡及抑制肝细胞p53的表达。     

One-stage human acellular nerve allograft reconstruction for digital nerve defects

Human acellular nerve allografts have a wide range of donor origin and can effectively avoid nerve injury in the donor area. Very little is known about one-stage reconstruction of digital nerve defects. The present study observed the feasibility and effectiveness of human acellular nerve allograft in the reconstruction of 5-cm digital nerve defects within 6 hours after injury. A total of 15 cases of nerve injury, combined with nerve defects in 18 digits from the Department of Emergency were enrolled in this study. After debridement, digital nerves were reconstructed using human acellular nerve allografts. The patients were followed up for 6–24 months after reconstruction. Mackinnon-Dellon static two-point discrimination results showed excellent and good rates of 89%. Semmes-Weinstein monofilament test demonstrated that light touch was normal, with an obvious improvement rate of 78%. These findings confirmed that human acellular nerve allograft for one-stage reconstruction of digital nerve defect after hand injury is feasible, which provides a novel trend for peripheral nerve reconstruction.     

Lamellar bodies in Purkinje nerve cells experimentally induced by electric field

Rabbits reared from mating to the age of almost two months outdoors in a substation in an electric (E)-field of 14 kV/m (undisturbed field, 50 Hz AC) only gained about half the weight of their controls, either protected by a Faraday's cage or being kept outside measurable E-field. The Purkinje nerve cells of cerebellum showed important alterations of the endoplasmic reticulum with disintegration of Nissl bodies, disappearance of hypolemmal cisterns and formation of numerous lamellar bodies. Concomitantly, there was a reduction in number of microtubules and increase in neurofilaments. These structural neuronal changes were reflected by changes in the behaviour of the rabbits which become slow in movements.     

Mechanism of histopathological changes of nerve cells experimentally induced by chronic alcohol poisoning

Experimental alcoholism was produced in rats by supplying them with 15% ethanol as the only source of liquid for a whole year. Histopathological examination revealed that Purkinje cells and granule cells in the cerebellum mainly showed such changes as decrease of ER, ribosomes and severe atrophy of the nerve cells. It might be speculated that these changes were caused by the disturbance of protein synthesis in the nerve cells induced by chronic alcohol effect.     

香丹注射液诱导人脐血间充质干细胞向神经细胞的分化

背景：选择一种高效、细胞损伤低的诱导人脐血间充质干细胞分化为神经细胞的方法是其应用于临床的前提。 目的：拟采用传统中药香丹注射液联合生长因子诱导脐血间充质干细胞向神经细胞方向分化,并且与单纯生长因子的诱导作用进行比较。 设计、时间及地点：免疫细胞化学水平的细胞观察实验,于2006-09/2008-04在潍坊医学院组织胚胎学教研室完成。 材料：人脐血标本取自潍坊市妇幼保健院,香丹注射液的有效成分为丹参素。 方法：采用密度梯度离心法分离人脐血单个核细胞,贴壁法筛选出人脐血间充质干细胞,体外扩增,以免疫荧光方法检测表面抗原。分为2组进行诱导分化,香丹注射液联合生长因子诱导组采用30 g/L香丹注射液联合表皮生长因子和碱性成纤维细胞生长因子诱导脐血间充质干细胞向神经细胞方向分化,并且与单纯生长因子诱导组进行比较。 主要观察指标：采用免疫细胞化学方法和免疫荧光方法检测诱导前后神经元特异性标志（神经元核抗原、β-TubulinⅢ）和神经胶质纤维酸性蛋白的表达。 结果：香丹注射液联合生长因子诱导法对脐血间充质干细胞损伤作用小,诱导效率高。脐血间充质干细胞经香丹注射液诱导后出现类似神经细胞的形态改变,胞体呈椭圆形,伸出长突起。免疫组织化学和免疫荧光方法鉴定显示,诱导后的细胞能特异性表达神经元核抗原和β-TubulinⅢ,而神经胶质纤维酸性蛋白阳性细胞较少。香丹注射液联合生长因子诱导组的 β-TubulinⅢ和神经元核抗原的阳性细胞率显著高于生长因子诱导组（P < 0.05）,神经胶质纤维酸性蛋白阳性细胞率明显低于生长因子诱导组（P < 0.05）。 结论：香丹注射液联合生长因子诱导对脐血间充质干细胞损伤作用小,诱导效率高,优于生长因子诱导法。且体外诱导后细胞主要分化为神经元样细胞,而非星形胶质细胞。     

他克莫司与肌源性干细胞联合应用对去细胞异体神经支架移植后神经再生和修复作用的影响

背景：肌源性干细胞作为种子细胞用于制备组织工程化人工神经已经被越来越多的学者所接受。他克莫司不仅具有抗免疫排斥的效果,还具有强大的促进神经再生和修复的作用。那么能否将两项因素与去细胞异体神经支架形成一个桥接体,既能抑制异体移植的免疫反应,又能有效促进损伤神经的再生与修复？ 目的：采用理化联合处理方法制备去细胞异体神经支架,探讨肌源性干细胞与免疫抑制剂他克莫司联合应用对去细胞异体神经支架移植后神经再生及功能恢复的影响。 方法：SD大鼠坐骨神经经脱细胞处理后形成异体神经桥接体。用100 μL微量注射器将含他克莫司与肌源性干细胞的凝胶注入异体神经支架,用以修复大鼠的坐骨神经缺损。32只成年SD大鼠,随机分为4组,每组8只。切断其左侧坐骨神经造成10 mm 的缺损。他克莫司+肌源性干细胞组、肌源性干细胞组、他克莫司组以注射植入后的异体神经进行桥接;对照组仅注入透明质酸凝胶。术后8,12周进行坐骨神经指数和神经电生理测定。术后12周进行大体观察,神经组织学和超微结构观察。 结果和结论：在同一时点他克莫司+肌源性干细胞组坐骨神经功能指数、坐骨神经运动传导速度恢复率、移植体及远段有髓纤维计数均优于其他3组(P < 0.05)。各组神经移植体粗细基本正常,表面大量血管分布,与周围组织轻度粘连;他克莫 司+肌源性干细胞组较其他3组再生神经纤维更加密集、排列规则整齐;移植体许旺细胞大量增殖,移植体中央及远段内的有髓神经纤维密度、直径高于肌源性干细胞组、他克莫司组,微束之间结缔组织少,接近于正常。说明肌源性干细胞和他克莫司联合应用促进去细胞异种神经移植的神经再生与功能恢复的效果优于单独应用。肌源性干细胞和他克莫司在周围神经损伤修复中是一对具有协同作用的因子。 关键词：周围神经损伤;异体神经移植;神经再生;肌源性干细胞;他克莫司 doi:10.3969/j.issn.1673-8225.2010.03.003