神经干细胞移植防治骨骼肌失神经肌萎缩的电生理研究

目的探讨采用神经干细胞移植的方法防治骨骼肌失神经萎缩的可行性。方法采用机械分离的方法从孕14～16 d的SD孕鼠中获取神经干细胞,并于神经元限定性培养基中进行传代培养,制备神经干细胞单细胞悬液。采用切断右侧胫神经的方法建立腓肠肌失神经支配的动物(SD大鼠)模型。将108只SD大鼠按注射药物的不同随机分为3组,每组36只大鼠。实验组:将神经干细胞悬液注射到切断的胫神经远端。损伤组:注射等量的生理盐水。对照组:注射等量的细胞培养液。术后8、12周采用HRP逆行示踪技术检测失神经骨骼肌重获神经再支配的情况,并应用肌肉电生理方法对重获神经再支配的骨骼肌进行功能评价。结果术后8、12周实验组用电刺激细胞移植部位的腓肠肌,均可引出肌肉收缩活动;且随着时间的延长,单次收缩的波幅、速度,和强直收缩的时间和强直收缩波幅的恢复率均进一步得到改善。对照组和损伤组均未能引出肌肉活动。结论神经干细胞移植能够实现失神经骨骼肌的神经再支配,并且能够与骨骼肌建立起功能性突触连接,有效预防骨骼肌的萎缩。     

5-溴脱氧尿嘧啶核苷标记示踪脂肪成体干细胞体内成骨分化的研究

目的 观察经成骨诱导后的脂肪成体干细胞(ADASCs)在体内的成骨分化行为.方法 分离培养兔ADASCs,用5-溴脱氧尿嘧啶核苷(BrdU)标记,并通过免疫组织化学方法检查标记结果.将标记的ADASCs体外成骨诱导培养2周后与脱钙骨基质(DBM)复合,共培养7d后植入自体肌袋内.术后8周取材,固定、脱钙、包埋后切片染色,镜下观察.结果 大量ADASCs被标记,标记率达(82.3±8.6)%;体外标记后的ADASCs可以向成骨方向分化;切片苏木素-伊红(HE)染色示体内有新生骨形成,切片BrdU免疫组织化学染色示新生骨处有阳性细胞.结论 ADASCs经体外成骨诱导后植入体内能够继续维持成骨分化,并进一步形成新生骨.     

胞嘧啶脱氨酶基因修饰神经干细胞及其表达的离体实验研究

目的 探讨胞嘧啶脱氨酶(cytimidine deaminase，CD)基因修饰神经干细胞及其基因表达。方法 通过构建真核表达质粒pCMVCD，限制性内切酶消化鉴定后，采用Lipofectamine 2000脂质体介导法转染新生大鼠室管膜下区神经干细胞(Neural stem cells，NSCs)，G418筛选阳性克隆，加入不同浓度的5-氟胞嘧啶(5-Flourocytosine，5-FC)，MTT比色法测定NSCs的生存率。结果 本实验成功地培养并鉴定了神经干细胞，并将CD基因成功地转染了神经干细胞，G418阳性NSCs对低浓度5-FC高度敏感。结论 CD基因修饰神经干细胞的离体实验研究为干细胞治疗研究提供依据。     

Serial in vivo MR tracking of magnetically labeled neural spheres transplanted in chronic EAE mice.

Neural stem cell (NSC) transplantation has been shown to attenuate the severity of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Central to the future success of NSC transplantation in MS is the ability of transplanted cells to migrate from the site of transplantation to relevant foci of disease. Using magnetically labeled mouse neurospheres and human embryonic stem cell (hESC)-derived neurospheres, we applied serial magnetic resonance imaging (MRI) to assess the biodynamics of transplanted cell migration in a chronic mouse EAE model. Magnetic labeling did not affect the in vitro and in vivo characteristics of cells as multipotential precursors. Cell migration occurred along white matter (WM) tracts (especially the corpus callosum (CC), fimbria, and internal capsule), predominantly early in the acute phase of disease, and in an asymmetric manner. The distance of cell migration correlated well with clinical severity of disease and the number of microglia in the WM tracts, supporting the notion that inflammatory signals promote transplanted cell migration. This study shows for the first time that hESC-derived neural precursors also respond to tissue signals in an MS model, similarly to rodent cells. The results are directly relevant for designing and optimizing cell therapies for MS, and achieving a better understanding of in vivo cell dynamics and cell-tissue interactions.     

Brain stem tuberculoma in adult patients: Diagnosis and treatment

A consecutive series of six adult patients ranging in age from 29 to 53 years is presented. The clinical and radiological features in each patient are described. Attention is drawn to the features demonstrated on computed axial tomography. In only one patient, the first encountered, was surgical excision undertaken and histological verification obtained. One patient died before any form of treatment could be instituted. The remaining four patients were treated with antituberculous chemotherapy alone and their progress monitored by sequential computed tomography. The excellent response and good outcome in this conservatively treated group are documented.     

Unilateral Meesmann‘s dystrophy

We report the first case of a unilateral microcysticMeesmann‘s epithelial dystrophy, observed in the left cornea of a 43-year-old patient. The diagnosis was verified histologically by the unilateral occurrence of a ’peculiar substance‘ within epithelial cysts and within the cytoplasm of the corneal epithelial cells. In an attempt to resolve thepatient‘s frequent corneal erosions, we performed an autologous stem cell transplantation from the non-involved right eye. The results are, so far, satisfactory. This revised version was published online in August 2006 with corrections to the Cover Date.     

Collection of blood mononuclear cells by leukapheresis for transplantation in a yucatan miniature swine model

A large animal model is needed to evaluate new apheresis technologies. These technologies include novel methods of harvesting the blood mononuclear cell population which contains the hematopoietic stem cells needed to restore hematopoiesis in recipients of hematopoietically lethal therapy and the use of cytokines to provide a safe and predictable method of manipulating these circulating hematopoietic stem cells. We describe the methods used to collect mononuclear cells by leukapheresis from Yucatan miniature swine. These animals are of sufficient size to tolerate the procedures and have many physiologic and hematologic similarities to man. They are of good temperament and are easily trained. Long-term venous access was obtained using single lumen silicone rubber catheters placed in the inferior vena cava. The animals were apheresed while fully awake using a Haemonetics Model V50 machine and a modified lymphocyte collection protocol. The procedure was highly efficient for the collection of mononuclear cells and a 10 pass procedure yielded a product which contained 19.7 × 10⁹ mononuclear cells, 10.7 × 10⁹ granulocytes, and 17 ml of erythrocytes in a volume of approximately 100 ml. This product can be cryopreserved and used for subsequent transplantation. The content of four apheresis procedures provides hematopoietic reconstitution of lethally irradiated swine on a time scale equivalent to transplantation of optimal numbers of bone marrow cells. © 1992 Wiley-Liss, Inc.     

人HLA半相合混合脐血在SCID小鼠移植的实验研究

目的：探讨不同供者来源脐血混合移植的可行性和植入特性。方法：分别将两份人HLA半相合混合脐血或单份脐血输入经亚致量照射后的严重联合免疫缺陷（SCID）小鼠，观察两组脐血在SCID小鼠体内的植入状况及多系造血重建特性。结果：混合脐血和单份脐血移植均可在受鼠体内植入，形成供－受混合嵌合体，并能重建多系造血，存活率和植入率无统计学意义（P＞0．05）。用多聚酶链反应－序列特异性寡核苷酸（PCR－SSO）探针检测人HLA－DQB1基因发现，HLA半相合混合脐血移植可有1份或2份脐血植入，其中造血祖细胞含量和体外克隆形成能力高者，更易于植入，造血重建特性与单一脐血移植比较无统计学意义。结论：HLA半相合人混合脐血可同时在SCID小鼠体内植入，形成来自供－受三者的多嵌合状态，并能重建造血及免疫功能。     

骨膜间质干细胞移植时机的实验和临床研究

目的 研究骨膜间质干细胞异体移植时机。方法 根据骨缺损内纤维组织生长时间的不同，将从大鼠骨膜分离培养的细胞分别分批植入，在植入的第2、3、4和8周时动态观察其成骨量，即骨小梁体积比；另将幼儿骨膜间质干细胞悬液，移植于26例长管状骨干骨折2周后的缺损内，观察骨折临床愈合的时间。结果 大鼠骨缺损后2周以内移植干细胞其骨小梁体积比大，愈合快，2周以后与对照组比无显著差异；临床应用9个月内病人骨折缺损愈合15例(57．7％)，骨不愈合者11例(42．3％)。结论 大鼠骨缺损2周以内移植干细胞其疗效好，大鼠实验和临床上骨缺损2周以后移植骨膜间质干细胞不能明显促进骨缺损愈合。     

稳定增殖神经干细胞的实验研究

目的探讨稳定、可靠建立神经干细胞体外增殖的方法,并对增殖培养的细胞进行鉴定。方法获取胚胎大鼠的脑组织,通过加入神经生长因子和采用保留细胞联系技术操作,使脑组织中的神经干细胞在体外克隆增殖并稳定传代。以免疫荧光方法对增殖克隆的神经干细胞球进行鉴定。结果神经干细胞不断增殖形成神经干细胞球且神经干细胞能快速稳定传代增殖。培养的细胞为神经干细胞特异性巢蛋白(nestin)染色阳性细胞。结论在神经生长因子的作用下,利用保留细胞联系技术操作,神经干细胞可以在体外快速、稳定克隆增殖并传代。