微囊化人头皮毛乳头细胞诱导小鼠耳毛囊再生的研究

目的 观察人头皮毛乳头细胞海藻酸钠-多聚赖氨酸-海藻酸钠（alginate-polylysine-alginate，APA）微囊是否具备诱导小鼠耳部毛囊再生的功能；寻找理想的微囊直径。方法 以APA微囊包裹体外分离培养的人头皮毛乳头细胞；将毛乳头细胞微囊移植至小鼠耳部皮下，6周后局部取材行组织学检查；共聚焦显微镜下观察葡聚糖-荧光素在APA微囊中的扩散速度和扩散方式，并对比相同时间、不同直径的APA微囊中葡聚糖-荧光素的强度，综合分析确定最佳的微囊直径。结果 组织学检查显示：移植部位皮下有密集的同心圆状毛囊结构形成，其数量、大小、分化程度等与对照组明显不同。荧光素以同心圆状、逐层渗透的方式向APA微囊中扩散；相同时间内荧光强度比较：小囊组〉中囊组〉大囊组。结论 微囊化毛乳头细胞具备诱导毛囊再生的生理功能；微囊理想的直径是400μm。     

大鼠门静脉分支结扎后肝再生的实验研究

目的 采用手术方法结扎门静脉分支，观察对侧肝脏的再生状态。方法 健康Wistar雄性大鼠60只，随机均分成结扎组和假结扎组，在乙醚麻醉下行门静脉左支结扎和假结扎手术，分别在术后第1、2、3、7及14d用乙醚麻醉动物，心脏采血检测血清ALT值，取出肝脏后称重量；在光镜下观察肝组织的病理变化，并计数肝细胞核分裂指数；采用免疫组化的方法计数增殖细胞核抗原（PCNA）指数；在电镜下观察肝细胞超微结构变化。结果 ①血清ALT值结扎组与假结扎组比较在术后第1d升高（P〈0．01），但在第2d开始恢复正常；②肝脏重量两组间差异无统计学意义（P〉0．05）；③肝细胞核分裂指数结扎组与假结扎组比较，术后第1～3d明显升高，第2d达高峰，第3d有所下降，但仍高于假结扎组（P〈0．01），以后逐渐恢复正常；④PCNA阳性细胞计数结扎组与假结扎组比较，术后第1～3d明显增多，第2d达高峰，第3d有所减少，但仍高于假结扎组（P〈0．01），以后逐渐恢复正常。结论 ①大鼠门静脉左支结扎后，引起未结扎侧肝细胞的活跃再生，再生后的肝脏可恢复原来的重量；②大鼠75％肝叶的门静脉分支结扎不影响肝功能，是安全可行的；③大鼠门静脉分支结扎可以作为研究肝脏再生动物模型的方法。     

角膜损伤后神经再生的实验研究

应用神经组织化学技术观察了兔角膜NA能神经及AchE阳性神经在角膜损伤后的再生,证实术后1月,两种神经均有再生轴突进入植片;术后3月可见交界区和植床内神经密度明显增加;同时,对术后两种神经再生的功能意义进行了讨论。     

An Evolutionarily Conserved Region in the Second lntron of the Human Nestin Gene Directs Gene Exmession to CNS Progenitor Cells and to Early Neural Ciest Cells

Central nervous system (CNS) progenitor cells transiently proliferate in the embryonic neural tube and give rise to neurons and glial cells. A characteristic feature of the CNS progenitor cells is expression of the intermediate filament nestin and it was previously shown that the rat nestin second intron functions as an enhancer, directing gene expression to CNS progenitor cells. In this report we characterize the nestin enhancer in further detail. Cloning and sequence analysis of the rat and human nestin second introns revealed local domains of high sequence similarity in the 3' portion of the introns. Transgenic mice were generated with the most conserved 714 bp in the 3' portion of the intron, or with the complete, 1852 bp, human second intron, coupled to the reporter gene lacZ. The two constructs gave a very similar nestin-like expression pattern, indicating that the important control elements reside in the 714 bp element. Expression was observed starting in embryonic day (E)7.5 neural plate, and at E10.5 CNS progenitor cells throughout the neural tube expressed lacZ. At E12.5, lacZ expression was more restricted and confined to proliferating regions in the neural tube. An interesting difference, compared to the rat nestin second intron, was that the human intron at E10.5 mediated lacZ expression also in early migrating neural crest cells, which is a site of endogenous nestin expression. In conclusion, these data show that a relatively short, evolutionarily conserved region is sufficient to control gene expression in CNS progenitor cells, but that the same region differs between rodents and primates in its capacity to control expression in neural crest cells.     

Automatic assessment of levodopa-induced dyskinesias in daily life by neural networks.

We developed an objective and automatic procedure to assess the severity of levodopa-induced dyskinesia (LID) in patients with Parkinson's disease during daily life activities. Thirteen patients were continuously monitored in a home-like situation for a period of approximately 2.5 hours. During this time period, the patients performed approximately 35 functional daily life activities. Behavior of the patients was measured using triaxial accelerometers, which were placed at six different positions on the body. A neural network was trained to assess the severity of LID using various variables of the accelerometer signals. Neural network scores were compared with the assessment by physicians, who evaluated the continuously videotaped behavior of the patients off-line. The neural network correctly classified dyskinesia or the absence of dyskinesia in 15-minute intervals in 93.7, 99.7, and 97.0% for the arm, trunk, and leg, respectively. In the few cases of misclassification, the rating by the neural network was in the class next to that indicated by the physicians using the AIMS score (scale 0-4). Analysis of the neural networks revealed several new variables, which are relevant for assessing the severity of LID. The results indicate that the neural network can accurately assess the severity of LID and could distinguish LID from voluntary movements in daily life situations.     

Ventrally emigrating neural tube cells migrate into the developing vestibulocochlear nerve and otic vesicle

Virtually all cell types in the inner ear develop from the cells of the otic vesicle. The otic vesicle is formed by the invagination of non-neural ectodermal cells known as the otic placode. We investigated whether a recently described cell population, originating from the ventral part of the hindbrain neural tube known as the ventrally emigrating neural tube (VENT) cells, also contributes cells to the otic vesicle. The ventral hindbrain neural tube cells were labeled with the fluorescent vital dye DiI or replication-deficient retroviruses containing the LacZ gene in chick embryos on embryonic day 2, after the emigration of neural crest from this region. One day later, the labeled cells were detected only in the hindbrain neural tube. Shortly thereafter, the labeled cells began to appear in the eighth (vestibulocochlear) cranial nerve and otic vesicle. From embryonic day 3.5-5, the labeled cells were detected in the major derivatives of the otic vesicle, i.e. the endolymphatic duct, semicircular canals, utricle, saccule, cochlea, and vestibulocochlear ganglion. That the emigrated cells originated from the ventral part of the hindbrain neural tube was confirmed by focal application of DiI impregnated filter paper and with quail chimeras. It is concluded that, in addition to the otic placode cells, the otic vesicle also contains the ventrally emigrating neural tube cells, and that both cell populations contribute to the structures and cell types in the inner ear. It is well known that inductive signals from the hindbrain are required for the morphogenesis of the inner ear. The migration of the hindbrain neural tube cells into the otic vesicle raises the possibility that the inductive effect of the hindbrain might be mediated, at least in part, by the ventrally emigrating neural tube cells and that, therefore, a mechanism exists that involves cells rather than diffusible molecules only.     

SCHWANN CELLS AND THE REGROWTH OF AXONS IN THE MAMMALIAN CNS: A REVIEW OF TRANSPLANTATION STUDIES IN THE RAT VISUAL SYSTEM

1. We have used peripheral nerve transplants or cultured Schwann cells grafted in association with different types of polymer to study axonal regrowth in the rat visual system. In some instances the glia were co-grafted with fetal tectal tissue. 2. The studies have two main aims: (i) to determine whether retinal axons can be induced to regrow at a site distant from their cell soma, that is, after damage to the brachial region of the optic tract; (ii) to determine whether retinal axons exposed to Schwann cells retain the ability to recognize their appropriate target neurons in CNS tissue. 3. In brachial lesion studies, Schwann cells were placed in the lesion site in association with nitrocellulose papers, within polycarbonate tubes in the presence or absence of a supporting extracellular matrix (ECM), or within polymer hydrogel scaffolds. Autologous sciatic nerve grafts were also used. Immuno-histochemical studies revealed the presence of regenerating axons within all polymer bridges. Regrowth of retinal axons was also seen, however, growth was not extensive and was limited to the proximal 1–1.5 mm of the implants. 4. In target innervation experiments, two surgical paradigms were developed. In one experiment, a segment of sciatic nerve was autografted onto the transected optic nerve in adult rats and the distal end of each graft was placed adjacent to fetal tectal (target) tissue implanted into the frontal cortex. To date, we have not been able to demonstrate selective recognition of target regions within tectal transplants by retinal axons exiting the sciatic nerve implants. 5. In the second experiment, Schwann cells were mixed with fetal tectal cells and co-grafted to the midbrain of newborn host rats. Schwann cells altered the characteristic pattern of host retinal growth into tectal grafts; in some cases axons were induced to grow away from appropriate target areas by nearby co-grafted Schwann cells. 6. In summary, Schwann cell/polymer scaffolds may provide a useful way of promoting the regrowth of damaged axons in the CNS, however: (i) in adults, at least, their effectiveness is reduced if they are located at a distance from the cell bodies giving rise to regenerating axons; (ii) in some circumstances exposure to a peripheral glial environment may affect the capacity of regenerating axons to recognize appropriate target cells in the CNS neuropil.     

人参皂甙Rg1对大鼠局灶性脑缺血脑组织神经干细胞增殖的影响

目的 观察人参皂甙Rg1对大鼠局灶性脑缺血脑组织神经干细胞增殖的影响,探讨其在脑保护及抗衰老作用中的可能机制.方法 取成年雄性Wistar大鼠,用线栓法制作大鼠大脑中动脉闭塞(MCAO)模型,采用脉冲标记法,经腹腔注射5′-溴脱氧尿苷(BrdU)标记处于增殖状态的神经干细胞,用免疫组织化学单标及双标免疫荧光技术观察人参皂甙Rg1对局灶性脑缺血后BrdU和巢蛋白(nestin)的免疫活性及nestin/BrdU双标阳性细胞的影响,镜下观察脑内神经干细胞的分布,并计数行定量分析.结果 局灶性脑缺血后大鼠室管膜下区及海马齿状回有nestin、BrdU及nestin/BrdU双标免疫阳性细胞分布.应用人参皂甙Rg1后,上述各部位阳性细胞数明显增多,与缺血组之间有显著性差异(P＜0.01).结论 人参皂甙Rg1具有促使神经干细胞增殖的能力,这可能是其神经保护及抗衰老作用的机制之一.