不同胚龄人神经干细胞的分离和培养

目的 探索不同胚龄人神经干细胞分离和培养的适宜条件。方法 在多种培养条件下，采用无血清培养和单细胞克隆技术，从7周和13周人胚脑中分离和培养神经干细胞，应用免疫荧光染色予以鉴定。结果 从两个不同胚龄的人胚脑中分离出具有自我更新和多分化潜能的神经干细胞，未包被组7周的人胚脑除高密度县浮培养法生长不良，其它密度及培养法均有克隆球形成；13周的人胚脑仅中等密度悬浮培养法及高密度贴壁培养法有克隆球形成；包被组各种密度培养均生长不良。结论 人胚脑中存在具有自我更新能力和多分化潜能的神经干细胞，随着胚龄增大，培养难度逐渐加大，高密度贴壁培养法适宜各胚龄神经干细胞的分离培养。     

小鼠胚胎神经干细胞的长期培养和分化

目的：探索小鼠胚胎神经干细胞的体外培养和分化条件,为神经干细胞的深入研究奠定基础。方法：无菌条件下分离小鼠胚胎脑皮质,制成单细胞悬液,种植在N2培养基中培养,培养基中加入20ng/ml的碱性成纤维细胞生长因子（每次实验用1－2只小鼠胚胎,实验重复8次）。采用机械方法传代,常规方法冻存和复苏。用免疫细胞化学方法对培养的细胞进行鉴定。结果：成功培养出小鼠的神经干细胞,神经干细胞呈悬浮状态生长,形成典型的神经球。细胞表达巢蛋白和波形蛋白2种神经干细胞的标志物。细胞在胎牛血清的诱导下,可分化成神经元、星型胶质细胞和少突胶质细胞,它们所占的比例分别为7％、85％－90％和2％－4％。结论:鼠的神经干细胞可以在体外稳定地培养和传代,为细胞治疗提供了一个理想的细胞来源。     

成年脊髓损伤大鼠脊髓神经干细胞的体外培养及分化研究

目的 探讨大鼠脊髓损伤后脊髓神经干细胞的分离培养方法及分化情况.方法 采用Allen法制作大鼠脊髓损伤模型,利用无血清培养和单细胞克隆技术在成年脊髓损伤7 d大鼠脊髓中分离具有单细胞克隆能力的神经干细胞,并进行培养鉴定.结果 从成年脊髓损伤7 d大鼠脊髓中成功分离出神经干细胞,该细胞具有连续克隆能力,可传代培养,表达神经巢蛋白抗原.分化后的细胞表达神经元细胞、星形胶质细胞和少突胶质细胞的特异性抗原.结论 致伤7 d的成年大鼠脊髓组织体外町培养出神经十细胞,并分化为神经无细胞、星形胶质细胞和少突胶质细胞,有可能参与脊髓损伤的修复过程.     

Vigorous neuronal differentiation of amplified and grafted basic fibroblast growth factor-responsive neurospheres derived from neuroepithelial stem cells

Neuroepithelial stem cells (NESCs) have emerged as a possible donor material aimed at neural transplantation for the repair of damaged neural circuitry, particularly because of their propensity to differentiate into neurons. We previously ascertained in vitro that NESCs derived from rat early embryos could be amplified in culture containing basic fibroblast growth factors (bFGF), and that neurospheres grown for 7 days in the culture had a strong tendency to differentiate into neurons. In this report, we analyze immunohistochemically the biological nature of bFGF-responsive neurospheres derived from NESCs. We first succeeded in amplifying the number of NESCs from the mesencephalic neural plate of embryonic day 10 Wistar rats with the addition of bFGF. Grown neurospheres were labeled with bromodeoxyuridine (BrdU) in vitro and were stereotactically transplanted into the right striatum of the normal adult Wistar rat. Two weeks after transplantation, a viable graft in the host brain was observed. While many BrdU/Hu double positive cells were seen in the graft, and a few BrdU/nestin double positive cells were also seen, no BrdU/GFAP double positive cells could be identified. These results suggested that bFGF-responsive neurospheres derived from NESCs demonstrated a propensity to differentiate into neurons in the adult brain environment. Furthermore, following in vitro amplification of the original stem cell number with bFGF, the grown neurospheres preserved their propensity to differentiate vigorously into neurons. NESCs are thus suggested as a feasible candidate for intracerebral grafting donor materials aimed at reconstruction of damaged neural circuits.     

体外琼脂克隆培养对胎肝干细胞分化的影响

目的 探讨和建立体外扩增胚胎肝干细胞并抑制其分化的培养条件及方法.方法 胶原酶结合机械消化法制备14 d胎龄大鼠胎肝干细胞悬液.将细胞随机分为两组.一组接种至Ⅰ型胶原包被的培养板中,常规贴壁培养.另一组细胞则接种至软琼脂培养基中进行悬浮培养.倒置显微镜下观察两组细胞的生长.培养2周后收集两组细胞,电镜观察两组细胞超微结构的差异,流式细胞仪检测其CD90.1+、CD49F+等干细胞标志物表达特征,碱性磷酸酶染色检测其分化状态的差异.结果 悬浮培养的胎肝干细胞具有高核浆比、胞浆内细胞器不发达等超微结构特征,并高表达CD90.1、CD49F等干细胞标志物,碱性磷酸酶染色阳性;而常规贴壁培养的胎肝干细胞则在培养过程中显示出显著的分化特征.结论 琼脂克隆培养的胎肝干细胞较有血清贴壁培养分化程度低,在琼脂克隆培养条件下能增殖形成具有显著干细胞特征的克隆球.     

大鼠嗅鞘细胞无血清上清液诱导脊髓神经干细胞分化的研究

[目的]观察大鼠嗅神经鞘细胞上清液对脊髓神经干细胞共培养发生的诱导分化作用。[方法]采用差速贴壁法获得较高纯度的嗅鞘细胞，分时段Mr丌法检测细胞活性，选取最佳状态细胞无血清培养后取上清液，与3代纯化后的神经干细胞共培养，观察分化过程，免疫荧光法鉴定诱导结果。[结果]MTT法分6个时段检测纯化后的嗅鞘细胞，发现9d及12d细胞活性最高。使用无血清嗅鞘细胞上清液与脊髓神经干细胞共培养发现诱导作用明显，向神经元样细胞及胶质细胞分化的比例分别达到53％和42％。[结论]嗅鞘细胞在体外培养的不同时间段活性不同，无血清的嗅鞘细胞上清有明显诱导神经干细胞向成熟神经元分化的作用。     

Long-term nonpassaged EGF-responsive neural precursor cells are stem cells

We have screened lines of nonpassaged epidermal growth factor-responsive neurospheres from embryonic striatum and brainstem. They have been maintained in defined medium with epidermal growth factor over a period of 2 years and remained in an undifferentiated state to this date. Since isolation from the brain 2 years ago, these nonpassaged epidermal growth factor responsive neurospheres have shown active proliferation and self-renewal capacity. When subplated on a poly-D-lysine coated surface, they resumed differentiation within 24 hours. The differentiation process of the nonpassaged epidermal growth factor responsive neurosphere appeared to recapitulate the neural development in the brain. Many cells migrated, extending radial processes while expressing nestin and S100 in the early 7-day subplating culture. They continued to differentiate into major neural types in 14-day subplating culture, including fibrous and cytoplasmic astrocytes, oligodendrocytes, and serotonin, γ-aminobutyric acid, and a small number of tyrosine hydroxylase-positive neurons. The nonpassaged epidermal growth factor-responsive neurospheres in many ways resemble hemopoietic cells. Both are proliferative, possess the potential of indefinite self-renewal, yet multipotent, and are capable of resuming the differential pathway. The nonpassaged epidermal growth factor responsive neurospheres meet the criteria of stem cells and have been found to be a useful model to study the development in vitro.     

Proliferation, migration, and differentiation of human neural stem/progenitor cells after transplantation into a rat model of traumatic brain injury

OBJECT: Cultures containing human neural stem and progenitor cells (neurospheres) have the capacity to proliferate and differentiate into the major phenotypes of the adult brain. These properties make them candidates for therapeutic transplantation in cases of neurological diseases that involve cell loss. In this study, long-term cultured and cryopreserved cells were transplanted into the traumatically injured rat brain to evaluate the potential for human neural stem/progenitor cells to survive and differentiate following traumatic injury. METHODS: Neural stem/progenitor cell cultures were established from 10-week-old human forebrain. Immunosuppressed adult rats received a unilateral parietal cortical contusion injury, which was delivered using the weight-drop method. Immediately following the injury, these animals received transplants of neural stem/progenitor cells, which were placed close to the site of injury. Two or 6 weeks after the procedure, these animals were killed and their brains were examined by immunohistochemical analysis. At both 2 and 6 weeks postoperatively, the transplanted human cells were found in the perilesional zone, hippocampus, corpus callosum, and ipsilateral subependymal zone of the rats. Compared with the 2-week time point, an increased number of HuN-positive cells was observed at 6 weeks. In addition, at 6 weeks post-injury/transplantation, the cells were noted to cross the midline to the contralateral corpus callosum and into the contralateral cortex. Double labeling demonstrated neuronal and astrocytic, but not oligodendrocytic differentiation. Moreover, the cortex appeared to provide an environment that was less hospitable to neuronal differentiation than the hippocampus. CONCLUSIONS: This study shows that expandable human neural stem/progenitor cells survive transplantation, and migrate, differentiate, and proliferate in the injured brain. These cells could potentially be developed for transplantation therapy in cases of traumatic brain injury.     

Neurite growth capability of rat fetal neuronal cells against matured CNS myelin in vitro

Reconstruction of neurocircuits by transplanted cells is expected to become an effective therapy for brain damage. In order to establish the transplantation therapy, it is necessary to find transplantable cells capable of reconstructing the lesioned neurocircuitry. We have reported that the younger neuronal cells such as neural stem cells are useful transplant materials because of their vigorous capacity for forming abundant neurites. On the other hand, it was reported that myelin-associated neurite growth inhibitor prevents neurite regeneration. In this study, we used rat fetal neuronal cells to examine the neurite growth capacity in the presence of mature CNS myelin. Crude CNS myelin was prepared from the brains of adult Wistar rats using previously described procedures. Testing wells were precoated with poly-L-lysine and additionally by over-night drying of a suspension containing 0, 5, 10, 15, or 20 microg/cm2 of the crude myelin protein. On embryonic days 10, 12, 15, and 17 (E10, E12, E15, and E17) embryos were surgically removed, mesencephalic neural plates were dissected out from the E10 embryos, and midbrain cells were taken from the E12, E15, and E17 embryos. The neural plates and midbrain cells were placed on the myelin-coated wells. After 24 h of culture (72 h in the case of neural plates), the number of surviving cells and the length of the neurites were examined immunocytochemically using anti-neurofilament (NF) antibody. Neurite length was measured by image analyzer Luzex-F. The mesencephalic neural plate was able to grow neurites even on 20 microg/cm2 central myelin. Almost the same number of midbrain cells attached themselves to the wells without myelin in every culture obtained from various stages of embryos. The number of cells attached on the myelin-coated wells decreased with the concentration of myelin. The number of NF-positive cells was higher in cultures of materials obtained from older embryos than in cultures obtained from younger embryos. The younger cells grew longer neurites than the older cells in the myelin noncoated wells. Neurite growth was inhibited strongly when the concentration of the central myelin was 10 microg/cm2 or greater, but on the 5 microg/cm2 myelin, the younger the cells were, the longer neurites they had. When the length of the longest neurites in one field of the image analyzer was further examined in the same way, the younger the cells were, the longer their axons grew on 0 and 5 microg/cm2 myelin. Thus, CNS myelin was seen to be a significant inhibitor of the recovery of injured neural tissue of the adult CNS. Younger cells grew longer neurites than older cells on CNS myelin, and so it was suggested that neural stem cells or younger neurons may serve as tissue for transplantation therapy.     

长期培养人胚神经干细胞对兔脊髓损伤的修复作用

[目的]观察长期培养人胚神经干细胞（hNSCs）的体外生长特性与转染EGFP基因后移植治疗兔脊髓横切损伤模型在体内的生物学活性及对神经结构修复和功能恢复的影响。[方法]体外分离、培养并鉴定hNSCs，用逆转录病毒介导的增强绿色荧光蛋白基因（EGFP）进行转染；制备兔T9全横断脊髓损伤模型；观察hNSCs移植对脊髓损伤后神经结构修复和功能恢复的影响。[结果]从胎龄10～20周的新鲜人胚脑皮层中成功分离出神经干细胞，该细胞具有连续克隆传代能力，诱导分化后表达分化细胞的特异抗原。本实验室已成功连续培养10个月（17代），转染EGFP基因后，仍保持未分化状态，能够自我更新形成新的神经球；移植入兔SCI模型后，hNSCs能在体内存活、迁移、分化并增殖。与对照组相比，hNSCs移植组明显促进了脊髓神经的再生、结构的修复和下肢运动功能的恢复。[结论]hNSCs移植促进了脊髓损伤后神经结构的修复和功能的恢复，是治疗急性脊髓损伤的一种有效方法。     

Pluripotent fates and tissue regenerative potential of adult olfactory bulb neural stem and progenitor cells

Neural stem cells and progenitor cells reside in the adult olfactory bulb (OB) core of mouse, rat, and human. Adult rodent OB core cells have the capacities for self-renewal and multipotency and form neurospheres. The differentiation fates of these neurosphere-forming cells were studied in vitro and in vivo. Adult OB neurospheres were comprised of stem cells and neuronal and glial progenitor cells. OB neurospheres in co-culture with primary embryonic striatal neurons and cortical neurons generated cells with morphological and neurochemical phenotypes of striatal and cortical neurons, respectively. Transplanted OB cells, delivered as dissociated cells or as intact neurospheres, dispersed, survived for long-term, extended neurites, migrated, expressed neuronal or glial markers, and formed synapses with host neurons when placed into the environment of the nonlesioned and lesioned central nervous system (CNS). Grafted cells in the CNS also showed angiogenic capacity by forming blood vessels. In a model of spinal motor neuron degeneration, adult OB neurosphere cells transplanted into lesioned spinal cord adopted phenotypes of motor neurons and had a robust potential to become oligodendrocytes. OB core cells in co-culture with skeletal myoblasts generated skeletal muscle cells. Chimeric skeletal muscle was formed when mouse OB neurospheres were transplanted into rat skeletal muscle. Within skeletal muscle, adult OB neurosphere cells became myogenic progenitor cells to form myotubes de novo. We conclude that the adult mammalian OB is a source of pluripotent neural stem cells and progenitor cells that have the potential to become, in a context-dependent manner, specific types of cells for regeneration of tissues in brain, spinal cord, and muscle.     

成体神经干细胞培养方法的建立

目的 探讨体外建立培养成体神经干细胞(MSC)的方法.方法 从6周龄大鼠脑组织中分离神经干细胞,用神经干细胞培养液[DMEM/F12培养液添加1%N2、2%B27、20 μg/L表皮生长因子(EGF)和20μg/L碱性成纤维细胞生长因子(bFGF)]使其稳定增殖,10%胎牛血清诱导其贴壁分化.倒置显微镜下观察神经干细胞形态学变化;流式细胞仪检测神经干细胞表面标记物巢蛋白(Nestin)、神经元特异性烯醇酶(NSE,成熟神经元的特异性标志)、半乳糖脑苷脂(Galc-C,成熟少突胶质细胞的标记物)的表达.结果 分离所得细胞能在体外传代培养,流式细胞仪检测显示Nestin阳性细胞为97.6%,细胞经胎牛血清诱导分化后能形成NSE、Galc-C阳性细胞.结论 采用无血清的神经干细胞培养液能培养出具有分化潜能的成体神经干细胞.     

Choukroun’SPRF对体外培养人脂肪干细胞增殖及成骨分化的影响

目的：观察自体富血小板纤维蛋白Choukroun＇s PRF（Choukroun＇s platelct-rich fibrin）对体外培养人脂肪来源干细胞（adipose-derived stem cells,ADSCs）增殖及成骨分化能力的影响。方法：取吸脂术者自愿捐献的脂肪组织分离培养ADSCs,采用Choukroun法制备自体PRF备用,观察细胞生长情况。取第3代ADSCs分别向骨细胞、脂肪细胞、神经球细胞定向诱导分化鉴定,并行细胞表面抗原CD29、CD45、CD90流式检测鉴定。将第3代ADSCs分别采用含PRF的普通培养基（PRF组Ⅰ）和不含PRF的普通培养基（对照组Ⅰ）进行培养,观察细胞生长情况,培养1天、3天、5天、7天后采用CCK-8试剂检测细胞增殖活性。另外分别采用含PRF的成骨诱导培养基（PRF组Ⅱ）、不含PRF的成骨诱导培养基（对照组Ⅱ）及不合PRF的普通培养基（空白组）进行培养,第7天、14天、21天、28天行碱性磷酸酶活性（ALP活性）检测;诱导细胞培养后第7天、14天天各组分别行von Kossa染色观察钙结节形成情况。结果：第3代ADSCs倒置显微镜下观察大多呈梭形,向骨细胞、脂肪细胞、神经干细胞定向诱导鉴定均为阳性,流式检测鉴定CD29、CD90为阳性,CD45为阴性。CCK-8法示PRF组Ⅰ的0D值均大于对照组Ⅰ,两组比较差异均有统计学意义（P〈0．01）。ALP活性检测示PRF组Ⅱ第7天、14天、21天、28天细胞活性较对照组Ⅱ均大,两组比较差异均有统计学意义（P〈0．01）。PRF组Ⅱ成骨诱导7天后vonKossa染色阳性;14天后阳性细胞增多,对照组Ⅱ诱导7天未见钙结节,14天见少量阳性钙结节,空白组培养14天未见黑色钙结节。结论：Choukroun’sPRF明显促进脂肪干细胞增殖及成骨分化,为骨组织工程提供了新的技术。PRF与干细胞共同培养可能还有许多潜在的临床及生物工程应用价值,值得进一步研究。     

Long-term culture of Japanese human embryonic stem cells in feeder-free conditions

Human pluripotent embryonic stem cells (hESCs) have great promise for research into human developmental biology, development of cell therapies for the treatment of diseases, toxicology, and drug discovery. Traditionally, undifferentiated hESCs are maintained on mouse embryonic fibroblasts (MEFs), which impede the clinical applications of hESCs. Here we have examined the long-term stability of the Japanese hESC line (KhES-1) in feeder-free culture. KhES-1 cells were cultured with MEF conditioned medium (CM) and different doses of basic fibroblast growth factor (bFGF) in six-well-plates of which the surface was coated with Matrigel. KhES-1 cells were maintained for at least 40 passages. In this culture system, the cells maintained stable proliferation rates and steadily expressed Oct-4, Nanog, and alkaline phosphatase. In addition, KhES-1 cells maintained without direct feeder contact formed embryonic bodies with expression of markers from the three germ layers. Here we demonstrated that Japanese human embryonic stem cells KhES-1 were cultured long term in a feeder-free method, while retaining pluripotency in vitro.     

Properties of secondary and tertiary human enteric nervous system neurospheres

Advances in enteric nervous system (ENS) stem cell biology have raised the possibility of treating Hirschsprung's disease with ENS stem/progenitor cell (ENSPC) transplantation. This study aimed to expand ENSPC numbers by the growth and redissociation of neurospheres and assess their differential potential.

Methods

Human ENS neurospheres were cultured as previously described and redissociated to generate secondary and tertiary neurospheres. Neurospheres were assessed for the presence of neuronal (PGP9.5), glial (S100), and stem cell (p75, nestin markers). The degree of immunofluorescence was quantified using the ImageJ program. Secondary/tertiary neurospheres were transplanted into mouse distal colon grown in tissue culture.

Results

Secondary/tertiary neurospheres could be generated with exponentially increasing numbers. Tertiary neurospheres showed a significant increase in the proportion of p75 staining but a significant decrease in the proportion of S100 staining. After transplantation, secondary/tertiary neurosphere-derived cells positive for PGP9.5 and S100 could be identified.

Conclusions

It is possible to exponentially expand neurosphere and therefore ENSPC numbers by repeated dissociation and culture. There is a loss of S100-positive cells in secondary/tertiary neurospheres, but the ENSPCs remain capable of differentiating into neurons and glia when transplanted into an embryonic gut environment.     

神经生长因子对神经干细胞增殖、分化和凋亡的影响

目的 观察不同剂量神经生长因子(NGF)对神经干细胞增殖、分化和凋亡的影响.方法 从孕15 d鼠胚胎脑室下区组织分离神经干细胞,培养于DMEM/F12+B27细胞基础培养液中,加入碱性成纤维细胞生长因子(bFGF)作为阳性对照组,实验组分别加入10、20、50μg/L的NGF,无添加成分作为阴性对照组.用噻唑蓝(MTT)比色分析法测定细胞增殖能力;倒置显微镜下观察细胞增殖、分化,鼠抗胶质纤维酸性蛋白(GFAP)、鼠抗神经元特异性烯醇化酶(NSE)免疫细胞化学染色鉴定;流式细胞仪检测细胞凋亡率.结果 10 d MTT比色显示,10μg/L NGF组(0.150±0.025)与阳性对照组(0.158±0.017)比较,差异无统计学意义(P＞0.05),与阴性对照组(0.128±0.015)比较,差异有统计学意义(P＜0.05).分化实验显示:随着NGF的浓度递增,NSE阳性率分别为(15.21±1.27)%、(20.64±2.61)%、(24.20±3.10)%,GFAP阳性率分别为(27.98±2.35)%、(28.94±2.84)%、(30.79±3.23)%,神经干细胞分化的比率增高[FNsz(+)=3.47、FGFAP(+)=3.47,PGFAP(+)＜0.05].流式细胞仪检测示20、50μg/L NGF组细胞凋亡率明显增高,l0μg/L NGF组细胞增殖指数(PI)较其他实验组高.结论 合适浓度NGF能促进神经干细胞的增殖.随神经生长因子浓度的增高,促神经分化作用增强.随着NGF浓度的增加,其促NSCs凋亡作用增强.

Abstract：

Objective To evaluate the influence of different doses of nerve growth factor (NGF) on the proliferation, differentiation and apoptosis of embryonic rat neural stem cells. Methods The neural stem cells were isolated from subventricular zone of rat embryonic brains and cultured in serum-free medium DMEM/F12 with B27. NGF with different doses of 10, 20, 50 μg/L were added into the different experimental groups. The proliferation ability of the cells was measured by methyl thiazol tetrazolium (MTT) assay at 10th day. All cells were collected in plates at 7th day. Streptvidin-peroxidase immunocytochemistry was used to detect the expression of neuron specific enolase (NSE) and glial fibrillary acidic protein (GFAP) , and the number of NSE( + ) and GFAP( + ) cells was counted. The apoptosis rat at 5th day after culture was assessed. Results There was no significant difference in the the number of neurospheres between 10 μg/L NGF group (0. 150 ±0. 025) and positive control group (0. 158 ±0. 017), but there was significant difference between 10 μg/L NGF group and blank control group (0. 128 ±0. 015). In 10, 20,50 μg/L N GF groups, the rate of NSE ( + ) cells was ( 15. 21 ± 1.27) %, (20. 64 ± 2. 61 )%, (24. 20 ± 3. 10) %, and that of GFAP( + ) cells was (27.98 ± 2. 35 ) %, ( 28.94 ± 2. 84 ) %, ( 30. 79 ± 3.23 ) % respectively. The rate of cells differentiation was increased with the dose of NGF added[( FNsE (+ ) = 3.47,FGFAp( + ) = 3. 47 ,PGFAP( + ) ＜ 0. 05)]. Flow cytometry revealed that in 20, 50 μg/L NGF groups the apoptosis rate was significantly increased as compared with other groups. The proliferation index (PI) in 10 μg/L NGF group was higher than other groups. Conclusion NGF at a rational dose can promote the proliferation and differentiation of human NSC. Low dose of NGF mainly facilitates the proliferation of NSCs, and high dose of NGF shows obvious influence on proliferation of NSC.     

人胚嗅球嗅鞘细胞及人胚鼻粘膜嗅鞘细胞的分离培养与纯化

[目的]采用差速贴壁法及免疫组化对人胚嗅粘膜OECs及人胚嗅球OECs进行体外纯化培养,探讨建立嗅粘膜OECs及嗅球OECs体外培养的方法.[方法]对差速贴壁后的人胚嗅粘膜OECs及嗅球OECs分别交替应用含13％胎牛血清DMEM - F12培养基进行原代培养.观察嗅鞘细胞的形态学变化,采用p75NTR和GFAP免疫细胞化学染色进行鉴定和纯度检测.[结果]人胚嗅粘膜及人胚嗅球均可培养出嗅鞘细胞,嗅粘膜嗅鞘细胞形态多呈双极、三极,伴有细长的突起.p75NTR和GFAP染色均呈阳性反应,体外培养时人胚嗅球嗅鞘细胞纯度比人胚嗅粘膜嗅鞘细胞高.[结论]差速贴壁法可以分离培养出人胚嗅粘膜嗅鞘细胞及人胚嗅球嗅鞘细胞.     

Feasibility of using early mesencephalic neural plate for intracerebral grafting

The purpose of this study was to elucidate the biological significance and the possibility of intracerebral grafting of neuroepithelial stem cells derived from the mesencephalic neural plate. Immunohistological studies of embryonic day 10.5 (E10.5) Wister rats revealed strong nestin expression in the mesencephalic part of the neural plate. Mesencephalic neural plates removed from E10.5 rats were processed to either tissue or cell dissociation culture. They were cultured in vitro under various conditions and were analyzed 7 days after the primary culture. When they were cultured as a tissue, cell proliferation and differentiation into neurons extending long neurites were obvious in a serum-free medium, in a medium containing 3% serum, and in a medium containing 20 ng/ml epidermal growth factor. On the other hand, in a medium containing 10 ng/ml basic fibroblast growth factor (bFGF), both vigorous cell proliferation and sphere formation were recognized. Furthermore, marked neurite growth was rarely seen in this culture. When they were plated in a dissociation culture, cell proliferation and neurosphere generation were also recognized only in a medium containing bFGF, depending on the initial cell concentration. The spheres, generated 7 days after the primary cell culture, were positively stained by nestin. These data suggested that bFGF was able to amplify the stem cell population present in the mesencephalic neural plate derived from early embryos. This might make it possible to obtain a large number of stem cells as donor material for neural transplantation on demand.     

基质金属蛋白酶抑制剂对胶质瘤细胞和胶质瘤干细胞体外侵袭抑制作用的比较

目的 观察基质金属蛋白酶(MMP)抑制剂对常规贴壁培养的人脑胶质瘤细胞和胶质瘤干细胞侵袭能力的影响及两者间的差异.方法 应用一种三维胶原凝胶侵袭模型,将常规贴壁培养的胶质瘤细胞与无血清、悬浮细胞球条件下培养的胶质瘤干细胞(1. 0×104～1.5×104个/样本)分别接种于该模型中,加入不同浓度的MMP抑制剂GM6001(0、25、50、75、100 μmol/L)培养4 d,通过观察各组细胞侵袭距离及其差异,判断该抑制剂对两种细胞侵袭能力的影响及差异.结果 两组细胞对GM6001的抑制作用表现出不同程度的剂量依赖性,75μmol/L GM6001对常规培养的胶质瘤细胞侵袭抑制最明显,而25 μmol/L GM6001对胶质瘤干细胞的侵袭抑制最显著.结论 MMP抑制剂在体外对胶质瘤细胞具有侵袭抑制作用,胶质瘤干细胞显示出对MMP抑制剂更高的敏感性.     

Promotion of neurite outgrowth from fetal hippocampal cells by TNF-alpha receptor 1-derived peptide

Cytokines such as tumor necrosis factor-alpha (TNF-alpha), FasL, and TNF-related apoptosis-inducing ligand (TRAIL) induce apoptosis or inflammation through binding to their specific receptors, TNFR1, Fas, and DR5, respectively. We have previously reported ligand-binding and cell death-inhibiting synthetic peptides, which were designed based on the crystal structure of a ligand-receptor complex and the homology of the amino acid sequence among the death receptor family members. Here we show that, among these death receptor-derived peptides, the TNFR1-derived peptide specifically arrested cell proliferation and promoted cell adhesion of fetal rat (E16) hippocampal cells, and promoted neurite outgrowth from hippocampus-derived neurospheres cultured with the addition of the peptide or cultured on a peptide-coated surface. Furthermore, among these death receptor-derived peptides, marked neurite outgrowth was observed only when the neurospheres were cultured on a TNFR1-derived peptide-conjugated covalently cross-linked alginate gel. The neurites from the neurospheres positively immunostained with an antibody against neurofilaments. These results suggest that the TNFR1-derived peptide promotes neuronal differentiation of the hippocampal neural stem cells and the TNFR1-derived peptide-conjugated covalently cross-linked alginate gel may be a useful material for assisting neural stem cell transplantation.