神经营养因子和生长因子等对培养的成人视网膜神经细胞的影响

目的　探讨成人视网膜神经细胞体外培养条件 ,脑源性神经营养因子 (BDNF)、神经营养素 (NT 4 )、表皮生长因子 (EGF)、成纤维细胞生长因子 (FGF)、诱导分化因子全反式视黄酸 (RA)等对成人视网膜神经细胞生长、增殖、凋亡的影响及调控机制。方法　胰蛋白酶消化结合机械吹打分离成人视网膜神经细胞 ,在培养基中加入或不加入BDNF、NT 4、EGF、FGF、RA。根据细胞形态、生长方式及免疫细胞化学特征确定细胞类型。比较各组中神经元的数目、转录调控因子c fos、c jun及细胞凋亡调控因子Bcl 2、Bax表达水平。结果　与对照组相比 ,BDNF、FGF处理组存活的神经元特异性烯醇酶、Thy1.1抗体和Bcl 2、c fos及c jun表达阳性细胞数也增多 (均P <0 0 1) ,培养的视网膜神经细胞在体外存活时间可长达 8个月 ;RA处理组c fos、c jun阳性细胞数较对照组增多 (均P <0 0 1) ;而NT 4、EGF处理组各项指标与对照组比较 ,差异无显著意义 (均P >0 0 5 )。结论　BDNF、FGF、RA能显著提高体外培养的成人视网膜神经细胞的存活 ,其机制可能涉及上调转录调控因子c fos、c jun及凋亡抑制因子Bcl 2的表达 ,或下调凋亡促进因子Bax的表达。但EGF、NT 4对体外培养的视网膜神经细胞存活状态无明显改善作用。     

生长因子对培养的胚胎视网膜神经细胞增殖与凋亡的影响

目的 探讨表皮生长因子（epidermal growth factor,EGF）、成纤维细胞生长因子（fibroblast growth factor,FGF）及胎牛血清对体外培养的人胚胎视网膜神经细胞 生长、增殖、分化和凋亡等的影响及可能的机制。 方法 采用胎牛血清培养人胚胎视网膜神经细胞，加入或不加入EGF、FGF。通过神经元特异稀醇化酶（neuron specific enolase, NSE）、视网膜神经节细胞特异的Thy1.1抗体、胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)免疫组织化学染色、倒置显微镜及扫描电镜观察鉴定细胞成分；细胞生长曲线及溴脱氧尿苷（bromodeoxyuridine, BrdU）掺入测定细胞增殖率；原位末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸切口末端标记（Tdt-mediated dUTP nick end labelling, TUN EL）检测细胞凋亡；c-fos、c-jun及bcl-2、bax免疫组织化学染色判断转录调控因子及细胞凋亡调控因子表达水平，计数阳性细胞并进一步作统计分析。 结果 经过EGF、FGF培养的人胚胎视网膜细胞总数增加，细胞倍增时间缩短，BrdU掺入率增加，凋亡细胞数减少，其 中NSE、Thy1.1阳性细胞数明显增加。同时，c-fos、c-jun及bcl-2阳性细胞数也增加。 结论 生长因子EGF、FGF可通过上调转录因子c-fos、c-jun及细胞凋亡抑制因子bcl-2表达促进体外培养的人胚胎视网膜细胞存活与增殖。 （中华眼底病杂志，2003，19：113-116）     

神经营养因子与生长因子在体外对人视网膜神经节细胞之作用

目的 研究神经营养因子(neurotrophic factor)与生长因子(growth factor)在体外对人视网膜神经节细胞(retinal ganglion cell,RGC)生存的作用。 方法 从捐献的尸体眼球分离并培养RGC。RGC之鉴定系根据形态学与免疫细胞化学研究。将各种神经营养因子与生长因子分别加入培养液。对RGC计数,并与未加任何因子的对照组相比较。 结果 未加任何因子的培养中不见或仅有极少量的RGC。加有神经营养蛋白-3(neurotrophin-3,NT-3)、神经生长因子(nerve growth factor,NGF)、表皮生长因子(epidermal growth factor,EGF)与血小板源性生长因子(plate derived growth factor, PDGF)的培养亦同。加有下列因子的培养中有较多的RGC出现,计数为(细胞数/每10个显微镜野)：脑源性神经营养因子(brain derived neurotrophic factor,BDNF)：4.08;睫状神经营养因子(ciliary neurotrophic factor,CNTF)：1.23;神经营养蛋白-4/5(neurotrophin-4/5,NT-4/5)：2.63;碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)：2.65。 结论 BDNF、NT-4/5、bFGF与CNTF在体外能改善人RGC之生存,而NGF、NT-3、EGF与PDＧF则不能。 (中华眼底病杂志, 1999, 15: 149-152)     

永生化胚胎视网膜干细胞的体外诱导分化

目的：研究R71882永生化视网膜干细胞系细胞的体外诱导分化。方法：对R71882细胞系细胞进行体外培养扩增，利用含有表皮生长因子(epidermal growth factor，EGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)及白血病抑制因子(leukemia inhibitory factor，LIF)等生长因子的DMEM／F12培养液对第43代细胞进行体外诱导分化，免疫荧光细胞化学鉴定诱导后细胞的抗原表达。结果：诱导后的R71882细胞系细胞增殖速度减慢，细胞表达微管相关蛋白2(microtubule-associated protein 2，MAP2)、胶质源性纤维酸性蛋白(glial fibrillary acid protein，GFAP)、蛋白激酶C-α(protein kinase C-α，PKC-α)、Thy1．1及Calretinin，同时部分细胞仍表达巢蛋白(Nestin)，但未见视紫红质(Rhodopsin)表达。结论：R71882细胞是一种多能视网膜干细胞。在EGF及bFGF等多种生长因子的共同作用下，该细胞系中部分细胞仍能表达未分化细胞标记物Nestin，部分细胞开始向神经元、神经胶质细胞及双极细胞、神经节细胞和无长突细胞等视网膜终末细胞方向分化。     

生长因子对人视网膜神经胶质细胞增殖的作用

目的：研究生长因子对培养的人视网膜神经胶质细胞增殖的作用。方法：在培养的人视网膜神经胶质细胞中分别加入表皮生长因子(epidermal growth factor, EGF,0.5～100.0ng/ml)和神经生长因子(nerve growth factor,NGF,0.05～10.0ng/ml),培养3天后用MTT法测量细胞增殖情况。结果：EGF(0.5～100.0ng/ml)和NGF(0.05～10.0ng/ml)均能刺激人视网膜神经胶质细胞的增殖,其EC₅₀(half effective concentration,半数有效浓度)分别为17.0ng/ml和 0.7ng/ml。结论：EGF和NGF均能刺激人视网膜神经胶质细胞的增殖,它们可能在增殖性玻璃体视网膜病变(proliferative vitreoretinopathy,PVR）等增殖性病变中起一定作用。 (中华眼底病杂志,1998,14:33-34) (中华眼底病杂志,1998,14:33-34)     

培养的胚胎视网膜神经细胞Ca2+分布与Ca2+通道特征

目的 检测分析培养的胚胎早期视网膜神经细胞游离钙（Ca^2 )分布与钙（Ca^2 ）通道特征。方法 体外培养11－15周胎儿视网膜神经细胞，在含Ca^2 与不含Ca^2 的Hepes缓冲液中与钙荧光指示剂Fluo3孵育染色，同时加入或不加入异博定、佩尔地平或地塞米松，共聚焦显微镜观察记录游离Ca^2 分布，以及受不同浓度K^ 刺激后Ca^2 通道开放与Ca^2 转移情况。结果 培养的11－15周胎儿视网膜神经元及神经胶质细胞受K^ 刺激后均出现明显的Ca^2 转移与再分布，在缺乏细胞外Ca^2 的情况下，细胞浆内钙库仍可迅速释放Ca^2 并转移至细胞核内。异博定、佩尔地平及地塞米松能够抑制细胞外Ca^2 进入视网膜神经细胞内。结论 胚胎早期视网膜神经元及神经胶质细胞存在L型Ca^2 通道，并已基本发育成熟。     

增生性玻璃体视网膜病变发病机制的研究进展

增生性玻璃体视网膜病变(proliferative vitreoretinopathy，PVR)是在孔源性视网膜脱离或视网膜脱离复位术后，由于视网膜色素上皮细胞(RPE)及神经胶质细胞的增生和收缩，造成牵拉性视网膜脱离的病变。PVR的病理特征是细胞增生，RPE一旦开始增殖，就产生细胞生长因子，而这些细胞生长因子反过来又刺激细胞增殖。PVR的过程有多种细胞及因子的参与，参与PVR增生的细胞主要是RPE和视网膜神经胶质细胞；涉及PVR的生长因子有：PDGF、EGF、VEGF、TGF、FGF、IGF、HGF等。     

APP17肽对糖尿病视网膜神经元表达NT-3和星形胶质细胞增殖的影响

目的　观察糖尿病早期视网膜神经元表达 NT- 3和星形胶质细胞增殖情况 ,并观察 APP17肽的作用。方法　用链脲佐菌素 (streptozotocin,STZ)复制 Wistar大鼠糖尿病模型 ,于模型建立 2周后 ,皮下注射 APP17肽 ,1月后处死大鼠取视网膜 ,进行神经免疫组化 ,观察 APP17肽对糖尿病早期视网膜神经元中神经营养因子 3(neurotrophin- 3,NT- 3)和星形胶质细胞中胶质原纤维酸性蛋白 (glial fibrillary acidi protein,GFAP)表达的影响。结果　同正常对照组相比模型组 (DM组 )视网膜神经元 NT- 3阳性细胞数明显减少 ,但APP17+DM组阳性细胞数多于 DM组 (P<0 .0 1) ,并且对照组与 APP17+DM组中星形胶质细胞 GFAP阳性数少于 DM组 (P<0 .0 1)。结论　在糖尿病视网膜病变早期存在视网膜神经细胞 NT- 3表达减少及星形胶质细胞增生 ,APP17肽可明显改善早期糖尿病大鼠视网膜神经元的表达 ,提示对神经视网膜具有保护作用     

SD大鼠视网膜神经细胞培养上清液对胚胎干细胞体外分化的诱导作用

目的 探讨视网膜神经细胞培养的上清液对胚胎干细胞（embryonic stem cells, ES）体外分化的诱导作用。 方法 收集SD大鼠视网膜神经细胞培养上清液，抽滤后按2∶3比例与DMEM培养液混合，用该混合液进行ES 细胞的诱导分化，每天倒置相差显微镜观察ES细胞的生长及分化情况,对诱导分化后的细胞进行神经丝蛋白(nellcofilament protein，NFP)免疫组织化学检查。 结果 加入了视网膜神经细胞培养上清液的ES细胞生长出类似神经细胞突起样结构，NFP免疫组织化学染色阳性。 结论 SD大鼠视网膜神经细胞培养的上清液具有诱导ES细胞向神经细胞分化的作用。 (中华眼底病杂志, 2002, 18: 134-136)     

阿魏酸对培养视网膜神经细胞增殖活性的影响

目的 研究阿魏酸促进视网膜神经细胞活性的可能机制,为治疗退行性视网膜病变提供依据。方法 以7个月人胚胎视网膜、新生小牛视网膜和生后4个月小鼠视网膜为研究对象,以细胞培养、MTT法和氚标记胸苷掺入法等为研究手段,检测阿魏酸、脑源性神经营养因子(BDNF)和葛根素作用72h后,视网膜神经细胞增殖和DNA合成变化。结果 阿魏酸对3种视网膜神经细胞均具有促增殖作用,细胞活性强度呈现剂量依赖性,对于小鼠视网膜神经细胞,阿魏酸在15．6—1000．0mg／L内均具有促细胞增殖作用;对于新生小牛和7个月人胚胎视网膜神经细胞,阿魏酸在125．0～1000．0mg／L内具有促细胞增殖作用。对7个月人胚胎视网膜神经细胞,阿魏酸在62．5—1000．0mg／L内具有促DNA合成作用,最佳效应浓度为500．0mg／L;BDNF在12,5～50．0μg/L内具有促细胞增殖作用,最佳效应浓度为50．0μg／L;葛根素未见明显促增殖效应。结论 阿魏酸对3种视网膜神经细胞的促增殖作用明显强于BDNF。阿魏酸能有效提高视网膜神经细胞活性,有望成为一种新的防治退行性视网膜病变的有效成分。     

In vivo expression of neurotrophins and neurotrophin receptors is conserved in adult porcine retina in vitro

PURPOSE. To characterize and compare the expression of neurotrophins (NTs) and their receptors within adult porcine retinal ganglion cells (RGCs) in vivo and in vitro. METHODS. The distribution of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and -4 (NT-4), and their high-affinity receptors TrkA, TrkB, TrkC and low-affinity receptor p75, was analyzed in adult porcine retinal sections by immunohistochemistry. In addition, adult porcine retinas were dissociated and cultured in four different conditions: control, semipure RGCs, supplemented with BDNF, or seeded on Müller glia feeder layers. Double immunostaining was performed with antibodies to NTs or their receptors combined with neurofilament antibody to identify RGCs in culture. RESULTS. In vivo, immunolabeling of NGF was very faint, BDNF was especially prominent in RGCs and inner nuclear layer cells, NT-3 stained widespread nuclei, and NT-4 was undetectable. TrkA immunoreactivity was visible in the nerve fiber layer, TrkB staining was within RGC bodies, TrkC was undetectable, and p75 was widely expressed across the retina, within the Müller glia. Expression of neurotrophins and their receptors was maintained in all four models of adult RGCs in vitro, showing that expression was not influenced by substrate or culture conditions. We observed prominent staining of TrkA within growth cones, and a clear expression of p75 within a subpopulation of RGCs in vitro. CONCLUSIONS. These findings demonstrate that the expression of NTs and their receptors within adult porcine RGCs is maintained in vitro, under conditions of limited interaction with neighboring neurons and deprived of afferent inputs and target tissue. TrkA may be involved in regeneration of nerve terminals.     

Basic fibroblast and epidermal growth factors stimulate survival in adult porcine photoreceptor cell cultures

PURPOSE. To investigate the effects of basic fibroblast and epidermal growth factor (FGF2 and EGF, respectively) on the survival and phenotypic expression of photoreceptors isolated from adult mammalian retinas. METHODS. Primary cultures highly enriched in photoreceptors were prepared from adult domestic pig retinas and maintained in chemically defined medium. Cell culture composition was characterized through the use of specific antibody markers of retinal neurons, and neuronal survival was quantified through viability assays as a function of time in the presence or absence of different doses of FGF2 and EGF. Western blot analysis of phosphotyrosine residues was used to monitor activation of FGF2 and EGF signaling pathways. RESULTS. Reproducible survival of adult pig rod and cone photoreceptors was obtained for approximately 2 weeks in vitro, with the continued expression of rod opsin, recoverin, S-antigen, cone arrestin, and neuron-specific enolase. Purity of cultures was routinely more than 95% photoreceptors, with a rod-cone ratio of 2:3.1. Photoreceptor survival was stable for the initial week, decreasing slowly during the second, with rapid cell loss occurring thereafter. In the presence of FGF2 (20 ng/mL), the percentage of photoreceptor survival during the second week in culture was statistically significantly different, at least two times higher than in control experiments. Photoreceptor survival correlated directly with increasing concentrations of FGF2, and also of EGF. Combined treatment with FGF2 and EGF did not induce higher survival than either factor alone. There was no detectable selective loss of rods or cones in the experimental model. Phosphotyrosine immunoblots after stimulation of cultures with FGF2 and EGF revealed time-dependent appearance of multiple immunoreactive bands. CONCLUSIONS. The adult pig photoreceptor culture in the current study exhibits reproducible neuronal survival in vitro and represents a useful novel experimental system for the study of potential neuroprotective effects and signaling pathways of neurotrophic factors such as FGF2 and EGF in fully adult higher mammalian retina.     

人视网膜祖细胞和脑神经干细胞的体外培养和诱导分化研究

目的比较人视网膜祖细胞和脑神经干细胞的体外分化潜能。方法分离8-12周人胎儿神经视网膜和脑皮质、纹状体神经干细胞,进行无血清体外培养;采用光镜和免疫细胞化学或荧光免疫细胞化学染色方法,分别观察在无血清和10％胎牛血清培养条件下,两种来源的神经干细胞分化后的细胞特性。结果两种来源的神经干细胞,均能在体外有或无血清培养条件下增殖并分化。视网膜祖细胞不但表达视网膜祖细胞标志物Pax-6,也可表达神经干细胞标志物．巢蛋白（Nestin）、成熟神经元标志物-微管相关蛋白2（Map2）、星形胶质细胞标志物-胶质纤维酸性蛋白（GFAP）、节细胞标志物Thy-1和视杆细胞标志物视紫红质（Rhodopsin）;脑神经干细胞也能表达这些特异性细胞标志物。血清诱导分化时,视网膜祖细胞较难贴壁,贴壁细胞球伸出少而短的突起,单个细胞形态不清;而脑神经干细胞球易贴壁并伸出较长突起交织成网,大量细胞从细胞球中沿突起徙出,单个细胞形态清晰。结论人胎儿视网膜祖细胞和脑神经干细胞体外培养均具有向神经元、胶质细胞及视网膜终末细胞分化的能力;两种干细胞在进行血清诱导分化时,细胞的贴壁、迁移能力及分化后的细胞形态均存在差异。（中华腠科杂志,2006,42：901．907）     

Comparison of the proliferation and differentiation ability between adult rat retinal stem cells and cerebral cortex-derived neural stem cells

Recent studies have demonstrated that retinal stem cells (RSCs) and stem cells of the central nervous system both exhibited the abilities of self-renewal, proliferation and differentiation into multilineage. In the present study, we compared the proliferation and differentiation abilities between RSCs and cerebral corticex-derived neural stem cells (CNSCs) of adult rats. Stem cells isolated from pigmented ciliary margins of eyes and cerebral cortical tissues of adult rats were cultured in 96-well plates that contained serum-free medium with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). In contrast to RSCs, which stopped proliferating after the 8th week, the total cell count of neurospheres in CNSCs increased twofold at the 5th week and more than fourfold at the 10th week after in vitro culture. In contrast, RSCs stopped proliferating after 8 weeks of culture. After adding 2% fetal calf serum and withdrawing EGF and bFGF from the culture medium, the percentages of nestin-positive cells(20.6 +/- 2.7%), microtubule-associated-protein-2-positive neurons (33.2 +/- 3.9%) and glial-fibrillary-acidic-protein-positive glial cells(51.3 +/- 6.2%) in the differentiated CNSCs were significantly higher than those in the differentiated RSCs (10.2 +/- 1.9, 22.3 +/- 1.3 and 44.6 +/- 5.1%, respectively; p < 0.05). We also found that the combination of transforming growth factor beta type III with retinoic acid played an important role in the induction of CNSCs to differentiate into opsin-positive cells. Our data demonstrated that CNSCs displayed a higher ability of proliferation and retinal lineage. This report also offers an alternative protocol of cell reproduction for producing retinal cells.     

联合共培养系统诱导骨髓间充质干细胞分化为视网膜色素上皮样细胞

目的 探讨添加脑源性神经生长因子（BDNF）、表皮生长因子（EGF）、碱性成纤维细胞生长因子（bFGF）的培养基联合人类视网膜色素上皮细胞（HRPECs）共培养对骨髓间充质干细胞（BMSCs）定向诱导分化的影响.方法 实验研究.实验分三组：HRPECs＋BDNF、EGF、bFGF＋BMSCs共培养组、BDNF、EGF、bFGF＋BMSCs共培养组和对照组（单独BMSCs）.第一组取第3代HRPECs接种在双层培养板的上层,将第3代人BMSCs接种于下层培养板中,在双层六孔板的每孔中加入混合有20 ng/mlbFGF、20 ng/ml EGF、20 ng/ml BDNF及10%胎牛血清（FBS）的DMEM-LG培养液（需做免疫细胞化学染色应同时在下层放入18 mm×18 mm盖玻片进行细胞爬片）.第二组取第3代人BMSCs接种在六孔培养板中,每孔中加入含20 ng/ml bFGF、20 ng/ml EGF、20 ng/ml BDNF及10%FBS的DMEM-LG培养液.第三组将第3代人BMSCs接种于六孔培养板中,加入10%FBS的DMEM-LG培养液.在倒置相差显微镜下观察细胞形态学变化.2周后停止培养,采用免疫细胞化学染色法和RT-PCR检测角蛋白18、RPE65蛋白存诱导细胞中的表达.数据采用Holm-Sidak法进行分析.结果 诱导2周后,第一组BMSCs细胞呈圆形、类圆形、不规则形、短棒状外观,细胞内有色素颗粒形成,其他两组没有类似改变.三组间免疫细胞化学染色法检测RPE65蛋白、角蛋白18,光密度值结果显示第一组和第二组间、第一组和第三组间差异有统计学意义（RPE65：t=37.416、36.236,P＜0.05;角蛋白18：t=38.611、37.532,P＜0.05）.而第二组和第三组间差异没有统计学意义（RPE65：t=1.180,P＞0.05;角蛋白18：t=1.079,P＞0.05）.RT-PCR检测相对mRNA表达量,结果显示第一组和第二组间、第一组和第三组间差异有统计学意义（RPE65/β-actin：t=176.110、174.820,P＜0.05;角蛋白18/β-actin：t=243.230、241.560,P＜0.05）.而第二组和第三组间差异无统计学意义（RPE65/β-actin：t=1.283.P＞0.05;角蛋白18/β-actin：t=1.670,P＞0.05）.结论 利用BDNF、EGF、bFGF联合HRPECs共培养可以使BMSCs分化为视网膜色素上皮样细胞.