联合应用EGF和NGF对成年大鼠海马神经干细胞分化的影响

目的探讨联合应用表皮生长因子(EGF)和神经生长因子(NGF)对成年大鼠海马神经干细胞(NSCs)分化的影响。方法用含碱性成纤维生长因子(bFGF)、表皮生长因子、B27的无血清细胞培养技术体外培养成年大鼠海马神经干细胞,单克隆培养细胞行Nestin免疫细胞化学染色,诱导分化1w后细胞行GFAP和NSE免疫细胞化学染色;根据培养基中所加营养因子的不同将第4代细胞分为4组培养:EGF组、EGF+NGF组、NGF组、对照组,此4组细胞培养1w后行NSE免疫细胞化学染色,计数阳性细胞比例后进行统计学分析。结果单克隆培养后克隆球表达Nestin,诱导分化1w后细胞表达NSE、GFAP。与空白对照组相比,EGF组、NGF组和EGF+NGF组细胞分化为神经元的比例较高(P<0.05),其中EGF+NGF组细胞的比例最高。结论单独或联合应用EGF、NGF可以促进成年大鼠海马神经干细胞向神经元分化。     

葛根素对骨髓间充质细胞的诱导分化

目的探索葛根素体外诱导大鼠骨髓间充质细胞(Bone Marrow Stromal Cells MSCs)分化为神经元和胶质细胞的可行性,为中药在细胞定向分化中的应用提供理论和实验依据.方法用贴壁法分离纯化SD大鼠骨髓间充质细胞.培养传至第5代,诱导组以2.5mg/ml的葛根素预诱导24h后,用17.5mg/ml的葛根素无血清培养基诱导,未诱导组加入等量培养基,24 h后相差显微镜观察形态变化,免疫细胞化学染色鉴定诱导后的细胞神经元特异性稀醇化酶(neuronspecific enolase NSE),神经胶质纤维酸性蛋白(glial fibrillary acidic protein GFAP)蛋白的表达情况,MTT检测不同浓度葛根素诱导后细胞的活力,流式细胞仪及RT-PCR检测诱导前后细胞中NSE、GFAP的表达情况.结果诱导18h后BMSCs胞体收缩,有突起伸出,24 h后突起增多呈网状.免疫细胞化学染色,NSE阳性表达率为(53.3±4.3)%,GFAP阳性表达为(64.5±5.2)%,流式细胞仪检测诱导24 h后的细胞NSE及GFAP表达量均较未诱导组升高,RT-PCR检测诱导后细胞表达NSE、GFAP,未诱导的细胞则不表达.结论葛根素可诱导大鼠骨髓间充质细胞在体外分化为神经元和神经胶质细胞.     

EGF培养条件下NGF与BDNF对大鼠海马神经干细胞向神经元分化的差异

目的探讨在表皮生长因子(EGF)培养条件下,相同浓度神经生长因子(NGF)与脑源性神经生长因子(BDNF)对成年大鼠海马神经干细胞向神经元分化比例的差异。方法用含碱性成纤维生长因子(bFGF)、EGF、B27的无血清细胞培养技术体外培养成年大鼠海马神经干细胞,单细胞克隆后行Nestin免疫细胞化学染色,诱导分化1周,行GFAP和NSE免疫细胞化学染色;根据培养液中所加营养因子的不同将单细胞克隆传代细胞分为5组培养:EGF组、NGF组、BDNF组、EGF+NGF组、EGF+BDNF组,此5组细胞培养1周,进行NSE免疫细胞化学染色,计数阳性细胞比例后进行统计学分析。结果:单细胞克隆培养后克隆球细胞表达Nestin,诱导分化1周,细胞表达NSE、GFAP;与EGF组、NGF组、BDNF组相比,EGF+NGF组和EGF+BDNF组细胞分化为神经元的比例较高(P<0.05),其中EGF+BDNF组细胞的比例最高。结论在EGF培养条件下,BDNF促进成年大鼠海马神经干细胞向神经元分化的能力高于NGF。     

黄连素诱导大鼠骨髓间质干细胞分化为神经元样细胞

目的:黄连素体外定向诱导SD大鼠骨髓间质干细胞分化为神经元样细胞。方法:用全骨髓细胞悬液体外扩增和纯化骨髓间质干细胞。选用第5代以后骨髓间质干细胞进行诱导分化,用含10 μg/L碱性细胞生长因子(bFGF)的完全培养液预诱导24 h,后更换含黄连素的无血清DMEM诱导骨髓间质干细胞分化为神经元样细胞。免疫组化鉴定神经元烯醇酶(NSE)、神经丝蛋白(NF)、胶质纤维酸性蛋白(GFAP)的表达。结果:大鼠骨髓间质干细胞体外扩增第5代后细胞形态达到均一,成梭形。加黄连素诱导1h-8 h,间质干细胞胞体逐渐增大并伸出细长突起,形似神经细胞。免疫组化显示诱导的神经元样细胞NSE、NF表达阳性,GFAP阴性。结论:黄连素可诱导骨髓间质干细胞分化为神经元样细胞。     

人脐带间充质干细胞诱导分化为神经元样细胞

目的 探讨人脐带间充质干细胞(hUC-MSCs)体外诱导向神经细胞分化的潜能.方法 用生长因子bFGF和EGF联合全反式维甲酸(RA)、中药单体熊果酸、新生大鼠皮层神经元原代无血清培养的上清液3种方法,诱导hUC-MSCs向神经细胞分化,观察形态改变,用免疫细胞化学法检测神经细胞相关的标记物Nestin、Musashi-1和Tubulin、GFAP、O4.结果 hUC-MSCs经细胞因子、2mg*L的熊果酸诱导后,细胞由成纤维状逐渐变成双极、多极或锥形,胞体缩小;有些细胞伸出突起,随时间推移逐渐伸长,有些可形成次级突起,相互连结形成网状.另有一些胞体呈球形,突起较短.新生大鼠皮层神经元原代无血清培养的上清液诱导hUC-MSCs先形成Nestin和Musashi-1阳性的神经球,再经历上述形态变化,分化形成神经元样细胞.hUC-MSCs经诱导14 d后,有少量细胞表达Tubulin、GFAP、O4.结论 hUC-MSCs在体外特定条件下,可诱导分化为神经细胞,作为神经系统疾病细胞移植治疗的备选来源.     

灯盏花素注射液对骨髓间充质干细胞的诱导分化

目的探索灯盏花素注射液体外诱导大鼠骨髓间充质细胞(BMSCs)分化为神经元和胶质细胞的可行性。方法贴壁法分离纯化SD大鼠骨髓间充质细胞。第4代细胞行表型鉴定后,用灯盏花素注射液诱导,每6h倒置相差显微镜观察形态变化,免疫细胞化学染色鉴定诱导后细胞的神经元特异性稀醇化酶(NSE)、神经胶质纤维酸性蛋白(GFAP)的表达情况,四甲基偶氮唑盐(MTT)检测不同浓度灯盏花素注射液诱导后细胞的活力,流式细胞术及RT-PCR检测诱导前后细胞中NSE、GFAP mRNA的表达变化。结果BMSCs表型鉴定为CD44+、CD54+、CD34-,诱导18h后BMSCs胞体开始收缩,有突起伸出,24h后突起增多形成网络结构。免疫细胞化学染色,NSE阳性表达率为(48.7±3.4)%,GFAP阳性表达为(56.8±4.2)%,流式细胞仪检测诱导24h后的细胞NSE及GFAP蛋白表达量均较未诱导组升高,RT-PCR检测诱导后细胞表达NSE、GFAP mRNA,未诱导的细胞则不表达。结论灯盏花素注射液可诱导大鼠骨髓间充质细胞在体外分化为神经元和神经胶质细胞。     

不同血清浓度对bFGF联合EGF诱导BMSCs向神经细胞分化的影响

目的:探讨不同血清浓度条件下碱性成纤维生长因子(basic fibroblast growth factor,bFGF)联合表皮生长因子(epidermal growth factor,EGF)诱导骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)向神经细胞分化方向的影响。方法:全骨髓贴壁法体外分离培养BMSCs,流式细胞法检测BMSCs表面骨髓基质标志CD90和造血细胞标志CD45,MTT法绘制第3(P3)代BMSCs生长曲线。取P4代细胞分为3组给予不同诱导条件,分别给予含20 ng/ml的bFGF和20 ng/ml EGF的无胎牛血清(A组)、10 ml/L胎牛血清(B组)及100 ml/L胎牛血清(C组)的DMEM/F-12培养液进行诱导,倒置显微镜观察细胞的形态变化,诱导6 d时免疫组织化学法检测细胞内神经元特异性烯醇化酶(NSE)及胶质纤维酸性蛋白(GFAP)的表达,台盼蓝染色检测各组细胞的活力。结果:BM-SCs经3-4次传代培养后形态大致呈单一的长梭形或扁平形,流式细胞鉴定显示CD90阳性,CD45阴性;细胞生长曲线近似呈"S"形;诱导后可见细胞胞体收缩,呈双极、多极并伸出突起;诱导6 d时3组活细胞率分别为:86.57%、95.29%、97.32%;免疫组化各组均见NSE、GFAP表达,3组NSE阳性率高于GFAP,A组NSE阳性率高于B、C组,差异具有统计学意义(P<0.01),C组NSE阳性率高于B组,差异具有统计学意义(P<0.01),A组GFAP阳性率低于B、C组,差异具有统计学意义(P<0.01),C组GFAP阳性率高于B组,差异具有统计学意义(P<0.01)。结论:bFGF联合EGF可诱导BMSCs向神经细胞分化,在无血清条件下趋向于向神经元分化,在高浓度血清条件下趋向胶质细胞分化。     

bFGF和EGF对胚胎神经干细胞分化为神经元及神经胶质细胞的影响

目的：观察碱性成纤维生长因子（bFGF）和表皮生长因子（EGF）对体外培养胚胎神经管神经干细胞生长和分化的影响。方法：从孕12天大鼠胚胎神经管分离神经干细胞，进行原代培养，分为bFGF组、EGF组、bFGF＋EGF组及对照组：培养过程中观察干细胞的生长，培养2小时做nestin染色鉴定神经干细胞，培养第5天用免疫组化方法检测培养细胞神经元特异烯醇化酶（NSE）和胶质纤维酸性蛋白（GFAP）的表达，以观察神经干细胞分化为神经元及神经胶质细胞的状况。结果：取材细胞大部分为nestin免疫阳性细胞；各实验组均可促进培养细胞的生长和分 化。免疫组化中，EGF使神经干细胞增殖成团，增加GFAP的表达（P＜0．01）；bFGF能明显增加NSE及GFAP的表达（P＜0．01）；两种因子联合应用，神经元和神经胶质细胞均比对照组增多（P＜0．01）。结论：EGF和bFGF两类生长因子均能促进胚胎神经干细胞的生长，在分化方面，EGF倾向于诱导干细胞增并向着胶质细胞分化，bFGF则诱导干细胞分 成更多的神经元。     

EGF和bFGF诱导骨髓间充质干细胞向神经细胞分化后对Tau和神经元特异性烯醇化酶表达的影响

目的：观察表皮细胞生长因子（BGF）、碱性成纤维生长因子（bFGF）体外诱导骨髓间充质干细胞（MSCs）分化为神经细胞后,神经元微管相关蛋白Tau和神经元特异性烯醇化酶（NSE）的表达。方法：取P4代MSCs分为EGF、bFGF和EGF＋bFGF及对照组;免疫细胞化学法检测Tau蛋白和NSE表达,ELISA法分析各组细胞Tau蛋白含量。结果：免疫细胞化学检测显示EGF＋bFGF811Tau蛋白和神经元特异性烯醇化酶NSE阳性细胞率最高,bFGF组、EGF组和对照组依序递减;各组Tau蛋白含量结果与Tau蛋白阳性细胞率一致。结论：EGF和bFGF均可促进MSCs向神经细胞分化,并表达Tau蛋白和NSE,二者具有协同作用。     

麝香多肽体外诱导成年大鼠和人骨髓间充质干细胞定向分化为神经元的研究

目的:观察麝香多肽体外定向诱导成年大鼠和人骨髓间质干细胞(Bonemarrowmesenchymalstemcells,BMMSCs)分化为神经元的能力。方法:采用含100-150mg/L麝香多肽的无血清L-DMEM培养基诱导成年大鼠和人BMMSCs分化为神经元,免疫组化鉴定神经元烯醇化酶(NSE)、神经丝蛋白(NF)、胶质纤维酸性蛋白(GFAP)、神经巢蛋白(nestin)的表达。结果:成年大鼠和人BMMSCs在加入含麝香多肽的无血清L-DMEM培养基诱导后,BMMSC胞体收缩,突起伸出,形似神经元;免疫组化显示诱导出的神经元样细胞NSE、NF、nestin表达阳性,GFAP阴性。结论:麝香多肽可以在体外诱导成年大鼠和人BMMSCs定向分化为神经元。     

The effects of transmural pressure on prostacyclin release from porcine endocardial endothelial cells – comparison with vascular endothelial cells

 We assessed the effects of pressure on the release of prostacyclin (PGI₂) from cultured endocardial endothelial cells (EECs) and vascular endothelial cells (VECs). EECs were harvested from the right ventricle (RV) and left ventricle (LV) of porcine hearts, and VECs from pulmonary artery (PA), aorta (Ao) and coronary artery (CA). Confluent EECs and VECs were incubated for 30 min under various pressures (0, 50, 100, 150 mmHg) and PGI₂ release from each cell was measured. Pressure-induced PGI₂ release from LV-EECs was larger than that from RV-EECs. Pressure also increased PGI₂ release from both PA- and Ao-VECs, but not from CA-VECs. These findings suggest that endocardium can produce PGI₂ in response to pressure and PGI₂ released into the coronary blood from the ventricle may play an important role in the prevention of myocardial ischemia. Received: 13 August 1996 / Received after revision: 13 December 1996 / Accepted: 17 January 1997     

Regulation of neuroinflammation by B cells and plasma cells

The remarkable success of anti‐CD20 B cell depletion therapies in reducing the burden of multiple sclerosis (MS) disease has prompted significant interest in how B cells contribute to neuroinflammation. Most focus has been on identifying pathogenic CD20⁺ B cells. However, an increasing number of studies have also identified regulatory functions of B lineage cells, particularly the production of IL‐10, as being associated with disease remission in anti‐CD20–treated MS patients. Moreover, IL‐10–producing B cells have been linked to the attenuation of inflammation in experimental autoimmune encephalomyelitis (EAE), the animal model of MS. In addition to IL‐10–producing B cells, antibody‐producing plasma cells (PCs) have also been implicated in suppressing neuroinflammation. This review will examine regulatory roles for B cells and PCs in MS and EAE. In addition, we speculate on the involvement of regulatory PCs and the cytokine BAFF in the context of anti‐CD20 treatment. Lastly, we explore how the microbiota could influence anti‐inflammatory B cell behavior. A better understanding of the contributions of different B cell subsets to the regulation of neuroinflammation, and factors that impact the development, maintenance, and migration of such subsets, will be important for rationalizing next‐generation B cell–directed therapies for the treatment of MS.     

肝脏组织工程学中的胚胎干细胞

肝脏组织工程是新近兴起的一门学科 ,其目的是经过培养形成有独立代谢功能的肝脏器官。其中 ,种子细胞来源是关键 ,胚胎干细胞由于不存在来源短缺及异种排斥问题 ,是理想的选择 ,现在许多学者进行了由胚胎干细胞向肝细胞的转化研究 ,并与人工材料 ,基因调控等技术结合 ,取得了显著的进展 ,这些研究解决了许多理论和实践上的问题 ,促进了肝脏组织工程学的发展     

In vitro apoptosis effects of GnRHII on endometrial stromal cells from patients with endometriosis

Purpose: To study the effect of gonadotropin-releasing hormone II (GnRHII) on the cell apoptosis of ectopic, eutopic and normal endometrial stromal cells cultured in vitro from endometriosis patients, and to provide theoretical basis for exploring new treatments for endometriosis (EMs). Methods: Ectopic, eutopic and normal endometrial stromal cells were isolated, cultured and identified in vitro, then treated with different concentrations of GnRHII (0, 10^-10 M, 10^-8 M and 10^-6 M). Cell apoptosis was detected by Hoechst staining and flow cytometry. Results: GnRHII increased apoptosis in ectopic, eutopic and normal stromal cells in a dosage-dependent manner (P<0.05), and apoptosis of ectopic stroma cells was significantly higher than that of eutopic and normal cells (P<0.05); apoptosis in eutopic and normal cells had no different (P>0.05). Conclusion: GnRHII can significantly induce apoptosis in ectopic, eutopic and normal endometrial stromal cells from patients with endometriosis, especially to the ectopic.     

二氢杨梅素对实验性结肠炎小鼠调节性B细胞的影响

目的 探讨二氢杨梅素(DHM)对实验性结肠炎小鼠的作用及其对调节性B细胞(Breg)的影响.方法 将小鼠随机分为正常组、DSS(葡聚糖硫酸钠)组、DSS+DHM组、DSS+mesalazine组(造模采用4％DSS连续饮用7 d).7 d后,每天分别给予灌胃水(0.2 mL/d)、DHM(40 mg/kg·d)和美沙拉...     

Transplantation of olfactory ensheathing cells promotes partial recovery in rats with experimental autoimmune encephalomyelitis

Objective: This study was to investigate the efficacy of olfactory ensheathing cell (OEC) transplantation on experimental autoimmune encephalomyelitis (EAE). Methods: EAE models were established by guinea pig spinal cord homogenate (GPSCH) immunization in Lewis rats. OECs were purified and cultured from the olfactory nerve layer of SD rats, and then transplanted to the EAE models through the vena caudalis (Group A) or into the lateral cerebral ventricle (Group B). Neurological function scores and body weights were daily recorded following transplantation, and histological analysis was performed to assess the pathological changes in EAE rats. Results: Cultured cells mainly exhibited bipolar or tripolar morphology, and the majority of these cells were positive for NGFR p75 staining. Neurological function scoring and the body weight measurement showed that, OEC transplantation could significantly improve the performance of EAE rats, and similar results were observed for the transplantation through the vena caudalis and into the lateral cerebral ventricle. Moreover, the transplanted OECs accumulated to the lesions in the brains of EAE rats, in spite of the different transplantation approaches. However, no significant differences in histopathology (HE and LFB staining) were observed between the OEC-transplanted groups and the control group. Conclusion: OEC transplantation could exert beneficial effects in the treatment of EAE, no matter which the cells were transplanted through the vena caudalis or into the lateral cerebral ventricle. Our findings might provide evidence for the clinical treatment of multiple sclerosis with cell transplantation.     

激活素A和骨形成蛋白-4诱导人羊膜上皮细胞转分化为心肌样细胞的实验研究

近年来羊膜上皮细胞(AECs)被认为是心肌细胞移植较好的种子细胞之一。研究表明,激活素A(ActivinA)和骨形成蛋白-4(BMP-4)能够诱导人胚胎干细胞向心肌细胞分化。本研究通过体外分离、培养并鉴定人AEC后,采用Activin A和BMP-4作为诱导剂,观察它们能否诱导AEC向心肌样细胞转分化。应用定量RT-PCR、Western blot、免疫细胞化学等方法来检测基因及蛋白表达变化。结果显示,人AEC能够表达上皮细胞特异性蛋白角蛋白19(CK19)。经Activin A和BMP-4共同诱导培养14d后,AEC心肌特异性转录因子Nkx2.5和特异性结构蛋白α-actinin的mRNA表达水平明显升高,同时α-actinin的蛋白表达水平亦明显增加。本实验提示,ActivinA和BMP-4在体外能够诱导人AEC向心肌样细胞转分化,同时该诱导方法可能成为诱导AEC向心肌样细胞转分化的一个新方法。     

胚胎干细胞移植治疗帕金森病的研究进展

帕金森病(PD)是一种以中脑多巴胺神经元丢失为特征的中枢神经系统退行性疾病。近年来发现,胚胎干细胞(ESC)在体外可诱导分化为多巴胺能神经元,认为ES细胞是移植治疗PD较为理想的细胞,有着广泛的治疗前景。本文综述近年来治疗PD的临床研究、胚胎干细胞体外诱导产生多巴胺能神经元的方法及其应用进展。     

Miscarriage induced by adoptive transfer of dendritic cells and invariant natural killer T cells into mice

Unexpected fetal loss is one of the common complications of pregnancy; however, the pathogenesis of many miscarriages, particularly those not associated with infections, is unknown. We previously found that activated DEC‐205⁺ dendritic cells (DCs) and NK1.1⁺ invariant natural killer T (iNKT) cells are recruited into the myometrium of mice when miscarriage is induced by the intraperitoneal administration of α‐galactosylceramide (α‐GalCer). Here we demonstrate that the adoptive transfer of DEC‐205⁺ bone marrow‐derived DCs cocultured with α‐GalCer (DEC‐205⁺ BMDCs‐c/w‐α‐GalCer) directly induced marked fetal loss by syngeneic pregnant C57BL/6 (B6) mice and allogeneic mice (B6 (♀) × BALB/c (♂)), which was accompanied by the accumulation of activated iNKT cells in the myometrium. Further, the adoptive transfer of NK1.1⁺ iNKT cells obtained from B6 mice injected with α‐GalCer facilitated miscarriages in syngeneic Jα18^(?/?) (iNKT cell‐deficient) mice. These results suggest that DEC‐205⁺ DCs and NK1.1⁺ iNKT cells play crucial roles required for the initiation of fetal loss associated with stimulation by glycolipid antigens and sterile inflammation.     

Isolation of myeloid-derived suppressor cells subsets from spleens of orthotopic liver cancer-bearing mice by fluorescent-activated and magnetic-activated cell sorting: similarities and differences

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that commonly expand during tumor development and that play a critical role in suppression of immune responses. MDSCs can be classified into two groups: Mo-MDSCs and G-MDSCs. These cells differ in their morphology, phenotype, differentiation ability, and immunosuppressive activity, and inhibit immune responses via different mechanisms. Therefore, identifying an effective method for isolating viable Mo-MDSCs and G-MDSCs is important. Here, we demonstrated the differences and similarities between fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) in sorting G-MDSCs and Mo-MDSCs. Both MACS and FACS could obtain G-MDSCs and Mo-MDSCs with high viability and purity. A high yield and purity of G-MDSCs could be obtained both by using FACS and MACS, because G-MDSCs are highly expressed in the spleen of tumor-bearing mice. However, Mo-MDSCs, which comprise a small population among leukocytes, when sorted by MACS, could be obtained at much greater cell number, although with a slightly lower purity, than when sorted by FACS. In conclusion, we recommended using both FACS and MACS for isolating G-MDSCs, and using MACS for isolation of Mo-MDSCs.