Effects of co-engraftment of Schwann cells with neural stem cells into rats with Parkinson disease

Neural stem cells （NSCs） are effective in treating Parkinsonian animals. Although cell transplantation is considered a promising treatment for Parkinson disease （PD）, its clinical use has been limited to only a few patients. The major limiting factors of this therapy are difficulty in obtaining sufficient viable embryonic mesencephalic tissue and the controversial ethical and/or legal issues raised by the use of human fetal allografts. Some reports suggest that Schwann cells （SCs） can promote the proliferation of embryonic stem cells and the induction of dopaminergic neurons. Our research focused on potential curative effects of co-graft SCs with mesencephalic stem cells into Parkinsonian animals and elucidating the underlying mechanisms.     

体外定向诱导胚胎干细胞分化为内皮细胞及其与脐静脉内皮细胞的比较

目的：研究干细胞定向分化为内皮细胞的效率,并与人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)比较。方法：用两步法诱导干细胞HUES9分化为内皮细胞,前3 d在N2B27培养基中加入骨形态发生蛋白4 (25 ng/mL)和肝糖原合成激酶3β受体的选择性抑制剂CHIR99021(10 μmol/L)使细胞分化到中胚层状态,第4天开始用 VEGF165(200 ng/mL)和Forskolin(2 μmol/L)将细胞诱导为内皮细胞。观察分化第6天细胞形态,并与HUVECs比较。用 CD144作为内皮细胞表面标志物,流式细胞术检测分化效率及分化后细胞的身份。比较两种内皮细胞迁移和成管能 力。结果：分化后的内皮细胞与HUVECs在显微镜下形态一致。分化细胞表面标志物阳性率达到73.4%,重新培养后 的内皮细胞表面CD144阳性率达到86.6%,HUVECs为94.4%。分化后的内皮细胞与HUVECs均具有良好的迁移和成管 能力,但分化后的内皮细胞能力不如HUVECs强。运用SB431542后,能够提高分化后内皮细胞的迁移和成管能力。 结论：此两步法将干细胞分化为内皮细胞有较高的分化效率,可作为理想的分化方法。     

高葡萄糖诱导的与内皮细胞共培养的雪旺细胞的损伤作用

 目的 观察高葡萄糖对与内皮细胞（endothelial cells ,EC）共培养的雪旺细胞（Schwann cells ,SC）的损伤情况。方法 建立大鼠SC与EC的共培养模型,根据形态学和MTT选定25mmol/L高葡萄糖浓度和48h作用时间。将细胞分为正常葡萄糖SC与EC共培养组、高葡萄糖SC与EC共培养组及高葡萄糖SC组,通过形态学和MTT、凋亡率（流式细胞仪）和Casepase-3mRNA表达（Realtime PCR）观察细胞损伤。结果 形态学及MTT显示,与EC共培养的SC在高葡萄糖条件下,比正常葡萄糖共培养以及高葡萄糖单培养的SC存活率明显下降;SC凋亡率比正常葡萄糖共培养及高葡萄糖单培养的SC明显增高;Casepase-3mRNA表达较正常葡萄糖共培养组明显升高,但与高葡萄糖单培养组相比其增高尚无统计学显著性意义。结论 在高葡萄糖环境下,与EC共培养的SC损伤更明显,可能与两种细胞相互作用加重SC损伤有关。     

高葡萄糖对与内皮细胞共培养的雪旺细胞的损伤作用

目的观察高葡萄糖对与内皮细胞(endothelial cells,EC)共培养的雪旺细胞(Schwann cells,SC)的损伤情况。方法建立大鼠SC与EC的共培养模型,根据形态学和MTT选定25 mmol/L高葡萄糖浓度和48 h作用时间。将细胞分为正常葡萄糖SC与EC共培养组、高葡萄糖SC与EC共培养组及高葡萄糖SC组,通过形态学和MTT、凋亡率(流式细胞仪)和Casepase-3 mRNA表达(Real time PCR)观察细胞损伤。结果形态学及MTT显示,与EC共培养的SC在高葡萄糖条件下,比正常葡萄糖共培养以及高葡萄糖单培养的SC存活率明显下降;SC凋亡率比正常葡萄糖共培养及高葡萄糖单培养的SC明显增高;Casepase-3 mRNA表达较正常葡萄糖共培养组明显升高,但与高葡萄糖单培养组相比其增高尚无统计学显著性意义。结论在高葡萄糖环境下,与EC共培养的SC损伤更明显,可能与两种细胞相互作用加重SC损伤有关。     

Pharmacological effects of scorpin venom associating with activities of nerve cells and regulation of inflammatory cells

Objective To study scorpin venom from Buthus martensii pharmacological effects on activities of nerve cells and regulation of inflammatory cells. Methods We applied the rabbit biventer cervicis muscle, venom （0.1 mg/ml） abolished nerve-mediated twitches.This inhibition was established by prior incubation of the venom with the phospholipase A inhibitor. Venom produced dose-dependant contractions of the rat colon. Including tumor associated macrophage （TAM）, mast cell （MC） and eosinophil leucocyte （EL） densities and angiogenesis, as well as the relation of TAM, MC and EL densities and anglogenesis to tumor stage were investigated in specimens of 63 non-small cell lung carcinoma （NSCLC）. Results 33 the rabbit phrenic nerve diaphragm preparation displayed greater sensitivity to venom. In the rabbit biventer muscle, venom fraction inhibited responses to acetylcholine but not KC1, indicating activity at post-synaptic nicotinic receptors. Venom did not show direct muscle stimulation. Contractile responses were signifcantly inhibited by indomethacin （1 mM） or prior incubation of the venom. There was statistically significant correlation between tumor＇s stage and TAM and EL counts. MC count and NVES were found to be higher in early stages. Conclusion The differing results may be due to wide variations in methodologies which were used for demonstration of inflammatory cells and vessels and variations in the degree of activation and complexity of functions of these cells.     

鼻咽癌患者组织中Treg细胞和Th17细胞与TNM分期相关性研究

目的:探讨Treg细胞和Th17细胞在鼻咽癌TNM分期组织中的表达及相关性.方法:选取我院鼻咽癌患者50例,鼻咽炎患者50例作为对照.采用免疫组织化学法检测初诊鼻咽癌TNM分期组织和鼻咽炎组织中Treg细胞和Th17细胞的表达,并分析其相关性.结果:各组组织中Treg细胞和Th17细胞阳性表达,鼻咽癌各组均高于鼻咽炎,差异具有统计学意义(P＜0.05),并随着分期逐步升高,Ⅰ期与Ⅲ、Ⅳa、Ⅳb期分别比较,差异具有统计学意义(P＜0.05),但Ⅰ、Ⅱ期及Ⅳa、Ⅳb期分别比较,差异无统计学意义(P＞0.05);鼻咽癌患者组织中Treg细胞与Th17细胞的百分率呈正相关性,具有统计学意义(P＜0.05).结论:Treg细胞和Th17细胞的异常表达可能涉及鼻咽癌的发生和发展过程;亦可能与鼻咽癌恶性进展密切相关.     

丁羟茴醚诱导幼儿骨髓间质干细胞分化为神经细胞

目的:探讨幼儿骨髓间质干细胞诱导分化为神经干细胞及神经细胞的方法及生物学特征,为临床利用骨髓间质干细胞或/和造血干细胞治疗小儿脊髓栓系综合征及其它神经系统疾病奠定基础。方法:取健康幼儿骨髓分离纯化骨髓间质干细胞,在α-MEM培养基(含20%胎牛血清,2 mmol/L谷氨酰胺,100 U/ml盘尼西林,100 mg/ml链霉素,25 ng/ml两性霉素B)中进行无分化培养,将传代细胞用丁羟茴醚(ButylatedHydroxyanisole,BHA)诱导,诱导剂是无血清DMEM培养基:含2%二甲基亚砜(Dimethyl sulfoxide,DMSO)、150μMBHA。结果:骨髓间质干细胞经过7 d体外诱导,大约有55%的细胞发生形态学变化,最初是胞体变圆,随后延展出长长的神经丝并相互交织成网,免疫组化结果表明这些细胞beta-微管蛋白Ⅲ(β-tublinⅢ)和神经胶质原纤维酸性蛋白(glial fibrillary acidic protein,GFAP)表达阳性。结论:儿童骨髓间质干细胞在合适的诱导剂作用下体外可定向分化成神经元和神经胶质细胞。为小儿脊髓栓系综合征及其它神经系统疾病自体骨髓移植实验治疗奠定了基础。     

羊膜上皮细胞促进共培养神经干细胞存活及分化

目的：探讨羊膜上皮细胞是否能在体外促进胚胎脑神经干细胞的存活及分化。 方法：Neurosphere法分离、克隆E12~14 d Wistar大鼠脑组织的神经干细胞,同时从羊膜中分离羊膜上皮细胞。将神经干细胞与羊膜上皮细胞在不同条件下共培养,通过免疫组织化学法对羊膜上皮细胞及神经干细胞的分化进行检测。 结果：羊膜上皮细胞表达神经干细胞及神经元、神经胶质细胞表面抗原;与羊膜上皮细胞共培养的神经干细胞克隆的分化细胞总数、分化为神经元的百分率及神经元初级突起长度明显高于对照组。 结论：羊膜上皮细胞与神经干细胞具有一定的同源性;羊膜上皮细胞能促进体外培养的神经干细胞的分化,且主要向神经元分化,并促进神经元初级突起的生长。提示羊膜上皮细胞为神经干细胞提供促进其分化及存活适宜的微环境。     

骨髓间充质干细胞与胶质瘤细胞非接触共培养后相关生物学特性分析

目的在体外通过大鼠胶质瘤C6细胞与大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)非接触共培养,以探明BMSCs是否受到肿瘤微环境的作用获得肿瘤细胞的相关生物学特性。方法通过6孔板结合Transwell小室建立BMSCs和C6胶质瘤细胞的共培养体系,以共培养组为实验组,设单独培养的BMSCs为对照组;相差显微镜下观察培养后2组细胞形态学的改变;核干细胞因子(nucleostemin,NS)检测细胞增殖力的变化;荧光定量PCR和Western blot检测细胞中mdm2、p53mRNA水平及其蛋白的变化;免疫荧光法检测神经胶质细胞特异的胶质纤维酸蛋白(glial fibrillary acidic protein,GFAP)在细胞中的定位及表达。结果共培养后实验组细胞呈现类似胶质瘤细胞样的形态,表达神经胶质细胞特异的GFAP蛋白;NS蛋白反映的细胞增殖能力未发生改变;共培养后实验组p53mRNA水平明显低于对照组(P<0.01),而p53蛋白的表达水平显著高于对照组[(1.63±0.31)vs(0.85±0.12),P<0.05];实验组mdm2mRNA及...     

鼠胶质瘤细胞上清液诱导人骨髓间充质干细胞向神经元样细胞的分化

目的：分离培养人骨髓间充质干细胞(MSCs),探讨鼠胶质瘤细胞上清液对其向神经元样细胞的诱导分化。方法：利用Percoll梯度分离法,培养人MSCs。采用鼠胶质瘤细胞上清液对MSCs进行诱导分化。观察人MSCs经诱导后细胞的形态变化,采用免疫细胞化学方法检测神经元烯醇化酶(NSE)、神经丝蛋白(NF)、胶质纤维酸性蛋白(GFAP)的表达。 结果：诱导24 h后,细胞胞体收缩呈锥形或球形,有突起长出,细胞间突起相互连接,交错成网,为典型的神经元样形态。免疫细胞化学结果显示,经诱导培养后的神经元样细胞的胞体及部分突起NSE和NF染色呈强阳性表达,而GFAP 染色呈阴性。诱导组出现NSE阳性的细胞率为(79.5±3.2)%,而对照组出现NSE阳性的细胞率为(12.1±2.0)%,两组之间比较差异有显著性 (P<0.01)。诱导组出现NF阳性的细胞率为(41.2±2.4)%,而对照组为阴性。结论：鼠胶质瘤细胞上清液可以诱导MSCs向神经元样细胞分化。     

CIK细胞联合DC细胞过继性免疫治疗结直肠癌化疗患者免疫功能的影响

目的 探讨CIK细胞联合DC细胞过继性免疫治疗结直肠化疗患者免疫功能的影响.方法 选取2011年1月～2012年1月该院收治的58例结直肠癌患者作为研究组.另选50例同期在我院进行健康体检人群作为对照组,研究组患者均采用化疗联合CIK细胞与DC细胞回输的治疗方法.比较分析两组CD3+、CD4+、CD8+及CD4+/CD8+等T细胞亚群指标的变化情况.结果 与对照组比较,研究组患者治疗前CD3、CD4+及CD4+/CD8+等T细胞亚群指标均明显减低,CD8+明显提高(均P ＜0.05).与研究组患者治疗前相比,治疗后CD3、CD4+及CD4+/CD8+等T细胞亚群指标均明显提高,CD8+明显降(均P＜0.05).6例(10.34％)患者在细胞皮下回输过程中发生体温升高现象.在一定细胞因子的诱导下,外周血单核细胞可以培养出纯度较高的DC细胞.随着CIK细胞培养时间的延长,双阳性细胞的百分率不断上升.结论 采用CIK细胞联合DC细胞过继性免疫治疗结直肠化疗患者,能够有效的提高患者的免疫功能指标,且不良反应小.     

切应力作用下与血管平滑肌细胞联合培养的内皮细胞的形态学

目的：观察生理切应力作用下与血管平滑肌细胞联合培养的内皮细胞的抗应力能力。方法：应用内皮细胞与血管平滑肌细胞联合培养模型和流动腔系统,以倒置相差显微镜及电镜观察生理切应力作用下的联合培养的内皮细胞的形态及超微结构。结果：０．２ｍＮ／ｃｍ＾２和０．４ｍＮ／ｃｍ＾２切应力作用２４ｈ,绝大多数内皮细胞均沿切应力方向发生重排,细胞内肌动蛋白微丝形成与切应力方向平行的应力纤维。２４ｈ后,内皮细胞未见明显脱落。结论：联合培养条件下,内皮细胞重排程度与切应力大小有关。切应力越大,重排程度越高,提示内皮细胞抗应力能力增强。     

Expansive effects of aorta-gonad-mesonephros-derived stromal cells on hematopoietic stem cells from embryonic stem cells

Background Hematopoietic stem cells (HSCs) give rise to all blood and immune cells and are used in clinical transplantation protocols to treat a wide variety of refractory diseases, but the amplification of HSCs has been difficult to achieve in vitro. In the present study, the expansive effects of aorta-gonad-mesonephros (AGM) region derived stromal cells on HSCs were explored, attempting to improve the efficiency of HSC transplantation in clinical practice.Methods The murine stromal cells were isolated from the AGM region of 12 days postcoitum (dpc) murine embryos and bone marrow（BM）of 6 weeks old mice, respectively. After identification with flow cytometry and immunocytochemistry, the stromal cells were co-cultured with ESCs-derived, cytokines-induced HSCs. The maintenance and expansion of ESCs-derived HSCs were evaluated by detecting the population of CD34+ and CD34+Sca-1+cells with flow cytometry and the blast colony-forming cells (BL-CFCs), high proliferative potential colony-forming cells (HPP-CFCs) by using semi-solid medium colonial culture. Finally, the homing and hematopoietic reconstruction abilities of HSCs were evaluated using a murine model of HSC transplantation in vivo.Results AGM and BM-derived stromal cells were morphologically and phenotypically similar, and had the features of stromal cells. When co-cultured with AGM or BM stromal cells, more primitive progenitor cells (HPP-CFCs ) could be detected in ESCs derived hematopoietic precursor cells, but BL-CFC’s expansion could be detected only when co-cultured with AGM-derived stromal cells. The population of CD34+ hematopoietic stem/progenitor cells were expanded 3 times,but no significant expansion in the population of CD34+Sca-1+ cells was noted when co-cultured with BM stromal cells. While both CD34+ hematopoietic stem/progenitor cells and CD34+Sca-1+ cells were expanded 4 to 5 times respectively when co-cultured with AGM stromal cells. AGM region-derived stromal cells, like BM-derived stromal cells, could promote hematopoietic reconstruction and HSCs’ homing to BM in vivo.Conclusions AGM-derived stromal cells in comparison with the BM-derived stromal cells could not only support the expansion of HSCs but also maintain the self-renewal and multi-lineage differentiation more effectively. They are promising in HSC transplantation. Chin Med J 2005; 118(23):1979-1986     

Co-culture of mesenchymal stem cells with umbilical vein endothelial cells under hypoxic condition

By co-culturing humm mesenchymal stem cells (hMSCs) and human umbilical rein en-dothelial cells (HUVECs) under hypoxia and creating a microenvironment similar to that of trans-planted hMSCs for the treatment of avascular ni ANFH, the effect of hMSCs on survival, apoptosis, migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) under the hypoxic condition were investigated in vitro. hMSCs and HUVECs were cultured and identified in vitro. Three kinds of conditioned media, CdM-CdMNOR, CdM-CdMHYP and HUVEC-CdMHYP were pre-pared. HUVECs were cultured with these conditioned media under hypoxia. The survival rate, apop-tosis rate, migration and angiogenesis of HUVECs were respectively detected by CCK-8, flow cy-tometry, Transwell and tube formation assay. The content of SDF-1α, VEGF and IL-6 in CdM was determined by ELISA. Our results showed that hMSCs and HUVECs were cultured and identified successfully. Compared with MSC-CdMNOR and HUVEC-CdMHYP groups, the survival rate, migra-tion and angiogenesis of HUVECs in MSC-CdMHYP group were significantly increased while the apoptosis rate was declined (P<0.05). Moreover, the expression of SDF-1α, VEGF and IL-6 in MSC-CdMHYP group was up-regulated. Under hypoxia, the apoptosis of HUVECs was inhibited while survival, migration and angiogenesis were improved by co-culture of hMSCs and HUVECs. The underlying mechanism may be that hMSCs could secrete a number of cytokines and improve niche, which might be helpful in the treatment of femoral head necrosis.     

白癜风患者外周血黑素细胞抗原特异性T细胞、调节性T细胞及自然杀伤T细胞水平及意义

目的 探讨白癜风患者外周血黑素抗原特异性CD8+T细胞、调节性T细胞及自然杀伤T细胞的变化及意义。方法 采用流式细胞术对初诊123例白癜风患者（观察组）和30例正常人（对照组）外周血中黑素细胞抗原特异性T细胞（CD8+Melan-A+/Tyrosinase+CTL）、调节性T细胞（CD4+CD125+CD127-Tregs）及自然杀伤T细胞（CD3+TCRα24+β11+NKT）进行定量分析并进行比较。结果观察组CD8+Melan-A+/Tyrosinase+CTL水平显著高于对照组（P＜0.05）;CD4+CD125+CD127-Tregs、CD3+TCRα24+β11+NKT水平均低于对照组（均P＜0.05）。不同分期白癜风患者外周血各种T细胞表达水平比较中,CD8+Melan-A+/Tyrosinase+CTL在各期中均高于对照组（均P＜0.05）;CD4+CD125+CD127-Tregs、CD3+TCRα24+β11+NKT水平仅在进展期中均低于对照组（均P＜0.05）,稳定期则无统计学差异。3种细胞水平与患者的白斑面积明显相关（r=0.929、-0.982、-0.957,均P＜0.01）。结论CD8+Melan-A+/Tyrosinase+CTL、CD4+CD125+CD127-Tregs、CD3+TCRα24+β11+NKT水平的异常导致白癜风患者体内免疫调节功能失衡,与白癜风的发生、发展有密切关系。     

C6胶质瘤细胞对体外共培养大鼠神经干细胞增殖影响的初步研究

目的探索与C6胶质瘤细胞体外共培养后大鼠神经干细胞（Neural Stem Cells NSCs）的增殖变化。方法分别培养神经干细胞、星形胶质细胞和C6胶质瘤细胞。利用共培养池（cell culturetranswell inserts）建立两种细胞的体外共培养模型。应用相差显微镜及电镜观察各组共培养后NSCs的形态变化;共培养后NSCs MTT法作生长曲线。结果原代培养后获得神经干细胞及星形胶质细胞;与C6胶质瘤细胞共培养7天后的NSCs增殖较对照组快,电镜观察可见核质比增高,细胞器较发达。结论 C6胶质瘤细胞对体外共培养的大鼠NSCs的增殖有一定促进作用。     

小鼠胃体部黏膜内分泌细胞的超微结构及与主细胞、壁细胞的关系

目的 观察小鼠胃体部黏膜内分泌细胞的超微结构,及其与胃底腺主细胞、壁细胞的相互关系.方法 取成年小鼠胃体部黏膜在透射电镜下观察内分泌细胞及主细胞、壁细胞的超微结构.结果 根据胞质中颗粒超微结构的不同特点,将内分泌细胞分为Ⅰ、Ⅱ、Ⅲ型.三型细胞均可见与主细胞及壁细胞紧密相邻,内分泌细胞的胞膜局部呈"Ω"型凹陷.结论 基本...     

初发系统性红斑狼疮患者调节性T细胞和Th17细胞的检测及意义

目的：观察初发系统性红斑狼疮（SLE）患者外周血调节性T细胞（Treg）和Th17细胞的变化,探讨其临床意义。方法检测15例初发SLE患者（活动组11例,非活动组5例）及10例健康对照者（对照组）的外周血CD4＋CD25highTreg、Th17细胞占CD4＋的比例,分析两种细胞的比率与疾病活动指数（SLEDAI）的相关性。结果初发SLE患者外周血CD4＋CD25highTreg和Th17细胞的比例与对照组相比,差异无统计学意义（P＞0.05）,与SLEDAI无相关性（均P＞0.05）。CD4＋CD25highT细胞与Th17细胞的比率在活动组患者下降尤为明显（P＜0.05）,且与SLEDAI呈明显的负相关（P＜0.05）。结论初发的活动性SLE患者外周血Treg与Th17细胞的比率明显下降,并与疾病活动密切相关,两群细胞的失衡可能在SLE发病机制中起重要的作用。     

CD3AK细胞联合BCG对膀胱癌细胞的体外杀伤活性研究

目的探讨CD3AK细胞对膀胱癌细胞的体外杀伤活性,以及CD3AK细胞与BCG联合作用于膀胱癌细胞的效应.方法采用抗CD3单抗和IL-2共同刺激健康人外周血单个核细胞诱导出CD,AK细胞;以流式细胞仪测定细胞表型;用MTT法测定CD,AK以及CDaAK联合BCG对膀胱癌细胞系(T24)的杀伤活性.结果CD3AK细胞为异质细质细胞群,其细胞类型主要是CD8 细胞;在效靶比为10:1及20:1时,培养4 d的CD:,AK细胞BCG联合作用于T24细胞的杀伤率高于二者分别对T24细胞的杀伤率.结论CD3AK细胞是一种高效的抗肿瘤效应细胞,对膀胱癌细胞具有较强的杀伤作用;CD3AK细胞与BCG对膀胱细胞呈协同杀伤作用.CD3AK细胞在膀胱癌过继免疫治疗中具有重要的临床应用前景.