恒河猴精原干细胞的筛选、培养与鉴定

目的:建立恒河猴精原干细胞的筛选和培养方法。方法:采用改良的二步酶消化法分离恒河猴睾丸细胞,用改进的差异贴壁筛选法纯化恒河猴精原干细胞,用添加了胶质细胞源神经营养因子(GDNF)、可溶性GFRα1和bFGF的DMEM/F12无血清培养基和小鼠胚胎成纤维细胞饲养层培养恒河猴精原干细胞,并通过形态观察、标志基因的免疫细胞化学分析和半定量RT-PCR分析鉴定培养细胞的干细胞活性。结果:分离纯化的恒河猴精原干细胞在添加了3种生长因子的培养基中能形成较大的干细胞克隆,并表达精原干细胞的标志基因。结论:本研究初步建立了恒河猴精原干细胞的培养体系,为进一步开展相关研究奠定基础。     

以支持细胞为饲养层培养冻存后大鼠精原干细胞研究

目的：研究冻存的精原干细胞体外分离培养的生物学特性,为今后冻存精原干细胞的批量培养和长期利用提供依据。方法：取7～8d的sD雄性大鼠,分离睾丸组织,用两步酶消化法和差速时间贴壁法,分离精原干细胞和支持细胞。以二甲基亚砜（DMSO）作冷冻保护液,将分离到的精原干细胞放置于液氮中冻存;利用体外培养体系,进行支持细胞的培养和饲养层的制备;然后将复苏的精原干细胞接种到支持细胞饲养层上培养,并利用碱性磷酸酶鉴定精原干细胞。结果：用两步酶消化法和差速贴壁法能分离到纯度较高的精原干细胞和支持细胞,冻存的SSCs解冻后能在支持细胞饲养层上贴壁、生长、增殖,碱性磷酸酶活性鉴定呈阳性。结论：冻存后精原干细胞在体外支持细胞饲养层上呈集落样生长,并能长时间维持细胞集落形态及数目,可为体外研究药物或毒物干扰精原干细胞提供细胞模型。     

人骨髓间充质干细胞向肝系细胞分化的实验研究

目的探讨肝细胞生长因子(hepatocyte growth factor,HGF)和碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)诱导人骨髓间充质干细胞在体外向肝系细胞分化的可能性。方法人骨髓间充质干细胞来自成人骨髓,采用密度梯度离心法分离和反复贴壁法纯化。为有效地促进其向肝细胞分化,本实验使用了HGF和bFGF联合诱导的方法,于0、7、14、21、28d采用细胞免疫荧光法检测肝细胞表面标志甲胎蛋白(alpha_fetoprotein,AFP),细胞角蛋白(cytokeratin 18,CK_18)和细胞角蛋白(cytokeratin 19,CK_19),同时采用Periodic Acid_Schiff(PAS)染色检测诱导细胞糖原合成及贮存功能,并留取不同时段的培养上清液,用放射免疫荧光法(radioimmunoassay,RIA)法检测AFP的分泌量。结果在诱导14d后可观察到诱导细胞变成短梭形和多角形,随诱导时间延长多角形细胞增多,并可形成肝细胞样细胞集落。第14天时AFP,CK_18表达阳性,第21天诱导细胞糖原染色阳性,第28天CK_19表达阳性。培养上清液中14d时,检测到AFP的分泌,浓度为0.1ng/ml;17d时分泌量最高,为0.4ng/ml;21d时下降至0.3ng/ml。结论HGF和bFGF能够诱导人骨髓间充质干细胞在体外分化为肝细胞,这将为肝脏疾病的细胞替代治疗提供新的细胞来源。     

关于改良差速贴壁法原代培养大型成年哺乳动物绵羊肌源性干细胞的研究

目的探讨改良差速贴壁法原代培养成年大型哺乳类动物绵羊肌源性干细胞(muscle-derived stem cells,MDSC)的可行性。方法改良差速贴壁法分离培养原代绵羊MDSC,并通过免疫荧光检测及western blotting法检测干细胞抗原-1(stem cell antigen,Sca-1),Desmin及CD 34表达情况。Western blotting法检测贴壁细胞(pp)1~pp6的Desmin表达情况来反应纯化情况。结果利用改良差速贴壁法可以成功分离培养出绵羊MDSC,该干细胞Sca-1,Desmin及CD 34表达均阳性,贴壁慢的细胞Desmin表达增高,在pp6中表达最高。结论改良差速贴壁法可以成功分离培养原代绵羊MDSC,且可扩增至20代。     

大鼠骨髓间充质干细胞的分离培养及鉴定

[目的]建立分离纯化、培养、扩增骨髓间充质干细胞(Mesenchymal stem cell,MSCs)鉴定的方法.[方法]采用密度梯度离心及贴壁培养相结合的方法分离、培养、扩增MSCs,并用流式细胞仪检测其表面标志CD34、CD44、CD105. [结果]培养48 h后即可见呈集落生长的细胞,72 h后可见少量贴壁MSCs细胞,7 d后贴壁细胞明显增多,梭形栅栏样分布,融合度达70%以上.第3代原代培养细胞表面标志CD44+、CD105+在95%左右,CD34的表达率甚低.[结论]联合应用密度梯度离心法及贴壁筛选法,可以获得纯度较高的骨髓MSCs     

神经生长因子对神经干细胞分化的影响

【目的】探讨不同神经生长因子对神经干细胞(neural stemcells,NSCs)分化特性的影响。【方法】自10～14周人胚胎脑分离培养海马神经干细胞,分别用表皮生长因子(human epidermal growth factor,EGF)和碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF),或单独用bFGF扩增神经干细胞,取第3代神经干细胞球,用含小剂量bFGF(0.5 ng)的无血清基础培养基诱导分化,体外诱导分化7 d后应用免疫荧光方法检测β_Ⅲ微管蛋白(β_ⅢTubulin,Tuj1)和神经胶质纤维酸性蛋白(glial fibriliary acidic protein,GFAP)表达情况。【结果】不同的生长因子培养的神经干细胞分化特性很不相同,EGF+bFGF培养的NSCs既可分化为神经元,又可分化为星形胶质细胞,其分化比率分别为38.8%和57.3%;bFGF培养的NSCs主要分化为神经元,比率达81.2%,未检测星形胶质细胞。【结论】不同神经生长因子扩增的神经干细胞分化特性很不相同,本实验为神经干细胞分化的研究提供了良好的实验平台。     

用VEGF165和bFGF诱导单个核细胞分化成内皮祖细胞的试验研究

目的研究从健康人外周血中提取单个核细胞，用VEGF165和bFGF诱导细胞分化成内皮祖细胞，并分析细胞形态、成集落数量，细胞数量活性。方法选择健康人20例，用密度梯度离心法从外周血获取单个核细胞，用生长因子VEGF165和bFGF诱导细胞分化，7d后将贴壁细胞进行分析。荧光显微镜鉴定FITC-UEA-I和DiI-acLDL双染色阳性细胞为正在分化的EPC；流式细胞仪检测CD34和KDR；MTT法检测细胞生长状态；并分析生长因子VEGF165和bFGF干预后细胞形态和成集落的数量。结果VEGF165和bFGF干预后EPC的细胞数目和集落数明显增加，细胞增殖活性明显增强。结论成人外周血中提取的单个核细胞，在VEGF165和bFGF作用下可培养为内皮祖细胞。     

新生大鼠海马神经干细胞的培养与传代

目的：探讨新生大鼠海马神经干细胞（Neural Stem Cells,NSCs）体外分离培养的条件和传代的新方法。方法：从新生大鼠海马中分离神经干细胞,采用无血清培养技术,在表皮生长因子（epidermalgrowthfactor,EGF）、碱性成纤维生长因子（basic fibroblastgrowth factor,bFGF）和B27联合作用下使其不断增殖克隆,采用神经小球分离试剂盒进行传代,利用免疫荧光染色的方法对培养的细胞进行鉴定。结果：从新生大鼠海马分离的细胞群可不断增殖形成细胞克隆球,并能稳定传代,呈nestin阳性表达,且具有多向分化能力。采用神经小球分离试剂盒传代的细胞球生长快,数量多,体积大,折光性好。结论：利用无血清技术和特定生长因子,可以使来源于新生大鼠海马的NSCs在体外扩增,利用大鼠神经小球分离试剂盒可以有效地进行传代,稳定高效地培养出NSCs。     

小鼠精原干细胞冷冻保存技术的研究

[目的]探讨精原干细胞(SSCs)冷冻复苏后与Sertoli支持细胞共培养的增殖分化能力。[方法]以7~8 d龄小鼠为材料,Percoll密度梯度离心,并将分离后的SSCs液氮冷冻,复苏后接种至Sertoli饲养层上,37℃下共培养,在倒置相差显微镜下观察细胞的生长特点,并对培养细胞进行C-Kit受体检测。[结果]增殖细胞的生长特点及形态学特征与文献报道精原干细胞的特征相符合。C-Kit受体显示细胞膜呈强阳性。这些均符合精原干细胞的生物学特征。[结论]冷冻复苏后精原干细胞在未加入细胞因子的Sertoli饲养层中能较长时间生长并分裂增殖。     

视黄酸和硷性成纤维细胞生长因子对神经干细胞增殖和分化的影响

目的:研究视黄酸(RA)、硷性成纤维细胞生长因子(bFGF)对体外培养的神经干细胞(NSCs)分裂增殖和分化的作用。方法:分离培养新生SD大鼠大脑皮层NSCs,采用不同组合配方的培养液培养细胞,利用MTT法检测其对NSCs存活和增殖的影响;利用免疫荧光鉴定和分化细胞的分类计数法,判定bFGF、RA等对NSCs分化的影响。结果:MTT实验表明,bFGF组、bFGF+RA组以及RA组均能促进NSCs增殖,其中bFGF作用最明显,bFGF+RA次之,RA最弱。RA处理组经诱导分化后产生的神经元是bFGF组及对照组的2～3倍。结论:bFGF对NSCs增殖及较长期存活具有重要的作用,同时抑制其分化;RA可以部分抵消bFGF的促有丝分裂和分化抑制作用,并促进NSCs向神经元方向分化。     

胶质细胞源性神经营养因子促进脊髓损伤后轴突再生的实验研究

目的探讨胶质细胞源性神经营养因子(GDNF)对轴突生长的促进作用。方法在体外培养胚胎大鼠脊神经后根神经节,随机分为两组,实验组加入GDNF,对照组只添加培养液,3天后用免疫荧光染色方法鉴定神经节轴突生长情况。在体内试验方面,36只成年SD大鼠建立脊髓半切损伤模型,随机分为两组,实验组移植含有GDNF的雪旺细胞微管,对照组仅移植雪旺细胞微管。移植术后2周、4周、6周取材,测量再生神经束的大小。结果 GDNF处理3天后,实验组脊髓后根神经节新生轴突的长度明显长于对照组,而且新生轴突的覆盖面积显著大于对照组。雪旺细胞微管移植后不同时间,含有GDNF的微管内再生神经束的截面积均显著大于对照组。结论 GDNF在体内外对轴突的生长都有显著的促进作用,有望成为治疗脊髓损伤的可选方法之一。     

胶质细胞源性神经营养因子促进脊髓损伤后轴突再生的实验研究

目的探讨胶质细胞源性神经营养因子（GDNF）对轴突生长的促进作用。方法在体外培养胚胎大鼠脊神经后根神经节,随机分为两组,实验纽加入GDNF,对照纽只添加培养液,3天后用免疫荧光染色方法鉴定神经节轴突生长情况。在体内试验方面,36只成年SD大鼠建立脊髓半切损伤模型,随机分为两组,实验组移植含有GDNF的雪旺细胞微管．对照组仅移植雪旺细胞微管。移植术后2周、4周、6周取材,测量再生神经束的大小。结果GDNF处理3天后,实验组脊髓后根神经节新生轴突的长度明显长于对照组,而且新生轴突的覆盖面积显著大于对照组。雪旺细胞微管移植后不同时间,含有GDNF的微管内再生神经柬的截面积均显著大于对照组。结论GDNF在体内外对轴突的生长都有显著的促进作用,有望成为治疗脊髓损伤的可选方法之一。     

In vitro effects of melatonin on colonization of neonate mouse spermatogonial stem cells

We have recently reported that antioxidant supplements enhance the efficacy of cryopreserved spermatogonial stem cells. Melatonin is considered a free radical scavenger which has direct and indirect antioxidant effects in in vitro and in vivo microenvironments. Due to the anti-apoptotic properties of melatonin, researchers have proposed that melatonin may improve the efficiency of spermatogonial stem cell (SSC) transplantation. However, the appropriate methodology which facilitates SSC proliferation remains to be determined. Identification of a proper propagation system is essential for the future application of SSCs in the field of infertility. The aim of the present study was to investigate the effects of melatonin on the colonization of SSCs. SSCs were isolated from the testes of three to six day old mice, and their purities were assessed by cytometry using Plzf antibody. Isolated testicular cells were cultured in the absence or presence of melatonin extract for two weeks. Suppression of differentiation and maintenance of spermatogonial stem cells was confirmed by alkaline phosphatase staining and immunocytochemistry using Plzf antibody. The number and diameter of the colonies of SSCs were assessed during the 7^th and 14^th days of culture, and the expression of Id4, Plzf, and C-kit were evaluated using real-time PCR at the end of the culture period. The survival rate of the cultured cells in the presence of melatonin was significantly higher than the control group. The number and diameter of colonies also increased in the cells treated with melatonin. The results of our study suggest that culture of SSCs with 100 μM melatonin supplementation can increase SSCs proliferation significantly.     

Low-dose ionizing irradiation triggers a 53BP1 response to DNA double strand breaks in mouse spermatogonial stem cells

The present study aims to examine the effect of low-dose ionizing irradiation on DNA double strand breaks (DSB) in mouse spermatogonial stem cells (SSCs) and reveal the underlying pathways for the DNA repair for DSB in SSCs. Eighteen one-month-old mice were divided into 6 groups and sacrificed separately at 45 minutes, 2 hours, 24 hours, 48 hours, and 72 hours after 0.1Gy X-ray irradiation (mice without receiving ionizing irradiation served as control). After perfusion fixation, testes were removed, sectioned, and followed by staining of γH2AX, 53BP1, Caspase 3, and promyelocytic leukemia zinc-finger (PLZF) for analysis among the different groups. The staining was observed by immunofluorescence visualized by confocal laser scanning. After low-dose irradiation, only 53BP1, but not Caspase3 or γH2AX was upregulated in PLZF positive SSCs within 45 minutes. The expression level of 53BP1 gradually decreased 24 hours after irradiation. Moreover, low-dose irradiation had no effect on the cell number and apoptotic status of SSCs. However other spermatogenic cells highly expressed γH2AX shortly after irradiation which was dramatically reduced following the events of DNA repair. It appears that low-dose ionizing irradiation may cause the DNA DSB of mouse spermatogenic cells. 53BP1, but not γH2AX, is involved in the DNA repair for DSB in SSCs. Our data indicates that 53BP1 plays an important role in the pathophysiological repair of DNA DSB in SSCs. This may open a new avenue to understanding the mechanisms of DNA repair of SSCs and male infertility.     

乳鼠心肌细胞原代培养方法

目的:探讨新生乳鼠心肌细胞原代培养的条件和方法,培养纯度高、存活时间长的心肌细胞。方法:参照Simpson等的方法加以改进,采用含0.05%EDTA的胰蛋白酶多次消化心肌,差速贴壁1.5 h纯化心肌细胞进行培养,培养过程在37℃、5%CO2条件下进行。用台盼蓝染色法检测培养细胞的存活率,倒置显微镜下观察细胞形态变化,记录3 d、6 d、9 d、12d心肌细胞搏动频率,测定细胞活力。结果:原代培养乳鼠心肌细胞生长良好,培养3 d、6 d、9 d、12 d时,用台盼蓝染色法检测心肌细胞的存活率均大于96%,培养12 d时仍为97.02%。在倒置显微镜下观察,培养48 h左右的细胞之间相互连接的并有细胞搏动。培养6 d、9 d、12 d的细胞与培养3 d的相比,活力增强,差异显著(P<0.05),从6 d开始,细胞活力并未显著增强,两两比较均无统计学差异(P>0.05)。结论:应用此方法培养的心肌细胞纯度高,存活时间长,是一种稳定、可靠的原代心肌细胞培养方法。     

EGF、bFGF对促进人毛乳头细胞生长及分化的影响

目的 探讨EGF、bFGF对促进人毛乳头细胞生长及分化的影响.方法 实验组培养基加入EFG、bFGF和3H-胸腺嘧啶,对照组培养基加入3H-胸腺嘧啶,采用闪烁计数器检测的方法,观察两组人毛乳头细胞的增殖情况,采用流式细胞仪检测两组人毛乳头细胞分化为成纤维细胞的情况.结果 实验组人毛乳头细胞放射性闪烁计数均数、成纤维细胞的特异性蛋白1（FSP1）阳性率与对照组相比有统计学差异（P＜0.05）.结论 EFG、bFGF能促进人毛乳头细胞的生长、增殖,并向成纤维细胞定向分化.     

Effect of a radical scavenger "water soluble protein" from broad beans (Vicia faba) on antioxidative enzyme activity in cellular aging

We isolated a free-radical scavenger "water soluble protein (WSP)" from broad beans. Hydrocortisone (HC) is known to inhibit superoxide generation and was used as the reference scavenger. WSP was examined for its effect on antioxidation in young (PDL 20, 25% of the maximum life span) and old (PDL 50, 62.5% of the maximum life span) human fibroblasts (TIG-1). Cells were treated with WSP or HC for 4 and 6 wk in young cells, and for 3 and 6 wk in old cells. The cytosolic superoxide dismutase activity in the cells treated with WSP or HC tended to decrease as compared with that in the non-treated cells (control) with the exception of WSP-treated young cells 4 wk after culturing. Young cells were equal in glutathione peroxidase activity to the control, but the activity level in WSP- or HC-treated young cells 6 wk after culturing was 10-50% lower than that in the control. Young and old cells treated with WSP or HC were superior to the control in catalase activity with the exception of HC-treated old cells. WSP- or HC-treated cells were higher in glutathione (GSH) concentration than the control with the exception of WSP-treated young cells 4 wk after culturing and HC-treated old cells 6 wk after culturing. Such increases in catalase activity and GSH concentration by WSP treatment may be related to the delay of cellular aging-dependent degeneration.