骨髓基质干细胞的分离纯化及培养

目的 建立骨髓基质干细胞(MSCs)良好的分离纯化和培养方法。方法 将小鼠骨髓基质干细胞自殷骨中分离,应用贴壁选择法结合细胞克隆挑选法进行分离纯化,应用细胞生长因子(EGF和PDGF-BB)刺激法进行MSCs的体外培养和传代,倒置显微镜下观察分离培养的细胞并照像记录。结果,培养获得了纯化的呈梭形成纤雏样细胞的骨髓基质干细胞。在生长因子EGF和PDGF-BB的共同作用下,传代MSCs生长旺盛,形态均一。结论 该方法是简便高效的骨髓基质干细胞的分离纯化和培养方法。     

大鼠表皮细胞的体外培养及纯化方法

根据表皮细胞和成纤维细胞对胰蛋白酶敏感性、贴壁时间及要求不同的,采用胰蛋白酶消化法和反复贴壁法相结合,能有效清除成纤维细胞的混合生长,获得纯化的表皮细胞。本方法经济、简便、实用。     

恒河猴皮肤干细胞的体外培养纯化与鉴定

探索恒河猴皮肤干细胞的体外培养及纯化条件,为进一步的研究奠定基础. 通过组织块培养法和消化培养法 在体外培养恒河猴表皮细胞,然后用Ⅳ型胶原吸附法吸附20 min,获得快吸附细胞. 对快吸附细胞进行克隆培养,并进行免疫细胞化学双标染色、RT PCR鉴定 β1 整合素和角蛋白15的表达,用流式细胞仪鉴定纯化前后的细胞中 β1 整合素和角蛋白15的阳性细胞比例,并通过透射电镜观察细胞的超微结构. 组织块培养法和消化培养法均可获得表皮细胞,Ⅳ型胶原纯化后的细胞胞体较小,饱满,核/浆比例大,细胞镶嵌状排列. 细胞克隆分析显示,细胞全克隆生长率高. 细胞免疫荧光显示,分选后的细胞显示 β1 整合素和角蛋白15阳性. RT PCR检查呈现 β1 整合素和角蛋白15的特异性片段. 流式细胞仪检查显示,纯化前的细胞中角蛋白15阳性细胞占总细胞中的比例为8％, β1 整合素阳性细胞的比例为10.7％;纯化后,角蛋白15阳性细胞的比例为89.4％, β1 整合素阳性细胞的比例为88.5％. 通过组织块培养法和消化培养法均可培养获得活性良好的表皮细胞,Ⅳ型胶原吸附法是一种简便、有效的皮肤干细胞分离方法,可以为进一步的眼表上皮替代重建眼表提供足量的高纯度的干细胞建立可靠的物质基础.     

人脐带间充质干细胞分离培养方法的研究

目的探讨人脐带间充质干细胞(MSCs)体外分离培养的最佳方法。方法无菌条件下采集早产儿(不足37周)和足月儿的脐带,分离MSCs,比较胎龄、脐带新鲜程度、分离方法和不同培养基对脐带MSCs原代培养过程的影响,通过免疫荧光法检测脐带MSCs表面标记物的表达情况,观察脐带MSCs的生物学特性。结果足月分娩,新鲜脐带,采用组织块平铺法和MesencultTM培养基,脐带MSCs原代培养成功率较高。相同条件下,早产儿脐带MSCs原代培养成功率低于足月分娩脐带。人脐带MSCs高表达CD44、CD90和CD29。结论筛选出一种人脐带MSCs体外分离培养的最佳方法。     

脂肪干细胞上清液培养雪旺细胞的实验研究

目的:研究应用脂肪干细胞上清液培养新生小鼠雪旺细胞的可行性。方法:取新生(出生5-7天)C57BL/6小鼠的坐骨神经,采用0.2%的复合胶原酶NB4消化法分离获取细胞,然后应用雪旺细胞条件培养基(SCCM)和C57BL/6小鼠的脂肪干细胞上清液(ADSC-CM)分别培养雪旺细胞。用0.2%复合胶原酶NB4差速分离纯化这两种方法培养的雪旺细胞,每48 h纯化1次,共进行2次纯化。应用P75免疫荧光染色方法鉴别两组P2代雪旺细胞并比较两组雪旺细胞的纯度和生长情况。结果:脂肪干细胞上清液培养的雪旺细胞纯化两次后,数量明显增多,其纯度与雪旺细胞条件培养基相比没有明显差异(P0.05)。结论:脂肪干细胞上清液可以较好的培养雪旺细胞,可以作为一种新的廉价方便的培养基代替雪旺细胞条件培养基来培养许雪旺细胞。     

人心肌肌钙蛋白T的纯化和单克隆抗体的制备

从人左室心肌中成功纯化心肌肌钙蛋白T(cTnT). 经匀浆, 70℃加热处理, 咪唑盐酸透析, DEAE-纤维素层析, 100g心肌获取cTnT 5mg, 纯度为97.6%. 同时采用脾内免疫法, 免疫Balb/C小鼠, 经细胞融合, 筛选, 克隆化得5株稳定分泌抗人cTnT单克隆抗体(McAb)的杂交瘤细胞(G₃, G₈, G₁₀, A₅, A₇), 4株为IgM, 1株为IgG, 染色体数目92～110条. 腹水效价为3.2×10^-6～1. 6×10^-7.     

大鼠精原干细胞的高效分离和纯化方法

精原干细胞是精子发生的基础,是永久分化成精子的克隆源,它既可以自我更新维持体内干细胞的数量,又可以增殖分化形成各阶段的生精细胞直至成熟精子。本文以22～25日龄Wistar-Iamichi大鼠为研究对象,利用两步酶消化法分离得到睾丸曲细精管细胞悬液,根据精原干细胞与曲细精管细胞悬液中体细胞(支持细胞及少量的管周细胞)及各级分化的生精细胞贴壁能力及对细胞外基质粘附力的不同,将大鼠精原干细胞进行纯化。经纯化后,5只大鼠的睾丸可以得到约3×10~5个精原干细胞,该精原干细胞在体外培养可形成克隆,并且该克隆可表达精原干细胞特异的标记基因GFRα1和CDH1。本文所介绍的高效分离和纯化大鼠精原干细胞的方法,操作简便,且得到的精原干细胞具有很高的活力和增殖能力,该方法为今后大鼠精原干细胞的长期培养及操作研究奠定了基础。     

骨髓基质干细胞向心肌样细胞分化的实验研究

目的探讨大鼠骨髓基质干细胞向心肌样细胞分化后超微结构和细胞因子表达的变化,为下一步细胞移植治疗扩张型心肌病提供理论依据。方法在体外扩增、定向诱导骨髓基质干细胞向心肌样细胞分化的基础上,电镜下观察骨髓基质干细胞诱导前后超微结构的改变,RT-PCR检测心肌特异性因子ANP、BNP、α-MHC、β-MHC的表达,并与原代培养的心肌细胞比较,观察二者之间的生物学异同。结果免疫组化法证实诱导后的骨髓基质干细胞向心肌样细胞分化,电镜下胞浆内可见糖原颗粒,肌原纤维排列与原代培养的心肌细胞相似;RT-PCR证实诱导后的骨髓基质干细胞表达心肌特异性因子ANP、BNP、α-MHC、β-MHC。结论骨髓基质干细胞经5-氮胞苷定向诱导后在超微结构和细胞因子的表达上类似于心肌细胞,已向心肌样细胞分化。     

兔角膜缘干细胞体外分离、培养和生物学鉴定的实验研究

通过体外培养兔角膜缘干细胞,观察其生物学特性,建立兔角膜缘干细胞的体外培养方法。方法0．25%胰蛋白酶消化角膜缘组织,用含15%胎牛血清的DMEM和F12(1:1)的培养液(DF)对兔角膜缘干细胞进行体外培养,形态学观察,培养的细胞早期使用AEl/AE3、晚期使用AE5角蛋白特异的单克隆抗体)作细胞免疫化学鉴定。结果:原代培养细胞48h后开始贴壁,部分细胞由圆形变为卵圆形或长梭形;10～14d形成单层,细胞呈圆形、卵圆形,类角膜上皮细胞;细胞传到第5代左右开始出现老化状态;免疫细胞化学染色:培养的细胞早期AEl/AE3呈阳性而少部分细胞AE5呈阳性,培养的细胞晚期AE5呈阳性。结论:本实验初步建立了一套兔角膜缘干细胞的体外培养方法。     

小鼠胚胎干细胞在六种培养体系的培养观察

目的　观察小鼠胚胎干细胞在六种培养体系中的生长情况。方法　小鼠胚胎干细胞 (ESD3细胞株 )在以下六种培养体系中培养 :1 .原代小鼠胚胎成纤维细胞 (MEF)有血清培养 ,2 .MEF无血清培养 ,3.SNL细胞有血清培养 ,4.LIF(白血病抑制因子 )有血清无饲养层培养 ,5.LIF无血清无饲养层培养 ,6.大鼠肝细胞 (BRL)条件培养基培养。经体外培养 1 0代后 ,观察其克隆形态 ,同时进行碱性磷酸酶检测并将ES细胞接种于裸小鼠皮下 ,观察ESD3的未分化状态和多潜能性。结果　六种培养体系培养的ESD3具有典型的ES细胞克隆形态 :巢状 (集落状 )隆起生长 ,边缘清楚 ,表面平滑 ,结构致密 ;AKP强阳性 ;裸小鼠体内形成了由多种组织构成的畸胎瘤。结论　六种培养体系均能支持ESD3生长 ,并能保持其未分化性和多潜能性 ,为ES细胞的应用研究奠定了良好的基础。     

心脏干细胞研究新进展

随着近年干细胞的研究发展,心脏中心脏干细胞(CSCs)被证实存在,并发现CSCs具有自我更新及多向分化能力,负责维持心脏内环境稳态,许多学者已经从CSCs着手研究治疗终末期心脏病的新途径。本文就CSCs以及其为基础的心脏病治疗进展作一综述,尤其对中药诱导CSCs在治疗心脏病中的前景进行展望。     

小熊猫精原干细胞的分选与培养

野生动物的保护手段主要包括就地保护、易地保护与离体保护。精原干细胞（SSCs）是雄性动物维持生殖能力的根本,既能通过自我更新产生新细胞,也能通过分化产生精子,在小熊猫（Ailurus fulgens）离体保护方面具有广阔的应用前景。动物睾丸中精原干细胞数量极少,分离纯化与体外培养对于其研究和应用至关重要。本研究选择整合素α6（ITGA6）蛋白作为精原干细胞分子标记,采用免疫磁珠分选（MACS）技术富集了3月龄小熊猫睾丸中的ITGA6阳性细胞。流式细胞术检测发现分选后ITGA6阳性细胞纯度可达74.27% ± 8.73%,显著高于分选前（32.60% ± 3.06%）。将分选后的细胞接种到层粘连蛋白包被的细胞培养板中,用含胶质细胞源性神经营养因子（GDNF）、表皮细胞生长因子（EGF）与成纤维细胞生长因子（bFGF）的培养基进行体外培养。培养10 d后,在显微镜下可观察到典型的精原干细胞集落,结合逆转录PCR（RT-PCR）和细胞免疫荧光染色发现这些细胞集落特异性表达精原干细胞分子标记蛋白ITGA6、早幼粒细胞白血病锌指蛋白（PLZF）和胸腺细胞分化抗原1（THY1）,同时也表达生殖细胞标记蛋白VASA和DAZL。本研究结果证实,ITGA6可作为小熊猫精原干细胞的分子标记用于细胞分选富集,同时初步建立的培养体系也为小熊猫精子发生机制与应用研究提供材料。     

Establishment of Alternative Culture Method for Spermatogonial Stem Cells Using Knockout Serum Replacement

Since spermatogonial stem cells (SSCs) are capable of both self-renewal and differentiation to daughter cells for subsequent spermatogenesis, the development of an efficient in vitro culture system is essential for studies related to spermatogenesis. Although the currently available system is serum-free and contains only chemically-defined components, it highly relies upon bovine serum albumin (BSA), a component with batch-to-batch quality variations similar to those of fetal bovine serum. Thus, we searched for an alternative BSA-free culture system that preserved the properties of SSCs. In this study, we utilized Knockout Serum Replacement (KSR) in the SSC culture medium, as a substitute for BSA. The results demonstrated that KSR supported the continuous growth of SSCs in vitro and the SSC activity in vivo without BSA, in a feeder-cell combination with mouse embryonic fibroblasts. The addition of BSA to KSR further facilitated cell cycle progression, whereas a transplantation assay revealed that the addition of BSA did not affect the number of SSCs in vivo. The combination of KSR with BSA also allowed the elimination of GFRA1 and FGF2, and the reduction of the GDNF concentration from 20 ng/ml to 5 ng/ml, while maintaining the growth rate and the expression of SSC markers. Furthermore, KSR was also useful with SSCs from non-DBA/2 strains, such as C57BL/6 and ICR. These results suggested that KSR is an effective substitute for BSA for long-term in vitro cultures of SSCs. Therefore, this method is practical for various studies related to SSCs, including spermatogenesis and germ stem cell biology.     

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.     

心脏干/祖细胞研究进展

心脏干/祖细胞(cardiac stem or progenitor cells, CSC/CPCs)的发现使人们认识到成体哺乳动物心脏是一个自我更新的器官, 同时也为心脏病的治疗提供一种优越的细胞类型.目前, 已分离鉴定出多种亚群的CSC/CPCs, 并对它们在心脏中赖以生存的生理微环境也有一定的认识.对不同亚群CSC/CPCs的表型、分化潜能及调节它们在体内静息、自我更新、迁移或分化等活动的分子机制进行综述, 讨论CSC/CPCs的来源并简要介绍胚胎干细胞(embryonic stem cells, ESCs)向心肌细胞的分化, 并对干细胞在心脏病治疗中的应用进行展望.     

Adult c-kitpos Cardiac Stem Cells Are Necessary and Sufficient for Functional Cardiac Regeneration and Repair

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Validation of the Cardiosphere Method to Culture Cardiac Progenitor Cells from Myocardial Tissue

Background

At least four laboratories have shown that endogenous cardiac progenitor cells (CPCs) can be grown directly from adult heart tissue in primary culture, as cardiospheres or their progeny (cardiosphere-derived cells, CDCs). Indeed, CDCs are already being tested in a clinical trial for cardiac regeneration. Nevertheless, the validity of the cardiosphere strategy to generate CPCs has been called into question by reports based on variant methods. In those reports, cardiospheres are argued to be cardiomyogenic only because of retained cardiomyocytes, and stem cell activity has been proposed to reflect hematological contamination. We use a variety of approaches (including genetic lineage tracing) to show that neither artifact is applicable to cardiospheres and CDCs grown using established methods, and we further document the stem cell characteristics (namely, clonogenicity and multilineage potential) of CDCs.

Methodology/Principal Findings

CPCs were expanded from human endomyocardial biopsies (n = 160), adult bi-transgenic MerCreMer-Z/EG mice (n = 6), adult C57BL/6 mice (n = 18), adult GFP⁺ C57BL/6 transgenic mice (n = 3), Yucatan mini pigs (n = 67), adult SCID beige mice (n = 8), and adult Wistar-Kyoto rats (n = 80). Cellular yield was enhanced by collagenase digestion and process standardization; yield was reduced in altered media and in specific animal strains. Heparinization/retrograde organ perfusion did not alter the ability to generate outgrowth from myocardial sample. The initial outgrowth from myocardial samples was enriched for sub-populations of CPCs (c-Kit⁺), endothelial cells (CD31⁺, CD34⁺), and mesenchymal cells (CD90⁺). Lineage tracing using MerCreMer-Z/EG transgenic mice revealed that the presence of cardiomyocytes in the cellular outgrowth is not required for the generation of CPCs. Rat CDCs are shown to be clonogenic, and cloned CDCs exhibit spontaneous multineage potential.

Conclusions/Significance

This study demonstrates that direct culture and expansion of CPCs from myocardial tissue is simple, straightforward, and reproducible when appropriate techniques are used.