[摘要] 目的 对比2种不同表面材质培养瓶培养的人脐带间充质干细胞（human umbilical cord mesenchymal stem cells, hUC-MSCs）的培养特性。 方法 采集10条新鲜脐带，每条脐带用组织块法分离脐带组织并贴壁培养12d，收集原代细胞，分别取2*106个细胞用225cm2高粘培养瓶（简称A组）和225cm2普通瓶子（简称B组）细胞培养72h至P1代,收集P1代细胞继续培养72h至P2代，实验重复五次，观察比较两组实验组的P1和P2代培养瓶的细胞镜下形态、P2代比较两组实验组细胞消化时长、细胞扩增曲线、检测表面标志物、三系分化潜能，分别收集细胞培养至P4代比较SA?β?半乳糖苷酶染色阳性率。结果 2组细胞形态均为扁平长梭形。 P2代A组细胞消化时间为（123.8 ±3.09）s，B组细胞消化时间（38.5 ±2.20）s,差异有统计学意义(P<0.001)。A组的P0到P2代细胞扩增倍数为（129.49±0.89）倍，显著高于B组（101.4±1.67）倍，差异有统计学意义(P<0.001)。A组SA?β?半乳糖苷酶染色阳性率（2.58±0.44）%显著低于B组（4.79±0.33）%，且均符合间充质干细胞的质量标准。结论 相同接种密度的条件下，高粘培养瓶比普通培养瓶扩增倍数更高，且细胞衰老水平更低，优化了培养体系和培养效率，提高了细胞质量。

[Abstract] Objective: To compare the culture characteristics of human umbilical cord mesenchymal stem cells (hUC-MSCs) cultured in two different surface-material flasks. Methods: Ten fresh umbilical cords were collected, and umbilical cord tissue was isolated using the tissue block method and cultured adherently for 12 days. Primary cells were collected, and 2×10^6 cells were respectively seeded into 225 cm2 high-adhesion flasks (Group A) and 225 cm2 ordinary flasks (Group B) for culture for 72 hours until passage 1 (P1). P1 cells were collected and further cultured for 72 hours until passage 2 (P2). The experiment was repeated five times. The morphology of cells at P1 and P2 in both groups was observed under a microscope, and P2 cells were compared for digestion duration, cell growth curves, surface marker expression, and trilineage differentiation potential. Cells were also cultured to P4 to compare the SA?β-galactosidase positivity rate. Results: Cells in both groups displayed a flat spindle-shaped morphology. The digestion time of P2 cells in Group A was (123.8 ± 3.09) seconds, while in Group B it was (38.5 ± 2.20) seconds, showing a statistically significant difference (P<0.001). The cumulative cell amplification from P0 to P2 in Group A was (129.49 ± 0.89)-fold, significantly higher than that in Group B [(101.4 ± 1.67)-fold, P<0.001]. The SA?β-galactosidase positivity rate in Group A was (2.58 ± 0.44)%, significantly lower than in Group B(4.79 ± 0.33)%, and both met the quality standards for mesenchymal stem cells. Conclusion: Under the same seeding density, high-adhesion flasks yield higher cell amplification, lower cell senescence, and optimize the culture system and efficiency, thereby improving cell quality.

目的 探讨骨髓间充质干细胞源性微泡(BMSC-MV)修复大鼠早发性卵巢功能不全的自噬机制。方法 大鼠骨髓分离培养骨髓间充质干细胞;超速离心法从骨髓间充质干细胞培养液中分离微泡;腹腔注射顺铂溶液制备早发性卵巢功能不全(POI)模型,制备后3 d尾静脉取血ELISA检测血清雌二醇(E2)及卵泡刺激素(FSH);尾静脉注射BMSC-MV移植治疗POI大鼠模型,移植后28 d尾静脉取血ELISA检测E2、FSH及抗苗勒管激素(AMH),同时取卵巢组织检测自噬相关蛋白LC3及P62。结果 模型对照组及微泡移植组在模型制备后3 d的E2 含量低于正常对照组,FSH 含量高于正常对照组(P<0.001);微泡移植组在移植后28 d的E2、AMH含量高于模型对照组(P<0.001),FSH含量低于模型对照组(P<0.001);微泡移植组的LC3较模型对照组表达升高,而P62表达降低(P<0.001)。结论 BMSC-MV介导自噬修复大鼠早发性卵巢功能不全。

Objective To investigate the autophagy mechanism of bone marrow mesenchymal stem cell-derived microvesicle (BMSC-MV) in repairing premature ovarian dysfunction in rats. Methods The whole bone marrow adherence method was used to isolate,culture and identify BMSCs of SD rats. Microvesicles were isolated from bone marrow mesenchymal stem cell by ultracentrifugation. Premature ovarian insufficiency (POI) model was prepared by intraperitoneal injection of cisplatin solution,and serum estradiol (E2) and follicular stimulating hormone (FSH) were detected by ELISA from tail vein 3 days after preparation. Rat model of POI was treated with BMSC-MV transplantation by tail vein. Blood from tail vein was collected 28 days after transplantation to detect E2,FSH and AMH by ELISA. Meanwhile,ovarian tissues were collected to detect autophagy-related proteins LC3 and P62. Results The E2 content of the model control group and the microvesicle transplantation group was lower than that of the normal control group,and the FSH content was higher than that of the normal control group (P<0.001). The content of E2 and AMH in the microvesicle transplantation group at 28 days after transplantation was higher than that in the model control group (P<0.001),and the content of FSH was lower than that in the model control group (P<0.001). Compared with the model control group,LC3 expression in the microvesicle transplantation group was increased,while P62 expression was decreased (P<0.001). Conclusion BMSC -MV mediate autophagy to repair premature ovarian insufficiency in rats.

目的 采用生物信息学方法预测低氧预处理人胎盘绒毛膜间充质干细胞环状RNAs相对应的miRNA及其靶基因,并分析靶基因所参与的生物学过程和信号通路。方法 用Arraystar公司的商业软件为环状RNAs预测其相对应的miRNAs,分别用targetScan7.1和mirdbV5数据库预测miRNAs的靶基因,并取两个预测结果的合集,应用在线网站http://www.geneontology.org和http://www.genome.ad.jp/kegg对靶基因进行功能富集分析和信号通路富集分析。结果 功能富集分析表明,circRNAs的靶基因主要涉及到细胞发育、细胞分化和细胞发育调控。东京基因和基因组百科全书信号通路富集分析表明肿瘤中转录失控和有丝分裂原激活蛋白激酶(MAPK)信号通路最有意义,而且分析发现MAPK信号通路为核心通路。本研究表明,低氧预处理使得间充质干细胞中部分circRNAs的表达量发生差异性变化。结论 低氧预处理人胎盘绒毛膜间充质干细胞环状RNAs同低氧预处理间充质干细胞的生物学特性变化密切有关,为了解低氧预处理影响间充质干细胞特性发生变化的分子机制提供新思路。

Objective To predict the miRNA and its target genes of circular RNAs in hypoxia- preconditioned human palcenta chorionic mesenchymal stem cells using bioinformatics, and analyze the biological process and signaling pathway. Methods Arraystar's commercial software was used to predict the corresponding miRNAs of circular RNAs. The target genes of miRNAs were predicted by targetScan7.1 and mirdbV5 databases respectively, and an intersection of two prediction results was obtained. The online databases http://www. geneontology.org and http://www.genome.ad.jp/kegg performed functional enrichment analysis and signal pathway enrichment analysis of target genes. Results Functional enrichment analysis indicated that the target genes of circRNAs mainly involved cell development, cell differentiation and cell development regulation. The signal enrichment analysis of the Tokyo Gene and Genome Encyclopedia indicates that transcriptional misregulation in cancer and mitogen-activated protein kinase (MAPK) signaling pathway are most meaningful, and the MAPK signaling pathway is found to be the core pathway. This study showed that hypoxic preconditioning caused significant changes in the expression of mesenchymal stem cell circRNAs. Conclusion The changes of circular RNAs in hypoxia-preconditioned human placental chorionic mesenchymal stem cell is closely related to the biological characteristics of hypoxia-preconditioned mesenchymal stem cells. This study provides a new idea for understanding the molecular mechanism of hypoxic preconditioning affecting the changes of biological characteristics in mesenchymal stem cells.

目的 探索不同浓度苯妥英钠(PHT)对大鼠牙周膜干细胞(rat periodontal ligament stem cells, rPDLSCs)粘附于牙根面的影响,为PHT应用于牙周重建提供一定的实验依据,为牙周炎的治疗提供了新思路。方法 提取大鼠rPDLSCs,培养并纯化。通过多项诱导分化、表面标记物鉴定后,使细胞在不同浓度PHT刺激条件下,与牙骨质片共同培养,检测粘附于牙骨质片上的细胞量并作比较。结果 20～80 mg/L 浓度范围内的PHT能够促进rPDLSCs的粘附数量,40 mg/L PHT组促进粘附的效果最强。实验组与对照组有显著统计学差异。结论 适合浓度的PHT可以促进rPDLSCs粘附于牙骨质表面,40 mg/L PHT组促进粘附的效果最强。

Objective To investigate the effects of PHT on attachment of rat periodontal ligament stem cells (rPDLSCs) to cementum chips, in order to provide a certain experimental basis for periodontal regeneration and new ideas for therapy of periodontitis. Methods To isolate rPDLSCs from SD rats, culture and purify them. To identify the cells by their apperance, induced multi-direction differentiation potential and cell surface markers. The rPDLSCs were cultured with cementum chips under the action of different concentrations of PHT. Then testing and comparing the amount of cells attached on the cementum chips in different groups. Results The concentraion among 20～80 mg/L of PHT can increase the number of attached cells. 40 mg/L PHT can promote the cells attachment mostly. Conclusion A proper concentration of PHT may promote rPDLSCs to attach to cementum chips′ surface, 40 mg/L PHT may promote the cell attachment mostly.

目的 探索内源性神经干细胞在大鼠海马可溶性因子中的体外发育归宿及分化鉴定。方法 显微镜下分离Wistar大鼠海马组织放置于低温DMEM/12培养基,低温振荡2小时后高速离心(15000 g),获取实验所用海马组织可溶性因子。取材出生1天的Wistar乳鼠海马中的内源性神经干细胞(endogenous neural stem cells, ENSCs),将ENSCs分别于含海马可溶性因子终浓度为0(对照组)、50、100、200、400 μl/mL的无血清DMEM/F12培养基中培养6天并每日观察,使用免疫细胞化学、Western Blot印记技术比较各组ENSCs中Nestin、CD133的表达量;同时计量并比较各组ENSCs成球个数,以探索在模拟颅内微环境情况下,ENSCs发育、归宿及分化。进一步于最适宜的海马可溶性因子终浓度中分化神经球,对分化的细胞行神经元特异性蛋白入(如:β-tubullin III、MAP2)及胶质细胞特异性蛋白(如:GFAP、S100及p75 NGFR)免疫细胞化学检测。结果 大鼠ENSCs在培养基中呈单细胞漂浮生长,球形; ENSCs于海马可溶性因子各实验分组中培养第2天呈细胞球状态,对照组中无细胞球形成(与100 μl/mL组比较,P₁=0.00),100 μl/mL组与对照组比较有统计学意义(P₁=0.00<0.05);至第6天,在100 μl/mL组中的细胞球数量明显多于其余各组(P₁'=P₂'=P₃'=P₄'=0.00)。在免疫细胞化学检测中,100 μl/mL组中细胞球表达干细胞高亲和蛋白Nestin、CD133阳性,Western Blot免疫印迹检测其中Nestin、CD133蛋白高于对照组。进一步分化试验中,细胞球呈贴壁生长的单细胞状态、有突起伸出、长梭形,免疫细胞化学检测分化的细胞表达胶质细胞特异性蛋白GFAP、S100、p75NGFR阳性,但不表达神经元特异性蛋白β-tubullin III与MAP2。结论 大鼠ENSCs在终浓度为100 μl/mL的HSF作用下,可促进 ENSCs的增殖分裂;ENSCs在同样浓度下的HSF中可进一步分化为表达GFAP、S100、p75NGFR阳性的胶质样细胞;100 μl/mL的HSFS是ENSCs的一种生理性诱导剂或参与促进ENSCs增殖、分化及通过细胞替代或因子分泌等机制修复神经损伤。

Objective The aim of this study was to explore induction and differentiation of endogenous neural stem cells(ENSCs) in the hippocampus soluble factors(HSF) from the hippocampus of adult Wistar rats by mimicking an intracranial microenvironment. Methods After Wistar rats sacrificed, the hippocampus tissue was obtained in cold DMEM/F12. After centrigued and filtered, the HSF was stored at -20℃. The ENSCs was obtained from the hippocampus tissue of a neonate Wistar rat. Collected the tissue, digested and obtained the ENSCs. After we observed the morphology, the ENSCs were cultured in different concentration (0、50、100、200、400 μl/mL) of HSF for 6 days, and compared the expression of Nestin and CD133 by immunocytochemistry. Meanwhile,we compared the Nestin and CD133 protein by western blot. And then we explored the optimal concentration of HSF by the numbers of all groups on the second and sixth day. Furthermore, we did the differentiated experiment using the same concentration of HSF. Results The number of neurospheres in the 100 μg/mL group was significantly higher than those in the other groups on the 6th day. Immunofluorescence revealed that the neurospheres from ENSCs in the 100 μg/mL group more highly expressed nestin and CD133 than control. This result was confirmed by western blot analysis. The neurospheres can differentiate into glia-like cells in 100 μg/mL HSF and 1% FBS expressing GFAP, S100 and P75 NGFR by immunofluorescence. Conclusion These data indicated that HSF alone, mimicking a destination of ENSCs in vitro, could induce and differentiate neurospheres from ENSCs, as a new method to get NSCs and glia-like cells differentiated from ENCs to repair the diseases of center nervous system.