miR-26a修饰的人牙周膜干细胞成骨分化能力的研究

目的:研究人牙周膜干细胞(hPDLSCs)在转染miR-26a后成骨分化的促进效果。方法:从因正畸拔除的无龋坏、无牙周疾病离体牙的牙根中部牙周膜组织中分离、胶原酶消化,进行人牙周膜干细胞的体外培养。使用脂质体2000进行miRNA转染,采用激光共聚焦观察转染效果;MTT方法检测转染后的细胞活力;成骨诱导后使用实时定量PCR技术检测miRNA修饰的hPDLSCs成骨分化相关基因的表达;采用BCIP/NBT、天狼星红、茜素红S分别对碱性磷酸酶、胶原以及钙化进行染色观察。结果:采用脂质体2000能够成功转染hPDLSCs,且转染效率较高;转染后的细胞活力有所下降,但仍在80%以上;转染后的细胞在经成骨诱导分化过程中骨钙素(OCN)和骨桥蛋白(OPN)基因表达显著上调,同时碱性磷酸酶活性、胶原分泌以及钙化能力均得到显著提升。结论:miR-26a可以用于修饰hPDLSCs以提高其成骨分化能力。     

两种Wnt 通路抑制剂对牙周膜干细胞作用的比较研究

目的:本实验主要探讨Wnt通路抑制剂XAV-939相比DKK1在牙周膜干细胞增殖及矿化中的作用差异。方法:酶消化法培养牙周膜干细胞,鉴定后,用CCK8试剂盒检测XAV-939和DKK1对牙周膜干细胞增殖能力的影响,茜素红染色及定量检测XAV-939和DKK1对牙周膜干细胞成骨分化能力的影响,q RT-PCR检测DKK1和XAV-939对牙周膜干细胞Wnt通路相关基因GSK-3β和β-catenin及成骨分化相关基因ALP,DSPP,BSP,OCN,RUNX-2的影响。结果:XAV-939和DKK1都可以通过抑制Wnt通路来抑制牙周膜干细胞的增殖及成骨分化。当没有外源性Wnt蛋白刺激时,XAV-939作为Wnt通路抑制剂的抑制作用要强于DKK1,而加入外源性Wnt蛋白后,XAV-939与DKK1的作用效果相当。结论:XAV-939对比DKK1,具有更为广泛而稳定的抑制效果。XAV-939可以作为高效的Wnt通路抑制剂应用于未来关于牙周膜干细胞和Wnt通路相关实验研究中。     

补肾益气活血方及其单味药对人牙周膜干细胞增殖分化的影响

摘要 目的：探讨自拟补肾益气活血方及其单味药当归、补骨脂对体外培养的人牙周膜干细胞（hPDLSCs）增殖和相关成骨基因表达的影响。方法：分离培养得到hPDLSCs,选取第3代hPDLSCs,细胞增殖试剂盒（CCK-8）检测不同浓度（0 g/mL、1×10^-7 g/mL、1×10^-5 g/mL、1×10^-3 g/mL）补肾益气活血方和当归、补骨脂对hPDLSCs增殖的影响,并确定最佳作用浓度和最佳作用时间;通过定量聚合酶链式反应（qPCR）检测Runt相关转录因子2（Runx2）、碱性磷酸酶（ALP）、骨桥蛋白（OPN）、骨钙蛋白（OCN）相关成骨基因的表达水平,通过茜素红染色观察细胞成骨分化情况。结果：与0 g/mL相比,补肾益气活血方各浓度在第一天和第三天时均能显著促进细胞增殖（P<0.05）,且浓度为1×10^-5 g/mL在第三天、第五天时效果均最为显著（P<0.05）;当归同样在浓度为1×10^-5 g/mL、第三天及第五天时效果均最为显著（P<0.05）;补骨脂则仅在第一天时能显著促进细胞增殖（P<0.05）。与空白对照组比较,1×10^-5 g/mL的补肾益气活血方、当归、补骨脂均能显著提升成骨相关Runx2、OCN、OPN、ALP的表达（P<0.05）,且补肾益气活血方的上调效果最为显著。茜素红染色检测显示,补肾益气活血方可增加矿化结节,促进作用最为显著。结论：补肾益气活血方可促进hPDLSCs的增殖和骨向分化,在治疗慢性牙周炎中有望发挥更大作用。     

淫羊藿苷对体外牙周膜细胞增殖、成骨分化以及成骨和破骨因子表达的影响

目的:明确淫羊藿苷对于体外分离培养的人牙周膜细胞的增殖、成骨分化以及成骨和破骨因子表达的影响。方法:原代分离培养人牙周膜细胞,实验组细胞加入0.01 mg/L淫羊藿苷溶液。采用MTT法检测牙周膜细胞的增殖速率,采用碱性磷酸酶活性检测试剂盒评估牙周膜细胞的成骨分化能力,分别通过real-time PCR和Western印迹检测成骨相关的OCN、Runx2、BMP2以及破骨相关的RANKL的基因和蛋白表达。结果:MTT结果揭示,淫羊藿苷显著提高了人牙周膜细胞的增殖速率(P0.05),也显著促进了碱性磷酸酶活性的提升(P0.05),揭示其对于牙周膜细胞的成骨分化具有促进作用;real-time PCR和Western印迹结果均表明,淫羊藿苷能够上调OCN、Runx2和BMP2的基因和蛋白表达(P0.05),并下调RANKL的基因和蛋白表达(P0.05)。结论:淫羊藿苷能够促进体外牙周膜细胞的增殖、成骨分化潜能和成骨功能活性。本研究有望为牙周炎治疗以及基于组织工程技术的牙周组织的损伤修复提供新思路。     

在海藻酸钠凝胶上诱导骨髓间充质干细胞分化为成骨细胞

通过在海藻酸钠凝胶上诱导bMSCs向成骨细胞分化,探讨其对骨髓间充质干细胞（bone mesenchymal stem cells, bMSCs）的生物学效应。采用MTT、甲苯胺蓝染色、von Kossa染色和RT-PCR分别检测细胞的增殖、生长形态、诱导后细胞的钙化结节和成骨相关基因的表达。实验组bMSCs生长状况良好、细胞增殖迅速,与对照组的增殖无差异;bMSCs成集落样生长明显,集落中央细胞重叠生长形成钙化结节;培养至12d,实验组和对照组的成骨相关基因,包括碱性磷酸酶、I型胶原和骨钙素,均为阳性表达,但实验组的表达量高于对照组。海藻酸钠凝胶能够促进bMSCs向成骨细胞的分化,是良好的骨组织工程支架材料。     

丹酚酸B促进胎鼠大脑皮质神经干细胞增殖、突起生长和分化

丹参是一种常用于治疗脑卒中的传统中草药,丹酚酸B是其有效成份之一.但人们对丹酚酸B如何影响神经干细胞的生长还了解甚少.本研究用MTT、流式细胞术、免疫荧光和RT-PCR技术检测丹酚酸B对胎鼠大脑皮质神经干细胞增殖、突起生长和分化的作用.结果显示,20和40μg/mL丹酚酸B对促进神经干细胞增殖的作用相似,适量的丹酚酸B可促进神经干细胞增殖并形成较多的神经干细胞球,其促增殖作用通过提高G2/S期细胞的数量而实现.丹酚酸B还可促进神经干细胞球发出较多的突起并分化出较多的成熟神经元.分化开始时,tau,GFAP和nestin mRNA均有表达.但分化后的细胞与对照组比较,tau mRNA表达增多,而GFAP mRNA表达减少.说明外源性的丹酚酸B具有促进神经干细胞增殖并向神经元分化的作用,有望成为一种获取更多神经干细胞和促进神经干细胞分化的药物.     

牙源性干细胞复合微渠多孔羟基磷灰石支架成骨性能研究*

目的: 探讨牙源性干细胞复合微渠多孔羟基磷灰石支架(grooved porous hydroxyapatite scaffolds, HAG支架)的成骨性能,为骨缺损修复治疗提供新手段。方法: 从健康成人第三磨牙中提取牙周膜干细胞(periodontal ligament stem cells, PDLSCs)及牙髓干细胞(dental pulp stem cells, DPSCs)分别接种于HAG支架上,进行多向分化鉴定及碱性磷酸酶(alkaline phosphatase,ALP)活性测定;并通过CCK-8检测细胞增殖能力;逆转录聚合酶链反应(qRT-PCR)检测骨形态发生蛋白2(bone morphogenetic protein 2, BMP-2)、骨钙素(osteocalcin, OCN)和骨桥蛋白(osteopontin, OPN)等成骨相关基因的表达。体内研究中将搭载PDLSCs和DPSCs的HAG支架移植到裸鼠的背部皮下,8周后取材,组织切片后采用苏木精-伊红(HE)染色观察新骨形成,提取组织蛋白采用Western blot检测ALP、OCN等成骨相关蛋白的表达。结果: 体外研究中DPSCs复合HAG支架组的细胞增殖能力、ALP活性,以及成骨相关基因ALP、BMP2、OCN等的表达均高于PDLSCs复合HAG支架组。体内研究中HE染色显示,PDLSCs复合HAG支架组及DPSCs复合HAG支架组均较空白HAG支架组有更多细胞生长区、纤维细胞增生及骨基质形成,且DPSCs复合HAG支架组的骨基质面积更大,成纤维细胞数量更多;PDLSCs复合HAG支架组及DPSCs复合HAG支架组成骨相关蛋白的表达量均高于空白HAG组,且DPSCs复合HAG支架组中ALP蛋白表达量显著高于PDLSCs复合HAG支架组。结论: PDLSCs、DPSCs复合HAG支架在体内外均表现出良好的成骨性能,其中DPSCs复合HAG支架的成骨性能更为优异。     

骨髓间充质干细胞分化为心肌细胞过程中Notch表达的研究

目的研究骨髓间充质干细胞分化为心肌细胞过程中Notch表达的研究。方法用密度梯度离心法分离培养犬骨髓间充质干细胞,按照酶法及差速贴壁法分离培养心肌细胞。观察干细胞增殖及传代情况。单独培养的干细胞为对照组,实验组将骨髓间充质干细胞与心肌细胞共培养,用RT-PCR、免疫细胞化学、MTT等方法检测干细胞分化为心肌细胞的情况,及干细胞在增殖与分化为心肌细胞过程中Notch信号系统的表达情况。结果骨髓间充质干细胞呈梭形、旋涡样生长,增殖及传代能力强,并可诱导分化为心肌样细胞,免疫荧光示心肌细胞标志物的表达。RT-PCR及免疫细胞化学显示实验组有Notch信号通路受体及配体的表达,而对照组表达微弱。结论骨髓间充质干细胞在增殖及分化过程中存在Notch信号通路,在干细胞分化为心肌细胞过程中Notch信号系统的表达上调。     

人参皂甙Rg1对体外人胃癌细胞增殖的抑制作用及机制

本研究用不同浓度人参皂甙Rg1作用人胃癌BGC-823细胞24 h、48 h和72 h,采用MTT法、流式细胞术及半定量RT-PCR检测GS-Rg1对胃癌细胞的增殖抑制作用、细胞周期分布时相和p16~(INK4a)、p21~(WAF1)表达水平的影响,以探讨人参皂甙Rg1对人胃癌BGC-823细胞增殖的抑制作用及机制。结果表明,随着作用时间和浓度的增加,人参皂甙Rg1对胃癌细胞增殖抑制作用逐渐增强(P<0.05),G_0/G_1期细胞比例增加,G_2/S期细胞比例下降,p16~(INK4a)、p21~(WAF1)基因水平上调。上述结果提示人参皂甙Rg1能抑制体外培养的胃癌BGC-823细胞增殖,其机制可能与上调肿瘤细胞内细胞周期蛋白依赖激酶抑制因子p16~(INK4a)及p21~(WAF1)mRNA的表达有关。     

人嗅黏膜神经干细胞体外培养及氟西汀对其增殖和神经发生的影响

人嗅黏膜神经干细胞是新近发现的可用于神经精神疾病研究的良好材料,同时,抗抑郁治疗促进脑部神经干细胞增殖及向神经细胞转化。然而,嗅黏膜神经干细胞体外培养方法及抗抑郁药对其增殖及神经发生的影响尚不清楚。该研究采用组织块培养获取嗅细胞,神经球培养法纯化神经干细胞,Nestin免疫荧光验证神经干细胞特异性。加入促神经分化培养基诱导神经干细胞向神经细胞分化,MAP2(microtubule associated protein 2)、TUJ1(β3-tubulin)免疫荧光验证神经细胞特异性。采用CCK-8和蛋白质分子印记技术分别检测抗抑郁药对嗅黏膜神经干细胞增殖及分化的影响。结果显示,体外培养嗅黏膜神经干细胞呈球状聚集;Nestin、MAP2、TUJ1免疫荧光阳性;抗抑郁药促进嗅黏膜神经干细胞增殖、促进其向神经细胞转化,TUJ1表达上升。综上所述,抗郁药能够促进神经干细胞增殖及神经发生,可能为其抗抑郁治疗的机制之一。     

Oestrogen retains human periodontal ligament stem cells stemness in long‐term culture

Objectives

During long‐term culture, loss of stemness is observed which greatly restricts the application of human periodontal ligament stem cells (hPDLSCs) in tissue regeneration. Oestrogen (E2) was found to significantly enhance the proliferation and osteogenic differentiation capacity in mesenchymal stem cells. Therefore, in this study, we investigated effects of E2 on hPDLSCs stemness in long‐term culture.

Materials and methods

Effects of E2 on hPDLSCs stemness were systematically evaluated. To characterize underlying the mechanisms, its effects on PI3K/AKT signalling pathway were determined.

Results

Our results showed that E2 was able to enhance the proliferation, modify cell cycle, up‐regulate stemness‐related genes expression, promote osteogenic differentiation and elevate the positive rate of CD146 and STRO‐1 over 10 passages in hPDLSCs. Importantly, PI3K/AKT signing pathway might play a role in these effects.

Conclusions

These findings suggest that E2 retains hPDLSCs stemness in long‐term culture, which might enhance its application in tissue engineering.     

Ginsenoside Rg1 facilitates neural differentiation of mouse embryonic stem cells via GR-dependent signaling pathway

Ginsenoside Rg1, a steroidal saponin of high abundance in ginseng, possesses the neuroprotective effects. In this study, we tried to explore the effect of Rg1 on promoting differentiation of mouse embryonic stem (ES) cells towards the neuronal lineage and its potential role involved in glucocorticoid receptor (GR) activation. Rg1 treatment induced a remarkable increase in the population of neuron-like cells in a time-dependent manner. More than 80% of Rg1-treated embryoid bodies (EBs) differentiated into neuron-like cells on d 8 + 10. Furthermore, the gradually increased protein expression of neurofilament (NEFM) and β-tubulin III (a neuronal specific protein) was determined. GR expression gradually increased during the differentiation course. RU486, an antagonist of GR, could efficiently block the neurogenesis-promoting activity of Rg1. On the other side, Rg1 stimulated the phosphorylation of ERK1/2 and Akt at different time points through GR activation-dependent mechanisms. Treatment of both U0126 (an inhibitor of MEK) and LY294002 (an inhibitor of PI3 K), hampered the neuronal differentiation induced by Rg1. Meantime, U0126 further decreased Rg1-induced p-Akt expression. In conclusion, Rg1 possesses the effects on inducing differentiation of mouse ES cells into neurons in vitro via the GR-MEK-ERK1/2-PI3 K-Akt signaling pathway.     

Protective effect of ginsenoside Rg3 on lung injury in diabetic rats

Ginsenoside has been used to treat diabetes, while ginsenoside Rg3 is the main active ingredient component of ginseng and is used to study its effects on lung tissue damage in diabetic rats. In this paper, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry were applied to detect the proliferation and apoptosis of BEAS-2B cells treated with different concentrations of Rg3. The inflammatory response and pathological change in the lung tissue of diabetic rats treated with Rg3 were evaluated by enzyme-linked immunosorbent assay, quantative real-time polymerase chain reaction, and hematoxylin and eosin staining immunohistochemistry. Meanwhile, PI3K and MAPK signaling pathway proteins in lung tissue were determined by Western blot analysis. The results showed that ginsenoside Rg3 had no significant influence on the proliferation and apoptosis of BEAS-2B cells. Ginsenoside Rg3 can inhibit inflammatory response and promote the activation of PI3K and MAPK signaling pathways to prevent damages of lung tissues induced by hyperglycemia. The protective effect provided by ginsenoside Rg3 indicates that ginsenoside Rg3 is a potential drug for the treatment of diabetes.     

A feasibility study of an in vitro differentiation potential toward insulin-producing cells by dental tissue-derived mesenchymal stem cells

Dental tissue-derived mesenchymal stem cells have been proposed as an alternative source for mesenchymal stem cells. Here, we investigated the differentiation ability toward insulin producing cells (IPCs) of human dental pulp stem cells (hDPSCs) and human periodontal ligament stem cells (hPDLSCs). These cells expressed mesenchymal stem cell surface markers and were able to differentiate toward osteogenic and adipogenic lineages. Upon 3 step-IPCs induction, hDPSCs exhibited more colony number than hPDLSCs. The mRNA upregulation of pancreatic endoderm/islet markers was noted. However, the significant increase was noted only for PDX-1, NGN-3, and INSULIN mRNA expression of hDPSCs. The hDPSCs-derived IPCs expressed PRO-INSULIN and released C-PEPTIDE upon glucose stimulation in dose-dependent manner. After IPCs induction, the Notch target, HES-1 and HEY-1, mRNA expression was markedly noted. Notch inhibition during the last induction step or throughout the protocol disturbed the ability of C-PEPTIDE release upon glucose stimulation. The results suggested that hDPSCs had better differentiation potential toward IPCs than hPDLSCs. In addition, the Notch signalling might involve in the differentiation regulation of hDPSCs into IPCs.     

人参皂苷Rb1、Rg1、Re对白血病细胞株KG1α增殖的影响

目的:探讨人参皂苷Rb1、Rg1、Re对急性髓系白血病细胞株(KG1α)增殖的影响.方法:取对数生长期KG1α细胞,分设人参皂苷Rb1、Rg1、Re组和常规培养组,以MTT比色法检测作用24h、48h、72h时对KG1α细胞增殖抑制作用,并计算Rb1的IC_(50)值,以此浓度为工作浓度,设常规培养组和处理组,台盼蓝计数法观察对KG1α细胞增殖的影响;流式细胞术测定细胞周期分布的变化.结果:MTT、台盼蓝计数显示人参皂苷单体Rb1、Rg1可抑制KG1α细胞增殖,呈浓度依赖性,以Rb1抑制效应最佳,于作用48h抑制率最高.台盼蓝计数显示人参皂苷单体Rb1-120μmol/L作用48h时抑制率达50.22%;流式细胞术结果提示,与对照组比较,Rb1-120μmol/L组G_2/M期KG1α细胞比例增加(P<0.05).结论:Rb1可抑制KG1α细胞体外增殖,其增殖抑制作用与将KG1α细胞阻滞于G_2/M期有关.     

肌间隙移植明胶海绵获取成体干细胞

通过移植生物材料到小鼠体内而建立一种新的干细胞获取方法.移植明胶海绵到小鼠后肢的大腿内侧肌间隙,12天后分离明胶海绵中的迁移细胞.海绵来源干细胞形态、增殖能力、多向分化能力与骨髓源干细胞相似,但海绵中成纤维细胞集落形成单位的比例(82.2±10.6/105)远远高于骨髓中的比例(43.7±7.4/106)(P＜0.05...