黄芪多糖对低氧环境中BMSCs成脂分化的影响

探讨黄芪多糖(APS)对低氧环境中骨髓间充质干细胞(BMSCs)成脂诱导分化的影响。采用四甲基偶氮唑盐(Methyl thiazolyl tetrazolium,MTT)法筛选促进BMSCs增殖的最佳APS浓度,干预不同氧浓度下(3%、6%、10%和20%)成脂诱导培养的BMSCs,通过油红O染色观察细胞内脂滴形成,Real-time PCR和Western blotting检测成脂相关基因过氧化物酶体增殖物激活受体γ_2(PPAR-γ_2)和脂蛋白脂肪酶(LPL)的mRNA和蛋白水平。结果表明,与对照组相比,40μg/mL APS能显著促进不同氧浓度下BMSCs的增殖(P0.05);含有40μg/mL APS的成脂诱导剂能提升低氧环境中BMSCs内脂滴含量及PPAR-γ_2和LPL的蛋白和mRNA水平,在氧浓度为10%时其促进作用较显著(P0.05),差异有统计学意义。40μg/mL APS具有促进低氧环境中BMSCs增殖和成脂诱导分化的作用,其促分化作用与细胞培养的氧环境相关。     

RUNX2对BMP9诱导的间充质干细胞C3H10T1/2成骨分化的影响

目的:研究和确认RUNX2在骨形态发生蛋白9(BMP9)诱导的间充质干细胞C3H10T1/2成骨分化中的作用。方法:通过Western blot、RT-PCR、荧光素酶活性分析检测BMP9对RUNX2表达的影响;分别在过表达RUNX2和RNA干扰抑制RUNX2表达的情况下,利用碱性磷酸酶(ALP)活性测定和染色、钙盐沉积实验,免疫细胞化学和裸鼠皮下异位成骨实验分析RUNX2对于BMP9诱导的间充质干细胞成骨分化的影响。结果:BMP9可以促进RUNX2的表达;RUNX2体外可促进BMP9诱导的C3H10T1/2的ALP活性和钙盐沉积,却抑制了OCN表达,RUNX2还可促进BMP9诱导的裸鼠皮下异位成骨;而在降低RUNX2表达后,BMP9诱导的C3H10T1/2细胞的ALP活性、钙盐沉积、OCN表达和裸鼠皮下异位成骨均受到抑制。结论:RUNX2可以促进BMP9诱导的间充质干细胞C3H10T1/2细胞成骨分化。     

机械敏感离子通道Trpm7在LIPUS促进牙髓间充质干细胞成骨分化中的作用机制的研究

该文主要研究低强度脉冲超声(low-intensity pulsed ultrasound,LIPUS)促进牙髓间充质干细胞(dental pulp mesenchymal stem cells,DPSCs)成骨分化,以及瞬时受体电位M7(transient receptor potential melastatin 7,Trpm7)在其中发挥的作用。培养人DPSCs,流式细胞术检测其表面分子标志表达,阿利新蓝、茜素红及油红O染色检测其成软骨、成骨和成脂分化能力。ALP活性、ALP染色和茜素红染色观察LIPUS促成骨分化的能力。实时定量PCR检测LIPUS处理组与对照组成骨分化相关基因OPN、OCN、RUNX2表达的差异以及不同时间点两组Trpm7 m RNA表达水平的变化。ALP活性检测LIPUS及不同浓度Trpm7抑制剂2-氨基乙酯二苯基硼酸(2-aminoethoxydiphenyl borate,2-APB)对成骨分化能力的影响。实验分为对照组、LIPUS组、LIPUS+二甲基亚砜(DMSO)组和LIPUS+2-APB组,ALP和茜素红染色观察各组成骨分化能力,Western blot检测各组OPN、OCN和RUNX2的蛋白表达。结果显示,成功培养DPSCs,LIPUS处理后ALP和茜素红阳性染色明显增多,ALP活性增强(P0.01);OPN、OCN、RUNX2的m RNA表达水平显著增加(P0.05),LIPUS处理第2天和第5天Trpm7的m RNA表达水平有明显升高(P0.05)。2-APB作用后明显下调ALP活性(P0.01)。LIPUS组、LIPUS+DMSO组与对照组相比,ALP和茜素红阳性染色以及OPN、OCN与RUNX2的蛋白表达均显著增加,而LIPUS+2-APB组较于LIPUS+DMSO组,ALP和茜素红染色以及OPN、OCN与RUNX2的蛋白表达明显降低。该研究结果提示,LIPUS能够促进DPSCs的成骨分化,且Trpm7在这一过程中发挥着重要作用。     

Ghrelin联合三氧化二砷对骨髓间充质干细胞增殖与成骨分化的影响

该文主要探究Ghrelin对三氧化二砷(As2O3)导致的骨髓间充质干细胞(BMSCs)增殖和成骨分化的影响。BMSCs设为对照组、As2O3组、Ghrelin组和联合(As2O3+Ghrelin)组。MTT法检测细胞增殖能力;成骨诱导的第7天和第14天,Real-time PCR及Western blot分别检测成骨相关因子OPN、ALP、RUNX2的mRNA及蛋白表达;第21天,茜素红染色分析钙盐沉积情况。结果显示,细胞增殖能力Ghrelin组>对照组>联合组>As2O3组。与对照组比,As2O3组各因子表达均显著下调(P<0.05),Ghrelin组第14天OPN蛋白表达无显著变化,其余因子均上调(P<0.05);联合组与As2O3组比,第14天OPN基因表达和第7天ALP蛋白表达无显著差异,其余均显著上调(P<0.05)。钙盐沉积:Ghrelin组>对照组>联合组>As2O3组。提示0.5μmol/L As2O3抑制BMSCs增殖和成骨分化,600 ng/mL Ghrelin增强细胞增殖和成骨分化;且Ghrelin能减弱As2O3导致的BMSCs增殖和成骨分化抑制作用。     

柚皮苷和木犀草素联合应用对大鼠BMSCs诱导成骨过程中Wnt/β-cantein通路相关基因表达的影响

摘要 目的：探讨柚皮苷和木犀草素联合应用对大鼠骨髓间充质干细胞（BMSCs）诱导成骨过程中Wnt/β-cantein通路相关基因表达的影响。方法：取大鼠股骨中提取的BMSCs,分别建立A组（空白组）、B组给予柚皮苷溶液10 μmol/L,C组给予木犀草素溶液5 μmol/L;D组给予骨碎补总黄酮溶液10 mg/mL;E组给予柚皮苷-木犀草素混合溶液配伍比为10 μmol/L：5 μmol/L,并诱导其向成骨细胞分化。应用碱性磷酸酶（ALP）染色及分光光度法检测第7 d各组细胞ALP活性。应用实时荧光定量聚合酶链反应（qRT-PCR）检测第7 d各组细胞Wnt/β-cantein通路相关基因及成骨基因的表达。结果：B组、C组、D组、E组细胞562 nm波长下光密度（OD）值显著高于A组,E组细胞562 nm波长下OD值最高,显著高于B组、C组、D组（P＜0.05）。B组、C组、D组、E组细胞ALP、骨钙素（OCN）、Runt相关转录因子2（RUNX2）基因表达水平显著高于A组,E组ALP、OCN、RUNX2基因表达水平最高,显著高于B组、C组、D组（P＜0.05）。B组、C组、D组、E组细胞β-catenin、Cyclin D1基因表达水平显著高于A组;B组、E组LEF-1基因表达水平显著高于A组;E组β-catenin、LEF-1、Cyclin D1基因表达水平最高,显著高于B组、C组、D组（P＜0.05）。结论：柚皮苷和木犀草素均具有促进大鼠BMSCs增殖和诱导其成骨向分化的作用,柚皮苷和木犀草素联合应用诱导大鼠BMSCs增成骨作用最强,其主要机制与Wnt/β-cantein通路激活有关。     

当归多糖减轻5-氟尿嘧啶对骨髓基质细胞成骨分化的抑制

该实验探讨当归多糖(ASP)对改善5-氟尿嘧啶(5-FU)所致人骨髓基质细胞成骨与成脂分化失衡的作用。人骨髓基质细胞株HS-5体外培养分为:对照组、ASP组、5-FU组、5-FU+ASP组和5-FU+LiCl组。CCK-8检测细胞抑制率,流式细胞术检测细胞凋亡;成骨与成脂诱导分化实验检测细胞成骨与成脂分化能力,Western blot检测Runx2、PPARγ和β-catenin蛋白表达,RT-PCR检测Runx2、OCN、BMP-2、Osterix、PPARγ和β-catenin mRNA表达。结果表明,与对照组相比5-FU作用HS-5细胞后细胞增殖抑制、凋亡率增加,成骨分化能力减弱、成脂分化能力增强,分化相关信号β-catenin蛋白和mRNA表达降低;相比5-FU组,ASP预处理可减少细胞凋亡;恢复细胞成骨分化能力,成骨相关因子Runx2、OCN、BMP-2和Osterix表达升高;降低细胞成脂分化能力,成脂相关因子PPARγ表达减少;β-catenin信号分子表达增加。结果提示,当归多糖可维持5-FU作用后骨髓基质细胞朝成骨方向分化的能力,其机制可能与激活Wnt/β-catenin信号通路有关。     

过表达miR-155抑制BMP9诱导间充质干细胞C3H10T1/2成骨分化

目的:研究过表达miR-155对BMP9诱导间充质干细胞C3H10T1/2成骨分化的影响。方法:(1)用重组腺病毒Ad-BMP9(BMP9)诱导C3H10T1/2细胞成骨分化,定量PCR(qPCR)检测miR-155的表达,RT-PCR检测Runx2和ALP的表达。(2)miR-155和BMP9共同处理C3H10T1/2细胞,qPCR检测miR-155的表达,ALP活性和染色检测早期成骨能力。(3)miR-155和BMP9共同处理C3H10T1/2细胞,诱导分化14d茜素红S染色检测晚期成骨能力。(4)miR-155和BMP9共同处理C3H10T1/2细胞,qPCR检测成骨分化相关基因Runx2、OSX、COL1A1、ALP、OCN和OPN的表达。(5)miR-155和BMP9共同处理C3H10T1/2细胞,Western blot检测p-Smad1/5/8、OCN和OPN蛋白水平的表达。(6)qPCR和Western blot分别检测HIF1α和VEGF的mRNA表达水平和蛋白质表达水平。(7)应用荧光素酶报告基因对miR-155的靶基因进行筛选和验证。结果:在BMP9诱导C3H10T1/2细胞成骨分化过程中,过表达miR-155降低ALP活性及染色;减少钙盐沉积;成骨分化相关基因Runx2、OSX、COL1A1、ALP、OCN和OPN表达降低;抑制p-Smad1/5/8、OCN和OPN蛋白水平的表达;HIF1α和VEGF的mRNA和蛋白表达水平减少。在对靶基因的检测中,过表达miR-155可以抑制HIF1α蛋白水平的表达,但对其mRNA水平无明显影响。结论:miR-155过表达减弱BMP9诱导间充质干细胞C3H10T1/2成骨分化,可能是通过抑制Smad/BMP信号通路发挥作用,也有可能是通过抑制靶基因HIF1α的表达来发挥作用。     

牙龈卟啉单胞菌感染通过Wnt通路调节牙周膜干细胞的成骨分化

目的观察牙龈卟啉单胞菌(P.gingivalis)感染通过Wnt通路调节牙周膜干细胞(PDLSCs)成骨分化的作用。方法培养原代PDLSCs,分为常规处理的对照组、P.gingivalis感染的P.gingivalis组和P.gingivalis感染并用Wnt3a处理的P.gingivalis+Wnt3a组,成骨诱导后茜素红染色并检测A_(405)值,Western blot检测Wnt通路分子的蛋白表达量,碱性磷酸酶(ALP)试剂盒检测ALP活力,PCR检测成骨标志基因Runt相关转录因子2(Runx2)、骨钙素(OCN)的mRNA表达量。结果与对照组比较,P.gingivalis组Wnt3a、β-catenin、p-GSK-3β的蛋白表达水平(0.33±0.07)、(0.27±0.08)、(0.44±0.09)以及成骨诱导后A_(405)值(0.55±0.08)、ALP活力(20.14±6.54)U/mL和Runx2、OCN的mRNA表达量(0.45±0.09)、(0.51±0.07)均明显减少;与P.gingivalis组比较,P.gingivalis+Wnt3a组成骨诱导后A_(405)值(0.89±0.15)、ALP活力(29.44±5.26)U/mL及Runx2、OCN的mRNA表达量(0.89±0.17)、(0.81±0.18)均明显增加。结论 P.gingivalis感染能够抑制PDLSCs的成骨分化,抑制Wnt通路是可能的分子机制。     

尿酸影响人骨髓间充质干细胞成骨分化过程中BMP-2 表达

目的:探讨在人骨髓间充质干细胞(h BMSCs)成骨分化过程中,不同浓度尿酸(UA)对骨形态形成蛋白-2(BMP-2)表达的影响。方法:以全骨髓贴壁培养法分离h BMSCs,将生长状态良好的第3代h BMSCs分为5组,分别为空白对照组(加入完全培养基)和成骨诱导组(加入成骨诱导液及含0 mmol/L、0.2 mmol/L、0.4 mmol/L、0.8 mmol/L尿酸的完全培养基)。连续干预诱导14d后,用倒置显微镜观察细胞形态的变化,通过观察茜素红染色情况及检测碱性磷酸酶(ALP)活性进行成骨情况的检测。RT-PCR技术检测各组细胞BMP-2 mR NA的表达情况。结果:第3代h BMSCs大多为形态单一的长梭形,呈旋涡状生长;干预诱导后的细胞逐渐变成不规则的立方形,局部形成团块状结节,以含尿酸浓度为0.8 mmol/L的成骨诱导培养基最为显著。连续干预14d后,空白对照组茜素红染色为阴性,而各成骨诱导组细胞茜素红染色结果为阳性,提示干预诱导后的细胞为成骨细胞。碱性磷酸酶活性随尿酸浓度的增加和干预时间的延长而增强(P<0.05)。RT-PCR检测结果显示,空白对照组无BMP-2 mR NA的表达。成骨诱导组随培养基中尿酸浓度的增加,BMP-2 mR NA表达逐渐增强,呈浓度依赖性(P<0.05)。结论:尿酸上调h BMSCs向成骨细胞分化过程中BMP-2 mR NA的表达。     

去卵巢骨质疏松山羊骨髓基质细胞成骨分化能力的研究

目的：研究在构建的去卵巢骨质疏松山羊动物模型中,骨髓基质细胞（MSCs）的生物学特性以及其成骨能力。方法：建立去卵巢骨质疏松山羊动物模型,使用全骨髓法获取去卵巢骨质疏松山羊（实验组）和正常山羊（对照组）MSCs,流式细胞仪检测实验组和对照组细胞周期及增殖指数（PI）;地塞米松诱导21d时油红O染色,观察成脂分化比例;成骨诱导液诱导14d,碱性磷酸酶（ALP）染色、检测ALP表达量。结果：对照组PI高于实验组;地塞米松诱导后实验组脂肪细胞比例明显高于对照组;成骨诱导第7d,对照组ALP的表达量明显高于实验组。结论：去卵巢骨质疏松山羊的MSCs增殖和成骨分化能力都降低,可能与骨质疏松症的发病机理有关。     

植物启动子的诱导模序

启动子是位于基因 5′端并负责该基因转录的DNA序列。诱导性启动子中有一些对伤害、真菌侵染、紫外线照射、激素等做出应答反应的顺式作用元件 ,被称为模序。已在植物启动子中鉴定出许多与诱导表达相关的模序 ,如伤害诱导模序 ,真菌诱导模序 ,植物激素诱导模序和光诱导模序等。这些模序作为启动子的顺式元件对各种诱导因子做出反应、调控基因的表达。     

Interaction of induced and uninduced cells during primary induction

Summary UsingTriturus pyrrhogaster embryos, the effects of uninduced cells on the differentiation of induced cells were investigated. The inducing stimulus was given to the presumptive ectoderm of early gastrulae by treatment with protein sooution from guinea pig bone-marrow. Mesodermal induction was evoked in the ectodermal explants. After the treatment, some of the ectodermal explants were cut into pieces 1/8 of their original size and combined with untreated presumptive ectoderm. Mesodermal tissues were differentiated in the combined explants too, but the mesodermal tissues evoked in these combined ectodermal explants were different in their regional characters from these in uncombined explants; dorsal structures, such as notochrod and muscle, were observed predominatly in the latter, whereas the dominant structures observed in the former were ventral ones, such as mesothelium and mesenchyme. The shifting of the regional characters in the combined explants was regarded as the result of an unknown effect from the uninduced cells.     

Efficient plant regeneration of garlic (Allium Sativum L.) by root-tip culture

Summary Root apices from in vitro cultured garlic (Allium sativum) cloves of cvs. ABEN and GT96-1 were used as axenic explants for organogenic callus production and plant regeneration experiments. Explants cultured in media based on those of Chu and co-workers (N6) or Murashige and Skoog (MS) could induce organogenic callus after 8 wk culture in darkness. Both media were supplemented with 2,4-dichlorophenoxyacetic acid (2.2–4.5 μM), alone or combined with 6-furfurylaminopurine (kinetin, 2.3–4.6 μM). Shoots started to grow 3 wk after culturing in the presence of light and the addition to culture media of 4.4 μM N⁶-benzyladenine. Plants capable of producing microbulbs regenerated 6 wk later. Up to 170 plants g⁻¹ FW callus were obtained when culturing was initiated in MS medium supplemented with 4.6 μM kinetin and 4.5 μM 2,4-dichlorophenoxyacetic acid.     

CnFL,a FLORICAULA/LEAFY homolog in Chrysanthemum nankingense is dramatically upregulated in induced shoot apical meristems

A FLORICOULA/LEAFY (FLO/LFY) homolog CnFL gene was isolated from the flower bud of a short-day Chrysanthemum nankingense plant during the flowering induction period. The sequence of CnFL contained a 1236 bp open reading frame that encoded a putative protein of 412 amino acids, which shared 68.67% homology with FLO and 60.23% homology with LFY. The spatial expression patterns of CnFL were analyzed using quantitative real-time PCR in different tissues and the apical meristem during short-day flowering induction. The results indicated that CnFL was highly expressed in the flower buds while its expression was also detected in the stems, young leaves, and vegetative apical meristem. During the period of flowering induction, CnFL expression increased remarkably and reached its highest levels after 15 days of induction. The expression of CnFL in the apical shoot after short-day flowering induction indicates that CnFL regulation is controlled primarily by photoperiodicity.     

The effect of heparin upon differentiation of ventral halves of frog gastrulae

Summary Culture of ventral halves of frog early gastrulae in heparin results in differentiation of dorsal tissues, e.g., brain, muscle, nephric tubules, and heparin stimulates the accumulation of labeled DNA and RNA.     

Callus induction and plantlet regeneration from mature cotyledonary segments of groundnut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

We evaluated the efficiency of callus induction and plantlet regeneration from mature cotyledonary segments of groundnut cultivars VRI-2 and VRI-3. Callus cultures were induced from mature tissues using NAA and IAA in combination with KIN or BAP. Maximum induction was recorded with 3.0 mg/L IAA and 1.0 mg/L BAP. However, green, compact, and nodular calli were obtained in 2.5 mg/L of IAA or NAA combined with 1.0 mg/L of either BAP or KIN. Fresh and dry weights were highly influenced by auxin concentration. Compact and nodular calli were then transferred to shoot induction media. The highest mean number of shoots was observed in 3.0 mg/L BAP plus 0.5 mg/L IAA. Finally, the resulting plantlets were rooted with IBA and NAA.     

Biochemical analysis of a blood meal-induced Aedes aegypti glutamine synthetase gene

Glutamine synthetase (GS) in the mosquito, Aedes aegypti, is induced in the midgut following a blood meal. Mosquito GS message is detected as soon as 1 h post-blood feeding and remains stable for 18 h. Using a PCR product encoding mosquito GS, a λgt10 adult female mosquito cDNA library was screened. A cDNA clone, pCl5A2, encoding the full translation product of mosquito GS was isolated and sequence analyses performed. Mosquito GS cDNA is 2.5 kb in length and its putative translation product shares all the conserved regions characteristic of the GS gene family, including the presumed ATP biding site. Glutamine synthetase activity in the mosquito midgut is highest at 18 h post-blood feeding. Activity can be detected over a broad pH range, from 6.0 to 7.5. Unlike other cellular GS enzymes, mosquito GS is not active in the presence of ATP. Very low dosages (0.05 mM) of L-methionine S-sulfoximine are sufficient to partially inhibit mosquito GS activity. Inhibition of GS disrupts the normal formation of the midgut peritrophic matrix, suggesting that GS enzyme might be involved in the initial pathway of chitin synthesis. The unique expression pattern and inducible nature of the mosquito GS gene make it an interesting candidate for studying promoter function. Additionally, the blood meal activation of the GS gene makes this a potentially valuable tool in mosquito transformation studies.     

Cross-induction of systemic induced resistance between an insect and a fungal pathogen in Austrian pine over a fertility gradient

Evidence for cross-induction of systemic resistance or susceptibility in plant–fungus–herbivore interactions is mostly derived from herbaceous model systems and not perennial woody plants. Furthermore, the effects of environmental variables such as soil fertility on these tripartite interactions are generally unknown. This study examined cross-induction of systemic resistance in Pinus nigra (Austrian pine) to infection by Sphaeropsis sapinea (a fungal pathogen), or feeding by Neodiprion sertifer (European pine sawfly), by prior induction with either S. sapinea or N. sertifer, over a fertility gradient. In a replicated 3-year study, cross-induction of systemic induced resistance (SIR) was found to be both asymmetric within a single year and variable between years. Prior induction with insect defoliation induced SIR to subsequent fungal challenge in 2006 but not in 2005. In 2005, a fertility-independent negative systemic effect of the fungal infection on herbivore growth was detected while herbivore survival was affected by a significant interaction between induction treatment and fertility level in 2006. Prior infection by the fungus induced SIR against the same fungus in both years regardless of fertility levels. This is the first report of whole-plant SIR against a defoliating insect induced by a fungal pathogen and vice versa, under variable nutrient availability, in a conifer or any other tree. Alieta Eyles and Rodrigo Chorbadjian contributed equally to the paper.     

Role of the FGF and MEK signaling pathway in the ascidian embryo

In the ascidian embryo, a fibroblast growth factor (FGF)-like signal from presumptive endoderm blastomeres between the 32-cell and early 64-cell stages induces the formation of notochord and mesenchyme cells. However, it has not been known whether endogenous FGF signaling is involved in the process. Here it is shown that 64-cell embryos exhibit a marked increase in endogenous extracellular signal-regulated kinase (ERK/MAPK) activity. The increase in ERK activity was reduced by treatment with an FGF receptor 1 inhibitor, SU5402, and a MEK (ERK kinase/MAPKK) inhibitor, U0126. Both drugs blocked the formation of notochord and mesenchyme when embryos were treated at the 32-cell stage, but not at the 2- or 110-cell stages. The dominant-negative form of Ras also suppressed notochord and mesenchyme formation. Both inhibitors suppressed induction by exogenous basic FGF. These results suggest that the FGF signaling cascade is indeed necessary for the formation of notochord and mesenchyme cells during ascidian embryogenesis. It is also shown that FGF signaling is required for formation of the secondary notochord, secondary muscle and neural tissues, and at least ERK activity is necessary for the formation of trunk lateral cells and posterior endoderm. Therefore, FGF and MEK signaling are required for the formation of various tissues in the ascidian embryo.     

The degrees of polymerization and N-acetylation of chitosan determine its ability to elicit callose formation in suspension cells and protoplasts of Catharanthus roseus

Partially and fully deacetylated chitosan fragments and oligomers were compared for their potency to elicit formation of the 1.3--glucan callose in suspension-cultured cells and protoplasts of Catharanthus roseus (line 385). Chitosan oligomers induced little callose formation, while callose synthesis increased with the degree of polymerization of chitosan up to several thousand corresponding to a molecular mass near 10⁶ Da. At a comparable degree of polymerization, partially N-acetylated chitosan fragments were less effective. Colloidal chitin and chitin oligomers induced only trace callose synthesis in protoplasts. These results indicate that the primary interaction involved the amino groups of chitosan and numerous negative charges at the surface of the plasma membrane with spacing in the nanometer range and occurring regularly over micrometer stretches. Charged phospholipid head-groups may fulfill these requirements. The resulting alteration of membrane fluidity may lead to the changes in ion transport known to be associated with the induction of callose formation.Abbreviations DP degree of polymerization - FDA fluorescein diacetate - PE pachyman equivalents