基因壳聚糖纳米粒表面修饰和转染研究

目的：研究递送基因纳米粒表面修饰对体外基因转染的影响。方法：利用末端活化的聚乙二醇(PEG)制备PEG化基因壳聚糖纳米粒;通过两端活化的PEG将糖蛋白配基连接到纳米粒表面,完成肝靶向纳米粒的制备;用透射电镜观察表面修饰对纳米粒粒径大小、粒子形态的影响;使用蛋白质测定试剂盒测算纳米粒表面蛋白连接量;利用体外转染实验考察表面修饰对纳米粒转染活性的影响;用倒置荧光显微镜观察并用流式细胞仪测定转染结果。结果：纳米粒PEG化使转染效率大幅度升高,半乳糖基牛血清白蛋白(Galn—BSA)使体系的转染效率比PEG化纳米粒略有下降,但比不经修饰的纳米粒转染活性高。壳聚糖纳米粒的表面PEG化能提高纳米粒的体外稳定性,从而提高体外转染效率,并适合于进行冷冻干燥。结论：长循环壳聚糖基因递送纳米粒在基因治疗研究中可能会发挥重要作用。     

基因壳聚糖纳米粒表面修饰和转染研究

目的:研究递送基因纳米粒表面修饰对体外基因转染的影响.方法:利用末端活化的聚乙二醇(PEG)制备PEG化基因壳聚糖纳米粒;通过两端活化的PEG将糖蛋白配基连接到纳米粒表面,完成肝靶向纳米粒的制备;用透射电镜观察表面修饰对纳米粒粒径大小、粒子形态的影响;使用蛋白质测定试剂盒测算纳米粒表面蛋白连接量;利用体外转染实验考察表面修饰对纳米粒转染活性的影响;用倒置荧光显微镜观察并用流式细胞仪测定转染结果.结果:纳米粒PEG化使转染效率大幅度升高,半乳糖基牛血清白蛋白(Galn-BSA)使体系的转染效率比PEG化纳米粒略有下降,但比不经修饰的纳米粒转染活性高.壳聚糖纳米粒的表面PEG化能提高纳米粒的体外稳定性,从而提高体外转染效率,并适合于进行冷冻干燥.结论:长循环壳聚糖基因递送纳米粒在基因治疗研究中可能会发挥重要作用.     

壳聚糖纳米粒用作基因递送载体的初步研究

目的初步研究基因壳聚糖纳米粒的性质和转染活性。方法用复凝聚法制备纳米粒;用透射电镜观察形态;用纳米粒度分析仪测定粒径、多分散度和zeta电位;用荧光分光光度法测定基因包封率;用凝胶阻滞分析和荧光扫描测定基因在纳米粒中的位置;用体外基因转染实验定性评价纳米粒的转染活性。结果纳米粒形态多呈球形,平均粒径为218.9 nm,多分散度为0.276,zeta电位为+21.2 mV;基因包封率为99.6%;凝胶阻滞分析和荧光扫描表明基因几乎全部被包裹在纳米粒内部,表面吸附很少;体外基因转染实验表明基因壳聚糖纳米粒能够转染人胚胎肾细胞(HEK293)和肝癌细胞(HepG2),基因能够在这两种细胞中表达。结论壳聚糖纳米粒能将基因递送到细胞内并且基因能够表达,因此可以用作基因药物载体。     

慢病毒载体介导的bFGF基因转染兔BMSCs的实验研究

摘要： 目的 观察在体外培养条件下, 慢病毒载体介导的碱性成纤维细胞生长因子 （bFGF） 基因转染对兔骨髓基质干细胞 （BMSCs） 生物学特性的影响。方法 采用密度梯度离心法及贴壁筛选法获取 BMSCs; 利用慢病毒载体将 bFGF 基因转染到 BMSCs 中, 根据转染条件分为 bFGF 转染组、 空病毒组、 未转染组, 转染后分别对 3 组细胞的形态 bFGF mRNA 及蛋白表达、 细胞增殖情况、 细胞周期及碱性磷酸酶 （ALP） 活性进行观察。结果 采用密度梯度离心法和贴壁筛选法, 成功获得高纯度的 BMSCs。载有 bFGF 基因的慢病毒转染 BMSCs 后, 细胞形态无明显变化, 而 bFGF mRNA 与蛋白表达均明显增强、 细胞增殖曲线上移、 增殖期细胞比例增大、 ALP 活性明显增高, 与空病毒组及未转染组比较, 差异均有统计学意义 （P＜0.05)。结论 利用慢病毒载体将兔 bFGF 基因导入体外培养的 BMSCs, 目的基因获得稳定表达, 并且自身过表达的bFGF 可以促进BMSCs 的增殖与成骨分化。     

转化生长因子β对成骨细胞增殖、BMP-2及Cbfal基因表达的影响

目的：通过转化生长因子β（TGF-β）对体外成骨细胞增殖、骨形态蛋白-2（BMP-2）及核心结合因子1（Cbfal）基因表达的影响,探讨TGF-β对骨代谢影响的可能机制。方法：不同浓度的TGF（0、0.01、100ng/ml）刺激体外培养的大鼠成骨细胞,采用噻唑蓝（MTT）法检测细胞增殖能力,用半定量RT-PCR法检测成骨细胞BMP-2、Cbfal基因的表达。结果：TGF-β可明显促进成骨细胞增殖（P〈0.05）,并促进成骨细胞BMP-2、Cbfal基因的表达（P〈0.01）,具有浓度依赖性。结论：TGF-β可促进成骨细胞的增殖、分化及成熟,可能是通过增强BMP-2、Cbfal基因的表达来实现的。     

中心组合设计法优化载基因壳聚糖纳米粒的最佳转染制备区域

目的：采用中心组合设计法优化载基因壳聚糖纳米粒的最佳转染制备区域。方法采用复凝聚法制备载质粒基因的壳聚糖纳米粒,选择壳聚糖浓度和质粒基因浓度作为实验考察因素,应用两因素五水平中心组合设计优化最佳转染制备区域,优化指标选择平均粒径和基因转染率。通过透射电镜观察纳米粒的形态;通过动态光散射和电泳光散射技术分别测量纳米粒的粒径和Zeta电位;通过凝胶电泳分析考察质粒在纳米粒制备过程中的稳定性;通过倒置荧光显微镜观察质粒基因在细胞内的表达;通过流式细胞技术测定纳米粒的转染效率。结果成功优化了载基因壳聚糖纳米粒的最佳转染制备区域。优选条件下制备的纳米粒大多呈球形,纳米粒平均粒径为217．6 nm,粒径多分散系数为0．241,表明粒径分布较窄。纳米粒zeta电位为＋22．4 mV,表明纳米粒表面带有正电荷,可以增加纳米粒混悬液的稳定性。凝胶电泳分析结果表明质粒基因在纳米粒制备过程中没有遭到破坏。纳米粒的细胞转染效率比较高,能够高效地将绿色荧光蛋白质粒基因递送到细胞内,并且基因表达产生绿色荧光蛋白。结论本研究建立的数学模型具有良好的预测性。在优化的制备区域内制备的载基因壳聚糖纳米粒的转染性能比较理想。     

载碱性成纤维细胞生长因子质粒纳米粒的制备及体外转染活性研究

目的:制备壳聚糖载bFGF基因纳米粒,并对其体外性质及转染成纤维细胞效率进行考察。方法:以复凝集法制备载绿色荧光蛋白(EGFP)与人碱性成纤维细胞生长因子(bFGF)融合蛋白EGFP-bFGF质粒(pEGFP/bFGF)的壳聚糖纳米粒CS-pEGFP/bFGF,透射电镜、纳米粒度电位仪测定纳米粒形态、粒径和表面电位,凝胶阻滞实验分析质粒与壳聚糖结合情况,细胞增殖实验考察载bFGF质粒转染后对细胞增殖的影响,荧光分光光度计及荧光显微镜观测纳米粒转染成纤维细胞后细胞对EGFP的表达情况。结果:壳聚糖纳米粒可将质粒pEGFP/bFGF成功转入成纤维细胞中并能有效降低转染产生的细胞毒性,细胞自分泌的bFGF能促进成纤维细胞的增殖。结论:载bFGF纳米粒具有提高转染效率、降低毒性及促进成纤维细胞增殖的作用。     

高糖环境中吡格列酮对MC3T3-E1成骨细胞的作用及其机制

目的观察高糖环境下吡格列酮对MC3T3-E1成骨细胞的作用并探讨其可能机制。方法高糖(22.5 mmol·L~(-1))环境培养MC3T3-E1细胞,分为对照组,吡格列酮2.5、5、10μmol·L~(-1)组,干预24、48 h。检测细胞增殖活性、凋亡率、骨钙素和碱性磷酸酶(ALP)分泌水平,以及过氧化物酶体增殖物激活受体γ(PPARγ)、成骨因子runt相关基因2(Runx2)和骨形态蛋白2(BMP-2)mRNA的表达水平。并分析PPARγ、Runx2的表达与骨钙素、ALP、BMP-2的相关性。结果在相同干预时限,吡格列酮各组MC3T3-E1成骨细胞增殖活性、骨钙素和ALP的分泌水平、Runx2 mRNA和BMP-2 mRNA的表达均低于对照组,凋亡率和PPARγmRNA的表达高于对照组(P<0.05)。随吡格列酮浓度增加,细胞增殖活性、骨钙素和ALP的分泌、Runx2 mRNA和BMP-2 mRNA的表达均降低,而细胞凋亡率和PPARγmRNA的表达增高(P<0.05)。与干预24 h相比,干预48 h时相同浓度吡格列酮组细胞增殖活性,骨钙素和ALP的分泌水平,PPARγ、Runx2、BMP-2 mRNA的表达或无变化或略增加。结论高糖环境下吡格列酮对成骨细胞有损害作用,促进PPARγ表达、抑制Runx2的表达可能为其作用机制之一。     

Smad6信号干扰对MSCs骨向分化的影响

目的研究Smad6信号干扰对骨形态发生蛋白2（BMP-2）诱导的骨髓间充质干细胞（MSCs）骨向分化的促进效应。方法培养小鼠MSCs,用BMP-2诱导骨向分化。细胞分为3组：A组细胞用携带绿色荧光蛋白（GFP）的Smad6重组RNA干扰载体转染;B组细胞用空白载体转染;C组细胞作为对照。结果病毒转染后GFP在MSCs中有效表达,病毒转染效率达98．5％。与B组比较,Smad6RNA干扰显著提高了A组细胞AIP活性和骨钙素水平（P〈0．01）,而C组ALP活性和骨钙素水平均显著低于其他2组（P〈0．01）。茜素红染色显示,A组矿化结节数目显著多于B组（P〈0．05）,而c组无矿化结节形成。结论Smad6RNA干扰可有效促进BMP-2诱导的MSCs骨向分化,该研究为骨组织工程中骨缺损修复提供了一个极具价值的手段。     

Bone morphogenetic protein-2 and -4 (BMP-2 and -4) mediates fraxetin-induced maturation and differentiation in human osteoblast-like cell lines

Fraxetin (7,8-dihydroxy-6-methoxy coumarin), a coumarin derivative, was investigated for its effects on differentiation of osteoblasts. By means of alkaline phosphatase (ALP) activity and osteocalcin ELISA assay, we have shown that fraxetin exhibits a significant induction of differentiation in two human osteoblast-like cell lines, MG-63 and hFOB. Alkaline phosphatase and osteocalcin are phenotypic markers for early-stage differentiated osteoblasts and terminally differentiated osteoblasts, respectively. Our results indicated that fraxetin stimulated osteoblast differentiation at various stages (from osteoprogenitors to terminally differentiated osteoblasts). Induction of differentiation by fraxetin was associated with increased bone morphogenetic protein-2 (BMP-2) and BMP-4 productions. Addition of purified BMP-2 and BMP-4 proteins did not increase the upregulation of ALP activity and osteocalcin secretion by fraxetin, whereas the BMPs antagonist noggin blocked both fraxetin and BMP-2 and BMP-4 mediated ALP activity and osteocalcin secretion enhancement, indicating that BMP-2 and BMP-4 productions are required in fraxetin-mediated osteoblast maturation and differentiation. These findings are novel and may be important in the treatment and prevention of osteoporosis.     

Magnetic nanoparticles modified-porous scaffolds for bone regeneration and photothermal therapy against tumors

For effectively treating tumor related-bone defects, design and fabrication of multifunctional biomaterials still remain a great challenge. Herein, we firstly fabricated magnetic SrFe₁₂O₁₉ nanoparticles modified-mesoporous bioglass (BG)/chitosan (CS) porous scaffold (MBCS) with excellent bone regeneration and antitumor function. The as-produced magnetic field from MBCS promoted the expression levels of osteogenic-related genes (OCN, COL1, Runx2 and ALP) and the new bone regeneration by activated BMP-2/Smad/Runx2 pathway. Moreover, the SrFe₁₂O₁₉ nanoparticles in MBCS improved the photothermal conversion property. Under the irradiation of near-infrared (NIR) laser, the elevated temperatures of tumors co-cultured with MBCS triggered tumor apoptosis and ablation. As compared with the pure scaffold group, MBCS/NIR group possessed the excellent antitumor efficacy against osteosarcoma via the hyperthermia ablation. Therefore, the multifunctional MBCS with excellent bone regeneration and photothermal therapy functions has a great application for treating the tumor-related bone defects.     

Comparison of ganglioside expression between human adipose- and dental pulp-derived stem cell differentiation into osteoblasts

Human adipose-derived stem cells (hADSCs) and dental pulp-derived stem cells (hDPSCs) have been considered alternative sources of adult stem cells because of their potential to trans-differentiate into multiple cell lineages. This study investigated the possible role of gangliosides in the osteoblast differentiation of hADSCs and hDPSCs. First, we investigated characterization of hADSCs and hDPSCs using FACS analysis. Mesenchymal stem cell specific markers, CD44 and CD105, were expressed but not hematopoetic markers, CD45 and CD117 in both of hADSCs and hDPSCs. High-performance thin-layer chromatography analysis showed that increased gangliosides were associated with differentiation of hADSCs and hDPSCs into osteoblasts. RT-PCR analysis confirmed that osteoblast specific genes, ALP, BMP-2, collagen were expressed in differentiated osteoblasts, however, the another osteoblast specific gene, osteocalcin, was not expressed. When hADSCs and hDPSCs were cultured under osteoblast-differentiation conditions, alkaline phosphatase (ALP) activity was increased in comparison to hADSCs and hDPSCs. Furthermore, specifically both ALP activity and ganglioside expression increased more in hDPSCs-derived osteoblasts than hADSCs-derived osteoblasts. These results suggest that gangliosides play a more important role in regulating the osteoblast-differentiation of hDPSCs compared to hADSCs.     

由BMP-4介导的肌母细胞分化为骨母细胞通路的改变

目的研究骨形态发生蛋白4(BM P-4)在诱导骨形成过程中的作用机制。方法通过构建pC I-neo-BM P-4重组质粒并研究BM P-4在改变肌母细胞的表型往骨母细胞转化过程中的具体作用,肌球蛋白重链(MHC)作为肌细胞分化的标志物而碱性磷酸酶(ALP)作为骨母细胞分化的标志物。结果结果表明在未分化的肌母细胞形态呈星状,不表达MHC,而肌母细胞或转染了pC I-neo质粒的肌母细胞则表现为分化现象,细胞形态互相融合,形成细长多核的肌管结构,同时伴有高水平的MHC表达,但是未检测到BM P-4蛋白或ALP的表达。相反,转染了BM P-4序列质粒的细胞表现为分化现象,但是不形成肌管结构,也没有MHC的高表达,而是表达BM P-4和ALP。结论BM P-4在细胞内的表达可以使肌母细胞在培养状态下往骨母细胞转化。     

Myricetin induces human osteoblast differentiation through bone morphogenetic protein-2/p38 mitogen-activated protein kinase pathway

Myricetin (3,3',4',5,5',7-hexahydroxyflavone), a flavonoid compound, is present in vegetables and fruits. By means of alkaline phosphatase (ALP) activity, osteocalcin, and type I collagen enzyme-linked immunosorbent assay (ELISA), we have shown that myricetin exhibits a significant induction of differentiation in MG-63 and hFOB human osteoblasts. Alkaline phosphatase and osteocalcin are phenotypic markers for early-stage differentiated osteoblasts and terminally differentiated osteoblasts, respectively. Our results indicate that myricetin stimulates osteoblast differentiation at various stages, from maturation to terminally differentiated osteoblasts. Induction of differentiation by myricetin is associated with increased bone morphogenetic protein-2 (BMP-2) production. The BMP-2 antagonist noggin blocked myricetin-mediated ALP activity and osteocalcin secretion enhancement, indicating that BMP-2 production is required in myricetin-mediated osteoblast maturation and differentiation. Induction of differentiation by myricetin is associated with increased activation of SMAD1/5/8 and p38 mitogen-activated protein kinases. Cotreatment of p38 inhibitor SB203580 inhibited myricetin-mediated ALP upregulation and osteocalcin production. In conclusion, myricetin increased BMP-2 synthesis, and subsequently activated SMAD1/5/8 and p38 MAPK, and this effect may contribute to its action on the induction of osteoblast maturation and differentiation, followed by an increase of bone mass.     

2,3-二氢-7-甲氧基-4H-1-苯并吡喃-4-异烟腙抗骨质疏松作用的体外活性研究

目的：在已有的研究基础上,从我们合成的一系列化合物中筛选出具有抗骨质疏松活性的化合物I（2,3-二氢-7-甲氧基-4H-1-苯并吡喃-4-异烟腙）,对其体外活性进行研究。方法：以雌二醇（E2）作为阳性对照,以细胞增殖作用、碱性磷酸酶（ALP）活性和骨钙素（OCN）分泌为指标考察该化合物对成骨细胞系MC3T3-E1细胞的增殖和分化的影响。结果：化合物I在10^-6mol/L浓度时对体外培养的细胞有较好的促进作用,显著增加细胞数量（125.73%,与对照组比较P〈0.05）,提高ALP活性（108.49%,与对照组比较P〈0.05）,且能够促进骨钙素分泌（109.00%,与对照组比较P〈0.05）,以上作用均可被雌激素受体（ER）阻断剂tamoxifen阻断。结论：化合物I具有促进成骨细胞增殖分化的作用,其机制可能与ER激动有关。     

Dual effects of heparin on BMP-2-induced osteogenic activity in MC3T3-E1 cells

Heparin displays several types of biological activities by binding to various extracellular molecules, including pivotal roles in bone metabolism. We have previously reported that heparin competitively inhibits the binding activity of bone morphogenic protein-2 (BMP-2) to BMP and the BMP receptor (BMPR) and suppresses BMP-2 osteogenic activity. In the present study, we examined whether heparin affects osteoblast differentiation induced by BMP-2 at various time points in vitro. We found that 72 h of treatment with heparin inhibited alkaline phosphatase (ALP) activity. However, 144 h of treatment enhanced the ALP activity in BMP-2-stimulated MC3T3-E1 cells. Although heparin decreased the phosphorylation of Smad1/5/8 after 0.5 h of culture, prolonged periods of culture with heparin enhanced the Smad phosphorylation. In addition, 72 h of treatment with heparin enhanced the mRNA expression of runx2 and osterix in BMP-2-stimulated MC3T3-E1 cells. Furthermore, the mRNA expression of BMP antagonists and inhibitory Smads induced by BMP-2 was preferentially blocked by heparin at the 24 and 48 h time points. These findings indicate biphasic effects of heparin on BMP-2 activity and suggest that heparin has complex effects on the BMP-2 osteogenic bioactivities. Prolonged culture with heparin stimulated BMP-2-induced osteogenic activity via down-regulation of BMP-2 antagonists and inhibitory Smads.