树突状细胞对海藻酸钙纳米胶囊的吞噬作用与功能诱导

本研究采用量子点标记方法,证实了人外周血来源树突状细胞对海藻酸钙纳米胶囊的吞噬作用,并对其体外成熟诱导和接受偶联有牛血清白蛋白的纳米胶囊刺激之后的自体T淋巴细胞免疫作用进行了探讨。实验结果表明,树突状细胞在吞噬海藻酸钙纳米胶囊后被诱导成熟,而且偶联有牛血清白蛋白的纳米胶囊可诱导自体T淋巴细胞增殖,显示该海藻酸钙纳米胶囊可能成为具有载体功能的癌症细胞免疫治疗佐剂。     

海藻酸钙对骨质疏松症大鼠骨骼肌SDF-1含量和骨密度的影响

摘要 目的：探讨海藻酸钙对骨质疏松症大鼠骨骼肌基质细胞衍生因子-1（Stromal Cell-derived Factor-1,SDF-1）含量和骨密度的影响。方法：骨质疏松症大鼠（n=48）随机平分为三组-模型组、尼尔雌醇组与海藻酸钙组,在建模后1周后三组分别给予双蒸水、0.1 mg/100 g尼尔雌醇与37.5 mg/mL海藻酸钙/枸杞多糖凝胶微球水溶液灌胃治疗,1 次/d,检测大鼠骨骼肌SDF-1含量和骨密度变化情况。结果：（1）尼尔雌醇组与海藻酸钙组给药第4周与第8周的血清钙离子含量高于模型组（P<0.05）,磷离子含量低于模型组（P<0.05）,尼尔雌醇组与海藻酸钙组对比差异有统计学意义（P<0.05）;（2）尼尔雌醇组与海藻酸钙组给药第4周与第8周的骨骼肌SDF-1含量低于模型组（P<0.05）,海藻酸钙组低于尼尔雌醇组（P<0.05）;（3）尼尔雌醇组与海藻酸钙组给药第4周与第8周的腰椎和股骨骨密度高于模型组（P<0.05）,海藻酸钙组低于尼尔雌醇组（P<0.05）;（4）尼尔雌醇组与海藻酸钙组给药第4周与第8周的股骨最大载荷、最大应力高于模型组(P<0.05),海藻酸钙组高于尼尔雌醇组（P<0.05）;（5）海藻酸钙组造血细胞数量较多,骨皮质结构较完整,致密均匀粗壮,小梁数目明显增多,骨髓腔变小。结论：海藻酸钙在骨质疏松症大鼠的应用能抑制骨骼肌SDF-1的释放,有助于提高骨密度,改善骨生物力学指标,提高血清钙离子含量,降低磷离子含量。     

低温胁迫下董棕（Garyota urens L.）幼苗叶肉细胞内Ca2+水平及细胞超微结构的变化

用焦锑酸钙沉淀的电镜细胞化学方法,研究了低温胁迫下董棕（Garyota urensL.） 幼苗叶肉细胞内Ca2+水平的变化。研究结果表明,未经低温处理的董棕幼苗叶肉细胞,焦锑 酸钙沉淀颗粒大量出现在液泡和细胞间隙中,细胞壁中也可见少量沉淀,而细胞基质中则看 不到焦锑酸钙沉淀;经2 ℃ 48 h低温处理后,细胞基质和细胞膜上焦锑酸钙沉淀增加,而液泡和细胞间隙中的焦锑酸钙沉淀则显著减少,并且超微结构已初步显示出寒害的特征,叶绿体外膜部分破损,类囊体片层稀疏且排列不规则,光合速率明显下降等;经2℃ 120 h低温处理后,细胞间隙内的焦锑酸钙沉淀极少,有的也紧贴在细胞外壁上,而细胞基质和细胞膜上则分布有非常多的焦锑酸钙沉淀,在核基质和液泡中也可见到少量的焦锑酸钙沉淀,并且超微结构遭到了显著破坏,叶绿体结构完全被破坏,核膜与液泡膜严重破损,内部结构模糊,细胞只表现为呼吸作用,不进行光合作用。表明Ca2+的区域性分布的变化与植物抗寒性存在一定关系。     

固定化微环境对酿酒酵母代谢的影响

本文报道不同载体固定化酵母的某些代谢行为和细胞形态。观察到以海藻酸钙凝胶固定酵母,其发酵液总挥发酸量、乙酸量分别比自然细胞减少25．8％和50．O％,而丁二酸量高出自然细胞36．5％。以海藻酸钙,聚乙烯醇凝胶固定酵母,其氨基氮的利用率比自然细胞分别提高31．1％和34．1％,在固定化前后、酵母菌对各种a-氨基酸的利用速度亦都发生明显的变化。电镜观察,酿酒酵母的细胞膜内陷、形成“凹池”。     

低温胁迫下董棕（Garyota urens L.）幼苗叶肉细胞内Ca^2＋水平及细胞超微结构的变化

用焦锑酸钙沉淀的电镜细胞化学方法,研究了低温胁迫下董棕(Caryota urens L.)幼苗叶肉细胞内Ca^2 水平的变化。研究结果表明,未经低温处理的董棕幼苗叶肉细胞,焦锑酸钙沉淀颗粒大量出现在液泡和细胞间隙中,细胞壁中也可见少量沉淀,而细胞基质中则看不到焦锑酸钙沉淀;经2℃ 48h低温处理后,细胞基质和细胞膜上焦锑酸钙沉淀增加,而液泡和细胞间隙中的焦锑酸钙沉淀则显著减少,并且超微结构已初步显示出寒害的特征,叶绿体外膜部分破损,类囊体片层稀疏且排列不规则,光合速率明显下降等;经2℃ 120h低温处理后,细胞间隙内的焦锑酸钙沉淀极少,有的也紧贴在细胞外壁上,而细胞基质和细胞膜上则分布有非常多的焦锑酸钙沉淀,在核基质和液泡中也可见到少量的焦锑酸钙沉淀,并且超微结构遭到了显著破坏,叶绿体结构完全被破坏,核膜与液泡膜严重破损,内部结构模糊,细胞只表现为呼吸作用,不进行光合作用。表明Ca^2 的区域性分布的变化与植物抗寒性存在一定关系。     

低温胁迫下董棕(Garyota urens L.)幼苗叶肉细胞内Ca~(2+)水平及细胞超微结构的变化

用焦锑酸钙沉淀的电镜细胞化学方法 ,研究了低温胁迫下董棕 (GaryotaurensL .)幼苗叶肉细胞内Ca2 + 水平的变化。研究结果表明 ,未经低温处理的董棕幼苗叶肉细胞 ,焦锑酸钙沉淀颗粒大量出现在液泡和细胞间隙中 ,细胞壁中也可见少量沉淀 ,而细胞基质中则看不到焦锑酸钙沉淀 ;经 2℃ 48h低温处理后 ,细胞基质和细胞膜上焦锑酸钙沉淀增加 ,而液泡和细胞间隙中的焦锑酸钙沉淀则显著减少 ,并且超微结构已初步显示出寒害的特征 ,叶绿体外膜部分破损 ,类囊体片层稀疏且排列不规则 ,光合速率明显下降等 ;经 2℃ 1 2 0h低温处理后 ,细胞间隙内的焦锑酸钙沉淀极少 ,有的也紧贴在细胞外壁上 ,而细胞基质和细胞膜上则分布有非常多的焦锑酸钙沉淀 ,在核基质和液泡中也可见到少量的焦锑酸钙沉淀 ,并且超微结构遭到了显著破坏 ,叶绿体结构完全被破坏 ,核膜与液泡膜严重破损 ,内部结构模糊 ,细胞只表现为呼吸作用 ,不进行光合作用。表明Ca2 + 的区域性分布的变化与植物抗寒性存在一定关系。     

固定化米根霉发酵制L-乳酸

采用海藻酸钙包埋法固定化米根霉（Ｒｈｉｚｏｐｕｓｏｒｙｚａｅ）,菌体在颗粒表面形成一层菌丝膜,有利于氧气和其它营养物质的传递;三相流化床生物反应器结构简单、动力消耗低、反应器内物质混合均匀、氧传递量大于固定化米根霉的需氧量,非常适合好氧的固定化米根霉发酵。利用它进行重复使用固定化米根霉的间歇发酵或连续发酵制备Ｌ 乳酸,整个过程一般可持续两周以上。固定化米根霉的产酸速率达１６～１８ｇ／Ｌ ｂｅａｄ．ｈｒ,得率为７０～８０％,反应器生产能力约为传统搅拌罐的３倍。采用海藻酸钙包埋法固定化米根霉在三相流化床生物反应器中进行发酵可以有效地提高Ｌ 乳酸的生产效率,具有良好的工业应用前景。     

固定化简单节杆菌细胞用于醋酸可的松转化的研究——固定化载体对转化作用的影响

海藻酸钙包埋简单节杆菌细施回收率达89％。将凝胶用磷酸缓冲液溶解后测定活力证明固定化过程对细胞活力没有显著影响,但转化高浓度底物时,有效活力很低。将菌种接八聚丙烯酰胺凝胶后进行培养、活化,制得了高活力的固定化细胞,可用于转化高浓度的底物。测得产物醋酸泼尼松在海藻酸钙和聚丙烯酰胺凝胶中与溶液间的分配系数分别为1．29和 O．69。作者认为产物的亲凝胶性可能是阻碍海藻酸钙凝胶包埋细胞进行转化反应的重要因素。     

固定化细胞合成酯类载体的研究

本文以海藻酸钠、聚乙烯醇为材料包埋固定化细胞。建立了聚乙烯醇水凝胶的固定化方法,并与海藻酸钙凝胶剂进行了比较。该凝胶在产酯活性、机械强度、使用寿命、贮存稳定性等方面均优于后者。电镜观察也表明该凝胶适于包埋固定化细胞。     

固定化酵母细胞产增香酯的研究

本工作在研究固定化己酸菌的基础上,进一步用海藻酸钙固定化酵母细胞,用活细胞计数法探讨了固定化酵母细胞的存活状态及增殖情况,从促进产酯能力上对固定化酵母和游离酵母进行了比较,并将其应用于白酒发酵之中与已酸菌液共同发酵生成己酸乙酯,可以使普通白酒的主体香气成份提高2.5倍,因此,认为固定化酵母细胞产增香酯在改进白酒质量上将很有前途。     

Preparation of alginate nanocapsules containing turmeric oil

To encapsulate turmeric oil, a model oily compound, with an alginate biopolymer coating, alginate nanocapsules were prepared in a three-step procedure using emulsification, crosslinking with calcium chloride, and solvent removal. The type of solvent, concentration of turmeric oil, sonication, and oil/alginate mass ratio affected the characteristics of the nanocapsules in terms of average size, zeta potential, morphology, loading capacity, and stability at 4 °C and 25 °C. Dissolution of turmeric oil in ethanol and presence of Tween 80^® in the formulation were found to be optimal in the preparation process. An increase in the oil concentration or oil/alginate mass ratio resulted in an increase in the average size of the nanocapsules. To obtain uniform-sized nanocapsules, sonication is required. In addition, alginate nanocapsules show good physical stability in long-term storage at 4 °C and data on loss of oil in key steps in the process may facilitate improvement in the procedure to produce an increased loading capacity.     

Fatty acid impurities in alginate influence the phenol tolerance of immobilized Escherichia coli

Summary A short time after the immobilization of Escherichia coli in calcium alginate substantial modifications of the fatty acid patterns of the cells were observed. This effect could be related to lipid impurities in the commercial alginate product used, which could be taken up, at least in part by the microorganisms. The impurities were mainly free fatty acids but sterols were also detected. Immobilization of the cells in alginate material extracted by chloroform or ethanol decreased the tolerance of the cells to phenol as compared with cells immobilized in raw alginate. This effect was diminished if the immobilized cells were exogenously supplied with palmitic acid, which is the main constituent of the fatty acids extracted from alginate. These results indicate that not only fatty acids but also other ingredients of commercial alginate have physiological effects on cells entrapped in this gel material. Offprint requests to: H.-J. Rehm     

生物响应酸性磷酸酶浓度的载药聚电解质的体外控释分析

研制一种可响应酸性磷酸酶浓度变化的聚电解质胶囊,(PAH/PSS-β-甘油磷酸酯)胶囊,在分析胶囊的理化性质的基础上对其阿霉素药物包封和体外控释行为进行研究.通过层层组装的方法,制备囊壁含有酸性磷酸酶底物β-甘油磷酸酯的空壳胶囊和囊壁不含酸性磷酸酶底物的对照空壳胶囊;用电镜测定胶囊的大小和形态:用MTT方法分析胶囊的生物相容性.通过药物浓度梯度法进行胶囊的阿霉素药物包封并测定其包封率.将酸性磷酸酶标准品、分泌酸性磷酸酶的HepG2细胞株分别与载药阿霉素胶囊和载药阿霉素对照胶囊作用,观察阿霉素胶囊的药物控释情况和对肿瘤细胞生长的影响.空壳(PAH/PSS-β-甘油磷酸酯)胶囊粒径多在200-300 nm之间,胶囊浓度≤250μtg/mL时生物相容性良好,对阿霉素的包封率迭68.12%;载药胶囊组和对照组分别与酸性磷酸酶标准品作用,至48 h时分别释放出载药量的38%和15%,两者差异具有显著的统计学意义(P<0.05);载药胶囊组较载药对照组对HepG2细胞株的生长抑制作用明显增加,24 h HepG2细胞凋亡相差7.59%(13.73 Vs 6.14),有明显统计学意义(P<0.05).囊壁含有酸性磷酸酶底物的载药聚电解质胶囊,可在体外响应酸性磷酸酶浓度变化,具有药物控释性状,为临床上有酸性磷酸酶升高的良、恶性疾病的药物控释治疗提供了一种新的方法,其应用前景值得进一步探讨.     

EWS fli-1 antisense nanocapsules inhibits ewing sarcoma-related tumor in mice

EWS Fli-1, a fusion gene resulting from a t(11;22) translocation is found in 90% of both Ewing's sarcoma and primitive neuroectodermal tumor (PNET). In the present study, we show that recently developed polyisobutylcyanoacrylate nanocapsules with an aqueous core were able to encapsulate efficiently high amounts of phosphorothioate oligonucleotides (ODN) directed against EWS Fli-1 chimeric RNA. Release of these ODN in serum medium was shown to be biphasic which was explained by the presence of two types of nanocapsules able to release ODN with different kinetics. In addition, nanocapsules were found to provide protection of these oligonucleotides from the degradation in serum. These ODN nanocapsules permitted to obtain inhibition of Ewing sarcoma-related tumor in mice after intratumoral injection of a cumulative dose as low as 14.4 nanomoles. This new type of non viral vector shows great potential for in vivo administration of oligonucleotides.     

Optimization of cardiac cell seeding and distribution in 3D porous alginate scaffolds

Cardiac tissue engineering has evolved as a potential therapeutic approach to assist in cardiac regeneration. We have recently shown that tissue-engineered cardiac graft, constructed from cardiomyocytes seeded within an alginate scaffold, is capable of preventing the deterioration in cardiac function after myocardial infarction in rats. The present article addresses cell seeding within porous alginate scaffolds in an attempt to achieve 3D high-density cardiac constructs with a uniform cell distribution. Due to the hydrophilic nature of the alginate scaffold, its >90% porosity and interconnected pore structure, cell seeding onto the scaffold was efficient and short, up to 30 min. Application of a moderate centrifugal force during cell seeding resulted in a uniform cell distribution throughout the alginate scaffolds, consequently enabling the loading of a large number of cells onto the 3D scaffolds. The percent cell yield in the alginate scaffolds ranged between 60-90%, depending on cell density at seeding; it was 90% at seeding densities of up to 1 x 10(8) cells/cm(3) scaffold and decreased to 60% at higher densities. The highly dense cardiac constructs maintained high metabolic activity in culture. Scanning electron microscopy revealed that the cells aggregated within the scaffold pores. Some of the aggregates were contracting spontaneously within the matrix pores. Throughout the culture there was no indication of cardiomyocyte proliferation within the scaffolds, nor was it found in 3D cultures of cardiofibroblasts. This may enable the development of cardiac cocultures, without domination of cardiofibroblasts with time.     

Synthesis and characterization of bovine serum albumin-coated nanocapsules loaded with indocyanine green as potential multifunctional nanoconstructs

We have synthesized and characterized bovine serum albumin (BSA)-coated polymeric nanocapsules (NCs) loaded with indocyanine green (ICG), an FDA-approved near infrared chromophore. Poly(allylamine) hydrochloride was electrostatically crosslinked with phosphate anions to form nanoconstructs encapsulating ICG. BSA was conjugated onto the polymeric NCs via glutaraldehyde. BSA-coated ICG-containing nanocapsules (BSA-ICG NCs) were characterized by FTIR and optical spectroscopy, SEM, and zeta-potential measurements. Using normal human endocervical epithelial cells, we demonstrate the effectiveness of BSA-ICG NCs for intracellular optical imaging in vitro. These nanoconstructs may potentially serve as a multifunctional platform for combined optical imaging, phototherapy, and drug delivery.     

Structure-activity relationship of alginate oligosaccharides in the induction of cytokine production from RAW264.7 cells

Guluronate and mannuronate oligomers with various degree of polymerization were prepared from polyguluronate (PG) and polymannuronate (PM) with an alginate lyase from a Pseudoalteromonas sp., and their activities to induce cytokine secretion from mouse macrophage cell line RAW264.7 cells were examined. Enzymatically depolymerized unsaturated alginate oligomers induced tumor necrosis factor (TNF)-alpha secretion from RAW264.7 cells in a structure-depending manner, while the activities of saturated alginate oligomers prepared by acid hydrolysis were fairly low or only trace levels. These results suggest that unsaturated end-structure of alginate oligomers was important for the TNF-alpha-inducing activity. Among the unsaturated guluronate (G3-G9) and mannuronate (M3-M9) oligomers, G8 and M7 showed the most potent activity, respectively. Bio-Plex assay revealed that interleukin (IL)-1alpha, IL-1beta, and IL-6 secretion from RAW264.7 cells were also induced by unsaturated alginate oligomers with similar structure-activity relationship profiles as seen in TNF-alpha, and the most potent activities were observed with G8 and M7. These results suggest that G8 and M7 may have the most suitable molecular size or entire structural conformation as stimulant for cytokine secretion. Since antibodies to Toll-like receptor (TLR)2 and TLR4 effectively inhibited the G8- and M7-induced production of TNF-alpha, these alginate oligomers may stimulate innate immunity through the pattern recognition receptors on macrophages similar to microbial products.     

Hepatocyte behavior within three-dimensional porous alginate scaffolds

A potential approach to facilitate the performance of implanted hepatocytes is to enable their aggregation and re-expression of their differentiated function prior to implantation. Here we examined the behavior of freshly isolated rat adult hepatocytes seeded within a novel three-dimensional (3-D) scaffold based on alginate. The attractive features of this scaffold include a highly porous structure (sponge-like) with interconnecting pores, and pore sizes with diameters of 100-150 microm. Due to their hydrophilic nature, seeding hepatocytes onto the alginate sponges was efficient. DNA measurements showed that the total cell number within the sponges did not change over 2 weeks, indicating that hepatocytes do not proliferate under these culture conditions. Nearly all seeded cells maintained viability, according to the MTT assay. Within 24 h post-seeding, small clusters of viable cells, were seen scattered within the sponge. More than 90% of the seeded cells participated in the aggregation; the high efficiency is attributed to the non-adherent nature of alginate. The spheroids had smooth boundaries and by day 4 in culture reached an average diameter of 100 microm, which is at the same magnitude of the sponge pore size. The cells appeared to synthesize fibronectin which was deposited on the spheroids. No laminin or collagen type IV were detected in the deposit. The 3-D arrangement of hepatocytes within the alginate sponges promoted their functional expression; within a week the cells secreted the maximal albumin secretion rate of 60 microg albumin/10(6) cells/day. Urea secretion rate did not depend on cell aggregation and was similar to that obtained when hepatocytes were cultured on collagen type I coated dishes (100 microg/10(6) cells/day). Our studies show that alginate sponges can provide a conducive environment to facilitate the performance of cultured hepatocytes by enhancing their aggregation.     

TAT Peptide-Conjugated Magnetic PLA-PEG Nanocapsules for the Targeted Delivery of Paclitaxel: <Emphasis Type="Italic">In Vitro</Emphasis> and Cell Studies

Paclitaxel (PTX) and organophilic iron oxide nanocrystals of 7 nm average size were co-encapsulated in the oily core of poly(lactide)-poly(ethyleneglycol) (PLA-PEG) nanocapsules in order to develop magnetically responsive nanocarriers of PTX. The nanocapsules were prepared by a solvent displacement technique and exhibited satisfactory drug and iron oxide loading efficiency, high colloidal stability, and sustained drug release properties. Drug release also proved responsive to an alternating magnetic field. Magnetophoresis experiments showed that the magnetic responsiveness of the nanocapsules depended on their SPION content. The PTX-loaded nanocapsules exhibited comparable to free PTX cytotoxicity against the A549 lung cancer cell line at 24 h of incubation but higher cytotoxicity than free drug at 48 h of incubation. The conjugation of a cysteine-modified TAT peptide (HCys-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-NH₂) on the surface of the nanocapsules resulted to highly increased uptake of nanocapsules by cancer cells, as well as to profound improvement of their cytotoxicity against the cancer cells. The results obtained justify further investigation of the prospects of these multifunctional PLA-PEG nanocapsules as a targeted delivery system of paclitaxel.