新生鼠胰岛干细胞分离、培养和分化的实验研究

目的　探索新生鼠胰岛干细胞体外分离鉴定、培养以及体外分化的方法。方法　用胶原酶消化新生大鼠胰腺 ,将消化的组织碎片在pH7.4～ 7.6条件下经含血清RPMI 164 0及无血清RPMI164 0 (添加bFGF ,EGF ,N 2 )培养液中培养 ,观察形成类胰岛样细胞团的全过程。用胰岛素释放实验检测胰岛功能 ,免疫荧光法检测nestin的表达。结果　胰腺消化碎片培养 3 6h内 ,可见nestin阳性细胞贴壁 ,添加bFGF ,EGF ,N2后nestin阳性细胞快速增长 ,18～ 2 4d形成类胰岛样细胞团 ,并可表达胰岛素。结论　胰腺细胞中nestin阳性细胞具有胰岛干细胞特点 ,经体外培养可获得类胰岛样细胞团     

槐耳清膏治疗肝癌的实验研究

目的　探讨槐耳清膏对人胎脐静脉内皮细胞的作用 ,以及其对裸鼠皮下接种肝癌细胞形成肝癌的抑制作用及其可能机制。方法　用不同浓度的槐耳清膏作用于人胎脐静脉血管内皮细胞 ,观察其对细胞的增殖能力、迁移能力、附壁能力及血管生成的影响 ,同时观察其对裸鼠皮下接种肝癌细胞形成肝癌过程的影响。结果　槐耳清膏≥ 2mg/ml时可显著降低肿瘤组织的血管内皮细胞增殖能力 ,减少血管形成 ,抑制血管内皮细胞的迁移、黏附 ,从而降低微血管密度。槐耳清膏 ( 3g/kg) MMC( 5 0 0 μg/kg)同时作用于裸鼠的肝癌组织 ,其抑瘤作用最强 ,后依次为MMC组 ,槐耳清膏 ( 3g/kg)组。结论　槐耳清膏对肝癌有抑制作用 ,其可能机制是作用于血管内皮细胞 ,影响血管内皮细胞的增殖能力、迁移能力、附壁能力及血管生成 ,从而抑制肝癌组织的血管生成 ,降低肝癌组织的微血管密度而发挥抑制肝癌生长的作用     

血瘀证兔血清对培养的血管内皮细胞活性因子基因表达的影响

【目的】探索血瘀证形成和发展过程中血管内皮细胞活性因子内皮素和一氧化氮 (NO)改变的分子机理。【方法】用半定量RT -PCR方法 ,观察血瘀证兔模型血清对体外培养的血管内皮细胞内皮素 (ET)和组成型NO合成酶 (constitutivenitricoxidesynthase ,cNOS)基因表达的影响。【结果】血瘀证动物模型血清导致体外培养的内皮细胞中ET - 1mRNA表达升高 (P <0 .0 1) ,cNOSmRNA表达下降 (P <0 .0 1) ,两者呈显著负相关 (r=- 0 .857,P <0 .0 1)。【结论】血管内皮细胞中ET基因高表达及cNOS基因低表达导致的二者平衡失调在血瘀证形成和发展过程中起着重要作用。     

喉鳞癌微血管密度与颈淋巴结转移及病理临床分期的相关性研究

目的研究喉鳞癌微血管密度与颈淋巴结转移及病理临床分期的相关性。方法采用抗Ⅷ因子相关抗原的抗体标记肿瘤血管内皮细胞并测定喉鳞癌组织微血管密度。结果①颈淋巴结转移组喉鳞癌的微血管密度显著高于非转移组(32.1518±6.489∶18.0672±4.983,P<0.01);②喉鳞癌微血管密度大于均数组则颈淋巴结转移率显著增高(P<0.01);③病理临床分期晚期组喉鳞癌的微血管密度明显高于早期组(P<0.01)。结论喉鳞癌微血管密度可作为预测其颈淋巴结转移的一项指标。     

Time-Dependent Alginate/Polyvinyl Alcohol Hydrogels as Injectable Cell Carriers

Alginate hydrogels have been widely utilized as cell carriers due to their simplicity for fabricating cell-immobilized gel beads or 3-dimentional porous scaffolds, biocompatibility and non-toxicity to cells. Generally alginate hydrogels have been produced by contacting alginate solution with CaCl₂ as a cross-linking agent. However, the major disadvantages of this system are that the gelation rate is too fast and hard to control, and the prepared alginate gels cannot be injected. Injectable alginates have been prepared by using CaSO₄ or CaCO₃ as a cross-linking agent. However, the gelation rate of alginate with CaCO₃ is slow owing to the low solubility of CaCO₃ in water, while that with CaSO₄ is too fast to form uniform gels. In this study, we prepared injectable alginate/polyvinyl alcohol (PVA) blend hydrogels with controllable gelation rate by using CaSO₄ as a cross-linking agent and Na₂HPO₄ as a cross-linking retardation agent. The gelation rate could be controlled by adjusting CaSO₄/Na₂HPO₄ ratio in the solution. The alginate and PVA showed good compatibility in aqueous solutions or gels. The gelation rate of alginate increased with increasing Na₂HPO₄ and decreasing CaSO₄ concentrations, as expected. The PVA itself in the alginate/PVA blend did not affect the gelation rate. All alginate/PVA hydrogels demonstrated some extraction of PVA, but the extraction extent was not much even after 7 days immersion in water. The alginate/PVA hydrogels were examined for their in vitro cell compatibility by the culture of chondrocytes (human chondrocyte cell line) in the gels up to 28 days. The cells were grown almost linearly in the alginate/PVA hydrogels with higher PVA compositions showing better cell growth. The GAG contents from the cells in the hydrogels did not show dramatic changes with culture time, however, they also increased gradually in the alginate/PVA hydrogels with higher PVA composition. The PVA in the hydrogels seemed to play positive roles for the growth and activity of chondrocytes. The alginate/PVA hydrogels with controllable gelation rate are expected to be applicable as injectable cell carriers.     

Adsorption of matrix metalloproteinases onto biomedical polymers: a new aspect in biological acceptance

Matrix metalloproteinases (MMPs) are zinc-dependent enzymes involved in the remodelling of connective tissues during the development and wound healing. Moreover, two MMPs, Gelatinase A (MMP-2) and Gelatinase B (MMP-9), are also present in body fluids such as blood and urine and, therefore, they can be in contact with implanted biomaterials and can be adsorbed onto their surface. In order to test this hypothesis disks of different polymers (polystyrene (PS), polyvinyl chloride (PVC), poly(D,L-lactide) (PLA), polymethyl methacrylate (PMMA) and poly(2-hydroxyethyl methacrylate) (PHEMA)) have been exposed to human plasma and adsorbed proteins have been eluted and analyzed. Using Western blot and substrate zymography analysis, we observed that both MMP-2 and MMP-9 adsorbed onto the surfaces of all the polymers, especially hydrophilic ones (PMMA and PHEMA) and PLA, in both the active and inactive forms. Furthermore, we observed that adhesion of human granulocyte neutophils to PMMA, the polymer that adsorbed the higher quantity of MMP-2 and MMP-9 compared to the others, was reduced by more that 50% by the presence of a gelatinase inhibitor. This data suggest a surprising role of these absorbed enzymes in the adhesion of neutrophil onto some polymeric biomaterials surface and, therefore, in the setting of inflammation.     

Patterning Collagen/Poloxamine-Methacrylate Hydrogels for Tissue-Engineering-Inspired Microfluidic and Laser Lithography Applications

The ability to pattern semi-synthetic collagen/poloxamine-methacrylate hydrogels into straight-channel flow circuits and sub-millimeter-sized rectangular blocks for tissue-engineering applications was evaluated. Endothelial cells, grown on the surface of flat collagen/poloxamine-methacrylate hydrogels, proliferated, expressed ICAM-1 (but not VCAM-1) and began to detach after 6 days. Seeding endothelial cells onto the lumen surface of straight collagen/poloxamine-methacrylate flow channels increased ICAM-1 and VCAM-1 expression, and exposure to laminar shear stress (0.3–10 dyn/cm²) was unable to attenuate activation on the relatively few cells that were able to withstand flow associated ablation. The enrichment of poloxamine-methacrylate at the lumen surface during fabrication likely caused the decrease in cell attachment and increased activation. To micropattern more complex structures, confocal microscopy UV laser lithography was used to selectively cross-link a HepG2-containing pre-polymer solution of collagen/poloxamine-methacrylate. Turbidity (caused by suspended cells and the incomplete miscibility of collagen and poloxamine-methacrylate) scattered the UV laser energy and necessitated the optimization of exposure times with respect to cross-linking extent and cell viability. Free radical diffusion beyond the bounds of the initial photopattern reduced the resolution of the structures and created a weakly cross-linked periphery around the original pattern. Over time, HepG2 cells migrated towards the less cross-linked periphery and proliferated, creating a non-uniform distribution of cells.     

Stem-cell culture on patterned bio-functional surfaces

Bio-functional surfaces have been created by printing proteins on antifouling surfaces in a customised geometry. Human umbilical cord neural stem cells incubated on the samples readily attach to the protein defined domains, where they have been monitored during 21 days of culture. The stability of the pattern varies with the density of cells anchored to the microstamped proteins. Highly packed cell patterned domains favoured non-differentiated mode, while low-density areas allowed the spreading out of the cells and differentiation. Tailoring the geometry (pattern size and distances) enables improving the monitoring of the stem cells' developmental processes. The biocompatible surfaces can serve as a model to study processes accompanying stem cell neural lineage commitment.     

Uptake of Enzymatically-Digested Hyaluronan by Liver Endothelial Cells in Vivo and in Vitro

Intravenously-injected hyaluronan (HA) is distributed into liver in which endothelium is a site of uptake and degradation of HA. The role and fate of HA have been widely investigated; however, effects of size and dose of HA on its metabolism have not been well documented yet. To investigate these effects, we prepared fluorescein-labeled HAs, according to the modified methods described by de Belder and Wik, which were enzymatically digested. The 90 kDa fluorescein-labeled HA gradually accumulated in a liver that was distributed into the endothelium; however, 10 kDa or less HA did not. Cell fractionation and flow cytometry further demonstrated the cell of uptake in the liver is an endothelial cell, both in vivo and in vitro. Interestingly, the largest uptake by liver endothelial cells in vitro was observed in 10 kDa HA, even though which did not accumulate in liver in vivo. These results suggest that the result observed with 10 kDa HA in vivo is due to the rapid excretion in urine. Thus, inhibiting of the digestion or suppressing of the urinary excretion would enhance uptake of HA in vivo. These ideas may help to deliver drugs or genes targeting to liver endothelium.