组胺对皮肤肿瘤坏死因子受体分布的影响的实验研究

目的　探讨人皮肤角质细胞TNFR的分布和炎症介质组胺对其分布的影响。方法　依据Tel lides成熟实验模型 ,将人皮肤移植于免疫缺陷小鼠上 ,分别注入等量 10ml组胺或生理盐水 ,然后制成共聚焦荧光显微镜和电镜标本观察。结果　正常皮肤TNFR2阳性反应细胞较少 ,TNFR1阴性细胞数量较多 ,阳性反应主要见于角质细胞的胞质和膜 ;组胺刺激后角质细胞质和膜的TNFR1消失 ,主要分布在角质细胞间隙。结论　组胺可使角质细胞质和膜上TNFR1消失 ,移位于细胞间隙     

Study on Preparation of Temperature-sensitive Injectable Hydrogel

To get a sort of new scaffold material for soft tissue reconstruction, we have prepared XLHA-PNIPAAm and XLHA-MC injectable hydrogels through blending crosslinked HA(XLHA)and two temperature-sensitive materials differed in degradation poly(N-isopropylacrylamide)(PNIPAAm)and methylcellulose(MC),respectively. We tested the injectablility, enzymatic biodegradability,temperature-sensitivity,structure cytotoxicity and hemolysis of the two injectable hydrogels. Our research has successfully obtained the preparation condition of XLHA-PNIPAAm injectable hydrogel, and verified that adding non-degradable material PNIPAAm can postpone the degradation of HA more effectively than degradable material MC. PNIPAAm prepared with 5 kGy dose radiation,MBAAm/NIPAAm(M/M)=0.015, monomer concentration=3% produced XLHA-PNIPAAm with slowest enzymatic biodegradability. DSC results showed that temperature-sensitivity of the XLHA-PNIPAAm was more stable than that of XLHA-MC. Two composite hydrogels were qualified in cytotoxicity and hemolysis tests and the biocompatibility of XLHA-PNIPAAm hydrogel showed better than XLHA-MC hydrogel.     

脱细胞基质水凝胶促组织再生的研究进展

脱细胞基质水凝胶是组织或器官通过物理、化学和酶解等手段去除其细胞的内容物,只保留细胞骨架结构和细胞外基质等成分,以实现促细胞黏附、增殖和分化并为其生长创造良好微环境的天然高分子生物材料。近年来,由于其良好的细胞相容性、生物可降解性和诱导组织再生能力,脱细胞基质水凝胶在组织修复、再生医学领域备受关注。首先,介绍该水凝胶的基本特点和材料特性,包括其内部的组成成分和结构、组织特异性以及潜在的免疫排斥反应;然后,从细胞水平的培养、临床前的研究和临床上的应用等三方面,重点阐述脱细胞基质水凝胶在组织工程学中的研究应用;最后,展望脱细胞基质材料运用的优势和需要克服的缺陷。总之,作为构建工程化组织以及修复组织缺损的新型生物活性材料,脱细胞基质水凝胶具有广阔的应用前景。     

Effect of Recombinant Human Keratinocyte Growth Factor (rHuKGF) on the Immunopathogenesis of Intestinal Graft-Vs.-Host Disease Induced Without a Preconditioning Regimen

We studied the effect of rHuKGF on acute, lethal graft- vs.-host disease (GVHD) in the C57BL/6-->(C57BL/6 X DBA/2)F(1)-hybrid model. rHuKGF-treated recipients did not develop intestinal GVHD despite elevated levels of intestinal NO and TNF alpha, did not develop endotoxemia, and did not die. LPS augmented serum TNF alpha release and intestinal NO production, but did not induce intestinal epithelial cell apoptosis, a phenomenon associated with acute GVHD. These data suggest that KGF prevents the development of acute lethal GVHD by protecting epithelial cell injury mediated by TNF-alpha, NO, and other potential cytotoxic factors. We noted a moderate reduction in intestinal KGFR mRNA expression in untreated GVH mice on day 8, when IFN-gamma mRNA levels were highest. This reduction in KGFR mRNA levels was not seen in recipients of IFN-gamma gene knockout grafts, suggesting that IFN-gamma may be involved in reducing KGFR mRNA expression in the intestine. A similar reduction in intestinal KGFR mRNA expression was also seen in rHuKGF-treated recipients, suggesting that rHuKGF does not mediate its protective effect by maintaining KGFR at control levels. KGF-treatment also redirected the cytokine response in acute GVH mice from Th1 to a mixed pattern of both Th1 and Th2 cytokines. This was associated with histopathologic changes resembling chronic GVHD.     

用于组织工程构建的表皮角质形成细胞的生物学特性

目的　研究体外培养的人表皮角质形成细胞的生物学特性 ,为构建组织工程皮肤提供技术参数。方法　用Dispase/胰蛋白酶法消化分离人表皮角质形成细胞 ,进行原代及传代培养。计算原代细胞获得率 ,观察各代细胞形态学改变 ,描记细胞生长曲线 ,计算群体倍增时间 (PDT) ,免疫组织化学方法检测细胞角蛋白表达。结果　原代表皮角质形成细胞获得率为 ( 0 .72 6± 0 .3 48)× 10 6 /cm2 ,表皮角质形成细胞体外可培养至第 7代 ,第 2代的PDT最短 ,为( 46.5 7± 2 .2 5 )h ,第 4代的细胞角蛋白表达仍为阳性。结论　综合细胞增殖速率、细胞数量及细胞功能的维持各因素 ,考虑第 3、4代的表皮角质形成细胞是构建组织工程皮肤最佳的种子细胞     

Vascularization Around Poly(tetrafluoroethylene) Mesh with Coating of Gelatin Hydrogel Incorporating Basic Fibroblast Growth Factor

For successful mesh hernia treatment with medical meshes, it is important to induce angiogenesis and fibroplasia around the site of the mesh implanted. The objective of this study is to combine a mesh with a gelatin hydrogel for basic fibroblast growth factor (bFGF) release and evaluate the angiogenic activity in vivo. The MotifMesh^® (MM) of poly(tetrafluoroethylene) was treated with corona discharge to make the surface hydrophilic. This corona discharge treatment increased the bonding strength between the gelatin hydrogel coated and the mesh surface. When implanted into the back subcutis of mice, the MM coated with the gelatin hydrogel incorporating bFGF showed significant angiogenesis around the implanted site, in contrast to the MM alone and that without gelatin hydrogel or bFGF incorporation. It is concluded that the coating of hydrogel incorporating bFGF is a promising technology to give the mesh angiogenic properties.     

Effect of the Addition of a Labile Gelatin Component on the Degradation and Solute Release Kinetics of a Stable PEG Hydrogel

Abstract

Characterization of the degradation mechanisms and resulting products of biodegradable materials is critical in understanding the behavior of the material including solute transport and biological response. Previous mathematical analyses of a semi-interpenetrating network (sIPN) containing both labile gelatin and a stable cross-linked poly(ethylene glycol) (PEG) network found that diffusion-based models alone were unable to explain the release kinetics of solutes from the system. In this study, degradation of the sIPN and its effect on solute release and swelling kinetics were investigated. The kinetics of the primary mode of degradation, gelatin dissolution, was dependent on temperature, preparation methods, PEGdA and gelatin concentration, and the weight ratio between the gelatin and PEG. The gelatin dissolution rate positively correlated with both matrix swelling and the release kinetics of high-molecular-weight model compound, FITC-dextran. Coupled with previous in vitro studies, the kinetics of sIPN degradation provided insights into the time-dependent changes in cellular response including adhesion and protein expression. These results provide a facile guide in material formulation to control the delivery of high-molecular-weight compounds with concomitant modulation of cellular behavior.     

正常人表皮细胞老化过程中生物学特性研究

目的　研究表皮细胞体外增殖与老化规律 ,为选择合适的组织工程化皮肤种子细胞提供依据。方法　取正常年轻人表皮细胞进行传代培养 ,以不同代龄细胞为实验对象 ,采用形态学观察、群体倍增时间(PDT)、免疫细胞化学及 β 半乳糖苷酶染色等一系列方法 ,检测表皮细胞老化规律。 结果　体外单层培养 9代 ,P2 (第 2代 )的PDT最短 ,前 5代增殖能力较强 ,P5(第 5代 )以后PDT明显延长 ,P8细胞不再增殖 ;随着细胞的连续传代培养 ,SA β Gal表达呈现从弱 (在年轻细胞中占 9% )到强 (在老化细胞中占 6 5 % )的趋势。 结论　体外培养第 1～第 5代表皮细胞可作为构建组织工程化皮肤的种子细胞。     

rhbFGF转染人皮肤成纤维细胞的研究

将重组人碱性成纤维细胞生长因子(rhbFGF)基因克隆入真核表达载体pEGFP-N1质粒增强型绿色荧光蛋白(EGFP)基因上游，并在其5′端加上白介素-4的信号肽序列，形成融合基因，利用脂质体转染原代培养的成纤维细胞，荧光显微镜和蛋白质印迹杂交检测rhbFGF-EGFP融合蛋白的表达，细胞计数法观察bFGF对细胞生长的影响。结果表明成功构建了pEGFP-rhbFGF质粒，并经脂质体介导有效地转入原代培养的人皮肤成纤维细胞内。用G418筛选纯化转基因后的细胞能够向胞外分泌rhbFGF活性物质，明显促进成纤维细胞自身的增殖。表明bFGF基因转染能够稳定有效地促进成纤维细胞的增殖。     

Intratracheal Administration of Recombinant Human Keratinocyte Growth Factor Promotes Alveolar Epithelial Cell Proliferation during Compensatory Lung Growth in Rat

Keratinocyte growth factor (KGF) is considered to be one of the most important mitogens for lung epithelial cells. The objectives of this study were to confirm the effectiveness of intratracheal injection of recombinant human KGF (rhKGF) during compensatory lung growth and to optimize the instillation protocol. Here, trilobectomy in adult rat was performed, followed by intratracheal rhKGF instillation with low (0.4 mg/kg) and high (4 mg/kg) doses at various time-points. The proliferation of alveolar cells was assessed by the immunostaining for proliferating cell nuclear antigen (PCNA) in the residual lung. We also investigated other immunohistochemical parameters such as KGF, KGF receptor and surfactant protein A as well as terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Consequently, intratracheal single injection of rhKGF in high dose group significantly increased PCNA labeling index (LI) of alveolar cells in the remaining lung. Surprisingly, there was no difference in PCNA LI between low and high doses of rhKGF with daily injection, and PCNA LI reached a plateau level with 2 days-consecutive administration (about 60%). Our results indicate that even at low dose, daily intratracheal injection is effective to maintain high proliferative states during the early phase of compensatory lung growth.     

几种生物材料种植人成纤维细胞的比较

目的 通过人成纤维细胞种植，比较几种形式的聚酯材料，以及脱细胞生物组织基质的细胞亲和性，为组织工程血管及瓣膜研究选择支架材料。方法 ①聚氨酯(PU)、第3代聚烷酸酯[PHAs，分子表达式为p(3HB—co—3HH)]、PHAs／PU共混物分别涂在载波片上制成薄膜，将人成纤维细胞种植其上，培养3天。HE染色后于显微镜下每片取相同4点计数，得出平均值。②分别在PHAs／PU片、脱细胞牛心包片、脱细胞猪肺动脉瓣(简称猪瓣)种植人成纤维细胞，PHAs／PU片培养7天，后两者培养3天。用HE染色、扫描电镜分别观察细胞生长情况。结果 ①3组薄膜片上细胞平均数分别为(PU)196、(PHAs)124、(PHAs／PU)160。②扫描电镜显示PHAs／PU片表面约50％有细胞覆盖，细胞聚合成粗大束带状结构，牛心包表面紧密覆盖着厚实的鳞甲状细胞结合体，猪瓣瓣叶表面覆盖紧密联结的板状细胞结合体。③HE染色显示PHAs／PU片和牛心包片、猪瓣瓣叶单侧有联结较完整的细胞层覆盖，局部有3—4层重叠而成，牛心包片和猪瓣瓣叶上细胞与基质联结紧密。结论 ①PHAs混入PU后细胞亲和性显著提高，适宜作血管及补片支架材料；②脱细胞牛心包组织和猪瓣细胞亲和性明显好于合成材料，适合作血管、补片及带瓣通道组织工程支架。     

羧甲基壳聚糖温敏凝胶对人牙周膜细胞骨向分化的影响

目的将羧甲基壳聚糖温敏凝胶作用于人牙周膜细胞,通过观察细胞的碱性磷酸酶活性来确定促进其骨向分化的最佳浓度,为羧甲基壳聚糖温敏凝胶应用于牙周组织再生领域提供理论依据。方法人牙周膜细胞原代及传代培养。将灭菌后的羧甲基壳聚糖（CMCS）和甘油磷酸盐（GP）按一定比例配制成2％、10％、15％、20％、50％5个浓度梯度的羧CMCS温敏凝胶。采用动力学方法（ALP法）检测不同浓度的CMCS温敏凝胶浸提液对NIH3T3细胞骨向分化的影响,测定加药后24h、48h、72h的吸光度值（OD值）,并进行统计学分析。结果24h后,实验组间及与对照组相比较,差异无统计学意义（P〉O．05）。加样48h、72h后,15％浓度组OD值最高,其次分别为10％、2％浓度组,差异均有统计学意义（P〈O．05）;15％浓度组OD值与20％、50％浓度组相比较,差异无统计学意义（P〉O．05）。结论羧甲基壳聚糖温敏凝胶在2％～50％浓度范围内均可有效促进人牙周膜细胞的增殖和骨向分化,浓度为15％时促进效果最佳,越接近最佳浓度,促进细胞骨向分化的效果越明显。     

Mechanical and structural response of a hybrid hydrogel based on chitosan and poly(vinyl alcohol) cross-linked with epichlorohydrin for potential use in tissue engineering

The development and characterization of a hybrid hydrogel based on chitosan (CS) and poly(vinyl alcohol) (PVA) chemically cross-linked with epichlorohydrin (ECH) is presented. The mechanical response of these hydrogels was evaluated by uniaxial tensile tests; in addition, their structural properties such as average molecular weight between cross-link points (M_crl), mesh size (D_N), and volume fraction (v_s) were determined. This was done using the equivalent polymer network theory in combination with the obtained results from tensile and swelling tests. The films showed Young’s modulus values of 11?±?2?MPa and 9?±?1?MPa for none irradiated and ultraviolet (UV) irradiated hydrogels, respectively. The cell viability was assessed using Calcein AM and Ethidium homodimer-1 assay and environmental scanning electron microscopy. The 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan thiazolyl blue formazan (MTT Formazan assay) results did not show cytotoxic effects; this was in good agreement with nuclear magnetic resonance and fourier transform infrared spectroscopies; their results did not show traces of ECH. This indicated that after the crosslinking process, there was no free ECH; furthermore, any possibility of ECH release in the construct during cell culture was discarded. The CS-PVA-ECH hybrid hydrogel allowed cell growth and extracellular matrix formation and showed adequate mechanical, structural, and biological properties for potential use in tissue engineering applications.     

羧甲基壳聚糖温敏凝胶对人牙周膜细胞增殖活性的影响

目的通过观察羧甲基壳聚糖温敏凝胶作用于人牙周膜细胞的增殖活性来确定最佳作用浓度,为羧甲基壳聚糖温敏凝胶应用于牙周组织工程提供理论依据。方法人牙周膜细胞原代及传代培养。将灭菌后的羧甲基壳聚糖（CMCS）和甘油磷酸盐（GP）按一定比例配制成不同浓度梯度的羧CMCS温敏凝胶。采用四甲基偶氮唑盐（MTT）法检测其对人牙周膜细胞增殖的作用,测定加药后48h、72h、96h的吸光度值（OD值）,并进行统计学分析。结果加样培养48h时,15％浓度组0D值高于其它4个实验组及对照组;加样72h、96h后,5个实验组0D值均高于对照组;15％浓度组OD值高于它4个实验组;10％、20％浓度组OD值高于2％、50％浓度组;以上差异均有统计学意义。结论羧甲基壳聚糖温敏凝胶在2％～50％浓度范围内均可有效促进人牙周膜细胞的增殖,浓度为15％时促进效果最佳,越接近最佳浓度,促进增殖和分化的效果越明显。     

Neurite Outgrowth on a DNA Crosslinked Hydrogel with Tunable Stiffnesses

Mechanical cues arising from extracellular matrices greatly affect cellular properties, and hence, are of significance in designing biomaterials. In this study, a DNA crosslinked hydrogel was employed to examine cellular responses of spinal cord neurons to substrate compliances. Using DNA as crosslinkers in polymeric hydrogel formation has given rise to a new class of hydrogels with a number of attractive properties (e.g., reversible gelation and controlled crosslinking). Here, it was demonstrated that by varying length of crosslinker, monomer concentration, and level of crosslinking, DNA gel stiffnesses span from ∼100 Pa to 30 kPa. Assessment of neurite outgrowth on functionalized DNA gels showed that although primary dendrite length is not significantly affected, spinal cord neurons extend more primary dendrites and shorter axons on stiffer gels. Additionally, a greater proportion of neurons have more primary dendrites and shorter axons on stiffer gels. There is a pronounced reduction in focal adhesion kinase (FAK) when neurons are exposed to stiffer substrates, suggesting its involvement in neuronal mechanosensing and neuritogenesis in response to stiffness. These results demonstrate the importance of mechanical aspects of the cell–ECM interactions, and provide guidance for the design of mechanical properties of bio-scaffolds for neural tissue engineering applications.     

Influence of soluble PEG-OH incorporation in a 3D cell-laden PEG-fibrinogen (PF) hydrogel on smooth muscle cell morphology and growth

We have been able to control hydrogel compliance and cell spreading in a three-dimensional (3D) cell-laden system (hydrogel) using soluble PEG-OH. This was accomplished by encapsulating smooth muscle cells (SMCs) into poly(ethylene glycol)-fibrinogen (PEG-fibrinogen or PF) with poly(ethylene glycol)-diol (PEG-OH) as a macromolecular leachant. The cell-encapsulating hydrogels were prepared with three concentrations of soluble PEG-OH having a mass of 10 kDa (1, 5 and 10% w/v). Rheology was used to measure the elastic (storage) component of the complex shear modulus of these hydrogels, while quantitative morphometrics were used to characterize SMC morphology. PF hydrogel with a higher amount of PEG-OH displayed a lower storage modulus and a higher elongated cell morphology of SMCs. Structural changes of PF hydrogels mainly owing to gelation-induced phase separation imparted by the soluble PEG-OH in 3D cell-laden hydrogels dramatically affected both the properties of the hydrogel network including the modulus as well as cell spreading.     

含人发角蛋白和胶原的真皮类似物的研究

目的　以大鼠为动物模型 ,探讨由人发角蛋白和胶原组成的真皮类似物植入体内后真皮重建过程中的形态学变化。方法　将含人发的活性皮肤替代物植入大鼠皮下 ,于术后不同时间取出植入物及其周围组织 ,作组织学和免疫组化观察。结果　术后 4天 ,为急性炎症期 ,同时可见有约 1/2的内皮细胞以及成纤维细胞等 ;术后第 7天 ,植入物内可见大量的血管及其他迁入的细胞 ;第 3周人发开始降解 ,出现胶原纤维 ;6周后 ,人发碎裂降解 ,胶原纤维粗大 ,集结成束 ,与真皮胶原无明显区别。结论　组织学的结果显示人发可以作为真皮基质来修复皮肤缺损     

APCP对人胃腺癌细胞HGC-27生长及血管内皮生长因子的影响

目的体外应用CD73(5’-核酸酶)特异性抑制剂APCP(alpha,beta-methylene adenosine-5’- diphosphate,5’-α,β-亚甲基-二磷酸腺苷),探讨其对人胃腺癌细胞HGC-27细胞生长及其对血管内皮生长因子VEGF-C、VEGF-D表达的影响。方法体外培养HGC-27细胞达指数生长,设正常组和APCP不同浓度干预组,倒置显微镜观察各组细胞生长状态,MTT法检测各组细胞活性,免疫组化法观测两组细胞VEGF-C、VEGF- D的表达及其积分光密度。结果同正常组相比,随药物浓度增加,APCP各干预组细胞在生长方面表现为生长缓慢,胞浆皱缩,细胞界线模糊,细胞数量减少(P<0.05);各干预组细胞内VEGF-C、VEGF-D的表达低于正常对照组(P<0.05)。结论APCP抑制HGC-27细胞生长,细胞数量减少,降低细胞内VEGF-C、VEGF-D的表达。     

骨髓间充质干细胞复合温敏水凝胶支架的制备

背景：壳聚糖及其衍生物制备的支架对细胞迁移和神经轴突再生有重要作用。壳聚糖及其衍生物的组织相容性好,易使干细胞在其表面附着生长,在神经组织工程具有较为广阔的应用前景。 目的：制备适宜骨髓间充质干细胞生长的壳聚糖/壳聚糖季铵盐/甘油磷酸钠温敏性水凝胶细胞支架,观察骨髓间充质干细胞在细胞支架中的生长情况。 方法：将壳聚糖进行季铵盐化改性处理,通过傅里叶变换红外光谱分析谱检测确定其生成。实验以壳聚糖与壳聚糖季铵盐配比为8∶1成功制备出较为稳定的壳聚糖/壳聚糖季铵盐/甘油磷酸钠温敏性温敏水凝胶细胞支架,观察成胶情况,并进行生物安全性检测。 结果与结论：实验在傅里叶变换红外光图谱上发现了季铵基基团的特征峰。细胞毒性实验显示,水凝胶浸提液干预的大鼠骨髓间充质干细胞无毒性。急性全身毒性实验显示,浸提液对大鼠体质量增加无明显影响,支架生物安全性较好。扫描电镜观察显示,骨髓间充质干细胞在细胞支架中能正常的生长和增殖。结果证实,实验成功制备了壳聚糖/壳聚糖季铵盐/甘油磷酸钠温敏性水凝胶细胞支架,适合骨髓间充质干细胞生长和增殖。