冻存乳猪肝细胞的胶原凝胶培养研究

目的 探索乳猪肝细胞的冻存以及培养条件和方法。方法 采用两步法分离乳猪肝细胞．液氮冻存4个月后复苏．与鼠尾胶原液混合后接种培养,或将肝细胞接种于胶原被覆的培养瓶中．然后被覆上第2层胶原培养．观察培养细胞的形态、白蛋白mRNA的表达、尿素合成及天冬氨酸氮基转移酶(AST)的漏出量等。结果 冻存肝细胞的活性维持较好．并成功地被固定于混合凝胶或三明治形凝胶中,较好地保持了尿素合成以及白蛋白mRNA表达能力。结论 此冻存条件适合于猪肌细胞的长期保存,胶原凝胶可为肝细胞提供更接近体内的培养环境,较好地维持了肝细胞的功能和形态学特征。     

采用温敏性壳聚糖水凝胶体外构建组织工程化软骨的实验研究

目的 验证自行制备的温敏性壳聚糖水凝胶作为软骨细胞支架材料构建组织工程化软骨的可行性.方法 以壳聚糖、β-甘油磷酸钠和羟乙基纤维素为原料,制备温敏性壳聚糖水凝胶,通过SD大鼠肌内注射植入,进行组织相容性检测.在此基础上,将其与软骨细胞复合构建组织工程化软骨,观察软骨细胞在壳聚糖水凝胶中的存活情况,并于体外培养3周后,作相关组织形态学检测.结果 制备的壳聚糖水凝胶具有温度敏感性,即室温时为液态,37℃时10～15min可发生交联反应成为固态凝胶.SD大鼠肌内注射不同时间点组织相容性检测表明,该材料具有良好的组织相容性,植入体内2、4周时有少量炎性细胞浸润,6周时材料降解明显,8周时已经基本降解.采用该材料构建的组织工程化软骨体外培养3周后取材,通过组织学检查,HE染色可观察到软骨陷窝样结构,甲苯胺蓝染色、番红O染色及免疫组化染色结果显示软骨细胞具有分泌细胞外基质的功能.结论 本研究自行制备的温敏性壳聚糖水凝胶材料具有良好的组织相容性,将其与软骨细胞复合后,可以在体外再造组织工程化软骨,是一种有广泛应用前景的软骨组织工程支架材料.     

小肠黏膜下层的制备及其在心肌组织工程中的应用

目的 制备小肠黏膜下层生物材料,并以其为支架材料体外构建工程化心肌组织.方法 将猪小肠黏膜下层以0.25%胰酶和0.5%SDS各处理24h,制备出脱细胞小肠黏膜下层.用材料试验机沿平行或垂直于管腔的方向进行单轴拉伸,检测其力学性能,同时检测其细胞毒性和组织相容性,然后将3日龄新生大鼠心肌细胞接种于小肠黏膜下层支架上,体外构建工程化心肌组织薄片,倒置显微镜下观察.经Masson组织化学染色、肌节型肌动蛋白免疫组化染色和透射电镜观察对获得的心肌薄片进行评价.结果 小肠黏膜下层的力学性能良好,已经完全脱去细胞,无细胞毒性,组织相容性良好;接种12h后,心肌细胞开始在小肠黏膜下层上搏动,2d后心肌细胞-支架复合物(工程化心肌组织薄片)开始自发搏动,可搏动14d.构建的工程化心肌组织薄片由多层心肌细胞构成并有大量胞外胶原.结论 成功制备出力学性能和生物相容性良好的小肠黏膜下层生物支架材料,并以其为支架成功构建出工程化心肌组织薄片.     

夹层胶原"三明治"法冻存复苏肝细胞的实验研究

目的 研究胶原、表皮生长因子对新鲜分离及冻仔复苏后Sprague-Dawley（SD）大鼠肝细胞活率、形态和功能的影响,期望建立一种较为完善的肝细胞体外培养及冻存复苏体系,为肝细胞移植、生物人工肝及相关肝细胞研究获取充足有效的肝细胞来源提供必须的技术支持和理论依据。方法 采用胶原酶二步原位循环肝脏灌注法分离SD大鼠肝细胞,测定不同条件培养及冻存复苏后各组肝细胞的活性及功能。结果 双层胶原及双层胶味＋表皮生长因子组细胞活率、蛋白质含量及细胞功能均优于普通组（P〈0．05）。结论 双层胶原体外培养体系更接近于肝细胞体内生长环境,能有效的减少各种理化因素对肝细胞的损伤,有利于肝细胞在体外长期保存,是一种较为理想的培养及冻存复苏体系。     

应用胶原壳聚糖体外构建组织工程心脏瓣膜的初步研究

 目的探讨应用胶原壳聚糖支架体外构建组织工程心脏瓣膜的可行性.方法观察材料结构特点,并行兔皮下包埋实验,分别于4、6、8、12周观察材料的生物相容性和降解率.将犬主动脉壁间质细胞和内皮细胞种植于壳聚糖胶原支架上,观察其形态并测定细胞合成胶原和前列环素的功能.结果胶原壳聚糖膜呈网孔状结构,孔径(103±17)μm.皮下包埋实验显示材料生物相容性好,完全降解时间为12周.种植实验示细胞在材料表面生长良好,并具有合成前列环素(62.3±3.2 pg/mlvs10.7±2.1 pg/ml,P＜0.05)的功能.结论以胶原壳聚糖膜为支架体外构建组织工程心脏瓣叶,种植细胞不仅能在材料表面生长,还能合成细胞间质和血管活性物质,初步提示应用本组材料和方法构建组织工程心脏瓣叶是可行的.     

肝脏原位注射新生大鼠肝细胞构建的工程化肝组织对大鼠肝纤维化的影响

目的观察原位注射新生大鼠肝细胞构建的工程化肝组织对实验性肝纤维化大鼠血清和肝组织学的影响,探讨其抗肝纤维化的作用。方法用四氯化碳制成实验性肝纤维化造模大鼠,肝脏原位多点注射工程化肝组织,3周后测定血清肝功能并行肝组织学检查。结果原位注射组大鼠肝功能较单纯假手术组大鼠有改善,且具有显著差异（P〈0．05）,HE染色提示纤维化明显改善。结论肝脏原位多点注射新生大鼠肝细胞的工程化肝组织能改善大鼠肝纤维化进程,有一定抗肝纤维化作用。     

用MDCK细胞为种子细胞体外构建组织工程化肾小管片层的实验研究

目的以MDCK细胞为种子细胞、胶原凝胶复合Matrigel为支架材料,采用组织工程技术体外构建组织工程化肾小管片层,观察不同浓度Matrigel以及静态拉伸作用对MDCK细胞在三维支架中极性重建并形成小管样结构的影响。方法将培养的MDCK细胞与添加不同浓度Matrigel的液态Ⅰ型胶原混合,在12孔板内铸模形成MDCK细胞-胶原片层。通过相差显微镜、HE染色、免疫荧光染色等方法对细胞的生长情况及形成的三维肾小管结构进行比较和鉴定。在此实验的基础上,对片层施加静态拉伸力,观察静态拉伸作用对细胞生长、小管样结构形成的影响。结果随培养时间的延长,各实验组片层内的MDCK细胞逐渐增殖、黏附、聚集。单纯以胶原为支架材料的片层内MDCK形成囊腔状结构;添加Matrigel的胶原片层内MDCK细胞可形成管腔样结构,且低浓度Matrigel的胶原片层内细胞存活率高。静态拉伸作用能够刺激细胞的生长、管腔样结构的形成。结论本研究应用组织工程技术,以MDCK细胞为种子细胞、添加Matrigel的液态Ⅰ型胶原为支架材料,培养过程中施加静态拉伸力,体外成功构建出组织工程化肾小管胶原组织片层,肾脏小管上皮细胞可在该支架材料中完成极性重建的自组装过程。     

壳聚糖编织纤维体内外降解及生物相容性研究

目的了解编织壳聚糖纤维的表征、降解及生物相容性。方法通过扫描电镜及密度、膨胀率、拉伸强度测定分析纤维的特征,黏度分析法测量鸡卵清溶菌酶对其降解作用,大鼠肌肉植入试验了解其体内吸收情况,大鼠骨髓基质干细胞培养了解其细胞毒性。结果纤维编织后直径2000±63μm,表面光滑,纤维密度1.093±0.270g/cm3,膨胀率2.30±0.21,拉伸强度80.18±1.56MPa。加入溶菌酶2h内特性黏度下降约1/2,以后渐趋稳定。植入肌肉1周后炎症反应明显,随时间延长反应减退直至消失,4周时吸收不明显,12周大部分吸收。细胞生长情况良好,毒性试验阴性。结论壳聚糖编织纤维具有一定强度、良好的生物可降解性及生物相容性,适合构建骨或者软骨组织工程支架。     

纤维蛋白胶对干细胞心肌梗死修复移植影响的研究

目的:应用可注射性纤维蛋白胶(fibrin glue,FG)液态支架材料联合人胎盘间充质干细胞 ( placent mesenchymal stem cells,PMSCs) 进行心肌内注射,观察其对细胞滞留、存活的影响,探讨其作为可注射性心肌组织工程支架的可行性.方法: (1)分离、培养、扩增人PMSCs备用;(2)建立大鼠急性心肌梗死模型并注射FG,观察其在心肌梗死区域存留及降解情况;(3)大鼠急性心肌梗死模型随机分为4组,对照组(PBS组)、FG组、PMSCs PBS组、PMSCs FG组, 对移植24 h后细胞滞留、4周后细胞的存活及局部新生血管的密度进行测量.结果:(1)FG组织相容性好,心肌内降解时间1周左右.(2)PMSCs FG组细胞24 h滞留率显著增高,4周存活细胞数量显著增高、心肌梗死区域新生血管密度显著提高(P<0.01).结论: 可注射性纤维蛋白胶可以提高移植细胞在心肌梗死部位的滞留及存活,发挥微环境调控作用并刺激心肌梗死部位微血管生成,是良好的可注射性心肌组织工程支架材料.     

高原环境体外猪肝细胞原代培养的初步研究

目的：初步了解在高原环境条件下体外培养猪肝细胞的生长、增殖与功能特征。方法：采用已适应本地环境的长白仔猪分离肝细胞并进行体外原代培养,观察细胞形态、细胞数量及培养上清中总蛋白、白蛋白和尿素水平变化。结果：细胞接种后4h开始贴壁,8h开始增殖,4d细胞数量明显增多,6d生长满瓶,培养上清中总蛋白、白蛋白和尿素水平均呈现显著升高。结论：体外培养猪肝细胞生长、增殖及其生物学功能良好,可以作为构建高原体外生物型人工肝脏的生物材料。     

Determination of fibrin glue with antibiotics on collagen production in colon anastomosis

BACKGROUND/AIM: Fibrin glue is used as a matrix for local application of antibiotics. The aim of this study was to determine whether application of fibrin glue in combination with antibiotics can strengthen collagen production, prevent dehiscence of colon anastomoses due to infection, and reduce frequency of mortality and morbidity comparing to the control group and the group with fibrin glue application. METHODS: The adult male Wistar rats divided into three groups were used in the experiment. The group 1 was the control one (after partial colon resection, colonic anastomoses performed were not treated), while to the group 2 and the group 3 were applied fibrin glue and fibrin glue with antibiotics (clindamycin and ceftriaxon) on the site of anastomoses, respectively. Quality of colonic anastomoses were estimated by means of determination of collagen (L-hydroxyproline) amount in the collon wall with anastomoses and histological analysis of this colon segment using light and electronic microscope on the days 5, 7 and 13 postoperatively. RESULTS: The highest morbidity rate was registered in the group 1 (30%), then in the group 2 (13.3%) and the lowest one in the group 3 (3.33%; p < 0.05 vs group 1). Mortality rate was significantly higher in the group 1 than in the group 3 (20% and 0%, respectively; p < 0.05). In the postoperative course, the highest concentrations of collagen in the colon wall on the site of anastomoses, which was confirmed by both light and electronic microscopy, were found in the group 3. CONCLUSION: The application of fibrin glue with antibiotics on colon anastomoses reduces the number of dehiscence, provides good mechanical protection and shorten the time of anastomoses healing.     

重组人肝再生增强因子可预防免疫损伤性肝纤维化的形成

观察重组人肝再生增强因子（ｒｈＡＬＲ）对免疫 务性肝纤维化的预防作用。建立人血白蛋白免疫损伤性大鼠肝纤维化模型。在造模的同时子ｒｈＡＬＲ腹腔注射。观察大鼠存活率,观察大鼠存活率,血清ＡＬＴ、ＡＳＴ、ＬＤＨ水平,肝组织胶原蛋白含量及肝脏病理学改变。结果：ｒｈＡＬＲ可提高人血白蛋白损伤生肝纤维化大鼠存活率氏肝纤维化大 ＡＬＴ、ＡＳＴ、ＬＤＨ水平,病理检查显示ｒｈＡＬＲ有明显减轻大鼠肝纤维化形成的作用;     

重组人肝再生增强因子对实验性肝纤维化血清透明质酸浓度的影响

为了解重组人肝再生增强因子（hALR)对大鼠实验性肝纤维化形成过程中血清透明质酸浓度的影响，建立了四氯化碳中毒性及人血白蛋白免疫损伤性两种大鼠肝纤维化模型，在造模的同时给予不同剂量hALR治疗，并在不同的时间点留取大量血清标本，测定透明质酸浓度。结果表明，在两种模型中hALR两组大鼠血清透明质酸浓度在造模过程中的不同阶段均明显低于模型组，高剂量hALR组大鼠血清透明质酸浓度均明显低于低剂量组，上述结果提示，重组人肝再生增强因子可降低实验性肝纤维化大鼠血清透明质酸含量。     

正常与粥样硬化血管壁荧光光谱机理研究

本文研究了离体正常与粥样硬化人主动脉壁的荧光光谱。用荧光分光光度计，以308nm为激发波长，分别记录市售动脉各种已知成分及动脉壁提取成分的荧光光谱。用紫外-可见吸收分光光度计测人血红蛋白吸收光谱、动脉壁经超声后上清液吸收光谱。对荧光光谱和吸收光谱进行分析，结果显示，正常和粥样硬化动脉荧光光谱分别以弹性蛋白和胶原蛋白为主构成，418nm波谷是由于血红蛋白再吸收形成的。     

骨髓间质干细胞移植对肝纤维化模型大鼠治疗效果

目的探讨骨髓间质干细胞(MSCs)对肝纤维化模型大鼠肝功能及肝纤维化的改善情况。方法将39只健康雌性SD大鼠分为对照组(n=14)和肝纤维化模型组(n=25)。对照组给予普通食水喂养;肝纤维化模型组给予四氯化碳液体橄榄油混合液皮下注射及乙醇喂养,建立大鼠肝纤维化模型。建模成功后将存活的大鼠随机分为对照组、模型组和移植组(每组各9只)。取健康SD大鼠骨髓,利用全骨髓贴壁的方法原代培养骨髓MSCs,然后将MSCs通过大鼠尾静脉对肝纤维化模型大鼠进行移植。移植后4周,检测血清白蛋白、转氨酶、胆红素、Ⅳ型胶原;HE和Masson染色观察肝纤维化程度。结果 MSCs移植后4周,移植组和模型组大鼠肝的纤维组织增生较对照组大鼠重,差异有统计学意义(P<0.01);但移植组大鼠肝的纤维组织增生较模型组大鼠显著减轻,差异有统计学意义(P<0.05)。模型组和移植组的肝功能较对照组差,差异有统计学意义(P<0.01);但与模型组比较,移植组大鼠血清白蛋白显著增高,胆红素、转氨酶、Ⅳ型胶原显著降低,差异有统计学意义(P<0.05)。结论肝纤维化模型大鼠骨髓MSCs移植4周后肝功能改善,肝纤维化程度显著减轻。     

Effect of in vitro culture on a chondrocyte-fibrin glue hydrogel for cartilage repair

Research in tissue engineering has been focused on articular cartilage repair for more than a decade. Some pioneristic studies involved the use of hydrogels such as alginate and fibrin glue which still possess valuable potential for cartilage regeneration. One of the main issues in cartilage tissue engineering is represented by the ideal maturation of the construct, before in vivo implantation, in order to optimize matrix quality and integration. The present study was focused on the effect of in vitro culture on a fibrin glue hydrogel embedding swine chondrocytes. We performed an evaluation of the immunohistochemical and biochemical composition and of the biomechanical properties of the construct after 1 and 5 weeks of culture. We noticed that chondrocytes survived in the fibrin glue gel and enhanced their synthetic activity. In fact, DNA content remained stable, while all indices of cartilage matrix production increased (GAGs content, immunohistochemistry for collagen II and safranin-o staining). On the other hand, the biomechanical properties remained steady, indicating a gradual substitution of the hydrogel scaffold by cartilaginous matrix. This demonstrates that an optimal preculture could provide the surgeon with a better engineered cartilage for implantation. However, whether this more mature tissue will result in a more efficient regeneration of the articular surface still has to be evaluated in future investigations.     

壳聚糖水凝胶局部注射对心梗的治疗作用研究

目的 观察心梗部位局部注射壳聚糖水凝胶的促血管生成及心肌保护作用.方法 将19只Wistar大鼠随机分为壳聚糖水凝胶注射组、单纯心肌梗死模型组、PBS注射组.结扎大鼠冠状动脉致心肌梗死30min后,心肌缺血处注射壳聚糖水凝胶.术后1、2、4周,分别处死动物进行组织学、免疫组织化学检查.结果 壳聚糖水凝胶注射1、2周后有明显存留,4周后已降解吸收,无明显残留,但注射后明显提高了心梗部位的心室壁厚度,梗死部位的血管密度明显增加,存活的心肌数量增多,结缔组织增生程度减轻.结论 心梗部位注射壳聚糖水凝胶可明显促进血管增生,有利于梗死部位心肌细胞的存活,适宜作为可注射性组织工程化心肌的支架材料.     

Healing of meniscal tissue by cellular fibrin glue: an in vivo study

Menisci represent fundamental structures for the maintenance of knee homeostasis, playing a key role in knee biomechanics. However, their intrinsic regenerative potential is poor. As a consequence, when a lesion occurs and the meniscus is partially removed by surgery, knee mechanics is subject to dramatic changes. These have been demonstrated to lead often to the development of early osteoarthritis. Therefore, menisci should be repaired whenever possible. In the last decades, tissue engineering approaches have been advocated to improve the reparative processes of joint tissues. In this study, the bonding capacity of an articular chondrocytes-fibrin glue hydrogel was tested as a biologic glue to improve the bonding between two swine meniscal slices in a nude mouse model. The composites were wrapped with acellular fibrin glue and implanted in subcutaneous pouches of nude mice for 4 weeks. Upon retrieval, a firm gross bonding was observed in the experimental samples while none of the control samples, prepared with acellular fibrin glue at the interface, presented any sign of bonding. This was consistent with the histological and scanning electron microscope findings. In particular, a fibrocartilaginous tissue was found at the interface between the meniscal slices, partially penetrating the native meniscus tissue. In order to overcome the lack of regenerative properties of the meniscus, the rationale of using cellular fibrin glue is that fibrin provides immediate stability while carrying cells in the site of lesion. Moreover, fibrin gel is recognized as an optimal scaffold for cell embedding and for promoting fibrocartilaginous differentiation of the cells which synthesize matrix having healing property. These results demonstrated the potential of this model for improving the meniscal bonding. However, further orthotopic studies in a large animal model are needed to evaluate its potential for clinical application.     

In vivo distribution of Tc-99m labeled recombinant tissue-type plasminogen activator in control and thrombus-bearing rats

In vivo distribution of Tc-99m labeled recombinant tissue-type plasminogen activator (Tc-99m-rt-PA) was studied in control rats and thrombus-bearing rats. To compare fibrin binding in vivo with that in vitro, Tc-99m-rt-PA binding to fibrin gel in vitro was also imaged. Rapid blood clearance and accumulation into the liver and kidneys were observed in both control and thrombus-bearing rats. Accumulation in the stomach, which indicates instability of labeled rt-PA in vivo, was very low until two hours after injection. Tc-99m-rt-PA accumulation in the clots was higher than that in skeletal and heart muscles, although it was lower than in blood, liver, and kidneys. Administration of aprotinin, an antifibrinolytic agent, significantly prolonged clot accumulation of Tc-99m-rt-PA at 30 minutes after injection. These results suggest that fibrinolysis is responsible for the low rt-PA concentration in the clots. A scintigram of a thrombus-bearing rat demonstrated increased radioactivity at the clot forming site. On the other hand, Tc-99m-labeled human albumin, which was used as a control, was not accumulated in the clot. Tc-99m-rt-PA binding to fibrin gel in vitro was clearly imaged. By comparison, in vivo fibrin binding of Tc-99m-rt-PA was much lower than in vitro. The reasons for low thrombus uptake in vivo may be: 1. biochemical inactivation of extrinsically administered rt-PA by t-PA inhibitor. 2. fibrinolysis by rt-PA activated plasminogen. Overcoming these limitations will enable Tc-99m-rt-PA to reach the stage of clinical trials.