3D生物打印的肝结构体植入兔纤维化肝的研究

通过将三维(3D)生物打印的类肝水凝胶结构体,经肝表面贴覆法植入纤维化肝兔模型,探讨改善肝纤维化以及形成类肝组织结构的可行性。采用兔离体肝脏胶原酶消化法提取兔原代肝细胞,并采用四氯化碳(CC1₄)皮下注射诱导兔肝纤维化模型。选取30只肝纤维化兔,随机分为实验组、对照组、假手术组,每组10只。3D生物打印兔肝细胞-海藻酸钠-明胶3D水凝胶网格状结构体片段,经肝表面贴覆法植入兔纤维化肝,为实验组。植入16 d后,检测兔肝功能生化指标及肝脏组织病理学变化,观察肝纤维化的发展程度,以及类肝组织结构形成情况。按照设计的参数,打印出含有兔原代肝细胞的网格状水凝胶结构体。通过LIVE/DEAD双荧光染色观察,打印后肝结构体的细胞存活率为82%±3%。兔肝细胞水凝胶支架体内植入16天后,可见每个植入物的大部分支架材料尚未降解,未降解的植入物与肝脏贴合紧密,融合生长。通过检测兔肝功能生化指标及植入物之下的肝脏组织病理学观察,实验组兔肝功能生化指标分别为:ALT(90.26±13.05)U/L,AST(75.37±13.45)U/L,γGT(16.62±6.72)U/L,ALB(32.48±4.43)g/L,与对照组、假手术组之间比较均P>0.05。实验组、对照组、假手术组的肝纤维化程度病理学检查评分结果分别为2.95±0.50、3.11±0.58、3.02±0.62,各组之间比较均P>0.05。兔肝细胞-3D水凝胶支架的植入,使肝功能生化指标及兔肝脏纤维化程度稍有改善,但各组之间比较均无统计学意义。组织学观察结果表明,实验组植入物中肝细胞均匀分布,未见细胞变性及死亡,可形成类肝组织结构。3D生物打印的肝结构体具有再造肝样组织片段和实现部分肝功能的潜力,将在基础医学和临床之间搭建桥梁,为肝脏再生打下基础。     

Studies on the Pathogenesis of Experimental Chronic Active Hepatitis in Rabbits: I. Induction of the Disease and Protective Effect of Allogeneic Liver Specific Proteins

By long-term immunization intraperitoneally over 143 weeks with human liver specific proteins (HLP) together with complete Freund''s adjuvant a chronic active hepatitis (CAH) was produced in rabbits, and the protective effect of allogeneic liver specific proteins (RLP) against the development of liver lesions was tested. The animals (white New Zealands) were divided into 6 groups. Immunization with HLP caused in all animals the development of CAH or liver cirrhosis, depending on the antigen dose. An autoimmunity against liver antigens, induced by HLP, persisted by immunization with RLP. The combined application of HLP and RLP (native and aggregate free, respectively) caused the development of histologically demonstrable liver lesions in only 13% of the animals. Underlying the pathogenesis of the experimental CAH is, according to the existing results, a cellular cytotoxicity. This concept is supported by the protective effect of RLP on cellular immune reactions against RLP and the induction of liver lesions. The importance of an antibody mediated cellular cytotoxicity for the pathogenesis of the experimental CAH has to be clarified by further experiments.     

控制性深低温对家兔血液流变性的影响

目的 通过建立低温家兔模型,探讨低温对家兔血液流变性的影响及其作用机制.方法 20只家兔随机分为2组,每组10只,分别设为对照组和降温组.所有家兔经3%戊巴比妥钠麻醉作气管切开插管后接呼吸机控制呼吸,然后静脉注入芬太尼、哌库溴胺加深麻醉;采用体表降温法将降温组家兔体温降至(25±1)℃,维持3 h.实验组于降温前、深低温、复温开始、复温至37℃时各采动脉血测定血液流变学各项指标.结果 降温组在鼻咽温度从36℃降至25℃后,全血黏度高、中、低切及全血还原黏度低切与降温前比较明显升高(P＜0.05),且低温持续过程中变化不大;复温后高、中、低切全血黏度及全血还原黏度低切显著降低(P＜0.05),与对照组比较,无统计学差异(P＞0.05).降温、低温及复温过程中血浆黏度和HCT变化不明显.结论 低温对家兔血液流变性有明显的影响,主要表现在各种切变率下全血黏度明显升高.     

Morphological and Autoradiographic Studies on the Corneal and Limbal Epithelium of Rabbits

The investigation was centered on the morphological features of the conjunctiva–cornea transition (limbus) of the rabbit eye and the proliferative behavior of its epithelium. The eyes were processed for examination with light and electron microscopy, as well as for autoradiography after intravitreal injection of [³H]thymidine ([³H]TdR). At the sites of extraocular muscle insertion, the vascularization of the stroma extended to the peripheral cornea, and the limbal epithelium was thin with its basal stratum made up by clear cuboidal cells. In between the muscle insertions, the cuboidal clear cells, as well as the stroma blood vessels, were scarce. At the light microscope level, the basement membrane was distinct in the cornea but not in the limbus or the conjunctiva. Autoradiographs demonstrated that, at the limbus, the basal cells migrated very quickly to the suprabasal region and remained there up to the 28‐day interval. Labeled cells were identified in all epithelial layers of the cornea, including the basal one, at 21 and 28 days but not in the limbal basal clear cells. The rate of renewal of conjunctival epithelium was similar to that observed for the transition with scarce clear cells. The high‐resolution autoradiographs demonstrated that the basal cuboidal clear limbal cells exhibit a quick renewal and that they are not label‐retaining cells. These latter ones were detected all over the corneal epithelium and in the suprabasal layers of the limbus up to 28 days, in physiological conditions, without the need of stimulation by damage to the corneal epithelium. Anat Rec, 291:191–203, 2008. © 2008 Wiley‐Liss, Inc.     

热休克预处理对家兔肝缺血-再灌注损伤的影响

目的 :探讨热休克预处理对肝缺血 再灌注损伤的影响。方法 :随机将家兔分为对照组和热休克预处理组 ,复制肝脏缺血 再灌注模型 ,分别于缺血前、缺血 45min、再灌注 45min三次取血 ,赖氏法检测血清谷丙转氨酶 (ALT)活性。结果 :热休克预处理组缺血前的ALT活性稍高于对照组 ,但是缺血后及再灌注后显著低于对照组。结论 :热休克预处理对肝细胞有轻微的损伤作用 ,但是对缺血后和再灌注后损伤均有显著的保护作用。     

环磷酰胺对家兔血液流变性的影响

观察抗肿瘤药物环磷酰胺（ＣＴＸ）对家兔血液流变性的影响。结果显示：ＣＴＸ给药组（１０只）多项血液流变学指标明显异常，与正常对照组（８只）比较具有显著性差异（Ｐ＜０．０５～０．００１），标志着ＣＴＸ有引起高粘滞血症的作用。提示：恶性肿瘤病人应用ＣＴＸ后，可加重原有的高粘滞血症。这一结果可作为肿瘤化疗并用活血化瘀及抗凝治疗的依据     

Studies on the Induction of Antibody Synthesis against Sulfanilic Acid in Rabbits.

The present experiments (carried out in rabbits) have demonstrated that antibodies directed against new antigenic determinants introduced in rabbit serum albumin (RSA) through the coupling with sulfanilic acid (Sulf) were able to enhance (or'help') a primary antibody response to Sulf₁₁RSA. This enhancement phenomenon was shown to be specific for Sulf-carrier conjugates.     

Studies on the Induction of Antibody Synthesis against Sulfanilic Acid in Rabbits.

The following three results emerged from the present experiments concerning the induction of antibody synthesis against sulfanilic acid: 1) the anti-Sulf antibody response in rabbits seems to be much more carrier-dependent when Sulf is coupled to heterologous serum protein compared to homologous (autologous) serum protein; 2) anti-NAD antibodies (antibodies directed against the new antigenic determinants introduced into the carrier through the coupling with hapten) were able to induce a secondary response to Sulf₁₁RSA in rabbits primarily stimulated with Sulf₁₀BGG; and 3) anti-NAD antibodies were able to induce an immune response to Sulf₁₁RSA in rabbits made unresponsive to Sulf₁₁RSA by i.v. injections of soluble Sulf₁₁RSA.     

Studies on the Relationships of IgA to Human Liver IgA Deposition in Non-alcoholic Liver Diseases

An investigation was conducted to clarify the relationships of IgA to the human liver. Immunocytochemical studies were performed on biopsy specimens from patients with cirrhosis and chronic hepatitis without any apparent history of alcohol abuse. The results showed that 1) a large amount of IgA is associated with the sinusoidal surface of hepatocytes, endothelial cells and Kupffer cells, 2) this IgA contains J chain and can form a complex with secretory component, and 3) this mainly belongs to the IgAl subclass, 4) IgA in vesicles within hepatocytes and Kupffer cells is always associated with acid phosphatase activity, and 5) IgA containing vesicles within ductular epithelial cells always lack such enzyme activity. We conclude that 1) the IgA bound to the surface of hepatocytes, sinus endothelial cells and Kupffer cells is polymeric IgAl uncom-plexed with SC, and 2) this IgA occasionally enters these cells, and may be degraded in the lysosomes. 3) Polymeric IgA combines with SC in the ductular epithelium and may be secreted into bile. These findings suggest that J chain-linked polymeric IgA bound to the surface of hepatocytes and Kupffer cells has a certain pathological significance in liver diseases and might be involved in the clearance of excess IgA from the circulation. Acta Pathol Jpn 39: 363 372, 1989.     

Pathogenesis of Rinderpest Virus Infection in Rabbits II. Effect of Rinderpest Virus on the Immune Functions of Rabbits

Rinderpest virus infection was shown to induce marked suppression of both humoral antibody response and cell-mediated immunity in rabbits. The virus exhibited a suppressive effect on primary antibody response as indicated by a decrease in numbers of plaque-forming cells (immunoglobulin [Ig]M) and hemagglutinating antibody titers of both IgM and IgG types to sheep red blood cells, whereas there was no detectable effect of the virus on the production of memory cells. Virus-induced suppression of cell-mediated immunity was demonstrated by a decreased rate of proliferative response of peripheral lymphocytes to phytohemagglutinin stimulus and by a depression of delayed-type skin reactions to purified protein derivative. Such suppressive effects were indicated to persist for 14 days or longer. Alteration in phagocytic activity of the reticuloendothelial system was not observed. The relevance of the virus-induced histological lesions in the lymphoid tissues to the virus-induced immunosuppression was discussed.     

高压氧对兔脑膜微循环的影响

目的观察高压氧变化对兔脑膜微循环的影响。方法在兔大脑顶叶部位颅骨开窗，暴露局部软脑膜，然后将兔置于高压氧舱内，加压的同时给予持续常压面罩吸氧，分别于加压吸氧前及加压吸氧后0．13、0．16、0．19、0．22及0．25MPa时进行脑膜微循环检测。结果随氧分压逐渐增高，血管径逐渐收缩，血流速逐渐减慢，血流量逐渐减少，特别是在0．25MPa时上述变化明显，血管收缩了24．3％（P〈0．05）、血流量下降了33．3％（P〈0．05）。3例（27％）出现了毛细血管周围的渗出或出血现象。结论高压氧压力在0．2MPa时脑膜微循环血流量最佳，为合理制订高压氧治疗方案提供了实验依据。