生物血管基质材料的抗原表达及力学特性

了解胰蛋白酶处理前后版纳微型猪近交系血管的抗原表达及力学特性，为异种移植及猪血管用于血管组织工程基质材料提供资料，取猪颈动脉，胰蛋白酶预处理猪血管前后不同时相点取标本石蜡包埋，采用组织形态学方法结合计算机图像分析，对猪血几何形态及纤维结构进行形态定量学研究，分别测定胰蛋白酶作用前后猪动脉压力直径关系，冰冻切片，用亲和免疫组化法检测α-gal抗原表达。结果表明α-gal抗原仅表达于血管内皮细胞，胰蛋白酶预处理血管后，未见α-agl抗原表达，血管内细胞包括内皮细胞，平滑肌细胞已去除，HE染色未见纤维断裂，血管无明显形变，处理前后血管顺应性相差不显著，提示经胰蛋白酶预处理的猪血管抗原性大大降低，其力学特性相差不显著，可用于血管基质材料。     

人血管平滑肌细胞种植构建工程生物血管的实验研究

了解猪血管去细胞后平滑肌细胞种植情况,为猪血管用于血管组织工程提供资料。取猪颈动脉,生物酶预处理猪血管,在自行设计制作的新型动力性生物反应器中,用原代培养的人平滑肌细胞种植在去细胞血管基质材料内,HE染色及银染检测平滑肌细胞种植效果。结果表明生物酶预处理血管后,HE染色及银染检测可见血管腔平滑肌细胞形态正常,沿血管长轴分布,提示经生物酶预处理的猪血管人平滑肌细胞能成功种植,可望构建实用的组织工程血管。     

脂肪间充质干细胞分化为内皮细胞并体外构建组织工程心脏瓣膜

背景：组织工程心脏瓣膜是利用组织工程技术将种子细胞种植于瓣膜支架上所构建的一种人工瓣膜,目前国内外研究主要集中于种子细胞来源及支架选择上。 目的：探讨人脂肪间充质干细胞体外向内皮细胞诱导分化后的细胞作为种子细胞,脱细胞猪主动脉瓣膜作为支架体外构建组织工程心脏瓣膜的可行性。 方法：利用吸脂术采集脂肪组织,分离、培养脂肪间充质干细胞,流式细胞仪鉴定细胞表型;免疫细胞化学方法及RT-PCR检测细胞分化标志物;应用Triton X-100联合胰蛋白酶的方法制备脱细胞猪主动脉瓣支架,将体外培养扩增的诱导分化后的内皮细胞种植于支架上构建组织工程心脏瓣膜,光镜及电镜下观察组织工程心脏瓣膜的组织学结构。 结果与结论：脂肪组织分离培养的脂肪间充质干细胞向内皮细胞诱导分化后表达CD31、CD34、CD144、Ⅷ因子和内皮型一氧化氮合成酶等内皮细胞特异性抗原;脱细胞猪主动脉瓣膜支架脱细胞完全,弹力纤维及胶原纤维保持完整;构建的组织工程心脏瓣膜可见支架上排列连续的单细胞层。提示脂肪间充质干细胞在体外向内皮细胞诱导分化后已初步具有内皮细胞功能,在脱细胞猪主动脉瓣膜支架上生长良好,可以在体外初步构建组织工程心脏瓣膜。     

心脏瓣膜组织工程支架的制备及细胞种植

为了探讨心脏瓣膜组织工程支架的制备及细胞种植 ,我们将新鲜猪心瓣膜用胰蛋白酶及 DNA酶消化去除细胞 ,光镜和电镜观察其结构 ,并将人脐静脉内皮细胞株、猪颈动脉内皮细胞和狗肌成纤维细胞滴种在脱细胞猪心瓣膜上 ,HE和免疫组化染色观察。结果显示胰蛋白酶联合 DNA酶消化去除了所有细胞 ,而瓣膜的三维结构保持完好。种植的人内皮细胞几乎完全覆盖了瓣膜的表面 ,猪内皮细胞在瓣膜上呈斑块状生长 ,狗肌成纤维细胞不但生长于瓣膜表面 ,且渗透到基质内部生长。这表明 ,胰蛋白酶联合 DNA酶消化是一种较为理想的猪瓣膜脱细胞方法 ;人和猪血管内皮细胞及狗肌成纤维细胞均能在猪瓣膜支架上较好地黏附和生长。     

应用脱细胞血管基质构建组织工程血管的初步研究

目的： 采用脱细胞处理的猪胸主动脉血管基质作为支架材料，接种人脐带血管内皮细胞，构建组织工程血管。方法: 以新鲜猪胸主动脉为原材料， 1% Triton X-100为脱细胞试剂，制备脱细胞血管基质；采用冷冻干燥和热交联法对脱细胞血管基质进行改性，并进行组织学观察及力学性能测定。用人脐带内皮细胞经培养和扩增后，再与所制备的脱细胞血管基质进行复合培养，光学显微镜及扫描电镜观察内皮细胞生长状况。结果: 1% Triton X-100处理84h的猪胸主动脉，既能完全脱除血管中细胞，同时又完整保留血管基质的三维结构；对脱细胞血管基质冷冻干燥24 h，120 ℃下热交联12 h，能有效提高材料的机械强度，断裂强度可达到1.70 MPa。扫描电镜下可见，脱细胞血管基质与内皮细胞复合培养7 d，已形成典型血管内膜样结构。结论: 经改性后的脱细胞血管基质与内皮细胞具有良好的相容性，用其作为支架材料与内皮细胞复合培养，有望应用于构建组织工程化血管。     

兔骨髓内皮祖细胞在组织工程血管构建中的实验研究

建立体外分离、培养及鉴定兔骨髓血内皮祖细胞(endothelial progenitor cell,EPCs)的方法,并探讨其在血管组织工程构建过程中的功能。采用密度梯度离心法分离单个核细胞,经培养鉴定为EPCs后作为种子细胞接种于人纤维连接蛋白包被(FN)的组织工程血管支架上,加入血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)进行体外诱导培养,同时设置未包被纤维连接蛋白及未添加血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)的培养方法作为对照组,体外培养10 d后,对构建的组织工程血管进行鉴定分析。分离培养的骨髓单个核细胞呈典型的"铺路石样"外观。经免疫荧光检测、细胞吞噬功能鉴定为内皮祖细胞;种植细胞10 d后结果显示:加入纤维连接蛋白和血管内皮生长因子的血管支架可见细胞种植密度明显高于对照组,扫描电子显微镜观察到,血管内腔面较为完整的覆盖内皮细胞。HE染色显示:内皮细胞在血管支架上成活并较为均匀;免疫组化结果显示分化为成熟血管内皮细胞并表达VEGFR-2、vWF、CD34。兔骨髓单个核细胞体外培养可以诱导分化为内皮祖细胞,血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)和纤维连接蛋白(FN)的组合更有利于内皮祖细胞在血管支架上增殖和分化,为人工血管制备创造了条件。     

兔骨髓内皮祖细胞在组织工程血管构建中的实验研究

建立体外分离、培养及鉴定兔骨髓血内皮祖细胞（endothelial progenitor cell,EPCs）的方法,并探讨其在血管组织工程构建过程中的功能。采用密度梯度离心法分离单个核细胞,经培养鉴定为EPCs后作为种子细胞接种于人纤维连接蛋白包被（FN）的组织工程血管支架上,加入血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）进行体外诱导培养,同时设置未包被纤维连接蛋白及未添加血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）的培养方法作为对照组,体外培养10 d后,对构建的组织工程血管进行鉴定分析。分离培养的骨髓单个核细胞呈典型的＂铺路石样＂外观。经免疫荧光检测、细胞吞噬功能鉴定为内皮祖细胞;种植细胞10 d后结果显示：加入纤维连接蛋白和血管内皮生长因子的血管支架可见细胞种植密度明显高于对照组,扫描电子显微镜观察到,血管内腔面较为完整的覆盖内皮细胞。HE染色显示：内皮细胞在血管支架上成活并较为均匀;免疫组化结果显示分化为成熟血管内皮细胞并表达VEGFR-2、vWF、CD34。兔骨髓单个核细胞体外培养可以诱导分化为内皮祖细胞,血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）和纤维连接蛋白（FN）的组合更有利于内皮祖细胞在血管支架上增殖和分化,为人工血管制备创造了条件。     

脂肪源干细胞向血管内皮细胞的分化

背景：脂肪来源干细胞在体内储备丰富,体外增殖快速,具有多向分化潜能,是目前组织工程种子细胞的研究热点。近年来越来越多的研究表明脂肪干细胞在一定条件下可被诱导分化为内皮细胞,促进血管生成。 目的：观察兔脂肪干细胞体外分离培养及诱导分化为血管内皮细胞的生物学特性。 方法：取兔附睾处脂肪,采用胶原酶消化分离获得脂肪干细胞,体外培养至第3代后加入血管内皮细胞生长因子与碱性成纤维细胞生长因子联合诱导分化,对诱导前后细胞进行形态学观察、生长曲线测定、免疫组织化学染色及流式细胞仪表型检测。 结果与结论：兔脂肪干细胞生长旺盛,第3代兔脂肪干细胞呈成纤维细胞样,生长曲线呈“S”型,15代以内细胞形态未见明显变化。免疫荧光法检测Vimentin阳性,流式细胞仪检测CD44表达阳性,CD31表达阴性;诱导后细胞CD31表达阳性,CD44表达阴性。第3代兔脂肪干细胞向血管内皮细胞诱导分化21 d显微镜下呈铺路石样形态,血管内皮细胞第Ⅷ因子相关抗原染色细胞阳性,透射电镜下可见W-P小体。提示脂肪干细胞在体外可诱导分化为血管内皮细胞,可为组织工程血管提供理想的种子细胞。     

应用猪主动脉脱细胞基质制备新型组织工程血管支架:生物相容性及力学性能评价(英文)

背景:组织工程血管构建的关键依赖于理想的支架。猪血管作为组织工程血管构建材料已有广泛应用,但其较高的免疫原性及较差的力学强度限制了该材料作为组织工程支架的应用。目的:应用猪主动脉脱细胞基质制备一种新的具有良好机械性能及生物相容性的组织工程血管支架。方法:对猪主动脉进行脱细胞处理和热交联改性制备脱细胞血管基质支架,采用苏木精-伊红染色及生物力学分析评估其脱细胞效果及血管基质的力学性能。将人脐静脉血管内皮细胞接种于脱细胞血管基质支架中进行体外培养,评估其生物相容性。结果与结论:用1%的TritonX-100溶液处理猪主动脉84h可完全脱除血管细胞,同时不破坏血管基质结构;经真空下120℃热交联处理12h,脱细胞基质的拉伸断裂强度得到明显提高,达到1.70MPa。在该改性血管基质支架上接种人脐带静脉内皮细胞体外培养7d,扫描电镜显示内皮细胞呈现典型的血管内皮层状结构。表明猪主动脉经过脱细胞处理能够维持血管基质完整,冷冻干燥和真空热交联处理可有效提高其拉伸强度,且对血管内皮细胞具有良好的相容性。     

内皮祖细胞作为组织工程瓣膜内皮种子细胞的可行性研究

目的 探讨利用外周血内皮祖细胞（EPC8）制备组织工程瓣膜的可行性。方法 分离人外周血EPCs，采用酶-去垢剂法去除新鲜猪主动脉瓣细胞制备去细胞瓣膜支架，将培养的人外周血EPCs接种到去细胞瓣膜上。结果 经酶-去垢剂法去除新鲜猪主动脉瓣细胞后，细胞成分全部去除，纤维支架保存完好。去细胞处理后瓣膜无明显细胞毒性。人外周血EPCs与去细胞瓣膜共孵育2周后，细胞紧贴瓣膜表面生长形成一层连续的单细胞层，初步生物力学测定示去细胞前与再内皮化后，瓣膜力学特性无明显改变。结论 外周血分离培养扩增得到的EPCs能够再内皮化去细胞猪主动脉瓣构建组织工程瓣膜，外周血EPCs是组织工程瓣膜内皮种子细胞的新的来源。     

Distribution of Human Leukocyte Antigen-ABC and -D/DR Antigens in the Unfixed Human Testis

ABSTRACT: The distribution of the major histocompatibility complex (MHC) antigens in the unfixed human testicle was studied by indirect immunofluorescence. Three murine monoclonal antibodies to the common determinants of class I MHC antigens (human leukocyte antigen [HLA]-ABC) and three against class II MHC antigens (HLA-D/DR antigens), respectively, were utilized. No class I MHC antigens were identified on developing testicular germ cells including spermatozoa, but interstitial cells between the seminiferous tubules (including Leydig cells) and blood vessel endothelium expressed the antigen. Class II MHC antigens were not found on any cells within the seminiferous tubules. However, the class II antigen was identified on dendritic-like cells between the seminiferous tubules and on vessel endothelium, although its expression was expectedly limited. These findings indicate that human testicular germ cells express minimal or no MHC antigens.     

Class II antigens on canine T lymphocytes

A panel of crossreactive anti-human, -mouse and -rat MHC class II monoclonal antibodies (Mabs) was used to examine MHC class II antigen expression on canine T lymphocytes by cytofluorometry. The presence of MHC class II antigens was demonstrated on activated T lymphoblasts as well as on non-stimulated peripheral blood T lymphocytes. A number of anti-MHC class II Mabs reacted only with activated T lymphoblasts. Immunoprecipitation studies confirmed the Ia-like or MHC class II molecular character of the antigens on canine T cells. The expression of MHC class II antigens on peripheral blood T lymphocytes appeared to be not induced by stimulation of the T cells, as purified T lymphocytes of specific pathogen free dogs reacted with anti-MHC class II Mabs. Moreover, the study indicates that MHC class II antigen expression is present in the neonatal thymus. Lectin stimulated and allogeneically stimulated T lymphoblasts showed a stronger expression of MHC class II antigens in comparison with non-stimulated T cells.     

An antigenic determinant common to lymphocytes and Langerhans cells of the guinea pig

A mouse anti-guinea pig monoclonal antibody, designated MSgp 2, was derived by the fusion of NS-1 myeloma cells with mouse splenocytes hyperimmunized with guinea pig B cells. MSgp 2 reacts with an antigen present on the majority of lymphocytes. An unexpected finding was the expression of the MSgp 2 antigen on epidermal Langerhans cells, as defined by cell morphology, expression of MHC class II antigen and presence of Birbeck granules in positive cells. It is suggested that the tissue distribution of MSgp 2 antigen on lymphocytes of the lymph node could indicate a role for this determinant in cell migration.     

Behaviour of guinea pig T cells stimulated by antigen, allo-antigen and mitogen

The expression of major histocompatibility (MHC) antigens on guinea pig T cells was used as a functional marker for lymphocyte activation. Antigen-stimulated lymphocytes were recovered from guinea pigs responding to the contact sensitizer DNFB, and isolated T cells were then phenotyped using a new antiguinea pig monoclonal antibody, MSgp7. The level of expression of MHC class II, as defined by the monclonal antibody, MSgp8, was increased on T cells recovered 4 days after sensitization, as compared with unsensitized controls. The value of this experiment was extended by measuring MHC class II expression on T cells stimulated in vitro by the mitogen concanavalin A, where a clear increase in MSgp8 binding was also observed. Confirmation of the specificity of MSgp8 for guinea pig MHC class II antigens was achieved by studying the inhibitory capacity of this antibody on an MHC class II restricted mixed leucocyte reaction. The combination of antibodies MSgp7 and MSgp8 with flow cytometry could be applied to other guinea pig experimental models to quantitate the expression of MHC class II antigens on T cells to determine their putative value in disease manifestation.     

Immunohistochemical study of the expression of major histocompatibility complex (MHC)-determined antigens in renal tissue of MRL/lpr mice

Expression of MHC antigens in renal tissue of MRL/lpr(H-2K) mice were demonstrated by monoclonal antibodies against class I(KK) and class II(IAK) antigens and ABC immunoperoxidase method. The expression of KK antigen in glomeruli, tubules and vessels of kidney and IAK antigen in glomeruli tubules was stronger in mice fed with beef tallow diet after gamma-interferon treatment than in the control group. This result suggests that using of gamma-interferon may enhance the presentation of MHC antigens in renal tissue of MRL/lpr mice. In mice raised with Menhaden fish oil diet after gamma-interferon treatment, however, the enhancement of expression of IAK antigen was detected only in renal tubules. In comparing with mice fed with beef tallow after gamma-interferon injection, the expression of KK antigen in glomeruli, vessels and IAK in the dendritic cells of renal interstitium was weaker in mice fed with fish oil. This result indicates that fish oil can more or less inhibit the expression of MHC antigens in renal tissue of mice. The mechanism of the inhibitory action of fish oil remains to be elucidated.     

Cytotoxicity of Tumour-Bearer Serum to D23 Hepatoma Cells Derived from Solid Tumours, Ascites or in Vitro Cultures

The complement-dependent cytoloxicity of antibodies in tumour-bearer serum (TBS) from rats carrying the chemically induced D23 hepatoina was investigated. Target cells were D23 cells from solid tumours (D23sol), from ascites tumours (D23asc), or from in vitro growing cell cultures (D23cc). The D23asc and D23cc cells were not lysed when used as target cells in the assay, although they evoke cylotoxic antibodies when growing in vivo. The D23 ascites cells became susceptible to complement-dependent lysis after trypsin treatment. This was, however, not due to unmasking of target antigens, since untreated D23 ascites cells absorbed cytoloxic antibodies as efficiently as trypsinized cells. No increase in susceptibility to complement-dependent lysis was observed after trypsin treatment of D23cc cells. Absorption of cytotoxic antibodies with D23cc cells showed no or very low antigen expression on the surface of these cells. They did, however, contain the antigen(s), since 3 m KCI extracts of the D23cc cells could inhibit the complement-dependent cytoloxicity of D23sol TBS against D23sol cells. From these data it was concluded that the susceptibility of hepatoma cells for antibody-mediated complement lysis is not only correlated with antigen expression, as was the case with the in-vitro-cultivated cells, but is also dependent on increased lylic susceptibility after trypsin treatment of the cells.     

Class II MHC antigens in the rat digestive system. Normal distribution and induced expression after interferon-gamma treatment in vivo

The normal and interferon-gamma induced expression of class II MHC antigens was investigated immunohistologically in the digestive system of LEW rats. In the normal state class II molecules were present in interstitial dendritic cells, B lymphocytes and epithelial cells. Epithelial class II expression was restricted to enterocytes in certain portions of the small intestine and to some duct epithelia in salivary glands. After continuous intravenous infusion of interferon-gamma (IFN-gamma) for 3 days, class II MHC antigens were induced in large vessel endothelium and in the surface epithelia of the tongue, oesophagus and proventricle. In the gastric glands class II molecules appeared in mucous neck cells and in parietal cells, while adjacent mucous surface cells and chief cells did not acquire class II reactivity. All enterocytes, including the previously negative colonic epithelium, were induced to express class II antigens. In the salivary glands class II antigens appeared in all duct epithelia. Serous acinar cells were induced in the parotids, but in the submandibular glands and in the pancreas the serous gland epithelium stayed negative. Our study thus shows that the effects of IFN-gamma on class II MHC antigen expression in vivo depend on the differentiation pathway of the individual cell. The normal distribution in rats suggest that class II MHC antigens may play a role in peptide transport across specialized epithelia. It remains to be determined whether such a function is enhanced after IFN treatment.     

豚鼠肠系膜淋巴管平滑肌细胞的培养

目的 :培养豚鼠肠系膜淋巴管平滑肌细胞。方法 :肠鼠肠系膜淋巴管用胰蛋白酶消化二次 ,第一次消化 15min ,除去肠系膜、淋巴管外膜等组织 ,第二次消化中膜的平滑肌 ,3 0～ 45min后 ,吸出未消化的组织 ,获得平滑肌细胞。常规培养、传代。结果 :培养细胞经光镜、电镜和荧光显微镜可观察到平滑肌的典型的形态和结构。结论 :所培养细胞为平滑肌细胞 ,此法可应用于管径较小的淋巴管和血管平滑肌细胞的培养。