用新生大鼠肝细胞体外构建工程化肝组织

目的:在供体肝短缺的情况下,构建可植入的工程化肝组织具有重要的临床意义.实验尝试以新生大鼠肝细胞为种子细胞体外构建工程化肝组织,以期进一步的体内植入.方法:实验于2007-04/08在解放军总医院普通外科研究所完成.①实验材料:SPF级、出生24 h以内的雄性SD新生大鼠.②实验过程:采用胰酶消化法获取新生大鼠肝细胞;以2×L-DMEM液(添加地塞米松10 μg/L、表皮生长因子20 μg/L、肝细胞生长因子40 μg/L及胰岛素0.04 U/mL)与液态鼠尾胶原等比例混合;再将肝细胞与胶原凝胶复合构建细胞/凝胶复合物,接种于培养板进行培养.③实验评估:培养后第1,3,5,7,9天,采用相差显微镜观察、四甲基偶氮唑盐比色法、苏木精-伊红染色和免疫组织化学染色分别对工程化组织的生长情况及组织形态特征进行观察,并对工程化组织的白蛋白合成功能及尿素的代谢水平进行评价.结果:①肝细胞与胶原凝胶复合后,细胞均匀的分布在复合物中.在整个培养过程中,肝细胞保持着稳定的细胞形态.②四甲基偶氮唑盐检测显示肝细胞活性在生长初期平缓下降,直至第5天时仍然保持75%的细胞活性,之后快速下降.③体外培养5 d后,相差显微镜下观察显示肝细胞在胶原凝胶中呈三维立体生长,并保持肝细胞胞体圆形,核大而圆的特异形态;免疫组织化学证实这些肝细胞抗白蛋白抗体染色呈强阳性.④对培养上清中白蛋白和尿素的含量测定表明胶原凝胶中的肝细胞在培养初期保持着稳定的代谢合成功能.结论:用新生大鼠肝细胞及胶原构建出一种有功能的工程化肝组织模型,这种模型可以应用于今后的工程化肝组织研究.     

脊髓组织工程的体外构建

目的:观察神经干细胞在聚乳酸-羟基乙酸支架上的生长和分化行为,探讨组织工程体外构建人工脊髓修复脊髓损伤。方法:实验于2005-10/2006-06在南京医科大学第一附属医院中心实验室完成。①在聚乳酸-羟基乙酸支架(孔径200～300μm,孔隙率90%,聚乳酸/聚羟基乙酸=75∶25;山东医疗器械研究所)上种植孕14d的胚胎大鼠脑细胞,培养7d。②将培养7d的聚乳酸-羟基乙酸支架置入含体积分数为0.05的胎牛血清和含脑源性神经营养因子20μg/L的DMEM/F12培养基中继续培养5d,诱导其向神经元分化。③用倒置相差显微镜和扫描电镜观察细胞在聚乳酸-羟基乙酸支架上的生长和分化行为;应用免疫组织化学鉴定聚乳酸-羟基乙酸支架上培养的细胞。结果:①神经干细胞生长形态:神经干细胞克隆球充满聚乳酸-羟基乙酸支架孔隙,其形状近似孔隙的不规则几何形状。②神经干细胞超微结构:诱导分化后,大量神经干细胞长出的神经突触跨越支架孔隙的三维空间结构,布满支架的表面和孔隙,并彼此建立了突触联系。③诱导分化前后的聚乳酸-羟基乙酸石蜡切片免疫组织化学鉴定结果:诱导分化前后免疫组织化学鉴定分别为巢蛋白和微管相关蛋白2抗体阳性,说明诱导分化前是神经干细胞,诱导分化后有神经元形成。结论:聚乳酸-羟基乙酸适合神经干细胞的生长和分化,其孔隙结构规范调节了神经干细胞生长增殖的空间排列,而脑源性神经营养因子调控了神经干细胞的分化方向,可以利用该支架的空间结构和细胞因子适度调控神经干细胞在聚乳酸-羟基乙酸上的生长和分化构建脊髓组织。     

体外构建组织工程骨-软骨复合组织

背景：设计一体化、具有过渡结构的双层支架材料,复合软骨细胞、骨髓间充质细胞,有利于新生的骨与软骨组织之间形成良好界面。目的：模仿自然骨一软骨基质构建复合支架,以软骨细胞和骨髓间充质干细胞为种子细胞,体外观察复合组织的成软骨及成骨能力。方法：制备明胶一硫酸软骨素一透明质酸及明胶一陶瓷化骨多孔复合支架,构建自然骨一软骨基质复合支架,复合兔软骨细胞与骨髓间充质干细胞,分未成骨诱导与成骨诱导两组培养,并进行MTT、糖胺多糖含量、碱性磷酸酶活性检测,以及苏木精一伊红染色检测。结果与结论：未成骨诱导与成骨诱导两组骨髓间充质干细胞增殖及糖胺多糖含量差异无显著性意义。未成骨诱导组碱性磷酸酶活性缓慢上升,成骨诱导组诱导后碱性磷酸酶活性迅速上升,14d时达到稳定状态。两组苏木精一伊红染色结果无明显区别,均已形成含有双层组织的类似骨一软骨样组织,其间可见未降解支架形态,但由于基质形成不完善及支架未完全降解,此种结构不成熟,细胞分布不均匀,支架内部可见散在无细胞区域。证实采用两种细胞与双层结构的支架经体外分层复合能够形成组织工程骨软骨复合组织。     

组织工程软骨细胞体外培养技术要点

通过组织工程技术，采用自身软骨细胞体外增殖构建软骨来修复关节软骨损伤，在临床上已成为一种有效的新方法。近年来，有关体外软骨细胞培养技术的研究较多。通过对入选文献进行分析、整理，对影响体外构建软骨的因素包括培养空间、应力、细胞密度和氧张力等方面进行分析，体外采用三维多条件联合培养软骨细胞可以改善构建软骨的生物品质。但是影响软骨细胞生长的因素众多复杂，在体外如何更好地构建软骨，有待进一步研究。     

In vitro uptake of SCH 27899 (evernimicin) by rat alveolar macrophages

The in vitro uptake of [(14)C]evernimicin ([(14)C]SCH 27899) by primary cultures of rat alveolar macrophages and hepatocytes was determined. Both cell populations exhibited linear rates of uptake. However, the initial rate of drug uptake by alveolar macrophages was about threefold higher than that by hepatocytes. These findings demonstrate that [(14)C]evernimicin is taken up by rat alveolar macrophages, supporting the likelihood that the drug is able to reach sites of infection.     

Activation of adenylate cyclase by cholera toxin in rat liver homogenates.

The effect of cholera toxin on adenylate cyclase from rat liver has been studied in a broken cell preparation. The activation of the enzyme in this in vitro preparation requires the addition of nicotinamide adenine dinucleotide (NAD) to the incubation medium and the presence of cell components other than the membrane-bound adenylate cyclase. Once the activation of the cyclase is produced, the effect persists despite repeated washing or solubilization of the enzyme. The effect can be obtained with concentrations of cholera toxin as low as 0.4 nM after 15 min of incubation at 22 degrees C, and stimulation can be detected after only 5 min of incubation at 37 degrees C. The activation of the enzyme is still apparent after at least 2 h at 0 degrees C. Preincubation with choleragenoid in vitro does not interfere with this effect of the toxin. Animals pretreated by an intravenous injection of cholera toxin do not respond to the in vitro addition of cholera toxin and NAD to the same extent as untreated animals; i.e., the effects overlap to suggest that the in vitro effect is the same as that in vivo. Responses to isoproterenol, glucagon, and NaF were also similar in the in vitro broken cell-activated system, as previously reported for the enzyme activated in vivo.     

可降解组织工程心肌材料聚氨酯的体外评价

背景:体外构建组织工程化心肌是当今医学领域的研究热点,支架材料的选择和设计是心肌组织工程的关键环节,但目前仍未找到理想的心肌支架材料.目的:对新型可降解材料聚氨酯进行体外评价,初步探讨其作为心肌组织工程支架的可行性.方法:以赖氨酸基二异氰酸酯为硬段,以赖氨酸为扩展链合成新型聚氨酯(PU-Lys).在拉力机上测试材料的缝合强度和拉伸强度;在37℃,pH=7.4的磷酸盐缓冲溶液中检测聚氨酯的降解性能;采用细胞培养MTT法、细胞形态学观察方法,分析该聚氨酯的细胞毒性.结果与结论:力学性能检测得出该聚氨酯的拉伸强度为(8.1±0.1)MPa,缝合强度为(12.2±0.8)N;体外降解8周后质量损失为(13.1±0.3)%;MTT比色法结果显示细胞毒性为0～1级;细胞形态学观察显示L929细胞在聚氨酯材料浸提液中呈梭形或三角形,贴壁良好.提示此种新型解聚氨酯具有良好的力学性能和降解性,细胞相容性良好,符合组织工程心肌支架材料的应用要求.     

以3种材料为载体体外构建组织工程软骨

背景:纤维胶原蛋白被公认是菲常理想的载体,但其胶性易流动,降解较快,且通透性有待置疑.而海绵状胶原蛋白多微孔,三维立体结构,吸附性好,临时基质与细胞基质相近(Ⅱ型胶原),作者所查文献中作为骨髓间充质干细胞载体用于软骨形成的报道较少.目的:对比观察骨髓间充质干细胞在纤维蛋白海绵、生物蛋白海绵、明胶海绵载体中软骨形成的能力和可行性.设计、时间及地点:体外对比观察实验,于2004 12/2008-08在海南省人民医院中心实验室完成.材料:纤维胶原蛋自海绵由美国Sigma公司提供,生物蛋白海绵(主要成分为Ⅱ型胶原,并吸附定量的碱性成纤维细胞生长因子)由辽宁绿谷制药有限公司提供,明胶海绵由南京金陵制约有限公司提供.方法:抽取猪髂骨骨髓血,用密度梯度离心法分离出有核细胞,在含转化生长因子β 1的DMEM培养液中扩增骨髓间充质干细胞,将第3代骨髓间充质干细胞以载体内多点注射和表面点滴法植于Ⅱ型纤维胶原蛋白海绵、生物蛋白海绵及明胶海绵3种载体上,继续以含有转化生长凶子β 1的DMEM培养液体外培养.主要观察指标:观察细胞的生长情况,于4周取材进行大体组织观察,组织学分析及超微结构观察.结果:骨髓间充质干细胞存体外分离后可在转化生长因子β 1培养液下扩增.纤维胶原蛋白海绵组可见少量的软骨基质与细胞:生物蛋白海绵组可见较多的基质与软骨细胞,细胞生长活跃,明胶海绵组可见少量散在的胶原基质,但无细胞生长.生物蛋白海绵组软骨细胞阳性数量多于纤维胶原蛋白海绵组(P<0.01),两组都明显多于明胶海绵组(P<0.01).结论:骨髓间充质干细胞在生物蛋白海绵载体中软骨形成能力最强,纤维蛋白海绵次之,而明胶海绵与细胞生长不同步,软骨形成能力最差.     

Metabolism of antisense oligonucleotides in rat liver homogenates

Phosphorothioate antisense oligodeoxynucleotides are novel therapeutic agents designed to selectively and specifically inhibit production of various disease-related gene products. In vivo pharmacokinetic experiments indicate that these molecules are widely distributed in many species, with the majority of oligomers accumulating within liver and kidney. To better understand the metabolism of these agents, we studied the stability of several phosphorothioate oligodeoxynucleotides, their congeners, and second generation oligomer chemistries in rat liver homogenates. To examine metabolism, background nuclease activity was characterized in whole liver homogenates by using ISIS 1049, a 21-mer phosphodiester oligodeoxynucleotide. Nuclease activity could readily be detected in liver homogenates. Under optimized conditions, the predominant enzymatic activity was 3'-exonucleolytic and could be influenced by pH and ionic conditions. However, in addition to 3' exonucleases, 5' exo- and endonuclease activities were also observed. Our data indicate that metabolism of phosphorothioate oligodeoxynucleotides was more complex than that of phosphodiesters for many reasons, including phosphorothioate oligodeoxynucleotide inhibition of nucleases and the presence of R(p) and S(p) stereoisomers. The rate of phosphorothioate metabolism also appeared to be influenced by sequence, with pyrimidine-rich compounds being metabolized to a greater extent than purine-rich oligomers. Other factors affecting stability included oligomer chemistry and length. Concomitant experiments performed in rats dosed systemically with the same compounds mimic the activities seen in vitro and suggest that this liver homogenate system is a valuable model with which to study the mechanism of metabolism of antisense oligonucleotides.     

In vitro stimulation by long-acting thyroid stimulator of thyroid glucose oxidation and 32P incorporation into phospholipids

Long-acting thyroid stimulator (LATS) increased glucose oxidation and (32)P incorporation into phospholipids in in vitro experiments with dog thyroid slices. The time course of the response was different from that obtained with thyroid-stimulating hormone (TSH), but was very similar to the delayed effect observed in vivo. During a 45 min incubation, TSH, but not LATS increased glucose oxidation, whereas in longer experiments up to 6 hr, both substances augmented (14)CO(2) production. Amounts of pooled human gamma globulin equivalent to LATS were inactive. Although TSH stimulated (32)P incorporation into phospholipids during a 2 hr incubation, LATS was ineffective. In longer incubations, from 4(1/2) to 8 hr, LATS did increase (32)P incorporation. The stimulatory effect of LATS was not abolished by anti-TSH antibody capable of neutralizing human TSH. Effects of LATS were also obtained with beef and pig thyroid slices. In addition to stimulation of glucose oxidation in dog thyroid slices, LATS occasionally also stimulated glucose oxidation in dog spleen and liver slices. Despite a 54-fold increase in LATS concentration, a satisfactory dose-response curve could not be demonstrated when (14)CO(2) production was measured.     

基于超声识别HIFU致生物组织变性

目的 探讨以超声筛选最佳特征向量，结合广义回归神经网络（GRNN）识别强度聚焦超声（HIFU）致生物组织变性的方法。方法 采用HIFU以不同剂量对300个新鲜离体猪肉组织样本进行辐照，获得变性及未变性样本各150个。于辐照前后采集超声声像图，经减影处理获得超声减影图像；以灰度-梯度共生矩阵法及灰度差分统计法提取18个特征参数，经P值显著性检测法及欧氏距离法筛选获得最佳特向量。以300个样本中的198组为训练样本，102组为测试样本。识别训练样本后，以P值显著性检测法剔除的特征向量和欧氏距离最小的2个特征向量为最佳特征向量的对照组，将其分别输入GRNN，以识别组织变性；计算特征向量结合GRNN对测试样本的正确识别率和总识别率。结果 最佳特征向量为梯度分布不均匀性和灰度分布不均匀性，其结合GRNN的总识别率分别为90.20%、91.18%，以2个最佳特征组合并结合GRNN后总识别率为98.04%。P值显著性检测法剔除的特征向量为平均值、对比度，其结合GRNN的总识别率分别为48.04%、75.49%，2以2个最佳特征组合并结合GRNN的总识别率为79.41%。欧氏距离最小的特征向量为能量、小梯度优势，结合GRNN的总识别率分别为88.24%、89.22%，以2个最佳特征组合并结合GRNN的总识别率为89.22%。最佳特征向量组合结合GRNN可明显提高对变性组织的识别率。结论 基于超声减影图像，以灰度分布不均匀性、梯度分布不均匀性与GRNN结合，均可提高对HIFU辐照所致组织变性的识别率；以2个最佳特征组合结合GRNN识别效果更佳。     

Three methods compared for isoamylase separation in tissue homogenates

Using homogenates of autopsy tissue, we compared three widely available techniques for separating amylase isoenzymes: wheat-germ inhibition (WI), and electrophoresis on cellulose acetate (CA) or agarose (AG). WI separated amylase into two isoforms, CA into seven (three pancreatic and four salivary), and AG into nine (five pancreatic and four salivary). CA and WI had similar isoamylase detection limits (8-10 U/L) and similar imprecision in measuring percent S-type vs P-type isoamylase (within-run SD 1-2%), and they demonstrated a linear response to added S or P isoamylase. In contrast, the AG method had higher detection limits (10-15 U/L), greater imprecision (within-run SD 3%), and showed a nonlinear response to added S or P isomylase. We conclude that CA and WI have essentially equivalent assay attributes, superior to AG, but that CA resolves more amylase isoforms than WI.     

In vitro suppression of programmed cell death of B cells by tissue inhibitor of metalloproteinases-1.

Cellular pathways for induction of programmed cell death (PCD) have been identified, but little is known about specific extracellular matrix processes that may affect apoptosis along those pathways. In this study, a series of Burkitt's lymphoma (BL) cell lines were assayed for their expression of tissue inhibitor of metalloproteinases (TIMP)-1. Results indicate that TIMP-1-positive BL lines show resistance to cold-shock-induced apoptosis. Furthermore, recombinant TIMP-1, but not TIMP-2 or a synthetic metalloproteinase inhibitor (BB-94), confers resistance to apoptosis induced by both CD95-dependent and -independent (cold shock, serum deprivation, and gamma-radiation) pathways in TIMP-1-negative BL lines. TIMP-1 suppression of PCD is not due to metalloproteinase inhibition, as reduction and alkylation of the TIMP-1 did not abolish this activity. Retroviral induction of TIMP-1 not only resulted in cell survival but also in continued DNA synthesis for up to 5 d in the absence of serum, while controls underwent apoptosis. This resistance to apoptosis is reversed by anti-TIMP-1 antibodies, demonstrating that secreted TIMP-1 is active in blocking apoptosis. Furthermore, TIMP-1 upregulation induced expression of Bcl-XL but not Bcl-2 as well as decreased NF-kappaB activity as compared with controls. These results demonstrate that TIMP-1 suppresses apoptosis in B cells and suggests a novel activity for TIMP-1 in tissue homeostasis.