多株消化道肿瘤细胞在硝酸纤维素膜界面生长的初步研究

目的观察多株消化道肿瘤细胞在硝酸纤维素膜界面上的生长情况,探讨硝酸纤维素膜作为细胞培养载体材料的可行性。方法将大肠癌细胞、胃癌细胞、胰腺癌细胞、肝癌细胞株等肿瘤细胞接种在硝酸纤维素膜界面培养,培养结束后在光学显微镜以及扫描电镜下观察细胞形态,免疫组化法检测肿瘤细胞标志物,对比检测实验组和六孔板对照组培养液上清中两者乳酸脱氢酶漏出率差异,评价硝酸纤维素膜的细胞相容性。结果膜染色透明处理后,在光学显微镜下可以观察到膜表面细胞保持较好的增殖状态和细胞形态;肿瘤细胞主要标志物均呈阳性表达;硝酸纤维素膜及膜上细胞的扫描电镜表征：该膜呈现孔径均一的海绵样空间立体结构,膜表面生长的细胞呈不规则形态且有伪足形成。结论硝酸纤维素膜具有良好的细胞相容性,可以作为一种新的细胞生长载体材料加以应用。     

聚乳酸-聚乙醇酸共聚物与心肌样细胞生物相容性的研究

目的: 探讨聚乳酸-聚乙醇酸(PLGA)与骨髓间充质干细胞(BM-MSCs)衍生的心肌样细胞的生物相容性,为构建心肌组织工程及其进一步体内回植提供研究基础。方法: 以密度梯度离心法体外分离培养SD大鼠的BM-MSCs,将培养的第3 代细胞用终浓度为10 μmol/L的5-氮杂胞苷和0.1μmol/L的血管紧张素Ⅱ诱导分化。将诱导后的心肌样细胞接种到PLGA材料上, 用沉淀法测定细胞的黏附力和黏附率;MTT比色法检测细胞的增殖并绘制增殖曲线;扫描电镜观察细胞在PLGA材料上的形态学特征及细胞与该材料的相容性。结果: 心肌样细胞在PLGA支架材料上贴附、生长良好,可分泌细胞外基质,其黏附、增殖能力与单纯的细胞相比无显著统计学差异。结论: PLGA与心肌样细胞具有良好的生物相容性,可作为心肌样细胞的理想载体构建心肌组织工程。     

胚胎干细胞用于肝组织工程研究的初步评价

目的在生物材料上进行胚胎干细胞（embryonic stem cell,ESc）的三维培养和肝细胞定向诱导分化,评价其用于构建肝组织工程的可行性。方法胰酶消化生物反应器悬浮旋转培养5天的拟胚体（embryoid bodies,EBs）,将细胞悬液与细胞外基质Matrigel 1∶1混合接种于聚乳酸（poly-L-lactic acid,PLLA）和聚乙醇酸（polyglycolic acid,PGA）共聚合三维支架内,用系列诱导剂定向诱导肝细胞分化。镜下动态观察ES细胞的培育和生长情况,并用HE染色、糖原染色、免疫荧光染色检测组织结构形态、肝细胞功能及其肝细胞标志物白蛋白（ALB）的表达。结果显微镜和扫描电镜见ESc可在聚合支架材料上三维立体状态生长,随培养时间不断增殖,交织成网状。HE染色提示形成了致密的类肝组织样结构。糖原染色和免疫荧光染色结果提示形成的类肝组织表达Alb,并有大量糖原存在。结论可在PLLA/PGA三维支架材料及Matrigel上培养ESc和定向肝细胞诱导分化,长时间培养后可形成类肝样组织。     

丝素蛋白/壳聚糖复合纳米纤维膜心脏补片的生物相容性研究

目的 探讨丝素蛋白(SF)/壳聚糖(CS)复合纳米纤维膜对小鼠脂肪间充质干细胞（AD-MSCs）黏附和生长的影响，评估该材料用作心脏补片的可行性。 方法 通过静电纺丝技术制备纤维素纳米纤维底板，用层层自组装技术(LBL)，将SF和CS组装到纤维素纳米纤维表面，得到改性的SF/CS复合纳米纤维膜，用ζ- 电位和X射线光电子能谱(XPS)测定材料的表征。分离培养携带绿色荧光蛋白（GFP）和荧光素酶（Fluc）双重报告基因的小鼠AD-MSCs细胞，并接种于SF/CS复合纳米纤维膜上共培养，用激光共聚焦显微镜(CFM)观察细胞黏附和生长形态，扫描电子显微镜(SEM)观察超微结构，生物发光成像(BLI)及MTT比色法评估AD-MSCs细胞的数量和活性。结果 XPS测定的结果提示SF/CS复合纳米纤维膜构建成功。SEM观察可见纳米纤维直径均一，具有良好的三维多孔结构，细胞在SF/CS复合纳米纤维膜上增殖活跃，形态和生长状态良好。CFM、BLI及MTT比色法的结果提示细胞与SF/CS复合纳米纤维膜共培养后呈三维分布，增殖状态好。 结论 对纤维素纳米纤维用LBL进行改性后可明星促进AD-MSCs的三维生长，SF/CS复合纳米纤维膜是可用于组织工程心肌补片的理想材料。     

大肠癌细胞在硝酸纤维素膜上的培养及染色的观察

目的 寻找一种既适于细胞培养和光镜下观察,而且具有良好柔韧度及化学稳定性、可替代玻片作为实验室细胞培养载体的支持介质.方法 硝酸纤维素膜经前期处理后,用于培养细胞和病理染色并与常规方法培养细胞的比较.结果 肉眼观察:染色后的NC膜平展,透明,有浅紫红色的背景.镜下观察:NC膜透明,膜上培养的细胞轮廓清晰,形态规整.细胞形态、数量与对照组玻片培养的细胞无明显差异.HE染色的细胞胞核为蓝色,胞浆红色,膜背景着色较浅.结论 经过处理后的NC膜,对细胞无毒副作用,可用于细胞培养.在NC膜上养成的细胞依然保持细胞的正常形态及数量,具有良好的透光性,可满足光镜观察需求.     

构建组织工程化人工食管模型的实验研究

用曲拉通X-100将猪主动脉进行脱细胞处理。将体外扩增培养的犬新生食管上皮细胞接种于猪脱细胞血管基质（ACTM）上，通过光镜及扫描电镜观察ACTM是否存在细胞成分以及食管黏膜上皮细胞能否生长于其上。结果：酶化学除垢剂法能使猪主动脉细胞脱落，基质三维结构变疏松。体外扩增的犬食管黏膜上皮细胞可以种植在猪ACTM上并能生长增殖。认为猪ACTM与犬食管上皮细胞具有良好的生物相容性，能结合形成人工食管移植体。     

中空纤维生物反应器的建立及其功能的初步鉴定

目的构建生物反应器并评估其生物学功能。方法采用自主建立的永生化HepG2细胞系为生物材料,以中空纤维构建生物反应器,观察肝细胞在生物反应器内生长情况,同时进行肝细胞功能检测,以肝衰竭患者血浆置换出的废弃血浆作为试验因素,试验性行生物人工肝治疗。结果该系统内肝细胞生长良好,每个生物反应器培养72h后细胞数量达7．60×10^8个,肝细胞活力保持90％以上。AST、LDH低水平波动;利多卡因转化实验良好;对细胞培养液进行细菌培养监测及支原体检测,均未发现异常。应用肝衰竭患者血浆500ml培养肝细胞6h,总胆红素平均下降98．6μmol／L。结论建立的永生化肝细胞系应用中空纤维构建生物反应器,肝细胞功能良好,经进一步改进有望应用于生物人工肝治疗。     

人工肝生物材料--猪肝细胞的研究进展

目前,生物人工肝细胞材料研究的主要来源有人肝细胞、肝肿瘤细胞株及异种动物肝细胞。由于人肝细胞来源不足以及肝肿瘤细胞株潜在的致瘤性,使异种动物肝细胞逐渐成为国内外生物人工肝研究的主要对象,其中猪肝细胞已成为生物人工肝应用最多的肝细胞。近年来,在大量实验研究基础上,猪肝细胞用于生物人工肝的研究已经取得较大进展。     

3-羟基丁酸-3-羟基己酸共聚酯材料促进小鼠胚胎干细胞分化为心肌细胞的实验研究

目的:观察3-羟基丁酸-3-羟基己酸共聚酯(PHBHHx)能否促进多能干细胞(m ESC)的存活、增殖以及分化,为心肌梗死的治疗提供新的治疗策略。方法:培养m ESC,传代培养至3代后与PHBHHx材料制成的生物薄膜共同培养72h,CCK-8试剂盒测定m ESC的活性及增殖,DAPI染色观察细胞,电镜观察m ESC生长。将小鼠m ESC向心肌细胞定向分化,15d后免疫荧光和流式细胞术定量测定心肌肌钙蛋白(c Tn T)。结果:CCK-8活性检测显示PHBHHx膜培养m ESC可以促进细胞更好的增殖;电镜扫描显示,m ESC在PHBHHx膜上能够粘附生长,细胞形态正常。加入干细胞分化培养基使干细胞向心肌细胞定向分化15d后,通过免疫荧光显示心肌细胞特异性标志蛋白c Tn T表达阳性;流式细胞术测得PHBHHx膜培养比传统的细胞培养方式能够促进细胞更好的分化,测得的c Tn T表达量明显增加。结论:PHBHHx膜与m ESC有较好的组织相容性,并且能够促进m ESC的增殖和分化。     

应用胶原膜构建组织工程心脏瓣膜的初步研究

目的 :探讨应用胶原膜构建组织工程心脏瓣膜的可行性。方法 :扫描电镜观察材料结构特点。材料兔皮下包埋实验 ,分别于 4,6 ,8,12周观察材料的生物相容性和降解率。培养犬主动脉瓣间质细胞、主动脉壁间质细胞和皮肤成纤维细胞 ,对照其生长曲线、平滑肌α肌动蛋白表达和扫描电镜特点。将犬主动脉壁间质细胞和内皮细胞种植于胶原膜上 ,观察其形态并测定细胞合成前列环素的功能。结果 :胶原膜呈网孔状结构 ,孔径 10 7μm。皮下包埋实验显示材料生物相容性好 ,体内降解时间为 12周。犬主动脉瓣间质细胞和主动脉壁间质细胞平滑肌α肌动蛋白均为部分阳性表达 ,细胞内有大量粗面内质网 ,生长曲线相似。种植实验示细胞在材料表面生长良好 ,并具有合成、分泌前列环素 （84.2 pg/ m L vs0 .4pg/ m L ,P<0 .0 5 ）的功能。结论 :以胶原膜为支架体外构建组织工程心脏瓣膜细胞不仅能在材料表面生长 ,还能合成、分泌血管活性物质 ,是具有“生理功能 "的组织工程心脏瓣膜。     

细粒棘球蚴生发层细胞系的建立

目的：为获得能在体外稳定繁殖的细粒棘球蚴细胞系。方法：用研磨法将绵羊源细粒棘球蚴生发层处理得分散的生发细胞，将其在含10％—20％小牛血清的RPMI1640培养基在24孔培养板和包被胶原的24孔细胞培养板上交替培养至14代，随后在常规24孔培养板上连续传代培养；用倒置显微镜观察细胞形态及生长繁殖情况；将培养细胞接种BALB／c小鼠以观察其形成继发性包囊的能力；用ELISA法研究细胞系抗原的免疫学特征。结果：生发层细胞已在体外连续培养75代以上，生长良好；细胞系细胞呈圆形，具有形成合胞体乃至类组织块 的趋势，在4℃冰箱中至少可保存半个月，在液氮中至少可保存10个月；将细胞接种BALB／c小鼠后3个月至7个月，解剖未见细粒棘球蚴包囊生长; 细胞系抗原可和抗生发层体抗原、原头节可溶性抗原、囊液抗原的小鼠血清及人工感染原头节的阳性小鼠血清起反应。结论: 细粒棘球蚴生发层细胞系已经建立。     

Ultrastructure of hamster bronchiolar epithelium in freeze fracture replicas

The morphology of the plasma membrane of ciliated and nonciliated bronchiolar epithelial cells of the hamster lung was studied using freeze fracture electron microscopy and transmission electron microscopy of thin sections. Tight junction structure of bronchiolar epithelial cells was evaluated with respect to the total number of junctional strands, and the organization of tight junctional fibrils. Tight junctional complexes seen on the surfaces of nonciliated cells had from 4 to 9 junctional strands (mean: 6.0±0.1). Ciliated cells displayed 4 to 10 tight junctional strands (mean: 6.9±0.1). Nonciliated bronchiolar epithelial cell (NC) membranes showed aggregates of 6 nm particles organized into rectilinear arrays of 2 to 30 particles. These particle clusters were seen predominantly at the basal pole of cells in close association with areas of intercellular interdigitation. NC cells also displayed membrane areas of particle clearing and aggregation consistent with intermediate phases of membrane fusion and exocytosis.     

Characterization of the IGF axis in a rat liver-derived epithelial cell line

We have used the techniques of chemical cross-linking, Western blotting and immunoprecipitation-ligand blotting to demonstrate that insulin-like growth factor binding protein-2 (IGFBP-2) is associated with plasma membranes of an epithelial cell line derived from rat liver as well as being secreted into the medium by these cells. We demonstrate that these cells secrete IGF-I, but not IGF-II into serum free medium. Evidence from signalling, cell proliferation and cross-linking experiments indicate that these cells also express cell surface IGF-I receptors. Dose-response experiments indicate an enhanced biological activity of the IGF-I analogue des (1-3) IGF-I compared to wild-type IGF-I in both acute signalling experiments and longer-term (24 h) mitogenic assays. As this IGF-I analogue has lower affinity for IGFBPs, we believe that in this cell culture system, activity of IGF-I may be attenuated in the long and short term by the accumulation of IGFBP-2 in conditioned medium and by the presence of IGFBP-2 associated with the cell membrane and/or ECM.     

Characterization of one newly established esophageal cancer cell line CSEC from a high-incidence area in China

SUMMARY.  The Chaoshan littoral is located in a high-incidence area of esophageal cancer in the south of China. In this study, a new esophageal cancer cell line CSEC was established from a 47-year-old female Chinese patient in this district. The biological characters of the cultured cells were investigated, including morphology, ultrastructure, growth kinetic features, tumorigenicity, expression of tumor-associated antigen and cytogenetic features. CSEC cell line grew continuously with a doubling time of 39.5 h and had been passaged over 80 times. The CSEC cells possessed features of squamous epithelial cells with cytokeratin indicated by immunohistochemical staining and tonofilaments and desmosomes revealed by electron microscopy. Tumorigenicity to severe combined immunodeficient mice was confirmed and the tumors developed revealed well-differentiated squamous cell carcinoma, similar to the origin tumor from which the cell line derived. The cytogenetic analysis demonstrated hypertetraploid karyotypes. Chromosome structure aberrations were common and complicated. Immunohistochemical staining showed that CSEC cells were infected with HPV and over-expressed p53. In summary, the CSEC cell line is a well-differentiated esophageal squamous cell carcinoma cell line from a high-incidence area in southern China. It may provide a useful model for the pathogenesis and therapeutic research of esophageal squamous cell carcinoma.     

人肝细胞系5-羟色胺自分泌系统的研究

目的通过对人肝癌细胞系和人正常肝细胞系的研究,探讨肝细胞是否具有合成5-HT的功能。方法以人肝癌细胞系HepG2和人肝细胞系7702为研究对象,采用ELISA方法检测细胞培养上清液中的5-HT水平,采用荧光定量PCR法检测细胞表达5-HT合成的关键酶及5-HT受体。结果两种细胞系细胞上清液均可检测到5-HT;均可表达多种类型的5-HT受体,均表达合成5-HT的关键酶色氨酸羟化酶（TPH）及运载体（SERT）。结论人肝癌细胞系HepG2和人肝细胞系7702均表达合成5-HT的关键酶和受体,说明肝细胞具有5-HT自分泌系统,5-HT可能以自分泌或者旁分泌的形式发挥作用。     

肝癌干细胞逃避5-氟尿嘧啶单药杀伤可能是造成化疗失败的重要原因

目的通过观察5-氟尿嘧啶(5-FU)处理人肝癌细胞系BEL-7402和SMMC-7721,分析细胞生物学特性的变化。方法采用Cell Counting Kit-8法检测5-FU作用前后肝癌细胞系BEL-7402和SMMC-7721增殖能力的变化;采用流式细胞法检测细胞周期组成变化和细胞群体中肿瘤干细胞标志物EpCAM、ABCG2和ICAM-1阳性细胞的比例变化。结果 10μg.ml-15-FU作用48h后,BEL-7402和SMMC-7721细胞增殖均明显受抑制(P<0.05);两细胞系实验组静息期(G0/G1期)细胞比例较对照组均明显升高(P<0.05);两细胞系中肿瘤干细胞标志物阳性细胞比例均明显升高(P<0.05)。结论肝癌细胞系BEL-7402及SMMC-7721中的肿瘤干细胞能够逃避一定剂量5-FU的杀伤作用。肝癌干细胞逃避5-FU单药杀伤可能是造成临床化疗失败的重要原因。     

Early effects of high doses of retinol (vitamin A) on the in situ cellular metabolism in rat liver

Abstract: Understanding of the possible toxicity associated with hypervitaminosis A becomes increasingly important in view of the popularity of vitamin A supplementation. Hypervitaminosis A for many years may eventually lead to hepatocellular damage. In the present study, rats were treated for 7 days with high doses of retinol to study the early effects on the metabolism of different types of liver cells using (enzyme) histochemistry, immunohistochemistry and electron microscopy. Excessive intake of vitamin A activates Kupffer cells and induces accumulation of lipid droplets in fat-storing cells as well as proliferation of these cells. Moreover, it affects the metabolic heterogeneity in the liver lobules, but does not lead to apparent cell damage. Based on the changes in marker enzymes for different metabolic processes, it is concluded that the capacity for breakdown of purines, the antioxidant capacity, the potential for phagocytosis and the regulation of ammonia levels were largely decreased. Increased alkaline phosphatase activity in hepatocytes pointed to an activated process of transport of retinol esters over the bile canalicular membrane. The possible causes of these metabolic changes have been described in the discussion.     

Tissue localization and kinetics of pit cells or large granule lymphocytes in the liver of rats treated with biological response modifiers

The numbers of Kupffer cells (macrophages) and pit cells (large granular lymphocytes) were counted by light and electron microscopy in perfusion-fixed liver sinusoids. After a single intravenous injection of the biological response modifiers zymosan, Propionibacterium acnes and OK-432, a 4- to 6-fold increase in the number of pit cells and a 2- to 4-fold increase in the number of Kupffer cells were observed within a period of 4 to 7 days. The mechanisms of the pit cell accumulation were further studied by the metaphase arrest method and by selective irradiation of the liver, or of the rest of the body, with a dose of 8.5 Gy. Stimulated pit cells showed mitotic activity in the liver and to a lesser extent in peripheral blood. The zymosan-induced increase in pit cell number was inhibited by irradiation of the liver but not by irradiation of the rest of the body (with shielded liver). It is concluded that the hepatic pit cell population, which has been shown to have natural tumoricidal activity, can be induced to expand by biological response modifiers, and local division of preexisting cells contributes significantly to this expansion.     

肝大部切除大鼠所致的IETM对血浆与肝组织IL-1β的影响

目的 探讨肝大部分切除(PH)诱导的肠源性内毒素血症(IETM)对血浆及残余肝组织IL-1β的影响或可能存在的关系.方法 102只健康雄性成年wistar大鼠随机分为正常对照(NC)组、假手术(SO)组和肝大部切除(PH)组,在不同时间点检测血浆内毒素(ET)、IL-1β、ALT的水平以及残余肝组织IL-1β的蛋白表达...     

体外培养的肝癌细胞株与正常肝细胞株蛋白质的差异表达

目的:运用SELDI蛋白质芯片技术分析体外培养的肝癌细胞株(HepG2)与正常肝细胞株(L02)蛋白质表达差异．方法:在体外培养HepG2和L02细胞株,收获细胞,将细胞用细胞裂解液裂解后,采用SELDI蛋白质芯片技术用IMAC3 及WCX2芯片检测HepG2、L02的蛋白质谱．结果:体外培养的肝癌细胞株与正常肝细胞株的蛋白质存在差异表达,IMAC3芯片共捕获61个蛋白,发现差异峰7个,与 L02细胞相比,其中3个差异蛋白在肝癌细胞中高表达,4个差异蛋白在肝癌细胞中低表达．WCX2芯片共捕获91个蛋白, 发现差异峰14个,其中3个差异蛋白在肝癌细胞中高表达,11 个差异蛋白在肝癌细胞中低表达．结论:SELDI蛋白芯片技术检测肝癌细胞株与正常肝细胞株蛋白质的差异表达方法简便,敏感性高,重复性好．