纳米细菌的分离培养和保存方法探讨

目的 以钙化的胎盘组织为例,寻求在钙化组织中分离纳米细菌的最佳方法,以便更多疑似与纳米细菌感染有关的疾病得以准确分离出纳米细菌菌株,并探讨培养和保存纳米细菌的最适宜的条件.方法 (Ⅰ)胎盘钙化组织标本分别用盐酸脱矿与超声振荡脱矿的方法分离纳米细菌,计算其分离阳性率.(2)分离出的纳米细菌分别用细胞培养箱与细菌培养箱培养1个月,利用分光光度计记录两种条件下纳米细菌浓度的变化,并描绘生长曲线.(3)分别用4、- 20与- 80℃冰箱保存钙化组织和纳米细菌,记录纳米细菌分离和复苏的生长状况并绘制生长曲线.结果 (1)钙化组织用盐酸脱矿更易分离得到纳米细菌.(2)细胞与细菌培养环境下纳米细菌的生长速度并无明显差别.(3)4℃保存钙化组织和纳米细菌对于其分离和复苏都要优于- 20℃和- 80℃.结论 对钙化组织进行盐酸脱矿可以更好的分离出纳米细菌,并且可以在细菌培养箱内培养纳米细菌,新鲜钙化组织标本和纳米细菌可以短时间保存在4℃.     

与钙化相关的骨基质蛋白研究的现状与展望

生物矿化（biomineralization）不仅包括正常情况下骨骼和牙齿的钙化也包括各种疾病时的钙化,如血管钙化、胎盘钙化,还包括珊瑚和贝类以及纳米细菌等不同物种的钙化。研究表明与骨骼钙化的相关的蛋白不仅发现于骨骼的钙化中,也同样发现于其他组织的钙化过程和其他物种的矿化过程中,因此本文就与骨骼钙化的相关蛋白做一综述,以期为进一步研究纳米细菌自身的矿化机制,探讨其引起的相关疾病的致病机制以及为进一步实验筛选合适的标志蛋白。     

对纳米细菌在牙结石形成过程中的作用的初步研究

目的探讨牙结石中是否含有纳米细菌及其在牙结石形成过程中的作用。方法ELISA法、免疫荧光染色、透射电镜方法、化学成分分析。结果ELISA检测结果显示20例牙周病患者的龈沟液和牙结石分离培养物中阳性结果分别为2例、16例。通过免疫荧光染色、透射电镜方法检测并观察到牙结石及牙结石分离培养物中均存在纳米细菌,化学成分分析证明了纳米细菌分泌晶体成分同牙结石主要化学成分相同。结论本研究证实了牙结石中存在纳米细菌,并且纳米细菌作为矿化中心参与牙结石形成。     

一株胞内合成纳米磁性颗粒菌株的筛选及鉴定

【目的】从聊城东昌湖湖水中分离纯化出一株可合成纳米磁性颗粒的菌株,将其命名为TZ-1。【方法】对该菌株进行形态学研究、分子生物学鉴定,将TZ-1菌株合成的纳米磁性颗粒进行提取纯化,并对菌体和纳米磁性颗粒进行透射电镜(transmission electron microscope,TEM)观察、扫描电镜(Scanning electron microscope,SEM)元素分析,对纳米磁性颗粒进行X射线衍射(X-ray diffraction,XRD)分析。【结果】经鉴定TZ-1属于伯克霍尔德氏菌属(Burkholderia sp.)。透射电镜下菌体为杆状,易聚集,有明显的单生鞭毛,有荚膜,在TEM下观察菌体内部有两种电子致密颗粒,较小颗粒分布在菌体细胞膜附近,近似多边形,大小约为60 nm,较大颗粒分布在菌体内部,大小约为180 nm,表面有膜包裹。扫描电镜(SEM)下细胞为杆状,大小与TEM下测量结果一致。SEM下对磁性颗粒进行元素分析,主要为Fe、P、O。根据TEM、SEM、XRD结果推测菌体可合成纳米磁性颗粒。【结论】分离纯化出的菌株TZ-1可合成纳米磁性颗粒,磁性颗粒X射线衍射结果分析知TZ-1合成的纳米磁性颗粒为单斜晶体,主要成分为Fe3(PO4)2·8H2O和Fe3O4。     

肺微血管内皮细胞的原代培养

目的:建立简单高效获取大鼠肺微血管内皮细胞(pulmonary microvascular endothelial cells,PMVECs)的培养方法.方法:(1)组织块贴壁法培养大鼠PMVECs;(2)光镜下观察细胞的形态;(3)扫描电镜和透射电镜分别观察细胞表面及内部的结构特征;(4)免疫荧光法鉴定PMVECs.结果:(1)获得的PMVECs长满后呈典型的铺路石或鹅卵石状;(2)扫描电镜下可见内皮细胞表面存在微绒毛等特殊结构;(3)透射电镜观察可见细胞浆内存在韦伯潘力氏小体(Webel Palade body,WPB);(4)免疫组化结果显示细胞浆中有Ⅷ因子相关抗原存在.结论:改良的组织块法培养肺微血管内皮细胞是一种简单、快捷,有效的方法.     

全氟辛烷磺酸降解菌的分离与鉴定

目的筛选和分离降解全氟辛烷磺酸(PFOS)的细菌,并进行初步鉴定和降解效率分析。方法采集全氟化工厂周边土壤,利用PFOS为唯一碳源的无机盐培养基进行驯化和富集培养,分离降解PFOS的细菌;通过形态观察、生理生化特性检测和基于16SrRNA基因序列的系统发育分析对分离菌株进行鉴定;采用GC-MS检测其降解PFOS的能力。结果分离获得3株能降解PFOS的细菌YSB1、YSB2和YSB3,初步鉴定分属于鞘酯菌属(Sphingobiumsp.)、中华根瘤菌属(Sinorhizobiumsp.)和苍白杆菌属(Ochrobactrumsp.)。菌株YSB1、YSB2和YSB3在以PFOS为唯一碳源生长的无机盐培养基中生长良好,其对PFOS最大降解率分别为18.4%、10.1%和14.1%。结论自然界存在较丰富的降解持久性有机污染物PFOS的微生物资源。     

生殖支原体在泌尿生殖道中感染的研究

目的 :采用培养、电镜技术 ,对吉林地区部分非淋菌性尿道 (宫颈 )炎 (NGU)高危人群泌尿生殖道分泌物标本进行分离鉴定 ,以探讨MG感染在STD的作用和地位。方法 :吉林地区NGU高危人群受检者 14 1例 ,宫颈或尿道口内拭子分泌物标本接种于 5mlSP 4培养基 37℃培养 ,对初代分离阳性株再行电镜负染 ,观察其形态特征 ,并与正常对照组 6 5例进行比较。结果 :14 1例标本中培养出 6株MG ,分离率为4 2 6 % ,正常对照组 6 5例 ,培养出 1株 ,分离率为 1 5 3% (1/ 6 5 ) ,差异无显著性 (χ2 =0 3439P =0 0 5 8>0 0 5 )。结论 :采用培养方法可在NGU高危人群和正常人群中分离出MG株 ,其分离株具有生长时间长(30～ 5 0d) ,需特殊培养基 ,分离培养生长条件高 ,分离率低。     

纳米细菌对人成骨细胞C_3H_(10)的细胞毒作用

目的比较纳米细菌(nanobacteria,NB)与羟基磷灰石(hydroxyapatite,HA)对成骨细胞的细胞毒作用,进一步探讨引起细胞毒作用的机制。方法实验分为正常对照组、HA组、NB组,其中HA组和NB组悬液浓度均为2麦氏浊度(M),对照组只加培养基,分别作用成骨细胞,通过CCK-8试剂盒检测其对细胞的增殖抑制作用;采用Hoechest33258荧光染色和Annexin V-FITC/PC双标法流式细胞仪(flow cotymetry)检测细胞凋亡率;倒置显微镜和透射电镜观察细胞形态的变化;Western blot法检测钙化相关蛋白BMP-2、凋亡相关蛋白Bax的表达。结果 CCK-8结果显示:NB对人成骨细胞C3H10有细胞增殖抑制作用,而HA则没有。Hoechst33258荧光染色和双标法流式细胞仪结果显示:NB对人成骨细胞C3H10主要通过促进细胞凋亡发挥细胞毒作用,而HA对人成骨细胞C3H10无细胞毒性作用。在倒置显微镜下观察,作用48 h后的人成骨细胞C3H10形态无明显变化。透射电镜结果显示:NB可引起人成骨细胞C3H10发生凋亡,出现凋亡小体;而HA组和对照组无明显变化。Western blot结果显示:NB组的钙化相关蛋白BMP-2和凋亡相关蛋白Bax均高于对照组;而HA组和对照组比较,HA组钙化相关蛋白BMP-2高于对照组,而凋亡相关蛋白Bax无明显变化。结论与对照组比较,纳米细菌能引起人成骨细胞C3H10细胞毒性作用,主要是通过促进细胞凋亡发挥细胞毒作用,能促进钙化相关蛋白BMP-2的表达,凋亡相关蛋白Bax的表达升高也预示着纳米细菌可促进细胞发生凋亡;而羟基磷灰石对人成骨细胞C3H10无细胞毒性作用,可促进钙化相关蛋白BMP-2的表达,但不能促进人成骨细胞C3H10发生凋亡。     

贴壁法分离培养大鼠骨髓间充质干细胞的生物学特性

目的建立一种简便有效的体外分离纯化及培养扩增大鼠骨髓间充质干细胞(MSCs)的方法。研究MSCs的生物学特性,为血管组织工程提供理想的种子细胞。方法贴壁培养法分离纯化大鼠MSCs体外培养和连续传代,在倒置显微镜下连续观察细胞的形态变化;利用MTT法测定MSCs的生长曲线;行免疫组化方法鉴定MSCs膜抗原;分别加成骨、成脂肪诱导剂后MSCs体外培养1到3周,分别做碱性磷酸酶(ALP)、VonKossa染色及油红O染色,观察细胞形态变化、成骨及成脂肪分化结果。结果MSCs体外培养生长状况良好,呈均一的成纤维细胞样,表达波形蛋白(Vimentin)、α-平滑肌肌动蛋白(α-SMA),不表达层粘连蛋白(Laminin)、CD34、VIII因子相关抗原(VIII)。经体外诱导后具有多向分化潜能。结论贴壁培养法能有效分离纯化大鼠MSCs,用此方法培养的细胞生长稳定,增殖能力活跃,具有MSCs的一般生物学特性,为其成为血管组织工程理想的种子细胞提供了进一步的支持。     

应用琼脂糖微包埋方法有氧培养胃黏膜菌群

目的采用琼脂糖微包埋技术分离胃黏膜菌群。方法应用琼脂糖微包埋技术在有氧条件下,通过过滤、荧光计数等方法按1∶10比例包埋单个细菌,在脑心浸液培养基(brian heart infusion,BHI)培养48h。提取细菌基因组DNA,通过16SrRNA序列分析、BLAST比对及构建进化树鉴定菌株。结果从胃黏膜标本中分离出37个菌株,分属12个菌种。其中Brevibacillus agri、Bacillus flexus、Micrococcus antarcticus和Microbacterium oleivorans四种细菌首次从胃黏膜标本中分离到。结论微包埋技术能够用于胃黏膜菌群培养,为进一步优化标准培养条件、分离出更多菌种提供理论和实验基础。     

四种显微技术观测大鼠颌下腺细胞与丝素-壳聚糖体外共培养的形态学特点

目的采用倒置显微镜、扫描电镜（scanning electron microscopy,SEM）、荧光显微镜和激光共聚焦显微镜（（laser scanning confocal microscopy,LSCM））技术对大鼠颌下腺细胞（rat submandibular gland cells,RSMGs）与丝素-壳聚糖（silk fibroin-chitosan,SFCs）的体外复合培养进行形态学观察。为观测、评估种子细胞在三维支架的内部生长情况提供技术支持。方法取0～8 d龄SD大鼠的颌下腺,对大鼠颌下腺细胞进行原代培养、分离纯化并传代;用抗细胞角蛋白单克隆抗体（CK8）及淀粉酶抗体的免疫细胞化学染色鉴定细胞来源。选取传至第二代的对数生长期的RSMGs作为种子细胞,选取SFCs共混膜（5×5×2）mm作为支架材料构建组织工程化涎腺样结构。将种子细胞与支架材料复合培养并分别于倒置显微镜、SEM、荧光显微镜和LSCM下观察二者复合生长情况。结果倒置显微镜可以直接观察活细胞与支架复合生长情况,方法简单易行。SEM可以较精确的展示细胞支架复合生长的表面超微结构。经过荧光染料的着色,荧光显微镜和LSCM都可以观察到支架上锚定的种子细胞。荧光显微镜可见细胞核的荧光信号均匀的分布在支架孔隙内。LSCM通过层扫描及三维重建技术对较厚的标本获取图像;并可以通过旋转图像,从不同角度观察细胞支架复合物的三维剖面或整体结构,得到更为准确的定位信息。结论四种显微技术均可应用于RSMGs与SFCs体外共培养的形态学观测。LSCM的三维重建技术结合荧光染料标记可以较好地获得RSMGs与SFCs复合生长的情况,有着较广泛的应用价值。     

人羊膜与人脱细胞羊膜形态结构的对比观察

目的：对比观察人羊膜（human amniotic membrane,HAM）脱细胞处理前后的形态结构变化,为人脱细胞羊膜（human acellu-lar amniotic membrane,HAAM）作为良好的生物支架材料提供依据。方法：取健康剖宫产孕妇的胎盘,剥离获取HAM,行HE染色、透射电镜（transmission electron microscope,TEM）、上皮面与基质面扫描电镜（scanning electron microscope,SEM）检测;将HAM经物理和胰蛋白酶等脱细胞处理后获得HAAM,亦行HE染色、TEM、上皮面与基质面SEM检测;最后将检测结果进行对比观察。结果：通过HE染色表明HAM的细胞成分去除干净;TEM断面观察HAAM表明其富含大量密集的呈点状、线状及条索状的纤维成分;SEM观察表明HAAM的上皮面与基质面呈现不同的三维结构,未见胶原纤维和网状纤维断裂。结论：HAM经脱细胞处理制备的HAAM,既去除了可引起移植排斥反应的细胞成分,又保存了完整的三维结构,为良好的生物支架材料。     

Ultrastructural visualization of the internalization of low density lipoprotein by human placental cells

Low density lipoproteins (LDL) were conjugated to colloidal gold to visualize the route for internalization of LDL in the cultured cells of human term placenta. Cells were obtained from placental villi (caesarian section) by a standard trypsin-DNase dispersion method followed in some cases by a Percoll gradient centrifugation step. Employing electron microscopy it was observed that after 3 days of culture, cells obtained by trypsin-DNase dispersion were a mixture of macrophages, mononucleated cells and large multinucleated cells. When the cells were incubated for 3 days after the Percoll purification, essentially multinucleated cells identical to the syncytiotrophoblast were present. The number of LDL receptor was increased by preincubation in medium with lipoprotein depleted serum. In binding experiments cells incubated at 4 degrees C for 2 h with medium containing gold LDL conjugates showed gold LDL attached to the plasma membrane without characteristic localization. After incubation with gold LDL at 37 degrees C for various times, the three cellular types showed ligand internalization. Gold LDL endocytosis involved first coated pits but also uncoated plasmalemmal invaginations. Then gold LDL was further observed in coated and non coated vesicles, smooth walled endosomes, multivesicular bodies and tubular vesicles. Lastly free gold particles were observed in lysosome like dense bodies. These results prove the internalization of gold LDL conjugates by human cultured placental cells, particularly by syncytiotrophoblast like multinucleated cells. This accumulation of LDL (the major cholesterol carrying protein in humans) is recognised to be responsible for the exogenous cholesterol supply indispensable to the progesterone biosynthesis and cellular growth of the placenta.     

Ultrastructural visualization of the internalization of low density lipoprotein by human placental cells

Summary Low density lipoproteins (LDL) were conjugated to colloidal gold to visualize the route for internalization of LDL in the cultured cells of human term placenta. Cells were obtained from placental villi (caesarian section) by a standard trypsin-DNase dispersion method followed in some cases by a Percoll gradient centrifugation step. Employing electron microscopy it was observed that after 3 days of culture, cells obtained by trypsin-DNAse dispersion were a mixture of macrophages, mononucleated cells and large multinucleated cells. When the cells were incubated for 3 days after the Percoll purification, essentially multinucleated cells identical to the syncytiotrophoblast were present. The number of LDL receptor was increased by preincubation in medium with lipoprotein depleted serum. In binding experiments cells incubated at 4° C for 2 h with medium containing gold LDL conjugates showed gold LDL attached to the plasma membrane without characteristic localization. After incubation with gold LDL at 37° C for various times, the three cellular types showed ligand internalization. Gold LDL endocytosis involved first coated pits but also uncoated plasmalemmal invaginations. Then gold LDL was further observed in coated and non coated vesicles, smooth walled endosomes, multivesicular bodies and tubular vesicles. Lastly free gold particles were observed in lysosome like dense bodies. These results prove the internalization of gold LDL conjugates by human cultured placental cells, particularly by syncytiotrophoblast like multinucleated cells. This accumulation of LDL (the major cholesterol carrying protein in humans) is recognised to be responsable for the exogenous cholesterol supply indispensable to the progesterone biosynthesis and cellular growth of the placenta.     

正常人和肝病患者血中“致密体”及其与纳米细菌的关系

目的研究正常和肝病患者血液中圆球体样微生物的生物学特性,初步确定纳米细菌与该种可滤过物的关系。方法将正常人和肝病患者培养阴性的血培养物进行细菌L型、穿菌和厌氧菌培养。同时取沉淀用透射电镜观察。L型培养液用0．45μm和0．22μm滤器过滤,接种RPMI 1640培养基,细胞培养条件下培养45d,用鼠抗纳米细菌单克隆抗体8D10免疫组化和钙盐染色进行纳米细菌鉴定。血浆纳米细菌培养阳性者12000×g离心后,进行普通和L型细菌培养。结果36／39患者和60／60健康对照血培养液中呈现类似于L型的巨型体、圆球体、原生小体的不明微生物。电镜观察圆球体内为电子致密样物质,周围未见细胞壁结构。电镜和光镜下可见其粘附在红细胞上或存在于红细胞内。滤过后培养物钙盐染色有5／39阳性,但纳米细菌免疫组化染色均阴性。纳米细菌阳性的培养物转种,未见一般细菌、细菌L型和上述血中“致密体”。结论血培养中致密体样微生物与纳米细菌无关,可能在维持机体正常免疫功能上具有一定意义。     

Regulation of growth hormone secretion in human trophoblastic cells in culture

In this study, we have cultured in vitro purified trophoblastic cells from first-trimester and term human placenta. These cells were obtained by specific enzymatic digestion and centrifugation through a Percoll gradient. Using 2 specific monoclonal antibodies, the pituitary 22-kD growth hormone (GH) and the placental GH variant were assayed in the culture medium by radioimmunoassay. After 48 h of culture, only the placental GH variant was measured in the medium corresponding to first-trimester placenta (3.4 ng/24 h/10(5) cells). Surprisingly, an immunoactivity pattern of pituitary GH type was found in 3 out of 5 media conditioned with term placenta cells, while GH immunoactivity was very low, around the detection level, in the 2 others. These secretions are not modified with the time in culture and the state of differentiation of the cells from cytotrophoblast to syncytiotrophoblast. Neither in early nor in term placenta does the addition of GH-releasing factor (10(-6) M in the culture medium) stimulate the secretion of pituitary 22-kD GH or placental GH variant.     

胎儿生长受限模型及其胎盘的病理学观察

目的证实胎儿生长受限大鼠模型的建立方法;观察胎儿生长受限(FGR)大鼠胎盘的组织形态及超微结构特征。方法健康Wistar雌鼠24只,按妊娠先后顺序随机分为两组:正常对照组(正常组)、模型组(烟酒处理组),采用烟酒混合因素建立大鼠FGR模型;比较两组胎鼠的体重、鼻臀长度、体重系数及FGR发生率,两组胎盘组织行HE染色,并应用光镜和电镜观察其病理变化。结果①模型组胎仔平均体重、鼻臀长度、体重系数较正常组分别减轻46.0%、21.9%、35.0%(P0.01)。对照组FGR发生率仅为12.5%,模型组FGR发生率为79.3%,显著高于对照组(P0.01)。②模型组胎盘形态学明显改变。结论通过烟酒干预的方法,成功建立缺血缺氧性FGR孕鼠模型。烟酒可导致胎盘形态结构的改变,这种病理改变是造成FGR胎盘功能减退的形态学基础。     

猪主动脉脱细胞血管基质制备

目的研究脱细胞血管基质的制备方法。方法采用胰蛋白酶、低渗溶液、化学除垢剂法处理猪胸主动脉来制备脱细胞血管基质,标本作苏木素-伊红染色,大体、光镜及扫描电镜、透射电镜观察。结果经该法处理的血管细胞全部脱除,胶原纤维、弹性纤维无断裂,细胞外基质保持完好。结论酶、低渗溶液、化学除垢剂联合法是制备脱细胞血管基质的较好方法。     

Spreading of vascular endothelial cells in culture: Spatial reorganization of cytoplasmic fibers and organelles

The three-dimensional organization and fine structure of cytoplasmic components within whole non-embedded bovine aortic endothelial cells were examined during their attachment and spreading in tissue culture. Cells were cultured directly on Formvar-coated gold grids, fixed in glutaraldehyde and osmium tetroxide, critical point dried and examined by transmission electron microscopy (TEM) using stereoscopic methods, and by scanning electron microscopy (SEM). Reorganization of cytoplasmic structures during cell spreading occurred in four sequential stages: (1) spreading of the plasma membrane and unstructured cytoplasmic matrix; (2) spreading of cytoplasmic fiber systems (microtubules, microfilament bundles and microtrabecular system); (3) alignment of microfilament bundles and formation of radial tracts of microtubules; and (4) centripetal movement of organelles along radial tracts. These stages observed by TEM correlated with progressive degrees of cell flattening as visualized by SEM. These studies demonstrate that a characteristic reorganization of intracellular fiber systems and organelles accompanies the spreading of endothelial cells in culture.