【作者】 郭亚楠；

【导师】 张德纯；

【作者基本信息】 重庆医科大学 ， 病原生物学， 2012， 博士

【摘要】 背景：芬兰科学家Kajander教授及其团队最早从哺乳动物细胞培养过程发现细胞的空泡样变化，在排除了一切可能的微生物污染的情况下对其进行分析，发现了一种可以自我复制矿化且直径只有50-800nm的微生物，命名为纳米细菌（Nanobacteria NB）并申请了专利1。利用傅里叶变换色散光谱分析（FTIR）表明纳米细菌外壳的主要成分为羟磷灰石，所以又称为纳米矿化颗粒（CNPs）。有科学家在透射电镜下观察早期钙化胎盘组织发现纳米矿化颗粒的存在，但未能分离培养出纳米细菌。目的：从胎盘钙化组织和相应胎儿脐带血中分离培养出与钙化相关的纳米细菌，观察其生长特性，探讨胎盘钙化发生的微生物感染证据以及胎盘钙化与胎儿纳米细菌感染的关系。方法：以临床所取36份胎盘钙化组织作为研究对象，透射电镜观察钙化组织中的纳米细菌。而后依次利用1mol/L盐酸脱矿，Tris中和，生理盐水稀释，14,000g高速离心，最后通过0.22μm细菌滤器过滤的方法进行分离。滤液过滤到有棉塞试管中，加入含有10%胎牛血清的RPMI1640细胞培养基，调整pH7.4，放入37℃细胞培养箱,5％CO2，95％空气条件下培养。观察细菌生长情况并依据沉淀类型分类。OD650波长下记录纳米细菌菌液相对浓度OD值，制作生长曲线。胎牛血清（FBS）和生理盐水作为试剂对照,无钙化的正常胎盘组织作为实验对照，用RPMI1640培养基作为空白对照。同时对存在胎盘钙化的胎儿取脐带血，对纳米细菌进行分离培养，方法同上。结果：透射电镜下观察钙化胎盘组织标本，可见胎盘绒毛组织和钙化斑块之间存在椭圆形纳米颗粒，其直径约为50-500nm，每个颗粒都被具有不同电子密度的薄外壳包围，其中有些颗粒可见分裂，与国内外文献报道相似。实验组中有28例可见白色沉淀完全或部分粘附在试管底部，对照组未见沉淀产生，差异具有显著性（P <0.01）。OD650分光光度计下记录得到纳米颗粒生长曲线与其他细菌类似。脐带血分离培养的结果与相应钙化组织胎盘中纳米细菌培养的结果正相关,有12例实验组培养物可见白色沉淀物，对照组未见（P <0.01）。结论：通过纳米细菌分离培养的方法可以从钙化胎盘组织中得到具有自我复制矿化功能的纳米颗粒，且存在胎盘钙化的胎儿也较易感染此纳米颗粒。.背景：进一步对培养所得白色沉淀物进行形态学和基因组学的鉴定。由于纳米细菌形态微小，有的还不足100nm，因此许多科学家认为它并不存在遗传基因，也不属于生物范畴。Kajander教授向GenBank中提交了纳米细菌特异的16SrRNA基因序列X98418，因此后续科学家多依据这个序列对分离培养出的纳米细菌进行种属鉴定。目的：利用透射电镜，扫描电镜，茜素红染色和16SrRNA基因分析的方法对分离培养得到的纳米细菌进行进一步鉴定。方法：①透射电镜观察：纳米细菌14,000g高速离心沉淀，戊二醛前固定，四氧化二锇后固定，梯度脱水，环氧树脂包埋，制作超薄切片，双重电子染色，双蒸水洗涤，干燥后在透射电镜下观察拍照。②扫描电镜观察：纳米细菌沉淀利用戊二醛固定,常规脱水，临界点干燥，喷金，在扫描电镜下观察拍照。③茜素红染色：培养标本涂片固定，冲洗，2％茜素红染液染，0.2％淡绿水溶液复染，O.5％醋酸水溶液冲洗,乙醇脱水，烘干后用中性树胶封片，高倍镜下观察拍照。④16SrRNA基因序列分析：提取纳米细菌基因组，设计纳米细菌16SrRNA基因序列特异性引物，PCR扩增其特异性序列，凝胶电泳观察并回收目的片段，送检进行基因序列分析，结果与GenBank比对。根据比对结果设计新发现纳米细菌16SrRNA基因特异性引物，对胎盘钙化组织进行16SrRNA基因序列分析，其结果与新发现纳米细菌进行比对，验证提取过程有无污染菌。再将所得菌株与GenBank中所收录纳米细菌相关16SrRNA基因做系统进化分析，绘制系统进化树。结果：①透射电镜下可见菌液中的纳米细菌呈椭圆形，直径在200-500nm，有或没有高电子密度的外壳包绕在外。②扫描电镜下可见纳米细菌形态微小，单个颗粒直径在200nm左右，或聚集成微米大小团块。③茜素红染色可见纳米细菌染成红色细小颗粒。④16SrRNA基因分析发现胎盘钙化组织中分离培养得到纳米细菌新种属，分别与纳米细菌X98418相比有93%和81%相似性，两个种属纳米细菌的16SrRNA基因测序结果提交GenBank得到基因编号JN029830与JF823648。钙化胎盘组织中纳米细菌基因扩增分析可知其与先前分离培养菌液中的纳米细菌是同一种菌。与GenBank中其他纳米细菌种属进行对比，发现这两个种属的纳米细菌与先期Kajander教授提交的Nanobacterium sanguineum(X98418)亲缘关系最近。结论：胎盘钙化组织中分离培养得到的纳米细菌是两个新的纳米细菌种属，与前期Kajander教授提交的Nanobacterium sanguineum具有较近的亲缘关系。背景：纳米细菌诱导钙化形成的机制还不明确，也没有相关实验研究证明它可以促进钙化相关蛋白的分泌。由于纳米细菌的主要成分是羟基磷灰石，而这种成分被证实可以促使骨骼形成同时促进钙化相关蛋白的分泌。因此，作者初步分析钙化相关蛋白--骨形态发生蛋白2（Bone morphogenetic protein BMP-2）与骨桥蛋白（Osteopontin OPN）在钙化胎盘组织中的分泌以及与纳米细菌感染之间的关系。目的：探讨纳米细菌感染对胎盘组织中骨形态发生蛋白2（BMP-2）与骨桥蛋白（OPN）两种钙化相关蛋白分泌的影响。方法：分别取钙化胎盘组织纳米细菌分离培养阳性组，阴性组以及正常胎盘组的胎盘组织标本作为实验对象。①免疫组化的方法分析不同实验组织BMP-2与OPN两种蛋白表达的差异：制作组织切片，HE染色，观察钙化。常规脱蜡水化，血清封闭，分别孵育BMP-2和OPN蛋白单克隆抗体4℃过夜，生物素化二抗作用，DAB显色，苏木素复染，显微镜下观察拍照。②Western Blot：SDS－PAGE电泳的方法分离出钙化与非钙化组织中BMP-2与OPN两种蛋白，化学发光，显影，定影，软件半定量分析目的条带的灰度值，比较BMP-2与OPN两种钙化相关蛋白在不同组织中的表达差异。结果：免疫组化结果可见钙化胎盘组织中纳米细菌分离培养阳性组与阴性组BMP-2与OPN的表达均高于正常胎盘组织(P<0.01)。Western Blot结果分析可知BMP-2与OPN在钙化胎盘组织中纳米细菌分离阳性组和阴性组的表达高于正常胎盘组织(P<0.01)。但两组之间表达差异不明显（P>0.05）结论：钙化组织BMP-2与OPN两种蛋白的表达确有增加，但纳米细菌感染对于促进胎盘组织的钙化机制仍不明确。背景：纳米细菌的特殊组成成分、大小和复制方式使得其分离培养与保存方法一直没有统一。为了使更多疑似与纳米细菌感染有关的疾病可以分离培养出纳米细菌，作者以胎盘钙化组织为例，探讨钙化组织中纳米细菌的分离技术。同时，通过对纳米细菌不同的培养和保存条件的实验研究，来探讨纳米细菌的培养与保存的最佳条件和方法，有助于纳米细菌的阳性培养和后续基因组计划的实施。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，探究培养和保存纳米细菌最适宜的方法和条件。方法：方法①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4周，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。③4℃保存钙化组织和纳米细菌菌液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℃。更多还原

【Abstract】 Background：Finnish scientist Kajander and his team found mammaliancell vacuolar changes with no possible microbial contamination in cellculture but self-calcification bacteria of a diameter of only50-800nm. Theynamed it Nanobacteria (NB) and applied for a patent. Fourier transformdispersive spectroscopy (FTIR) showed that the main component ofnanobacteria shells is hydroxyapatite, so NB also known as CalcifyingNanoparticles (CNPs). Indian scientists found CNPs existed in earlycalcified placental tissues under TEM, but they failed to isolate and culturenanobacteria.Objective: Isolate and cultivate nanobacteria from placental calcification tissues and observe their growth characteristics. Investigatethe microbial infection evidence on placental calcification. Meanwhile,preliminary discussion the relationship between placental calcification andfetal nanobacteria infection。Methods：Calcified placental tissue samples were collected from36confirmed PC cases. They were observed under transmission electronmicroscopy (TEM). All the samples were decalcified in1mol/L HCl,neutralized with1mol/L Tris, centrifuging at14,000g and filtered with0.22μm Millipore filters to isolate nanobacteria. The filtered liquids weremixed with cell culture medium supplemented with10%fetal bovineserum (FBS), adjusted PH to7.4, and cultured in a37oC incubator with5%CO2and95%air. Growth of nanobacteria were observed and classified.FBS or normal saline was used as vehicle controls; and normal placentaltissues which were decalcified and cultured under the same conditions wereused as negative controls; RPMI1640medium as a blank control.Meanwhile isolated and cultured nanobacteria from fetal umbilical cordblood which have placental calcification.Results：Under transmission electron microscopy, the fixed calcifiedplacental samples showed nanoparticles with oval-shape and different sizesranging from50nm to500nm in diameter. Each particle was wrapped by ashell with different electron-dense，some could be seen in the process ofself-dividing as descripted by other scientists.28PC samples showed white precipitation complete or partly adhered to the bottom of glass tubes, whilenegative groups showed nothing deposited. OD650measurements duringculturing indicated that NB grew in a similar way as other bacteria.Therewere positive correlation between nanobacteria isolated from placentalcalcification tissues and corresponding fetal umbilical cord blood，whichwere12blood samples showed white precipitation.Conclusion：Self-replicating calcifying nanoparticles could be isolatedfrom calcified placental tissues with the methods descript for nanobacteria.The fetuses with placental calcification are more susceptible to thenanobacteria. Background：Further identify morphology and genomics of thecultured white precipitate. Nanobacteria are so small, some less than100nm, that many scientists believe it does not have the genes, or not a lifeform. Since professor Kajander submitted the Nanobacteria-specific16SrRNA gene sequence X98418to GenBank, many scientists have triedto identify newly isolated nanobacteria based on this sequence.Objective: Transmission electron microscopy (TEM), scanningelectron microscopy (SEM), alizarin red calcium staining and analysis of16srRNA gene were used to get a further identification of the isolatednanobacteria.Methods：①For TEM observation: Nanobacteria were centrifuged at14,000g, fixed in glutaraldehyde, post-fixed in osmic acid, dehydrated inethanol, embedded in epoxy, dual electronic stained, double distilled waterwashed, dried and observed with TEM and photographed.②For SEMobservation: Nanobacteria were fixed in Glutaraldehyde, routinedehydrated, critical point drying, sputter-coated with gold, then observedwith SEM and photographed.③Alizarin red staining: Cultured samplessmears were fixed,2%alizarin red dye solution stained,0.2%aqueous lightgreen counterstained,0.5%aqueous acetic acid washed, ethanol dehydrated and mounted with neutral resin after drying, observed under the oilmicroscope and photographed.④16SrRNA gene sequence analysis:Extracted nanobacterial genome, designed nanobacterial16SrRNA genesequence-specific primers, amplified its specific sequence, examined andrecovered the target fragment in gel electrophoresis, analyzed the geneticsequence results in GenBank. Then，designed the16SrRNA gene primersof newly discovered nanobacteria, amplified the16SrRNA gene sequenceof placental calcification and compared the results to the newly discoverednanobacteria.Results：①Under TEM, nanobacteria showed oval-shaped particleswith a diameter with200-500nm, having high electron-dense shells orsingle solid structure nanobacteria without shells.②Under SEM,nanobacteria showed small single particles with a diameter of about200nm,or gathered into micron size clumps.③Alizarin red staining showednanobacteria stained red particles.④16SrRNA gene analysis revealed twonew Nanobacteria species which isolated and cultured from placentalcalcified tissues. The16SrRNA gene sequencing results were submitted toGenBank and their access number are JN029830and JF823648, which arerespectively of93%and81%similarity compared with the nanobacteriastrains (GenBank accession: X98418).Conclusion： The16SrRNA gene sequencing of NB isolated fromplacental calcification tissues indicated the NB are novel nanoparticles. Background: Mechanism of nanobacteria induced calcification isunclear. There is no relevant studies have shown that it can promote thesecretion of calcification-related proteins. Nanobacteria are composited ofhydroxyapatite which is proven to promote bone formation and simutalecalcification-related protein secretion. The authors preliminary analyze therelationship between secretion of bone morphogenetic protein2(BMP-2)、osteopontin (OPN) in calcification placental tissue and nanobacteriainfections.Objective: To investigate the influence of nanobacteria infection onsecretion of protein BMP-2and OPN in calcified placental tissues.Methods: Placenta calcified tissues and normal placental tissues wereincluded in the study.①Immunohistochemistry (IHC) to analyze theprotein expression differences of different tissues: production of tissuesections, conventional dewaxing, hydration, serum closed, OPN andBMP-2monoclonal antibody incubation at4℃overnight, biotinylatedsecondary antibody reaction, DAB coloration and observation undermicroscope.②Western Blot analysis: SDS-PAGE electrophoresis methodto isolate BMP-2and OPN proteins in calcified and non-calcified tissue, chemiluminescence, developing and fixing, semi-quantitative analysis thegray value of target band by software. Comparison the expressiondifferences of calcification-related proteins OPN and BMP-2in differenttissues.Results: The IHC results indicate the expression of BMP-2and OPNin calcification placental tissues were higher than in normal placentaltissues (P<0.01). Western Blot analysis shows the expression of OPN andBMP-2in placental tissue of calcification is higher than in normal placentaltissue (P<0.01).Conclusion: Nanobacteria infection promotes the expression of OPNand BMP-2and the calcification of placenta. Background：Because of the special composition and replication wayof nanobacteria, there are no unified approaches to isolate、culture andpreserve nanobacteria. In order to let more nanobacteria infection-relateddiseases detected, we took placenta calcified tissues for example to explorethe best separation technology of nanobacteria in the calcified tissues.Studying the culture and preservation methods of nanobacteria, andfounding the best conditions and methods could help nanobacteria positivecultivation and genome project.Objective: To find an optimum method to isolate nanobacteria incalcified tissues and a most suitable condition to culture and observenanobacteria, we took placental calcified tissues for example to do thisresearch wishing these methods can be done with other calcified tissues aswell.Methods①Calcified placental tissues were demineralized separatelywith hydrochloric acide and ultrasonic vibration to isolate nanobacteriafrom the samples, cultured and compared their positivity rates.②Nanobacteria were cultured separately in cells and bacteria cultureconditions and concentration was recorded with spectophoto fluorometer ③Fresh calcified tissues and cultured nanobacteria were preserved indifferent low temperature conditions，recovered and concentration wasrecorded.Results①Nanobacteria were more easy to obtain in HCldemineralized group.②Nanobacteria could grow in bacteria cultureconditions as well as in cell culture conditions.③Fresh samples andnanobacteria could preservation in4℃condition for several weeks.Conclusion Nanobacteria could be isolated with HCl, cultured inbacteria cultured conditions and preserved in4℃for a short time.更多还原