Isolation, Cultivation And Identification Of Nanobacteria In Placental Calcification And Preliminary Study Of Calcified-Related Proteins

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.