Isolation And Identification Of Nanobacteria And Study Its Calicification Mechanism

Objective: To isolate and identify nanobacteria from placentalcalcification tissues and its Umbilical cord blood,cultured with breastcancer MDA—MB—231cells, observe the changes in cells, expression ofcalcification related proteins such as BMP-2,OPN and poptosis relatedproteins such as Bcl-2,Bax,to discuss the mechanism of calcificationcaused by NB infection.Methods:20cases of calcified placental tissues， umbilical cordblood, and normal placental tissues were collected for NB culture. NBwere detected with Gram staining and alizarin red staining,observed withtransmission electron microscope(TEM) and scanning electronmicroscope(SEM) to identifiy,after that three groups were chosen for thenext studies: control group, NB group and nHA group. The toxic effect onMDA-MB-231breast cancer cells were detected by CCK-8after12h、24h、48h. meanwhile, the activity of LDH and ALP were surveyed after12h、24h、48h、72h, respectively. And72h later, the apoptotic rates of each groupwere measured by FCM, and the ultra structure of MDA-MB-231were observed by TEM, The expression of BMP-2,OPN,Bcl-2,Bax weremeasured by Western blot,all datas analysised by GraphPad Prism5, P＜0.05P <0.05was statistically significant.Reasults:65%(13/20)calcified placental tissues and80%(16/20)cordblood specimens can be obtained NB:Gram-negative, Alizarin Redpositive, under electron microscope,80-500nm, spherical with burr edge.The isolation rate of nanobacteria in the umbilical cord blood(80%) andcalcified placental tissue were higher than in the normal placentaltissues(10%), there is a significant difference χ2were12.07and9.09, P<0.01.While there was no significant difference in the positive isolationrate of NB between the umbilical cord blood and calcified placental tissues（χ2=1.33,P＞0.05）. CCK-8revealed that the inhibition of NB on breastcancer cells is clearly higher than nHA group and normal group in24h,48hand72h(P<0.001). There were statistical differences in LDH activity leveland ALP activity level between between NB group and normal group in24h，48h and72h，while there were statistical differences in LDH activitylevels between NB group and nHA group only in24h,48h,and statisticaldifferences in ALP activity levels between NB group and nHA group in24h,48h,and72h. After72h, the apoptotic rates of breast cancer cells NBgroup were obviously higher than that in nHA group(P<0.001). The TEMobservation showed that MDA-MB-231cell in NB group had cytoplasmiccavity sample variable，karyopyknosis and apoptotic bodies. The nHA group didn’t see obvious anomaly. The expression of BMP-2in NB groupis higher than normal, t=5.52, P <0.01, there are significant differences,higher than that of nHA group, but without statistical significance; OPN,Bax were expressed higher than that of normal group and nHA group, P <0.001, significant difference; The expression of Bcl-2reduced, comparedwith normal group, t=6.12, P <0.01, there are significant differences,below the nHA group, but no statistical significance.Conclusion: Placental calcification is associated with infection of NB,calcification mechanism may be generated in the NB secrete certainsubstances inducing apoptosis, or NB components other than the nHA shellinduced cell apoptosis, cell apoptosis rate is greater than the organismremoval rate, causing local calcium phosphorus metabolism disorders, suchas combination of NB core substances such as formation ofbiomineralization, further lead to calcification.