The Study Of Mechanism About Human Placenta Calcification Induced By Nanobacteria

PART Ⅰ The Morphology observation, Isolation, andCultivation of Nanobacteria and the Expression of relationProteinBackground：The nanobacteria, found by Kajander etal. in bovine serum,were a risk factor of stone and calcification disease. In recent years, theresearches indicated that nanobacteria were related to stones, inflammation,atherosclerosis and other diseases. The nanobacteria were isolated andcultured from the human saliva, teeth, dental plaque, dental calculus, dentalpulp stone, kidney stones, gallstones, bladder, urinary calculi, prostatestones, the atherosclerotic plaque and chronic prostatitis successfully.Furthermore, there is no tissue specificity about nanobacteria, because thenanobacteria were isolated culture from different tissues and cellsrespectively, such as blood, saliva and articular cavity effusion. However,the placenta calcification (placental calcification, PC) associated with thenanobacteria has not been reported. Calcification usually occurs in late pregnancy, and the bacteria is the most common obstetric pathologiccalcification. Serious level Ⅲ calcification can lead to fetal dysfunction,fetal distress, fetal growth restriction, fetal abnormalities, aspirationpneumonia and a series of pregnancy complications. To date, placentacalcification mechanism is not clear, the relation of placenta calcificationand the nanobacteria has not been reported. The aim of this paper is toexplore the relationship of nanobacteria and placenta calcification, and itsmechanism of action.Objective:1. to seek the direct evidence of nanobacteria relation to theplacenta calcification;2. to compare the nanobacteria morphology andnanobacteria positive rate of various kinds of tissues (placenta calcifiedtissue, umbilical cord blood and gallstones);3, to discuss the method aboutnanobacteria quick proliferation;4, to study the relationship of calcificationrelated proteins (OPN, BMP-2), apoptosis related proteins (Bax, Fas) andinflammatory factor (IL-6) between nanobacteria and calcification ofplacenta.Methods:1, to select the fresh calcification placenta tissue to section andstain; to observe the nanobacteria morphology by the transmission electronmicroscope（TEM）and scanning electron microscope (SEM) directly, withthe normal tissues of the same placenta as negative control.2, to isolate andcultivate the nanobacteria from the calcification of placenta and umbilicalcord blood of the same pregnant women, and to compare the nanobacteriamorphology and nanobacteria positive rate.3, to compare the nanobacteriamorphology and nanobacteria positive rate from different pregnant womenthe calcification of placenta and normal placental tissue.4, to compare thenanobacteria morphology and nanobacteria positive rate of the calcificationplacenta tissue and gallstones from different pregnant women.5, to compare different proliferation methods for nanobacteria culture andobserve the turbidity value changes. Sixth，OPN, BMP-2, Bax and Fasproteins were measured by Western Blot and immunohistochemicalmethods in calcific placenta group (NB+) and normal placenta (NB-),respectively.6, we assayed the IL-6concentration of calcific placentagroup (NB+) and normal calcific placenta (NB-) blood homogenate withELISA method and detected the concentration of ALP by automaticbiochemical analyser.Results:1. The nanobacteria were found by TEM in the freshcalcification placenta tissue, which were80-500nm in diameter, and hadcoccoid, coccobacillar, or bacillar form surrounded by a thin shell withdifferent electron density or burrs. But there were no nanobacteria in thenormal placenta tissue.2. The nanobacteria were isolated and cultured from20cases calcification placenta tissues and umbilical cord bloodsuccessfully, which was white precipitate and attached the bottom of thetube entirely or partly. The nanobacteria positive rates were65%and80%,respectively. There is no statistical difference (p>0.05) and theirmorphology are very similar.3. We isolated20normal placenta tissues andthe umbilical cord blood, and the nanobacteria positive rate were10%and20%, respectively. Compared with calcifical placenta tissue group, thenanobacteria positive rate was lower significantly (P<0.01). But there wasno difference about nanobacteria morphology.4. The20cases of gallstoneswere isolated, and the nanobacteria was white precipitation as the sameisolated from the placenta tissue. But its density was stronger than theplacenta tissue and the nanobacteria positive rate was90%, which washigher than that of calcifical placenta tissue. And its morphology wassimilar as that of placenta tissue.5. Dividing method represented that the nanobacteria proliferation was faster than that of changing culture mediumdirectly. The turbidity value increases1times every6-10days.6.Theexpressions of OPN, BMP-2, Bax and Fas proteins and the concentrationsof IL-6, ALP in calcific placenta group (NB+) were all higher than those ofnormal placenta (NB-) remarkably (P<0.01).Conclusion: Nanobacteria had obvious relation to the placentacalcification. And the nanobacteria’s shape was consistent with literaturereported. Meanwhile, there was no tissue specificity about nanobacteriainfection. No matter placenta tissue or umbilical cord blood and gallstones,the nanobacteria morphology was similar. The nanobacteria of calcificplacenta tissue maybe result from the same as the umbilical cord blood.Exchang bottle mathod can extend nanobacteria proliferation. Moreover,nanobacteria, apoptosis, flammatory factors and ALP maybe promote theplacenta calcificated. The nanobacteria were a factor above all. Part Ⅱ Cytotoxicity and Mechanism induced bynanobacteria and nano-hydroxyapatites in humanchoriocarcinoma cells JAR, Osteoblast C3H10and breastcancer MDA-MB-231cellsBackground: The correlation of nanobacteria and placenta calcificationwas proved at the level of the tissue in partⅠ. And the nanobacteria canmake the calcification related proteins and apoptosis related proteins up-regulation. According to the literatures, nanobacteria can inhibit theproliferation of fibroblasts. Nano-hydroxyapatite can cause tumor cellapoptosis, but it had a good compatibility with normal fibroblasts andosteoblasts. There was a problem that could nanobacteria make normalosteoblast and tumor cells cytotoxic effects? The aim of this study was toexplore the cytotoxicity and inflammatory effects in osteoblast C3H10cells,breast cancer MDA-MB-231cells and choriocarcinoma JAR cells inducedby nanobacteria and nanohydroxyapatite.Objective: To compare the cytotoxicity about nanobacteria andnano-hydroxyapatites on human choriocarcinoma cells and OsteoblastC3H10, breast cancer MDA-MB-231cells for further clarifying themechanism and the relationship cause of the cytotoxic effects.Methods: The growing well osteoblast C3H10, breast cancerMDA-MB-231cells and JAR carcinoma cells were treated with NB andHA, respectively. Then the cell proliferation inhibition rate can bedetermined by CCK8accordong to manipulate. The hoechest22358fluorescence staining and flow cytometry instrument were used for thedetermination of cell apoptosis rate. We used inverted microscope andelectron microscope to observe the change of cell morphology, and westernblot to determinate the expression of BMP-2, Bax, Fas and OPN in threekinds of cells.Results: Under the inverted microscope, after a48h treatment, the NBand HA were swallowed by breast cancer MDA-MB-231cells, but therewere no obvious changes in human choriocarcinoma cells and OsteoblastC3H10. Comparing to control group, a large number of vacuoles appeared inthe cell plasma, the effect of NB was more potent than HA in breast cancerMDA-MB-231cells. CCK8results showed that the NB and HA can inhibitcell proliferation after a48h treatment for tumour cells. NB effect was stronger than HA with time and the concentration dependence. But forOsteoblast C3H10, there was no cytotoxicity in HA group. By Hoechest22358fluorescence staining and flow cytometry, the apoptosis rates weredetermined. And NB effect was stronger than HA for tumour cells.Westernblot results indicated that OPN, BMP-2, Bax, Fas expression of NB andHA were higher than control group, and NB effect was stronger than HAfor tumour cells.TEM results showed that NB can cause cell apoptosis andthe mitochondria swelling, nucleus pycnosis, cell apoptosis. Thecharacteristics of the cell apoptosis were visible in the humanchoriocarcinoma JAR cells, Osteoblast C3H10and breast cancerMDA-MB-231cells. Furthermore, in the JAR cells, the cell dissolved and alarge number of autophagies occured, but in the HA group and the controlgroup, there were no obvious changes. In the Osteoblast C3H10cells,cytotoxic effect occured only in the NB group, and there were no changesin the HA group, compared with the control group.Conclusion: Compared with control group, nanobacteria andhydroxyapatite can cause cytotoxic effect, and whether humanchoriocarcinoma JAR cells and Osteoblast C3H10or breast cancerMDA-MB-231cells, cytotoxic effects of nanobacteria were stronger thannanohydroxyapatite. NB played a key role of promoting apoptosis. Theexpressions of OPN, BMP-2, Bax and Fas were more potent thanHA.Western blot showed that the expression of Bax and Fas in NB groupobviously stronger than that in HA group. The mechanism of apoptosismay be activated via FasL binding to the FasR signaling pathway. PART Ⅲ The preliminary discussion of placenta calcificationin SD rats induced by Nanobacteria andNano-hydroxyapatitesBackground: In the partⅠ,we found that nanobacteria associated withplacenta calcification and cell apoptosis at the level of tissue. In the partⅡ,we compared the cytotoxicity in choriocarcinoma JAR cells, osteoblastC3H10cells and human breast cancer MDA-MB-231cells induced by thenanobacteria and nano-hydroxyapatite, respectively. NB not only inducedtumor cell apoptosis, but also caused normal osteoblast apoptosis and death.Though, only did HA cause apoptosis of tumor cells. This showed thecytotoxicity induced by NB was not just the effect of hydroxyapatite, andalso relation with the secrete proteins and proliferation of nanobacteria. Inaddition, bacteria and nano-hydroxyapatite can make calcificationassociated protein OPN and BMP-2up-regulation. Then, did thenanobacteria and nano-hydroxyapatite directly cause the placentacalcification? We study this point by SD rats model.Objective: To study the nanobacteria function that can directly lead toSD rat placenta calcification by comparing the effects of SD rat placentatissue attacked on nanobacteria and hydroxyapatite.Methods:28SD rats were choosed,7males and21females, folded bythe male and female1:3. When the females SD rats were fertilized anddivided into three groups (PBS group, HA group and NB group). On the12th day after conception, we injected200μl PBS fluid,2MCF HA fluidand2MCF NB fluid into the SD rats’ uterus, respectively. And injecting again on the14th day. On the21th day after conception, we took out theSD rat placenta to observe the changes and to carry out electronmicroscope slices, immunohistochemical staining, westernblot assay andnanobacteria culture.Results: Comparing with three groups of SD rats placental tissue, therewere no obvious calcification by macro-observation. Nanobacteria culturesof three groups were all negative. The nanoparticles and the changes of cellmorphology were not been observed under TEM. And there were nosignificant differences about the expressions of apoptosis related proteins(Bax) and calcification (BMP-2) in three groups.Conclusion: The placenta has no obvious calcification in three groups(PBS, HA and NB) of SD rats. There were also no significant differences ofprotein expression (Bax and BMP-2) and morphological structure. On theone hand, the time of HA and NB interacted with SD rat placenta was short,and not forming calcification. The results showed that NB was just a factorfor inducing apoptosis or death and associated with calcification, but not adirect resean of placenta calcification.