本文選題：納米細菌 + 胎盤鈣化��； 參考：《重慶醫(yī)科大學》2012年博士論文

【摘要】：背景：芬蘭科學家Kajander教授及其團隊最早從哺乳動物細胞培養(yǎng)過程發(fā)現(xiàn)細胞的空泡樣變化，在排除了一切可能的微生物污染的情況下對其進行分析，發(fā)現(xiàn)了一種可以自我復制礦化且直徑只有50-800nm的微生物，命名為納米細菌（Nanobacteria NB）并申請了專利1。利用傅里葉變換色散光譜分析（FTIR）表明納米細菌外殼的主要成分為羥磷灰石，所以又稱為納米礦化顆粒（CNPs）。有科學家在透射電鏡下觀察早期鈣化胎盤組織發(fā)現(xiàn)納米礦化顆粒的存在，但未能分離培養(yǎng)出納米細菌。 目的：從胎盤鈣化組織和相應胎兒臍帶血中分離培養(yǎng)出與鈣化相關的納米細菌，觀察其生長特性，探討胎盤鈣化發(fā)生的微生物感染證據(jù)以及胎盤鈣化與胎兒納米細菌感染的關系。 方法：以臨床所取36份胎盤鈣化組織作為研究對象，透射電鏡觀察鈣化組織中的納米細菌。而后依次利用1mol/L鹽酸脫礦，Tris中和，生理鹽水稀釋，14,000g高速離心，最后通過0.22μm細菌濾器過濾的方法進行分離。濾液過濾到有棉塞試管中，加入含有10%胎牛血清的RPMI1640細胞培養(yǎng)基，調整pH7.4，放入37℃細胞培養(yǎng)箱,5％CO2，95％空氣條件下培養(yǎng)。觀察細菌生長情況并依據(jù)沉淀類型分類。OD650波長下記錄納米細菌菌液相對濃度OD值，制作生長曲線。胎牛血清（FBS）和生理鹽水作為試劑對照,無鈣化的正常胎盤組織作為實驗對照，用RPMI1640培養(yǎng)基作為空白對照。同時對存在胎盤鈣化的胎兒取臍帶血，對納米細菌進行分離培養(yǎng)，方法同上。 結果：透射電鏡下觀察鈣化胎盤組織標本，可見胎盤絨毛組織和鈣化斑塊之間存在橢圓形納米顆粒，其直徑約為50-500nm，每個顆粒都被具有不同電子密度的薄外殼包圍，其中有些顆�？梢姺至�，與國內(nèi)外文獻報道相似。實驗組中有28例可見白色沉淀完全或部分粘附在試管底部，對照組未見沉淀產(chǎn)生，差異具有顯著性（P 0.01）。OD650分光光度計下記錄得到納米顆粒生長曲線與其他細菌類似。臍帶血分離培養(yǎng)的結果與相應鈣化組織胎盤中納米細菌培養(yǎng)的結果正相關,有12例實驗組培養(yǎng)物可見白色沉淀物，對照組未見（P 0.01）。 結論：通過納米細菌分離培養(yǎng)的方法可以從鈣化胎盤組織中得到具有自我復制礦化功能的納米顆粒，且存在胎盤鈣化的胎兒也較易感染此納米顆粒。. 背景：進一步對培養(yǎng)所得白色沉淀物進行形態(tài)學和基因組學的鑒定。由于納米細菌形態(tài)微小，有的還不足100nm，因此許多科學家認為它并不存在遺傳基因，也不屬于生物范疇。Kajander教授向GenBank中提交了納米細菌特異的16SrRNA基因序列X98418，因此后續(xù)科學家多依據(jù)這個序列對分離培養(yǎng)出的納米細菌進行種屬鑒定。 目的：利用透射電鏡，掃描電鏡，茜素紅染色和16SrRNA基因分析的方法對分離培養(yǎng)得到的納米細菌進行進一步鑒定。 方法：①透射電鏡觀察：納米細菌14,000g高速離心沉淀，戊二醛前固定，四氧化二鋨后固定，梯度脫水，環(huán)氧樹脂包埋，制作超薄切片，雙重電子染色，雙蒸水洗滌，干燥后在透射電鏡下觀察拍照。②掃描電鏡觀察：納米細菌沉淀利用戊二醛固定,常規(guī)脫水，，臨界點干燥，噴金，在掃描電鏡下觀察拍照。③茜素紅染色：培養(yǎng)標本涂片固定，沖洗，2％茜素紅染液染，0.2％淡綠水溶液復染，O.5％醋酸水溶液沖洗,乙醇脫水，烘干后用中性樹膠封片，高倍鏡下觀察拍照。④16SrRNA基因序列分析：提取納米細菌基因組，設計納米細菌16SrRNA基因序列特異性引物，PCR擴增其特異性序列，凝膠電泳觀察并回收目的片段，送檢進行基因序列分析，結果與GenBank比對。根據(jù)比對結果設計新發(fā)現(xiàn)納米細菌16SrRNA基因特異性引物，對胎盤鈣化組織進行16SrRNA基因序列分析，其結果與新發(fā)現(xiàn)納米細菌進行比對，驗證提取過程有無污染菌。再將所得菌株與GenBank中所收錄納米細菌相關16SrRNA基因做系統(tǒng)進化分析，繪制系統(tǒng)進化樹。 結果：①透射電鏡下可見菌液中的納米細菌呈橢圓形，直徑在200-500nm，有或沒有高電子密度的外殼包繞在外。②掃描電鏡下可見納米細菌形態(tài)微小，單個顆粒直徑在200nm左右，或聚集成微米大小團塊。③茜素紅染色可見納米細菌染成紅色細小顆粒。④16SrRNA基因分析發(fā)現(xiàn)胎盤鈣化組織中分離培養(yǎng)得到納米細菌新種屬，分別與納米細菌X98418相比有93%和81%相似性，兩個種屬納米細菌的16SrRNA基因測序結果提交GenBank得到基因編號JN029830與JF823648。鈣化胎盤組織中納米細菌基因擴增分析可知其與先前分離培養(yǎng)菌液中的納米細菌是同一種菌。與GenBank中其他納米細菌種屬進行對比，發(fā)現(xiàn)這兩個種屬的納米細菌與先期Kajander教授提交的Nanobacterium sanguineum(X98418)親緣關系最近。 結論：胎盤鈣化組織中分離培養(yǎng)得到的納米細菌是兩個新的納米細菌種屬，與前期Kajander教授提交的Nanobacterium sanguineum具有較近的親緣關系。 背景：納米細菌誘導鈣化形成的機制還不明確，也沒有相關實驗研究證明它可以促進鈣化相關蛋白的分泌。由于納米細菌的主要成分是羥基磷灰石，而這種成分被證實可以促使骨骼形成同時促進鈣化相關蛋白的分泌。因此，作者初步分析鈣化相關蛋白--骨形態(tài)發(fā)生蛋白2（Bone morphogenetic protein BMP-2）與骨橋蛋白（Osteopontin OPN）在鈣化胎盤組織中的分泌以及與納米細菌感染之間的關系。 目的：探討納米細菌感染對胎盤組織中骨形態(tài)發(fā)生蛋白2（BMP-2）與骨橋蛋白（OPN）兩種鈣化相關蛋白分泌的影響。 方法：分別取鈣化胎盤組織納米細菌分離培養(yǎng)陽性組，陰性組以及正常胎盤組的胎盤組織標本作為實驗對象。①免疫組化的方法分析不同實驗組織BMP-2與OPN兩種蛋白表達的差異：制作組織切片，HE染色，觀察鈣化。常規(guī)脫蠟水化，血清封閉，分別孵育BMP-2和OPN蛋白單克隆抗體4℃過夜，生物素化二抗作用，DAB顯色，蘇木素復染，顯微鏡下觀察拍照。②Western Blot：SDS－PAGE電泳的方法分離出鈣化與非鈣化組織中BMP-2與OPN兩種蛋白，化學發(fā)光，顯影，定影，軟件半定量分析目的條帶的灰度值，比較BMP-2與OPN兩種鈣化相關蛋白在不同組織中的表達差異。 結果：免疫組化結果可見鈣化胎盤組織中納米細菌分離培養(yǎng)陽性組與陰性組BMP-2與OPN的表達均高于正常胎盤組織(P0.01)。Western Blot結果分析可知BMP-2與OPN在鈣化胎盤組織中納米細菌分離陽性組和陰性組的表達高于正常胎盤組織(P0.01)。但兩組之間表達差異不明顯（P0.05） 結論：鈣化組織BMP-2與OPN兩種蛋白的表達確有增加，但納米細菌感染對于促進胎盤組織的鈣化機制仍不明確。 背景：納米細菌的特殊組成成分、大小和復制方式使得其分離培養(yǎng)與保存方法一直沒有統(tǒng)一。為了使更多疑似與納米細菌感染有關的疾病可以分離培養(yǎng)出納米細菌，作者以胎盤鈣化組織為例，探討鈣化組織中納米細菌的分離技術。同時，通過對納米細菌不同的培養(yǎng)和保存條件的實驗研究，來探討納米細菌的培養(yǎng)與保存的最佳條件和方法，有助于納米細菌的陽性培養(yǎng)和后續(xù)基因組計劃的實施。 目的：以鈣化的胎盤組織為例，尋求在鈣化組織中分離納米細菌的最佳方法，探究培養(yǎng)和保存納米細菌最適宜的方法和條件。 方法：方法①胎盤鈣化組織標本分別用鹽酸脫礦與超聲振蕩脫礦的方法分離納米細菌，計算其分離陽性率。②分離出的納米細菌分別用細胞培養(yǎng)箱與細菌培養(yǎng)箱培養(yǎng)4周，利用分光光度計記錄兩種條件下納米細菌濃度的變化，并描繪生長曲線。③分別用4℃，-20℃與-80℃冰箱保存鈣化組織和納米細菌，記錄納米細菌分離和復蘇的生長狀況并繪制生長曲線。 結果：①鈣化組織用鹽酸脫礦更易分離得到納米細菌。②細胞與細菌培養(yǎng)環(huán)境下納米細菌的生長速度并無明顯差別。③4℃保存鈣化組織和納米細菌菌液對于其分離和復蘇都要優(yōu)于-20℃和-80℃ 結論：對鈣化組織進行鹽酸脫礦可以更好的分離出納米細菌，并且可以在細菌培養(yǎng)箱內(nèi)培養(yǎng)納米細菌，新鮮鈣化組織標本和納米細菌可以短時間保存在4℃。
[Abstract]:Background: Professor Kajander and his team, Finland scientist, and his team first discovered cell vacuoles from mammalian cell culture, analyzed it in the absence of all possible microbial contamination, and found a microorganism that could be self replicating mineralization with a diameter of only 50-800nm, named nanoscale (Nanobacte RIA NB) and application for patent 1., the Fourier transform dispersive spectroscopy (FTIR) was used to show that the main component of the nanoscale shell is hydroxyapatite, so it is also called nano mineralized particle (CNPs).
Objective: to isolate and cultivate nanometallic bacteria related to calcification from placental calcification and fetal umbilical cord blood, to observe its growth characteristics, to explore the evidence of microorganism infection in placental calcification, and to investigate the relationship between placental calcification and fetal nanoscale bacterial infection.
Methods: 36 placental calcification tissues were taken as the research object, and the nanoscale in calcified tissue was observed by transmission electron microscope. Then, 1mol/L hydrochloric acid demineralization, Tris neutralization, saline dilution, 14000g high speed centrifugation were used. Finally, the method was separated through the filter of 0.22 m bacteria filter. Filter filtrate was filtered to the test tube with cotton plug, and the filter solution was added to the test tube with cotton plug. In the RPMI1640 cell culture medium containing 10% fetal bovine serum, the pH7.4 was adjusted, the cell culture box at 37 C and the 5%CO2,95% air were cultured. The growth of the bacteria was observed and the relative concentration of nanoscale bacterial liquid was recorded at the.OD650 wavelength of the precipitation type, and the growth curve was made. The fetal bovine serum (FBS) and the physiological saline were used as the reagents. The normal placental tissue without calcification was used as the experimental control, and the RPMI1640 medium was used as a blank control. At the same time, the umbilical cord blood of the fetus with placental calcification was taken from the umbilical cord blood, and the nanoscale bacteria were isolated and cultured.
Results: an elliptical placental tissue specimen was observed under transmission electron microscopy. There was an elliptical nano particle between placental villus and calcified plaque. The diameter of the nanoparticles was about 50-500nm. Each particle was surrounded by a thin shell with different electron densities. Some of the particles were visible and split. In the experimental group, 28 cases were available. The white precipitation was completely or partially adhered to the bottom of the test tube, and the control group had no precipitation. The difference was significant (P 0.01).OD650 spectrophotometer. The growth curve of nanoparticles was similar to that of other bacteria. The results of umbilical cord blood isolation and culture were positively related to the results of nanoscale culture in the corresponding calcified tissue. There were 12 cases. White sediment was observed in the culture samples, but not in the control group (P 0.01).
Conclusion: the nano particles with self replicating mineralization can be obtained from the calcified placenta by the method of isolation and culture of nanoscale, and the fetus of placental calcification is also easier to infect the nanoparticles.
Background: to further identify the morphology and genomics of the cultured white precipitates. Because the morphology of the nanometers is small and some are less than 100nm, many scientists believe that it does not exist and does not belong to Professor.Kajander, a biological category, to submit the specific 16SrRNA gene sequence X98 of nanoscale bacteria to GenBank. 418, therefore, subsequent scientists based on this sequence were used to identify species of nanomaterials isolated and cultured.
Objective: to further identify the nanomaterials isolated and cultured by transmission electron microscopy, scanning electron microscopy, alizarin red staining and 16SrRNA gene analysis.
Methods: (1) transmission electron microscopy: nanometrobacterium 14000g high speed centrifuge precipitation, glutaraldehyde fixed, four oxidation two osmium fixed, gradient dehydration, epoxy resin embedding, ultra-thin section, double electron staining, double water washing and observation under transmission electron microscope. 2. Scanning electron microscope observation: nanometroms precipitation using glutaraldehyde Fixed, dehydrated, critical point drying, spray gold, observation and photograph under scanning electron microscope. 3. Alizarin red staining: culture specimen smear fixed, rinse, 2% alizarin red dye dye, 0.2% light green water solution, O.5% acetic acid water solution rinse, ethanol dehydration, after drying with neutral gum seal, high times mirror observation taking pictures. (4) 16SrRNA gene sequence Analysis: the genome of nanoscale bacteria was extracted, the specific primers of the nanoscale 16SrRNA gene sequence were designed, the specific sequence was amplified by PCR, the target fragments were observed and recovered by gel electrophoresis, the gene sequence analysis was carried out and the results were compared with the GenBank. According to the comparison results, the specific primers of the nanoscale 16SrRNA gene were designed, and the placental calcium was found. 16SrRNA gene sequence analysis was carried out in the chemical tissue, and the results were compared with the new nanoscale bacteria. The results showed that there were pollution-free bacteria in the extraction process, and then the 16SrRNA gene related to nanbacteria in GenBank was analyzed, and the phylogenetic tree was plotted.
Results: under transmission electron microscopy, the nanometers in the bacteria were elliptical, the diameter of the bacteria was in 200-500nm, and the outer shell with or without high electron density was around. 2. Under the scanning electron microscope, the morphology of nanoscale was small, the diameter of the single particle was about 200nm, or the micromass mass was aggregated. 3. The alizarin red staining showed that the nanoscale bacteria were dyed red. 16SrRNA gene analysis found that the new species of nanoscale were isolated and cultured in placental calcification, and 93% and 81% were similar to nano bacteria X98418 respectively. The 16SrRNA gene sequencing results of two species of nanoscale were submitted to GenBank to obtain the nanoscale gene gene in JN029830 and JF823648. calcified placenta. The amplification analysis showed that the nanoscale bacteria in the previously isolated culture bacteria were the same bacteria. Compared with the other nanoscale species in GenBank, the nanoscale of the two species was found to be closest to the Nanobacterium sanguineum (X98418) submitted by Professor Kajander.
Conclusion: the isolation and culture of nanoscale in placental calcification is two new species of nanoscale, which is closely related to Nanobacterium sanguineum submitted by Professor Kajander in the earlier period.
Background: the mechanism of nanoscale induced calcification is not clear, and no related experimental studies have shown that it can promote the secretion of calcium related proteins. As the main component of the nanoscale is hydroxyapatite, this component has been proved to promote bone formation and promote the secretion of calcium related proteins. The relationship between calcium related protein - bone morphogenetic protein 2 (Bone morphogenetic protein BMP-2) and osteopontin (Osteopontin OPN) in calcified placental tissue and with nanoscale bacterial infection.
Objective: To investigate the effect of nanomaterials infection on the secretion of bone morphogenetic protein 2 (BMP-2) and osteopontin (OPN) two calcification associated proteins in placenta.
Methods: the positive group, the negative group and the placental tissue of the normal placenta group were taken as the experimental objects, respectively. (1) the difference of the expression of two proteins between BMP-2 and OPN in different experimental tissues was analyzed by immunohistochemistry: tissue section, HE staining, calcification, routine dewaxing, and serological seal Closed, incubating BMP-2 and OPN protein monoclonal antibodies at 4 C overnight, biotinylated two anti action, DAB coloration, hematoxylin redyeing, and microscopic observation under microscope. (2) Western Blot:SDS PAGE electrophoresis method was used to separate two kinds of protein of BMP-2 and OPN in calcified and non calcified tissues, chemiluminescence, development and fixing, and the software semi quantitative analysis of the target strip Compare the expression of two calcification related proteins between BMP-2 and OPN in different tissues.
Results: the results of immunohistochemistry showed that the expression of BMP-2 and OPN in the positive group and negative group in the calcified placenta tissue and negative group were higher than the normal placental tissue (P0.01).Western Blot results. It was found that the expression of BMP-2 and OPN in the positive and negative groups in the calcified placenta tissue was higher than that of the normal placental tissue (P0.01). However, the difference of expression between the two groups was not obvious (P0.05)
Conclusion: the expression of BMP-2 and OPN two proteins in calcified tissue is increased, but the mechanism of nanomaterials infection on promoting placenta calcification is still unclear.
Background: the special components, size and replication of nanoscale have not been unified. In order to isolate and culture more suspected diseases related to nanoscale bacterial infection, the authors take placental calcification as an example to explore the separation technology of nanoscale in calcified tissue. The best conditions and methods for the culture and preservation of nanoscale bacteria are discussed by the experimental study of the different culture and preservation conditions of nanoscale. It is helpful to the positive culture of nanoscale and the implementation of the follow-up genome project.
Objective: to seek the best method of separating nanoscale in calcified tissue by taking calcified placenta as an example, and to explore the most suitable methods and conditions for the cultivation and preservation of nanometers.
Methods: (1) the separation of nanbacteria by demineralization by hydrochloric acid and demineralization by ultrasonic oscillation was used to calculate the isolation positive rate. (2) the nanometers were cultured in cell culture box and bacterial culture box for 4 weeks, and the changes in the concentration of nanoscale under two conditions were recorded by spectrophotometer. The calcified tissue and nanoscale were stored at 4, -20 and -80, respectively. The growth conditions of nanoscale isolation and resuscitation were recorded and the growth curve was drawn.
Results: (1) there is no obvious difference in the growth rate of nanoscale by the demineralization of calcic tissue with hydrochloric acid. (2) the growth rate of nanoscale in cell and bacteria culture environment is not obviously different. (3) the preservation of calcified tissue and nanoscale bacterial liquid at 4 degrees is better than -20 C and -80 C for its separation and recovery.
Conclusion: the demineralization of hydrochloric acid in calcified tissue can better separate the nanoscale bacteria, and the nanoscale bacteria can be cultured in the bacterial culture box. The fresh calcified tissue specimen and the nanoscale can be kept at 4 C for a short time.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R378

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