本文關(guān)鍵詞：納米細(xì)菌的分離鑒定及其鈣化機(jī)制的研究　出處：《重慶醫(yī)科大學(xué)》2013年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 納米細(xì)菌 胎盤鈣化 凋亡 納米羥基磷灰石

【摘要】：目的：從鈣化胎盤組織及其臍帶血標(biāo)本中分離鑒定納米細(xì)菌（Nanobacteria，NB），經(jīng)鑒定的NB作用于乳腺癌MDA-MB-231細(xì)胞，觀察其對細(xì)胞的影響，并測定細(xì)胞內(nèi)與鈣化相關(guān)的骨形態(tài)蛋白-2（Bonemorphogenetic protein2，BMP-2），骨橋蛋白（osteopontin，OPN）以及與凋亡相關(guān)的Bcl-2蛋白和Bax蛋白的表達(dá)，最終探討NB導(dǎo)致鈣化的可能機(jī)制。 方法：收集孕婦胎盤鈣化組織及其臍帶血20例，并以相同條件下取得的20例正常孕婦胎盤組織作對照組，分離培養(yǎng)NB；用革蘭染色、茜素紅染色、掃描電鏡和透射電鏡進(jìn)行形態(tài)學(xué)鑒定。經(jīng)鑒定NB與納米羥基磷灰石（Nanohydroxyapatite，nHA）均以2麥?zhǔn)蠞岫龋∕），分別作用于乳腺癌MDA-MB-231細(xì)胞，經(jīng)24h、48h、72h后通過CCK-8試劑盒檢測其對細(xì)胞的抑制作用；經(jīng)12h、24h、48h、72h，，取上清經(jīng)全自動生化分析儀通過檢測LDH、ALP的活性，反映其損傷和鈣化情況；72h后經(jīng)流式細(xì)胞儀(FCM)測定凋亡率；透射電鏡(TEM)觀察細(xì)胞超微結(jié)構(gòu)變化；提取細(xì)胞總蛋白，Western blot的方法測定細(xì)胞中鈣化相關(guān)蛋白BMP-2，OPN，以及線粒體凋亡相關(guān)蛋白Bcl-2，Bax的表達(dá)情況。所有數(shù)據(jù)采用GraphPad Prism5進(jìn)行雙因素或單因素方差分析并繪圖，P＜0.05有統(tǒng)計學(xué)意義。 結(jié)果：培養(yǎng)沉淀物符合文獻(xiàn)描述的NB特性。油鏡下臍帶血和鈣化胎盤組織分離的NB為球狀或桿狀，多成球形，形態(tài)相似；茜素紅染色結(jié)果表明，臍帶血NB較多且聚集成簇；透射電鏡和掃描電鏡下NB呈球形，大小為80~500nm，并可見毛刺結(jié)構(gòu)。NB分離率：臍帶血為80%（16/20），鈣化胎盤組織為65%（13/20），與正常胎盤組織10%(2/20)比較，χ2分別為12.07，9.09，P均＜0.01，有統(tǒng)計學(xué)意義；臍帶血與鈣化胎盤組織比較，差異無統(tǒng)計學(xué)意義（χ2=1.33，P＞0.05）。2M濃度的NB和nHA作用于乳腺癌細(xì)胞后CCK-8顯示，NB組24h,48h,72h對細(xì)胞的抑制作用均強(qiáng)于nHA組和正常對照組，P＜0.05，有統(tǒng)計學(xué)意義；NB組ALP、LDH活性在24h,48h,72h時均高于正常對照組，P＜0.05,有統(tǒng)計學(xué)意義，24h,48h,72h均高于nHA組，但僅在24h,48h有統(tǒng)計學(xué)差異；72h后NB組細(xì)胞凋亡率高于nHA組,P＜0.001；透射電鏡下觀察，NB組可以看到胞質(zhì)空泡樣變,核固縮,以及明顯的凋亡小體形成，nHA組未見明顯異常；72h后NB組：BMP-2的表達(dá)高于正常，t=5.52,P＜0.01，有顯著性差異，高于nHA組，但無統(tǒng)計學(xué)意義；OPN，Bax的表達(dá)量均高于正常組和nHA組，P＜0.001，均有顯著性差異；Bcl-2的表達(dá)降低，與正常組相比，t=6.12,P＜0.01，有顯著性差異，低于nHA組，但無統(tǒng)計學(xué)意義。 結(jié)論：胎盤鈣化與NB感染有關(guān)，鈣化產(chǎn)生的機(jī)制可能是HA及NB的其他組成成分或代謝產(chǎn)物在一定程度上抑制細(xì)胞生長，增強(qiáng)Bax蛋白的表達(dá)，促進(jìn)細(xì)胞的凋亡，細(xì)胞凋亡速率大于機(jī)體清除速率，局部鈣、磷代謝紊亂，殘留物質(zhì)結(jié)合吞噬細(xì)胞、NB等形成生物礦化的核心，導(dǎo)致鈣化的發(fā)生發(fā)展；鈣化的發(fā)生也可加重局部的鈣、磷代謝紊亂，進(jìn)而促進(jìn)凋亡的發(fā)生，鈣化凋亡互為因果，相互促進(jìn)。
[Abstract]:Objective: to isolate and identify nanobacterium-nb#en0# from calcified placenta and umbilical cord blood samples, and to identify the effect of NB on breast cancer MDA-MB-231 cells. The effect of BMP on cells was observed, and bone morphogenetic protein (BMP-2), which was related to calcification, was determined. Osteopontinin (OPN) and the expression of apoptosis-related Bcl-2 and Bax proteins, and finally to explore the possible mechanism of calcification induced by NB. Methods: 20 cases of placental calcification tissue and umbilical cord blood of pregnant women were collected, and 20 cases of normal placenta tissues obtained under the same conditions were used as control group. The morphology was identified by Gram staining, alizarin red staining, scanning electron microscope and transmission electron microscope. NB and nano-hydroxyapatite were identified. NHAs were treated with 2 McClurian turbidimetric motif on breast cancer MDA-MB-231 cells for 48 h after 24 h. After 72 hours, the inhibitory effect on cells was detected by CCK-8 kit. The activity of LDHH ALP was detected by automatic biochemical analyzer after 12 h ~ 24 h ~ 48 h ~ 72 h, and the damage and calcification were reflected by automatic biochemical analyzer. After 72 hours, the apoptosis rate was measured by flow cytometry (FCM). The ultrastructure of the cells was observed by TEM. The calcification-associated protein BMP-2OPN and mitochondrial apoptosis-related protein Bcl-2 were determined by Western blot. The expression of Bax. All the data were analyzed by GraphPad Prism5 or univariate ANOVA (P < 0. 05). Results: the culture sediment was in accordance with the NB characteristics described in the literature. NB isolated from umbilical cord blood and calcified placenta under oil microscope was spherical or rod-shaped, mostly spherical in shape and similar in shape. The results of alizarin red staining showed that there were more NB in cord blood and gathered into clusters. NB was spherical under transmission electron microscope and scanning electron microscope with a size of 80 ~ 500nm.The separation rate of NB from umbilical cord blood was 80 / 16 / 20). The calcified placenta tissue was 65 / 20 / 20. Compared with normal placenta tissue, 蠂 ~ 2 was 12.07 / 9.09% (P < 0.01), and there was significant difference between them (蠂 ~ 2 = 12.07 / 9.09, P < 0.01). There was no significant difference between umbilical cord blood and calcified placental tissue (蠂 2: 1.33 P > 0.05? M NB and nHA) on CCK-8 of breast cancer cells. The inhibitory effect of NB group at 24 h, 48 h and 72 h on cells was stronger than that of nHA group and normal control group (P < 0.05). The activity of LDH in NB group was significantly higher than that in control group at 24 h, 48 h and 72 h, respectively (P < 0.05). The activity of LDH in NB group was significantly higher than that in nHA group at 48 h and 72 h. But there was statistical difference only at 24 h or 48 h. After 72 hours, the apoptosis rate of NB group was higher than that of nHA group (P < 0.001). Under the transmission electron microscope, the cytosolic vacuolation, nuclear pyknosis and the formation of apoptotic bodies were not found in the NHA group. After 72 hours, the expression of BMP-2 in NB group was higher than that in normal group (P < 0.01), which was higher than that in nHA group, but had no statistical significance. The expression of nHA Bax was significantly higher than that of normal group and nHA group (P < 0.001). The expression of Bcl-2 was significantly lower than that of the normal group (P < 0.01), but there was no significant difference between the nHA group and the control group. Conclusion: placental calcification is related to NB infection. The mechanism of calcification may be that HA and other components or metabolites of NB inhibit cell growth to some extent and enhance the expression of Bax protein. Promote cell apoptosis, the rate of apoptosis is greater than the rate of body clearance, local calcium, phosphorus metabolism disorder, residual substance binding to NB phagocytes formed the core of biomineralization, leading to the occurrence and development of calcification; The occurrence of calcification can also aggravate the disorder of calcium and phosphorus metabolism and promote the occurrence of apoptosis.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R378.99

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