纳米细菌的分离鉴定及其钙化机制的研究

发布时间：2018-01-12 22:04

本文关键词：纳米细菌的分离鉴定及其钙化机制的研究　出处：《重庆医科大学》2013年硕士论文　论文类型：学位论文

【摘要】：目的：从钙化胎盘组织及其脐带血标本中分离鉴定纳米细菌（Nanobacteria，NB），经鉴定的NB作用于乳腺癌MDA-MB-231细胞，观察其对细胞的影响，并测定细胞内与钙化相关的骨形态蛋白-2（Bonemorphogenetic protein2，BMP-2），骨桥蛋白（osteopontin，OPN）以及与凋亡相关的Bcl-2蛋白和Bax蛋白的表达，最终探讨NB导致钙化的可能机制。方法：收集孕妇胎盘钙化组织及其脐带血20例，并以相同条件下取得的20例正常孕妇胎盘组织作对照组，分离培养NB；用革兰染色、茜素红染色、扫描电镜和透射电镜进行形态学鉴定。经鉴定NB与纳米羟基磷灰石（Nanohydroxyapatite，nHA）均以2麦氏浊度（M），分别作用于乳腺癌MDA-MB-231细胞，经24h、48h、72h后通过CCK-8试剂盒检测其对细胞的抑制作用；经12h、24h、48h、72h，，取上清经全自动生化分析仪通过检测LDH、ALP的活性，反映其损伤和钙化情况；72h后经流式细胞仪(FCM)测定凋亡率；透射电镜(TEM)观察细胞超微结构变化；提取细胞总蛋白，Western blot的方法测定细胞中钙化相关蛋白BMP-2，OPN，以及线粒体凋亡相关蛋白Bcl-2，Bax的表达情况。所有数据采用GraphPad Prism5进行双因素或单因素方差分析并绘图，P＜0.05有统计学意义。结果：培养沉淀物符合文献描述的NB特性。油镜下脐带血和钙化胎盘组织分离的NB为球状或杆状，多成球形，形态相似；茜素红染色结果表明，脐带血NB较多且聚集成簇；透射电镜和扫描电镜下NB呈球形，大小为80~500nm，并可见毛刺结构。NB分离率：脐带血为80%（16/20），钙化胎盘组织为65%（13/20），与正常胎盘组织10%(2/20)比较，χ2分别为12.07，9.09，P均＜0.01，有统计学意义；脐带血与钙化胎盘组织比较，差异无统计学意义（χ2=1.33，P＞0.05）。2M浓度的NB和nHA作用于乳腺癌细胞后CCK-8显示，NB组24h,48h,72h对细胞的抑制作用均强于nHA组和正常对照组，P＜0.05，有统计学意义；NB组ALP、LDH活性在24h,48h,72h时均高于正常对照组，P＜0.05,有统计学意义，24h,48h,72h均高于nHA组，但仅在24h,48h有统计学差异；72h后NB组细胞凋亡率高于nHA组,P＜0.001；透射电镜下观察，NB组可以看到胞质空泡样变,核固缩,以及明显的凋亡小体形成，nHA组未见明显异常；72h后NB组：BMP-2的表达高于正常，t=5.52,P＜0.01，有显著性差异，高于nHA组，但无统计学意义；OPN，Bax的表达量均高于正常组和nHA组，P＜0.001，均有显著性差异；Bcl-2的表达降低，与正常组相比，t=6.12,P＜0.01，有显著性差异，低于nHA组，但无统计学意义。结论：胎盘钙化与NB感染有关，钙化产生的机制可能是HA及NB的其他组成成分或代谢产物在一定程度上抑制细胞生长，增强Bax蛋白的表达，促进细胞的凋亡，细胞凋亡速率大于机体清除速率，局部钙、磷代谢紊乱，残留物质结合吞噬细胞、NB等形成生物矿化的核心，导致钙化的发生发展；钙化的发生也可加重局部的钙、磷代谢紊乱，进而促进凋亡的发生，钙化凋亡互为因果，相互促进。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R378.99

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