人工合成抗菌肽Pac525对种植体周围炎病原菌抑制作用的研究

发布时间：2018-10-12 08:05

【摘要】：目的：本实验选用前期实验已证实对牙龈卟啉单胞菌、具核梭杆菌及血链球菌这三种种植体周围炎病原菌有不同程度抑制作用的人工合成抗菌肽Pac525，通过活菌计数绘制其对这三种菌的kill-time曲线，以期进一步发现其在不同时间、不同药物浓度下Pac525对这三种菌的抑制作用；建立活菌生物膜，扫描电镜观察其生长状况；采用结晶紫法测定Pac525对这三种菌完整生物膜形成的药物最低抑制生物膜浓度(MBIC)和药物最低清除已形成生物膜浓度(MBRC)。 方法： 一、抗菌肽Pac525对种植体周围炎致病菌的kill-time 依据Pac525的氨基酸序列，人工合成该抗菌肽。本实验采用活菌计数法研究Pac525对牙龈卟啉单胞菌、具核梭杆菌及血链球菌的kill-time。 二、纯钛表面牙龈卟啉单胞菌、具核梭杆菌及血链球菌的菌斑生物膜的建立。 为模拟口腔环境，用唾液对光滑钛片预处理（37℃，24小时），使其表面形成获得性膜。将钛片放入24孔板中，PBS冲洗，每孔加入1500μl BHI液体培养基和500μl菌液，37℃恒温培养24~48小时，脱水干燥，扫描电镜观察其生物膜。 三、抗菌肽Pac525对种植体周围炎致病菌菌斑生物膜的影响 MBIC的测定：在96孔板上分别建立三种菌的生物膜，加入200μl Pac525药液培养基和20μl1×107cfu/ml菌液，37℃恒温培养24~48小时，PBS冲洗，甲醛固定，结晶紫染色，无菌去离子水冲洗，乙醇复吸，酶标仪测定吸光度。根据公式计算其MBIC。使用SPSS17.0统计软件进行统计学分析。 MBRC的测定：在96孔板上加入200μlBHI液体培养基和20μl1×107CFU/ml菌液37。C恒温培养24~48小时，吸出上清和浮游菌，PBS冲洗，加入200μl Pac525药液培养基24~48小时，吸出上清和浮游菌，PBS冲洗，甲醛固定，结晶紫染色，无菌去离子水冲洗，乙醇复吸，酶标仪测定吸光度。根据公式计算其MBRC。使用SPSS17.0统计软件进行统计学分析。 结果： 一、抗菌肽Pac525对种植体周围炎致病菌的kill-time 通过kill-time曲线我们可以看出，随着时间的改变，不同浓度的Pac525均显示出了一定程度的抑菌及杀菌功效。对于血链球菌，0.5h不同浓度的Pac525即显示出不同程度的抑菌作用，其中2MIC（minimum inhibitory concentration,最小抑菌浓度）和4MIC尤为明显，1h时4MIC已杀死全部血链球菌,3.5h时MIC的杀菌作用趋于平稳，1h2MIC杀菌作用趋于平稳，均保持在较高水平。对于具核梭杆菌，4h时不同浓度的Pac525即显示出不同程度的抑菌作用，8h时2MIC已杀死全部具核梭杆菌，此时MIC的杀菌作用也趋于稳定。对于牙龈卟啉单胞菌，4h时2MIC已杀死全部牙龈卟啉单胞菌，8h时MIC也已杀死全部牙龈卟啉单胞菌。结果表明Pac525对菌的杀死作用随药物的浓度和时间呈正相关。通过kill-time曲线可以得出这种作用趋势。 二、抗菌肽Pac525对种植体周围炎致病菌菌斑生物膜作用结果 经单因素方差分析（P＜0.05）PAC525对血链球菌MBIC50为1mg/ml，为其MIC的8倍。PAC525对具核梭杆菌MBIC50为0.125mg/ml，为其MIC的16倍。PAC525对牙龈卟啉单胞菌MBIC500.5mg/ml，，为其MIC的8倍。但是MBRC并未测出来。 结论： 1.抗菌肽Pac525对种植体周围炎致病菌牙龈卟啉单胞菌、具核梭杆菌及血链球菌的抑菌作用随时间和药物的浓度呈正相关改变。 2.抗菌肽Pac525对三种细菌的菌斑生物膜均有破坏作用。MBIC为MIC的8~16倍。 3.MBRC50并未测出，这极有可能与其早期抑菌有关。
[Abstract]:Objective: In this experiment, we selected the synthetic antimicrobial peptide Pac525 with different degree of inhibition to the pathogenic bacteria in the three implants, such as Pseudomonas sp., Bacillus cereus and Streptococcus sanguis. The kill-time curve of these three kinds of bacteria was plotted by living bacteria count. In order to find out the inhibitory effect of Pac525 on the three kinds of bacteria at different time and different drug concentrations, the growth status of living bacteria biofilm and scanning electron microscope were observed. The lowest inhibitory biofilm concentration (MBIC) and the lowest drug clearance of the drug formed by Pac525 for the three strains of intact biofilm were determined by crystallization violet method (MBRC). Methods: One, antibacterial peptide Pac525 was used to kill pathogenic bacteria in peri-implant inflammation. l-time according to the amino acid sequence of Pac525, The antibacterial peptide was artificially synthesized. The experiment was carried out by using live bacteria counting method. ill-time. di, pure titanium surface gum base, pseudomonas, Bacillus cereus and blood chain Establishment of a plaque biofilm for cocci. To simulate the oral environment, pre-treat a smooth titanium sheet with saliva (37.degree. C., 24 (h) forming an acquired film on the surface thereof, placing the titanium sheet in a 24-well plate, washing with PBS, adding 1500 & mu; l of BHI liquid culture medium and 500 & mu; l of bacteria liquid per hole, culturing at 37 & deg; C for 24-48 hr, and removing The biofilm was observed by water drying and scanning electron microscope. The antibacterial peptide Pac525 The effect of MBIC on dental plaque biofilm was determined: three kinds of biofilm were established on 96-well plates, 200 ul Pac525 solution and 20. mu. l/ 107cfu/ ml of bacteria liquid were added, and 37 鈩

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