Wang Hongyan, Tan Lisi, Liu Junchao, Li Qian, Pan Yaping, Zhong Ming
Department of Periodontology, School of Stomatology, China Medical University & Liaoning Institute of Dental Research, Shenyang 110002, China.
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Zhonghua Kou Qiang Yi Xue Za Zhi. 2014 Jan;49(1):15-20.
To investigate the effects of serum from smoking individuals or non-smoking individuals with periodontitis on Porphyromonas gingivalis (Pg) internalizing KB cells, and the expression of matrix metalloproteinase(MMP)-1, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1) in the culture supernatant of KB cells.
The venous blood of 20 periodontitis patients' (10 smoking and 10 non-smoking) was extracted under the informed consent and centrifuged for serum. The smoking-individual serum (Y group) and non-smoking-individual (N group) serum were added to the model of Pg internalizing KB cells for 12 hours, plated on brain-heart infusion (BHI) and incubated anaerobically at 37 °C for 5 days. The colony forming units (CFU) of cell-invasive bacteria were estimated by colony counting. MMP-1, MMP-9 and TIMP-1 protein levels in culture supernatant were determined by enzyme-linked immunosorbent assay(ELISA) in the two groups following co-culture of Pg with KB cells for 12 hours.
The CFU were (11.2 ± 1.1)×10(4), (12.6 ± 1.2)×10(4), (44.7 ± 1.3)×10(4) CFU/ml when adding 200, 400, 800 µl Y-group serum to the model of Pg co-culture with KB cells and when the serum was extracted from N group, the CFU were (33.6 ± 1.4)×10(4),(38.9 ± 1.1)×10(4), (11.2 ± 1.2)×10(4) CFU/ml respectively. When 200, 400, 800 µl Y group-serum was added to co-culture fluid of Pg internalizing KB cells, the concentrations of MMP-1 secreted from KB cells were (107.2 ± 21.5), (165.9 ± 20.2), (434.4 ± 48.0) µg/L respectively, the concentrations of MMP-9 were (3.99 ± 0.29), (4.21 ± 0.61), (5.62 ± 0.47) µg/L respectively, the concentrations of TIMP-1 were (401.3 ± 12.7), (418.3 ± 28.5), (637.3 ± 37.3) µg/L. When the serum (200, 400, 800 µl) extracted from N group, the concentration of MMP-1 and MMP-9 secreted by KB cell were (77.6 ± 10.8), (84.7 ± 10.2) and (98.2 ± 9.7) µg/L and (3.84 ± 0.52), (4.02 ± 0.68), (4.25 ± 0.37) µg/L, respectively. The concentration of TIMP-1 were (67.3 ± 26.9) , (89.4 ± 22.7) and (78.2 ± 16.5) µg/L secreted by KB cells in the course of Pg internalized KB cell. With the increasing of Y group-serum, the more MMP-1, MMP-9 and TIMP-1 were secreted by KB cells(P < 0.05). When 800 µl Y group-serum was added compared with N group-serum to the Pg co-culture with KB model, the more MMP-1, MMP-9 and TIMP-1 were secreted by KB cells(P < 0.05), when 400 µl Y group-serum was added compared with N group-serum to the Pg co-culture with KB model, the more MMP-1 and TIMP-1 were secreted by KB cells (P < 0.05).
The smoking-serum might enhance Pg internalizing KB cells and enhance the expression of MMP-1, MMP-9 and TIMP-1 secreted from KB cells. The local microenvironment of smoking individual may contribute to the recurrence and progression of chronic periodontitis.
研究吸烟个体或患牙周炎的非吸烟个体的血清对牙龈卟啉单胞菌（Pg）内化KB细胞的影响，以及KB细胞培养上清液中基质金属蛋白酶（MMP）-1、MMP-9、金属蛋白酶组织抑制剂-1（TIMP-1）的表达。
在获得知情同意的情况下，抽取20例牙周炎患者（10例吸烟者和10例非吸烟者）的静脉血并离心获取血清。将吸烟个体血清（Y组）和非吸烟个体血清（N组）加入Pg内化KB细胞模型中作用12小时，接种于脑心浸液（BHI）上，在37℃厌氧培养5天。通过菌落计数估算细胞侵袭性细菌的菌落形成单位（CFU）。在Pg与KB细胞共培养12小时后，用酶联免疫吸附测定（ELISA）法测定两组培养上清液中MMP-1、MMP-9和TIMP-1蛋白水平。
向Pg与KB细胞共培养模型中分别加入200、400、800μl Y组血清时，细胞侵袭性细菌的CFU分别为（11.2±1.1）×10⁴、（12.6±1.2）×10⁴、（44.7±1.3）×10⁴CFU/ml，而血清取自N组时，CFU分别为（33.6±1.4）×10⁴、（38.9±1.1）×10⁴、（11.2±1.2）×10⁴CFU/ml。向Pg内化KB细胞的共培养液中加入200、400、800μl Y组血清时，KB细胞分泌的MMP-1浓度分别为（107.2±21.5）、（165.9±20.2）、（434.4±48.0）μg/L，MMP-9浓度分别为（3.99±0.29）、（4.21±0.61）、（5.62±0.47）μg/L，TIMP-1浓度分别为（401.3±12.7）、（418.3±28.5）、（637.3±37.3）μg/L。当加入取自N组的血清（200、400、800μl）时，KB细胞分泌的MMP-1和MMP-9浓度分别为（77.6±10.8）、（84.7±10.2）、（98.2±9.7）μg/L和（3.84±0.52）、（4.02±0.68）、（4.25±0.37）μg/L，TIMP-1浓度分别为（67.3±26.9）、（89.4±22.7）、（78.2±16.5）μg/L。随着Y组血清量增加，KB细胞分泌的MMP-1、MMP-9和TIMP-1越多（P<0.05）。在Pg与KB细胞共培养模型中，与加入N组血清相比，加入800μl Y组血清时，KB细胞分泌的MMP-1、MMP-9和TIMP-1更多（P<0.05）；与加入N组血清相比，加入400μl Y组血清时，KB细胞分泌的MMP-1和TIMP-1更多（P<0.05）。
吸烟血清可能增强Pg内化KB细胞及KB细胞分泌MMP-1、MMP-9和TIMP-1的表达。吸烟个体的局部微环境可能促使慢性牙周炎复发和进展。
