侵入细胞内的牙龈卟啉单胞菌影响人牙周膜细胞的增殖及成骨分化

Effects of intracellular Porphyromonas gingivalis on proliferation and osteogenic differentiation of human periodontal ligament cells in vitro

目的探究活体牙龈卟啉单胞菌(P.gingivalis)侵入人牙周膜细胞(HPDLCs)后,对细胞增殖性能及成骨向分化作用的影响。方法将P.gingivalis与HPDLCs以感染复数(MOI)为10、100共培养90 min、8、24 h后,利用细胞侵袭实验比较不同时间及MOI值下,P.gingivalis对HPDLCs的侵袭效能。用CFDA-SE(Carboxyfluorescein diacetate,succinimidyl ester,C29H19NO11)标记HPDLCs,加入P.gingivalis后共培养8、24、48、72 h,流式细胞仪检测细胞增殖性能的变化。将SYTO9标记的P.gingivalis与HPDLCs共培养24 h后,流式细胞荧光分选技术(Fluorescence activated Cell Sorting,FACS)分选出被感染细胞,利用实时荧光定量PCR对Runx2基因进行检测,并且对分选出的细胞进行矿化诱导,分别于7、14、21 d,进行茜素红染色检测HPDLCs矿化结节形成。结果 P.gingivalis与HPDLCs共培养90 min后,即可进入细胞内,共培养24 h后,侵袭效能达到最大值。被P.gingivalis感染后,细胞的Runx2基因表达明显下调,且其矿化结节的形成明显低于对照组。结论侵袭进入HPDLCs内的活体P.gingivalis,未对细胞的增殖产生明显的影响,但可以明显抑制细胞的成骨分化作用,这可能是通过下调Runx2的表达来实现的。

Objective To investigate the ability of Porphyromonas gingivalis to invade human periodontal ligament cells （hPDLCs） and the effect of intracellular P. gingivalis on cell proliferation and osteogenic differentiation in vitro. Methods The invasion ability of P. gingivalis in hPDLCs was tested using an antibiotic protection assay at the multiplicity of infection （MOI） of 10 and 100. The proliferation of the infected cells was detected using a CFDA-SE kit, and the cells were sorted by fluorescence-activated cell sorting （FACS） followed by alizarin red staining for detecting mineralization nodules deposition;real-time PCR was used to examine the expression of Runx2 mRNA in the cells. Results P. gingivalis actively invaded hPDLCs, and the internalized P. gingivalis was able to resist antibiotic treatment. The cells infected by P. gingivalis exhibited no significant suppression of cell proliferation, but showed significantly lowered capacity for osteogenic differentiation, down-regulated RUNX2 mRNA expression, and reduced mineral deposition. Conclusion Intracellular P. gingivalis does not significantly affect the proliferation of hPDLCs but inhibits osteogenic differentiation of the cells.