Establishing A Co-culture Model Of Nasopharyngeal Commensal Bacteria And Nasopharyngeal Epithelial Cells To Study The Characteristics Of Bacterial Invasion

Objective:Nasopharyngeal carcinoma(NPC)is a malignant tumor with high incidence in south China.In Guangdong and Guangxi regions,the incidence and mortality of NPC are the highest in the world.The etiology of NPC is complex:genetic predisposition,Epstein-Barr virus(EB virus,EBV)and environmental carcinogenes are the main reasons.The pathogenesis of NPC is still not very clear.Recent studies have found that the imbalance of normal flora in human may initiate tumorigenesis.Epigenetic changes caused by a bacterial infection is closely related to tumorigenesis.Nasopharyngeal commensal bacteria may be involved in the latent infection of EBV and the carcinogenic process of nasopharyngeal epithelium.In this study,aimed to build a simple model of NPC cells co-cultured with bacteria for exploring the association of bacterial infection and NPC.In addition,we explored the characteristics of invasion and ultrastructural changes of nasopharyngeal carcinoma cells after co-cultured,which might provid a theoretical basis for the subsequent investigation of the bacterial load and structural changes in nasopharyngeal carcinoma biopsies.Methods:Firstly,we gained a certain amount of bacteria by the colony counting method.After that,we treated nasopharyngeal epithelial cells(HONE1,CNE2,NP69)respectively with bacteria under a gradient of multiplicity of infection(MOI)for 12 hours.The status of cells were observed under an optical microscope to determine a tolerable range of MOI.Gentamicin protection assay was perfromed to prove the invasive capability of bacteria into the nasopharyngeal epithelial cells and further explore the characteristics of bacterial invasion,such as the time dependence of the colony-forming units(CFU)formed by invaded bacteria;the relationship between the intracellular invasion of bacteria and MOI;and the trend of the number of bacteria invading cells over time.Using fluorescence in situ hybridization(FISH)based on the universal probe EUB338 targeted to 16S ribosomal RNA(rRNA)gene to determine intracellular bacteria of nasopharyngeal epithelial cells.In addition,we observed the intracellular bacteria and ultrastructural changes in nasopharyngeal carcinoma cells using transmission electron microscope(TEM)after co-cultured for 2 hours.Results:1.We treated HONE1 and NP69 with bacteria for 12 hours with a series of MOI,respectively.We found that cells keep at a good status when MOI less than 250,but cells died significantly when MOI more than 500.2.The characteristics of human nasopharyngeal epithelial cells invaded by bacteria:2.1 The intracellular CFU in HONEl was increased when MOI increased under 2 hours treatment with bacteria under a MOI less than 200(P<0.05).2.2 After co-cultured with bacteria,the changes of intracellular CFU in HONE1 cells over time were observed:from 0 to 1 hour,the intracellular CFU increased significantly(P<0.05);from 1 hour to 4 hours,the intracellular CFU no significantly increased(P>0.05);6 hours compared to 4 hours,the intracellular CFU significantly increased(P<0.05).2.3 The internalization ability of NPC cells(HONE1,CNE1,CNE2)were greater than non-malignant nasopharyngeal epithelial cell line(NP69),with statistically significance(P<0.05).Staphylococcus epidermidis can obviously invade to nasopharyngeal epithelial cells(HONE1).3.The detection of intracellular bacteria by FISH and ultrastructural observation3.1 FISH technique based on the universal probe EUB338 detects the intracellular bacteria in nasopharyngeal carcinoma cells treated for 2 hours(MOI=100)with a clear signal,but not in non-treated groups;After Staphylococcus aureus tread HONE1 for 2 hours(MOI=100),the different steps of internalizing bacterial cells by epithelial cells could be seen by TEM:Staphylococcus aureus adhered to the surface microvilli of HONE1 cells;HONE1 showed membrane retracted,and retraction of vesicles wrapped bacteria;the bacteria were lysed by digestive organelles.3.2 The ultrastructural changes of HONE1 cells were observed after treated with Staphylococcus aureus and Staphylococcus epidermidis for 2 hours(MOI =100)by TEM:1)It have been showed in the cytoplasm of HONE1 cells a varying degrees accumulation of glycogen near the intracellular bacteria;2)Compared with the non-treated cells,a small amount of lipid droplets distributed in the cytoplasm;3)In contrast to the non-treated group,the swelling of mitochondria and atrophy of golgi have been found.Conclusions:1.Nasopharyngeal epithelial cells could tolerate a range of MOI(less than 250)when co-cultured with bacteria,the MOI range from 0 to 200 are relatively suitable for co-culture.2.Nasopharyngeal epithelial cells can internalize bacteria.The internalization ability of NPC cells were greater than NP69,suggesting that bacteria have a high rate of invasion in human nasopharynx.3.The ultrastructural changes,such as accumulation of glycogen and distribution of a small amount of lipid droplets,swelling of mitochondria and atrophy of golgi have been showed in the cytoplasm of nasopharyngeal epithelial cells after co-cultured with bacteria.4.The characteristics of co-culture between nasopharyngeal epithelial cells and bacteria will provide a simple cell model for further molecular biological study.