Mucus secretions from a conditionally reprogrammed primary endocervical cell culture

Abstract

Objective: To determine if the secretions collected from a conditionally reprogrammed primary endocervical cell culture are suitable surrogates for mucus studies.

Design: Experimental.

Setting: University research center.

Animal(s): Female rhesus macaque (n = 2).

Intervention(s): None.

Main outcome measure(s): Quantitative proteomic analysis using tandem mass tag mass spectrometry liquid chromatography/tandem mass spectrometry.

Result(s): We identified 3,047 proteins, common proteins present in both primary endocervical cell cultures and the mucus of rhesus macaques. We found a 71% overlap in the top 500 most prevalent proteins in the samples. Cell culture secretions contained many essential mucus proteins, including MUC5B, the primary mucin of the endocervix.

Conclusion(s): Similarities in secreted proteins suggest that conditionally reprogrammed primary endocervical cells could be used to study mucus secretion in vitro.

Keywords: Endocervix; mucus; primate; proteome.

Figure 1:

Study design. Mucus and tissue were collected from the endocervix of fresh necropsy specimens of the rhesus macaque. Tissue samples underwent conditional reprogramming with irradiated Swiss mouse fibroblasts 3T3J2 and Rho protein kinase (ROCK) inhibitor Y-27632 and were differentiated to produce mucus. Mucus from both conditionally reprogrammed endocervical cells (CRECs) and rhesus macaque were trypsin digested, labeled with tandem mass tags, separated with high pH reverse phase/low pH reverse phase liquid chromatography, and analyzed with Thermo Orbitrap Fusion (SPS MS3). Proteomic analysis workbench (PAW)/Comet pipeline provided peptide ID and protein inference; edgeR (Bioconductor) was used for differential expression testing. =

Figure 2:

There are 564 proteins with increased relative abundance in conditionally reprogrammed endocervical cells (CRECs) and 602 proteins with increased relative abundance in whole mucus based on an edgeR exact test Benjamini-Hochberg corrected false discovery rate<0.05. DE 1⁄4 differentially expressed; CPM 1⁄4 counts per million; C/M 1⁄4 CREC intensity/mucus intensity.