Primary human GECs were isolated from healthy human gingival tissue samples from patients To examine the expression of PARs mRNA, total RNA was isolated from GECs (grown to Table 1.<\/strong> Oligonucleotide sequences used for RT-PCR\n <\/p>\n P. gingivalis<\/em> ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, P. gingivalis<\/em> supernatants were diluted in cell culture medium and their concentration expressed After stimulation, total RNA was extracted using an RNeasy Kit (Qiagen, Valencia, Table 2.<\/strong> Oligonucleotide sequences used for QRT-PCR\n <\/p>\n Cells were washed in phosphate-buffered saline (PBS), incubated for 30\u00c2\u00a0min at 4\u00c2\u00a0\u00c2\u00b0C All data are shown as the mean?\u00c2\u00b1?the standard deviations (SD). QRT-PCR data are expressed Gingival epithelial cell culture Primary human GECs were isolated from healthy human gingival tissue samples from patients undergoing third molar extraction at the Dental Department, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, China. Written informed consent was obtained from all individuals participating in this study. The study were evaluated and approved by […]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[],"tags":[],"class_list":["post-26346","post","type-post","status-publish","format-standard","hentry"],"_links":{"self":[{"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/posts\/26346","targetHints":{"allow":["GET"]}}],"collection":[{"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/comments?post=26346"}],"version-history":[{"count":0,"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/posts\/26346\/revisions"}],"wp:attachment":[{"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/media?parent=26346"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/categories?post=26346"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/tags?post=26346"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}
\n undergoing third molar extraction at the Dental Department, Sir Run Run Shaw Hospital,
\n School of Medicine, Zhejiang University, China. Written informed consent was obtained
\n from all individuals participating in this study. The study were evaluated and approved
\n by the Ethics Committee of the Affiliated Sir Run Run Show Hospital of Zhejiang University
\n School of Medicine (20131120). Fresh gum tissue was placed into D-Hanks containing
\n 300 U\/ml penicillin G and 300\u00c2\u00a0?g\/ml streptomycin and incubated at 4\u00c2\u00a0\u00c2\u00b0C. Within 1\u00c2\u00a0h,
\n tissue was prepared to obtain epithelial cells. Briefly, the tissue was cut into small
\n pieces (1\u00c2\u00a0mm?\u00c3\u2014?1\u00c2\u00a0mm), treated with a solution of 25\u00c2\u00a0% dispase II (Sigma\u00e2\u20ac\u201cAldrich, St
\n Louis, MO, USA) and incubated for 18\u00c2\u00a0h at 4\u00c2\u00a0\u00c2\u00b0C. After incubation, the epidermal layer
\n of human keratinocytes was lifted from the dermis and placed into a 15\u00c2\u00a0ml sterile
\n centrifuge tube containing 2\u00c2\u00a0ml trypsin\u00e2\u20ac\u201cEDTA. The tissue was incubated at 37\u00c2\u00a0\u00c2\u00b0C for
\n approximately 10\u00c2\u00a0min. Subsequently, isolated GECs were seeded into T-75 flasks (BD
\n Biosciences) at a cell density of approximately 3?\u00c3\u2014?10
6<\/sup>
\n cells per flask in 10\u00e2\u20ac\u201c15\u00c2\u00a0ml serum-free keratinocyte medium (keratinocyte-SFM) to which
\n supplements were added according to the manufacturer\u00e2\u20ac\u2122s instructions (Gibco BRL, Life
\n Technologies, Rockville, MD, USA). Fluids in the flasks were exchanged for fresh complete
\n medium and gassed with 5\u00c2\u00a0% CO
2<\/sub>
\n every 2\u00e2\u20ac\u201c3 days. Cells were passaged when 75\u00e2\u20ac\u201c80\u00c2\u00a0% confluence was reached.\n <\/p>\nReverse transcription-PCR (RT-PCR) analysis for the determination of PARs expression<\/h4>\n
\n 70\u00c2\u00a0% confluence) using TRIzol\u00c2\u00ae reagent (Gibco BRL, Life Technologies, Rockville, MD,
\n USA) according to the manufacturer\u00e2\u20ac\u2122s suggested protocol. The synthesis of the first
\n strand cDNA and RT-PCR were performed using a PromeScript\u00c2\u00ae RT-PCR Kit (Takara Biotechnology
\n Co., Ltd, Dalian, China). The primers for PAR-1, PAR-2, PAR-3, PAR-4 and ?-actin were
\n synthesized by Sangon Biotech Co., Ltd (Shanghai, China; Table\u00c2\u00a01). For amplification of PAR-1, PAR-2 and ?-actin products, PCR was performed for 30\u00c2\u00a0cycles.
\n The first cycle included a denaturation step of 5\u00c2\u00a0min at 94\u00c2\u00a0\u00c2\u00b0C. Cycles 2\u00e2\u20ac\u201c30 had a
\n denaturation step of 30\u00c2\u00a0s at 94\u00c2\u00a0\u00c2\u00b0C, 30\u00c2\u00a0s of annealing at 60\u00c2\u00a0\u00c2\u00b0C and 45\u00c2\u00a0s of elongation
\n at 72\u00c2\u00a0\u00c2\u00b0C. The last cycle included an elongation step of 10\u00c2\u00a0min at 72\u00c2\u00a0\u00c2\u00b0C. For PAR-3
\n and PAR-4 amplification, PCR was performed for 30\u00c2\u00a0cycles. The first cycle included
\n a denaturation step of 5\u00c2\u00a0min at 94\u00c2\u00a0\u00c2\u00b0C. Cycles 2\u00e2\u20ac\u201c30 had a denaturation step of 30\u00c2\u00a0s
\n at 94\u00c2\u00a0\u00c2\u00b0C, 30\u00c2\u00a0s of annealing at 65\u00c2\u00a0\u00c2\u00b0C and 45\u00c2\u00a0s of elongation at 72\u00c2\u00a0\u00c2\u00b0C. The last cycle
\n included an elongation step of 10\u00c2\u00a0min at 72\u00c2\u00a0\u00c2\u00b0C. DNA products and molecular weight
\n marker DL1,000\u00e2\u201e\u00a2 DNA Marker (Takara Biotechnology Co., Ltd, Dalian, China) were separated
\n in 1.5\u00c2\u00a0% agarose gel, after which the gels were stained with GelRed\u00e2\u201e\u00a2 and visualized
\n under UV light.<\/p>\nBacteria culture and supernatants collection<\/h4>\n
\n USA) and anaerobically cultured (80\u00c2\u00a0%\u00c2\u00a0N
2<\/sub>
\n , 10\u00c2\u00a0%\u00c2\u00a0H
2<\/sub>
\n and 10\u00c2\u00a0% CO
2<\/sub>
\n ) in a brain\u00e2\u20ac\u201cheart infusion (BHI; Oxoid) agar plate containing 5\u00c2\u00a0% defibrinated sheep
\n blood enriched with 5\u00c2\u00a0g\/l yeast extract, 5\u00c2\u00a0mg\/l hemin and 10\u00c2\u00a0mg\/l menadione (Sigma\u00e2\u20ac\u201cAldrich,
\n Dorset, UK) at 37\u00c2\u00a0\u00c2\u00b0C for up to 5\u00c2\u00a0weeks. Liquid cultures were prepared by inoculation
\n of bacterial colonies (3\u00e2\u20ac\u201c4 days old) from blood agar plates into 10\u00c2\u00a0ml BHI broth supplemented
\n with 5\u00c2\u00a0g\/l yeast extract, 5\u00c2\u00a0mg\/l hemin and 10\u00c2\u00a0mg\/l menadione and incubated for 24\u00c2\u00a0h.
\n Ten percent inoculum was transferred to 90\u00c2\u00a0ml of the same medium and incubated for
\n 6\u00c2\u00a0days. After this culture period, bacteria were harvested by centrifugation at 10,000?g<\/em> for 15\u00c2\u00a0min at 4\u00c2\u00a0\u00c2\u00b0C and supernatants were collected, filter-sterilized over a 0.2\u00c2\u00a0mm
\n filter and stored at ?80\u00c2\u00a0\u00c2\u00b0C until use. Before treatment, aliquots of the supernatant
\n were used for pre-incubation (10\u00c2\u00a0min) with 1\u00c2\u00a0mmol\/l of the serine and cysteine protease
\n inhibitor tosyl-L<\/small>-lysine chloromethylketone (TLCK; Sigma\u00e2\u20ac\u201cAldrich, St Louis, MO, USA), which inhibits
\n gingipains 15], 21].\n <\/p>\nCharacterization of bacterial culture supernatants and treatment<\/h4>\n
\n as the total bacterial protein (mg\/ml) present in the cell cultures. The protein concentration
\n was determined with a BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Absorbance
\n was measured at 562\u00c2\u00a0nm on a SpectraMax\u00c2\u00ae Plus plate reader. Protease activity was measured
\n with a Protease Assay\u00e2\u201e\u00a2 Kit (G-Biosciences, St Louis, MO, USA) 22]. The absorbance of the dye-labeled peptide was measured at 570\u00c2\u00a0nm for determination
\n of the protease activity. Chemically stabilized trypsin (MSG-Trypsin\u00e2\u201e\u00a2) was supplied
\n with the kit as a general protease standard. GECs were grown to 80\u00c2\u00a0% confluence and
\n stimulated with either 50\u00c2\u00a0?g\/ml culture supernatant protein from P. gingivalis<\/em> supernatants or TLCK-preincubated supernatants, for 6\u00c2\u00a0h 22]. Unstimulated GEC medium served as a control for the stimulation experiments. Each
\n stimulation experiment was performed in triplicate and cells from two to five different
\n donors were tested.\n <\/p>\nQuantitative real-time RT-PCR (QRT-PCR)<\/h4>\n
\n CA, USA) and reverse-transcribed using a SuperScript\u00c2\u00ae RT-PCR Kit (Takara, Tokyo, Japan).
\n Quantitative real-time RT-PCR was performed using the Applied Biosystems 7500 PCR
\n machine and SYBR Premix Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer\u00e2\u20ac\u2122s
\n instructions. PCRs were carried out in 96-well plates in a total volume of 20\u00c2\u00a0?l,
\n including 1\u00c2\u00a0?l cDNA and 0.8\u00c2\u00a0?l primers (10\u00c2\u00a0?M; Table\u00c2\u00a02). Sample expression was normalized against that of the housekeeping gene ?-actin,
\n which was included in each QPCR run. PCR controls were performed using water instead
\n of cDNA. All reactions were carried out in duplicate. At each time point, the expressions
\n of the selected mRNAs in cells incubated with P. gingivalis<\/em> supernatants or TLCK-preincubated supernatants were calculated relative to the housekeeping
\n gene ?-actin (?C<\/em>t<\/sub>
\n ) for each sample and then expressed relative to untreated cells at the same time
\n point using the 2
-??<\/sup>C<\/sup><\/em>t<\/sup>
\n method 23].<\/p>\nFlow cytometry<\/h4>\n
\n with PBS containing 20\u00c2\u00a0% heat-inactivated normal human serum, washed again and then
\n incubated for 30\u00c2\u00a0min at 4\u00c2\u00a0\u00c2\u00b0C with 15 nM of specific monoclonal antibodies (mAb) to
\n human PAR-1\u00e2\u20ac\u201c4 (PE-conjugated; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).
\n Flow cytometry analyses were performed on a FACScan (BD Biosciences, San Jose, CA,
\n USA). Control cells incubated with PE-conjugated nonspecific antibodies obtained from
\n the same manufacturers were used to set the threshold for the fluorescence parameter,
\n such that the fraction of cells with positive fluorescence was 2.5\u00c2\u00a0% of the total
\n cells. The percentage of PAR-1\u00e2\u20ac\u201c4 positive cells was determined from the fraction of
\n cells in the sample incubated with specific antibodies that exceeded the threshold
\n for the fluorescence signal intensity obtained with the control sample.\n <\/p>\nStatistical analyses<\/h4>\n
\n as C<\/em>t<\/sub>
\n (cycle threshold), ?C<\/em>t<\/sub>
\n (C<\/em>t<\/sub>
\n PAR mRNA \u00e2\u20ac\u201c C<\/em>t<\/sub>
\n ?-actin mRNA) and relative quantification (RQ; expressed as fold change). The fold
\n changes of PARs mRNA expression were calculated using the 2
-??<\/sup>C<\/sup><\/em>t<\/sup>
\n method 23]. Student\u00e2\u20ac\u2122s t<\/em>-test was used for comparison between two groups. SPSS version 19.0 (SPSS Inc., Chicago,
\n IL, USA) was used for statistical analysis and P<\/em>???0.05 was considered statistically significant.\n <\/p>\n","protected":false},"excerpt":{"rendered":"