Clinical Trial Details
— Status: Recruiting
Administrative data
| NCT number |
NCT00167050 |
| Other study ID # |
9461700634 |
| Secondary ID |
|
| Status |
Recruiting |
| Phase |
N/A
|
| First received |
September 12, 2005 |
| Last updated |
November 25, 2005 |
| Start date |
July 2005 |
| Est. completion date |
June 2006 |
Study information
| Verified date |
June 2005 |
| Source |
National Taiwan University Hospital |
| Contact |
Fung-Rong Hu, MD |
| Phone |
886-2-23123456 |
| Email |
fungrong[@]ha.mc.ntu.edu.tw |
| Is FDA regulated |
No |
| Health authority |
Taiwan: Department of Health |
| Study type |
Observational
|
Clinical Trial Summary
Superior limbic keratoconjunctivitis (SLK) was first described in detail as a clinical
entity by Frederick Theodore in 1963. The clinical picture of SLK is well documented, but
the etiology is still unknown. This project will be conducted into through two parts: one is
to investigate the presentation of chemokine receptors on mast cell and matrix
metalloproteinases on fibroblasts by immunohistochemistry method from the pathological
specimens of SLK patients who received conjunctiva resection as the treatment. The other
part is to investigate the mRNA level of those chemokine receptors via reverse transcription
- polymerase chain reaction from the conjunctiva collecting form SLK patients.
Description:
1. Specimens collection:
We will collect the specimens in two different parts. One part is to collect all the
paraffinized block of the SLK patients to get fifteen copies of 5-μm section of each
paraffinized block from the Department of Pathology. All the slides will be
deparaffinized and rehydration for further immunohistochemistry (IHC) stain . Primary
antibodies for IHC stain include chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4, CCR1.
CCR3, CCR4, CCR5, CD30L) and matrix metalloproteinases (MMP-1, -2, -3, -9).
Another part of this study is to collect the conjunctiva of SLK patients and normal
control patients. This part of the clinical study is sent for IRB approval
concomitantly. We will collect the conjunctiva of SLK patients when they receive
conjunctival resection as the treatment in the following one year. And the normal
control conjunctiva will be obtained while the patients come to our hospital for
cataract surgery or retinal surgery with redundant conjunctiva noted after peritomy.
The fresh conjunctiva will be stored in -80℃ before RNA isolation. All the tissue will
be processed into RNA and reverse transcripted into cDNA. Polymerase chain reaction
will be done with the primers, including CXCR1, CXCR2, CXCR3, CXCR4, CCR1, CCR3, CCR4,
CCR5, and CD30L.
2. Preparation of RNA and cDNA:
Total RNA will be extracted from the conjunctiva of SLK and control groups with Trizol
reagent (Life, Gaithersburg, MD, USA). One microgram of total RNA from each sample will
be annealed for 5 min at 70℃ with 500 ng oligo(dT) (Fermentas, Hanover, MD, USA) and
reverse transcribed to cDNA by 200u RevertAid™ M-MuLV Reverse Transcriptase (Fermentas,
Hanover, MD, USA) per 20 µl reaction for 1 hr at 42℃. The reaction will be stopped by
heating for 10 min at 70℃.
3. Polymerase chain reaction (PCR):
PCR will be performed on the resultant cDNA from each sample with specific primers for
human chemokines, chemokine receptors and glyceraldehydes-3-phosphate dehydrogenase
(GAPDH). The amplification will be performed with a thermocycler. The 25-μl reaction
mixture consists of 2 μl cDNA, 1 μl sense and antisense primer, 12.5 μl 2x PCR mix.
Conditions for amplifying each chemokine and chemokine receptor are as follows:
denaturation, 1 min at 94℃, and elongation, 3 min at 72℃. The annealing temperature and
the cycle of annealing for chemokines and chemokine receptors will fit the requested
temperature and the appropriate number of annealing cycles of each specific primer. At
the end of amplification, the reaction mixture will be heated for 10 min at 72℃ and
then cooled to 4℃. A 10-μl sample of each PCR product will be separated by performing
gel electrophoresis on 2% agarose containing ethidium bromide (Sigma, St Louis, MO,
USA). The 2% agarose gel will be analyzed under ultraviolet light against the DNA
molecular length markers. The internal control of each sample is human GAPDH.
4. Immunohistocytometry (IHC):
A pre-determined optimal dilution of the primary antibody specifically against mast cell
tryptase, associated chemokines, chemokine receptors, MMPs, and inflammatory cytokines will
be used for IHC. All IHC staining will be performed at room temperature. Sections are then
deparaffinized in xylene and made hydrophilic by immersion in a graded series of ethanol
dilutions (100, 95, 80, and 60%). All slides are immersed in antigen retrieval solution
(Dako ,S3307, SA, USA) and autoclaved for 10 min. Sections are quenched with 3% fresh H2O2
for 10 min to inhibit endogenous tissue peroxidase activity that might interfere with the
result of staining, and rinsed with 1x PBS for 5 min twice. Sections are further incubated
in blocking serum solution for 30 min and then incubated at 37℃ for 1 h with primary
antibody in a humid chamber. After washing with 1x PBS for 5 min twice, the sections are
then incubated with biotinylated secondary antibody solution for 30 min. The horseradish
peroxidase-conjugated streptavidin-biotin complex is subsequently spread evenly over the
sections and incubated for 30 min. The sections are washed with 1x PBS for 5 min twice, then
incubated with diaminobenzidine (DAB) perioxidase substrate solution for 15 min followed by
wash with 1x PBS. Finally, the section are counterstained with an adequate volume of
hematoxylin, and then mounted.
