The present study was approved by the Ethics Committee at our institute (Clinical Research Review Board of the Kitasato Institute; reference number: KMEO B13-113) and abides by the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. All participants gave written informed consent.
To study the expression of collagen genes in the SSCT of HD and non-HD patients, SSCT specimens were obtained from patients with CTS during carpal tunnel release (CTR). A total of 110 patients underwent CTR. Given that the body mass index (BMI) is a reported risk factor for the formation of CTS , we excluded 46 patients who were overweight or obese (BMI ≥ 25 kg/m). All patients underwent diagnostic neurophysiological tests comprising electromyography and nerve conduction studies based on the American Association of Electrodiagnostic Medicine standards and received confirmed diagnosis of CTS . We further excluded patients with a history of thyroid disease, rheumatoid arthritis, osteoarthrosis, degenerative joint disease, flexor tendinitis, sarcoidosis, amyloidosis, peripheral nerve disease, or traumatic injuries to the ipsilateral arm based on their medical charts. Finally, 64 patients with CTS (47 non-HD and 17 HD) were included in this study.
In our study of the pathological role of B2M in patients with CTS, two men and four women with an age range of 52–71 years [mean ± standard deviation (SD), 66.8 ± 7.5 years] were excluded because they had a history of thyroid disease, rheumatoid arthritis, osteoarthrosis, degenerative joint disease, flexor tendinitis, sarcoidosis, amyloidosis, peripheral nerve disease, or traumatic injuries to the ipsilateral arm.
SSCT cell culture
SSCT specimens were treated with 20 ml of 0.1% type I collagenase derived from clostridium histoluticum (Wako Pure Chemicals, Osaka, Japan) for one day at 37°C. SSCT cells were maintained in culture for two weeks in α-MEM with 10% fetal bovine serum (FBS) and 20 mg/ml basic fibroblast growth factor (bFGF). To eliminate any effects of 10% FBS and bFGF on collagen gene expression, cells were washed twice with α-MEM and the medium was replaced α-MEM containing 0.5% FBS. Three hours after replacing the medium, SSCT cells were stimulated in α-MEM with 0.5% fetal bovine (vehicle) or 1 or 10 µg/ml B2M.
SSCT cells were homogenized with TRIzol Reagent using a homogenizer. The mixture was centrifuged to obtain the supernatant, from which total RNA was subsequently extracted by Direct-zolTM RNA Micro Prep (Zymo Research, Irvine, CA) based on the manufacturer's protocol. The SuperScriptIII First-Strand Synthesis Super Mix kit (Life Technologies, ThermoFisher, Waltham, MA) was then used to conduct cDNA synthesis, with the purified total RNA as the template. Primers used in the PCR assay are listed in Table 1. To examine COL1A1 and COL3A1 mRNA expression, real-time PCR analysis (MiniOpticon, Bio Rad, USA) was conducted using PCR Master Mix (Takara SYBR® PreMix Ex Taq II). mRNA expression levels were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and analyzed using the delta-delta CT method.
Shapiro-Wilk's test was used to ascertain whether the data were normally distributed. In the comparison of collagen expression in the SSCT of HD and non-HD patients, variables that were not normally distributed were analyzed using the Mann-Whitney test. In the study of the pathological role of B2M in patients with CTS, data were compared by one-way repeated measures ANOVA and pairwise comparison with Bonferroni adjustment. P < 0.05 determined in SSPS software v19.0 (IBM, USA) was used to indicate statistical significant.