High performance liquid chromatography (HPLC) 방법을 통한 치수 내 calcitonin gene-related peptide (CGRP)의 정량적 검출

Abstract

[한글]

Calcitonin gene related peptide(CGRP)는 37개의 amino acid 를 가진 peptide이다. 이는 가장 강력한 혈관 확장제로 알려져 있으며, neurotransmitter 혹은 neuromodulator로 작용한다.

본 연구는 교정 목적으로 발거가 예정된 사람의 상, 하악 소구치 7개를 대상으로 치수 염증을 유발시킬 목적으로 협면에 유해자극을 가한 후, 자극을 가하지 않은 군(n=7)과 CGRP 양을 정량적으로 비교함으로써 외부 자극에 대한 초기 치수 반응을 관찰해 보고자 하였다.

치근단공이 완전히 닫힌 성숙한 영구치를 대상으로 하였으며, 유해 자극은 국소마취 하에서 치아의 협면 중앙부에 치아의 장축에 평행하게 직경 1mm의 diamond bur를 이용하여 coolant없이 약간의 압력을 주어 국소적으로 열이 발생되도록 하였다. 발치는 자극을 준 후 15분 후에 시행하였다. CGRP 분석 및 정량은 High performance liquid chromatography 방법으로 Shimadzu 사의 LC-10A VP series를 사용하였다.

실험 결과, 유해 자극을 가한 군(median 123.69, interquartile range 178.74)에서 자극을 가하지 않은 군(median 76.83, interquartile range 18.74)에서보다 CGRP 양이 높게 나타났으며, 이는 통계학적으로 유의성 있게 나타났다 (p=0.0033).

[영문]I. Objectives

The purpose of this study was to develop methodology for the detection and identification of calcitonin gene related peptide (CGRP) in human dental pulp and to quantify this substance in induced pulpal inflammation using reverse-phase high performance liquid chromatography (RP-HPLC).

Ⅱ. Materials and Methods

ⅰ) Human CGRP standard preparation α-CGRP (#C0167, Sigma-Aldrich, St. Louis, MO, USA) was used for standard. Human CGRP standard stock was loaded on HPLC device with different doses (0.1, 0.05, 0.025㎍/㎕). The data was used to confirm when CGRP was detected and to make a standard curve.

ⅱ) Tissue management for extraction of neuropeptide

14 human first or second premolars without caries, restorations or periodontal disease which were going to be extracted for orthodontic reason were used in this study. Traumatic injury for pulpal inflammation was induced on buccal surface of the crown by preparing a groove longitudinally with a high speed diamond bur (1.0mm diameter) with a depth of about 1.5mm without coolant. 15 minutes after grooving they were extracted atraumatically and placed in liquid nitrogen (-196℃) immediately. The tooth was split longitudinally and pulp tissue was gained. This step was done within 1 minute for prevention of peptide degeneration. The pulp tissue was weighed and 0.5M acetic acid was added to the tissue 8ml/g and boiled for 10 minutes. It was centrifuged at 5300rpm for 20 minutes at 4℃. The supernatant was transferred to another tube and diluted with 0.5M acetic acid until the total volume was about 200㎕. Then it was filtered through Millipore GS filter (Millex(r), Billerica, Massachusetts, USA). The resultant was loaded 50㎕ each on HPLC device for 10 minutes. Wavelength for detecting CGRP was 215nm.

ⅲ) High performance liquid chromatography

Reverse-phase HPLC was done using LC-10A series (Shimadzu. Co., Japan). Delta-Pak C18 5UM 300A 3.9 x 150mm (Waters, Tokyo, Japan) was used as a column. HPLC grade 30:70 acetonitrile : DW were used as a solvent. Y axis expresses absorbent unit (mAU) and X axis expresses time (minute) (Fig.2~7).

Ⅲ. Results

The peak of human CGRP standard was detected at 3.6 minute. This peak was shown to be dose-dependent and time-constant.

In experimental group CGRP was also detected at 3.6 minute. Fig 2 ~ 4. shows the standard curve of CGRP standard. CGRP amounts of both normal and experimental group were calculated using the standard curve (Fig 6, 7).

According to Wilcoxon rank sum test, CGRP was significantly higher in induced pulp inflammation (median 123.69, interquartile range 178.74) than normal pulp (median 76.83, interquartile range 18.74). P value was 0.0033.

Ⅳ. Conclusion

Calcitonin gene related peptide (CGRP) could be detected and quantified successfully by high performance liquid chromatography (HPLC) device in both normal and induced pulpal inflammation.

Further study should be directed to substantiate the early pulpal reaction against the external stimulus using this technique.