자기장 저속 냉동보관법을 이용한 쥐 치아 치주인대세포의 활성도 검사 = EVALUATION OF THE VIABILITY OF PERIODONTAL LIGAMENT CELL IN RAT TEETH USING SLOW CRYOPRESERVATION METHOD WITH MAGNETIC FIELD

국문 초록 (Abstract)

본 연구의 목적은 흰쥐 상악 대구치를 발거하여 자기장 저속 냉동보관법을 이용하여 냉동 시 치주인대세포의 활성도 및 세포 사멸도를 MTT검색법과 TUNEL검사를 이용하여 측정하고자 하였다.
4주령의 암컷 Sprague-Dawley계 된 쥐의 상악 좌우 제1,2대 구치를 발거하여 각군 당 12개의 쥐 치아를MTT검색에 이용하였고 6개의 치아를 TUNEL 검사에 이용하였다. 실험군은 5개군으로 대조군은 즉시 발치군이며 4℃냉장고에서 1주일간 보관한 냉장군, 발치 후 동해방지제 처리과정을 거쳐 -196℃의 액화질소에 넣어 급속 냉동한 액화질소군, 21.7 ㎃, 60 ㎐, 1 G의 자기장을 이용하여 -0.3℃/min 의 속도로 -20℃까지 냉동 후 -196℃로 급속 냉동한 자기장군, -0.3℃/min의 속도로 -20℃까지 냉동 후 -196℃에 급속 냉동한 저속 냉동군으로 나누었다. 보존액은 F medium을 사용했으며 동해방지제로 10% dimethyl sulfoxide (DMSO)를 사용하였다. 치근면을 단위면적으로 표준화하기 위해 MTT측정값을 Eosin 염색 후 530 ㎜에서 측정한 흡광도 값으로 나누었다. TUNEL 검사 시 각 조직슬라이드에서 400배 크기의 현미경 시야에서 임의로 세 부분을 지정하여 정상 세포수와 양성 세포수를 세어 그 비율을 계산하여 각 실험군 당 평균치를 구하였다. 통계 분석을 위해 one way ANOVA를 시행하였으며 사후검정으로 Scheffe와 Tukey HSD방법을 썼으며 결과는 다음과 같다.
MTT검색에 의한 흡광도를 Eosin염색 후 측정한 흡광도로 나눈 값에서는 자기장군은 즉시 발치군보다 낮은 세포활성을 보였고 (p < 0.05) 액화질소군, 저속 냉동군과는 통계적으로 유의성 있는 차이를 보이지 않았다. 그러나 자기장군은 액화질소군, 저속 냉동군과 함께 냉장군보다는 높은 세포 활성도를 보였다 (p<0.05).TUNEL검사 결과도 자기장군은 즉시 발치군보다 치주인대의 세포사멸도가 높았으나 (p < 0.05) 저속 냉동군과 액화 질소군과는 통계적으로 유의한 차이를 보이지 않았다. 자기장군은 냉장군보다 세포사멸도가 낮았으며 냉장군은 모든 군 중에서 세포 사멸도가 가장 높았다 (p<0.05).

다국어 초록 (Multilingual Abstract)

The purpose of this study was to evaluate the viability of periodontal ligament cell in rat teeth using slow cryopreservation method with magnetic field through MTT assay and TUNEL test. For each group, 12 teeth of 4 weeks old white female Sprague-Dawley rat were used for MTT assay, and 6 teeth in TUNEL test. The Maxillary left and right, first and second molars were extracted as atraumatically as possible under tiletamine anesthesia. The experimental groups were groupl (immediately extraction), group 2 (cold preservation at 4℃ for 1 week), group 3 (rapid Cryopreservation in liquid nitrogen), group 4 (slow cryopreservation with magnetic field of 1 G), and group 5 (slow cryopreservation). F medium was used as preservation medium and 10% DMSO as cryoprotectant. After preservation and thawing, the MTT assay and TUNEL test were processed. One way ANOVA and Scheffe method were performed at the 95 % level of confidence. The value of optical density obtained after MTT analysis was divided by the value of eosin staining for tissue volume standardization. In both MTT assay and TUNEL test, it had showed no significant difference among group 3,4, and 5. And group 3 had showed higher viability of periodontal ligament cell than group 2.
From this study, slow cryopreservation method with magnetic field can be used as one of cryopreservation methods.

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