Effects of Warming Acupuncture on Ligament Recovery Following Injury = 손상된 인대의 회복에 대한 온침의 효과

국문 초록 (Abstract)

목적 : 온침은 인대의 손상을 포함한 물리학적 병변과 여러 가지 질병에 대한 치료로 한의학에서 사용되어 왔다. 하지만 세포학적, 분자학적 기전에 대한 근거는 거의 알려져 있지 않다. 이에 족관절 염좌를 유발한 쥐의 모델에서 손상된 인대에 대한 온침의 효과에 대하여 조사하였다.
방법 : 우측 뒷발 족관절 염좌를 유발한 쥐들을 4주와 8주 후 각각에 대하여 매주 1회 6주간 환측 족관절 전외측 부위(GB40, 구허)에 온침을 시행하였다. 인대조직을 박리하여 Western blotting and Immunofluorescence, RT-PCR에 의하여 단백을 분석하기위해 사용하였다. Hoechst 핵 염색법은 각 세포수를 측정하는데, Masson's trichrome염색법은 콜라겐 섬유를 시각화 하는데 사용하였다.
결과 : 온침 자극을 시행한 인대 조직에서 phospho-Erk1/2 활성 수준은 단지 손상만 유발된 군에 비하여 증가하였다. 또한 phospho-Erk1/2는 Cdc2 kinase에 의해 세포주기를 진행하는데 있어서 중요한 것으로 알려진 cyclin B1 단백질의 발현과 양의 상관관계로 증가하였다. Hoechst 핵 염색법으로 확인된 섬유아세포 핵의 수는 온침을 시행한 군이 손상만 유발된 대조군 보다 증가하였다.
온침을 시행한 군이 손상만 유발된 대조군에 비하여 type III 콜라겐 mRNA와 단백질의 발현이 증가하였다. 하지만, type I 콜라겐 강도는 type III 콜라겐 보다 적었으며, 온침에 의한 명확한 변화는 관찰되지 않았다. Masson's Trichrome 염색 결과는 온침 시행군에서 콜라겐 섬유의 시각적 강도가 단지 손상만 유발된 대조군보다 증가하였다.
족관절 염좌에 의해 유사한 손상 및 회복단계를 거칠 것으로 생각되는 인접한 건 조직에서 phospho-Erk1/2 및 cyclin B1/Cdc2 단백질의 양적변화를 조사한 결과 그 양적 증가에 있어 인대보다 소폭으로 증가하는 양상을 보여, 온침의 효과가 건조직보다 인대조직에 더 효과적으로 나타났다.
결론 : 본 실험결과는 족관절 염좌후 4주, 8주 후에 온침의 자극이 치유과정에 효과적인 것으로 보여진다. 현재의 쥐 염좌 모델은 온침에 의한 조직재생의 근원을 세포학적, 분자학적 기전으로 설명하는데 대하여 유용할 것으로 생각된다.

다국어 초록 (Multilingual Abstract)

Object : Warming acupuncture has been used in the oriental medicine for the treatment of diverse diseases and physical disorders including ligament damages. However, supportive evidences at cellular and molecular levels are largely unknown. Thus, possible healing effects of warming acupuncture on injured ligament were investigated using the rat model.
Methods : The rats were given sprain injury on the right hind ankle, GB40 (Quixu) and 4 or 8 weeks later, warming acupuncture was given to the injury site on a weekly basis for 6 weeks. The ligament tissue was dissected and used for protein analysis by Western blotting and immunofluorescence staining, and also for mRNA analysis by RT-PCR methods. Hoechst nuclear staining was used for individual cell counting, and collagen fibers were visualized by Masson's trichrome staining method.
Results : Active form of phospho-Erk1/2 kinase was increased in the ligament from warming acupuncture group compared with the injury only group. Increases in phospho-Erk1/2 protein levels positively correlated with increased cyclin B1 protein, known to be important for the cell cycle progression mediated by cell division cycle 2 (Cdc2). Also, the total cell number which was identified by Hoechst nuclear staining was significantly increased in the ligament from warming acupuncture group compared with injury only group. Type III collagen mRNA and protein levels in the ligament were elevated in warming acupuncture group compared with injury only group. Yet, the signals of type I collagen was much lower than type III collagen, and any clear changes by warming acupuncture were not observed. Msson's trichrome staining further visualized intense collagen fibers in the ligament with warming acupuncture. Comparative analysis in the tendon tissues of the ankle given sprain injury showed no increases in phospho-Erk1/2 and moderate increase in cyclin B1 protein levels, indicating that warming acupuncture was more effective in the ligament than in the tendon tissues.
Conclusions : The present data suggest that warming acupuncture stimulation given 4 weeks or 8 weeks after ligament injury may have a promoting effect on the healing process. The present rat ankle sprain model would be useful for elucidating cellular and molecular mechanisms underlying the tissue regeneration by warming acupuncture.

목차 (Table of Contents)

• 국문초록 = 1
• Ⅰ. Introduction = 3
• Ⅱ. Materials and methods = 6
• 1. Experimental materials= 6
• 1) Experimental animals = 6
• 2) Warming acupuncture = 6
• 2. Experimental methods = 6
• 1) Animals groups = 6
• 2) Ankle sprain injury of the ligament and tendon in the rats= 7
• 3) Warming acupuncture and prolotherapy = 8
• 4) Treatment protocol = 9
• 5) Histological analysis = 9
• 6) Western blot analysis = 12
• 7) RT-PCR = 13
• 3. Statistical analysis = 14
• Ⅲ. Results = 15
• 1. Effects of warming acupuncture on Erk1/2 kinase levels in the injured ligament = 15
• 2. Effects of warming acupuncture on Cdc2/cyclin B1 protein expression in the injured ligament = 19
• 3. Changes in collagen protein expression in the ligament after warming acupuncture = 23
• 4. Masson's trichrome staining of the ligament tissue = 25
• 5. Effects of warming acupuncture on the tendon tissues = 27
• Ⅳ. Discussion = 33
• Ⅴ. Conclusions = 44
• References = 46
• Abstract = 53