제대혈 유래 중간엽 줄기 세포를 이용한 피부 창상 치료시 세포투여 운반체에 따른 창상 치유 효과의 비교

Abstract

[한글]

창상치유 촉진을 위한 치료법으로 세포를 창상에 직접 뿌리는(spray) 세포치료 방법이 일부 임상에 적용되고 있으나 이 방법은 창상 밖으로 세포가 흐르거나 효과적으로 세포가 창상에 부착되기 어려운 단점이 있다. 보다 안정적이고 효과적으로 세포를 창상에 공급할 수 있는 세포치료법을 개발하고자 다양한 세포전달 방법을 이용 창상치유 정도를 비교 분석함으로써 세포치료 시 가장 효율성 있는 방법을 정립하고자 하였다.생후 4주 된 마우스(ICR) 배부에 8mm punch를 이용하여 전층 피부 결손을 만들고 인체 제대혈 유래 중간엽 줄기세포(MSC)를 각기 다른 5가지 방법 혹은 운반체를 이용 투여하였다. 투여방법으로는 Cell suspension법, Local injection법, Collagen GAG matrix(Terudermis？) seeding법, 세포고정 효과가 높은 Fibrin(Tissucol○R) mix법, 창상에 습윤한 환경을 가장 잘 유지시켜주는 Hydrogel(Intrasite○R) mix법을 이용하였다. 각 실험군은 4 x 105 개의 중간엽 줄기세포를 운반체와 더불어 투여하였고 대조군에는 운반체만을 동량 넣은 후, Tegaderm○R을 이용하여 창상이 마르지 않도록 유지하면서 2주간 창상치유의 정도를 Digital imaging program을 이용하여 측정하였다. 각 군에서 7일과 14일째에 조직을 채취하여 진피 층의 신생혈관의 갯수를 측정하였고 Paracrine effect를 확인하기 위해 TGF-β, VEGF의 발현 정도를 면역염색 및 Optical density를 측정을 통하여 분석하였다. Anti-human nuclei Ab를 이용 MSC의 생존 여부를 검증하였다. 각 군간의 통계학적 분석은 T-test, paired T-test, ANOVA, repeated ANOVA를 사용하였다.창상치유율은 3일째부터 9일째 사이 모두 급격히 증가하였으나 Local injection군에서만 통계적으로 유의성을 보였다(p<0.05). 창상의 크기가 원래 크기의 절반으로 치유된 날짜인 HT50(Time to heal 50% wounds)은 Local injection군 6.1일, Collagen matrix군 6.4일, Fibrin glue군 6.7일, Hydrogel군 7.1일, Suspension군 7.2일로 Local injection군에서 가장 창상치유가 빨랐으며 Suspension군이 가장 늦었다. 14일째에 신생 혈관의 생성은 Collagen matrix군, Suspension군, Hydrogel군 순으로 증가 되어 있었고 조직내 TGF-β의 양은 Fibrin glue군, Suspension군, Local injection군, Collagen matrix군, Hydrogel군 순이었으나 통계학적으로는 Suspension군과 Local injection군 만이 대조군에 비해 유의한 차이(p<0.05)를 보였다. VEGF의 양은 Collagen matrix군, Suspension군, Local injection군, Fibrin glue군, Hydrogel군 순으로 증가되어 있었며 Collagen marix군, Suspension군, Hydrogel군은 대조군에 비해 통계학적으로 유의한 차이(p<0.05)를 보였다.세포 치료시 창상치유 속도가 가장 빠른 것은 Local injection군이었고 Suspension군은 중간엽 줄기세포가 투여 후 잘 생착하지 못하고 씻겨버리기 때문에 그 효과가 적은 것으로 사료되었다. Collagen matrix군은 새로운 진피의 역할을 하여 창상의 수축을 줄이기 때문에 창상이 치유되는 속도는 상대적으로 늦었으나 혈관 개수나 VEGF는 가장 증가되어 있었다. 이는 Collagen matrix가 3차원적 구조체 역할을 함으로써, 중간엽 줄기세포가 생착하여 활동하는 것을 돕고 신생혈관 생성을 촉진시키는 VEGF를 증가시켜 창상치유를 촉진하는 것으로 여겨진다. 따라서 창상수축을 통한 빠른 창상의 치유를 위해서는 Local injection의 방법으로 MSC를 투여하는 것이 유리하나 화상과 같이 넓은 부위의 창상 치유의 과정에서는 Collagen matrix에 세포를 seeding하는 것이 효과적일 것으로 생각된다.

[영문]

The method of spraying stem cells directly on wound site has been applied in certain clinical settings. This however has some disadvantages such as leakage of stem cells out of the wound or difficulty of stem cells adhering to the wound surface. Therefore, the authors of this study have attempted to establish the most effective and stable method of delivering stem cells by comparing various modes of cell delivery.Full thickness skin defect was artificially made on each dorsal aspect of mouse (gestational age 4weeks) (ICR) trunk by using an 8mm punch. Human umbilical cord derived mesenchymal stem cell (MSC) were delivered into these skin defects by 5 different methods or by using a carrier. Method of delivery include Suspension(spray) method, Local injection method, collagen GAG matrix(Terudermis？) seeding method, method mixing with Fibrin glue (Tissucol？) that is highly effective in fixating cells, and method mixing with Hydrogel (Intrasite？).Each experiment group was treated with 4x105 /ml of MSC along with a carrier, while the control group was inserted with only the carrier. The wound was covered with Tegaderm？ in order to prevent it from drying. The extent of wound healing was assessed by quantifying with a Digital imaging program. Tissue sampling was done from each group on day 7 and day 14 to measure the amount of newly formed blood vessels. Expression rates of TGF-β and VEGF was measured in order to confirm paracrine effects. This was done by analyzing the data obtained through immunohistochemical staining procedures and measuring the optical density with an optical densitometry. The viability of the MSC was substantiated by quantitatively measuring the anti-human nuclei Ab. For our statistical analysis, T-test, paired T-test, ANOVA, repeated ANOVA was employed.All experiment group had a steep wound healing rate between day 3 and day 9, but only the group with Local injection method had statistical significance(p<0.05). The time when the wound size decreased to half its size was defined as HT50. HT50 was 6.1 days in the Local injection group, 6.4 days in collagen matrix group, 6.7 days in the fibrin glue group, 7.1days in Hydrogel group, and 7.2days in the Suspension group respectively. The Local injection group had the fastest healing rate while Suspension group had the slowest. At day 14, new vessel formation rate increased from Collagen matrix group, Suspension group, to the Hydrogel group. Likewise, the amount of TGF-β was in decreasing order from Local injection method group, Collagen matrix group, to Hydrogel group but only the Suspension group and the Local injection group had statistical significance(p<0.05) when compared with the control group. The amount of VEGF was in decreasing order from Collagen matrix group, Suspension group, Local injection group, Fibrin glue group to Hydrogel group and Collagen matrix group, Suspension group and Hydrogel group had stastistical significance(p<0.05) when compared with the control group.The most effective mode of delivering stem cell to wound during cell therapy was Local injection method and the least was the Suspension method perhaps because the MSC cannot be fixed on the wound surface and sloughs off. The Collagen matrix group served as a new dermis, which in turn acts against contraction. Therefore, wound closure was delayed relative to other groups. However, the number of new blood vessels or the amount of releasing VEGF was increased the most among others. This may be explained by the fact that the Collagen matrix acts as a three dimensional structure which facilitates the MSC to fix itself and function more actively. Further, it also increases the VEGF, which in turn stimulates new vessel formation. Hence, an enhanced effect of wound healing. Therefore, wound that needs rapid closure through contracture might benefit from delivering the MSC using the Local injection method, but in situations of vast raw surface wounds such as in burns, seeding of cells into Collagen matrix is thought to be effective.