아텔로콜라겐 지지체, 유전자 치료, 배양된 수핵세포를 이용한 조직 공학적 추간판의 재생 = Tissue Engineering of the Intervertebral Disc with Cultured Nucleus Pulposus Cells Using Atelocollagen Scaffold and Gene Therapy

다국어 초록 (Multilingual Abstract)

Study Design: This is an in-vitro experiment using rabbit intervertebral disc (IVD) cells and growth factors.
Objectives: We wanted to determine the effect of types I,and II atelocollagen and growth factor gene therapy for matrix regeneration of rabbit IVD cells.
Summary of the Literature Review: Adenovirus-medicated growth factor gene therapy is efficient for matrix regeneration of the IVD. Atellocollagen has provided a favorable environment for matrix synthesis. However, a combined approach using gene and cell therapy in an atelocollagen scaffold has not yet been attempted.
Materials and Methods: Rabbit IVD cells were transduced with Ad/TGF-β1 and Ad/BMP-2. The cells were then implanted to the atelocollagen scaffold. The [methyl-3H]thymidine incorporation for DNA synthesis and the [35S]sulfur incorporation for proteoglycan synthesis were measured. RT-PCR was performed for assessing the aggrecan, collagen type I, collagen type II and osteocalcin mRNA expressions.
 Results: The rabbit IVD cells with Ad/TGF-β1 and that were cultured in type I atelocollagen showed a 130% increase in new proteoglycan synthesis, while the rabbit IVD cells with Ad/TGF-β1 and that were cultured in type II atelocollagen showed a 180% increase in new proteoglycan synthesis (p<0.05). The rabbit IVD cells with Ad/BMP-2 and that were cultured in type I atelocollagen showed a 70% increase in new proteoglycan synthesis, while the rabbit IVD cells with Ad/BMP-2 and that were cultured in type II atelocollagen showed a 95% increase (p<0.05). Rabbit IVD cells with Ad/TGF-β1 and Ad/BMP-2 and that were cultured in type I and II atelocollagen demonstrated increased collagen type I and II mRNA expressions without an osteocalcin mRNA expression (p<0.05).
Conclusion: Cell and gene therapy in an atelocollagen scaffold provided a efficient mechanism for chondrogenic matrix regeneration of rabbit IVD cells.

국문 초록 (Abstract)

연구 계획: 토끼의 추간판 세포와 제5형 아데노바이러스(Ad) transforming growth factor beta-1(TGF-β1), 아데노바이러스 bone morphogenetic protein-2 (BMP-2), 아텔로콜라젠을 이용한 실험적 연구목적: 제1, 2형 아텔로콜라젠과 성장 인자 유전자 치료의 토끼 추간판 세포의 기질 생성 반응을 알아보았다.
선행문헌의 요약: 아데노바이러스를 이용한 성장 인자 유전자 치료는 추간판 세포의 기질 재생에 효과적이다. 아텔로콜라젠은 추간판 세포의 기질 합성에 유리한 환경을 제공한다. 하지만 아텔로콜라젠 지지체에 유전자 치료를 병행하는 방법은 시도된 바가 없었다.
대상 및 방법: 토끼의 추간판 세포를 배양하였다. Ad/TGF-β1, Ad/BMP-2 아텔로콜라젠을 생산하였다. 추간판 세포를 Ad/TGF-β1, Ad/BMP-2에 감염시키고 이 세포를 아텔로콜라젠 지지체에 배양하였다. DNA생성은 [methyl-3H] Thymidine을 이용하였고 새로운 당단백 생성은 [35S]Sulfur를 이용하였다. 또한 전사 수준에서의 추간판세포 관련 유전자의 변화를 확인하고자 RT-PCR을 통한 aggrecan, 제 1, 2형 교원질, osteocalcin mRNA발현을 조사하였다.
결과: 제1형 아텔로콜라젠에 배양되고 Ad/TGF-β1로 유전자 전달된 추간판 세포군은 대조군에 비해 130% 신생 당단벡 생성증가가 있었고 제 2형 아텔로콜라젠에 배양되고 Ad/TGF-β1로 유전자 전달된 세포군은 180%의 신생 당단백 생성 증가가 있었다. (p<0.05) 제 1형 아텔로콜라젠에 배양되고 Ad/BMP-2로 유전자 전달된 추간판 세포군은 대조군에 비해 70% 신생 당단벡 생성증가가 있었고 제2형 아텔로콜라젠에 배양되고 Ad/BMP-2로 유전자 전달된 세포군은 95%의 신생 당단백 생성 증가가 있었다. (p<0.05) 제 1, 2형 아텔로콜라젠에 배양되고 Ad/TGF-β1, Ad/BMP-2로 유전자 전달된 세포군은 제 1, 2형 교원질 mRNA발현증가가 있었으나 (p<0.05) 어떠한 경우에도 osteocalcin mRNA발현은 없었다.
결론: 추간판 세포를 아텔로콜라젠 지지체에서 배양하고, Ad/TGF-β1, Ad/BMP-2으로 유전자 치료를 시행하여 효과적으로 연골형 기질을 생성을 유도할 수 있었다.

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