Effects of hUCB-derived MSCs and small intestinal submucosa hydrogel in combined radiation-wound = 인간 제대혈 유래 중간엽 줄기세포와 소장점막하조직 하이드로겔의 방사선 복합 피부 손상에서의 효과

다국어 초록 (Multilingual Abstract)

Angiogenesis is one of the critical processes for proper tissue regeneration. However, the angiogenesis is downregulated in irradiated body because of the radiation inhibits angiogenesis by directly affect to endothelial system and bone marrows. The mesenchymal stem cell (MSC) is a promising agent for impaired wound regeneration but its effect is limited in radiation combined injuries. Extracellular matrix scaffolds have been studied as a cell culture scaffold or transplant cell carrier to enhance the therapeutic ability of the MSC.
In this study, small intestinal submucosa (SIS) gel was made from commercially available porcine SIS derived extracellular matrix sheet and the humane umbilical cord blood-derived mesenchymal stem cell (hUCB-MSC) was cultured on the SIS gel. The effect of SIS gel on the hUCB-MSC culture and the representative angiogenic factors in conditioned medium were assessed and its angiogenic effect was evaluated using irradiated human umbilical vein endothelial cells (HUVECs) in vitro. Proliferation of the hUCB-MSC was not enhanced but pluripotency of the hUCB-MSC were significantly increased. Angiogenic growth factor secretion such as HGF, VEGF and ANG-1 from the hUCB-MSC was significantly increased by SIS hydrogel. Conditioned medium of the hUCB-MSC on SIS hydrogel promoted chemotaxis of irradiated HUVECs predominantly rather than promote proliferation and HGF was a predominant chemoattractant of HUVECs.
Therapeutic effect of the hUCB-MSC and SIS gel on impaired wound model was assessed in the second study. Wound closing and histological evaluation were performed after the combination therapy of the hUCB-MSC and SIS gel was applied to excisional wound splinting model of whole body irradiated C57BL/6 mice. Wound healing and histologically mature skin regeneration of radiation combined full thickness skin wound were enhanced by the hUCB-MSC and SIS hydrogel composite treatment in the excisional wound splinting mice model compare to the hUCB-MSC treatment alone. The angiogenesis which is decreased by the radiation was recovered by the hUCB-MSC treatment and significantly enhanced by co-treatment of the hUCB-MSC and SIS hydrogel.
In conclusion, the angiogenic paracrine effect of the hUCB-MSC can be promoted by SIS hydrogel and its therapeutic effect in impaired wound healing through enhanced angiogenesis by recruitment of irradiated vascular endothelial cell.

국문 초록 (Abstract)

혈관 신생은 조직 재생을 위한 중요한 과정 중 하나이다. 하지만 방사선에 의한 혈관내피세포와 골수의 손상은 혈관신생을 감소시킨다. 중간엽 줄기세포는 치유지연 손상 피부의 재생에 있어서 기대되는 치료제이지만 방사선에 의한 치유지연 피부손상에 대해서는 그 효과가 제한적이다. 세포외기질 유래 지지체는 중간엽 줄기세포의 치료 능력을 향상할 수 있는 세포 배양 지지체 또는 이식 세포 운반체로서 연구되어 왔다.
본 연구에서는 시중에서 판매하는 돼지 소장점막하조직(Small Intestinal Submucosa, SIS)을 이용하여 SIS gel을 만들고 이 위에 인간 제대혈 유래 중간엽 줄기세포 (hUCB-MSC)를 배양 하였다. hUCB-MSC를 배양한 배지에서 대표적인 혈관신생인자의 발현을 평가하고, 그 효과를 방사선 조사된 인간제대정맥내피세포(human umbilical vein endothelial cell, HUVEC)를 사용하여 평가하였다. hUCB-MSC의 증식(proliferation)은 SIS gel에 의해 향상되지 않았지만, 전분화능(pluripotency)은 크게 증가 하였다. hUCB-MSC의 HGF, VEGF, ANG-1과 같은 혈관신생인자의 분비량도 SIS gel에 의해서 유의적으로 크게 증가하였다. hUCB-MSC를 SIS gel 위에서 배양한 배지는 방사선 조사에 의해 감소한 HUVEC의 증식을 증진시키지는 못하고, 유주 활성(chemotaxis)을 증진시키는 것으로 나타났으며 이는 HGF가 주된 화학 유인 인자로 역할을 한 결과로 나타났다.
본 연구에서는 전신 방사선 조사한 C57BL/6 마우스를 이용한 피부 절제 모델에 hUCB-MSC 및 SIS gel를 적용한 후 상처 치유 및 조직학적 평가를 하였다. 방사선 조사로 인해 지연된 피부 절제 상처의 치유가 hUCB-MSC와 SIS hydrogel 복합치료에 의해 촉진되었고 조직학적으로도 hUCB-MSC의 단독치료보다 성숙한 피부재생이 이뤄졌다. 방사선으로 인해 감소된 혈관 신생은 hUCB-MSC에 의해 회복되었고 이는 hUCB-MSC와 SIS hydrogel의 복합치료에 의해 더욱 증진되었다.
이 연구를 통하여 hUCB-MSC의 혈관신생인자의 분비가 SIS hydrogel에 의해서 증진되는 것을 확인하였고, hUCB-MSC와 SIS hydrogel의 복합치료는 방사선에 의해 치유가 지연된 피부손상에서 hUCB-MSC 단독 치료보다 나은 치료효과를 나타냈다. 이는 방사선에 의해 감소한 혈관 내피 세포의 화학 유주 활동을 회복시켜 손상부위의 혈관 내피 세포를 모으는 작용에 의한 혈관 신생 효과 증진에 있는 것으로 보인다.

목차 (Table of Contents)

• General Introduction 1
• Part I. Small Intestinal Submucosa Hydrogel Enhances Angiogenic Paracrine Action of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells 4
• 1.1. Introduction 5
• 1.2. Materials and Methods 7
• 1.2.1. Preparation of SIS hydrogel. 7
• 1.2.2. Culture of human UCB-MSCs (hUCB-MSCs). 9
• 1.2.3. RNA extraction, reverse transcription, and quantitative real-time polymerase chain reaction (qRT-PCR). 11
• 1.2.4. Collecting conditioned medium of hUCB-MSCs. 13
• 1.2.5. Enzyme-linked immunosorbent assay (ELISA). 13
• 1.2.6. Culture and irradiation of human umbilical vein endothelial cells (HUVECs). 13
• 1.2.7. Alamar blue assay. 14
• 1.2.8. Senescence-associated beta-galactosidase (SA-β-gal) assay. 15
• 1.2.9. Transwell migration assay. 15
• 1.2.10. Tube-like structure formation assay. 15
• 1.2.11. Statistical analysis. 16
• 1.3. Results 17
• 1.3.1. Proliferation of hUCB-MSCs on SIS gel or conventional culture plastic 17
• 1.3.2. SIS gel enhances pluripotency and angiogenic paracrine secretion of hUCB-MSCs. 19
• 1.3.3. hUCB-MSC and SIS composite cannot recover proliferation of irradiated HUVECs through a paracrine effect. 21
• 1.3.4. hUCB-MSC and SIS composite enhances migration of irradiated HUVECs through a paracrine effect. 24
• 1.3.5. hUCB-MSC and SIS composite enhances migration of irradiated HUVECs through HGF secretion. 26
• 1.3.6. hUCB-MSCs and SIS composite enhances the tube-like structure formation of irradiated HUVECs. 28
• 1.4. Discussion 30
• Part II. Mesenchymal Stem Cells and Small Intestinal Submucosa Hydrogel Composite Enhance Angiogenesis and Combined Radiation-Wound Healing in Mice 33
• 2.1. Introduction 34
• 2.2. Materials and Methods 36
• 2.2.1. Preparation of SIS hydrogel 36
• 2.2.2. Culture of human umbilical cord blood MSCs (hUCB-MSCs) 36
• 2.2.3. Animal 37
• 2.2.4. Radiation combined wound model 37
• 2.2.5. Wound analysis. 38
• 2.2.6. Histological examination. 38
• 2.2.7. Western Blotting Analysis. 39
• 2.2.8. Statistical analysis. 39
• 2.3. Results 40
• 2.3.1. Full thickness skin wound healing is delayed by radiation 40
• 2.3.2. hUCB-MSC and SIS composite enhances combined radiation-wound healing in mice. 43
• 2.3.3. hUCB-MSC and SIS composite enhances skin structure regeneration in combined radiation-wound of mice. 45
• 2.3.4. hUCB-MSC and SIS composite enhances angiogenesis at the wound site 47
• 2.4. Discussion 51
• Conclusion 53
• References 55
• Abstract (in Korean) 69