B. METHODS
Ⅳ. DISCUSSION
Ⅳ
Ⅳ Ⅳ DISCUSSION
The present study demonstrated that hMSCs has a protective effect on dopaminergic neurons through anti-inflammatory actions. First, we demonstrated that in microglia and mesencephalic neuron co-cultures, hMSCs treatment prevented dopaminergic neuronal death by reducing LPS-induced release of the pro-inflammatory cytokines. Second, we confirmed that in rats, hMSCs injection significantly reduced LPS- induced dopaminergic neuronal loss in the SN.
Besides regenerative capacity of hMSCs, it has been known that hMSCs also possess immunoregulatory properties. Although the exact mechanism responsible for hMSC-mediated immunoregulation is not fully understood, in vitro studies suggested that hMSCs can not only inhibit nearly all cells participating in the immune response cell-cell contact-dependant mechanism, but also release a variety of soluble factors, which may be implicated in the immunosuppressive activity of hMSCs (Karussis et al. 2007; Krampera et al. 2006; Nauta and Fibbe 2007). Recent animal studies in a model of experimental autoimmune encephalomyelitis reported that hMSCs treatment showed a significantly milder disease and fewer relapses compared with control mice, with decreased number of inflammatory infiltrates, reduced demyelination, and axonal loss (Gerdoni et al. 2007; Zappia et al. 2005). Additionally, Guo et al.
(Gerdoni et al. 2007) reported that MSC transplantation decreased protein production and gene expression of inflammation cytokines as well as increased functional recovery from myocardial infarct. These studies suggest that anti-inflammation action of hMSCs might be one of underlying mechanisms for the tissue protective effect.
In the present study, hMSCs significantly decreased the release of inflammatory cytokine and
dopaminergic neuronal loss induced by LPS stimulation. In co-cultures of microglia and mesencephalic neurons, these anti-inflammatory actions of hMSCs actually led to a significant decrease (up to ~40%) in dopaminergic neuronal death induced by LPS stimulation.
Furthermore, hMSCs administration dramatically decreased the dopaminergic neuronal loss in the SN induced by LPS stimulation, which was clearly accompanied by attenuation of microglial activation and a reduction in the formation of TNF-α and iNOS. In addition to a variety of pleiotrophic mechanisms of hMSCs as a trophic mediator (Caplan and Dennis 2006), our present data suggest that the neuroprotective property of hMSCs through its anti-inflammatory behavior also works in animal model of PD.
A large body of experimental evidence indicates that inhibition of the inflammatory response can prevent degeneration of nigrostriatal dopaminergic neurons. For example, sodium salicylate, COX-2 inhibitor, or minocycline have been shown to significantly reduce striatal dopaminergic depletion and dopaminergic neuronal loss induced by MPTP or LPS-models (Aubin et al. 1998;
Du et al. 2001; He et al. 2001). A large cohort study of patients has shown that the risk of developing PD in regular nonsteroidal anti-inflammatory drugs (NSAID) users was decreased by up to 45% compared with those who take NSAIDs on a non-regular basis and higher exposure to NSAID demonstrates a trend toward a greater benefit (Chen et al. 2003), supporting the neuroprotective effects of NSAID in the development or progression of PD. A very recent epidemiological study also supports that NSAIDs are protective against PD, with a particularly strong protective effect evident among regular nonaspirin NSAID users (Wahner et al. 2007).
Therefore, the evidence demonstrating the neuroprotective effect of anti-inflammatory agents on the nigrostriatal dopamine system, in an experimental system or epidemiological study, has
revitalized interest in identifying inhibition of inflammation as a possible strategy in the treatment of PD.
Recent studies indicate that human hMSCs can be induced to differentiate into neuron-like cells (Mareschi et al. 2006; Pittenger et al. 1999; Woodbury et al. 2000). Additionally, hMSCs express an expression of several specific neuronal markers and transcriptional factors, of which a large proportion of the genes was participating in the neuro-dopaminergic system, suggesting that expression of neural gene as well as gene associated with the dopaminergic system is a widespread phenomenon of hMSCs (Blondheim et al. 2006). There have been a few reports about application of hMSCs in animal model of PD. Li et al. (Li et al. 2001) and Blondheim et al. (Blondheim et al. 2006) reported that using MPTP and 6-hydroxydopamine-treated PD models, respectively, hMSCs injected intrastriatally exhibited the phenotype of dopaminergic neurons. Along with possible transdifferention potency of hMSCs into dopaminergic phenotype, neuroprotective property of hMSCs on dopaminergic neurons through anti-inflammatory actions may raise the possibility of clinical application of hMSCs as a possible strategy in the treatment of PD. In addition to the molecular and cellular benefits of hMSCs, cell therapy with hMSCs has an advantage in clinical applications. hMSCs can be easily harvested from self bone marrow, cultured in vitro, and administered to patients via various roots including intravenous, intraarterial, intrathecal, or intralesional infusion. In contrast with embryonic stem cell therapy, there is no immunological rejection, and cell therapy with hMSCs is free from ethical issues.
Importantly, regarding the safety of hMSCs application, our group recently documented that cell therapy with hMSCs in patients with multiple system atrophy and ischemic stroke is feasible and safe (Bang et al. 2005; Lee et al. 2007).
In summary, the present study demonstrated that hMSCs have a neuroprotective effect on dopaminergic neurons via an anti-inflammatory mechanism mediated by the modulation of microglial activation. Along with various trophic effect and transdifferentiational potency, anti-inflammatory mechanism of hMSCs could have major therapeutic implication in the treatment of PD.
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성체 성체
성체 성체 중간엽 중간엽 중간엽 중간엽 줄기세포의 줄기세포의 줄기세포의 항염증 줄기세포의 항염증 항염증 항염증 작용을 작용을 작용을 작용을 통한 통한 통한 통한 도파민
도파민
도파민 도파민 신경세포의 신경세포의 신경세포의 신경세포의 보호 보호 보호 보호 효과 효과 효과 효과
김 유 정
아주대학교 대학원 의학과 (지도교수: 이필휴)
파킨슨 씨 병은 흑색질 안에 도파민 신경세포의 감소가 원인이 되는 퇴행성 뇌질환이다. 이미 여러 연구에서 신경염증이 파킨슨 씨 병의 발병에 중요한 역할을 함이 증명되었다. 현 우리 실험에서는 성체 중간엽 세포가 신경염증을 완화하는 효과를 확인하였다. 우리는 lipopolysaccharide (LPS)로 실헌 동물 모델과 세포 모델을 만들어 염증을 유도한 후 성체 중간엽 줄기세포가 항염증 메커니즘을 통하여 도파민 신경세포를 보호하는 효과를 갖는지 확인하였다. 성체 중간엽 줄기세포를 처리하였을 때 LPS만 처리한 개체군과 비교하여 LPS에 의해 유도된 전구염증 사이토카인의 증가와 미세아교세포의 활성이 유의미하게 감소함을 알 수 있었다.
또한 미세아교세포와 중뇌 도파민 신경세포의 co-culture system에서 성체 중간엽 줄기세포를 처리한 경우 LPS로 인한 도파민 신경세포의 감소가 현저히 줄어듦을
확인하였다. 그리고 동물 실험을 통하여 성체 중간엽 줄기세포를 주입하였을 때 성체 중간엽 줄기세포를 주입한지 7, 14일이 지난 후 TH-immunopositive (TH-ip)를 표현하는 세포가 증가함을 확인하였으며 이는 LPS 개체군에 비하여 그 양이 대략 두 배 이상이었고 흑색질에서 미세아교세포의 활성의 감소도 수반하였다. 그리고 흑색질 안의 사이토카인 정량 실험 결과, 성체 중간엽 줄기세포를 주입한 개체군은
LPS에 의해 증가된 TNF-α와 iNOS mRNA의 발현을 조절억제 되었고, 성체 중간엽
줄기세포가 주입된지 4시간과 3일 후의 개체군에서 TNF-α와 iNOS의 발현 역시 LPS만 투여된 개체군에 비교하여 유의미하게 감소됨을 관찰하였다.
본 연구는 성체 중간엽 줄기세포가 항염증 메커니즘을 통하여 미세아교세포의 활성을 조절함으로 도파민 신경세포를 보호하는 것을 확인하였고, 이는 다양한 trophic effect와 transdifferentiation 가능성과 함께 성체 중간엽 줄기세포의 항염증 메커니즘이 파킨슨 씨 병의 치료에 주요하게 사용될 수 있음을 시사한다.
핵심어
핵심어
핵심어핵심어: 성체 중간엽 줄기세포, 항염증, 도파민 신경세포, 미세아교세포, 파킨슨씨병