[논문]조절 T 세포 유래 TGF-β1에 의한 췌장섬세포의 기능 및 활성 증가

최봉금; 김사현

doi:10.15324/kjcls.2018.50.3.304

[국내논문] 조절 T 세포 유래 TGF-β1에 의한 췌장섬세포의 기능 및 활성 증가
Regulatory T Cells Promote Pancreatic Islet Function and Viability via TGF-β1 in vitro and in vivo 원문보기

Korean journal of clinical laboratory science : KJCLS = 대한임상검사과학회지, v.50 no.3, 2018년, pp.304 - 312

초록
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본 연구에서는 면역 억제 역할을 하는 것으로 알려져 있는 조절 T 세포 (regulatory T cell, Treg)의 새로운 생리학적 기능 대하여 확인해보고자 하였다. 시험관내나 동물실험에서 조절 T 세포가 분비하는 transforming growth factor ${\beta}1$ ($TGF-{\beta}1$)에 의하여 이식 직전까지 췌장섬세포의 생존률을 향상시키면서 동시에 혈당조절 기능이 향상될 수 있을 것이라는 가설이다. 이를 증명하기 위하여 마우스를 이용한 1형 당뇨병 모델을 제작한 뒤, 180 IEQ (islet equivalents)의 췌장섬세포를 동종간 이식하였다. 췌장섬세포는 이식 수술 시행 전까지 48시간 동안 $4{\times}10^6$의 Treg 세포와 함께 배양하여 Treg 유래 $TGF-{\beta}1$에 충분히 노출시킨 뒤 사용하였다. Treg 단독군, 췌장섬세포 단독군 및 Treg/islet 동시 배양군에서 각각 $TGF-{\beta}1$, IL-6 및 인슐린 분비 수준의 변화를 측정하였다. Treg/islet 동시 배양군에서 IL-6와 인슐린 분비는 증가하였고 (P<0.0005, P<0.005), 췌장섬세포 단독군과 비교하여 생존율이 향상되었다(P<0.005). 또한, 이식 후, 동시 배양된 췌장섬세포는 1형 당뇨병 마우스 모델에서 혈당 수치를 보다 효율적으로 조절하였다. 이러한 결과는 Treg 세포가 $TGF-{\beta}1$ 분비를 통하여 췌장섬세포의 기능과 생존력을 향상시킬 수 있음을 시사한다.

Abstract ▼ AI-Helper

Regulatory T cells (Treg), known as immune-suppressors, may help modulate the immune response. In this study, we investigated the effect of Treg-derived $TGF-{\beta}1$ on pancreatic islet cell function in vitro and in vivo. One hundred eighty IEQ (islet equivalents) of pancreatic islets, the marginal amount to regulate blood glucose level after syngeneic islet transplantation in mouse type 1 diabetes (T1D) model, were co-cultured with $4{\times}10^6$ Treg cells for 48 hours. The changes in $TGF-{\beta}1$, interleukin-6 (IL-6), and insulin secretion levels were measured and analyzed among the Treg-only group, the islet-only group, and the Treg/islet co-cultured group. In the Treg/islet co-cultured group, IL-6 and insulin secretion levels were increased (P<0.0005, P<0.005) and islet viability was improved (P<0.005) compared with the islet-only group. Furthermore, after transplantation, the co-cultured islets regulated blood glucose levels efficiently in the T1D mouse model. These data suggest that Treg could improve islet functions and viability via the $TGF-{\beta}1$ secretion pathway (P<0.05~0.005), thus the use of Treg in islet transplantation should be explored further.

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문제 정의

In this study, it was suggested that nTreg might be helpful to improve the function and viability of pancreatic islet cells in vitro and in vivo study. First, we investigated whether islet cells could increase the secreting insulin level when they were exposed to nTreg-derived TGF-β1 in our Treg/islet co-culture system.

제안 방법

After collecting the supernatants, the soluble cytokines [transforming growth factor-β1 (TGF-β1), interleukin-6 (IL-6), insulin], and cell death index (fragmented and solubilized nucleosomal DNA) levels were determined using an enzyme-linked immunosorbent assay (ELISA) kits for TGF-β1, IL-6, and insulin (R&D Systems, Minneapolis, MN, USA), and for cell death (Roche Applied Science, Indianapolis, IN, USA).
65 mmol/L) and was produced by a single intraperitoneal (IP) injection of streptozotocin (STZ) (200 mg/kg) (Sigma) 2 days before transplantation. And recipient mice were classified into two groups, namely, islet transplantation (IT) without any intervention (control, N=6), and Treg co-cultured islet transplantation (TCIT, N=6). 180 IEQ of islets were implanted beneath the renal capsule of the left kidney in a syngeneic recipient C57BL/6.
To investigate the glucose regulation ability in vivo, IPGTT was performed on graft recipients from all two treatment groups at 21 days after transplantation. Briefly, after overnight fasting, unanesthetized mice were injected with 20% glucose solution (1 mg/kg body weight) by IP.
To investigate the functional responses of transplanted islets in vivo, we performed IPGTT test at 21 days after transplantation (Figure 4B). Fasted overnight mice were injected with 20% glucose solution (1 mg/kg body weight) intraperitoneally.
In order to evaluate the effect of Treg-derived TGF-β1 on islet cell function in vivo transplantation, 180 IEQ of syngeneic islet cells (IT group) or 180 IEQ islet cells which were co-cultured with Treg (TCIT group; using the trans-well culture system, islet and Treg were not contacted directly) were transplanted under the kidney capsule of T1D mice.

대상 데이터

Inbred C57BL/6 mice, aged 6 to 10 weeks, were purchased from Charles River Laboratories, Inc. (Wilmington, MA, USA) and fed standard rodent chow (PMI Nutrition International, St. Louis, MO, USA) in sterile cages in a barrier animal facility under a 12 h light/dark cycle. Selected C57BL/6 mice (6-8 week-old) were sacrificed, and their spleens were harvested to isolate Treg cell population.
To identify the function and survival of cultured islets in vivo, our study was conducted at Semyung University animal research facility following an Institutional Animal Care and Use Committee (IACUC)-approved protocol. Male inbred syngeneic C57BL/6 mice, aged 8 to 10 weeks, were used as donors and transplantation recipients. Diabetes (as defined as a blood glucose ＞16.

성능/효과

In conclusion, we demonstrate that Treg-produced TGF-β1 up-regulate IL-6 and insulin secretion levels from islet, while concomitantly inhibit islet cell death in vitro.

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