Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis | Protocol (Translated to Chinese)
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人多发性骨髓瘤（MM）细胞需要间质细胞和细胞外基质成分为存活和增殖的支持性微环境。我们建立了与植入人类骨髓瘤和间质细胞体内鸡胚模型来研究癌症药物对肿瘤的生长，侵袭和血管生成作用。
Cite this Article
Martowicz, A., Kern, J., Gunsilius, E., Untergasser, G. Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis. J. Vis. Exp. (99), e52665, doi:10. (2015).
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多发性骨髓瘤（MM），恶性浆细胞疾病，无法治愈的和新的药物都需要改善患者的预后。由于缺乏骨微环境和自动/旁分泌生长因子的人的MM细胞不易培养。因此，目前迫切需要建立适当的体外和体内培养系统来研究对人MM细胞新的治疗的作用。这里，我们提出一个模型，以在体外和体内生长的人类多发性骨髓瘤细胞在一个复杂的三维环境。 MM细胞系OPM-2和RPMI-8226被转染以表达该转基因GFP和培养在人类间质细胞和胶原的存在I型基质为三维球状体。此外，球状体接枝在鸡胚绒毛尿囊膜（CAM）和肿瘤生长通过立体声荧光显微镜监测。这两种型号允许新的治疗DRU研究GS在一个复杂的三维环境和肿瘤细胞块移植物在转基因特异性GFP-ELISA均化后进行量化处理。此外，主机和肿瘤细胞的侵袭进入下卧宿主组织的血管生成反应，可以每日通过立体显微镜监测和针对人肿瘤细胞（Ki-67的，CD138，波形蛋白）或宿主壁细胞覆盖血管免疫染色分析（结蛋白/ ASMA）。 总之，onplant系统允许研究在一个复杂的三维环境MM细胞生长和血管生成，使筛选靶向生存MM细胞的增殖和新的治疗的化合物。
根据奥地利法律，美国公共卫生服务禽流胚胎实验室动物福利办公室不被视为活的脊椎动物，直到实验动物福利hatching.The NIH办公室在这方面（HTTP提供了书面指导：// www.grants.nih.gov/grants/olaw/references/ilar91.htm和NIH出版号：06-4515）。
1.细胞培养和慢病毒转染培养的MM细胞系OPM-2，RPMI-8226，并从骨髓在RPMI1640培养基中的人类间质干细胞，补充有在存在10％牛胎儿小牛血清和100IU / ml青霉素，100微克/毫升链霉素和2mM谷氨酰胺的5％的CO 2在37℃。 转染5×10 6的HEK 293FT细胞病毒包装结构（9微克）的DNA和3微克pLenti6 / V5 dest中eGFP的向量通过使用30微升脂质体转染试剂转染培养基（10毫升）中。 12小时后取出转染中，加入10 mL的DMEM培养基含10％小牛血清和1％非必需氨基酸（NEAA）。
5天后，游泳细胞（1000 XG，5分钟），离心后收集HEK293FT细胞的上清液。通过实时PCR测定病毒滴度为28别处描述。 转染1×10 6个MM细胞与eGFP的慢病毒颗粒（1×10 5个颗粒）在24孔板中的完全生长培养基。 3天后，加入2微克/毫升瘟到培养基开始选择过程。对于市售的eGFP慢病毒，使用500微克/ ml新霉素的。
2周选择后，eGFP的簇表达MM细胞会出现;通过离心（1,000×g离心，5分钟）收集细胞，并扩大其作为OPM-2 的eGFP和RPMI-8226 eGFP的亚系进行实验（第2和3）。
2. 3D-多发性骨髓瘤球体模型寒意胶原I型解决方案和10×DMEM??冰。 混合1/10体积10×DMEM培养基进入胶原基质;添加NaOH水溶液（0.2 N），以中和酸性的胶原溶液以7.4的pH值;店胶原/冰介质溶液。 转基因混合MM细胞系（OPM-2或的eGFP RPMI-8226 绿色荧光蛋白 ; 250,000球体，）与人类间质细胞（50,000个细胞/球体， 即 30微升滴）。 在15ml试管（1000×g离心，5分钟）离心细胞混合物，冷制备胶原混合物（1mL）溶液加入到细胞沉淀拌匀（1,000微升尖）。 立即吸取30微升胶原/细胞混合物（用100μl尖端）在一个24孔板上无菌石蜡薄膜，并允许细胞/胶原混合物在37℃（ 见图1A）聚合30分钟。 覆盖的MM球体用含有1,10和100nM的硼替佐米1mL中培养基（参见图1B）。 之后，在37℃72小时温育的，文件球状体由荧光立体显微镜（ 见图1C）。 通过使用镊子具有宽扁钳口在反应管转移的每个球体的GFP的测量（ 见图1D）。
3. 3D多发性骨髓瘤异种移植模型中的CAM 孵化鸡蛋中为鸟卵的特别培养箱中在37℃和70％湿度下三天。 此后，打开蛋和转移胚胎灭菌用乙醇，方形，10厘米的塑料称量船与细胞培养板盖和孵化“ 前卵 ”用于进一步六天，使得CAM是能够开发（参见图2A）。 寒意胶原I型溶液和10×DMEM在冰上，混合1/10体积10×DMEM培养基到胶原基质，添加氢氧化钠（0.2 N），以中和酸性的胶原溶液以7.4的pH值;店胶原/冰介质溶液。 混合转基因MM细胞系（OPM-2 的eGFP或RPMI-8226 绿色荧光蛋白 ; 250,000球体，）与人类间质细胞（50,000个细胞/球体）。 离心细胞在15ml试管（1000×g离心，5分钟;对于每个试验化合物1小瓶），药物添加1mL中冷制备胶原混合物（在所需的工作浓度）的细胞沉淀并充分混合（1,000微升尖）。 放置胶原滴在6孔颈部30分钟上的封口膜（各30微升），以允许细胞外基质的聚合在37℃。 转移“onplants”来自步骤3.6与使用镊子到CAM的未处理表面（2厘米的距离胚胎）的9日龄鸡胚（4- onplants每个鸡胚，参见图2B） 经过5天的体内生长在蛋孵化器在37℃和70％的湿度，文件异种移植物通过荧光立体显微镜（ 见图2C）。受低温的冰箱5小时安乐死鸡胚胎在4℃。 由眼科剪刀取出移植与下层组织CAM和镊子与宽阔平坦的下颌。它们用于GFP的测量（第4节， 见图2D）或血管和/或侵入的肿瘤细胞（第5）免疫组化分析。 通过ELISA 4.绿色荧光蛋白定量的蛋白质传送每个MM球体或切除异种移植入0.5毫升RIPA缓冲液含有200微克/毫升蛋白酶抑制剂。 均质球体/异种移植物在冰上组织匀浆器。 执行三个冷冻/解冻周期在液氮和37℃水浴中。 离心机匀浆在4℃下20分钟（12,000克）和存储上清液。 稀释样品中的ELISA试剂盒的测定缓冲液（200微升）1:20。测量GFP的水平通过使用生物素化抗GFP抗体商业GFP ELISA试剂盒，根据制造商的协议。
5. Immunohi对血管和入侵肿瘤细胞stochemical分析切除修复与移植CAM面积在4％多聚甲醛O / N在4℃。 放置固定异种移植到嵌入盒，并将它们与增加的梯度醇系（50％，70％，80％，95％的乙醇，二甲苯和石蜡;每个步骤60分钟）转移至组织包埋站。 部异种移植物（5微米）通过使用台式旋转切片机。烘烤在56℃下在载玻片O / N石蜡切片。
Deparaffinize部分由递减梯度酒精串联双蒸水（混合二甲苯，95％，80％，70％，50％的乙醇，双蒸水;每个步骤10分钟）。 在水浴（95℃，20分钟）与抗原修复液进行抗原修复（柠檬酸盐缓冲液; pH值7.0;体积100微升）。 阻断内源性过氧化物酶活性用100μl3％的H 2 O 2 /甲醇30分钟。 在含PBS块段10％胎牛血清45分钟（体积100微升）。 染色1小时，用100μl初级抗体（1微克/毫升）稀释在含有1％胎牛血清在RT PBS中。 洗涤3次在PBS中后，孵育1与生物素化第二抗体（0.1微克/毫升）在含有1％胎牛血清在RT PBS中。 洗涤3次在PBS中后由抗生物素蛋白/生物素复合物（ABC）和二氨基联苯胺（DAB）底物溶液，根据制造商的说明进行显色反应。
5.11。停止反应通过转移部分，以双蒸水，染液用苏木精和安装部分用合成封固剂。 Subscription Required. Please recommend JoVE to your librarian.
Representative Results
在目标化合物的3D多发性骨髓瘤球体测定体外分析 由于在体外原代人MM细胞培养的限制，我们建立了新的三维体外培养模型为人类MM细胞系，利用细胞外生长基质和支持原代人骨髓间充质细胞从骨髓的（ 图1A，B）。 EGFP转基因MM细胞系允许可视化和MM肿瘤块的量化的三维生长球状体之后。两个MM细胞系OPM-2 的eGFP和RPMI-8226 的eGFP中培养3天，在增加浓度的硼替佐米和肿瘤（1- 100纳米）的存在下通过GFP的上立体荧光显微镜的表达进行观察（ 图1C） 。肿瘤细胞团由GFP-ELISA（ 图1球体的同质化和测量绿色荧光蛋白含量量化后D）。
在目标化合物的鸡胚中多发性骨髓瘤异种移植物的体内分析 三日龄鸡胚胎中培养前卵 6天，并在用于MM细胞（ 图2A）的接枝天9。 EGFP转基因骨髓瘤细胞（OPM-2 EGFP）与人骨髓间充质细胞嵌入到胶原蛋白I型细胞外基质成分。目标物质的硼替佐米的混合物在1纳摩尔（ 图2B）局部施用。每个动物4“onplants”接枝于鸡胚的绒毛膜尿囊膜（CAM）。经过5天的MM异种移植物形成，可以通过EGFP的表达被可视化的肿瘤。与对照组相比，MM细胞的异种移植物中的硼替佐米抑制生长。移植物显示较少绿色的MM肿瘤细胞块（ 图2C）。从单次移植的MM稀土元素不同的动物（N = 12）切下，均质化之后，由绿色荧光蛋白ELISA测量。在直接比较对照硼替佐米处理的异种移植物有一个显著降低骨髓瘤细胞块（ 图2D）。
在血管生成反应和骨髓瘤侵异种移植在鸡胚体内分析 周围onplants的血管生成反应可通过立体显微镜进行观察。异种移植物的血管形成在药物处理的异种移植物（1纳摩尔Plitidepsin， 图3）中的显著降低。血管生成应答通过如由Ribatti 等人 21所述的用于明胶海绵测定计数血管出芽成onplant量化。 对于入侵分析，异种移植物被切除与相邻的CAM区。异种移植物固定，石蜡包埋和切片制备（ 图4 </STRONG&）。切片用针对ASMA /结蛋白，以检测覆盖鸡血管壁细胞的抗体，并与针对人Ki-67的，波形蛋白和CD138来检测增殖和侵入的人类肿瘤细胞鸡宿主组织中（ 图4）的抗体。
图1. 3D多发性骨髓瘤细胞球体。 OPM-2 的eGFP和RPMI-8226 eGFP的球状体与原代人骨髓间充质细胞和胶原I型作为细胞外基质组分（B）的球体（A）的代培养用培养基和硼替佐米的各浓度（1- 100纳米）。（C）的 MM球状体生长3天并通过立体荧光显微镜拍照。横道500微米。（D）单SPHeroids在裂解缓冲液之后，测定GFP的ELISA分析。单个球体的绿色荧光蛋白的浓度，计算（5例，平均值±SEM）。星号表示P值&0.05;公司=控制; BZB =硼替佐米。
图2.多发性骨髓瘤异种移植模型。 （一）在发育9天前OVO鸡胚胎用于移植实验。（B）OPM-2 EGFP和RPMI-8226 EGFP的混合与原代人骨髓间充质细胞，胶原蛋白I型是细胞外基质成分， 1纳米硼替佐米。后凝固球状体（n = 4时的）被移植对鸡胚的绒毛膜尿囊膜第（℃）5天后的MM移植在CAM可以通过EGFP的表达被可视化。异种移植可以每天通过立体荧光显微镜拍照。横道500微米。（D）的单MM异种移植物被切除与下层的CAM组织，在裂解缓冲液，并在绿色荧光蛋白ELISA测量。单个肿瘤的GFP浓度计算（12例，平均值±SEM）。星号表示P值&0.05; BZB =硼替佐米。
记录在案移植对鸡胚的CAM五天后血管瘤图3.分析。MM异种移植物（OPM-2 EGFP）。相比于对照移植物，treatm耳鼻喉科与plitidepsin（1nMol，N = 10）导致表面上生长的肿瘤是由CAM组织血管少。血管生长到onplants（红色）被计为在血管类型的图形模型描绘。横道1毫米。
骨髓瘤细胞浸润图4.分析。MM异种移植物（OPM-2 EGFP）与下层CAM宿主组织（硼替佐米治疗与控制）的免疫组化分析。增殖人MM细胞染色阳性，Ki-67的，CD138和波形和入侵的细胞群宿主组织。鸡血管沾满壁画细胞标记阿斯玛（大血管和动脉）和结（毛细血管）。 Magnificati200X上，星号表示凸轮的血管。
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Discussion
的新的治疗剂用于难治性MM的发展需要较少耗时且昂贵的体内系统评估人类MM细胞的对药物的敏感性。迄今为止，只有少数的体内系统可用于新的抗骨髓瘤治疗的临床前评价。他们都有自己的化合物库29的大规模筛选的限制。 目前最好的模型为人类MM细胞是高度免疫缺陷小鼠7,13,30和火鸡胚胎29。既SCID小鼠和禽流胚胎异种移植模型可用于研究的MM的生物学和测试新的治疗的化合物。但是，鼠系统有一些局限性，包括自交系基因型，技术要求高的程序，长周期的观察，成本高。 在这项研究中，一种新的三维球体和禽流异种移植模型，提出研究人类MM细胞生物学，涉及人类MES的存在enchymal干细胞和细胞外基质作为支持部件。 MM细胞强烈地依赖于它们各自的微环境，即间质来源的生长因子，细胞因子和ECM成分的存在下存活和增殖31-33。此外，药物可能是低效或显示改变的活性时的骨髓瘤细胞是由它们的局部微环境31,34的保护。 相较于鼠模型中，所描述的三维体外和体内模型系统并不昂贵，快速，容易处理。此外，3D的MM移植球状体到鸡胚允许骨髓瘤诱导的血管生成的分析。我们的系统的一个限制是MM细胞生长的时间短，因此，转移的分析禽流骨头是不可能的。我们的模型的另一个局限性是我们与药物可能没有反映全身应用和药品营业额/修改肝酶CAM局部应用工作。在此外，约50％的存活率，直到接枝观察由于前卵鸡胚胎的生长条件。关于内皮标志为IHC分析，在人类和小鼠部用于染色的内皮细胞标记物最不特定于血管鸡。因此，我们建议染色壁画细胞标志物结蛋白/ ASMA或凝集素注射到鸡胚35。并不是所有可用人骨髓瘤细胞系将增长到侵入鸡宿主组织并显示肤浅的增长。这将导致损坏的组织切割切片在切片机之后。此外，特别照顾（选择抗生素），应采取转染的细胞没有时间内失去了绿色荧光蛋白转基因表达，由于永久遗传重组或DNA甲基化过程。 总之，人类的MM细胞我们的鸡胚胎移植瘤模型与基质的支持提供了一个可重复和可预测的体内模型STU镝MM细胞生长和血管生成。这说明MM模型可以促进体内的抗MM药物筛选过程更快，有助于缩短开发时间和新药的成本。随着进一步的改进步骤，例如骨替代材料，复杂的ECM基质和细胞因子系统可能用于测试治疗剂还对患者样品得到改善。这是个性化癌症医学和个体难治MM患者药敏试验的先决条件。 Subscription Required. Please recommend JoVE to your librarian.
Disclosures
作者有没有竞争经济利益
Catalog Number
RPMI-8226 cells
STR profiled
OPM-2 cells
STR profiled
Human mesenchymal stem cells&
PC-C-12974
HEK293FT cells&
Invitrogen
RPMI1640 Medium
Sigma Aldrich
Fetal Bovine Serum& HyClone
ThermoScientific
SH30070.03
L-Glut- Pen- Strep solution
DMEM Medium
Sigma Life Sciences
Transfection Medium/Opti-MEM&
eGFP lentiviral particles
GeneCopoeia
LPP-EGFP-LV105
Ready to use viral particles
pLenti6/V5Dest6 eGFP vector
Invitrogen
PN 35-1271
from authors
ViralpowerTM packaging mix&
Invitrogen
P/N 35-1275
Transfection reagent/ Lipofectamin 2000
Invitrogen
Blasticidin
Invitrogen
Collagen-Type1& Rat Tail
BD Biosciences
DMEM powder
Life Technologies
plitidepsin
bortezomib
LKT Lab., Inc.
SPF-white hen eggs
Charles River
Fertilized& white Leghorn& chicken eggs
Plastic weighing boats
Art.Nr. 1-1125
for ex-ovo culture
Petridish square (Lids)
for ex-ovo culture
RIPA Buffer (10x)
Cell Signaling
Protease Inhibitor Tablets
11 836 170 001
Complete Mini EDTA-free
Cell Biolabs, Inc.
Histocette II
Ethanol absolut
20,821,321
Roti-Histol
Art.Nr.6640.4
SuperFrost Microscope Slides
R. Langenbrinck&
Labor- u. Medizintechnik
DakoCytomation Wash Buffer 10x
DakoCytomation
Target Retrieval Solution (10x)& pH 6,1
m-a-hu ASMA clone 1A4
m-a-hu CD138 clone MI15
m-a-hu Vimentin clone V9
m-a-hu Desmin clone D33
m-a-hu Ki67& clone MIB-1&&
biotinylated goat- anti-mouse IgG
Vector Laboratories Inc.
Vectastain Elite ABC Kit
Vector Laboratories Inc.
FAST DAB Tablet Set.
Sigma Biochemicals
Mayer&s haemalaun solution
1,092,490,500
Roti Histokitt
Art.Nr.6638.2
Bench top rotary microtome
Thermo Electron, Shandon Finesse ME+
Tissue embedding station
Leica, TP1020
Egg-Incubator
Stereo fluorescence microscope equipped with an connected with a digital camera (Olympus E410) and flexible cold light&
Olympus, SZX10
Ultra Turrax&
Homogenizer
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Get cutting-edge science videos from JoVE sent straight to your inbox every month.PMCID: PMC2816838Effect of Antisense TGF-&#x003b2;1 Oligodeoxynucleotides in Streptozotocin-Induced Diabetic Rat Kidney, ,* ,* , ,&#x02020; and
&#x02021;*Department of Pathology, Keimyung University School of Medicine, Daegu, Korea.&#x02020;Department of Internal Medicine, Keimyung University School of Medicine, Daegu, Korea.&#x02021;Kidney Institute, Keimyung University School of Medicine, Daegu, Korea.Corresponding author.Address for correspondence: Kwan Kyu Park, M.D. Department of Pathology, Keimyung University School of Medicine, 194 Dongsan-dong, Jung-gu, Daegu 700-712, Korea. Tel: +82.53-250-7465, Fax: +82.53-250-7852, Email:
This article has been
other articles in PMC.Transforming growth factor (TGF)-&#x003b2;1 is an important fibrogenic factor that is involved in the pathogenesis of diabetic nephropathy. We evaluated the effect of circular antisense TGF-&#x003b2;1 oligodeoxynucleotides (ODNs) on the TGF-&#x003b2;1 expression in the rat mesangial cell culture and in streptozotocin (STZ)-induced diabetic rats. Circular antisense TGF-&#x003b2;1 ODNs were found to be stable in rat serum, significantly decreasing TGF-&#x003b2;1 mRNA expression compared with linear antisense ODNs in the rat mesangial cell culture. Circular antisense TGF-&#x003b2;1 ODNs were introduced into the tail vein of normal rats using hemagglutinating virus of Japan (HVJ)-liposome-mediated gene transfer method and were confirmed to be delivered effectively into the kidney, liver, lungs, and spleen. To inhibit the overexpression of TGF-&#x003b2;1 in diabetic kidneys, we introduced circular antisense TGF-&#x003b2;1 ODNs into the STZ-induced diabetic rats. On day 13 after circular antisense TGF-&#x003b2;1 ODNs injection, TGF-&#x003b2;1 mRNA and protein expression markedly decreased and urinary TGF-&#x003b2;1 excretion rate also dropped in the circular antisense TGF-&#x003b2;1 ODNs-treated diabetic rats. These results suggest that circular antisense TGF-&#x003b2;1 ODNs may be a useful tool for developing new therapeutic application for progressive diabetic nephropathy.Keywords: Transforming Growth Factors, Diabetes Mellitus, Oligonucleotides, StreptozotocinDiabetic nephropathy is the most common cause of end stage renal disease in developed countries and a major cause of morbidity and mortality in patients with diabetes. It is characterized by structural abnormalities including hypertrophy of both glomerular and tubular elements, increase in the thickness of glomerular basement membranes, and progressive accumulation of extracellular matrix components. It also results in functional alterations including the early increase in the glomerular filtration rate with intraglomerular hypertension, subsequent proteinuria, systemic hypertension, and eventual loss of renal function (-). The development of irreversible renal change in diabetes mellitus such as glomerulosclerosis and tubulointerstitial fibrosis results ultimately in renal failure.It has been established that transforming growth factor (TGF)-&#x003b2;1 is a critical cytokine involved in diabetes-induced extracellular matrix accumulation in both cell culture and animal models of diabetic kidney disease. Investigations of glomerular mesangial cells and proximal tubular cells have demonstrated that high glucose media induce the enhanced expression of TGF-&#x003b2;1 mRNA and protein and the biosynthesis of collagen and other extracellular matrix constituents (-). Several in vivo studies have also reported that TGF-&#x003b2;1 expression is elevated in diabetic kidneys (-). TGF-&#x003b2; is a multifunctional cytokine that plays an important role in healing wounds and repairing tissues. In mammals the cytokine has three isoforms i.e., TGF-&#x003b2;1, 2, and 3, whose biological properties are nearly identical. Among the three isoforms, TGF-&#x003b2;1 is known to contribute mostly to the pathogenesis of tissue fibrosis of organs such as kidney, liver, lungs, and heart and scarring of superficial tissues. TGF-&#x003b2;1 plays a central role in tissue repair by stimulating the balanced expression of extracellular matrix such as collagen, fibronectin, and matrix proteoglycans. However, persistent injury with sustained autoinduction of TGF-&#x003b2;1 overrides normal termination signals and leads to the continuous production of TGF-&#x003b2;1 and
thus resulting in tissue fibrosis (, ). Therefore, anti-TGF-&#x003b2;1 or anti-fibrotic therapies have been attempted by administering various reagents: anti-TGF-&#x003b2;1 antibody, interferon-&#x003b1;, anti-oxidants such as &#x003b1;-tocopherol, and decorin (-).In diabetic kidney models, anti-TGF-&#x003b2;1 antibodies significantly suppressed renal hypertrophy and expression of genes encoding extracellular matrix components (). Therapy with antisense TGF-&#x003b2;1 oligodeoxynucleotides (ODNs) decreased TGF-&#x003b2;1 production and reduced hyperglycemia-induced proximal tubular cell hypertrophy in vitro and partially prevented the increase in kidney weight and extracellular matrix expression (). Anti-TGF-&#x003b2;1 therapies on diabetic models to date suggested that blockade of the biological actions of TGF-&#x003b2;1 may be useful in preventing matrix accumulation at the early stage of diabetic nephropathy.In recent years, gene therapy has been tried actively with antisense ODNs to block the aberrant gene expression implicated in many diseases. Antisense ODNs can work through several potential mechanisms involving hybridization with mRNA and function of RNase H that has the ability to degrade the target message (). However, biological instability is the first problem to consider when delivering ODNs to cells. Unmodified phosphodiester backbone ODNs are rapidly degraded in the biological fluid by a combination of both endo- and exonucleases. To overcome this problem, a variety of chemically modified ODNs have been developed: phosphorothioates, methylphosphonates, and phosphoramidate analogues (-). Nonetheless, most of the modified ODNs exhibited problems as well. Phosphorothioate ODNs have non-sequence related toxicity, while methylphosphonate ODNs show resistance to RNase H (, ). Compared with natural DNA/RNA complexes, phosphoramidate oligonucleotides are weak activators of RNase H (). Given these problems, circular antisense ODNs only structurally modified for stability against nucleases have been developed recently (). These were in contrast with those of other chemically modified ODNs, designed only through structural modification.The aim of the present study was to investigate the effect of circular antisense TGF-&#x003b2;1 ODNs on the TGF-&#x003b2;1 expression in the rat mesangial cell culture and in streptozotocin (STZ)-induced diabetic rats.Synthesis of circular antisense TGF-&#x003b2;1 ODNsCircular antisense TGF-&#x003b2;1 ODNs were synthesized as described previously (, ). The selection of antisense ODNs for TGF-&#x003b2;1 was performed using the DNAsis program (Hitach Software, San Bruno, CA, U.S.A.). The sequences of ODN for rat TGF-&#x003b2;1 mRNA (755 to 800 of the rat TGF-&#x003b2;1 mRNA sequence, GenBank, accession X52498) are as follows:Antisense, 5'-TGTGTGTGATGTCTTTGGTTTTGTCATAGATTTCGTTGTTGCGGTC-3';Sense, 5'-GACCGCAACAACGCAATCTATGACAAAACCAAAGACATCACACACA-3'.To form a closed structure, both antisense ODNs and sense ODNs were modified. The 5' end of ODNs has 8 bases of sequence of 5'-GATCACGT-3', while the 3' end of ODNs has 4 bases of sequence of 5'-ACGT-3'. The modified 58-nucleotides (nt) ODNs were custom-synthesized by Bionics (Seoul, Korea). The linear 58-nt ODNs were joined by the complementary 4 base sequences (5'-GATC-3') at the 5' ends. Briefly, 60 &#x000b5;g of linear antisense TGF-&#x003b2;1 ODNs were incubated in a final volume of 100 &#x000b5;L containing 1&#x000d7; ligase buffer and 3,500 U T4 DNA ligase (Takara, Otsu, Japan) at 16 for 24 hr. The formation of circular ODNs (116-nt) was confirmed by electrophoresis on a 6% denaturing polyacrylamide gel containing 7 M urea.Stability of circular antisense TGF-&#x003b2;1 ODNsCircular antisense TGF-&#x003b2;1 ODNs and linear antisense ODNs were incubated at 37 for 24 hr with rat serum which was not inactivated by heat to preserve nuclease activity. The serum was added to ODNs in a 50% volume of reaction mixture. ODNs were then extracted with phenol/chloroform (1:1, v/v) and examined by electrophoresis on a 6% denaturing polyacrylamide gel containing 7 M urea.Cell cultureRat mesangial cells (RMCs) were obtained from a culture of glomeruli isolated from male Sprague-Dawley (SD) rats weighing 100 to 150 g using differential sieving methods as previously described (). Isolated glomeruli were cultured in RPMI 1640 (Gibco BRL, Gaithersburg, MD, U.S.A.) complete medium containing 100 U/mL penicillin, 100 &#x000b5;g/mL streptomycin, 0.25 &#x000b5;g/mL amphotericin B, 10 mg/L insulin, 6.7 &#x000b5;g/L sodium selenite, 5.5 mg/L transferrin, and 20% heat-inactivated fetal calf serum (FCS; HyClone, Logan, UT, U.S.A.) at 37 in a humidified 5% CO2 atmosphere. The identity of mesangial cells was confirmed by phase contrast microscopy according to the morphologic criteria (). RMCs between the 10th and 15th passage were used for cell studies.In vitro transfection of circular antisense TGF-&#x003b2;1 ODNs and antisense studyRMCs were seeded at 2&#x000d7;104 cells/well in 8-well chamber slides (Nunc, Inc., Naperville, IL, U.S.A.) and cultured in RPMI 1640 complete medium for 24 hr. RMCs were serum-starved for 48 hr in RPMI 1640 containing 0.5% FCS and transfected with fluorescein-labeled-circular antisense TGF-&#x003b2;1 ODNs. Circular antisense TGF-&#x003b2;1 ODNs were labeled using Label IT&#x000 Fluorescein Nucleic Acid Labeling Kit (Panvera Corp., Madison, WI, U.S.A.). Commercially available cationic liposomes (LipofectAMINE PLUS&#x02122; R Gibco BRL, Gaithersburg, MD, U.S.A.) were used to facilitate the transfection of ODNs into RMCs. Briefly, 0.5 &#x000b5;g of ODNs in DMEM (Gibco BRL, Gaithersburg, MD, U.S.A.) were mixed with the Plus reagent and incubated at room temperature for 15 min. The ODNs-Plus reagent were mixed with 2 &#x000b5;g of LipofectAMINE reagent and subsequently incubated at room temperature for 15 min. The cells were washed with serum-free DMEM and then 0.03 mL/well of serum-free DMEM was added to the cells. The ODNs-liposome complexes were added drop by drop to each well and incubated at 37 for 5 hr. Cells were then replaced with RPMI 1640 containing 17% FCS and incubated further at 37 for 24 hr. Finally, the RMCs were washed three times with phosphate-buffered saline (PBS) and fixed in methanol at 4 for 10 min. The cells were mounted and observed through a fluorescence microscope. The effect of antisense ODNs on TGF-&#x003b2;1 mRNA expression in RMCs was investigated. Cells were cultured at 2&#x000d7;105 cells/well in 6-well cell culture plates (Nunc, Inc., Naperville, IL, U.S.A.) and circular antisense (0.5 &#x000b5;g or 1 &#x000b5;g), linear antisense (0.5 &#x000b5;g or 1 &#x000b5;g), and circular sense (1 &#x000b5;g) TGF-&#x003b2;1 ODNs were introduced selectively into the cells as described above.Preparation of HVJ-liposomeThe hemagglutinating virus of Japan (HVJ)-liposomes were prepared as described previously () with minor modifications. HVJ was grown in chorioallantoic fluid of 10-day-old embryonated chicken eggs at 35.5. HVJ was collected by centrifugation at 3,000 rpm for 10 min and suspended with balanced salt solution (BSS; 140 mM NaCl, 5.4 mM KCl, 10 mM Tris-HCl, pH 7.5). HVJ was purified by another centrifugation at 12,000 rpm for 1 hr, resuspended with BSS, and stored at 4 until it was used. Egg yolk phosphatidyl choline (ePC; Sigma, U.S.A.), dioleoyl phosphatidyl ethanolamine (DOPE; Avanti Polar Lipid, Birmingham, AL, U.S.A.), egg yolk sphingomyelin (eS Sigma, U.S.A.), bovine brain phosphatidyl serine (bPS; Sigma, U.S.A.), and cholesterol (C Sigma, U.S.A.) were each dissolved in chloroform, mixed in a weight ratio of 1.6:3:1.5:1.3:1.5, and then dried with a rotary evaporator. The dried lipid mixture was hydrated in 200 &#x000b5;L of BSS containing 10 &#x000b5;g of the ODNs, mixed with vigorous agitation, and filtered to form liposomes. The liposomes were mixed with HVJ that had been inactivated by ultraviolet irradiation. The mixture was incubated at 4 for 10 min and then at 37 for 60 min with gentle agitation. Free HVJ was removed from the HVJ-liposome mixture by sucrose density gradient centrifugation. The HVJ-liposome suspensions were maintained at 4 until it was used.In vivo transfection of circular antisense TGF-&#x003b2;1 ODNsTo estimate the delivery efficiency of the circular antisense TGF-&#x003b2;1 ODNs by intravenous administration, fluorescein-labeled-circular antisense TGF-&#x003b2;1 ODNs were injected into the tail vein of normal 6-week-old male SD rats by the HVJ-liposome mediated gene transfer method. Kidney, liver, lungs, and spleen were removed 24 hr after injection. Four-&#x000b5;m thick cryostat sections of unfixed snap-frozen specimens were observed through a confocal laser scanning microscope (TCS/SPII, Leica, Wetzlar, Germany).STZ-induced diabetic ratsDiabetes was induced in 21 male SD rats (180-210 g) by a single intraperitoneal injection of 70 mg/kg STZ (Sigma, U.S.A.) dissolved in 10 mM sodium citrate buffer (pH 4). Blood glucose was measured using reagent strips (MediSense, Bedford, MA, U.S.A.) 48 hr after STZ administration. Animals with blood glucose &#x0003e;15 mM were included in this study. All of the animals had unlimited access to standard rat food and water. STZ-induced diabetic rats were separated into two groups. One group (ODNs-treated diabetic rats) was treated with circular antisense TGF-&#x003b2;1 ODNs and the other group (untreated diabetic rats) was not. Once hyperglycemia was detected, HVJ-liposome mediated circular antisense TGF-&#x003b2;1 ODNs were injected into the tail vein of the diabetic rat the following day. Each group was sacrificed on day 1, 5, and 13 after injection of ODNs. Normal rats were sacrificed on day 13. Rats were anesthetized with ethyl ether and the kidneys were removed, immediately frozen in liquid nitrogen, and stored at -70 for subsequent RNA extraction. Portions of tissues were fixed in neutral buffered formalin for immunohistochemical staining. At the end of each experimental periods, individually collected urine was centrifuged at 3,000 rpm at 4 for 10 min. The supernatant was collected and stored at -70 until it was used.RNA Isolation and RT-PCRTotal RNA was extracted from cultured RMCs and frozen kidneys with RNAzol B (TEL-TEST, Friendswood, TX, U.S.A.) according to the manufacturer's instructions. The purity and quantity of the RNA preparation were determined by measuring the optical densities at 260 and 280 nm. Total RNA was reverse transcribed with oligo-d(T)15 primers and M-MLV reverse transcriptase (Promega, Madison, WI, U.S.A.). Aliquots of cDNA were amplified by PCR using primer sets specific to rat TGF-&#x003b2;1 and a constitutively expressed housekeeping gene, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as the control. The primers used for PCR are as follows:Rat TGF-&#x003b2;1 upstream CCTGCTGCTTTCTCCCTCAACC, downstream CTGGCACTGCTTCCCGAATGTC;Rat GAPDH upstream GTGGACATTGTTGCCATCAACG, downstream GAGGGAGTTGTCATATTTCTCG.The reaction was run for 25 cycles with a cycling parameter of 30 sec at 95, 60 sec at 60, and 120 sec at 72. PCR products were visualized on a 2% agarose gel. The amounts of TGF-&#x003b2;1 mRNA were measured by densitometer and expressed relative to the densities of GAPDH.Immunohistochemical staining of TGF-&#x003b2;1Paraffin-embedded sections obtained from the kidneys were deparaffinized with xylene and graded ethanol solutions and incubated in 3% H2O2 in methanol at room temperature for 20 min to block endogenous peroxidase activity. They were then washed with PBS for 10 min, incubated at 37 for 30 min in 0.1% trypsin, and washed with PBS for 5 min three times. Incubation with the first antibody (Rabbit anti-porcine TGF-&#x003b2;1 Cell Science, Norwood, MA, U.S.A.) was performed at 37 for 1 hr. After three serial washes with PBS, the sections were processed by an indirect immunoperoxidase technique using a commercial kit (LSAB DAKO, Carpinteria, CA, U.S.A.), counterstained with Mayer's hematoxylin, and examined under light microscope.TGF-&#x003b2;1 ELISATotal TGF-&#x003b2;1 in rat urine was determined using a sandwich ELISA kit (TGF-&#x003b2;1 Emax ImmunoAssay S Promega, Madison, WI, U.S.A.), following the instructions provided by the manufacturer (). Acid activation of urine samples was required to convert latent TGF-&#x003b2;1 into active form and record detectable levels of total TGF-&#x003b2;1. Samples were activated with 1N HCl at room temperature for 30 min, followed by neutralization with 1N NaOH. Samples were plated on microtiter plates coated with anti-TGF-&#x003b2;1 monoclonal antibody and incubated at room temperature for 3 hr with shaking. After vigorous washing, wells were incubated overnight with polyclonal anti-TGF-&#x003b2;1 at 4 and washed. They were then incubated with antibody conjugated with horseradish peroxidase at room temperature for 3 hr with shaking. Following additional washes, color was developed by adding peroxidase substrate in 3,3',S,S'-tetramethyl benzidine solution. A standard curve was constructed using serial dilutions of human TGF-&#x003b2;1. TGF-&#x003b2;1 levels in the samples were determined from the standard curve.Statistical analysisThe data are presented as mean&#x000b1;standard error (SE). The statistical difference between means was determined using the Student's t- p&#x was considered significant.Synthesis of circular antisense TGF-&#x003b2;1 ODNsCircular antisense TGF-&#x003b2;1 ODNs (116-nt) synthesized by T4 DNA ligase from linear ODNs (58-nt) and analyzed by denaturing polyacrylamide gel electrophoresis had slower mobility compared with linear ODNs ().Photograph of stained 6% denaturing polyacrylamide gel showing relative mobilities of linear and circular antisense TGF-&#x003b2;1 oligodeoxynucleotides (ODNs). (A) Synthesis of circular antisense TGF-&#x003b2;1 ODNs from linear ODNs: lane 1, 58-nt linear ...Stability of circular antisense TGF-&#x003b2;1 ODNsThis test was performed to prove the stability of ODNs against nucleases in serum. Linear antisense TGF-&#x003b2;1 ODNs were completely digested after incubation for 24 hr in rat serum. However, circular antisense TGF-&#x003b2;1 ODNs remained considerably intact after incubation for 24 thus exhibiting markedly improved stability compared with linear antisense ODNs ().In vitro uptake and localization of circular antisense TGF-&#x003b2;1 ODNsTo examine the transfection efficiency and localization of circular antisense TGF-&#x003b2;1 ODNs, fluorescein-labeled-circular antisense TGF-&#x003b2;1 ODNs were transfected to RMCs. The cells were then evaluated by fluorescence microscopy. Most of the RMCs exhibited strong fluorescence in the cytoplasm and nucleus ().Fluorescence microscopy of rat mesangial cells transfected with circular antisense TGF-&#x003b2;1 oligodeoxynucleotides (ODNs) in cationic liposomes. Fluorescein-labeled-circular antisense TGF-&#x003b2;1 ODNs were observed in both the nuclei and the cytoplasm ...Comparison of the effects of circular antisense ODNs and linear antisense ODNs on TGF-&#x003b2;1 mRNA expression of RMCsTo examine the effect of antisense ODNs, circular antisense ODNs (0.5 &#x000b5;g or 1 &#x000b5;g), linear antisense ODNs (0.5 &#x000b5;g or 1 &#x000b5;g), and circular sense TGF-&#x003b2;1 ODNs (1 &#x000b5;g) were introduced selectively into the RMCs. Total RNA was isolated from the transfected cells and RT-PCR was performed. Compared with the transfection of circular sense TGF-&#x003b2;1 ODNs (1 &#x000b5;g) and liposome alone, the transfection of circular antisense TGF-&#x003b2;1 ODNs reduced TGF-&#x003b2;1 mRNA levels. Furthermore, the effect of circular antisense ODNs on the ablation of the TGF-&#x003b2;1 mRNA content was stronger than that of the linear antisense ODNs in the same amount of ODNs. These results indicated that circular antisense TGF-&#x003b2;1 ODNs were more effective than linear antisense ODNs in reducing TGF-&#x003b2;1 mRNA expression ().Effect of circular antisense oligodeoxynucleotides (ODNs) on TGF-&#x003b2;1 mRNA expression in rat mesangial cells (RMCs). RMCs were serum-starved for 48 hr and subsequently transfected with circular antisense ODNs (1 &#x000b5;g)+cationic liposome (4 ...In vivo uptake and localization of circular antisense TGF-&#x003b2;1 ODNsTo evaluate the efficiency of circular antisense TGF-&#x003b2;1 ODNs via intravenous administration, fluorescein-labeled-circular antisense TGF-&#x003b2;1 ODNs were introduced into the tail vein of normal SD rats using the HVJ-liposome transfer method. The kidney, liver, lungs, and spleen were removed 24 hr after the tail vein injection of ODNs and observed through a confocal laser scanning microscope. Most of the tubular epithelial cells exhibited strong fluorescence in the cytoplasms and nuclei (). In addition, results confirmed that ODNs were delivered mostly into liver, lungs, and spleen ().Confocal microscopy of rat organs transfected with fluorescein-labeled-circular antisense TGF-&#x003b2;1 ODNs in HVJ-liposome by intravenous systemic administration method (A: kidney, B: liver, C: lung, D: spleen, &#x000d7;200).Blood glucose in STZ-induced diabetic rats shows the parameters measured during the induction of diabetes. Both circular antisense TGF-&#x003b2;1 ODNs-treated diabetic rats and untreated diabetic rats had slightly higher blood glucose level on day 13 after ODNs injection compared to days 1 and 5. Treatment with circular antisense TGF-&#x003b2;1 ODNs had little effect on the blood glucose level of diabetic rats, however.Values for body weight and blood glucose in STZ-induced diabetic ratsEffects of circular antisense ODNs on TGF-&#x003b2;1 mRNA and protein expression in STZ-induced diabetic ratsThe effect of circular antisense ODNs on TGF-&#x003b2;1 mRNA and protein expression in diabetic kidneys was determined by RT-PCR and immunohistochemical study. Circular antisense TGF-&#x003b2;1 ODNs-treated and untreated diabetic rats were studied on days 1, 5, and 13 after the introduction of ODNs. On day 13 after ODNs injection, TGF-&#x003b2;1 mRNA levels were decreased significantly in circular antisense TGF-&#x003b2;1 ODNs-treated diabetic rats compared with untreated diabetic rats (0.82&#x000b1;0.05 vs. 1.32&#x000b1;0.01; p&#x) (). Furthermore, circular antisense TGF-&#x003b2;1 ODNs treatment markedly inhibited TGF-&#x003b2;1 protein expression in diabetic kidneys (). On days 1 and 5 after ODNs injection, TGF-&#x003b2;1 was strongly expressed in the glomerular endothelial cells and tubular epithelial cells of the untreated diabetic kidneys (), while circular antisense TGF-&#x003b2;1 ODNs-treated diabetic kidneys () showed mild TGF-&#x003b2;1 protein expression in the tubular epithelial cells. On day 13 after ODNs injection, TGF-&#x003b2;1 was markedly expressed in the peritubular capillaries, glomerular endothelial cells, and tubular epithelial cells of the untreated diabetic kidneys compared with the circular antisense TGF-&#x003b2;1 ODNs-treated diabetic kidneys ().RT-PCR analysis of the effect of circular antisense oligodeoxynucleotides (ODNs) on TGF-&#x003b2;1 mRNA expression in diabetic kidneys on days 1, 5, and 13 after injection of ODNs into diabetic rats. Lane 1, lane 2, on day 1, untreated ...Immunohistochemical staining of TGF-&#x003b2;1. On day 1 after injection of circular antisense TGF-&#x003b2;1 oligodeoxynucleotides (ODNs) into diabetic rats, ODNs-treated diabetic kidney (B), on day 5, ODNs-treated diabetic kidney (D), on day 13, ODNs-treated ...Effect of circular antisense ODNs on urinary TGF-&#x003b2;1 excretion rate of diabetic ratsUrinary excretion rate of total TGF-&#x003b2;1 (latent plus active fractions) markedly increased in both untreated and circular antisense TGF-&#x003b2;1 ODNs-treated diabetic rats. On days 1 and 5 after ODNs injection, the urinary TGF-&#x003b2;1 excretion rates of circular antisense TGF-&#x003b2;1 ODNs-treated diabetic rats were not significantly different from that of the untreated diabetic rats. In contrast, on day 13 after ODNs injection, the urinary TGF-&#x003b2;1 excretion rate of circular antisense TGF-&#x003b2;1 ODNs-treated diabetic rats showed approximately 25% reduction compared with that of the untreated diabetic rats ().Effect of circular antisense oligodeoxynucleotides (ODNs) on urinary TGF-&#x003b2;1 excretion rate. Collected urine samples on days 1, 5, and 13 after injection of ODNs into diabetic rats were assayed by a TGF-&#x003b2;1 ELISA (normal: open bars, diabetes: ...Much attention has been focused on exploring mechanisms related to the development of diabetic nephropathy. Among the mechanisms, TGF-&#x003b2;1 has been implicated as an etiologic cause in its progression. TGF-&#x003b2;1 is a key mediator of the hyperglycemia-induced panoramic effects on cell growth and extracellular matrix accumulation (, -, , ). In addition, this cytokine has powerful fibrogenic potential because of its capability for simultaneous actions such as stimulation of matrix synthesis, inhibition of matrix degradation, and modulation of matrix receptor expression to facilitate cell matrix interactions (). Thus, TGF-&#x003b2;1 has been considered as a therapeutic target in fibrotic disease such as diabetic nephropathy and other chronic kidney diseases, and anti-TGF-&#x003b2;1 therapies have also been performed (-).More recently, gene therapy using antisense ODNs as one of the anti-TGF-&#x003b2;1 therapies has been investigated and applied to renal disease (, , ). In the period of early gene therapy, a major problem was the rapid degradation of phosphodiester antisense ODNs by nuclease both in vitro and in vivo. To improve the biological instability of antisense ODNs, chemical modifications of the phosphate backbone or bases were attempted and ODNs such as phosphorothioate, methylphosphate, and phosphoramidate analogues have been developed (). In particular, phosphorothioate (PS)-ODNs have been studied mainly in animal models of kidney disease where one of the non-bridging oxygens in the phosphodiester backbone is replaced with a sulfur atom. However, PS-ODNs were found to have non-sequence-specific effects because of their polyanionic nature (, ).Circular antisense TGF-&#x003b2;1 ODNs used in the present work were synthesized from linear antisense ODNs, retaining the integrity of the phosphodiester backbone of the ODNs for its stability to nuclease attack. Theoretically, these ODNs could resist exonuclease attack with excellent binding affinity, sequence specificity, and ability to activate RNase H since they possess no 5' or 3'end. In this study, it was shown that circular antisense ODNs in rat serum were less digested than linear ODNs. The ablation of the TGF-&#x003b2;1 mRNA was more marked in the circular antisense ODNs than in the linear antisense ODNs treated RMCs. These results indicate that circular antisense TGF-&#x003b2;1 ODNs are superior to linear antisense ODNs. Furthermore, circular antisense TGF-&#x003b2;1 ODNs markedly suppressed the levels of TGF-&#x003b2;1 mRNA and protein in diabetic kidneys. Its specific activity in blocking TGF-&#x003b2;1 expression was demonstrated by the observed absence of difference in the level of GAPDH mRNA. An enhanced stability of the circular antisense TGF-&#x003b2;1 ODNs could enable less frequent dosing because of its longer duration of action, thereby reducing potential unwanted side-effects from fewer degradation metabolites.Various routes for the efficient transfer of ODNs into the kidney have been tried in experimental models. Akagi et al. () reported transfection of ODNs via renal artery in the anti-Thy glomerulonephritis model. Isaka et al. () introduced retrograde transfection of ODNs via the ureter in the unilateral ureter obstruction model. Han et al. () reported systemic infusion of ODNs through implanted osmotic pump in diabetic mice. All these works should be performed with minor-surgery for administration and would be quite troublesome in case of repetitive administration. Morishita et al. () attempted systemic administration via the tail vein for insulin vector transfer, resulting in the expression of insulin vector in the liver and spleen. Others reported that circulating ODNs could reach and accumulate in the kidney (, ). With those reports taken into consideration, the present study used intravenous administration to transfer ODNs to the kidney. After the intravenous administration, circular antisense TGF-&#x003b2;1 ODNs appeared in the liver, lungs, spleen, and kidney. In the kidneys, they appeared mainly in the tubular epithelial cells. In addition, they inhibited effectively the expression of TGF-&#x003b2;1 mRNA and protein in diabetic kidneys. These results are consistent with the notion that systemic administration of antisense TGF-&#x003b2;1 ODNs is effective in reducing TGF-&#x003b2;1 expression (). The study also employed HVJ-liposome method for the effective delivery of ODNs into kidney cells because of the several advantages of this delivery system, such as rapid translocation to the nucleus and stability of the transfected ODNs in the nucleus ().In the long-term diabetic state, TGF-&#x003b2;1 may perpetuate the disease process by inducing persistent hyperglycemia. In this regard, it seems necessary to perform further studies on the long-term blockade of progression of diabetic nephropathy and renal failure through treatment with circular antisense TGF-&#x003b2;1 ODNs.In conclusion, the effect of circular antisense TGF-&#x003b2;1 ODNs designed for stability against nucleases was superior to that of the linear antisense ODNs on TGF-&#x003b2;1 mRNA expression in RMCs. Circular antisense TGF-&#x003b2;1 ODNs suppressed efficiently the expression of TGF-&#x003b2;1 in the mRNA and protein levels by intravenous administration combined with HVJ-liposome in STZ-induced diabetic rats. These findings suggest that circular antisense TGF-&#x003b2;1 ODNs may be a useful tool for developing new therapeutic application for progressive diabetic nephropathy.A part of the technique used in this experiment was supported by Welgene biopharmaceuticals Inc.. This work was supported by Korean Research Foundation Grant (KRF-005).1. Ziyadeh FN, Goldfarb S. 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[]Articles from Journal of Korean Medical Science are provided here courtesy of Korean Academy of Medical Sciences
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