Carcassonne External Circuitry - 27Jun12

专利名称：冷冻探针
专利类型：发明专利
发明人：J·克罗南塔勒,M·阿德勒申请号：CN202010716460.X 申请日：20200723
公开号：CN112274190A
公开日：
20210129
专利内容由知识产权出版社提供
摘要：发明的冷冻探针包括头部(20)，低温流体经由毛细管(22a)供应到头部(20)。

软管(18)用于低温流体的去除，其中毛细管(22a)延伸穿过其腔(21)。

毛细管(22a)包括由牵引元件(26)桥接的柔性区段(25)。

以该方式，获得可良好操纵的冷冻探针11，其可容易和非常大程度地弯曲且仍传递对于样本提取所必需的张力。

申请人：厄比电子医学有限责任公司
地址：德国蒂宾根
国籍：DE
代理机构：中国专利代理(香港)有限公司
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专利名称：用于改善软膜侧支循环和治疗血液凝固障碍的方法与组合物
专利类型：发明专利
发明人：C·森,C·瑞克,S·罗伊,G·克里斯托佛里迪斯
申请号：CN201380040067.1
申请日：20130607
公开号：CN104507468A
公开日：
20150408
专利内容由知识产权出版社提供
摘要：本发明提供了促进受试者的动脉生成的方法。

实施例包括以下方法，这些方法包括：向该受试者给予有效剂量的生育三烯酚；在该受试者中导致脑血管侧支循环的血管中的金属蛋白酶组织抑制剂金属肽酶抑制剂1(TIMP1)增加；减弱基质金属蛋白酶-2(MMP2)的活性；由此促进动脉生成。

申请人：俄亥俄州立大学
地址：美国俄亥俄
国籍：US
代理机构：中国国际贸易促进委员会专利商标事务所
代理人：李程达
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丝裂原活化蛋白激酶信号通路在骨桥蛋白介导黄韧带骨化中的作用李学斌;许政;周盛源;刘晓东;王智清;许国峰;陈雄生【摘要】BACKGROUND:Mitogen-activated protein kinase (MAPK) signaling pathway is an important signal transduction system.Our precious animal experiments have shown that osteopontin can mediate the ossification of the ligamentum flavum.OBJECTIVE:To investigate the role of MAPK signaling pathway in osteopontin-induced ossification of the ligamentum flavum.METHODS:The ligamentum flavum specimens obtained from 16 cases undergoing thoracic/lumbar posterior decompression surgery were divided into ossification and non-ossification groups (n=8 per group).The expression of osteopontin and its receptors CD44 and integrin was observed by immunohistochemical staining.The activation of phosphorylation in MAPK signaling pathway was detected by western blot assay.The MAPK signaling pathway was blocked by SB203580 or U0126 blocker alone to observe the induction of osteopontin.RESULTS AND CONCLUSION:Osteopontin and its receptor CD44 were expressed in the ossification group,but not in the non-ossififcation group.However,the expression of integrin was not detected in the ossification group.The expression levels of alkaline phosphatase and osteocalcin in the ligamentum flavum were significnatly increased under the induction of osteopontin (P ＜ 0.05),and osteopontin could activate the phosphorylation of P38 and ERK1/2 in the MAPK signaling pathway (P ＜0.05),but the phosphorylation of JNK was not obvious.p38 phosphorylation blocked with SB203580 blocker could significantly inhibit the osteopontin-induced osteoblast differentiation of ligament flavum cells (P ＜ 0.05),while U0126 blocker had no obvious effect.These results indicate that p38 in MAPK signaling pathway is a key molecule in osteopontin-mediated ossification of the ligamentum flavum.%背景:丝裂原活化蛋白激酶(MAPK)信号通路是生物体内重要的信号转导系统之一.课题组前期动物实验提示骨桥蛋白可介导黄韧带骨化的发生.目的:观察MAPK信号通路在骨桥蛋白介导黄韧带骨化中的作用.方法:将16例患者后路椎板切除手术中取得黄韧带标本分为黄韧带骨化以及非骨化两组,每组8例.通过免疫组织化学染色观察骨桥蛋白及其受体CD44、整合素的表达;利用骨桥蛋白对黄韧带细胞进行诱导,并利用蛋白印记方法检测丝裂素活化蛋白激酶信号通路分子的磷酸化激活情况,随后再利用SB203580和U0126阻滞剂分别阻断丝裂素活化蛋白激酶相应信号通路观察骨桥蛋白的诱导情况.结果与结论;①骨桥蛋白及其受体CD44及整合素的表达:骨化组的标本中均检测到骨桥蛋白及其受体CD44的表达,但未检测到整合素的表达,非骨化组中则未见到骨桥蛋白及其受体的表达;②MAPK信号通路变化:在骨桥蛋白的诱导作用下黄韧带细胞中骨化指标碱性磷酸酶及骨钙素表达增加(P＜0.05),同时骨桥蛋白能激活丝裂素活化蛋白激酶信号通路中的p38及ERKI/2信号分子的磷酸化(P＜0.05),但JNK氨基末端激酶的磷酸化激活并不明显.利用SB203580阻滞剂阻断p38的磷酸化可明显阻断骨桥蛋白诱导的黄韧带细胞的骨向分化(P＜0.05),而U0126阻滞剂阻断效果不明显;③结果证实,MAPK信号通路在骨桥蛋白介导黄韧带骨化中其重要作用,p38是诱导黄韧带细胞骨向分化的关键因子.【期刊名称】《中国组织工程研究》【年(卷),期】2017(021)028【总页数】6页(P4469-4474)【关键词】组织构建;组织工程;黄韧带骨纯症;骨桥蛋白;丝裂素活化蛋白激酶信号通路;黄韧带细胞;国家自然科学基金【作者】李学斌;许政;周盛源;刘晓东;王智清;许国峰;陈雄生【作者单位】解放军第二军医大学附属上海长征医院脊柱三科,上海市200003;解放军第二军医大学附属上海长征医院脊柱三科,上海市200003;解放军第二军医大学附属上海长征医院脊柱三科,上海市200003;解放军第二军医大学附属上海长征医院脊柱三科,上海市200003;解放军第二军医大学附属上海长征医院脊柱三科,上海市200003;解放军第二军医大学附属上海长征医院脊柱三科,上海市200003;解放军第二军医大学附属上海长征医院脊柱三科,上海市200003【正文语种】中文【中图分类】R3180.引言.Introduction黄韧带骨化症(ossification of the ligamentum flavum，OLF)是一种特发于人脊柱黄韧带上的异位骨化病变[1]。

doi:10.3969/j.issn.1000⁃484X.2018.09.009TNF⁃α通过调控KLF4抑制口腔鳞癌细胞CAL27增殖龚靖淋　唐朝贤　刘一秀　杨　鑫　张玉莲(重庆大学附属肿瘤医院/重庆市肿瘤研究所/重庆市肿瘤医院,重庆400030) 中图分类号　R739.85 文献标志码　A 文章编号　1000⁃484X (2018)09⁃1321⁃05作者简介:龚靖淋,女,硕士,主要从事头颈肿瘤方面的研究,E⁃mail:cqgjl86@㊂通讯作者及指导教师:张玉莲,女,硕士,主要从事头颈肿瘤方面的研究,E⁃mail:zhangyulian2005@㊂[摘　要]　目的:探讨肿瘤坏死因子⁃α(TNF⁃α)通过上调口腔鳞癌细胞CAL27中Krüppel 样因子4(KLF4)表达对细胞增殖的影响㊂方法:用不同浓度的TNF⁃α(0㊁2.5㊁5㊁10㊁20ng /ml)处理CAL27细胞24h,免疫印迹试验(Western blot)检测KLF4表达变化;用20ng /ml TNF⁃α处理细胞,分别于24h㊁48h㊁72h 用噻唑蓝(MTT)法检测细胞增殖变化;构建过表达KLF4的CAL27细胞,MTT 检测其对细胞增殖的影响;MTT 检测沉默KLF4(siKLF4)及siKLF4联合TNF⁃α处理对细胞增殖能力的影响㊂结果:MTT 检测发现TNF⁃α可抑制细胞增殖;TNF⁃α可呈剂量依赖性地诱导CAL27细胞中KLF4表达;Western blot 和qPCR 检测显示成功构建过表达KLF4的CAL27细胞;过表达KLF4可抑制细胞增殖;沉默KLF4可部分恢复TNF⁃α对CAL27细胞增殖的抑制作用㊂结论:TNF⁃α可诱导口腔鳞癌细胞CAL27中KLF4表达增加,KLF4可能参与了TNF⁃α对CAL27细胞增殖的抑制作用㊂[关键词]　TNF⁃α;KLF4;口腔癌;增殖;CAL27Effect of TNF⁃αon proliferation of oral squamous cell carcinoma CAL27cells via regulating KLF4expressionGONG Jing⁃Lin ,TANG Chao⁃Xian ,LIU Yi⁃Xiu ,YANG Xin ,ZHANG Yu⁃Lian .Chongqing University Cancer Hospital &Chongqing Cancer Institute &Chongqing Cancer Hospital ,Chongqing 400030,China[Abstract ]　Objective :To investigate the effect of TNF⁃αon cell proliferation by up⁃regulating KLF4expression in oralsquamous cell carcinoma cell line CAL27.Methods :CAL27cells were treated with different concentrations of TNF⁃α(0,2.5,5,10,20ng /ml)for 24hours,and the expression of KLF4protein was detected by Western blot;the cell proliferation of CAL27treated with 20ng /ml TNF⁃αwas detected by MTT assay at 24h,48h and 72h,respectively;the effect of KLF4overexpression,siKLF4and siKLF4combined with TNF⁃αon cell proliferation was detected by MTT assay.Results :The expression of KLF4in CAL27cells was induced by TNF⁃αin a dose⁃dependent manner;the CAL27cells overexpressing KLF4were constructed successfully;the proliferation of CAL27cells was decreased by treating with 20ng /ml TNF⁃αand overexpression of KLF4,respectively;silencing KLF4could restore theinhibitory effect of TNF⁃αon CAL27cells.Conclusion :TNF⁃αcan increase KLF4expression in oral squamous cell carcinoma cellsCAL27,and KLF4may be involved in the inhibition of proliferation of CAL27cells by TNF⁃α.[Key words ]　TNF⁃α;KLF4;Oral carcinoma;Proliferation;CAL27 口腔癌是头颈部最常见的恶性肿瘤,近年来医学和生命科学取得了高速发展,但患者预后却未得到改善[1]㊂癌细胞在侵袭之前常常表现为异常基因表达,并在疾病进展过程中分化为间充质细胞样状态[2]㊂另外,持续地细胞增殖是恶性肿瘤细胞的另一个突出特征㊂细胞的增殖和分化平衡控制着上皮细胞的发育和稳态,对癌症的病理状态起着决定性的作用[3]㊂这些细胞过程通常由肿瘤抑制基因调控,而在癌症发展过程中抑癌基因常常处于失活状态,揭示抑癌基因功能作用对于了解口腔癌进展具有重要意义㊂近年来研究发现,KLF4是KLF 家族中的一个多功能转录因子[4,5],在细胞增殖㊁分化㊁凋亡㊁炎症反应㊁抗病毒免疫等过程中发挥着重要功能[6⁃9]㊂包括口腔上皮在内的复层鳞状上皮中,KLF4㊁KLF5分别在有丝分裂后角化基底细胞和增殖性低分化基底细胞中表达,它们严格控制着上皮细胞的增殖分化平衡[10]㊂KLFs 可通过与靶基因启动子结合进而转录调控靶基因表达,先前研究证实,KLF4表达缺失与口腔癌细胞的去分化相关,并常常在侵袭性口腔癌细胞中低表达[10⁃12],表明KLF4表达缺失与肿瘤进展密切相关㊂研究发现,TNF⁃α现作为一种具有多种生物学活性的多效性细胞因子能够促进乳腺癌细胞中CD44㊁KLF4表达进而调控细胞增殖[13,14],但在口腔鳞癌中TNF癌能否通过KLF4调控细胞增殖仍然未知㊂在本研究中,采用不同浓度TNF⁃α(0㊁1㊁5㊁10㊁20ng/ml)处理口腔鳞癌CAL27细胞24h,Western blot检测TNF⁃α对KLF4表达的影响;MTT法分别检测20ng/ml TNF⁃α和过表达KLF4对CAL27增殖的影响;沉默KLF4联合TNF⁃α作用于CAL27细胞,进一步研究KLF4对TNF⁃α诱导CAL27增殖抑制的影响,旨在揭示TNF⁃α治疗口腔鳞癌潜能及KLF4表达在口腔鳞癌中的作用,为更有效治疗口腔鳞癌提供新靶点㊂1　材料与方法1.1　主要材料　DMEM(Dulbecco′s modified eagle medium)培养基㊁胎牛血清㊁青链霉素㊁胰蛋白酶均购自Gibco公司,TNF⁃α购自PeproTech公司,KLF4抗体购自Cell Signaling Technology(CST)公司,甘油醛⁃3⁃磷酸脱氢酶(Glyceraldehyde⁃3⁃phosphate de⁃hydrogenase,GAPDH)购自碧云天公司,羊抗兔和羊抗鼠二抗购自北京全式金公司,RNA提取试剂盒RNAiso plus Reagent㊁RNA逆转录试剂盒PrimeScriptTM RT reagent Kit with gDNA Eraser㊁qPCR试剂盒SYBR®Premix Ex Taq TM均购自TaKaRa,四甲基偶氮唑蓝(Methylthiazolyldiphenyl⁃tetrazolium bromide,MTT)和二甲基亚砜(Dimethyl sulphoxide,DMSO)购自Sigma㊂1.2　方法1.2.1　细胞培养　人口腔鳞状细胞癌细胞CAL27购自中科院细胞库,CAL27在添加有10%胎牛血清㊁1%青链霉素的DMEM培养基中,置于37℃恒温㊁5%CO2饱和湿度的培养箱传代培养,取对数期细胞加入0㊁2.5㊁5㊁10㊁20ng/ml浓度的TNF⁃α处理24h用于提取蛋白进行Western blot实验检测㊂1.2.2　细胞转染　用RNAiso plus Reagent提取细胞总RNA并逆转录成cDNA,用特异引物行PCR扩增KLF4cDNA,并克隆至pcDNA3.1中,构建pcDNA3.1⁃KLF4载体,用pcDNA3.1空载作为空白对照㊂KLF4cDNA扩增引物:F:5⁃CCTCGAGGACCT⁃TCTGGGCCCCCACATTAATG⁃3和R:5⁃CCGCGG⁃CCGCACTACGTGGGATTTAAAAGTG⁃3㊂KLF4siRNA (5⁃AUCGUUGAACUCCUCGGUCUCUCUC⁃3)购自上海吉玛,用Lipofectamine2000将pcDNA3.1⁃KLF4和KLF4siRNA转染至细胞,进行提取蛋白㊁mRNA 及增殖能力检测㊂1.2.3　qPCR检测实验　取对数生长期细胞,分离提取总RNA,DEPC水溶解后全波长酶标仪测RNA 浓度,按照逆转录试剂盒说明书进行反转录得cDNA,实时荧光定量PCR仪检测KLF4的mRNA相对表达量,以GAPDH为内参,用2-ΔΔCt法来计算KLF4相对表达量㊂KLF4引物F:5⁃ACAAAGAGT⁃TCCCATCTCAA⁃3和R:5⁃GTAGTGCCTGGTCATTTC⁃3;GAPDH引物F:5⁃GCTCCTCCTGTTCGACAGTCA⁃3和R:5⁃ACCTTCCCCATGGTGTCTGA⁃3㊂1.2.4　免疫印迹试验(Western blot)　收集处理后细胞,冷PBS洗涤,后重悬于RIPA裂解液,冰上于摇床摇动孵育30min,裂解完后4℃,12000r/min 离心10min,取上清液用BCA法测蛋白浓度,取30μg蛋白配成上样缓冲液,经10%SDS⁃PAGE胶电泳分离㊁转膜和5%脱脂奶粉封闭后,用KLF4㊁GAPDH一抗及HRP偶联的二抗进行孵育,ECL曝光液于曝光仪(Bio⁃Rad)上曝光显影㊂用Image J软件进行灰度分析,测定KLF4相对表达㊂1.2.5　MTT检测细胞增殖能力　收集对数生长期细胞,用含10%胎牛血清和1%青链霉素的DMEM 培养基调整细胞浓度为2×104ml-1,每孔200μl接种于96孔板中,分别于24h㊁48h㊁72h时,每孔加入20μl MTT溶液,继续于细胞培养箱孵育4h,然后弃上清每孔加入150μl DMSO,于酶标仪上振荡10min使结晶充分溶解,测定490nm处各孔吸光度值㊂1.3　统计学处理　实验数据用x±s表示,采用SPSS22.0进行统计学分析,两组数据采用独立样本t 检验,多组差异比较采用单因素方差分析,组间比较采用LSD⁃t检验,以P<0.05为差异有统计学意义㊂2　结果2.1　TNF⁃α对口腔鳞癌细胞CAL27增殖的影响　用20ng/ml的TNF⁃α处理CAL27细胞后,MTT检测24h㊁48h㊁72h其对细胞增殖的影响,结果发现,与TNF⁃α(0ng/ml)组相比,在72h时TNF⁃α(20ng/ml)可显著抑制CAL27细胞增殖能力(P< 0.05)(图1)㊂2.2　TNF⁃α对口腔鳞癌细胞CAL27中KLF4表达的影响　口腔鳞癌细胞CAL27经不同浓度的TNF⁃α(0㊁2.5㊁5㊁10㊁20ng/ml)处理24h后,Western blot检测发现,随着TNF⁃α浓度增加,CAL27细胞中KLF4表达水平呈浓度依赖逐渐增加(P<0.05)(图2A㊁B)㊂2.3　构建过表达KLF4的CAL27细胞　为证明TNF⁃α对CAL27细胞增殖的抑制作用是通过上调KLF4表达介导的,本研究构建了过表达KLF4的CAL27细胞株,经qPCR 和Western blot 检测发现,与pcDNA3.1组相比,转染pcDNA3.1⁃KLF4后CAL27细胞中KLF4的蛋白和mRNA 表达均显著增加(0.94±0.06㊁2.03±0.09,t 1=17.454,P 1=0.000;1.02±0.11㊁4.05±0.26,t 2=18.590,P 2=0.000)(图3)㊂2.4　过表达KLF4对口腔鳞癌细胞增殖的影响　在CAL27细胞中过表达KLF4后,利用MTT 检测过表达KLF4对细胞增殖的影响,结果发现,与pcDNA3.1相比,在48h㊁72h 时过表达KLF4可显著抑制CAL27细胞增殖能力(0.51±0.03㊁0.39±0.02,t 48h =5.765,P 48h =0.005;0.95±0.05㊁0.56±0.04,t 72h =10.550,P 72h =0.001)(图4)㊂图1　TNF⁃α抑制CAL27细胞增殖Fig.1　TNF⁃αinhibits proliferation of CAL27cellsNote:*.P <0.05compared with TNF⁃α(0ng /ml)at 72h.图2　TNF⁃α诱导CAL27细胞中KLF4表达Fig.2　TNF⁃αupregulated expression of KLF4in CAL27cellsNote:A.Western blot was used to detect the expression of KLF4inCAL27cells after treating with different concentration of TNF⁃α;B.The expression level of KLF4in CAL27cells after treating with different concentration of TNF⁃α,*.P <0.05,compard with 0ng /mlgroup.图3　构建过表达KLF4的CAL27细胞Fig.3　Construction of CAL27cells overexpressing KLF4Note:A.The expression of KLF4protein in CAL27was detected byWestern blot;B.The relative expression of KLF4protein and mRNA in CAL27after transfecting with plasmids;*.P <0.05compared with pcDNA3.1group.图4　过表达KLF4抑制CAL27细胞增殖Fig.4　KLF4inhibits proliferation of CAL27cellsNote:*.P <0.05compared to pcDNA3.1group at 48h or 72h.图5　沉默KLF4逆转了TNF⁃α对口腔鳞癌细胞增殖抑制作用Fig.5　Silencing KLF4reversed the inhibitory effect ofTNF⁃αon proliferation of oral squamous cell car⁃cinomaNote:A.The expression of KLF4protein in CAL27was detected byWestern blot after transfection of siKLF4;B.The relativeexpression of KLF4protein and mRNA in CAL27after transfecting siKLF4;C.The proliferation of CAL27cells was detected by MTT;*.P <0.05compared to Control group,#.P <0.05compared to TNF⁃α(20ng /ml)group.2.5　沉默KLF4逆转了TNF⁃α对口腔鳞癌细胞增殖抑制作用　为进一步证实,TNF⁃α是通过上调KLF4表达来发挥促进CAL27细胞增殖作用的,本研究用siRNA沉默KLF4,然后再联合20ng/ml的TNF⁃α处理细胞,分别用MTT检测24h㊁48h㊁72h 时各组细胞吸光值㊂经qPCR和Western blot检测发现,与Control组相比,转染siKLF4后CAL27细胞中KLF4蛋白和mRNA表达均显著下调(1.26±0.17㊁0.53±0.04,t1=7.24,P1=0.014;0.99±0.06㊁0.32±0.07,t2=12.59,P2=0.000)(图5A㊁B)㊂MTT 实验结果发现,与Control组相比,沉默KLF4可明显促进细胞增殖(P<0.05);与TNF⁃α组相比,沉默KLF4联合TNF⁃α处理可恢复TNF⁃α对细胞增殖的抑制作用(P<0.05)㊂上述结果表明,TNF⁃α可能部分通过上调KLF4来发挥对CAL27细胞增殖的抑制作用㊂3　讨论口腔鳞状细胞癌(SCC)是我国常见的致命性恶性肿瘤之一,也是全球癌症相关死亡的主要原因之一[15]㊂口腔鳞癌的病因包括使用烟草制品㊁饮酒以及人乳头瘤病毒(HPV)感染等[16]㊂目前,手术切除联合放化疗是首选治疗策略,但疗效仍不理想,其5年生存率较差[16,17]㊂迄今为止,已经寻找到了口腔癌的一些分子标记物,但标记物明确的预后或诊断意义尚未确定,因此,寻求新的治疗手段及治疗靶标对于开发新的有效口腔癌治疗策略至关重要[18]㊂在肿瘤进展过程中,癌细胞主要通过激活癌基因和/或失活抑癌基因来促进肿瘤恶性进展㊂据研究报道,KLF4可通过调控细胞周期在细胞转化和细胞增殖中发挥重要作用[16];在多种类型晚期肿瘤中KLF4表达降低,且其表达丧失可导致癌细胞上皮间质转化,强烈刺激肿瘤恶性进展[18]㊂最近发现,在高转移胰腺癌细胞中KLF4表达上调,胰腺癌细胞中高表达的KLF4可通过降低p27表达来促进细胞增殖[19]㊂此外,KLF4在口腔癌中同样具有促癌作用,KLF4可促进口腔鳞癌细胞侵袭[12,20,21]㊂据研究报道,在低分化和进展期口腔癌中KLF4表达下调并抑制细胞分化[10]㊂此外,李文文等[20]研究也发现,KLF4对口腔鳞癌细胞增殖有抑制作用㊂本研究也发现,在口腔鳞癌细胞中过表达KLF4后,细胞增殖能力显著下降,提示KLF4与口腔鳞癌发展有关㊂TNF⁃α作为肿瘤坏死因子在体内具有多种生物学作用,TNF⁃α在抵抗病原菌感染及抗肿瘤中起着重要作用,是迄今为止抗癌活性最强的细胞因子,但当其超过一定量时便会反过来与其他炎性因子一起促进癌症发生发展及引发多种病理性损伤[21,22]㊂研究发现,TNF⁃α在口腔癌患者血清中高表达,且与患者预后有关[23];TNF⁃α可调控基质金属蛋白酶促口腔癌细胞侵袭转移[24]㊂此外,TNF⁃α也可促进乳腺癌细胞中CD44㊁KLF4表达进而诱导细胞增殖[13,14],TNF⁃α是否能够通过促进口腔鳞癌细胞CAL27中KLF4表达进而促进细胞增殖仍然未知㊂本研究中我们发现,TNF⁃α可呈剂量依赖性地促进CAL27细胞中KLF4表达;20ng/ml的TNF⁃α处理CAL27可抑制细胞增殖,CAL27细胞过表达KLF4后增殖能力显著降低,而沉默KLF4可部分恢复TNF⁃α对CAL27细胞增殖抑制作用㊂综上所述, 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Published:July 13,2011/bcSynthesis and Evaluation of an Anti-MLC1ÂAnti-CD90Bispecific Antibody for Targeting and Retaining Bone-Marrow-Derived Multipotent Stromal Cells in Infarcted MyocardiumC.William Gundlach IV,†Amy Caivano,†Maria da Graca Cabreira-Hansen,†Amir Gahremanpour,†Wells S.Brown,‡Yi Zheng,†Bradley W.McIntyre,‡James T.Willerson,†Richard A.F.Dixon,†Emerson C.Perin,†and Darren G.Woodside*,††The Texas Heart Institute at St.Luke ’s Episcopal Hospital,Houston,Texas 77225,United States‡The University of Texas MD Anderson Cancer Center,1515Holcombe Boulevard,Houston,Texas 77030,United States ’INTRODUCTIONStem cell therapy is a promising therapeutic modality for re-storing cardiac function in cardiovascular disease.1,2The preclinical findings that bone-marrow-derived multipotent stromal cells (BMMSCs)e ffect tissue repair 3and have immunomodulatory activity 4have led to the clinical testing of these cells in the treat-ment of a variety of diseases.5Furthermore,in a pig model of myocardial infarction,BMMSC engraftment and di fferentiation have been correlated with improved heart function.6However,the e ffectiveness of this therapy may be limited by low rates of cellular retention and engraftment in areas of cell delivery,even when cells are applied directly to the myocardium.7,8One approach to address this issue is to target stem cells to ischemic tissue by creating a bispeci fic antibody that can bind to antigens speci fic for the stem cell and to the target tissue.This approach has been used in cancer therapy to target immune e ffector cells to tumor cells,9and catumaxomab,a first-generation bispeci fic antibody,has been approved recently in Europe fortreating malignant ascites.10Lee and colleagues 11have applied this methodology to cardiac stem cell therapy by generating a bispeci fic antibody construct to direct hematopoietic stem cells to infarcted myocardium in rodents.11,12In this construct,the tissue-targeting arm uses a monoclonal antibody (mAb)speci fic for myosin light chain 1(MLC1)(mAb MLM508),which is found within the interstitial tissue of damaged hearts 13and serves as an antigen marker for injured myocardium.14À16The other arm of the construct for binding hematopoietic stem cells is com-posed of a mAb to CD45,11,12which is a common leukocyte antigen 17found on CD34+hematopoietic stem cells.18In the present study,we have created a novel bispeci fic antibody for targeting BMMSCs to injured myocardium.The tissue-targeting arm of our reagent comprises the mAb MLM508describedReceived:June 13,2011Revised:July 11,2011ABSTRACT:A key issue regarding the use of stem cells in cardiovascular regenerative medicine is their retention in target tissues.Here,we have generated and assessed a bispeci fic antibody heterodimer designed to improve the retention of bone-marrow-derived multipotent stromal cells (BMMSC)in cardiac tissue damaged by myocardial infarction.The hetero-dimer comprises an anti-human CD90monoclonal antibody (mAb)(clone 5E10)and an anti-myosin light chain 1(MLC1)mAb (clone MLM508)covalently cross-linked by a bis-arylhy-drazone.We modi fied the anti-CD90antibody with a pegy-lated-4-formylbenzamide moiety to a molar substitution ratio(MSR)of 2.6and the anti-MLC1antibody with a 6-hydrazinonicotinamide moiety to a MSR of 0.9.The covalent modi fications had no signi ficant deleterious e ffect on mAb epitope binding.Furthermore,the binding of anti-CD90antibody to BMMSCs did not prevent their di fferentiation into adipo-,chondro-,or osteogenic lineages.Modi fied antibodies were combined under mild conditions (room temperature,pH 6,1h)in the presence of a catalyst (aniline)to allow for rapid generation of the covalent bis-arylhydrazone,which was monitored at A 354.We evaluated epitope immunoreactivity for each mAb in the construct.Flow cytometry demonstrated binding of the bispeci fic construct to BMMSCs that was competed by free anti-CD90mAb,verifying that modi fication and cross-linking were not detrimental to the anti-CD90complementarity-determining region.Similarly,ELISA-based assays demonstrated bispeci fic antibody binding to plastic-immobilized recombinant MLC1.Excess anti-MLC1mAb competed for bispeci fic antibody binding.Finally,the anti-CD90Âanti-MLC1bispeci fic antibody construct induced BMMSC adhesion to plastic-immobilized MLC1that was resistant to shear stress,as measured in parallel-plate flow chamber assays.We used mAbs that bind both human antigens and the respective pig homologues.Thus,the anti-CD90Âanti-MLC1bispeci fic antibody may be used in large animal studies of acute myocardial infarction and may provide a starting point for clinicalstudies.above,which reacts with both pig and human MLC1.19Because BMMSCs do not express CD4520[the antigen used in the construct made by Lee and colleagues],11,12the stem-cell binding arm of our antibody was composed of mAb5E10,which recognizes the phenotypic cell surface marker CD90(Thy-1)21found on BM-MSCs.mAb5E10recognizes both pig and human BMMSCs.22 To generate an anti-CD90Âanti-MLC1bispecific antibody construct,we used methods that labeled one mAb with a6-hydra-zinonicotinamide moiety via a conventional NHS ester.The second mAb in the bispecific construct was modified with a pegylated 4-formylbenzamide moiety,via a pentafluorophenyl ester.For-mation of the bis-arylhydrazone,which is readily monitored by UVÀvis spectroscopy,was rapid and precluded formation of homodimeric antibody products,which can occur with the use of other protein cross-linking chemistries.Herein,we describe the methodology involved in creating our bispecific antibody and the evaluation of its potential use as a BMMSC-targeting agent.’EXPERIMENTAL PROCEDURESS-HyNic Modification of Anti-MLC1Antibody.Anti-MLC1 (mouse anti-myosin light chain1antibody[MLM508,IgG2a]; Abcam,Cambridge,MA)was concentrated to3.5mg/mL and exchanged into modification buffer(100mM sodium phosphate, 150mM sodium chloride,pH7.4)by using a spin column(Zeba, Thermo Fisher Scientific,Rockford,IL)according to the man-ufacturer’s instructions.A portion of the resulting solution(30μL, 2.7mg/mL)was mixed with S-HyNic(0.8μL,35.5mM;Solulink, San Diego,CA)in anhydrous dimethylformamide(DMF; Sigma-Aldrich,St.Louis,MO).After incubation at room tem-perature for2h,the reaction mixture was applied to a spin column equilibrated with coupling buffer(100mM sodium phosphate, 150mM sodium chloride,pH6.0).HyNic-MLM508(1,35μL, 2.0mg/mL)was stored at4°C.4FB(PEG)4-PFP Modification of Anti-CD90Antibody.Anti-CD90(mouse anti-human CD90antibody[5E10,IgG1]);BD Pharmingen,San Diego,CA)was concentrated to3.5mg/mL and subjected to buffer exchange as above.An aliquot of the resulting solution(25μL,3.5mg/mL)was mixed with(4FB-(PEG)4-PFP(Solulink)(0.5μL,6.9mM)in anhydrous DMF. After incubation at room temperature for2h,the reaction mix-ture was applied to a spin column equilibrated with coupling buffer. 4FB(PEG)4-5E10(2,30μL,2.2mg/mL)was stored at4°C.Measurement of Molar Substitution Ratio.An aliquot of modified antibody(3.3μL)was mixed with either2-hydrazino-pyridine dihydrochloride(2-HP)or4-nitrobenzaldehyde(4-NB) (both from Sigma-Aldrich)(6.7μL,0.5mM in100mM MES, pH6.0).The mixture was incubated at37°C for0.5h,and the absorbance at either390nm(4-NB)or354nm(2-HP)was re-corded.UVÀvis and absorbance spectra were measured with a Nanodrop1000(Nanodrop Products,Wilmington,DE).The MSR was calculated by using Beer’s law and an extinction coefficient of24000(4-NB)or18000(2-HP).ELISA Procedure.ELISA assays were performed to determine the functional activity of HyNic-MLM508either alone or as part of the bispecific antibody construct.Briefly,recombinant human MLC1(Abcam,20μg/mL)in TBS was immobilized onto wells of a96-well plate(BD Falcon353228)overnight at4°C.BSA (1%w/v)was added for1h at room temperature as a blocker. The wells were washed3times with PBS-T(0.1mL,10mM sodium phosphate,150mM NaCl,0.1%Tween-20(v/v),pH 7.2)containing1%FBS(v/v),and control antibodies or bispecific reagent were incubated in the wells for1h at room temperature in PBS-T containing1%FBS(v/v).In competition experiments, wells were incubated with the excess MLM508for0.5h,followed by0.5h with the bispecific reagent.The wells were washed with PBS-T containing1%FBS(v/v),and the secondary antibodyÀHRP conjugate(Biosource International,Camarillo,CA;1:500 dilution from commercial stock in PBS-T containing1%FBS(v/v)) was incubated at room temperature for1h.For measurements of HyNic-MLM508bound to MLC1,GAM-HRP was utilized.For measurements of the bispecific antibody binding to MLC1,anti-IgG1(the isotype of mAb5E10)was used.Wells were washed4 times with PBS-T containing1%FBS(v/v).Then the1-Step Turbo TMB ELISA reagent(Thermo Fisher Scientific)was added to the wells.The colorimetric reaction was stopped with 1.5M H2SO4,and the absorbance at450nm was recorded on a Safire2(Tecan,Durham,NC)plate reader.Pig-Bone-Marrow-Derived Multipotent Stromal Cells. BMMSCs were isolated on the basis of their ability to adhere to plastic surfaces.Bone marrow aspirates from male pigs were collected into50mL conical tubes through a cell strainer(70μm mesh).Cells were counted and diluted in PBS to a final concen-tration of107/mL.The mononuclear fraction was isolated by density-dependent cell separation(Ficoll, 1.077g/mL,GE Healthcare,Uppsala,Sweden).After two washes with PBS,the mononuclear cells were plated in175cm2flasks at(0.5À1)Â106cells per cm2in R minimal essential medium(R MEM,In-vitrogen,Carlsbad,CA)containing ribonucleosides supplemen-ted with10%FBS(v/v)(Atlanta Biologicals,Lawrenceville, GA).One day later,fresh medium was added,and nonadherent cells were removed.At70%confluence,cells were harvested with 0.25%trypsin(Invitrogen)/1mM EDTA and further seeded at 103cells per cm2.The medium was changed every3À4days,and BMMSCs were further passaged at70%confluency.BMMSCs between passages3À6were used in this study.Bone-Marrow-Derived Multipotent Stromal Cell Differen-tiation Assays.Chondro-,osteo-,and adipogenesis differen-tiation assays were performed as previously described.20,23Briefly, monolayer cultures for osteogenesis and adipogenesis assays were initiated by seeding2Â103cells in six-well tissue culture plates in R MEM.At70%confluence,adipogenesis was induced by incubation with R MEM containing10mg/mL insulin,10% FBS(v/v),1mM dexamethasone,0.5mM methylisobutylxanthine, and100mM indomethacine for3d,followed by3d in adipogenic maintenance medium(R MEM containing10mg/mL insulin and 10%(v/v)FBS).This alternating treatment was repeated until full adipogenic differentiation was obtained(approximately2 weeks).Oil Red staining of neutral lipids was used to denote adipocytes.Osteogenesis was induced in70%confluent monolayers by incubation of BMMSCs with medium containing50mg/mL ascorbate2-phosphate,0.1mM dexamethasone,and10mM β-glycerol phosphate and10%FBS(v/v)for21days.The pres-ence of osteoblasts was evaluated by the accumulation of intra-cellular alkaline phosphatase(Vector Red alkaline phosphatase substrate kit,Sigma-Aldrich).For chondrogenic differentiation,2Â105cells were centri-fuged at450g for5min in15mL conical centrifuge tubes.The cell pellets were incubated for21days in differentiation medium (40mg/mL proline,100mg/mL sodium pyruvate,10ng/mL TGF-β3,0.1mM dexamethasone,50mg/mL ascorbate2-phos-phate).To demarcate glycosaminoglycans,Alcian blue staining was applied to cell pelletsfixed in4%(w/v)paraformaldehyde.Flow Cytometry.For analysis of 4FB(PEG)4-5E10binding toBMMSCs,cells (2Â105)were incubated with 4FB(PEG)4-5E10for 0.5h at 4°C in PBS.After two washes in PBS containing 1%FBS (v/v),cells were incubated with GAM-IgG-FITC conjugate and analyzed on a LSRII (BD Biosciences,San Jose,CA).For analysis of bispecific antibody binding to BMMSCs,cells were treated as above except that secondary antibody treatment was GAM-IgG2a-FITC (BD Biosciences)to detect the MLM508antibody.Formation of Anti-MLC1ÂAnti-CD90Bispecific Anti-body.HyNic-MLM508(1,27.5μL)and 4FB(PEG)4-5E10(2,25μL)were combined with aniline (200mM,2.6μL)in con-jugation buffer to give a final aniline concentration of 10mM.Reac-tion progress was monitored by measuring the UV Àvis spectrum of 2μL aliquots of the reaction mixture.After room-temperature incubation for 2.5h,the reaction mixture was applied to a spin column equilibrated with 100mM sodium phosphate,150mM sodium chloride,pH 7.2.The anti-MLC1Âanti-CD90bispecific antibody mixture (3,60μL,1.3mg/mL)was stored at 4°C.Parallel-Plate Flow Experiments.Recombinant human MLC1(20μg/mL in 0.1M NaHCO 3,pH 9.5)was immobilized overnight at 4°C onto 24mm Â50mm slides cut from 15mm Â100mm polystyrene Petri dishes.The slides were washed with PBS,blocked with 2%(w/v)BSA for 2h at room temperature,and assembled into a parallel-plate flow chamber.Control wellswere coated with BSA (2%,w/v)only.Pig BMMSCs were either untreated or treated with bispecific antibody (5μg/mL)for 0.5h at 4°C before washing and resuspension in running buffer (10nM Tris,103mM NaCl,24mM NaHCO 3,5.5mM glucose,5.4mM KCl,2mg/mL BSA,pH 7.4).Then the BMMSCs (2.0Â106cells)were injected into the flow chamber and allowed to settle on the slides for 10min.An increasing linear gradient of shear flow was pulled over the adherent cells for 300s with the use of a computer-controlled syringe pump (Harvard Apparatus,Holliston,MA),and the number of adherent cells remaining was recorded by digital microscopy.Shear stress calculations were determined every 20s.The shear stress in dyn/cm 2is defined as (6μQ )/(wh 2):μis the viscosity of the medium (0.007);Q is the flow rate in cm 3/s;w is the width of the chamber (0.3175cm);h is the height of the chamber (0.01524cm).The number of cells attached was recorded by digital microscopy (VI-470charge-coupled device video camera,Optronics Engineering,Goleta,CA)at 20Âon an inverted Nikon DIAPHOT-TMD microscope every 20s and was plotted against time,as previously described.24’RESULTSS-HyNic Modification of Anti-MLC1mAb MLM508.mAbMLM508(anti-MLC1)was modified by treatment with S-HyNic,yielding HyNic-MLM508(1,Figure1A).To determine theFigure 1.S-HyNic modi fication of anti-MLC1antibody and retention of epitope speci ficity.(A)Reaction of anti-MLC1antibody (mAb MLM508)with S-HyNic.The reaction products not shown include excess S-HyNic,N -hydroxysuccinimide (NHS),and unreacted mAb.S-HyNic and NHS were removed by bu ffer exchange before further testing and generation of the bispeci fic reagent.Modi fied antibodies are represented with one modi fication per molecule for simplicity.DMF is dimethylformamide;MES is 2-(N -morpholino)ethanesulfonic acid.(B)Reaction scheme followed to measure the molar substitution ratio (MSR).A portion of HyNic-MLM508was incubated with 4-nitrobenzaldehyde (4-NB)to generate 4NB-HyNic-MLM508,which absorbs at 390nm.During incubation at pH 6,the alkylhydrazone hydrolyzes in situ to produce the free hydrazine.The calculated MSR was 0.9.(C)ELISA analysis of HyNic-MLM508binding to immobilized substrate recombinant human MLC1.Modi fied MLM508binding was detected with GAM-HRP.All antibodies were used at 10μg/mL.Results are presented as average A 450(SD from triplicate wells.One of three experiments performed is shown.molar substitution ratio (MSR),4-NB was added to a portion of buffer-exchanged HyNic-MLM508to generate 4NB-HyNic-MLM508(Figure 1B)with an absorbance at 390nm.Thecalculated MSR was 0.9,with a mass recovery of 86%.To de-termine whether the HyNic modification of MLM508(1)altered epitope recognition of MLC1,we performed an ELISA assay utilizing purified recombinant human MLC1as a substrate.The binding of HyNic-MLM508(10μg/mL)to immobilized MLC1protein was measured relative to the binding of unmodified MLM508(10μg/mL)by ELISA,and the optical density values were within 1standard deviation (Figure 1C).Thus,modifying MLM508with S-HyNic had only a minimal effect on MLM508interactions with MLC1.Effects of CD90mAb on Bone-Marrow-Derived Multi-potent Stromal Cell Differentiation.The anti-CD90compo-nent of the bispecific antibody construct binds BMMSCs.To ensure that the binding of the CD90antigen on BMMSCs did not affect the differentiation ability of BMMSCs,we performed osteo-,adipo-,and chrondogenic differentiation assays in the presence of the anti-CD90mAb or an isotype control mAb (Figure 2).Anti-CD90was used at 10μg/mL,which was the saturating concen-tration as shown by flow cytometric analysis (data not shown).Under culture conditions for differentiation,BMMSCs showed the ability to differentiate along osteo-,adipo-,and chrondogenic lineages in the presence of the anti-CD90antibody,indicating that binding of the CD90surface antigen did not adversely affectcell differentiation (Figure 2).Figure 2.Di fferentiation capacity of BMMSCs treated with anti-CD90antibody (mAb 5E10).BMMSCs were treated with anti-CD90mAb 5E10(A ÀC)or isotype control mAb (D ÀF).Di fferentiation of cells into adipogenic (A,D;arrows indicate neutral lipid accumulation),osteogenic (B,E),and chondrogenic (C,F)lineages was examined.Figure 3.Modi fication of anti-CD90antibody (mAb 5E10)with 4FB(PEG)4-PFP and retention of antibody-binding speci ficity.(A)Reaction of mAb 5E10with 4FB(PEG)4-PFP.Reaction products not shown include excess 4FB(PEG)4-PFP,penta fluorophenol,and unreacted antibody.Modi fied antibodies are represented with one modi fication per molecule for simplicity.4FB(PEG)4-PFP and penta fluorophenol were removed by bu ffer exchange before generation of the bispeci fic reagent and further use.DMF is dimethylformamide.(B)Reaction scheme to determine the molar substitution ratio (MSR).A portion of 4FB(PEG)4-5E10was incubated with 2-hydrazinopyridine dihydrochloride (2-HP)to form 2HP-4FB(PEG)4-5E10,which absorbs at 354nm.The calculated MSR was 2.6.(C)Flow cytometric analysis of 4FB-5E10and 4FB(PEG)4-5E10binding to pig BMMSCs.Unmodi fied 5E10(solid,black),4FB(PEG)4-5E10(solid,light gray),4FB-5E10(solid,dark gray),and IgG1control antibody (dotted,black)binding to BMMSCs was detected with GAM-FITC (10μg/mL).4FB-5E10was synthesized in the same fashion as 2(MSR =3.0)using S-4FB.The histograms are representative of three experiments.4FB(PEG)4-PFP Modification of Anti-CD90mAb 5E10.Anti-CD90mAb 5E10was modified by treatment with 4FB-(PEG)4-PFP,yielding 4FB(PEG)4-5E10(2,Figure 3A).To de-termine the MSR,2-HP was incubated with a portion of 4FB-(PEG)4-5E10to form 2HP-4FB(PEG)4-5E10(Figure 3B).On the basis of absorbance at 354nm,we calculated an MSR of 2.6,and mass recovery was 75%.To determine whether 4FB(PEG)4modification of 5E10affected its ability to bind to CD90on the surface of pig BMMSCs,we compared the binding of 4FB-(PEG)4-5E10to BMMSCs with that of unmodified 5E10by flow cytometric analysis (Figure 3C).The mean fluorescent intensity of 4FB(PEG)4-5E10(10μg/mL)binding to BMMSCs in vitro was similar to that of unmodified 5E10(10μg/mL)(19722vs 22029,respectively).This finding is in contrast to previous attempts to label mAb 5E10with 4FB lacking the (PEG)4spacer,which inhibited 5E10interactions with CD90(Figure 3C).Generation of Bispecific Antibody.To generate the bispe-cific antibody,HyNic-MLM508(1)was combined with 4FB-(PEG 4)-5E10(2)in a 1:1molar ratio in the presence of 10mM aniline 25as a catalyst (Figure 4).The progress of the reaction was monitored over time by UV Àvis spectroscopy,measuring A 354from the bis-arylhydrazone (Figure 5A,rectangle).The reaction reached a maximum A 354within 1h,indicating that the reaction was complete (Figure 5B).Aniline was removed from the re-action mixture by buffer exchange of the bispecific antibody into PBS (pH 7.2)for storage.We analyzed a sample of the product (3)by nonreducing SDS ÀPAGE (Figure 6).As shown by den-sitometry,the reaction product comprised 61%mAb monomers,25%dimers,and 14%multimers.Antigen Specificity of the Bispecific Antibody.Individual modification of MLM508and 5E10did not result in the loss of antigen-binding activity (Figures 1C and 3C).However,to target BMMSCs to ischemic tissue,each arm of the bispecific antibody must remain functional after heterodimer formation.Thus,we tested the bispecific antibody reagent (3,Figure 7)to verify that the immunoreactivity of each half of the heterodimer was not reduced in the cross-linked species.First,we used an ELISA toverify that the bispecific antibody mixture could bind purified re-combinant MLC1.We coated the wells with BSA (0.1%w/v)as a control or with MLC1(10μg/mL);unbound sites were blocked with BSA (0.1%w/v).HRP-conjugated goat anti-mouse IgG1(the isotype of anti-CD90mAb 5E10)was used as the detection antibody.Several control assays were performed to rule out nonspecific binding of the bispecific antibody to immobilized MLC1.No binding was observed when the bispecific antibody was incubated in wells coated with BSA only (Figure 7B).In addition,IgG2a (the isotype of MLM508)did not bind to immobilized MLC1,serving as a control for the MLM508arm of the bispecific antibody.Furthermore,nonspecific binding of mAb 5E10to immobilized MLC1was not observed.Im-portantly,we saw significant binding ofthe bispecific antibodyFigure 4.Reaction of two independently modi fied antibodies (HyNic-MLM508and 4FB(PEG)4-5E10)to form a bispeci fic construct mixture.The reactant-modi fied antibody mixtures are represented with one modi fication per molecule and without unmodi fied antibody for sim-plicity.Reaction products not shown include unmodi fied antibodies,1,2,heterodimers bearing multiplecross-links,and multimers.Figure 5.Generation of the bispeci fic antibody as monitored by UV Àvis spectroscopy.(A)Bis-arylhydrazone (rectangle)generated in the reaction of 4FB(PEG)4-5E10with HyNic-MLM508(see Figure 4).(B)The progress of the cross-linking reaction shown in Figure 4was monitored by the absorbance of the bis-arylhydrazone at 354nm (dashed,t =0;solid,t =1h).A 354plateaued 1h after initiation.Figure 6.Analysis of reaction products by nonreducing SDS ÀPAGE:lane 1,unmodi fied MLM508(2μg);lane 2,unmodi fied mAb 5E10(2μg);lane 3,unmodi fied MLM508and 5E10combined (2μg each,4μg total);lane 4,bispeci fic antibody reactant products (4μg).Asterisk indicates bispeci fic antibody.to immobilized MLC1,and this binding was inhibited by preincuba-tion of the ELISA wells with free,unmodified MLM508(Figure 7B).We used flow cytometry to test the anti-CD90portion of the bispeci fic construct.Pig BMMSCs expressing CD90were treated with individual unmodi fied antibodies,the bispeci fic antibody mixture,and excess unmodi fied anti-CD90followed by the bispe-ci fic antibody mixture.No nonspeci fic binding of MLM508to BMMSCs was observed (data not shown).Bispeci fic antibody binding to BMMSC was detected with goat-anti-mouse IgG2a-FITC (Figure 7C).Excess unmodi fied anti-CD90inhibited the bispeci fic antibody from binding to BMMSCs by more than 90%(Figure 7C).Functional Activity of the Bispecific Antibody.Parallel-plate flow chambers were used to test whether the bispecific agent we generated could increase the binding of pig BMMSCs to immobilized MLC1.MLC1was immobilized in the cham-ber,and BMMSCs pretreated with bispecific antibody or left untreated (as controls)were perfused into the flow chamber.At 2.5dyn/cm 2,40%of the bispecific antibody-treated BMMSCs remained adherent to immobilized MLC1substrate,whereas the percent of cells adhered in the control chambers was negligible (Figure 8).Even at high shear levels (15dyn/cm 2),10%of the bispecific antibody-treated BMMSCs were stillattached (data not shown).Figure 7.Antigen speci ficity of the bispeci fic antibody product (3).(A)Bispeci fic antibody (BiAb)highlighting the isotypes of the mAb of each respective arm.(B)ELISA analysis of BiAb binding to plastic immobilized recombinant human MLC1.MLC1was immobilized at 10μg/mL.After blocking with BSA,primary antibody was added at 10μg/mL.In the case of “excess MLM508”,unmodi fied MLM508was added to the ELISA wells (20μg/mL)0.5h before adding primary antibody.HRP-conjugated secondary antibody speci fic for IgG1(the isotype of 5E10)was used for detection.Data are presented as average A 450(SD from triplicate wells.One of three representative experiments is shown.(C)BiAb binding to pig BMMSCs as detected with anti IgG2a-FITC.To demonstrate speci fic BiAb binding,BMMSCs were pretreated with saturating levels of unlabeled mAb 5E10(10μg/mL)for 1h before adding BiAb.Histograms are representative of three experiments.’DISCUSSIONDeveloping an e ffective method for increasing the retention of transplanted stem cells in the injured myocardium would im-prove the bene fits of cell therapy after myocardial infarction.Here,we describe the generation of a novel bispeci fic antibody designed to target BMMSCs to infarct tissue.The arms of the bispeci fic antibody are composed of the anti-CD90mAb 5E10and the anti-MLC1mAb MLM508.The bis-arylhydrazone cross-linking chemistry we used in generating the construct allows for rapid quanti fication of antibody modi fications,real-time non-destructive monitoring of bispeci fic antibody generation,and the assurance of heterodimer formation.Finally,our bispeci fic anti-body product retained immunoreactivity of both arms of the construct (anti-CD90and anti-MLC1)and mediated the reten-tion of BMMSCs to MLC1under stringent adhesion conditions.The bispeci fic antibody approach has been used successfully to target human CD34+hematopoietic stem cells to ischemic myo-cardial tissue.11,12In these studies,the bispeci fic reagent com-prised an anti-CD45x anti-MLC1antibody.This approach is not suitable for use in targeting BMMSCs because CD45is not ex-pressed on this cell population.20We used the anti-CD90(Thy-1)mAb 5E10in the heteroconjugate,as CD90is a glycophosphatidy-linositol-linked cell surface glycoprotein expressed on the surface of BMMSCs.20Although primarily used in the past as a pheno-typic cell marker,CD90has now been shown to regulate several cell surface signaling receptors.21Before using the anti-CD90mAb 5E10as one arm of the bispeci fic antibody,we showed that the binding of mAb 5E10did not adversely a ffect BMMSC di ffer-entiation into adipogenic,chondrogenic,or osteogenic lineages (Figure 2).Furthermore,modi fication of mAb 5E10with the extended linker 4FB(PEG)4-PFP,at an MSR of 2.6,did not a ffect the binding of the mAB to CD90expressed on the cell surface,thus maintaining antibody functionality (Figure 3).Previously,we had modi fied mAb 5E10with S-4FB (the same moiety without the PEG extension,introduced via a NHS-ester),to an MSR of 3.0,but this modi fication inhibited mAb 5E10from binding CD90as indicated by flow cytometry experiments (Figure 3C).This indicates that the extended linker (4FB(PEG)4)was superiorin maintaining functionality after modi fication.Notably,in addition to human CD90,mAb 5E10binds pig CD90and thus is suitable for use in large animal studies.In the present study,an antibody to MLC1was used as the arm that binds antigens exposed within ischemic tissue.Because ischemia damages the myocyte cell membrane 26and exposes intracellular contents to the extracellular environment,27MLC1can be found within the interstitial tissue of damaged hearts.13Futhermore,MLC1is not cleaved by caspases 28that are active after myocar-dial apoptosis,29and circulating MLC1can be detected as early as 6h after an infarction.14À16We used S-HyNic to modify ML-M508(anti-MLC1).This modi fication allowed for the rapid determination of the MSR of MLM508by its incubation with 4-nitrobenzaldehyde,as the resulting bis-arylhydrazone adsorbs at 390nm.Modifying MLM508with S-HyNic at an MSR of 0.9did not a ffect the binding of MLM508to MLC1(Figure 1).Many methods are available for generating bispeci fic antibo-dies,including the method of chemical cross-linking of puri fied mAbs to generate a heteroconjugate.30In the method used in our study,a covalent cross-link was formed between partners when a 6-hydrazinonicotinamide moiety reacted with a 4-formylbenza-mide moiety,yielding a bis-arylhydrazone.These groups are readily introduced into proteins by using conventional NHS-ester or penta fluorophenyl ester reaction with free amines,presumably lysine side chains.We chose this approach because of its advantages in cross-linking speci ficity and the ability to readily quantify both mAb modi fications and the extent of cross-link formation.One common protein Àprotein cross-linking approach is to modify one partner with a thiol-reactive group,such as a malemide or pyridyl disul fide,and introduce free thiols into the other partner.When the two modi fied proteins are mixed,covalent intermole-cular bonds are formed.A signi ficant disadvantage of this method is the formation of homodimers.The thiol modi fied protein can form disul fide dimers and because of this,thiol-modi fied protein typically must be used immediately after production.Our approach of using two functional groups that do not naturally occur in proteins and are not self-reactive allows for storage of modi fied proteins before use and avoids homodimer formation.Additionally,the distinct chromophore of the bis-arylhydrazone cross-link allows for rapid monitoring of reactions by UV Àvis spectroscopy.When a low-volume UV Àvis spectro-meter is used,small cross-linking reactions (10μL)may be conducted and monitored in a nondestructive and nondilutive manner,making this approach suitable for using di fficult-to-obtain proteins.Several elegant strategies are now available to generate bispe-ci fic antibodies for clinical development,involving both whole IgG and Ig fragments such as Fab ’s and scFv ’s.31However,the majority of these strategies are recombinant protein based,which requires mAb sequence information and long lead times for development.An approach to generating bispeci fic antibodies that can be utilized even for antibodies that are only available com-mercially is advantageous because several di fferent mAb can be tested before a candidate for clinical development can be de-veloped and further evaluated.For in vivo studies,the bispeci fic antibody reaction product will be puri fied on an anti-IgG2a a ffinity column.This will result in the removal of free monomeric IgG1(the anti-CD90arm).Then the reagent will be incubated with BMMSCs,which will bind only the antibodies in the mixture that contain anti-CD90.Free antibody (e.g.,monomeric anti-MLC1)will be washed away from the cells,leaving only bispeci fic reagent boundto cells before transplantation.Figure 8.Bispeci fic antibody-induced retention of BMMSCs on MLC1under conditions of shear stress.Parallel-plate flow chambers were coated with MLC1or BSA as a control.Cells were either treated with the bispeci fic antibody (BiAb,5μg/mL)or untreated (controls)and then loaded onto the substrate and incubated for 0.5h at room temperature.Cell detachment was measured as shear stress increased.Data represent the average percent of cell adhesion.Error bars indicate the data range (n =2).。

第四军医大学硕士学位论文BALB/c小鼠溃疡性结肠炎远段结肠Cajal间质细胞分布及超微结构变化的研究姓名：***申请学位级别：硕士专业：内科学（儿科）指导教师：***20070501缩略语表缩略语英文全称中文全称英文缩写英文全称中文全称ICC interstitial cells of Cajal Cajal间质细胞UC ulcerativecolitis 溃疡性结肠炎DSS dextran sulfate sodium 葡聚糖硫酸钠CM circular muscle layer 环肌层LM longitudinallayer纵肌层muscleENS enteric nervous system 肠神经系统plexus 肌间神经丛MYP myentericplexus 粘膜下神经丛SMP submucosalAch acetylcholine 乙酰胆碱VIP vasoactive intestinal peptide 血管活性肠肽P P物质SP substanceGAS gastrin 促胃液素（胃泌素）CCK cholecystokinin 胆囊收缩素MT motilin 胃动素SS somatostatin 生长抑素IC-MY myenteric plexus ICC 肠肌丛Cajal间质细胞IC-SM submucosal plexus ICC 粘膜下丛Cajal间质细胞IC-DMP deep muscular plexus ICC 深肌层Cajal间质细胞ICC 肌内Cajal间质细胞IC-IM intramuscularcell 平滑肌细胞mucleSMC smoothSCFstem cell factor 干细胞因子 MCGF mast cell growth factor 肥大细胞生长因子SW slow wave慢波 BERbasic electrical rhythm 基本电节律 DGIM disordersof gastrointestinal motility 胃肠动力疾病 GER gastroesophagealreflux 胃食管反流 LESlower esophageal sphincter 食管下括约肌 AOCAchalasia of cardia 贲门失弛缓症 IHPS infantilehypertrophic pyloric stenosi 婴儿肥厚性幽门狭窄 IGPidiopathic gastric perforation 特发性胃穿孔 CIIPChronic idiopathic intestinal pseudo-obstruction 慢性假性肠梗阻 NEC necrotizingenterocolitis 坏死性小肠结肠炎 CIA congenitalintestinal atresia 先天性肠闭锁 HD Hirschsprung’sdisease 先天性巨结肠 STC slowtransit constipation 慢传输型便秘 CD Crohn’sdisease 克罗恩病 MMIHS Megacystismicrocolon intestinal hypoperistalsis syndrome 巨膀胱－小结肠－肠蠕动迟缓综合征 ARM anorectalmalformations 肛门直肠畸形 IASA internal anal sphincter肛门内括约肌弛缓不能独创性声明秉承学校严谨的学风与优良的科学道德，本人声明所呈交的论文是我个人在导师指导下进行的研究工作及取得的研究成果。

专利名称：科罗索酸在制备抗肿瘤药物中的应用专利类型：发明专利
发明人：凌昌全,辛海量,徐燕丰,苏永华,盛佳钰申请号：CN200910057252.7
申请日：20090513
公开号：CN101884636A
公开日：
20101117
专利内容由知识产权出版社提供
摘要：本发明涉及医药技术领域，具体涉及科罗索酸在制备抗肿瘤药物中的应用，辅以药学上可接受的辅料，可制备成各种口服、注射、外用制剂，具有较好的开发应用前景。

本发明人在发现其具有较强的抗肿瘤作用的基础上，还进一步阐明其抗肿瘤作用的机理为由阻滞肿瘤细胞于S期及诱导细胞凋亡介导。

申请人：中国人民解放军第二军医大学
地址：200433 上海市杨浦区翔殷路800号
国籍：CN
代理机构：上海浦一知识产权代理有限公司
代理人：潘诗孟
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专利名称：取得关于嗜酸性粒细胞性呼吸道炎症的信息的装置及方法
专利类型：发明专利
发明人：长谷川武宏
申请号：CN201410513445.X
申请日：20140929
公开号：CN104515858A
公开日：
20150415
专利内容由知识产权出版社提供
摘要：本发明提供取得关于嗜酸性粒细胞性呼吸道炎症的信息的计算机系统。

此计算机系统含处理器，及存储器。

存储器含用于在处理器的控制下，在计算机系统上运行指定的操作的软件指令。

指定的操作包括：测定受试者的样品中的选自CXCL9及CCL27的至少1种标志物的量的步骤；以及基于得到的测定值，取得受试者中的关于嗜酸性粒细胞性呼吸道炎症的信息的步骤。

申请人：希森美康株式会社
地址：日本兵库县
国籍：JP
代理机构：中国国际贸易促进委员会专利商标事务所
代理人：罗菊华
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