CN116284391A - 使用胰岛干细胞和抗体治疗糖尿病 - Google Patents
使用胰岛干细胞和抗体治疗糖尿病 Download PDFInfo
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Abstract
本发明涉及使用胰岛干细胞和抗体治疗糖尿病。本发明提供了分离的胰岛干细胞具有分泌胰岛素的功能。同时制备获得了特异性针对CD3的单克隆抗体，该单克隆抗体具有激活T细胞的功能，同时将该抗体与胰岛干细胞联用后能够有效的抑制小鼠体内免疫机制降低血糖值，可以用于制备治疗糖尿病的药物组合物，具有极好的应用前景。
Description
技术领域
本申请涉及生物领域，更具体的涉及使用胰岛干细胞和抗体治疗糖尿病。
背景技术
糖尿病是一种慢性疾病，当胰脏不能产生足够的胰岛素或者当机体不能有效利用胰岛素时就会产生高血糖症，最终导致多器官损坏，神经系统和血管受累尤重。糖尿病发生受多种因素的影响，包括遗传、衰老和不良生活方式等。近年来，随着人们生活水平的提高和以高能量、高脂肪和高动物性食物为特征的膳食结构的改变，我国糖尿病的发病率呈快速上升趋势。其中糖尿病分为1型糖尿病和2型糖尿病。其中，1型糖尿病是一种由多种病因引起的针对胰岛β细胞的自身免疫性疾病，需应用外源性胰岛素控制血糖，目前尚无根治办法。干细胞是一类具有自我复制能力的多潜能细胞，具有在一定条件下分化为各种细胞、组织、器官的功能。由于糖尿病的发病机制主要为胰岛功能损害造成胰岛素绝对或相对分泌不足，因此，通过体内或体外诱导干细胞定向分化为具有胰岛素分泌能力的细胞，重建胰岛功能，有望彻底治愈1型糖尿病。
目前干细胞治疗1型糖尿病的研究可分为胚胎干细胞(ESC)和成体干细胞(ASC)。ASC存在于胎儿和成人组织及器官中，是已分化组织中的未分化细胞，具有自我更新能力。虽然ASC可能不具有ESC那样的全能性，但在一定条件下，ASC可跨越传统胚层概念的界限，分化为其他胚层来源的细胞，这种特性称为干细胞的可塑性，也有人称之为横向分化或者转分化。目前在1型糖尿病中研究较多的ASC是间充质干细胞、造血干细胞、胰岛干细胞。
胰岛干细胞也成为了胰腺干细胞，是胚胎发育中没有进行终末分化的胰腺细胞，能分化为各种胰腺细胞，成为糖尿病治疗的重要干细胞来源。Peck等成功地从人和小鼠的胰腺导管上皮分离了干细胞，在体外培养条件下能快速增殖，并失去导管上皮细胞特异性的表型，该细胞在合适的条件下可分化形成诱导的各种分泌细胞，具有多向分化潜能，被称为胰腺干细胞。国外也有人将小鼠胰腺组织制成单细胞悬液，用添加正常小鼠血清的低糖EHAA培养基对其进行培养，直到生长出单层上皮样的细胞，这些干细胞具有很强的自我更新能力及分化潜能。Ramiya等长期(超过3年)培养了胰腺导管中的干细胞，在整个培养过程中，它们均保持着分化为胰岛细胞的潜能。BonnerWeir等对分离于人胰腺组织的胰管细胞进行培养，成功诱导胰腺干细胞分化为既含胰管细胞又含胰岛细胞的细胞群，这些细胞群能分泌胰岛素，其分泌量随着糖浓度的增高而增多，他们用免疫组化等方法证明了这些细胞群含有胰岛的所有细胞类型。很显然，扩增及诱导胰腺干细胞分化是获得β细胞替代物更直接的途径。
胰岛β细胞主要功能是分泌胰岛素。胰岛素是由前体分子胰岛素原(proinsulin)通过型I、II型内肽酶(PC1/3、PC2)及羧基肽酶-H(CHP)裂解产生的。胰岛素原加工产生胰岛素(insulin)和C肽有以下两种途径，同时产生多种中间产物。其中的一个途径是，胰岛素原在PC2的作用下，在第65位氨基酸C端剪切成前胰岛素65-66，而后在CPH酶的作用下将第64和65位氨基酸剪切掉，然后在PC3和CPH共同作用下剪切成成熟的胰岛素和C肽；另一途径是，胰岛素在PC1/3的作用下，在第32位氨基酸C端剪切成前胰岛素32-33，然后在CPH酶的作用下将第31和32位氨基酸剪切掉，然后在PC2和CPH共同作用下剪切成成熟的胰岛素和C肽。因此，提供胰岛β细胞可以有效的用于糖尿病的治疗。
此前的研究表明，T细胞的功能与胰岛β细胞的自身免疫破坏直接相关。抗CD3的单克隆抗体与T细胞表明CD3结合，具有调剂T细胞功能的作用。因此，CD3是治疗1型糖尿病的作用靶点，通过抗CD3单克隆抗体的治疗，实现对胰岛β细胞的保护作用。目前上市的CD3单克隆抗体主要是teplizumab，而国内目前有效的单克隆抗体还不够多，提供可选择的类型也不够丰富。因此，需要开展继续的研究。
发明内容
本发明克服现有技术的缺陷，提供一种特异性针对CD3的单克隆抗体。
更进一步的，所述单克隆抗体为CD3-5F7。
在一些实施方式中，所述单克隆抗体包括：如SEQ ID NO:1所示的轻链可变区以及如SEQ ID NO:2所示的重链可变区。
进一步的，本发明的轻链可变区和重链可变区可以在保持抗体活性不变的情形下，进行一个或多个氨基酸的替换。其中“氨基酸”表示天然存在或非天然存在的α-氨基酸。术语“氨基酸”用在本申请中可以包括天然存在的氨基酸和非天然存在的氨基酸。天然存在的氨基酸包括丙氨酸(三字母密码：A1a，单字母密码：A)，精氨酸(Arg，R)，天冬酰胺(Asn，N)，天冬氨酸(Asp，D)，半胱氨酸(Cys，c)，谷氨酰胺(G1n，Q)，谷氨酸(G1u，E)，甘氨酸(G1y，G)，组氨酸(His，H)，异亮氨酸(I1e，I)，亮氨酸(Leu，L)，赖氨酸(Lys，K)，甲硫氨酸(Met，M)，苯丙氨酸(Phe，F)，脯氨酸(Pro，P)，丝氨酸(Ser，S)，苏氨酸(Thr，T)，色氨酸(Trp，W)，酪氨酸(Tyr，Y)，和缬氨酸(Va1，V)。非天然存在的氨基酸包括但不限于α-氨基己二酸，氨基丁酸，瓜氨酸，高瓜氨酸，高亮氨酸，高精氨酸，羟基脯氨酸，正亮氨酸，吡啶基丙氨酸，肌氨酸等等。
进一步的，单克隆抗体还可以与SEQ ID NO:1所示的轻链可变区以及如
SEQ ID NO:2所示的重链可变区保持90％以上的同源性，并且保持单克隆抗体的活性。术语“同源性”或“同一性”或“相似性”是指两个肽之间或两个核酸分子之间的序列相似性。同源性和同一性中的每一个可以通过比较每个序列中的位置来确定，所述序列为了比较目的而进行比对。当所比较的序列中的等同位置被相同的碱基或氨基酸占据时，则所述分子在此位置是相同的；当等同位置被相同或相似氨基酸残基(例如在空间和/或电子性质方面相似的)占据时，则所述分子可称作在此位置是同源的(相似的)。同源性/相似性或同一性的百分比表示是指，在所比较的序列共有的位置处的相同或相似的氨基酸的数目的函数。在比较两个序列中，残基(氨基酸或核酸)的缺失或额外残基的存在，也会降低同一性和同源性/相似性。
在一些实施方式中，所述单克隆抗体还包含抗体恒定区序列。
在一些实施方式中，所述恒定区序列选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE、IgD任何其中之一恒定区的序列。
进一步的，本发明提供了CD3单克隆抗体在激活T细胞中的应用。
进一步的，本发明还提供了CD3单克隆抗体在制备治疗糖尿病的药物组合物中的用途。
进一步的，本发明还提供了胰岛干细胞以及由胰岛干细胞分化制备而成的胰岛样细胞在制备治疗糖尿病的药物组合物中的用途。
进一步的，本发明还提供了CD3单克隆抗体联合胰岛干细胞或者由胰岛干细胞分化制备而成的胰岛样细胞在制备治疗糖尿病的药物组合物中的用途。
药物组合物和药品套装“药物组合物”是指用于人或动物的药物制剂。该药物组合物包含本发明的人源化单克隆抗体或其抗原结合片段以及载体、稳定剂和/或赋形剂的合适制剂。本发明提供包含本发明的单克隆抗体或其抗原结合片段的药物制剂。为了制备药物组合物或无菌组合物，让抗体或其抗原结合片段与可药用载体或赋形剂混合。可通过与生理学上可接受的载体、赋形剂或稳定剂混合，来制备呈例如冻干粉、浆液、水溶液剂或混悬剂形式的治疗及诊断药物的制剂。
该组合物含有药学上有效量的本发明单抗以及药学上可接受的载体。本文所用的术语“药学上可接受的”是指当分子本体和组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其它不良反应。本文所用的“药学上可接受的载体”应当与本发明的突变蛋白相容，即能与其共混而不会在通常情况下大幅度降低药物组合物的效果。可作为药学上可接受的载体或其组分的一些物质的具体例子是糖类，如乳糖、葡萄糖和蔗糖；淀粉，如玉米淀粉和土豆淀粉；纤维素及其衍生物，如羧甲基纤维素钠、乙基纤维素和甲基纤维素；西黄蓍胶粉末；麦芽；明胶；滑石；固体润滑剂，如硬脂酸和硬脂酸镁；硫酸钙；植物油，如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油；多元醇，如丙二醇、甘油、山梨糖醇、甘露糖醇和聚乙二醇；海藻酸；乳化剂，如Tween；润湿剂，如月桂基硫酸钠；着色剂；调味剂；压片剂、稳定剂；抗氧化剂；防腐剂；无热原水；等渗盐溶液；和磷酸盐缓冲液等。
本发明的药物组合物可根据需要制成各种剂型，并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。
合适的给药途径包括胃肠外给药(例如肌内、静脉内或皮下给药)及口服给药。可按多种常规方式给予用于药物组合物或用于实践本发明方法的抗体，这些方法例如有经口摄取、吸入、局部施用或经皮肤、皮下、腹膜内、胃肠外、动脉内或静脉内注射。在一个实施方式中，静脉内给予本发明的结合化合物。在另一个实施方式中，皮下给予本发明的结合化合物。或者，人们可以以局部而非全身方式(通常为长效制剂或缓释制剂)给予抗体，例如经由将抗体直接注射到作用位点。此外，人们可以在靶向药物递送系统中给予抗体。
治疗当用“给予”和“治疗”提及动物、人、实验对象、细胞、组织、器官或生物液时，是指将外源性药物、治疗剂、诊断剂或组合物与动物、人、受治疗者、细胞、组织、器官或生物液接触。“给予”和“治疗”可指例如治疗方法、药动学方法、诊断方法、研究方法和实验方法。治疗细胞包括让试剂与细胞接触以及让试剂与流液接触，其中所述流液与细胞接触。“给予”和“治疗”还意味着例如通过试剂、诊断剂、结合组合物或通过其他细胞对细胞进行体外和离体治疗。
有益效果
本发明提供了分离的胰岛干细胞具有分泌胰岛素的功能。同时制备获得了特异性针对CD3的单克隆抗体，该单克隆抗体具有激活T细胞的功能，同时将该抗体与胰岛干细胞联用后能够有效的抑制小鼠体内免疫机制降低血糖值，可以用于制备治疗糖尿病的药物组合物，具有极好的应用前景。
附图说明
图1单抗特异性鉴定结果图
图2各组治疗后的血糖值
具体实施方式
下面将结合实施例对本公开的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本公开，而不应视为限制本公开的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。
实施例1CD3单克隆抗体的研制
将小鼠CD3重组蛋白(商品编号G5F8FCC9C13A94，华安生物)浓度调整为1mg/mL,取0.5mL与等量的福氏完全佐剂混合、乳化,注射于Wistar大鼠腹腔，每只大鼠0.25mL。2周后，同样剂量的CD3与等量的福氏不完全佐剂混合、乳化,加强免疫。融合前3d，尾静脉注射100μgCD3蛋白。将SP2/0骨髓瘤细胞用完全RPMI1640培养液在培养瓶中进行扩大培养。选择生长旺盛、活力大于95％的骨髓瘤细胞，收集于50mL离心管中，1000r/min离心15min。弃上清液，沉淀细胞用无血清RPMI1640培养液洗涤2次，1000r/min离心15min。用无血清RPMI1640培养液重悬末次沉淀细胞，计数活细胞，配成1×106细胞/mL，以备融合使用。
免疫大鼠脾细胞悬液的制备无菌条件下取免疫大鼠脾脏，置培养皿中的200目不锈钢网上。加少量无血清RPMI1640培养液，用注射器平端将脾脏轻轻压过钢网，取出钢网，用滴管轻轻吹吸分散细胞。再以RPMI1640培养液洗涤一次，1000r/min离心10min。沉淀细胞重悬于50mL无血清RPMI1640培养液，计数淋巴细胞，以备融合使用。
细胞融合取制备好的骨髓瘤细胞与脾淋巴细胞，以1：10比例在50mL离心管中混合，1000r/min离心10min。弃尽上清，轻轻叩松留存管内的沉淀细胞。将细胞、50％PEG及无血清RPMI1640培养液均置于37℃水浴箱中预温。将50％PEG0.8mL于1min内逐滴加入细胞沉淀中，37℃水浴箱中静置1min。逐渐加入预温的无血清RPMI1640培养液30mL,轻轻摇动试管，5min内加完。在室温中以1000r/min离心10min。弃上清，沉淀细胞用10mL含小牛血清的HAT培养液重新悬浮。滴加于预先铺有饲养细胞的96孔培养板中，每孔100μL。置37℃、5％CO2孵箱培养。
选择鉴定为阳性的35个阳性孔采用有限稀释法进行杂交瘤细胞的克隆化。于96孔平底微量培养板制备巨噬细胞培养层，用一块板作阳性孔杂交瘤细胞的克隆化。收集并计数各阳性孔中的细胞。分别制备细胞数目为20、10、5/mL的细胞悬液。分别加入96孔培养板，每孔100μL。培养板置37℃、5％CO2孵箱培养。1～2周可观察到克隆生长。检测上清中的抗体，将阳性孔中杂交瘤细胞再次选出培养并作克隆化。重复4次，选取阳性反应最强，传代稳定的杂交瘤细胞CD3-1H3和CD3-5F7进行后续实验。
单抗的制备：每只大鼠腹腔内注射0.5mL降植烷。7d后于不同大鼠腹腔内分别注射1×106杂交瘤细胞CD3-1H3和CD3-5F7，2周后大多数可产生实体瘤和腹水。用注射器抽出腹水，在4℃以2000r/min离心15min，间接ELISA法检测收集腹水的效价，。将腹水加等量PBS，于搅拌下逐滴加入等体积的饱和硫酸铵。冰浴30min，2500r/min,30min，使其充分沉淀。将所得沉淀物以1mLPBS溶解至装入透析袋。对PBS充分透析、除盐换液3次，至萘氏试剂测透析外液为无黄色。将透析袋内样品取少许作适当倍数稀释后，以紫外分光度计测蛋白含量。单克隆抗体的进一步纯化按照ProteinAAgrose说明书进行，使得纯化后的抗体浓度都保持在1mg/mL。
实施例2CD3-5F7单克隆抗体的特性鉴定
利用AMC传感器，纯化出来的CD3-5F7抗体用PBST稀释到10ug/ml，CD3重组蛋白用PBST进行梯度稀释。运行流程：缓冲液1(PBST)中平衡60s，CD3-5F7抗体溶液中固化抗体300s，缓冲液2(PBST)中孵育180s，抗原溶液中结合420s，缓冲液2中解离1200s，用10mMpH1.69GLY溶液及缓冲液3(PBST)进行传感器再生，输出数据。(KD表示平衡解离常数即亲和力)，表1即为所得亲和力检测数据。
表1亲和力检测数据
| 抗体名称 | KD(nM) |
| CD3-5F7 | 4.26±0.12 |
从表1的结果可以看出，CD3-5F7具有较好的结合特异性，其解离常数仅有4.26nM，结合效果较好。
实施例3CD3-5F7单抗特异性鉴定
包被人重组CD3、小鼠重组CD3、BSA、人血清、小鼠血清，大肠杆菌裂解液的酶标板，加入1:3000稀释的CD3-5F7抗体。同时设阴性对照孔。酶标仪测定A450nm。P/N样本孔吸光度值(S)/阴性对照孔吸光度值(N)≥2.1判断为阳性。结果如图1所示。
从图1的ELISA体系的特异性鉴定结果可以看出，CD3-5F7单抗针对小鼠重组CD3以及小鼠血清的P/N值显著大于2.1，其中针对小鼠重组CD3达到了(10.53±0.34)，针对其他物质的检测结果为阴性，说明该体系能特异检测小鼠重组CD3以及小鼠血清中的CD3，也说明CD3-5F7单抗具有较好的用于区分小鼠CD3与其他物质。
实施例4抗CD3单克隆抗体CD3-5F7刺激T细胞活化及转化的活性检测
取正常小鼠外周血，用肝素钠抗凝，生理盐水2倍稀释后小心加入等体积的淋巴细胞分离液(ρ＝1.077g/mL)，2500g离心20分钟，取中间单个核细胞层，生理盐水洗2遍，用含10％FCS的RPMI1640培养液制备成细胞密度为1×109/L，加入96孔板，100μl/wall。加入相应浓度的CD3抗体CD3-5F7(2μg/ml)、PBS对照，设三个复孔，并设PBS阴性对照孔，于5％CO2、37℃培养60小时。加3H-TdR(1mCi/ml)0.5μCi/wall，继续培养16小时。收集细胞并于液体闪烁仪上计数cpm值。计算刺激指数(SI)＝(实验组cpm值-本底值)/(阴性对照组cpm值-本底值)。结果如表2所示。
表2刺激指数
| 各组 | SI |
| CD3抗体CD3-5F7 | 18.9±1.0* |
| PBS对照 | 0.14±0.02 |
3H-TdR掺入实验结果显示，抗CD3抗体CD3-5F7在体外可以激活T淋巴细胞，其刺激指数为19.3。PBS对照刺激活性与CD3相比差异显著(P<0.01)。
在实验浓度的抗体刺激后，收集细胞，分析激活后的细胞中CD8+CD25+细胞亚群的变化。结果显示，抗CD3抗体CD3-5F7刺激后的细胞中CD8+CD25+细胞由PBS对照组的(3.50±0.12)％增加到(15.2±1.14)％，显示出较好的刺激效果。
实施例5胰腺干细胞(胰岛干细胞)制备及转分化为胰岛样细胞团及鉴定
将新生5d的SD大鼠处死，分离胰腺，剪成1mm×1mm小碎块，加入三四倍体积1g/LⅣ型胶原酶(pH7.4)37℃水浴振荡消化，收集各次消化浊液，离心，沉淀细胞接种于含10％BSA(胎牛血清)DMEM/F12培养液中。37℃5％CO2培养箱48h后弃除漂浮物，贴壁细胞用0.25％胰酶-EDTA消化，将所得悬浮细胞接种于完全培养基中。每4天换液1次，纯化和扩增。倒置显微镜下动态观察细胞并摄片，另将部分细胞培养于放置有盖波片的培养皿中培养。通过CK-19和PDX-1免疫细胞化学染色证实了在CK19染色后胞浆染成棕色呈阳性。PDX-1染色细胞质和细胞核均未着色，细胞核被苏木精染成蓝色，呈阴性，说明分离制备得到了胰腺干细胞(胰岛干细胞)。
当胰腺干细胞(胰岛干细胞)增殖至培养瓶底的80％-90％时，更换诱导分化培养基：DMEM/F12、BSA2g/L、10％、5％、2％FBS、谷氨酰胺2mmmol/L、胰岛素+转铁蛋白+硒1g/L、角质细胞生长因子10μg/L、激动肽-410nmol/L、尼克酰胺10mmmol/L，每隔三四天换液，定期于倒置显微镜下观察。诱导4周后，细胞团继续增大，增多，并逐渐汇聚成圆形或椭圆形的胰岛样结构。
天然胰岛的分离纯化与鉴定：SD大鼠腹腔注射水合氯醛麻醉后，无菌条件下开腹，经胰胆管逆行灌注Ⅴ型胶原酶(1g/L)取出胰腺组织，静置于37℃水浴中消化10分钟，用非连续密度梯度离心法(四种不同浓度的Ficoll:25％、23％、20％、11％)纯化获得胰岛，经DTZ染色检测胰岛纯度较好。
诱导后胰岛细胞与天然胰岛功能学的比较：将转分化的胰岛样细胞团与纯化后的胰岛经Hank’s液洗涤3次,分别转移至高糖(16.7mmol/L)无血清RPML-1640培养基中,每孔50个胰岛,于37℃、体积分数为0.05的CO2培养箱内培养2h，收集培养液，大鼠胰岛素的测定采用放射免疫法。结果如表3所示。
表3胰岛素分泌量
| 各组 | 分泌量(pkat/L) |
| 分化胰岛 | 28.6±1.2* |
| 天然胰岛 | 720.4±22.4 |
从表3可以看出，采用分化胰岛经过高糖诱导后，也能够产生胰岛素的分泌。虽然不如天然胰岛高，但是由于培养的干细胞具有无限扩增可能，因此，可以用于大规模使用。
实施例6胰岛样细胞以及胰岛干细胞和/或CD3单克隆抗体CD3-5F7治疗I型糖尿病实验
链脲菌素制备大鼠糖尿病模型:采用完全随机法将30只SD大鼠分为链脲菌素注射组24只,正常对照组6只。用0.1mol/L柠檬酸-柠檬酸钠缓冲液(pH4.4)将链脲菌素配制成2％注射液。空腹12h后,链脲菌素注射组腹腔注射70mg/kg体质量的链脲菌素,正常对照组腹腔注射70mg/kg体质量的0.1mol/L柠檬酸-柠檬酸钠缓冲液(pH4.4)。大鼠分别于注射前、注射后48h、第5天和第8天空腹断尾采血,血糖仪测定全血血糖浓度。选取连续2次血糖浓度高于16.7mmol/L的大鼠作为糖尿病模型。
采用完全随机法将已成模的糖尿病大鼠分为4组:
实验组1：胰岛移植组5只,进行肾被膜下胰岛样细胞团移植,每只大鼠植入胰岛数(3000±100)个；
实验组2：胰岛干细胞移植组5只,肾被膜下移植未进行诱导的胰腺干细胞(胰岛干细胞),细胞数约2×106个；
模型对照组：糖尿病对照组5只,肾被膜下移植不含细胞的培养液。
空白对照组：正常小鼠组5只,肾被膜下移植不含细胞的培养液。
实验组3：CD3-5F7单抗联合胰岛移植组5只，进行肾被膜下胰岛样细胞团移植,每只大鼠植入胰岛数(3000±100)个；同时，单抗按照剂量为50μg/只，每周给药2次，共给药3周。
实验组4：CD3-5F7单抗胰岛干细胞移植组5只,肾被膜下移植未进行诱导的胰腺干细胞(胰岛干细胞),细胞数约2×106个；同时，单抗按照剂量为50μg/只，每周给药2次，共给药3周。
5组大鼠均于移植前测定空腹血糖浓度均大于16.7mmol/L,在移植后的第2周和第8周分别测空腹血糖1次。结果如图2所示。
从图2的结果可以看出，模型组的血糖浓度始终保持在高值运行，比空白组的值显著高。从实验组1和实验组2的2周血糖和8周血糖值可以看出，胰岛移植组大鼠移植后2周内,血糖浓度均有所降低,但仍高于正常血糖浓度范围,移植后血糖浓度迅速上升,可看出诱导的胰岛样细胞团植入大鼠体内可迅速发挥降糖作用,但是维持时间短,免疫排斥反应影响了其长期发挥功能导致之后一直维持高血糖状态。干细胞移植组大鼠移植后血糖处于较高水平,移植后第8周血糖显著下降,提示胰腺干细胞在体内经历了向胰岛β细胞分化的过程之后维持血糖于较低水平。实验组3和实验组4联合CD3单克隆抗体CD3-5F7使用后，能够刺激小鼠体内产生免疫调节，维持免疫耐受，导致胰岛不被免疫排斥。使得胰岛移植组的降糖效果得到显著的提高。特别是实验组4中，采用二者联合使用后，使得CD3-5F7单抗联合胰岛干细胞移植一起使用后在第8周，血糖浓度只有(4.75±0.29)mmol/L，与空白组基本接近，具有较好的治疗效果。结果显示，抗CD3单克隆抗体CD3-5F7治疗可有效逆转糖尿病。
需要说明的是，在本文中，诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者终端设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者终端设备所固有的要素。在没有更多限制的情况下，由语句“包括……”或“包含……”限定的要素，并不排除在包括所述要素的过程、方法、物品或者终端设备中还存在另外的要素。此外，在本文中，“大于”、“小于”、“超过”等理解为不包括本数；“以上”、“以下”、“以内”等理解为包括本数。
需要说明的是，尽管在本文中已经对上述各实施例进行了描述，但并非因此限制本发明的专利保护范围。因此，基于本发明的创新理念，对本文所述实施例进行的变更和修改，或利用本发明说明书及附图内容所作的等效结构或等效流程变换，直接或间接地将以上技术方案运用在其他相关的技术领域，均包括在本发明的专利保护范围之内。
Claims (7)
1.一种特异性针对CD3的单克隆抗体CD3-5F7，其特征在于所述单克隆抗体包括如SEQID NO:1所示的轻链可变区以及如SEQ ID NO:2所示的重链可变区。
2.如权利要求1所述的CD3的单克隆抗体CD3-5F7在制备刺激T细胞活化的药物中的用途，所述活化是体外活化，是非治疗目的。
3.如权利要求1所述的CD3的单克隆抗体CD3-5F7在制备治疗糖尿病的药物组合物中的用途。
4.如权利要求1所述的CD3的单克隆抗体CD3-5F7和胰岛干细胞在制备治疗糖尿病的药物组合物中的用途，其中胰岛干细胞是商业化购买的，或者是分离制备得到的。
5.如权利要求3或4所述的用途，其特征在于所述的药物组合物中含有药学上可接受的载体。
6.如权利要求5所述的用途，其特征在于药学上可接受的载体是糖类，淀粉，或纤维素。
7.如权利要求6所述的用途，其进一步的含有抗氧化剂和防腐剂。
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