CN110623982A - 卵巢表面上皮细胞的3d-emt免疫活性制剂及制备与应用 - Google Patents
卵巢表面上皮细胞的3d-emt免疫活性制剂及制备与应用 Download PDFInfo
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Abstract
本发明涉及一种卵巢表面上皮细胞的3D‑EMT免疫活性制剂及制备与应用，在2ml的冻存管中保存有经160Gy X射线辐照修饰的卵巢表面上皮细胞的3D‑EMT微球活性制剂，微球数为5×103个,球直径为195±25μm，冻存液为人的AB血浆，总体积为1ml，于－196℃液氮中保存。其制备方法在无菌下分五步：1.分离卵巢表面上皮细胞，2.卵巢表面上皮细胞的贴壁扩大培养，3.卵巢表面上皮细胞在无血清动态悬浮培养体系中诱导转化成3D‑EMT微球，球直径为195±25μm，4.3D‑EMT微球接受X射线辐照修饰，5.3D‑EMT微球免疫活制剂的制备。本发明制剂具有为免疫衰老/免疫缺陷引起的包括艾滋病、进展期恶性肿瘤、慢性感染、老年免疫功能低下等相关疾病开创全新的药物制剂应用。
Description
技术领域
本发明涉及生物医学工程技术，特别是一种用无血清动态悬浮法诱导卵巢表面上皮细胞发生上皮间质转化(Epithelial-mesenchymal transition,EMT)和间质上皮转化(Mesenchymal-epithelial transition,MET)而成的3D-EMT微球，射线辐照修饰制备为多能免疫活性制剂及制备与应用。
背景技术
卵巢表面上皮细胞(Ovarian surface epithelium cell,OSEC)是一类表型不确定，分化特点不明显，受环境变化刺激可向多个方向分化的细胞，是包被于卵巢表面的单层的立方形或长方形，具有间质细胞特性的上皮细胞。卵巢表面上皮先前被称为“生发上皮”，人们曾经错误地认为它可以产生新的生殖细胞。早期胚胎发育过程中卵巢表面上皮来源于体腔上皮，这些体腔上皮在皮质的间叶细胞中增殖；成年妇女的卵巢表面上皮细胞是单层的上皮细胞，分布于卵巢的皮质表面。这些细胞并不是扁平或方形的细胞而是不确定的表型较少分化的单层细胞。具有接受不同环境信号刺激后分化成不同类型细胞的能力,因其与腹膜具有相同的胚胎起源。因此，有着和腹膜间质细胞相同的特征。由于卵巢具有定期排卵的生理功能，需要上皮-间质转化(EMT)和间质-上皮转化(MET)的特殊生物学过程来参与卵巢表面上皮的修复。卵巢排卵后，启动EMT，使卵巢表面上皮细胞转化为间充质样细胞获得迁移能力，来修复破溃的组织，这一过程中DNA损伤修复若出现异常，OSEC的化生异常或过度增殖都会引起细胞的恶性增殖和转化而导致肿瘤的发生。EMT是正常卵巢表面上皮细胞的生物学特性。而在胸腺(Thymus)的器官发育和老化的过程中也存在着EMT/MET的发生，胸腺的组织结构的进化与退化中，胸腺上皮细胞(Thymic epithelial cells,TECs)可以在上皮样与间质样细胞之间发生相互转化(EMT/MET)进而促使胸腺退化功能丧失或者促使进化，胸腺功能得以恢复。
胸腺是机体免疫系统的主要中枢免疫器官，是免疫细胞发生、分化和成熟的重要场所。胸腺位于胸骨柄后方的前纵隔上部，腺体后面附着于心包及大血管前面，由不对称的左、右两叶组成，其形状不一，有时呈短粗肥厚或长扁条状，胸腺的大小和结构随着年龄的不同具有明显的差异。胸腺在胚胎期的第20周发育成熟，是发育最早的免疫器官，到出生时胸腺约重15～20g，以后逐渐增大，到青春期可达30～40g，青春期后，胸腺随年龄增长而逐渐萎缩退化，到老年时，经历EMT之后，基本被脂肪组织所取代。随着胸腺的逐渐萎缩，功能的衰退，细胞免疫力逐渐下降，对感染和肿瘤的监视功能也减低，因此在发育早期摘除胸腺，会带来严重的免疫功能缺陷。外源性胸腺相关的组织或细胞移植，虽然在一定程度上有望纠正这一免疫缺陷，但由于外源性胸腺上皮细胞(TECs)对宿主固有组织的淋巴祖细胞的发育，分化存在不适应，选择输出的淋巴细胞可能会导致机体特异性自身免疫性疾病或其他免疫失调，从而诱发人类白细胞抗原(Human leukocyte antigen，HLA)/主要组织相容性复合物(Majorhistocompatibility complex，MHC)不相容事件的发生。事实上，只有通过内源性地更新中枢免疫器官，才能为免疫老化或免疫缺陷相关疾病患者找到一种切实可行且安全有效的治疗途径。基于卵巢表面上皮细胞的3D-EMT多能免疫活性制剂在免疫缺陷动物(Balb/Cnu/nu)及免疫老化的灵长类模型中重置更新中枢免疫微环境的能力，为免疫缺陷病及免疫老化相关疾病探索切实可行的临床治疗途径，以期解决当前治疗这些免疫缺陷相关疾病所面临的困境。
发明内容
本发明正是为了克服目前在治疗免疫缺陷引起的相关疾病所面临的困境，开创性的诱导卵巢表面上皮细胞在无血清动态悬浮培养体系中，诱导其处于EMT/MET转化中间过渡态，制备卵巢表面上皮细胞的3D-EMT多能免疫活性制剂能够为免疫衰老/免疫缺陷引起的包括艾滋病(AIDS)、进展期恶性肿瘤、慢性感染、老年免疫功能低下等相关疾病开创一种全新的药物制剂的应用。
卵巢表面上皮细胞，是包被于卵巢表面的单层的立方形或长方形，具有间质细胞特性的上皮细胞。当卵巢不排卵时，OSEC就会处在静止的间质状态，而同时具有上皮细胞和间质细胞的特性。我们利用卵巢表面上皮细胞具有EMT/MET的特性，及在胸腺的组织结构演化与老化过程中，胸腺的上皮细胞将经历EMT/MET的转化这一共同特性和3D的细胞微环境可以有效增强机体免疫治疗反应活性。
本发明的任务是这样实现的：在无血清动态悬浮培养体系中，诱导卵巢表面上皮细胞为3D-EMT多能免疫活性制剂，160GyX射线辐照修饰3D-EMT多能免疫活性制剂，在2ml的冻存管中保存,3D-EMT微球数为5×103个,微球直径为195±25μm，冻存液为人的AB血浆，总体积为1ml，于－196℃液氮中保存待用。
上述射线修饰3D-EMT多能免疫活性制剂的制备方法：其操作是在无菌条件下分为以下五个步骤制备：1.分离卵巢表面上皮细胞，2.卵巢表面上皮细胞的贴壁扩大培养，3.卵巢表面上皮细胞在无血清动态悬浮培养体系中诱导转化成3D-EMT微球，微球直径为195±25μm，4.3D-EMT微球接受160Gy X射线辐照修饰，5.射线辐照修饰3D-EMT微球免疫活性制剂的制备。
本发明卵巢表面上皮细胞的3D-EMT微球免疫活性制剂在制备工程化药物中的应用，制备对免疫缺陷/免疫衰老引起的包括艾滋病(AIDS)、老年免疫功能低下、慢性感染引起的相关疾病开创一种全新的药物制剂的应用。
本发明的卵巢表面上皮细胞的3D-EMT免疫活性制剂在制备工程化药物中的应用，制备对抑制进展期恶性肿瘤的发展，作为一种全新的广谱抗肿瘤制剂的应用。
本发明的3D-EMT微球免疫活性制剂的使用，选择在免疫缺陷/免疫衰老引起的包括艾滋病(AIDS)、老年免疫功能低下、慢性感染和肿瘤的宿主，注射部位选择皮下注射，剂量为5×103个3D-EMT微球,首次免疫注射后，2周后再进行第二次免疫注射，之后每周注射1次加强，总共4次注射。
附图说明以下是本发明说明书附图的图面说明：
图1是本发明卵巢表面上皮细胞贴壁常规培养与3D-悬浮培养形成EMT球的动态比较：E-上皮部分，M-间质转化部分。
图2是本发明卵巢表面上皮细胞3D-EMT球经160GyX射线的辐照修饰改良后制备为EMT多能免疫活性制剂(ETSB)的形态及增殖活性的检测：3D-EMT球失去了增殖能力。
图3是本发明卵巢表面上皮细胞贴壁常规培养(Control)，贴壁常规培养细胞经过辐照修饰改良(2D-CB)和3D-EMT球经过辐照修饰改良(3D-EMT)。三种细胞的转录组差异表达基因(DEG)的表达量热图：Control,2D-CB和3D-ETSB三组的主要差异表达基因聚类热图比较。
图4是本发明各制剂组对免疫缺陷鼠治疗前后中枢免疫器官的活化更新情况的检测，(A)各制剂组对免疫缺陷鼠治疗前后胸腔MRI活体扫描中枢免疫器官的更新情况，3D-EMT微球免疫活性制剂(ETSB)组宿主治疗后的中枢免疫器官明显更新。(B)是本发明各制剂组对免疫缺陷鼠治疗后，通过活体胸腔探查小鼠中枢免疫器官胸腺的更新情况，ETSB组宿主的中枢免疫器官胸腺显著更新活化。
图5是本发明(A)各制剂组对免疫缺陷鼠免疫治疗后各组更新的中枢免疫器官胸腺的体积大小统计分析。(B)是本发明是各制剂组对免疫缺陷鼠免疫治疗后更新的中枢免疫器官胸腺的MHC和HLA基因表达量的检测，各组小鼠均不表达HLA,说明免疫缺陷鼠的胸腺得到了内源性的更新。
图6是本发明使获得免疫更新小鼠对(A)肺癌，(B)乳腺癌，(C)胃癌的肿瘤模型疗效比较，ETSB组的最终无瘤生存率均达到80％以上，具有很好的抗肿瘤效应。
图7是本发明对免疫更新荷瘤宿主肿瘤转移指数的作用比较，ETSB组的荷瘤小鼠的肺与淋巴结的肿瘤转移指数趋向0％。
图8是本发明(A)各种制剂对免疫老化的恒河猴免疫治疗后的外观形态学观察，ETSB组的恒河猴外观形态更年轻化。(B)各制剂对免疫老化的恒河猴免疫治疗后胸腔活体MRI扫描成像，ETSB组的中枢免疫器官胸腺仍然存在:箭头为中枢免疫器官胸腺的位置。
图9是本发明的各制剂组恒河猴外周血淋巴细胞CD3和CD45RA的流式检测，3D-ETSB组的Naive-T细胞比例较对照组显著增高，表明ETSB制剂组重新启动老化的中枢免疫功能.
图10是本发明的各制剂组的恒河猴的外周血淋巴细胞TREC的检测(T细胞受体重排删除环T cell receptor rearrangement excision circles，TREC)，ETSB组的表达量较对照组显著增高，表明ETSB制剂组重新启动老化的中枢免疫功能。
本发明与现有技术相比具有以下优点：
1.该3D-EMT多能免疫生物活性制剂采用射线辐照修饰卵巢表面上皮细胞3D-EMT微球制作，一方面该制剂的细胞有同时处于EMT/MET转化过渡态的各类型细胞，与胸腺发育过程中胸腺上皮细胞的EMT/MET转化调控胸腺的进化与退化，能够内源性的调控免疫缺陷宿主退化的中枢免疫和延缓免疫老化宿主的免疫退化；另一方面活化的中枢免疫微环境，能够调控全身的免疫微环境，可以抵抗包括HIV和肿瘤新生物在内的多种外来生物学负担；
2.X射线辐照对卵巢表面上皮细胞3D-EMT微球的免疫原性起到持久的保护作用，长效刺激机体，使机体的免疫微环境的到持续更新，激发更强的抵抗外来生物学负担的免疫功能；
3.该生物制剂制备简便，成本低廉，具有大规模工程化培养的可能，为免疫缺陷和老化引起的相关疾病、进展期恶性肿瘤的治疗提供潜在的理想资源。
具体实施方式本发明以下将结合实施例和说明书附图作进一步详述：
实施例1.一种卵巢表面上皮细胞的3D-EMT免疫活性制剂，是在2ml的冻存管中保存有经160Gy X射线辐照修饰的卵巢表面上皮细胞的3D-EMT微球活性制剂，3D-EMT微球数为5×103个,微球直径为195±25μm，冻存液为人的AB血浆，总体积为1ml，于－196℃液氮中保存待用。
实施例2.卵巢表面上皮细胞的3D-EMT免疫活性制剂的制备：其操作是在无菌条件下分为以下五个步骤制备：(1)分离卵巢表面上皮细胞；(2)卵巢表面上皮细胞的贴壁扩大培养；(3)卵巢表面上皮细胞在无血清动态悬浮培养体系中诱导转化成3D-EMT微球，微球直径为195±25μm；(4)3D-EMT微球接受160Gy X射线辐照修饰；(5)射线辐照修饰3D-EMT微球免疫活性制剂的制备：收集3D-EMT微球装入2ml冻存小管，每支3D-EMT微球数为5×103个,微球直径为195±25μm，冻存液为人的AB血浆，总体积为1ml，于－196℃液氮中贮存，解冻后直接使用。
制备操作过程详述如下：
(1)分离卵巢表面上皮细胞：取卵巢组织标本，在医院经过供者知情同意，放入无菌生理盐水中，用电动移液器吸取无菌的1×PBS溶液，冲洗卵巢组织。用无菌手术剪剥离卵巢组织，并剪碎，放入装有胰酶的BD管中，放入37℃的培养箱中，30min后，从培养箱中取出BD管，来回晃动几次，取出没有消化的组织，放入另一个新的装有胰酶的BD管中，继续消化,在BD管中加入含有胎牛血清的培养基，终止消化,1200rpm离心3min,弃掉上清，用含10％FBS的培养基重悬分离出来的卵巢表面上皮细胞，加入培养皿中，放入37℃的细胞培养箱中培养分离出来的卵巢表面上皮细胞。
(2)卵巢表面上皮细胞的贴壁扩大培养：显微镜下观察细胞的贴壁情况，避免剧烈晃动，根据细胞的生长状况，进行换液和传代，取对数生长期细胞用于诱导重塑筛选处于EMT状态的卵巢上皮细胞，并留一部分进行保存备用；
(3)卵巢表面上皮细胞在无血清动态悬浮培养体系中诱导转化成3D-EMT微球：取上述对数生长期的卵巢表面上皮细胞，用无血清DMEM/F12培养基重悬于低粘附的培养皿中，置于37℃，5％CO2的培养箱中培养,每隔两天换一次液，重新置于37℃，5％CO2的培养箱中培养,细胞均贴壁之后，换液，将培养皿放在60rpm的水平摇床(37℃，5％CO2培养箱)上，根据细胞状态进行加液或换液，10天之后，收集细胞，低速离心，无血清DMEM/F12重悬，分到新的培养皿中，37℃，5％CO2培养箱中培养，培养皿再放回摇床上，形成3D-EMT细胞微球(参见说明书附图1)，低速离心，收集细胞微球；
(4)3D-EMT微球接受160Gy X射线辐照修饰：通过160Gy X射线辐照卵巢表面上皮细胞3D-EMT微球，建立高稳定型的射线辐照修饰化3D-EMT免疫活性制剂(参见说明书附图2)；
(5)射线辐照修饰3D-EMT微球免疫活性制剂的制备：收集上述辐照后3D-EMT免疫活性制剂，球直径为195±25μm(参见说明书附图2)，装入2ml冻存小管,每支5×103个射线辐照修饰的3D-EMT微球，冻存液为人的AB血浆，总体积为1ml，于－196℃液氮中贮存，解冻后直接使用。
实施例3.卵巢表面上皮细胞的3D-EMT免疫活性制剂的多能型鉴定：将卵巢表面上皮细胞贴壁常规培养(Control)，贴壁常规培养细胞经过辐照修饰改良(2D-CB)和3D-EMT球经过辐照修饰改良(3D-EMT)这三种细胞通过转录组的测序及生物信息分析，对Control,2D-CB和3D-ETSB三组的主要差异表达基因进行聚类热图比较(参见说明书附图3)。
实施例4.卵巢表面上皮细胞的3D-EMT免疫活性制剂在免疫缺陷宿主Balb/Cnu/nu小鼠免疫接种的方案：(1)取6周龄的免疫缺陷Balb/Cnu/nu实验小鼠随机分成3组，分别是Control(生理盐水)，CB(正常细胞生物制剂组)，ETSB(卵巢表面上皮细胞逆转的3D活性生物制剂)，接种各制剂之前，先对其进行胸腔核磁共振(MRI)的扫描，观测其中枢免疫器官原基的发育情况。(2)在各组小鼠的左侧背部皮下分别在Day1、day14、day21、day28分别注射各制剂ETSB的注射剂量为5×103个3D-EMT微球，CB的注射剂量为5×105个细胞，Control的注射剂量为0.1ml的生理盐水，共四次。(3)治疗完成后2周，再对其进行胸腔核磁共振(MRI)的扫描(参见说明书附图4A)。(4)进一步通过胸腔探查各组的中枢免疫器官胸腺的发育情况(参见说明书附图4B)。
实施例5.卵巢表面上皮细胞的3D-EMT免疫活性制剂免疫治疗后的免疫缺陷宿主更新的胸腺的体积大小的统计分析(参见说明书附图5A)，与对照组相比有统计学意义(*P＜0.05，***P＜0.001)。
实施例6.卵巢表面上皮细胞的3D-EMT免疫活性制剂免疫治疗后的免疫缺陷宿主更新的胸腺组织的MHC和HLA的表达情况通过RT-PCR进行检测(参见说明书附图5B)，均不表达HLA，说明胸腺获得内源性更新。
实施例7.卵巢表面上皮细胞的3D-EMT免疫活性制剂免疫治疗后的免疫缺陷宿主对外来生物学负担的抵抗，在获得免疫更新的小鼠分别建立肺癌(A549)，乳腺癌(MDA-231)和胃癌(BCG-803)肿瘤模型，结果显示各肿瘤模型的无瘤生存率均达到80％以上(参见说明书附图6A-C)。
实施例8.卵巢表面上皮细胞的3D-EMT免疫活性制剂免疫治疗后的免疫缺陷宿主对外来生物学负担的抵抗,肿瘤模型中ETSB组宿主的肿瘤转移指数趋向0％，(参见说明书附图7)。与对照组相比，结果有统计学意义。
实施例9.卵巢表面上皮细胞的3D-EMT免疫活性制剂在非人灵长类恒河猴的免疫治疗，可见ETSB组的恒河猴与对照组相比，外观形态更年轻化(参见说明书附图8A)。
实施例10.卵巢表面上皮细胞的3D-EMT免疫活性制剂在非人灵长类恒河猴的免疫治疗，我们对治疗完成的恒河猴进行了胸腔MRI扫描，可见ETSB组的中枢免疫器官胸腺仍然存在，而对照组的均萎缩退化(参见说明书附图8B)。
实施例11.卵巢表面上皮细胞的3D-EMT免疫活性制剂在非人灵长类恒河猴的免疫治疗,采集恒河猴的外周血,对外周血淋巴细胞进行CD3和CD45RA的流式检测，3D-ETSB组的Naive-T细胞比例较对照组明显上升，表明ETSB制剂组重新启动老化的中枢免疫功能(参见说明书附图9)。
实施例12.卵巢表面上皮细胞的3D-EMT免疫活性制剂在非人灵长类恒河猴的免疫治疗，采集恒河猴的外周血，对其进行TREC的检测，ETSB组的表达量较对照组显著增高(参见说明书附图10)，表明ETSB制剂组重新启动老化的中枢免疫功能。
Claims (4)
1.一种卵巢表面上皮细胞的3D-EMT免疫活性制剂，其特征在于：是在2ml的冻存管中保存有经160Gy X射线辐照修饰的卵巢表面上皮细胞的3D-EMT微球活性制剂，3D-EMT微球数为5×103个,微球直径为195±25μm，冻存液为人的AB血浆，总体积为1ml，于－196℃液氮中保存待用。
2.一种卵巢表面上皮细胞的3D-EMT免疫活性制剂的制备方法，其特征在于：其操作是在无菌条件下分为以下五个步骤制备：
(1)分离卵巢表面上皮细胞:
取卵巢组织标本，在医院经过供者知情同意，放入无菌生理盐水中，用无菌的1×PBS溶液，冲洗卵巢组织,剥离卵巢组织，并剪碎，放入装有胰酶的BD管中，放入37℃的培养箱中，30min后，取出BD管，来回晃动几次，取出没有消化的组织，放入另一个新的装有胰酶的BD管中，继续消化,在BD管中加入含有胎牛血清的培养基，终止消化,1200rpm离心3min,弃掉上清，用含10％FBS的培养基重悬分离出来的卵巢表面上皮细胞，加入培养皿中，放入37℃的细胞培养箱中培养分离出来的卵巢表面上皮细胞；
(2)卵巢表面上皮细胞的贴壁扩大培养：
显微镜下观察细胞的贴壁情况，避免剧烈晃动，根据细胞的生长状况，进行换液和传代，取对数生长期细胞用于诱导重塑筛选处于EMT状态的卵巢表面上皮细胞，并留一部分进行保存备用；
(3)卵巢表面上皮细胞在无血清动态悬浮培养体系中诱导转化成3D-EMT微球，微球直径为195±25μm：
取上述对数生长期的卵巢表面上皮细胞，用无血清DMEM/F12培养基重悬于低粘附的培养皿中，置于37℃，5％CO2的培养箱中培养,每隔两天换一次液，重新置于37℃，5％CO2的培养箱中培养,细胞均贴壁之后，换液，将培养皿放在60rpm的水平摇床(37℃，5％CO2培养箱)上，根据细胞状态进行加液或换液，10天之后，收集细胞，低速离心，无血清DMEM/F12重悬，分到新的培养皿中，37℃，5％CO2培养箱中培养，培养皿再放回摇床上，形成3D-EMT细胞微球，低速离心，收集细胞微球；
(4)3D-EMT微球接受160Gy X射线辐照修饰：
通过160Gy X射线辐照卵巢表面上皮细胞3D-EMT微球，建立高稳定型的射线辐照修饰化3D-EMT免疫活性制剂；
(5)射线辐照修饰3D-EMT微球免疫活制剂的制备：收集3D-EMT微球装入2ml冻存小管，每支3D-EMT微球数为5×103个,微球直径为195±25μm，冻存液为人的AB血浆，总体积为1ml，于－196℃液氮中贮存，解冻后直接使用。
3.根据权利要求1一种卵巢表面上皮细胞的3D-EMT免疫活性制剂在制备工程化药物中的应用，其特征在于：对免疫缺陷/免疫衰老引起的包括艾滋病(AIDS)、老年免疫功能低下、慢性感染相关疾病产生直接作用而开创一种全新的药物制剂的应用。
4.根据权利要求1一种卵巢表面上皮细胞的3D-EMT免疫活性制剂在制备工程化药物中的应用，其特征在于：对抑制进展期恶性肿瘤的发展，作为一种新型广谱抗肿瘤药物制剂的应用。
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