CN117025502A - 前列腺前体样细胞、细胞制剂及其制备方法和应用 - Google Patents
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Abstract
本发明提供了一种前列腺前体样细胞、细胞制剂及其制备方法和应用,包括以下步骤:S0:提供前列腺原代细胞;S1:对所述S0中的前列腺原代细胞使用重编程培养基进行退分化培养以获得表达阳性标志物CD90、CD73、CD44和SOX9中至少一种的前列腺前体样细胞,其中,所述重编程培养基包括基础培养基、抑制剂和生长因子;本发明前列腺前体样细胞能够在体外实现持续培养,又能够不表达MHC二类分子,在移植前列腺前体样细胞后不需要服用抗排药,可以减少对人身体的伤害。
Description
本申请要求2022年05月10日提交的申请号为2022105036786,发明名称为“生物制剂、细胞衍生物及制备方法和应用”的中国专利申请的优先权。上述申请的内容以引用方式被包含于此。
技术领域
本发明涉及生物技术技术领域,尤其涉及一种前列腺前体样细胞、细胞制剂及其制备方法和应用。
背景技术
慢性前列腺炎症/慢性盆腔疼痛综合征(Chronic prostatitis/Chronic pelvicpain syndrome,CP/CPPS)和前列腺增生(Benign prostatic hyperplasia,BPH)属于男科疾病,对患者生活质量造成的影响巨大。慢性前列腺炎症治疗主要分药品治疗和非药物治疗,主要包括:1,抗生素,是临床上治疗慢性前列腺炎主要方式;2,α受体阻滞剂,消除排尿时前列腺内尿液返流,减少慢性前列腺炎的发生几率;3,高频透热治疗。目前治疗前列腺增生的一线药物是α受体阻剂和5α还原酶抑制剂。但是疾病产生的机制以及有效的治疗方式,相关研究一直进展缓慢,其中很重要的一个原因是前列腺正常细胞无法在体外长期培养,造成研究工作无法持续开展。因此,有必要开发新型的前列腺前体样细胞及细胞制剂以使前列腺前体样细胞的持续培养。
发明内容
本发明的目的在于提供一种前列腺前体样细胞、细胞制剂及其制备方法和应用,能够使得肝内胆管前体样细胞能够连续传代,同时,在移植前列腺前体样细胞后不需要服用抗排药,可以减少对人身体的伤害。
为实现上述目的,本发明提供了一种前列腺前体样细胞的制备方法,包括以下步骤:
S0:提供前列腺原代细胞;
前列腺原代细胞获得过程如下:
提供成熟的前列腺组织;使用无菌PBS缓冲液对成熟的前列腺组织进行清洗和灭菌处理后,使用由Ⅰ型胶原酶、无菌PBS缓冲液和中性蛋白酶消化液组成的3毫升细胞消化液在37摄氏度下对组织消化90分钟,从而获取原代细胞悬液。接着,使用70微米的无菌筛网对所述原代细胞悬液在无菌PBS缓冲液的辅助下进行筛网分选,收集滤液并去除粘液和未消化的组织,以完成筛网分选。然后,将得到的滤液离心并去除上清后,向得到的沉淀物内加入红细胞溶解平衡液进行重悬后再次离心并重复上述过程直至再次离心后在细胞沉淀中观察不到红细胞为止,以完成红细胞裂解去除,从而得到前列腺原代细胞。
值得注意的是,在前列腺原代细胞制备过程,Ⅰ型胶原酶占无菌PBS缓冲液的体积百分比为1%,中性蛋白酶占无菌PBS缓冲液的体积百分比为5%。每次离心的速率均为1000g,离心时间均为3分钟。
S1:对所述S0中的前列腺原代细胞使用重编程培养基进行退分化培养以获得表达阳性标志物CD90、CD73、CD44和SOX9中至少一种的前列腺前体样细胞,其中,所述重编程培养基包括基础培养基、抑制剂和生长因子。
可选的,还包括:
S2:将所述前列腺原代细胞放入重编程培养基中进行退分化培养,直至所述前列腺前体样细胞的融合度不低于80%,使用胰酶消化液对所述前列腺前体样细胞进行消化处理,以得到前列腺前体样细胞。
可选的,所述重编程培养基还包括Wnt信号通路激动剂、营养补充剂和缓冲液。
可选的,所述抑制剂包括抑制TGF-β信号通路的ROCK激酶抑制剂和A83-01抑制剂。
可选的,以占所述基础培养基的含量计,所述ROCK激酶抑制剂的含量为5-20μM,所述A83-01抑制剂的含量为1-5μM。
可选的,所述生长因子包括EGF、bFGF、Noggin和R-spondin1。
可选的,以占所述基础培养基的含量计,EGF的含量为10-50ng/mL,所述bFGF的含量为10-50ng/m,所述Noggin的含量为1-10ng/mL,所述R-spondin1的含量为10-30ng/mL。
可选的,所述前列腺前体样细胞不表达CD34、CD45、HLA-DR、HLA-DP和HLA-DQ。
本发明还提供了一种所述的前列腺前体样细胞的应用,所述前列腺前体样细胞用于制备治疗前列腺炎或前列腺增生的前列腺前体样细胞制剂,使用所述前列腺前体样细胞制剂干预体内动物模型后考察对前列腺炎的影响。
可选的,所述动物模型包括药物诱导的前列腺炎或前列腺增生模型。
本发明还提供了一种前列腺前体样细胞制剂,包括所述的表达阳性标志物CD90、CD73、CD44和SOX9中至少一种的前列腺前体样细胞,以及药学上可接受的载体。
本发明还提供了一种前列腺前体样细胞制剂的应用,使用所述的前列腺前体样细胞制剂干预体内动物模型。
本发明的有益效果在于:
本发明通过重编程培养基培养得到表达阳性标志物CD90、CD73、CD44和SOX9中至少一种的前列腺前体样细胞,不表达MHC二类分子,因此在移植前列腺前体样细胞后不需要服用抗排药,可以减少对人身体的伤害,该前列腺前体样细胞能够在体外实现持续培养。
附图说明
图1为本发明提供的前列腺前体样细胞的光学显微下的形态示意图;
图2为本发明中前列腺前体样细胞分别经过重编程培养基和基础培养基扩增培养后其中的基底细胞标志物含量对照图;
图3为本发明中前列腺前体样细胞分别经过重编程培养基和基础培养基扩增培养后其中的腔细胞标志物含量对照图;
图4为本发明中前列腺前体样细胞分别经过重编程培养基和基础培养基扩增培养后其中的干细胞标志物含量对照图;
图5为本发明中前列腺前体样细胞分别经过重编程培养基和基础培养基扩增培养后其中的前列腺特异性标志物含量对照图;
图6为本发明实施例的前列腺前体样细胞的基因表达标志物CD44的情况示意图;
图7为本发明实施例的前列腺前体样细胞的基因表达标志物CD73的情况示意图;
图8为本发明实施例的前列腺前体样细胞的基因表达标志物HLA-DRPQ的情况示意图;
图9为本发明实施例的前列腺前体样细胞的基因表达标志物SOX9的情况示意图;
图10为本发明实施例的前列腺前体样细胞的基因表达标志物CD90的情况示意图;
图11为本发明实施例的前列腺前体样细胞的基因表达标志物CD34的情况示意图;
图12为本发明实施例的前列腺前体样细胞的基因表达标志物CD45的情况示意图;
图13为本发明中大鼠前列腺炎模型建立过程示意图;
图14为假手术组、空白对照组和前列腺前体样细胞治疗组采用Elisa检测法检测IL-6表达水平示意图;
图15为假手术组、空白对照组和前列腺前体样细胞治疗组采用Elisa检测法检测IL-1a表达水平示意图;
图16为本发明中假手术组、空白对照组和前列腺前体样细胞治疗组中分别对前列腺病例切片HE染色的荧光照片;
图17为本发明中前列腺增生模型病理结果。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合本发明的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明的一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。除非另外定义,此处使用的技术术语或者科学术语应当为本发明所属领域内具有一般技能的人士所理解的通常意义。本文中使用的“包括”等类似的词语意指出现该词前面的元件或者物件涵盖出现在该词后面列举的元件或者物件及其等同,而不排除其他元件或者物件。
慢性前列腺炎,又叫慢性盆腔疼痛综合征(Chronic prostatitis/Chronicpelvic pain syndrome,CP/CPPS)是泌尿外科、男科常见疾病之一,以尿路症状和慢性疼痛障碍为主要征象,占前列腺炎的90~95%。CP/CPPS涉及复杂的病理生理机制,包括隐性感染、免疫、炎症、神经、内分泌、前列腺内尿反流、氧化应激和心理因素等,且不同因素之间经常交叉影响。药物疗法主要包括:1,抗生素,抗生素药物具有脂溶性高,与蛋白结合率低,分子量小以及离解度高等特点,阿奇霉素等抗生素药物具有高水平的组织穿透力,并且对细菌和支原体均有较高的活性,是临床上治疗慢性前列腺炎主要方式;2,α受体阻滞剂,使用受体阻滞剂让患者紧张的膀胱颈和前列腺组织变得松弛,从而消除排尿时前列腺内尿液返流,减少慢性前列腺炎的发生几率;3,高频透热治疗,前列腺组织在高频热的作用下,会发生血管扩张、代谢废物以及渗出物等现象,这会在一定程度上加快患者体内物质的排出。非药物治疗包括盆底物理治疗、肌筋膜触发点释放、针灸、心理支持、电身体冲击波治疗、局部热疗等。
良性前列腺增生症(Benign Prostate Hyperplasia,BPH)是老年男性常见病,主要表现为尿频,尿急,排尿困难呈进行性加重,排尿无力,尿程缩短,尿不尽或淋漓。随着我国人口的老龄化,近年来其发病率呈上升趋势。前列腺增生的目前分为组织学前列腺增生和临床前列腺增生,组织学前列腺增生是一个生理老化过程。临床前列腺增生为病理型,表现为:前列腺体积增大,膀胱出口梗阻特征。前列腺增生发生的两个重要因素是:高龄和具有功能的睾丸,睾丸分泌的雄激素,转化为双氢睾酮后发挥作用,男性外生殖器的发育、青春期前列腺的生长、老年的前列腺增生等均需双氢睾酮的存在。雄激素还能刺激产生生长因子,前列腺可产生多种生长因子,包括表皮生长因子(EGF),成纤维细胞生长因子(FGF),β-转换生长因子(TGF-β)等,生长因子与生长抑制因子的相对平衡,前列腺才能正常发育,生长。BPH的治疗,主要有三种方式:1,药物治疗:5α-还原酶抑制剂能抑制双氢睾酮的产生,可以抑制前列腺组织持续增殖;前列腺受体α1拮抗剂(α受体阻滞剂),对排尿困难的症状有缓解作用,但是药物治疗仅对轻度前列腺增生患者有一定的疗效,中重度患者的药物治疗疗效明显降低。中、重度前列腺增生患者需要手术治疗才能解除梗阻;2,开放手术包括:耻骨上经膀胱前列腺切除术、耻骨后前列腺切除术、耻骨后保留尿道前列腺切除术,开放手术存在创伤大、并发症多、术后恢复慢等缺陷;微创手术包括:经尿道前列腺切除,激光治疗,微波治疗等,创伤小恢复快,但是可能会有病情的反复,老年前列腺增生患者由于年龄过大,一般同时患有多种疾病,可能不具备手术条件。
总体来说,慢性前列腺和前列腺增生目前临床尚未有突破性的进展,还缺乏针对老年男性较好的临床治疗方式。
前列腺是男性性器官,分泌前列腺液占精液成分的30%,前列腺管由假复层上皮细胞排列而成,主要有三种细胞类型:1)分泌腔细胞,以细胞角蛋白CK8/CK18、雄激素受体(AR)和前列腺特异性抗原(PSA)为标志;2)基底细胞,以CK5/CK14/p63为标志;3)罕见的神经内分泌细胞。
基底细胞表现为双重性,即能够产生管腔和基底细胞系,表明基底细胞具有潜能,也有研究表明,在人成熟前列腺中,基底细胞和腔细胞都含有干细胞活性。多能基底干细胞可以促进前列腺基底细胞、管腔细胞和神经内分泌细胞谱系的分化。
人前列腺前体样细胞的分离、持续培养的技术困难,目前的研究尚未完全攻克。目前对于前列腺上皮干样细胞的研究,主要以模型大鼠和小鼠的前列腺为主,将鼠前列腺组织消化分离后,经过流式分选获得CD44和CD133阳性的细胞为前列腺干样细胞,并在体外进行培养。
前列腺上皮由基底细胞、分泌管腔细胞和罕见的神经内分泌细胞组成,由于前列腺上皮在小鼠和大鼠体内经过反复的雄激素剥夺和恢复后具有退化和再生的能力,可以作为研究前列腺干细胞生物学的细胞来源。研究人员通过体内肾包膜下组织再生、细胞分裂模型和体外类器官形成等分析方式,证明了前列腺基底和管腔上皮中都存在前列腺干样细胞。
多能基底干细胞可以促进前列腺基底细胞、管腔细胞和神经内分泌细胞谱系的分化,特别是在出生后早期发育期间。多能基底干细胞仅位于尿道旁壁龛,并以定向迁移的方式产生上皮祖细胞。而成年小鼠前列腺再生过程中,基底和腔细胞谱系是独立维持的。干细胞相关标志物(CD117、CD133、CD44、Trop2、CD49f、Sca1等)均存在于基底细胞和腔细胞。
前列腺炎症药物治疗需要的时间长,起效慢,都只能暂时缓解患者症状,多种治疗方式联合可取得疗效稍强于单一疗法,但是不良反应发生率和所需费用也相应增加,但是无论哪种治疗方式,都不能根治前列腺炎症,现代医学目前仍然无法明确慢性前列腺炎/慢性盆腔疼痛综合征(CP/CPPS)具体的发病机制,且对于CP/CPPS的管理和治疗未达到预期的疗效。
治疗前列腺增生的药物中,a受体阻剂起效快,对于不同位置的不同程度的前列腺肥大都有效,但只对症状比较轻、前列腺体积比较小的患者有良好的疗效,而且可能引起体位性低血压,5a还原酶抑制剂可以缩小前列腺体积,逆转疾病的进展,改善症状,但起效相对慢,一般需要连续服药2-3个月以上才起效,此外可能有勃起功能障碍、性欲低下等症,植物药治疗前列腺
增生症的机制目前还不明确,目前仍然缺乏高质量、大规模、安慰剂对照的长程临床试验来进一步检验植物药疗法的有效性和安全性。
针对现有技术存在的问题,本发明的实施例提供了一种前列腺前体样细胞的制备方法,包括以下步骤:
S0:提供前列腺原代细胞。
前列腺原代细胞制备过程如下:
提供成熟的前列腺组织;使用无菌PBS缓冲液对成熟的前列腺组织进行清洗和灭菌处理后,使用由Ⅰ型胶原酶、无菌PBS缓冲液和中性蛋白酶消化液组成的3毫升细胞消化液在37摄氏度下对组织消化90分钟,从而获取原代细胞悬液。接着,使用70微米的无菌筛网对所述原代细胞悬液在无菌PBS缓冲液的辅助下进行筛网分选,收集滤液并去除粘液和未消化的组织,以完成筛网分选。然后,将得到的滤液离心并去除上清后,向得到的沉淀物加入红细胞溶解平衡液进行重悬后再次离心并重复上述过程直至再次离心后在细胞沉淀中观察不到红细胞为止,以完成红细胞裂解去除。
值得注意的是,在前列腺原代细胞制备过程,Ⅰ型胶原酶占无菌PBS缓冲液的体积百分比为1%,中性蛋白酶占无菌PBS缓冲液的体积百分比为5%。每次离心的速率为1000g,离心时间为3分钟。
其中,Ⅰ型胶原酶来源于默克。中性蛋白酶来源于源叶生物。
S1:对所述S0中的前列腺原代细胞使用重编程培养基进行退分化培养以获得表达阳性标志物CD90、CD73、CD44和SOX9中至少一种的前列腺前体样细胞,其中,所述重编程培养基包括基础培养基、抑制剂和生长因子。
优选地,所述基础培养基为DMEM/F12培养基。
一种实施例中,所述的前列腺前体样细胞的制备方法,还包括:S2:将所述前列腺原代细胞放入重编程培养基中进行退分化培养,直至所述前列腺前体样细胞的融合度不低于80%,使用胰酶消化液对所述前列腺前体样细胞进行消化处理,以得到前列腺前体样细胞。可以通过该实施例的前列腺前体样细胞的制备方法,能够在体外持续传代培养。
一种实施例中,所述重编程培养基还包括Wnt信号通路激动剂、营养补充剂和缓冲液。
优选地,所述Wnt信号通路激动剂为CHIR99021,以占所述基础培养基的含量计,所述Wnt信号通路激动剂的含量为3μM。
优选地,所述营养补充剂包括N2(1X)和B27(1X)。所述N2(1X)的含量为培养基终体积的0.5%。所述B27(1X)的含量为培养基终体积的1%。
一种实施例中,所述抑制剂包括抑制TGF-β信号通路的ROCK激酶抑制剂和A83-01抑制剂。
一种实施例中,所述ROCK激酶抑制剂为Y-27632。以占所述基础培养基的含量计,所述ROCK激酶抑制剂的含量为5-20μM,所述A83-01抑制剂的含量为1-5μM。其中,所述A83-01作为Alk3/4/5的抑制剂来抑制TGF-β信号通路,防止前列腺前体样细胞增殖受阻。
一种实施例中,所述生长因子包括EGF、bFGF、Noggin和R-spondin1。以占所述基础培养基的含量计,所述EGF的含量为10-50ng/mL,所述bFGF的含量为10-50ng/mL,所述Noggin的含量为1-10ng/mL,所述R-spondin1的含量为10-30ng/mL。其中,所述生长因子Noggin,为BMP-4拮抗剂,拮抗BMP活性可以使干细胞增殖,同时保持其未分化状态。所述生长因子R-spondin1可调节Wnt/β–catenin信号通路,可用于维持前列腺前体样细胞增殖。
一种实施例中,所述前列腺前体样细胞不表达CD34、CD45、HLA-DR、HLA-DP和HLA-DQ。所述CD34、CD45、HLA-DR、HLA-DP和HLA-DQ属于MHC二类分子,本发明中得到的前列腺前体样细胞不表达MHC二类分子,因此在移植前列腺前体样细胞后不需要服用抗排药,可以减少对人身体的伤害,本发明的前列腺前体样细胞在治疗前列腺炎的安全性更高,应用范围更广泛,患者接受度更强。
本发明还提供了一种前列腺前体样细胞,通过所述的前列腺前体样细胞的制备方法制备得到,并能特异性表达阳性标志物CD90、CD73、CD44和SOX9中的至少一种。
本发明还提供了一种所述的前列腺前体样细胞的应用,所述前列腺前体样细胞用于制备治疗前列腺炎或前列腺增生的前列腺前体样细胞制剂,使用所述前列腺前体样细胞制剂干预体内动物模型后考察对前列腺炎的影响。
一种实施例中,所述动物模型包括药物诱导的前列腺炎或前列腺增生模型。
本发明还提供了一种前列腺前体样细胞制剂,包括所述的表达阳性标志物CD90、CD73、CD44和SOX9中至少一种的前列腺前体样细胞,以及药学上可接受的载体。
本发明还提供了一种前列腺前体样细胞制剂的应用,使用所述的前列腺前体样细胞制剂干预体内动物模型。
以下通过具体的实施例进行详细说明:
实施例
一、起始组织性质以及来源合法性声明:
以临床良性前列腺增生手术后切除的组织或者供体捐献的正常前列腺组织为来源,获取阳性表达CD90、CD73、CD44和SOX9的人前列腺前体样细胞。
具体的,所述前列腺组织经病理检查显示为正常前列腺组织。
具体的,所述临床良性前列腺增生手术后切除的组织或者供体捐献的正常前列腺组织为来源于年龄不超过70岁的患者的手术样本,患者经医学检查无传染性病毒感染,患者在术前6个月内未使用过类固醇激素药物。患者在术前对手术样本的获取目的充分知情,并签署了知情同意书。
二、前列腺原代细胞获取
提供成熟的前列腺组织;
使用无菌PBS缓冲液对所述成熟的前列腺组织进行清洗和灭菌处理后,使用由Ⅰ型胶原酶、无菌PBS缓冲液和中性蛋白酶消化液组成的3毫升细胞消化液在37摄氏度下对组织消化90分钟,从而获取原代细胞悬液。接着,使用70微米的无菌筛网对所述原代细胞悬液在无菌PBS缓冲液的辅助下进行筛网分选,收集滤液并去除粘液和未消化的组织,以完成筛网分选。然后,将得到的滤液离心并去除上清后,向得到的沉淀物加入红细胞溶解平衡液进行重悬后再次离心并重复上述过程直至再次离心后在细胞沉淀中观察不到红细胞为止,以完成红细胞裂解去除,得到前列腺原代细胞。所述前列腺原代细胞包括基底细胞、腔细胞和干细胞。
值得注意的是,在前列腺原代细胞制备过程,Ⅰ型胶原酶占无菌PBS缓冲液的体积百分比为1%,中性蛋白酶占无菌PBS缓冲液的体积百分比为5%。每次离心的速率为1000g,离心时间为3分钟。
三、前列腺前体样细胞体外培养
重编程培养基组分包括:以占重编程培养基的体积计,基础培养基DMEM/F12(来源于武汉普诺赛生命科技有限公司),含量为20ng/mL的上皮细胞生长因子EGF,含量为50ng/mL的碱性成纤维细胞生长因子bFGF,含量为培养基终体积的0.5%营养补充剂N2(1X),含量为培养基终体积的1%B27营养补充剂(1X),含量为5-20μM的ROCK激酶抑制剂Y-27632,含量为3μM的Wnt信号通路激动剂CHIR99021,含量为1-5μM的TGF-β信号抑制剂A8301,含量为1-10ng/mL的促进胆管前体样细胞的增殖的Noggin和含量为10-30ng/mL的R-spondin1。
将前列腺前体样细胞以10000个/平方厘米的接种面积置于6孔板中,每孔加2毫升重编程培养基进行退分化培养,直至前列腺前体样细胞的融合度不低于80%后,使用胰酶消化液对前列腺前体样细胞进行1-5分钟的消化后,再继续使用重编程培养基进行扩增培养,扩增培养又可称为传代培养,前列腺前体样细胞可持续传代。
本发明还提供了前列腺前体样细胞在光学显微镜下的形态示意图,具体地,可参见图1,其中,图1为本发明提供的前列腺前体样细胞的光学显微下的形态示意图;从图1中可以看出,前列腺前体样细胞,呈现多边形,聚集生长,可见通过该实施例中的重编程培养基能够体外扩增前列腺前体样细胞。
四、前列腺前体样细胞分别通过重编程培养基和基础培养基培养,并对标志物结果分析
1.重编程培养基和基础培养基分别对前列腺前体样细胞进行体外培养
重编程培养基培养组:
将前列腺前体样细胞以10000个/平方厘米的接种面积置于6孔板中,每孔加2毫升重编程培养基进行退分化培养,直至前列腺前体样细胞的融合度不低于80%后,使用胰酶消化液对前列腺前体样细胞进行1-5分钟的消化后,再继续使用重编程培养基进行扩增培养。
基础培养基培养组:将前列腺前体样细胞以10000个/平方厘米的接种面积置于6孔板中,每孔加2毫升基础培养基进行退分化培养,使用胰酶消化液对前列腺前体样细胞进行1-5分钟的消化后,再继续使用基础培养基进行扩增培养。
2.重编程培养基和基础培养基培养的前列腺前体样细胞中标志物含量表达情况
图2为本发明中前列腺前体样细胞分别经过重编程培养基和基础培养基扩增培养后其中的基底细胞标志物含量对照图;图3为本发明中前列腺前体样细胞分别经过重编程培养基和基础培养基扩增培养后其中的腔细胞标志物含量对照图;图4为本发明中前列腺前体样细胞分别经过重编程培养基和基础培养基扩增培养后其中的干细胞标志物含量对照图;图5为本发明中前列腺前体样细胞分别经过重编程培养基和基础培养基扩增培养后其中的前列腺特异性标志物含量对照图。
其中,图2、图3、图4和图5中每个标志物均对应两根矩形柱,矩形柱表示标志物的相对含量,并且左侧矩形柱对应的是在基础培养基扩增培养的结果,右侧对应的是在重编程培养基扩增培养的结果。
图2中的基底细胞标志物包括CK14、CK5和P63,从图2中可以看出,上述标志物在重编程培养基扩增培养的结果均高于在基础培养基扩增培养的结果,可见,前列腺前体样细胞在重编程培养基中较之在基础培养基中能够扩增培养更多的基底细胞。
图3中的腔细胞标志物包括CK18、CK8和AR,从图3中可以看出,上述标志物在重编程培养基扩增培养的结果均高于在基础培养基扩增培养的结果,可见,前列腺前体样细胞在重编程培养基中较之在基础培养基中能够扩增培养更多的基底细胞。
图4中的干细胞标志物包括CD44、CD49b、CD133、CD117、Trop2、CD491、Sca-1和p63在重编程培养基扩增培养的结果均高于在基础培养基扩增培养的结果,可见,前列腺前体样细胞在重编程培养基中较之在基础培养基中能够扩增培养更多的具有干性特征细胞。
图5中的前列腺特异性标志物包括pbsn和MLX3-1,从图5中可以看出,上述标志物在重编程培养基扩增培养的结果均低于在基础培养基扩增培养的结果,可见,重编程培养基可以维持前列腺前体细胞区去分化状态,不表达成熟前列腺组织细胞标记物。
五、对前列腺前体样细胞进行流式检测
图6为本发明实施例的前列腺前体样细胞的基因表达标志物CD44的情况示意图;图7为本发明实施例的前列腺前体样细胞的基因表达标志物CD73的情况示意图;图8为本发明实施例的前列腺前体样细胞的基因表达标志物HLA-DRPQ的情况示意图;图9为本发明实施例的前列腺前体样细胞的基因表达标志物SOX9的情况示意图;图10为本发明实施例的前列腺前体样细胞的基因表达标志物CD90的情况示意图。
使用重编程培养基对前列腺前体样细胞进行传代培养后,吸弃重编程培养基后用无菌PBS缓冲液润洗,然后用胰酶消化液对前列腺前体样细胞进行消化处理后再进行离心处理,其中,离心处理的转速为200g,离心处理的时间为5分钟,离心处理结束后收集细胞沉淀物;向细胞沉淀物中加入100微升染色缓冲液重悬细胞至流式管中,再分别加入5微升待测流式抗体孵育20分钟后每管用400微升染色缓冲液重悬后进行表面抗体流式检测,流式检测结果见图6、图7、图8、图9和图10。
参照图8,标志物HLA-DRPQ的阴性峰的阳性率为%,阴性峰的阴性率大于98%说明前列腺前体样细胞阴性表达该标志物,因此,本发明的胃粘膜上皮前体样细胞阴性表达HLA-DR、HLA-DP和HLA-DQ。图6、图7、图9和图10,标志物CD44的阳性峰的阳性率为99.7%,标志物CD73的阳性峰的阳性率为99.8%,标志物SOX9的阳性峰的阳性率为87.9%,标志物CD90的阳性峰的阳性率为74.6%,阳性峰的阳性率介于70-99%说明前列腺前体样细胞阳性表达该标志物,因此,本发明的前列腺前体样细胞阳性表达CD44、CD73、SOX9和CD90。
六、前列腺前体样细胞制剂的制备
使用重编程培养基对前列腺前体样细胞进行传代培养后,吸弃重编程培养基后用无菌PBS缓冲液润洗,然后用胰酶消化液对前列腺前体样细胞进行消化处理后再进行离心处理,其中,离心处理的转速为200g,离心处理的时间为5分钟,离心处理结束后收集细胞沉淀物。将细胞沉淀物用生理盐水注射液(石家庄四药)重悬后,计数细胞密度,并用生理盐水注射液定容至需要的细胞密度。
七、前列腺前体样细胞制剂在动物适应症模型中的应用,并进行效果论证:
一,大鼠急性前列腺炎模型
1.本实施例提供了大鼠急性前列腺炎模型的建模方法,并使用前列腺前体样细胞对大鼠前列腺炎模型进行干预,考察前列腺前体样细胞对前列腺炎的作用。
本实施例使用购自北京维通利华的雄性性成熟的10-12周龄野生型SD大鼠进行建模。大鼠的体重平均达到300g。每组3只大鼠。
大鼠前列腺炎模型的建立:大鼠腹腔注射10%水合氯醛,一至两分钟后,将其平卧于超净台上,与阴茎根部上方2~3cm正中线两侧皮下各注射0.1ml盐酸利多卡因,做1~2cm纵行切口,逐层切开皮肤筋膜,到达肌肉组织是上皮肌肉向下切一小口,出现踏空感后,使用止血钳进入腹腔钝性分离机组织提起膀胱其后方找到颜色较深的前列腺两侧叶向两侧分别注射3%卡拉胶,30~50微升,注射完成后将膀胱和前列腺回纳腹腔逐层缝合切口建模一周后,各组大鼠进行测控实验,模型建立过程可参见图13,图13为本发明中大鼠前列腺炎模型建立过程示意图。
将大鼠前列腺炎模型随机分为假手术组(3只)、空白对照组(3只)和细胞治疗组(3只),
假手术组,大鼠腹腔注射10%水合氯醛,一至两分钟后,将其平卧于超净台上,与阴茎根部上方2~3cm正中线两侧皮下各注射0.1ml盐酸利多卡因,做1~2cm纵行切口,逐层切开皮肤筋膜,到达肌肉组织是上皮肌肉向下切一小口,出现踏空感后,使用止血钳进入腹腔钝性分离机组织提起膀胱其后方找到颜色较深的前列腺两侧叶向两侧分别注射3%卡拉胶,30~50微升,注射完成后将膀胱和前列腺回纳腹腔逐层缝合切口,不进行细胞注射治疗,仅进行前列腺炎造模。
空白对照组,不进行操作,与实验组和假手术组动物同时入组,一直饲养。
前列腺前体样细胞治疗组:在建模同时对细胞治疗组大鼠的两侧叶各注射50ul的前列腺前体样细胞注射1E+06 50ul+50ul matrigel基质胶;建模后一周,对细胞治疗组大鼠的两侧叶再次各注射50ul的前列腺前体样细胞注射1E+0650ul+50ul matrigel基质胶。
2.动物实验血液炎症指标
收集各组大鼠外周血100ul,分离血清,Elisa检测法IL-1a、IL-6表达水平。实验前基础值和实验后不同时间点及终点(术前、7d、21d)。其中,所述IL-1a、IL-6属于炎性分子。
图14为假手术组、空白对照组和前列腺前体样细胞治疗组采用Elisa检测法检测IL-6表达水平示意图;图15为假手术组、空白对照组和前列腺前体样细胞治疗组采用Elisa检测法检测IL-1a表达水平示意图。
从图14和图15中可以看出,前体样细胞治疗组相对炎性细胞浸润较空白对照组显著下降。炎性细胞理解为发生炎症反应相关的细胞。
3.前列腺炎病理分析(HE染色)
手术21天对假手术组、空白对照组和前列腺前体样细胞治疗组内的大鼠分别取样做前列腺病例切片,并进行HE染色,HE染色具体如下:
(1)石蜡切片脱蜡至水
依次将切片放入二甲苯Ⅰ10min-二甲苯Ⅱ10min-无水乙醇Ⅰ5min-无水乙醇Ⅱ5min-95%酒精5min-90%酒精5min-80%酒精5min-70%酒精5min-蒸馏水洗。
(2)苏木素染细胞核
切片入Harris苏木素染3-8min,自来水洗,1%的盐酸酒精分化数秒,自来水冲洗,0.6%氨水返蓝,流水冲洗。
(3)伊红染细胞质
切片入伊红染液中染色1-3min。
(4)脱水封片
将切片依次放入95%酒精I 5min-95%酒精II 5min-无水乙醇Ⅰ5min-无水乙醇Ⅱ5min-二甲苯Ⅰ5min-二甲苯Ⅱ5min中脱水透明,将切片从二甲苯拿出来稍晾干,中性树胶封片。
(5)显微镜镜检,图像采集分析
图16为本发明中假手术组、空白对照组和前列腺前体样细胞治疗组中分别对前列腺病例切片HE染色的荧光照片。
从图16中可知,假手术组前列腺组织腺体完整,无炎症细胞浸润,空白对照组可见不等量的组织变性,前列腺腺体水肿或增生,间质内炎性细胞大量浸润。
综上,假手术组前列腺组织腺体完整,无炎症细胞浸润,空白对照组可见不等量的组织变性,前列腺腺体水肿或增生,间质内炎性细胞大量浸润,前体样细胞治疗组相对炎性细胞浸润较空白对照组显著下降。
二、大鼠前列腺增生模型
建模过程:丙酸睾酮,50mg每公斤,隔天一次皮下注射,注射3周。
假手术组:本实施例使用购自北京维通利华的雄性10-12周龄野生型SD大鼠进行建模。大鼠的体重平均达到300g,每组3只大鼠,按照50mg每公斤剂量,皮下注射丙酸睾酮,共注射2次,间隔1天。不进行细胞给药治疗。
空白对照组:不进行操作,与实验组和假手术组动物同时入组,一直饲养。
前列腺前体样细胞治疗组:大鼠腹腔注射10%水合氯醛,一至两分钟后,将其平卧于超净台上,与阴茎根部上方2~3cm正中线两侧皮下各注射0.1ml盐酸利多卡因,做1~2cm纵行切口,逐层切开皮肤筋膜,到达肌肉组织是上皮肌肉向下切一小口,出现踏空感后,使用止血钳进入腹腔钝性分离机组织提起膀胱其后方找到颜色较深的前列腺两侧叶,前列腺前体样细胞1E+06/50ul与50ul matrigel基质胶混匀后,注射到前列腺两侧叶;并且按照50mg每公斤剂量,皮下注射丙酸睾酮。
间隔一天,再次按照50mg每公斤剂量,皮下注射丙酸睾酮。
建模后一周,对细胞治疗组大鼠的两侧叶再次各注射1E+06/50ul的前列腺前体样细胞l+50ul matrigel基质胶。
术后21天取样病理切片,并进行HE染色;
图17为本发明中前列腺增生模型病理结果;如图17所示,假手术组前列腺组织腺体完整,无炎症细胞浸润,空白对照组可见组织变性,前列腺腺体增生,前体样细胞治疗组组织变性和腺体增生较空白对照组显著下降。
可见,假手术组前列腺组织腺体完整,无炎症细胞浸润,空白对照组可见组织变性,前列腺腺体增生,前体样细胞治疗组组织变性和腺体增生较空白对照组显著下降。
综上,根据对前列腺前体样细胞培养后的表面标志物检测,发现该前列腺前体样细胞标志物为阳性表达CD90、CD73、CD44和SOX9的细胞,并且在体外培养时外观呈现典型的前体样细胞特征。可以用于后续的前列腺炎症和增生的机制研究,可以进行体外细胞层面的各种实验。
而且培养获得的前列腺前体样细胞,在进行动物实验时,前列腺炎模型中,细胞回输治疗后,可以显著降低血液炎性因子IL-6以及IL-2a的水平,和前列腺组织炎症的程度。并且,在前列腺增生模型中,细胞回输后可以使前列腺增生改善。
并且,由于前列腺前体样细胞表面不表达于免疫排斥反应相关的蛋白CD34、CD45、HLA-DR、HLA-DP和HLA-DQ,因此可以实现前列腺前体样细胞的异体回输,以临床良性前列腺增生手术后切除的组织或者供体捐献的正常前列腺组织为来源,取前列腺组织消化后得到的原代前列腺细胞作为种子细胞,使用重编程培养基进行体外培养,获得阳性表达CD90、CD73、CD44和SOX9的前列腺前体样细胞,并且进行体外扩增建立细胞种子库。在用于治疗临床前列腺炎、前列腺增生时,可直接原位注射前列腺前体样细胞。
虽然在上文中详细说明了本发明的实施方式,但是对于本领域的技术人员来说显而易见的是,能够对这些实施方式进行各种修改和变化。但是,应理解,这种修改和变化都属于权利要求书中所述的本发明的范围和精神之内。而且,在此说明的本发明可有其它的实施方式,并且可通过多种方式实施或实现。
Claims (13)
1.一种前列腺前体样细胞的制备方法,其特征在于,包括以下步骤:
S0:提供前列腺原代细胞;
S1:对所述S0中的前列腺原代细胞使用重编程培养基进行退分化培养以获得表达阳性标志物CD90、CD73、CD44和SOX9中至少一种的前列腺前体样细胞,其中,所述重编程培养基包括基础培养基、抑制剂和生长因子。
2.根据权利要求1所述的前列腺前体样细胞的制备方法,其特征在于,还包括:
S2:将所述前列腺原代细胞放入重编程培养基中进行退分化培养,直至所述前列腺前体样细胞的融合度不低于80%,使用胰酶消化液对所述前列腺前体样细胞进行消化处理,以得到前列腺前体样细胞。
3.根据权利要求2所述的前列腺前体样细胞的制备方法,其特征在于,所述重编程培养基还包括Wnt信号通路激动剂、营养补充剂和缓冲液。
4.根据权利要求1所述的前列腺前体样细胞的制备方法,其特征在于,所述抑制剂包括抑制TGF-β信号通路的ROCK激酶抑制剂和A83-01抑制剂。
5.根据权利要求4所述的前列腺前体样细胞的制备方法,其特征在于,以占所述基础培养基的含量计,所述ROCK激酶抑制剂的含量为5-20μM,所述A83-01抑制剂的含量为1-5μM。
6.根据权利要求1所述的前列腺前体样细胞的制备方法,其特征在于,所述生长因子包括EGF、bFGF、Noggin和R-spondin1。
7.根据权利要求6所述的前列腺前体样细胞的制备方法,其特征在于,以占所述基础培养基的含量计,所述EGF的含量为10-50ng/mL,所述bFGF的含量为10-50ng/mL,所述Noggin的含量为1-10ng/mL,所述R-spondin1的含量为10-30ng/mL。
8.根据权利要求1所述前列腺前体样细胞的制备方法,其特征在于,所述前列腺前体样细胞不表达CD34、CD45、HLA-DR、HLA-DP和HLA-DQ。
9.一种前列腺前体样细胞,其特征在于,通过权利要求1至8中任一项所述的前列腺前体样细胞的制备方法制备得到,并能特异性表达阳性标志物CD90、CD73、CD44和SOX9中的至少一种。
10.一种如权利要求9所述的前列腺前体样细胞的应用,其特征在于,所述前列腺前体样细胞用于制备治疗前列腺炎或前列腺增生的前列腺前体样细胞制剂,使用所述前列腺前体样细胞制剂干预体内动物模型后考察对前列腺炎的影响。
11.根据权利要求10所述的前列腺前体样细胞的应用,其特征在于,所述动物模型包括药物诱导的前列腺炎或前列腺增生模型。
12.一种前列腺前体样细胞制剂,其特征在于,包括如权利要求9所述的表达阳性标志物CD90、CD73、CD44和SOX9中至少一种的前列腺前体样细胞,以及药学上可接受的载体。
13.一种前列腺前体样细胞制剂的应用,其特征在于,使用如权利要求12中所述的前列腺前体样细胞制剂干预体内动物模型。
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