CN114540278A - 微血管体外培养方法及培养液 - Google Patents
微血管体外培养方法及培养液 Download PDFInfo
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- CN114540278A CN114540278A CN202210305895.4A CN202210305895A CN114540278A CN 114540278 A CN114540278 A CN 114540278A CN 202210305895 A CN202210305895 A CN 202210305895A CN 114540278 A CN114540278 A CN 114540278A
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Abstract
本发明属于生物医药领域,具体涉及微血管体外培养方法及培养液。本发明的微血管体外培养方法,具体包括分离、过滤和培养步骤,采用短时循环消化的方法从组织中分离微血管,减少了原代微血管细胞在酶溶液中的处理时间,降低了酶溶液对细胞的伤害,提高细胞活力。本发明还提供了一种微血管体外培养液,使得微血管可以在体外普通培养皿的硬基底表面以管状形式生长,同时保留同一组织来源的周细胞围绕在内皮细胞周围一起生长,更好地模拟体内微血管,并且可以在体外长时间培养。
Description
技术领域
本发明属于生物医药领域,涉及生物医学基础研究、药理研究、药物筛选和评价以及再生医学研究及临床应用领域,具体涉及微血管体外培养方法及培养液。
背景技术
微血管又称毛细血管,是分布于各种组织和器官中连通小动脉和小静脉之间的细小血管,其分支众多连通成网络,也被称为终末血管床。微血管在发育和组织再生过程中发挥着重要作用,很多疾病的发生伴随着微血管结构和功能的失调。微血管内皮细胞周围有周细胞包围,其嵌入内皮细胞基膜中,在促进微血管发育、稳定微血管结构、调控微血管功能等方面发挥重要作用。
动物模型是研究微血管疾病的重要工具,但是,由于动物和人在基因、生理和病理等方面的差异,在动物模型研究中得出的科学结论或药物实验结果,在人体中不一定可行。因此,人的微血管模型具有重要的研究意义和市场价值。
现有的微血管培养模型主要使用商用培养液,如EGM2MV,所使用的细胞包括多个组织来源的不同细胞类型,例如,来自人脐带静脉血管的内皮细胞、血液来源的血管内皮细胞、动脉血管内壁的血管内皮细胞、肾脏来源的内皮细胞、大脑来源的周细胞、诱导多能干细胞定向分化产生的血管内皮细胞、周细胞等等,这些方法各有其缺点,比如:
1、血管细胞具有异质性,不同器官的血管细胞具有各自特定的生物学特征,来自脐带静脉、动脉血管等大血管的内皮细胞难以代替微血管的内皮细胞,由诱导多能干细胞分化生成的血管细胞难以反映某一特定组织或器官的微血管生物学特征。
2、微血管内皮细胞和周细胞的共培养模型是研究二者相互作用,以及微血管稳定性的重要工具。在现有的微血管内皮细胞和周细胞共培养模型中,内皮细胞和周细胞分别来自不同的器官,例如将来自脐带静脉血管的内皮细胞和来自大脑的周细胞共培养,由于血管细胞的异质性,不同器官来源的内皮细胞和周细胞的共培养模型难以准确反映微血管的组织特异性。
3、现有的常用方法中,血管内皮细胞在普通培养皿的二维表面上以单层细胞片的形式生长,丢失了体内原有的管状形态。
4、现有的常用方法中,血管内皮细胞只有在水凝胶(如Matrigel、fibrin或胶原)表面或内部才能形成管状结构,但是很难在体外长期培养。在水凝胶表面形成的微血管仅能维持几天时间,水凝胶内部所形成的管状微血管结构一般最多培养两周时间。体外培养时间是限制体外微血管模型的一个重要因素。
5、利用水凝胶形成微血管的方法成本较高,三维微血管模型难以利用常规的免疫染色方法进行表征和检测,难以进行高通量筛选实验。
发明内容
根据现有技术上存在的缺陷,结合目前的研究前沿,本发明提供了微血管体外培养方法及培养液。
本发明是采用以下的技术方案实现的:
本发明提供了一种微血管体外培养方法,具体包括以下步骤:
步骤(1)、分离:将组织块剪碎,置于胶原酶溶液中消化,分离胶原酶溶液中的细胞悬液离心,将获得的细胞沉淀重悬于PBS溶液中;将剩余组织块再次加入胶原酶溶液继续消化,收集细胞悬液并离心,离心后重悬;重复以上步骤,直至组织块消化完为止,离心收集所有细胞沉淀,重悬于PBS溶液中;
步骤(2)、过滤:将步骤(1)分离得到的细胞悬液,用细胞筛网进行过滤,红细胞和单细胞以及死细胞会通过筛网,而微血管段留在筛网上,用PBS溶液反向冲洗,收集微血管段;
步骤(3)、培养:将步骤(2)获得的微血管段,置于微血管培养液进行培养。
具体的,所述步骤(3)中所述微血管培养液包括如下成分:DMEM/F12培养液、1-20%胎牛血清、100U/ml青霉素、100μg/ml链霉素、1-20mM nicotinamide、1-10mM NAC、10-100μM Vitamin C、1-10μM glutathione、1-20μg/ml insulin、1-20μg/ml transferrin、10-100nM Sodium Selenite、5-50ng/ml血管内皮生长因子VEGF、0.01-20μM Wnt/β-catenin信号通路激活剂、0.01-20μM TGFβ/Smad信号通路抑制剂、0.1-20μM ROCK抑制剂。
其中,所述步骤(3)中所述微血管培养液包括如下成分:DMEM/F12培养液、1-5%胎牛血清、100U/ml青霉素、100μg/ml链霉素、1-10mM nicotinamide、1-5mM NAC、10-50μMVitamin C、1-5μM glutathione、5-15μg/ml insulin、1-10μg/ml transferrin、10-50nMSodium Selenite、5-15ng/ml血管内皮生长因子VEGF、0.01-20μM Wnt/β-catenin信号通路激活剂、0.01-20μM TGFβ/Smad信号通路抑制剂、0.1-20μM ROCK抑制剂。
作为本发明的一种情况,所述Wnt/β-catenin信号通路激活剂包括Chir99021、R-Spondin-1、Wnt-1蛋白、Wnt-3a蛋白、Wnt-7a蛋白、Wnt-9b蛋白、Laduviglusibtrihydrochloride、CHIR-99021HCl、SB415286、AZD1080、AR-A014418、LY2090314、BIO-acetoxime、9-ing-41、BRD0705、KY19382、SB216763、CHIR-98014、TWS119、Tideglusib、6-bromoindirubin-3-oxime、AZD2858、TDZD-8、Indirubin-3'-oxime、CP21R7、1-Azakenpaullone、IM-12、5-Bromoindole、Wnt agonist 1、Methyl Vanillate、WAY-316606、Foxy-5、SKL2001、IQ-1。
进一步地,所述TGFβ/Smad信号通路抑制剂包括A83-01、SB431542、SD-208、GW788388、TP0427736HCl、RepSox、LY2109761、Ophiopogonin D、SB505124、SIS3、SIS3 HCl、BIBF-0775、LY3200882、Galunisertib、A77-01、Vactosertib、Halofuginone、BMS-986260、LY364947、Oxymatrine、Pirfenidone、Hypaconitine、SB525334、ITD-1、TP-008、SM16、R-268712、SD-208、GW788388、BIO-013077-01。
进一步地,所述ROCK抑制剂包括Y27632、Y-27632 2HCl、ZINC00881524、Thiazovivin、Fasudil、Fasudil HCl、Hydroxyfasudil、Hydroxyfasudil HCl、GSK429286A、RKI-1447、GSK269962A HCl、Azaindole 1、Netarsudil 2HCl、Ripasudil、Ripasudil freebase、Ripasudil hydrochloride dihydrate、Y-39983HCl、H-1152dihydrochloride、Belumosudil、Rho-Kinase-IN-1、ROCK2-IN-2、Chroman 1、Chroman 1dihydrochloride、SB-772077B dihydrochloride、Verosudil、ZINC00881524、Y-33075dihydrochloride、Y-33075、SR-3677。
具体的,所述步骤(1)中组织块在胶原酶溶液中消化的时间为5~15分钟。
具体的,所述步骤(3)中培养条件为37℃恒温,95%湿度,5%CO2。
本发明还提供一种微血管体外培养液,所述微血管体外培养液成分如上述。
其中,所述培养液用于哺乳动物各种组织和器官中的微血管体外培养,组织和器官类型包括但不限于皮下或器官的疏松结缔组织、大脑、眼、心脏、肺脏、肾脏、肝脏、胸腺、脾、胰腺、胃、肠组织、肌肉、骨髓、脐带。
与现有技术相比,本发明的有益效果:
1、在现有的方法中,微血管在普通培养皿的二维表面上只能以单层细胞片状结构生长,而本发明建立的方法可以使得微血管在普通培养皿的二维表面上以管状形式生长,形成网络结构,与体内微血管更为相似。
2、现有的微血管共培养模型一般使用不同来源的内皮细胞和周细胞,或者由多能干细胞分化而来的血管细胞,而本发明建立的方法可以使得来源于同一组织或器官的微血管内皮细胞和周细胞共同生长,与体内微血管更为相似,更能反映组织或器官特异性。
3、现有的体外微血管培养模型一般最多维持2周时间,而本发明建立的方法可以在体外培养微血管长达8周时间,而且在至少前4周时间内保持较高水平的微血管生长状态,大大延长了体外培养时间,可以进行更多的体外实验干预和检测项目。
4、现有的三维微血管模型一般需要用到水凝胶,成本高昂,而本发明建立的方法在普通培养皿表面就可以实现三维微血管培养,成本低廉。
5、现有的商用微血管培养液中含有多种生长因子,价格高昂,而本发明建立的方法大大减少了生长因子的使用,降低了成本,适于大规模推广应用。
附图说明
附图用来提供对本发明的进一步理解,并且构成说明书的一部分,与本发明的实施例一起用于解释本发明,并不构成对本发明的限制。
在附图中:
图1为实施例1中从组织中分离微血管的流程示意图;
图2为实施例1中获得原代微血管段的表征;其中,图2(1)为原代微血管段的明场照片,图内标尺为100μm;图2(2)和图2(3)为原代微血管段长度统计;
图3为实施例1中商用对照组培养获得的微血管明场和共聚焦显微镜成像照片,分别为图3(1)和图3(2),DAPI标记细胞核,CD31标记内皮细胞,图内标尺均为100μm;
图4为实施例1中空白对照组和C组培养获得的微血管明场照片,分别为图4(1)和图4(2);其中C代表加有Chir99021的空白对照组培养液;图内标尺均为100μm;
图5为实施例1中不同组的微血管培养一周后的激光共聚焦显微镜成像照片,DAPI标记细胞核,CD31标记内皮细胞;其中,C、A、C+A、C+Y、C+A+Y分别代表加有Chir99021、A83-01、Chir99021和A83-01混合、Chir99021和Y27632混合、上述三种混合的空白对照组培养液,Medium-X代表实验组;DAPI标记细胞核,CD31标记内皮细胞,图内标尺均为100μm;
图6为实施例1中不同组的微血管培养一周后的的长度和分支节点统计柱状图,分别为图6(1)、图6(2);其中,X代表实验组,***P<0.001(双尾学生t检验);
图7为实施例1中EGM2MV和Medium-X培养液中微血管示意图;
图8、图9和图10为实施例2原代分离的新鲜微血管、在EGM2MV培养液和Medium-X培养液中培养1周后的微血管差异表达基因的GO功能富集分析和热图分析;其中,V代表原代分离得到的微血管、E代表商用对照组培养获得的微血管;
图11为实施例2中qRT-PCR验证多种基因的表达情况柱状图,***P<0.001;
图12为实施例3中原代微血管在含有VEGF的培养液中加入不同小分子进行培养一周后的激光共聚焦成像照片,其中,VEGF、VEGF+C、VEGF+CA、VEGF+CY和VEGFCAY分别代表VEGF对照组、C组、CA组、CY组、CAY组;DAPI标记细胞核,CD31标记内皮细胞,图内标尺均为100μm;
图13为实施例3中不同组的微血管培养1周后的的长度和分支节点统计柱状图,分别为图13(1)、图13(2);其中,***P<0.001(双尾学生t检验);
图14为实施例3中不同组的微血管差异表达基因的热图分析;
图15为实施例3中,VEGF+C组相比VEGF对照组上调基因的生物学过程的GO功能富集分析;
图16为实施例3中CA相比于C,以及CAY相比于CY上调基因的生物学过程的GO功能富集分析;
图17为实施例4中各组的微血管在体外培养7、14、21、28、56天的激光共聚焦成像照片;DAPI标记细胞核,CD31标记内皮细胞,图内标尺均为100μm;
图18为实施例4中各组微血管在体外培养不同时间点的长度和分支节点数量统计柱状图,分别为图18(1)、图18(2);其中,***P<0.001(双尾学生t检验);
图19为实施例5中培养1周后的微血管经过免疫染色和激光共聚焦成像照片,所用抗体分别为LYVE1(图19(1))、vWF(图19(2))、CD31(图19(3)~图19(6))、ZO1(图19(3))、NG2(图19(4))、PDGFRβ(图19(5))、SMA(图19(6)),DAPI标记细胞核,图内标尺均为100μm;
图20为实施例6中微血管在含有不同A83-01浓度(1、0.1、0.2、0.5、1、2μM)培养液中培养2周后的激光共聚焦成像照片;DAPI标记细胞核,CD31标记内皮细胞,图内标尺均为100μm;
图21为实施例6中微血管在体外培养不同时间点的长度和分支节点数量统计柱状图,分别为图21(1)、图21(2);其中,***P<0.001(双尾学生t检验);
图22为实施例7中各组微血管在体外培养14天后经过免疫染色和激光共聚焦成像照片;DAPI标记细胞核,CD31标记内皮细胞,NG2标记周细胞,图内标尺均为100μm;
图23为实施例7中各组微血管的微血管长度和周细胞覆盖率统计;
图24为实施例8中微血管在含有不同Wnt/β-catenin信号通路的激活剂培养液中的微血管长度统计柱状图;
图25为实施例9中微血管在含有不同TGFβ/Smad信号通路的抑制剂培养液中的微血管长度统计柱状图;
图26为实施例10中微血管在含有不同ROCK的抑制剂培养液中的微血管长度统计柱状图。
具体实施方式
为了使本发明目的、技术方案更加清晰明了;便于理解,下面结合附图1~26,对本发明作进一步详细说明。下述实施例中所述实验方法,如无特殊说明,均为常规方法;实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行;所述试剂和材料,如无特殊说明,均可从商业途径获得。
一、检测方法:
1、微血管长度统计和分支节点统计:血管长度和分支节点的统计利用ImageJ软件进行;
2、差异表达蛋白质Gene Ontology(GO)功能富集分析:利用Blast2GO对目标蛋白质集合进行GO注释,过程大致可以归纳为序列比对(Blast)、GO条目提取(Mapping)、GO注释(Annotation)和补充注释(Annotation Augmentation)等四个步骤。然后通过Fisher精确检验方法对差异表达蛋白质进行GO功能富集分析。
3、qRT-PCR验证:上下游引物的序列如下:
Apln(AAGCCCAGAACTTCGAGGAC;GGCAGCATATTTCCGCTTCTG),
Dll4(AGTGTACTCCCGCACTAGCC;CGATGCCTCGGTAGGTAATCC),
Kcne3(ACAGATCGCAGAGTCAGATCAC;TGATTGTCTGGCCCTGTTCC),
Car2(GCTGGAATGTGTGACCTGGA;CCCAGCTGCAGGGTCATTTT),
Plxnd1(ATCGCCCAGGCCTTCATAGAT;TCTTCCGGTACTCGGGGAT),
Pcdh12(CAGCAGGTCTGAAGTGGGAG;GTAGCATCGTGCTTACCGGA),
Wnt5a(TGGGCACATTTCCACGCTAT;TGTCCTTGAGAAAGTCCCGC),
Fzd1(GCCTCACAACCAGTCCACAA;TGCTTTACAAATGCCACTCGG),
Cxcl12(AGCCTTAAACAAGAGGCTCAAG;TGAGGGTGGATCTCGCTCTT),
Mmp9(GGATCCCCCAACCTTTACCAG;AAGGTCAGAACCGACCCTACA);
对照基因GAPDH(GGAGAAGGCCGGGGCCCACTTGAA;GCATGGACTGTGGTCATGAGCCCTTCCAC)。
二、下列实施例采用的材料如下:
1、组织:
大鼠皮下疏松结缔组织;
2、培养液:
EGM2MV培养液、DMEM/F12培养液;
在本发明Medium-X培养液的成分和用量的范围,均出现了较明显效果。Medium-X培养液中Wnt/β-catenin信号通路激活剂选择CHIR99021,TGFβ/Smad信号通路抑制剂选择A83-01,ROCK抑制剂选择Y27632;同时对Medium-X培养液的浓度进行优选,选用效果最突出的,作为实施例,组成如下:
DMEM/F12培养液、2%胎牛血清、100U/ml青霉素、100μg/ml链霉素、5mMnicotinamide、1mM NAC、50μM Vitamin C、3μM glutathione、10μg/ml insulin、7.5μg/mltransferrin、40nM Sodium Selenite、10ng/ml血管内皮生长因子VEGF、2μM CHIR99021、0.2μM A83-01、5μM Y27632;
空白对照组培养液成分如下:相比Medium-X培养液,缺少VEGF、CHIR99021、A83-01、Y27632;
VEGF对照组培养液成分如下:相比Medium-X培养液,缺少CHIR99021、A83-01、Y27632。
3、消化酶溶液:
消化酶溶液含有2mg/ml胶原酶、5mg/ml BSA、5μM Y27632,用DMEM培养液配制,经过0.22μm无菌滤膜过滤除菌。
4、培养条件:
所有培养都在37℃恒温细胞培养箱中进行,95%湿度,5%CO2。
实施例1.分离微血管并培养
步骤(1)、分离:将组织块用剪刀剪成约2-10mm的小块,置于胶原酶溶液中,在37℃环境中进行消化。为了减少酶消化过程对微血管的损伤,采用短时循环消化的方法,即每10分钟左右从胶原酶溶液中取出细胞悬液,离心后获得细胞沉淀,重悬于PBS溶液中。每次取出细胞悬液后,再向组织块中加入胶原酶溶液继续消化。重复以上步骤,直至组织块消化完为止,离心收集所有细胞沉淀,重悬于PBS溶液中。
步骤(2)、过滤:将上述分离得到的细胞悬液,用30μm孔径的细胞筛网进行过滤,红细胞和单细胞以及一些死细胞会通过筛网,而微血管段留在筛网上,用PBS溶液反向冲洗,收集微血管段,在普通培养皿中进行培养。所获得的微血管段如图1、图2所示。
步骤(3)、培养:将步骤(2)获得的微血管段分成若干组,其中实验组使用微血管专用培养液(Medium-X)进行培养;与实验组不同的是,空白对照组采用的培养液不含有VEGF、CHIR99021、A83-01和Y27632;C组、A组、CA组、CY组、CAY组分别采用加有Chir99021、A83-01、Chir99021和A83-01混合、Chir99021和Y27632混合、上述三种混合的对照组培养液。
将C组和对照组培养2周时间,所得微血管生长情况如图4所示,可以看出对照组中的细胞都分化成为脂肪细胞,细胞内含有大量的脂滴;而C组则几乎没有脂肪细胞,微血管以管状形式生长。
将空白对照组、实验组和C组、A组、CA组、CY组、CAY组的微血管培养1周时间,进行激光共聚焦显微镜成像,结果如图5所示。所获得的微血管进行长度和分支节点统计,结果如图6所示,可以看出含有三种小分子(CAY)的培养液中的微血管比含有一种或两种小分子的培养液中的微血管有显著增加的微血管总长度和分支节点数量,在CAY培养液中加入VEGF(即Medium-X)后,微血管的总长度和分支节点数量进一步显著增加。
商用对照组采用商用EGM2MV培养液中培养1周时间,所得微血管生长情况如图3所示,与实验组培养所得微血管形貌相比(图5和图7),可以看出本发明的培养液可以更好地维持微血管的管状形态,与体内的微血管更为相似。
实施例2.原代培养的微血管转录组分析
对实施例1中原代分离的新鲜微血管、在EGM2MV培养液和Medium-X培养液中培养1周后的微血管进行差异表达基因的GO功能富集分析和热图分析,分析结果如图8~10所示。
经过GO功能富集分析发现,如图8所示,在EGM2MV培养液中下调而在Medium-X培养液中上调(V>E<X)的基因主要涉及血管新生、血管形成、血管细胞增殖和迁移等重要生物学过程。如图9所示,在EGM2MV中上调(V<E>X)的基因主要涉及氧化、脂质代谢等等。经过热图分析发现一些血管细胞特异的关键基因在Medium-X中都上调表达。
另外,通过qRT-PCR验证10个关键基因的表达情况,包括Apln、Dll4、Kcne3、Car2、Plxnd1、Pcdh12、Wnt5α、Fzd1、Cxcl12、Mmp9,如图11所示,可以看出血管新生相关的基因在Medium-X中特异上调。
实施例3.Chir99021、A83-01和Y27632的作用机理的转录组学分析
将实施例1的步骤(2)中获得的原代微血管在不同培养液中进行培养1周,VEGF对照组中加入VEGF,并与加入C(Chir99021)、A(A83-01)和Y(Y27632)的不同组合的培养液培养结果相比表,对获得的微血管特征进行表征,所得结果如图12、图13,可以看出单独加入C就可以大大增加微血管总长度和分支节点,加入A后使得出现了一些较宽的血管束,加入Y后可以进一步提高微血管总长度和分支节点数量。
图14(1)中比较了VEGF和VEGF+C组的一些基因表达差异,表示加入C后促进了血管新生和内皮细胞特异性基因表达;图14(2)中比较了在都含有VEGF的情况下,加入C和CA,以及CY和CAY的基因表达差异,表示加入A之后促进了血管功能和增殖相关的基因表达差异;CD34是内皮细胞祖细胞的一个生物标志物,分析发现C和A都会促进CD34的基因表达。
图15所示,VEGF+C组相比于VEGF组上调基因的GO分析气泡图,可以看出Chir99021可以特异上调血管新生和血管分支形成相关的基因。
图16所示,分析了VEGF+CA组相比于VEGF+C组,以及VEGF+CAY组相比于VEGF+CY组上调的基因的GO分析气泡图,可以看出,在加入了A83-01后,血管功能相关的特异上调,参与抗凝和脂质转运相关的生物学过程。
实施例4.微血管长期培养
将实施例3中的各组培养时间延长,分别培养56天,并于7、14、21、28、56天时间点进行激光共聚焦成像,观察形貌,结果如图17所示,可以看出,虽然加入Chir99021后可以显著增加微血管长度和分支节点,但是微血管稳定性较差,从第二周开始就急剧退化;只有包含了VEGF+CAY的培养液(Medium-X)是最优的培养条件,可以在四周内维持较高水平的微血管,甚至可以培养微血管到8周时间。
对各个实验组的微血管在体外培养不同时间点的长度和分支节点数量统计,结果如图18所示。
实施例5.微血管生物标志物表达情况
将原代微血管用Medium-X中培养1周后,进行免疫染色和激光共聚焦成像,结果如图19所示,可以看出,极少有表达LYVE1的淋巴管细胞(1),说明本发明方法可以特异扩增微血管细胞,微血管表达较强的内皮细胞标志物vWF(2)和内皮细胞间连接蛋白ZO1(3),另外还有较强的周细胞标志物表达,包括NG2(4)、PDGFRβ(5)和SMA(6)。
实施例6.A83-01浓度对微血管内皮细胞和周细胞的影响
与实施例1中实验组培养液不同的是,将原代微血管用含有不同浓度A83-01的培养液培养2周时间,将0、0.1、0.2、0.5、1、2μM浓度A83-01的各组获得微血管进行激光共聚焦成像,并对长度和分支节点数量进行统计,结果分别如图20和图21所示。可以看出,随着A83-01浓度升高,微血管长度和分支节点显著增加,0.5μM A83-01到达最高水平,大于0.5μM的实验组(1和2μM A83-01)的微血管长度和分支节点没有显著区别。然而,与之相反的是,随着A83-01浓度升高,NG2+周细胞数量急剧下降,0.2μM及更高浓度A83-01的实验组中几乎看不到NG2+周细胞。
实施例7.A83-01浓度对微血管周细胞的影响
将原代微血管在CY组培养液中培养14天(图22(1)、图23(1)-(3));在CAY组培养液中培养14天(图22(2));先在CAY组培养液中培养7天,然后换成CY培养液继续培养7天(图22(3)、图23(1)-(3))后,经免疫染色和激光共聚焦显微镜成像,并进行微血管长度和周细胞覆盖率统计。可以看出,在不含A83-01的CY培养液中存在大量NG2+周细胞,而在加入A83-01后的CAY培养液中,几乎看不到NG2+周细胞,中途换液去除A83-01后,可以保留一定数量的NG2+周细胞。
实施例8.Wnt/β-catenin信号通路的其他激活剂的作用
将原代微血管在Medium-X中进行培养1周时间,并将Wnt/β-catenin信号通路的激活剂Chir99021替换为其他分子,比较不同分子的作用(图24)。可以看出不同Wnt/β-catenin信号通路的激活剂都对微血管的生长有促进作用,按照不同分子的特性,其浓度范围在0.01-20μM之间。
实施例9.TGFβ/Smad信号通路的其他抑制剂的作用
将原代微血管在Medium-X中进行培养1周时间,并将TGFβ/Smad信号通路的抑制剂A83-01替换为其他分子,比较不同分子的作用(图25)。可以看出不同TGFβ/Smad信号通路的抑制剂都对微血管的生长有促进作用,按照不同分子的特性,其浓度范围在0.01-20μM之间。
实施例10.ROCK的其他抑制剂的作用
将原代微血管在Medium-X中进行培养1周时间,并将ROCK抑制剂Y27632替换为其他分子,比较不同分子的作用(图26)。可以看出不同ROCK抑制剂都对微血管的生长有促进作用,按照不同分子的特性,其浓度范围在0.1-20μM之间。
当然,以上仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种微血管体外培养方法,其特征在于,具体包括以下步骤:
步骤(1)、分离:将组织块剪碎,置于胶原酶溶液中消化,分离胶原酶溶液中的细胞悬液离心,将获得的细胞沉淀重悬于PBS溶液中;将剩余组织块再次加入胶原酶溶液继续消化,收集细胞悬液并离心,离心后重悬;重复以上步骤,直至组织块消化完为止,离心收集所有细胞沉淀,重悬于PBS溶液中;
步骤(2)、过滤:将步骤(1)分离得到的细胞悬液,用细胞筛网进行过滤,红细胞和单细胞以及死细胞会通过筛网,而微血管段留在筛网上,用PBS溶液反向冲洗,收集微血管段;
步骤(3)、培养:将步骤(2)获得的微血管段,置于微血管培养液进行培养。
2.根据权利要求1所述的微血管体外培养方法,其特征在于,所述步骤(3)中所述微血管培养液包括如下成分:DMEM/F12培养液、1-20%胎牛血清、100U/ml青霉素、100μg/ml链霉素、1-20mM nicotinamide、1-10mM N-Acetyl-L-cysteine(NAC)、10-100μM Vitamin C、1-10μM glutathione、1-20μg/ml insulin、1-20μg/ml transferrin、10-100nM SodiumSelenite、5-50ng/ml血管内皮生长因子VEGF、0.01-20μM Wnt/β-catenin信号通路激活剂、0.01-20μM TGFβ/Smad信号通路抑制剂、0.1-20μM ROCK抑制剂。
3.根据权利要求2所述的微血管体外培养方法,其特征在于,所述步骤(3)中所述微血管培养液包括如下成分:DMEM/F12培养液、1-5%胎牛血清、100U/ml青霉素、100μg/ml链霉素、1-10mM nicotinamide、1-5mM NAC、10-50μM Vitamin C、1-5μM glutathione、5-15μg/ml insulin、1-10μg/ml transferrin、10-50nM Sodium Selenite、5-15ng/ml血管内皮生长因子VEGF、0.01-20μM Wnt/β-catenin信号通路激活剂、0.01-20μM TGFβ/Smad信号通路抑制剂、0.1-20μM ROCK抑制剂。
4.根据权利要求2或3任一项所述的微血管体外培养方法,其特征在于,所述Wnt/β-catenin信号通路激活剂包括Chir99021、R-Spondin-1、Wnt-1蛋白、Wnt-3a蛋白、Wnt-7a蛋白、Wnt-9b蛋白、Laduviglusib trihydrochloride、CHIR-99021HCl、SB415286、AZD1080、AR-A014418、LY2090314、BIO-acetoxime、9-ing-41、BRD0705、KY19382、SB216763、CHIR-98014、TWS119、Tideglusib、6-bromoindirubin-3-oxime、AZD2858、TDZD-8、Indirubin-3'-oxime、CP21R7、1-Azakenpaullone、IM-12、5-Bromoindole、Wnt agonist 1、MethylVanillate、WAY-316606、Foxy-5、SKL2001、IQ-1。
5.根据权利要求2或3所述的微血管体外培养方法,其特征在于,所述TGFβ/Smad信号通路抑制剂包括A83-01、SB431542、SD-208、GW788388、TP0427736HCl、RepSox、LY2109761、Ophiopogonin D、SB505124、SIS3、SIS3 HCl、BIBF-0775、LY3200882、Galunisertib、A77-01、Vactosertib、Halofuginone、BMS-986260、LY364947、Oxymatrine、Pirfenidone、Hypaconitine、SB525334、ITD-1、TP-008、SM16、R-268712、SD-208、GW788388、BIO-013077-01。
6.根据权利要求2或3所述的微血管体外培养方法,其特征在于,所述ROCK抑制剂包括Y27632、Y-27632 2HCl、ZINC00881524、Thiazovivin、Fasudil、Fasudil HCl、Hydroxyfasudil、Hydroxyfasudil HCl、GSK429286A、RKI-1447、GSK269962A HCl、Azaindole 1、Netarsudil 2HCl、Ripasudil、Ripasudil free base、Ripasudilhydrochloride dihydrate、Y-39983HCl、H-1152dihydrochloride、Belumosudil、Rho-Kinase-IN-1、ROCK2-IN-2、Chroman 1、Chroman 1dihydrochloride、SB-772077Bdihydrochloride、Verosudil、ZINC00881524、Y-33075 dihydrochloride、Y-33075、SR-3677。
7.根据权利要求1-6任一项所述的微血管体外培养方法,其特征在于,所述步骤(1)中组织块在胶原酶溶液中消化的时间为5~15分钟。
8.根据权利要求7所述的微血管体外培养方法,其特征在于,所述步骤(3)中培养条件为37℃恒温,95%湿度,5%CO2。
9.一种微血管体外培养液,其特征在于,所述微血管体外培养液成分如权利要求2或3所述。
10.根据权利要求9所述的微血管体外培养液,其特征在于,所述培养液用于哺乳动物各种组织和器官中的微血管体外培养,组织和器官类型包括但不限于皮下或器官的疏松结缔组织、大脑、眼、心脏、肺脏、肾脏、肝脏、胸腺、脾、胰腺、胃、肠组织、肌肉、骨髓、脐带。
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