CN112430672B - 一种人类角膜内皮细胞的鉴定方法 - Google Patents
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Abstract
本发明公开了一种人类角膜内皮细胞的鉴定方法,具体是将待测细胞和对照角膜基质细胞的cDNA进行实时荧光定量PCR扩增,检测NCAM1、ALCAM、CDH2、ACTA2基因的表达水平,通过将对照角膜基质细胞的各基因表达量定为1,计算待测细胞中各基因的相对表达量RQ,从而判断待测细胞是否为人角膜内皮细胞。本发明通过NCAM1、ALCAM、CDH2、ACTA2四种标志基因组合可对体外培养HCECs进行角膜基质细胞污染及HCECs成纤维细胞转化程度进行鉴定,指导体外HCECs培养工艺参数的优化及对制备最终HCECs产品进行鉴定,保证体外培养HCECs具有CEC细胞特定功能。
Description
技术领域
本发明属于生物医学技术领域,具体涉及一种人类角膜内皮细胞的鉴定方法。
背景技术
人角膜由三个细胞层组成,即上皮细胞、基质细胞和内皮细胞。其中内皮细胞是4μm厚的单层细胞,位于角膜内侧,人角膜内皮通过泵和漏屏障功能的组合调节房水流向角膜基质,从而保持角膜透明度。当人角膜内皮细胞(HCECs)的增殖能力受到严重限制,对角膜内皮的严重损害导致角膜内皮功能障碍,并最终导致角膜透明度的丧失。目前,角膜移植是治疗角膜内皮功能障碍的唯一治疗选择,培养的HCECs已被认为是供体角膜的替代组织来源,但细胞培养的技术障碍来自于HCECs容易受到基质角膜细胞和成纤维细胞转化污染,也称为内皮-间质转化,进而引起细胞功能的改变。因此,开发有效鉴定人角膜内皮细胞的方法很有必要。
发明内容
针对上述现有技术的不足,本发明提供了一种人类角膜内皮细胞的鉴定方法。
发明人通过对分离培养的HCECs与角膜基质细胞进行基因转录组高通量测序分析,筛选得到与基质细胞有显著性差异、且与角膜功能相关的8种基因(ACTA2、CDH2、ALCAM、TGFB1I1、COL9A2、LTBP2、PCP4、NCAM1),同时对制备得到的多批次HCECs进行质量研究,通过体外生物学评价方法并结合动物实验对HCECS进行有效性评价,最终确定HCECs生物学特性及动物实验有效性相关的四种标志基因(NCAM1、ALCAM、CDH2、ACTA2),可用于指导体外HCECs培养工艺参数的优化及对制备的最终HCECs细胞产品进行鉴定。
本发明采用的技术方案如下:
一种人类角膜内皮细胞的鉴定方法,包括以下步骤:
步骤1,提取待测细胞和角膜基质细胞的总RNA并将其反转录为cDNA;
步骤2,以步骤1中得到的cDNA为模板进行实时荧光定量PCR扩增,检测NCAM1、ALCAM、CDH2、ACTA2基因的表达水平;
步骤3,根据步骤2的检测结果,将角膜基质细胞的各基因表达量定为1,计算待测细胞中各基因的相对表达量RQ,如果log(RQ)同时满足NCAM1≥0.6、ALCAM≥1.0、CDH2≥2.0、ACTA2≤-1.0,则说明待测细胞为人角膜内皮细胞;
步骤2中所采用的扩增引物如下:
NCAM1:上游引物cctcccaccaaccatcatct,下游引物tttcttgatgccccggatct,
ALCAM:上游引物ctgcaggaggttgaaggact,下游引物ggctggctttggaaaacctt,
CDH2:上游引物ccatcattgccatcctgctc,下游引物gtttggcctggcgttcttta,
ACTA2:上游引物aagatcctgactgagcgtgg,下游引物ttctccttgatgtcccggac。
有益效果:本发明通过NCAM1、ALCAM、CDH2、ACTA2四种标志基因组合可对体外培养HCECs进行角膜基质细胞污染及HCECs成纤维细胞转化程度进行鉴定,指导体外HCECs培养工艺参数的优化及对制备最终HCECs产品进行鉴定,保证体外培养HCECs具有CEC细胞特定功能。
附图说明
图1为本发明中差异基因分析图。
图2为本发明中CD56(NCAM1)、CD166(ALCAM)两种基因表达的蛋白在角膜组织切片的免疫荧光检测结果。
图3为实施例1中的细胞形态图。
具体实施方式
发明人通过对分离培养的HCECs与角膜基质细胞进行基因转录组高通量测序分析,筛选得到与基质细胞有显著性差异、且与角膜功能相关的8种基因(ACTA2、CDH2、ALCAM、TGFB1I1、COL9A2、LTBP2、PCP4、NCAM1),同时对制备得到的多批次HCECs进行质量研究,通过体外生物学评价方法并结合动物实验对HCECS进行有效性评价,最终确定HCECs细胞生物学特性及动物实验有效性相关的四种标志基因(NCAM1、ALCAM、CDH2、ACTA2),可用于指导体外HCECs培养工艺参数的优化及对制备的最终HCECs产品进行鉴定。具体过程如下:
步骤1,首先对发明人在前期分离培养的HCECs与角膜基质细胞进行基因转录组高通量测序,筛选出差异基因、同时对差异基因进行富集分析,富集分析结果见图1,筛选得到与基质细胞有显著性差异,同时与角膜功能相关的8种基因,基因列表见表1。
表1转录组测序分析筛选标志基因列表
geneName | Category | Description | Count | Up | Down |
ACTA2 | BP | muscle contraction | 106 | 73 | 33 |
CDH2 | BP | blood vessel morphogenesis | 178 | 107 | 71 |
ALCAM | BP | chemotaxis | 143 | 79 | 64 |
TGFB1I1 | BP | regulation of cellular response to growth factor stimulus | 82 | 46 | 36 |
COL9A2 | BP | skeletal system development | 109 | 55 | 54 |
LTBP2 | CC | extracellular matrix | 189 | 102 | 87 |
PCP4 | BP | positive regulation of nervous system development | 140 | 57 | 83 |
NCAM1 | BP | chemotaxis | 143 | 79 | 64 |
步骤2,对发明人前期制备的7批次HCECs、1批次角膜基质细胞、1批次角膜间质化细胞进行标志基因检测,检测结果见表2,对HCECs基因表达量对分析,NCAM1、ALCAM、CDH2、ACTA2基因组合能较好区分角膜基质细胞、角膜间质化细胞。
表2 HCEC细胞、角膜基质细胞、角膜间质化细胞标志基因检测
步骤3,对步骤2中的样本同时进行细胞表型检测,NCAM1、ALCAM、CDH2、ACTA2基因表达量与HCECs、角膜基质细胞、角膜间质化细胞具有表型相关性,与对照角膜基质细胞的基因表达量对比,统计7批次HCECs NCAM1、ALCAM、CDH2、ACTA2基因相对表达量(RQ),将7批次log(RQ)求均值后-SD作为HCECs质量标准,即NCAM1≥0.6、ALCAM≥1.0、CDH2≥2.0、ACTA2≤-1.0。结果见表3。
表3 NCAM1、ALCAM、CDH2、ACTA2基因表达量
通过以上结果,最终确定HCECs生物学特性及动物实验有效性相关的四种标志基因(NCAM1、ALCAM、CDH2、ACTA2),可用于指导体外HCECs培养工艺参数的优化及对制备的最终HCECs产品进行鉴定,如果待测细胞同时满足相对基因表达量log(RQ)值NCAM1≥0.6、ALCAM≥1.0、CDH2≥2.0、ACTA2≤-1.0,则说明该细胞为人类角膜内皮细胞。
其中,待测细胞和对照角膜基质细胞总RNA的提取和反转录为cDNA的步骤可以按照本领域的常规操作方法进行,本发明没有特别的要求。
其中,实时荧光定量PCR检测NCAM1、ALCAM、CDH2、ACTA2基因的表达水平的方法可以按照本领域的常规操作方法进行,本发明没有特别的要求。
所述扩增中用到的引物可以根据人类NCAM1、ALCAM、CDH2、ACTA2基因的mRNA序列及引物设计的规则来设计,优选情况下,所述引物包括如表4所示的上游引物和下游引物。
表4 HCECs鉴定引物
基因名称 | 上游引物 | 下游引物 |
NCAM1 | cctcccaccaaccatcatct(SEQ ID NO.1) | tttcttgatgccccggatct(SEQ ID NO.2) |
ALCAM | ctgcaggaggttgaaggact(SEQ ID NO.3) | ggctggctttggaaaacctt(SEQ ID NO.4) |
CDH2 | ccatcattgccatcctgctc(SEQ ID NO.5) | gtttggcctggcgttcttta(SEQ ID NO.6) |
ACTA2 | aagatcctgactgagcgtgg(SEQ ID NO.7) | ttctccttgatgtcccggac(SEQ ID NO.8) |
同时为了证明NCAM1、ALCAM基因能够区分角膜内皮及角膜基质细胞,对CD56(NCAM1)、CD166(ALCAM)两种基因表达的蛋白在角膜组织切片进行免疫荧光检测,如图2所示,结果显示D56(NCAM1)、CD166(ALCAM)为角膜基质细胞不表达,角膜内皮细胞高表达。
下面结合附图和具体实施例对本发明作进一步详细说明,但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。实施例中未注明具体条件的实验方法及未说明配方的试剂均为按照本领域常规条件。
实施例1
一种人类角膜内皮细胞的鉴定方法,该方法主要包括如下步骤:
1、待测细胞和对照角膜基质细胞的总RNA提取。
取100万待测细胞和对照角膜基质细胞离心后的细胞沉淀,分别加入1mL TRIZOL试剂(life technologies,货号15596018),用移液枪反复吹打30次使细胞完全裂解。根据TRIZOL试剂的说明书提取总RNA。用微量分光光度计检测RNA浓度后取1μg RNA,按照DNaseⅠ试剂盒(Thermo Scientific,货号EN0521)的说明书所述步骤进行基因组DNA的消化,以防止基因组DNA的干扰。
2、总RNA的反转录。
取上述去除基因组DNA后的总RNA,按照RevertAid First Stand cDNA SynthesisKit试剂盒(Thermo Scientific,货号K1622)说明书所述步骤进行反转录。得到的cDNA进行10倍稀释后即为实时荧光定量PCR的模板。
3、待测细胞的标志基因检测。
根据SuperRealPreMix Plus(SYBR Green)试剂盒(天根,货号FP205-02)说明书所述步骤进行NCAM1、ALCAM、CDH2、ACTA2基因的表达量检测。
具体来说,20μL反应体系包括:DEPC H2O 6.2μL,2×SuperReal PreMixPlus 10μL,10μM上游引物0.6μL,10μM下游引物0.6μL,50×ROX Reference Dye 2μL,待测样品cDNA0.6μL。
具体的PCR反应程序为:95℃15min;95℃10sec,62℃30sec,40个循环。
对多批次样本的相对基因表达量(RQ)检测,检测结果各基因log10RQ均值–SD为基准,根据上述鉴定标准,NCAM1≥0.6、ALCAM≥1.0、CDH2≥2.0、ACTA2≤-1.0,则可判定该人角膜内皮细胞纯度符合要求。
采用细胞形态检查及动物实验对上述结果进行验证,取样本编码1、2为基因相对表达量检测结果符合要求的人角膜内皮细胞,样本编码3为相对表达量检测结果不符合要求人角膜内皮细胞。
1.细胞形态检查
对四种标志基因检测符合要求的多批次细胞进行细胞形态检查,检测结果显示NCAM1、ALCAM、CDH2基因表达越高、ACTA2基因表达越低的样本,结果如图3所示,与间质化细胞相比,HCECs表现出典型多边形细胞形态。
2.将样本编码1、2、3培养角膜内皮细胞用于治疗角膜内皮功能不全,动物实验结果显示:角膜浑浊程度及浑浊面积角膜浑浊从边缘开始逐渐恢复,经NCAM1、ALCAM、CDH2、ACTA2基因鉴定符合要求角膜内皮细胞在动物体内呈现出角膜内皮细胞相关功能。
综上所述,NCAM1、ALCAM、CDH2、ACTA2基因表达水平符合设定质控要求的角膜内皮细胞样本,进行细胞形态检查及动物实验,细胞形态表现典型的角膜内皮样细胞,动物实验结果显示可用于替代角膜组织移植。因此,通过四种标志基因质量控制检测,能够获得质量较好的角膜内皮细胞。
序列表
<110> 江苏艾尔康生物医药科技有限公司
<120> 一种人类角膜内皮细胞的鉴定方法
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
cctcccacca accatcatct 20
<210> 2
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tttcttgatg ccccggatc 19
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ctgcaggagg ttgaaggact 20
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ggctggcttt ggaaaacctt 20
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ccatcattgc catcctgctc 20
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gtttggcctg gcgttcttta 20
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
aagatcctga ctgagcgtgg 20
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ttctccttga tgtcccggac 20
Claims (1)
1.一种人类角膜内皮细胞的鉴定方法,其特征在于:包括以下步骤:
步骤1,提取待测细胞和对照角膜基质细胞的总RNA并将其反转录为cDNA;
步骤2,以步骤1中得到的cDNA为模板进行实时荧光定量PCR扩增,检测NCAM1、ALCAM、CDH2、ACTA2基因的表达水平;
步骤3,根据步骤2的检测结果,将角膜基质细胞的各基因表达量定为1,计算待测细胞中各基因的相对表达量RQ,如果log(RQ)同时满足NCAM1 ≥ 0.6、ALCAM ≥1.0 、CDH2 ≥2.0、ACTA2 ≤ -1.0,则说明待测细胞为人角膜内皮细胞;
步骤2中所采用的扩增引物如下:
NCAM1:上游引物cctcccaccaaccatcatct,下游引物tttcttgatgccccggatct,
ALCAM:上游引物ctgcaggaggttgaaggact,下游引物ggctggctttggaaaacctt,
CDH2:上游引物ccatcattgccatcctgctc,下游引物gtttggcctggcgttcttta,
ACTA2:上游引物aagatcctgactgagcgtgg,下游引物ttctccttgatgtcccggac。
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