CN113699183A - 一种标记及追踪cd133阳性室管膜细胞的重组质粒、构建方法及鉴定方法 - Google Patents
一种标记及追踪cd133阳性室管膜细胞的重组质粒、构建方法及鉴定方法 Download PDFInfo
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Abstract
本发明属于生物工程领域，具体涉及一种标记及追踪CD133阳性室管膜细胞的重组质粒、构建方法及鉴定方法。所述重组质粒是以质粒pCAG‑cre为载体，在质粒pCAG‑cre限制性酶切位点SpeI和EcoRI之间插入CD133‑promoter2基因得到的CD133‑promoter2‑Cre重组质粒，所述CD133‑promoter2基因序列为SEQ ID NO:1所示。当本发明提供的重组质粒在活体内成功电转染到ROSA26‑LacZ小鼠的CD133阳性室管膜细胞中，通过β‑gal免疫荧光染色可以检测到转染成功的CD133阳性的室管膜细胞和它们的子代细胞，从而可以观察到CD133阳性的室管膜细胞的迁移路径和分化情况。
Description
技术领域
本发明属于生物工程领域，具体涉及一种标记及追踪CD133阳性室管膜细胞的重组质粒、构建方法及鉴定方法。
背景技术
在成体哺乳动物的大脑中，神经干细胞（NSCs）存在于许多地方，例如海马齿状回、嗅球、室管膜下区等，其中在前脑中生成神经元和胶质细胞的NSCs分布的区域主要是脑室区（VZ）与室下区（SVZ）。紧临室管膜细胞的SVZ是由具有迁移特性的成神经细胞，星形胶质细胞样形态的NSCs和快速分裂及短暂扩增的NSCs组成，室管膜细胞因与SVZ临近，被认为具有神经干细胞的属性。CD133/Prominin-1是细胞表面含5次跨膜结构的糖蛋白分子，在分离小鼠NSCs时被发现，也表达在多种组织干细胞和肿瘤干细胞中。通常CD133标记的室管膜细胞广泛分布于脑室的室管膜下区和SVZ中，正常情况下CD133阳性的室管膜细胞是处于静息状态（qNSC），当机体遭受损伤或受到信号通路调控时，CD133阳性室管膜细胞会从静息状态被激活而具有NSCs属性，形成短暂扩增细胞（TAC）并迅速分化为成神经母细胞并沿喙侧迁移流迁移到其它部位（如嗅球），表明体内静息状态的室管膜细胞仍保留细胞分裂的能力，在受到损伤时被激活成具有NSCs属性并产生新的神经元。NSCs如何平衡增殖和分化这个问题不仅是神经生物学今后发展的关键问题，而且与脑肿瘤形成、中枢神经系统畸形、神经衰老和中风等临床疾病高度相关。因此可靠的追踪CD133阳性室管膜细胞的方法在研究如何防止和治疗中风、神经衰老、胶质细胞瘤等疾病的发生中具有重要的科学和临床意义。
目前检测CD133阳性室管膜细胞的方法是通过CD133抗体进行免疫荧光染色标记，但是无法追踪室管膜细胞及其子代细胞的迁移路径以及分化的情况。
发明内容
本发明的目的是为了克服现有技术存在的缺点和不足，而提供一种标记及追踪CD133阳性室管膜细胞的重组质粒、构建方法及鉴定方法。
本发明所采取的技术方案如下：一种标记及追踪CD133阳性室管膜细胞的重组质粒，所述重组质粒是以质粒pCAG-cre为载体，在质粒pCAG-cre限制性酶切位点SpeI和EcoRI之间插入CD133-promoter2基因得到的CD133-promoter2-Cre重组质粒，所述CD133-promoter2基因序列为SEQ ID NO:1所示。
如上所述的标记及追踪CD133阳性室管膜细胞的重组质粒的构建方法，包括以下步骤：
（1）人工合成带有酶切位点SpeI和EcoRI序列的CD133-promoter2产物；
（2）将带有酶切位点SpeI和EcoRI序列的CD133-promoter2产物经SpeI和EcoRI双酶切，回收纯化带有CD133-promoter2基因的目的片段；
（3）将质粒pCAG-cre经SpeI和EcoRI双酶切，回收纯化酶切后的质粒pCAG-cre片段；
（4）将步骤（2）得到的带有CD133-promoter2基因的目的片段和步骤（3）得到的质粒pCAG-cre片段酶连组装；
（5）将步骤（4）酶连组装得到的产物转移到受体菌或细胞中进行转化克隆，繁殖筛选得到阳性克隆株，表达提取得到CD133-promoter2-Cre重组质粒。
步骤（5）中，步骤（4）酶连组装得到的产物转移到DH5α感受态细菌中，培养后涂于含氨苄青霉素的平板上培养，然后调取阳性克隆接种于含氨苄青霉素的培养液中培养，然后提取得到重组质粒。
一种鉴定转染有如上所述的标记及追踪CD133阳性室管膜细胞的重组质粒的ROSA26-LacZ小鼠的CD133阳性室管膜细胞其子代细胞的方法，通过β-gal免疫荧光染色检测。
本发明的有益效果如下：当本发明提供的重组质粒在活体内成功电转染到ROSA26-LacZ小鼠的CD133阳性室管膜细胞中，通过β-gal免疫荧光染色可以检测到转染成功的CD133阳性的室管膜细胞和它们的子代细胞，从而可以观察到CD133阳性室管膜细胞的迁移路径和分化情况。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，根据这些附图获得其他的附图仍属于本发明的范畴。
图1为重组质粒CD133-promoter2-Cre的基因测序的比对：图1和2显示来自CD133-promoter 2-Cre的样品1和2的反向互补的DNA序列，而3显示来自 CD133- promoter 2的原始DNA序列。
图2为用限制酶将重组 CD133-promoter 2-Cre质粒酶切后通过琼脂糖凝胶电泳鉴定，其中，1和2显示的为253-bp的CD133-promoter 2片段，3和4显示的为4.17-kbp的启动子和1.7-kbp的Cre片段，5显示的为重组CD133-promoter 2-Cre的4.429-kbpPCR片段。
图3为使用ROSA26-LacZ作为报告基因小鼠的Cre -LoxP介导的重组方案图。在重组之前，floxed Stop序列在其启动子的控制下表达，而LacZ是沉默的，重组后，Cre介导的重组使floxed Stop序列被删除，所以表达LacZ的细胞表示CD133阳性室管膜细胞及其子代细胞。
图4中，（D~R）为在3V的室管膜或SVZ层中观察到的CD133阳性细胞（D, G, J, M和P）和红色标记的Sox2（E）或GFAP（H）或Nestin（K）或β-gal（N, Q）。图F，I，L，Q，R分别是D和E，G和H，J和K，M和N，P和Q的合并。在图像L中，黄色箭头指示的CD133/Nestin双标记的细胞是绿色箭头显示的CD133阳性细胞（J）和红色箭头显示Nestin阳性细胞（K）的合并。在图像O中，黄色箭头显示的β-gal/CD133双阳性细胞，绿色箭头（M）显示的CD133阳性细胞和红色箭头显示β-gal阳性细胞（N）的合并。在图像R中，黄色箭头显示的β-gal/Cre双阳性细胞是绿色箭头（P）显示的CD133阳性细胞和红色箭头显示的β-gal阳性细胞（Q）的合并。注：3V，第三脑室。图像D〜O的标尺为100μm，P〜R的标尺为50μm。
图5为6-OHDA注射后β-gal标记的CD133阳性细胞子代细胞的迁移和分化。（A，B）白色箭头表示6-OHDA注射后3天的β-gal阳性细胞在6-OHDA注射侧和溶剂注射侧的3V或Aq的室管膜或SVZ层中的分布。（C）6-OHDA注射后的第7天（D7），白色箭头表示的β-gal阳性细胞向SN移动。在图像A〜C的右下白框区域中由白色箭头表示的是X-gal阳性细胞的高倍放大图片。（D）三重标记，证实在6-OHDA注射后第7天（D7），β-gal阳性细胞（红色）在TH阳性神经元（绿色）周围迁移，Hoechst阳性细胞核显示为蓝色。图像E是在图像G的小白框中由黄色箭头表示的三重标记的细胞的高倍放大图。注意：3V，第三脑室；Aq，中脑水管；SN，黑质。（F〜M）β-gal阳性细胞（F和J，红色），NeuN阳性神经元或TH阳性神经元（G和K，绿色）和Hoechst阳性细胞核（H和L，蓝色），6-OHDA注射后第14或21天SNpc中观察到（D14和D21）。在图像I和M中，黄色箭头指示β-gal/NeuN/Hoechst或β-gal/TH/Hoechst三标记的细胞，红色箭头（F或J）显示β-gal阳性细胞合并，绿色箭头（G或K）表示NeuN阳性神经元或TH阳性神经元，蓝色箭头（H或L）显示Hoechst阳性核。对于图像F〜I，标尺为100μm，对于图像I〜M，标尺为50μm。
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，下面将结合附图对本发明作进一步地详细描述。
一、材料
1.菌株，质粒
DH5α 天跟
pCAG-cre 上海同济大学实验动物中心惠赠
2.实验动物
ROSA26-LacZflox/flox报告基因小鼠（上海同济大学实验动物中心惠赠）经本校实验动物中心传代，鉴定基因型。选用4月龄雄性小鼠，体重25-30 g。SPF级4月龄B6小鼠作为质粒活体内电转染的对照小鼠，体重25-30g。
3.主要试剂和仪器
二、实验方法
1. 重组质粒CD133-Promoter2-Cre构建
1.1．根据CD133基因的promoter2序列（SEQ ID NO:1）生工构建，加有酶切位点SpeI（ACTAGT）和EcoRI（GAATTC）序列。
1.2．酶切
将生工构建产物CD133-promoter2和质粒pCAG-cre经SpeI和EcoRI双酶切，1%琼脂糖凝胶分离纯化酶切产物。
CD133-promoter2产物酶切体系和反应条件如下：
10X FastDigest buffer 2ul
CD133-promoter2产物2ul（1ug）
FastDigest Spe I 1 ul
FastDigest EcoRI 1ul
去离子水 14ul
总体系20ul，37度水浴反应30min，80度灭活5min
质粒pCAG-cre酶切体系、反应条件同上。
酶切产物经1%琼脂糖凝胶电泳鉴定，琼脂糖凝胶回收试剂盒回收纯化目的片段。
1.3．酶连
目的基因CD133-promoter2和质粒pCAG-cre约按3:1摩尔比混合于20ul反应体系中，20度连接3h。
20ul反应体系如下：
目的基因CD133-promoter2 x ul
pCAG-cre 3x ul
T4 DNA Ligase 0.5ul（2.5U）
10X T4 DNA Ligase buffer 2ul
ddH2O 补足到20ul。
1.4．转化克隆
在冰上取连接产物10ul加入DH5α感受态细菌中，冰浴30min。
42℃热激活90s，再冰浴1min。
加入800ul LB培养液，37℃振荡培养1h。
取适量菌液涂于含氨苄青霉素的LB平板上，37℃过夜（12~14h）培养。
调取阳性克隆接种于10ml 含氨苄青霉素的LB的培养液中，37℃振荡培养到适当浓度。
参照质粒小提试剂盒提取质粒，将提出的质粒送至擎科生物公司测序。
1.5．去内毒素质粒大提
将1ml构建成功的甘油菌接种到500ml含含氨苄青霉素的LB的培养液中，37℃振荡培养到适当浓度，参照质粒大提去内毒素试剂盒进行抽提。
1.6. 通过DNAMAN（Lynnon Corporation，USA）软件将测序结果与CD133-Promoter2原始序列进行比对。
．ROSA-LacZ reporter小鼠鉴定及给药
侧脑室定位注射质粒并活体内电转染：腹腔注射5％水合氯醛使B6或ROSA26-LacZflox/flox报道基因小鼠（25〜30g）完全麻醉，脑立体定位注射CD133-Promoter2-Cre重组质粒（4μl，1μg/μl）到右侧LV（AP -1.0mm，ML -0.35mm，DV -2.25mm），随后，使用BTX ECM830电转仪对鼠脑进行连续6次电击，每次电转600毫秒75V，间隔时间200毫秒，将CD133-Promoter2-Cre重组质粒电转染至表达CD133的室管膜细胞中。接着在同侧SN注射6-OHDA。手术后第3,7,14,21,28或35天，处死小鼠，取脑冰冻切片做免疫荧光染色。
．免疫荧光染色
小鼠麻醉、开胸，4％冷多聚甲醛溶液灌注固定，快速剥取脑组织，并浸入4℃ 4％多聚甲醛溶液中后固定8 h，30％蔗糖0.01mol/LPBS溶液中脱水处理48 h，待组织沉底用OCT包埋，进行脑组织冠状位连续冰冻切片，切片厚度40 μm，共收集六套连续冠状切片，每套中的切片间距为240 μm。为检测BrdU掺入的增殖细胞，切片预先在96℃的抗原修复液（0.01 mol/L柠檬酸钠溶液，pH 6.0）中修复6 min，含0.1% Triton X-100的PBS溶液漂洗两次，之后在2N盐酸溶液中变性60 min， 0.1mol/L的硼酸盐缓冲液（pH 8.5）中和10 min，漂涤3次后浸入含3％TritonX-100，1％牛血清白蛋白（Bovine serum albumin, BSA）及一抗的溶液中4℃孵育过夜。一抗包括：β-gal抗体（1:3200；Cell Signal Technology，USA）；酪氨酸羟化酶（tyrosine hydroxylase，TH）抗兔多克隆抗体（1: 400，Merck Millipore，USA），CD133抗大鼠抗体（1:100，eBioscience，USA），BrdU抗鼠单克隆抗体（1:500，Novus，USA）。次日，脑切片再于室温下加入合适的荧光二抗孵育1.5 h，二抗包括：DyLightTM594驴抗小鼠IgG（1: 400；Jackson，USA），DyLightTM488山羊抗兔IgG（1: 400；EarthOX，USA）。PBS漂洗三次后，用含Hoechst的封片剂封片。对照实验采用逐步省去抗体或以正常血清替代，结果为阴性。
三、实验结果
1.C D133阳性室管膜细胞性质的鉴定
为了进一步追踪 SVZ源性 NPCs 的子代并确认SNpc中新产生的TH阳性神经元的来源，构建了含有 CD133（prominin-1）mP2启动子的Cre重组质粒。重组 CD133-promoter2-Cre 的测序结果如SEQ ID NO:2和SEQ ID NO:3所示，与CD133 - promoter 2原始序列对比，其中限制性酶切位点SpeI和EcoRI之间均含有与CD133 - promoter 2原始序列一致的基因序列（图 1）。来自琼脂糖凝胶电泳的结果显示，通过限制酶消化目的片段的长度与理论值相一致（图 2）。一旦 CD133-promoter 2-Cre 质粒在活体内电转染到ROSA26-LacZ小鼠的室管膜细胞中，在CD133 -promoter 2的控制下成功激活Cre ，被 LoxP位点靶定着的转录终止子被切除，便可诱导 LacZ表达（图 3）。电转染后在不同的时间点通过β-gal免疫荧光染色可以检测到转染成功的CD133阳性的室管膜细胞和它们子代细胞。我们的研究结果表明， CD133阳性室管膜细胞主要位于3V内衬的室管膜或SVZ层，它们并不与Sox2（神经前体细胞的另一个标记）或GFAP共标记。但是其中一小部分CD133阳性室管膜细胞可以与Nestin共标记（图4D-L），表明CD133阳性室管膜细胞可分化成Nestin阳性NPCs。此外，在6-OHDA注射后第3天，12±3.29％的CD133阳性室管膜细胞可以与β-gal共标记，表明β-gal阳性细胞是CD133阳性室管膜细胞的子代细胞（图4M-O），同时几乎所有的β-gal阳性细胞与Cre共标，表明β-gal阳性细胞具备Cre重组酶活性，体内电转成功（图4P-R）。
2. 6-OHDA 对 β-gal阳性细胞迁移和分化的影响
6-OHDA注射后第3天，大量β-gal标记的CD133阳性细胞及其子代主要出现在6-OHDA注射侧的3V和Aq的室管膜或SVZ层中（图5A，B）。6-OHDA注射后的第7天，观察到6-OHDA注射侧中的一些β-gal阳性细胞突出进入脑室邻近实质区域，例如来自3V和Aq的室管膜或SVZ层的SN（图5C）
6-OHDA注射后第14天或第21天，我们发现6-OHDA注射侧向SN迁移的这些β-gal阳性细胞的8.24±1.21％或3.23±0.64％分化成NeuN或TH阳性神经元（图5F-M），显示CD133阳性室管膜细胞的子代细胞可能在6-OHDA注射的情况下进入神经源性分化程序。此外，在用空质粒处理的对照组中没有观察到β-gal阳性细胞。
以上所揭露的仅为本发明较佳实施例而已，当然不能以此来限定本发明之权利范围，因此依本发明权利要求所作的等同变化，仍属本发明所涵盖的范围。
序列表
<110> 温州医科大学
<120> 一种标记及追踪CD133阳性室管膜细胞的重组质粒、构建方法及鉴定方法
<160> 3
<170> SIPOSequenceListing 1.0
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<212> DNA
<213>人工合成
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gtgagtatgt ttaaggaatc ctttccatta cggcggcccc atacctaggt ccccgtccgg 60
gacagaggaa gccgcaacgg gtccccccgg gcacccgggc ctttctcctg cctcccgcca 120
cgtccgaggg tccggccgca gcgccgcctg agcccctccg cggccggcag tgggaggcgg 180
gctctccgaa agccgtcgcg gtggtcccag aagccgggtc ataaataatt cacgagccag 240
ggtctggcga gct 253
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<212> DNA
<213> 人工合成
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tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 60
aaagcgggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg cccgcagtgt 120
tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 180
gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 240
cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa 300
aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 360
tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 420
tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 480
gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 540
atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa 600
taggggttcc gcgcacattt ccccgaaaag tgccacctgg gtcgacattg attattgact 660
agtgtgagta tgtttaagga atcctttcca ttacggcggc cccataccta ggtccccgtc 720
cgggacagag gaagccgcaa cgggtccccc cgggcacccg ggcctttctc ctgcctcccg 780
ccacgtccga gggtccggcc gcagcgccgc ctgagcccct ccgcggccgg cagtgggagg 840
cgggctctcc gaaagccgtc gcggtggtcc cagaagccgg gtcataaata attcacgagc 900
cagggtctgg cgagctgaat tctgagccgc caccatggcc aatttactga ccgtacacca 960
aaatttgcct gcattaccgg tcgatgcaac gagtgatgag gtcgcaagaa cctgatgaca 1020
tcagct 1026
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gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc 60
ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa 120
gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat 180
gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata 240
gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca 300
tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag 360
gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc 420
agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc 480
aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata 540
ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta 600
gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgggtcgac 660
attgattatt gactagtgtg agtatgttta aggaatcctt tccattacgg cggccccata 720
cctaggtccc cgtccgggac agaggaagcc gcaacgggtc cccccgggca cccgggcctt 780
tctcctgcct cccgccacgt ccgagggtcc ggccgcagcg ccgcctgagc ccctccgcgg 840
ccggcagtgg gaggcgggct ctccgaaagc cgtcgcggtg gtcccagaag ccgggtcata 900
aataattcac gagccagggt ctggcgagct gaattctgag ccgccaccat ggccaattta 960
ctgaccgtac accaaaattt gcctgcatta ccggtcgatg caacgagtga tgaggtcgca 1020
agaacctgat gacatcgcca 1040
Claims (4)
1. 一种标记及追踪CD133阳性室管膜细胞的重组质粒，其特征在于：所述重组质粒是以质粒pCAG-cre为载体，在质粒pCAG-cre限制性酶切位点SpeI和EcoRI之间插入CD133-promoter2基因得到的CD133-promoter2-Cre重组质粒，所述CD133-promoter2基因序列为SEQ ID NO:1所示。
2.如权利要求1所述的标记及追踪CD133阳性室管膜细胞的重组质粒的构建方法，其特征在于，包括以下步骤：
（1）人工合成带有酶切位点SpeI和EcoRI序列的CD133-promoter2产物；
（2）将带有酶切位点SpeI和EcoRI序列的CD133-promoter2产物经SpeI和EcoRI双酶切，回收纯化带有CD133-promoter2基因的目的片段；
（3）将质粒pCAG-cre经SpeI和EcoRI双酶切，回收纯化酶切后的质粒pCAG-cre片段；
（4）将步骤（2）得到的带有CD133-promoter2基因的目的片段和步骤（3）得到的质粒pCAG-cre片段酶连组装；
（5）将步骤（4）酶连组装得到的产物转移到受体菌或细胞中进行转化克隆，繁殖筛选得到阳性克隆株，表达提取得到CD133-promoter2-Cre重组质粒。
3.根据权利要求2所述的标记及追踪CD133阳性室管膜细胞的重组质粒的构建方法，其特征在于：步骤（5）中，步骤（4）酶连组装得到的产物转移到DH5α感受态细菌中，培养后涂于含氨苄青霉素的平板上培养，然后调取阳性克隆接种于含氨苄青霉素的培养液中培养，然后提取得到重组质粒。
4.一种鉴定转染有如权利要求1所述的标记及追踪CD133阳性室管膜细胞的重组质粒的ROSA26-LacZ小鼠的CD133阳性室管膜细胞其子代细胞的方法，其特征在于：通过β-gal免疫荧光染色检测。
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