CN112980845A - 一种用于敲低人cbs基因的载体、制剂及其制备方法和应用 - Google Patents
一种用于敲低人cbs基因的载体、制剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种用于敲低人CBS基因的载体、制剂及其制备方法和应用，涉及生物制药技术领域，所述载体的制备方法包括：依据人CBS基因序列，设计引物，用于PCR扩增目的基因，具体序列见SEQ ID NO.1；对GV102载体中的限制性酶切位点BamH I和HindⅢ进行酶切，形成线性化GV102载体，所述GV102载体顺序为：hU6‑MCS‑CMV‑GFP‑SV40‑Neomycin；将上两步的产物进行连接构建GV102‑CBS载体。本发明制备的载体能够显著抑制人CBS基因的表达，达到抑制人甲状腺癌细胞增长、降低癌细胞迁移率的目的，可用于制备治疗人甲状腺癌的制剂之中。
Description
技术领域
本发明涉及生物制药技术领域，具体涉及一种用于敲低人CBS基因的载体、制剂及其制备方法和应用。
背景技术
甲状腺癌（thyroid carcinoma）是内分泌系统最常见的恶性肿瘤，属于甲状腺上皮的肿瘤，它包括四种病理类型，分别是乳头状癌、滤泡状癌、未分化癌和髓样癌。除髓样癌外，大约90%的甲状腺癌起源于滤泡上皮细胞。乳头状癌为最常见的恶性程度较低、预后较好的一种甲状腺癌。目前国内外对于甲状腺癌治疗的手段主要包括外科手术、促甲状腺素抑制治疗、生物治疗和中药治疗。但是还没有通过制备敲低胱硫醚β合成酶(CBS)基因的相关药物来治疗甲状腺癌的报道。
胱硫醚β合成酶(CBS)是一种细胞质中的同源四聚体，由63KD的亚基组成，是一种磷酸吡哆醛(pyridoxalphosphate,PLP)依赖性酶。人类CBS基因编码一种551个氨基酸的蛋白质，由四个63-kDa亚基组成的人CBS活性形式。每个亚单位由三个结构域组成，N端结构域与辅因子血红素结合，这是蛋白质成功折叠和组装所必需的。催化结构域包含另一种辅因子PLP 的结合位点。碳末端调节结构域包含两个CBS基序(CBS1和CBS2)，它们二聚形成贝特曼结构域。这个域名负责亚单位四聚化并含有变构激活剂S-腺苷甲硫氨酸的结合位点。在天然的四元结构中，底物进入催化核心的途径被碳末端调节基序封闭，SAM的结合诱导构象变化，从而改善底物进入催化位点的途径。CBS参与了有助于内源性硫化氢(H2S)产生的脱硫反应。CBS主要通过控制半胱氨酸（Hcy）和H2S代谢发挥多种生物学功能，包括线粒体生物能学、氧化还原稳态、DNA 甲基化和蛋白质修饰。CBS 的去调控和相关的Hcy 和/或H2S水平的改变导致心血管、免疫和中枢神经系统的广泛病理紊乱，并导致疾病的发展。
发明内容
为了解决上述问题，本发明提供一种用于敲低人CBS基因的shRNA，所述shRNA的具体序列见SEQ ID NO.1。
本发明还提供一种用于敲低人CBS基因的载体，所述载体包含本发明所述shRNA。
本发明还提供一种用于敲低人CBS基因的载体的制备方法，其特征在于，包括以下步骤：
S1：依据人CBS基因序列，设计引物，用于PCR扩增目的基因，具体序列见SEQ IDNO.1；
具体序列如下：CAAGGTCGGGCAGGAAGAG,TAATACGACTCACTATAGGG
S2：对GV102载体中的限制性酶切位点BamH I和Hind Ⅲ进行酶切，形成线性化GV102载体，所述GV102载体顺序为：hU6-MCS-CMV-GFP-SV40-Neomycin；
S3：将步骤S1和步骤S2的产物进行连接构建GV102-CBS载体。
本发明还提供一种用于敲低人CBS基因的制剂，所述制剂含有本发明所述载体。
本发明还提供一种本发明所述载体在制备治疗人甲状腺癌的制剂中的应用。
本发明的有益效果：
本发明制备的载体能够显著抑制人CBS基因的表达，达到抑制人甲状腺癌细胞增长、降低癌细胞迁移率的目的，可用于制备治疗人甲状腺癌的制剂之中。
除了上面所描述的目的、特征和优点之外，本发明还有其它的目的、特征和优点。下面将参照图，对本发明作进一步详细的说明。
附图说明
构成本申请的一部分的附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。
图1是本发明实施例的为CBS过表达质粒和敲低质粒转染进人甲状腺癌细胞后，通过Western blot检测CBS蛋白表达结果；
图2是本发明实施例的通过EDU检测细胞增殖能力图；
图3是本发明实施例的通过平板克隆检测细胞的克隆能力；
图4是本发明实施例的划痕实验；
图5为本发明实施例的为软琼脂实验；
图6为本发明实施例的细胞迁移实验；
图7为本发明实施例的细胞侵袭实验；
图8为本发明实施例的通过TUNEL实验检测细胞的凋亡；
图9为本发明实施例的裸鼠成瘤实验中肿瘤的大小；
图10为本发明实施例的裸鼠成瘤实验中裸鼠肿瘤的增长趋势；
图11为本发明实施例的裸鼠成瘤实验中裸鼠肿瘤重量；
图12为本发明实施例的裸鼠成瘤实验的肿瘤抑制率。
其中，Control为野生型细胞，Mock为过表达对照细胞，CBS为基因过表达组细胞，sh-Scb为敲低对照细胞，sh-CBS为基金敲低组细胞，DAPI是一种能够与DNA强力结合的荧光染料，用于显微镜观测，Merge是指两张图片融合。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
主要试剂、药品及样品：
人甲状腺癌细胞TPC-1、ARO购自南京科佰生物技术有限公司。
转染试剂Lipofectamine 3000购自Thermo Fisher公司；
遗传霉素G418、嘌呤霉素购自索莱宝公司；
MTS购自美国Sigma公司；
细胞增殖检测试剂盒购自锐博生物科技有限公司；
细胞凋亡检测试剂盒购自碧云天生物技术有限公司；
Transwell小室购自Corning公司。
主要仪器设备：
荧光倒置显微镜（型号：ECLIPSE Ti），购自Nikon公司。
实施例1制备敲低人CBS基因的载体
一种用于敲低人CBS基因的载体，其制备方法包括：
S1：依据人CBS基因序列，设计引物，用于PCR扩增目的基因，具体序列见SEQ IDNO.1；
S2：对GV102载体中的限制性酶切位点BamH I和Hind Ⅲ进行酶切，形成线性化GV102载体，所述GV102载体顺序为：hU6-MCS-CMV-GFP-SV40-Neomycin；
S3：将步骤S1和步骤S2的产物进行连接构建GV102-CBS载体。
S4：PCR鉴定，扩增人CBS基因，进行琼脂糖凝胶电泳。
S5：阳性克隆测序，鉴定GV102-CBS载体中人CBS基因正确性；
实施例2 制备过表达CBS基因的载体
一种用于过表达人CBS基因的载体，其制备方法包括：
S6：依据人CBS基因序列，设计引物，用于PCR扩增目的基因，具体序列见SEQ IDNO.2；
具体序列如下：CGAGCTCAAGCTTCGAATTCCGCCACCATGCCTTCTGAGACCCCCCAGG
S7：对GV230载体(载体顺序：CMV-MCS-EGFP-SV40-Neomycin)中的限制性酶切位点EcoRI和BamHI 进行酶切，形成线性化GV230载体；
S8：将步骤S6和步骤S7中产物进行连接构建GV230-CBS载体；
S9：PCR鉴定，扩增人CBS基因，进行琼脂糖凝胶电泳；
S10：阳性克隆测序，鉴定GV230-CBS载体中人CBS基因正确性。
实施例3
通过药敏试验确定最佳遗传霉素G418和嘌呤霉素浓度，即能够完全杀死细胞的最低G418和嘌呤霉素浓度；
具体为：
消化人甲状腺癌细胞TPC-1、ARO，并接种于96孔培养板，接种量为1×104个/孔；肿瘤细胞接种于96孔培养板后，第二天每孔分别加入100μL含有G418或嘌呤霉素的培养基，G418梯度设置具体为：100µg/mL、200µg/mL、300µg/mL、400µg/mL、500µg/mL、600µg/mL、700µg/mL、800µg/mL、900µg/mL、1mg/mL；嘌呤霉素梯度设置具体为：1µg/mL、2µg/mL、3µg/mL、4µg/mL、5µg/mL、6µg/mL、7µg/mL、8µg/mL、9µg/mL、10µg/mL；加入含有 G418或嘌呤霉素的培养基后，定期观察细胞培养情况，获得能够完全杀死细胞的最低G418浓度和嘌呤霉素浓度；
结果表明，针对上述2种人甲状腺癌细胞系，最优G418浓度均为 600µg/mL，最优嘌呤霉素浓度均为2µg/mL；
利用实施例1和实施例2制备的CBS敲低和过表达载体转染细胞，具体过程为：
分别消化上述2种人甲状腺癌细胞系，并接种6孔培养板，接种量3×105个/孔，接种后置于5%CO2、37℃培养箱内过夜；第二天转染前，将完全培养基换成DMEM基础培养基；
取一个EP管作为A管，加入100μL Optimem，然后加入3.75µL lip3000，轻轻混匀；另取一EP管作为B管，加入100μL Optimem，然后加入2.5μg质粒，再加5µLP3000，混匀；
将B管的液体加入A管混匀，孵育5min；
从培养箱内取出已接种细胞的6孔培养板，每孔分别加入上述混合物；
轻轻摇晃混匀溶液，放入培养箱内继续培养，6小时后换成完全培养基继续培养；48h后向瞬转的CBS敲低及敲低对照、过表达及过表达对照载体的细胞中加2µg/mL嘌呤霉素或600μg/mLG418，以后每3天换一次液，以去除被药物杀死的肿瘤细胞，在显微镜下观察转染效果，直至未转染细胞全部杀死，转染细胞不断增殖铺满整个孔板底面，并在荧光显微镜下可见绿色荧光表达即得到稳定株。
CBS 蛋白表达水平检测：
分别提取肿瘤细胞的野生型（即空白对照）、过表达对照、过表达、敲低对照和敲低CBS基因后肿瘤细胞中的总蛋白，对其浓度进行测定后，进行免疫印迹检测，以确定细胞中CBS蛋白的表达水平。
CBS蛋白表达水平检测如图1所示，从图中可以看出，过表达组细胞中的CBS蛋白表达水平较对照组有大幅度明显提高，敲低组细胞中的CBS蛋白表达水平较对照组明显偏低，表明本发明所制备的载体能较好地过表达或敲低了人甲状腺癌细胞中的CBS基因。
实施例4
（1）采用EDU法确定CBS过表达或敲低后对人甲状腺癌细胞增殖的影响，具体过程如下：
EDU标记（12孔板操作）：用细胞培养基按1000:1的比例稀释EdU溶液（试剂A），制备适量50μM EdU培养基；每孔加入300µL 50μM EdU培养基孵育2h，弃培养基；PBS清洗细胞2次，每次5min。
细胞固定化：每孔加入150µL细胞固定液（即含4%多聚甲醛的PBS）室温孵育30min，弃固定液；每孔加入150µL 2mg/mL甘氨酸，脱色摇床孵育5min，弃甘氨酸溶液；每孔加入300µL PBS，清洗1次，5min，弃PBS；（加强）每孔加入100µL渗透剂（0.5%Triton-X100的PBS）脱色摇床孵育 10min，PBS清洗1次，5min。
Apollo染色：每孔加入300µL的1×Apollo染色反应液（按顺序配，现用现配，30min用完），避光，室温，脱色摇床孵育30min，弃染色反应液；
Apollo染色反应液配制：1.5mL去离子水1407µL
Apollo 反应缓冲液（B）75µL
Apollo 催化剂溶液（C）15µL
Apollo 荧光染料溶液（D）4.5µL
Apollo 缓冲添加剂（E）13.5mg
加入300µL渗透剂（0.5%Triton-X 100 的PBS）脱色摇床清洗2次，每次10min，弃渗透剂；（加强）每孔每次加入300µL甲醇清洗1-2 次，每次5min，PBS洗1次，每次5min。
DNA染色：用去离子水按100:1的比例稀释试剂F，制备适量1×Hoechst33342反应液，避光保存；每孔加入300µL1×Hoechst33342反应液，避光，室温，脱色摇床孵育30min，弃染色反应液；每孔每次加入300µLPBS 洗3次，每次5min；每孔加300µLPBS保存，拍照，计数细胞增值率。
结果如图2所示。从图中可以看出，CBS基因过表达的肿瘤细胞其细胞增殖率显著高于对照组肿瘤细胞，CBS基因敲低的肿瘤细胞其细胞增值率显著低于对照组肿瘤细胞，这表明在肿瘤细胞中敲低CBS基因为抑制肿瘤细胞生长（因为专利中不允许使用彩图，所以图中不能清除的看到此结论）。
（2）采用平板克隆法确定CBS过表达或敲低后对人甲状腺癌细胞克隆能力的影响，
具体过程如下：
消化人甲状腺癌细胞并重悬计数，六孔板中每孔加约500个细胞，放5%CO2、37℃细胞培养箱中，两周后待其长出肉眼可见克隆停止培养，将细胞用PBS洗一遍，加甲醇固定10min，吸走甲醇，用0.2%结晶紫染色30min，用PBS洗去多余结晶紫，拍照。
结果如图3所示。从图中可以看出，CBS基因过表达的肿瘤细胞克隆团数量及大小显著高于对照组肿瘤细胞，CBS基因敲低的肿瘤细胞克隆团数量及大小显著低于对照组肿瘤细胞，这表明在肿瘤细胞中过表达CBS基因可以促进肿瘤细胞的克隆，敲低CBS基因为抑制肿瘤细胞克隆。
（3）采用划痕法确定CBS过表达或敲低后对人甲状腺癌细胞迁移能力的影响，
具体过程如下：
分别在六孔板内铺不同处理组细胞，待细胞融合度达到100%。用中枪尖沿直尺划线，实时观察稳定株，分别取0、12、24小时拍照。迁移率(100%)=[(A - B)/A]×100，其中A表示0h时的划痕宽度，B为24h时的宽度。结果如图4所示。从图中可以看出，CBS基因过表达的肿瘤细胞迁移率增加，CBS基因敲低的肿瘤细胞迁移率降低。
（4）采用软琼脂集落形成法确定CBS过表达或敲低后对人甲状腺癌细胞增殖的影响，
具体过程如下：
1）用双蒸水配1.2%、0.7%两个浓度的琼脂糖溶液，高压灭菌，放60℃干燥箱以免凝固；
2)同时将细胞离心，用含20% FBS的2×DMEM重悬细胞并计数，最终六孔板每孔加10000个细胞；
3)将1.2%琼脂糖与2×DMEM(含血清平和双抗)等体积混合，六孔板中每孔加2mL，室温下待凝固作为底层琼脂；
4)0.6%琼脂糖与2×DMEM等体积混合(于50mL离心管中)，每孔加2mL，迅速加入细胞悬液，混匀，切勿产生气泡，在室温放置20min使细胞琼脂凝固，然后在上层加1mL DMEM培养基，防止干；
5)置于5%CO2、37℃培养箱，两周后显微镜下拍照。
结果如图5所示。从图中可以看出，CBS基因过表达的肿瘤细胞集落数增加，CBS基因敲低的肿瘤细胞集落数减少。
实施例5
为检测上述实施例4中过表达PCNP基因对肿瘤细胞迁移的影响，发明人做了transwell检测实验，相关过程介绍如下。
（1）采用Transwell法确定CBS过表达或敲低后对肿瘤细胞迁移的影响，
具体过程如下：
将小室置于24孔板中，小室下层加入600µL含20%血清的培养基，上层加入200µL不含血清的培养基，每孔4×104个细胞，37℃培养箱内孵育 24h；取出培养板，弃去培养基，每孔加75%酒精固定15min，PBS洗2次；
结晶紫染色10min，自来水冲洗，将结晶紫洗去，用棉签将小室上层擦干净，用刀片轻轻刮下薄膜放于载玻片上，用中性树胶固定，100×镜下拍照。
（2）采用Invasion法确定CBS过表达或敲低后对肿瘤细胞侵袭的影响，
具体过程如下：
具体操作同（1），不同的是小室上有基质胶，每孔6×104个细胞。迁移结果如图6所示，CBS基因过表达肿瘤细胞与对照组相比，迁移能力明显增强，CBS基因敲低肿瘤细胞与对照组相比，迁移能力明显降低；侵袭结果如图7所示，CBS基因过表达肿瘤细胞与对照组相比，侵袭能力明显增强，CBS基因敲低肿瘤细胞与对照组相比，侵袭能力明显降低，因此表明在人甲状腺癌细胞中敲低CBS可以降低细胞迁移和侵袭。
实施例6
为检测上述实施例4中过表达或敲低CBS基因对人甲状腺癌细胞凋亡的影响，发明人做了TUNEL检测实验，相关过程介绍如下。
采用TUNEL法确定CBS过表达或敲低后对肿瘤细胞凋亡的影响，
具体过程如下：
将细胞消化计数，铺至24孔培养板中，接种量为5×104个/孔，于5% CO2、37℃培养箱内孵育；待细胞贴壁12小时后，用PBS将细胞清洗1次；用4%多聚甲醛固定30min；PBS清洗1次；加入含0.1%Triton-X 100的PBS，冰浴孵育2min；PBS洗2次，配250µL Tunel检测液：10µLTdT酶+240µL荧光标记液；在样品上加50µL Tunel检测液，37℃避光孵育60min；PBS洗3次；DAPI染色：5%BSA 1:1000稀释，3min；每孔加200µLPBS保存，荧光显微镜下观察，拍照，计数细胞凋亡率。
结果如图8所示。从图中可以看出，CBS基因过表达的肿瘤细胞其细胞凋亡率显著低于对照组肿瘤细胞,CBS基因敲低的肿瘤细胞其细胞凋亡率显著高于对照组肿瘤细胞，这表明在肿瘤细胞中敲低CBS可以促进肿瘤细胞的凋亡。
实施例7
为证明在体内敲低人甲状腺癌细胞中CBS基因能够抑制人甲状腺癌的生长，进行了裸鼠成瘤实验，相关过程介绍如下。
采用裸鼠成瘤实验确定CBS敲低在体内抑制人甲状腺癌细胞生长，具体过程如下：
将细胞消化计数，用PBS重悬细胞配制成1×107个/mL的细胞悬液，采用1mL注射器取细胞悬液0.1mL注射于裸鼠腋下；之后每天测量肿瘤体积，给小鼠称重，观察肿瘤变化，连续测量4周；4周后将小鼠处死，从腋下取出肿瘤，称重并拍照，计算抑瘤率。
结果如图9所示，图9为注射细胞4周后从裸鼠腋下取出的肿瘤图片，可见CBS敲低后肿瘤体积明显减小；图10为28天内肿瘤体积变化，结果表明与对照相比，CBS敲低后肿瘤体积增速明显减慢；图11为肿瘤取出后测得的瘤重，CBS 敲低后肿瘤重量明显减轻；图12是根据瘤重统计得出的抑瘤率，可见CBS敲低对肿瘤形成有明显抑制作用。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，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。
SEQUENCE LISTING
<110> 河南大学
<120> 一种用于敲低人CBS基因的载体、制剂及其制备方法和应用
<130> 1
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
caaggtcggg caggaagagt aatacgactc actataggg 39
<210> 2
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cgagctcaag cttcgaattc cgccaccatg ccttctgaga ccccccagg 49
Claims (5)
1.一种用于敲低人CBS基因的shRNA，其特征在于，所述shRNA的具体序列见SEQ IDNO.1。
2.一种用于敲低人CBS基因的载体，其特征在于，所述载体包含权利要求1所述shRNA。
3.一种用于敲低人CBS基因的载体的制备方法，其特征在于，包括以下步骤：
S1：依据人CBS基因序列，设计引物，用于PCR扩增目的基因，具体序列见SEQ ID NO.1；
S2：对GV102载体中的限制性酶切位点BamH I和Hind Ⅲ进行酶切，形成线性化GV102载体，所述GV102载体顺序为：hU6-MCS-CMV-GFP-SV40-Neomycin；
S3：将步骤S1和步骤S2的产物进行连接构建GV102-CBS载体。
4.一种用于敲低人CBS基因的制剂，其特征在于，所述制剂含有权利要求2所述载体。
5.一种权利要求2所述载体在制备治疗人甲状腺癌的制剂中的应用。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107630027A (zh) * | 2017-09-14 | 2018-01-26 | 吴东栋 | 一种过表达pcnp基因的药物的制备及用途 |
| US20200276322A1 (en) * | 2017-10-25 | 2020-09-03 | The Administrators Of The Tulane Educational Fund | Peptide compositions and methods of use thereof |
-
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107630027A (zh) * | 2017-09-14 | 2018-01-26 | 吴东栋 | 一种过表达pcnp基因的药物的制备及用途 |
| US20200276322A1 (en) * | 2017-10-25 | 2020-09-03 | The Administrators Of The Tulane Educational Fund | Peptide compositions and methods of use thereof |
Non-Patent Citations (2)
| Title |
|---|
| DONGDONG WU ET AL.: "Exogenous Hydrogen Sulfide Regulates the Growth of Human Thyroid Carcinoma Cells", 《OXIDATIVE MEDICINE AND CELLULAR LONGEVITY》 * |
| 叶洋等: "气体信号分子硫化氢在肿瘤发生发展与治疗中的作用研究进展", 《江苏大学学报(医学版)》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113621632A (zh) * | 2021-08-06 | 2021-11-09 | 河南大学 | 一种敲低cbs基因的方法及其在制备治疗人脑胶质瘤的药物中的应用 |
| CN113621632B (zh) * | 2021-08-06 | 2023-06-06 | 河南大学 | 一种敲低cbs基因的方法及其在制备治疗人脑胶质瘤的药物中的应用 |
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