CN112626031B - 一种基因修饰的牙髓干细胞及其制备方法和应用 - Google Patents
一种基因修饰的牙髓干细胞及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种基因修饰的牙髓干细胞及其制备方法和应用,属于脊髓损伤治疗技术领域,所述牙髓干细胞过表达骨桥蛋白、胰岛素样生长因子1和睫状神经营养因子;制备方法包括:1)将骨桥蛋白、胰岛素样生长因子1和睫状神经营养因子的基因克隆到穿梭质粒pUC57中,获得重组质粒pUC57‑Syn;2)将所述重组质粒pUC57‑Syn和骨架质粒pKAd5f11pES‑PmeI分别酶切、Gibson组装后的组装产物电转染细胞后获得重组腺病毒Ad‑OIC;3)将所述重组腺病毒Ad‑OIC感染牙髓干细胞获得基因修饰的牙髓干细胞。本发明提供的基因修饰的牙髓干细胞能够改善脊髓损伤炎症微环境,达到治疗脊髓损伤的效果。
Description
技术领域
本发明属于脊髓损伤治疗技术领域,尤其涉及一种基因修饰的牙髓干细胞及其制备方法和应用。
背景技术
脊髓损伤(SCI)患者会发生永久性的机体运动和感觉功能障碍,且临床上缺乏行之有效的治疗方法。脊髓损伤按照病理生理可分为原发性损伤和继发性损伤,原发性损伤是由各种外力对脊髓造成的直接损伤,在发生瞬间已经形成,表现为血管损伤及神经细胞、胶质细胞等大量死亡。继发性损伤则是由于这些细胞死亡所导致的缺血再灌注、炎症反应、离子紊乱、毒性物质的释放等构成更为严重的损伤过程。继发性损伤对SCI疾病加重至关重要,也是目前各种临床干预措施的重点。及早针对继发性损伤进行干预,能有效减缓SCI的恶化,减轻脊髓损伤状况以及远期的致残程度,利于SCI患者神经功能的后期恢复。
脊髓损伤后,多种免疫细胞和免疫分子被活化,这些炎症细胞(如巨噬细胞,中性粒细胞等)及炎症因子(如TNF-α及IL-1β)产生毒性作用,抑制脊髓内残余神经元再生。同时,损伤微环境中的多种分子如髓鞘相关的抑制蛋白(Nogo,MAG,Omgp)、硫酸软骨素蛋白聚糖(CSPGs)及轴突导向分子(semaphorins,ephrins)等,也可通过不同的信号途径抑制神经元的轴突再生。因此,控制损伤后炎性环境,构建适宜神经元再生修复的微环境对SCI治疗至关重要。
细胞治疗是脊髓损伤修复的有效途径,目前应用于治疗脊髓损伤的主要是干细胞,包括胚胎干细胞、间充质干细胞、神经干细胞/神经前体细等;但是治疗效果并不理想。
发明内容
有鉴于此,本发明的目的在于提供一种基因修饰的牙髓干细胞及其制备方法和应用;本发明以牙髓干细胞(DPSCs)作为重塑脊髓损伤微环境的载体细胞,通过在牙髓干细胞中过表达OPN、IGF-1和CNTF基因,改善脊髓损伤炎症微环境,促进神经修复和再生,实现改善脊髓损伤后的机体运动功能,达到治疗脊髓损伤的效果。
为了实现上述发明目的,本发明提供了以下技术方案:
本发明提供了一种基因修饰的牙髓干细胞,所述牙髓干细胞过表达骨桥蛋白、胰岛素样生长因子1和睫状神经营养因子。
优选的,所述骨桥蛋白、胰岛素样生长因子1和睫状神经营养因子的基因的核苷酸序列分别如SEQ ID No.1~SEQ ID No.3所示。
本发明提供了所述的牙髓干细胞的制备方法,包括以下步骤:
1)将骨桥蛋白、胰岛素样生长因子1和睫状神经营养因子的基因克隆到穿梭质粒pUC57的多克隆位点,获得重组质粒pUC57-Syn;
2)将所述重组质粒pUC57-Syn和骨架质粒pKAd5f11pES-PmeI分别酶切、Gibson组装后的组装产物电转染细胞后获得重组腺病毒Ad-OIC;
3)将所述重组腺病毒Ad-OIC感染牙髓干细胞获得基因修饰的牙髓干细胞。
优选的,步骤1)中所述骨桥蛋白的基因克隆至穿梭质粒pUC57的BglII和SalI酶切位点之间;所述睫状神经营养因子和胰岛素样生长因子1的基因顺次连接后克隆至穿梭质粒pUC57的KpnI和AgeI酶切位点之间。
优选的,在步骤3)所述重组腺病毒Ad-OIC感染牙髓干细胞前,转染HEK293细胞进行扩增。
优选的,步骤3)中所述重组腺病毒Ad-OIC与牙髓干细胞的数量比例为100~150:1。
本发明提供了所述基因修饰的牙髓干细胞在制备治疗脊髓损伤的药物中的应用。
优选的,所述药物包括所述基因修饰的牙髓干细胞能够接受的辅料。
本发明的有益效果:本发明提供的基因修饰的牙髓干细胞,以牙髓干细胞(DPSCs)作为重塑脊髓损伤微环境的载体细胞,并采用在脊髓损伤修复过程中具有明确作用的基因——骨桥蛋白(OPN)基因、胰岛素样生长因子1(IGF-1)基因和睫状神经营养因子(CNTF)基因对DPSCs进行修饰;本发明提供的基因修饰的牙髓干细胞移植后能够在脊髓原位存活较长时间;经过移植本发明提供的基因修饰的牙髓干细胞后,可显著减少脊髓损伤、脊髓水肿及瘢痕形成,促进行为学恢复与膀胱排尿功能恢复。本发明提供的基因修饰的牙髓干细胞能够作为治疗脊髓损伤的药物,具有良好的治疗效果。
附图说明
图1为使用SacI限制性内切酶对重组质粒pUC57-Syn切割后的电泳结果;
图2为使用PmeI酶切骨架质粒pKAd5f11pES-PmeI,使用PacI酶切pUC57-Syn电泳结果;
图3为使用EcoRV和HindIII酶切重组腺病毒Ad-OIC的电泳鉴定结果图;其中(A)为实际酶切条带,1:pKAd5f11p-Syn#1EcoRV;2:pKAd5f11p-Syn#3EcoRV;3:pKAd5f11p-Syn#5EcoRV;4:pKAd5f11p-Syn#1HindIII;5:pKAd5f11p-Syn#3HindIII;6:pKAd5f11p-Syn#5HindIII;(B)为理论酶切条带,1:pKAd5f11p-Syn EcoRV,2:pKAd5f11p-Syn HindIII;
图4为Ad-OIC病毒感染DPSC细胞后,western blot检测细胞蛋白表达结果;其中,OPN为骨桥蛋白;IGF-1为胰岛素样生长因子1;CNTF:睫状神经营养因子;GAPDH为内标蛋白;
图5为DPSC细胞脊髓原位立体定位注射活体成像示意图;
图6为DPSC-Null、DPSC-OIC细胞脊髓原位治疗后磁共振影像学拍摄;图中为T2加权项下脊髓损伤恢复情况;其中PBS为脊髓损伤后原位注射PBS治疗组;DPSC-Null为脊髓损伤后原位注射感染对照病毒Ad.Null治疗组;DPSC-OIC为脊髓损伤后原位注射感染三基因过表达病毒Ad.OIC治疗组;
图7为脊髓损伤小鼠治疗后BMS行为学评分情况;PBS为脊髓损伤后原位注射PBS治疗组;DPSC-Null为脊髓损伤后原位注射感染对照病毒Ad.Null治疗组;DPSC-OIC为脊髓损伤后原位注射感染三基因过表达病毒Ad.OIC治疗组;
图8为脊髓损伤小鼠治疗后膀胱体积测量情况;PBS为脊髓损伤后原位注射PBS治疗组;DPSC-Null为脊髓损伤后原位注射感染对照病毒Ad.Null治疗组;DPSC-OIC为脊髓损伤后原位注射感染三基因过表达病毒Ad.OIC治疗组。
具体实施方式
本发明提供了一种基因修饰的牙髓干细胞,所述牙髓干细胞过表达骨桥蛋白、胰岛素样生长因子1和睫状神经营养因子。本发明对所述牙髓干细胞的制备方法没有特殊限定,采用本领域常规的牙髓干细胞培养方法即可。在本发明中,所述牙髓干细胞优选的来源于健康人正常完整的第三磨牙。本发明优选的从所述第三磨牙的牙根中分离出牙髓组织,将所述牙髓组织消化培养获得所述牙髓干细胞。
在本发明中,所述骨桥蛋白、胰岛素样生长因子1和睫状神经营养因子的基因的核苷酸序列优选的分别如SEQ ID No.1~SEQ ID No.3所示;具体如下:
骨桥蛋白基因(OPN):
atgagaattgcagtgatttgcttttgcctcctaggcatcacctgtgccataccagttaaacaggctgattctggaagttctgaggaaaagcagctttacaacaaatacccagatgctgtggccacatggctaaaccctgacccatctcagaagcagaatctcctagccccacagacccttccaagtaagtccaacgaaagccatgaccacatggatgatatggatgatgaagatgatgatgaccatgtggacagccaggactccattgactcgaacgactctgatgatgtagatgacactgatgattctcaccagtctgatgagtctcaccattctgatgaatctgatgaactggtcactgattttcccacggacctgccagcaaccgaagttttcactccagttgtccccacagtagacacatatgatggccgaggtgatagtgtggtttatggactgaggtcaaaatctaagaagtttcgcagacctgacatccagtaccctgatgctacagacgaggacatcacctcacacatggaaagcgaggagttgaatggtgcatacaaggccatccccgttgcccaggacctgaacgcgccttctgattgggacagccgtgggaaggacagttatgaaacgagtcagctggatgaccagagtgctgaaacccacagccacaagcagtccagattatataagcggaaagccaatgatgagagcaatgagcattccgatgtgattgatagtcaggaactttccaaagtcagccgtgaattccacagccatgaatttcacagccatgaagatatgctggttgtagaccccaaaagtaaggaagaagataaacacctgaaatttcgtatttctcatgaattagatagtgcatcttctgaggtcaattaa(SEQ ID No.1)。
胰岛素样生长因子1(IGF-1):
atgattacacctacagtgaagatgcacaccatgtcctcctcgcatctcttctacctggcgctgtgcctgctcaccttcaccagctctgccacggctggaccggagacgctctgcggggctgagctggtggatgctcttcagttcgtgtgtggagacaggggcttttatttcaacaagcccacagggtatggctccagcagtcggagggcgcctcagacaggcatcgtggatgagtgctgcttccggagctgtgatctaaggaggctggagatgtattgcgcacccctcaagcctgccaagtcagctcgctctgtccgtgcccagcgccacaccgacatgcccaagacccagaaggaagtacatttgaagaacgcaagtagagggagtgcaggaaacaagaactacaggatgtag(SEQIDNo.2)。
睫状神经营养因子(CNTF):
atggctttcacagagcattcaccgctgacccctcaccgtcgggacctctgtagccgctctatctggctagcaaggaagattcgttcagacctgactgctcttacggaatcctatgtgaagcatcagggcctgaacaagaacatcaacctggactctgcggatgggatgccagtggcaagcactgatcagtggagtgagctgaccgaggcagagcgactccaagagaaccttcaagcttatcgtaccttccatgttttgttggccaggctcttagaagaccagcaggtgcattttaccccaaccgaaggtgacttccatcaagctatacatacccttcttctccaagtcgctgcctttgcataccagatagaggagttaatgatactcctggaatacaagatcccccgcaatgaggctgatgggatgcctattaatgttggagatggtggtctctttgagaagaagctgtggggcctaaaggtgctgcaggagctttcacagtggacagtaaggtccatccatgaccttcgtttcatttcttctcatcagactgggatcccagcacgtgggagccattatattgctaacaacaagaaaatg(SEQIDNo.3)。
在本发明中,使用牙髓干细胞(DPSCs)作为重塑脊髓损伤微环境的载体细胞;牙髓干细胞是由神经嵴衍生的一类外胚层MSCs,所述牙髓干细胞除表达其他中胚层来源间充质干细胞的标志分子(如CD29、CD105等)外,同样表达神经干细胞标志分子(如巢蛋白、胶质原纤维酸性蛋白等)。与骨髓来源间充质干细胞相比,牙髓干细胞分泌更多的神经修复相关生长因子,更适合作为脊髓损伤修复的种子细胞。同时,所述牙髓干细胞具有比其他类型干细胞更好的抗炎作用,这对改善脊髓损伤炎症微环境具有重要意义。
在本发明中,为进一步增强治疗效果,采用三种在脊髓损伤修复过程中具有作用的基因:骨桥蛋白(OPN)基因、胰岛素样生长因子1(IGF-1)基因、睫状神经营养因子(CNTF)对所述牙髓干细胞进行修饰。在本发明中,所述骨桥蛋白(OPN)是在各种组织中广泛分布的细胞因子及黏附蛋白;在脊髓损伤后,骨桥蛋白对胶质细胞能起到趋化作用;在中枢神经损伤后,局部骨桥蛋白的过表达能够诱导干细胞的定向迁移,促进神经再生。在本发明中,所述胰岛素样生长因子1(IGF-1)是中枢神经系统具有保护作用的神经细胞因子。脊髓损伤后,IGF-1能够减少神经递质兴奋性毒性,同时减少神经元的凋亡与坏死促进脊髓运动神经元的存活。所述睫状神经营养因子(CNTF)对神经细胞的增殖、分化、凋亡等都有着非常重要的作用,所述睫状神经营养因子无论是全身还是局部应用,都有促进损伤脊髓的修复作用,通过减少神经细胞凋亡、促进轴突再生进而改善损伤后运动功能得以实现。
本发明还提供了所述的牙髓干细胞的制备方法,包括以下步骤:1)将骨桥蛋白、胰岛素样生长因子1和睫状神经营养因子的基因克隆到穿梭质粒pUC57的多克隆位点,获得重组质粒pUC57-Syn;2)将所述重组质粒pUC57-Syn和骨架质粒pKAd5f11pES-PmeI分别酶切、Gibson组装后的组装产物电转染细胞后获得重组腺病毒Ad-OIC;3)将所述重组腺病毒Ad-OIC感染牙髓干细胞获得基因修饰的牙髓干细胞。
在本发明中,将骨桥蛋白、胰岛素样生长因子1和睫状神经营养因子的基因克隆到穿梭质粒pUC57的多克隆位点,获得重组质粒pUC57-Syn。在本发明中,所述骨桥蛋白的基因优选的克隆至穿梭质粒pUC57的BglII和SalI酶切位点之间;所述睫状神经营养因子和胰岛素样生长因子1的基因顺次连接后优选的克隆至穿梭质粒pUC57的KpnI和AgeI酶切位点之间。本发明对所述穿梭质粒pUC57的来源没有特殊限定,采用本领域常规市售产品即可。本发明对所述重组质粒pUC57-Syn的制备方法没有特殊限定,采用本领域常规的人工合成方法即可。
本发明在获得所述重组质粒pUC57-Syn后,分别使用PacI酶和PmeI酶将所述重组质粒pUC57-Syn和骨架质粒pKAd5f11pES-PmeI酶切,并进行混合回收和Gibson组装后电转染细胞后获得重组腺病毒Ad-OIC。在本发明中,所述骨架质粒pKAd5f11pES-PmeI优选的购自Addgene公司,货号为#122698。在本发明中,所述Gibson组装后的组装产物优选的电转染至TOP感受态细胞中,所述TOP感受态细胞优选的购自北京华越洋生物科技有限公司。在本发明中,上述两种质粒酶切、混合回收和Gibson组装具体操作优选的如下:取使用PacI酶切重组质粒pUC57-Syn,回收3581bp条带;使用PmeI酶切骨架质粒pKAd5f11pES-PmeI,回收32626bp条带。使用Zymoclean Large Fragment DNA Recovery Kit试剂盒将上述两条带混合回收,将回收的全部32626bp条带和回收的3581bp条带总量的1/7混合进行回收,最终产物溶解于EB中。取混合回收产物使用HiFi DNAAssembly Master Mix进行组装获得组装产物。
在本发明中,所述电转染的具体操作包括将所述重组质粒pUC57-Syn、骨架质粒pKAd5f11pES-PmeI的组装产物取8μl与100μl TOP感受态细胞混合后进行电转染。在本发明中,所述混合优选的在冰上操作,所述电转染优选的在电转杯中进行;所述电转染的参数设置优选的如下:200Ω、2.5kV、25μF,立即脉冲一次。本发明在所述电转染结束后,立即将电转染后的体系与LB培养基混合后培养。在本发明中,所述LB培养基优选的预热至37℃;所述培养的温度优选为36~38℃,更优选为37℃;所述培养的转速优选为220~270rpm,更优选为250rpm;所述培养的时间优选为50~70min,更优选为60min。本发明在所述培养后,优选的进行重组腺病毒Ad-OIC的鉴定,所述鉴定优选为酶切鉴定,所述鉴定用酶优选的采用EcoRV和HindIII酶。本发明对所述酶切的具体条件没有特殊限定,采用本领域常规的酶切条件即可。在本发明中,EcoRV酶切的产物在9045bp,7637bp,5936bp,4545bp,3075bp,2617bp,2052bp,1238bp出现条带;HindIII酶切的产物在8009bp,5590bp,5322bp,5262bp,5209bp,4597bp,2081bp出现条带。
本发明在所述鉴定后,优选的将鉴定正确的重组腺病毒感染HEK293细胞进行扩增。在本发明中,所述HEK293细胞优选的购自ATCC细胞库。在本发明中,所述感染优选的按照1:100浓度滴度感染HEK293细胞。本发明在所述感染后,将感染后的细胞进行反复冻融,然后进行氯化铯密度梯度离心收集重组腺病毒Ad-OIC。在本发明中,所述氯化铯密度梯度离心优选的进行两次,第一次氯化铯密度梯度离心,氯化铯的密度分别为0.705g/ml、0.503g/ml和0.387g/ml,所述氯化铯密度梯度离心的转速优选为35000rpm,所述氯化铯密度梯度离心的温度优选为4℃,所述氯化铯密度梯度离心的时间优选为60~80min,更优选为70min;在本发明中,所述氯化铯密度梯度离心优选的采用SW40的转头的13.2ml离心管进行。本发明在第一次氯化铯密度梯度离心后,收集病毒条带后,进行第二次氯化铯密度梯度离心,所述第二次氯化铯密度梯度离心的条件与第一次氯化铯密度梯度离心一致,在此不再赘述;本发明在所述氯化铯密度梯度离心后,优选的将收集到的病毒条带透析后保存,所述透析优选的采用10000MWCO Slide-A-Lyzer透析卡(购于赛默飞世尔科技(中国)有限公司)进行,所述保存的保存液优选为含10%(体积分数)甘油的Hank’s液。
本发明在获得所述重组腺病毒Ad-OIC后,将所述重组腺病毒Ad-OIC感染牙髓干细胞获得基因修饰的牙髓干细胞。在本发明中,所述重组腺病毒Ad-OIC与牙髓干细胞的数量比例优选为100~150:1。
本发明还提供了所述基因修饰的牙髓干细胞在制备治疗脊髓损伤的药物中的应用。在本发明中,所述药物优选的还包括所述基因修饰的牙髓干细胞能够接受的辅料,例如细胞保存液或细胞重悬液。在本发明中,所述基因修饰的牙髓干细胞移植后能够在脊髓原位存活较长时间,具有减少脊髓损伤、脊髓水肿及瘢痕形成,促进行为学恢复与膀胱排尿功能恢复的作用。
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
重组腺病毒-OIC(Ad-OIC)的制备和纯化
将人OPN、IGF-1、CNTF基因(核苷酸序列分别如SEQ ID No.1~SEQ ID No.3所示)克隆到穿梭质粒pUC57的多克隆位点,获得重组质粒pUC57-Syn;其中OPN克隆至BglII和SalI酶切位点之间,CNTF与IGF-1顺次连接后克隆至KpnI和AgeI酶切位点之间;根据克隆后的重组质粒pUC57-Syn的核苷酸序列对其进行人工合成,获得重组质粒pUC57-Syn。使用SacI限制性内切酶对pUC57-Syn质粒切割后,电泳结果如图1所示,出现1175bp的条带和5.1kb的条带,证明pUC57-Syn质粒构建成功。
取使用PacI酶切8μl重组质粒pUC57-Syn,回收3581bp条带(胶重:0.21g);使用PmeI酶切2μl骨架质粒pKAd5f11pES-PmeI(购自Addgene公司,货号#122698),回收32626bp条带(胶重:0.31g)。使用Zymoclean Large Fragment DNA Recovery Kit试剂盒将上述两条带混合回收,32626bp全加,3581bp条带加1/7,最终产物溶解于20μl EB中。取6μl混合回收产物使用HiFi DNAAssembly Master Mix进行组装。组装产物8ul加入到1管TOP感受态细胞(100μl)(购于北京华越洋生物科技有限公司)中(冰上操作),将混合液转移到电转杯中,将电转仪的参数设置为:200Ω、2.5kV、25μF,立即脉冲一次,之后迅速取出电转杯,并立即添加1ml LB培养基(预热至37℃)重悬细胞,将细胞悬液转移到1.5ml的Ep管中,37℃,250rpm振摇1h。使其进行同源重组,EcoRV和HindIII酶切鉴定重组腺病毒Ad-OIC是否构建成功,结果如图3所示:EcoRV酶切的产物在9045bp,7637bp,5936bp,4545bp,3075bp,2617bp,2052bp,1238bp出现条带;HindIII酶切在8009bp,5590bp,5322bp,5262bp,5209bp,4597bp,2081bp出现条带。图A为实际酶切条带,与图B理论酶切条带相比,条带出现位置相同,说明重组腺病毒Ad-OIC构建成功。
将鉴定正确的重组腺病毒质粒Ad-OIC感染HEK293细胞(购自ATCC细胞库),使其在细胞内产生重组腺病毒。普通显微镜观察细胞形态改变,9天可见噬斑形成,并逐渐扩大,直至12d出现完全病变。收集细胞和上清,反复冻融3次后收集上清,获得重组腺病毒Ad-OIC。使用重组腺病毒质粒Ad-OIC感染HEK293细胞,进行病毒扩增。经大量扩增后,反复冻融细胞及上清3次。之后,采用氯化铯密度梯度离心纯化。首次离心使用0.705g/ml、0.503g/ml、0.387g/ml氯化铯,使用SW40的转头的13.2ml离心管,转速设置35000rpm,4℃离心1小时10分钟。吸出病毒条带后,进行二次离心。将离心后的病毒条带加入透析卡透析后,保存于10%(体积百分数)甘油的Hank’s液中备用。
使用Particle Metrix公司纳米历经跟踪分析仪(NTA)和有限稀释法(MedianTissue Culture Infective Dose,TCID50)分别测定腺病毒Ad-OIC的颗粒滴度(ViralParticle Units per Milliliter,vp/ml)和感染滴度(Infection Units perMilliliter,IU/ml)。结果如表1所示。
表1重组腺病毒Ad-OIC的纯度和滴度测定结果
病毒名称 | 颗粒滴度(vp/ml) | 感染滴度(IU/ml) |
Ad-OIC | 1×10<sup>11</sup> | 3×10<sup>10</sup> |
实施例2
重组腺病毒Ad-OIC感染DPSC后修饰基因表达情况鉴定。
在本发明中,所述牙髓干细胞(DPSC)取自健康人正常完整的第三磨牙;截牙冠后,从牙根中分离出牙髓组织并在37℃条件下,用胶原酶和中性蛋白酶消化40min;使用氯化钠注射液清洗1次,1500rpm离心5min后去除液体;将上述组织置于75cm2培养瓶,培养至第14天,获得细胞记为P0代DPSC。
设置空白对照组和阴性对照组,空白对照组以等体积的PBS替换重组腺病毒Ad-OIC感染DPSC,阴性对照组以等体积的腺病毒Ad.Null(未重组OPN、IGF-1和CNTF基因)替换重组腺病毒Ad-OIC感染DPSC。
本发明在DPSC细胞感染Ad-OIC后继续培养72小时,通过Wsetern Blot法检测OPN、IGF-1及CNTF蛋白表达的变化。结果如图4所示,与对照组(感染对照病毒Ad.Null)相比,实验组DPSC-OIC中OPN、IGF-1及CNTF蛋白表达均升高。
实施例3
基因修饰的牙髓干细胞DPSC脊髓原位移植后存活时间鉴定
(1)Ad-GFP.Luc双荧光病毒感染DPSC示踪模型构建:
为了检测基因修饰的牙髓干细胞DPSC脊髓原位移植后存活时间,使用Ad-GFP.Luc双荧光病毒感染DPSC(实施例1中制备获得的基因修饰的牙髓干细胞),构建示踪细胞模型。本发明所述Ad-GFP.Luc,是将EGFP基因和Luciferase基因通过T2A连接后,构建到pshuttle-cmv载体的多克隆位点,经Kana+筛选获得阳性克隆pshuttle-cmv-EGFP-T2A-luciferase,并经测序鉴定正确。按照Adeasy系统,将pshuttle-cmv-EGFP-T2A-luciferasePmeI线性化后,电转BJ5183-Adeasy-1感受态细胞,同源重组获得重组腺病毒质粒pAd-GFP.Luc。pAd-GFP.Luc经PacI线性化后经脂质体转染HEK293细胞,包装获得重组腺病毒Ad-GFP.Luc。Ad-GFP.Luc双荧光病毒感染DPSC(实施例1中制备获得的基因修饰的牙髓干细胞)48h后,使用荧光显微镜对其荧光GFP信号进行检测。约有80%细胞表达GFP蛋白。说明细胞模型构建成功,满足实验需要。
(2)Ad-GFP.Luc双荧光病毒感染DPSC示踪细胞脊髓原位移植:
为了检测基因修饰的牙髓干细胞DPSC脊髓原位移植后存活时间,将示踪DPSC细胞立体定位注射于小鼠脊髓原位。麻醉小鼠后使用手术剪暴露小鼠锥体,在打开小鼠脊柱椎板并固定脊柱后,按照每位点1×105个将细胞注射于脊髓原位(损伤头、尾端的左右两侧共四位点)。注射结束后,缝合肌肉、皮下组织及皮肤,并定期使用碘酒消毒。
(3)小鼠活体成像检测细胞存活时间
分别在细胞移植后3、7、15天,给小鼠注射200μl,10mg/ml Luciferase发光底物,并使用Berthold Technologies的动物活体成像仪检测小鼠脊髓原位荧光强度。荧光检测结果见图5;结果说明,DPSC在小鼠原位移植后至少可存在15天以上。
实施例4
脊髓损伤小鼠DPSC-OIC原位移植治疗后疗效检测
在脊髓原位移植DPSC-OIC细胞后,原位移植方法参见实施例3中的步骤(2),设置空白对照组PBS处理和阴性对照组DPSC-Null处理,PBS处理为脊髓损伤后原位注射PBS;DPSC-Null处理为脊髓损伤后原位注射感染对照病毒Ad.Null;DPSC-OIC为脊髓损伤后原位注射感染三基因过表达病毒Ad.OIC。
本发明对三个处理组的小鼠进行了脊髓MRI成像检测、BMS行为学评分等以对疗效进行评估。
(1)DPSC-OIC细胞对脊髓损伤疗效的影像学分析
按照80mg/kg腹腔注射1%戊巴比妥钠对治疗10天后小鼠进行麻醉。之后,使用MRI对脊髓软组织进行评价。结果如图6所示,DPSC-OIC治疗后,脊髓损伤区域水肿减轻,瘢痕形成较少。结果显示,与对照病毒相比,DPSC-OIC有较好疗效。
(2)DPSC-OIC细胞对脊髓损伤疗效的行为学评价
对治疗后小鼠按照BMS评分原则,对小鼠行走步态恢复情况进行评价(结果见图7)。结果显示,与对照病毒相比,DPSC-OIC治疗组小鼠步态评分好转,且有统计学意义(p<0.01)。
(3)DPSC-OIC细胞治疗后,小鼠自主排尿功能恢复分析
为检测DPSC-OIC治疗后对小鼠自主排尿功能恢复的影响,对治疗后小鼠膀胱体积进行测定。结果如图8所示,移植DPSC或DPSC-OIC后,小鼠排尿功能较PBS组均有好转。
由上述实施例可知,DPSC移植小鼠后可在脊髓原位较长时间存活;经过DPSC-OIC治疗后,可减少脊髓损伤、脊髓水肿及瘢痕形成,促进行为学恢复与膀胱排尿功能恢复。总之,本发明提供的基因修饰的牙髓干细胞能够有效的应用于脊髓损伤的细胞治疗。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 中国人民解放军军事科学院军事医学研究院
<120> 一种基因修饰的牙髓干细胞及其制备方法和应用
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atgagaattg cagtgatttg cttttgcctc ctaggcatca cctgtgccat accagttaaa 60
caggctgatt ctggaagttc tgaggaaaag cagctttaca acaaataccc agatgctgtg 120
gccacatggc taaaccctga cccatctcag aagcagaatc tcctagcccc acagaccctt 180
ccaagtaagt ccaacgaaag ccatgaccac atggatgata tggatgatga agatgatgat 240
gaccatgtgg acagccagga ctccattgac tcgaacgact ctgatgatgt agatgacact 300
gatgattctc accagtctga tgagtctcac cattctgatg aatctgatga actggtcact 360
gattttccca cggacctgcc agcaaccgaa gttttcactc cagttgtccc cacagtagac 420
acatatgatg gccgaggtga tagtgtggtt tatggactga ggtcaaaatc taagaagttt 480
cgcagacctg acatccagta ccctgatgct acagacgagg acatcacctc acacatggaa 540
agcgaggagt tgaatggtgc atacaaggcc atccccgttg cccaggacct gaacgcgcct 600
tctgattggg acagccgtgg gaaggacagt tatgaaacga gtcagctgga tgaccagagt 660
gctgaaaccc acagccacaa gcagtccaga ttatataagc ggaaagccaa tgatgagagc 720
aatgagcatt ccgatgtgat tgatagtcag gaactttcca aagtcagccg tgaattccac 780
agccatgaat ttcacagcca tgaagatatg ctggttgtag accccaaaag taaggaagaa 840
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taa 903
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gagctggtgg atgctcttca gttcgtgtgt ggagacaggg gcttttattt caacaagccc 180
acagggtatg gctccagcag tcggagggcg cctcagacag gcatcgtgga tgagtgctgc 240
ttccggagct gtgatctaag gaggctggag atgtattgcg cacccctcaa gcctgccaag 300
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accgaaggtg acttccatca agctatacat acccttcttc tccaagtcgc tgcctttgca 360
taccagatag aggagttaat gatactcctg gaatacaaga tcccccgcaa tgaggctgat 420
gggatgccta ttaatgttgg agatggtggt ctctttgaga agaagctgtg gggcctaaag 480
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Claims (8)
1.一种基因修饰的牙髓干细胞,其特征在于,所述牙髓干细胞过表达骨桥蛋白、胰岛素样生长因子1和睫状神经营养因子。
2.根据权利要求1所述的牙髓干细胞,其特征在于,所述骨桥蛋白、胰岛素样生长因子1和睫状神经营养因子的基因的核苷酸序列分别如SEQ ID No.1~SEQ ID No.3所示。
3.权利要求1或2所述的牙髓干细胞的制备方法,包括以下步骤:
1)将骨桥蛋白、胰岛素样生长因子1和睫状神经营养因子的基因克隆到穿梭质粒pUC57的多克隆位点,获得重组质粒pUC57-Syn;
2)将所述重组质粒pUC57-Syn和骨架质粒pKAd5f11pES-PmeI分别酶切、Gibson组装后的组装产物电转染细胞后获得重组腺病毒Ad-OIC;
3)将所述重组腺病毒Ad-OIC感染牙髓干细胞获得基因修饰的牙髓干细胞。
4.根据权利要求3所述的制备方法,其特征在于,步骤1)中所述骨桥蛋白的基因克隆至穿梭质粒pUC57的BglII和SalI酶切位点之间;所述睫状神经营养因子和胰岛素样生长因子1的基因顺次连接后克隆至穿梭质粒pUC57的KpnI和AgeI酶切位点之间。
5.根据权利要求3所述的制备方法,其特征在于,在步骤3)所述重组腺病毒Ad-OIC感染牙髓干细胞前,转染HEK293细胞进行扩增。
6.根据权利要求3所述的制备方法,其特征在于,步骤3)中所述重组腺病毒Ad-OIC与牙髓干细胞的数量比例为100~150:1。
7.权利要求1或2所述基因修饰的牙髓干细胞、权利要求3~6任意一项所述的制备方法制备获得的基因修饰的牙髓干细胞在制备治疗脊髓损伤的药物中的应用。
8.根据权利要求7所述的应用,其特征在于,所述药物包括所述基因修饰的牙髓干细胞能够接受的辅料。
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