用于诱导多潜能性干细胞的人羊水细胞培养及逆转录病毒包装平台的建立
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摘要
基因治疗作为一种全新的疾病治疗方法,有着广泛的应用前景。随着分子病理学和分子生物学等相关学科的发展,基因治疗所针对的疾病也从早期的单基因遗传病扩展到肿瘤、病毒性疾病和心血管病等严重威胁人类健康的疾病。
在目前广泛采用的体外基因治疗策略中,靶细胞的选择是一个关键因素。干细胞由于具有自我更新与多向分化潜能的特性,因此是一种理想的基因治疗靶细胞。2006年出现的将几种转录因子导入成体细胞将其诱导成为具有干细胞特性的多潜能性干细胞(iPSC)的新技术,更是使利用患者来源的成体细胞诱导成为干细胞后进行自体化基因治疗成为可能。
因此,本室拟利用产前诊断过程中的遗传病患儿羊水细胞,通过逆转录病毒将其诱导成为iPS细胞,建立不同疾病特异性的iPS细胞系,进而利用我室的核糖体基因区打靶载体进行基因治疗研究。本研究作为这一研究方向的前期准备,着重建立羊水细胞的培养和筛选技术平台,同时建立用于诱导的逆转录病毒包装体系,为后续的诱导多潜能性干细胞奠定基础。
本研究的实验结果显示,在获取人羊水细胞后,用机械法去除成纤维样细胞和上皮样细胞的方法,能较好的纯化出羊水中的短梭形细胞。利用携带Slc7a1受体基因的慢病毒感染纯化后的短梭形细胞,使其表达Slc7a1受体,从而获得了可用于病毒感染诱导的羊水细胞。同时,为获得诱导所需的逆转录病毒,本研究成功利用Plat-E包装细胞包装出了可用于细胞感染的逆转录病毒。为检测病毒感染能力,用包装好的pMX-GFP对照逆转录病毒感染携带Slc7a1受体的人羊水细胞,48h后在倒置显微镜下可以观察到人羊水细胞内有GFP荧光表达,证明用Plat-E包装细胞包装的逆转录病毒可以成功感染携带Slc7a1受体的人羊水细胞,为诱导多潜能干细胞提供了前提基础。
本研究成功建立了用于诱导iPS细胞的人羊水细胞纯化和筛选技术条件,并建立了利用Plat-E细胞包装逆转录病毒的技术平台,为我室后续的iPS细胞诱导工作提供了基础。
Gene therapy, as a new method for curing disease, has a promising prospect. Along with the development of molecular pathology, molecular biology and other subjects in biomedical field, diseases which gene therapy aims at are from single gene inheritance diseases to those severely threatening human health, such as tumor, viral diseases and cardiovascular diseases.
For in vitro gene therapy, it's important to choose proper target cells. Stem cells have the potential to self renew and differentiate into many cell types, so they are the ideal target cells in gene therapy. In 2006, a new technology emerges that transfers some transcription factors to the somatic cells to induce these cells to the pluripotent state. This new technology makes it possible to utilize the patients' somatic cells that are induced to the pluripotent stem cells in autoallergic gene therapy.
Our lab plans to use the amniotic fluid cells from the infants who have genetic diseases to be induced by retrovirus. In this way, we can establish different kinds of disease-specific iPS cell lines, then these cell lines and our lab's human ribosomal DNA targeting vector can be used together in the gene therapy research. This study is the preparation, and aims at establishing the platform for human amniotic fluid cell culturing and retrovirus packaging for induction.
The experiment results show that after getting the human amniotic fluid, through inoculation and cultivation, cells adhered to grow and after 6 days the cell cluster appeared, then the adherent epithelial-like cells and long fibroblast-like cells were mechanically removed, leaving the short and spindle-like cells. The cells were infected by lentivirus encoding Slc7al receptor, thus these cells could also express Slc7a1. In this way, we got the cells suitable for induction. At the same time, in order to get the retrovirus for induction, we successfully utilized Plat-E to package the retrovirus vectors encoding exogenous inducing factors. For detecting the infection ability, pMX-GFP retrovirus, as a control, infected the human amniotic fluid cells expressing Slc7a1. After 48h, GFP could be objected in the cells, showing that retrovirus packaged by Plat-E could infect the human amniotic fluid cells expressing Slc7a1.
This study successfully established the purification and screening technical conditions of human amniotic fluid cells, and the platform for retrovirus packaging by Plat-E, providing a basis for the later research.
在目前广泛采用的体外基因治疗策略中,靶细胞的选择是一个关键因素。干细胞由于具有自我更新与多向分化潜能的特性,因此是一种理想的基因治疗靶细胞。2006年出现的将几种转录因子导入成体细胞将其诱导成为具有干细胞特性的多潜能性干细胞(iPSC)的新技术,更是使利用患者来源的成体细胞诱导成为干细胞后进行自体化基因治疗成为可能。
因此,本室拟利用产前诊断过程中的遗传病患儿羊水细胞,通过逆转录病毒将其诱导成为iPS细胞,建立不同疾病特异性的iPS细胞系,进而利用我室的核糖体基因区打靶载体进行基因治疗研究。本研究作为这一研究方向的前期准备,着重建立羊水细胞的培养和筛选技术平台,同时建立用于诱导的逆转录病毒包装体系,为后续的诱导多潜能性干细胞奠定基础。
本研究的实验结果显示,在获取人羊水细胞后,用机械法去除成纤维样细胞和上皮样细胞的方法,能较好的纯化出羊水中的短梭形细胞。利用携带Slc7a1受体基因的慢病毒感染纯化后的短梭形细胞,使其表达Slc7a1受体,从而获得了可用于病毒感染诱导的羊水细胞。同时,为获得诱导所需的逆转录病毒,本研究成功利用Plat-E包装细胞包装出了可用于细胞感染的逆转录病毒。为检测病毒感染能力,用包装好的pMX-GFP对照逆转录病毒感染携带Slc7a1受体的人羊水细胞,48h后在倒置显微镜下可以观察到人羊水细胞内有GFP荧光表达,证明用Plat-E包装细胞包装的逆转录病毒可以成功感染携带Slc7a1受体的人羊水细胞,为诱导多潜能干细胞提供了前提基础。
本研究成功建立了用于诱导iPS细胞的人羊水细胞纯化和筛选技术条件,并建立了利用Plat-E细胞包装逆转录病毒的技术平台,为我室后续的iPS细胞诱导工作提供了基础。
Gene therapy, as a new method for curing disease, has a promising prospect. Along with the development of molecular pathology, molecular biology and other subjects in biomedical field, diseases which gene therapy aims at are from single gene inheritance diseases to those severely threatening human health, such as tumor, viral diseases and cardiovascular diseases.
For in vitro gene therapy, it's important to choose proper target cells. Stem cells have the potential to self renew and differentiate into many cell types, so they are the ideal target cells in gene therapy. In 2006, a new technology emerges that transfers some transcription factors to the somatic cells to induce these cells to the pluripotent state. This new technology makes it possible to utilize the patients' somatic cells that are induced to the pluripotent stem cells in autoallergic gene therapy.
Our lab plans to use the amniotic fluid cells from the infants who have genetic diseases to be induced by retrovirus. In this way, we can establish different kinds of disease-specific iPS cell lines, then these cell lines and our lab's human ribosomal DNA targeting vector can be used together in the gene therapy research. This study is the preparation, and aims at establishing the platform for human amniotic fluid cell culturing and retrovirus packaging for induction.
The experiment results show that after getting the human amniotic fluid, through inoculation and cultivation, cells adhered to grow and after 6 days the cell cluster appeared, then the adherent epithelial-like cells and long fibroblast-like cells were mechanically removed, leaving the short and spindle-like cells. The cells were infected by lentivirus encoding Slc7al receptor, thus these cells could also express Slc7a1. In this way, we got the cells suitable for induction. At the same time, in order to get the retrovirus for induction, we successfully utilized Plat-E to package the retrovirus vectors encoding exogenous inducing factors. For detecting the infection ability, pMX-GFP retrovirus, as a control, infected the human amniotic fluid cells expressing Slc7a1. After 48h, GFP could be objected in the cells, showing that retrovirus packaged by Plat-E could infect the human amniotic fluid cells expressing Slc7a1.
This study successfully established the purification and screening technical conditions of human amniotic fluid cells, and the platform for retrovirus packaging by Plat-E, providing a basis for the later research.
引文
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