表达双报告基因的慢病毒颗粒的制备及体外标记小鼠间充质干细胞

发布时间：2018-07-27 16:36

【摘要】：间充质干细胞(mesenchymal stem cells,MSCs)是一类具有高度自我更新和多向分化潜能的成体干细胞,由于取材方便、体外能大量扩增,近年成为干细胞领域的研究热点之一。MSCs主要来源于骨髓和脂肪组织,其次也可从脐血、外周血、胎盘、肺、羊水、肌肉、网膜等组织获得。它能够向血管、心肌、骨、脂肪、神经和皮肤等多个胚层组织分化,是组织工程的理想种子细胞。在各种原因所致的严重创伤下许多受损的组织和脏器需要及时的修复和替代,间充质干细胞的可塑性使其有可能成为治疗多种疾病的有力手段。 MSCs的免疫原性低,不表达MHC-Ⅱ类分子和CD80、CD86等协同刺激分子。研究表明,MSCs能够提供细胞因子和生长因子支持造血干细胞的扩增和成熟。另外,它能够抑制T细胞增殖和免疫细胞因子的产生,具有免疫调节作用,能够诱导免疫耐受,可用于造血干细胞移植和自身免疫性疾病的治疗。本研究应用胶原酶消化骨片的方法成功分离小鼠MSCs,并在体外进行培养。这种方法获得的细胞有均一的表型,不表达造血和内皮的标记分子CD11b、CD31、CD45、CD34,表达细胞粘附分子CD29、CD44、CD105和干细胞标志Sca-1。其具有贴壁性,能够向脂肪、成骨和软骨诱导分化,符合MSCs的鉴定标准。体外稳定高效的标记细胞,对研究其在体内的分布、迁移、增殖及分化有重要意义。在传统的动物实验中,往往需要处死大批动物后才能获得实验结果,很难直观、活体、动态地连续观察细胞的分布迁移情况,限制了对细胞,尤其是早期细胞的分布、迁移、增殖、分化、持续时间和治疗效果的研究和评价。因此,建立便于检测的细胞标记方法具有非常重要的意义。目前,实验室研究常用的一些体外细胞标记方法有荧光染料标记方法、荧光报告基团及生物发光方法和放射性核素标记法等。红色荧光蛋白(red fluorescent protein,RFP)/萤火虫荧光素酶双标技术(firefly luciferase,Fluc)就是将化学发光和荧光成像技术结合起来,化学发光技术是用荧光素酶(Luciferase)基因标记细胞或DNA,而荧光技术则采用荧光报告基团(GFP、RFP,Cyt及Dyes等)进行标记,然后利用一套非常灵敏的光学检测仪器,直接监控活体生物体内的细胞活动和基因行为。该技术在干细胞学研究领域中的应用,迄今在国内的报道还为数不多,其不仅继承了RFP或Fluc单标技术的优势,而且突破了后者的局限性,结合活体成像技术,可以直接监测干细胞在动物体内的生物学行为,是研究细胞输注后在实验动物体内分布和分化的强有力手段,在医学和生物医学研究领域中具有越来越广泛的应用前景。慢病毒系统能够有效地感染各类细胞,实现外源基因在细胞内的稳定表达,对双报告基因在细胞中的表达具有重要意义。本研究通过构建萤火虫荧光素酶和红色荧光蛋白双报告基因表达的慢病毒载体,转染293T细胞后,纯化获得具有高滴度和感染活性的慢病毒颗粒,转导特定细胞系(Hela),采用荧光显微镜和荧光素酶报告基因检测试剂盒检测RFP和Fluc双报告基因的表达。采用酶切和测序的方法证实rfp和fluc基因成功连入目的载体,所构建pLenti-Fluc-RFP慢病毒重组质粒序列符合预期。获得的慢病毒滴度测定为1×107TU/ml。将病毒转导Hela细胞系后,荧光显微镜下可见大量RFP表达阳性细胞,荧光发光检测仪也能通过检测发光单位证实转导细胞具有较高的荧光素酶活性。慢病毒载体的成功构建为研究间充质干细胞在体内的动态分布奠定了基础。在再生医学及组织工程中MSCs虽然得到了广泛应用,但其在临床应用中的安全性仍不是很清楚。目前有许多关于MSCs与肿瘤关系的研究,其能否转化成恶性肿瘤细胞及在体内外对肿瘤细胞增殖的影响,各研究结果不同。而且目前关于MSCs的许多生物学特性及分子调控机制尚不十分清楚,对其体内分布、归巢、增殖和定植的研究尚处于初级阶段,有待进一步探索。MSCs移植的方法和最佳时机等还不确定。经体外分离、培养的干细胞是一类具有多向分化能力的细胞,其是否存在基因突变,植入机体后是否有癌变的可能,对于MSCs的横向分化潜能,也有科学家提出质疑。由于目前所进行的临床研究较少,病例数较少,其有效性和安全性等问题仍有待进一步观察。目前已有的研究工作表明循环的骨髓间充质干细胞可以迁延到不同的部位,如骨髓、肺、肝、脾、心脏、脑部、胰岛等;经静脉注射后骨髓间充质干细胞最初的分布器官是肺、肝、肾,特别在注射后第一天就发现骨髓间充质干细胞大部分滞留于肺泡,而注射七天后才发现在肌肉、心脏、脑和脾的分布;在机体组织受损伤时,骨髓MSCs可经骨髓动员自发到达损伤部位,并在局部微环境诱导下分化为特异的组织细胞参与自身修复;动员循环中的骨髓间充质干细胞迁延到特定组织或器官的相关机制尚没有统一的说法,但一般认为涉及多种分子及信号通路的相互作用。本研究应用制备的表达双报告基因系统的慢病毒颗粒体外标记感染小鼠MSCs,经检测感染后的MSCs能够稳定表达RFP和Fluc报告基因,且形态均一,呈成纤维样生长,通过细胞表型和体外诱导分化鉴定证实细胞一般生物学特性未受病毒影响,同时结合流式细胞分选技术使RFP/Fluc双标表达率接近100%,为后期体外大量扩增培养细胞,采用活体成像技术监测稳定感染细胞在小鼠体内深部组织的分布情况的实验奠定基础。
[Abstract]:Mesenchymal stem cells (MSCs) is a kind of adult stem cells with high self-renewal and pluripotent differentiation potential. Because of the convenience of taking materials, it can be expanded in large quantities in vitro. In recent years,.MSCs has become one of the hot spots in the field of stem cells..MSCs is mainly derived from bone marrow and fat tissue. Secondly, it can also be derived from umbilical blood, peripheral blood, placenta, lung, sheep. Water, muscle, omentum and other tissues are obtained. It can differentiate into blood vessels, myocardium, bone, fat, nerve and skin. It is the ideal seed cell for tissue engineering. Under the severe trauma caused by various causes, many damaged tissues and organs need to be repaired and replaced in time. The plasticity of mesenchymal stem cells may make it possible. A powerful means to treat a variety of diseases.
MSCs has low immunogenicity, does not express MHC- class II molecules and CD80, CD86 and other synergistic stimulators. The study shows that MSCs can provide cytokines and growth factors to support the expansion and maturation of hematopoietic stem cells. In addition, it can inhibit the proliferation of T cells and the production of immune cell factors, which can induce immune regulation and can induce immune tolerance. It is used for hematopoietic stem cell transplantation and autoimmune diseases.
This study used collagenase to digest bone slices successfully and successfully isolated MSCs in mice and cultured in vitro. This method has a homogeneous phenotype that does not express CD11b, CD31, CD45, CD34 of hematopoietic and endothelial markers, CD29, CD44, CD105, and stem cell markers, which are adhered to the adherence of CD44, CD105 and stem cells, and can be directed to fat. Osteogenesis and cartilage differentiation were in accordance with the identification criteria of MSCs.
Stable and efficient labelled cells in vitro are of great significance for studying their distribution, migration, proliferation and differentiation in the body. In traditional animal experiments, it is often necessary to kill a large number of animals to obtain experimental results. It is difficult to visualized, live, dynamically and continuously observe the distribution and migration of the cells, and limit the cells, especially the early cells. Research and evaluation of distribution, migration, proliferation, differentiation, duration and therapeutic effect. Therefore, it is of great significance to establish a method for detecting cell markers. At present, some methods commonly used in laboratory study are fluorescent dye labeling, fluorescence report groups and bioluminescence methods and radionuclides. Red fluorescent protein (RFP) / fluorescein double standard (firefly luciferase, Fluc) is a combination of chemiluminescence and fluorescence imaging, and chemiluminescence is used to mark cell or DNA with luciferase (Luciferase) gene, while fluorescence technology uses fluorescence report group (GFP, RFP, C). YT and Dyes, etc., are labeled, and then use a very sensitive optical instrument to directly monitor cell activity and gene behavior in living organisms. The application of this technology in the field of dry cytology has not been reported so far in China, not only inheriting the advantages of RFP or Fluc single standard technology, but also breaking through the latter The limitation, combined with living imaging technology, can directly monitor the biological behavior of stem cells in the animal body. It is a powerful means to study the distribution and differentiation of the cells after the cell infusion in the experimental animals. It has a more and more extensive application prospect in the field of medical and biomedical research. The slow virus system can effectively infect various kinds of fine. The stable expression of foreign genes in cells is of great significance to the expression of double reporter genes in cells.
In this study, the lentivirus vector expressed by fluorescein and red fluorescent protein gene was constructed. After transfecting 293T cells, the lentivirus particles with high titer and infection activity were purified, and the specific cell line (Hela) was transduced. The double reports of RFP and Fluc were detected by fluorescence microscopy and fluoro enzyme reporter gene detection kit. RFP and fluc genes were successfully linked to the target carrier by enzyme digestion and sequencing, and the sequence of the recombinant plasmid of pLenti-Fluc-RFP lentivirus was expected. The acquired lentivirus titer was 1 * 107TU/ml. and a large number of RFP positive cells were found under fluorescence microscopy. The light detector can also prove that the transduced cells have high luciferase activity by detecting the luminescent units. The successful construction of the lentivirus vector has laid the foundation for the study of the dynamic distribution of mesenchymal stem cells in the body.
Although MSCs has been widely used in regenerative medicine and tissue engineering, the safety of its clinical application is still not very clear. There are many studies on the relationship between MSCs and tumor, whether it can be transformed into malignant tumor cells and the effects on the proliferation of tumor cells in vivo and in vivo, and the results are different for MSCs. Many biological characteristics and molecular regulatory mechanisms are still not very clear. The research on its distribution, homing, proliferation and colonization is still in the primary stage. It is still uncertain that the methods and the best time for.MSCs transplantation are still to be further explored. The possibility of canceration after the gene mutation is implanted into the body. There is also a challenge for the lateral differentiation potential of MSCs. The number of cases is less, the number of cases is less, its effectiveness and safety remain to be further observed. To move to different parts, such as bone marrow, lung, liver, spleen, heart, brain, islet, and so on. After intravenous injection, the initial distribution organ of bone marrow mesenchymal stem cells is lung, liver, kidney, and the bone marrow mesenchymal stem cells were found most of the pulmonary alveolus on the first day after injection, and the distribution of muscle, heart, brain and spleen was found seven days after injection. When the body tissue is damaged, bone marrow MSCs can reach the site of injury spontaneously through bone marrow mobilization, and differentiate into specific tissue cells in local microenvironment to participate in self repair. There is no unified statement on the mechanism of mobilizing bone marrow mesenchymal stem cells in the circulation to specific tissues or organs. The interaction of species and signal pathways.
In this study, the lentivirus particles expressed by the prepared double reporter gene system were labeled with MSCs in vitro. After detection of infected MSCs, the expression of RFP and Fluc reporter genes were stable, and the morphology was homogeneous and fibroid growth. The general biological characteristics of the cells were confirmed to be unaffected by the virus by the phenotype and differentiation of the cells in vitro. At the same time, combined with flow cytometry, the expression rate of RFP/Fluc double standard was close to 100%, which was a large number of cultured cells in the later period, and the living body imaging technique was used to monitor the distribution of the stable infected cells in the deep tissues of the mice.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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