LIF、bFGF真核表达载体的构建及其在BMSCs中的稳定表达
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摘要
周围神经损伤的修复与再生是外科领域至今尚未解决的问题之一。随着基础医学、生命科学和显微外科技术的发展,周围神经损伤后相关神经元的保护、断裂神经干的修复、再生神经轴突重新长入相应靶器官有效支配等方面均有许多深入研究,临床应用也取得了一定的效果。然而,由于多种因素的影响和现代医学技术的限制,周围神经损伤修复后的总体疗效目前仍不尽人意。因此,损伤神经的合理修复、再生轴突的生长速度和重新长入相应靶器官有效支配等方面一直是周围神经外科领域的研究热点。
在十九世纪,Waller首先描述了周围神经损伤后的变性过程。这一过程称为华勒变性(Wallerian dengenerantion),它是在周围神经损伤后,近端神经元胞体和远端轴突所发生的一系列反应。包括炎症反应、生长锥形成延伸、选择性粘附、细胞外基质作用、神经营养因子上调、神经递质合成、神经元重塑、细胞运动等方面,其中神经营养因子(neurotrophic factor, NTFs)在这个过程中起了重要的作用。
骨髓间充质干细胞(BMSCs)是具有自我更新和多向分化潜能的成体干细胞,近年来发现其可分化为神经细胞,具有潜在的修复神经损伤的能力。本实验目的是:体外将LIF、bFGF基因转染培养BMSCs后,通过试验来确定转染后的BMSCs是否表达具有促进周围神经再生和减少再生神经周围瘢痕形成的LIF、bFGF。
研究内容包括:
(1)通过RT-PCR获得LIF、bFGF的cDNA;
(2)采用贴壁法应用Ficoll分离液在体外分离培养大鼠BMSCs,观察其一般生物学特性,并结合其具有多向分化潜能及表面标志物来给予鉴定;
(3)将连于LIF、bFGF基因片段的表达载体转染于BMSCs,通过RT-PCR技术、免疫组化、Westernblot进行鉴定其蛋白表达水平及活性。
结果显示:(1)成功获取LIF、bFGF的cDNA基因片段;(2)从一周龄大鼠骨髓血内成功分离出BMSCs,其形态为呈成纤维细胞样生长,并可以向成脂、成骨细胞转化,其表面标记物CD44(+)、CD45(-);(3)成功获取了稳定表达LIF、bFGF的BMSCs,并通过RT-PCR技术、Westernblot进行了鉴定。
研究结论:通过脂质体将连于LIF、bFGF基因片段的真核表达载体分别和共同转染于体外培养的BMSCs能使其在BMSCs内获得稳定的表达。为进行动物试验奠定了基础。
It is an important task to promote peripheral nerve regeneration after injury in neuroscience. Recent technical progress of molecular biology、cell biology and microsurgery results in better outcome, but not all patients have satisfactory results. It is a nodus how to improve the recovery after nerve injury in peripheral nerve surgery.
The objective task is to solve that bioprotein factor simply applied cure to peripheral nerve injury and surgery couldn’t satisfy the patient’s recovery of the never injury. To reconstitute the part of function and improve the past of never injury, the long time recovery make people suffering. The ideal of this experiment is to rebuilt the peripheral never injury and promote the recovery of never injury.
Our experimental study were divied into three parts
Part I: Construction of eukaryotic expression vector pcDNA3.1-LIF、pcDNA3.1-bFGF LIF/bFGF gene was cloned from pregnant uterine deciduas/placenta tissues by using RT-PCR method and was inserted into pcDNA3.1 to construct eukaryotic expression vector consisting of LIF/bFGF gene. Identified by restriction enzyme analysis and PCR amplification.
Part II: MSCs(mesenchymal stem cells) derived from rat bone marrow were isolated and culture expanded efficiently in vitro and its biological features Ficoll separating medium was added to rat bone marrow mixed liquor that should be centrifuge at 2000rmp×15min soon after it was taken out, then absorb the white layer. Wash the cells twice with PBS and seed cells at a density of 1×106cells per well (6-well format) in 1ml of 15% DMEM of FBS medium. Incubate cells at 37℃in a CO2 incubator. Passage cell was seeded at a density 2～4×104/ml.
Bone marrow mesenchymal stem cells (BMSCs) is one kind of adult stem cells that have self-renewal and multilineage differentiation potential. It has recently been reported that BMSCs can differentiate into neural cells, opening new frontiers in therapy for nerve injury. The morphology of BMSCs was observed with invert-microscope constantly. BMSCs' curve of growth was depicted and cell cycle was analyzed by flowing cytometry. BMSCs were induced to differentiate into osteoblasts and adipocytes in induction medium containing desamethasone、β-Glycerophosphate and horse serum. Then differentiated cells were identificated by AKP and oil O stain. Results showed BMSCs could be isolated and proliferated by density gradient centrifugation adherent method in vitro. The appearance of BMSCs were fibroblast-like cells and its growth curves was like shape of S. Cell cycle analysis showed that most of BMSCs were in G1/G0 phase. BMSCs can be differentiated into osteoblasts and adipocytes in vitro.
Part III: LIF/bFGF was successfully expressed in BMSCs
Transfected by Lipofectmine into BMSCs. The eukaryotic expression vector pcDNA3.1-LIF/pcDNA3.1-bFGF, which can be expressed stablely in BMSCs, has been successfully constructed. LIF/bFGF expression was analyzed using Western blot and RT-PCR. LIF/bFGF was successfully expressed in BMSCs.
Conclude: LIF/bFGF gene was cloned from pregnant uterine deciduas/ placenta tissues by using RT-PCR method and was inserted into pcDNA3.1 to construct eukaryotic expression vector consisting of LIF/bFGF gene. The recombinant vector pcDNA3.1-LIF/ pcDNA3.1- bFGF was identified by restriction enzyme analysis and PCR amplification. Then transfected by Lipofectmine into BMSCs. LIF/bFGF expression was analyzed using Western blot and RT-PCR. LIF/bFGF was successfully expressed in BMSCs. The eukaryotic expression vector pcDNA3.1-LIF/bFGF, which can be expressed stablely in BMSCs, has been successfully constructed.
在十九世纪,Waller首先描述了周围神经损伤后的变性过程。这一过程称为华勒变性(Wallerian dengenerantion),它是在周围神经损伤后,近端神经元胞体和远端轴突所发生的一系列反应。包括炎症反应、生长锥形成延伸、选择性粘附、细胞外基质作用、神经营养因子上调、神经递质合成、神经元重塑、细胞运动等方面,其中神经营养因子(neurotrophic factor, NTFs)在这个过程中起了重要的作用。
骨髓间充质干细胞(BMSCs)是具有自我更新和多向分化潜能的成体干细胞,近年来发现其可分化为神经细胞,具有潜在的修复神经损伤的能力。本实验目的是:体外将LIF、bFGF基因转染培养BMSCs后,通过试验来确定转染后的BMSCs是否表达具有促进周围神经再生和减少再生神经周围瘢痕形成的LIF、bFGF。
研究内容包括:
(1)通过RT-PCR获得LIF、bFGF的cDNA;
(2)采用贴壁法应用Ficoll分离液在体外分离培养大鼠BMSCs,观察其一般生物学特性,并结合其具有多向分化潜能及表面标志物来给予鉴定;
(3)将连于LIF、bFGF基因片段的表达载体转染于BMSCs,通过RT-PCR技术、免疫组化、Westernblot进行鉴定其蛋白表达水平及活性。
结果显示:(1)成功获取LIF、bFGF的cDNA基因片段;(2)从一周龄大鼠骨髓血内成功分离出BMSCs,其形态为呈成纤维细胞样生长,并可以向成脂、成骨细胞转化,其表面标记物CD44(+)、CD45(-);(3)成功获取了稳定表达LIF、bFGF的BMSCs,并通过RT-PCR技术、Westernblot进行了鉴定。
研究结论:通过脂质体将连于LIF、bFGF基因片段的真核表达载体分别和共同转染于体外培养的BMSCs能使其在BMSCs内获得稳定的表达。为进行动物试验奠定了基础。
It is an important task to promote peripheral nerve regeneration after injury in neuroscience. Recent technical progress of molecular biology、cell biology and microsurgery results in better outcome, but not all patients have satisfactory results. It is a nodus how to improve the recovery after nerve injury in peripheral nerve surgery.
The objective task is to solve that bioprotein factor simply applied cure to peripheral nerve injury and surgery couldn’t satisfy the patient’s recovery of the never injury. To reconstitute the part of function and improve the past of never injury, the long time recovery make people suffering. The ideal of this experiment is to rebuilt the peripheral never injury and promote the recovery of never injury.
Our experimental study were divied into three parts
Part I: Construction of eukaryotic expression vector pcDNA3.1-LIF、pcDNA3.1-bFGF LIF/bFGF gene was cloned from pregnant uterine deciduas/placenta tissues by using RT-PCR method and was inserted into pcDNA3.1 to construct eukaryotic expression vector consisting of LIF/bFGF gene. Identified by restriction enzyme analysis and PCR amplification.
Part II: MSCs(mesenchymal stem cells) derived from rat bone marrow were isolated and culture expanded efficiently in vitro and its biological features Ficoll separating medium was added to rat bone marrow mixed liquor that should be centrifuge at 2000rmp×15min soon after it was taken out, then absorb the white layer. Wash the cells twice with PBS and seed cells at a density of 1×106cells per well (6-well format) in 1ml of 15% DMEM of FBS medium. Incubate cells at 37℃in a CO2 incubator. Passage cell was seeded at a density 2～4×104/ml.
Bone marrow mesenchymal stem cells (BMSCs) is one kind of adult stem cells that have self-renewal and multilineage differentiation potential. It has recently been reported that BMSCs can differentiate into neural cells, opening new frontiers in therapy for nerve injury. The morphology of BMSCs was observed with invert-microscope constantly. BMSCs' curve of growth was depicted and cell cycle was analyzed by flowing cytometry. BMSCs were induced to differentiate into osteoblasts and adipocytes in induction medium containing desamethasone、β-Glycerophosphate and horse serum. Then differentiated cells were identificated by AKP and oil O stain. Results showed BMSCs could be isolated and proliferated by density gradient centrifugation adherent method in vitro. The appearance of BMSCs were fibroblast-like cells and its growth curves was like shape of S. Cell cycle analysis showed that most of BMSCs were in G1/G0 phase. BMSCs can be differentiated into osteoblasts and adipocytes in vitro.
Part III: LIF/bFGF was successfully expressed in BMSCs
Transfected by Lipofectmine into BMSCs. The eukaryotic expression vector pcDNA3.1-LIF/pcDNA3.1-bFGF, which can be expressed stablely in BMSCs, has been successfully constructed. LIF/bFGF expression was analyzed using Western blot and RT-PCR. LIF/bFGF was successfully expressed in BMSCs.
Conclude: LIF/bFGF gene was cloned from pregnant uterine deciduas/ placenta tissues by using RT-PCR method and was inserted into pcDNA3.1 to construct eukaryotic expression vector consisting of LIF/bFGF gene. The recombinant vector pcDNA3.1-LIF/ pcDNA3.1- bFGF was identified by restriction enzyme analysis and PCR amplification. Then transfected by Lipofectmine into BMSCs. LIF/bFGF expression was analyzed using Western blot and RT-PCR. LIF/bFGF was successfully expressed in BMSCs. The eukaryotic expression vector pcDNA3.1-LIF/bFGF, which can be expressed stablely in BMSCs, has been successfully constructed.
引文
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