重组质粒载体pEGFP-N2-SMDF体外转染小鼠肌源性干细胞及其鉴定

发布时间：2019-05-16 16:02

【摘要】：【研究背景】肌源性干细胞（muscle-derived stem cells, MDSCs）是一类新近发现的成体干细胞，可以从动物的骨骼肌中分离获得，具有干细胞的自我更新和多向分化的潜能。MDSCs在体内/外分化成肌肉细胞时不需要特定的刺激，但让MDSCs分化成骨骼肌细胞以外的其它谱系细胞时，可能需要特殊的生长因子刺激或将其置于适宜的环境中。越来越多的研究表明，MDSCs在特定组织的发育、组织再生及修复和基因治疗方面的巨大潜能。有证据显示，MDSCs能够促进肌纤维的再生，增强再生肌组织的功能，并具有分化为神经外膜组织细胞和具有雪旺细胞（Schwann cells,SCs）表型细胞的潜能，为神经组织工程研究中寻找雪旺细胞替代物带来了希望。Neu基因调节素-1（neuregulin-1,NRG1）是一种由膜携带或分泌的多肽，属于表皮生长因子（Epidermal Growth Factor,EGF）家族，主要是由神经元产生，能与ErbB酪氨酸激酶受体相结合，引起广泛的生物学效应。根据其N端结构域的不同可将其分为Ⅰ、Ⅱ、Ⅲ3种亚型。最近的研究表明轴突中NRG1Ⅲ型（sensory and motor neuron-derivedfactor,SMDF）的水平是髓鞘形成的一个关键指标。它不仅能促进雪旺细胞的增殖、分化，而且还调节轴突的髓鞘化及髓鞘的厚度。SMDF的分泌在胚胎期和新生期达到高峰，而成年后的表达明显减少。在周围神经损伤后，，雪旺细胞及神经支配的靶器官—肌组织中均可见其表达，然而其表达量不足以支持成年轴突完成再髓鞘化过程，仅能形成包绕轴突的薄髓鞘和变短的节间长度。也有研究显示，雪旺细胞自身可以产生NRG1（Ⅲ型α2同源体），以自分泌或旁分泌的形式促进雪旺细胞增殖，并参与到清除病变轴突和髓鞘的过程中。周围神经损伤后，雪旺细胞分化和增殖是轴突再生的必备条件，而SMDF是髓鞘厚度的关键调节因子。因此，提高周围神经损伤处SMDF蛋白水平为雪旺细胞的髓鞘化过程提供了一个潜在的治疗策略。【研究目的】（1）采用新生小鼠坐骨神经培养纯化后的雪旺细胞条件培液，诱导小鼠MDSCs向雪旺细胞方向分化，为组织工程研究寻找新的雪旺细胞替代物；（2）克隆SMDF基因和构建SMDF真核表达载体，为进一步的功能研究提供有力的工具；（3）鉴定转染pEGFP-N2-SMDF的MDSCs，研究SMDF基因在mRNA和蛋白水平的表达，进一步证实构建的载体能有效的转导目的基因；（4）证实SMDF基因可促进类雪旺细胞的增殖、分化的，进一步验证SMDF蛋白的功能，为研究周围神经损伤后SCs髓鞘化过程及分子机制奠定了实验基础。【研究方法】（1）应用改良的Preplate技术，从新生小鼠骨骼肌中分离MDSCs，利用抗体Desmin和Sca-1对分离得到的MDSCs进行免疫细胞化学鉴定；（2）从新生小鼠坐骨神经分离纯化雪旺细胞，利用抗体P75NTR、S-100β进行鉴定并收集雪旺细胞的条件培液用以诱导MDSCs分化为类雪旺细胞；（3）利用Western Blot对不同组织中SMDF蛋白的表达情况进行分析，利用免疫组织化学的方法鉴定SMDF在DRG中的表达；（4）针对小鼠SMDF基因mRNA全序列设计引物，Trizol法提取新生小鼠DRG总RNA，通过RT-PCR得到SMDF基因编码区全序列，利用DNA重组技术将该基因片段重组到pEGFP-N2真核表达质粒上，构建重组真核表达载体pEGFP-N2-SMDF，并通过PCR、酶切及测序的方法进行鉴定；（5）应用脂质体介导的方法将真核表达载体pEGFP-N2-SMDF和空质粒pEGFP-N2转染体外培养的MDSCs，通过PCR、Western-blot方法检测转染前后SMDF基因和蛋白的表达情况。（6）采用雪旺细胞条件培液诱导转染SMDF基因的MDSCs分化为类雪旺细胞，并采用免疫细胞化学的方法进行鉴定。【结果】（1）应用改良的Preplate技术，从小鼠骨骼肌中分离得到MDSCs，能在体外稳定增殖传代，Desmin和Sca-1免疫细胞化学染色阳性；（2）从新生小鼠坐骨神经中分离纯化得到雪旺细胞，将收集的雪旺细胞条件培液加入MDSCs中，成功诱导出能表达雪旺细胞特异性标记物P75NTR、S-100β的细胞；（3）将小鼠DRG中的SMDF基因克隆至真核表达载体pEGFP-N2上，双酶切、PCR和DNA测序鉴定结果表明，插入的序列与GenBank上小鼠SMDF基因CDS序列完全符合；（4）pEGFP-N2-SMDF真核表达载体转染MDSCs后，荧光显微镜下可见绿色荧光蛋白的表达；PCR和Western-blot结果显示，重组载体pEGFP-N2-SMDF转染后的细胞中SMDF mRNA水平和蛋白水平的表达明显高于空质粒组及未转染组细胞，而后两者间比较无统计学差异；转染SMDF基因的MDSCs，经雪旺条件培液诱导后可分化为类雪旺细胞，且S-100β的阳性率明显高于直接诱导的类雪旺细胞。【结论】（1）采用雪旺细胞条件培液，能够将新生小鼠骨骼肌中分离得到的MDSCs诱导分化为类雪旺细胞，为组织工程神经研究找到了新的种子细胞替代物；（2）成功将重组的真核表达载体pEGFP-N2-SMDF转染至MDSCs中，转染后的细胞中SMDF的mRNA水平和蛋白水平都显著增高，同时证实SMDF基因可促进类雪旺细胞的增殖、分化，为进一步研究周围神经损伤后雪旺细胞髓鞘化过程及分子机制奠定了实验基础
[Abstract]:[Study Background] Muscle-derived stem cells (MDSCs) are a new type of adult stem cells, which can be isolated from the skeletal muscle of the animal and have the potential of self-renewal and multi-directional differentiation of the stem cells. The MDSCs do not need a specific stimulus when the MDSCs differentiate into muscle cells in vivo/ out, but may require special growth factors to stimulate or place them in a suitable environment. An increasing number of studies have shown that MDSCs can promote the regeneration of muscle fibers, enhance the function of the regenerated muscle tissue, and have the potential to differentiate into the outer membrane histiocytes and Schwann cells (SCs) phenotype cells. Increasing the level of the SMDF protein at peripheral nerve injury provides a potential therapeutic strategy for the treatment of Schwann cells. [Study objective] (1) The induced mouse MDSCs were induced to differentiate in the direction of Schwann cells, and the new Schwann cell replacement was found for tissue engineering. (2) The SMDF gene was cloned and the SMDF eukaryotic expression vector was constructed to provide a powerful tool for further functional research. (3) The MDSCs transfected with pEGFP-N2-SMDF were identified, the expression of the SMDF gene in the mRNA and protein level was studied, and the constructed vector was further proved to be effective for transduction of the target gene. (4) The SMDF gene was confirmed to promote the proliferation and differentiation of the Schwann cells and further verify the function of the SMDF protein. The invention discloses a method for preparing a recombinant eukaryotic expression vector pEGFP-N2-SMDF by using an improved Preplate technique to separate MDSCs from the skeletal muscle of a newborn mouse, and carrying out immunocytochemical identification on the MDSCs obtained by the separation by using the antibody Desmin and the Sca-1; (2) separating and purifying the Schwann cells from the sciatic nerve of the newborn mouse, and identifying and collecting the conditions of the Schwann cells to induce the MDSCs to be differentiated into a class of Schwann cells by using the antibody P75NTR and the S-100 antigen; (3) analyzing the expression of the SMDF protein in different tissues by using the Western Blot; (3) using the Western Blot to analyze the expression of the SMDF protein in the different tissues; (4) extracting the whole sequence of the SMDF gene coding region by using the Western Blot; and (4) the recombinant eukaryotic expression vector pEGFP-N2-SMDF is constructed by using the DNA recombination technology to extract the whole sequence of the SMDF gene coding region by the RT-PCR, and the recombinant eukaryotic expression vector pEGFP-N2-SMDF is constructed by using the DNA recombination technology to obtain the whole sequence of the SMDF gene coding region. and (6) inducing MDSCs that transfect the SMDF gene into a class of Schwann cells by using a Schwann cell culture solution, and adopting a method of immunocytochemistry Line identification.[Results] (1) The modified Preplate technique was used to separate and purify the MDSCs from the skeletal muscle of the mouse, and the cells were chemically stained for Desmin and Sca-1. (2) The Schwann cells were isolated and purified from the sciatic nerve of the newborn mice, and the collected Schwann cell conditioned medium was added to the MDSCs to successfully induce the expression of the Schwann cell-specific marker P75NTR and S-100 antigen. (3) The SMDF gene in the DRG of the mouse was cloned into the eukaryotic expression vector pEGFP-N2, and the expression of the green fluorescent protein was observed under the fluorescence microscope after the expression of the pEGFP-N2-SMDF eukaryotic expression vector was transfected into the MDSCs. The results of the PCR and the Western-blot showed that the expression of the SMDF mRNA in the cells transfected with the recombinant vector pEGFP-N2-SMDF was confirmed by PCR and Western-blot. Schwann cells were found in Schwann cells.[Conclusion] (1) Schwann cells were used to induce MDSCs to differentiate into Schwann cells, and a new seed cell replacement was found for the study of tissue engineering. (2) The recombinant eukaryotic expression vector pEGFP-N2-SMDF was successfully transfected into MDSCs, and the mRNA level and protein level of SMDF in the transfected cells increased significantly, and the SMDF gene was confirmed to promote the proliferation and differentiation of Schwann cells.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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