神经浸出液诱导脂肪干细胞向雪旺细胞分化的研究与应用
发布时间：2019-05-22 04:58
【摘要】：周围神经损伤在临床中比较常见,由于成熟的神经元不能分裂增殖,因此,外周神经损伤后的修复效果很不理想。随着组织工程学的发展,人们用组织工程神经代替自体神经修复外周神经损伤并取得了较好效果。组织工程神经的构建主要包含种子细胞、支架材料和神经营养因子三个方面,其中理想的种子细胞是构建人工神经的前提和基础。雪旺细胞(Schwann cells,SCs)在神经再生中起重要作用,是修复神经损伤理想的种子细胞。但是SCs体外培养困难,增殖速度慢,且培养过程中容易受到成纤维细胞污染,不能满足神经修复的要求。脂肪源性干细胞(Adipose stem cells,ADSCs)是具备多向分化潜能的干细胞,在特定环境下可以向SCs分化,同时,ADSCs还具有来源广泛、取材简便、并发症少、增殖速度快、扩增稳定等优点,因此可以成为组织工程神经理想的种子细胞来源。然而目前常用的诱导ADSCs向SCs分化的方法都存在一定的弊端。本研究应用共培养原理,利用外周神经分泌的神经营养因子来诱导ADSCs向SCs分化,以期简化操作程序,节约诱导成本,为解决临床上修复外周神经损伤过程中的种子细胞难题提供新思路,整个实验分为五个部分。1.大鼠ADSCs的体外分离培养和鉴定无菌条件下取出3周龄SD大鼠两侧腹股沟脂肪,采用Ⅰ型胶原酶消化法获得单细胞悬液,并培养在含10%FBS的DMEM培养基中。通过骨向和脂向分化以及流式细胞仪检测细胞表面Marker对所培养的细胞进行鉴定。结果显示成骨诱导后ADSCs形成典型的黑色钙结节,茜素红染色呈现红色;成脂诱导后逐渐出现脂滴,油红O染色为红色。流式检测结果显示:CD44和CD90呈阳性表达,CD31和CD45呈阴性。以上结果证实,分离到的细胞为ADSCs。2.ADSCs体外特性研究本部分的目的是掌握ADSCs的体外分化特性,为选择合适的传代次数和适宜的接种密度提供理论基础。将ADSCs培养至12代,然后诱导第3、6、9和12代的ADSCs向成骨和成脂分化,并通过茜素红和油红O染色对诱导结果进行检测。同时选取第3代ADSCs用两种不同的密度(5×105/cm2和1×103/cm2)进行接种,再用Western-blot检测第3、6、9、12代以及两种不同传代密度的ADSCs的BMP-2的表达,并检测第1-12代ADSCs的碱性磷酸酶(Alkaline phosphatase,ALP)活性。结果证实,第3、6、9、12代的ADSCs均可被诱导成骨细胞和脂肪细胞,但随着传代次数的增加,ADSCs成脂分化潜能逐渐降低,成骨能力未见明显差异。第12代以及以1×103/cm2密度培养的ADSCs的BMP-2呈阳性表达;随着传代次数的增加,第1-12代的ADSCs ALP活性逐步增加,1×103/cm2密度培养的ADSCs的ALP活性较5×105/cm2接种的ALP活性显著增加。ADSCs在体外连续传代或者以较低密度接种时可导致衰老并自发分化成骨。因此ADSCs的体外研究应选择3-5代,并以5×105/cm2的密度进行传代较为适宜。3.神经浸出液的制备及对PC12细胞神经样分化的影响无菌采集的大鼠坐骨神经通过剪碎、浸泡、过滤制备出神经浸出液,待用。为了初步验证本实验室制备的神经浸出液具有促进前体细胞向神经样细胞分化的潜能,本研究选择PC12细胞作为前体细胞模型,将制备好的神经浸出液代替PC12细胞的培养基,观察PC12细胞轴突分化情况。对神经浸出液培养第3、6、9 d的PC12细胞轴突长度进行测量统计,并检测神经元标志物β3-tubulin和MAP-2的表达。结果显示,神经浸出液培养的PC12细胞明显长出突起,第3、6、9 d的轴突平均长度分别达到28.07±1.76μm、58.14±2.60μm、170.43±10.08μm;神经元标志物β3-tubulin和MAP-2蛋白呈阳性表达,初步证实神经浸出液具有诱导前体细胞神经样分化的潜能。4.神经浸出液诱导ADSCs向SCs分化取培养至第3代的ADSCs,用神经浸出液代替正常培养基,培养5 d后用免疫荧光和Western-blot检测SCs特异标志蛋白S-100和胶质原纤维酸性蛋白(glial fibrillary acidic protein,GFAP)的表达,以证实诱导效果。结果表明神经浸出液诱导后的ADSCs形态发生明显改变,胞体由宽大扁平样转变为两极或多极,部分胞体变小,呈梭形,出现2~3个长突起,类似成熟的SCs。免疫荧光和Western-blot检测结果均显示SCs标志物S-100和GFAP呈阳性表达,表明神经浸出液能够诱导ADSCs向SCs分化。5.神经浸出液诱导的ADSCs对大鼠坐骨神经缺损的修复作用将ADSCs与胶原凝胶复合并注入支架材料中制备成组织工程神经移植物作为A组,复合诱导后的ADSCs制备成组织工程神经移植物为B组,植入大鼠1cm坐骨神经损伤处,并用未复合细胞的支架材料组(C组)和自体神经移植组(D组)作为对照。修复大鼠神经损伤,2个月后通过电镜观察、电生理以及图像分析等方法进行神经再生评价。各组结果均显示D组和B组对大鼠坐骨神经损伤的修复效果最好,B组与D组间无显著差异,都显著高于A组,C组最差。表明诱导后的ADSCs对神经损伤的修复效果要优于未经诱导的ADSCs。
[Abstract]:Peripheral nerve injury is common in the clinic, because mature neurons can not split and proliferate, the repair effect after peripheral nerve injury is not ideal. With the development of the tissue engineering, people use the tissue engineering nerve to replace the autologous nerve to repair the peripheral nerve injury and achieve a better effect. The construction of the tissue engineering nerve mainly includes three aspects of seed cell, scaffold material and neurotrophic factor, wherein the ideal seed cell is the premise and foundation for constructing artificial nerve. Schwann cells (SCs) play an important role in nerve regeneration. However, that in vitro culture of the SCs is difficult, the proliferation rate is slow, and the culture process is easy to be polluted by the fibroblasts, and the requirement of nerve repair cannot be met. Adipose stem cells (ADSCs) are stem cells with multi-directional differentiation potential, and can be differentiated to SCs in a specific environment, and the ADSCs also have the advantages of wide source, simple materials, less complications, high proliferation speed, stable amplification and the like. So that the seed cell source of the tissue engineering nerve ideal can be formed. However, the commonly used methods to induce ADSCs to differentiate into SCs have some disadvantages. In this study, the co-culture principle was applied to induce ADSCs to differentiate into SCs by using the neurotrophin factor secreted by peripheral nerve, in order to simplify the operation procedure, save the induction cost and provide a new way to solve the problem of seed cell in the process of repairing peripheral nerve injury. The whole experiment is divided into five parts. The rat ADSCs were isolated and cultured in vitro, and the inguinal fat on both sides of the 3-week-old SD rats were taken out under sterile conditions. The single cell suspension was obtained by a collagenase digestion method of type I and cultured in a DMEM medium containing 10% FBS. The cultured cells were identified by bone-to-fat differentiation and flow cytometry to detect cell surface Marker. The results showed that ADSCs form a typical black calcium nodule after osteogenic induction, and the red staining of the red blood was observed after the formation of fat, and the oil red O was stained with red. The results showed that CD44 and CD90 were positive and CD31 and CD45 were negative. The above results confirm that the isolated cells are ADSCs.2. The purpose of the in vitro characterization of ADSCs is to master the in vitro differentiation of ADSCs and to provide a theoretical basis for selecting the appropriate number of passages and the appropriate seeding density. The ADSCs were cultured to 12 passages, and the ADSCs of the 3rd, 6th, 9th and 12th generation were induced to differentiate into osteogenesis and adipogenesis, and the induced results were tested by the red and oil red O staining. The expression of BMP-2 of ADSCs of 3,6,9,12 and two different passaging densities was detected by Western-blot and the alkaline phosphatase (ALP) activity of ADSCs of the 1st to 12th generation was detected by Western-blot. The results showed that ADSCs of 3rd, 6th, 9th and 12th generation could be induced into osteoblasts and adipocytes, but with the increase of the number of passages, the potential of ADSCs was gradually decreased, and the osteogenic ability was not significantly different. The ADSCs of the 12th generation and the ADSCs cultured at a density of 1-103/ cm2 showed a positive expression; with the increase of the number of passages, the ALP activity of the ADSCs of the 1st-12th generation was gradually increased, and the ALP activity of the ADSCs cultured at a density of 1-103/ cm2 was significantly increased by 5-105/ cm2. ADSCs can lead to aging and spontaneously differentiate into bone at a continuous passage in vitro or at a lower density. Therefore, the in vitro study of ADSCs should be 3-5 generations, and the passage is more appropriate at a density of 5 to 105/ cm2. The preparation of the nerve leaching solution and the effect on the nerve-like differentiation of the PC12 cells are prepared by cutting, soaking, and filtering to prepare the nerve leach solution for later use. In order to preliminarily verify the potential of the neural extract prepared by the laboratory to promote the differentiation of the precursor cells to the neural-like cells, the PC12 cells were selected as the precursor cell model, and the prepared nerve leachate was replaced with the culture medium of the PC12 cells, and the axon differentiation of the PC12 cells was observed. The axon length of PC12 cells from 3,6 and 9 days was measured and the expression of 3-tubelin and MAP-2 were detected. The results showed that the average length of axon in the cultured PC12 cells was 28.07-1.76. m, 58.14-2.60. m, 170.43-10.08. m The potential of the neural-like differentiation of the precursor cells was preliminarily confirmed. The expression of S-100 and glial fibrillary acidic protein (GFAP) of SCs-specific marker protein S-100 and glial fibrillary acidic protein (GFAP) was detected by immunofluorescence and Western-blot. The results showed that the morphology of ADSCs after the induction of nerve extract was obviously changed, and the body was transformed from a large flat to a two-pole or a multi-pole, and some of the cells were small, in the form of a shuttle, with 2 to 3 long protrusions, similar to the mature SCs. The results of both immunofluorescence and Western-blot showed that the S-100 and GFAP of the SCs were positive, indicating that the neural extract can induce ADSCs to differentiate into SCs. the ADSCs induced by the nerve extract and the ADSCs are combined with the collagen gel and injected into the stent material to prepare the tissue engineering nerve graft as a group A, and the compound-induced ADSCs are prepared into a group B of the tissue engineering nerve graft, 1 cm of sciatic nerve injury was implanted in the rat, and the group (group C) and the autograft group (group D) were used as the control for the group of stent material (group C) and the autograft group (group D) of the uncomplexed cells. Nerve regeneration was evaluated by electron microscopy, electrophysiology and image analysis after 2 months in the repair of nerve injury in rats. The results showed that the effect of group D and group B on the repair of sciatic nerve injury in rats was the best. There was no significant difference between group B and group D. The results showed that the effect of ADSCs on the nerve injury was better than that of the uninduced ADSCs.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.3
本文编号：2482695
[Abstract]:Peripheral nerve injury is common in the clinic, because mature neurons can not split and proliferate, the repair effect after peripheral nerve injury is not ideal. With the development of the tissue engineering, people use the tissue engineering nerve to replace the autologous nerve to repair the peripheral nerve injury and achieve a better effect. The construction of the tissue engineering nerve mainly includes three aspects of seed cell, scaffold material and neurotrophic factor, wherein the ideal seed cell is the premise and foundation for constructing artificial nerve. Schwann cells (SCs) play an important role in nerve regeneration. However, that in vitro culture of the SCs is difficult, the proliferation rate is slow, and the culture process is easy to be polluted by the fibroblasts, and the requirement of nerve repair cannot be met. Adipose stem cells (ADSCs) are stem cells with multi-directional differentiation potential, and can be differentiated to SCs in a specific environment, and the ADSCs also have the advantages of wide source, simple materials, less complications, high proliferation speed, stable amplification and the like. So that the seed cell source of the tissue engineering nerve ideal can be formed. However, the commonly used methods to induce ADSCs to differentiate into SCs have some disadvantages. In this study, the co-culture principle was applied to induce ADSCs to differentiate into SCs by using the neurotrophin factor secreted by peripheral nerve, in order to simplify the operation procedure, save the induction cost and provide a new way to solve the problem of seed cell in the process of repairing peripheral nerve injury. The whole experiment is divided into five parts. The rat ADSCs were isolated and cultured in vitro, and the inguinal fat on both sides of the 3-week-old SD rats were taken out under sterile conditions. The single cell suspension was obtained by a collagenase digestion method of type I and cultured in a DMEM medium containing 10% FBS. The cultured cells were identified by bone-to-fat differentiation and flow cytometry to detect cell surface Marker. The results showed that ADSCs form a typical black calcium nodule after osteogenic induction, and the red staining of the red blood was observed after the formation of fat, and the oil red O was stained with red. The results showed that CD44 and CD90 were positive and CD31 and CD45 were negative. The above results confirm that the isolated cells are ADSCs.2. The purpose of the in vitro characterization of ADSCs is to master the in vitro differentiation of ADSCs and to provide a theoretical basis for selecting the appropriate number of passages and the appropriate seeding density. The ADSCs were cultured to 12 passages, and the ADSCs of the 3rd, 6th, 9th and 12th generation were induced to differentiate into osteogenesis and adipogenesis, and the induced results were tested by the red and oil red O staining. The expression of BMP-2 of ADSCs of 3,6,9,12 and two different passaging densities was detected by Western-blot and the alkaline phosphatase (ALP) activity of ADSCs of the 1st to 12th generation was detected by Western-blot. The results showed that ADSCs of 3rd, 6th, 9th and 12th generation could be induced into osteoblasts and adipocytes, but with the increase of the number of passages, the potential of ADSCs was gradually decreased, and the osteogenic ability was not significantly different. The ADSCs of the 12th generation and the ADSCs cultured at a density of 1-103/ cm2 showed a positive expression; with the increase of the number of passages, the ALP activity of the ADSCs of the 1st-12th generation was gradually increased, and the ALP activity of the ADSCs cultured at a density of 1-103/ cm2 was significantly increased by 5-105/ cm2. ADSCs can lead to aging and spontaneously differentiate into bone at a continuous passage in vitro or at a lower density. Therefore, the in vitro study of ADSCs should be 3-5 generations, and the passage is more appropriate at a density of 5 to 105/ cm2. The preparation of the nerve leaching solution and the effect on the nerve-like differentiation of the PC12 cells are prepared by cutting, soaking, and filtering to prepare the nerve leach solution for later use. In order to preliminarily verify the potential of the neural extract prepared by the laboratory to promote the differentiation of the precursor cells to the neural-like cells, the PC12 cells were selected as the precursor cell model, and the prepared nerve leachate was replaced with the culture medium of the PC12 cells, and the axon differentiation of the PC12 cells was observed. The axon length of PC12 cells from 3,6 and 9 days was measured and the expression of 3-tubelin and MAP-2 were detected. The results showed that the average length of axon in the cultured PC12 cells was 28.07-1.76. m, 58.14-2.60. m, 170.43-10.08. m The potential of the neural-like differentiation of the precursor cells was preliminarily confirmed. The expression of S-100 and glial fibrillary acidic protein (GFAP) of SCs-specific marker protein S-100 and glial fibrillary acidic protein (GFAP) was detected by immunofluorescence and Western-blot. The results showed that the morphology of ADSCs after the induction of nerve extract was obviously changed, and the body was transformed from a large flat to a two-pole or a multi-pole, and some of the cells were small, in the form of a shuttle, with 2 to 3 long protrusions, similar to the mature SCs. The results of both immunofluorescence and Western-blot showed that the S-100 and GFAP of the SCs were positive, indicating that the neural extract can induce ADSCs to differentiate into SCs. the ADSCs induced by the nerve extract and the ADSCs are combined with the collagen gel and injected into the stent material to prepare the tissue engineering nerve graft as a group A, and the compound-induced ADSCs are prepared into a group B of the tissue engineering nerve graft, 1 cm of sciatic nerve injury was implanted in the rat, and the group (group C) and the autograft group (group D) were used as the control for the group of stent material (group C) and the autograft group (group D) of the uncomplexed cells. Nerve regeneration was evaluated by electron microscopy, electrophysiology and image analysis after 2 months in the repair of nerve injury in rats. The results showed that the effect of group D and group B on the repair of sciatic nerve injury in rats was the best. There was no significant difference between group B and group D. The results showed that the effect of ADSCs on the nerve injury was better than that of the uninduced ADSCs.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.3
【参考文献】
相关期刊论文 前1条
1 张磊;肖扬;吴震东;陈磊;应志豪;陈子高;;cAMP/PKA信号通路介导人脂肪干细胞成骨分化的体外试验[J];临床骨科杂志;2010年05期
，本文编号：2482695
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