鹿茸多肽修复周围神经损伤的应用研究及辽西地区周围神经损伤的流行病学初步调查
发布时间：2018-09-12 07:48
【摘要】：目的:本实验拟以鹿茸多肽(Pilose Antler Polypeptides,PAP)和大鼠肌源性干细胞(muscle-derived stem cell,MDSC)为研究对象,探讨鹿茸多肽促进肌源性干细胞向类雪旺细胞分化及其作用机制;运用微囊化技术包裹使用含鹿茸多肽的血清处理过的肌源性干细胞,同透明质酸钠凝胶载体、硅胶导管组构建人工神经,通过动物实验来评估鹿茸多肽及肌源性干细胞促进神经缺损修复的效果。材料与方法:实验一大鼠肌源性干细胞的分离、培养及鉴定应用酶对大鼠的骨骼肌进行消化,再通过差速贴壁法和胰酶消化法得到纯化的MDSC。使用倒置显微镜观察纯化后MDSC的生长状况、形态学特点;同时进行Desmin免疫组化鉴定。实验二鹿茸多肽促进肌源性干细胞向类雪旺细胞分化的实验研究10只Wistar大鼠分为给药组及对照组,根据公式计算鹿茸多肽用量,每天两次喂药给药。抽动脉血,离心后取上清液,制备成含鹿茸多肽血清及空白血清;将第一部分实验获MDSC分为A、B、C、D4组,培养基内加入不同物质,A组:培养基中加入鹿茸多肽含药血清;B组:培养计中加入鹿茸多肽溶液;C组:培养基中加入空白血清;D组:为正常细胞培养基。培养21天,观察A、B、C、D4组细胞的形态特征;通过四甲基偶氮唑盐(Methyl thiazolyl tetrazolium,MTT)法检测细胞增殖情况;通过免疫组化技术对诱导细胞进行雪旺细胞特异性标记物鉴定。实验三鹿茸多肽处理肌源性干细胞在周围神经损伤修复中应用的实验研究将第一部分实验获得的肌源性干细胞标记为N组细胞;第二部分实验获得的类雪旺细胞标记为M组细胞。通过微囊化技术分别制备出M组微囊化细胞及N组微囊化细胞。实验大鼠行坐骨神经缺损造模,平均分成四组,以硅胶管为导管,透明质酸钠为载体,A组加入M组微囊化细胞,B组为自体神经移植组,C组未加入任何细胞,D组加入N组微囊化细胞,术后1、2、3、6、9、12周分别观察各组大鼠的足跟溃疡时间、步态情况。4、8、12周计算坐骨神经指数(sciatic nerve function index,SFI)数值。12周观察再生后神经纤维数目及其直径,测量修复神经动作电位的幅度和传导速度,通过电镜观察再生神经微观结构。结果:1.改良传统肌源性干细胞培养方法,可以在短时间内培养出纯度较高、数量较多的肌源性干细胞;培养的细胞desmin染色、Sca-1抗体染色阳性。2.实验成功获得了PAP含药血清;使用不同培养液对肌源性干细胞进行诱导分化,培养72h后观察发现A组所培养细胞多呈双极梭形,少量细胞呈三角形,相邻细胞之间以突起相连,胞核较大;B组所培养细胞,在细胞培养板内出现大量脱壁、漂浮,倒置相差显微镜下可以观察到细胞皱缩,细胞损害明显;C组、D组的细胞基本保持长梭形的外观,逐渐向细长发展。MTT检测结果提示诱导细胞在第4-5天时达到增殖最为明显;PAP含药血清诱导21天的细胞,免疫组化显示S100,GFAP,和P75阳性表达,证明诱导所得细胞为大鼠类雪旺细胞。3.在动物实验中发现术后9周A、B、D组术侧足底部红肿和溃疡情况均有不同程度好转。C组术侧足底部红肿和溃疡改善不明显;术后12周时A、B、D组术侧足底的红肿和溃疡情况基本消失,C组术侧足底的红肿和溃疡仍存在。12周时A组再生神经外形似正常神经;B组移植神经断端连接处无瘢痕组织形成,但神经与周围组织轻度粘连;C组植入的导管内有再生神经,但再生神经比较纤细;D组同样也可见再生神经,比C组稍粗。12周时A组坐骨神经指数等指标优于C组、D组,差异有统计学意义(P0.05);与B组接近,差异没有统计学意义(P0.05)。术后12周A组、B组再生神经可见雪旺氏细胞排列紧密,D组再生神经S-100免疫反应阳性的雪旺氏细胞数量相对较少,C组再生神经S-100免疫反应阳性的雪旺氏细胞数量最少,且条带状分布不明显。透射电镜观察A组、B组再生的神经髓鞘、轴突结构优于另外两组。结论:1.应用显微外科技术、混合酶及差速贴壁培养方法可以简便快捷地分离出肌源性干细胞。2.鹿茸多肽含药血清可明显促进肌源性干细胞的体外增殖和向类雪旺细胞分化,提示鹿茸多肽具有开发为促进神经损伤修复新药的潜能。3.鹿茸多肽和肌源性干细胞联合应用可促进周围神经缺损的修复,两者可应用于周围神经组织工程学研究。目的:分析辽西地区周围神经损伤患者流行病学特点,为预防周围神经损伤提供参考。材料与方法:采取统一调查表,通过对2014年1月至2014年12月期间辽西地区五市七家三甲医院收治周围神经损伤住院患者1410例进行回顾性分析,将患者分成5个年龄组:童年(0-6周岁);少年(7-17周岁);青年(18-40周岁);中年(41-65周岁);老年(大于65周岁),统计受伤时间、年龄、文化程度、职业、受伤地点、致伤原因、受伤部位、合并损伤、是否手术及并发症。对数据进行统计学分析。结果:1年时间内,共收治周围神经损伤住院患者1410例,男性患者1105名,女性患者305名,男女比列为3.62:1;年龄组中18岁至40岁青壮年为高发病组,为51.06%;文化程度中以小学文化和中学文化发病率高,二者无明显区别;致伤因素中工作受伤48.94%,交通受伤28.72%,其中不同性别原因不同,女性患者主要以交通致伤为主,而男性患者主要以工作受伤为主。结论:处于16岁至40岁,且文化程度不高的青壮年为周围神经损伤高危人群;工作中切割伤为主要致伤因素;加强意识,严格生产管理是预防周围神经损伤的重点。
[Abstract]:AIM: To investigate the effect of pilose antler polypeptides (PAP) and muscle-derived stem cells (MDSC) on the differentiation of myogenic stem cells into Schwann-like cells (SCs) and its mechanism, and to encapsulate the treated muscle with serum containing pilose antler polypeptides by microencapsulation technique. Material and Methods: Isolation, culture and identification of rat myogenic stem cells, digestion and recanalization of rat skeletal muscles by enzyme were studied. Purified MDSCs were obtained by super-differential adhesion method and trypsin digestion method.The growth and morphological characteristics of purified MDSCs were observed by inverted microscope and identified by Desmin immunohistochemistry.In experiment 2,10 Wistar rats were divided into treatment group and control group. According to the formula, the dosage of velvet antler polypeptide was calculated and given twice a day. Arterial blood was drawn and supernatant was taken after centrifugation to prepare serum containing velvet antler polypeptide and blank serum. Peptide solution; C group: blank serum was added into culture medium; D group: normal cell culture medium. The morphological characteristics of A, B, C, D4 cells were observed after 21 days of culture; the proliferation of cells was detected by MTT method; and the specific markers of Schwann cells were identified by immunohistochemistry. Experimental study on the application of myogenic stem cells treated with Sanlu antler polypeptide in peripheral nerve injury repair Sciatic nerve defect model was established in rats. The rats were divided into four groups. Silica gel tube was used as catheter, sodium hyaluronate was used as carrier, group A was added with microencapsulated cells in group M, group B was used with autologous nerve transplantation, group C was not added with any cells, group D was added with microencapsulated cells in group N. The time of heel ulcer was observed at 1, 2, 3, 6, 9 and 12 weeks after operation. Sciatic nerve function index (SFI) was calculated at 4, 8, and 12 weeks. The number and diameter of regenerated nerve fibers were observed at 12 weeks. The amplitude and conduction velocity of action potential were measured. The microstructure of regenerated nerve was observed by electron microscope. Results: 1. Modified traditional culture method of myogenic stem cells could be used in short time. Myogenic stem cells with high purity and large quantity were cultured in time. The cultured cells were stained with desmin and positive for Sca-1 antibody. Corneal, adjacent cells were connected by protuberances, and the nucleus was larger. Cells in group B were desquamated and floated in a large number. Cell shrinkage and cell damage were observed under inverted phase contrast microscope. Cells in group C and group D kept the appearance of long spindle and gradually developed to slender. The cells in group A, group B and group D were found to be reddish and ulcerated at the foot base 9 weeks after operation. The cells in group C were reddish and swollen at the foot base. At 12 weeks postoperatively, the swelling and ulcer of the operation side of the foot in group A, B and D disappeared, and the swelling and ulcer of the operation side of the foot in group C still existed. Nerve regeneration was found in group D, which was slightly thicker than that in group C. At 12 weeks, sciatic nerve index in group A was better than that in group C, and there was significant difference between group D and group B (P 0.05). The number of S-100 immunoreactive Schwann cells in group C was the least, and the distribution of S-100 immunoreactive Schwann cells in group A and B was not obvious. Myogenic stem cells can be isolated easily and quickly by adherent culture. 2. Velvet antler polypeptide containing serum can significantly promote the proliferation and differentiation of myogenic stem cells into Schwann-like cells in vitro, suggesting that velvet antler polypeptide has the potential to be developed as a new drug for nerve injury repair. 3. The combination of velvet antler polypeptide and myogenic stem cells can promote the proliferation of myogenic stem cells. Objective: To analyze the epidemiological characteristics of peripheral nerve injury patients in Western Liaoning and provide reference for the prevention of peripheral nerve injury. Materials and Methods: A unified questionnaire was used to investigate the effects of peripheral nerve injury on peripheral nerve tissue engineering. 1410 inpatients with peripheral nerve injury were divided into five age groups: childhood (0-6 years old); juvenile (7-17 years old); youth (18-40 years old); middle age (41-65 years old); old age (over 65 years old). The injury time, age, education level, occupation, place of injury, cause of injury, site of injury, combined injury were counted. Results: 1 410 inpatients with peripheral nerve injury were admitted in one year, including 1 105 male patients and 305 female patients. The ratio of male to female was 3.62:1. The incidence of peripheral nerve injury was 51.06% among the young and middle-aged aged from 18 to 40 years old. There was no significant difference between the two groups. 48.94% of the injuries were caused by work and 28.72% were caused by traffic. The main causes were traffic injuries in female patients and work injuries in male patients. Cutting is the main injury factor; strengthening consciousness and strict production management are the key points to prevent peripheral nerve injury.
【学位授予单位】：辽宁中医药大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R745
本文编号：2238371
[Abstract]:AIM: To investigate the effect of pilose antler polypeptides (PAP) and muscle-derived stem cells (MDSC) on the differentiation of myogenic stem cells into Schwann-like cells (SCs) and its mechanism, and to encapsulate the treated muscle with serum containing pilose antler polypeptides by microencapsulation technique. Material and Methods: Isolation, culture and identification of rat myogenic stem cells, digestion and recanalization of rat skeletal muscles by enzyme were studied. Purified MDSCs were obtained by super-differential adhesion method and trypsin digestion method.The growth and morphological characteristics of purified MDSCs were observed by inverted microscope and identified by Desmin immunohistochemistry.In experiment 2,10 Wistar rats were divided into treatment group and control group. According to the formula, the dosage of velvet antler polypeptide was calculated and given twice a day. Arterial blood was drawn and supernatant was taken after centrifugation to prepare serum containing velvet antler polypeptide and blank serum. Peptide solution; C group: blank serum was added into culture medium; D group: normal cell culture medium. The morphological characteristics of A, B, C, D4 cells were observed after 21 days of culture; the proliferation of cells was detected by MTT method; and the specific markers of Schwann cells were identified by immunohistochemistry. Experimental study on the application of myogenic stem cells treated with Sanlu antler polypeptide in peripheral nerve injury repair Sciatic nerve defect model was established in rats. The rats were divided into four groups. Silica gel tube was used as catheter, sodium hyaluronate was used as carrier, group A was added with microencapsulated cells in group M, group B was used with autologous nerve transplantation, group C was not added with any cells, group D was added with microencapsulated cells in group N. The time of heel ulcer was observed at 1, 2, 3, 6, 9 and 12 weeks after operation. Sciatic nerve function index (SFI) was calculated at 4, 8, and 12 weeks. The number and diameter of regenerated nerve fibers were observed at 12 weeks. The amplitude and conduction velocity of action potential were measured. The microstructure of regenerated nerve was observed by electron microscope. Results: 1. Modified traditional culture method of myogenic stem cells could be used in short time. Myogenic stem cells with high purity and large quantity were cultured in time. The cultured cells were stained with desmin and positive for Sca-1 antibody. Corneal, adjacent cells were connected by protuberances, and the nucleus was larger. Cells in group B were desquamated and floated in a large number. Cell shrinkage and cell damage were observed under inverted phase contrast microscope. Cells in group C and group D kept the appearance of long spindle and gradually developed to slender. The cells in group A, group B and group D were found to be reddish and ulcerated at the foot base 9 weeks after operation. The cells in group C were reddish and swollen at the foot base. At 12 weeks postoperatively, the swelling and ulcer of the operation side of the foot in group A, B and D disappeared, and the swelling and ulcer of the operation side of the foot in group C still existed. Nerve regeneration was found in group D, which was slightly thicker than that in group C. At 12 weeks, sciatic nerve index in group A was better than that in group C, and there was significant difference between group D and group B (P 0.05). The number of S-100 immunoreactive Schwann cells in group C was the least, and the distribution of S-100 immunoreactive Schwann cells in group A and B was not obvious. Myogenic stem cells can be isolated easily and quickly by adherent culture. 2. Velvet antler polypeptide containing serum can significantly promote the proliferation and differentiation of myogenic stem cells into Schwann-like cells in vitro, suggesting that velvet antler polypeptide has the potential to be developed as a new drug for nerve injury repair. 3. The combination of velvet antler polypeptide and myogenic stem cells can promote the proliferation of myogenic stem cells. Objective: To analyze the epidemiological characteristics of peripheral nerve injury patients in Western Liaoning and provide reference for the prevention of peripheral nerve injury. Materials and Methods: A unified questionnaire was used to investigate the effects of peripheral nerve injury on peripheral nerve tissue engineering. 1410 inpatients with peripheral nerve injury were divided into five age groups: childhood (0-6 years old); juvenile (7-17 years old); youth (18-40 years old); middle age (41-65 years old); old age (over 65 years old). The injury time, age, education level, occupation, place of injury, cause of injury, site of injury, combined injury were counted. Results: 1 410 inpatients with peripheral nerve injury were admitted in one year, including 1 105 male patients and 305 female patients. The ratio of male to female was 3.62:1. The incidence of peripheral nerve injury was 51.06% among the young and middle-aged aged from 18 to 40 years old. There was no significant difference between the two groups. 48.94% of the injuries were caused by work and 28.72% were caused by traffic. The main causes were traffic injuries in female patients and work injuries in male patients. Cutting is the main injury factor; strengthening consciousness and strict production management are the key points to prevent peripheral nerve injury.
【学位授予单位】：辽宁中医药大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R745
【参考文献】
相关期刊论文 前1条
1 曾祥一;王伟;孙亮;张力;曾令达;;睫状神经营养因子和丹参注射液体外诱导肌源性干细胞向神经元样细胞的分化[J];中国组织工程研究与临床康复;2009年27期
，本文编号：2238371
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