默克尔细胞局部注射联合MEBO外用对大鼠足底创面神经再生的实验研究

发布时间：2018-01-20 09:21

本文关键词： 默克尔细胞 MEBO 创面触觉神经再生　出处：《遵义医学院》2017年硕士论文　论文类型：学位论文

【摘要】：目的:通过对S D大鼠默克尔细胞(merkel cell,MCs)的提取、分离、培养及鉴定,观察局部注射MCs联合美宝湿润烧伤膏(MEBO)外用对大鼠足底创面神经再生的影响。方法:1)取新生SD大鼠(1~3d)足垫、须垫及背部皮肤,采用酶消化法分离提取MCs,接种于多聚赖氨酸(PLL)包被的培养瓶内,通过细胞免疫组织化学法测定细胞角蛋白CK20的表达,qPCR检测MCs中Piezo2 mRNA的表达,ELISA法检测培养第2d、4d、6d、8d、10d细胞分泌CGRP情况。2)建立SD大鼠足底创面模型,按照干预方式的不同分为如下五组:A组(MCs注射+MEBO组);B组(MCs局部注射组);C组(MEBO外用组);D组(阴性对照组);E组(鼠神经生长因子注射阳性对照组)。细胞组用MCs 1×106个/mL分别注射于创面基底中央及创缘3、6、9、12点标记处,24小时后追加1次,每次每个部位25μL;MEBO组按相应分组给予MEBO外用,每日换药一次,直至创面愈合。阳性及空白对照组分别予等体积鼠神经生长因子及PBS注射。观察创面愈合时间,计算各组创面各时间点愈合率,棉拭子检测足底感觉恢复情况,分别于干预后7d、14d、21d三个时相各组分别随机抽取6只大鼠,切取组织,制作石蜡切片,免疫荧光染色观察新生皮肤神经纤维生长情况。3)每组大鼠均于造模后第7d、14d、21d时用棉拭子检测大鼠足底缩足反射,记录、统计缩足次数并计算百分比。结果:1)采用中性蛋白酶和胰酶依次消化SD大鼠足底及须垫部位所取皮肤组织可得到较多MCs,以CK20做细胞免疫化学染色可见MCs呈阳性反应。2)实验中应用中性蛋白酶及胰酶双酶消化法可得到MCs,以Ham,s F-12培养基为基础培养基,加入20ng/ml bFGF可使MCs稳定增殖;qPCR结果示MCs中Piezo2 mRNA的表达量为:0.0270±0.0042;酶联免疫吸附试验(ELISA)方法检测检测MCs上清液中降钙素基因相关肽(CGRP)在培养第2、4、6、8、10天浓度随培养时间的延长逐渐增加,培养第6d到8d时增加速率更快,间接提示默克尔细胞增殖情况。3)创面愈合方面,大体观察肿胀程度及炎性反应程度无明显区别,总愈合时间上应用MEBO的两组创面较其他实验组提前,A组~E组愈合时间分别为14.33±0.69天、16.44±0.86天、14.83±0.71天、17.44±0.98天、16.27±0.67天。在同时间点创面愈合率上,A组和C组优于其他对照组(P0.05),到21d时各组创面均全部愈合。在缩足反射比较上,除E组外,A组较其他三组反射阈值更低、对刺激更敏感(P0.05),提示默克尔细胞联合MEBO应用有促进大鼠足底创面神经再生的作用。4)创面新生皮肤免疫荧光染色显示示A组在神经纤维数量较多、排列有序,优于阴性对照或单一实验处理组,与阳性对照组(E组)相近。结论:1)中性蛋白酶及胰酶双酶消化法是获取大鼠MCs的有效方法,在加入bFGF的Ham,s F-12培养基的体系中培养的MCs大量增殖。2)MCs局部注射联合MEBO外用可缩短创面愈合时间、促进创面皮肤神经的再生。
[Abstract]:Objective: to isolate, culture and identify the MCS of S D rat Merkel cells. To observe the effect of local injection of MCs and MEBO on nerve regeneration of plantar wound in rats. MCs were isolated and extracted by enzyme digestion and inoculated in the culture flask coated with polylysine. The expression of cytokeratin CK20 was detected by immunohistochemistry. QPCR was used to detect the expression of Piezo2 mRNA in MCs. Elisa was used to detect the expression of Piezo2 mRNA in MCs. SD rat plantar wound model was established after 10 days of CGRP secretion. According to the different intervention methods, SD rats were divided into five groups as follows: group A: MCs were injected with MEBO. Group B: MCs were injected locally; Group C: Mebo for external use; Group D (negative control group); In group E (nerve growth factor injection positive control group), the cells were injected with MCs 1 脳 106 / mL at 12 points in the basal center of the wound and in the margin of the wound. After 24 hours, each site was added once with 25 渭 L of each site. The MEBO group was divided into two groups according to the corresponding group: MEBO was given for external use once a day until the wound healed. The positive group and the blank control group were injected with equal volume nerve growth factor and PBS respectively. The wound healing time was observed. The healing rate of wound was calculated at each time point, and the recovery of plantar sensation was detected by cotton swab. Six rats were randomly selected from each group on the 7th day, 14d and 21d after intervention, and the tissues were removed. Paraffin sections were made, and the growth of newborn skin nerve fibers was observed by immunofluorescence staining. 3) every group of rats were tested with cotton swab for foot contraction reflex on the 7th day, 14d and 21d after model making, and recorded. Results: MCs was obtained by neutral protease and trypsin digestion in turn. The positive reaction of MCs was observed by cytoimmunochemical staining of CK20. 2) in the experiment, MCs could be obtained by neutral protease and trypsin double enzyme digestion, and Ham could be obtained. S F-12 medium was the base medium, adding 20ng / ml bFGF could make MCs proliferate stably. QPCR results showed that the expression of Piezo2 mRNA in MCs was 0.0270 卤0.0042; Enzyme linked immunosorbent assay (Elisa) was used to detect calcitonin gene-related peptide (CGRP) in the supernatant of MCs. The concentration of 10 days increased gradually with the extension of culture time, and the increase rate increased more rapidly from the 6th to 8th day of culture, which indirectly indicated the proliferation of Merkel cells. 3) the wound healing. There was no significant difference in the degree of swelling and inflammatory reaction between the two groups. The total healing time of the two groups treated with MEBO was earlier than that of the other experimental groups. The healing time of group A and E was 14.33 卤0.69 days and 16.44 卤0.86 days, 14.83 卤0.71 days and 17.44 卤0.98 days respectively. At the same time, the wound healing rate of group A and group C was better than that of group A and group C, and all the wounds were healed by 21 days. Except group E, the reflex threshold of group A was lower than that of the other three groups, and it was more sensitive to stimulation (P0.05). It was suggested that the combination of Merkel cells and MEBO could promote the nerve regeneration in the plantar wound of rats. 4) Immunofluorescence staining showed that the number of nerve fibers in group A was more than that in group A, and the arrangement of nerve fibers in group A was orderly. It is superior to the negative control group or the single experimental group, and is similar to the positive control group (E group). Conclusion the neutral protease and trypsin double enzyme digestion method is an effective method to obtain MCs in rats. The proliferation of MCs cultured in Hams F-12 medium supplemented with bFGF. The local injection of MCs combined with MEBO could shorten the wound healing time. Promote the regeneration of wound skin nerve.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R622

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