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  本文關(guān)鍵詞： 默克爾細(xì)胞 MEBO 創(chuàng)面 觸覺 神經(jīng)再生　出處：《遵義醫(yī)學(xué)院》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:通過對S D大鼠默克爾細(xì)胞(merkel cell,MCs)的提取、分離、培養(yǎng)及鑒定,觀察局部注射MCs聯(lián)合美寶濕潤燒傷膏(MEBO)外用對大鼠足底創(chuàng)面神經(jīng)再生的影響。方法:1)取新生SD大鼠(1~3d)足墊、須墊及背部皮膚,采用酶消化法分離提取MCs,接種于多聚賴氨酸(PLL)包被的培養(yǎng)瓶內(nèi),通過細(xì)胞免疫組織化學(xué)法測定細(xì)胞角蛋白CK20的表達(dá),qPCR檢測MCs中Piezo2 mRNA的表達(dá),ELISA法檢測培養(yǎng)第2d、4d、6d、8d、10d細(xì)胞分泌CGRP情況。2)建立SD大鼠足底創(chuàng)面模型,按照干預(yù)方式的不同分為如下五組:A組(MCs注射+MEBO組);B組(MCs局部注射組);C組(MEBO外用組);D組(陰性對照組);E組(鼠神經(jīng)生長因子注射陽性對照組)。細(xì)胞組用MCs 1×106個/mL分別注射于創(chuàng)面基底中央及創(chuàng)緣3、6、9、12點標(biāo)記處,24小時后追加1次,每次每個部位25μL;MEBO組按相應(yīng)分組給予MEBO外用,每日換藥一次,直至創(chuàng)面愈合。陽性及空白對照組分別予等體積鼠神經(jīng)生長因子及PBS注射。觀察創(chuàng)面愈合時間,計算各組創(chuàng)面各時間點愈合率,棉拭子檢測足底感覺恢復(fù)情況,分別于干預(yù)后7d、14d、21d三個時相各組分別隨機抽取6只大鼠,切取組織,制作石蠟切片,免疫熒光染色觀察新生皮膚神經(jīng)纖維生長情況。3)每組大鼠均于造模后第7d、14d、21d時用棉拭子檢測大鼠足底縮足反射,記錄、統(tǒng)計縮足次數(shù)并計算百分比。結(jié)果:1)采用中性蛋白酶和胰酶依次消化SD大鼠足底及須墊部位所取皮膚組織可得到較多MCs,以CK20做細(xì)胞免疫化學(xué)染色可見MCs呈陽性反應(yīng)。2)實驗中應(yīng)用中性蛋白酶及胰酶雙酶消化法可得到MCs,以Ham,s F-12培養(yǎng)基為基礎(chǔ)培養(yǎng)基,加入20ng/ml bFGF可使MCs穩(wěn)定增殖;qPCR結(jié)果示MCs中Piezo2 mRNA的表達(dá)量為:0.0270±0.0042;酶聯(lián)免疫吸附試驗(ELISA)方法檢測檢測MCs上清液中降鈣素基因相關(guān)肽(CGRP)在培養(yǎng)第2、4、6、8、10天濃度隨培養(yǎng)時間的延長逐漸增加,培養(yǎng)第6d到8d時增加速率更快,間接提示默克爾細(xì)胞增殖情況。3)創(chuàng)面愈合方面,大體觀察腫脹程度及炎性反應(yīng)程度無明顯區(qū)別,總愈合時間上應(yīng)用MEBO的兩組創(chuàng)面較其他實驗組提前,A組~E組愈合時間分別為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。在同時間點創(chuàng)面愈合率上,A組和C組優(yōu)于其他對照組(P0.05),到21d時各組創(chuàng)面均全部愈合。在縮足反射比較上,除E組外,A組較其他三組反射閾值更低、對刺激更敏感(P0.05),提示默克爾細(xì)胞聯(lián)合MEBO應(yīng)用有促進大鼠足底創(chuàng)面神經(jīng)再生的作用。4)創(chuàng)面新生皮膚免疫熒光染色顯示示A組在神經(jīng)纖維數(shù)量較多、排列有序,優(yōu)于陰性對照或單一實驗處理組,與陽性對照組(E組)相近。結(jié)論:1)中性蛋白酶及胰酶雙酶消化法是獲取大鼠MCs的有效方法,在加入bFGF的Ham,s F-12培養(yǎng)基的體系中培養(yǎng)的MCs大量增殖。2)MCs局部注射聯(lián)合MEBO外用可縮短創(chuàng)面愈合時間、促進創(chuàng)面皮膚神經(jīng)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 鹵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 鹵0.69 days and 16.44 鹵0.86 days, 14.83 鹵0.71 days and 17.44 鹵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.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R622

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