【摘要】：目的:本實驗擬以鹿茸多肽(Pilose Antler Polypeptides,PAP)和大鼠肌源性干細(xì)胞(muscle-derived stem cell,MDSC)為研究對象,探討鹿茸多肽促進(jìn)肌源性干細(xì)胞向類雪旺細(xì)胞分化及其作用機(jī)制;運用微囊化技術(shù)包裹使用含鹿茸多肽的血清處理過的肌源性干細(xì)胞,同透明質(zhì)酸鈉凝膠載體、硅膠導(dǎo)管組構(gòu)建人工神經(jīng),通過動物實驗來評估鹿茸多肽及肌源性干細(xì)胞促進(jìn)神經(jīng)缺損修復(fù)的效果。材料與方法:實驗一大鼠肌源性干細(xì)胞的分離、培養(yǎng)及鑒定應(yīng)用酶對大鼠的骨骼肌進(jìn)行消化,再通過差速貼壁法和胰酶消化法得到純化的MDSC。使用倒置顯微鏡觀察純化后MDSC的生長狀況、形態(tài)學(xué)特點;同時進(jìn)行Desmin免疫組化鑒定。實驗二鹿茸多肽促進(jìn)肌源性干細(xì)胞向類雪旺細(xì)胞分化的實驗研究10只Wistar大鼠分為給藥組及對照組,根據(jù)公式計算鹿茸多肽用量,每天兩次喂藥給藥。抽動脈血,離心后取上清液,制備成含鹿茸多肽血清及空白血清;將第一部分實驗獲MDSC分為A、B、C、D4組,培養(yǎng)基內(nèi)加入不同物質(zhì),A組:培養(yǎng)基中加入鹿茸多肽含藥血清;B組:培養(yǎng)計中加入鹿茸多肽溶液;C組:培養(yǎng)基中加入空白血清;D組:為正常細(xì)胞培養(yǎng)基。培養(yǎng)21天,觀察A、B、C、D4組細(xì)胞的形態(tài)特征;通過四甲基偶氮唑鹽(Methyl thiazolyl tetrazolium,MTT)法檢測細(xì)胞增殖情況;通過免疫組化技術(shù)對誘導(dǎo)細(xì)胞進(jìn)行雪旺細(xì)胞特異性標(biāo)記物鑒定。實驗三鹿茸多肽處理肌源性干細(xì)胞在周圍神經(jīng)損傷修復(fù)中應(yīng)用的實驗研究將第一部分實驗獲得的肌源性干細(xì)胞標(biāo)記為N組細(xì)胞;第二部分實驗獲得的類雪旺細(xì)胞標(biāo)記為M組細(xì)胞。通過微囊化技術(shù)分別制備出M組微囊化細(xì)胞及N組微囊化細(xì)胞。實驗大鼠行坐骨神經(jīng)缺損造模,平均分成四組,以硅膠管為導(dǎo)管,透明質(zhì)酸鈉為載體,A組加入M組微囊化細(xì)胞,B組為自體神經(jīng)移植組,C組未加入任何細(xì)胞,D組加入N組微囊化細(xì)胞,術(shù)后1、2、3、6、9、12周分別觀察各組大鼠的足跟潰瘍時間、步態(tài)情況。4、8、12周計算坐骨神經(jīng)指數(shù)(sciatic nerve function index,SFI)數(shù)值。12周觀察再生后神經(jīng)纖維數(shù)目及其直徑,測量修復(fù)神經(jīng)動作電位的幅度和傳導(dǎo)速度,通過電鏡觀察再生神經(jīng)微觀結(jié)構(gòu)。結(jié)果:1.改良傳統(tǒng)肌源性干細(xì)胞培養(yǎng)方法,可以在短時間內(nèi)培養(yǎng)出純度較高、數(shù)量較多的肌源性干細(xì)胞;培養(yǎng)的細(xì)胞desmin染色、Sca-1抗體染色陽性。2.實驗成功獲得了PAP含藥血清;使用不同培養(yǎng)液對肌源性干細(xì)胞進(jìn)行誘導(dǎo)分化,培養(yǎng)72h后觀察發(fā)現(xiàn)A組所培養(yǎng)細(xì)胞多呈雙極梭形,少量細(xì)胞呈三角形,相鄰細(xì)胞之間以突起相連,胞核較大;B組所培養(yǎng)細(xì)胞,在細(xì)胞培養(yǎng)板內(nèi)出現(xiàn)大量脫壁、漂浮,倒置相差顯微鏡下可以觀察到細(xì)胞皺縮,細(xì)胞損害明顯;C組、D組的細(xì)胞基本保持長梭形的外觀,逐漸向細(xì)長發(fā)展。MTT檢測結(jié)果提示誘導(dǎo)細(xì)胞在第4-5天時達(dá)到增殖最為明顯;PAP含藥血清誘導(dǎo)21天的細(xì)胞,免疫組化顯示S100,GFAP,和P75陽性表達(dá),證明誘導(dǎo)所得細(xì)胞為大鼠類雪旺細(xì)胞。3.在動物實驗中發(fā)現(xiàn)術(shù)后9周A、B、D組術(shù)側(cè)足底部紅腫和潰瘍情況均有不同程度好轉(zhuǎn)。C組術(shù)側(cè)足底部紅腫和潰瘍改善不明顯;術(shù)后12周時A、B、D組術(shù)側(cè)足底的紅腫和潰瘍情況基本消失,C組術(shù)側(cè)足底的紅腫和潰瘍?nèi)源嬖凇?2周時A組再生神經(jīng)外形似正常神經(jīng);B組移植神經(jīng)斷端連接處無瘢痕組織形成,但神經(jīng)與周圍組織輕度粘連;C組植入的導(dǎo)管內(nèi)有再生神經(jīng),但再生神經(jīng)比較纖細(xì);D組同樣也可見再生神經(jīng),比C組稍粗。12周時A組坐骨神經(jīng)指數(shù)等指標(biāo)優(yōu)于C組、D組,差異有統(tǒng)計學(xué)意義(P0.05);與B組接近,差異沒有統(tǒng)計學(xué)意義(P0.05)。術(shù)后12周A組、B組再生神經(jīng)可見雪旺氏細(xì)胞排列緊密,D組再生神經(jīng)S-100免疫反應(yīng)陽性的雪旺氏細(xì)胞數(shù)量相對較少,C組再生神經(jīng)S-100免疫反應(yīng)陽性的雪旺氏細(xì)胞數(shù)量最少,且條帶狀分布不明顯。透射電鏡觀察A組、B組再生的神經(jīng)髓鞘、軸突結(jié)構(gòu)優(yōu)于另外兩組。結(jié)論:1.應(yīng)用顯微外科技術(shù)、混合酶及差速貼壁培養(yǎng)方法可以簡便快捷地分離出肌源性干細(xì)胞。2.鹿茸多肽含藥血清可明顯促進(jìn)肌源性干細(xì)胞的體外增殖和向類雪旺細(xì)胞分化,提示鹿茸多肽具有開發(fā)為促進(jìn)神經(jīng)損傷修復(fù)新藥的潛能。3.鹿茸多肽和肌源性干細(xì)胞聯(lián)合應(yīng)用可促進(jìn)周圍神經(jīng)缺損的修復(fù),兩者可應(yīng)用于周圍神經(jīng)組織工程學(xué)研究。目的:分析遼西地區(qū)周圍神經(jīng)損傷患者流行病學(xué)特點,為預(yù)防周圍神經(jīng)損傷提供參考。材料與方法:采取統(tǒng)一調(diào)查表,通過對2014年1月至2014年12月期間遼西地區(qū)五市七家三甲醫(yī)院收治周圍神經(jīng)損傷住院患者1410例進(jìn)行回顧性分析,將患者分成5個年齡組:童年(0-6周歲);少年(7-17周歲);青年(18-40周歲);中年(41-65周歲);老年(大于65周歲),統(tǒng)計受傷時間、年齡、文化程度、職業(yè)、受傷地點、致傷原因、受傷部位、合并損傷、是否手術(shù)及并發(fā)癥。對數(shù)據(jù)進(jìn)行統(tǒng)計學(xué)分析。結(jié)果:1年時間內(nèi),共收治周圍神經(jīng)損傷住院患者1410例,男性患者1105名,女性患者305名,男女比列為3.62:1;年齡組中18歲至40歲青壯年為高發(fā)病組,為51.06%;文化程度中以小學(xué)文化和中學(xué)文化發(fā)病率高,二者無明顯區(qū)別;致傷因素中工作受傷48.94%,交通受傷28.72%,其中不同性別原因不同,女性患者主要以交通致傷為主,而男性患者主要以工作受傷為主。結(jié)論:處于16歲至40歲,且文化程度不高的青壯年為周圍神經(jīng)損傷高危人群;工作中切割傷為主要致傷因素;加強(qiáng)意識,嚴(yán)格生產(chǎn)管理是預(yù)防周圍神經(jīng)損傷的重點。
[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.
【學(xué)位授予單位】：遼寧中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R745

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 曾祥一;王偉;孫亮;張力;曾令達(dá);;睫狀神經(jīng)營養(yǎng)因子和丹參注射液體外誘導(dǎo)肌源性干細(xì)胞向神經(jīng)元樣細(xì)胞的分化[J];中國組織工程研究與臨床康復(fù);2009年27期

，

本文編號：2238371