【摘要】：周?chē)窠?jīng)損傷在臨床中比較常見(jiàn),由于成熟的神經(jīng)元不能分裂增殖,因此,外周神經(jīng)損傷后的修復(fù)效果很不理想。隨著組織工程學(xué)的發(fā)展,人們用組織工程神經(jīng)代替自體神經(jīng)修復(fù)外周神經(jīng)損傷并取得了較好效果。組織工程神經(jīng)的構(gòu)建主要包含種子細(xì)胞、支架材料和神經(jīng)營(yíng)養(yǎng)因子三個(gè)方面,其中理想的種子細(xì)胞是構(gòu)建人工神經(jīng)的前提和基礎(chǔ)。雪旺細(xì)胞(Schwann cells,SCs)在神經(jīng)再生中起重要作用,是修復(fù)神經(jīng)損傷理想的種子細(xì)胞。但是SCs體外培養(yǎng)困難,增殖速度慢,且培養(yǎng)過(guò)程中容易受到成纖維細(xì)胞污染,不能滿足神經(jīng)修復(fù)的要求。脂肪源性干細(xì)胞(Adipose stem cells,ADSCs)是具備多向分化潛能的干細(xì)胞,在特定環(huán)境下可以向SCs分化,同時(shí),ADSCs還具有來(lái)源廣泛、取材簡(jiǎn)便、并發(fā)癥少、增殖速度快、擴(kuò)增穩(wěn)定等優(yōu)點(diǎn),因此可以成為組織工程神經(jīng)理想的種子細(xì)胞來(lái)源。然而目前常用的誘導(dǎo)ADSCs向SCs分化的方法都存在一定的弊端。本研究應(yīng)用共培養(yǎng)原理,利用外周神經(jīng)分泌的神經(jīng)營(yíng)養(yǎng)因子來(lái)誘導(dǎo)ADSCs向SCs分化,以期簡(jiǎn)化操作程序,節(jié)約誘導(dǎo)成本,為解決臨床上修復(fù)外周神經(jīng)損傷過(guò)程中的種子細(xì)胞難題提供新思路,整個(gè)實(shí)驗(yàn)分為五個(gè)部分。1.大鼠ADSCs的體外分離培養(yǎng)和鑒定無(wú)菌條件下取出3周齡SD大鼠兩側(cè)腹股溝脂肪,采用Ⅰ型膠原酶消化法獲得單細(xì)胞懸液,并培養(yǎng)在含10%FBS的DMEM培養(yǎng)基中。通過(guò)骨向和脂向分化以及流式細(xì)胞儀檢測(cè)細(xì)胞表面Marker對(duì)所培養(yǎng)的細(xì)胞進(jìn)行鑒定。結(jié)果顯示成骨誘導(dǎo)后ADSCs形成典型的黑色鈣結(jié)節(jié),茜素紅染色呈現(xiàn)紅色;成脂誘導(dǎo)后逐漸出現(xiàn)脂滴,油紅O染色為紅色。流式檢測(cè)結(jié)果顯示:CD44和CD90呈陽(yáng)性表達(dá),CD31和CD45呈陰性。以上結(jié)果證實(shí),分離到的細(xì)胞為ADSCs。2.ADSCs體外特性研究本部分的目的是掌握ADSCs的體外分化特性,為選擇合適的傳代次數(shù)和適宜的接種密度提供理論基礎(chǔ)。將ADSCs培養(yǎng)至12代,然后誘導(dǎo)第3、6、9和12代的ADSCs向成骨和成脂分化,并通過(guò)茜素紅和油紅O染色對(duì)誘導(dǎo)結(jié)果進(jìn)行檢測(cè)。同時(shí)選取第3代ADSCs用兩種不同的密度(5×105/cm2和1×103/cm2)進(jìn)行接種,再用Western-blot檢測(cè)第3、6、9、12代以及兩種不同傳代密度的ADSCs的BMP-2的表達(dá),并檢測(cè)第1-12代ADSCs的堿性磷酸酶(Alkaline phosphatase,ALP)活性。結(jié)果證實(shí),第3、6、9、12代的ADSCs均可被誘導(dǎo)成骨細(xì)胞和脂肪細(xì)胞,但隨著傳代次數(shù)的增加,ADSCs成脂分化潛能逐漸降低,成骨能力未見(jiàn)明顯差異。第12代以及以1×103/cm2密度培養(yǎng)的ADSCs的BMP-2呈陽(yáng)性表達(dá);隨著傳代次數(shù)的增加,第1-12代的ADSCs ALP活性逐步增加,1×103/cm2密度培養(yǎng)的ADSCs的ALP活性較5×105/cm2接種的ALP活性顯著增加。ADSCs在體外連續(xù)傳代或者以較低密度接種時(shí)可導(dǎo)致衰老并自發(fā)分化成骨。因此ADSCs的體外研究應(yīng)選擇3-5代,并以5×105/cm2的密度進(jìn)行傳代較為適宜。3.神經(jīng)浸出液的制備及對(duì)PC12細(xì)胞神經(jīng)樣分化的影響無(wú)菌采集的大鼠坐骨神經(jīng)通過(guò)剪碎、浸泡、過(guò)濾制備出神經(jīng)浸出液,待用。為了初步驗(yàn)證本實(shí)驗(yàn)室制備的神經(jīng)浸出液具有促進(jìn)前體細(xì)胞向神經(jīng)樣細(xì)胞分化的潛能,本研究選擇PC12細(xì)胞作為前體細(xì)胞模型,將制備好的神經(jīng)浸出液代替PC12細(xì)胞的培養(yǎng)基,觀察PC12細(xì)胞軸突分化情況。對(duì)神經(jīng)浸出液培養(yǎng)第3、6、9 d的PC12細(xì)胞軸突長(zhǎng)度進(jìn)行測(cè)量統(tǒng)計(jì),并檢測(cè)神經(jīng)元標(biāo)志物β3-tubulin和MAP-2的表達(dá)。結(jié)果顯示,神經(jīng)浸出液培養(yǎng)的PC12細(xì)胞明顯長(zhǎng)出突起,第3、6、9 d的軸突平均長(zhǎng)度分別達(dá)到28.07±1.76μm、58.14±2.60μm、170.43±10.08μm;神經(jīng)元標(biāo)志物β3-tubulin和MAP-2蛋白呈陽(yáng)性表達(dá),初步證實(shí)神經(jīng)浸出液具有誘導(dǎo)前體細(xì)胞神經(jīng)樣分化的潛能。4.神經(jīng)浸出液誘導(dǎo)ADSCs向SCs分化取培養(yǎng)至第3代的ADSCs,用神經(jīng)浸出液代替正常培養(yǎng)基,培養(yǎng)5 d后用免疫熒光和Western-blot檢測(cè)SCs特異標(biāo)志蛋白S-100和膠質(zhì)原纖維酸性蛋白(glial fibrillary acidic protein,GFAP)的表達(dá),以證實(shí)誘導(dǎo)效果。結(jié)果表明神經(jīng)浸出液誘導(dǎo)后的ADSCs形態(tài)發(fā)生明顯改變,胞體由寬大扁平樣轉(zhuǎn)變?yōu)閮蓸O或多極,部分胞體變小,呈梭形,出現(xiàn)2~3個(gè)長(zhǎng)突起,類(lèi)似成熟的SCs。免疫熒光和Western-blot檢測(cè)結(jié)果均顯示SCs標(biāo)志物S-100和GFAP呈陽(yáng)性表達(dá),表明神經(jīng)浸出液能夠誘導(dǎo)ADSCs向SCs分化。5.神經(jīng)浸出液誘導(dǎo)的ADSCs對(duì)大鼠坐骨神經(jīng)缺損的修復(fù)作用將ADSCs與膠原凝膠復(fù)合并注入支架材料中制備成組織工程神經(jīng)移植物作為A組,復(fù)合誘導(dǎo)后的ADSCs制備成組織工程神經(jīng)移植物為B組,植入大鼠1cm坐骨神經(jīng)損傷處,并用未復(fù)合細(xì)胞的支架材料組(C組)和自體神經(jīng)移植組(D組)作為對(duì)照。修復(fù)大鼠神經(jīng)損傷,2個(gè)月后通過(guò)電鏡觀察、電生理以及圖像分析等方法進(jìn)行神經(jīng)再生評(píng)價(jià)。各組結(jié)果均顯示D組和B組對(duì)大鼠坐骨神經(jīng)損傷的修復(fù)效果最好,B組與D組間無(wú)顯著差異,都顯著高于A組,C組最差。表明誘導(dǎo)后的ADSCs對(duì)神經(jīng)損傷的修復(fù)效果要優(yōu)于未經(jīng)誘導(dǎo)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.
【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S852.3

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本文編號(hào)：2482695