【摘要】： 迄今為止,周圍神經(jīng)缺損的修復(fù)仍然是臨床外科領(lǐng)域尚待解決的疑難問題之一。如果兩斷端距離過遠則影響再生軸突的生長。臨床上常用神經(jīng)移植進行修補,雖然傳統(tǒng)的自體神經(jīng)移植方法效果最好,但因為材料來源的限制而制約了它的應(yīng)用范圍;而同種異體神經(jīng)移植又由于免疫排斥、移植成功率低等原因而難以推廣。20世紀(jì)以來,神經(jīng)組織工程研究的發(fā)展,尤其是由支架材料和細胞外基質(zhì)、種子細胞以及誘導(dǎo)和促進生長的因子等幾個部分組成的“組織工程神經(jīng)”的構(gòu)建為人們尋找神經(jīng)代用品修復(fù)周圍神經(jīng)缺損帶來了新的希望。 Schwann細胞是神經(jīng)組織工程中最重要、也是最常使用的種子細胞。但由于自體Schwann細胞來源有限且不容易增殖;而異體的Schwann細胞又會引起免疫排斥反應(yīng),因此將Schwann細胞化的“組織工程神經(jīng)”應(yīng)用于臨床的研究仍遇到難以逾越的障礙。骨髓基質(zhì)細胞(bone marrow stromal cells MSCs)因取材方便,來源廣泛,在一定培養(yǎng)條件下可迅速大量擴增,具有自我更新、活躍增殖和多向分化潛能,且自體移植克服了倫理和免疫排斥等問題,作為一種理想的種子細胞脫穎而出。 近年來一些在體外的研究證明MSCs在給予合適的誘導(dǎo)條件下,可以表達神經(jīng)元的標(biāo)記。這些研究表明MSCs在誘導(dǎo)分化后形態(tài)上發(fā)生了類似神經(jīng)元的形態(tài)變化并且可以表達神經(jīng)細胞的特異性蛋白,所以這些研究者認為是MSCs向神經(jīng)細胞發(fā)生了轉(zhuǎn)分化。但是也有持不同觀點的報道,有研究者認為MSCs在體內(nèi)的分化是與宿主的神經(jīng)系細胞發(fā)生融合的過程。有的研究認為MSCs并沒有真正的分化,而細胞形態(tài)的改變僅是由于誘導(dǎo)劑的化學(xué)性刺激的作用引起細胞的收縮和細胞骨架的塌陷。因此,目前關(guān)于MSCs是否有潛能分化成神經(jīng)系的細胞是當(dāng)今干細胞研究領(lǐng)域中的一個熱點問題。 我們以前的研究證明了大鼠的MSCs移植到坐骨神經(jīng)受損的部位后,在體內(nèi)有一部分能夠分化為Schwann細胞樣細胞并且表達S100蛋白,MSC移植到體內(nèi)是有利于神經(jīng)的再生。為了進一步分析MSC在向Schwann細胞分化的過程中Schwann細胞標(biāo)記蛋白S100蛋白是否真正發(fā)生了表達變化,我們的實驗探討大鼠骨髓基質(zhì)細胞在體外誘導(dǎo)的過程中,MSCs的形態(tài)變化以及表達Schwann細胞的標(biāo)記蛋白S100B蛋白的變化。 我們從成年SD大鼠的股骨和脛骨中分離培養(yǎng)MSCs。采用MTT法檢測細胞的活力,FCM檢測細胞周期和CD分子等方法研究MSCs的特性。結(jié)果證明MSCs在體外易擴增,傳1—8代以內(nèi)的細胞增殖能力無明顯變化。未經(jīng)誘導(dǎo)的MSCs大部分處于G_0/G_1期,G_0/G_1期的細胞所占百分比平均高于80%;骨髓基質(zhì)細胞CD29(+),CD11b(-),CD90(+)。 本實驗應(yīng)用復(fù)合誘導(dǎo)因子BME,RA,FSK,bFGF,PDGF,HRG體外連續(xù)誘導(dǎo)MSCs向Schwann細胞樣細胞分化。誘導(dǎo)分化后采用免疫熒光細胞化學(xué)染色,流式細胞儀,Western Blot和逆轉(zhuǎn)錄PCR方法分別檢測檢測Schwann細胞的標(biāo)記蛋白S100蛋白及其mRNA的表達。結(jié)果發(fā)現(xiàn)誘導(dǎo)分化后的MSCs形態(tài)上發(fā)生了類似Schwann細胞樣細胞的變化。MSCs在誘導(dǎo)過程中S100的mRNA有上升的趨勢;通過免疫熒光方法觀察到誘導(dǎo)后MSCs表達S100蛋白增強;我們又通過Western Blot和流式細胞儀進一步定量檢測到MSCs表達S100蛋白增高。 又因為大多數(shù)的研究觀察了細胞形態(tài)以及幾種神經(jīng)相關(guān)蛋白的免疫反應(yīng),也有報道用基因組學(xué)方法分析MSCs誘導(dǎo)過程中基因譜的表達變化。目前尚未見報道用蛋白質(zhì)組學(xué)方法分析MSCs在誘導(dǎo)過程中蛋白譜的表達變化。我們進一步利用蛋白質(zhì)組學(xué)技術(shù)分析MSCs向Schwann細胞樣細胞誘導(dǎo)分化過程中蛋白譜的表達變化。我們采用2-DE技術(shù)分離未誘導(dǎo)和誘導(dǎo)分化過程中MSCs總蛋白,應(yīng)用基質(zhì)輔助激光解吸電離飛行時間質(zhì)譜得到相應(yīng)的肽質(zhì)量指紋圖譜,搜索數(shù)據(jù)庫分析差異蛋白質(zhì)點。我們所得到的MSCs蛋白譜有792±23個蛋白點,通過PDQuest軟件分析未誘導(dǎo)MSCs蛋白譜和誘導(dǎo)分化過程中MSCs蛋白譜,初步分析發(fā)現(xiàn)有74個蛋白質(zhì)的表達發(fā)生了明顯的變化(p＜0.05),其中43個蛋白表達上調(diào),31個蛋白表達下調(diào)。我們通過質(zhì)譜分析并進行網(wǎng)上數(shù)據(jù)庫搜索匹配,初步分析發(fā)現(xiàn)這些蛋白主要包括骨架和結(jié)構(gòu)蛋白(tubulin alpha,vimentin,brain-specific alpha actinin 1 isoform等)、生長因子類的蛋白(ciliary neurotropic factor,brain-derived neurotrophic factor等)、代謝相關(guān)蛋白及酶類(UDP-glucose dehydrogenase,eph and elk-related kinase等)、伴侶蛋白(heat shockprotein等)、受體類蛋白(Laminin receptor 1,Ly49 stimulatory receptor 3等)、細胞周期蛋白(p27,RAS p21 protein activator 1等)、鈣結(jié)合蛋白(nucleobindin 1,non musclecaldesmon等)以及其它蛋白等。 總之,我們的實驗結(jié)果表明MSCs在誘導(dǎo)分化后形態(tài)上發(fā)生了類似Schwann細胞樣細胞的變化。未經(jīng)誘導(dǎo)分化的MSCs表達少量S100蛋白,MSCs在這種體外條件誘導(dǎo)分化的過程中表達S100蛋白有明顯的增加,而且在誘導(dǎo)分化過程中S100mRNA的表達有上升的趨勢。 MSCs體外條件誘導(dǎo)分化過程中有較多蛋白表達發(fā)生變化;其中與神經(jīng)細胞和神經(jīng)膠質(zhì)細胞相關(guān)蛋白:BDNF,CNTF,ILGF,pleiotrophin,FGF,GFAP,synaptophysin等在MSCs體外條件誘導(dǎo)分化過程中表達明顯上調(diào);本研究從蛋白質(zhì)水平為MSCs體外向Schwann細胞樣細胞條件誘導(dǎo)提供了新的研究資料。
[Abstract]:So far, the repair of peripheral nerve defects remains one of the most difficult problems to be solved in the field of clinical surgery. If the distance between the two ends is too far, it affects the growth of the regenerative axon. It is difficult to promote the development of neural tissue engineering research since the.20 century, especially the structure of "tissue engineering nerve" composed of scaffold materials and extracellular matrix, seed cells, and inducing and promoting growth factors. It provides new hope for people to search for nerve substitutes to repair peripheral nerve defects.
Schwann cells are the most important and most often used seed cells in the neural tissue engineering. But because the source of autologous Schwann cells is limited and it is not easy to proliferate, the allogenic Schwann cells will cause immune rejection. Therefore, the application of the "tissue engineering nerve" to the clinical study of the Schwann cell is still difficult to overcome. Bone marrow stromal cells MSCs (bone marrow stromal cells) has a wide range of sources, which can be expanded rapidly in a certain culture condition, with self renewal, active proliferation and multidirectional differentiation potential, and autologous transplantation overcomes the problems of ethical and immune rejection. As an ideal seed cell, it stands out.
In recent years, some in vitro studies have shown that MSCs can express the markers of neurons under the appropriate induction conditions. These studies have shown that MSCs has morphologically similar morphologic changes in neurons after induction of differentiation and can express specific proteins in neural cells, so these researchers think that MSCs sends to nerve cells. But there are different points of view, but there are different points of view. Some researchers believe that the differentiation of MSCs in the body is the process of fusion with the host's nerve cells. Some studies think that MSCs does not really differentiate, and the change of cell morphology is only due to the effect of inducer chemical stimulation to induce cell contraction and cell bone. It is a hot topic in the field of stem cell research that MSCs has potential to differentiate into neural lineage.
Our previous studies have shown that after MSCs transplantation in rats to the injured part of the sciatic nerve, part of the body can differentiate into Schwann cell like cells and express the S100 protein. The transplantation of MSC to the body is beneficial to the regeneration of the nerve. In order to further analyze the Schwann cell marker protein S10 in the process of MSC to Schwann fine cell differentiation. The change of expression of 0 protein is true. In our experiment, the morphologic changes of MSCs and the expression of S100B protein in Schwann cells during the induction of rat bone marrow stromal cells were investigated.
We isolated and cultured from the femur and tibia of adult SD rats and cultured MSCs. to detect cell viability by MTT method. FCM detected cell cycle and CD molecular methods to study the properties of MSCs. The results showed that MSCs was easily amplified in vitro, and the cell proliferation ability within 1 to 8 generations was not significantly changed. The uninduced MSCs was mostly in G_0/G_1 phase, G_0/G_1. The percentage of cells in the period was higher than 80%, bone marrow stromal cells CD29 (+), CD11b (-), CD90 (+).
This experiment used compound inducer BME, RA, FSK, bFGF, PDGF, HRG to induce MSCs to differentiate into Schwann cell like cells in vitro. Immunofluorescent cytochemical staining, flow cytometry, Western Blot and reverse transcription PCR were used to detect the expression of marker protein and expression of Schwann fine cell after induction of differentiation. The morphology of MSCs after induction of differentiation was similar to that of Schwann cell like cells. The mRNA of S100 increased in the induction process, and the MSCs expression of S100 protein was enhanced by immunofluorescence. We also quantified the MSCs expression S100 protein by Western Blot and flow cytometry.
Because most studies have observed the cell morphology and the immune responses of several nerve related proteins, it is also reported that the genomics method is used to analyze the changes in the expression of gene spectrum during the induction of MSCs. There is no report of proteomic analysis of the changes in the expression of egg white spectrum during the induction of MSCs. Analysis of the protein expression changes during the induction of differentiation of MSCs to Schwann cell like cells by white matter technology. We used 2-DE technology to separate MSCs total protein in the process of uninduced and induced differentiation. The corresponding peptide mass fingerprints were obtained by using matrix assisted laser desorption ionization time of flight mass spectrometry, and the differential protein was searched by database to analyze the differential protein. The MSCs protein spectrum has 792 + 23 protein points, and the PDQuest software is used to analyze the uninduced MSCs protein spectrum and the MSCs protein spectrum during the induction of differentiation. The preliminary analysis shows that the expression of 74 proteins has changed significantly (P < 0.05), of which 43 protein tables are up and 31 proteins are down regulated. Analysis and matching of online database search, preliminary analysis found that these proteins mainly include skeleton and structural proteins (tubulin alpha, vimentin, brain-specific alpha actinin 1 isoform, etc.), the protein of growth factor class (ciliary neurotropic factor, brain-derived neurotrophic), metabolic related proteins and enzymes Ose dehydrogenase, Eph and Elk-related kinase etc., chaperone protein (heat shockprotein, etc.), receptor class proteins (Laminin receptor 1, Ly49 stimulatory), cyclin protein (1, etc.), calcium binding protein (1, etc.), and other proteins.
In conclusion, our experimental results showed that the morphology of MSCs was similar to that of Schwann cell like cells after the induction of differentiation. The expression of S100 protein was obviously increased during the induction of differentiation without the MSCs expression of a small amount of S100 protein in the undifferentiated MSCs, and the expression of S100mRNA in the induced differentiation process was higher. The trend of rising.
The expression of more protein in the process of MSCs induced differentiation in vitro was changed, and the expression of BDNF, CNTF, ILGF, pleiotrophin, FGF, GFAP, synaptophysin in the process of differentiation in vitro was obviously up-regulated in the process of induction and differentiation of MSCs in vitro; this study was from protein level to Schwann cell like in vitro. Cell condition induction provides new research data.
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2006
【分類號】：R329

【共引文獻】

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7 彭錫嘉,劉少章,葉劍;少突膠質(zhì)細胞與視神經(jīng)再生[J];創(chuàng)傷外科雜志;2002年03期

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8 李宜炯;體外雪旺細胞與血管內(nèi)皮細胞共培養(yǎng)的實驗研究[D];河北醫(yī)科大學(xué);2011年

9 李家華;不同途徑給予外源性神經(jīng)生長因子NGF對骨折愈合影響的實驗研究[D];安徽醫(yī)科大學(xué);2011年

10 楊會云;甲狀腺素對成年期甲狀腺功能減退大鼠前額葉SNARE和syt-1表達的影響[D];安徽醫(yī)科大學(xué);2011年

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