【摘要】：隨著嗓音外科學(xué)的不斷發(fā)展，嗓音外科醫(yī)生可在顯微鏡下通過(guò)激光等方法切除聲帶不良病變，但術(shù)中易造成聲帶固有層損傷，導(dǎo)致固有層細(xì)胞外基質(zhì)成分的含量及分布發(fā)生改變從而形成瘢痕。如何減少瘢痕的形成，一直是困擾研究人員的重要問(wèn)題。既往有學(xué)者通過(guò)在動(dòng)物聲帶內(nèi)注射各種干細(xì)胞，如骨髓間充質(zhì)干細(xì)胞、脂肪基質(zhì)干細(xì)胞及胚胎干細(xì)胞等修復(fù)聲帶損傷，然而骨髓間充質(zhì)干細(xì)胞取材時(shí)患者較為痛苦，不宜反復(fù)取材，脂肪基質(zhì)干細(xì)胞雖易取材，但其組織來(lái)源與聲帶組織結(jié)構(gòu)差異大，胚胎干細(xì)胞的應(yīng)用仍存在倫理方面的問(wèn)題等，因此限制了這些細(xì)胞在臨床上的應(yīng)用。 近年來(lái)研究人員已證實(shí)在高等動(dòng)物體內(nèi)廣泛存在著間充質(zhì)干細(xì)胞，喉黏膜因其范圍廣，取材相對(duì)方便，且此部位細(xì)胞的組織來(lái)源與聲帶組織結(jié)構(gòu)最為接近，不存在倫理方面的問(wèn)題，因此我們?cè)O(shè)想喉黏膜中是否同樣存在間充質(zhì)干細(xì)胞。若能成功分離此種細(xì)胞，并將其用于喉部組織的缺損修復(fù)，將會(huì)為聲帶損傷的患者帶來(lái)新的希望。因此前期實(shí)驗(yàn)中劉陽(yáng)已成功從人喉黏膜中培養(yǎng)出具有快速增殖能力及多向分化潛能的間充質(zhì)干細(xì)胞，命名為喉黏膜間充質(zhì)干細(xì)胞(LM-MSCs)。本實(shí)驗(yàn)的研究目的是將LM-MSCs應(yīng)用于動(dòng)物實(shí)驗(yàn)，觀(guān)察LM-MSCs在受損聲帶內(nèi)的存活情況及轉(zhuǎn)歸，并在形態(tài)及組織學(xué)水平觀(guān)察其對(duì)聲帶損傷的修復(fù)作用。 1目的 探討犬喉黏膜中是否存在間充質(zhì)干細(xì)胞，對(duì)其進(jìn)行分離培養(yǎng)及鑒定，將其植入犬受損聲帶，觀(guān)察LM-MSCs在聲帶內(nèi)的存活及轉(zhuǎn)歸，并評(píng)估其對(duì)聲帶損傷的修復(fù)作用。 2方法 2.1犬喉黏膜間充質(zhì)干細(xì)胞的分離培養(yǎng)及鑒定 犬處死后取材，獲取正常會(huì)厭舌側(cè)黏膜，利用消化培養(yǎng)法獲得黏膜固有層細(xì)胞，利用成脂、成骨、成軟骨誘導(dǎo)液對(duì)其多向分化的能力進(jìn)行研究，通過(guò)MTT實(shí)驗(yàn)、平板克隆形成試驗(yàn)觀(guān)察LM-MSCs的生物學(xué)特性及通過(guò)流式細(xì)胞儀對(duì)LM-MSCs表面標(biāo)志物進(jìn)行檢測(cè)分析。 2.2犬聲帶激光損傷模型的建立 中華田園犬5只，其中4只在支撐喉鏡下挑起會(huì)厭顯露雙側(cè)聲帶，局部噴少量利多卡因，半導(dǎo)體激光雙側(cè)對(duì)稱(chēng)性切除聲帶膜部中后1/3處，深度達(dá)甲杓肌，雙側(cè)對(duì)稱(chēng)，1只犬未損傷作為對(duì)照組。術(shù)后觀(guān)察雙側(cè)聲帶的大體愈合情況，如充血、水腫、損傷表面的不規(guī)則性，有無(wú)萎縮及瘢痕形成。分別于術(shù)后4d、2w、4w及8w聲帶取材，左側(cè)聲帶行石蠟切片(厚度約5μm)，HE染色觀(guān)察聲帶損傷處炎性細(xì)胞侵潤(rùn)及纖維組織增生情況， Massontrichrome染色、Elastin VG染色、Alcian blue染色分別觀(guān)察膠原纖維、彈力纖維、透明質(zhì)酸的分布與含量變化，右側(cè)聲帶行冰凍切片(厚度約5μm)并行纖維連接蛋白(Fibronectin)免疫組化染色觀(guān)察fibronectin的含量變化。 2.3喉黏膜間充質(zhì)干細(xì)胞在聲帶急性損傷修復(fù)中作用的實(shí)驗(yàn)研究 2.3.1喉黏膜間充質(zhì)干細(xì)胞對(duì)聲帶大體愈合情況的影響 取第3代LM-MSCs，Dil熒光染料標(biāo)記后備用，中華田園犬10只，支撐喉鏡下半導(dǎo)體激光損傷雙側(cè)聲帶膜部中后1/3，深度達(dá)甲杓肌，損傷后即行聲帶注射術(shù)，左側(cè)聲帶注射0.2ml LM-MSCs與膠原的混合物(約含2×106個(gè)細(xì)胞)作為干細(xì)胞組，右側(cè)聲帶僅注射0.2ml膠原作為對(duì)照組。對(duì)10只犬隨機(jī)分組，1-6號(hào)犬分別于術(shù)后2w、4w及8w在支撐喉鏡下觀(guān)察受損聲帶愈合的情況，如充血、水腫、損傷表面的不規(guī)則性，有無(wú)萎縮及瘢痕形成。2.3.2喉黏膜間充質(zhì)干細(xì)胞對(duì)聲帶固有層結(jié)構(gòu)的影響 將犬處死取材，行冰凍切片后，并行免疫組化染色觀(guān)察fibronectin的含量變化，石蠟切片HE染色觀(guān)察聲帶固有層炎性細(xì)胞侵潤(rùn)及纖維組織增生情況，并行Masson trichrome染色、EVG染色、Alcian blue染色觀(guān)察聲帶固有層中膠原纖維、彈力纖維及透明質(zhì)酸(HA)的排列及含量改變。2.3.3喉黏膜間充質(zhì)干細(xì)胞在聲帶內(nèi)存活的情況 1-6號(hào)犬聲帶行冰凍切片后，用Hochest熒光染料襯染聲帶組織細(xì)胞核15min，PBS沖洗切片，熒光顯微鏡下觀(guān)察植入的LM-MSCs在聲帶內(nèi)存活及分布的情況，觀(guān)察植入的LM-MSCs隨時(shí)間的推移存活數(shù)量的變化。2.3.4喉黏膜間充質(zhì)干細(xì)胞在聲帶內(nèi)的轉(zhuǎn)歸 術(shù)后4w及8w分別處死2只犬(7-10號(hào))觀(guān)察干細(xì)胞在聲帶內(nèi)的轉(zhuǎn)歸，冰凍切片后行免疫熒光Vimentin染色及Smooth musle actin染色，二抗帶FITC熒光，熒光顯微鏡下觀(guān)察植入的LM-MSCs在聲帶組織中轉(zhuǎn)化為成纖維細(xì)胞及肌成纖維細(xì)胞的能力，植入的LM-MSCs本身呈紅色熒光，若在藍(lán)光的激發(fā)下同時(shí)可呈綠色熒光，證明其可轉(zhuǎn)化為成纖維細(xì)胞或肌成纖維細(xì)胞。 3結(jié)果 3.1喉黏膜間充質(zhì)干細(xì)胞的分離培養(yǎng)及鑒定 原代培養(yǎng)的LM-MSCs在3d左右貼壁生長(zhǎng)，起初成短梭狀，隨后細(xì)胞伸展?jié)u成長(zhǎng)梭狀，與成纖維細(xì)胞形態(tài)相似，胞核大。在體外分別經(jīng)成脂、成骨和成軟骨誘導(dǎo)分化后，油紅O染色陽(yáng)性，茜素紅染色陽(yáng)性，成軟骨微團(tuán)II型膠原免疫組化染色DAB顯色可觀(guān)察到陽(yáng)性信號(hào)。培養(yǎng)的LM-MSCs增殖能力強(qiáng)，增殖過(guò)程中無(wú)停滯期，接種后第3d開(kāi)始處于對(duì)數(shù)生長(zhǎng)期，第7d達(dá)到生長(zhǎng)峰值，第8d進(jìn)入生長(zhǎng)平臺(tái)期。平板克隆形成試驗(yàn)結(jié)果示LM-MSCs有較高的克隆形成率約19.7%。通過(guò)流式細(xì)胞儀對(duì)LM-MSCs進(jìn)行表面標(biāo)志物的分析， LM-MSCs高表達(dá)間充質(zhì)的表面分子標(biāo)志(CD29-77.6%、CD44-75.5%、CD90-97.3%、CD105-38.3%)，不表達(dá)造血系的表面分子標(biāo)志(CD34-1.8%、CD45-0.9%)。3.2犬聲帶激光損傷模型的建立 術(shù)后4d支撐喉鏡下雙側(cè)聲帶充血、水腫，肉芽組織形成，表面不規(guī)則，組織切片HE染色示上皮未被覆固有層，損傷處可見(jiàn)大量炎性細(xì)胞侵潤(rùn)，固有層與聲帶肌層界限不清，各種ECM成分不易檢出。術(shù)后2w喉鏡下見(jiàn)聲帶充血較前減輕，但仍有水腫，受損表面不規(guī)則，HE染色示新生上皮被覆受損部位，炎性細(xì)胞數(shù)量較前減少，大量纖維組織增生；ECM改變(根據(jù)切片IOD值檢測(cè))：fibronectin含量增加，膠原纖維增粗含量增加，排列紊亂，彈力纖維含量減少且變細(xì)，排列不齊，HA含量下降。術(shù)后4w喉鏡可見(jiàn)雙側(cè)聲帶充血水腫基本消失，受損處表面不規(guī)則，萎縮形成，HE染色示固有層無(wú)炎性細(xì)胞侵潤(rùn)，且被致密的纖維組織所替代；ECM改變：fibronectin含量持續(xù)增加，膠原含量增加，排列紊亂，彈力纖維含量略下降，且排列不齊，HA含量下降。術(shù)后8w喉鏡可見(jiàn)雙側(cè)聲帶無(wú)明顯充血水腫，損傷表面不規(guī)則，局部萎縮，瘢痕修復(fù)；ECM改變：fibronectin含量略上升，膠原纖維呈粗大條索狀，含量增加排且列紊亂，彈力纖維含量趨于穩(wěn)定，HA含量下降。 3.3喉黏膜間充質(zhì)干細(xì)胞在聲帶急性損傷修復(fù)中作用的實(shí)驗(yàn)研究 3.3.1喉黏膜間充質(zhì)干細(xì)胞對(duì)聲帶大體愈合情況的影響 術(shù)后2w可觀(guān)察到雙側(cè)聲帶水腫，稍充血，表面不規(guī)則，且有肉芽形成，干細(xì)胞組聲帶肉芽形成較少，且聲帶表面較對(duì)照組規(guī)則；術(shù)后4w時(shí)雙側(cè)聲帶充血、水腫基本消失，表面稍不規(guī)則，肉芽組織不明顯，萎縮形成，對(duì)照組聲帶表面不規(guī)則性及萎縮較干細(xì)胞組明顯；術(shù)后8w時(shí)雙側(cè)聲帶無(wú)充血、水腫，無(wú)新生肉芽組織形成，聲帶損傷局部可見(jiàn)萎縮、瘢痕形成，對(duì)照組聲帶瘢痕較干細(xì)胞組大，且萎縮明顯。3.3.2喉黏膜間充質(zhì)干細(xì)胞對(duì)聲帶固有層結(jié)構(gòu)的影響 犬處死后聲帶取材并行組織切片觀(guān)察，2w時(shí)干細(xì)胞組聲帶固有層內(nèi)炎性細(xì)胞侵潤(rùn)及纖維組織增生要小于對(duì)照組；雙側(cè)聲帶fibronectin含量持續(xù)上升，干細(xì)胞組含量略低于對(duì)照組；雙側(cè)聲帶膠原含量增加，干細(xì)胞組的膠原纖維含量稍低于對(duì)照組，且排列上較對(duì)照組規(guī)則；雙側(cè)聲帶彈力纖維含量均減少，排列紊亂，干細(xì)胞組含量高于對(duì)照組；HA含量呈下降趨勢(shì)，干細(xì)胞組含量略高于對(duì)照組。4w時(shí)雙側(cè)聲帶炎性細(xì)胞侵潤(rùn)基本消失，纖維組織增生，干細(xì)胞組增生小于對(duì)照組；雙側(cè)聲帶fibronectin上升，干細(xì)胞組含量低于對(duì)照組；膠原纖維呈條索狀，含量持續(xù)增加，干細(xì)胞組較對(duì)照組含量低，排列較規(guī)則；彈力纖維含量繼續(xù)下降，纖維直徑變細(xì)，干細(xì)胞組含量稍高于對(duì)照組；HA含量下降，干細(xì)胞組含量略高于對(duì)照組。8w時(shí)雙側(cè)聲帶觀(guān)察不到炎性細(xì)胞侵潤(rùn)，纖維組織增生明顯；雙側(cè)聲帶fibronectin上升，對(duì)照組含量高于干細(xì)胞組；雙側(cè)聲帶膠原呈粗大條索狀，含量增加，對(duì)照組較干細(xì)胞組排列紊亂；彈力纖維含量趨于穩(wěn)定，干細(xì)胞組彈力纖維直徑較對(duì)照組粗。HA含量持續(xù)降低，干細(xì)胞組含量高于對(duì)照組。 3.3.3喉黏膜間充質(zhì)干細(xì)胞在聲帶內(nèi)的存活情況 干細(xì)胞注射術(shù)后2w、4w及8w熒光顯微鏡下觀(guān)察到植入的LM-MSCs在聲帶固有層內(nèi)存活，2w時(shí)固有層內(nèi)有大量紅色熒光標(biāo)記的細(xì)胞，提示此時(shí)有較多數(shù)量的移植細(xì)胞存活，4w時(shí)植入的LM-MSCs存活數(shù)量較2w時(shí)明顯減少，8w時(shí)仍可觀(guān)察到存活的LM-MSCs，但數(shù)量較前明顯減少。 3.3.4喉黏膜間充質(zhì)干細(xì)胞在聲帶內(nèi)的轉(zhuǎn)歸 Vimentin免疫熒光染色，4w時(shí)熒光顯微鏡下一些呈紅色熒光的LM-MSCs在藍(lán)光的激發(fā)下同時(shí)可發(fā)出綠色熒光，提示植入的LM-MSCs可轉(zhuǎn)化為成纖維細(xì)胞，同樣的方法，8w時(shí)也可觀(guān)察到一些植入的LM-MSCs轉(zhuǎn)化為成纖維細(xì)胞。Smooth mucle actin免疫熒光染色，熒光顯微鏡下可見(jiàn)4w時(shí)植入的LM-MSCs在藍(lán)光的激發(fā)下同時(shí)可發(fā)出綠色熒光，提示植入的LM-MSCs可轉(zhuǎn)化為肌成纖維細(xì)胞，同樣的方法，8w時(shí)未能觀(guān)察到植入的LM-MSCs轉(zhuǎn)化為肌成纖維細(xì)胞。 4結(jié)論 4.1本實(shí)驗(yàn)利用消化培養(yǎng)法成功培養(yǎng)并鑒定了犬喉黏膜間充質(zhì)干細(xì)胞，證明了其具有向脂肪細(xì)胞、成骨細(xì)胞及軟骨細(xì)胞分化的能力。犬喉黏膜間充質(zhì)干細(xì)胞具有有較快的生長(zhǎng)增殖速度和較高的克隆形成能力，表達(dá)間充質(zhì)干細(xì)胞表面標(biāo)志物。 4.2支撐喉鏡下應(yīng)用半導(dǎo)體激光造成犬聲帶膜部損傷，術(shù)后喉鏡下可見(jiàn)2w時(shí)雙側(cè)聲帶充血水腫，，4w時(shí)出現(xiàn)萎縮，8w時(shí)瘢痕形成，組織學(xué)示固有層內(nèi)膠原含量持續(xù)上升，排列紊亂，提示大量瘢痕組織形成，彈力纖維含量下降示聲帶組織的彈性下降，HA含量降低示聲帶振動(dòng)能力及抑制膠原無(wú)序沉積的能力下降，fibronectin含量增加示組織纖維化程度加重，以上結(jié)果表明本實(shí)驗(yàn)成功的建立了犬聲帶損傷模型，為后續(xù)損傷修復(fù)實(shí)驗(yàn)提供了可靠的動(dòng)物模型。 4.3.1喉黏膜間充質(zhì)干細(xì)胞聲帶植入后，充血水腫較對(duì)照組輕。干細(xì)胞組聲帶表面較對(duì)照組規(guī)則，萎縮程度小，瘢痕形成少。 4.3.2組織切片示喉黏膜間充質(zhì)干細(xì)胞能降低固有層中膠原的沉積及無(wú)序排列，提高彈力纖維與HA含量以保證聲帶的有效振動(dòng)，且具有抑制fibronectin含量上升以減少固有層纖維化的功能，以上結(jié)果表明喉黏膜間充質(zhì)干細(xì)胞可通過(guò)調(diào)節(jié)細(xì)胞外基質(zhì)成分的合成，改變微環(huán)境修復(fù)聲帶損傷。 4.3.3熒光顯微鏡下植入的喉黏膜間充質(zhì)干細(xì)胞主要分布于固有層中，術(shù)后2w、4w及8w均可觀(guān)察到植入的LM-MSCs在聲帶內(nèi)存活，但隨著時(shí)間的推移，其存活數(shù)量逐漸減少。 4.3.4熒光顯微鏡下植入的喉黏膜間充質(zhì)干細(xì)胞在受損聲帶固有層內(nèi)轉(zhuǎn)化為成纖維細(xì)胞及肌成纖維細(xì)胞，提示LM-MSCs可通過(guò)直接轉(zhuǎn)化為固有層自身細(xì)胞參與聲帶的損傷修復(fù)。
[Abstract]:With the continuous development of voice surgery, voice surgeons can remove vocal cord defect by laser under microscope. However, it is easy to cause damage to the lamina propria during operation, resulting in changes in the content and distribution of extracellular matrix components in lamina propria, thus forming scars. How to reduce the formation of scars has been a problem for researchers. Previous scholars have repaired vocal cord injury by injecting various kinds of stem cells, such as bone marrow mesenchymal stem cells, adipose-derived mesenchymal stem cells and embryonic stem cells, into the vocal cords of animals. However, bone marrow mesenchymal stem cells (BMSCs) are more painful and should not be taken repeatedly. Because of the great difference of vocal cord structure and ethical problems in the application of embryonic stem cells, the clinical application of these cells is limited.
In recent years, researchers have confirmed the existence of mesenchymal stem cells in a wide range of animals, laryngeal mucosa because of its wide range, relatively easy to obtain materials, and this part of the cell tissue source and vocal cord tissue structure is the closest, there is no ethical issues, so we assume that laryngeal mucosa is also the existence of mesenchymal stem cells. Successful isolation of these cells and their use in repairing the defect of laryngeal tissue will bring new hope to the patients with vocal cord injury. Therefore, Liu Yang has successfully cultured mesenchymal stem cells with rapid proliferation and multi-differentiation potential from human laryngeal mucosa in previous experiments, named as laryngeal mucosal mesenchymal stem cells (LM-MSCs). The aim of this study was to apply LM-MSCs to animal experiments to observe the survival and prognosis of LM-MSCs in the damaged vocal cords, and to observe the repair effect of LM-MSCs on the damaged vocal cords at morphological and histological levels.
1 purposes
To investigate the existence of mesenchymal stem cells (MSCs) in the laryngeal mucosa of dogs, isolate, culture and identify them, implant them into the damaged vocal cords of dogs, observe the survival and prognosis of LM-MSCs in the vocal cords, and evaluate the repair effect of LM-MSCs on the damaged vocal cords.
2 method
Isolation, culture and identification of 2.1 dog laryngeal mucous mesenchymal stem cells
After the dogs were sacrificed, the lingual mucosa of normal epiglottis was obtained. The lamina propria cells of normal epiglottis were obtained by digestion and culture. The multidirectional differentiation ability of the cells was studied by lipogenesis, osteogenesis and cartilage induction fluid. The biological characteristics of LM-MSCs and the surface markers of LM-MSCs were observed by MTT test, plate cloning test and flow cytometry. Test and analysis.
Establishment of a laser injury model for 2.2 canine vocal cords
Four of the five Chinese idyllic dogs were exposed bilateral vocal cords by supporting laryngoscope, and a small amount of lidocaine was sprayed locally. Semiconductor laser bilateral symmetrical resection of the middle and posterior 1/3 part of the vocal cord membranes was performed in a depth of arytenoid muscle and bilateral symmetry. One of the dogs was used as control group. The general healing of bilateral vocal cords, such as congestion, edema and lesion, was observed after operation. Four, two, four and eight weeks after the operation, paraffin sections of the left vocal cords (about 5 microns thick) were performed. Inflammatory cell invasion and fibrous tissue hyperplasia were observed by HE staining, Massontrichrome staining, Elastin VG staining and Alcian blue staining respectively. Distribution and content changes of force fibers and hyaluronic acid were observed by frozen section of right vocal cord (about 5 microns thick) and immunohistochemical staining of fibronectin.
Experimental study on the effect of 2.3 laryngeal mucous mesenchymal stem cells on acute vocal cord injury
Effect of 2.3.1 laryngeal mucous mesenchymal stem cells on gross healing of vocal cords
The third generation of LM-MSCs were labeled with Dil fluorescent dye. Ten Chinese idyllic dogs were used as stem cells. Semiconductor laser assisted laryngoscopy was used to injure the middle and posterior 1/3 of the bilateral vocal fold membranes, and the depth reached to the arytenoid muscle. After injury, the vocal cord was injected with 0.2ml LM-MSCs and collagen mixture (containing about 2 *106 cells) into the left vocal fold. 0.2 ml collagen was used as the control group. Ten dogs were randomly divided into groups. The healing of the injured vocal cords was observed under the laryngoscope at 2w, 4W and 8W after operation, such as congestion, edema, irregularity of the injured surface, atrophy and scarring. 2.3.2 laryngeal mucosal mesenchymal stem cells affected the structure of the lamina propria of the vocal cords.
Immunohistochemical staining was used to observe the changes of fibronectin content, paraffin section HE staining was used to observe the invasion of inflammatory cells and fibrous tissue proliferation in the lamina propria, Masson trichrome staining, EVG staining and Alcian blue staining were used to observe the collagen fibers, elastic fibers and hyaluronan in the lamina propria. The arrangement and content of acid (HA) alter.2.3.3 laryngeal mucous mesenchymal stem cells in the vocal fold memory.
After frozen section of the vocal cords of dogs 1-6, the nuclei of vocal cords were stained with Hochest fluorescent dye for 15 minutes, and then washed with PBS. The survival and distribution of the implanted LM-MSCs in the vocal cords were observed under fluorescence microscope. The survival number of the implanted LM-MSCs was observed with time. 2.3.4 laryngeal mucosal mesenchymal stem cells were transferred into the vocal cords.
Two dogs (No. 7-10) were sacrificed 4 and 8 weeks after operation to observe the prognosis of stem cells in vocal cords. Immunofluorescence Vimentin staining and Smooth musle actin staining were performed on frozen sections. FITC fluorescence was used as the second antibody. The ability of implanted LM-MSCs to transform into fibroblasts and myofibroblasts in vocal cords was observed under fluorescence microscope. It is red fluorescent and green fluorescent when stimulated by blue light, which proves that it can be transformed into fibroblasts or myofibroblasts.
3 Results
Isolation, culture and identification of 3.1 laryngeal mucosa mesenchymal stem cells
The primary cultured LM-MSCs adhered to the wall and grew at about 3 days, initially in a short spindle shape, then gradually expanded into spindle shape, similar to fibroblasts with large nuclei. After adipogenesis, osteogenesis and chondrogenesis induction in vitro, oil red O staining was positive, alizarin red staining was positive, and type II collagen immunohistochemical staining of cartilage microclusters was feasible with DAB staining. LM-MSCs had strong proliferative ability and no stagnation in the process of proliferation. They began to grow logarithmically on the 3rd day after inoculation, reached the peak on the 7th day and entered the growth Plateau on the 8th day. The results of plate cloning test showed that LM-MSCs had a high clone formation rate of 19.7%. LM-MSCs were labeled by flow cytometry. The surface molecular markers of LM-MSCs were overexpressed (CD29-77.6%, CD44-75.5%, CD90-97.3%, CD105-38.3%) and did not express hematopoietic surface molecular markers (CD34-1.8%, CD45-0.9%).
Four days after the operation, bilateral vocal cord congestion, edema, granulation tissue formation, irregular surface, tissue sections HE staining showed that the epithelium was not covered with lamina propria, a large number of inflammatory cells invasion, lamina propria and vocal cord muscular layer boundaries were not clear, various ECM components were difficult to detect. HE staining showed that the number of inflammatory cells decreased and a large number of fibrous tissues proliferated. ECM changes (according to IOD value of slices) showed that fibronectin content increased, collagen fiber thickening content increased, arranged disorderly, elastic fiber content decreased and thinned, arranged irregularly, and HA content decreased. The laryngoscopy showed that bilateral vocal cord congestion and edema disappeared, the damaged surface was irregular, atrophy formation, HE staining showed that lamina propria had no inflammatory cell invasion, and was replaced by dense fibrous tissue; ECM changes: fibronectin content continued to increase, collagen content increased, arranged disorderly, elastic fiber content decreased slightly, and arranged irregularly, under the HA content. Eight weeks after operation, there was no obvious congestion and edema in bilateral vocal cords, irregular surface, local atrophy, and scar repair. ECM changes: fibronectin content increased slightly, collagen fibers showed a large strip, content increased in disorder, elastic fibers content tended to stabilize, HA content decreased.
Experimental study on the effect of 3.3 laryngeal mucous mesenchymal stem cells on acute vocal cord injury
Effect of 3.3.1 laryngeal mucous mesenchymal stem cells on gross healing of vocal cords
Two weeks after operation, bilateral vocal cord edema, slightly hyperemia, irregular surface, and granulation formation were observed. In stem cell group, vocal cord granulation formation was less, and the surface of vocal cord was more regular than that in control group; 4 weeks after operation, bilateral vocal cord congestion, edema disappeared, slightly irregular surface, granulation tissue was not obvious, atrophy formation, and vocal cord surface irregularity and atrophy in control group. 8 weeks after operation, bilateral vocal cords had no congestion, edema, no new granulation tissue formation, local vocal cord injury can be seen atrophy, scar formation, control group vocal cord scar larger than stem cell group, and atrophy was obvious. 3.3.2 laryngeal mucosal mesenchymal stem cells on the structure of the lamina propria of vocal cords
The inflammatory cell invasion and fibrous tissue hyperplasia in the lamina propria of the vocal cords in the stem cell group were less than those in the control group at 2 weeks after the death of the dogs. The fibronectin content in the bilateral vocal cords continued to increase, while the collagen content in the stem cell group was slightly lower than that in the control group. The content of HA in the stem cell group was higher than that in the control group, and the content of HA in the stem cell group was slightly higher than that in the control group. The content of fibronectin in the lateral vocal cord increased, while that in the stem cell group was lower than that in the control group; collagen fibers were striped, and the content of collagen fibers in the stem cell group was lower than that in the control group, and the arrangement of collagen fibers was more regular; the content of elastic fibers continued to decrease, the diameter of fibers became thinner, the content of HA in the stem cell group was slightly higher than that in the control group; the content of At 8th week, inflammatory cells were not observed in bilateral vocal cords, and fibronectin in bilateral vocal cords was increased, and the content of collagen in bilateral vocal cords was higher than that in stem cell group. The crude.HA content of the irradiated group decreased, while the stem cell group content was higher than that of the control group.
Survival of 3.3.3 laryngeal mucosa mesenchymal stem cells in vocal cords
The survival of LM-MSCs in the lamina propria was observed under fluorescence microscope at 2, 4 and 8 weeks after stem cell injection. A large number of red fluorescent labeled cells were observed in the lamina propria at 2 weeks, suggesting that a large number of transplanted cells survived at this time. The survival of LM-MSCs implanted at 4 weeks was significantly less than that at 2 weeks, and the survival of LM-MSCs was still observed at 8 weeks. The number decreased significantly.
3.3.4 laryngeal mucosa mesenchymal stem cells in vocal cord
Vimentin immunofluorescence staining showed that some LM-MSCs with red fluorescence could emit green fluorescence under the fluorescence microscope at 4 W under the excitation of blue light, suggesting that implanted LM-MSCs could be transformed into fibroblasts. Similarly, some implanted LM-MSCs could be transformed into fibroblasts at 8 w. Smooth mucle actin immunofluorescence staining showed that some implanted LM-MSCs could be transformed into fibroblasts. Under fluorescence microscope, the implanted LM-MSCs could emit green fluorescence at the same time when stimulated by blue light, suggesting that the implanted LM-MSCs could be transformed into myofibroblasts. Similarly, the implanted LM-MSCs could not be transformed into myofibroblasts at the 8th week.
4 Conclusion
4.1 The canine laryngeal mucosal mesenchymal stem cells were successfully cultured and identified by digestion culture method, which proved that the canine laryngeal mucosal mesenchymal stem cells have the ability to differentiate into adipocytes, osteoblasts and chondrocytes. The canine laryngeal mucosal mesenchymal stem cells have a faster growth and proliferation rate and higher clonal formation ability, and express the surface markers of mesenchymal stem cells. Things.
4.2 Semiconductor laser was used to injure the membranes of the vocal cords in dogs under supportive laryngoscope. Bilateral vocal cords congestion and edema were observed under laryngoscope 2 weeks after operation and 4 weeks after operation.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R767.4

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