喉黏膜间充质干细胞在声带急性损伤修复中作用的实验研究

发布时间：2018-09-14 18:06

【摘要】：随着嗓音外科学的不断发展，嗓音外科医生可在显微镜下通过激光等方法切除声带不良病变，但术中易造成声带固有层损伤，导致固有层细胞外基质成分的含量及分布发生改变从而形成瘢痕。如何减少瘢痕的形成，一直是困扰研究人员的重要问题。既往有学者通过在动物声带内注射各种干细胞，如骨髓间充质干细胞、脂肪基质干细胞及胚胎干细胞等修复声带损伤，然而骨髓间充质干细胞取材时患者较为痛苦，不宜反复取材，脂肪基质干细胞虽易取材，但其组织来源与声带组织结构差异大，胚胎干细胞的应用仍存在伦理方面的问题等，因此限制了这些细胞在临床上的应用。近年来研究人员已证实在高等动物体内广泛存在着间充质干细胞，喉黏膜因其范围广，取材相对方便，且此部位细胞的组织来源与声带组织结构最为接近，不存在伦理方面的问题，因此我们设想喉黏膜中是否同样存在间充质干细胞。若能成功分离此种细胞，并将其用于喉部组织的缺损修复，将会为声带损伤的患者带来新的希望。因此前期实验中刘阳已成功从人喉黏膜中培养出具有快速增殖能力及多向分化潜能的间充质干细胞，命名为喉黏膜间充质干细胞(LM-MSCs)。本实验的研究目的是将LM-MSCs应用于动物实验，观察LM-MSCs在受损声带内的存活情况及转归，并在形态及组织学水平观察其对声带损伤的修复作用。 1目的探讨犬喉黏膜中是否存在间充质干细胞，对其进行分离培养及鉴定，将其植入犬受损声带，观察LM-MSCs在声带内的存活及转归，并评估其对声带损伤的修复作用。 2方法 2.1犬喉黏膜间充质干细胞的分离培养及鉴定犬处死后取材，获取正常会厌舌侧黏膜，利用消化培养法获得黏膜固有层细胞，利用成脂、成骨、成软骨诱导液对其多向分化的能力进行研究，通过MTT实验、平板克隆形成试验观察LM-MSCs的生物学特性及通过流式细胞仪对LM-MSCs表面标志物进行检测分析。 2.2犬声带激光损伤模型的建立中华田园犬5只，其中4只在支撑喉镜下挑起会厌显露双侧声带，局部喷少量利多卡因，半导体激光双侧对称性切除声带膜部中后1/3处，深度达甲杓肌，双侧对称，1只犬未损伤作为对照组。术后观察双侧声带的大体愈合情况，如充血、水肿、损伤表面的不规则性，有无萎缩及瘢痕形成。分别于术后4d、2w、4w及8w声带取材，左侧声带行石蜡切片(厚度约5μm)，HE染色观察声带损伤处炎性细胞侵润及纤维组织增生情况， Massontrichrome染色、Elastin VG染色、Alcian blue染色分别观察胶原纤维、弹力纤维、透明质酸的分布与含量变化，右侧声带行冰冻切片(厚度约5μm)并行纤维连接蛋白(Fibronectin)免疫组化染色观察fibronectin的含量变化。 2.3喉黏膜间充质干细胞在声带急性损伤修复中作用的实验研究 2.3.1喉黏膜间充质干细胞对声带大体愈合情况的影响取第3代LM-MSCs，Dil荧光染料标记后备用，中华田园犬10只，支撑喉镜下半导体激光损伤双侧声带膜部中后1/3，深度达甲杓肌，损伤后即行声带注射术，左侧声带注射0.2ml LM-MSCs与胶原的混合物(约含2×106个细胞)作为干细胞组，右侧声带仅注射0.2ml胶原作为对照组。对10只犬随机分组，1-6号犬分别于术后2w、4w及8w在支撑喉镜下观察受损声带愈合的情况，如充血、水肿、损伤表面的不规则性，有无萎缩及瘢痕形成。2.3.2喉黏膜间充质干细胞对声带固有层结构的影响将犬处死取材，行冰冻切片后，并行免疫组化染色观察fibronectin的含量变化，石蜡切片HE染色观察声带固有层炎性细胞侵润及纤维组织增生情况，并行Masson trichrome染色、EVG染色、Alcian blue染色观察声带固有层中胶原纤维、弹力纤维及透明质酸(HA)的排列及含量改变。2.3.3喉黏膜间充质干细胞在声带内存活的情况 1-6号犬声带行冰冻切片后，用Hochest荧光染料衬染声带组织细胞核15min，PBS冲洗切片，荧光显微镜下观察植入的LM-MSCs在声带内存活及分布的情况，观察植入的LM-MSCs随时间的推移存活数量的变化。2.3.4喉黏膜间充质干细胞在声带内的转归术后4w及8w分别处死2只犬(7-10号)观察干细胞在声带内的转归，冰冻切片后行免疫荧光Vimentin染色及Smooth musle actin染色，二抗带FITC荧光，荧光显微镜下观察植入的LM-MSCs在声带组织中转化为成纤维细胞及肌成纤维细胞的能力，植入的LM-MSCs本身呈红色荧光，若在蓝光的激发下同时可呈绿色荧光，证明其可转化为成纤维细胞或肌成纤维细胞。 3结果 3.1喉黏膜间充质干细胞的分离培养及鉴定原代培养的LM-MSCs在3d左右贴壁生长，起初成短梭状，随后细胞伸展渐成长梭状，与成纤维细胞形态相似，胞核大。在体外分别经成脂、成骨和成软骨诱导分化后，油红O染色阳性，茜素红染色阳性，成软骨微团II型胶原免疫组化染色DAB显色可观察到阳性信号。培养的LM-MSCs增殖能力强，增殖过程中无停滞期，接种后第3d开始处于对数生长期，第7d达到生长峰值，第8d进入生长平台期。平板克隆形成试验结果示LM-MSCs有较高的克隆形成率约19.7%。通过流式细胞仪对LM-MSCs进行表面标志物的分析， LM-MSCs高表达间充质的表面分子标志(CD29-77.6%、CD44-75.5%、CD90-97.3%、CD105-38.3%)，不表达造血系的表面分子标志(CD34-1.8%、CD45-0.9%)。3.2犬声带激光损伤模型的建立术后4d支撑喉镜下双侧声带充血、水肿，肉芽组织形成，表面不规则，组织切片HE染色示上皮未被覆固有层，损伤处可见大量炎性细胞侵润，固有层与声带肌层界限不清，各种ECM成分不易检出。术后2w喉镜下见声带充血较前减轻，但仍有水肿，受损表面不规则，HE染色示新生上皮被覆受损部位，炎性细胞数量较前减少，大量纤维组织增生；ECM改变(根据切片IOD值检测)：fibronectin含量增加，胶原纤维增粗含量增加，排列紊乱，弹力纤维含量减少且变细，排列不齐，HA含量下降。术后4w喉镜可见双侧声带充血水肿基本消失，受损处表面不规则，萎缩形成，HE染色示固有层无炎性细胞侵润，且被致密的纤维组织所替代；ECM改变：fibronectin含量持续增加，胶原含量增加，排列紊乱，弹力纤维含量略下降，且排列不齐，HA含量下降。术后8w喉镜可见双侧声带无明显充血水肿，损伤表面不规则，局部萎缩，瘢痕修复；ECM改变：fibronectin含量略上升，胶原纤维呈粗大条索状，含量增加排且列紊乱，弹力纤维含量趋于稳定，HA含量下降。 3.3喉黏膜间充质干细胞在声带急性损伤修复中作用的实验研究 3.3.1喉黏膜间充质干细胞对声带大体愈合情况的影响术后2w可观察到双侧声带水肿，稍充血，表面不规则，且有肉芽形成，干细胞组声带肉芽形成较少，且声带表面较对照组规则；术后4w时双侧声带充血、水肿基本消失，表面稍不规则，肉芽组织不明显，萎缩形成，对照组声带表面不规则性及萎缩较干细胞组明显；术后8w时双侧声带无充血、水肿，无新生肉芽组织形成，声带损伤局部可见萎缩、瘢痕形成，对照组声带瘢痕较干细胞组大，且萎缩明显。3.3.2喉黏膜间充质干细胞对声带固有层结构的影响犬处死后声带取材并行组织切片观察，2w时干细胞组声带固有层内炎性细胞侵润及纤维组织增生要小于对照组；双侧声带fibronectin含量持续上升，干细胞组含量略低于对照组；双侧声带胶原含量增加，干细胞组的胶原纤维含量稍低于对照组，且排列上较对照组规则；双侧声带弹力纤维含量均减少，排列紊乱，干细胞组含量高于对照组；HA含量呈下降趋势，干细胞组含量略高于对照组。4w时双侧声带炎性细胞侵润基本消失，纤维组织增生，干细胞组增生小于对照组；双侧声带fibronectin上升，干细胞组含量低于对照组；胶原纤维呈条索状，含量持续增加，干细胞组较对照组含量低，排列较规则；弹力纤维含量继续下降，纤维直径变细，干细胞组含量稍高于对照组；HA含量下降，干细胞组含量略高于对照组。8w时双侧声带观察不到炎性细胞侵润，纤维组织增生明显；双侧声带fibronectin上升，对照组含量高于干细胞组；双侧声带胶原呈粗大条索状，含量增加，对照组较干细胞组排列紊乱；弹力纤维含量趋于稳定，干细胞组弹力纤维直径较对照组粗。HA含量持续降低，干细胞组含量高于对照组。 3.3.3喉黏膜间充质干细胞在声带内的存活情况干细胞注射术后2w、4w及8w荧光显微镜下观察到植入的LM-MSCs在声带固有层内存活，2w时固有层内有大量红色荧光标记的细胞，提示此时有较多数量的移植细胞存活，4w时植入的LM-MSCs存活数量较2w时明显减少，8w时仍可观察到存活的LM-MSCs，但数量较前明显减少。 3.3.4喉黏膜间充质干细胞在声带内的转归 Vimentin免疫荧光染色，4w时荧光显微镜下一些呈红色荧光的LM-MSCs在蓝光的激发下同时可发出绿色荧光，提示植入的LM-MSCs可转化为成纤维细胞，同样的方法，8w时也可观察到一些植入的LM-MSCs转化为成纤维细胞。Smooth mucle actin免疫荧光染色，荧光显微镜下可见4w时植入的LM-MSCs在蓝光的激发下同时可发出绿色荧光，提示植入的LM-MSCs可转化为肌成纤维细胞，同样的方法，8w时未能观察到植入的LM-MSCs转化为肌成纤维细胞。 4结论 4.1本实验利用消化培养法成功培养并鉴定了犬喉黏膜间充质干细胞，证明了其具有向脂肪细胞、成骨细胞及软骨细胞分化的能力。犬喉黏膜间充质干细胞具有有较快的生长增殖速度和较高的克隆形成能力，表达间充质干细胞表面标志物。 4.2支撑喉镜下应用半导体激光造成犬声带膜部损伤，术后喉镜下可见2w时双侧声带充血水肿，，4w时出现萎缩，8w时瘢痕形成，组织学示固有层内胶原含量持续上升，排列紊乱，提示大量瘢痕组织形成，弹力纤维含量下降示声带组织的弹性下降，HA含量降低示声带振动能力及抑制胶原无序沉积的能力下降，fibronectin含量增加示组织纤维化程度加重，以上结果表明本实验成功的建立了犬声带损伤模型，为后续损伤修复实验提供了可靠的动物模型。 4.3.1喉黏膜间充质干细胞声带植入后，充血水肿较对照组轻。干细胞组声带表面较对照组规则，萎缩程度小，瘢痕形成少。 4.3.2组织切片示喉黏膜间充质干细胞能降低固有层中胶原的沉积及无序排列，提高弹力纤维与HA含量以保证声带的有效振动，且具有抑制fibronectin含量上升以减少固有层纤维化的功能，以上结果表明喉黏膜间充质干细胞可通过调节细胞外基质成分的合成，改变微环境修复声带损伤。 4.3.3荧光显微镜下植入的喉黏膜间充质干细胞主要分布于固有层中，术后2w、4w及8w均可观察到植入的LM-MSCs在声带内存活，但随着时间的推移，其存活数量逐渐减少。 4.3.4荧光显微镜下植入的喉黏膜间充质干细胞在受损声带固有层内转化为成纤维细胞及肌成纤维细胞，提示LM-MSCs可通过直接转化为固有层自身细胞参与声带的损伤修复。
[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.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R767.4

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