蠶絲人工韌帶復(fù)合骨髓基質(zhì)干細(xì)胞的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-11-17 14:46
【摘要】：目的 利用自制的蠶絲人工韌帶支架，多種方法對(duì)表面結(jié)構(gòu)進(jìn)行改性，觀察對(duì)比不同分組間種子細(xì)胞粘附、生長(zhǎng)情況，為組織工程化人工韌帶支架材料與種子細(xì)胞復(fù)合提供一種理想的方法。 方法 蠶絲人工韌帶支架材料，采用胰酶、碳酸鈉聯(lián)合脫膠，稱重測(cè)韌帶脫膠率，以EDC（1-乙基-3-(3-二甲基氨丙基)碳二亞胺）/NHS（N-羥基琥珀酰亞胺）作為交聯(lián)劑進(jìn)行膠原的交聯(lián)，酶標(biāo)儀法測(cè)其交聯(lián)度。采用熒光顯微鏡、掃描電鏡觀察蠶絲脫膠及與交聯(lián)膠原復(fù)合后的表面結(jié)構(gòu)變化。 把韌帶支架分為試驗(yàn)組、參照組和空白對(duì)照組。參照組蠶絲人工韌帶與膠原結(jié)合形成蠶絲-膠原結(jié)合韌帶支架，不做交聯(lián)處理；試驗(yàn)組采用EDC/NHS對(duì)蠶絲-膠原結(jié)合韌帶支架材料進(jìn)行交聯(lián)；空白組未作任何處理。兔的骨髓基質(zhì)干細(xì)胞（Marrowmesenchymal stem cells，MSCs）全血貼壁法培養(yǎng)至第八代，通過(guò)CD34、CD44、CD50免疫組化及流式細(xì)胞儀進(jìn)行干細(xì)胞檢測(cè)，調(diào)整細(xì)胞密度為1×106/L，分別種植于三種支架材料上，對(duì)三種材料表面培養(yǎng)的細(xì)胞進(jìn)行了熒光顯微鏡、掃描電鏡及酶標(biāo)儀的檢測(cè)。比較不同分組間細(xì)胞粘附、增殖生長(zhǎng)情況。 結(jié)果 采用胰酶、碳酸鈉聯(lián)合脫膠，能將絲膠基本去除，脫膠率29%左右。當(dāng)EDC濃度為3g/L時(shí)，交聯(lián)度最大。熒光顯微鏡下未脫膠蠶絲表面比較完整；脫膠蠶絲結(jié)構(gòu)變得蓬松，表面光滑，絲素纖維暴露。SEM觀察，未脫膠蠶絲表面粗糙不平，絲膠包繞在絲素纖維周圍；脫膠完全的蠶絲表面很光滑，可見(jiàn)單根的絲素纖維。熒光倒置顯微鏡及SEM下復(fù)合膠原后的韌帶可見(jiàn)，膠原呈一薄層分布在韌帶表面，呈不規(guī)則的線狀突起，色澤較白。交聯(lián)后膠原-蠶絲韌帶成蜂窩狀。 全血貼壁法體外培養(yǎng)的BMSC，檢測(cè)CD34陰性、CD44、CD50、STRO-1陽(yáng)性，流式細(xì)胞儀檢測(cè)CD34+細(xì)胞占10%左右；CD44+占42%左右，表現(xiàn)為干細(xì)胞特性。熒光顯微鏡下，未交聯(lián)膠原-蠶絲韌帶細(xì)胞數(shù)目較少，細(xì)胞未見(jiàn)舒展；交聯(lián)后的膠原-蠶絲人工韌帶，蠶絲表面粗糙，細(xì)胞粘附率較高，舒展充分，蠶絲之間的間隙模糊。SEM下見(jiàn)，交聯(lián)后的蠶絲人工韌帶與細(xì)胞貼合緊密，細(xì)胞伸展充分，有較多的觸角伸出，明顯優(yōu)于其它組。經(jīng)單因素方差分析，試驗(yàn)組與參照組、空白對(duì)照組比較P＜0.05，差異有統(tǒng)計(jì)學(xué)意義。 結(jié)論 1.骨髓基質(zhì)干細(xì)胞能夠向多種組織細(xì)胞分化，在實(shí)驗(yàn)中表現(xiàn)出良好的增殖能力，是種子細(xì)胞的理想選擇。 2.膠原蛋白具有良好的生物相容性，是一種良好的蠶絲表面修飾劑。EDC毒性很低，可作為良好的交聯(lián)劑。 3.經(jīng)過(guò)EDC/NHS交聯(lián)的蠶絲-膠原結(jié)合韌帶支架材料，能夠顯著提高細(xì)胞黏附率。 意義 隨著人們物質(zhì)文化水平的不斷提高和體育事業(yè)的蓬勃發(fā)展，韌帶損傷病例呈現(xiàn)快速增長(zhǎng)的趨勢(shì)。韌帶損傷后，往往需要韌帶重建手術(shù)治療。目前韌帶移植材料有①自體韌帶；②異體韌帶；③人工韌帶，都存在來(lái)源少，免疫排斥及傳播疾病的缺陷。組織工程人工韌帶的發(fā)展，為治療韌帶損傷提供了另一條途徑。骨髓間充質(zhì)干細(xì)胞能夠向多種組織細(xì)胞分化，是組織工程人工韌帶理想的種子細(xì)胞。蠶絲具備良好的生物相容性和細(xì)胞親和力，可作為良好的組織工程支架材料，由于材料表面過(guò)于光滑、致密，，種子細(xì)胞不能與蠶絲韌帶復(fù)合。本實(shí)驗(yàn)通過(guò)對(duì)蠶絲韌帶進(jìn)行改性，克服了這一難題，為組織工程化人工韌帶的發(fā)展提供有力的實(shí)驗(yàn)支持。
[Abstract]:Purpose The surface structure was modified by using a self-made artificial ligament support, and the adhesion and growth of the seed cells in different groups were observed. The method The method for preparing the silk artificial ligament support material adopts the combination of the pancreatin and the sodium carbonate for degumming, and the degumming rate of the ligament is measured, and the EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide)/ NHS (N-hydroxy-amber-imine) is used as the crosslinking agent. the crosslinking of the collagen, the enzyme, The cross-linking degree of the cross-linked collagen was measured by means of a fluorescence microscope and a scanning electron microscope. The cross-linking collagen was combined with the cross-linked collagen. The posterior surface structure changes. The ligamentum support is divided into two groups. In group, reference group and blank control group, the silk-collagen combined ligament stent was formed with the combination of the artificial ligament of the reference group and the collagen, and the cross-linking treatment was not performed; the test group used the EDC/ NHS to carry out the cross-linking of the silk-collagen-binding ligament support material. The bone marrow stromal cells (MSCs) of the rabbit were cultured to the eighth generation by means of the whole blood-adhering method of the bone marrow stromal cells (MSCs), and the stem cells were detected by CD34, CD44, CD50 immunohistochemistry and flow cytometry, and the density of the cells was 1-106/ L. the three kinds of support materials are not planted, and the cells cultured on the surface of the three materials are used for fluorescence microscopy, The detection of the scanning electron microscope and the microplate reader. The comparison of the different groups cell The result is that pancreatin and sodium carbonate are used for degumming, can basically remove the sericin, and the degumming rate is about 29%. The cross-linking degree is the largest when the concentration of EDC is 3g/ L. The surface of the unglued silk under the fluorescence microscope is relatively complete; the degummed silk knot The structure becomes fluffy, the surface is smooth, and the silk fibroin fiber is exposed. The SEM observation shows that the surface of the non-degummed silk is not rough, the silk glue bag is wound around the silk fibroin fiber, and the degumming is complete. The surface of the silk is smooth, and a single silk fibroin fiber can be seen. The fluorescence inversion microscope and the ligament of the composite collagen under the SEM are visible, and the collagen is distributed in a thin layer in the ligament. a surface, an irregular linear protrusion, a color, After cross-linking, the collagen-silk ligament was made into a honeycomb shape. The BMSC, which was cultured in vitro by the whole blood-attached method, was used to detect CD34 +, CD44, CD50 and STRO-1, and the CD34 + cells were detected by flow cytometry. CD44 + accounted for about 42%, and expressed as a stem cell characteristic. Under the fluorescence microscope, the number of uncrosslinked collagen-silk ligament cells is small and the cells are not stretched; the cross-linked collagen-silk artificial ligament, the surface of the silk, the cells, the adhesion rate is high, the stretching is sufficient, the gap between the silk is blurred, The show is full, with more tentacles extending, which is obviously superior to other groups. The single-factor analysis of variance, the test group and the reference group, the blank control Conclusion 1. The bone marrow stromal stem cells can differentiate into many kinds of tissue cells. and it is an ideal choice for seed cells. The collagen has good biocompatibility, A good surface modification agent for silk. The toxicity of EDC is very low, which can be used as a good cross-linking agent. 3. EDC /N HS-crosslinked silk-collagen combined with the ligament stent material can significantly improve the cell adhesion rate. The continuous improvement of the level of material culture and the construction of the sports cause In the development of the ligament, there is a trend of rapid growth in the case of ligament damage. The ligament is often used for the reconstruction of the ligament after the ligament is damaged. The presence of an autograft of the ligamentum; an allogenic ligament; an artificial ligament, both of which are of low origin, immune, The development of artificial ligament of tissue engineering provides another way for the treatment of ligament damage. The bone marrow mesenchymal stem cells can be differentiated into a plurality of tissue cells and are the ideal seed cells for tissue engineering artificial ligaments. The silk has good biocompatibility and cell affinity, and can be used as a good The tissue engineering scaffold material has the advantages that the surface of the material is too smooth and dense, and the seed cell cannot be compounded with the silk ligament.
【學(xué)位授予單位】：泰山醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329.2
本文編號(hào)：2338162
[Abstract]:Purpose The surface structure was modified by using a self-made artificial ligament support, and the adhesion and growth of the seed cells in different groups were observed. The method The method for preparing the silk artificial ligament support material adopts the combination of the pancreatin and the sodium carbonate for degumming, and the degumming rate of the ligament is measured, and the EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide)/ NHS (N-hydroxy-amber-imine) is used as the crosslinking agent. the crosslinking of the collagen, the enzyme, The cross-linking degree of the cross-linked collagen was measured by means of a fluorescence microscope and a scanning electron microscope. The cross-linking collagen was combined with the cross-linked collagen. The posterior surface structure changes. The ligamentum support is divided into two groups. In group, reference group and blank control group, the silk-collagen combined ligament stent was formed with the combination of the artificial ligament of the reference group and the collagen, and the cross-linking treatment was not performed; the test group used the EDC/ NHS to carry out the cross-linking of the silk-collagen-binding ligament support material. The bone marrow stromal cells (MSCs) of the rabbit were cultured to the eighth generation by means of the whole blood-adhering method of the bone marrow stromal cells (MSCs), and the stem cells were detected by CD34, CD44, CD50 immunohistochemistry and flow cytometry, and the density of the cells was 1-106/ L. the three kinds of support materials are not planted, and the cells cultured on the surface of the three materials are used for fluorescence microscopy, The detection of the scanning electron microscope and the microplate reader. The comparison of the different groups cell The result is that pancreatin and sodium carbonate are used for degumming, can basically remove the sericin, and the degumming rate is about 29%. The cross-linking degree is the largest when the concentration of EDC is 3g/ L. The surface of the unglued silk under the fluorescence microscope is relatively complete; the degummed silk knot The structure becomes fluffy, the surface is smooth, and the silk fibroin fiber is exposed. The SEM observation shows that the surface of the non-degummed silk is not rough, the silk glue bag is wound around the silk fibroin fiber, and the degumming is complete. The surface of the silk is smooth, and a single silk fibroin fiber can be seen. The fluorescence inversion microscope and the ligament of the composite collagen under the SEM are visible, and the collagen is distributed in a thin layer in the ligament. a surface, an irregular linear protrusion, a color, After cross-linking, the collagen-silk ligament was made into a honeycomb shape. The BMSC, which was cultured in vitro by the whole blood-attached method, was used to detect CD34 +, CD44, CD50 and STRO-1, and the CD34 + cells were detected by flow cytometry. CD44 + accounted for about 42%, and expressed as a stem cell characteristic. Under the fluorescence microscope, the number of uncrosslinked collagen-silk ligament cells is small and the cells are not stretched; the cross-linked collagen-silk artificial ligament, the surface of the silk, the cells, the adhesion rate is high, the stretching is sufficient, the gap between the silk is blurred, The show is full, with more tentacles extending, which is obviously superior to other groups. The single-factor analysis of variance, the test group and the reference group, the blank control Conclusion 1. The bone marrow stromal stem cells can differentiate into many kinds of tissue cells. and it is an ideal choice for seed cells. The collagen has good biocompatibility, A good surface modification agent for silk. The toxicity of EDC is very low, which can be used as a good cross-linking agent. 3. EDC /N HS-crosslinked silk-collagen combined with the ligament stent material can significantly improve the cell adhesion rate. The continuous improvement of the level of material culture and the construction of the sports cause In the development of the ligament, there is a trend of rapid growth in the case of ligament damage. The ligament is often used for the reconstruction of the ligament after the ligament is damaged. The presence of an autograft of the ligamentum; an allogenic ligament; an artificial ligament, both of which are of low origin, immune, The development of artificial ligament of tissue engineering provides another way for the treatment of ligament damage. The bone marrow mesenchymal stem cells can be differentiated into a plurality of tissue cells and are the ideal seed cells for tissue engineering artificial ligaments. The silk has good biocompatibility and cell affinity, and can be used as a good The tissue engineering scaffold material has the advantages that the surface of the material is too smooth and dense, and the seed cell cannot be compounded with the silk ligament.
【學(xué)位授予單位】：泰山醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329.2
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