骨髓間充質(zhì)干細胞復(fù)合溫敏性水凝膠三維微環(huán)境體外培養(yǎng)體系的構(gòu)建與評價
發(fā)布時間：2018-11-24 12:15
【摘要】：目的：以溫敏性水凝膠作為載體,以骨髓間充質(zhì)干細胞為種子細胞,構(gòu)建三維微環(huán)境培養(yǎng)體系；評價間充質(zhì)干細胞與溫敏性水凝膠三維支架的相容性；探討間充質(zhì)干細胞在三維微環(huán)境下分裂增殖與分化的細胞生物學(xué)特性。為開展相關(guān)的組織工程及應(yīng)用基礎(chǔ)研究提供實驗依據(jù),建立實驗基礎(chǔ)。 方法：1.優(yōu)化三維微環(huán)境支架材料：以殼聚糖、β-甘油磷酸鈉為原料,羥乙基纖維素為交聯(lián)劑制備溫敏性水凝膠。將殼聚糖、β-甘油磷酸鈉、羥乙基纖維素按不同體積比例混合,分為六個實驗組,評價其成膠時間及硬度。2.建立穩(wěn)定的骨髓間充質(zhì)干細胞資源：從新生SD乳鼠骨髓中分離BMSCs,在體積分數(shù)10%胎牛血清的低糖DMEM中培養(yǎng),細胞達到培養(yǎng)瓶瓶底面積70%,進行傳代培養(yǎng)至第四代,后續(xù)各代BMSCs的傳代時間穩(wěn)定在4～5d。各代細胞檢測證實保持多向分化潛能和低分化狀。3.選取生長良好的第3代MSCs為種子細胞,溫敏性水凝膠為支架,10%FBS的L-DMEM培養(yǎng)2W,并加入心肌組織裂解液誘導(dǎo)5d,構(gòu)建三維微環(huán)境培養(yǎng)體系,倒置相差顯微鏡下觀察細胞的生長情況,并行HE染色、A0染色,掃描電子顯微鏡下觀察,評價三維微環(huán)境中細胞、溫敏性水凝膠支架之間的相互作用。 結(jié)果：1.骨髓間允質(zhì)干細胞接種于水凝膠上24h后細胞仍維持球形,48h內(nèi)生長較慢,72h球形細胞開始增多,附著于溫敏性水凝膠支架的表面上。連續(xù)培養(yǎng)2W,取細胞-支架共培養(yǎng)物行蘇木素-伊紅染色,鏡下可見大量圓形細胞沿著支架表面生長,部分細胞可跨越于支架之間；掃描電子顯微鏡下,可見大量細胞維持圓形、均質(zhì)性好、分裂能力強,粘附于支架上,部分細胞表面可見以胞吐方式分泌的膜被顆粒。2.以心肌組織裂解液誘導(dǎo)5d,HE染色可見細胞呈長梭形并包被支架材料,掃描電鏡下可觀測到細胞開始由圓形變?yōu)殚L梭形,細胞之間出現(xiàn)牽拉。 結(jié)論：通過優(yōu)化溫敏性水凝膠各組分體積比,可構(gòu)建具有良好孔徑率的三維支架；骨髓間充質(zhì)干細胞與支架有良好的生物相容性,可保持低分化狀態(tài)和活躍的分裂增殖能力；在特定的三維微環(huán)境培養(yǎng)體系中,能夠較好的模擬體內(nèi)組織結(jié)構(gòu),促進骨髓間充質(zhì)干細胞的分化。
[Abstract]:Objective: To construct a three-dimensional micro-environment culture system based on the temperature-sensitive hydrogel as a carrier, and to evaluate the compatibility of the mesenchymal stem cells and the temperature-sensitive hydrogel three-dimensional scaffold. To study the cell biological characteristics of the cell proliferation and differentiation of mesenchymal stem cells in three-dimensional micro-environment. To provide the experimental basis for carrying out the relevant research on the tissue engineering and application, and to establish the experimental basis. The method 1. Optimizing the three-dimensional micro-environment support material: preparing the temperature-sensitive water by using the chitosan, the chitosan-sodium glycerophosphate as the raw material and the hydroxyethyl cellulose as a cross-linking agent; mixing the chitosan, the chitosan-sodium glycerophosphate and the hydroxyethyl cellulose in different volume proportions, and dividing into six experimental groups to evaluate the gel time and the hardness of the two experimental groups;. 2. Stable bone marrow mesenchymal stem cell resources were established: BMSCs were isolated from the bone marrow of neonatal SD rats and cultured in low-sugar DMEM containing 10% fetal bovine serum. The cells reached 70% of the bottom area of the flask, and the cells were subcultured to fourth generation. The passage time of subsequent generation of BMSCs was stable at 4 ~ 5d. The detection of each generation cell confirms that the multi-directional differentiation potential and the low differentiation state are maintained. and 3, selecting the third generation MSCs with good growth as the seed cells, the temperature-sensitive hydrogel as the support, the L-DMEM of the 10% FBS for 2W, and adding the myocardial tissue lysis liquid to induce 5d, constructing a three-dimensional micro-environment culture system, observing the growth of the cells under the inverted phase-difference microscope, and parallel HE staining, A0, the interaction between the cells in the three-dimensional micro-environment and the temperature-sensitive hydrogel support is evaluated under the observation of the dyeing and scanning electron microscope, action. Results: 1. The cells of the bone marrow mesenchymal stem cells were inoculated on the hydrogel for 24 h, the cells remained spherical, the growth of the cells was slower in the 48h, the growth of the 72h spherical cells began to increase, and the cells were attached to the warm-sensitive hydrogel branch. The surface of the rack is continuously cultured for 2W, and the cell-support coculture is subjected to the hematoxylin-eosin staining, a large number of circular cells can be seen under the mirror to grow along the surface of the support, and the part of the cells can be crossed between the supports; under the scanning electron microscope, a large number of cells can be seen to be round, and the homogeneity is good; a membrane that is strong in division, adheres to a stent, and can be seen to be secreted in a cellular manner on the surface of a portion of the cell. The cells were stained with myocardial tissue lysing liquid for 5d. The cells of HE staining were found to be in a long-shuttle shape and coated with the stent material. The cells were observed under the scanning electron microscope from the circular to the long-shuttle-shaped, and the cells Conclusion: The three-dimensional scaffold with good aperture ratio can be constructed by optimizing the volume ratio of each component of the temperature-sensitive hydrogel, and the bone marrow mesenchymal stem cells have good biocompatibility with the support, and the low-differentiation state and the activity can be maintained. in a specific three-dimensional micro-environment culture system, the body tissue structure can be well simulated, the bone marrow is promoted,
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329
本文編號：2353675
[Abstract]:Objective: To construct a three-dimensional micro-environment culture system based on the temperature-sensitive hydrogel as a carrier, and to evaluate the compatibility of the mesenchymal stem cells and the temperature-sensitive hydrogel three-dimensional scaffold. To study the cell biological characteristics of the cell proliferation and differentiation of mesenchymal stem cells in three-dimensional micro-environment. To provide the experimental basis for carrying out the relevant research on the tissue engineering and application, and to establish the experimental basis. The method 1. Optimizing the three-dimensional micro-environment support material: preparing the temperature-sensitive water by using the chitosan, the chitosan-sodium glycerophosphate as the raw material and the hydroxyethyl cellulose as a cross-linking agent; mixing the chitosan, the chitosan-sodium glycerophosphate and the hydroxyethyl cellulose in different volume proportions, and dividing into six experimental groups to evaluate the gel time and the hardness of the two experimental groups;. 2. Stable bone marrow mesenchymal stem cell resources were established: BMSCs were isolated from the bone marrow of neonatal SD rats and cultured in low-sugar DMEM containing 10% fetal bovine serum. The cells reached 70% of the bottom area of the flask, and the cells were subcultured to fourth generation. The passage time of subsequent generation of BMSCs was stable at 4 ~ 5d. The detection of each generation cell confirms that the multi-directional differentiation potential and the low differentiation state are maintained. and 3, selecting the third generation MSCs with good growth as the seed cells, the temperature-sensitive hydrogel as the support, the L-DMEM of the 10% FBS for 2W, and adding the myocardial tissue lysis liquid to induce 5d, constructing a three-dimensional micro-environment culture system, observing the growth of the cells under the inverted phase-difference microscope, and parallel HE staining, A0, the interaction between the cells in the three-dimensional micro-environment and the temperature-sensitive hydrogel support is evaluated under the observation of the dyeing and scanning electron microscope, action. Results: 1. The cells of the bone marrow mesenchymal stem cells were inoculated on the hydrogel for 24 h, the cells remained spherical, the growth of the cells was slower in the 48h, the growth of the 72h spherical cells began to increase, and the cells were attached to the warm-sensitive hydrogel branch. The surface of the rack is continuously cultured for 2W, and the cell-support coculture is subjected to the hematoxylin-eosin staining, a large number of circular cells can be seen under the mirror to grow along the surface of the support, and the part of the cells can be crossed between the supports; under the scanning electron microscope, a large number of cells can be seen to be round, and the homogeneity is good; a membrane that is strong in division, adheres to a stent, and can be seen to be secreted in a cellular manner on the surface of a portion of the cell. The cells were stained with myocardial tissue lysing liquid for 5d. The cells of HE staining were found to be in a long-shuttle shape and coated with the stent material. The cells were observed under the scanning electron microscope from the circular to the long-shuttle-shaped, and the cells Conclusion: The three-dimensional scaffold with good aperture ratio can be constructed by optimizing the volume ratio of each component of the temperature-sensitive hydrogel, and the bone marrow mesenchymal stem cells have good biocompatibility with the support, and the low-differentiation state and the activity can be maintained. in a specific three-dimensional micro-environment culture system, the body tissue structure can be well simulated, the bone marrow is promoted,
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329
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