本文選題：骨髓基質(zhì)細(xì)胞 + 神經(jīng)元樣細(xì)胞��； 參考：《河北醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 目的:腦損傷后不可逆性的神經(jīng)元網(wǎng)絡(luò)破壞和膠質(zhì)細(xì)胞脫失,阻礙了腦組織結(jié)構(gòu)的重建,造成相應(yīng)神經(jīng)功能缺損。神經(jīng)組織工程技術(shù)的發(fā)展為中樞神經(jīng)系統(tǒng)損傷的修復(fù)提供了新思路、新方法。組織工程的關(guān)鍵是種子細(xì)胞和生物材料接合構(gòu)建三維空間復(fù)合體。殼聚糖(C)與β-甘油磷酸鈉(GP)按一定比例混合得到一種溫敏型水凝膠,具有生物相容性好、無毒性、可降解等特點(diǎn)。當(dāng)它注射到體內(nèi)后,在溫度、pH值等因素作用下完成溶膠-凝膠轉(zhuǎn)變,使操作簡(jiǎn)單、創(chuàng)傷小、塑型方便,是良好的支架材料。本實(shí)驗(yàn)將體外誘導(dǎo)的神經(jīng)元樣細(xì)胞與C/GP進(jìn)行聯(lián)合培養(yǎng),觀察細(xì)胞的生長(zhǎng)狀況,以了解C/GP對(duì)神經(jīng)元樣細(xì)胞生長(zhǎng)、增殖、分化的影響及與神經(jīng)元樣細(xì)胞的生物相容性,探索以C/GP為載體與神經(jīng)元樣細(xì)胞制成組織工程材料的可行性,為體外誘導(dǎo)的神經(jīng)元的腦內(nèi)移植尋找較合適的可注射性載體提供實(shí)驗(yàn)依據(jù)。 方法:1骨髓基質(zhì)細(xì)胞的培養(yǎng):50-60g的SD大鼠,麻醉處死,分離雙側(cè)脛骨和腓骨,暴露并沖洗骨髓腔,用貼壁培養(yǎng)法,分離出骨髓基質(zhì)細(xì)胞(marrow stromal cell,MSCs)并連續(xù)傳代、擴(kuò)增,獲取更純化的MSCs。 2誘導(dǎo)分化神經(jīng)元:取第3代MSCs,加入0.2mg/ml的三磷酸胞苷二鈉(CTP),每天觀察細(xì)胞變化,約10天左右可見較多的神經(jīng)元樣細(xì)胞。做神經(jīng)元特異性烯醇化酶(NSE)、神經(jīng)膠質(zhì)纖維酸性蛋白(GFAP)的免疫組化染色鑒定細(xì)胞。 3溫敏型水凝膠的制備:稱取脫乙酰度91%的殼聚糖(CS)200mg溶于9mlHCl溶液中,稱取β-甘油磷酸鈉(β-GP)560mg溶于1ml去離子水中。消毒后室溫下按不同體積比逐滴將β-GP溶液滴入不斷攪拌著的CS溶液中,分別測(cè)混合液的PH值及370C條件下的凝固時(shí)間(以凝膠在容器內(nèi)倒置90°不流動(dòng)、不變形為凝膠形成的標(biāo)準(zhǔn)),選擇最適混合比。 4神經(jīng)元在C/GP凝膠中的培養(yǎng)與觀察:將培養(yǎng)的神經(jīng)元樣細(xì)胞制成密度為2×10~5/ml細(xì)胞懸液與C/GP混合,接種于96孔培養(yǎng)板,每孔20μl細(xì)胞懸液加C/GP復(fù)合物10μl,設(shè)為實(shí)驗(yàn)組;每孔取20μl細(xì)胞懸液加10μl完全培養(yǎng)基,設(shè)為細(xì)胞對(duì)照組;每孔取10μlC/GP復(fù)合物加完全培養(yǎng)基20μl,設(shè)為C/GP對(duì)照組。每日換液1次,并在倒置顯微鏡下觀察神經(jīng)元三維培養(yǎng)的生長(zhǎng)情況。 5 MTT法細(xì)胞活性檢測(cè)及生長(zhǎng)曲線的繪制:實(shí)驗(yàn)組、細(xì)胞對(duì)照組分別在24h后每組取5孔細(xì)胞、C/GP對(duì)照組取1孔(測(cè)實(shí)驗(yàn)組OD值時(shí)調(diào)0用)加入MTT液,倒置顯微鏡下觀察細(xì)胞形態(tài)及分布,酶標(biāo)儀測(cè)吸光度(OD值),連續(xù)觀察7天繪制生長(zhǎng)曲線。對(duì)兩組的OD值作t檢驗(yàn),進(jìn)行統(tǒng)計(jì)學(xué)分析。 結(jié)果:1分離的MSCS呈圓形,胞體透亮。接種24h后,少量細(xì)胞貼壁,72h后大部分細(xì)胞貼壁,形態(tài)為梭形、圓形、多角形,8-10d細(xì)胞達(dá)到90%融合,細(xì)胞呈長(zhǎng)梭形排列呈柵欄狀,傳代后的MSCS貼壁較快約24h完全貼壁,3-7d基本鋪滿瓶底。 2加誘導(dǎo)劑后3d細(xì)胞突起逐漸伸長(zhǎng)呈單極或多極同時(shí)胞體變圓,光暈明顯,隨時(shí)間的延長(zhǎng)不同細(xì)胞突起之間有交叉連接現(xiàn)象,10d左右神經(jīng)元樣細(xì)胞數(shù)量最多。免疫組化呈NSE、GFAP染色陽(yáng)性表達(dá)。 3可注射水凝膠的物理性狀:殼聚糖稀鹽酸溶液呈淡黃色透明粘稠液體,pH值為5.65, 56%的甘油磷酸鈉水溶液呈無色透明液體,pH值為8.91。殼聚糖與β-甘油磷酸鈉的體積比不同,C/GP溶液的PH值及37℃凝固時(shí)間亦不同(表1)。其中按3:2體積比混合的C/GP、PH為7.2,置于37℃細(xì)胞培養(yǎng)箱中膠凝時(shí)間約為10min,較為理想。 4神經(jīng)元細(xì)胞在C/GP凝膠中三維培養(yǎng):24h后倒置顯微鏡下觀察發(fā)現(xiàn)大部分細(xì)胞保持球形,定植在凝膠中,隨時(shí)間的延長(zhǎng)變形細(xì)胞增多,以貼近培養(yǎng)板底面的細(xì)胞變形更為明顯,胞體呈圓形、多角形,并伸出較長(zhǎng)的突起,隨時(shí)間的延長(zhǎng)突起可交叉連接,視角聚集到凝膠的不同層面可見神經(jīng)元細(xì)胞呈三維分布。 5 MTT法細(xì)胞活性檢測(cè)及生長(zhǎng)曲線的繪制: MTT染色后,倒置顯微鏡下觀察可見活細(xì)胞質(zhì)內(nèi)有棕黑色結(jié)晶顆粒沉積,間接顯示活細(xì)胞數(shù)量、形態(tài)及分布。通過測(cè)定OD值繪制兩條生長(zhǎng)曲線均為“S”型,經(jīng)過1-2d的潛伏期后進(jìn)入對(duì)數(shù)生長(zhǎng)期,第3-5d為對(duì)數(shù)生長(zhǎng)期,一周后細(xì)胞數(shù)量減少,實(shí)驗(yàn)組的OD值較對(duì)照組略低。 6統(tǒng)計(jì)學(xué)分析:兩組間差異比較用t檢驗(yàn)得出P0.05,兩組無明顯統(tǒng)計(jì)學(xué)差異,說明神經(jīng)元樣細(xì)胞在C/GP凝膠中生長(zhǎng)狀態(tài)良好。 結(jié)論:1 MSCS具有強(qiáng)大的增殖能力,在一定條件下能分化為神經(jīng)元樣細(xì)胞,誘導(dǎo)的神經(jīng)元性質(zhì)穩(wěn)定,可作為良好的種子細(xì)胞。 2 CTP在體外可誘導(dǎo)MSCs分化為神經(jīng)元樣細(xì)胞 3殼聚糖鹽酸溶液與β-甘油磷酸鈉按一定體積比(3:2)室溫混合后可以得到PH值為7.2的溶膠狀混合物,37℃時(shí)約10min可發(fā)生溶膠-凝膠轉(zhuǎn)變,變成凝膠狀,即有利于必要的注射操作的完成又利于收斂細(xì)胞。 4神經(jīng)元在C/GP凝膠中生長(zhǎng)良好,與在完全培養(yǎng)基中生長(zhǎng)無明顯區(qū)別,是較為理想的支架材料。
[Abstract]:Objective: the damage of the irreversible neuron network and the loss of glial cells after brain injury hinders the reconstruction of the brain tissue and causes the corresponding neural function defect. The development of neural tissue engineering provides new ideas and new methods for the repair of central nervous system damage. The key of tissue engineering is the joint of seed cells and biomaterials. A three dimensional space complex is constructed. Chitosan (C) and beta glycerphosphate sodium (GP) are mixed to a certain temperature sensitive hydrogel, which has the characteristics of good biocompatibility, non-toxic and biodegradable. When it is injected into the body, the sol-gel transition is completed under the action of temperature and pH, which makes the operation simple, small wound and convenient molding. Good scaffolding material. In this experiment, the cultured neuron like cells were co cultured with C/GP in vitro to observe the growth of cells in order to understand the effect of C/GP on the growth, proliferation, differentiation and biocompatibility with neuron like cells, and to explore the tissue engineering materials made of C/GP as the carrier and neuron like cells. It is feasible to provide experimental evidence for in vitro induced neuronal transplantation in the brain to find more suitable injectable vectors.
Methods: 1 the culture of bone marrow stromal cells: 50-60g SD rats, anesthesia was executed, bilateral tibia and fibula were separated, and the bone marrow cavity was exposed and washed. The bone marrow stromal cells (marrow stromal cell, MSCs) were separated from the bone marrow stromal cells, and the bone marrow stromal cells (stromal cell, MSCs) were isolated and continuously passaged and amplified to obtain a more purified MSCs..
2 induced differentiation neurons: third generation MSCs, 0.2mg/ml three phosphate cytidine two sodium (CTP), the cell changes were observed every day and more neuron like cells were observed for about 10 days. Neuron specific enolase (NSE) and immunohistochemical staining of glial fibrillary acidic protein (GFAP) were used to identify the cells.
The preparation of 3 thermosensitive hydrogels: the chitosan (CS) 200mg of deacetylation degree was dissolved in 9mlHCl solution, and sodium beta glycerphosphate (beta -GP) 560mg was dissolved in 1ml deionized water. After disinfection, the beta -GP solution was dripped by drop by drop in CS solution at different volume ratio at room temperature, and the pH and 370C conditions of the mixture were determined respectively. The best mixing ratio was chosen as the time (in which the gel was inverted 90 degrees in the container without flowing, not being deformed into gel forming standard).
The cultivation and observation of 4 neurons in C/GP gel: the cultured neuron like cells were made into 2 x 10~5/ml cell suspension and mixed with C/GP, inoculated on 96 hole culture plate, 20 mu L cell suspension per pore plus 10 mu C/GP compound, and set as experimental group; 20 mu L cell suspension and 10 mu L complete medium were taken per pore, and the cell control group was set up with 10 mu lC/ per pore. The GP complex was added to the medium of 20 l for C/GP control. 1 times a day, the growth of three dimensional neurons was observed under inverted microscope.
5 MTT method cell activity detection and growth curve drawing: the experimental group, the cell control group took 5 hole cells in each group after 24h, the C/GP control group took 1 holes (the experimental group O time adjustment 0) added MTT solution, the inverted microscope observed the cell morphology and distribution, the enzyme labeling instrument measured the absorbance (o value), and observed the growth curve for 7 days for 7 days. The OD of the two groups The value was tested by T, and the statistical analysis was carried out.
Results: 1 the separation of MSCS was round and the cell body was bright. After inoculation of 24h, a small number of cells were adhered to the wall, and most of the cells were adhered to the wall after 72h. The morphology of the cells was spindle, round and polygonal, and the 8-10d cells reached 90% fusion. The cells showed a long shuttle form of railing, and the MSCS adherent after the passage was almost 24h completely adhered to the wall, and 3-7d was basically paved with the bottom of the bottle.
After 2 addition of inducer, the protuberances of 3D cells gradually elongated to be unipolar or multipolar at the same time and turn round, and the halo was obvious. There was a cross connection between different cell protrusions with time. The number of neuron like cells around 10d was the most. The immunohistochemical staining was NSE and GFAP staining was positive.
3 the physical properties of the injectable hydrogel: the chitosan dilute hydrochloric acid solution shows a light yellow transparent viscous liquid, the pH value is 5.65, the 56% glycerol phosphate water solution is colorless transparent liquid, the pH value is different from the volume ratio of 8.91. chitosan to the sodium beta glycerol phosphate, the pH value of the C/GP solution and the solidification time of the sodium glycerphosphate are different (Table 1). The 3:2 volume ratio is mixed. C/GP and PH were 7.2, and the gelation time was about 10min at 37 C cell incubator.
The 4 neuron cells were cultured in C/GP gel in three dimensions. After 24h, the cells remained spherical, and the cells were colonized in the gel. The cells were increased with time. The cell deformation was more obvious at the bottom of the culture plate, and the cell body was round and polygonal, and extended the protuberance with the extension of time. The junction of neurons is seen in three dimensions.
5 MTT cell activity detection and the drawing of the growth curve: after MTT staining, the deposition of brown black crystalline particles in the living cytoplasm was observed under the inverted microscope, and the number, morphology and distribution of the living cells were indirectly displayed. By measuring the OD value, the two growth curves were all "S", after the incubation period of 1-2D into the logarithmic growth period, 3-5d For logarithmic growth phase, the number of cells decreased after one week, and the OD values in the experimental group were slightly lower than those in the control group.
6 statistical analysis: the difference between the two groups was compared with the P0.05 obtained by t test. There was no significant difference between the two groups, indicating that the neuron like cells grew well in the C/GP gel.
Conclusion: 1 MSCS has strong proliferative ability and can differentiate into neuron like cells under certain conditions. The induced neuron is stable and can be used as a good seed cell.
2 CTP can induce MSCs to differentiate into neuron like cells in vitro.
3 chitosan hydrochloric acid solution and sodium beta glycerphosphate mixed with a certain volume ratio (3:2) room temperature can get a pH value of 7.2 sol-gel mixture. At 37 C, about 10min can have a sol-gel transition and become gelatinous, which is beneficial to the completion of the necessary injection operation and the convergence of the cells.
4 neurons grew well in C/GP gel, and had no obvious difference from those grown in full medium, which is an ideal scaffold material.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R329

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