本文選題：組織工程 + 骨髓基質(zhì)干細胞��； 參考：《華北煤炭醫(yī)學院》2009年碩士論文

【摘要】：目的通過分離大鼠骨髓基質(zhì)干細胞(BMSCs),檢測其在特定培養(yǎng)條件下誘導為血管內(nèi)皮細胞的生物學特性,并將誘導培養(yǎng)的成骨細胞、血管內(nèi)皮細胞以及共培養(yǎng)的兩種細胞分別接種于I型膠原修飾的同種異體凍干脫鈣骨基質(zhì)支架材料,從而探討兩種細胞與支架材料復合構(gòu)建組織工程活性骨的可行性,最終為組織工程骨修復骨缺損提供實驗依據(jù)。 方法4周齡SD大鼠,進行BMSCs原代細胞培養(yǎng)。取生長狀態(tài)良好的第3代BMSCs進行實驗。分組如下:1)對照組:普通培養(yǎng)基組;2)實驗組:血管內(nèi)皮條件培養(yǎng)基組。倒置相差顯微鏡觀察細胞形態(tài);CD31 CD34免疫細胞化學染色、透射電鏡W-P小體鑒定血管內(nèi)皮細胞; MTT法檢測血管內(nèi)皮細胞的生長與增殖情況并繪制生長曲線;掃描電鏡觀察誘導的成骨細胞、血管內(nèi)皮細胞及兩種細胞共培養(yǎng)在I型膠原修飾的支架材料表面粘附、生長情況。 結(jié)果1. BMSCs接種后隨著時間的延長,貼壁細胞增加。細胞形態(tài)呈梭形、纖維母細胞樣;集落形成,其大小、形狀、密度不同,最終融合。細胞無接觸性抑制。 2.誘導后的血管內(nèi)皮細胞具有一定的分化能力,但增殖速度變得緩慢,CD31、CD34免疫細胞化學染色鑒定可見陽性表達;透射電鏡觀察細胞內(nèi)可見W-P小體。 3. MTT檢測細胞生長、增殖結(jié)果顯示:第1-2d,細胞數(shù)目均無明顯變化,為潛伏期;第3天起細胞進入對數(shù)生長期,數(shù)量開始增加;7天后,細胞進入到生長穩(wěn)定的平臺期。 4.同種異體凍干脫鈣骨基質(zhì)為天然的多孔樣結(jié)構(gòu)。誘導后的血管內(nèi)皮細胞在其表面平鋪、伸展、生長狀態(tài)良好。 5.誘導培養(yǎng)的成骨細胞、血管內(nèi)皮細胞以及共培養(yǎng)的兩種細胞分別接種于I型膠原修飾的同種異體凍干脫鈣骨基質(zhì),修飾后細胞在支架材料上的粘附率增加。 結(jié)論1.從骨髓中可分離得到較高純度BMSCs,體外培養(yǎng)增殖能力活躍,在含有VEGF、bFGF誘導因子誘導培養(yǎng)條件下,能定向分化為具有典型形態(tài)學特點的血管內(nèi)皮細胞,可作為骨組織工程血管化理想的細胞來源。 2.同種異體凍干脫鈣骨基質(zhì)與血管內(nèi)皮細胞有良好的細胞相容性,同種異體凍干脫鈣骨基質(zhì)有利于細胞的粘附、生長,是較為理想的骨組織工程支架材料。 3.血管內(nèi)皮細胞與支架材料的粘附率與細胞接種密度密切相關。當細胞接種密度為2×106/ml時,血管內(nèi)皮細胞與支架材料的粘附率達到最大。提示:細胞接種于支架時有最適接種密度,支架與細胞的粘附能力存在極限即最大細胞粘附數(shù)量。 4.支架材料經(jīng)I型膠原修飾后較修飾前所粘附的細胞數(shù)量多。提示:I型膠原是較為理想的支架材料表面修飾物。
[Abstract]:Objective to investigate the biological characteristics of rat bone marrow stromal stem cells (BMSCs) induced into vascular endothelial cells (VEC) under specific culture conditions. Vascular endothelial cells (VECs) and co-cultured cells were inoculated with type I collagen modified allogeneic decalcified bone matrix scaffolds to investigate the feasibility of constructing tissue-engineered active bone by combining two kinds of cells with scaffold materials. Finally, it provides experimental basis for tissue engineering bone to repair bone defect. Methods Primary BMSCs cells were cultured in 4 weeks old SD rats. The third generation BMSCs with good growth state was selected for experiment. The control group was divided as follows: control group: normal medium group 2) experimental group: vascular endothelial conditioned medium group. CD31 CD34 immunocytochemical staining was observed by inverted phase contrast microscope, vascular endothelial cells were identified by transmission electron microscope W-P corpuscles, the growth and proliferation of vascular endothelial cells were detected by MTT method and the growth curve was drawn. The adhesion and growth of osteoblasts, vascular endothelial cells and two kinds of cells co-cultured on the surface of collagen I scaffold were observed by scanning electron microscope (SEM). Result 1. After BMSCs inoculation, adherent cells increased with time. Cell morphology is fusiform, fibroblast-like, colony formation, its size, shape, density is different, finally fusion. There was no contact inhibition. 2. The induced vascular endothelial cells had the ability of differentiation, but the proliferation rate became slow. The positive expression of CD31and CD34 was detected by immunocytochemistry, and W-P corpuscles were observed by transmission electron microscope. 3. The results of MTT showed that the number of cells did not change obviously at the 1st to 2nd day, which was the latent period, and the cells entered the logarithmic growth period on the 3rd day, and the number began to increase 7 days later, the cells entered the stable stage of growth. 4. Allogeneic freeze-dried demineralized bone matrix is a natural porous structure. The induced vascular endothelial cells on the surface of the flat, stretch, good growth state. 5. The osteoblasts, vascular endothelial cells and co-cultured cells were inoculated into the allogeneic freeze-dried decalcified bone matrix modified by type I collagen, and the adhesion rate of the cells on the scaffold material was increased. Conclusion 1. BMSCs with high purity could be isolated from bone marrow. The proliferation of BMSCs was active in vitro. When cultured with VEGF bFGF inducing factors, BMSCs could be differentiated into vascular endothelial cells with typical morphological characteristics. It can be used as an ideal cell source for vascularization of bone tissue engineering. 2. The allogeneic freeze-dried decalcified bone matrix has good cytocompatibility with vascular endothelial cells. The allogeneic freeze-dried decalcified bone matrix is an ideal scaffold material for bone tissue engineering, which is conducive to cell adhesion and growth. 3. The adhesion rate of vascular endothelial cells to scaffold materials is closely related to the density of cell inoculation. When the cell density was 2 脳 106/ml, the adhesion rate of vascular endothelial cells to the scaffold was the highest. The results suggested that the optimal inoculation density was found when the cells were inoculated on the scaffold, and the adhesion capacity of the scaffold to the cells was limited, that is, the maximum number of cell adhesion. 4. The number of cells adhered to the scaffold after type I collagen modification was higher than that before modification. It is suggested that type I collagen is an ideal surface modifier for scaffolds.
【學位授予單位】：華北煤炭醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R329

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