【摘要】：目的: 研究脂肪干細胞向軟骨細胞培養(yǎng)誘導的方法;用絲素蛋白和殼聚糖作為三維多孔支架材料,研究它的制作方法和特點;探討用誘導后的軟骨細胞與絲素蛋白和殼聚糖支架材料體外構(gòu)建細胞-支架復合物的可行性。 方法: (1)采用貼壁分離方法從人體脂肪組織抽吸液中分離出脂肪干細胞,培養(yǎng)擴增,傳到第三代時在骨形態(tài)發(fā)生蛋白和堿性成纖維細胞生長因子的軟骨細胞誘導培養(yǎng)基中,誘導脂肪干細胞向軟骨細胞轉(zhuǎn)化。Ⅱ型膠原免疫組織化學檢測細胞特性。 (2)以絲素蛋白、殼聚糖為支架原料,蠶絲經(jīng)過溶解、脫膠、透析后得絲素蛋白溶液,與殼聚糖溶液混合溶解,得混合溶液。冷凍干燥法制成絲素蛋白/殼聚糖三維立體多孔支架材料,通過體積法計算支架孔隙率,通過掃描電鏡對支架的外觀和性能進行觀察和測量。 (3)將誘導后的細胞接種于消毒滅菌過的支架材料上,細胞與支架復合物聯(lián)合培養(yǎng)。掃描電鏡觀察細胞-支架復合物的外觀和內(nèi)部結(jié)構(gòu),以及細胞在支架里的生長情況。 結(jié)果: (1)脂肪組織通過消化得到脂肪干細胞,并且通過培養(yǎng)基培養(yǎng)長勢良好。三代的脂肪干細胞在骨形態(tài)發(fā)生蛋白和堿性成纖維細胞生長因子的軟骨誘導培養(yǎng)基中誘導培養(yǎng),細胞的形態(tài)發(fā)生改變,呈軟骨細胞的特點。由長梭形變?yōu)槎噙呅?Ⅱ型膠原免疫組織化學染色呈陽性。 (2)制作完成的絲素蛋白/殼聚糖三維多孔支架呈白色,呈海綿狀,可見纖維狀排列。支架材料表面均勻,柔軟有彈性,孔隙大小均勻,沒有明顯突起和凹陷。支架的孔隙率大于80%。以不同比例制作的支架以絲素蛋白和殼聚糖的比例5:5和4:6最為合適。 (3)誘導后的細胞接種在支架上在培養(yǎng)基中聯(lián)合培養(yǎng),掃描電鏡顯示細胞與支架黏附良好,細胞可以在支架材料里面貼附生長。 結(jié)論: (1)脂肪干細胞在軟骨培養(yǎng)基中,通常單獨應用一種細胞生長因子都可以誘導其成軟骨細胞。在本實驗中聯(lián)合應用骨形態(tài)發(fā)生蛋白14和堿性成纖維細胞生長因子兩種細胞生長因子較單獨應用骨形態(tài)發(fā)生蛋白能更好的誘導脂肪干細胞向軟骨細胞方向分化。細胞長的更好,更密集。 (2)蠶絲和殼聚糖兩種天然材料通過混合溶解得到的多孔三維立體支架材料經(jīng)測得空隙率大于80%,有利于細胞的滲透和營養(yǎng)物質(zhì)的交換。三維立體環(huán)境也可以給細胞提供適宜的微環(huán)境。所有絲素蛋白/殼聚糖支架可以作為軟骨組織工程的三維多孔支架。 (3)脂肪干細胞經(jīng)過誘導后的軟骨細胞可以很好的附著在絲素蛋白和殼聚糖支架上,細胞可以在表面和內(nèi)部生長。本實驗的細胞-支架復合物可以用來作為組織工程軟骨的實驗研究。
[Abstract]:Objective:
The methods for inducing the induction of adipose stem cells to chondrocytes were studied. The methods and characteristics were studied by using silk fibroin and chitosan as a three-dimensional porous scaffold material. The feasibility of constructing the cell scaffold complex in vitro with fibroin and chitosan scaffold materials was discussed.
Method:
(1) the fat stem cells were isolated from the sucking fluid of human adipose tissue by the method of adherent wall separation, and were cultured and amplified to be transmitted to the third generation of chondrocytes induced by bone morphogenetic protein and basic fibroblast growth factor to induce the transformation of fat stem cells to chondrocytes. Sex.
(2) the silk fibroin and chitosan were used as the scaffold. The silk fibroin protein solution was dissolved, degumbed and dialysated, and the chitosan solution was mixed and dissolved. The mixed solution was obtained. The freeze-drying method was used to calculate the three-dimensional porous scaffold of fibroin protein / chitosan. The scaffold porosity was calculated by the volume method. The appearance and appearance of the scaffold were examined by scanning electron microscope. The performance is observed and measured.
(3) the induced cells were inoculated on the sterilized scaffold material, and the cells were co cultured with the scaffold complex. The appearance and internal structure of the cell scaffold complex and the growth of the cells in the scaffold were observed by scanning electron microscope.
Result:
(1) adipose tissue is digested to obtain fat stem cells, and the growth potential is good by culture medium. The three generation of fat stem cells are induced in the cartilage induced medium of bone morphogenetic protein and basic fibroblast growth factor, and the morphological changes of the cells are characterized by the characteristics of chondrocytes. The long spindle is a polygon, II. The immuno histochemical staining of type collagen was positive.
(2) the three-dimensional porous scaffolds made of silk fibroin / chitosan were white, spongy and fibrous. The surface of the scaffold was uniform, soft and elastic, the size of the pores was uniform, no obvious protruding and depression. The ratio of scaffolds with different proportions of 80%. and 5:5 and 4:6 was the best ratio of silk fibroin and chitosan. It's right.
(3) the induced cell inoculation was co cultured on the scaffold in the culture medium. The scanning electron microscope showed that the cells and the scaffold adhered well, and the cells could attach the growth in the scaffold material.
Conclusion:
(1) in the cartilage medium, adipose stem cells usually use a single cell growth factor to induce their chondrocytes. In this experiment, the combination of bone morphogenetic protein 14 and basic fibroblast growth factor two cell growth factors can better induce adipose stem cells than the single use of bone morphogenetic protein. Chondrocytes differentiate in a more compact way.
(2) the porous three-dimensional scaffold material obtained by mixed solution of two kinds of silk and chitosan can be obtained through mixed dissolution of the porous three-dimensional scaffold material, the void ratio is greater than 80%, which is beneficial to the infiltration of cells and the exchange of nutrients. The three-dimensional environment can also provide the suitable microenvironment for the cells. All silk egg white / chitosan scaffolds can be used as cartilage tissue engineering. The three-dimensional porous scaffold.
(3) the chondrocytes of the adipose stem cells can be well attached to the silk fibroin and chitosan scaffold after the induction of the stem cells. The cells can grow on the surface and inside. The cell scaffold complex in this experiment can be used as an experimental study of tissue engineered cartilage.
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329

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