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  本文選題：骨髓間充質(zhì)干細胞 + 脂肪干細胞��； 參考：《第四軍醫(yī)大學》2008年碩士論文

【摘要】： 椎間盤退變?yōu)檠惩粗饕蛑?其退變主要是由于椎間盤細胞減少和細胞外基質(zhì)成份的改變,盡管并沒明確椎間盤內(nèi)細胞的性質(zhì),但是研究表明:髓核細胞與軟骨細胞在某些方面有很多共性。近年來研究表明:細胞移植可以達到組織功能上的修復,所以此方法最有希望成為治療椎間盤退變的最佳辦法。因為干細胞可以向軟骨樣細胞方面分化,所以大部分學者主要通過改變細胞外環(huán)境等來研究骨髓間充質(zhì)干細胞向軟骨樣細胞分化。最近研究表明:人體脂肪組織中的脂肪干細胞含量較豐富,而脂肪干細胞在不同的獲取技術下可分化為軟骨樣細胞,如轉基因和不同生長因子聯(lián)合誘導脂肪干細胞向軟骨樣紹胞分化技術。所以我們設計這個試驗就是為了探索比較體外培養(yǎng)的骨髓間充質(zhì)干細胞(Bone Marrow Mesenchymal Stem Cells,BMSCs)和脂肪干細胞(Adipose Stem Cells,ASCs)變化,以及堿性成纖維縐胞生長因子(Basic Fibroblast Growth Factor,BFGF)對其代謝的影響。 方法 1.抽取日本大耳白兔骨髓,用全骨髓培養(yǎng)法進行BMSCs的體外培養(yǎng)、擴增,取日本大耳白兔肩胛間的脂肪組織,在0.075%Ⅱ皎原酶溶液中下剪碎并消化1小時(37℃),所得的消化液過濾離心獲取脂肪干細胞,進行體外單層培養(yǎng)擴增,所收集的骨髓間充質(zhì)干細胞和脂肪干細胞分別用DMEM、DMEM/F12(2:1)、α—MEM培養(yǎng),倒置顯微鏡觀察骨髓間充質(zhì)干細胞和脂肪干細胞的形態(tài)改變,分別比較兔骨髓間充質(zhì)干細胞和脂肪干細胞的數(shù)量。 2.當細胞傳三代后,胰蛋白酶消化收集的脂肪干細胞和骨髓間充質(zhì)干細胞沉淀以2×10~5/ml接種型24cm~2的培養(yǎng)瓶里,用軟骨誘導培養(yǎng)基(Chondrogenic Medium,CM)進行誘導,CM的組成:1.25mg/ml牛血清白蛋白、10ng/ml TGF-β1、100nmol/L地塞米松、50μg/mL維生素C、100μg/mL丙酮酸鈉、40μg/mL脯氨酸、1%ITS-plus(10 mg/ml胰島素,6.7 mg/ml亞硒酸鈉,5.5 mg/ml轉鐵蛋白,2ng/ml乙醇胺)的DMEM/F12(2:1)培養(yǎng)液 3。兔骨髓間充質(zhì)干細胞和脂肪干細胞分為4組后分別進行誘導培養(yǎng)三周,即A組,BMSCs+CM;B組,BMSCs+CM+5ng/ml BFGF;C組,ASCs+CM;D組,ASCs+CM+5ng/mlBFGF,測定培養(yǎng)液上清羥脯氨酸含量和~(35)SO_4~(2-)基摻入量。 結果 1.α—MEM培養(yǎng)基比DMEM、DMEM/F12(2:1)制備的BMSCs和ASCs所需時間大大縮短。 2.單層培養(yǎng):單層培養(yǎng)的BMSCs和ASCs增殖較快貼壁較牢且生成細胞集落,然而在CM誘導后,細胞增殖較漫,貼壁不牢,且細胞沒有集落生成。 3.誘導后的BMSCs和ASCs的形狀趨向于類圓形,B組的細胞數(shù)量比A組的細胞多,D的細胞數(shù)量比C組的細胞多,B組比A組表達的羥脯氫酸增加、~(35)SO_4~(2-)攝入量增加,D組分別比C組表達羥脯復酸增加、~(35)SO_4~(2-)攝入量增加。而D組表達羥脯氫酸和~(35)SO_4~(2-)攝入量與B組表達羥脯氫酸和~(35)SO_4~(2-)攝入量無差別。 結論 α-MEM培養(yǎng)基比DMEM、DMEM/F-12更適合兔骨髓間充質(zhì)干細胞和脂肪干細胞的單層培養(yǎng),添加BFGF的軟骨誘導液可以增加誘導后兔骨髓間充質(zhì)干細胞和脂肪干細胞的代謝,脂肪干細胞可作為髓核組織工程種子細胞的替代物。
[Abstract]:The degeneration of intervertebral disc is one of the main causes of low back pain. Its degeneration is mainly due to the reduction of intervertebral disc cells and the changes in the components of the extracellular matrix. Although the nature of the intervertebral disc cells is not clear, the study shows that the nucleus pulposus cells have many similarities with the chondrocytes in some aspects. In recent years, the study showed that the cell transplantation could reach the group. This method is the most promising way to treat intervertebral disc degeneration. Because stem cells can differentiate into chondroid cells, most scholars mainly study the differentiation of bone marrow mesenchymal stem cells into chondroid cells by changing the extracellular environment. Recent research shows that human adipose tissue is in human body. The fat stem cells are rich in fat stem cells, and fat stem cells can differentiate into chondroid cells under different acquisition techniques, such as transgenic and different growth factors combined to induce adipose stem cells to differentiate into cartilage like cells. So we designed this experiment to explore bone marrow mesenchymal stem cells (Bone Ma) than in vitro culture. The changes in rrow Mesenchymal Stem Cells, BMSCs) and fat stem cells (Adipose Stem Cells, ASCs), and the effect of basic fibroblast crepe growth factor (Basic Fibroblast Growth) on its metabolism.
Method
1. the bone marrow of Japanese big ear white rabbit was extracted and cultured in vitro by full bone marrow culture. The fat tissue between the scapula of Japanese big ear was amplified, and the fat tissue was extracted from the scapula of Japanese big ear white rabbit. The fat stem cells were obtained by filtration and centrifugation in the 0.075% II clear enzyme solution and digested for 1 hours (37 degrees C). The bone marrow was cultured and amplified in vitro, and the collected bone marrow was collected. Mesenchymal stem cells and adipose stem cells were cultured with DMEM, DMEM/F12 (2:1) and alpha MEM respectively. The morphologic changes of bone marrow mesenchymal stem cells and fat stem cells were observed by inverted microscope, and the number of rabbit bone marrow mesenchymal stem cells and fat stem cells were compared respectively.
2. when the cells were passed on the three generation, the trypsin digested fat stem cells and bone marrow mesenchymal stem cells were precipitated in the culture bottle of 2 x 10~5/ml inoculated 24cm~2, and the cartilage induced medium (Chondrogenic Medium, CM) was induced, and the composition of CM was: 1.25mg/ml bovine serum white egg white, 10ng/ml TGF- beta 1100nmol/L dexamethasone, 50 mu g/mL vitamin. C, 100 mu g/mL sodium pyruvate, 40 micron proline, 1%ITS-plus (10 mg/ml insulin, 6.7 mg/ml sodium selenite, 5.5 mg/ml transferrin, 2ng/ml ethanolamine) DMEM/F12 (2:1) culture
3. rabbit bone marrow mesenchymal stem cells and fat stem cells were divided into 4 groups to be induced and cultured for three weeks, namely, group A, BMSCs+CM; group B, BMSCs+CM+5ng/ml BFGF; C group, ASCs+CM; D group, ASCs+CM+5ng/mlBFGF, determine the content of hydroxyproline and ~ (35) SO_4~ (2-) content in the culture liquid supernatant.
Result
1. a - MEM medium than in DMEM, DMEM/F12 (2:1) prepared by BMSCs and ASCs required time shortened.
2. single layer culture: the proliferation of BMSCs and ASCs in monolayer culture was fast and cell colonies were fast, but after CM induction, the proliferation of cells was more diffuse, the adhesion was not strong, and the cells were not formed.
3. the shape of BMSCs and ASCs tended to be round, the number of cells in group B was more than that in the A group, and the number of D cells was more than that in the C group. The intake of hydroxyl proline was increased in the B group, and the intake of ~ (35) SO_4~ (2-) was increased. (35) SO_4~ (2-) and hydroxyproline acid intake and expression of ~ (35) B group SO_4~ (2-) were no difference.
conclusion
The basal cell culture of rabbit bone marrow mesenchymal stem cells (MSCs) and adipose stem cells (adipose stem cells) is more suitable for the culture of rabbit bone marrow mesenchymal stem cells and adipose stem cells. The addition of BFGF cartilage inducer can increase the metabolism of rabbit bone marrow mesenchymal stem cells and fat stem cells, and fat stem cells can be used as substitutes for seed cells of nucleus pulposus tissue engineering. The DMEM/F-12 is more suitable for the culture of rabbit bone marrow mesenchymal stem cells and adipose stem cells.

【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R329

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本文編號：1773280