本文選題：脂肪間充質干細胞 + 髓核細胞　； 參考：《第四軍醫(yī)大學》2010年碩士論文

【摘要】： 椎間盤(Intervertebral Disc, IVD)由軟骨終板、髓核以及纖維環(huán)組成,髓核(Nucleus pulposus)是椎間盤結構與功能的核心,包含髓核細胞和細胞外基質。人類椎間盤在生命中“第二個十年”就開始退變,出現(xiàn)細胞凋亡、基質成分改變、纖維環(huán)松弛等病理變化。而椎間盤退變是一系列脊柱退行性疾病發(fā)生的前提和始動環(huán)節(jié),特別是下腰痛發(fā)生的首要因素。如若可以控制椎間盤退變進程,就可以從一方面防止這些疾病的發(fā)生。組織工程技術是利用種子細胞結合載體、支架材料重建組織器官、修復病變的方法,這一技術的成功應用,可以從根本上解決椎間盤退變的問題。目前多選用間充質干細胞作為種子細胞,在體外以適宜的方法誘導其分化為髓核樣細胞。復合生長因子誘導以及細胞共培養(yǎng)誘導在業(yè)界均有一定研究,但二者對于脂肪干細胞向髓核樣細胞分化的效果有何不同,尚需探索。另外,本實驗把處于軟骨組織工程領域內(nèi)研究熱點的殼聚糖基可注射支架與誘導后的髓核樣細胞復合,評估其是否具備良好的相容性并維持髓核樣細胞的分化表型、保證正常的細胞外基質分泌,為“可注射支架復合髓核樣細胞微創(chuàng)修復退變椎間盤”這一臨床應用構想進行體外實驗。 實驗一選取2月齡新西蘭兔,從肩胛間脂肪組織提取脂肪干細胞并提取髓核細胞,分別進行體外單層培養(yǎng)擴增,倒置顯微鏡觀察細胞形態(tài);脂肪干細胞傳3代后,流式細胞術鑒定細胞表面抗原;將3代脂肪干細胞分為兩組,一組以成髓核誘導培養(yǎng)基誘導分化2周,另一組與髓核細胞在Transwell培養(yǎng)板中共培養(yǎng)2周,比較兩組細胞在蛋白聚糖、Ⅱ型膠原mRNA表達水平上的差異。 實驗二以兔脂肪干細胞作為種子細胞,采用實驗一中成髓核誘導培養(yǎng)基誘導為髓核樣細胞;以氯化殼聚糖(C)與β-甘油磷酸鈉(GP)構建溫敏型可注射支架。將細胞與支架混合,37℃成膠后,在成髓核誘導培養(yǎng)基中培養(yǎng)3周,通過AO-PI染色法和掃描電鏡觀測細胞存活情況,PR-PCR檢測蛋白聚糖、Ⅱ型膠原mRNA表達水平。 結果證明,生長因子誘導與共培養(yǎng)誘導對細胞在蛋白聚糖、Ⅱ型膠原mRNA表達水平上均無統(tǒng)計學差異,僅蛋白聚糖表達水平前者略高;髓核樣細胞在溫敏型殼聚糖支架上生存狀況良好,培養(yǎng)3周后細胞功能狀態(tài)(蛋白聚糖、Ⅱ型膠原mRNA表達水平)較復合培養(yǎng)前的髓核樣細胞有一定提高,其蛋白聚糖表達水平差異有統(tǒng)計學意義。
[Abstract]:Intervertebral disc (IVD) consists of endplate of cartilage, nucleus pulposus and fibrous ring. Nucleus pulposus is the core of the structure and function of intervertebral disc, including nucleus pulposus cells and extracellular matrix. In the second decade of human intervertebral disc degeneration, apoptosis, matrix composition changes, fiber ring relaxation and other pathological changes. Intervertebral disc degeneration is the premise and initiation of a series of spinal degenerative diseases, especially the primary factor of low back pain. If the process of disc degeneration can be controlled, these diseases can be prevented on the one hand. Tissue engineering is a method to reconstruct tissues and organs and repair lesions by using seed cell and carrier. The successful application of this technique can fundamentally solve the problem of disc degeneration. Mesenchymal stem cells are often used as seed cells to induce them to differentiate into nucleus pulposus like cells in vitro. The effects of co-culture and co-culture on the differentiation of adipose stem cells into nucleus pulposus cells have been studied in the industry, but it is still necessary to explore the difference between them in the differentiation of adipose stem cells into nucleus pulposus like cells. In addition, chitosan based injectable scaffolds in cartilage tissue engineering were combined with induced nucleus pulposus like cells to evaluate their compatibility and maintain the differentiation phenotype of nucleus pulposus cells. To ensure normal extracellular matrix secretion, in vitro experiments were carried out for the clinical application of "injectable scaffold combined with minimally invasive repair of degenerative intervertebral disc with nucleus pulposus cells". In experiment one, adipose stem cells were extracted from interscapular adipose tissue and nucleus pulposus cells were extracted from 2-month-old New Zealand rabbits. The third generation of adipose stem cells were divided into two groups: one group was induced to differentiate into two groups on myeloblastic medium for 2 weeks, the other group was co-cultured with nucleus pulposus cells on Transwell culture plate for 2 weeks, and the two groups were compared in proteoglycan. The difference of mRNA expression level of type 鈪，

本文編號：2054209