本文選題：脂肪間充質(zhì)干細(xì)胞 + 髓核細(xì)胞�。� 參考：《第四軍醫(yī)大學(xué)》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(jié)構(gòu)與功能的核心,包含髓核細(xì)胞和細(xì)胞外基質(zhì)。人類椎間盤在生命中“第二個十年”就開始退變,出現(xiàn)細(xì)胞凋亡、基質(zhì)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(shù)是利用種子細(xì)胞結(jié)合載體、支架材料重建組織器官、修復(fù)病變的方法,這一技術(shù)的成功應(yīng)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(zhì)干細(xì)胞作為種子細(xì)胞,在體外以適宜的方法誘導(dǎo)其分化為髓核樣細(xì)胞。復(fù)合生長因子誘導(dǎo)以及細(xì)胞共培養(yǎng)誘導(dǎo)在業(yè)界均有一定研究,但二者對于脂肪干細(xì)胞向髓核樣細(xì)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(lǐng)域內(nèi)研究熱點的殼聚糖基可注射支架與誘導(dǎo)后的髓核樣細(xì)胞復(fù)合,評估其是否具備良好的相容性并維持髓核樣細(xì)胞的分化表型、保證正常的細(xì)胞外基質(zhì)分泌,為“可注射支架復(fù)合髓核樣細(xì)胞微創(chuàng)修復(fù)退變椎間盤”這一臨床應(yīng)用構(gòu)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(xì)胞并提取髓核細(xì)胞,分別進行體外單層培養(yǎng)擴增,倒置顯微鏡觀察細(xì)胞形態(tài);脂肪干細(xì)胞傳3代后,流式細(xì)胞術(shù)鑒定細(xì)胞表面抗原;將3代脂肪干細(xì)胞分為兩組,一組以成髓核誘導(dǎo)培養(yǎng)基誘導(dǎo)分化2周,另一組與髓核細(xì)胞在Transwell培養(yǎng)板中共培養(yǎng)2周,比較兩組細(xì)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(xì)胞作為種子細(xì)胞,采用實驗一中成髓核誘導(dǎo)培養(yǎng)基誘導(dǎo)為髓核樣細(xì)胞;以氯化殼聚糖(C)與β-甘油磷酸鈉(GP)構(gòu)建溫敏型可注射支架。將細(xì)胞與支架混合,37℃成膠后,在成髓核誘導(dǎo)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(xì)胞存活情況,PR-PCR檢測蛋白聚糖、Ⅱ型膠原mRNA表達水平。 結(jié)果證明,生長因子誘導(dǎo)與共培養(yǎng)誘導(dǎo)對細(xì)胞在蛋白聚糖、Ⅱ型膠原mRNA表達水平上均無統(tǒng)計學(xué)差異,僅蛋白聚糖表達水平前者略高;髓核樣細(xì)胞在溫敏型殼聚糖支架上生存狀況良好,培養(yǎng)3周后細(xì)胞功能狀態(tài)(蛋白聚糖、Ⅱ型膠原mRNA表達水平)較復(fù)合培養(yǎng)前的髓核樣細(xì)胞有一定提高,其蛋白聚糖表達水平差異有統(tǒng)計學(xué)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