本文選題：干細胞 + 脂肪組織�。� 參考：《中國醫(yī)科大學》2008年碩士論文

【摘要】： 實驗目的 探討從大鼠的脂肪組織中分離、培養(yǎng)脂肪干細胞(ADSCs)的方法及鑒定方法。并對在單層培養(yǎng)條件下用外源性轉化生長因子TGF-β1定向誘導ADSCs分化為軟骨細胞的可行性進行初步的探討研究。為軟骨組織工程研究中的種子細胞的選擇提供一個新的思路。 實驗方法 Wistar雄性大鼠處死,取腹股溝區(qū)及睪丸脂肪墊等處脂肪組織,清除小血管和結締組織充分剪碎,加入0.15%的Ⅰ型膠原酶消化,離心后收集細胞置于孵箱中培養(yǎng)。2-3天換液一次去除未貼壁細胞,待貼壁細胞生長融合至80-90%時傳代培養(yǎng)。應用倒置相差顯微鏡觀察各代ADSCs形態(tài)學的改變,并用免疫熒光法檢測傳代ADSCs表面抗原CD29表達。取第3代生長良好的脂肪干細胞,分成兩組:一組用含有TGF-β_1的培養(yǎng)基定向誘導培養(yǎng),另一組為對照組(空白組)用普通培養(yǎng)基繼續(xù)培養(yǎng),誘導兩周后行Ⅱ型膠原免疫組化染色進行檢測鑒定。 結果 1、脂肪干細胞的形態(tài)特點:原代培養(yǎng)的脂肪干細胞24小時左右即可見細胞貼壁,多數貼壁細胞呈小圓形,折光性強,其中散在少量的梭形細胞或多角細胞。3d后梭形細胞逐漸增多,可見核分裂相。5d后細胞呈團簇狀生長,并形成集落。約7d左右細胞融合超過90%時進行傳代,傳代后細胞形態(tài)較為均一,呈長梭形,排列緊密,類似成纖維細胞。 2、脂肪干細胞的鑒定:免疫熒光法檢測脂肪干細胞(ADSCs)。結果顯示細胞表面抗原CD29呈陽性表達,從而證實所培養(yǎng)細胞為干細胞來源。 3、ADSCs向軟骨細胞的定向分化:實驗組細胞生長速度變慢,誘導第2天即可見少數單個細胞由梭形變?yōu)槎噙呅?多角形,可見細長偽足,核周出現黑色顆粒,隨誘導時間延長,形態(tài)發(fā)生如前變化的細胞逐漸增多。誘導第14天左右部分細胞開始變圓,細胞邊界不清,核略呈偏位,核周顆粒密集,細胞逐漸聚集成團。 4、誘導后細胞Ⅱ型膠原免疫組化檢測:實驗組在TGF-β1向軟骨表型定向誘導后14d,Ⅱ型膠原免疫組化染色結果顯示有部分細胞呈陽性,可見棕黃色顆粒分布于細胞胞漿內而對照組則為陰性。 結論 1、表面抗原CD29表達陽性,證實大鼠脂肪組織中存在干細胞來源細胞。 2、從大鼠脂肪組織中可以分離出脂肪干細胞(ADSCs),并且細胞在體外培養(yǎng)條件下生物學性狀保持穩(wěn)定。 3、外源性轉化生長因子TGF-β定向分化、定向誘導連續(xù)培養(yǎng)14d后,Ⅱ型膠原免疫組織化學染色呈陽性反應,表現出來軟骨細胞的部分特性。證實脂肪干細胞具有向軟骨細胞定向分化的能力。
[Abstract]:Experimental purpose To study the method and identification of adipose stem cells isolated and cultured from adipose tissue of rats. The feasibility of inducing ADSCs to differentiate into chondrocytes by transforming growth factor TGF- 尾 1 in monolayer culture was studied. It provides a new idea for the selection of seed cells in cartilage tissue engineering. Experimental method The male Wistar rats were killed, the fat tissues in the inguinal region and testis fat pad were removed, the small vessels and connective tissues were cut and digested with 0.15% type I collagenase. After centrifugation, the collected cells were cultured in incubators for 2 to 3 days. The unadherent cells were removed once for 2-3 days. When the adherent cells were fused to 80-90%, the cells were subcultured. The morphologic changes of ADSCs were observed by inverted phase contrast microscope and the expression of ADSCs surface antigen CD29 was detected by immunofluorescence. The third generation of adipose stem cells with good growth was divided into two groups: one group was cultured on the medium containing TGF- 尾 1, the other group was the control group (blank group) and the control group (blank group). Two weeks after induction, type 鈪，

本文編號：1930156