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

【摘要】： 實(shí)驗(yàn)?zāi)康?探討從大鼠的脂肪組織中分離、培養(yǎng)脂肪干細(xì)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(zhuǎn)化生長因子TGF-β1定向誘導(dǎo)ADSCs分化為軟骨細(xì)胞的可行性進(jìn)行初步的探討研究。為軟骨組織工程研究中的種子細(xì)胞的選擇提供一個新的思路。 實(shí)驗(yàn)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(jié)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(xì)胞置于孵箱中培養(yǎng)。2-3天換液一次去除未貼壁細(xì)胞,待貼壁細(xì)胞生長融合至80-90%時(shí)傳代培養(yǎng)。應(yīng)用倒置相差顯微鏡觀察各代ADSCs形態(tài)學(xué)的改變,并用免疫熒光法檢測傳代ADSCs表面抗原CD29表達(dá)。取第3代生長良好的脂肪干細(xì)胞,分成兩組:一組用含有TGF-β_1的培養(yǎng)基定向誘導(dǎo)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(dǎo)兩周后行Ⅱ型膠原免疫組化染色進(jìn)行檢測鑒定。 結(jié)果 1、脂肪干細(xì)胞的形態(tài)特點(diǎn):原代培養(yǎng)的脂肪干細(xì)胞24小時(shí)左右即可見細(xì)胞貼壁,多數(shù)貼壁細(xì)胞呈小圓形,折光性強(qiáng),其中散在少量的梭形細(xì)胞或多角細(xì)胞。3d后梭形細(xì)胞逐漸增多,可見核分裂相。5d后細(xì)胞呈團(tuán)簇狀生長,并形成集落。約7d左右細(xì)胞融合超過90%時(shí)進(jìn)行傳代,傳代后細(xì)胞形態(tài)較為均一,呈長梭形,排列緊密,類似成纖維細(xì)胞。 2、脂肪干細(xì)胞的鑒定:免疫熒光法檢測脂肪干細(xì)胞(ADSCs)。結(jié)果顯示細(xì)胞表面抗原CD29呈陽性表達(dá),從而證實(shí)所培養(yǎng)細(xì)胞為干細(xì)胞來源。 3、ADSCs向軟骨細(xì)胞的定向分化:實(shí)驗(yàn)組細(xì)胞生長速度變慢,誘導(dǎo)第2天即可見少數(shù)單個細(xì)胞由梭形變?yōu)槎噙呅?多角形,可見細(xì)長偽足,核周出現(xiàn)黑色顆粒,隨誘導(dǎo)時(shí)間延長,形態(tài)發(fā)生如前變化的細(xì)胞逐漸增多。誘導(dǎo)第14天左右部分細(xì)胞開始變圓,細(xì)胞邊界不清,核略呈偏位,核周顆粒密集,細(xì)胞逐漸聚集成團(tuán)。 4、誘導(dǎo)后細(xì)胞Ⅱ型膠原免疫組化檢測:實(shí)驗(yàn)組在TGF-β1向軟骨表型定向誘導(dǎo)后14d,Ⅱ型膠原免疫組化染色結(jié)果顯示有部分細(xì)胞呈陽性,可見棕黃色顆粒分布于細(xì)胞胞漿內(nèi)而對照組則為陰性。 結(jié)論 1、表面抗原CD29表達(dá)陽性,證實(shí)大鼠脂肪組織中存在干細(xì)胞來源細(xì)胞。 2、從大鼠脂肪組織中可以分離出脂肪干細(xì)胞(ADSCs),并且細(xì)胞在體外培養(yǎng)條件下生物學(xué)性狀保持穩(wěn)定。 3、外源性轉(zhuǎn)化生長因子TGF-β定向分化、定向誘導(dǎo)連續(xù)培養(yǎng)14d后,Ⅱ型膠原免疫組織化學(xué)染色呈陽性反應(yīng),表現(xiàn)出來軟骨細(xì)胞的部分特性。證實(shí)脂肪干細(xì)胞具有向軟骨細(xì)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