本文選題：軟骨組織工程　切入點：脂肪干細胞　出處：《山東大學》2010年碩士論文

【摘要】： 目的 由于軟骨的組織學特點及其無神經和血管的營養(yǎng),在正常的生理環(huán)境中,關節(jié)軟骨缺損的修復是很困難的。應用生物相容性聚合物與細胞復合構建組織功能性軟骨以及刺激誘導軟骨形成都是修復軟骨缺損的方法。在軟骨再生中,自體軟骨細胞是很有用的,但是其作用卻受制于終末分化的軟骨細胞增殖能力和纖維性軟骨形成等因素。因此,在軟骨組織工程中,在干細胞的應用方面,開展了許多研究。近來,脂肪源性干細胞(adipose-derived stem cells, ADSCs)因其具有自我更新能力、長時間的細胞生存能力以及多向分化潛能而成為關節(jié)透明軟骨修復和再生的種子細胞。另外,ADSCs還具有能夠在局麻下從身體不同部位大量獲取的優(yōu)勢。本實驗研究的目的是通過一種創(chuàng)新的脂肪干細胞、軟骨細胞和滑膜細胞共培養(yǎng)方法,誘導兔脂肪干細胞向軟骨細胞方向分化,通過各項指標的檢測,在實踐中檢驗該共培養(yǎng)方法的可行性,并對實驗結果進行評價。 材料和方法 1.取3-4月齡健康新西蘭大白兔,3%戊巴比妥耳緣靜脈麻醉,無菌取出腎周脂肪組織,I型膠原酶酶解法分離出脂肪源性干細胞(adipose-derived stem cells, ADSCs),進行體外原代培養(yǎng),傳代后差速貼壁培養(yǎng)純化。流式細胞儀鑒定細胞表面標志CD49d,CD105,CD34,CD106的表達。 2.無菌切取3-4月齡新西蘭大白兔膝關節(jié)非負重區(qū)關節(jié)軟骨及關節(jié)內滑膜,將軟骨剪成1—3mm3薄片,先以0.25%胰蛋白酶消化30-35分鐘,再以0.1%的Ⅱ型膠原酶消化6-8小時,過濾、離心收集、計數(shù),以2×105個／ml的細胞密度接種于培養(yǎng)瓶內；將滑膜剪碎,4mg／m1Ⅰ型膠原酶消化、過濾、離心收集、計數(shù)后,以1×105個／ml的細胞密度接種于細胞培養(yǎng)瓶中。將兩種細胞分別體外常規(guī)培養(yǎng)與擴增,第三代細胞用于實驗。 3.取第三代的ADSCs,根據(jù)誘導方式不同分為三組：Ⅰ組空白對照組,不加任何誘導；Ⅱ組軟骨細胞誘導組滴加軟骨細胞誘導液(組成：TGF-β1 10n g／ml、bFGF-25ng／ml、Vc 50μg/ml、Dexamethasone10-7M／L)；Ⅲ組共培養(yǎng)組取35mm×10mm培養(yǎng)皿培養(yǎng)的第三代脂肪干細胞、軟骨細胞和滑膜細胞,將三個皿放入一個200mm×20mm培養(yǎng)皿中,成“品”字形擺放,在大皿中加入培養(yǎng)基(加10%胎牛血清)培養(yǎng)。收集三組中的脂肪干細胞,提取mRNA,反轉錄酶-聚合酶鏈鎖反應(RT-PCR)產物電泳及熒光定量PCR檢測SOX-9、Aggrecan、TypeⅡcollagen的表達。 結果 1.細胞形態(tài)學改變倒置相差顯微鏡觀察發(fā)現(xiàn),原代細胞接種后24小時可見少量細胞貼壁,呈短梭形。48小時后多數(shù)細胞已貼壁并開始伸展,分裂,出現(xiàn)由單個細胞分裂形成的集落。換液2-3次之后,大多數(shù)漂浮細胞被清除。5-7天后,細胞逐漸分裂、增殖,形成多個細胞克隆。傳代細胞貼壁快,7-8天形成單層,細胞核大,胞漿內顆粒多,呈平行或漩渦狀生長。 2.ADSCs免疫表型流式測定流式細胞學分析顯示,CD49d和CD105均呈陽性表達,CD34和CD106均呈陰性表達。 3.RT-PCR電泳及熒光定量PCR檢測：在分組培養(yǎng)后的第7天和第14天,三個目的基因中只有SOX-9、Aggrecan表達,第21天,三個目的基因都有表達。共培養(yǎng)組SOX-9和TypeⅡcollagen的表達水平均高于誘導組,且有統(tǒng)計學差異；兩組在Aggrecan的表達水平上無統(tǒng)計學差異。 結論 1.ADSCs適合成為軟骨組織工程的種子細胞。取材方便,Ⅰ型膠原酶酶解法分離可以獲取大量脂肪干細胞,體外培養(yǎng)性能穩(wěn)定,易于傳代擴增,在體外增殖多代之后仍具有完好的細胞特異性標志,并繼續(xù)保持分化的能力。 2.軟骨細胞和滑膜細胞可分泌多種生長因子如：轉化生長因子TGF-β、胰島素樣生長因子IGF等。這些因子是公認的間充質干細胞(MSCs)軟骨分化誘導因子,這些可溶性因子可能在ADSCs軟骨形成過程中發(fā)揮了重要作用。 3.本實驗所采用的脂肪干細胞、軟骨細胞和滑膜細胞共培養(yǎng)方法,可以使軟骨細胞和滑膜細胞分泌的多種生長因子對ADSCs進行誘導,使其向軟骨細胞方向分化,并具有軟骨細胞表型,誘導效果優(yōu)于外源性轉化生長因子TGF-β的誘導。
[Abstract]:Purpose



It is difficult to repair articular cartilage defects in normal physiological environment due to the histological characteristics of cartilage and the nutrition of nerve and blood vessels . In the regeneration of cartilage , many researches have been carried out on the repair and regeneration of cartilage cells . The aim of this experiment is to induce the differentiation of rabbit adipose - derived stem cells into chondrocytes from different parts of the body under local anaesthesia .



Materials and Methods



1 . The adipose - derived stem cells ( ADSCs ) were isolated from 3 - 4 month old healthy New Zealand white rabbits , 3 % Penobarbituric acid vein , and the adipose - derived stem cells ( ADSCs ) were isolated by collagenase method . The expression of CD49d , CD105 , CD34 and CD106 was identified by flow cytometry .



2 . The articular cartilage and intraarticular synovium of the knee joint of 3 - 4 month old New Zealand white rabbit knee joint were cut into 1 - 3 mm3 thin slice , digested with 0.25 % trypsin for 30 - 35 minutes , digested with 0.1 % collagenase type II collagenase for 6 - 8 hours , filtered , collected by centrifugation , counted , and inoculated into culture bottle at a density of 2 脳 105 cells / ml ;
The synovium was broken , 4 mg / ml I collagenase was digested , filtered and collected by centrifugation . After counting , the cells were inoculated into the cell culture flask at a density of 1 脳 105 cells / ml . Two kinds of cells were cultured and amplified separately in vitro and the third generation cells were used for the experiment .



3 . The third generation ADSCs were divided into three groups according to the induction method : group 鈪，

本文編號：1710798