大鼠骨髓來(lái)源與前交叉韌帶來(lái)源MSCs體外生物學(xué)特性比較研究
發(fā)布時(shí)間：2019-02-20 21:55
【摘要】：目的比較大鼠骨髓來(lái)源與前交叉韌帶(anterior cruciate ligament,ACL)來(lái)源的MSCs體外增殖及成骨、成軟骨、成脂分化潛能的差異。方法取SPF級(jí)6周齡雄性BN大鼠10只,體質(zhì)量200~220 g,無(wú)菌條件下分別取大鼠骨髓及ACL,貼壁法培養(yǎng)獲取BMSCs與ACL來(lái)源MSCs并進(jìn)行傳代,倒置相差顯微鏡下觀察細(xì)胞形態(tài)變化。取生長(zhǎng)良好的第3代細(xì)胞,流式細(xì)胞儀檢測(cè)細(xì)胞免疫表型CD34、CD45、CD90和CD29表達(dá);細(xì)胞計(jì)數(shù)試劑盒8(cell counting kit 8,CCK-8)法檢測(cè)細(xì)胞體外增殖能力,克隆形成實(shí)驗(yàn)檢測(cè)細(xì)胞體外克隆形成能力;行成骨、成軟骨及成脂體外誘導(dǎo)多向分化能力檢測(cè);實(shí)時(shí)熒光定量PCR檢測(cè)成骨[ALP、骨鈣蛋白、RUNX2、BMP-2、分泌性磷蛋白1(secreted phosphoprotein 1,Spp1)]、成軟骨[Ⅱ型膠原α1(collagen typeⅡα1,Col2α1)、軟骨聚糖蛋白(Aggrecan,Acan)、Sox9]及成脂[過(guò)氧化物酶體增殖物激活受體γ2(peroxisome proliferator activated receptorγ2,PPARγ2)、CCAAT/增強(qiáng)子結(jié)合蛋白α]相關(guān)基因mRNA相對(duì)表達(dá)量。結(jié)果第3代ACL來(lái)源MSCs與BMSCs形態(tài)相似,均表現(xiàn)為貼壁生長(zhǎng)的長(zhǎng)梭形細(xì)胞。流式細(xì)胞儀檢測(cè)示,兩種細(xì)胞均表達(dá)CD29、CD90,基本不表達(dá)CD45及CD34。CCK-8法檢測(cè)示,ACL來(lái)源MSCs的吸光度(A)值(1.11±0.08)顯著高于BMSCs(0.78±0.05),差異有統(tǒng)計(jì)學(xué)意義(t=3.599,P=0.023);ACL來(lái)源MSCs的細(xì)胞集落數(shù)[(53.00±5.51)個(gè)/孔]亦明顯多于BMSCs[(30.67±4.84)個(gè)/孔](t=3.045,P=0.038)。經(jīng)成骨、成軟骨、成脂體外誘導(dǎo)培養(yǎng)21d后,BMSCs及ACL來(lái)源MSCs均表現(xiàn)為茜素紅、甲苯胺藍(lán)及油紅O染色陽(yáng)性。實(shí)時(shí)熒光定量PCR檢測(cè)示,ACL來(lái)源MSCs的BMP-2、Spp1、Col2α1、Acan、Sox9及PPARγ2 mRNA相對(duì)表達(dá)量顯著高于BMSCs,差異均有統(tǒng)計(jì)學(xué)意義(P0.01)。結(jié)論 ACL來(lái)源MSCs比BMSCs具有更強(qiáng)的體外增殖能力,在相同體外條件下更易向軟骨分化,是一種有潛力的促進(jìn)腱骨愈合的種子細(xì)胞。
[Abstract]:Objective to compare the proliferation, osteogenesis, cartilage formation and adipogenic potential of MSCs derived from rat bone marrow and anterior cruciate ligament (anterior cruciate ligament,ACL) in vitro. Methods 10 male BN rats of SPF grade 6 weeks old, weighing 200 ~ 220g, were harvested from bone marrow of rats under aseptic condition and cultured with ACL, adherent method to obtain MSCs derived from BMSCs and ACL. The morphologic changes of cells were observed under inverted phase contrast microscope. The expression of CD34,CD45,CD90 and CD29 was detected by flow cytometry. Cell count kit 8 (cell counting kit 8 CCK-8) was used to detect the ability of cell proliferation in vitro, clone formation test to detect the ability of cell clone formation in vitro, osteogenesis, cartilage formation and fat-forming ability to induce multidirectional differentiation in vitro. Osteogenesis [ALP, osteocalcin, RUNX2,BMP-2, secreted phosphoprotein 1 (Spp1)], chondrogenic cartilage (type 鈪，
本文編號(hào)：2427324
[Abstract]:Objective to compare the proliferation, osteogenesis, cartilage formation and adipogenic potential of MSCs derived from rat bone marrow and anterior cruciate ligament (anterior cruciate ligament,ACL) in vitro. Methods 10 male BN rats of SPF grade 6 weeks old, weighing 200 ~ 220g, were harvested from bone marrow of rats under aseptic condition and cultured with ACL, adherent method to obtain MSCs derived from BMSCs and ACL. The morphologic changes of cells were observed under inverted phase contrast microscope. The expression of CD34,CD45,CD90 and CD29 was detected by flow cytometry. Cell count kit 8 (cell counting kit 8 CCK-8) was used to detect the ability of cell proliferation in vitro, clone formation test to detect the ability of cell clone formation in vitro, osteogenesis, cartilage formation and fat-forming ability to induce multidirectional differentiation in vitro. Osteogenesis [ALP, osteocalcin, RUNX2,BMP-2, secreted phosphoprotein 1 (Spp1)], chondrogenic cartilage (type 鈪，
本文編號(hào)：2427324
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