【摘要】：目的平板及微球培養(yǎng)純化的前軟骨干細(xì)胞(PSCs),對(duì)其進(jìn)行動(dòng)態(tài)壓應(yīng)力刺激及添加甲狀腺激素相關(guān)肽(PTHrP)干預(yù),進(jìn)一步揭示前軟骨干細(xì)胞的增殖、分化規(guī)律及其調(diào)控機(jī)制,為選擇有效干預(yù)措施治療骺板發(fā)育異常、修復(fù)及重建骺板等提供理論及實(shí)驗(yàn)依據(jù)。方法取材新生SD大鼠股骨干骺端軟骨,免疫磁珠分選純化,分別進(jìn)行二維及三維分組培養(yǎng),隨機(jī)分組后適宜壓應(yīng)力(90mmg)及添加不同濃度PTHrP干預(yù)下連續(xù)培養(yǎng),并設(shè)對(duì)照組(不干預(yù)),細(xì)胞計(jì)數(shù)及cck-8法檢測(cè)細(xì)胞增殖情況,免疫組化法測(cè)定三維培養(yǎng)軟骨細(xì)胞成軟骨特異性細(xì)胞外基質(zhì)Ⅱ型膠原(CollagenⅡ),RT-PCR法測(cè)定CollagenⅡ、聚集蛋白聚糖(Aggrecan)、PTHrP、X型膠原、TGF-β1等。根據(jù)實(shí)驗(yàn)結(jié)果評(píng)價(jià)應(yīng)力及PTHrP對(duì)前軟骨干細(xì)胞生物學(xué)性狀的影響。結(jié)果1免疫磁珠分選培養(yǎng)出生長(zhǎng)狀態(tài)良好的前軟骨干細(xì)胞,免疫組化、免疫熒光鑒定顯示細(xì)胞穩(wěn)定表達(dá)相對(duì)特異性標(biāo)志物FGFR-3與PCNA;適宜參數(shù)壓應(yīng)力刺激與PTHrP干預(yù)組細(xì)胞培養(yǎng)至5代以上仍狀態(tài)較好,而對(duì)照組細(xì)胞致5代已老化;2細(xì)胞計(jì)數(shù)及cck-8法檢測(cè)細(xì)胞增殖能力有效干預(yù)組較對(duì)照組增大(P㩳0.05)其中應(yīng)力刺激與PTHrP干預(yù)有協(xié)同效應(yīng)。3 RT-PCR檢測(cè)結(jié)果顯示有效刺激實(shí)驗(yàn)組CollagenⅡ、aggrecan、Sox9表達(dá)較空白對(duì)照組升高,X型膠原降低,(P㩳0.05)90mmg壓力刺激組PTHrP、TGF-β1、IGF-1表達(dá)水平增強(qiáng)。(P㩳0.05)結(jié)論壓應(yīng)力刺激與體外PTHrP干預(yù)均能影響PSCs的增值和分化。其中90mmg壓應(yīng)力間斷刺激組細(xì)胞增殖作用明顯,終末分化時(shí)間延長(zhǎng),可能與壓應(yīng)力導(dǎo)致細(xì)胞內(nèi)PTHrP表達(dá)增高有關(guān);小劑量(0.1nmol/L)PTHrP干預(yù)對(duì)細(xì)胞增殖有促進(jìn)作用且延遲其終末分化,Sox9可能為其靶點(diǎn);壓應(yīng)力刺激及PTHrP干預(yù)對(duì)PSCs的增殖和細(xì)胞外基質(zhì)的分泌影響有協(xié)同作用。
[Abstract]:Objective to investigate the proliferation, differentiation and regulation mechanism of prechondrocyte stem cells (PSCs) by dynamic compressive stress stimulation and (PTHrP) intervention by plate and microspheres culture. To select effective intervention measures for the treatment of epiphyseal plate abnormal development, repair and reconstruction of epiphyseal plate to provide theoretical and experimental basis. Methods the femoral metaphyseal cartilage of newborn SD rats was collected and purified by immunomagnetic beads. The samples were cultured in two and three dimensional groups respectively. After randomly grouping, it was suitable for compression stress (90mmg) and continuous culture with different concentrations of PTHrP. Cell count and cck-8 method were used to detect the proliferation of chondrocytes in the control group, and Collagen 鈪，

本文編號(hào)：2251910