本文選題：微小RNA- + 殼聚糖　； 參考：《中國修復重建外科雜志》2017年10期

【摘要】：目的探討核定位信號肽偶聯(lián)核激酶底物短肽(nucleus localization signal linked nucleic kinase substrate short peptide,NNS)修飾殼聚糖(chitosan,CS)(~(NNS)CS),介導人微小RNA-140(micro RNA-140,miR-140)基因轉(zhuǎn)染,對體外培養(yǎng)的兔關(guān)節(jié)軟骨細胞的作用。方法將重組質(zhì)粒GV268-miR-140和空質(zhì)粒GV268分別與~(NNS)CS復合形成~(NNS)CS/pDNA納米復合物。取新生新西蘭大耳白兔膝關(guān)節(jié)軟骨,采用胰蛋白酶和膠原酶聯(lián)合消化法分離培養(yǎng)原代軟骨細胞。取第2代軟骨細胞分為3組:正常細胞對照組(A組)、~(NNS)CS/GV268空質(zhì)粒轉(zhuǎn)染組(B組)、~(NNS)CS/GV268-miR-140轉(zhuǎn)染組(C組),B、C組細胞分別以~(NNS)CS/GV268及~(NNS)CS/GV268-miR-140納米復合物瞬時轉(zhuǎn)染。轉(zhuǎn)染后,實時熒光定量PCR(real-time fluorescent quantitative PCR,RT-qPCR)檢測外源mi R-140的表達;AnnexinⅤ-FITC/PI雙染色法及MTT法分別檢測外源miR-140對軟骨細胞凋亡及增殖活力的影響;RT-qPCR檢測軟骨細胞中Sox9、聚集蛋白聚糖(Aggrecan)、組蛋白去乙�；�4(histone deacetylase 4,Hdac4)基因表達。結(jié)果 RT-qPCR檢測示,C組外源miR-140表達水平較A、B組明顯上調(diào)(P0.05)。與A、B組比較,C組軟骨細胞凋亡率明顯降低,細胞增殖活力明顯增加,細胞內(nèi)Sox9、Aggrecan基因相對表達量明顯上調(diào)、Hdac4基因相對表達量明顯下調(diào)(P0.05);A、B組間以上指標比較,差異均無統(tǒng)計學意義(P0.05)。結(jié)論 ~(NNS)CS可攜帶外源基因進入軟骨細胞并高效表達,高表達的miR-140能提高體外培養(yǎng)軟骨細胞的生物活性,為其用于治療軟骨損傷性疾病提供了實驗依據(jù)。
[Abstract]:Objective to investigate the effect of nuclear localization signal peptide coupled nuclear kinase substrates localization signal linked nucleic kinase substrate short peptiden (NNSN) on rabbit articular chondrocytes cultured in vitro, and to mediate the transfection of human minimal RNA-140 micro RNA-140 miR-140 gene into chitosan chondrocytes. Methods the recombinant plasmids GV268-miR-140 and empty plasmids GV268 were combined with NNSCS to form the NNSN CSP / pDNA nanocomplexes. The articular cartilage of newborn New Zealand rabbits was isolated and cultured by trypsin and collagenase digestion. The second passage of chondrocytes were divided into three groups: normal control group (group A) was transfected with NNSCSP / GV268 empty plasmid, group B was transfected with NNSCSP / GV268-miR-140, group C was transfected with NNSCSP / GV268-miR-140 and group C was transiently transfected with NNSCSP / GV268-miR-140 nanocomplex, respectively. After transfection, Real-time fluorescent quantitative PCR RT-qPCR The expression of exogenous mi R-140 was detected by Annexin 鈪，

本文編號：2027587