本文選題：低氧誘導因子α　切入點：終板軟骨細胞　出處：《中國修復重建外科雜志》2017年03期 　論文類型：期刊論文

【摘要】：目的探討低氧誘導因子1α(hypoxia-inducible factor 1α,HIF-1α)基因在大鼠終板軟骨細胞中的表達情況及其與終板軟骨細胞凋亡的關系。方法取8只10周齡SPF級雄性SD大鼠L1~5腰椎終板軟骨組織,采用酶消化法分離培養(yǎng)終板軟骨細胞并傳代,取第3代細胞進行實驗。首先將終板軟骨細胞分為兩組,分別于20%O_2常氧條件下(A組)以及0.5%O_2低氧下條件(B組)培養(yǎng);培養(yǎng)24 h,倒置相差顯微鏡觀察細胞形態(tài),流式細胞儀檢測細胞凋亡率,實時熒光定量PCR檢測HIF-1α基因表達,Western blot檢測凋亡蛋白HIF-1α、Bax、Bcl-2表達。然后,構建Lipofectamin~(TM)2000載體試劑和HIF-1αsi RNA/Lipofectamin~(TM)2000載體混合劑分別轉染終板軟骨細胞后,于0.5%O_2低氧條件下培養(yǎng)(分別為C、D組)。培養(yǎng)24 h后,倒置相差顯微鏡下觀察細胞形態(tài),行HIF-1α免疫熒光染色觀察,流式細胞儀檢測細胞凋亡率,實時熒光定量PCR檢測HIF-1α、Ⅱ型膠原、蛋白多糖、SOX9基因表達,Western blot檢測凋亡蛋白HIF-1α、Bax、Bcl-2表達。結果培養(yǎng)24 h后,A、B組均見少量空泡壞死細胞;細胞凋亡率比較差異無統(tǒng)計學意義(t=1.026,P=0.471);B組HIF-1α基因及蛋白相對表達量顯著高于A組(t=22.672,P=0.015;t=18.396,P=0.013);兩組Bax、Bcl-2蛋白表達比較,差異均無統(tǒng)計學意義(t=0.594,P=0.781;t=1.251,P=0.342)。低氧培養(yǎng)24 h后,D組空泡壞死細胞較C組增多,且可見大量HIF-1α陽性終板軟骨細胞;與C組相比,D組細胞凋亡率顯著增高(t=27.143,P=0.002),D組HIF-1α、Ⅱ型膠原、蛋白多糖、SOX9基因表達較C組顯著降低(t=21.097,P=0.015;t=34.829,P=0.002;t=18.673,P=0.022;t=31.949,P=0.007),HIF-1α、Bcl-2蛋白相對表達量較C組均顯著降低(t=37.648,P=0.006;t=16.729,P=0.036),而Bax蛋白表達較C組提高(t=25.583,P=0.011)。結論缺氧條件下,終板軟骨細胞中HIF-1α表達上調,以提高細胞耐氧性;HIF-1α可抑制終板軟骨細胞在低氧環(huán)境中發(fā)生凋亡。
[Abstract]:Objective to investigate the expression of hypoxia-inducible factor 1 偽 (HIF-1 偽) gene in rat endplate chondrocytes and its relationship with endplate chondrocyte apoptosis. The endplate chondrocytes were isolated and subcultured by enzyme digestion method. The third passage of chondrocytes was carried out. Firstly, the endplate chondrocytes were divided into two groups: group A (group A) and group B (group B under hypoxia). After 24 h culture, cell morphology was observed by inverted phase contrast microscope, apoptosis rate was detected by flow cytometry, HIF-1 偽 gene expression was detected by real-time fluorescence quantitative PCR and the apoptotic protein HIF-1 偽 Baxbumin Bcl-2 was detected by Western blot. Lipofectamin~(TM)2000 vector reagent and HIF-1 偽 si RNA/Lipofectamin~(TM)2000 vector mixture were constructed and transfected into endplate chondrocytes respectively, and cultured under 0. 5% O\ -2 hypoxia (group C ~ (2) D respectively). After 24 h culture, cell morphology was observed under inverted phase contrast microscope and observed by HIF-1 偽 immunofluorescence staining. Apoptosis rate was detected by flow cytometry, HIF-1 偽, type 鈪，

本文編號：1601605