中华骨科杂志
中華骨科雜誌
중화골과잡지
CHINESE JOURNAL OF ORTHOPAEDICS
2014年
9期
936-944
,共9页
杜清峰%张海宁%王英振%吕成昱%刘英魁
杜清峰%張海寧%王英振%呂成昱%劉英魁
두청봉%장해저%왕영진%려성욱%류영괴
软骨细胞%慢病毒属%软骨蛋白聚糖类%RNA干扰
軟骨細胞%慢病毒屬%軟骨蛋白聚糖類%RNA榦擾
연골세포%만병독속%연골단백취당류%RNA간우
Chondrocytes%Lentivirus%Aggrecans%RNA interference
目的 探讨慢病毒介导的agggrecanase-2 shRNA对类风湿关节炎患者软骨细胞aggrecan的影响.方法 术中切取类风湿关节炎患者关节软骨,通过胰酶+Ⅱ型胶原酶两步消化法消化软骨块并培养,取第2～3代软骨细胞.以慢病毒为载体将aggrecanase-2 shRNA5～8转染进入软骨细胞,观察转染后细胞的生长变化;以荧光定量PCR检测转染后第2、5、10天aggrecanase-2 mRNA水平的变化及aggrecan mRNA水平的变化,以循环阈值(cycle threshold,Ct)表示;用免疫组织化学方法检测aggrecan蛋白水平的变化,以积分光密度(integral optical density,IOD)表示,筛选最佳抑制序列.结果 以慢病毒为载体介导aggrecanase-2 shRNA转染软骨细胞后对细胞生长速度及形态无明显影响.加入病毒液200μl、100μl、50μl后感染复数分别为100、50、25,转染效率分别为90％、60％、30％.转染后aggrecanase-2mRNA的表达水平明显下降,特别是mRNA5,由开始的0.876 3±0.115 6下降至0.069 9±0.015 1 (P＜ 0.05);aggrecan mRNA水平显著上调,由开始的0.992 1±0.201 3增加至3.049 2±0.278 2(P＜ 0.05);aggrecan蛋白表达水平显著提高,由开始的496.160 5±225.673 7增加至4 525.433 0±1 131.813 0(P＜0.05);最佳抑制序列为aggrecanase-2 shRNA5.结论 以慢病毒为载体介导aggrecanase-2shRNA转染骨关节炎患者软骨细胞可有效干扰aggrecanase-2 mRNA表达,相应地增加aggrecan表达,是一种保护aggrecan的有效途径.
目的 探討慢病毒介導的agggrecanase-2 shRNA對類風濕關節炎患者軟骨細胞aggrecan的影響.方法 術中切取類風濕關節炎患者關節軟骨,通過胰酶+Ⅱ型膠原酶兩步消化法消化軟骨塊併培養,取第2～3代軟骨細胞.以慢病毒為載體將aggrecanase-2 shRNA5～8轉染進入軟骨細胞,觀察轉染後細胞的生長變化;以熒光定量PCR檢測轉染後第2、5、10天aggrecanase-2 mRNA水平的變化及aggrecan mRNA水平的變化,以循環閾值(cycle threshold,Ct)錶示;用免疫組織化學方法檢測aggrecan蛋白水平的變化,以積分光密度(integral optical density,IOD)錶示,篩選最佳抑製序列.結果 以慢病毒為載體介導aggrecanase-2 shRNA轉染軟骨細胞後對細胞生長速度及形態無明顯影響.加入病毒液200μl、100μl、50μl後感染複數分彆為100、50、25,轉染效率分彆為90％、60％、30％.轉染後aggrecanase-2mRNA的錶達水平明顯下降,特彆是mRNA5,由開始的0.876 3±0.115 6下降至0.069 9±0.015 1 (P＜ 0.05);aggrecan mRNA水平顯著上調,由開始的0.992 1±0.201 3增加至3.049 2±0.278 2(P＜ 0.05);aggrecan蛋白錶達水平顯著提高,由開始的496.160 5±225.673 7增加至4 525.433 0±1 131.813 0(P＜0.05);最佳抑製序列為aggrecanase-2 shRNA5.結論 以慢病毒為載體介導aggrecanase-2shRNA轉染骨關節炎患者軟骨細胞可有效榦擾aggrecanase-2 mRNA錶達,相應地增加aggrecan錶達,是一種保護aggrecan的有效途徑.
목적 탐토만병독개도적agggrecanase-2 shRNA대류풍습관절염환자연골세포aggrecan적영향.방법 술중절취류풍습관절염환자관절연골,통과이매+Ⅱ형효원매량보소화법소화연골괴병배양,취제2～3대연골세포.이만병독위재체장aggrecanase-2 shRNA5～8전염진입연골세포,관찰전염후세포적생장변화;이형광정량PCR검측전염후제2、5、10천aggrecanase-2 mRNA수평적변화급aggrecan mRNA수평적변화,이순배역치(cycle threshold,Ct)표시;용면역조직화학방법검측aggrecan단백수평적변화,이적분광밀도(integral optical density,IOD)표시,사선최가억제서렬.결과 이만병독위재체개도aggrecanase-2 shRNA전염연골세포후대세포생장속도급형태무명현영향.가입병독액200μl、100μl、50μl후감염복수분별위100、50、25,전염효솔분별위90％、60％、30％.전염후aggrecanase-2mRNA적표체수평명현하강,특별시mRNA5,유개시적0.876 3±0.115 6하강지0.069 9±0.015 1 (P＜ 0.05);aggrecan mRNA수평현저상조,유개시적0.992 1±0.201 3증가지3.049 2±0.278 2(P＜ 0.05);aggrecan단백표체수평현저제고,유개시적496.160 5±225.673 7증가지4 525.433 0±1 131.813 0(P＜0.05);최가억제서렬위aggrecanase-2 shRNA5.결론 이만병독위재체개도aggrecanase-2shRNA전염골관절염환자연골세포가유효간우aggrecanase-2 mRNA표체,상응지증가aggrecan표체,시일충보호aggrecan적유효도경.
Objective To investigate the effects of aggrecanase-2 knockdown in chondrocytes from rheumatoid arthritis patient by shRNA infection.Methods Cartilage harvested from rheumatoid arthritis patients who underwent total knee arthroplasty was digested by pancreatin and type Ⅱ collagen enzyme to obtain chondrocytes.Then chondrocytes were cultured and passaged to second or third generation.After aggrecanase-2 shRNA5,aggrecanase-2 shRNA6,aggrecanase-2 shRNA7,aggrecanase-2 shRNA8 infection,growth and morphological changes of the chondrocytes were examined.To select the best target sequence,mRNA expression of aggrecanase-2 and aggrecan was detected by RT-qPCR assay on day 2,5,10,represented with Ct (Cycle threshold) value.Expression of aggrecan protein was detected by immunocytochemistry,represented with IOD (integral optical density) value.Results aggrecanase-2 knockdown had no obvious effects on the morphology and growth of the chondrocytes.MOI (multiplicity of infection) was 100,50,25,and infection efficiency was 90％,60％,30％ with the corresponding viral load of 200 μl,100 μl,50 μl after transfection.mRNA expression of aggrecanase-2 was suppressed significantly,especially the group of shRNA5 which suppressed aggrecanase-2 expression from 0.876 3±0.115 6 to 0.069 9±0.015 1 (P ＜ 0.05).mRNA and protein expression of aggrecan were significantly upregulated after infection.mRNA expression of aggrecan increased from 0.992 1±0.201 3 to 3.049 2±0.278 2 (P ＜ 0.05) and protein expression of aggrecan increased from 496.160 5± 225.673 7 to 4 525.433 0±1 131.813 0 (P ＜ 0.05).Conclusion aggrecanase-2 suppression in chondrocytes by lentivirius infection is an effective method to protect the expression of aggrecan.
