转染白细胞介素1受体拮抗剂与转化生长因子β1软骨细胞和致炎软骨块共培养后炎性因子的变化

Change of inflammatory cytokines after co-culture of inflamed cartilage blocks and chondrocytes transfected by interleukin-β1 receptor antagonist gene and transforming growth factor-β1 gene in vitro

目的 观察重组人白细胞介素1受体拮抗剂（IL-1Ra）和转化生长因子β1（TGF-β1）双基因在体外对兔骨关节炎（OA）的治疗效果.方法 取新西兰大白兔关节软骨经酶消化分离原代培养软骨细胞,并采用特异性细胞外基质Ⅱ型胶原免疫细胞染色进行鉴定.软骨细胞分为转染IL-1Ra组（A组）、转染TGF-β1组（B组）、转染IL-1Ra＋TGF-β1双基因组（C组）、未转染组（D组）、空白组（E组）.A、B、C 3组均以LipofectamineTM 2000 Reagent脂质体为转染媒介.各组加入软骨块与软骨细胞共培养,除E组外,其余各组均加入20 ng IL-1β,转染共培养后分别在12、24 h、2、4、6 d通过荧光显微镜观察软骨细胞转基因的表达.于共培养第2,4、6天后应用放射免疫分析（RIA）分别检测各组IL-1β和肿瘤坏死因子-α（TNF-α）的含量.结果 成功分离培养软骨细胞,Ⅱ型胶原免疫细胞染色显示细胞胞质染成棕黄色.胞核基本不着色.荧光显微镜下A、B、C组均有绿色荧光蛋白的表达,转染后24 h,3组细胞的转染率最高分别为（16.16＋2.71）%、（16.54±2.91）%、（17.20±2.39）%,第2天后随时间的延长转染率逐渐降低.同时间点IL-1β水平D组＞B组＞A组＞C组＞E组;而第2、6天TNF-α水平D组＞A组＞B组＞C组＞E组,第4天E组＞A组＞B组＞C组＞D组,其中A组虽然略高于B组,但差异没有统计学意义,其余各组间差异均有统计学意义.A、B、C组的IL-1β和TNF-α在第6天的水平明显低于第2、4天,D、E组IL-1β水平不随时间的延长而改变,TNF-α水平D组在第4天时最低,E组则最高.结论 转染IL-1Ra和TGF-β1基因均有降低炎性因子水平的作用.联合应用转染IL-1Ra和TGF-β1基因可能控制炎性反应,为体外OA的基因治疗提供实验基础.

Objective To evaluate the in vitro effects of recombinant human interluekin-β1 receptor antagonist（IL-1Ra） gene and transforming growth factor-β1 （TGF-β1） gene on rabbit osteoarthritis （OA）.Methods Articular cartilages were extracted from mature New Zealand rabbits and by enzyme digestion,isolated for chondrocytes which were then identified with specific extracellular matrix collagen type Ⅱ stained immunocytochemistry.The chondrocytes were divided into IL-1Ra-transfected group （group A）, TGF-β1？transfected group （group B） , combined IL-1Ra- and TGF-β1-transfected group （group C） , untransfected group （group D） and the blank control group （group E）.LipofectamineTM 2000 Reagent was used as the vehicle for transfection among groups A, B and C.All the groups of chondrocytes were co-cultured with fragmented articular cartilages and added with 20 ng IL-β 1？expect for group E.The transgenic expression of chondrocytes was detected under fluorescence microscope at 12h,24h,2d,4d and 6 d after transfection and co-culture.In addition, radioimmunoassay （RIA） was used to determine the levels of IL-1βand TNF-α in each group at 2 d, 4 d and 6 d after transfection and co-culture.Results The chondrocytes were successfully isolated and cultured.Collagen type Ⅱ stained immunocytochemistry showed the brownish - yellow cytoplasm and unstained chromophobic nuclei.Under fluorescence microscope, the expression of enhanced green fluorescent protein was observed in groups A, BandC, which peaked at 24 hours after transfection （16.16±2.71）% vs （16.54±2.91）% vs （17.20±2.39）% and gradually declined 2 d later.At any time spots, the IL-1βevel was highest in group D, followed by group B, group A, group C, and group E.The level of TNF-a in each group was ordered by group D＞group A＞group B＞group C＞group E on days 2 and 6, and by group E＞group A＞group B＞group C＞group D on day 4.The level of TNF-α in group A was slightly higher than that of group B, but the difference was not statistical significance.There were statistical difference among the other groups.The expressions of IL-1βand TNF-α in groups A, B and C were significantly lower on day 6 than those on days 2 and 4.The level of IL-1βin groups D and E did not change with time, while the level of TNF -α was the lowest in group D and highest in group E on day 4.ConclusionsTransfection with IL-1Ra or TGF-β1 can reduce the level of inflammatory cytokines.Combined use of IL-1Ra and TGF-β1 genes may show control of inflammatory response and provide evidences for gene therapy of osteoarthritis.