中华生物医学工程杂志
中華生物醫學工程雜誌
중화생물의학공정잡지
CHINESE JOURNAL OF BIOMEDICAL ENGINEERING
2011年
1期
9-13
,共5页
张平%刘斌%蔡道章%钟志宏%潘永谦%张振山
張平%劉斌%蔡道章%鐘誌宏%潘永謙%張振山
장평%류빈%채도장%종지굉%반영겸%장진산
白细胞介素1受体拮抗蛋白质%转化生长因子β1%软骨细胞%骨关节炎%转染
白細胞介素1受體拮抗蛋白質%轉化生長因子β1%軟骨細胞%骨關節炎%轉染
백세포개소1수체길항단백질%전화생장인자β1%연골세포%골관절염%전염
Interleukin 1 receptor antagonist protein%Transforming growth factor betal%Chondrocytes%Osteoarthritis%Transfection
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.
目的 觀察重組人白細胞介素1受體拮抗劑(IL-1Ra)和轉化生長因子β1(TGF-β1)雙基因在體外對兔骨關節炎(OA)的治療效果.方法 取新西蘭大白兔關節軟骨經酶消化分離原代培養軟骨細胞,併採用特異性細胞外基質Ⅱ型膠原免疫細胞染色進行鑒定.軟骨細胞分為轉染IL-1Ra組(A組)、轉染TGF-β1組(B組)、轉染IL-1Ra+TGF-β1雙基因組(C組)、未轉染組(D組)、空白組(E組).A、B、C 3組均以LipofectamineTM 2000 Reagent脂質體為轉染媒介.各組加入軟骨塊與軟骨細胞共培養,除E組外,其餘各組均加入20 ng IL-1β,轉染共培養後分彆在12、24 h、2、4、6 d通過熒光顯微鏡觀察軟骨細胞轉基因的錶達.于共培養第2,4、6天後應用放射免疫分析(RIA)分彆檢測各組IL-1β和腫瘤壞死因子-α(TNF-α)的含量.結果 成功分離培養軟骨細胞,Ⅱ型膠原免疫細胞染色顯示細胞胞質染成棕黃色.胞覈基本不著色.熒光顯微鏡下A、B、C組均有綠色熒光蛋白的錶達,轉染後24 h,3組細胞的轉染率最高分彆為(16.16+2.71)%、(16.54±2.91)%、(17.20±2.39)%,第2天後隨時間的延長轉染率逐漸降低.同時間點IL-1β水平D組＞B組＞A組＞C組＞E組;而第2、6天TNF-α水平D組＞A組＞B組＞C組＞E組,第4天E組＞A組＞B組＞C組＞D組,其中A組雖然略高于B組,但差異沒有統計學意義,其餘各組間差異均有統計學意義.A、B、C組的IL-1β和TNF-α在第6天的水平明顯低于第2、4天,D、E組IL-1β水平不隨時間的延長而改變,TNF-α水平D組在第4天時最低,E組則最高.結論 轉染IL-1Ra和TGF-β1基因均有降低炎性因子水平的作用.聯閤應用轉染IL-1Ra和TGF-β1基因可能控製炎性反應,為體外OA的基因治療提供實驗基礎.
목적 관찰중조인백세포개소1수체길항제(IL-1Ra)화전화생장인자β1(TGF-β1)쌍기인재체외대토골관절염(OA)적치료효과.방법 취신서란대백토관절연골경매소화분리원대배양연골세포,병채용특이성세포외기질Ⅱ형효원면역세포염색진행감정.연골세포분위전염IL-1Ra조(A조)、전염TGF-β1조(B조)、전염IL-1Ra+TGF-β1쌍기인조(C조)、미전염조(D조)、공백조(E조).A、B、C 3조균이LipofectamineTM 2000 Reagent지질체위전염매개.각조가입연골괴여연골세포공배양,제E조외,기여각조균가입20 ng IL-1β,전염공배양후분별재12、24 h、2、4、6 d통과형광현미경관찰연골세포전기인적표체.우공배양제2,4、6천후응용방사면역분석(RIA)분별검측각조IL-1β화종류배사인자-α(TNF-α)적함량.결과 성공분리배양연골세포,Ⅱ형효원면역세포염색현시세포포질염성종황색.포핵기본불착색.형광현미경하A、B、C조균유록색형광단백적표체,전염후24 h,3조세포적전염솔최고분별위(16.16+2.71)%、(16.54±2.91)%、(17.20±2.39)%,제2천후수시간적연장전염솔축점강저.동시간점IL-1β수평D조＞B조＞A조＞C조＞E조;이제2、6천TNF-α수평D조＞A조＞B조＞C조＞E조,제4천E조＞A조＞B조＞C조＞D조,기중A조수연략고우B조,단차이몰유통계학의의,기여각조간차이균유통계학의의.A、B、C조적IL-1β화TNF-α재제6천적수평명현저우제2、4천,D、E조IL-1β수평불수시간적연장이개변,TNF-α수평D조재제4천시최저,E조칙최고.결론 전염IL-1Ra화TGF-β1기인균유강저염성인자수평적작용.연합응용전염IL-1Ra화TGF-β1기인가능공제염성반응,위체외OA적기인치료제공실험기출.
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.
