逆转录病毒载体介导的白细胞介素-1受体拮抗剂和转化生长因子-β1联合基因体外转染兔膝关节软骨细胞表达的研究  

In vitro transfection of interleukin-lRa and transforming growth factor-βl mediated with retrovirus PLNCX2 into knee articular chondocytes of rabbits

作　　者：向川[1] 刘君[2] 卫小春[1] 耿俊海[1] 

机构地区：[1]山西医科大学第二医院骨科,太原030001 [2]太原市中心医院骨科

出　　处：《中华实验外科杂志》2013年第1期125-128,F0003,共5页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30500515、30371440）;山西省自然科学基金资助项目（2006021045、20041117）

摘　　要：目的运用重组逆转录病毒载体介导白细胞介素-1受体拈抗剂（IL-1Ra）和转化生长因子-β1（TGF-β1），分别和共同体外转染兔膝关节软骨细胞，观察其在关节软骨细胞中的表达及其对软骨细胞生物学性状的影响。方法构建含有目的基因的表达载体PLNCX2-IL-1Ra-GFP和PLNCX2-TGF-β1-RFP，分别和联合体外转染到兔膝关节软骨细胞，采用NO检测试剂盒检测基因转染对软骨细胞的影响，酶联免疫吸附试验（ELISA）检测细胞培养上清液中hlL-IRa和hTGF-β1的表达。结果成功构建真核表达载体PLNCX2-IL-1Ra-GFP和PLNCX2-TGF-β1-RFP并稳定转染软骨细胞；培养液NO含量检测基因转染组[hlL-1Ra：（92．15±5．36）μmol／L．；hTGF-β1：（89．71±5．43）μmol／L；hlL-1Ra＋hTGF-βl：（94．93±4．88）μmol／L]均比空载体组（60．19±4．68）μmol／L和空白组（57．23±4．29）μmol／L高，差异有统计学意义（P〈0．05），基因转染组间差异无统计学意义（P〉0．05），非基因转染组间差异无统计学意义（P〉0．05）；ELISA检测发现基因转染组均有一定量hlL-1Ra和hTGF-β1表达，空白组与空载体组均无基因表达，基因转染组与非基因转染组间比较差异有统计学意义（P〈0．05），单基因转染组和联合基因转染组问差异无统计学意义（P〉0．05）。结论逆转录病毒载体PLNCX2介导的IL-1Ra和TGF-β1能有效的转染到兔膝关节软骨细胞并获得一定程度表达，转染后的软骨细胞增生活跃。Objective A study of using interleukin （IL）- 1 Ra and transforming growth factor-βl （ TGF-β1 ） transfect knee articular chondrocytes of the rabbit in vitro respectively and commonly, and observing expression of IL- IRa and TGF-β1 in the knee articular chondrocytes of the rabbit. Methods To use expressed carrier PLNCX2-IL-1Ra-GFP and PLNCX2-TGF-β1-RFP with aim gene transfection the knee articular chondrocytes of the rabbit respectively and commonly, to detect the effect of the gene transfection on articular chondrocytes by NO-detecting kit and the expression of hIL- 1 Ra and hTGF-β1 in cell culture supemate by Elisa method. Results Enzyme digestion identification showed that PLNCX2-IL-1Ra-GFP and PLNCX2-TGF-β1-RFP were constructed successfully. After transfecting articular chondrocytes, using immunofluorescence inverted microscope can detect the green fluorescence in the TGF-β1 group, the red fluorescence in the IL-1Ra group and the green and red fluorescence in the same cell of the cotransfected group, which tested the transfection are successful. In the culture medium, the NO content-test shows that transfected group [ hIL-1Ra： （92. 15±5.36） μmoL/L;hTGF-β1 ：（89. 71 ±5.43） μmol/L;hlL-1Ra ＋ hTGF-β1 ： （94. 93 ± 4. 88 ） μmol/L ] is more than non-transfected group （ 60. 19 ± 4. 68 ）, （ 57.23 ± 4. 29） μmol/L and there was no statistical significance among transfected groups （P 〉 O. 05 ） and non- transfected group （P 〉 O. 05 ）, there was statistical significance between transfected group and non-trans- fected group （P 〈 0. 05）. Detecting expression of hIL-1 Ra and hTGF-β1 is found in the transfected group by enzyme linked immunosorbent assay （ELISA）, there was no expression in the non-transfected group and the nude carrier group. There was statistical significance between the transfected group and the non-transfected group （P 〈 0. 05）, no statistical significance between the single gene group and the cotransfected gene group （P 〉 0.

关 键 词：逆转录病毒 白细胞介素-1Ra 转化生长因子-Β1 基因转染 软骨细胞 

分 类 号：R6[医药卫生—外科学]