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

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. 05 ）. Conclusion IL-1Ra and TGF-β1 mediated by retrovirus PLNCX2 can successfully transfect knee articular chondrocytes of the rabbit respectively and commonly and get stably expression, meanwhile the transfected cell have an active life, which provide IL-1Ra and TGF-β1 respectively and commonly treating OA with theoretical basis.