以慢病毒为载体介导aggrecanase-2 shRNA转染类风湿关节炎患者软骨细胞对aggrecanase-2 mRNA表达的干扰作用

Effect of aggrecanase-2 shRNA transfecting chondrocytes mediated by slow virus on interference of the expression of aggrecanase-2 mRNA in RA patients

目的研究以慢病毒为载体介导aggrecanase-2 shRNA转染类风湿关节炎(RA)患者软骨细胞对aggrecanase-2 mRNA表达的干扰作用。方法回顾性分析2015~2016年确诊为活动期或者是中晚期的RA患者30例,并在关节置换术中切除患者膝关节的软骨块,冲洗软骨块,并使用0.25%胰酶和0.20%的Ⅱ型胶原酶模拟体温在36.8℃的恒温水浴箱内让其自行消化25~35 min,离心,取其沉淀并加入一定量的10.00%胎牛血清的DMEM培养基内培养,最终取其培养的第2~3代软骨组织。将设计好的慢病毒载体介导的aggrecanase-2 shRNA转染进RA患者的软骨细胞中。按照慢病毒剂量,将细胞分为200μl(A组)、100μl(B组)、50μl(C组)。其中,A组分为空白对照组、阴性对照组、aggrecanase-2 shRNA1组(shRNA1组)、aggrecanase-2 shRNA2组(shRNA2组)、aggrecanase-2 shRNA3组(shRNA3组)、aggrecanase-2 shRNA4组(shRNA4组)。各孔加入促转染剂约3~7μm/ml,其中阴性对照为含杂乱序列的病毒液,空白对照组则用培养基来代替病毒。利用PCR技术定量检测转染之后由各种转染复数转染的aggrecanase-2和aggrecanase mRNA在各种的时间段内的表达系数。结果加入慢病毒之后的A、B、C三组RA患者的感染复数分别为100、50、25,转染效率分别为91.21%、59.98%、31.09%。以慢病毒为载体介导aggrecanase-2 shRNA转染的RA患者的软骨细胞的aggrecanase-2 mRNA的表达水平明显有所降低,差异具有统计学意义(P<0.05)。随着时间的增加,各组aggrecan蛋白表达水平出现明显的变化,其中shRNA4组升高最为明显,差异具有统计学意义(P<0.05)。结论以慢病毒为载体介导的aggrecanase-2 shRNA转染的RA患者的软骨细胞中的aggrecanase-2 mRNA的表达受到明显的抑制,但是其对RA患者的软骨组织没有实质上和功能上的伤害;同时其还可以通过抑制aggrecanase-2 mRNA的表达来促进aggrecan蛋白的表达,从而起到保护软骨组织退化的作用。

Objective To study effect of aggrecanase-2 shRNA transfecting chondrocytes mediated by slow virus on interference of the expression of aggrecanase-2 mRNA in RA patients. Methods 30 patients diagnosed as RA in activity or middle or late stage were retrospectively analyzed in our hospital from 2015 to 2016. Articular cartilage of patients＇ knee were cut during joint replacement, which was flushed with 0.25% trypsin and 0.20% type II collagenase in constant 36.8℃temperature water bath box for 25 - 35 min which temperature imitated with boby temperature. After centrifugation, the precipitation was cultured with a certain amount of 10.00% fetal bovine serum DMEM culture medium, and we took the cultivation of Cartilage tissue from second to third. We put the shRNA of aggrecanase-2 that was mediated by slow virus vector into RA patients＇cartilage cells. According to the dose of lentivirus, the cells were divided into 200 μl （group A）, 100 μl （group B） and 50 μl （C group）. Among them, A group was divided into blank control group, negative control group, aggrecanase-2 shRNA1 group （shRNA1 group）, aggrecanase-2 shRNA2 group （shRNA2 group）, aggrecanase-2 shRNA3 group （shRNA3 group）, aggrecanase-2 shRNA4 group （Group）. The transfection agent was added to each pore for about 3-7 μm/ml, of which the negative control was viral fluid containing chaotic sequences, while the blank control group used the culture medium instead of the virus. We detected quantitatively the expression coefficient of aggrecanase-2 and aggrecanase mRNA with PCR detection in various time periods after transfection by a variety of transfection to complex. Results After the addition of lentivirus, the number of A, B and C three groups of RA patients were 100, 50, 25 respectively, and the transfection efficiency was 91.21%, 59.98% and 31.09% respectively. The expression level of aggrecanase-2 mRNA in the chondrocytes of RA patients transfected with aggrecanase-2 shRNA mediated by lentivirus was significantly decreased, suggesting that the difference was statistically significant （ P 〈0.05）. With the increase of time, the expression level of aggrecan protein in each group changed obviously, and the increase of shRNA4 group was the most obvious, suggesting that the difference was statistically significant （ P 〈0.05）. Conclusion The expression of mRNA aggrecanase-2 is significantly inhibited in aggrecanase-2 transfected RA patients with slow virus vector that is mediated by shRNA, but it has no substantial and functional damage to cartilage tissue in RA patients; at the same time it also can promote the expression of mRNA aggrecan protein and polysaccharide by inhibiting the expression of mRNA aggrecanase-2, so as to protect the cartilage degradation.