| Rheumatoid arthritis(RA)is a systemic autoimmune disease mainly involving joint synovial inflammation and cartilage destruction.Although the mortality rate of animal RA is not high,it is difficult to cure,resulting in a reduction in breeding efficiency.A large number of studies have shown that small interfering RNA(siRNA)can effectively treat related diseases by inhibiting the expression of target genes in the body.Bone marrow mesenchymal stem cells(BMSCs)are widely used in the treatment of immune diseases due to their multi-directional differentiation and homing properties,and they can participate in immune regulation from multiple links and promote damage repair.In this study,using the RA rat model,on the basis of exploring the relationship between tumor necrosis factor alpha(TNF-?)expression and the occurrence and development of RA,siRNA transfection in vivo was used to inhibit TNF-? overexpression,combined with BMSCs transfection in vivo,to explore feasibility and mechanism of siRNA gene combined with BMSCs transplantation for RA.1.Rheumatoid arthritis rat model of optimization and partial mechanism researchIn the experiment,two models of collagen induction(CIA group)and cold water bath+collagen induction(cold water bath+CIA group)were used to prepare the rat RA model.The CIA group was injected intradermally with chicken type ? collagen and complete Freund's adjuvant emulsion into the back and tail roots,the cold water bath+CIA group was injected collagen in the same way as before,supplemented by continuous 1w cold water swimming.At the same time,a normal control(control group)was set up,injecting intradermally normal saline.According to the indexes of growth and development,behavior,imaging and pathology of rats,the preparation scheme of stable RA model was selected.By monitoring the serum IL-1? and TNF-? of model rats,matrix metalloproteinase-9(MMP-9)and tissue metalloproteinase inhibitor-1(TIMP-1)in ankle bone tissue,some of the pathogenic mechanisms of RA were analyzed to explore the significance of TNF-? in the occurrence and development of RA.The results showed that the weight of the model rats increased slowly,and the cold water bath+CIA group was more significant;the toes were swollen seriously,and the ankle and knee joints were stiff and could not be bent.The spleen index of the model rats was significantly higher than that of the normal group(P<0.01),and the cold water bath+CIA group was significantly higher than that of the CIA group(P<0.01);The forced swimming fixed time of the model rats was higher than that of the control group(P<0.01);The knee joints of rats in the cold water bath+CIA group showed typical synovitis lesions of RA.The concentration of serum IL-1? and TNF-? in the model rats at the 4th and 6th week of modeling was significantly higher than that of the control group(P<0.01),and at the 6th week the cold water bath+CIA group was significantly higher than the CIA group;Compared with the control group,the expression of MMP-9 protein in the model rats increased,and the expression of TIMP-1 protein decreased;The difference between the CIA group and the control group was significant(P<0.05),and the difference between the cold water bath group and the control group was extremely significant(P<0.01).It indicated that the induction of type II collagen supplemented by cold water swimming successfully prepared a stable RA rat model;and the serum TNF-? increased as the disease progressed,and TNF-? was an important factor in the development of RA.2.Experimental study of TNF-?-siRNA interfering with TNF-? expression in BMSCsThe results,that the TNF-? expression in the serum of RA model rats increased significantly,suggested that it is expected to treat RA by inhibiting the body's TNF-?secretion.This experiment first stimulated bone marrow mesenchymal stem cells(BMSCs)to express TNF-? with different concentrations of lipopolysaccharide(LPS)in vitro,and established an in vitro cell model of RA inflammation,and then used siRNA interference technology to inhibit TNF-? expression,and then screened out the best siRNA sequence for TNF-?.The results of LPS stimulation of BMSCs showed that the amount of TNF-? in the cell supernatant and lysate of the 10 ?g/mL LPS group was significantly higher than that of the untreated BMSCs group(P<0.01).By comparing the fluorescence intensity,120 nM siRNA and 1.5 ?l transfection reagent were selected as the optimal transfection conditions for siRNA.After BMSCs transfection with siRNA,the expression levels of TNF-? in the cell supernatants of LPS+BMSCs+siRNA494,LPS+BMSCs+siRNA504,LPS+BMSCs+siRNA564 groups were respectively 278.71±6.25 pg/mL,247.96 ± 5.72 pg/mL,283.21±5.56 pg/mL,all significantly lower than the LPS+BMSCs group(P<0.01),in which the expression of TNF-? in the LPS+BMSCs+siRNA504 group was not only significantly lower than the LPS+BMSCs group but also significantly lower than the untreated BMSCs group(P<0.01),all three candidate siRNA sequences can inhibit the secretion of TNF-? by BMSCs,and the siRNA504 group has the best effect.It is prompted that 10?g/mL LPS stimulated BMSCs to successfully simulate the inflammatory response of RA;siRNA504 is the best interference sequence of TNF-?.3.TNF-?-siRNA joint BMSCs research on the treatment of rheumatoid arthritis modelTNF-?-siRNA504 in vivo transfection combined with BMSCs transplantation was used to treat RA model rats,Comprehensively analyzing the rats' weight changes,toe swelling,forced swimming,IL-1? and TNF-? levels in serum,MMP-9 and TIMP-1 protein expression and other indicators,to explore the therapeutic effect of gene alone therapy,cell alone therapy and gene combined cell therapy on RA.The results showed that the weight gain of all the treatment groups was significantly higher than that of the PBS group(P<0.01),of which the body weight gain was the most obvious in the TNF-?-siRNA504+BMSCs group;The toe swelling value from low to high was followed by TNF-?-siRNA504+BMSCs,TNF-?-siRNA504,BMSCs,methotrexate,PBS treatment group;The fixed time of forced swimming in all treatment groups were significantly lower than that in the PBS group(P<0.01),and TNF-?-siRNA504+BMSCs group was significantly lower than other treatment groups(P<0.01);The concentrations of IL-1? and TNF-? in the serum of rats decreased significantly after 4 weeks of treatment,and the TNF-?-siRNA504+BMSCs group respectively decreased to 27.13 ± 2.83 pg/mL,209.85±5,83 pg/mL,extremely significantly lower than other treatment groups(P<0.01);Compared with PBS group rats,the expression of MMP-9 protein in each treatment group was significantly reduced(P<0.01),BMSCs,methotrexate,TNF-?-siRNA504,TNF-?-siRNA504+BMSCs treatment group decreased sequentially,while TIMP-1 protein expression increased sequentially.It is prompted that all three treatment methods have therapeutic effects on RA,but TNF-?-siRNA504 combined with BMSCs transplantation is superior to TNF-?-siRNA504 and BMSCs alone treatment,suggesting that in vivo gene transfection combined with cell transplantation can be used as an important treatment strategy for RA. |
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