| Rheumatoid arthritis(RA)is a kind of systemic autoimmune diseases[1],the onset of arthritis are exist in animals,especially the harm of chickens,pigs,cattle,serious[2].Arthritis,although the fatality rate is not high,due to its once the disease is difficult to heal,the resulting action inconvenience,livestock poultry retardation,reduce or even eliminate economic value,thus bring serious loss[3]farms.Existing findings would be of interleukin 1 receptor(IL-1R)gene transfection into rats,inhibited the interleukins beta(IL-1 beta),RA symptoms of rats can be improved significantly.Between bone marrow mesenchymal stem cells(BMSCs)unique biological characteristics and involved in many links of immune regulation,have the function of the repair damage,and therefore is widely applied to the cells in the treatment of autoimmune disease.This experiment using the method of type ? collagen induced preparation RA model rats,preliminarily RA immune mechanism of the disease.Study the pathogenesis of RA and IL-1 beta expression quantity changes,the relationship between beta and IL-1 small interfering RNA(SiRNA)in vivo transfection combined with BMSCs transplantation in the treatment of RA model rats,the BMSCs combined effect of gene therapy for the treatment of RA.1 arthritis rat model of preparation and mechanism researchThe tail root scatter injection of chicken type ? collagen and completely after's adjuvant emulsion preparation method of rheumatoid arthritis rats model,with toes swelling of rats after building,forced swimming,DR-X-ray imaging,viscera index,joint pathological tests,such as screen success RA rat model.Based on the successful model of rats serum IL-1 beta,spleen tissue Treg Foxp3 tumor-specific markers and Treg cells on the surface of TGF-beta 1 and apoptosis related molecular PD 1,discusses its significance in the development of regulatory T cells in RA.Toes swelling,according to the results of the CIA built modules within 14 days of joint swelling,joint part rats can not load the crawling difficulties.Relative to the CIA model AA model group arthritis course of 14 days before the onset nasty,swelling rapidly but shorter duration,21 days began to fade;Weight,according to the results of AA model combined the CIA rats of model group than normal rats compared weight growth has slowed,the CIA model rat slow more obvious;Viscera index,according to the results of the CIA and AA group rats spleen of body mass index were 2.91±0.07,2.59±0.06 significantly(p<0.01)higher than that of normal control group 2.2510.10,the thymus of body mass index were 2.2710.05,1.8510.08 significantly(p<0.01)higher than that of normal control group 1.49±0.05;Forced swimming test results show that the CIA model combined AA model group rats forced swimming fixed time from building,1 week,2 weeks,3 weeks after 4 weeks were very significant(p<0.01)higher than normal group,the struggle of the time significantly(p<0.01)lower than the normal control group;it also shows how nuclear giant cell granuloma formation,namely AA model group rats synovial hyperplasia;Beta test results showed that serum IL-1 AA model group and the model group rats 2 weeks after building in serum IL-1 beta content were 18.49± 1.70 pg/mL and 36.98±0.97 pg/mL significantly(p<0.01)higher than that of normal control group(16.23±0.44 pg/mL),AA model group and the model group rats serum IL-1 beta 4 weeks after building content were 141.91±0.41 pg/mL and42.07±0.61 pg/mL significantly(p<0.01)higher than that of normal control group(14.91 ± 0.41),TGF-PD-1 beta 1 and Foxp3 mRNA test results show that the CIA model rat PD 1,TGF-beta 1,the relative expression of Foxp3 were 0.12±0.079,0.12±0.059 and 0.23±0.089,significantly lower than that of control group(p<0.01),AA model rat PD-1,TGF-beta 1,the relative expression of Foxp3 were 0.45±0.082,0.48±0.067 and 0.74±0.027,significantly lower than that of control group(p<0.01),at the same time,the CIA model rats spleen PD 1,the TGF-beta 1,the expression of Foxp3 significantly(p<0.01)lower than AA rat model.Prompt scatter the tail root injection of chicken type II collagen and completely after's adjuvant emulsion RA rat model was established successfully,and the model of rat serum IL-1 in the expression of beta amount rise with RA course of the disease.The occurrence of RA may be related to reducing the number of regulatory T cells or functional flaws.2 IL-1 beta siRNA disturb BMSCs expression the IL-1 beta in the experimental studyRA model rats serum IL-1 beta content compared with normal rats have obvious rise,use of lower animals' IL-1 beta production technology,prevent inflammation process,this experiment using LPS stimulation BMSCs in vitro high expression of IL-1 beta,simulated RA inflammation in the body,using siRNA interference technology interfere with BMSCs in vitro expression of IL-1 beta,through the protein level and gene level to detect the expression of IL-1 beta changes,select the best IL-1 beta siRNA suppressor genes.LPS stimulation induced BMSCs experimental results showed that LPS-BMSCs with BMSCs group on the amount of IL-1 beta in the qing dynasty were 335.29±5.17 pg/mL and 121.76±3.10 pg/mL,extremely significant differences(p<0.01);Pyrolysis of LPS-BMSCs and BMSCs group of IL-1 beta were 176.65±6.33 pg/mL and 78.52±3.24 pg/mL,there is also a very significant difference(p<0.01).The LPS-BMSCs IL-1 beta expression quantity is significantly higher than that of BMSCs;Screening of siRNA experimental results showed that the optimal transfection efficiency of normal not transfection group without fluorescence under the fluorescent microscope,the 100 100nMsiRNA with 0.5 ul transfection reagent in other combinations in comparison with the combination of the fluorescence,the brightest,the maximum number of highest transfection efficiency;SiRNA interference after ELISA detection results show that the supernatant of LPS-IL-1 beta expression of BMSCs group was 347.84±6.17 pg/mL,significantly(p<0.01)than the control group was 119.38±2.92 pg/mL;LPS+ SiRNA157 +BMSCs,LPS +SiRNA238 + BMSCs,the expression of LPS+SiRNA788+BMSCs were 126.21±2.25 pg/mL,52.66±1.72 pg/mL and 101.83±3.65 pg/mL were significantly lower than that of LPS-BMSCs group(p<0.01),LPS+SiRNA238+BMSCs group of IL-1 beta expression was significantly lower than that of LPS-BMSCs group(p<0.01)and significantly lower than that of control group(p<0.01);Cracking of LPS-IL-1 beta expression of BMSCs group was 178.29±2.18 pg/mL significantly(p<0.01)than the control group was 72.35±2.26 pg/mL,LPS + SiRNA 157 +BMSCs,LPS+SiRNA238+ BMSCs,the expression of LPS+SiRNA788+ BMSCs were 69.71 ± 1.97 pg/mL,28.47±1.27 pg/mL and 79.63±3.21 pg/mL were significantly lower than that of LPS-BMSCs group(P<0.01),in which the LPS+SiRNA238+BMSCs group of IL-1 beta expression was significantly lower than that of LPS-BMSCs group(P<0.01)and significantly lower than that of control group(P<0.01).SiRNA interference after Western blot test results showed that three siRNA sequences(157,238,788)on the protein expression of IL-1 beta silence effect,transfection reagent set no statistical difference compared with control group,including grey value minimum SiRNA238 sequences,extremely significant difference compared with the control group(p<0.01).SiRNA interference after Q-PCR detection results show that the LPS + siRNA 157+ BMSCs,LPS+siRNA238+BMSCs,relative expression of LPS + siRNA788+ BMSCs were dropped to 0.92±0.079,0.28±0.015,0.97±0.174 significantly(P<0.01)lower than that of 2.74±0.15 of LPS+BMSCs,LPS+siRNA238+BMSCs also significantly(P<0.01)lower than the normal control group of 1.00±0.124.Prompt LPS stimulation BMSCs after successfully simulates the inflammatory reaction in the body to promote the IL-1 beta expression,both inside and outside the cell protein level,gene level region.perform IL-1 beta siRNA238 best interference.3.IL-1 beta siRNA joint BMSCs research on the treatment of rheumatoid arthritis model in ratsThis experiment by IL-1 small interference RNA beta in vivo transfection combined with BMSCs transplantation in the treatment of RA model rats,growth situation,toes swelling in rats,forced swimming,joint pathological slices,such as the content of serum IL-1 beta testing,research BMSCs combined effect of gene therapy for the treatment of RA.Tumor-specific markers of mice spleen group after treatment of Treg Foxp3 and Treg cells TGF-beta 1 and apoptosis related molecules on the surface of the PD 1,continue to explore its significance in the development of regulatory T cells in RA.Treatment of rats in growth,according to the results of IL-1 beta SiRNA238 treatment group and beta SiRNA238 IL-1+BMSCs in treatment group increased the weight of the rat,toes swelling a PBS treatment group were significantly higher(p<0.01),IL-1 beta SiRNA238 + BMSCs treatment group increased more obviously;Forced swimming test,according to the results of IL-1 beta SiRNA238 treatment group and IL-1 beta SiRNA238 + BMSCs in treatment group in the rat forced swimming fixed time in 1 week after treatment were dropped to 156.02±4.58 s,106.45±7.97 s,significantly(p<0.01)lower than that of PBS treatment of 187.90±5.49 in the control group,2 weeks after treatment respectively dropped to 91.24±5.39 s,56.77±3.67 s,significantly(p<0.01)less than PBS treatment in the control group's 182.30±3.59 s,after treatment for 1 week,2 weeks IL-1 beta SiRNA238 + BMSCs fixed time treatment group significantly(p<0.01)lower than that of IL-1 beta SiRNA238 treatment group.l week after treatment,IL-1 beta SiRNA238 treatment group and IL-1 beta SiRNA238 + BMSCs forced swimming struggle time of the treatment group rats were 43.68±4.74 s,62.22±13.36 s significantly(p<0.01)higher than that of PBS treatment control group 13.62±2.17 s,1 week,2 weeks,3 weeks after treatment of IL-1 beta SiRNA238 +BMSCs treatment group rats struggle time significantly(p<0.01)higher than that of IL-I beta SiRNA238 treatment group.ELISA to detect serum IL-1 beta concentration,according to the results of treatment after 1 week IL-1 beta SiRNA238 treatment group rats and IL-1 beta SiRNA238 + BMSCs in the treatment group rats serum IL-1 beta concentrations were 44.34±2.19 pg/mL,31.27± 1.87 pg/mL significantly(p<0.01)lower than the PBS group of 69.15±0.55 pg/mL,2 weeks after treatment of IL-1 beta SiRNA238 treatment group rats and IL-1 beta SiRNA238 +BMSCs treatment group rats serum IL-1 beta concentrations were 31.76±1.59 pg/mL,20.21±0.83 pg/mL significantly(p<0.01)lower than the PBS group 65.44±1.94 pg/mL.And 1 week,2 weeks after treatment IL-1 beta SiRNA238 +BMSCs treatment group rats serum IL-1 beta concentrations were significantly higher than that of IL-1 beta SiRNA238 treatment group(p<0.01).Spleen tissues,TGF beta 1 and PD-1 Foxp3 mRNA testing results show that the beta SiRNA238 treatment group PD IL-1-1,TGF-betal,relative expression of Foxp3 significantly higher than that of PBS treatment control group(p<0.01),IL-1 beta SiRNA238 + BMSCs PD treatment group 1,TGF-beta 1,the relative expression of Foxp3 were 5.13±0.081,3.72±0.069,2.17±0.108 significantly higher than that of PBS treatment control group(p<0.01),IL-1 beta SiRNA238 + BMSCs in the treatment group rats spleen PD 1,TGF-beta 1,the expression of Foxp3 capacity significantly(p<0.01)higher than that of IL-1 beta SiRNA238 treatment group.The results suggest that BMSCs collaborative treatment can enhance IL-1 beta SiRNA238 in vivo transfection effect for the treatment of RA,the experiment for genes with cell therapy RA opened up a new way of thinking. |
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