IL-1β-siRNA联合骨髓间充质干细胞治疗类风湿性关节炎的实验研究

发布时间：2018-06-07 09:14

本文选题：大鼠 + 类风湿性关节炎　；参考：《扬州大学》2017年硕士论文

【摘要】：类风湿性关节炎(RA)是一种系统性的自身免疫性疾病,关节炎发病在家禽家畜中均有存在,尤其对鸡、猪、牛的危害较为严重。该病虽然致死率不高,但其一旦发病便难以痊愈,导致畜禽行动不便,出现发育迟缓,经济价值降低甚至淘汰,由此给养殖场带来严重损失。已有研究发现将白细胞介素1受体(IL-1R)基因转染进大鼠,抑制了白细胞介素β(IL-1β)的作用,大鼠RA症状可得到明显改善。骨髓间充质干细胞(BMSCs)生物学特性独特并且参与多环节的免疫调节,具有促进损伤的修复的作用,因而被广泛应用于免疫性疾病的细胞治疗。本实验利用Ⅱ型胶原蛋白诱导方法制备RA模型大鼠,初步探讨RA发病的免疫机制。研究RA的发病与IL-1β表达量变化的关系,并用IL-1β小干扰RNA(SiRNA)体内转染结合BMSCs移植治疗RA模型大鼠,研究BMSCs联合基因治疗对RA的治疗效果。1关节炎大鼠模型的制备及部分机制的研究采用尾根部散点注射鸡Ⅱ型胶原和完全复氏佐剂乳化液的方法制备类风湿性关节炎大鼠模型,实验组分为CIA、AA和正常组,造模后以大鼠足趾肿胀、强迫游泳、DR-X线成像、脏器指数、关节病理切片等进行检测,筛选出成功RA大鼠模型。通过对成功大鼠模型的血清中IL-1β、脾脏组织Treg的特异性标志物Foxp3及Treg细胞表面的TGF-β1以及凋亡相关分子PD-1的研究,探讨调节性T细胞在RA发生发展中的意义。结果显示,AA模型组和CIA模型组的大鼠体重增长速度下降,CIA模型鼠减缓更明显,CIA造模组14天内关节肿胀明显,21天后大鼠左右关节肿胀达到病程的最高峰,30天后部分大鼠关节僵直、畸形、表面出现皮肤出现结节。CIA组和AA组大鼠脾脏、胸腺占体重指数极显著高于正常对照组(p0.01);两模型组大鼠强迫游泳不动时间从造模后1周、2周、3周、4周均极显著高于正常组(p0.01),挣扎时间均极显著低于正常对照组(p0.01);关节滑膜组织切片显示,大鼠模型关节软骨有明显的糜烂或溃疡,滑膜、关节软骨及关节腔内有重度的混合型炎性细胞浸润,另外还可见有多核巨细胞即肉芽肿的形成。AA模型组和CIA模型组大鼠造模后2周血清中IL-1β含量极显著高于正常对照组(18.49±1.70pg/mL、36.98±0.97pg/mLvs16.23±0.44pg/mL;p0.01),两模型大鼠 PD-1、TGF-β1、Foxp3的相对表达量极显著低于对照组(p0.01),同时CIA模型大鼠脾脏中PD-1、TGF-β1、Foxp3的表达量极显著低于AA模型大鼠(p0.01)。提示采用尾根部散点注射鸡Ⅱ型胶原和完全复氏佐剂乳化液成功建立RA大鼠模型,且模型大鼠血清内IL-1β的表达量随RA病程而上升。RA的发生可能与调节性T细胞数量的减少或者功能缺陷有关。2 IL-1β-siRNA干扰BMSCs中IL-1β表达的实验研究针对RA模型大鼠血清中IL-1β的含量显著高于正常大鼠的实验结果,利用降低动物机体IL-1β分泌技术,阻止炎症反应进程,本实验利用LPS体外刺激BMSCs高表达IL-1β,模拟RA体内炎症反应,采用siRNA干扰技术体外干扰BMSCs表达IL-1β,通过ELISA法和Westernblot对IL-1β蛋白水平表达变化进行检测,Q-PCR对IL-1β基因水平的表达变化进行检测,筛选出最佳IL-1β SiRNA抑制基因。LPS刺激后,LPS-BMSCs组与BMSCs组细胞上清以和裂解物中的IL-1β量存在极显著差异(p0.01),通过荧光强度筛选siRNA的最佳转染条件为1OOnMsiRNA与0.5ul转染试剂的组合;siRNA干扰后,BMSCs培养上清中LPS-BMSCs组的IL-1β表达量极显著高于对照组(347.84±6.17pg/mL vs 119.38±2.92pg/mL;p0.01);LPS+SiRNA157、LPS+SiRNA238、LPS+SiRNA788 的表达量分别为 126.21±2.25pg/mL、52.66±1.72pg/mL、101.83±3.65 pg/mL均极显著低于LPS-BMSCs组(p0.01)。三条siRNA序列均可沉默IL-1β的蛋白表达,其中SiRNA238组的IL-1β表达极显著低于对照组(P0.01)。LPS+BMSCs+siRNA157、LPS+BMSCs+siRNA238、LPS+BMSCs+siRNA788 的 mRNA 相对表达量极显著低于 LPS+BMSCs(0.92±0.079、0.28±0.015、0.97±0.174vs2.74±0.15;p0.01),LPS+BMSCs+siRNA238同时也极显著低于正常对照组(p0.01)。提示LPS刺激BMSCs后成功模拟了体内炎性反应促进了 IL-β在胞内外的过表达,蛋白水平、基因水平检测筛选出IL-1β最佳干扰siRNA238。3.IL-1β-siRNA联合BMSCs对类风湿关节炎模型大鼠的治疗研究本实验通过IL-1β小干扰RNA体内转染结合BMSCs移植治疗RA模型大鼠,以大鼠生长情况、足趾肿胀、强迫游泳、关节病理切片、血清中IL-1β的含量等进行检测,研究BMSCs联合基因治疗对RA的治疗效果。对治疗后老鼠脾脏组织Treg的特异性标志物Foxp3及Treg细胞表面的TGF-β1以及凋亡相关分子PD-1进行检测,继续探讨调节性T细胞在RA发生发展中的意义。IL-1β SiRNA238治疗组和IL-1βSiRNA238+BMSCs治疗组大鼠的体重增长、足趾肿胀较PBS治疗组都极显著升高(p0.01),IL-1βSiRNA+BMSCs治疗组升高更明显。IL-1β SiRNA治疗组和IL-1β SiRNA+BMSCs治疗组大鼠强迫游泳不动时间在治疗后1周、2周极显著低于治疗对照组(p0.01)。治疗后1周、2周、3周IL-1β SiRNA238+BMSCs治疗组大鼠挣扎时间都极显著高于IL-1βSiRNA238治疗组(p0.01)。治疗后1周、2周IL-1β SiRNA治疗组大鼠和IL-1β SiRNA+BMSCs治疗组大鼠血清中IL-1β的浓度极显著低于PBS治疗对照组(p0.01),且治疗后1周、2周IL-1β SiRNA238+BMSCs治疗组大鼠血清中IL-1β的浓度都极显著高于IL-1β SiRNA238治疗组(p0.01)。IL-1β SiRNA238治疗组PD-1、TGF-β1、Foxp3的相对表达量极显著高于治疗对照组(p0.01),IL-1βSiRNA+BMSCs治疗组PD-1、TGF-β1、Foxp3的相对表达量极显著高于治疗对照组(p0.01),IL-1βSiRNA+BMSCs治疗组大鼠脾脏中PD-1、TGF-β1、Foxp3的表达量极显著高于IL-1β SiRNA治疗组(p0.01)。本实验结果提示,BMSCs协同治疗可以增强IL-1βSiRNA体内转染治疗RA的效果,该实验为基因联合细胞治疗RA开辟了新思路。
[Abstract]:Rheumatoid arthritis (RA) is a systematic autoimmune disease. Arthritis is found in poultry and domestic animals, especially in chickens, pigs and cattle. Although the mortality rate is not high, it is difficult to recover once the disease is fatal, which leads to the slow development of livestock and poultry, the decrease of economic value and even elimination of economic value. It has caused serious loss to the farm. It has been found that the gene of interleukin 1 receptor (IL-1R) is transfected into rats and the effect of IL-1 beta (IL-1 beta) is inhibited. The symptoms of RA in rats can be significantly improved. The biological characteristics of bone marrow mesenchymal stem cells (BMSCs) are unique and participate in multiple link immunomodulating, which can promote the repair of injury. It is widely used in the cell therapy of immune diseases. In this experiment, RA model rats were prepared by using type II collagen induction method. The immune mechanism of RA was preliminarily discussed. The relationship between the pathogenesis of RA and the changes of IL-1 beta expression was studied, and the IL-1 beta small interference RNA (SiRNA) in vivo transfection and BMSCs transplantation were used to treat the rat model of RA. Study on the effect of BMSCs combined gene therapy on RA, the preparation and part of the mechanism of.1 arthritis rat model, the rat model of rheumatoid arthritis was prepared by injection of chicken type II collagen and complete compound's adjuvant emulsification in the tail root. The experimental group was divided into CIA, AA and normal group. After the model, the rat toe was swelled and forced to be forced. Swimming, DR- X ray imaging, Viscera index, joint pathological section were detected, and a successful RA rat model was screened. Through the study of IL-1 beta in the serum of successful rat model, the specific marker of Treg in the spleen tissue, TGF- beta 1 on the surface of Treg cell and apoptosis related molecule PD-1, the development of regulatory T cells in the development of RA was discussed. The results showed that the weight growth rate of rats in the AA model group and the CIA model group decreased, the CIA model rat slowed down more obviously, the joint swelling in the CIA model group was obvious within 14 days, and the swelling of the left and right joints reached the peak in the course of the disease in 21 days. After 30 days, the joints of the rats were stiff and deformed, and the surface of the skin appeared in the.CIA group and the AA group of the spleen of the.CIA group. The body mass index of the thymus was significantly higher than that of the normal control group (P0.01), and the time of forced swimming in the two model group was significantly higher than that of the normal group (P0.01) at 1 weeks, 2 weeks, 3 weeks, and 4 weeks, and the struggle time was significantly lower than that of the normal control group (P0.01). There was a severe mixed inflammatory cell infiltration in the synovial membrane, articular cartilage and articular cavity, and the content of IL-1 beta in the serum of the.AA model group and the CIA model group was significantly higher than that of the normal control group (18.49 + 1.70pg/mL, 36.98 + 0.97pg/mLvs16.23 + 0.44pg/mL; P0.01), two in the 2 weeks. The relative expression of PD-1, TGF- beta 1 and Foxp3 in the model rats was significantly lower than that in the control group (P0.01), and the expression of PD-1, TGF- beta 1, Foxp3 in the CIA model rats was significantly lower than that of the AA model rats (P0.01). The expression of IL-1 beta in serum increases with the course of RA disease and the occurrence of.RA may be associated with the decrease in the number of regulatory T cells or functional defects related to the interference of.2 IL-1 beta -siRNA in BMSCs IL-1 beta expression in BMSCs, the content of IL-1 beta in serum of RA model rats is significantly higher than that of normal rats, and the secretion of IL-1 beta secretion in animal body is reduced. Technology, to prevent the process of inflammatory reaction, this experiment uses LPS to stimulate BMSCs high expression of IL-1 beta in vitro, to simulate the inflammatory reaction in RA, and to use siRNA interference technique to interfere with BMSCs to express IL-1 beta in vitro, to detect the expression of IL-1 beta protein by ELISA and Westernblot, and to detect the expression changes of the IL-1 beta gene level, screening and screening the expression of the IL-1 beta gene. After the best IL-1 beta SiRNA inhibitory gene.LPS stimulation, there was a very significant difference between the cell supernatant of the LPS-BMSCs group and the BMSCs group and the IL-1 beta in the lysate (P0.01). The optimum transfection condition of siRNA by fluorescence intensity was the combination of 1OOnMsiRNA and 0.5ul transfection reagents; siRNA interference, the expression of the beta expression in the BMSCs culture supernatant It was significantly higher than the control group (347.84 + 6.17pg/mL vs 119.38 + 2.92pg/mL; P0.01); LPS+SiRNA157, LPS+SiRNA238, LPS+SiRNA788, respectively, were 126.21 + 2.25pg/mL, 52.66 + 1.72pg/mL, and 101.83 + 3.65 pg/mL were significantly lower than LPS-BMSCs group (P0.01). The expression of beta was significantly lower than that of the control group (P0.01).LPS+BMSCs+siRNA157, and the relative expression of mRNA in LPS+BMSCs+siRNA238 and LPS+BMSCs+siRNA788 was significantly lower than that of LPS+BMSCs (0.92 + 0.079,0.28 + 0.015,0.97 + 0.174vs2.74 + 0.15; P0.01), and LPS+BMSCs+siRNA238 was also significantly lower than that of the normal control group (P0.01). The inflammatory response in vivo promotes the over expression of IL- beta in and out of the cell, protein level, and gene level detection and screening of the best interference of IL-1 beta siRNA238.3.IL-1 beta -siRNA combined with BMSCs in the treatment of rheumatoid arthritis rats. The experiment is carried out by IL-1 beta small interference RNA transfection in vivo and BMSCs transplantation for the treatment of RA model rats, and the rat is born in rats. Long condition, toes swollen, forced swimming, joint pathological section and the content of IL-1 beta in serum were detected, and the therapeutic effect of BMSCs combined gene therapy on RA was studied. The specific markers of Treg in the spleen tissue of mice after treatment, TGF- beta 1 and Treg cell PD-1 were detected, and the regulatory T continued to be explored. The significance of cells in the development of RA in the.IL-1 beta SiRNA238 treatment group and the IL-1 beta SiRNA238+BMSCs group was significantly higher than that of the PBS group (P0.01). The increase of the IL-1 beta SiRNA+BMSCs treatment group was more obvious in the.IL-1 beta SiRNA treatment group and the IL-1 beta group. After 1 weeks, 2 weeks was significantly lower than the treatment control group (P0.01). The struggle time of the rats in the IL-1 beta SiRNA238+BMSCs treatment group after 1 weeks, 2 weeks and 3 weeks was significantly higher than the IL-1 beta SiRNA238 treatment group (P0.01). The concentration of IL-1 beta in the serum of the IL-1 beta SiRNA treatment group and the IL-1 beta SiRNA+BMSCs treatment group of the IL-1 beta SiRNA treatment group was significantly lower than that of the PBS treatment for the 1 weeks after treatment. In the control group (P0.01), and 1 weeks after treatment, the concentration of IL-1 beta in the serum of IL-1 beta SiRNA238+BMSCs group was significantly higher than that of the IL-1 beta SiRNA238 treatment group (P0.01).IL-1 beta SiRNA238 treatment group PD-1, TGF- beta 1, and the relative expression of Foxp3 was significantly higher than that of the treatment control group. The expression level was significantly higher than that in the treatment control group (P0.01). The expression of PD-1, TGF- beta 1, Foxp3 in the IL-1 beta SiRNA+BMSCs group was significantly higher than that in the IL-1 beta SiRNA treatment group (P0.01). The results of this experiment suggest that the synergistic therapy of BMSCs can enhance the effect of IL-1 beta transfection in vivo. This experiment opens up a combination of gene combined cell therapy. A new way of thinking.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R593.22

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