RNA干扰阻断RANKL信号传导通路抑制破骨细胞前体转化的实验研究

发布时间：2018-06-16 06:33

本文选题：RANKL + RNA干扰　；参考：《四川大学》2007年博士论文

【摘要】： 【目的】细胞核因子κB受体活化因子配基(Ligand of receptor activator of NFκB，RANKL)在破骨细胞增殖、分化、活化和存活等一系列生理过程中起十分重要的作用。RNA干扰(RNA interference，RNAi)是一种序列特异性、转录后基因沉默的机制，目前，RNAi已应用于基因功能、疾病治疗的研究。用RNAi技术来抑制RANKL的表达将为骨质疏松等骨溶解性疾病提供新的治疗思路。本实验研究目的：(1)合成并筛选有效的靶向RANKL基因的小分子干扰RNA(siRNA)。(2)研究siRNA特异性抑制基因表达的时效性，研究RANKL基因沉默对成骨细胞功能的影响及对破骨细胞前体向破骨细胞转化的影响。(3)研究去卵巢大鼠骨组织内RANKL、OPG和骨密度的变化规律，为进一步实验做准备。【方法】(1)化学法合成4条siRNA，转染成骨细胞，RT-PCR检测RANKLmRNA，筛选出干扰效果最佳的序列用于下一步实验。(2)最佳siRNA转染成骨细胞，观察RANKL、OPG的mRNA、蛋白水平表达的时间规律，同时观察成骨细胞增殖能力、碱性磷酸酶活性、Ⅰ型胶原表达的变化；将转染的成骨细胞与破骨细胞前体共培养，观察对破骨细胞生成的影响。(3)建立去卵巢大鼠模型，于术后不同时间点取股骨髁，检测骨组织RANKL、OPG的mRNA、蛋白表达情况，，测量股骨髁的骨密度，观察RANKL、OPG表达与骨质疏松的关系。【结果】(1)合成的4条siRNA均可抑制成骨细胞RANKL的表达，RT-PCR结果显示siRNA4对RANKLmRNA的抑制率为89％，与空白对照组(无siRNA转染组)差异相比具有显著性(P＜0．05)。(2)最佳siRNA转染后1、2、3、5、7天，与空白对照组相比成骨细胞RANKLmRNA表达率分别为35．3％、11．1％、25．9％、49．0％、66．9％(P＜0．05)，转染后2天表达率最低；RANKL蛋白表达率分别为59．1％、39．5％、26．6％、40．0％、57．3％(P＜0．05)，转染后3天表达率最低。(3)最佳siRNA转染后1、2、3、5、7天，成骨细胞OPGmRNA、蛋白表达率较空白对照组降低，但差异不具有显著性(P＞0．05)；成骨细胞的增殖能力，碱性磷酸酶活性、Ⅰ型胶原分泌较空白对照组降低，差异也不具有显著性(P＞0．05)。(4)最佳siRNA转染后的成骨细胞与骨髓细胞共培养，破骨细胞的生成数量少于空白对照组，差异具有显著性(P＜0．05)(5)大鼠去卵巢6周后股骨髁骨密度开始降低，与假手术组相比差异均具有显著性(P＜0．05)。(6)大鼠去卵巢后RANKLmRNA水平在第4周达到高峰，蛋白水平在第6周达到高峰，此后均呈持续高表达，与假手术组相比，各时间点表达水平差异具有显著性(P＜0．05)；OPG mRNA水平在第4周达到高峰，蛋白水平在第2周达到高峰，此后均迅速下降，与假手术组相比，各时间点表达水平差异具有显著性(P＜0．05)。【结论】 (1)4条化学合成的靶向RANKL的siRNA能抑制成骨细胞RANKLmRNA的表达，5′→3′UCC CAU CGG GUU CCC AUA AdTdT是最有效的干扰序列。(2)有效的靶向RANKL的siRNA转染成骨细胞可明显降低RANKLmRNA、蛋白的表达，到转染后第7天仍有部分抑制作用存在。 (3)有效的靶向RANKL的siRNA转染成骨细胞后对OPGmRNA和蛋白的表达水平均无明显影响；对成骨细胞的增殖能力、ALP活性和Ⅰ型胶原表达也无明显影响。 (4)siRNA转染后的成骨细胞与骨髓细胞共培养，可显著抑制破骨细胞的生成。 (5)大鼠去卵巢后最早6周在股骨下端可以观察到骨密度的降低，股骨下端是检测骨密度敏感部位。 (6)RANKL表达持续增高，OPG表达短期内升高，迅速降低是绝经后骨质疏松的直接原因，为绝经后骨质疏松的基因治疗研究提供了理论依据。
[Abstract]:[Objective] the activation factor ligand of nuclear factor kappa B receptor (Ligand of receptor activator of NF kappa B, RANKL) plays an important role in the proliferation, differentiation, activation and survival of osteoclasts in a series of physiological processes, such as RNA interference, which is a mechanism of sequence Let heterosexual and post transcriptional gene silencing. The study of gene function, disease treatment. The use of RNAi to inhibit the expression of RANKL will provide new therapeutic ideas for osteolytic diseases such as osteoporosis. The purpose of this study was to synthesize and screen effective small molecular interference RNA (siRNA) of the effective target RANKL gene (siRNA). (2) study the aging of the expression of siRNA specific suppressor gene. The effect of RANKL gene silencing on osteoblast function and the effect of osteoclast precursor to osteoclast transformation. (3) study the changes of RANKL, OPG and bone density in the bone tissue of ovariectomized rats, and prepare for further experiments.
[method] (1) (1) chemical synthesis of 4 siRNA, transfection osteoblast, RT-PCR detection of RANKLmRNA, screening the best sequence of interference effect for the next experiment. (2) the best siRNA transfection osteoblast, observe the mRNA of RANKL, OPG, the time law of protein level expression, at the same time observe osteoblast proliferation, alkaline phosphatase activity, type I glue Changes in the original expression; co culture the transfected osteoblasts and osteoclast precursors to observe the effect of osteoclast formation. (3) the ovariectomized rat model was established, the femoral condyle was taken at different time points after the operation, the mRNA of the bone tissue, the mRNA of the OPG, the protein expression, the bone density of the femoral condyle were measured, the expression of RANKL, OPG and osteoporosis were observed. Relationship.
[results] (1) the 4 siRNA synthesized could inhibit the expression of RANKL in osteoblast. The RT-PCR results showed that the inhibition rate of siRNA4 to RANKLmRNA was 89%, compared with the blank control group (P < 0.05). (2) the best siRNA was transfected 1,2,3,5,7 days, compared with the blank control group, the expression rate of RANKLmRNA was compared with the blank control group. The expression rates of 35.3%, 11.1%, 25.9%, 49%, 66.9% (P < 0.05) were the lowest, and the expression rates of RANKL protein were 59.1%, 39.5%, 26.6%, 40%, 57.3% (P < 0.05), and the lowest siRNA expression rate after transfection. The best siRNA transfection was 1,2,3,5,7 days after the transfection, and the protein expression rate was less than that of the blank. The control group was lower, but the difference was not significant (P > 0.05); the proliferation ability of osteoblast, alkaline phosphatase activity, the secretion of type I collagen decreased compared with the blank control group, and the difference was not significant (P > 0.05). (4) the best siRNA transfected osteoblasts were co cultured with bone marrow cells, and the number of osteoclast cells was less than that of the blank control The difference was significant (P < 0.05) (5) the femoral condyle density of the femur began to decrease after 6 weeks of ovariectomy, and the difference was significant compared with that of the sham group (P < 0.05). (6) the level of RANKLmRNA in the ovariectomized rats reached the peak at fourth weeks, and the protein level reached the peak at sixth weeks, and then all showed continuous high expression, compared with the sham operation group. The level of time point expression level difference was significant (P < 0.05); the level of OPG mRNA reached the peak in fourth weeks, the protein level reached the peak in second weeks, and then decreased rapidly. Compared with the sham operation group, the difference of expression level in each time point was significant (P < 0.05).
[Conclusion]
(1) 4 chemically synthesized target RANKL siRNA can inhibit the expression of RANKLmRNA in osteoblasts, 5 'to 3' UCC CAU CGG GUU CCC AUA AdTdT is the most effective interference sequence. (2) effective targeting RANKL siRNA into osteoblasts can significantly reduce the expression of protein, and there is still some inhibition in the seventh days after the transfection.
(3) the effective siRNA transfection of RANKL to osteoblasts had no significant effect on the expression level of OPGmRNA and protein, and had no significant effect on the proliferation of osteoblasts, ALP activity and the expression of type I collagen.
(4) co culture of osteoblasts and bone marrow cells after siRNA transfection can significantly inhibit the formation of osteoclasts.
(5) bone density was observed at the lower end of the femur 6 weeks after ovariectomy, and bone mineral density was detected at the lower end of the femur.
(6) the expression of RANKL increased continuously, the expression of OPG increased in the short term, and the rapid decrease was the direct cause of postmenopausal osteoporosis, which provided a theoretical basis for the study of gene therapy for postmenopausal osteoporosis.
【学位授予单位】：四川大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R329.2

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