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  本文選題：RANKL + RNA干擾��； 參考：《四川大學》2007年博士論文

【摘要】： 【目的】細胞核因子κB受體活化因子配基(Ligand of receptor activator of NFκB，RANKL)在破骨細胞增殖、分化、活化和存活等一系列生理過程中起十分重要的作用。RNA干擾(RNA interference，RNAi)是一種序列特異性、轉錄后基因沉默的機制，目前，RNAi已應用于基因功能、疾病治療的研究。用RNAi技術來抑制RANKL的表達將為骨質疏松等骨溶解性疾病提供新的治療思路。本實驗研究目的：(1)合成并篩選有效的靶向RANKL基因的小分子干擾RNA(siRNA)。(2)研究siRNA特異性抑制基因表達的時效性，研究RANKL基因沉默對成骨細胞功能的影響及對破骨細胞前體向破骨細胞轉化的影響。(3)研究去卵巢大鼠骨組織內RANKL、OPG和骨密度的變化規(guī)律，為進一步實驗做準備。 【方法】(1)化學法合成4條siRNA，轉染成骨細胞，RT-PCR檢測RANKLmRNA，篩選出干擾效果最佳的序列用于下一步實驗。(2)最佳siRNA轉染成骨細胞，觀察RANKL、OPG的mRNA、蛋白水平表達的時間規(guī)律，同時觀察成骨細胞增殖能力、堿性磷酸酶活性、Ⅰ型膠原表達的變化；將轉染的成骨細胞與破骨細胞前體共培養(yǎng)，觀察對破骨細胞生成的影響。(3)建立去卵巢大鼠模型，于術后不同時間點取股骨髁，檢測骨組織RANKL、OPG的mRNA、蛋白表達情況，，測量股骨髁的骨密度，觀察RANKL、OPG表達與骨質疏松的關系。 【結果】(1)合成的4條siRNA均可抑制成骨細胞RANKL的表達，RT-PCR結果顯示siRNA4對RANKLmRNA的抑制率為89％，與空白對照組(無siRNA轉染組)差異相比具有顯著性(P＜0．05)。(2)最佳siRNA轉染后1、2、3、5、7天，與空白對照組相比成骨細胞RANKLmRNA表達率分別為35．3％、11．1％、25．9％、49．0％、66．9％(P＜0．05)，轉染后2天表達率最低；RANKL蛋白表達率分別為59．1％、39．5％、26．6％、40．0％、57．3％(P＜0．05)，轉染后3天表達率最低。(3)最佳siRNA轉染后1、2、3、5、7天，成骨細胞OPGmRNA、蛋白表達率較空白對照組降低，但差異不具有顯著性(P＞0．05)；成骨細胞的增殖能力，堿性磷酸酶活性、Ⅰ型膠原分泌較空白對照組降低，差異也不具有顯著性(P＞0．05)。(4)最佳siRNA轉染后的成骨細胞與骨髓細胞共培養(yǎng)，破骨細胞的生成數量少于空白對照組，差異具有顯著性(P＜0．05)(5)大鼠去卵巢6周后股骨髁骨密度開始降低，與假手術組相比差異均具有顯著性(P＜0．05)。(6)大鼠去卵巢后RANKLmRNA水平在第4周達到高峰，蛋白水平在第6周達到高峰，此后均呈持續(xù)高表達，與假手術組相比，各時間點表達水平差異具有顯著性(P＜0．05)；OPG mRNA水平在第4周達到高峰，蛋白水平在第2周達到高峰，此后均迅速下降，與假手術組相比，各時間點表達水平差異具有顯著性(P＜0．05)。 【結論】 (1)4條化學合成的靶向RANKL的siRNA能抑制成骨細胞RANKLmRNA的表達，5′→3′UCC CAU CGG GUU CCC AUA AdTdT是最有效的干擾序列。(2)有效的靶向RANKL的siRNA轉染成骨細胞可明顯降低RANKLmRNA、蛋白的表達，到轉染后第7天仍有部分抑制作用存在。 (3)有效的靶向RANKL的siRNA轉染成骨細胞后對OPGmRNA和蛋白的表達水平均無明顯影響；對成骨細胞的增殖能力、ALP活性和Ⅰ型膠原表達也無明顯影響。 (4)siRNA轉染后的成骨細胞與骨髓細胞共培養(yǎng)，可顯著抑制破骨細胞的生成。 (5)大鼠去卵巢后最早6周在股骨下端可以觀察到骨密度的降低，股骨下端是檢測骨密度敏感部位。 (6)RANKL表達持續(xù)增高，OPG表達短期內升高，迅速降低是絕經后骨質疏松的直接原因，為絕經后骨質疏松的基因治療研究提供了理論依據。
[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.
【學位授予單位】：四川大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R329.2

【參考文獻】

相關期刊論文 前1條

1 孫建國,廖榮霞,陳正堂;RNA干涉分子機制研究進展[J];生物化學與生物物理進展;2002年05期

本文編號：2025734