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
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。
【Objective】Ligand of receptor activator of NFκB(RANKL) plays a important role in proliferation, differentiation, activation, survival of osteoclast. RNA interference(RNAi) is a sequence-specific, post-transcriptional gene silencing mechanism. Now RNAi has been adopted as a functional genomics tool and a gene therapy approach. Suppression of the expression of RANKL by RNAi is likely to have great impact as a therapeutic tool in osteolysis such as osteoporosis. The objective of this experiment include:(1) to synthesize and screen effective siRNA targeting RANKL.(2) To explore time-course changes of siRNA down regulation of RANKL mRNA and protein, and to observe the effect of RANKL gene silencing on the function of osteoblast and transformation of pre-osteoclast.(3) To investigate the rhythm of rankl and opg expression in bone and BMD of ovariectomized(OVX) S-D rat for the further experiment.
【Method】(1) Osteoblasts were transfected with 4 chemically synthesized siRNA. RANKL mRNA levels were analyzed by real time reverse transcription-polymerase chain reaction(RT-PCR). The optimal siRNA were selected for next experiment.(2) The optimal siRNA were transfected into osteoblasts, and the levels of mRNA and protein of RANKL and OPG were detected at different times. Proliferation, ALP activity and I type collagen expression of osteoblasts were observed at the same time. Osteoblasts transfected with siRNA and pre-osteoclasts were co-cultured, and the influence of osteoclastogenesis was investigated.(3) OVX model of S-D rats was established. Bone mineral density(BMD) and the levels of mRNA and protein of RANKL and OPG in condyles of femur were detected at different times. The relationship between the expression of RANKL and OPG and osteoporosis was investigated.
【Result】(1) 4 chemically synthesized siRNA can suppress the expression of RANKL mRNA in osteoblasts. The inhibitory rates of siRNA4 was 89%, and compared with the blank control group(no transfected group), significant statistical difference existed(P<0.05).(2) Compared with the blank control group, the expression rate of RANKL mRNA was 35.3%、11.1%、25.9%、49.0%、66.9%(P<0.05) respectively at 1st, 2nd, 3rd, 5th, 7th day after the optimal siRNA transfection, and the expression rate of RANKL protein was 59.1%、39.5%、26.6%、40.0%、57.3%(P<0.05) respectively at the sane time. The optimal silencing effect at mRNA and protein level was at 2nd and 3rd day post-transfection, respectively.(3) The expression rate of OPG mRNA and protein is lower than the blank control group at 1st, 2nd, 3rd, 5th, 7th day after the optimal siRNA transfection, however no statistical difference existed(p>0.05). Proliferation, ALP activity and I type collagen expression of osteoblasts were similar to the expression profile of OPG(P>0.05).(4) After transfected osteoblasts and bone marrow cells were co-cultured 7 days, the number of osteoclasts was less than the blank control group(P<0.05).(5) The femoral condyles BMD in OVX was significantly lower than that in sham control since 6 weeks after OVX(P<0.05).(6) The expression level of RANKL mRNA peaked at 4 weeks after OVX, and that of RANKL protein peaked at 6 weeks after OVX, then both maintain at a high level. Compared with sham control, statistical difference existed in the expression level at each time point after OVX(P<0.05). The expression level of OPG mRNA peaked at 4 weeks after OVX, and that of OPG protein peaked at 2 weeks after OVX, then both decreased rapidly. Compared with sham control, statistical difference existed in the expression level at each time point after OVX(P<0.05).
【Conclusion】
(1) 4 chemically synthesized siRNA targeting RANKL can suppress the expression of RANKL mRNA in osteoblasts, and 5'→3' UCC CAU CGG GUU CCC AUA is the most effective sequence.
(2) Effective siRNA targeting RANKL can suppress the expression of RANKL mRNA and protein in osteoblasts, and the suppression is up to 7 days.
(3) Effective siRNA targeting RANKL doesn't influence the expression of OPG mRNA and protein, and has no significant effect on proliferation, ALP activity and I type collagen expression of osteoblasts.
(4) Transfected osteoblasts are co-cultured with bone marrow cells, which can inhibit transformation of pre-osteoclast.
(5) The femoral condyles BMD in OVX is significantly lower than that in sham control since 6 weeks after OVX, and the femoral condyles BMD is sensitive to OVX.
(6) The expression level of RANKL maintains at a high level and that of OPG increases transiently and decreases rapidly, which is the direct reason of postmenopausal osteoporosis.
【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。
【Objective】Ligand of receptor activator of NFκB(RANKL) plays a important role in proliferation, differentiation, activation, survival of osteoclast. RNA interference(RNAi) is a sequence-specific, post-transcriptional gene silencing mechanism. Now RNAi has been adopted as a functional genomics tool and a gene therapy approach. Suppression of the expression of RANKL by RNAi is likely to have great impact as a therapeutic tool in osteolysis such as osteoporosis. The objective of this experiment include:(1) to synthesize and screen effective siRNA targeting RANKL.(2) To explore time-course changes of siRNA down regulation of RANKL mRNA and protein, and to observe the effect of RANKL gene silencing on the function of osteoblast and transformation of pre-osteoclast.(3) To investigate the rhythm of rankl and opg expression in bone and BMD of ovariectomized(OVX) S-D rat for the further experiment.
【Method】(1) Osteoblasts were transfected with 4 chemically synthesized siRNA. RANKL mRNA levels were analyzed by real time reverse transcription-polymerase chain reaction(RT-PCR). The optimal siRNA were selected for next experiment.(2) The optimal siRNA were transfected into osteoblasts, and the levels of mRNA and protein of RANKL and OPG were detected at different times. Proliferation, ALP activity and I type collagen expression of osteoblasts were observed at the same time. Osteoblasts transfected with siRNA and pre-osteoclasts were co-cultured, and the influence of osteoclastogenesis was investigated.(3) OVX model of S-D rats was established. Bone mineral density(BMD) and the levels of mRNA and protein of RANKL and OPG in condyles of femur were detected at different times. The relationship between the expression of RANKL and OPG and osteoporosis was investigated.
【Result】(1) 4 chemically synthesized siRNA can suppress the expression of RANKL mRNA in osteoblasts. The inhibitory rates of siRNA4 was 89%, and compared with the blank control group(no transfected group), significant statistical difference existed(P<0.05).(2) Compared with the blank control group, the expression rate of RANKL mRNA was 35.3%、11.1%、25.9%、49.0%、66.9%(P<0.05) respectively at 1st, 2nd, 3rd, 5th, 7th day after the optimal siRNA transfection, and the expression rate of RANKL protein was 59.1%、39.5%、26.6%、40.0%、57.3%(P<0.05) respectively at the sane time. The optimal silencing effect at mRNA and protein level was at 2nd and 3rd day post-transfection, respectively.(3) The expression rate of OPG mRNA and protein is lower than the blank control group at 1st, 2nd, 3rd, 5th, 7th day after the optimal siRNA transfection, however no statistical difference existed(p>0.05). Proliferation, ALP activity and I type collagen expression of osteoblasts were similar to the expression profile of OPG(P>0.05).(4) After transfected osteoblasts and bone marrow cells were co-cultured 7 days, the number of osteoclasts was less than the blank control group(P<0.05).(5) The femoral condyles BMD in OVX was significantly lower than that in sham control since 6 weeks after OVX(P<0.05).(6) The expression level of RANKL mRNA peaked at 4 weeks after OVX, and that of RANKL protein peaked at 6 weeks after OVX, then both maintain at a high level. Compared with sham control, statistical difference existed in the expression level at each time point after OVX(P<0.05). The expression level of OPG mRNA peaked at 4 weeks after OVX, and that of OPG protein peaked at 2 weeks after OVX, then both decreased rapidly. Compared with sham control, statistical difference existed in the expression level at each time point after OVX(P<0.05).
【Conclusion】
(1) 4 chemically synthesized siRNA targeting RANKL can suppress the expression of RANKL mRNA in osteoblasts, and 5'→3' UCC CAU CGG GUU CCC AUA is the most effective sequence.
(2) Effective siRNA targeting RANKL can suppress the expression of RANKL mRNA and protein in osteoblasts, and the suppression is up to 7 days.
(3) Effective siRNA targeting RANKL doesn't influence the expression of OPG mRNA and protein, and has no significant effect on proliferation, ALP activity and I type collagen expression of osteoblasts.
(4) Transfected osteoblasts are co-cultured with bone marrow cells, which can inhibit transformation of pre-osteoclast.
(5) The femoral condyles BMD in OVX is significantly lower than that in sham control since 6 weeks after OVX, and the femoral condyles BMD is sensitive to OVX.
(6) The expression level of RANKL maintains at a high level and that of OPG increases transiently and decreases rapidly, which is the direct reason of postmenopausal osteoporosis.
引文
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