RNAi靶向Atp6i特异性阻断大鼠破骨样细胞骨吸收功能
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摘要
破骨细胞(osteoclast,OC)是骨吸收的功能细胞,其功能异常往往导致骨重建平衡失调,是多种代谢性骨病的病理基础。OC功能异常活跃可出现骨组织的过量吸收,在骨质疏松症、Paget病、风湿性关节炎、骨巨细胞瘤及牙周肿瘤等疾病中是普遍存在的病理变化。通过应用雌激素、降钙素和二磷酸盐等药物,在一定程度上可以抑制OC的分化,从而抑制异常骨吸收,然而这些药物的靶向性低加之其毒副作用等问题一直困扰着临床。研究表明,OC的空泡型质子泵(vacuolar type H~+ adenosinetriphosphatase,v-ATPase)Atp6i基因的正常表达,对OC发挥骨吸收功能是非常重要的,对OC与成骨细胞(osteoblast,OB)共同维持骨组织代谢的动态平衡也是非常重要的。
为了探索无毒副作用、高效且高特异性的分子治疗方法和策略,我们针对大鼠Atp6i基因,采用慢病毒介导的RNA干扰(RNA interference,RNAi)技术,进行了靶基因转录后水平的特异性抑制研究。尝试制备一种通过RNAi抑制Atp6i表达的生物抑制剂,为临床治疗由于OC功能亢进所导致的骨破坏提供理论依据。
1设计、构建携带大鼠Atp6i基因小发卡RNA结构的慢病毒载体
首先应用基因工程技术,建立体外过表达外源大鼠Atp6i基因的293T细胞培养体系,在此细胞中筛选出高效、高特异性抑制Atp6i的小干扰RNA(short interferingRNA,siRNA)靶点,并制备成携带该靶点的慢病毒。设计5组(1-5#)大鼠Atp6i基因siRNA靶点,并制备出携带该靶点小发卡RNA(short hairpin RNA,shRNA)结构的慢病毒质粒(pGCL-GFP)即Atp6i-shRNA穿梭质粒,在过表达Atp6i基因融合蛋白(pEGFP-Cl-Atp6i携带)的293T细胞中,利用Western Blot方法筛选出了高效的RNA干扰Atp6i-shRNA(5号)穿梭质粒,以此为模版,包装成携带Atp6i-shRNA结构的慢病毒,然后制备出高滴度的Atp6i-shRNA慢病毒。
2大鼠破骨样细胞体外培养体系的建立
选用3-4周龄的Sprague-Dawley(SD)雄性大鼠,无菌条件下用α-MEM冲洗、收集股骨和胫骨骨髓细胞,用含有诱导剂1,25-(OH)_2D_3的全培养基[含20%胎牛血清、1×10~(-8)mol/L的1,25-(OH)_2D_3、氨苄青霉素100U/ml以及链霉素100μg/ml的α-MEM培养基]稀释到1.5×10~6个/ml,接种在预置玻片和骨片的24孔培养板中,37℃、5%CO_2及饱和湿度下培养,每2d换液一次,每次换总量的1/2。大鼠骨髓细胞在1,25-(OH)_2D_3诱导培养下,可于培养的第5d开始逐渐分化成具有典型形态、生化以及骨吸收特征的破骨样细胞(osteoclast-like cell,OCL)。培养8d时,成熟的OCL细胞数目达到最多,12d后开始出现空泡变性,并逐渐死亡。骨吸收陷窝于细胞诱导培养的第7d开始出现,第10-11d最多,之后细胞开始死亡,陷窝数目不再增加。因此于培养第6d开始对OCL的Atp6i基因进行RNAi实验最为合适。
3体外RNAi实验
本实验共分3组:转染组(Atp6i-shRNA慢病毒)、阴性对照组(阴性对照慢病毒)、空白对照组。分别将不同的慢病毒转染到体外诱导培养第6d的大鼠OCL中,于转染96h抽提各组OCL总RNA,采用实时定量聚合酶链反应(real time polymerase chainreaction)检测Atp6i基因表达量的变化。同时统计转染96h与OCL共培养的牛骨片上骨吸收陷窝的面积,分析Atp6i抑制后对OCL骨吸收的影响。
转染96h实时定量PCR检测发现,与阴性对照组以及空白对照组相比,转染组中Atp6i基因的mRNA水平明显减少(P＜0.01),有效抑制率为57.73%。同时转染组中骨陷窝的数量也明显减少(P＜0.01),有效抑制率为60.64%。
综上所述,本研究成功建立了体外诱导培养大鼠OCL体系,并构建了Atp6i-shRNA慢病毒,进行了体外抑制Atp6i基因表达的实验。研究表明,自行设计制备的Atp6i-siRNA慢病毒可以高效抑制体外培养的OCL的骨吸收功能,Atp6i基因对于破骨细胞维持正常的骨吸收功能是必需的。
The osteoclast(OC) is the functional cell in the bone absorption.Dys -function of OC often causes unbalances of bone reconstruction.Normal bone remodeling requires precise control over the rates of bone formation by osteoblast(OB) and degradation by OC.It is a common pathological change in bone diseases resulting from the OC dysfunction and over-activity of bone resorption,such as osteoporosis,Paget disease,giant cell tumor of bone, rheumatoid arthritis and tumor periodontal disease.The gene Atp6i of vacuolar type H~+ adenosine triphosphatase(v-ATPase),which is the specific and highest expression in the ruffled border membrane of activated OC,is essential for OC function,such as bone resorption and metabolism balance with normal function of osteoblast.Estrogen,calcitonin and diphosphonate have been used clinically to suppress differentiation of OC and its abnormal bone resorption. These medicines,however,often bother clinicians due to side-effects and non-specificity.
In order to seek nontoxicity,non side-effect and higher specific target molecular methods and strategy,we focus on post-transcration level inhibition of Atp6i gene in OC-like cell(OCL).Lentivirus mediated RNA interference(RNAi) technique has been used to knock-down the rat Atp6i gene in OCL specificly and to inhibit bone absorption.Biomedication could be made inhibiting Atp6i expression via RNAi,which is considered to be a medication method to control overactive osteoclast function in clinical therapeusis.
1 Design and construction of a lentivirusal vector driving shRNA for Atp6i
First of all,the lentivirus(pGCL-GFP) shuttle plasmid containing target Atp6i gene was constructed by gene engineering,and highly efficient short hairpin RNA(Atp6i-shRNA) shuttle vector was selected by Western Blotting from 293T cell which over-expressed Atp6i gene fusion protein(contained by pEGFP-C1-Atp6i).Five short interfering RNA(siRNA) targets had been desighed. Finally,Atp6i-shRNA(NO.5) shuttle vector was the best one used to make high titer Atp6i-shRNA lentivirus in the presence of packaging plasmid.
2 Establish a system for rat OCL culture in vitro
Three to four week-old male Sprague -Dawley(SD) rats were sacrificed and femurs and tibias were dissected free from adherent tissue.The marrow cells were washed withα-MEM and diluted at a density of 1.5×10~6 cells/ml on plates with either cover slips or bone slices in complete medium(α-MEM with 20% FBS,10nM of 1,25-(OH)_2D_3,100U/ml penicillin and 100ug/ml streptomycin).For stimulation of osteoclast formation,marrow cells were continuously incubated with 1,25-(OH)_2D_3.The medium were replaced half with fresh complete medium every two days.After 5 days in culture,mononuclear precursor and multinuc -leated OCL phenotype were verified by tartrate-resistant acid phosphatase (TRAP) staining and bone-resorptive capacity.The number of the muture OCL achieved the peak on the 8~(th) day of culture,and OCL was closed to death on the 12~(th) day.The resorption lacunae on bone slices were formed from the 7~(th) day of culture,and their number reached maximum at 10-11 days.Therefore, it was the best time to do the RNAi experiment of Atp6i of OCL on the 6~(th) day of culture.
3 RNAi experiments in vitro
Using the SD male rats of 3-4 weeks and making 1,25-(OH)_2D_3 as ininducer, it was to set up OCL in vitro,which was cultured with cattle femurs bone slices. The OCL was divided into 3 groups:transfection group(positive Atp6i-shRNA lentivirus),negative control group(negative Atp6i-shRNA lentivirus),and blank group(no siRNA).The Atp6i-shRNA lentivirus vector was transfected into the groups on the 6~(th) day marrow cells were cultured.Ninety-six hours after transfection,total RNA of the OCL of each group was harvest,used for real time polymerase chain reaction(PCR) analysis to detect the different expression levels of products of Atp6i gene.Quantities of the areas of absorption lacuna were also analyzed.
Ninety-six hours after Atp6i-shRNA lentivirus transfected,compared with negative groups and blank group,we can find the extinction level of products of real time PCR in transfection group was significantly reduced by 57.73% (P＜0.01),and the areas of absorption lacuna in transfection group was reduced by 60.6%(P＜0.01).
In summary,this study successfully constructed lentivirus Atp6i-shRNA vector and established system for rat OCL culture in vitro.We found that it efficiently suppressed the expression level to Atp6i gene of OCL,and bone absorptive function of OCL of rat was also identified.Atp6i gene is indispensable to maintain the bone absorption function of OC.
为了探索无毒副作用、高效且高特异性的分子治疗方法和策略,我们针对大鼠Atp6i基因,采用慢病毒介导的RNA干扰(RNA interference,RNAi)技术,进行了靶基因转录后水平的特异性抑制研究。尝试制备一种通过RNAi抑制Atp6i表达的生物抑制剂,为临床治疗由于OC功能亢进所导致的骨破坏提供理论依据。
1设计、构建携带大鼠Atp6i基因小发卡RNA结构的慢病毒载体
首先应用基因工程技术,建立体外过表达外源大鼠Atp6i基因的293T细胞培养体系,在此细胞中筛选出高效、高特异性抑制Atp6i的小干扰RNA(short interferingRNA,siRNA)靶点,并制备成携带该靶点的慢病毒。设计5组(1-5#)大鼠Atp6i基因siRNA靶点,并制备出携带该靶点小发卡RNA(short hairpin RNA,shRNA)结构的慢病毒质粒(pGCL-GFP)即Atp6i-shRNA穿梭质粒,在过表达Atp6i基因融合蛋白(pEGFP-Cl-Atp6i携带)的293T细胞中,利用Western Blot方法筛选出了高效的RNA干扰Atp6i-shRNA(5号)穿梭质粒,以此为模版,包装成携带Atp6i-shRNA结构的慢病毒,然后制备出高滴度的Atp6i-shRNA慢病毒。
2大鼠破骨样细胞体外培养体系的建立
选用3-4周龄的Sprague-Dawley(SD)雄性大鼠,无菌条件下用α-MEM冲洗、收集股骨和胫骨骨髓细胞,用含有诱导剂1,25-(OH)_2D_3的全培养基[含20%胎牛血清、1×10~(-8)mol/L的1,25-(OH)_2D_3、氨苄青霉素100U/ml以及链霉素100μg/ml的α-MEM培养基]稀释到1.5×10~6个/ml,接种在预置玻片和骨片的24孔培养板中,37℃、5%CO_2及饱和湿度下培养,每2d换液一次,每次换总量的1/2。大鼠骨髓细胞在1,25-(OH)_2D_3诱导培养下,可于培养的第5d开始逐渐分化成具有典型形态、生化以及骨吸收特征的破骨样细胞(osteoclast-like cell,OCL)。培养8d时,成熟的OCL细胞数目达到最多,12d后开始出现空泡变性,并逐渐死亡。骨吸收陷窝于细胞诱导培养的第7d开始出现,第10-11d最多,之后细胞开始死亡,陷窝数目不再增加。因此于培养第6d开始对OCL的Atp6i基因进行RNAi实验最为合适。
3体外RNAi实验
本实验共分3组:转染组(Atp6i-shRNA慢病毒)、阴性对照组(阴性对照慢病毒)、空白对照组。分别将不同的慢病毒转染到体外诱导培养第6d的大鼠OCL中,于转染96h抽提各组OCL总RNA,采用实时定量聚合酶链反应(real time polymerase chainreaction)检测Atp6i基因表达量的变化。同时统计转染96h与OCL共培养的牛骨片上骨吸收陷窝的面积,分析Atp6i抑制后对OCL骨吸收的影响。
转染96h实时定量PCR检测发现,与阴性对照组以及空白对照组相比,转染组中Atp6i基因的mRNA水平明显减少(P＜0.01),有效抑制率为57.73%。同时转染组中骨陷窝的数量也明显减少(P＜0.01),有效抑制率为60.64%。
综上所述,本研究成功建立了体外诱导培养大鼠OCL体系,并构建了Atp6i-shRNA慢病毒,进行了体外抑制Atp6i基因表达的实验。研究表明,自行设计制备的Atp6i-siRNA慢病毒可以高效抑制体外培养的OCL的骨吸收功能,Atp6i基因对于破骨细胞维持正常的骨吸收功能是必需的。
The osteoclast(OC) is the functional cell in the bone absorption.Dys -function of OC often causes unbalances of bone reconstruction.Normal bone remodeling requires precise control over the rates of bone formation by osteoblast(OB) and degradation by OC.It is a common pathological change in bone diseases resulting from the OC dysfunction and over-activity of bone resorption,such as osteoporosis,Paget disease,giant cell tumor of bone, rheumatoid arthritis and tumor periodontal disease.The gene Atp6i of vacuolar type H~+ adenosine triphosphatase(v-ATPase),which is the specific and highest expression in the ruffled border membrane of activated OC,is essential for OC function,such as bone resorption and metabolism balance with normal function of osteoblast.Estrogen,calcitonin and diphosphonate have been used clinically to suppress differentiation of OC and its abnormal bone resorption. These medicines,however,often bother clinicians due to side-effects and non-specificity.
In order to seek nontoxicity,non side-effect and higher specific target molecular methods and strategy,we focus on post-transcration level inhibition of Atp6i gene in OC-like cell(OCL).Lentivirus mediated RNA interference(RNAi) technique has been used to knock-down the rat Atp6i gene in OCL specificly and to inhibit bone absorption.Biomedication could be made inhibiting Atp6i expression via RNAi,which is considered to be a medication method to control overactive osteoclast function in clinical therapeusis.
1 Design and construction of a lentivirusal vector driving shRNA for Atp6i
First of all,the lentivirus(pGCL-GFP) shuttle plasmid containing target Atp6i gene was constructed by gene engineering,and highly efficient short hairpin RNA(Atp6i-shRNA) shuttle vector was selected by Western Blotting from 293T cell which over-expressed Atp6i gene fusion protein(contained by pEGFP-C1-Atp6i).Five short interfering RNA(siRNA) targets had been desighed. Finally,Atp6i-shRNA(NO.5) shuttle vector was the best one used to make high titer Atp6i-shRNA lentivirus in the presence of packaging plasmid.
2 Establish a system for rat OCL culture in vitro
Three to four week-old male Sprague -Dawley(SD) rats were sacrificed and femurs and tibias were dissected free from adherent tissue.The marrow cells were washed withα-MEM and diluted at a density of 1.5×10~6 cells/ml on plates with either cover slips or bone slices in complete medium(α-MEM with 20% FBS,10nM of 1,25-(OH)_2D_3,100U/ml penicillin and 100ug/ml streptomycin).For stimulation of osteoclast formation,marrow cells were continuously incubated with 1,25-(OH)_2D_3.The medium were replaced half with fresh complete medium every two days.After 5 days in culture,mononuclear precursor and multinuc -leated OCL phenotype were verified by tartrate-resistant acid phosphatase (TRAP) staining and bone-resorptive capacity.The number of the muture OCL achieved the peak on the 8~(th) day of culture,and OCL was closed to death on the 12~(th) day.The resorption lacunae on bone slices were formed from the 7~(th) day of culture,and their number reached maximum at 10-11 days.Therefore, it was the best time to do the RNAi experiment of Atp6i of OCL on the 6~(th) day of culture.
3 RNAi experiments in vitro
Using the SD male rats of 3-4 weeks and making 1,25-(OH)_2D_3 as ininducer, it was to set up OCL in vitro,which was cultured with cattle femurs bone slices. The OCL was divided into 3 groups:transfection group(positive Atp6i-shRNA lentivirus),negative control group(negative Atp6i-shRNA lentivirus),and blank group(no siRNA).The Atp6i-shRNA lentivirus vector was transfected into the groups on the 6~(th) day marrow cells were cultured.Ninety-six hours after transfection,total RNA of the OCL of each group was harvest,used for real time polymerase chain reaction(PCR) analysis to detect the different expression levels of products of Atp6i gene.Quantities of the areas of absorption lacuna were also analyzed.
Ninety-six hours after Atp6i-shRNA lentivirus transfected,compared with negative groups and blank group,we can find the extinction level of products of real time PCR in transfection group was significantly reduced by 57.73% (P＜0.01),and the areas of absorption lacuna in transfection group was reduced by 60.6%(P＜0.01).
In summary,this study successfully constructed lentivirus Atp6i-shRNA vector and established system for rat OCL culture in vitro.We found that it efficiently suppressed the expression level to Atp6i gene of OCL,and bone absorptive function of OCL of rat was also identified.Atp6i gene is indispensable to maintain the bone absorption function of OC.
引文
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