慢病毒介导RNA干扰沉默IL-1Ⅰ型受体在大鼠骨关节炎基因治疗中的初步研究
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摘要
背景
骨关节炎(osteoarthritis OA)是一种慢性、渐进性、退行性关节病变，是临床上最常见的关节疾病。据统计，在50岁以上人群中，OA的发病率仅次于心脏病列第二位。在有肢体发育畸形和关节创伤病史的病人中，其发病率更高。随着我国人口老龄化和创伤发生率增高，OA已成为导致老龄者和创伤病人病废的最主要疾病之一。目前研究显示，OA是力学因素和生物学因素作用下软骨合成和降解偶联失衡的结果。细胞因子学说目前并不能解释OA中软骨破坏的全部原因。但是，不论软骨的哪个部位以何种形式发生退变，都不能脱离IL-1等炎症介质的作用。因此，对IL-1以及IL-1受体的研究成为OA基础研究方面的一个热点。IL-1包括IL-1α、IL-1β两种亚型，其中IL-1β是细胞外IL-1主要的亚型。IL-1β可由多种细胞产生，如软骨细胞、滑膜细胞、巨噬细胞等。在OA中软骨细胞上IL-1β受体的表达要高于正常的软骨细胞。研究表明，IL-1β能刺激软骨细胞、成纤维细胞和滑膜细胞分泌前列环素2(PGE2)、胶原酶和MMPs，加速软骨基质中蛋白多糖和Ⅱ型胶原的降解，并抑制蛋白多糖和Ⅱ型胶原的合成，抑制Ⅱ型前胶原mRNA的表达，改变和破坏了软骨细胞周围环境，促进软骨细胞的凋亡。
IL-1受体分为80kD和68kD两种，前者称为IL-1RI，生物体内分布广泛，为穿膜蛋白，胞浆区含有丝氨酸和苏氨酸残基，当IL-1与IL-1RI结合后丝氨酸和苏氨酸很快被磷酸化，起信号转导的作用。目前体外的研究显示IL-1β通过与软骨细胞膜上的IL-1 RI，经丝裂素原激活的蛋白激酶(mitogen-activated protein kinases，MAPKs)途径和NF-κB(nuclear factor-κB)途径进行细胞内信号转导，引起MMPs的表达升高、软骨细胞凋亡等一系列反应。
本研究通过慢病毒介导的RNA干扰(RNAi)技术，抑制IL-1Ⅰ型受体的表达，阻断IL-1的作用，观察其在体外培养的软骨细胞以及大鼠OA模型中MMP-1表达的影响，并观察在模型中对关节软骨破坏的影响。评价这种治疗方法的有效性，并为进一步研究MMP-1升高的机制提供基础。
第一部分大鼠IL-1Ⅰ型受体有效RNA干扰片段的设计与筛选
目的设计并筛选大鼠IL-1Ⅰ型受体有效RNA干扰片段，为后续实验准备
材料与方法使用公用网站设计、筛选符合公开文献筛选参数的序列4条以及阴性对照序列1条，由上海吉凯基因技术有限公司合成。与pGCL-GFP质粒重组后，转染大肠杆菌感受态细胞。阳性克隆经过PCR鉴定以及DNA测序鉴定后，进行慢病毒小量包装。慢病毒滴度测定后，用Western blot方法在体外培养的软骨细胞中进行有效靶点的筛选。
结果PCR鉴定和DNA测序鉴定显示重组质粒中含有所设计的寡核苷酸单链，序列正确。转染siRNA-NC质粒的大鼠软骨细胞中IL-1RⅠ表达未受影响，而转染IL-1RⅠsiRNA-2号片段的大鼠软骨细胞中IL-1RⅠ蛋白质表达水平显著下降，提示小干扰RNA介导的IL-1RⅠ序列特异性基因沉默。
结论成功设计重组了大鼠IL-1RⅠsiRNA有效片段，并包装了慢病毒载体。
第二部分慢病毒介导的RNAi沉默IL-1Ⅰ型受体在体外培养的大鼠软骨细胞中的作用
目的在体外培养大鼠关节软骨细胞的基础上，验证第一部分中大鼠IL-1RⅠsiRNA在离体关节软骨细胞中对IL-1RⅠ和MMP-1的作用。
材料与方法使用80-100g SD大鼠，取正常关节软骨培养软骨细胞并鉴定。慢病毒包装和滴度测定等前期准备工作完成后，使用IL-1刺激软骨细胞，然后将慢病毒包装的IL-1RⅠRNA有效干扰片段感染软骨细胞。感染3天后首先观察报告基因GFP的表达情况，发现感染效率大于70％，收集总蛋白并进入后续Western blot的实验，测定IL-1RⅠ和MMP-1的表达情况。
结果通过细胞形态学观察、甲苯胺兰染色和Ⅱ型胶原染色，证明所培养的细胞确是软骨细胞。慢病毒介导的IL-1RⅠRNA干扰后，实验组细胞的IL-1RⅠ和MMP-1蛋白较对照组均有明显下降。
结论体外成功培养了大鼠关节软骨细胞。慢病毒介导的IL-1RⅠRNA干扰有效，并且IL-1RⅠRNA干扰对MMP-1的表达有明显抑制作用。
第三部分慢病毒介导的RNAi沉默IL-1Ⅰ型受体在大鼠骨关节炎模型中的作用
目的在制作大鼠骨关节炎模型的基础上，观察慢病毒介导的IL-1RⅠsiRNA对大鼠骨关节炎软骨破坏的影响以及对软骨组织中MMP-1表达的作用，初步观察这种治疗方法的作用，并为进一步的机制研究提供基础。
材料与方法采用前交叉韧带切断加内侧半月板部分切除的方法制作骨关节炎模型，根据软骨破坏程度的观察和软骨组织中MMP-1表达变化的研究，确认该模型可行性。然后在大鼠的骨关节炎模型中使用慢病毒介导的RNA干扰抑制自介素-1Ⅰ型受体，用Western blot法测定干预后大鼠关节软骨破坏程度的变化以及软骨组织中MMP-1的表达。
结果大体观察和病理切片观察发现大鼠行前交叉韧带切断加内侧半月板部分切除术后4周，即开始出现骨关节炎表现，同时MMP-1表达明显上调。而在进行慢病毒介导的RNA干扰抑制白介素-1Ⅰ型受体后，关节软骨的破坏程度明显下降，但是MMP-1的表达下降达不到统计学差异。
结论前交叉韧带切断加内侧半月板部分切除的方法制作大鼠骨关节炎模型是可行的。慢病毒介导的RNA干扰抑制白介素-1Ⅰ型受体对关节软骨有保护作用。MMP-1的表达可能受到包括IL-1在内的多种炎性因子的联合作用。
Background
Osteoarthritis (OA) is a chronic and degenerative disease of joint and is one of the most common joint disorders. According to statistics, the incidence of OA is the second in the people above 50 years old, only lower than that of heart disease. And the incidence is higher in the people suffering from limbs deformity or joint trauma. OA is becoming one of the principal diseases leading to disability with aging of population and high incidence of trauma. Previous research in the field of OA has characterized that OA is the result of disequilibrium between cartilage degeneration and synthesis controlled by the mechanic and biologic factors. The effect of inflammation mediators, such as interleukin-1 (IL—1), is necessary in the cartilage degeneration although cytokines is not enough. So, the study on IL—1 and IL—1 receptors is a hot point. IL-1 include IL-1αand IL-1βand the latter is principle in extracellular environment. IL-1βmay be synthesized by several kinds of cells, such as chondrocytes, synoviocytes and macrophages. According to the previous researches, IL—1βstimulates chondrocytes, fibroblasts and synoviocytes to synthesize prostaglandin E2 (PGE2), collagenases and matrix metalloproteinases (MMPs), and accelerates the degeneration of main compositions of cartilage, such as proteoglycan and type II collagen, depresses the mRNA expression of type II collagen, destroys the environment of chondrocytes and promotes their apoptosis.
There are two kinds of IL-1 receptors, 80kD and 68kD in molecular weight respectively and the former is called IL-1RI, expressing extensively in organisms. When coupled with IL-1, serine and threonine residues in IL—1RI are phosphorylated and IL-1RI become activated to signal transduction. It has been certificated that mitogen-activated protein kinases (MAPKs)and nuclear factor-κB(NF-κB) signaling pathways participate in the process IL-1β- induced MMP-1 express and chondrocytes apoptosis.
The primary goal of the present study is to observe, using lentivirus mediated RNA interference to repress the expression of IL-1RI, the effect on MMP-1 expression of IL—1 in both cultured rat chondrocytes and osteoarthritic rats. We also observed the effect of IL—1 on articular cartilage destruction in osteoarthritic rats and evaluated the validy of the treatment.
Part I Design and Screening of Valid siRNA of Rat IL-1R I
Objectives Design siRNA of rat IL-1R I and screen those valid for the following experiments.
Material and methods Design 4 gene sequences and 1 non-sence control gene sequence consistent with the screening feature described in previous articles using open websites, and the sequences were synthesized with the help of Genechem Company. The sequences were amplificated in Bacterium coli after recombinated with pGCL-GFP plasmid. After PCR and sequencing identification, the positive clones were packaged into lentivirus. Then we screen the valid targets by western blot in cultured rat chondrocytes after detect the virus titer.
Results PCR and DNA sequencing certificate that the oligonucleotides were correct. The protein level of IL—1R I in chondrocytes with transfection by siRNA-2# plasmid is depressed, while that of the chondrocytes with transfection by siRNA-NC plasmid is not influenced.
Conclusions We designed and recombinated valid siRNA of rat IL-1R I , and packaged lentivirus vector.
Part II The Effect of Lentivirus Mediated RNA Interference on MMP-1 Expression in Cultured Rat Chondrocytes
Objectives Culture rat chondrocytes and observe the effect on IL-1R I and MMP-1 expression of lentivirus mediated IL-1R I RNA interference in cultured rat chondrocytes.
Material and methods Harvest articular cartilage from 80～100g SD rats knee joints to carry out rat chondrocytes culture and identification. IL—1 stimulation up-regulates IL-1R I and lentivirus mediated siRNA transfection to the chondrocytes. If the infection ratio beyond 70% three days later through observing the expression of reporting gene GFP, Western blot is carried out to detect the expression of IL-1R I and MMP-1.
Results Through morphologic and immunocytochemistry identification, cultured chondrocytes were identified. Compared with non-sence control goup, IL-1R I and MMP-1 expression is much lower in 2#-siRNA group after lentivirus mediated RNA interference.
Conclusions We cultured rat chondrocytes successfully and lentivirus mediated IL-1R I RNA interference work well. IL-1R I RNA interference depress the expression of MMP-1 in cultured rat chondrocytes.
Part III The Effect of Lentivirus Mediated RNA Interference on MMP-1 Expression and Cartilage Destruction in Osteoarthritic Rat
Objectives Observe MMP-1 expression and cartilage destruction in osteoarthritic rats and observe the effect on MMP-1 expression and cartilage destruction of lentivirus mediated IL-1R I RNA interference in osteoarthritic rat. Evaluate the validity of the gene therapy and make foundation for further studies.
Material and methods Establish OA rat model by resecting of the anterior cruciate ligament (ACL) and anterior part of the right knee medial meniscus. Evaluate the validity of the model by observing the MMP-1 expression and cartilage destruction in osteoarthritic rats. Then repress IL-1R I expression by lentivirus mediated RNAi. Observe the cartilage destruction and detect MMP-1 expression in cartilage by Western blot.
Results There was osteoarthritic change in articular cartilage from 4 weeks after operation by macroscopic and microscopic observe. While, MMP-1 expression was up-regulated in articular cartilage from 2 weeks after operation. There was significant decrease in articular cartilage destruction in both macroscopic and microscopic observing. However, the differences of MMP-1 expression among the IL-1R I RNAi group and negative control groups did not reach statistical significance.
Conclusions It works to establish OA rat model by resecting of the anterior cruciate ligament (ACL) and anterior part of the right knee medial meniscus. Lentivirus mediated RNA interference on IL-1R I provide protect on articular cartilage in osteoarthritic rats. MMP-1 expression may be controlled by several inflammatory factors including IL-1.
骨关节炎(osteoarthritis OA)是一种慢性、渐进性、退行性关节病变，是临床上最常见的关节疾病。据统计，在50岁以上人群中，OA的发病率仅次于心脏病列第二位。在有肢体发育畸形和关节创伤病史的病人中，其发病率更高。随着我国人口老龄化和创伤发生率增高，OA已成为导致老龄者和创伤病人病废的最主要疾病之一。目前研究显示，OA是力学因素和生物学因素作用下软骨合成和降解偶联失衡的结果。细胞因子学说目前并不能解释OA中软骨破坏的全部原因。但是，不论软骨的哪个部位以何种形式发生退变，都不能脱离IL-1等炎症介质的作用。因此，对IL-1以及IL-1受体的研究成为OA基础研究方面的一个热点。IL-1包括IL-1α、IL-1β两种亚型，其中IL-1β是细胞外IL-1主要的亚型。IL-1β可由多种细胞产生，如软骨细胞、滑膜细胞、巨噬细胞等。在OA中软骨细胞上IL-1β受体的表达要高于正常的软骨细胞。研究表明，IL-1β能刺激软骨细胞、成纤维细胞和滑膜细胞分泌前列环素2(PGE2)、胶原酶和MMPs，加速软骨基质中蛋白多糖和Ⅱ型胶原的降解，并抑制蛋白多糖和Ⅱ型胶原的合成，抑制Ⅱ型前胶原mRNA的表达，改变和破坏了软骨细胞周围环境，促进软骨细胞的凋亡。
IL-1受体分为80kD和68kD两种，前者称为IL-1RI，生物体内分布广泛，为穿膜蛋白，胞浆区含有丝氨酸和苏氨酸残基，当IL-1与IL-1RI结合后丝氨酸和苏氨酸很快被磷酸化，起信号转导的作用。目前体外的研究显示IL-1β通过与软骨细胞膜上的IL-1 RI，经丝裂素原激活的蛋白激酶(mitogen-activated protein kinases，MAPKs)途径和NF-κB(nuclear factor-κB)途径进行细胞内信号转导，引起MMPs的表达升高、软骨细胞凋亡等一系列反应。
本研究通过慢病毒介导的RNA干扰(RNAi)技术，抑制IL-1Ⅰ型受体的表达，阻断IL-1的作用，观察其在体外培养的软骨细胞以及大鼠OA模型中MMP-1表达的影响，并观察在模型中对关节软骨破坏的影响。评价这种治疗方法的有效性，并为进一步研究MMP-1升高的机制提供基础。
第一部分大鼠IL-1Ⅰ型受体有效RNA干扰片段的设计与筛选
目的设计并筛选大鼠IL-1Ⅰ型受体有效RNA干扰片段，为后续实验准备
材料与方法使用公用网站设计、筛选符合公开文献筛选参数的序列4条以及阴性对照序列1条，由上海吉凯基因技术有限公司合成。与pGCL-GFP质粒重组后，转染大肠杆菌感受态细胞。阳性克隆经过PCR鉴定以及DNA测序鉴定后，进行慢病毒小量包装。慢病毒滴度测定后，用Western blot方法在体外培养的软骨细胞中进行有效靶点的筛选。
结果PCR鉴定和DNA测序鉴定显示重组质粒中含有所设计的寡核苷酸单链，序列正确。转染siRNA-NC质粒的大鼠软骨细胞中IL-1RⅠ表达未受影响，而转染IL-1RⅠsiRNA-2号片段的大鼠软骨细胞中IL-1RⅠ蛋白质表达水平显著下降，提示小干扰RNA介导的IL-1RⅠ序列特异性基因沉默。
结论成功设计重组了大鼠IL-1RⅠsiRNA有效片段，并包装了慢病毒载体。
第二部分慢病毒介导的RNAi沉默IL-1Ⅰ型受体在体外培养的大鼠软骨细胞中的作用
目的在体外培养大鼠关节软骨细胞的基础上，验证第一部分中大鼠IL-1RⅠsiRNA在离体关节软骨细胞中对IL-1RⅠ和MMP-1的作用。
材料与方法使用80-100g SD大鼠，取正常关节软骨培养软骨细胞并鉴定。慢病毒包装和滴度测定等前期准备工作完成后，使用IL-1刺激软骨细胞，然后将慢病毒包装的IL-1RⅠRNA有效干扰片段感染软骨细胞。感染3天后首先观察报告基因GFP的表达情况，发现感染效率大于70％，收集总蛋白并进入后续Western blot的实验，测定IL-1RⅠ和MMP-1的表达情况。
结果通过细胞形态学观察、甲苯胺兰染色和Ⅱ型胶原染色，证明所培养的细胞确是软骨细胞。慢病毒介导的IL-1RⅠRNA干扰后，实验组细胞的IL-1RⅠ和MMP-1蛋白较对照组均有明显下降。
结论体外成功培养了大鼠关节软骨细胞。慢病毒介导的IL-1RⅠRNA干扰有效，并且IL-1RⅠRNA干扰对MMP-1的表达有明显抑制作用。
第三部分慢病毒介导的RNAi沉默IL-1Ⅰ型受体在大鼠骨关节炎模型中的作用
目的在制作大鼠骨关节炎模型的基础上，观察慢病毒介导的IL-1RⅠsiRNA对大鼠骨关节炎软骨破坏的影响以及对软骨组织中MMP-1表达的作用，初步观察这种治疗方法的作用，并为进一步的机制研究提供基础。
材料与方法采用前交叉韧带切断加内侧半月板部分切除的方法制作骨关节炎模型，根据软骨破坏程度的观察和软骨组织中MMP-1表达变化的研究，确认该模型可行性。然后在大鼠的骨关节炎模型中使用慢病毒介导的RNA干扰抑制自介素-1Ⅰ型受体，用Western blot法测定干预后大鼠关节软骨破坏程度的变化以及软骨组织中MMP-1的表达。
结果大体观察和病理切片观察发现大鼠行前交叉韧带切断加内侧半月板部分切除术后4周，即开始出现骨关节炎表现，同时MMP-1表达明显上调。而在进行慢病毒介导的RNA干扰抑制白介素-1Ⅰ型受体后，关节软骨的破坏程度明显下降，但是MMP-1的表达下降达不到统计学差异。
结论前交叉韧带切断加内侧半月板部分切除的方法制作大鼠骨关节炎模型是可行的。慢病毒介导的RNA干扰抑制白介素-1Ⅰ型受体对关节软骨有保护作用。MMP-1的表达可能受到包括IL-1在内的多种炎性因子的联合作用。
Background
Osteoarthritis (OA) is a chronic and degenerative disease of joint and is one of the most common joint disorders. According to statistics, the incidence of OA is the second in the people above 50 years old, only lower than that of heart disease. And the incidence is higher in the people suffering from limbs deformity or joint trauma. OA is becoming one of the principal diseases leading to disability with aging of population and high incidence of trauma. Previous research in the field of OA has characterized that OA is the result of disequilibrium between cartilage degeneration and synthesis controlled by the mechanic and biologic factors. The effect of inflammation mediators, such as interleukin-1 (IL—1), is necessary in the cartilage degeneration although cytokines is not enough. So, the study on IL—1 and IL—1 receptors is a hot point. IL-1 include IL-1αand IL-1βand the latter is principle in extracellular environment. IL-1βmay be synthesized by several kinds of cells, such as chondrocytes, synoviocytes and macrophages. According to the previous researches, IL—1βstimulates chondrocytes, fibroblasts and synoviocytes to synthesize prostaglandin E2 (PGE2), collagenases and matrix metalloproteinases (MMPs), and accelerates the degeneration of main compositions of cartilage, such as proteoglycan and type II collagen, depresses the mRNA expression of type II collagen, destroys the environment of chondrocytes and promotes their apoptosis.
There are two kinds of IL-1 receptors, 80kD and 68kD in molecular weight respectively and the former is called IL-1RI, expressing extensively in organisms. When coupled with IL-1, serine and threonine residues in IL—1RI are phosphorylated and IL-1RI become activated to signal transduction. It has been certificated that mitogen-activated protein kinases (MAPKs)and nuclear factor-κB(NF-κB) signaling pathways participate in the process IL-1β- induced MMP-1 express and chondrocytes apoptosis.
The primary goal of the present study is to observe, using lentivirus mediated RNA interference to repress the expression of IL-1RI, the effect on MMP-1 expression of IL—1 in both cultured rat chondrocytes and osteoarthritic rats. We also observed the effect of IL—1 on articular cartilage destruction in osteoarthritic rats and evaluated the validy of the treatment.
Part I Design and Screening of Valid siRNA of Rat IL-1R I
Objectives Design siRNA of rat IL-1R I and screen those valid for the following experiments.
Material and methods Design 4 gene sequences and 1 non-sence control gene sequence consistent with the screening feature described in previous articles using open websites, and the sequences were synthesized with the help of Genechem Company. The sequences were amplificated in Bacterium coli after recombinated with pGCL-GFP plasmid. After PCR and sequencing identification, the positive clones were packaged into lentivirus. Then we screen the valid targets by western blot in cultured rat chondrocytes after detect the virus titer.
Results PCR and DNA sequencing certificate that the oligonucleotides were correct. The protein level of IL—1R I in chondrocytes with transfection by siRNA-2# plasmid is depressed, while that of the chondrocytes with transfection by siRNA-NC plasmid is not influenced.
Conclusions We designed and recombinated valid siRNA of rat IL-1R I , and packaged lentivirus vector.
Part II The Effect of Lentivirus Mediated RNA Interference on MMP-1 Expression in Cultured Rat Chondrocytes
Objectives Culture rat chondrocytes and observe the effect on IL-1R I and MMP-1 expression of lentivirus mediated IL-1R I RNA interference in cultured rat chondrocytes.
Material and methods Harvest articular cartilage from 80～100g SD rats knee joints to carry out rat chondrocytes culture and identification. IL—1 stimulation up-regulates IL-1R I and lentivirus mediated siRNA transfection to the chondrocytes. If the infection ratio beyond 70% three days later through observing the expression of reporting gene GFP, Western blot is carried out to detect the expression of IL-1R I and MMP-1.
Results Through morphologic and immunocytochemistry identification, cultured chondrocytes were identified. Compared with non-sence control goup, IL-1R I and MMP-1 expression is much lower in 2#-siRNA group after lentivirus mediated RNA interference.
Conclusions We cultured rat chondrocytes successfully and lentivirus mediated IL-1R I RNA interference work well. IL-1R I RNA interference depress the expression of MMP-1 in cultured rat chondrocytes.
Part III The Effect of Lentivirus Mediated RNA Interference on MMP-1 Expression and Cartilage Destruction in Osteoarthritic Rat
Objectives Observe MMP-1 expression and cartilage destruction in osteoarthritic rats and observe the effect on MMP-1 expression and cartilage destruction of lentivirus mediated IL-1R I RNA interference in osteoarthritic rat. Evaluate the validity of the gene therapy and make foundation for further studies.
Material and methods Establish OA rat model by resecting of the anterior cruciate ligament (ACL) and anterior part of the right knee medial meniscus. Evaluate the validity of the model by observing the MMP-1 expression and cartilage destruction in osteoarthritic rats. Then repress IL-1R I expression by lentivirus mediated RNAi. Observe the cartilage destruction and detect MMP-1 expression in cartilage by Western blot.
Results There was osteoarthritic change in articular cartilage from 4 weeks after operation by macroscopic and microscopic observe. While, MMP-1 expression was up-regulated in articular cartilage from 2 weeks after operation. There was significant decrease in articular cartilage destruction in both macroscopic and microscopic observing. However, the differences of MMP-1 expression among the IL-1R I RNAi group and negative control groups did not reach statistical significance.
Conclusions It works to establish OA rat model by resecting of the anterior cruciate ligament (ACL) and anterior part of the right knee medial meniscus. Lentivirus mediated RNA interference on IL-1R I provide protect on articular cartilage in osteoarthritic rats. MMP-1 expression may be controlled by several inflammatory factors including IL-1.
引文
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