人白细胞介素-10基因治疗对去卵巢大鼠实验性牙周炎发生的影响
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摘要
牙周炎是由牙菌斑中的微生物所引起的牙周支持组织的慢性感染性疾病,主要表现为牙周支持组织的炎症和牙槽骨吸收,最终导致牙松动及丧失。牙周致病菌是牙周病发生的必要条件,宿主防御机能在牙周病发生、发展中起十分重要的作用。越来越多的证据表明PMO是牙周炎的全身促进因子。但牙周炎与PMO的联系机制还未完全阐明,预防和治疗PMO导致的牙周组织破坏是牙周领域的研究热点和难点之一。
实验一细胞因子在去卵巢大鼠实验性牙周炎牙周组织中的表达
目的了解PMO在牙周炎发病中的作用机制。方法将24只雌性Sprague-Dawley(SD)大鼠随机分成4组,每组6只。1:假手术(SHAM)组:对大鼠实施去卵巢假手术;2:去卵巢组(OVX):切除双侧卵巢;3:假手术+实验性牙周炎(SHAM+EP)组:对大鼠实施去卵巢假手术,术后10w,采用丝线结扎左侧上颌第二磨牙牙颈部;4:去卵巢+实验性牙周炎组(OVX+EP):切除双侧卵巢,术后10w,采用丝线结扎左侧上颌第二磨牙牙颈部。丝线结扎后2w,过量麻醉药处死大鼠。观察各组大鼠的体重、骨密度、血清生化指标、牙槽骨吸收和牙周组织细胞因子的表达。结果卵巢切除导致大鼠牙槽骨吸收量增加;上调了根分叉区牙周膜IL-6、OPG、RANKL的表达,下调了IL-10的表达。结论卵巢切除导致大鼠实验性牙周炎牙周组织的破坏,可能与牙周组织中IL-6、OPG、RANKL表达增加,IL-10表达下降有关。
实验二尾静脉高压注射hIL-10质粒对去卵巢大鼠实验性牙周炎发生的影响
目的探讨全身性hIL-10基因治疗对去卵巢大鼠骨代谢、牙槽骨吸收的影响及其机制。方法将96只3月龄雌性SD大鼠随机分成4组,每组24只。1:SHAM+hIL-10组;2:SHAM+VECTOR;3:OVX+hIL-10组;4:OVX+VECTOR组。术后即刻将各组大鼠随机分成3小组,分别于术后12w、14w、18w,予以丝线结扎左侧上颌第二磨牙,建立实验性牙周炎模型。术后12w起,每周给予尾静脉加压注射hIL-10质粒(1、3两组)或空载体质粒(2、4两组)一次。丝线结扎后2w,处死大鼠。观察所表达的hIL-10对大鼠体重、骨密度、血清生化指标、牙槽骨吸收和牙周组织细胞因子表达的影响。结果尾静脉高压注射hIL-10质粒可在血清中表达,抑制血清TNF-α和TRAP 5b的升高(P﹤0.05)。但在牙周膜中IL-10表达量较少,与空载体组差异不显著;大鼠牙周膜的IL-1β和TNF-α阳性细胞数目不同程度地减少(P﹤0.05)。结论尾静脉高压注射hIL-10质粒并不能抑制去卵巢大鼠实验性牙周炎的牙槽骨吸收,对牙周膜IL-1β和TNF-α有抑制作用,对其他细胞因子的影响不明显。
实验三局部转染hIL-10基因对去卵巢大鼠实验性牙周炎发生的影响
目的观察口腔粘膜注射hIL-10质粒对大鼠骨代谢、牙槽骨吸收的影响及其机制。方法将24只3月龄雌性Sprague-Dawley(SD)大鼠随机分成4组,每组6只。1:SHAM+hIL-10组;2:SHAM+VECTOR组。假手术后12w,丝线结扎1、2组大鼠的左侧上颌第二磨牙(SHAM+hIL-10+EP),右侧不结扎作为对照(SHAM+hIL-10+C)。3:OVX+hIL-10组;4:OVX+VECTOR组。卵巢切除后12w,丝线结扎3、4组大鼠的左侧上颌第二磨牙(OVX+hIL-10+EP)。同时,在组1大鼠的双侧上颌第二磨牙腭侧牙龈粘膜下和组3大鼠左侧上颌第二磨牙腭侧牙龈粘膜下注射5μg hIL-10质粒和5μl脂质体复合物;在组2大鼠的双侧上颌第二磨牙腭侧牙龈粘膜下和组4大鼠的左侧上颌第二磨牙腭侧牙龈粘膜下注射5μg空载体质粒和5μl脂质体复合物。隔天注射1次,第7次注射后的24小时,处死大鼠。观察所表达的hIL-10对大鼠体重、骨密度、血清生化指标、牙槽骨吸收和牙周组织细胞因子表达的影响。结果hIL-10基因编码的蛋白可在牙周组织中表达;表达的IL-10蛋白可抑制牙槽骨吸收和根分叉区牙周膜IL-1β、IL-6、TNF-α、RANKL、MMP-8的表达。结论局部注射hIL-10质粒可抑制去卵巢大鼠实验性牙周炎的牙槽骨吸收,可能与牙周组织促炎因子的表达下降有关。
Periodontitis is a chronic infection of the supporting structures of teeth resulted from the accumulation of microorganism in dental plaque biofilm. Periodontitis is characterized by the inflammation of the supporting tissue of teeth and the alveolar absorption. Epidemiology studies demonstrated that periodontitis was the most common reason for teeth loss of the old. The role of systemic factors in the initiation and progression of periodontitis has been recognized. Considerable evidence has accumulated that postmenopausal osteoporosis (PMO) is a systemic contributor to periodontitis. The mechanism of the correlation between these two diseases still remains uncertain. In addition, the prevention and therapies of these disorders are the focuses of recent researches.
Experiment One: Expression of cytokines in periodontal tissues in ovariectomized rats with experimental periodontitis
Objective: To investigate the mechanism of PMO in the initiation and progression of periodontitis. Material and Methods: Twenty-four Sprague-Dawley (SD) female rats were randomly divided into 4 equal groups: Group I: Sham-operated (SHAM); Group II: ovariectomy (OVX); Group III: SHAM+experimental periodontitis (EP); Group IV: OVX+EP. Ten weeks after the surgery, the EPs were induced by placing silk ligatures around the cervix of the left upper second molars in Group III and IV. Two weeks after the ligation, the rats were sacrificed and the body weight, serum biomechanical marker, bone density, absorption of alveolar bone and the numbers of cytokine-positive cells in the periodontal ligament were measured to detect the effects of PMO on periodontal tissues. Results: Ovariectomy resulted in the loss of alveolar bone, upregrulated the expressions of IL-6, OPG, and RANKL and downregurated the expressions of IL-10 in the periodontal tissues of furcation. Conclusion: Ovariectomy resulted in the loss of alveolar bone could be mediated by the changed expressions of cytokines.
Experiment Two: Effect of the hIL-10 gene transfered by hydrodynamic tail vein injection on experimental periodontitis in ovariectomized rats
Objective: To investigate the effect of the hIL-10 gene transfered by hydrodynamic tail vein injection on bone metabolism, absorption of alveolar bone, and expressions of cytokines in periodontal tissues in ovariectomized rats. Material and Methods: Ninety-six SD female rats were randomly divided into 4 equal groups: Group I: SHAM+hIL-10; Group II: SHAM+Control vector (VECTOR); Group III: OVX+hIL-10; Group IV: OVX+VECTOR. Twelve weeks after surgery, the rats were transferred with hIL-10 or vector plasmids by hydrodynamic tail vein injection weekly. Seven days after the second, fourth and eighth injection, 8 rats were sacrificed from each group. Two weeks before being sacrificed, the left upper second molars were assigned to receive silk ligatures to induced experimental periodontitis. The body weight, serum biomechanical marker, bone density, absorption of alveolar bone and the numbers of cytokine-positive cells in the furcation were measured to detect the effects of hIL-10 gene therapy on bone metabolism, absorption of alveolar bone and the effect on the expression profile of cytokines in the periodontal tissue. Results: It was demonstrated that the expressed hIL-10 protein in serum after gene transfer downregulated the serum expression levels of TNF-αand TRAP 5b in ovariectomized rats (P<0.05). Although the difference of IL-10 positive cells in the furcation between transfer and vector groups was not significant statistically, the numbers of IL-1βand TNF-αpositive cells in the furcation decreased significantly (P<0.05). Conclusion: Although the numbers of IL-1βand TNF-αpositive cells in the furcation of ovariectomized rats were decreased, hIL-10 gene transferred by hydrodynamic tail vein injection had no significant effect on the absorption of alveolar bone.
Experiment Three: Effect of local hIL-10 gene transfer on experimental periodontitis in ovariectomized rats
Objective: To investigate the effect of local hIL-10 gene transfer on bone metabolism, absorption of alveolar bone, and expressions of cytokines in periodontal tissue in ovariectomized rats. Material and Methods: Twenty-four SD female rats were randomly divided into four groups. Group I: SHAM+hIL-10; Group II: SHAM+VECTOR; Group III: OVX+hIL-10; Group IV: OVX+VECTOR. Twelve weeks after SHAM or OVX operation, the experimental periodontitis were induced by placing 4-0 silk ligatures around the cervix of the left upper second molars. At the same time, the complex of 5μg hIL-10 plasmid with 5μl liposome for Group I and III or 5μg vector plasmid with 5μl liposome for Group II and IV were injected into the palatal gingiva of the upper second molars on left sides one time every two days. However, the palatal gingiva of the upper second molars on the right sides were also injected with the same complex as the left for Group I and Group II. Twenty hours after the seventh injection, the rats were sacrificed and the body weight, serum biomechanical marker, bone density, absorption of alveolar bone and the numbers of cytokine-positive cells in the furcation were measured to detect the effects of hIL-10 gene therapy on bone metabolism, absorption of alveolar bone and the expression of cytokines in the periodontal tissue. Results: It was demonstrated that significantly increased hIL-10 exporession was detected in the periodontal tissues after local gene delivery. It was also noted that after hIL-10 gene local delivery, the expression levels of IL-1β, IL-6, TNF-α, RANKL and MMP-8 were significantly downregulated in the furcation of ligatured teeth and the alveolar bone absorption was significantly decreased (P<0.05). Conclusion: The hIL-10 gene transferred by local injection had significant effect on the absorption of alveolar bone and the expression of cytokines including IL-1β, IL-6, TNF-α, RANKL and MMP-8 in periodontal tissues of experimental periodontitis in ovariectomized rats.
实验一细胞因子在去卵巢大鼠实验性牙周炎牙周组织中的表达
目的了解PMO在牙周炎发病中的作用机制。方法将24只雌性Sprague-Dawley(SD)大鼠随机分成4组,每组6只。1:假手术(SHAM)组:对大鼠实施去卵巢假手术;2:去卵巢组(OVX):切除双侧卵巢;3:假手术+实验性牙周炎(SHAM+EP)组:对大鼠实施去卵巢假手术,术后10w,采用丝线结扎左侧上颌第二磨牙牙颈部;4:去卵巢+实验性牙周炎组(OVX+EP):切除双侧卵巢,术后10w,采用丝线结扎左侧上颌第二磨牙牙颈部。丝线结扎后2w,过量麻醉药处死大鼠。观察各组大鼠的体重、骨密度、血清生化指标、牙槽骨吸收和牙周组织细胞因子的表达。结果卵巢切除导致大鼠牙槽骨吸收量增加;上调了根分叉区牙周膜IL-6、OPG、RANKL的表达,下调了IL-10的表达。结论卵巢切除导致大鼠实验性牙周炎牙周组织的破坏,可能与牙周组织中IL-6、OPG、RANKL表达增加,IL-10表达下降有关。
实验二尾静脉高压注射hIL-10质粒对去卵巢大鼠实验性牙周炎发生的影响
目的探讨全身性hIL-10基因治疗对去卵巢大鼠骨代谢、牙槽骨吸收的影响及其机制。方法将96只3月龄雌性SD大鼠随机分成4组,每组24只。1:SHAM+hIL-10组;2:SHAM+VECTOR;3:OVX+hIL-10组;4:OVX+VECTOR组。术后即刻将各组大鼠随机分成3小组,分别于术后12w、14w、18w,予以丝线结扎左侧上颌第二磨牙,建立实验性牙周炎模型。术后12w起,每周给予尾静脉加压注射hIL-10质粒(1、3两组)或空载体质粒(2、4两组)一次。丝线结扎后2w,处死大鼠。观察所表达的hIL-10对大鼠体重、骨密度、血清生化指标、牙槽骨吸收和牙周组织细胞因子表达的影响。结果尾静脉高压注射hIL-10质粒可在血清中表达,抑制血清TNF-α和TRAP 5b的升高(P﹤0.05)。但在牙周膜中IL-10表达量较少,与空载体组差异不显著;大鼠牙周膜的IL-1β和TNF-α阳性细胞数目不同程度地减少(P﹤0.05)。结论尾静脉高压注射hIL-10质粒并不能抑制去卵巢大鼠实验性牙周炎的牙槽骨吸收,对牙周膜IL-1β和TNF-α有抑制作用,对其他细胞因子的影响不明显。
实验三局部转染hIL-10基因对去卵巢大鼠实验性牙周炎发生的影响
目的观察口腔粘膜注射hIL-10质粒对大鼠骨代谢、牙槽骨吸收的影响及其机制。方法将24只3月龄雌性Sprague-Dawley(SD)大鼠随机分成4组,每组6只。1:SHAM+hIL-10组;2:SHAM+VECTOR组。假手术后12w,丝线结扎1、2组大鼠的左侧上颌第二磨牙(SHAM+hIL-10+EP),右侧不结扎作为对照(SHAM+hIL-10+C)。3:OVX+hIL-10组;4:OVX+VECTOR组。卵巢切除后12w,丝线结扎3、4组大鼠的左侧上颌第二磨牙(OVX+hIL-10+EP)。同时,在组1大鼠的双侧上颌第二磨牙腭侧牙龈粘膜下和组3大鼠左侧上颌第二磨牙腭侧牙龈粘膜下注射5μg hIL-10质粒和5μl脂质体复合物;在组2大鼠的双侧上颌第二磨牙腭侧牙龈粘膜下和组4大鼠的左侧上颌第二磨牙腭侧牙龈粘膜下注射5μg空载体质粒和5μl脂质体复合物。隔天注射1次,第7次注射后的24小时,处死大鼠。观察所表达的hIL-10对大鼠体重、骨密度、血清生化指标、牙槽骨吸收和牙周组织细胞因子表达的影响。结果hIL-10基因编码的蛋白可在牙周组织中表达;表达的IL-10蛋白可抑制牙槽骨吸收和根分叉区牙周膜IL-1β、IL-6、TNF-α、RANKL、MMP-8的表达。结论局部注射hIL-10质粒可抑制去卵巢大鼠实验性牙周炎的牙槽骨吸收,可能与牙周组织促炎因子的表达下降有关。
Periodontitis is a chronic infection of the supporting structures of teeth resulted from the accumulation of microorganism in dental plaque biofilm. Periodontitis is characterized by the inflammation of the supporting tissue of teeth and the alveolar absorption. Epidemiology studies demonstrated that periodontitis was the most common reason for teeth loss of the old. The role of systemic factors in the initiation and progression of periodontitis has been recognized. Considerable evidence has accumulated that postmenopausal osteoporosis (PMO) is a systemic contributor to periodontitis. The mechanism of the correlation between these two diseases still remains uncertain. In addition, the prevention and therapies of these disorders are the focuses of recent researches.
Experiment One: Expression of cytokines in periodontal tissues in ovariectomized rats with experimental periodontitis
Objective: To investigate the mechanism of PMO in the initiation and progression of periodontitis. Material and Methods: Twenty-four Sprague-Dawley (SD) female rats were randomly divided into 4 equal groups: Group I: Sham-operated (SHAM); Group II: ovariectomy (OVX); Group III: SHAM+experimental periodontitis (EP); Group IV: OVX+EP. Ten weeks after the surgery, the EPs were induced by placing silk ligatures around the cervix of the left upper second molars in Group III and IV. Two weeks after the ligation, the rats were sacrificed and the body weight, serum biomechanical marker, bone density, absorption of alveolar bone and the numbers of cytokine-positive cells in the periodontal ligament were measured to detect the effects of PMO on periodontal tissues. Results: Ovariectomy resulted in the loss of alveolar bone, upregrulated the expressions of IL-6, OPG, and RANKL and downregurated the expressions of IL-10 in the periodontal tissues of furcation. Conclusion: Ovariectomy resulted in the loss of alveolar bone could be mediated by the changed expressions of cytokines.
Experiment Two: Effect of the hIL-10 gene transfered by hydrodynamic tail vein injection on experimental periodontitis in ovariectomized rats
Objective: To investigate the effect of the hIL-10 gene transfered by hydrodynamic tail vein injection on bone metabolism, absorption of alveolar bone, and expressions of cytokines in periodontal tissues in ovariectomized rats. Material and Methods: Ninety-six SD female rats were randomly divided into 4 equal groups: Group I: SHAM+hIL-10; Group II: SHAM+Control vector (VECTOR); Group III: OVX+hIL-10; Group IV: OVX+VECTOR. Twelve weeks after surgery, the rats were transferred with hIL-10 or vector plasmids by hydrodynamic tail vein injection weekly. Seven days after the second, fourth and eighth injection, 8 rats were sacrificed from each group. Two weeks before being sacrificed, the left upper second molars were assigned to receive silk ligatures to induced experimental periodontitis. The body weight, serum biomechanical marker, bone density, absorption of alveolar bone and the numbers of cytokine-positive cells in the furcation were measured to detect the effects of hIL-10 gene therapy on bone metabolism, absorption of alveolar bone and the effect on the expression profile of cytokines in the periodontal tissue. Results: It was demonstrated that the expressed hIL-10 protein in serum after gene transfer downregulated the serum expression levels of TNF-αand TRAP 5b in ovariectomized rats (P<0.05). Although the difference of IL-10 positive cells in the furcation between transfer and vector groups was not significant statistically, the numbers of IL-1βand TNF-αpositive cells in the furcation decreased significantly (P<0.05). Conclusion: Although the numbers of IL-1βand TNF-αpositive cells in the furcation of ovariectomized rats were decreased, hIL-10 gene transferred by hydrodynamic tail vein injection had no significant effect on the absorption of alveolar bone.
Experiment Three: Effect of local hIL-10 gene transfer on experimental periodontitis in ovariectomized rats
Objective: To investigate the effect of local hIL-10 gene transfer on bone metabolism, absorption of alveolar bone, and expressions of cytokines in periodontal tissue in ovariectomized rats. Material and Methods: Twenty-four SD female rats were randomly divided into four groups. Group I: SHAM+hIL-10; Group II: SHAM+VECTOR; Group III: OVX+hIL-10; Group IV: OVX+VECTOR. Twelve weeks after SHAM or OVX operation, the experimental periodontitis were induced by placing 4-0 silk ligatures around the cervix of the left upper second molars. At the same time, the complex of 5μg hIL-10 plasmid with 5μl liposome for Group I and III or 5μg vector plasmid with 5μl liposome for Group II and IV were injected into the palatal gingiva of the upper second molars on left sides one time every two days. However, the palatal gingiva of the upper second molars on the right sides were also injected with the same complex as the left for Group I and Group II. Twenty hours after the seventh injection, the rats were sacrificed and the body weight, serum biomechanical marker, bone density, absorption of alveolar bone and the numbers of cytokine-positive cells in the furcation were measured to detect the effects of hIL-10 gene therapy on bone metabolism, absorption of alveolar bone and the expression of cytokines in the periodontal tissue. Results: It was demonstrated that significantly increased hIL-10 exporession was detected in the periodontal tissues after local gene delivery. It was also noted that after hIL-10 gene local delivery, the expression levels of IL-1β, IL-6, TNF-α, RANKL and MMP-8 were significantly downregulated in the furcation of ligatured teeth and the alveolar bone absorption was significantly decreased (P<0.05). Conclusion: The hIL-10 gene transferred by local injection had significant effect on the absorption of alveolar bone and the expression of cytokines including IL-1β, IL-6, TNF-α, RANKL and MMP-8 in periodontal tissues of experimental periodontitis in ovariectomized rats.
引文
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