独活寄生汤对骨性关节炎软骨细胞增殖与凋亡影响的实验研究
发布时间：2018-07-21 11:00
【摘要】：目的 探讨独活寄生汤对骨性关节炎大鼠关节软骨细胞增殖与凋亡的影响。 方法 1.健康2月龄清洁级SD大鼠144只,雌雄各半,适应性喂养1周,随机分为空白组(36只)和造模组(108只)。造模组在1,4,7天双膝关节腔注射4%木瓜蛋白酶复制骨性关节炎模型。造模成功后,抽签法随机分为模型组(36只)、治疗组(36只)和阳性组(36只)。空白组和模型组按照10ml·kg-1·d-1的量灌服0.9%生理盐水；治疗组按照9.3g.kg-1·d-1的药量灌服独活寄生汤；阳性组按照0.15g-kg-1d-1的药量灌服奥泰灵胶囊；每2周为1个疗程,中间休息2天,共4个疗程。 2.每2个疗程后,处死一批实验动物,切取胫骨平台内侧软骨组织于4%多聚甲醛中固定、石蜡包埋,HE染色观察软骨组织形态变化；免疫组化检测Cyclin D1、CDK4、 Rb、p16、Bc1-2、Bax、Cytochrome c、caspase-9及caspase-3蛋白表达情况；切取胫骨平台外侧软骨组织于电镜固定液中固定,用于观察软骨细胞超微结构变化；其余软骨组织迅速置入液氮中保存,用于检测Cyclin D1、CDK4、Rb及p16mRNA表达情况。 3.健康2月龄清洁级SD大鼠144只,雌雄各半,抽签法随机分为：不用药血清对照组(空白血清组,36只)和独活寄生汤临床等效剂量组(含药血清组,108只)。空白血清组按10ml·kg-1·d-1的量给予0.9%生理盐水；含药血清组按9.3g-kg-1-d-1的量给予独活寄生汤。连续7天灌胃,最后1天连续2次灌胃,中间间隔2h,采血前禁食12h,分别在末次灌胃1h、2h、3h后,在2%戊巴比妥钠2ml/kg麻醉下腹主动脉采血,制备含药血清。 4.健康4周龄清洁级SD大鼠20～30只,取膝关节的关节软骨,建立软骨细胞体外培养体系。第2代软骨细胞以1.0×104/孔接种于96孔板中,在含10%FBS的DMEM培养基中培养24h后,用不含FBS的DMEM培养液培养24h,同步化软骨细胞。分别加入含10%、15%、20%、30%四个浓度的不同采血时间点的含药血清和空白血清,培养24h、36h、48h、60h、72h后,MTT法检测软骨细胞的活性。 5.用10ng/ml IL-1β诱导第3代软骨细胞复制退变软骨细胞模型,采用倒置相差显微镜观察细胞形态变化和Ⅱ型胶原免疫组化进行鉴定。 6.采用(6)中确定的含药血清最佳干预条件,体外培养退变软骨细胞,分别干预24h,48h,72h后,收集软骨细胞。MTT法检测细胞增殖情况；流式细胞术检测细胞周期变化情况；Annexin V-FITC/PI染色流式检测细胞凋亡情况；JC-1染色流式检测含药血清干预后线粒体膜电位的改变情况; RT-PCR法检测Cyclin D1、CDK4、Rb、p16、Bcl-2、 Bax mRNA表达情况；Western Blot法检测Cyclin D1、CDK4、Rb、p16、Bcl-2、Bax蛋白表达情况；分光光度法检测caspase-9和caspase-3活化情况。 结果 1.膝关节X侧位片示,正常组关节间隙正常、关节面平整、关节边缘可见光滑锐利的线样致密影,软骨下骨密度均匀。模型组关节间隙明显变窄、关节面不平整,软骨破坏区边缘骨质增生硬化,关节边缘骨赘形成。HE染色和电镜观察结果表明独活寄生汤和奥泰灵均可以促进OA软骨的修复,促进软骨细胞增殖,两者无明显差别。独活寄生汤和奥泰灵均能促进Cyclin D1、CDK4、Rb mRNA和蛋白,抑制p16、Cytochrome c、 caspase-9及caspase-3mRNA和蛋白的表达,两者无显著性差异。 2.第3代软骨细胞Ⅱ型胶原免疫组织化学染色,软骨细胞胞浆被染成棕黄色,胞核不着色；阿利新蓝染色,软骨细胞核为浅蓝色,细胞质中可见不少浅蓝色的分泌颗粒和小泡。独活寄生汤含药血清干预软骨细胞增殖最佳采血时间点为2小时,最佳干预浓度为10%。采用10ng/ml IL-1β诱导的软骨细胞,与正常细胞相比,细胞体积变大,边缘出现一些指状突起,核也变大,胞膜和胞浆不清晰,细胞呈多边形改变,折光度下降；Ⅱ型胶原免疫组化显示软骨细胞胞浆棕黄色染色变浅。含药血清干预后,退变软骨细胞G0/G1期比例逐渐降低,S期比例和增殖指数逐渐增加；退变软骨细胞的凋亡率逐渐降低；线粒体膜电位逐渐降低；随着干预时间的延长,caspase-9和caspase-3活性均逐渐降低；含药血清能促进Cyclin D1、CDK4、Rb、Bcl-2mRNA和蛋白表达,抑制p16和Bax mRNA和蛋白的表达。 结论 1.体内外研究表明,独活寄生汤通过促进软骨细胞周期关键限制点G1/S期的切换,促进软骨细胞增殖。 2.体内外研究表明,独活寄生汤通过抑制线粒体凋亡通路的激活,抑制软骨细胞凋亡。
[Abstract]:Purpose
Objective To investigate the effect of live parasitic soup on the proliferation and apoptosis of articular chondrocytes in rats with osteoarthritis .
method
One week , 144 male SD rats were randomly divided into two groups : blank group ( 36 rats ) and model group ( 108 only ) . The model group was randomly divided into model group ( 36 rats ) , treatment group ( 36 rats ) and positive group ( 36 rats ) . The blank group and the model group were administered 0.9 % physiological saline in 10 ml 路 kg - 1 路 d -1 .
the treatment group is administered with a live parasitic soup according to the dosage of 9.3 g 路 kg - 1 路 d - 1 ;
The positive group was administered in the amount of 0.15 g - kg - 1d - 1 to the Oteling capsule ;
1 course of treatment every 2 weeks , 2 days in the middle and 4 courses of treatment .
2 . After every 2 treatment courses , a group of experimental animals were sacrificed , the inner cartilage of the tibial plateau was cut and fixed in 4 % polyformaldehyde , paraffin embedded and HE staining was used to observe the morphological changes of cartilage tissue ;
The expression of Cyclin D1 , Rb , p16 , Bc1 - 2 , Bax , bcl c , caspase - 9 and caspase - 3 protein were detected by immunohistochemistry .
The outer cartilage of the tibial plateau was excised and fixed in the electron microscope fixing solution , which was used to observe the ultrastructural changes of the chondrocytes .
The remaining cartilage tissues were rapidly placed in liquid nitrogen and used to detect the expression of Cyclin D1 , cd4 , Rb and p16mRNA .
3 . 144 male and female male SD rats were randomly divided into two groups : serum control group ( blank serum group , 36 rats ) and single live parasitic soup ( including serum group , 108 ) . the blank serum group was given 0.9 % physiological saline in the amount of 10ml 路 kg - 1 路 d - 1 ;
The drug - containing serum group was administered in a dose of 9.3 g - kg - 1 - d - 1 for 7 consecutive days . After the last 1 day for 2 consecutive times of gavage , the middle interval was 2 h , the blood was fasted for 12 h before blood sampling . After the last dose of 1 h , 2 h and 3 h , the blood was collected from the aorta of the lower abdominal aorta with 2 % sodium opental 2 ml / kg , and the serum was prepared .
4 . In healthy 4 - week - old SD rats , 20 - 30 healthy SD rats were cultured for 24 h in DMEM medium containing 10 % FBS for 24 h , and cultured for 24 h in DMEM medium containing 10 % FBS . After 24 h , 36 h , 48 h , 60 h and 72 h , the activity of chondrocytes was detected by MTT assay .
5 . Using 10 ng / ml IL - 1尾 to induce the replication and degeneration of chondrocytes in the third generation of chondrocytes , the morphological changes of cells and the immunohistochemical staining of type 鈪，
本文编号：2135308
[Abstract]:Purpose
Objective To investigate the effect of live parasitic soup on the proliferation and apoptosis of articular chondrocytes in rats with osteoarthritis .
method
One week , 144 male SD rats were randomly divided into two groups : blank group ( 36 rats ) and model group ( 108 only ) . The model group was randomly divided into model group ( 36 rats ) , treatment group ( 36 rats ) and positive group ( 36 rats ) . The blank group and the model group were administered 0.9 % physiological saline in 10 ml 路 kg - 1 路 d -1 .
the treatment group is administered with a live parasitic soup according to the dosage of 9.3 g 路 kg - 1 路 d - 1 ;
The positive group was administered in the amount of 0.15 g - kg - 1d - 1 to the Oteling capsule ;
1 course of treatment every 2 weeks , 2 days in the middle and 4 courses of treatment .
2 . After every 2 treatment courses , a group of experimental animals were sacrificed , the inner cartilage of the tibial plateau was cut and fixed in 4 % polyformaldehyde , paraffin embedded and HE staining was used to observe the morphological changes of cartilage tissue ;
The expression of Cyclin D1 , Rb , p16 , Bc1 - 2 , Bax , bcl c , caspase - 9 and caspase - 3 protein were detected by immunohistochemistry .
The outer cartilage of the tibial plateau was excised and fixed in the electron microscope fixing solution , which was used to observe the ultrastructural changes of the chondrocytes .
The remaining cartilage tissues were rapidly placed in liquid nitrogen and used to detect the expression of Cyclin D1 , cd4 , Rb and p16mRNA .
3 . 144 male and female male SD rats were randomly divided into two groups : serum control group ( blank serum group , 36 rats ) and single live parasitic soup ( including serum group , 108 ) . the blank serum group was given 0.9 % physiological saline in the amount of 10ml 路 kg - 1 路 d - 1 ;
The drug - containing serum group was administered in a dose of 9.3 g - kg - 1 - d - 1 for 7 consecutive days . After the last 1 day for 2 consecutive times of gavage , the middle interval was 2 h , the blood was fasted for 12 h before blood sampling . After the last dose of 1 h , 2 h and 3 h , the blood was collected from the aorta of the lower abdominal aorta with 2 % sodium opental 2 ml / kg , and the serum was prepared .
4 . In healthy 4 - week - old SD rats , 20 - 30 healthy SD rats were cultured for 24 h in DMEM medium containing 10 % FBS for 24 h , and cultured for 24 h in DMEM medium containing 10 % FBS . After 24 h , 36 h , 48 h , 60 h and 72 h , the activity of chondrocytes was detected by MTT assay .
5 . Using 10 ng / ml IL - 1尾 to induce the replication and degeneration of chondrocytes in the third generation of chondrocytes , the morphological changes of cells and the immunohistochemical staining of type 鈪，
本文编号：2135308
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