【作者】 吴广文；

【导师】 刘献祥；

【作者基本信息】 福建中医药大学 ， 中西医结合临床， 2014， 博士

【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。更多还原

【Abstract】 ObjectiveTo discuss the effect of Duhuo Jisheng Decoction (DHJSD) on osteoarthritis chondrocyte proliferation and apoptosis.Methods1. After one week of acclimation, healthy and clean, two-month-old Sprague Dawley rats of average gender (n=144) were randomly divided into blank group (n=36) and osteoarthritis model group (n=108). Osteoarthritis model was induced in double knees of rats by injecting0.2ml of4%papain solution on days1,4and7. After successfully establish the model, the animals were randomly divided into three groups:the model group (received an equivalent amount of saline only), the positive control group [received a clinical oral dose of Glucosamine Hydrochloride Capsules (0.15g/kg/day)] and the treatment group [received a clinical oral dose of DHJSD (9.3g/kg/day)]. The blank group also received an equivalent amount of saline only. Every treatment course includes two weeks. There are totally four courses.2. After every two courses, the animals were sacrificed and tibial plateau cartilage specimens were obtained. The morphological changes were observed under an optical microscope following staining with hematoxylin and eosin (H&E) and by transmission electron microscopy (TEM). The protein levels of Cyclin D1、CDK4、Rb、p16、Bcl-2、Bax、 Cytochrome c、Caspase-9and caspase-3were measured by immunohistochemistry. The mRNA levels of Cyclin D1、CDK4、 Rb and p16were measured by RT-PCR.3. A total of144two-month-old Sprague Dawley rats of average gender were randomly divided into two groups:the DHJSD group (n=108) treated with a dose of9.3g/kg/day DHJSD, which is the equivalent dosage for human in clinical use and the blank group (n=36) treated with an equivalent amount of saline only. All testing drugs and saline were administered via gastric gavage twice a day in the morning and afternoon for7consecutive days. The daily doses were given2h apart on the seventh day. Animals were anesthetized by intraperitoneal injection of2ml/kg2%pentobarbital sodium, and arterial blood in DHJSD group was collected from the abdominal aorta at1h,2h and3h after the final dose in DHJSD group, respectively and at2h in blank group.4. Cartilage was isolated from the knee joint of20～30health and clean, four-week-old SD rats, and used to establish a cultivation system of chondrocytes in vitro. The second-generation chondrocytes were seeded into96-well plates at a density of1.0x105cells/ml in0.1ml of medium. After synehronization, the cells were treated with10%、15%、20%、30%concentrations of different sampling time of DHJSD serum for24,36,48,60and72h, respectively. Cell viability was assessed by the MTT colorimetric assay.5. The degenerative chondrocyte model was established by exposing to10ng/ml IL-1β for24h, and verified by optical microscopy analyses and immunohistochemical staining of Collagen II analyses.6. After treated degenerative chondrocytes with the best intervention condition for24h,48h and72h, cell proliferation was detected by MTT assay and DNA staining followed by FACS analysis; cell apoptosis was detected by Flow Cytometry Analysis with Annexin V-FITC/PI Staining; mitochonrial membrane potential (ΔΨm) was measured by Flow Cytometry analysis with JC-1staining; Caspase-9,-3activation was analysesed by spectrophotometry; and the mRNA and protein expression levels of Cyclin D1, CDK4, Rb, p16, Bcl-2and Bax were measured by RT-PCR and Western Blot, respectively.Results1. The knee lateral X ray showed normal joint space, smooth articular surface, smooth lines dense shadow on joint edge and uniformity bone density in subchondral in normal group, while in model group, the joint space became narrow; the articular surface was not flat; on the edge of damage cartilage appeared bone hyperplasia and sclerosis; joint marginal appeared osteophyte formation. HE staining and transmission electron microscopy (TEM) results showed DHJSD and Glucosamine Hydrochloride Capsules could both promote OA cartilage repair and chondrocytes proliferation with no significant difference. DHJSD and Glucosamine Hydrochloride Capsules could both promote the mRNA and protein expression of Cyclin Dl, CDK4, Rb and inhibit p16, Cytochrome c, caspase-9, caspase-3mRNA and protein expression with no significant difference.2. Identification of the third generation chondrocytes with immunohistochemical staining of Collagen Ⅱ, the cytoplasm appeared brown and no coloration in the nucleus; with alcian blue staining, the nucleus were stained light blue, a lot of secretion particles and vesicles appeared in the cytoplasm. The best sampling time and concentration of DHJSD serum on promoting chondrocyte proliferation were2hour and10%, respectively. Compared with the normal chondrocytes,10ng/ml IL-1β induced chondrocytes became larger with some finger-like protrusions on the edge, and the cell membrane and cytoplasm was not clear. Meanwhile, the IL-1β-induced cells were polygonal changed, along with declining refraction. Besides, the immunohistochemical staining of Collagen II analyses showed that the brownish yellow staining in cytoplasm became shallow. After treated with DHJSD2h serum, the percentage of G0/G1phase cells was gradually decreased; however, the percentage of S phase cells and the proliferation index were increased. DHJSD2h serum treatment inhibited the apoptosis of degenerative chondrocytes, the loss of plasma membrane asymmetry (externalization of phosphatidylserine), collapse of mitochondrial membrane potential, and activation of caspase-9and caspase-3. DHJSD serum could promote the mRNA and protein expression of Cyclin D1, CDK4, Rb, Bcl-2and inhibit the mRNA and protein expression of p16, Bax.Conclusion1. DHJSD could promote osteoarthritis chondrocytes proliferation by promoting G1/S transition, in vivo and vitro.2. DHJSD could inhibit the mitochondrion-dependent pathway of apoptosis in osteoarthritis chondrocytes, in vivo and vitro.更多还原