柚皮苷对骨质疏松及骨质疏松骨折的影响及其作用机制的研究

发布时间：2018-05-05 12:38

本文选题：柚皮苷 + 破骨细胞　；参考：《天津医科大学》2015年硕士论文

【摘要】：目的：1.观察柚皮苷(Naringin, NG)对破骨细胞的影响。2.观察柚皮苷对去势大鼠骨质疏松的影响。3.观察柚皮苷对去势大鼠骨质疏松骨折的影响。方法：1.利用来自小鼠的破骨前体RAW264.7细胞株,采用含有浓度为100ng/mL的小鼠重组可溶性核因子KB受体活化因子配基(receptor activator of NF-κ B ligand, RANKL)的高糖DMEM (High glucose Dulbecco minimum essential medium)培养基,诱导培养实验用的成熟破骨细胞。利用TRAP染色与扫描电子显微镜观察破骨细胞形成的骨吸收陷窝的方法鉴定破骨细胞。流式细胞术检测破骨细胞的凋亡率,RT-PCR检测破骨细胞凋亡相关基因Bcl-2、BAX、 caspase-3以及基质金属蛋白酶9(MMP-9),组织蛋白酶K (Cath-K),抗酒石酸酸性磷酸酶(T RAP) mRNA表达情况。2.3月龄雌性S-D大鼠随机分为去势组(OVX),假手术组(SHAM),柚皮苷(40mg/kg、100mg/kg、200mg/kg)组与雌激素组(22.5μg/kg)阳性对照组,去势组手术切除大鼠双侧卵巢,假手术组只切开皮肤,不做卵巢切除。切除卵巢后3月开始灌胃给药,给药周期为60天。观察各组大鼠股骨骨密度,Micro-CT参数,血清生化指标,股骨力学性能与骨形态计量学,并进行组间比较。3.采用3月龄雌性SD大鼠行双侧卵巢切除,饲养3个月后手术造成大鼠右侧胫骨横行骨折,术后按照随机数字表法分为对照组(20只)、柚皮苷组(20只)雌激素组(20只)。柚皮苷组每天灌服柚皮苷100mg/kg/d;雌激素组灌服17-β雌二醇22.5(μg/kg/d;对照组给予等体积的等渗盐水皮下注射。术后分别于2,8周处死动物后,取出手术侧胫骨,行X线拍片进行评分；然后行双能X线吸收测定法骨折部位骨密度(BMD)；检测BMD后,采用Micro-CT对骨折部位进行定量分析,检测指标：骨体积(BV)、相对骨体积比(BV/TV)、平均骨小梁厚度(Tb.Th);酶联免疫吸附实验(Elisa)测定血清骨钙素与大鼠Ⅰ型胶原C端肽(CTX-1);三点弯曲力学实验测定骨折最大载荷,并进行统计学分析。结果：1.单用RANKL (100ng/ nl)诱导即可获得破骨细胞。柚皮苷可以降低TRAP阳性细胞数,减少骨吸收陷窝数目；促进破骨细胞的凋亡；柚皮苷干预组Bcl-2、 MMP-9、Cath-K、TRAP mRNA的表达降低,BAX与caspase-3 mRNA表达升高。2.用药60天,与去势组比较,中、高剂量柚皮苷组体重降低；雌激素与柚皮苷组骨密度,骨小梁数目增多；血钙降低,雌激素与骨钙素升高；大鼠Ⅰ型胶原C端肽(CTX-1)降低,对总胆固醇有调节作用,增加矿化率、提升股骨力学性能。3.术后第2周柚皮苷组X线评分、BMD、Tb.Th、骨钙素、最大载荷较对照组明显升高(P0.05); BV、BV/TV较对照组明显升高(P0.01)；血CTX-1较对照组明显降低(P0.01)；BV、最大载荷较雌激素组也升高(P0.05)；骨钙素较雌激素组明显升高(P0.01)；雌激素组X线评分、BMD、BV、BWTV,Tb.Th、最大载荷较对照组亦升高(P0.05)；雌激素组骨钙素、CTX-1较对照组降低(P0.05)；柚皮苷组X线评分、BMD、BWTV、Tb.Th、CTX-1与雌激素组比较差异无统计学意义(P0.05)。8周时柚皮苷组X线评分、BMD、BV、BV/TV、Tb.Th较对照组升高(P0.05)；而柚皮苷组与雌激素组差异无统计学意义；柚皮苷组骨钙素、最大载荷高于对照组与雌激素组(P0.05)结论：柚皮苷可减少破骨细胞数量、降低骨吸收功能,促进破骨细胞凋亡；其机制可能是通过降低BCL-2 mRNA表达,升高BAX mRNA表达,激活caspase-3从而使破骨细胞凋亡来实现的。柚皮苷可以提高去势大鼠BMD,增加骨小梁数量,改善骨代谢,提高股骨的力学性能；柚皮苷提升大鼠骨质疏松骨折部位BMD、BV、BV/TV、Tb.Th,改善骨代谢,从而提高骨折愈合最大载荷。
[Abstract]:Objective: 1. to observe the effect of Naringin (NG) on osteoclasts.2. observation on the effect of naringin on osteoporosis in ovariectomized rats.3. observation of the effect of naringin on osteoporotic fracture in ovariectomized rats. Methods: 1. using RAW264.7 cell lines from the osteoclast of mice and using a recombinant soluble nucleus containing a concentration of 100ng/mL in mice. The high sugar DMEM (High glucose Dulbecco) culture medium of the factor KB receptor activating factor ligand (receptor activator of NF- kappa B ligand, RANKL) is used to induce mature osteoclasts in the culture experiment. The method of identification of bone resorption lacunae from osteoclasts by scanning electron microscopy and scanning electron microscopy Cell apoptosis rate of osteoclasts was detected by flow cytometry. RT-PCR detection of osteoclast apoptosis related genes Bcl-2, BAX, Caspase-3, matrix metalloproteinase 9 (MMP-9), cathepsin K (Cath-K), and tartaric acid acid phosphatase (T RAP) mRNA expression of.2.3 month old female rats were randomly divided into castrated group, sham operation group, pomelo 40mg/kg (100mg/kg, 200mg/kg) group and estrogen group (22.5 mu g/kg) positive control group, the ovariectomized rats were operated on bilateral ovariectomized ovariectomized rats. The sham operation group only cut the skin and did not do the ovariectomy. After the resection of the ovary, the gavage was administered in March and the period of the Administration was 60 days. The femoral bone density, Micro-CT parameter, serum biochemical index, femur of the femur of the rats were observed. Mechanical properties and bone morphometry were compared between 3 month old female SD rats and 3 month old female rats were treated with bilateral ovariectomy. After 3 months of rearing, the right tibial transverse fracture was caused by operation. After the operation, the control group was divided into the control group (20 rats) and the naringenin group (20) (20). The naringin group was given naringin 100mg/kg every day. /d; estrogen group was given 17- beta estradiol 22.5 (mu g/kg/d); the control group was given equal volume of isosmotic saline subcutaneous injection. After the animals were killed at 2,8 weeks after the operation, the tibia of the surgical side was taken out and the X-ray film was scored; then the bone mineral density (BMD) of the fracture site was performed by double energy X-ray absorptiometry; after BMD, the fracture site was detected by Micro-CT, and the fracture site was carried out by Micro-CT. Quantitative analysis: bone volume (BV), relative bone volume ratio (BV/TV), mean bone Liang Houdu (Tb.Th); enzyme linked immunosorbent assay (Elisa) for determination of serum osteocalcin and rat type I collagen C end peptide (CTX-1); three point bending mechanical test was used to determine the maximum load of fracture and was statistically analyzed. Results: 1. only by RANKL (100ng/ NL) induction. Osteoclast can be obtained. Naringin can reduce the number of TRAP positive cells, reduce the number of bone resorption lacunae and promote the apoptosis of osteoclast; the expression of Bcl-2, MMP-9, Cath-K, TRAP mRNA in the naringin intervention group is reduced, the mRNA expression of BAX and caspase-3 mRNA.2. is increased for 60 days, and the high dose of naringin group is lower than the high dose of naringin group. Hormone and naringin group bone density, bone trabecular number increased, blood calcium decreased, estrogen and osteocalcin increased, rat type I collagen C end peptide (CTX-1) decreased, the total cholesterol had a regulatory effect, increase mineralization rate, increase the mechanical performance of femur after.3. second weeks of naringin group X ray score, BMD, Tb.Th, osteocalcin, the maximum load is more obvious than the control group Increased (P0.05), BV and BV/TV were significantly higher than that in the control group (P0.01); CTX-1 in blood was significantly lower than that in the control group (P0.01); BV, the maximum load was higher than that of the estrogen group (P0.05), and the osteocalcin was significantly higher than the estrogen group (P0.01); the X-ray score of the estrogen group, BMD, BV, BWTV, and the increase of the maximum load were also higher than the control group; the estrogen group osteocalcin was also increased. CTX-1 was lower than the control group (P0.05); the X - ray score of naringin group, BMD, BWTV, Tb.Th, CTX-1 and estrogen group had no significant difference (P0.05), the X - ray score of naringin group at the time of.8, BMD, BV, BV/TV, Tb.Th was higher than the control group, but there was no significant difference between the naringin group and the estrogen group; the naringin group osteocalcin, the maximum load Higher than the control group and the estrogen group (P0.05) conclusion: naringin can reduce the number of osteoclast, reduce the bone absorption function and promote osteoclast apoptosis. Its mechanism may be achieved by reducing the expression of BCL-2 mRNA, increasing the expression of BAX mRNA and activating the apoptosis of osteoclasts by activating caspase-3. Naringin can increase the BMD in the ovariectomized rat, and increase the BMD in the ovariectomized rat. The number of bone trabeculae improves bone metabolism and improves the mechanical properties of the femur. Naringin improves the fracture site of osteoporotic rats, BMD, BV, BV/TV, Tb.Th, and improves bone metabolism, thus improving the maximum load of fracture healing.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R683;R580

【参考文献】