本文選題：柚皮苷 + 破骨細(xì)胞��； 參考：《天津醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的：1.觀察柚皮苷(Naringin, NG)對(duì)破骨細(xì)胞的影響。2.觀察柚皮苷對(duì)去勢(shì)大鼠骨質(zhì)疏松的影響。3.觀察柚皮苷對(duì)去勢(shì)大鼠骨質(zhì)疏松骨折的影響。方法：1.利用來(lái)自小鼠的破骨前體RAW264.7細(xì)胞株,采用含有濃度為100ng/mL的小鼠重組可溶性核因子KB受體活化因子配基(receptor activator of NF-κ B ligand, RANKL)的高糖DMEM (High glucose Dulbecco minimum essential medium)培養(yǎng)基,誘導(dǎo)培養(yǎng)實(shí)驗(yàn)用的成熟破骨細(xì)胞。利用TRAP染色與掃描電子顯微鏡觀察破骨細(xì)胞形成的骨吸收陷窩的方法鑒定破骨細(xì)胞。流式細(xì)胞術(shù)檢測(cè)破骨細(xì)胞的凋亡率,RT-PCR檢測(cè)破骨細(xì)胞凋亡相關(guān)基因Bcl-2、BAX、 caspase-3以及基質(zhì)金屬蛋白酶9(MMP-9),組織蛋白酶K (Cath-K),抗酒石酸酸性磷酸酶(T RAP) mRNA表達(dá)情況。2.3月齡雌性S-D大鼠隨機(jī)分為去勢(shì)組(OVX),假手術(shù)組(SHAM),柚皮苷(40mg/kg、100mg/kg、200mg/kg)組與雌激素組(22.5μg/kg)陽(yáng)性對(duì)照組,去勢(shì)組手術(shù)切除大鼠雙側(cè)卵巢,假手術(shù)組只切開(kāi)皮膚,不做卵巢切除。切除卵巢后3月開(kāi)始灌胃給藥,給藥周期為60天。觀察各組大鼠股骨骨密度,Micro-CT參數(shù),血清生化指標(biāo),股骨力學(xué)性能與骨形態(tài)計(jì)量學(xué),并進(jìn)行組間比較。3.采用3月齡雌性SD大鼠行雙側(cè)卵巢切除,飼養(yǎng)3個(gè)月后手術(shù)造成大鼠右側(cè)脛骨橫行骨折,術(shù)后按照隨機(jī)數(shù)字表法分為對(duì)照組(20只)、柚皮苷組(20只)雌激素組(20只)。柚皮苷組每天灌服柚皮苷100mg/kg/d;雌激素組灌服17-β雌二醇22.5(μg/kg/d;對(duì)照組給予等體積的等滲鹽水皮下注射。術(shù)后分別于2,8周處死動(dòng)物后,取出手術(shù)側(cè)脛骨,行X線(xiàn)拍片進(jìn)行評(píng)分；然后行雙能X線(xiàn)吸收測(cè)定法骨折部位骨密度(BMD)；檢測(cè)BMD后,采用Micro-CT對(duì)骨折部位進(jìn)行定量分析,檢測(cè)指標(biāo)：骨體積(BV)、相對(duì)骨體積比(BV/TV)、平均骨小梁厚度(Tb.Th);酶聯(lián)免疫吸附實(shí)驗(yàn)(Elisa)測(cè)定血清骨鈣素與大鼠Ⅰ型膠原C端肽(CTX-1);三點(diǎn)彎曲力學(xué)實(shí)驗(yàn)測(cè)定骨折最大載荷,并進(jìn)行統(tǒng)計(jì)學(xué)分析。結(jié)果：1.單用RANKL (100ng/ nl)誘導(dǎo)即可獲得破骨細(xì)胞。柚皮苷可以降低TRAP陽(yáng)性細(xì)胞數(shù),減少骨吸收陷窩數(shù)目；促進(jìn)破骨細(xì)胞的凋亡；柚皮苷干預(yù)組Bcl-2、 MMP-9、Cath-K、TRAP mRNA的表達(dá)降低,BAX與caspase-3 mRNA表達(dá)升高。2.用藥60天,與去勢(shì)組比較,中、高劑量柚皮苷組體重降低；雌激素與柚皮苷組骨密度,骨小梁數(shù)目增多；血鈣降低,雌激素與骨鈣素升高；大鼠Ⅰ型膠原C端肽(CTX-1)降低,對(duì)總膽固醇有調(diào)節(jié)作用,增加礦化率、提升股骨力學(xué)性能。3.術(shù)后第2周柚皮苷組X線(xiàn)評(píng)分、BMD、Tb.Th、骨鈣素、最大載荷較對(duì)照組明顯升高(P0.05); BV、BV/TV較對(duì)照組明顯升高(P0.01)；血CTX-1較對(duì)照組明顯降低(P0.01)；BV、最大載荷較雌激素組也升高(P0.05)；骨鈣素較雌激素組明顯升高(P0.01)；雌激素組X線(xiàn)評(píng)分、BMD、BV、BWTV,Tb.Th、最大載荷較對(duì)照組亦升高(P0.05)；雌激素組骨鈣素、CTX-1較對(duì)照組降低(P0.05)；柚皮苷組X線(xiàn)評(píng)分、BMD、BWTV、Tb.Th、CTX-1與雌激素組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。8周時(shí)柚皮苷組X線(xiàn)評(píng)分、BMD、BV、BV/TV、Tb.Th較對(duì)照組升高(P0.05)；而柚皮苷組與雌激素組差異無(wú)統(tǒng)計(jì)學(xué)意義；柚皮苷組骨鈣素、最大載荷高于對(duì)照組與雌激素組(P0.05)結(jié)論：柚皮苷可減少破骨細(xì)胞數(shù)量、降低骨吸收功能,促進(jìn)破骨細(xì)胞凋亡；其機(jī)制可能是通過(guò)降低BCL-2 mRNA表達(dá),升高BAX mRNA表達(dá),激活caspase-3從而使破骨細(xì)胞凋亡來(lái)實(shí)現(xiàn)的。柚皮苷可以提高去勢(shì)大鼠BMD,增加骨小梁數(shù)量,改善骨代謝,提高股骨的力學(xué)性能；柚皮苷提升大鼠骨質(zhì)疏松骨折部位BMD、BV、BV/TV、Tb.Th,改善骨代謝,從而提高骨折愈合最大載荷。
[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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R683;R580

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

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