摘要
目的:通过研究补肾中药血清对离体SD大鼠成骨细胞中BMP2、BMP7、Smad1/5在细胞中活性的变化,探讨骨质疏松症病机的分子生物学机制;研究补肾益髓壮骨中药防治骨质疏松症的作用机理,从而为临床防治骨质疏松症提供科学的实验依据。
材料与方法:采用多次胶原酶消化法获取新生SD大鼠颅盖骨中的成骨细胞进行体外培养,并应用Gomori改良钙钴法对其进行碱性磷酸酶表达的鉴定。随机将实验用大鼠分为正常组、骨疏康颗粒对照组(骨疏康组)、补肾中药组(补肾组)、补中益气颗粒对照组(补脾组);对实验大鼠给药干预8天后,通过血清药理学的方法使用各组大鼠血清培养成骨细胞48小时,另设加入UPP通路的蛋白酶体抑制剂MG132作为对照组;应用MTT法检测离体成骨细胞的增殖率,并应用免疫组化SABC法检测成骨细胞中BMP2、BMP7、Smad1/5活性的表达。
结果:
1补肾中药血清作用于体外培养的成骨细胞48小时后可以明显促进成骨细胞的增殖(P< 0.01)。
2应用UPP通路蛋白酶体抑制剂MG132可以明显抑制成骨细胞的增殖(P<0.01)。
3成骨细胞碱性磷酸酶染色结果:成骨细胞经Gomori改良钙钴法染色和苏木精复染后,可见棕黑色细微颗粒,多数分布于细胞膜上以及周围,胞浆中也可见到少量颗粒。阴性对照组细胞内无棕黑色颗粒,细胞核内可见嗜苏木精之蓝色颗粒。
4成骨细胞BMP2免疫组化结果:与正常组比较,骨疏康组和补肾组表达水平明显上升(P<0.01);补脾组、MG132+正常组、MG132+骨疏康组、MG132+补肾组和MG132+补脾组表达水平明显降低(P<0.01)。与补肾组比较,各组的表达水平均明显降低(P<0.01)。
5成骨细胞BMP7免疫组化结果:与正常组相比,补肾组表达水平明显上升(P<0.01);骨疏康组、补脾组、MG132+正常组、MG132+骨疏康组、MG132+补肾组和MG132+补脾组表达水平明显降低(P<0.01)。与补肾组比较,各组表达水平均明显降低(P<0.01)。加入MG132的各含药血清组中,MG132+正常组表达水平最高,MG132+补肾组与MG132+正常组比较其阳性表达水平降低(P<0.01)。
6成骨细胞Smad1/5免疫组化结果:与正常组相比,补肾组、骨疏康组、补脾组、MG132+正常组、MG132+骨疏康组、MG132+补肾组和MG132+补脾组表达水平明显降低(P<0.01)。与补肾组比较,骨疏康组、补脾组、MG132+正常组、MG132+骨疏康组、MG132+补肾组和MG132+补脾组表达水平明显降低(P<0.01)。加入MG132的各含药血清组中,MG132+正常组表达水平最高,MG132+补肾组与MG132+正常组比较阳性表达水平降低(P<0.01);MG132+补肾组与MG132+骨疏康组、MG132+补脾组比较阳性表达水平上升(P<0.01)。
结论:
1采用多次胶原酶消化法获取大鼠成骨细胞进行体外培养的方法是可靠、简便、易行的,可以提供大量高纯度的成骨细胞用于科学研究使用。
2体外培养的新生大鼠颅盖骨中存在BMP2、BMP7和Smad1/5的活性表达,表明正常大鼠成骨细胞中存在BMP-Smad信号转导通路。
3补肾中药血清可以明显促进成骨细胞的增殖,明显升高成骨细胞中BMP2、BMP7和Smad1/5的表达水平,提示补肾益髓壮骨中药具有防治骨质疏松症的作用。
4应用UPP通路的蛋白酶体抑制剂MG132可以明显抑制成骨细胞的增殖,并且明显降低成骨细胞中BMP2、BMP7和Smad1/5的表达水平。
5应用补肾益髓壮骨中药血清具有干预蛋白酶体抑制剂MG132抑制成骨细胞增殖的作用,对体外培养成骨细胞中BMP2、BMP7和Smad1/5活性的表达有明显提升作用。
Purpose:
Through studying the impact of Kidney-tonifying Chinese herbs in osteoblasts to the act- ive change of BMP2,BMP7 and Smad1/5 in SD rats, explore the osteoporosis pathogenesis mechanism of molecular biology and study the mechanism of action of herbs prevention oste- oporosis to provide scientific experiment basis for the clinical prevention of osteoporosis.
Material and method: To get freshmen SD rats' cranial bones of osteoblast through co-llagenase digestion and cultivate it in vitro and identify the alkaline phosphatase expression with cobalt calcium of appraisal. The experimental rats were randomly divided into normal group, Gushukang parti- cle control group (Gushukang group), Kidney-tonifying Chinese herbs group (Kidney-tonif- ying group) and Tonifying the Middle and Replenishing group (tonifying spleen group). Eight days after the medicine intervention of experimental rat, the serum of each group's rats was used to cultivate the osteoblasts in rats for 48 hours.Besid- es, there were comparison groups with Protease inhibitors MG132 of UPP pathway. We tested the appreciation rate of vitro ost- eoblasts with MTT and the activity expressions of BMP2, BMP7, Smad1/5 with Immunohist- ochemistry SABC.
Results:
1 48 hours after the kidney medicine serum applied to the vitro osteoblasts, the vitro osteobla- sts can obviously promote the proliferation of osteoblasts (P < 0.01).
2 The application of UPP pathway inhibitor MG - 132 protease body can obviously inhibit the proliferation of osteoblast (P < 0.01).
3 The results of the dyeing of alkaline phosphatase: osteoblast via the improved Gomori Calc- uim Cobail method and the redyeing of hematoxylin, some brownish-black fine particles can be seen mostly on and around the epicytes, and a few in the endochylema. There are no brow- nish black particles with the negative control in cells. Some blue granules of Tropic hematox- ylin can be seen in the nucleolus.
4 The result of osteoblast BMP2 Immunohistochemistry: compared with the normal group, the expression level of Gushukang group and Kidney-tonifying group raises obviously (P < 0.01), tonifying spleen group,MG132+ normal group, MG132+ Gushukang group and MG132+ tonifying spleen group reduce significantly (P < 0.01). Compared with the Kidney- tonifying group, the expression of other groups reduce significantly (P < 0.01).
5 The result of osteoblast BMP7 Immunohistochemistry: compared with the normal group, the expression level of Kidney-tonifying group raises obviously (P < 0.01), the expression level of Gushukang group, tonifying spleen group,MG132+ normal group, MG132+ Gushu- kang group, MG132+ Kidney-tonifying group and MG132+ tonifying spleen group reduce significantly (P < 0.01). Compared with the Kidney-tonifying group, the expression of other groups reduce significantly (P < 0.01). Compare each group with herbs serum, the expression of MG132+ normal group was the highest, the Kidney-tonifying group was little reduce.
6 The result of osteoblast Smad1/5 Immunohistochemistry: compared with the normal group, the expression level of Gushukang group,Kidney-tonifying group, tonifying spleen group, MG132+ normal group, MG132+ Gushukang group, MG132+ Kidney-tonifying group and MG132+ tonifying spleen group reduce significantly (P < 0.01). Compared with the Kidney- tonifying group, the expression level of Gushukang group, tonifying spleen group,MG132+ normal group, MG132+ Gushukang group, MG132+ Kidney-tonifying group and MG132+ tonifying spleen group reduce significantly (P < 0.01). Compare each group with HERBS serum, the expression of MG132+ normal group was the highest. Compare each group with the Kidney-tonifying group, the expression level of MG132+ normal group raises obviously (P < 0.01), MG132+ Gushukang group and MG132+ tonifying spleen group reduce significa ntly (P < 0.01).
Conclusion:
1 The method to get rats' osteoblast with repeated collagenase digestion to cultivate it in vitro is reliable, simple and easy, which can provide a lot of highly puritied osteoblasts for scientif- ic research.
2 There are positive expressions of BMP2、BMP7and Smad1/5 in the newborn rats' cranial cultivated in vitro, which means that there are BMP-Smad signal transduction pathways in normal rats' osteoblast.
3 The kidney medicine serum can obviously promote the proliferation of osteoblast, increase significantly the expression levels of BMP2, BMP7, and Smad1/5 in osteoblast, which means that the HERBS of tonifying the kidney, marrow and bones can prevent and treat osteoporosis.
4 Application of protease inhibitors of UPP pathways MG132 could inhibit the proliferation of bone cells, decrease the expression levels of BMP2, BMP7 and Smad1/5 in osteoblasts.
5 Application of HERBS serum of tonifying the kidney, marrow and bones could inhibit the proliferation of osteoplasts by intervening the protease inhibitors MG132 and increase significantly the active expressions of BMP2, BMP7 and Smad1/5 in osteoblasts cultivated in vitro.
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