中药地龙提取液的促成骨作用及对实验性牙槽骨吸收疗效的研究

Therapeutic effects of Pheretima guillelmi on rat experimental model of alveolar bone resorption and mouse osteoblastic MC3T3-E1 cells

探讨了中药地龙水提液和醇提液有无促进成骨细胞增殖、分化和基质矿化的作用及对大鼠牙槽骨吸收疗效进行了研究.制备地龙的水相及醇相提取液,以小鼠成骨细胞株MC3T3-E1作为体外药效的试验模型,分别通过MTT法,流式细胞术,碱性磷酸酶活性测定,骨钙素检测和Vonkossa基质矿化染色法观察上述药物的促细胞增殖、分化、基质矿化作用.以实验牙位龈正中注射脂多糖建立的SD大鼠实验性牙槽骨吸收作为体内药效试验模型,通过组织切片形态学骨密度观察和抗酒石酸酸性磷酸酶染色评价上述药物对大鼠实验性牙槽骨吸收的疗效.结果发现:1mg.L-1水提液,0.01mg.L-1和1mg.L-1醇提液能显著促进MC3T3-E1细胞增殖.各浓度的水提液和醇提液均能使S期细胞百分率升高,G1期细胞百分率减少.0.01mg.L-1水提液和1mg.L-1醇提液作用于细胞的第1～3d内、100mg·L-1醇提液作用第7～9d内、100mg·L-1水提液作用第10～12d内可显著提高细胞骨钙素合成和分泌.0.01mg·L-1水提液和醇提液,1mg·L-1水提液可显著促进细胞体外基质矿化.地龙水提液和醇提液组的骨密度均有显著增高,破骨细胞数显著降低.阴性对照组至第30d时牙槽骨仍可见大量破骨细胞.结果表明:地龙水相和醇相提取物中均存在较高活性的促成骨细胞增殖、分化和基质矿化的物质,且对实验性牙槽骨吸收模型有一定治疗作用,可抑制其牙槽骨吸收,促进成骨活动使其修复重建.

To explore the effects of Phertima guillelmi（PG）on promoting the proliferation.differentiation and matrix mineralization of osteoblasts.and to understand the therapeutic effects of PG on rats suffering from alveolar bone resorption.PG aqueous and ethanol-extracts were prepared.MC3T3-E1 cell ling was chosen as an eperimental model in vitro.MTT viability analysis.flow cytometry.alkaling phosphatase detection.osteocalcin detection and Von kossa staining were utilized to investigate the proliferation.differentiation and matrix mineralization of cells.An E.coli lipopolyasaccharidc（E-LPS）induced experimental rat model with alveolar bone resprption was established as an experimental model in vivo.Bone mineral density measurements and tratrate resistant acid phosphatasc（TRAP）stained ostcoclast count were used to evaluate the therapeutic effects of PG in vivo.1 mg·L^-1 of PG aqueous-extracts,0.01and 1 mg·L^-1 of PG ethanol-extracts showed the proliferative promotion for MC3T3-El cells significantly.Each dosage of the two PG extracts incrcased the percentages of S-phase cells whereas the percentages of G-phase cels were ddecreased.0.01 mg·L^-1 of PG aqueous-extracts and 1 mg·L^-1 of the ethanol-extracts caused the enhancement of osteocalcin of the supernate during the 1-3 d,whilc 100 mg·L^-1 of the entanol-extracts acted during the 7-9 d and 100 mg·L^-1 of PG aqueous-extracts acted during the 9-12 d,0.01mg·L^-1 of two extracts and 1 mg·L^-1 of the aqueous-extracts raised the percentage of matrix minerlization area.BMD values of groups administrated with PG aqueous and ethanol-extractd were increased,while the number of osteoclasts in these groups were decreased.However,massive osteoclasts did not disappeared yet in the negative control group during the 30 d.There are some components in the PG that can promote the proliferation.differentiation and matrix mineralization of osteoblasts.The PG extracts showed a therapeutic effects on the alveolar bone resorption in the experimental rats through suppressing the alvolar bone resorption and promoting bone regeneration