万伤合剂对小鼠胫骨缺损模型愈合影响的研究
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摘要
目的：
探讨万伤合剂促进小鼠胫骨缺损愈合的体外、体内机制。
方法：
体外实验：万伤合剂浓缩液对IL-1β-luciferase(白介素1-β-荧光素酶)转基因小鼠腹腔巨噬细胞IL-1β表达的影响；大鼠含药血清对原代培养成骨细胞活性及上清的碱性磷酸酶表达的影响。体内实验：建立胫骨缺损模型,以micro CT(显微CT)动态研究胫骨缺损模型自然愈合过程；胫骨缺损模型小鼠分别给予万伤合剂、OPG(成骨生长肽)、万伤合剂加OGP、水,给药后检测血清碱性磷酸酶、离子钙、无机磷等生化指标；检测造模第10天,第20天血清必须氨基酸含量；应用免疫组化法与免疫印迹法检测胫骨缺损修复处VEGF(内血管内皮生长因子)的表达。同时检测了万伤合剂浓缩液内碱性磷酸酶、钙、磷及氨基酸浓度。
结果：
体外实验：万伤合剂组巨噬细胞的荧光值表达显著高于LPS(脂多糖)组(p<0.05)。大鼠含药血清对培养的成骨细胞的细胞活性无提高作用,而10%浓度单日连续给药的大鼠含药血清的碱性磷酸酶浓度显著大于对照组(p<0.05)。体内实验：在胫骨缺损的自然修复过程中,micro CT检查发现其骨矿物质含量、骨密度、三维校准骨小梁厚度均随时间逐渐增高趋势,三维校准骨小梁分离度随时间逐渐降低,骨小梁数目术后一直低于术前,且术后各时间点之间无明显差异,骨体积分数逐渐增高,骨表面积与体积比值于术后先升高后降低。对胫骨缺损小鼠模型研究发现给药组小鼠碱性磷酸酶表达水平显著低于对照组(分别为p<0.05；p<0.01；p<0.001)。而血清钙离子测定：万伤合剂组、OGP组的血钙水平较为稳定显著低于对照组,万伤合剂加OGP组术后血清钙离子浓度显著高于对照组,呈先升高后降低趋势。血清无机磷测定发现用药组血清无机磷显著低于对照组。血清氨基酸测定：用药组小鼠第20天血清精氨酸、亮氨酸、赖氨酸、脯氨酸含量均显著高于对照组,而第10天,上述指标无此变化。免疫组化及免疫印迹发现骨组织在用药后VEGF表达显著上调。
结论：
体外实验发现：万伤合剂可以通过上调IL—1β的表达起到促成骨、促矿化的作用；未发现大鼠含药血清对成骨细胞增殖有明显影响,但单日连续给药的大鼠血清可以有效促进成骨细胞上清碱性磷酸酶表达。体内实验发现：micro CT可以反映胫骨骨缺损模型的动态修复过程。万伤合剂能抑制骨折修复过程的溶骨反应,促进成骨反应,加快骨修复；可以提高促骨损伤修复所需的氨基酸含量,从而达到加快骨折愈合的效果。万伤合剂促进骨修复的过程与促进VEGF表达密切相关,可能通过增加骨缺损修复区域内的血管增生来实现。本研究为万伤合剂促进骨折修复提供了直接而客观的证据。
Objective:investigate the enhancement of mouse tibia injury healing in vitro and in vivo by Wanshangheji compound.
Material and Method:in vitro, Macrophages from L-1β-luciferase transgenic mice were challenged with Wangshangheji compound (WSHJ). Mouse primary osteoblastic cells were incubated with different type of medicated rat serum prepared with WSHJ. In vivo:A mouse tibia injury model was established. Micro CT was used for follow-up of healing in treatment-naive tibia injury mice. Serum alkaline phosphatase, ionized calcium (Ca2+) and inorganic phosphorus (IP) were measured at day20after different drugs administered. The essential amino acids in mouse serum at day10and day20were quantitated by HPLC. The alkaline phosphatase (Alp), ionized calcium, inorganic phosphorus and essential amino acids in WSHJ were measured as control. The expression of VEGF in the tibia tissue was detected by immunohistochemistry and western blot.
Result:WSHJ stimulated the expression of luciferase significantly (p<0.05) higher than LPS in the macrophages from L-1β-luciferase transgenic mice. There was no proliferation effect in medicated rat serum treated mouse primary osteoblasts. But the medium alkaline phosphatase level in10%rat medicated serum was significantly (p<0.05) higher than the control group. in vivo:Micro CT analysis showed an increasing trend in BMC,BMD and Calib.Tb.Th. after tibia injury while Calib.Tb.Sp. decreasing. Tb.N was low after tibia injury. But there was no any trend in different time points. BV/TV was increasing after the injury. BS/BV increased at first and decreased subsequently. All the administered groups showed a lower alkaline phosphatase level than the control group (p<0.05, p<0.01, p<0.001, respectively). Ca2+in the group of WSHJ and OGP groups were stable and significantly lower than the control. But the group of combination of WSHJ&OGP showed a higher level of Ca2+than the control. All the administered groups showed a lower IP level than the control. Serum essential amino acids test showed arginine, leucine, leucine and proline were significantly higher than in the control group at day20. But there were no apparent changes at day10. IH and WB showed an elevated VEGF expression of WSHJ administered group as compared to control.
Conclusion:
WSHJ up-regulated the expression of IL-1β to improves bone formation and mineral accretion. There was no evidence of proliferative effect in mouse primary osteoblasts incubated with rat WSHJ medicated serum. But Alp increased in the medium of osteoblasts incubated with10%WSHJ medicated (q.h×4) serum. Micro CT showed dynamic changes during the healing of tibia healing in vivo. WSHJ showed some inhibitory effect on the bone dissolving process. It promoted osteogenesis and accelerated healing progress. WSHJ increased the level of bone related amino acids. The accelerated healing effect of WSHJ was involved with the up-regulated expression of VEGF and increasing angiogenic activity at the bone injury site. This pilot study provided some direct and objective evidence of acceleration fracture healing effect of WSHJ.
探讨万伤合剂促进小鼠胫骨缺损愈合的体外、体内机制。
方法：
体外实验：万伤合剂浓缩液对IL-1β-luciferase(白介素1-β-荧光素酶)转基因小鼠腹腔巨噬细胞IL-1β表达的影响；大鼠含药血清对原代培养成骨细胞活性及上清的碱性磷酸酶表达的影响。体内实验：建立胫骨缺损模型,以micro CT(显微CT)动态研究胫骨缺损模型自然愈合过程；胫骨缺损模型小鼠分别给予万伤合剂、OPG(成骨生长肽)、万伤合剂加OGP、水,给药后检测血清碱性磷酸酶、离子钙、无机磷等生化指标；检测造模第10天,第20天血清必须氨基酸含量；应用免疫组化法与免疫印迹法检测胫骨缺损修复处VEGF(内血管内皮生长因子)的表达。同时检测了万伤合剂浓缩液内碱性磷酸酶、钙、磷及氨基酸浓度。
结果：
体外实验：万伤合剂组巨噬细胞的荧光值表达显著高于LPS(脂多糖)组(p<0.05)。大鼠含药血清对培养的成骨细胞的细胞活性无提高作用,而10%浓度单日连续给药的大鼠含药血清的碱性磷酸酶浓度显著大于对照组(p<0.05)。体内实验：在胫骨缺损的自然修复过程中,micro CT检查发现其骨矿物质含量、骨密度、三维校准骨小梁厚度均随时间逐渐增高趋势,三维校准骨小梁分离度随时间逐渐降低,骨小梁数目术后一直低于术前,且术后各时间点之间无明显差异,骨体积分数逐渐增高,骨表面积与体积比值于术后先升高后降低。对胫骨缺损小鼠模型研究发现给药组小鼠碱性磷酸酶表达水平显著低于对照组(分别为p<0.05；p<0.01；p<0.001)。而血清钙离子测定：万伤合剂组、OGP组的血钙水平较为稳定显著低于对照组,万伤合剂加OGP组术后血清钙离子浓度显著高于对照组,呈先升高后降低趋势。血清无机磷测定发现用药组血清无机磷显著低于对照组。血清氨基酸测定：用药组小鼠第20天血清精氨酸、亮氨酸、赖氨酸、脯氨酸含量均显著高于对照组,而第10天,上述指标无此变化。免疫组化及免疫印迹发现骨组织在用药后VEGF表达显著上调。
结论：
体外实验发现：万伤合剂可以通过上调IL—1β的表达起到促成骨、促矿化的作用；未发现大鼠含药血清对成骨细胞增殖有明显影响,但单日连续给药的大鼠血清可以有效促进成骨细胞上清碱性磷酸酶表达。体内实验发现：micro CT可以反映胫骨骨缺损模型的动态修复过程。万伤合剂能抑制骨折修复过程的溶骨反应,促进成骨反应,加快骨修复；可以提高促骨损伤修复所需的氨基酸含量,从而达到加快骨折愈合的效果。万伤合剂促进骨修复的过程与促进VEGF表达密切相关,可能通过增加骨缺损修复区域内的血管增生来实现。本研究为万伤合剂促进骨折修复提供了直接而客观的证据。
Objective:investigate the enhancement of mouse tibia injury healing in vitro and in vivo by Wanshangheji compound.
Material and Method:in vitro, Macrophages from L-1β-luciferase transgenic mice were challenged with Wangshangheji compound (WSHJ). Mouse primary osteoblastic cells were incubated with different type of medicated rat serum prepared with WSHJ. In vivo:A mouse tibia injury model was established. Micro CT was used for follow-up of healing in treatment-naive tibia injury mice. Serum alkaline phosphatase, ionized calcium (Ca2+) and inorganic phosphorus (IP) were measured at day20after different drugs administered. The essential amino acids in mouse serum at day10and day20were quantitated by HPLC. The alkaline phosphatase (Alp), ionized calcium, inorganic phosphorus and essential amino acids in WSHJ were measured as control. The expression of VEGF in the tibia tissue was detected by immunohistochemistry and western blot.
Result:WSHJ stimulated the expression of luciferase significantly (p<0.05) higher than LPS in the macrophages from L-1β-luciferase transgenic mice. There was no proliferation effect in medicated rat serum treated mouse primary osteoblasts. But the medium alkaline phosphatase level in10%rat medicated serum was significantly (p<0.05) higher than the control group. in vivo:Micro CT analysis showed an increasing trend in BMC,BMD and Calib.Tb.Th. after tibia injury while Calib.Tb.Sp. decreasing. Tb.N was low after tibia injury. But there was no any trend in different time points. BV/TV was increasing after the injury. BS/BV increased at first and decreased subsequently. All the administered groups showed a lower alkaline phosphatase level than the control group (p<0.05, p<0.01, p<0.001, respectively). Ca2+in the group of WSHJ and OGP groups were stable and significantly lower than the control. But the group of combination of WSHJ&OGP showed a higher level of Ca2+than the control. All the administered groups showed a lower IP level than the control. Serum essential amino acids test showed arginine, leucine, leucine and proline were significantly higher than in the control group at day20. But there were no apparent changes at day10. IH and WB showed an elevated VEGF expression of WSHJ administered group as compared to control.
Conclusion:
WSHJ up-regulated the expression of IL-1β to improves bone formation and mineral accretion. There was no evidence of proliferative effect in mouse primary osteoblasts incubated with rat WSHJ medicated serum. But Alp increased in the medium of osteoblasts incubated with10%WSHJ medicated (q.h×4) serum. Micro CT showed dynamic changes during the healing of tibia healing in vivo. WSHJ showed some inhibitory effect on the bone dissolving process. It promoted osteogenesis and accelerated healing progress. WSHJ increased the level of bone related amino acids. The accelerated healing effect of WSHJ was involved with the up-regulated expression of VEGF and increasing angiogenic activity at the bone injury site. This pilot study provided some direct and objective evidence of acceleration fracture healing effect of WSHJ.
引文
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