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[1]宋奕,丁道芳,李玲慧,等.股密葆含藥血清對(duì)成骨細(xì)胞分化成熟的影響及其作用機(jī)制[J].中醫(yī)正骨,2014,26(03):9-13.
　Song Yi*,Ding Daofang,Li Linghui,et al.Effect of GUMIBAO medicated serum on osteoblastic differentiation and maturation and its mechanism of action[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2014,26(03):9-13.
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股密葆含藥血清對(duì)成骨細(xì)胞分化成熟的影響及其作用機(jī)制()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第26卷
期數(shù):
2014年03期
頁(yè)碼:
9-13
欄目:
基礎(chǔ)研究
出版日期:
2014-03-30

文章信息/Info

Title:
Effect of GUMIBAO medicated serum on osteoblastic differentiation and maturation and its mechanism of action
作者:
宋奕1; 丁道芳2; 李玲慧3; 杜國(guó)慶3; 劉錦濤1; 詹紅生2; 姜宏1
1.江蘇省蘇州市中醫(yī)醫(yī)院,江蘇 蘇州 215009;
2.上海市中醫(yī)藥研究院,上海 200021;
3.上海中醫(yī)藥大學(xué),上海 201203
Author(s):
Song Yi*; Ding Daofang; Li Linghui; Du Guoqing; Liu Jintao; Zhan Hongsheng; Jiang Hong.
*Traditional Chinese Medicine Hospital of Suzhou city,Suzhou 215009,Jiangsu,China
關(guān)鍵詞:
股骨頭壞死 成骨細(xì)胞 股密葆 含藥血清 堿性磷酸酶 膠原Ⅰ型 Runt相關(guān)轉(zhuǎn)錄因子2 β連環(huán)素 轉(zhuǎn)錄因子4
Keywords:
Femur head necrosis;  Osteoblasts;  GUMIBAO;  Medicated serum;  Alkaline phosphatase;  Collagen TypeⅠ;  Runt-related transcription factor 2;  Beta catenin;  Transcription factor 4
摘要:
目的:探討股密葆含藥血清對(duì)成骨細(xì)胞分化成熟的影響及其作用機(jī)制。方法:將20只2月齡雌性SD大鼠隨機(jī)分成4組,對(duì)照組8只,高、中、低濃度股密葆組各4只。對(duì)照組按10 mL·kg-1體質(zhì)量以生理鹽水灌胃,高濃度組按10 mL·kg-1體質(zhì)量以2 430 mg·mL-1股密葆混懸液灌胃,中濃度組按10 mL·kg-1體質(zhì)量以1 215 mg·mL-1股密葆混懸液灌胃,低濃度組按10 mL·kg-1體質(zhì)量以607.5 mg·mL-1股密葆混懸液灌胃。每天灌胃1次,連續(xù)3 d。末次灌胃后1 h提取各組實(shí)驗(yàn)大鼠含藥血清,低溫保存?zhèn)溆谩?0只新生SD大鼠處死,取其顱骨,采用多次酶消化法分離出成骨細(xì)胞。將第1代成骨細(xì)胞分別在含有對(duì)照組大鼠血清及高、中、低濃度股密葆含藥血清的培養(yǎng)基中培養(yǎng)1周后,采用染色法檢測(cè)堿性磷酸酶表達(dá)情況,采用Western Blotting法檢測(cè)Ⅰ型膠原、Runt相關(guān)轉(zhuǎn)錄因子2、β連環(huán)素及轉(zhuǎn)錄因子4的蛋白表達(dá)情況。結(jié)果:①成骨細(xì)胞中堿性磷酸酶的表達(dá)。與對(duì)照組比較,各濃度股密葆含藥血清組堿性磷酸酶染色較深,其中中濃度組染色最深。②成骨細(xì)胞中Ⅰ型膠原蛋白的表達(dá)。對(duì)照組、高濃度組、中濃度組、低濃度組Ⅰ型膠原蛋白的表達(dá)量比較,差異有統(tǒng)計(jì)學(xué)意義[(1.172±0.047),(2.023±0.131),(2.792±0.737),(2.235±0.525),F=6.466,P=0.016]。組間兩兩比較,中濃度組、低濃度組高于對(duì)照組(P=0.003,P=0.022); 高濃度組與對(duì)照組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.052); 其余各組間比較,差異均無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。③成骨細(xì)胞中Runt相關(guān)轉(zhuǎn)錄因子2蛋白的表達(dá)。各組Runt相關(guān)轉(zhuǎn)錄因子2蛋白的表達(dá)量比較,差異有統(tǒng)計(jì)學(xué)意義[(1.968±0.263),(2.255±0.286),(2.675±0.181),(2.652±0.211),F=6.066,P=0.019]。組間兩兩比較,中濃度組、低濃度組高于對(duì)照組(P=0.007,P=0.008); 高濃度組與對(duì)照組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.180); 其余各組間比較,差異均無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。④成骨細(xì)胞中β連環(huán)蛋白的表達(dá)。各組β連環(huán)蛋白表達(dá)量比較,差異有統(tǒng)計(jì)學(xué)意義[(1.571±0.140),(2.264±0.265),(2.783±0.432),(2.860±0.576),F=6.980,P=0.013]。組間兩兩比較,中、低濃度組均高于對(duì)照組(P=0.005,P=0.004); 高濃度組與對(duì)照組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.061); 其余各組間比較,差異均無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。⑤成骨細(xì)胞中轉(zhuǎn)錄因子4蛋白的表達(dá)。各組轉(zhuǎn)錄因子4蛋白表達(dá)量比較,差異有統(tǒng)計(jì)學(xué)意義[(3.268±0.298),(3.941±0.360),(4.446±0.443),(4.186±0.249),F=6.431,P=0.016]。組間兩兩比較,高、中、低濃度組均高于對(duì)照組(P=0.044,P=0.003,P=0.012); 其余各組間比較,差異均無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。結(jié)論:適當(dāng)濃度股密葆含藥血清可促進(jìn)成骨細(xì)胞的分化,其作用機(jī)制或許與Wnt/β連環(huán)素信號(hào)通路的激活有關(guān)。
Abstract:
Objective:To explore the effect of GUMIBAO medicated serum on osteoblastic differentiation and maturation and its mechanism of action.Methods:Twenty two-month-old female SD rats were randomly divided into 4 groups,8 cases in control group and 4 cases in each of other groups.Rats in control group were intragastric administrated with normal saline(10 mL/kg),while other rats were intragastric administrated with GUMIBAO suspension(10 mL/kg)with dose of 2 430 mg/mL(high-concentration group),1 215 mg/mL(middle-concentration group)and 607.5 mg/mL(low-concentration group),once a day for consecutive 3 days.The medicated serum were extracted from rats one hour after the last intragastric administration and were reserved at low temperature.Then ten newborn SD rats were executed and the skulls were fetched out for separating osteoblasts by multiple enzyme digestion.The expression of alkaline phosphatase(ALP)were detected by staining,and the protein expression of collagen typeⅠ,Runt-related transcription factor 2,beta catenin and transcription factor 4 were detected by Western Blotting after the first-generation osteoblasts were cultured in medicated serum for 1 week.Results:The ALP staining in the GUMIBAO medicated serum groups with different concentration were darker than that of control group,and the ALP staining was the darkest in middle concentration group compared to other groups.There were statistical differences in the expression of collagen typeⅠbetween the groups((1.172+/-0.047),(2.023+/-0.131),(2.792+/-0.737),(2.235+/-0.525),F=6.466,P=0.016).Further pairwise comparison showed that the expression of collagen typeⅠin middle and low concentration group were higher than that of high concentration group(P=0.003,P=0.022).There were no statistical differences in the expression of collagen typeⅠbetween high concentration group and control group(P=0.052),while there were no statistical differences between rest groups(P>0.05).There were statistical differences in the protein expression of Runt-related transcription factor 2 between the groups((1.968+/-0.263),(2.255+/-0.286),(2.675+/-0.181),(2.652+/-0.211),F=6.066,P=0.019).Further pairwise comparison showed that middle and low concentration group surpassed the control group(P=0.007,P=0.008),and there were no statistical differences between high concentration group and control group(P=0.180),while there were no statistical difference between rest groups(P>0.05).There were statistical differences in the expression of beta catenin between the groups((1.571+/-0.140),(2.264+/-0.265),(2.783+/-0.432),(2.860+/-0.576),F=6.980,P=0.013).Further pairwise comparison showed that the expression of beta catenin in middle and low concentration group were higher than that of control group(P=0.005,P=0.004),and there were no statistical differences between high concentration group and control group(P=0.061),while there were no statistical differences between rest groups(P>0.05).There were statistical differences in the expression of transcription factor 4 between the groups((3.268+/-0.298),(3.941+/-0.360),(4.446+/-0.443),(4.186+/-0.249),F=6.431,P=0.016).Further pairwise comparison showed that the expression of transcription factor 4 in high,middle and low concentration group were higher than that of control group(P=0.044,P=0.003,P=0.012),while there were no statistical differences between rest groups(P>0.05).Conclusion:GUMIBAO medicated serum with suitable concentration can significantly promote osteoblastic differentiation and it may concerned the activation of Wnt/beta catenin signaling pathway.

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備注/Memo

備注/Memo:
基金項(xiàng)目:國(guó)家自然科學(xué)基金項(xiàng)目(81073114,81072830),江蘇省中醫(yī)藥管理局2010年度康緣中醫(yī)藥科技創(chuàng)新基金(HZ1013KY),蘇州市科技計(jì)劃項(xiàng)目(SYS201257)
通訊作者:姜宏 E-mail:[email protected]
更新日期/Last Update: 2014-03-20