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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:

第24卷

期數:

2012年07期

頁碼:

3-7

欄目:

基礎研究

出版日期:

2012-07-30

文章信息/Info

Title:

Study on the effect of TOUGU XIAOTONG capsule medicated serum on the expression of Cyclin D1 mRNA in Chondrocytes

作者:

葉蕻芝¹; 李西海¹; 陳加守²; 鄭春松³; 楊建萍²; 翁霞萍²; 曾宗鵬²; 鄭志²; 劉獻祥¹

1.福建中醫(yī)藥大學中西醫(yī)結合研究院,福建 福州 350108;
2.福建中醫(yī)藥大學 藥學院,福建 福州 350108;
3.福建省中西醫(yī)結合老年性疾病重點實驗室, 福建 福州 350108

Author(s):

YE Hong-zhi^*; LI Xi-hai; CHEN Jia-shou; ZHENG Chun-song; YANG Jian-ping; WONG Xia-ping; ZENG Zong-peng; ZHENG Zhi; LIU Xian-xiang.^*

Academy of Integrated Traditional Chinese Medicine with Western Medicine of Fujian University of Traditional Chinese Medicine,Fuzhou 350108,Fujian,China

關鍵詞:

骨關節(jié)炎 透骨消痛膠囊 細胞周期蛋白D1 軟骨細胞 含藥血清

Keywords:

Osteoarthritis;  TOUGU XIAOTONG capsule;  Cyclin D1;  Chondrocytes;  Medicated serum

摘要:

目的:觀察透骨消痛膠囊含藥血清對軟骨細胞CyclinD1 mRNA表達的影響,探討透骨消痛膠囊治療骨性關節(jié)炎的可能作用機 制。方法:采用抽簽法將16只12周齡雄性新西蘭大白兔隨機分為空白組、低劑量組、中劑量組、高劑量組,每組4只。低劑量組按 5 mL·kg^-1體質量以5 mg·mL^-1透骨消痛膠囊溶液灌胃,中劑量組按5 mL·kg^-1體質量以 10 mg·mL^-1透骨消痛膠囊溶液灌胃,高劑量組按5 mL·kg^-1體質量以20 mg·mL^-1透骨消 痛膠囊溶液灌胃,空白組按5 mL·kg^-1體質量以生理鹽水灌胃。每天灌胃2次,連續(xù)7 d,最后1 d連續(xù)2次灌胃,中間間 隔2 h。末次灌胃后3 h提取各組實驗兔含藥血清低溫保存?zhèn)溆谩��?只4周齡雄性新西蘭兔處死,取其膝關節(jié)軟骨,置入盛有Ⅱ型 膠原酶的培養(yǎng)皿中進行消化,建立軟骨細胞體外培養(yǎng)體系,并采用甲苯胺藍染色法鑒定細胞功能。將體外培養(yǎng)的第3代軟骨細胞隨 機分為空白組、低劑量組、中劑量組、高劑量組,并分別加入含空白血清、低劑量中藥血清、中劑量中藥血清、高劑量中藥血清 的DMEM培養(yǎng)液中繼續(xù)培養(yǎng)72 h后,采用MTT法檢測軟骨細胞的增殖情況,采用RT-PCR法檢測CyclinD1 mRNA的表達,并在透射電子顯 微鏡下觀察細胞形態(tài)。結果:Ⅱ型膠原酶消化法成功建立了軟骨細胞的體外培養(yǎng)體系,甲苯胺藍染色可見軟骨細胞內呈紫紅色異 染顆粒。干預72 h后,各組軟骨細胞光密度值比較,差異有統(tǒng)計學意義(F=6.209,P=0.004); 中劑量組、高劑量組軟骨細胞光密度 值高于空白組(P=0.005,P=0.002); 中劑量組、高劑量組軟骨細胞光密度值高于低劑量組(P=0.034,P=0.011)。各組軟骨細胞 CyclinD1 mRNA表達量比較,差異有統(tǒng)計學意義(F=8.262,P=0.001); 中劑量組、高劑量組軟骨細胞CyclinD1 mRNA表達量高于空 白組(P=0.002,P=0.001); 中劑量組、高劑量組軟骨細胞CyclinD1 mRNA表達量高于低劑量組(P=0.012,P=0.004); 中劑量組、高 劑量組可見較多處于分裂期的細胞。結論:透骨消痛膠囊的含藥血清能促進軟骨細胞G1期正性調節(jié)因子CyclinD1 mRNA的表達,從 而促進軟骨細胞增殖,這可能是透骨消痛膠囊在臨床上治療骨性關節(jié)炎具有較好療效的部分機理,而有關透骨消痛膠囊治療骨性 關節(jié)炎的具體作用機制還需作更進一步的深入研究,以便為臨床應用提供理論依據。

Abstract:

Objective:To observe the effect of TOUGU XIAOTONG capsule medicated serum on the expression of Cyclin D1 mRNA in chondrocytes,and explore the possible mechanism of TOUGU XIAOTONG capsule in the treatment of osteoarthritis(OA).Methods:Sixteen male New Zealand white rabbits of 12 weeks old were randomly divided into blank group,low-dose group,middle-dose group and high-dose group by drawing lots,4 cases in each group.Rabbits in the low-dose group were intragastric administrated with 5 mg·mL^-1 TOUGU XIAOTONG solution(5 mL· kg^-1),rabbits in the middle-dose group were intragastric administrated with 10 mg·mL^-1 TOUGU XIAOTONG solution(5 mL·kg^-1),rabbits in the high-dose group were intragastric administrated with 20 mg·mL^-1 TOUGU XIAOTONG solution(5 mL·kg^-1),and rabbits in the blank group were intragastric administrated with normal saline(5 mL·kg^-1).All the rabbits were intragastric administrated twice a day for 7 consecutive days,and they were intragastric administrated continuous 2 times with interval of 2 hours on the last day.Three hours after the last administration,medicated serums of experimental rabbits in every group were extracted and preserved in low temperature.Six male New Zealand white rabbits of 4 weeks old were executed and their knee articular cartilages were fetched and placed in the Petri dish with typeⅡcollagenase for digestion.The culture system of cartilage cell in vitro was built,meantime,cell function was evaluated through toluidine blue staining method.The third-generation cartilage cell cultured in vitro were randomly divided into blank group,low-dose group,middle-dose group and high-dose group,and they were placed in the DMEM culture fluid respectively added with blank serum,low-dose traditional Chinese medicine(TCM)serum,middle-dose TCM serum and high-dose TCM serum for further culture.Seventy-two hours later,the cartilage cell proliferation was detected by MTT method,Cyclin D1 mRNA expression was inspected through RT-PCR method,and the cell shape was observed under transmission electron microscope.Results:Culture system of cartilage cell in vitro was successfully built through collagenaseⅡdigestion and purple metachromatic granules were found in cartilage cell through toluidine blue staining method.There were statistical differences in optical density(OD)of cartilage cell among the 4 groups after 72 hours' intervention(F=6.209,P=0.004); the OD of cartilage cell in middle-dose group and high- dose group were higher than that of blank group(P=0.005,P=0.002); the OD of cartilage cell in middle-dose group and high-dose group were higher than that of low-dose group(P=0.034,P=0.011).There were statistical differences in protein expression of CyclinD1 mRNA in cartilage cell among the 4 groups(F=8.262,P=0.001); CyclinD1 mRNA in cartilage cell of middle-dose group and high-dose group were higher than that of blank group(P=0.002,P=0.001) and low-dose group(P=0.012,P=0.004); more chondrocytes in dividing phase were found in middle-dose group and high-dose group.Conclusion:TOUGU XIAOTONG capsule medicated serum can promote the proliferation of chondrocytes through promoting the expression of positive regulator CyclinD1 mRNA in G1 period,and it may be partial mechanisms of the better curative effect of TOUGU XIAOTONG capsule in the treatment of OA.Howerer,further research is needed to determin the concrete mechanism of action of TOUGU XIAOTONG capsule on purpose of providing theoretical basis for the clinic.

備注/Memo

備注/Memo:

2011-12-06收稿 2011-12-23修回
基金項目:國家自然科學基金(81102609),福建省自然基金(2011J05074),福建省自然科學基金重點項目(2010Y0032),福建省中醫(yī) 藥科研重點課題資助課題(WZZG0901)
通訊作者:劉獻祥 E-mail:[email protected]

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更新日期/Last Update: 2012-07-30