课题背景：课题是在辽宁省教育厅项目：壳寡糖及其衍生物促进骨缺损愈合作用的研究（20060153）；大连民族学院博士启动基金资助项目：人工细胞信号转导系统的构建和研究（20056106）资助下进行的。课题宗旨拟从分子水平上探讨壳寡糖及其衍生物促进骨形成的作用。

应用要点：壳寡糖和N-乙酰氨基葡萄糖分别在500，1 000 mg/L时促进成骨细胞增殖效果最为明显，氨基葡萄糖表现出双向调节作用，在低质量浓度时（10，100 mg/L）有促进作用，高质量浓度（> 500 mg/L）则表现出抑制作用；羧甲基壳寡糖在各质量浓度下的促进效果均较N-乙酰氨基葡萄糖和壳寡糖弱。

偏倚或不足：在细胞培养实验中，DMEM培养液中的酚红和血清以及所用的壳寡糖及其衍生物N-乙酰氨基葡萄糖、氨基葡萄糖、羧甲基壳寡糖也具有一定的还原性，可能对实验结果有一定的干扰。

摘要
背景：甲壳素、壳聚糖降解后得到的壳寡糖和单糖分子质量小、水溶性良好，具有大分子糖所不具备的更独特的生理生化活性。
目的：观察壳寡糖及其衍生物N-乙酰氨基葡萄糖、氨基葡萄糖、羧甲基壳寡糖对体外培养的成骨细胞MC3T3-E1的作用特点。
设计、时间及地点：对比观察实验，于2004-02/2005-07在中国海洋大学海洋生命学院生化实验室完成。
材料：小鼠颅骨成骨细胞株MC3T3-E1由北京协和医院细胞中心提供；N-乙酰氨基葡萄糖、壳寡糖、氨基葡萄糖、羧甲基壳寡糖为中国海洋大学海洋生命学院生化实验室自制。
方法：分别将N-乙酰氨基葡萄糖、氨基葡萄糖、壳寡糖、羧甲基壳寡糖以10，100，500，1 000，2 000 mg/L加入DMEM培养基中。将用4种糖培养的成骨细胞按试剂盒方法提取mRNA，进行mRNA差异显示分析。
主要观察指标：MTT法测定成骨细胞的增殖情况。倒置显微镜下观察成骨细胞的生长情况。分析4种糖的mRNA差异。
结果：N-乙酰氨基葡萄糖在5个质量浓度梯度均能促进成骨细胞增殖，1 000 mg/L时效果最为明显；壳寡糖在各质量浓度下都表现出较明显的促进细胞增殖作用，500 mg/L为最适质量浓度；氨基葡萄糖表现出双向调节作用，在10，100 mg/L时有促进作用，> 500 mg/L则表现出抑制作用；羧甲基壳寡糖在各质量浓度下均表现出一定的促进成骨细胞生长作用，但促进效果较N-乙酰氨基葡萄糖和壳寡糖弱。光镜观察结果与MTT测定法结果一致。mRNA差异显示4种糖无明显激活新基因的产生。
结论：壳寡糖及其衍生物对体外培养的成骨细胞均有不同程度的促进增殖作用，其中N-乙酰氨基葡萄糖和壳寡糖的效果优于氨基葡萄糖和羧甲基壳寡糖，作用中成骨细胞无新基因产生。
关键词：成骨细胞；壳寡糖；衍生物；mRNA差异显示；生物材料

金黎明，魏长征，田文杰，赵小菁，王雅玲，范圣第.壳寡糖及其衍生物对体外培养成骨细胞增殖的作用特点[J].中国组织工程研究与临床康复，2008，12(19):3637-3640 [www.zglckf.com/zglckf/ejournal/upfiles/08-19/19k-3637(ps).pdf]

Chitooligosaccharide and its derivatives promote the proliferation of osteoblasts in vitro

Abstract
BACKGROUND: Chitooligosaccharide (COS) and monosaccharide moleculars derived from chitin and chitosan have the superiority to macromolecular saccharide, owing to low weight and good water solubility.
OBJECTIVE: To observe the abilities of COS and its derivates, such as glucosamine (GluNH2), N-acetyl-glucosamine (NAG) and carboxymethyl-COS (CM-COS) to promote the proliferation of osteoblasts in vitro.
DESIGN, TIME AND SETTING: Controlled observation experiments were completed in the Biochemistry Laboratory, College of Marine Life Sciences, Ocean University of China from February 2004 to July 2005.
MATERIALS: Cell strain MC3T3-E1 of mice skull was offered by the Cell Center of Peking Union Medical College Hospital (China). NAG, COS, GluNH2 and CM-COS were prepared in the Biochemistry Laboratory, College of Marine Life Sciences, Ocean University of China (China).
METHODS: NAG, COS, GluNH2 and CM-COS were added into DMEM medium according to the concentrations of 10, 100, 500, 1 000 and 2 000 mg/L, respectively. The mRNA was extracted from the cultured osteoblasts. Then mRNA differential display analysis was applied to study the mechanism.
MAIN OUTCOME MEASURES: The proliferation of osteoblasts was studied by MTT method. The growth of osteoblasts was observed under inverted microscope. The mechanism was studied by mRNA differential display.
RESULTS: NAG at appropriate concentrations could promote the proliferation of osteoblasts, and the higher concentration (1 000 mg/L) had stronger effects. The promotion of COS was more obvious, and the most effective concentration was 500 mg/L. GluNH2 promoted the proliferation of osteoblasts on lower concentrations (10 and 100 mg/L) while higher concentrations (> 500 mg/L) indicated inhibiting function. CM-COS also showed the function of proliferation, but the effect was not prominent. The optical microscopy outcome was consistent with that of MTT method. The results of mRNA differential display showed that four samples had no obvious effect on activating new genes.
CONCLUSION: In some sense, COS and its derivates all can promote the proliferation of osteoblasts cultured in vitro. NAG and COS are more effective than GluNH2 and CM-COS. No gene production is found in osteoblasts.

Jin LM, Wei CZ, Tian WJ, Zhao XJ, Wang YL, Fan SD.Chitooligosaccharide and its derivatives promote the proliferation of osteoblasts in vitro.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(19):3637-3640
[www.zglckf.com/zglckf/ejournal/upfiles/08-19/19k-3637(ps).pdf]