镁合金经TRPM7激活PI3K/Akt通路调节p21-CDKs表达促进软骨细胞增殖的分子机制

Scaffolds for cartilage tissue engineering has not been made of metal materials. Previous study found that magnesium alloys could promote the proliferation of chondrocyte by regulating the expression of TRPM7, p-Akt, CDKs and p21. Based on previous research, we intend to explore the molecular mechanism of magnesium alloys in promoting the proliferation of chondrocyte. Through the cultivation of chondrocyte in the alginate hydrogel containing magnesium alloys and interfering the expression of TRPM7 and the inhibition of the PI3K/Akt pathway, we want to reveal the intrinsic links between TRPM7, PI3K/Akt signaling pathway, and p21-CDKs in magnesium alloys promoting chondrocyte proliferation in vitro. By interfering the expression of gene TRPM7 and Akt, chondrocyte labeled by PKH26 combined with alginate hydrogel containing magnesium alloys will be implanted into the cartilage defect model of the rabbits. Molecular imaging technique will be used to observe the changes of the chondrocyte; The molecular mechanism of magnesium alloys promoting chondrocyte proliferation through the regulation of p21-CDKs via PI3K/Akt signaling pathway activated by TRPM7 may be revealed using molecular biology methods. Ultimately, this study is expected to lay the foundation for the development of magnesium alloys scaffold of cartilage tissue engineering with excellent biological activity.

软骨组织工程材料尚未涉及金属。前期研究发现镁合金能通过影响TRPM7、p-Akt、CDKs、p21表达，促进软骨细胞增殖。本项目拟在前期研究基础上进一步探讨镁合金促进软骨细胞增殖的分子机制。软骨细胞在含镁合金藻酸盐凝胶中培养，干扰TRPM7基因表达，抑制PI3K/Akt通路，在体外采用分子生物学手段揭示TRPM7、PI3K/Akt通路、p21-CDKs等在镁合金促进软骨细胞增殖中的内在联系；分别影响TRPM7及Akt基因表达，应用PKH26标记软骨细胞后将软骨细胞镁合金藻酸盐凝胶复合体植入兔关节软骨缺损处，采用活体成像技术观察软骨细胞变化，在体内运用分子生物学方法诠释镁合金经TRPM7激活PI3K/Akt通路，调控p21-CDKs表达促进软骨细胞增殖的分子机制，为最终研制出具有优良生物学活性的镁合金软骨组织工程材料奠定基础。

软骨组织工程材料尚未涉及金属。前期研究发现镁合金能通过影响TRPM7、p-Akt、CDKs、.p21表达，促进软骨细胞增殖。本课题主要研究了四个方面的内容并顺利完成研究任务：（1）采用不同浓度的Mg2+与软骨细胞共培养技术，找到最适宜软骨细胞增殖的Mg2+浓度；（2）应用扫描电镜、元素图谱和X线衍射分析镁合金微球体的降解行为，阐明了镁合金微球体动态释放Mg2+的规律，量化与筛选出最适宜软骨细胞增殖的镁合金微球体；（3）证实镁合金通过TRPM7-PI3K/Akt-p21-CDKs信号通路促进软骨细胞增殖。阐明Mg2+经TRPM7激活PI3K/Akt信号通路，影响p21的表达，进而调控CDK2与CDK4表达，促进软骨细胞增殖及其胞外基质合成；（4）双相水凝胶支架作为组织工程支架对大鼠关节软骨及软骨下骨修复的体内体外研究；通过研究我们得出以下结论：（1）Mg2+可以促进软骨细胞的增殖。体外共培养实验结果显示促增殖作用与Mg2+浓度有关，10mM的Mg2+促进软骨细胞增殖的作用最强。Mg2+可以诱导软骨细胞合成软骨特异性的细胞外基质。体外共培养实验结果显示促软骨基质合成的作用与Mg2+浓度有关，10到15mM的Mg2+诱导软骨细胞合成细胞外基质的作用最强。（2）含镁合金微球的藻酸盐水凝胶与软骨细胞有着良好的相容性。直径150微米的镁合金微球在藻酸盐水凝胶中可以稳定降解，释放的Mg2+是软骨细胞增殖和分泌细胞外基质的最佳浓度。含镁合金微球的藻酸盐水凝胶构筑的软骨细胞三维培养体系可以抑制软骨细胞的去分化，其降解过程中释放的Mg2+可以促进软骨细胞的增殖和基质合成。（3）含镁合金微球的藻酸盐水凝胶三维培养体系可通过激活TRPM7促进PI3K/Akt表达，抑制p21后激活CDKs促进软骨细胞增殖和基质蛋白表达。从转录、翻译、细胞周期三个环节验证Mg2+是通过TRPM激活下游信号分子CDK、p21、AKT和PI3K的转录促进软骨细胞的增殖和基质蛋白的表达。（4）从转录、翻译、细胞周期三个环节验证Mg2+是通过TRPM激活PI3K/Akt通路调节p21-CDKs表达促进软骨细胞增殖和细胞外基质的合成。本研究结果表明含镁合金微球的藻酸盐水凝胶在治疗骨缺损方面具有一定的应用潜能。