Molecular analysis of the tissue-engineered and regenerated bones using the mesenchymal stem cells and gene-containing carriers

为了进一步分析细胞因子信号传导在骨再生中的机制和参与，首先测试人瘢痕疙瘩来源的成纤维细胞原代培养物对神经酰胺诱导的细胞凋亡的可能抗性。神经酰胺诱导成纤维细胞凋亡呈剂量依赖性和Banner依赖性;然而，瘢痕疙瘩来源的成纤维细胞通过FACS细胞分选仪显示G2期阻滞，并且通过WST-t分析显示细胞数量减少。当加入IGF-1时，瘢痕疙瘩成纤维细胞抵抗神经酰胺诱导的凋亡。这与IGF-1受体表达水平呈正相关。PI-3抑制剂。Wortmaninn预处理，通过IGF-1信号转导的细胞凋亡的抑制作用，显着取消。本实验一方面探讨IGF-1通过其受体参与后续的信号传导，并进行分子调控，另一方面以延长移植皮肤存活为代表，对细胞因子基因治疗等有效方式进行了试验。BALB/c小鼠和B6 D2 F1小鼠皮肤移植后，分别于移植后24小时和21天，将LIF质粒cDNA注射到皮肤移植供者体内，观察LIF的表达情况。LIF的信号转导成分gp 130与LIF一起沿着表达。B6 D2 F1与BALB/c同种异体皮肤移植后，可检测到IL-10的表达，提示LIF基因治疗对同种异体皮肤移植具有免疫耐受性。LIF通过激活骨细胞有效地促进骨形成。将成骨细胞因子（bFGF和BMP-2）和人骨髓间充质干细胞联合应用于颅骨缺损，在海绵载体下显示出显著的骨再生。

In order to further analyze the mechanisms and involvement of the cytokine signaling in the bone regeneration, first human keloid-derived fibroblast primary cultures were tested for the' possible resistance against induced apoptosis by ceramide. Ceramide normally induces the fibroblast apoptosis in dose-dependent and in the-dependent Banner; however, fibroblasts derived from keloid showed the G2 arrest by FACS cell sorter and confined by reduced cell number by WST-t assay. When IGF-1 added, keloid fibroblasts resist the ceramide-induced apoptosis. This was positively correlated with IGF-1 receptor expression level. PI-3 inhibitor. Wortmaninn pre-treatment, was markedly abolished inhibition of apoptosis via IGF-1 signaling. In this experiment, the IGF-1 involves in the subsequent signaling via its proper receptor and the molecularly regulated.On the other hand, on behalf of prolonged skin allograft survival, the effective modalities such as cytokine gene therapy were tested. In between BALB/c and B6D2F1 strain mice Leukemia Inhibitory Factor, LIF plasmid CDNA was injected into the skin allograft donor, LIF expressions were observed in 24 hours and 21 days post-transplantation. The gp130, signal-transduction component of LIF, was expressed along with LIF. When B6D2F1 to BALB/c skin allograftIng, helper T type2 cytokine (IL-10) was observed, thus immune tolerance was indicated for skin allografting in LIF gene therapy.In bone metabolism, LIF was tested in the cranial bone defect model. LIF was efficiently enhanced the bone formation via osteocyte activation. In combined use of the osteogenic cytokioea (bFGF and BMP-2) and human mesenchymal stem cells in the cranial bone defects demonstrated the significant boneregeneration with sponge carriers underneath.