KLD-12多肽/重组人BMP-2凝胶诱导兔BMSCs成骨活性的实验研究

EXPERIMENTAL STUDY ON OSTEOGENIC ACTIVITY OF RABBIT BONE MARROW MESENCHYMAL STEM CELLS INDUCED BY KLD-12 POLYPEPTIDE/RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN 2 GEL

目的探讨KLD-12多肽复合重组人BMP-2（recombinant human BMP-2, rhBMP-2）诱导兔BMSCs成骨活性的影响。方法取3月龄新西兰大白兔骨髓，采用密度梯度法分离培养BMSCs。取第3代BMSCs分别采用KLD-12多肽／rhBMP-2凝胶三维培养（实验组）和KLD-12多肽凝胶培养（对照组）。培养7d倒置相差显微镜下观察实验组细胞在凝胶内的形态；3、7、10、14、21d检测两组细胞培养基中ALP及骨钙蛋白含量；培养14d两组行I型胶原免疫荧光染色观察，实时荧光定量PCR检测I型胶原和骨钙蛋白基因相对表达量。结果倒置相差显微镜下观察，培养7d实验组及对照组凝胶内BMSCs呈圆形，分布均匀。两组培养3、7d时ALP表达量及骨钙蛋白含量比较差异无统计学意义（P〉0．05），10～21d实验组以上指标均明显高于对照组（P〈0．05）。培养14d。激光共聚焦显微镜观察示，实验组I型胶原免疫荧光染色阳性，且荧光强度较对照组高；实时荧光定量PCR检测实验组I型胶原、骨钙蛋白基因相对表达量均显著高于对照组，比较差异有统计学意义（t=15．902，P=O．000；t=12．998。P=0．000）。结论BMSCs在KLD-12多肽内正常生长并增殖，KLD-12多肽／rhBMP-2凝胶诱导BMSCs向成骨细胞分化的生物活性良好。

Objective To investigate the effect of KLD-12 polypeptide complexed with recombinant human bone morphogenetic protein 2 （rhBMP-2） on osteogenic activity of rabbit bone marrow mesechymal stem cells （BMSCs）. Methods Bone marrow was harvested from 3-month-old New Zealand white rabbit, and density gradient method was used to isolate and culture BMSCs. The third generation BMSCs were used for three-dimensional culture of KLD- 12 polypetide/rhBMP-2 in vitro （experimental group） and KLD-12 polypeptide （control group）. The morphology of the cells in the gel was observed by inverted phase contrast microscope at 7 days; alkaline phosphatase （ALP） and osteocalcin protein content were dectected at 3, 7, 10, 14, and 21 days; collagen type I immunofluorescence staining was done and real- time fuorescent quantitative PCR was performed to detect the relative expression of collagen type I and osteocalcin gene at 14 days. Results Under the inverted phase contrast microscope, the BMSCs in the gel of the experimental group and the control group showed circular growth, and the distribution was uniform at 7 days. There was no significant difference in the expressions of ALP and osteocalcin protein content between 2 groups at 3 and 7 days （P〉0.05）; the above indexes in experimental group were significantly higher than those in the control group at 10-21 days （P〈0.05）. Laser scanning confocal microscope observation showed that immunofluorescence staining for collagen type I was positive in the experimental group, and the expression was higher than that in the control group at 14 days. Real-time fluorescence quantitative PCR detection showed that the collagen type I and osteocalcin gene expressions were significantly higher than those in the control group （t=15.902, P=0.000; t=12.998, P=0.000）. Conclusion BMSCs can normally grow and proliferate in the KLD-12 polypeptide, and KLD-12 polypeptide/rhBMP-2 has good biological activity to induce BMSCs differentiation into osteoblasts.