降钙素相关肽对骨髓间充质干细胞生物学特征影响的实验研究

Experimental research on the effects of calcitonin gene related peptide on biological characteristics of bone marrow stem cells

目的 观察携带降钙素基因相关肽（CGRP）的慢病毒对细胞生存和分化的影响. 方法 培养大鼠骨髓间充质干细胞（MSCs）,携带CGRP的慢病毒载体转染MSCs（MSCsCGRP＋＋组）,空病毒载体转染MSCs作为对照组;应用流式细胞仪检测病毒转染率,酶联免疫吸附（ELISA）法测定CGRP蛋白表达,应用流式细胞仪及细胞免疫组化检测细胞表面标记物,胎盘蓝染色观察细胞存活能力,噻唑蓝（MTT）法检测细胞活力,β-半乳糖苷酶染色检测细胞衰老情况. 结果 携带CGRP的慢病毒转染MSCs后1d,荧光显微镜未观察到细胞有荧光,但ELISA测定MSCsCGRP＋ ＋组有少量CGRP表达,随着转染时间延长,细胞逐渐出现荧光,慢病毒转染MSCs后3d和4d,镜下可见强荧光表达,细胞转染率分别为（77.87％和79.58％）,ELISA检测MSCsCGRP＋＋组CGRP表达量较对照组[（19.53±0.50） pg/ml和（3.12±0.001） pg/ml]增加（t=48.964,P＜0.01）.慢病毒转染MSCs后3d,流式细胞仪检测MSCs仍高表达CD29和CD90,CD31的表达为24.75％,细胞转染后7d,CD31的表达为20.72％,与对照组6.63％比较CD31表达增加,vWF表达也随着转染时间延长表达逐渐增加;细胞转染后3d,MSCsCGRP＋/＋组及对照组均未见α-SMA表达,细胞转染14 d,在MSCsCGRP＋＋组α SMA染色仍阴性,对照组胞浆见α-SMA阳性表达;细胞转染后3d和7d,MTT及台盼蓝染色结果显示两组间细胞活力及存活情况均无差异性（3d细胞活力t=-0.253,3d细胞存活率t=-0.307,7d细胞活力t=0.290,7d细胞存活率t=-0.690,均P＞0.05）,β半乳糖苷酶检测细胞衰老程度在两组间差异也无统计学意义（3 d t=0.167,7 dt=0.141,均P＞0.05）,随着转染时间延长,细胞转染14d,MSCsCGRP＋＋组衰老程度较对照组降低（t=2.446,P＜0.05）. 结论 转染慢病毒后细胞生物活性无明显影响,细胞仍具有增殖分化能力,细胞转染分泌的CGRP能促进细胞向内皮分化,抑制细胞向平滑肌细胞分化,这为基因工程细胞疗法治疗血管增殖性疾病提供新的思路和理论及实验依据.

Objective To study the effect of the recombinant Lentivirus containing calcitonin gene related peptide （CGRP） gene on cells biological activity and differentiation of rat bone marrow stem cells（MSCs）.Methods Rat MSCs were isolated and cultured by granulocytes adherent.MSCs were transfected with Lenti-EGFP CGRP（MSCsCGRP＋/＋ group）,While MSCs were transfected with Lenti-EGFP as control group.Cell transfection rate was detected by flow cytometry,protein secretion in the above-mentioned MSCsCGRP＋ ＋ supernatant was detected using ELISA method.Cells surface markers weare detected by flow cytometry and immunohistochemistry.Trypan blue was used to examin the survive rate,β galactosidase staining was used to examin aging of MSCs transfection,and MTT was used to examine cell vitality.Results At first day after transfecting with Lenti-EGFP-CGRP,fluorescence was not observed by fluorescence microscope,but a small amount of CGRP protein was detected by ELISA in MSCsCGRP＋/＋ group,at 3 days and 4 days after transfecting with MSCs,strong fluorescence was observed by fluorescence microscope （the cell transfection rates were 77.87% and 79.58%）.The CGRP expression was significantly higher in MSCsCGRP＋ ＋ group than in control group [（19.53＆#177;0.50） pg/ml vs.（3.12＆#177;0.00） pg/ml,t=48.964,P〈0.01].At three days after transfection with MSCs,CD29 and CD90 expression were significantly higher,as compared with control group,CD31 expression was increased in MSCsCGRP＋ /＋ group.Seven days after transfection with MSCs,CD31 expression was significantly increased in MSCsCGRP＋ ＋ group,vWF expression was significantly increased in MSCsCGRP＋ ＋ group after MSCs were transfected with LentiEGFP CGRP for 14 days,but a SMA expression was decreased in MSCsCGRP＋ ＋group.At 3 days and 7 days after transfection with Lenti-EGFP-CGRP,the proliferation,survive and aging showed no difference in MSCsCGRP＋/＋group and in control group （the proliferation of cell：t=0.253,0.290the survive of cell t=-0.307,0.690,all P〉0.05）.At 14 days after transfection with Lenti-EGFP-CGRP,aging of cell were decreased in MSCsCGRP＋ ＋ group as compared with control group （t=2.446,P〈 0.05）.Conclusions After MSCs are transfected with Lenti EGFP-CGRP,biological characteristics of MSCs has no significant effects,there is still proliferation and differentiation activity.Cell secretion of CGRP can promote the endothelial cell differentiation,and inhibit the differentiation to smooth muscle cells.The CGRP modification of MSCs may play a role in the regulation of angiogenesis.