Transplantation of lentiviral-transfected endothelial progenitor cells for pulmonary hypertension and the interventional effect of astragalosides

doi:10.3969/j.issn.2095-4344.0475

Abstract

Abstract:

BACKGROUND: Activation of CD40 pathway negatively regulates the therapeutic effect of endothelial progenitor cells (EPCs), while inhibition of this pathway can enhance the biological function of the cells.
OBJECTIVE: To compare the therapeutic effects of CD40-silenced EPCs (EPCs shRNA-CD40) and conventional EPCs transplantation in an animal model of pulmonary hypertension, and to explore the interventional effect of astragalosides on EPCs transplantation in the treatment of pulmonary hypertension.
METHODS: Ninety SD rats were randomly divided into five groups: normal control group (n=24), model group (n=24), lentivirus transfection group (n=18), conventional transplantation group (n=18) and astragaloside group (n=6). Except the normal control group, the remaining four groups were given monocrotaline to induce pulmonary hypertension. Rats in the lentivirus transfection and conventional transplantation groups were given intravenous injection of Lv-shRNA-CD40-transfected EPCs and conventional EPCs respectively at 7, 14, 21 days after modeling (n=6 at each time point). Rats in the astragaloside group were given daily intraperitoneal injection of 80 mg/(kg•d) astragaloside within 1-21 days after modeling, and then Lv-shRNA-CD40-transfected EPCs were intravenously injected at 21 days after modeling. Hemodynamics, plasma endothelin-1 level and right ventricular hypertrophy index were detected at 28 days after modeling.
RESULTS AND CONCLUSION: (1) After modeling, right ventricular pressure, mean pulmonary arterial pressure and right ventricular hypertrophy index were all increased compared with the normal control group (P < 0.05), while these indices were then decreased significantly after EPCs transplantation (P < 0.05). With the increasing of transplantation time, there was an increasing trend in the right ventricular pressure, mean pulmonary arterial pressure and right ventricular hypertrophy index in the two EPCs transplantation groups, but this trend was not remarkable in the lentivirus transfection group. (2) After modeling, the level of endothelin-1 was increased significantly compared with the normal control group (P < 0.05), and then decreased after EPCs transplantation (P < 0.05). The level of endothelin-1 in the lentivirus transfection group was significantly lower than that in the conventional transplantation group at the same time point (P < 0.05). (3) A significant improvement in hemodynamics, plasma endothelin-1 level and right ventricular hypertrophy index was observed in the astragaloside group as compared with the lentivirus transfection group (P < 0.05). Given the above findings, CD40-silenced EPCs transplantation is more effective and durable than the conventional transplantation in the treatment of pulmonary hypertension, and moreover, astragaloside can enhance the therapeutic effect.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Hypertension, Pulmonary, Endothelial Cells, Lentivirus Infections, CD40 Ligand, Astragalus membranaceus, Tissue Engineering

CLC Number:

R394.2

Zhou Xiao-xiong, Wei Wei-chao, Sun Ce, Ye Tao-chun, Wang Song, Qing Li-jin, Wu Hui, Xian Shao-xiang. Transplantation of lentiviral-transfected endothelial progenitor cells for pulmonary hypertension and the interventional effect of astragalosides[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(9): 1425-1431.

Figures/Tables 2

2.1 内皮祖细胞的培养与鉴定原代培养48 h后，有少量细胞贴壁，多数为小的圆形细胞。随着培养时间延长，细胞体积增大，呈梭形。培养至第7天时，形成细胞集落，可见部分细胞由梭形变为菱形；荧光显微镜下双染色显示，DiI-Ac-LDL染色后细胞呈红色(图1A)，FITC-UEA-1染色后细胞呈绿色(图1B)，双染细胞叠加呈黄色(图1C)；流式细胞仪检测结果显示，细胞高表达CD133及FIK-1，阳性表达率分别为(68.6±5.9)%，(58.6±7.8)%，以上结果提示实验获得的为早期内皮祖细胞。 2.2 Lv-shRNA-CD40慢病毒载体的转染转染24 h后，荧光显微镜下可见转染成功的细胞发出绿色荧光，转染效率达85%以上；Western Blot检测显示，Lv-shRNA-CD40转染的内皮祖细胞CD40蛋白表达明显低于未转染内皮祖细胞(P < 0.05，图2)；PCR检测结果显示，Lv-shRNA-CD40转染的内皮祖细胞CD40基因表达明显低于未转染内皮祖细胞(P < 0.05，图3)，与Western Blot检测结果相同。 2.3 可溶性CD40L水平野百合碱注射后第7-21天，正常对照组可溶性CD40L水平变化不大，模型组可溶性CD40L水平不断升高，且野百合碱注射后第14，21天的可溶性CD40L水平均高于正常对照组(P < 0.05)，见图4，说明PAH伴随可溶性CD40L水平的升高，CD40通路参与了其发病及发展过程。 2.4 血流动力学检测结果与正常对照组比较，模型组野百合碱注射后的右心室压力及平均肺动脉压均升高(P < 0.05)，见表1，提示造模成功。与模型组比较，慢病毒转染组、常规移植组右心室压力与平均肺动脉压均明显下降(P < 0.05)，说明内皮祖细胞移植具有改善PAH右心室压力及平均肺动脉压的作用。常规移植组组内，随着移植内皮祖细胞时间的延长，右心室压力与平均肺动脉压逐渐升高，野百合碱注射后第7，14，21天移植细胞的大鼠右心室压力、平均肺动脉压比较差异有显著性意义(P < 0.05)，说明常规内皮祖细胞移植效果不稳定。慢病毒转染组组内，随着移植内皮祖细胞时间的延长，虽然右心室压力与平均肺动脉压逐渐升高，但野百合碱注射后第7，14，21天移植细胞的大鼠右心室压力、平均肺动脉压比较差异无显著性意义(P > 0.05)，说明慢病毒转染内皮祖细胞移植效果稳定。慢病毒转染组野百合碱注射后第7，14天移植细胞的大鼠右心室压力与平均肺动脉压均低于常规移植组(P < 0.05)。 2.5 血浆内皮素1水平检测结果与正常对照组比较，模型组大鼠血浆内皮素1水平升高(P < 0.05)；与模型组比较，慢病毒转染组、常规移植组血浆内皮素1水平降低(P < 0.05)；慢病毒转染组相同移植时间点的血浆内皮素1水平低于常规移植组(P < 0.05)，见图5。 2.6 右心室肥大指数检测结果与正常对照组比较，模型组野百合碱注射后的右心室肥大指数均升高(P < 0.05)，见图6。与模型组比较，慢病毒转染组、常规移植组右心室肥大指数均明显下降(P < 0.05)，说明内皮祖细胞移植具有改善右心室肥大指数的作用。常规移植组组内，随着移植内皮祖细胞时间的延长，右心室肥大指数逐渐升高，野百合碱注射后第7，14，21天移植细胞各时间点间比较差异有显著性意义(P < 0.05)，说明常规内皮祖细胞移植效果不稳定。慢病毒转染组组内，随着移植内皮祖细胞时间的延长，虽然右心室肥大指数有升高趋势，但野百合碱注射后第7，14，21天移植细胞各时间点间比较差异无显著性意义(P > 0.05)，说明慢病毒转染内皮祖细胞移植效果稳定。慢病毒转染组野百合碱注射后第7，14天移植细胞的大鼠右心室肥大指数均低于常规移植组(P < 0.05)。 2.7 黄芪总苷的干预效果黄芪总苷组右心室压力、平均肺动脉压、右心室肥大指数及血浆内皮素1水平均低于慢病毒转染组(P < 0.05)，见图7。"

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