蛋白酶Corin在骨髓间充质干细胞分化过程中的作用及机制研究
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Methods: we get BM-MSCs from normal fracture patients and identified by flow cytometry. BM-MSCs were differentiated into osteoblasts, adipogenic cells and chondroblasts in different inducer. Corin mRNA expression during the differentiation was analyzed by RT-PCR. We use quantitative real-time PCR to quantitative analyze corin mRNA. Finally, corin protein expression in the chondrogenic differentiation of BM-MSCs was analyzed by immunofluorescence staining.
increased with time. This indicated that corin may be involved in the differentiation
process.
Keywords: corin; BM-MSCs; differentiation; chondroblast ; staining
蛋白酶 Corin 在骨髓间充质干细胞分化过程中的作用及机制研究
中文摘要
（2）细胞迁移实验用于检测 corin 被抑制后的 BM-MSCs 迁移能力。
（3）细胞增殖实验检测 corin 被抑制后的 BM-MSCs 增殖能力。
（4）免疫组织化学方法检测 corin 基因被抑制后 BM-MSCs 成软骨分化能力的变
株病毒沉默效果最好，将这株病毒侵染 BM-MSCs 发现 corin mRNA 抑制了 80%以上，
而对照组 corin mRNA 仅沉默了 5%左右（p<0.01 vs. siCTRL）。
（2）抑制 BM-MSCs 中 corin mRNA 表达对 BM-MSCs 细胞的迁移及ofluorescence staining.
Conclusions:
The primary bone marrow mesenchymal stem cells were grow well, this BM-MSC
were passaged genetic stability.
BM-MSCs have strong differentiation characteristics, then it can be used as an ideal
BM-MSCs 向成软骨细胞诱导，检测软骨特异性蛋白及基因，发现其表达量均降低，
这说明 corin 基因被抑制后，BM-MSCs 成软骨分化能力减弱，由此，我们推断出 corin
参与了 BM-MSCs 的成软骨分化过程，这为临床疾病如骨关节炎等的治疗提供了一个
很好的入口。
关键词：Corin；慢病毒；基因沉默；成软骨分化
Results: We collect bone marrow from 8 normal fracture patients. All of that bone marrow was cultured primary BM-MSCs. Primary cells arranged in a helical or spiral shape.This BM-MSCs expressed CD44, CD73 and CD90 without expression of CD34 by flow cytometry detection. The 3 type of differentiation were explored by Alizarin Red staining, oil red O staining and Alcian blue staining, and we observed the specific substance formation.
Methods: We use calcium phosphate precipitation to prepared lentiviral, for inhibiting the expression of corin mRNA. Then filter the optimal lentiviral by qRT-PCR and Western blot. Transwell Migration Assay and CCK-8 were used to explore the proliferation and migration. Immunohistochemistry were used to detect the specific protein
Objective: Human corin is a type II transmembrane serine protease discovered in the heart. Corin plays an important role in the regulation of blood pressure and heart function. Corin mRNA and protein were also detected in other tissues such as kidney, bone and pregnant uterus. Recent studies have found that corin mRNA also expression in bone marrow mesenchymal stem cells （BM-MSCs）. To further explore the function of corin in BM-MSCs. In this study, we detect the corin gene and protein expression in the process of BM-MSCs differentiation. Aims to investigate the role of corin in BM-MSCs differentiation. For further study of the function of the type II transmembrane serine protease and provide the basis for the role of corin act in bone marrow cell metabolism.
V
英文摘要
蛋白酶 Corin 在骨髓间充质干细胞分化过程中的作用及机制研究
during the process of chondrogenic differentiation.
Results:
To detection corin mRNA in which HEK293-corin cells were infected by lentiviral,
Part II. The study of corin expression and function in the
chondrogenic differentiation of BM-MSCs
Objective: Osteoarthritis is one of the most common chronic diseases in the elderly. Its pathogenesis based on the characterized of cartilage chronic injury. Our previous study shows that corin mRNA expression was significantly increased during BM-MSCs differentiation into chondrocytes proceess. In order to investigate the relationship between corin with BM-MSCs chondrogenic differentiation, we silencing corin gene used RNA interference technology. To observe the changes in BM-MSC differentiation into chondrocytes process, thus inferred of the relationship between corin with MSC differentiation.
IV
蛋白酶 Corin 在骨髓间充质干细胞分化过程中的作用及机制研究
英文摘要
We found corin mRNA expression in the process of 3 type differentiation, the
expression of corin is gradually increased with time （p<0.01）.
作 者：祝劲松 指导老师：周海斌
III
英文摘要
蛋白酶 Corin 在骨髓间充质干细胞分化过程中的作用及机制研究
Role of corin in bone marrow mesenchymal stem cell differentiation Abstract
Part I. The expression of corin in bone marrow mesenchymal stem cell differentiation
cell in cell engineering.
We used RT-PCR and immunofluorescence detected corin expression during
chondrogenic differentiation, and we also found the expression of corin is gradually
（3）抑制 BM-MSCs 中 corin mRNA 表达影响了 BM-MSCs 向成软骨分化，检测
软骨特异性的硫酸化多糖和 II 型胶原蛋白的表达，发现其形成量明显减少，IPP 软件
分析 siCORIN 组和 siCTRL 组中图片蓝色及棕色区域面积，发现其面积均明显减少（硫
酸化多糖面积：18.9% vs. 60.6%, p<0.01; II 型胶原蛋白面积：11.4% vs. 39.5%, p<0.05）。
We observed the highest corin mRNA expression at 4 week during chondrogenic
differentiation. We also detect corin protein at 4 week after chondrogenic differentiation by
化，并用 Image Pro-Plus 软件分析 IHC 图片中阳性染色区域的平均光密度，评价正常
BM-MSCs 及干扰后 BM-MSCs 诱导后特异性指标的表达水平。
结果：
（1）利用 HEK293-corin 细胞筛选最佳病毒株，通过 qRT-PCR 和 Western blot 技
术检测病毒侵染后的 HEK293-corin 细胞中 corin mRNA 的表达，发现第 V2LHS_12562
同时通过 qRT-PCR 法检测软骨特异性基因 COL2A1 和 COMP 的表达，发现其表达量
也明显降低（*p<0.05；**p<0.01 vs. siCTRL）。
结论：
我们通过 RNA 干扰技术成功的将 BM-MSCs 中 corin 基因沉默，并且 corin 基因
沉默后的 BM-MSCs 细胞增殖及迁移能力未受明显影响，我们将 corin 表达下调的
increased with time. This indicated that corin may be involved in the differentiation
process.
Keywords: corin; BM-MSCs; differentiation; chondroblast ; staining
蛋白酶 Corin 在骨髓间充质干细胞分化过程中的作用及机制研究
中文摘要
（2）细胞迁移实验用于检测 corin 被抑制后的 BM-MSCs 迁移能力。
（3）细胞增殖实验检测 corin 被抑制后的 BM-MSCs 增殖能力。
（4）免疫组织化学方法检测 corin 基因被抑制后 BM-MSCs 成软骨分化能力的变
株病毒沉默效果最好，将这株病毒侵染 BM-MSCs 发现 corin mRNA 抑制了 80%以上，
而对照组 corin mRNA 仅沉默了 5%左右（p<0.01 vs. siCTRL）。
（2）抑制 BM-MSCs 中 corin mRNA 表达对 BM-MSCs 细胞的迁移及ofluorescence staining.
Conclusions:
The primary bone marrow mesenchymal stem cells were grow well, this BM-MSC
were passaged genetic stability.
BM-MSCs have strong differentiation characteristics, then it can be used as an ideal
BM-MSCs 向成软骨细胞诱导，检测软骨特异性蛋白及基因，发现其表达量均降低，
这说明 corin 基因被抑制后，BM-MSCs 成软骨分化能力减弱，由此，我们推断出 corin
参与了 BM-MSCs 的成软骨分化过程，这为临床疾病如骨关节炎等的治疗提供了一个
很好的入口。
关键词：Corin；慢病毒；基因沉默；成软骨分化
Results: We collect bone marrow from 8 normal fracture patients. All of that bone marrow was cultured primary BM-MSCs. Primary cells arranged in a helical or spiral shape.This BM-MSCs expressed CD44, CD73 and CD90 without expression of CD34 by flow cytometry detection. The 3 type of differentiation were explored by Alizarin Red staining, oil red O staining and Alcian blue staining, and we observed the specific substance formation.
Methods: We use calcium phosphate precipitation to prepared lentiviral, for inhibiting the expression of corin mRNA. Then filter the optimal lentiviral by qRT-PCR and Western blot. Transwell Migration Assay and CCK-8 were used to explore the proliferation and migration. Immunohistochemistry were used to detect the specific protein
Objective: Human corin is a type II transmembrane serine protease discovered in the heart. Corin plays an important role in the regulation of blood pressure and heart function. Corin mRNA and protein were also detected in other tissues such as kidney, bone and pregnant uterus. Recent studies have found that corin mRNA also expression in bone marrow mesenchymal stem cells （BM-MSCs）. To further explore the function of corin in BM-MSCs. In this study, we detect the corin gene and protein expression in the process of BM-MSCs differentiation. Aims to investigate the role of corin in BM-MSCs differentiation. For further study of the function of the type II transmembrane serine protease and provide the basis for the role of corin act in bone marrow cell metabolism.
V
英文摘要
蛋白酶 Corin 在骨髓间充质干细胞分化过程中的作用及机制研究
during the process of chondrogenic differentiation.
Results:
To detection corin mRNA in which HEK293-corin cells were infected by lentiviral,
Part II. The study of corin expression and function in the
chondrogenic differentiation of BM-MSCs
Objective: Osteoarthritis is one of the most common chronic diseases in the elderly. Its pathogenesis based on the characterized of cartilage chronic injury. Our previous study shows that corin mRNA expression was significantly increased during BM-MSCs differentiation into chondrocytes proceess. In order to investigate the relationship between corin with BM-MSCs chondrogenic differentiation, we silencing corin gene used RNA interference technology. To observe the changes in BM-MSC differentiation into chondrocytes process, thus inferred of the relationship between corin with MSC differentiation.
IV
蛋白酶 Corin 在骨髓间充质干细胞分化过程中的作用及机制研究
英文摘要
We found corin mRNA expression in the process of 3 type differentiation, the
expression of corin is gradually increased with time （p<0.01）.
作 者：祝劲松 指导老师：周海斌
III
英文摘要
蛋白酶 Corin 在骨髓间充质干细胞分化过程中的作用及机制研究
Role of corin in bone marrow mesenchymal stem cell differentiation Abstract
Part I. The expression of corin in bone marrow mesenchymal stem cell differentiation
cell in cell engineering.
We used RT-PCR and immunofluorescence detected corin expression during
chondrogenic differentiation, and we also found the expression of corin is gradually
（3）抑制 BM-MSCs 中 corin mRNA 表达影响了 BM-MSCs 向成软骨分化，检测
软骨特异性的硫酸化多糖和 II 型胶原蛋白的表达，发现其形成量明显减少，IPP 软件
分析 siCORIN 组和 siCTRL 组中图片蓝色及棕色区域面积，发现其面积均明显减少（硫
酸化多糖面积：18.9% vs. 60.6%, p<0.01; II 型胶原蛋白面积：11.4% vs. 39.5%, p<0.05）。
We observed the highest corin mRNA expression at 4 week during chondrogenic
differentiation. We also detect corin protein at 4 week after chondrogenic differentiation by
化，并用 Image Pro-Plus 软件分析 IHC 图片中阳性染色区域的平均光密度，评价正常
BM-MSCs 及干扰后 BM-MSCs 诱导后特异性指标的表达水平。
结果：
（1）利用 HEK293-corin 细胞筛选最佳病毒株，通过 qRT-PCR 和 Western blot 技
术检测病毒侵染后的 HEK293-corin 细胞中 corin mRNA 的表达，发现第 V2LHS_12562
同时通过 qRT-PCR 法检测软骨特异性基因 COL2A1 和 COMP 的表达，发现其表达量
也明显降低（*p<0.05；**p<0.01 vs. siCTRL）。
结论：
我们通过 RNA 干扰技术成功的将 BM-MSCs 中 corin 基因沉默，并且 corin 基因
沉默后的 BM-MSCs 细胞增殖及迁移能力未受明显影响，我们将 corin 表达下调的