Chinese Journal of Tissue Engineering Research ›› 2020, Vol. 24 ›› Issue (23): 3627-3635.doi: 10.3969/j.issn.2095-4344.2661
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Xue Junjie1, Li Jingyu2, Zhang Li1, Ren Chaochao1
Received:
2019-09-18
Revised:
2019-09-20
Accepted:
2019-10-26
Online:
2020-08-18
Published:
2020-04-25
Contact:
Ren Chaochao, MD, Associate chief physician, Department of Orthodontics, Beijing Stomatological Hospital, Capital Medical University, Beijing 100050, China
About author:
Xue Junjie, MD, Attending physician, Department of Orthodontics, Beijing Stomatological Hospital, Capital Medical University, Beijing 100050, China
Supported by:
CLC Number:
Xue Junjie, Li Jingyu, Zhang Li, Ren Chaochao. Expression of connexin 43 in cartilage and chondrocyte of osteoarthritis and construction of shRNA lentiviral vector targeting connexin 43[J]. Chinese Journal of Tissue Engineering Research, 2020, 24(23): 3627-3635.
2.1 实验动物数量分析 实验选用C57BL/6小鼠6只,实验过程无脱失,全部进入结果分析。 2.2 骨关节炎动物模型中实验侧关节较对照侧关节有更高的OARSI评分 见图2。镜下可见手术诱导膝骨关节炎4周后,实验侧与对照侧相比,关节软骨层明显变薄,粘合线崎岖不平,局部出现裂隙。根据OARSI对小鼠骨关节炎模型建立后关节炎严重程度的评分体系[10],OARSI评分较高则证明骨关节炎较严重,反之则证明骨关节炎病变程度较轻。对甲苯胺蓝染色后的小鼠双侧关节组织切片进行评分及统计,结果发现对照侧与实验侧关节评分有明显差异,实验侧的评分明显高于对照侧,说明膝关节前交叉韧带切断术可以有效诱导小鼠膝骨关节炎,并出现明显的骨关节炎表型。 "
2.5 Cx43、Cx37、Cx40、Cx45、Cx46在SW1353细胞中的表达 使用RT-PCR检测软骨细胞SW1353细胞株中Cx43、Cx37、Cx40、Cx45、Cx46基因的表达水平,结果显示SW1353细胞中Cx43基因的表达水平较Cx37、Cx40、Cx45、Cx46基因的表达水平高。见图5。实验结果显示:在基因水平上,SW1353细胞中编码Cx43的Gja1基因的表达水平较编码Cx37、Cx40、Cx45、Cx46的基因表达水平高(P < 0.01)。结果表明:与连接蛋白家族的其他缝隙连接蛋白的基因表达水平对比,Cx43在SW1353细胞中的表达水平较高,验证了SW1353细胞中Cx43的表达在连接蛋白家族中具有主导地位。 "
2.6 Cx43-shRNA慢病毒载体构建结果 慢病毒载体的测序鉴定结果显示:阳性克隆测序结果与目标序列完全一致,质粒构建成功。转染293T细胞36 h后,荧光显微镜下观察,质粒转染组各组均可见荧光蛋白表达,4个Cx43-shRNA慢病毒质粒Cx43-shRNA-1,Cx43-shRNA-2,Cx43-shRNA-3,Cx43-shRNA-4中,Cx43-shRNA-1荧光表达率最高,见图6。将转染36 h后的细胞进行裂解,获取RNA,反转录成cDNA,通过RT-PCR分析Cx43的相对表达量。相对于空载对照组Cx43-CON,其他组Cx43的表达量均明显下降,Cx43-shRNA-1组的Cx43表达量最低,表明Cx43-shRNA-1的抑制效率最高,其抑制率>75%,见图7。 "
2.7 SW1353细胞稳转细胞株的筛选及鉴定结果 用转染后抑制效率最高Cx43-shRNA-1慢病毒质粒转染SW1353细胞。转染48 h后,未转染的SW1353作为空白组,对照组和实验组分别取自复苏后的6株SW1353细胞样本,并在转染前后做自身对照,对照组为:SW1353-sh-con-1,SW1353-sh-con-2,SW1353-sh-con-3,SW1353-sh-con-4,SW1353-sh-con-5,SW1353-sh-con-6,感染了Cx43-shRNA-1慢病毒载体的SW1353细胞作为实验组:SW1353-sh-1-1,SW1353-sh-1-2,SW1353-sh-1-3,SW1353-sh-1-4,SW1353-sh-1-5,SW1353-sh-1-6。对上述细胞进行裂解,获取RNA,反转录成cDNA,通过RT-PCR分析Cx43在各组细胞中的的相对表达量,见图8;SW1353细胞稳转细胞株中Cx43蛋白的表达见图9。根据结果筛选出良好的稳转株为后续研究做准备。 "
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