过表达VEGF165和TGFβ1对鼠牙本质样组织再生的影响

上海口腔医学 ›› 2013, Vol. 22 ›› Issue (6): 649-654.

过表达VEGF165和TGFβ1对鼠牙本质样组织再生的影响

杨琳¹，杨海兵²，田成¹，王燕¹

(1.山东省口腔医院牙体牙髓病科，山东省口腔生物医学重点实验室，山东济南 250012；2.江苏省常州市第二人民医院口腔科, 江苏常州 213003)

收稿日期:2013-03-08 修回日期:2013-05-20 出版日期:2013-04-12 发布日期:2013-04-12
通讯作者: 王燕，Tel: 0531-88382623，E-mail:wangyan1965@sdu.edu.cn
作者简介:杨琳(1988-)，女，硕士，E-mail:linl_521@126.com
基金资助:
山东省科技发展计划项目(2010G0020230)；山东省自然科学基金(Y2006C47)

Effect of overexpressed VEGF165 and TGFβ1 on the regeneration of dentin-like tissue in rat

YANG Lin¹, YANG Hai-bing², TIAN Cheng¹, WANG Yan¹

1.Department of Endodontics, School of Stomatology, Shandong University, Shandong Provincial Key Laboratory of Oral Biomedicine. Jinan 250012, Shandong Province; 2. Department of Stomatology, Changzhou Second People’s Hospital. Changzhou 213003, Jiangsu Province, China

Received:2013-03-08 Revised:2013-05-20 Online:2013-04-12 Published:2013-04-12
Supported by:
Supported by Science and Technology Development Foundation of Shandong Province (2010G0020230) and Natural Science Foundation of Shandong Province (Y2006C47).

摘要/Abstract

摘要： 目的：探讨重组质粒pcDNA3.1hisA-VEGF165和pcDNA3.1hisA-TGFβ1稳定转染仓鼠卵巢细胞(CHO)后持续分泌的血管内皮细胞生长因子165(VEGF165)和转化生长因子β1 (transforming growth factor，TGFβ1)蛋白对大鼠体内牙本质样组织形成的影响。方法：脂质体介导重组质粒pcDNA3.1hisA-VEGF165和pcDNA3.1hisA-TGFβ1转染CHO细胞，经G418筛选，建立单克隆细胞株。反转录聚合酶链反应(RT-PCR)检测转染后CHO细胞中VEGF165和TGFβ1 mRNA的表达情况，酶联免疫吸附测定(ELISA)检测转染后上清中VEGF165和TGFβ1蛋白的表达水平。接种转染后的CHO细胞至胶原膜，选择24只Wistar大鼠双侧上颌第一磨牙为实验牙，将胶原膜置于人工穿髓孔处，ChemFlex玻璃离子充填窝洞。8周后处死动物取材，制备硬组织切片，甲苯胺蓝染色。采用SPSS17.0软件包对ELISA数据进行统计学分析。结果：RT-PCR和ELISA结果显示， CHO细胞转染后VEGF165和TGFβ1基因和蛋白稳定表达。甲苯胺蓝染色显示，转染pcDNA3.1hisA-VEGF165的CHO组中穿髓孔下方见毛细血管明显充血及炎症细胞浸润，未见硬组织形成；转染pcDNA3.1hisA-TGFβ1的CHO组中，穿髓孔下方可见少量深染矿化团块，周围见增生的牙髓成纤维细胞，偶见炎症细胞浸润，未见管状牙本质形成；转染pcDNA3.1hisA-VEGF165和pcDNA3.1hisA-TGFβ1的2种CHO组中，穿髓孔下方可见大量深染矿化团块，几乎完全封闭穿髓孔，未见结构规则的牙本质桥形成，团块下端成牙本质细胞呈柱状整齐排列；对照组未见硬组织形成。结论：TGFβ1能促进体内矿化组织形成，VEGF165和TGFβ1共同作用，能更好地促进体内矿化组织形成。

关键词: 血管内皮细胞生长因子, 转化生长因子β1, 转染, 矿化作用

Abstract: PURPOSE: To transfect recombinant vectors pcDNA3.1hisA-VEGF165 and pcDNA3.1hisA-TGFβ1 in Chinese hamster ovary cell (CHO) and investigate the effect of released VEGF165 and TGFβ1 proteins by CHO on the regeneration of dentin-like tissues. METHODS: The recombinant plasmids pcDNA3.1hisA-VEGF165 and pcDNA3.1hisA-TGFβ1 were transfected in CHO via liposome. After screen culture by G418，stable transfected CHO cell line was established. The levels of VEGF165 and TGFβ1 were evaluated by RT - PCR and ELISA. Then the cells were seeded on collagen membranes. The bilateral maxillary first molars of 24 Wistar rats were selected as experimental teeth and the collagen membranes were separately planted over the holes of artificial dental pulp exposure. The cavities were filled with ChemFlex finally. After 8 weeks, specimens from 24 rats were collected and dyed with toluidine blue. Statistical analysis was performed with SPSS 17.0 software package. RESULTS: The results of RT-PCR and ELISA showed that CHO stably expressed VEGF165 and TGFβ1 mRNAs and proteins after transfection and selection. The result of toluidine blue staining showed that in the group of CHO transfected with pcDNA3.1hisA-VEGF165, the blood capillaries were congestive and inflammatory cells infiltrated obviously under the mechanically exposed pulpal site, but no hard tissue regenerated. In the group of CHO transfected with pcDNA3.1hisA-TGFβ1, a spot of colored mineralization pellets under the mechanically exposed pulpal site surrounded with hyperplastic fibroblast were observed, no tubular dentin and sporadic inflammatory cells were detected. In the group of CHO transfected with pcDNA3.1hisA-VEGF165 and pcDNA3.1hisA-TGFβ1, generous colored mineralization pellets almost closed the mechanically exposed pulpal site and columnar odontoblast were arranged orderly, no regular dentin bridge was detected. The hard tissue was not detected in the control group. CONCLUSIONS: TGFβ1 could promote the formation of mineralization pellets in vivo, and VEGF165 and TGFβ1 could promote the formation of mineralization pellets better.

Key words: Vascular endothelial growth factor, Transforming growth factor-beta 1, Transfection, Mineralization

中图分类号:

R781

杨琳, 杨海兵, 田成, 王燕. 过表达VEGF165和TGFβ1对鼠牙本质样组织再生的影响[J]. 上海口腔医学, 2013, 22(6): 649-654.

YANG Lin, YANG Hai-bing, TIAN Cheng, WANG Yan. Effect of overexpressed VEGF165 and TGFβ1 on the regeneration of dentin-like tissue in rat[J]. Shanghai Journal of Stomatology, 2013, 22(6): 649-654.

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