[Effect of overexpressed VEGF165 and TGFβ1 on the regeneration of dentin-like tissue in rat].
OBJECTIVE
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. Supported by Science and Technology Development Foundation of Shandong Province (2010G0020230) and Natural Science Foundation of Shandong Province (Y2006C47).
Department of Endodontics, School of Stomatology, Shandong University, Shandong Provincial Key Laboratory of Oral Biomedicine. Jinan 250012, Shandong Province;Jiangsu Province, China.E-mail:linl_521@126.com.
Lin
L
Yang
Hai-bing
HB
Yang
Cheng
C
Tian
Yan
Y
Wang
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003804:Dentin; D005347:Fibroblasts; D005822:Genetic Vectors; D012333:RNA, Messenger; D051381:Rats; D017208:Rats, Wistar; D012038:Regeneration; D014162:Transfection; D053773:Transforming Growth Factor beta1; D042461:Vascular Endothelial Growth Factor A
D012333:RNA, Messenger; C508962:Tgfb1 protein, rat; D053773:Transforming Growth Factor beta1; D042461:Vascular Endothelial Growth Factor A; C467498:vascular endothelial growth factor A, rat