Construction Of PDX-1 Eukaryotic Expression Vector And Its Expression In Rat Bone Marrow Mesenchymal Stem Cells

Backgroud and Objective:The current conventional treatment of diabetes can not control all levels of blood glucose ideally, islet cell transplantation to cure diabetes has caused many scholars'concerning. However, the problems of scarcity of insulin-producing cells and immune rejection have limited the widespread clinical application of allogeneic islet cell transplantation.Bone marrow mesenchymal stem cells are a class of non-hematopoietic stem cells, which are present in adult marrow of high self-renewal and differentiation potential. Under the proper conditions, MSCs can differentiate into endoderm cells, such as pancreatic islet-like cells, fat cells and so on.MSCs derived convenient, easy isolation and culture, highly expansion capacity in vitro, stable genetic performance, ease of transfection and stable expression of foreign genes and autologous transfusion to avoid immune rejection. There is no ethical and moral issues like human embryonic stem cells faces. It is considered the ideal cells and seed cells for tissue engineering, gradually become a new source to find islet-like cells.Pancreatic duodenal homeobox-1(PDX-1) in human is also known as insulin promoter factor-1, which is a transcription factor expressed in the early stage of pancreas development. It not only for the dorsal and ventral mesoderm intestinal embryonic pancreas, plays an important role in growth and differentiation, also involved in islet development and maturation process of cell differentiation.In this study, we construct the eukaryotic expression vectors which containing PDX-1 gene, by technique of lipofectin transfecting, transfected the recombinant expression vectors into BMSCs. Then we screened the stable expressing cell lines, making preparations for the follow-up study to differentiate BMSCs into pancreatic islet-like cells and transplant for curing diabetes.Methods:1 Clone the cDNA of PDX-1 gene and constracte of the recombinant eukaryotic vectors1.1 A pairs of primers were designed according to PDX-1 gene sequences of GenBank database, RT-PCR, the PCR product was sequenced and checked through Blast tools after cloned into the pMD18-T vectors (Takara, P.R. China).1.2 The constraction of the recombinant eukaryotic vectorsAfter the fragment of PDX-1 was inserted into the Hindâ…¢-BamHI restriction sites of the eukaryotic expression vectors pEGFP-Cl and pcDNA3.1(+) (Invitrogen, USA) respectively, the recombinant eukaryotic vectors were identified by digestion and sequencing, and named the correct vectors pEGFP-Cl-PDX-1 and pcDNA3.1 (+)-PDX-1 respectively.2. In vitro culture of rat BMSCs and screen the stable transfected cells containing PDX-12.1 Adherent culture using whole bone marrow isolated, primary cultured BMSCs2.2 BMSCs surface marker identification and determination of growth curve2.3 Cell transfectionThe recombinant vectors were transfected into rat BMSCs by LipofectamineTM 2000.2.4 Cellular localization of EGFP-PDX-1 fusion proteinThe location of EGFP-PDX-1 proteins was determined by fluorescent microscope in 24 hour after transfection.2.5 Screening of the stably transfected pcDNA3.1 (+)-PDX-1 cells line by G418. RT-PCR was used to detect the expressions of PDX-1 gene before and after transfection.Results:1. Identification of total RNAThe purity and concentration of total RNA were assessed by spectrophotometry and the absobance ratio of A260/A280 was 1.85. The integrity of the RNA was verified by agarose gel electrophoresis and the luminance ratio of 28S rRNA to 18S rRNA was about 2:1. Therefore, the total RNA met the requirements of RT-PCR. Sequences analysis of PCR product showed that the cloned fragment was about 883 bp in length, which was consistent with the sequences submitted in GenBank. By Hindâ…¢and BamHI restriction enzyme digestion and DNA sequencing analysis, recombinant vector containing the size, orientation and reading frame were correct inserts.2. Isolation and primary culture rat BMSCs. Immunofluorescence showing CD29, CD44 positive, CD34 negative expression, consistent with characteristics of BMSCs. Cellular localization of the PDX-1 gene of rat BMSCs:after transfection with empty vector pEGFP-C1, cytoplasm and nucleus of BMSCs appeared green fluorescent under the high-power fluorescent microscope. However, green fluorescent could be mainly observed in cell nucleus in the group of BMSCs which transfected with pEGFP-C1-PDX-1.3. Screening and identification of the stable transfected cellsThe results of RT-PCR showed that in all three separate experiments the expression of the PDX-1 gene was only detected in the BMSCs transfected with pcDNA3.1(+)-PDX-1, which testified PDX-1 gene was successfully transferred into BMSCs and obtained the stable transfected cell line of pcDNA3.1 (+)-PDX-1.Conclusion:1. This study cloned mouse PDX-1 gene fragment and constructed the recombinant eukaryotic expression vectors.2. Through the fusion of green fluorescent protein eukaryotic expression vector transfection, proved the PDX-1 gene is located in the nucleus of the transfected BMSCs, screened the stable transfected cells of pcDNA3.1(+)-PDX-1, which make preparation for the further inducing its differentiate into islet-like cells, and to study its role in insulin regulation and make foundation for the transplantation of curing diabetes.