Construction Of Leptin Gene Eukaryotic Expression Vector And Its Transfection And Expression In Human Placenta-Derived Mesenchymal Stem Cells

The tissue defect caused by maxillofacial tumors surgery was usually repairedby flap transplantation.After the resection of the malignant tumor, chemotherapy andradiation therapy were usually used. This would cause skin flap secondary radiationdamage and delayed healing，the refractory wounds and the skin flap necrosis whichwere caused by chemotherapy and radiation therapy was the typical one kind ofwounds combined with radiation injury.One study suggested that leptin and human placenta-derived mesenchymal stemcells(HPMSCs) played an important role in the process of promoting woundhealing.Research shows,leptin plays a very important role in wound healing in thestage of inflammatory reaction, the hyperplasia of granulation tissue, angiogenesis,the epithelial regeneration, the contraction of wound and T lymphocyte theproliferation of T lymphocyte etc,so it may become a new type of drug forregulating and improving wound healing and trauma metabolism, but the half-life ofleptin is very short, only (9.4±3.0)minutes, so it is difficult to play a long-time rolein improving wound healing by local simple application. So we considered thatwhether it could be transfected into stem cells so as to get continuously expression,andthen the leptin could play a long-time role in improving wound healing. HPMSCs hasbecome a " new star" in the field of stem cell by the advantages of adequate source,safe and reliable,hematopoietic support, the characteristics of immune suppression,thenegative of teratogenic carcinoma,the wider range of multidirectional differentiation,application prospect of industrialization and other things.The purpose of this study wasto transfect leptin into HPMSCs, one side we hoped that it could get correctmodification and continuous high expression, on the other side,HPMSCs transfectedwith leptin could still keep the ability of multidirectional differentiation,which would lay a solid foundation for the subsequent research of promoting wound healing byHPMSCs transfected with leptin.We first separated and cultured the HPMSCs from full-term placenta amnioticmembrane, we separated and cultured the HPMSCs from human full-term placentaamniotic membrane through collagenase II digestion，verified proliferation ability ofthe HPMSCs by counting living cells，and drew its growth curve;we identified theinduced HPMSCs by oil red O.The results confirmed that the HPMSCs separated andcultured human full-term placenta amniotic membrane were spindle adherent cells withhigh proliferation ability and strong expression of CD29, CD105and CD73, withoutCD34;the HPMSCs could differentiate into osteoblast and adipose cell after it beinduced,through be dyed by oil red O and alizarin red respectively,the result werepositive.In this experiment we designed primer according to leptin gene sequence,extracted and amplified human leptin gene from fat tissue, inserted the leptin gene intothe eukaryotic expression vector pIRES2-EGFP, and got the recombinant plasmidpIRES2-EGFP-LEP, used restriction enzyme to identify the recombinant plasmid bydouble enzyme digestion.After identification we transfected the recombinant plasmid into HPMSCs,appraised its expression through reverse transcription-polymerase chainreaction(RT-PCR) and Western blotting, and identified multipotential differentiation ofthe HPMSCs had been transfected.The results confirmed: after RT-PCR amplification we got the specific fragmentlength for500bp or so, and after double enzyme cutting the recombinant plasmiddisplayed two segments about5.3KB and500bp, it showed that leptin gene hadalready been inserted into the eukaryotic expression vector pIRES2-EGFP, and therecombinant plasmid had been built successfully.The HPMSCs identified by RT-PCR and Western blotting after being transfectedshowed that the expression of leptin gene was positive,and the HPMSCs induced by osteoblast and adipose cell induced liquid were dyed by oil red O and alizarin redrespectively,the results were positive.This suggested that leptin gene was transfected into the HPMSCs successfullyand won the expression, at the same time after transfection the HPMSCs coulddifferentiate into osteoblast and adipose cell successfully, this confirmed the HPMSCsstill keep the multipotential differentiation, which kept the ability to promote woundhealing.Conclusions:1.Through enzyme digestion we could successfully eparated,screened,amplified,trained and identified the human placenta-derived mesenchymal stem cells in vitro,thismethod was convenient and feasible,by what we could got high purity and highproliferation ability HPMSCs.2.The enzyme identification of pIRES2-EGFP-LEP confirmed that we constructedthe pIRES2-EGFP-LEP with leptin gene successfully;the recombinant plasmidcontained enhanced green fluorescent genes which was conducive for screening andobservation after HPMSCs being transfected.3.Through the method of liposome we transfected leptin gene into HPMSCssuccessfully,which confirmed that we established stable transgenic expression systemof HPMSCs in vitro,it laid a foundation for subsequent animal experiment.4.HPMSCs transfected still keep the Multidirectional differentiation ability，and itconfirmed that the HPMSCs transfected still have the ability to promote woundhealing.