Role Of MiR-586 On Odontogenic And Angiogenic Potential In Dental Pulp Stem Cells Cocultured With Endothelial Progenitor Cells In Vitro

BackgroundIn the endodontics clinic, the treatment of endodontic diseases are always depended on the development of the RCT (Root canal therapy) technique and the auxiliary equipment. As the rapid development of regenerative medicine and tissue engineering, the treatment of endodontic diseases are gradually transfer the focus onto a concept of pulp regeneration. The researchers desired to make this concept come true with the help of tissue engineering techniques. They hope to achieve a success that functional brand new teeth can be regenerated and replanted them back in patients’ mouths. Tissue engineering is a kind of technology that integrated with the theory and technologies of medicine, bioscience, life science and engineering. And there are three important elements in the theory of tissue engineering, which contains seed cells, biomaterial scaffold and growth factors.Dental pulp stem cells (DPSCs) is the most common one that used by researchers on the study of pulp regeneration. Odontoblast, a significant kind of cells in the process of tooth development, can secrete dentin matrix. Odontoblast, as a cell in the final stage of mitosis, is difficult to isolated and cultured in vitro. Therefore, the first step to achieve pulp regeneration is to figure out how to regenerate odontoblast. One of the hotspot in this field is to induced the seed cells differentiated into odontoblast. In the process of mineralization, extracellular matrix plays a vital role and dentin sialophosphoprotein (DSPP) can be defined as a key factor. Studies found that DSPP can repairs the pulp injury and promote the process of mineralization. The mutation or knock down of DSPP will induce tooth hypoplasia. The study of DSPP has been the hot area of research in the field of pulp regeneration for a long time.Over the years, a great progress and exploration has been made on the aspect of regulatory mechanism in seed cells differentiation by researchers worldwide. However, many issues still remain to us become an obstacle to the study of pulp regeneration. In recent years, researchers realized that the non-coding small RNA, which has been overlooked in the past, plays a significant role in the life of organism. The non-coding RNAs can regulate the transcription and translation of coding RNAs. A new upsurge of research on these non-coding RNAs has been made among the scientists. MicroRNA (miRNA) is one of these non-coding RNAs. The length of miRNA is between 18 and 25nt. After a long process of shearing by nuclease, miRNA can be assembled into RISCs. According to the complementary way between miRNA and the 3’UTR of the target gene, the RISCs will degrade or suppress the translation of the target gene’s mRNA. As a result, the miRNA regulate the target gene. miRNAs widely exist in the cells of plants, nematodes and humans and have the capacity to regulate the gene expression on the translation level. This regulation ability of miRNA can be process even the complementation is partial. Researchers also found that there’re nearly 30% of genes can be regulated by miRNAs. A single miRNA may regulate multiple genes and also the mRNA of gene can be regulated by multiple miRNAs. miRNA, as a new found regulatory factor, widely regulated the expression of target genes and widely participated in the differentiation, proliferation and apoptosis of cells.A serious previous experiments have been done by the members of our research group. First, they made a prediction on the relation between miRNAs and their target genes in the process of mineralization in DPSCs by software and the website database. According to the result of prediction, they operated a dual-luciferase reporter gene assay to verify the binding of miRNAs and DSPP. Finally, they confirmed that miR-586 can bind with the 3’UTR of DSPP and the binding site has also been verified. The inhibition of miR-586 will increased the expression level of DSPP and eventually promote the induction of mineralization. Second, they constructed a lentivirus that carried VEGF and SDF-1 overexpress gene and transfected the lentivirus into EPCs successfully. The VEGF and SDF-1 overexpress EPCs was 3D-cocultured with DPSCs. Results indicated that the combined utilization of VEGF and SDF-1 can promote the odontogenic and angiogenic differentiation.ObjectivesAccording to the results of previous study by our research group, the objectives of this study is to construct a miR-586 inhibition lentivirus and stably transfected DPSCs with it. The stably transfected DPSCs, which have been screening by puromycin, was cocultured with Endothelial progenitor cells (EPCs). DPSCs/EPCs will be induce towards odontogenic and angiogenic differentiation, respectively. The cells will be harvested for detecting the odontogenic and angiogenic relative factors to observe whether miR-586 inhibition will promote the odontogenic differentiation of DPSCs/EPCs without losing or suppressing the angiogenesis capacity of EPCs. We hope this DPSCs (miR-586 inhibition)/EPCs model can be utilized in the further study of pulp regeneration.Chapter One The isolation and identification of DPSCs and EPCs1. Methodsa. We used modified tissue explant collagenase method to isolated and cultured DPSCs. The pulp tissue was obtain from the patients whose third molar or premolar have an extraction or orthodontic treatment need. Pulp tissue was pull out of the tooth and cultured in vitro.b. The characteristics of stem cells were tested by different kind of methods. First, limiting dilution analysis was utilized to purified DPSCs. Second, the stem cells markers(CD29,CD4,CD90) were detected by flow cytometry. Third, the multiple differentiation potential were also tested.c. The EPCs were gift from others. EPCs were recovered from liquid nitrogen and cultured regularly.d. The EPCs were also tested for the endothelial cells markers(CD31,CD105,KDRCD105), hematopoietic cell markers(CD45,CD14) and stem cells markers(CD34,CD133) by flow cytometry after subcultured.2. Resultsa. The morphology and the growth status of DPSCs were observe under microscope. The cells was spindle shape. The cells can be subcultured until cell confluence was approximately 80%. The passage cells were proliferated continually which will finally showing a tendency of multi-layer growth. Generally, DPSCs can be subcultured every 3 days.b. The flow cytometry results showed that the positive expression rate of mesenchymal stem cells markers CD29,CD44 and CD90 was 94.70%,100% and 99.90%, respectively, while the hemopoietic stem cells markers CD34 and CD45 was 0.16% and 0.47%, respectively in DPSCs.c. The primary cells cultured from the pulp tissue in our study have the multiple differentiation potential. The mineralized nodules can be found in DPSCs after induction by Alizarin Red staining. And DPSCs can also be inducted differentiated into adipose cells and cartilage cell.d. The passage 4 EPCs, which proliferated fast, were obtained after recovery and the showing a cobblestone-like appearance. EPCs can be subcultured every 2 days.e. The flow cytometry results showed that the positive expression rate of endothelial cells markers CD31, CD105 and KDRCD105 was 99.82%,98.91% and 92.11%, respectively, while the hemopoietic stem cells markers CD45 and CD 14 was 0.06% and 1.1%, respectively in EPCs. The positive expression rate of mesenchymal stem cells markers CD34 and CD133 was 7.51% and 0%, respectively in EPCs.Chapter Two DPSCs stably transfected with miR-586 inhibition lentivirus1. Methodsa. The miR-586 inhibition lentivirus vector were obtain from the Genechem corporation. The vector system were constructed by 3 kind of vectors which contains pHelper 1.0 vector, pHelper 2.0 vector and GV vector system. The GV vector system contains HIV basic and auxiliary elements. pHelper 1.0 vector contain gag. gene from HIV. pHelper 2.0 vector contains VSV-G gene. All these vectors were participant in the lentivirus packaging. And the lentivirus titer were also identified.b. Preliminary experiment of transfection by lentivirus. The gradient screening experiment with puromycin. The suitable concentration of puromycin were tested for the further screening of study.c. Confirm of multiplicity of infection (MOI). A gradient MOI and different condition of infection were designed to tested for the best MOI and condition of infection.d. The detection of miR-586 and its target gene after stably transfected by miR-586 inhibition lentivirus by qRT-PCR. Furthermore, western blot was also operated to tested for the expression level of DSPP.2. Resultsa. The harvest of miR-586 inhibition lentivirus was done and the first generation DNA sequencing technology was utilized to test the positive cloning sequence of the lentivirus. The results showed that the antisense oligonucleotides sequence can be paired with the sequence of the mature miR-586. The miR-586 inhibition lentivirus titer is 3E+8TU/ml.b. The suitable screening concentration of puromycin for DPSCs is 2μg/ml. DPSCs can be eliminated in 3 days.c. DPSCs can be transfected easily under the condition of Complete Medium +5μg/ml polybrene by miR-586 inhibition lentivirus. Transfection efficiency may over 90% when MOI is 50.d. DPSCs have a good status and morphology after transfection and screening. The proliferation of DPSCs are still fast after screening. The subcultured DPSCs were screening by puromycin again and the cells condition remain good. The qRT-PCR results showed that the miR-586 relative expression level in DPSCs(miR-586 inhibition) group was (0.3952±0.0357) times to DPSCs group. Compared to DPSCs group, the miR-586 expression level is significantly suppressed in DPSCs(miR-586 inhibition) group(P<0.05).And the miR-586 expression level is also significantly suppressed in DPSCs(miR-586 inhibition) group when compared with DPSCs (EV) group(P<0.05).Chapter Three The role of miR-586 inhibition in the odontogenic differentiation of DPSCs/EPCs1. Methodsa. miR-586 inhibition DPSCs cocultured with EPCs at ratio 1:1. The culture medium is also mix with 10% FBS DMEM and 10% FBS EGM-2 MV at ratio 1:1. Experiment group was designed into 5:DPSCs group, DPSCs(miR-586 inhibition) group, DPSCs/EPCs group, DPSCs(Empty Vector, EV)/EPCs group and DPSCs(miR-586 inhibition)/EPCs group which will abbreviated as DPSCs, DPSCs(586), DPSCs/EPCs, DPSCs(EV)/EPCs and DPSCs(586)/EPCs in the following description. These 5 groups will be induce towards odontogenic differentiation at different time points (3d,7d,14d).b. Some analysis will be run to detect the cells in these 5 groups after induction at different time points. First, MTT for decteting on proliferation of DPSCs. Second, Alizarin red staining. Then, the expression level of DSPP, DMP-1, ALP and BSP will be detected by qRT-PCR. At last, the protein expression level of DSPP will be detected with western blot.2. Resultsa. MTT showed that the OD value of DPSCs(miR-586 inhibition) group were statitcally higher than DPSCs (EV) group on day 3,5 and 7.b. Alizarin ren staining showed that the number and color of mineralized nodules in the three cocultured groups are more and darker than that in DPSCs group. The quantity of mineralized nodules in DPSCs(586)/EPCs are the most in 5 groups.c. qRT-PCR showed that the expression level of DSPP in DPSCs(586) was (3.2172±0.2280) times to DPSCs group(P<0.05) at day 3 after induction. The expression level of DSPP in DPSCs(586)/EPCs group was (4.4134±0.2756) times to DPSCs group(P<0.05) at day 3 after induction. The expression level of DSPP in DPSCs(586) group was (5.0632±0.0537) times to DPSCs group(P< 0.05), while the expression level of DSPP in DPSCs(586)/EPCs group was (9.1833±1.0052) times to DPSCs group(P<0.05) at day 7 after induction. The expression level of DSPP in DPSCs(586) group was (4.1587±0.3162) times to DPSCs group(P<0.05), while the expression level of DSPP in DPSCs(586)/EPCs group was (4.0150±0.5468) times to DPSCs group(P<0.05) at day 14 after induction.d. Western blot:Results showed that the protein expression level of DSPP in DPSCs(586)/EPCs and DPSCs(586) are higher than the other three groups at day 3 after induction. The protein expression level of DSPP in DPSCs(586)/EPCs and DPSCs(586) are also higher than the other three groups and the DPSCs(586)/EPCs group was the highest at day 7 after induction. The other three groups show a distinct protein band. At day 14 after induction, the situation was almost the same as that at day 7. DPSCs(586)/EPCs and DPSCs(586) groups were still the highest two among 5 groups. However, the protein band of DPSCs(586) was higher than that of DPSCs(586)/EPCs.Chapter Four The role of miR-586 inhibition in the angiogenic differentiation of DPSCs/EPCs1. Methodsa. miR-586 inhibition DPSCs cocultured with EPCs at ratio 1:1. The culture medium is also mix with 10% FBS DMEM and 10% FBS EGM-2 MV at ratio 1:1. Experiment group was designed into 4:EPCs, DPSCs/EPCs, DPSCs(EV)/EPCs and DPSCs(586)/EPCs group.b. Different analysis will be run to detect the angiogenic properties. First, Tube formation assay. Second, the expression level of CD34、VEGF、Flk-1 and SDF-1 will be detected by qRT-PCR. Third, the protein expression level of VEGF will be detected with western blot.2. Resultsa. Tube foemation assay:The vessel-like structure constructed in DPSCs/EPCs, DPSCs(EV)/EPCs and DPSCs(586)/EPCs groups are more stable than that in EPCs group. The junctional area of the vessel-like structure in the three coculture groups are much larger than that in the EPCs group. The Total tube length and the mean loop perimeter of the three cocultured groups are longer than that in EPCs group.b. qRT-PCR showed that the expression level of CD34, VEGF, Flk-1 and SDF-1 in the three cocultured groups are significantly higher than that in EPCs group(P< 0.05). There is no significant difference between the three cocultured groups on the expression level of CD34, VEGF, Flk-1 and SDF-1(P>0.05).c. Western blot:Results showed that the protein expression level of VEGF in three cocultured groups are higher than that in EPCs group. The protein bands of three cocultured groups did not show a distinct difference and the DPSCs group did not show a distinct band.Conclusions1. There will be a high success rate of isolation and cultivation of DPSCs when using the modified tissue explant collagenase method. The DPSCs in this study show a high expression rate of mesenchymal stem cells markers and show a low expression rate of the hemopoietic stem cells markers. EPCs used in this study have the potential to differentiate into mature endothelial cells after identified.2. DPSCs cocultured with EPCs are well-growth at ratio 1:1 and also with a 1:1 mixture medium with DMEM and EGM-2 MV.3. miR-586 inhibition lentivirus vector was successfully constructed. And the stably transfected miR-586 inhibition DPSCs was also successfully constructed. The miR-586 inhibition DPSCs model can be used for further study of odontogenic and angiogenic differentiation.4. miR-586 inhibition DPSCs/EPCs model was successfully constructed. This model can be used for the pulp regeneration research as a well-combined seed cells.5. The combination of miR-586 inhibition and EPCs can synergistically facilitate the odontogenic differentiation on DPSCs. EPCs cocultured with DPSCs can promote the odontogenic differentiation on DPSCs.6. miR-586 inhibition has indirectly facilitate the angiogenesis differentiation on DPSCs/EPCs. DPSCs cocultured with EPCs can promote the angiogenic differentiation on EPCs.7. The duration of vessel-like structure constructed in DPSCs/EPCs are longer than that in EPCs. And the junctional area of the vessel-like structure will formed a special complex in DPSCs/EPCs.