| Objective To study the effects of rat PDE5gene specific small interference RNA (PDE5-siRNA) and iNOS single/double gene expression in rat adipose-derived stem cells (ADSCs), and investigate the feasibility and effectiveness of ADSCs modified with PDE5-siRNA and iNOS gene for erectile dysfunction (ED) therapy.Methods Isolate and culture ADSCs from the adipose tissue of rat inguinal region by collagenase digestion. The cultured cells were identified not only by flow cytometer detection of mesenchymal stem cells (MSCs) related-immunophynotypes, but also by their capacities in the adipogenic, myogenic, endothelial and neurogenic differentiation. Then the fourth passage ADSCs were labeled with10μmol/L chloromethyl-benzamidodialkyl-carbocyanine (CM-Dil) and50μmol/L5-ethynyl-2’-deoxyuridine (EdU), and the labeling effects of CM-Dil and EdU on ADSCs were detected by flow cytometer and fluorescence microscope at7days,14days,21days and28days, respectively. The recombinant lentivirus vector expressing PDE5-siRNA (LV-PDE5-siRNA-GFP) and recombinant adenovirus vector carrying iNOS gene (Ad-iNOS-EGFP) were constructed, respectively. PDE5-siRNA and iNOS gene were cotransfected into ADSCs mediated by lentivirus and adenovirus. The experimental groups were designed as blank group, negative control group1(LV-NC-siRNA-GFP transfected group), negative control group2(Ad-NC-EGFP transfected group), negative control group3(LV-NC-siRNA-GFP and Ad-NC-EGFP cotransfected group), positive control group1(LV-PDE5-siRNA-GFP transfected group) and positive control group2(Ad-iNOS-EGFP transfected group) and experimental group (LV-PDE5-siRNA-GFP and Ad-iNOS-EGFP cotransfected group). The expression of PDE5and iNOS gene were examined with real-time fluorescence quantitative RT-PCR assay and western blot at3days,5days,7days,10days and14days, respectively. At the same time, the concentration of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) were assayed by nitrate reductase method and enzyme-linked immunosorbent assay (ELISA).Results The cultured cells expressed CD49d, CD73, CD90, CD105and CD106, lacked CD31, CD34and CD45. Adipogenic, myogenic, endothelial and neurogenic differentiation cells can be stained with Oil Red O, a-SMA and Desmin, vWF, NeuN and Nestin, respectively, and the negative control was negative. The expression of PDE5gene was downregulated at3days, reached the peak at5to7days, and decreased at10days, and remained higher later. Compared to positive control group1(LV-PDE5-siRNA-GFP transfected group), no significant difference was noted (P>0.05). The mRNA and protein expression of iNOS gene mediated by recombinant adenovirus vector worked at24hours, reached a peak at5to7days, decreased at10days, and lasted at least2weeks. The expression level of PDE5was down-regulated by more than70percent from3to14days, significantly lower than negative control (P<0.001). However, there was no significant difference between cotransfected group and positive control group2(Ad-iNOS-EGFP transfected group), P>0.05. The NO concentration of cotransfected group and positive control group2was elevated at3days, reached the peak at5to7days, decreased at10days, and kept at least14days, but no greater than positive control group2(P>0.05). At the same time, the concentration of cGMP in cotransfected group and positive control groups increased at3days, reached the peak at5to7days, decreased at10days, but still kept at a high level later. The concentration level of cotransfected group was much higher than positive control groups (P<0.01), but no significant difference was noted between positive control group1and group2(P>0.05). The results showed that the concentration of NO and cGMP in ADSCs were elevated at3days, reached the peak at5to7days, decreased at10days but increased later, and lasted at least14days.Conclusion PDE5-siRNA and iNOS gene mediated by lentivirus/adenovirus vector have successfully cotransfected and co-expressed in ADSCs. ADSCs modified by PDE5-siRNA and iNOS double gene could produce enough NO and more cGMP, which is more effective than single gene modified. Therefore, CM-Dil labeling ADSCs modified with PDE5-siRNA and iNOS genes could be the ideal seed cells in the future treatment of ED. PartⅠCulture and Identification of Rat Adipose-Derived Stem CellsObjective To isolate and culture rat ADSCs in vitro, and provide the available seed cells for PDE5-siRNA and iNOS gene modified stem cells in the future treatment of ED.Methods Obtain adipose tissue from rat inguinal region by surgical resection, and isolate and culture ADSCs by collagenase type I digestion method. The fourth generation cell growth curve was described with cell counting kit-8(CCK-8). Mesenchymal stem cells (MSCs) related-immunophynotypes CD31, CD34, CD45, CD49d, CD73, CD90, CD105and CD106were detected by flow cytometer detection. The characterization of ADSCs was also arrayed by inducing in the adipogenic, myogenic, endothelial and neurogenic differentiation, and identified with Oil Red O staining, a-SMA and Desmin staining,vWF staining, NeuN and Nestin staining, respectively.Results The growth curve of the fourth passage cultured cells was presented typical "S" shape. The cultured cells expressed CD49d, CD73, CD90, CD105and CD106, lacked CD31, CD34and CD45. The positive rate was (1.88±0.12)%,(4.05±0.13)%,(99.07±0.45)%,(4.68±0.23)%,(11.81±3.60)%,(0.35±0.04)%,(0.38±0.01)%and (0.54±0.33)%, respectively. Adipogenic, myogenic, endothelial and neurogenic differentiation cells can be stained with Oil Red O, a-SMA and Desmin, vWF, NeuN and Nestin, respectively. However, the negative control was negative.Conclusion ADSCs have been successfully isolated and cultured from rat adipose tissue, and could be used as ideal cell carrier for PDE5-siRNA and iNOS gene modified stem cells, provide the available seed cells for PDE5-siRNA and iNOS gene modified stem cells in the future treatment of ED. Part ⅡCell Tracker Labeling for Rat Adipose-Derived Stem CellsObjective To compare the labeling effects of CM-Dil and EdU on rat ADSCs, and seek the optimal stem cell labeling technique applied to ED therapy.Methods The fourth generation ADSCs were labeled with2μmol/L,4μmol/L,6μmol/L,8μmol/L,10μmol/L,12μmol/L,14μmol/L,16μmol/L,18μmol/L and20μmol/L CM-Dil solution. The optimal labeling concentration was screened by flow cytometer and fluorescence microscope. Then the CM-Dil positive cell rate was detected by flow cytometer and fluorescence microscope at7days,14days,21days and28days, respectively. In order to seek the optimal labeling time of EdU for ADSCs, ADSCs were labeled with50μmol/L EdU containing medium incubated for2h,6h,12h,24h,48h,72h and96h. EdU positive cell rate was detected by fluorescence microscope at7days,14days,21days and28days, respectively.Results The optimal labeling concentration of CM-Dil for ADSCs was10μmol/L, and the positive cell rate was near to100%.10μmol/L CM-Dil had no bad effects on the growth of ADSCs. The positive cell rate was higher at7days and14days (P>0.05), decreased at21days (P<0.05), but still kept more than80%at28days. The labeling efficiency of EdU reached the peak incubating for48hours, and decreased at72hours. The positive rate of EdU labeling cells was (87.1±1.8)%, and decreased at7days (P<0.01), and the positive rate was only (14.4±2.7)%at28days.Conclusion CM-Dil and EdU are suitable for ADSCs tracker labeling. However, CM-Dil gains much more advantages than EdU in long-term tracing, which may be an ideal and optimal ADSCs tracing technology in the future treatment of ED. Part ⅢConstruction and Identification of Recombinant Viral Vector Carrying Rat PDE5-siRNA and iNOS GenePartⅢ-AConstruction of Recombinant Lentivirus Vector Expressing RatPDE5-siRNAObjective Construct recombinant lentivirus vector expressing rat PDE5-siRNA (LV-PDE5-siRNA-GFP), and provide a good transfer media for PDE5-siRNA expression in ADSCs thereby.Methods The effective interfere sequence for PDE5(GCAGCCGAATTCTTTGATC) was obtained from our prophase research results. Double-stranded DNA Oligo containing PDE5-siRNA sequence was synthesized and connected with linearized pFU-GW-RNAi recombinant lentivirus vector by T4DNA ligase. There recombinant pFU-GW-PDE5-siRNA recombinant lentivirus vector was transformed into competent cells. The positive recombination was selected by polymerase chain reaction (PCR) and further identified by sequencing analysis.293T cells were infected by successfully constructed pFU-GW-PDE5-siRNA lentivirus vector together with pHelper1.0and pHelper2.0plasmid. Cellular supernatant of transfected293T cells was harvested, concentrated and purified at48hours. The lentivirus titer was determined by dilution method.Results The sequencing results showed that targeted PDE5-siRNA sequence GCAGCC-GAATTCTTTGATC was was consistent with pFU-GW-PDE5-siRNA lentivirus vector sequence. The lentivirus titer was1E+9TU/mL.Conclusion Recombinant lentivirus vector carrying rat PDE5-siRNA was successfully constructed and high lentivirus titer was obtained, which could be applied to further study. PartⅢ-BConstruction and Identification of Recombinant Adenovirus Vector Harboring Rat iNOS GeneObjective Construct recombinant adenovirus over-expression vector harboring enhanced green fluorescent protein (EGFP) and rat iNOS gene, and observe iNOS gene expression mediated by recombinant adenovirus in293T cells, which may provide reliable basis for the possibility of iNOS gene expression in ADSCs.Method The full-length iNOS gene was fished and amplified by PCR. pDC315-EGFP adenovirus vector was linearized with Age I digestion, then connected with iNOS gene. There recombinant pDC315-iNOS-EGFP adenovirus vector was transformed into competent cells and identified by restriction enzyme digestion, PCR and DNA sequencing.293T cells were transfected with confirmed pDC315-iNOS-EGFP plasmid. The expression of pDC315-iNOS-EGFP plasmid was detected by western blot assay. The recombinant adenovirus was generated by AdMax packaging system in HEK293cells. Raw adenovirus was harvested, concentrated and purified by Adeno-X Maxi purification kit. The lentivirus titer was determined by doubling dilution method. The iNOS gene recombinant adenovirus was identified by western blot in293T cells.Results pDC315-iNOS-EGFP adenovirus vector was successfully constructed and confirmed by restriction enzyme digestion, PCR, DNA sequencing and western blot assay. The adenovirus titer was1.2E+10PFU/mL. The iNOS/GFP fusion proteins were successfully expressed in293T cells observing by western blot at48hours.Conclusion Recombinant adenovirus over-expression vector harboring rat iNOS gene was successfully constructed and expressed in293T cells, which could be further applied to the modification of ADSCs. Part IVThe Expression of PDE5-siRNA and iNOS Gene in Rat Adipose-Derived Stem CellsObjective To compare the effects of rat PDE5-siRNA and iNOS single/double gene rat on ADSCs, and investigate the possibility and feasibility of ADSCs modified with PDE5-siRNAand iNOS gene for future ED therapy by grafting into corpus cavernosum.Methods The experiment groups were designed as blank group, negative control group1(LV-NC-siRNA-GFP transfected group), negative control group2(Ad-NC-EGFP transfected group), negative control group3(LV-NC-siRNA-GFP and Ad-NC-EGFP cotransfected group), positive control group1(LV-PDE5-siRNA-GFP transfected group) and positive control group2(Ad-iNOS-EGFP transfected group) and experimental group (LV-PDE5-siRNA-GFP and Ad-iNOS-EGFP cotransfected group). ADSCs were cotransfected with LV-PDE5-siRNA-GFP (MOI=70) and Ad-iNOS-EGFP (MOI=30). The apoptosis of transfected ADSCs was detected by Annexin V-PE/7-AAD apoptosis kit and flow cytometry at3days. The expressions of PDE5and iNOS gene in ADSCs were detected by real time RT-PCR and western blot assay at3days,5days,7days,10days and14days. Nitric oxide and cGMP were determined by Nitrate/Nitrite Assay Kit cultured with lOmM L-arginine containing medium for24hours and ELISA Kit cultured with10μM sodium nitroprusside containing medium for2minutes, respectively.Results The percentage of cotransfected ADSCs apoptosis was (3.04±0.58)%, and no significant difference was noted between the7groups (P>0.05). PDE5-siRNA worked at3days, reached a peak at5to7days, decreased at10days, and lasted at least14days. The expression level of PDE5was down-regulated by more than70percent from3to14days, significantly lower than negative control (P<0.001). iNOS gene began to express at24hours, reached a peak at4to5days, and decreased at10days. There was an overexpression of iNOS gene in ADSCs modified with iNOS (P<0.001). The level of NO was greatly higher than negative control (P<0.01), but no greater than iNOS single gene modified control (P>0.05). The concentration of cGMP was significantly greater than PDE5-siRNA/iNOS single gene modified control (P<0.01). The activation effects of PDE5-siRNA and iNOS double gene on NO/cGMP in ADSCs were kept at least2weeks.Conclusion PDE5-siRNA and iNOS gene were coexpressed in ADSCs, and produced enough NO and more cGMP thereby. Therefore, ADSCs modified with PDE5-siRNA and iNOS genes could be the ideal seed cells in the future treatment of ED by grafting into corpus cavernosum. |
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