| Background.Bioartificial kidney is an efficient therapy for end stage renal disease.With the progress in study of engineering kidney,there are several problems to be faced:1.how to obtain a number of engineering cells in relative short time in order to engineer an bioartificial kidney? 2.how to make the bioartificial kidney perform the functions of both bioartificial glomerulus and bioartificial tubule? As we know,seeding cells are the critical for tissue engineering and regenerative medicine and a enormous number of cells are usually needed for these purposes. So,there is necessary to obtain a plenty of seeding cells.However,there are many hurdels to be overcomed for in vitro cell proliferation before a number of engineering cells for bioartificial kidney are produced.Although Human Nanog gene acts as a gatekeeper of pluripotency and is an important self-renewal determinant in embryonic stem cells,it remains unknown whether human Nanog can enhance the cell proliferation of mature cells.In our study,we transfected human vascular endothelial cell and human kidney epithelial cell for engineering bioartificial kidney by human Nanog gene.Moreover,we engineered an bioartificial kiney nephron using transfected-vascular endothelial cells and transfected-epithelial nephronal cells and examined the abilities of reabsorption and transport for glucose and electrolyte after both transfected vascular endothelial cells and transfected epithelial nephronal cells were implanted into the luman of polysulfone hollow fibres.After this,the dispension and proliferation of both transfected cells were checked by various examinations.In order to test reabsorptive and endocrine functions,first,the confluency of mixedly implanting cells was examined,then the amount of the glucose and sodium reabsorbed and transported by each kind of bioartificial nephron was measured respectively.Methods. Cell culture,transfection and identification The human renal epithelial cell line HKC and human vascular endothelial cell line were transfected by the expression vector rAAV2-hNanog and the mock vector rAAV2-EGFP(Both are purchased from Beijing Ben-yuan Biotechnology co.),respectively.The rAAV2-hNanog transfected both ECV304 and HKC cell lines at the concentration of 10~3-10~4 vg/cell. So did the mock vector rAAV2-EGFP.Stable clones of ECV304-hNanog, HKC-hNanog,ECV304- EGFP and HKC-EGFP were selected and isolated.Then, the effect of hNanog on proliferation of the seeding cells by MTT assay and BrdU incorporation test,before this,the expression of hNanog gene in two transfected-seeding cells was examined by RT-PCR.The construction of bioartificial nephron After we obtained enough ECV304-hNanog and HKC-hNanog cells,the suspensions of two seeding cells were mixed homogenously at relative optimal ratio 1:1 because it was necessary for the bioartificial nephron with both the function of bioartificial glomerulus and that of bioartificial tubule.Thus,this device would probably more function as nephron than does a bioartificial tubule with only renal epithelial cell.In fact,we designed this tissue-engineered bioartificial nephron as possible to mimic the foundamental structure of physical nephron as we can.Therefore,this bioartificial nephron device containing ECV304- hNanog and HKC-hNanog can be properly named "bioartificial nephron".The inner surfaces of the hollow fibres were coated with 0.74mg/ml laminin(Sigma,USA)before seeding with mixed engineering cells.The influence of hNanog gene on the adhesion of two seeding cells to polysulfone hollow fibres In order to ascertain the effect of hNanog gene on the abilities of ECV304 and HKC to adhere to the hollow fibres,we observed the changes in the adhesion of ECV304 and HKC cell lines before and after the their transfection to adhere to the inner and outer surface of polysulfone hollow fibres. For this,we implanted two engineering cells and the mixed engineering cells containing a half ECV304 cells and a half HKC cells before and after their transfections onto the outer surface of the hollow fibres.Meanwhile,the densities of mixed two transfected cells were assessed by the hoechst33342,then the corresponding distribution of ECV304-hNanog and HKC-hNanog was showed by PKH26 and PKH67 cell linker dyes.Finally,the morphology of mixed transfected cells on the hollow fibres was observed under scanning electron microscope.The measurement of ability to transport and reabsorb for bioartificial nephron After 7 days of construction of bioartificial nephron,the evaluation of abilities to transport and reabsorb the glucose and sodium was measured, moreover,the measurement of leakage rates for UN and Cr and endocrine for bioartificial kidney nephrons in every group was performed.Results.Experssion of human Nanog in transfected seeding cells After transfection, the expression of exogenous human Nanog gene in stable clones of ECV304-hNanog and HKC-hNanog was confirmed by RT-PCR.Expression levels were normalized using an internal control Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)gene.It was obvious that the ECV304-hNanog and HKC-hNanog expressed hNanog,while there was no expression of hNanog in ECV304-GFP,HKC-EGFP and their corresponding parental seeding cells.Effect of human Nanog on cell proliferation After transfection of both engineering cells,the optical densities of two engineering cells modified by hNanog were measured at 8h,12h 18h,24h,30h,36h,42h,48h,the results displayed that hNanog can significantly enhance the growth of ECV304 and HKC cells compared with their controls,while there were no differences between the grouptransfected by mock vector rAAV2-EGFP and the parental group of two seeding cells(p＞0.05).In addition,BrdU incorporation test revealed that BrdU positive cells of ECV304-hNanog,HKC-hNanog were more than those of their negative contrals ECV304-EGFP,HKC-EGFP,ECV304 and HKC(ECV304-hNanog vs ECV304-EGFP,ECV304:158.00±13.71 vs 96.25±28.64,92.25±12.04 P＜0.01;HKC-hNanog vs HKC-EGFP,HKC:109.40±9.56 vs 93.75±10.44,90.50±8.66 P＜0.01). Influence of human Nanog on adhesion of ECV304 and HKC to polysulfone hollow fibres Before and after transfection,there was no obvious changes in adhesion of ECV304 and HKC to polysulfone hollow fibres which facilitate the construction of bioartificial nephron.Distributions of two transfected seeding cells on inner surface Both transfected seeding cells patchily distributed on the inner surface of polysulfone hollow fibres after implantation of mixing transfected seeding cells,among which the ECV304-hNanog labeled by PKH26 and HKC-hNanog labeled by PKH 67, we can see ECV304-hNanog labeled by PKH26 showed red colour and HKC-hNanog labeled by PKH 67 showed yellow.However,there were no labeling cells on the hollow fibres in control.Density of transfected seeding cells on inner surface After 5 days of implantation,the polysulfone hollow fibres from the filters of experimental and control groups were opened and treated with Hoechst33342 followed by the observation of distribution for two transfected engineering cells under fluorescence microscope,there were a number of sparkled cells and the mixed transfected cells grew closely together on the fibres of experimental group compared with the fibres in control panel,on the contrary,there were no cells adhesive to the intraluminal surface in control groups.Morphology of two transfected engineering cells on the hollow fibre When the bioartificial nephron containing transfected seeding cells were cultured in cell incubator for 7 days,the polysulfone hollow fibres from experimental and control groups were examined by the scanning electron microscopy.In experimental group,transfected seeding cells with many microvillus grew on the hollow fibre surface,but no cells attached to the hollow fibre in the controlAssessment of ability to transport,reabsorb and endocrine for transfected seeding cells in bioartificial nephron In order to assess functions of transport and reabsorption of engineering cells transfected by hNanog gene seeded onto the inner surface of the hollow fibres in bioartificial nephron,the measurements of leakage for UN,Cr,glucose and sodium were tested after 7 days of cell-seeding(n=4 each),data are expressed as mean±SEM.The medium containing 442mmol/l UN and 8.34mmol/l Cr were perfused into the inner lumin of bioartificial nephron,then the leakage rates of UN and Cr were measured for 90min.The results demonstrated the leakages of Cr and UN in blank-filter were 40.56±3.14%and 45.85±4.39%,respectively,but were 13.98±0.51%, 14.57±0.41%for transfected-HKC-filter,14.02±1.77%and 16.09±1.58 for transfected -compound-filter,13.37±2.18%and 15±2.14%for transfected-ECV304 -filter.The amount of reabsorption for glucose and electrolyte sodium of transfected -compound-nephron was by far more than those of blank nephron and transfected -ECV304 -nephron(p＜0.01),but was lesser than those of transfected-HKC- nephron due to the reabsorptive ability of ECV304 is much lower.In addition,the abilities to reabsorb glucose and sodium of transfected-compound nephron and transfected-HKC-nephron can be inhibited by phlorizin and ouabain respectively which elucidated that glucose reabsorption was facilitated by the sodium-dependent glucose transporter.Meanwhile,compared to the transfected-HKC-nephron and blank-nephron, the concentrations of endothelin(ET)and 6-keto-prostaglandinFlα(6-keto-PGFlα) in transfected -compound-nephron were much higher(p＜0.01),but is lower than that in transfected -ECV304-nephron(p＜0.05);the 6-keto-PGFlαconcentration in transfected -compound -nephron is much higher than those in transfected-ECV304-nephron and blank -nephron(p＜0.01),respectively,but was lower than that in transfected-HKC-nephron(p＜0.01).Conclusions.hNanog gene can improve the abilities of ECV304 and HKC to proliferate,which facilitated the construction of BAK,in addition,the bioartificial nephron by mixed implanation of two seeding cells transfected by hNanog had functions of both bioartificial glomerulus and bioartificial tubule.Thus,the study of bioartificial nephron will lay a good foundation for the BAK with compound functions. |
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