The Experimental Study On Re-construction Of Corneal Surface Layer By Using Human Amniotic Epithelial Cells Transfered With HTERT Gene Mediated By Lentiviral Vector

Engineering cornea epithelium surface layer need suitable seed cells.Researchers have found,Human amniotic epithelial cells(hAECs),with the characteristics of both embryonic stem cells and pluripotent stem cells,have the potential to differentiate into various tissue cells in particular condition.Being obtained easily and convenience,with no any ethical arguments,have much more chances to differentiate to corneal epithelial cells,So hAECs may be a new source of seed cells of tissue engineering cornea.But being terminally differentiated cells and with short duration in vitro,the hAECs were difficult to culture.This shortcoming restricted its application in cell therapy.In view of this,us used this fusion gene of destination gene hTERT and marker gene amplification green fluorescent protein(GFP) to transfect hAECs,which compositive with lentivirus vector,to prolong cells growth cycle,even immortalized them.next,utilizing this trans-gened hAECs to re-construct laminated epithelium and cornea surface layer,to investigate application value of the trans-gened hAECs as a new seed cell souce of engineering cornea epithelium surface layer.Part One The culture and biologic identification of human amniotic epithelial cells(hAECs) in vitro Objective:To study the biologic characteristics of the cultured human amniotic epithelial cells(hAECs) in vitro,and to investigate the culturing method and stem cell characteristics of hAECs;to establish experimental foundation for transfecting hAECs with hTERTgene and reconstructing corneal surface layer.Methods:1.We used enzyme digestion to isolate and culture hAECs and observed the adherence and growth of the cells,the morphologic changes of cultured AECs were observed by HE staining,inverted microscope and light microscope.2.The expression of cytokeratin CK7,CK8 and CK18 in AECs were detected by using immunohistochemical staining.The immunofluorescence was used to detect the expression of OCT-4 of stem cell factor in passaged hAECs.3.Stem cell surface molecule marker CD29,CD34,CD44,CD45,CD 105 were detected by flow cytometry in passaged hAECs.4.Using immunohistochemistry to study the expression of hTERT protein in passaged hAECsResults:1 hAECs could adhere and grow very well in vitro,the cells looked like slabstone, they could passage 7—8 generations in normal circumstances.2 The results of immunohistochemistry showed The hAECs expressed cytokeratin CK7,CK8,CK18 in cytoplasm,the result of immunofluorescence showed The expression of OCT-4 of stem cell factor in hAECs was positive too.3 Detection of stem cell surface molecule marker by the flow cytometry showed The expression of CD29,CD34 on hAECs were positive,The expression of CD44,CD45,CD 105 on hAECs were negative.4 The results of immunohistochemistry showed The expression of hTERT protein in hAECs were negative.Conclusions:1.hAECs could be isolated successfully in vitro,but the cell only could be cultivated for short term in normal circumstances. 2.the hAECs were simple epithelium cells,hAECs had some characteristics of embryonic stem cells.Part two Construction of eukaryotic high expression vector containing pLenti6/V5-DEST-hTERT-EGFPObjective:To construct eukaryotic high expressing vector containing pLenti6/V5-DEST-hTERT -EGFP gene using lentiviral vector,to establish experimental foundation for transfecting hAECs by pLenti6/V5-DEST -hTERT -EGFP.Methods:1 The construction and identification of gene-recombinated shuttle plasmid pD ONR221-hTERT-EGFPUsing the PCI-neo-hTERT plasmid as model,We obtained objective gene hTERT by PCR reactions.Through BP reaction,we directly cloned hTERT into pDONR221 to get pDONR221-hTERT.And using the pEGFP-N1 plasmid as model,We obtained production -linker-EGFP- through PCR reactions.Through ambi-enzyme-tomy and T4-DNA joining-enzyme reaction,the production attB1-hTERT-linker-EGFP-attB2 was obtained, then converted in Bacillus coli DH5α.The gene-recombinated shuttle plasmid vector pDONR221-hTERT-EGFP was identified by digestant,PCRreaction and sequencing.2 The construction and identification of recombination carrier pLenti6/V5-DEST-hTERT-EGFPRecombinant plasmid pLenti6/V5-DEST-hTERT-EGFP was obtained through LR recombinating reaction using recombinant plasmid vector pDONR221- hTERT-EGFP and carrier pLenti6/V5-DEST.Then,converted in Bacillus coli STB13 and cultured on LB flat plate containing ammine benzyl-and-mycin.The recombination carrier pLenti6/V5-DEST-hTERT-EGFP was identified by digestant,PCR reactions and sequencing.3 The package and collection of supernatant containing hTERT-EGFP viral particle The 293FT cells were transfected with pLenti6/V5-DEST-hTERT-EGFP plasmid through lipofectctamine reagent,the supematant were collected by filtration method and conservated in—80℃.Results:1 The result of construction and identification of lentiviral rudiment carder pDONR221-hTERT -EGFPOur study successfully amplificated attB1-hTERT-attB2 fragment which was as long as 3.4Kbp.hTERT directly cloned into lentiviral rudiment carder pDONR221 through BP reaction. The Results of sequencing and identification showed hTERT fragment had been successfully cloned into rudiment carrier pDONR221.2 1547site rite-directed mutagenesis oflentivirus rudiment carrier pDONR221-hTERT-EGFPSequencing results indicated the objective gene hTERT of lentiviral rudiment carrier had mutanted in 1547 site.Then,we designed basi-primer containing correctiful 1547 site,the rite-directed mutagenesis of lentivirus rudiment carrier pDONR221-hTER fixed-point was succeeded and was confirmed by sequencing.3 The result of the construction and identification of lentiviral recombinant carrier pLenti6/V5-DEST-hTERT-EGFPlentiviral recombinant plasmid pLenti6/V5-DEST-hTERT-EGFP can be obtained through LR Recombinating reaction.The sequencing of recombinant plasmid confirmed that the construction of lentiviral recombinant plasmid pLenti6/V5-DEST -hTERT-EGFP was correct.4 The result of supernatant containing hTERT-EGFP viral particleThe supernatant fluid cortaining hTERT-GFP viral particle were seccessfully collected through lipofectctamine reagent.Conclusion:1 We had successfully constructed lentiviral recombinant plasmid pLenti6/V5-DEST-hTERT-EGFP for the first time.Part Three Transfaction and Expression of lentiviral rudiment carrier pLenti6/V5-DEST-hTERT-EGFP in hAECs ⅠTransfer hAECs with pLenti6/V5-DEST-hTERT-EGFPObjective:To transfer hAECs with objective gene hTERT and marker gene EGFP by lentiviral vector, then amplify cells clone containing hTERT-EGFP gene.EGFP-positive rates and the change of transfected cell cycle were detected by flow cytometry examination,to establish experimental foundation for transfected cells transplantation in vivo.Methods:1 To culture human amniotic epithelium cells(hAECs) in vitro.2 To transfer human amniotic epithelium cells(hAECs) with objective gene hTERT and marker gene EGFP by lentiviral vector.2.1 Confirm the density of Blasticidin for filtrating.We cultured hAECs with DMEM containing different level Blasticidin(0ug/ml,lug/ml,3ug/ml,5ug/ml,7ug/ml,10ug/ml) for about 14 days,observing the grow status of cells everday.2.2 To transfer hAECs by Lentiviral vectorAt the time of reaching 50%confluence of cells,hAECs were transfered with pLenti6/V5-DEST-hTERT-EGFP viral particle,the cells contained two orifices for experiment,two orifices for no-load control group,two orifices for normal control group, stoped transfection after 24h.the resistant clones secreting the recombinant proteins were estabilished with Blasticidin.3 To observe the transient expression of transgene in the transfered hAECs with fluorescence microscopewe changed culture medium to DMEM without screening reagent and cultured cells sequentially after transfection.the expression of EGFP in hAECs was detected with fluorescence microscope respectively at the time of 12h,24h,48h,72h,96h after transfection4 To culture extendly the cell clonesThe cultured solution was changed with DMEM without screening reagent when the cell clones had appeared,then the cells were cultured for 1 week sequentially.the cells were trypsinized by trypsin/EDTA solution when the cells insoculated,then we extendly cultured the transfected hAECs..5 To detect EGFP-positive rates of the transfered hAECs by flow cytometry examinationTo collect the cells of nomal control hAECs and transient transfered hAECs groups of culturing 12h,24h,48h,72h,96h after transfection,then to examine marker gene EGFP in cells by flow cytometry and use cell-quest software to analyze EGFP-positive rates. 6 To examine the change of cell cycle by flow cytometryTo collect the hAECs cultured for 48h of stably transfected group,transfected empty plasmid group and normal control group with the density of 1×10~6 cells/ml,then to examine the cell cycle and calculate the proliferation index(PI) using flow cytometry.Results:1 The resistant clones were obtained by using DMEM containing 1ug/ml Blasticidin to culture for 10 days.2 after cultured for 12h,the transient transfered hAECs could be observed the expression of green fluorescent protein in cytoplasm,but the amount was litter,the intensity was thin, only 0-2 positive cells could be seen in every low-power field.After 24h,the amount and intensity of green fluorescent protein gradually enhanced,about 2-5 positive cells could be seen in every low-power field.48h later,about over 10 positive cells could be seen in every low-power field,there were no difference between 48h and 72h.the amount and intensity of positive cells gradually decreased after 72h,2-3 positive cells could be seen in every low-power field at 96h.adding lug/ml Blasticidin and screening for 10d,mono cell clones appeared.3 After transfected 2h,some transfected hAECs fell off.no obvious configuration difference have been found between transfered hAECs and normal hAECs.4 EGFP-positive rate of transient transfected 48h group was the highest and was 18.53%.Compared with that of 12h,24h,96h groups,the difference had statistical significance(P＜0.05).Compared with that of 72h groups,the difference hadn't statistical significance(P＞0.05).Dense green fluorescence could been detected in the stably transfected groups.But EGFP-positive cells were not detected in the normal control group and transfected empty plasmid group.5 The flow cytometry assay showed:Compared with normal control group,the cellular proportion of stabilly transfected hAECs group increased in S phase and G2/M phase significantly,and PI value increased,no distinct difference had been found between normal control group and no-load group.Conclusions:1 This is the first time for transfecting hAECs with objective gene hTERT and marker gene EGFP through Lentiviral vector 1,the transient expression and expression in transfected hAECs were all obtained.At the time of transfection 48h,the positive expression was highest and was 18.53%.2 After transfected with pLenti6/V5-DEST-hTERT-EGFP through Lentiviral Vector,the PI of hAECs increased obviously,growth cycle of cells prolonged obviously too.3 The results indicated the transfection through Lentiviral Vector had no influence to the cell appearance,differentiation and reproductive activity and other bionomics of hAECs,and showed that the Lentiviral Vector was a relative safeful gene transfectionⅡThe expression of pLenti6/VS-DEST-hTERT-EGFP in transfered hAECsObjective:To detect the expression of hTERT mRNA gene and hTERT protein in transfected hAECs by Real Time fluorescent quantitation PCR and Western blotting.The immunohistochemical staining was used to detect hTERT protein expression in transfected hAECs,The athymic mouse experiment was used to detect the oncogenicity of stabilly transfected cells and to research the biological safety of transfected hAECs.Methods:1 To detect the expression of hTERT mRNA gene in transfected cells by Real Time fluorescent quantitation-PCR1.1 The total RNA were abstracted from the cells cultured for 48h of normal control group,positive control group(HepG2 oncogenic cells),stably transfected group and transfected empty plasmid group with the density of 1×10~6 cells/ml.1.2 The Purity and integrality of total RNA were detected by nucleic acid protein detection.the ratios of 00260/0D280 more than 1.8 showed RNA was fairly pure and no protein pollution.1.3 The total RNA reverse- transcribed into cDNA by the PCR reactions.1.4 After designing primer of qRT-PCR,then,begin to qRT-PCR reaction.2 To detect the expression of hTERT Protein within the cells of four groups by using Westem blotting.3 The immunohistoehemieal staining was used to detect expression of hTERT Protein within the transfected hAECs.4 The athymic mouse experiment was used to detect the oncogenicity of stabilly transfected hAECs.Results:1 The results of Real Time fluorescent quantitation-PCRThe relative amount of hTERT mRNA in normal control group and no-load transfected control group are 0,the relative amount of positive control group(HepG2 oncogenic cells) is 450,stablly transfected group is 200,transient transfected group is 185,these results showed hTERT gene had been integrated in transfected hAECs on the level of mRNA and hTERT gene didn't express in normal hAECs and no-load transfected hAECs.2 The results of Western blotting.There was positive band of 314KD of hTERT protein in positive control group,not in normal control group,no-load transfected group and transfected group.3 The results ofimmunohistochemical stainingThe stablly transfected hAECs were stained to brown with immunohistochemical staining,but the cells of other groups were negative.4 The results of athymic mouse experimentAfter injected for two weeks,the mice injected HepG2 hepatoma carcinoma cells growed tumor in injection site and the tumor grew up along with the time,the mice injected transfected hAECs had't no change in the injection site.Conclusions:1 hTERT gene was confirmed to had integrated in gene of tablly transfected hAECs on levels of mRNA for the first time.2 The stabilly transfected hAECs with objective gene hTERT have not been found oncogenicity at the timePart Four Study on re-construction of corneal surface layer using transfered hAECsObjective:To study the subsistence of stabilly transfered hAECs on fresh corneal stroma.The fresh corneal stroma with transfered hAECs was transplanted onto the rabbit's corneal surface without limbal stem cells to reconstruct the corneal surface layer.To observe the survival of transfered h.AECs and the stability of ocular surface.GFP gene was used to detect the subsisitentce and distribution of transfered cells after transplantation in living cornea tissue.To establish experimental foundation for using stabilly transfected hAECs to re-establish the corneal surface layer.Methods:1 To creat Stem cell deficiency(SCD)models on 12 rabbits eyes2 To culture transfered human amniotic epithelium cells(hAECs) in vitro.3 To obtain fresh rabbit's corneal stroma4 The transfered hAECs were seeded onto fresh corneal stroma surface.negative control group was seeded no cells.After cultured 10 days by Millicell culture plate inserts.The transfered hAECs and fresh corneal stroma fixed and dehydrated,observed by HE staining4 The Stem cell deficiency(SCD) modols of New Zealand white rabbits were set up,and were divided into 2 groups:group A(normal control group,n=6):the stroma had not any cells,group B(pLenti6/V5-DEST-hTERT-EGFP group,n=6):the seed cells were hAECs transfered pLenti6/V5-DEST-hTERT-EGFP gene.5 Corneal epithelium reconstruction procedures were performed after creating Stem cell deficiency(SCD)models for 2 months.During the surgery process,a 7.0mm diameter of corneal recipient bed was made.The corneal stroma with transfected cells or without any cells were placed on the recipient bed and sutured interruptedly using 10-0 suture.6 The operated eyes were observed everyday since the first day after operation,the observation included corneal conjunctivalization,vascularization,and stromal opacity,and compared with each other for two months.7 The structure of corneal implant in two groups were detected by HE attaining,the expression of of cytokeratin CK8,CK18,CK12 were detected by using immunohistochemical staining.Results:1 The transfered hAECs could grow well on the rabbit's corneal stromal cartier,after cultured on Millicell culture plate inserts for 10 days,the stabilly transfered hAECs were stratified and became corneal-like stratified epithelial cells by using the air-lifting cultivation.2 during the observation period,six cornea of GroupA all became opaque and cloudy badlly.there were plentiful neovaseular around the grafts.five cornea of Group B maintained clear for 2 moulths,only one cornea rejected during observation Period.There were also a few neovaseular around the graft.3 Imunohistochemistry staining showed:In group B,,the expression of of cytokeratin CK8,CK18,CK12 in epithelial lays were positive..In group A,Positive brown cells couldn't be observed in all lays.Conclusions:1 The function of corneal surface layer using transfered hAECs was more stable than control group.2 The transfered hAECs with pLenti6/V5-DEST-hTERT-EGFP gene would be a new and excellent feed cells to reconstruct corneal surface layer.