Experimental Study On Construction Of Novel FGF And Its Impacts On Corneas And Vascular Endothelial Cells

Corneas locate at the foreside of eyeballs,liable to be influenced by wounds and inflammation. According to the statistics,corneal diseases rank secondly among ocular diseases causing blindness, and ocular wound is one of the most important reasons of stemma blindness. Along with the increase of intraocular surgery, corneal endothelial wounds increase, visional function are severely impaired.fibroblast growth factor, FGF possesses abroad biological activities on many types of cells rooting in mesoderm and exoderm, aFGF (acidic FGF) and bFGF(basic FGF) are two primary members, they have similar molecular structure. FGF is a normal and tiny polypeptide in body.bFGF plays an important role in corneal wound healing testified by many studies.From eighties in 20th century to now,there are a lot of exploitations and applications about bFGF,but there is a risk of inducing.neovascularization or tumor if it is used by great amount for a long time.Cornea is a transparent tissue without blood vessels,the procedure of its wound healing is very complicated. Corneal neovascularization may help protect from infections and play a role in wound healing, but its structure and function are not perfect, liable to cause plasma leakage,and then corneal edema,scar and so on,vision may be damaged severely. At present, the effect of inducing CNV influences the therapy of FGF on corneal diseases.So people hope to construct novel FGF. We had a try and got satisfied results in vivo and in vitro studies, presenting some helpful datas for searching an ideal remedy to protect and heal corneas.Our studies can be divided into four parts.First,construct a novel hbFGF, the modified hbFGF, m-hbFGF has biological activities and a weaker effect of promoting proliferation of vascular endothelial cells. Second, study the effect of promoting proliferation of three types of corneal cells in vivo.Third, study the effect of m-hbFGF on corneal wound healing and CNV.Fourth,study the expression of m-hbFGF in corneal endothelia and the protective effect on corneal endothlia.Part Ⅰ Study of constructing a novel hbFGFbFGF play a role in corneal wound healing, but there are risks of inducing CNV if used for a long time because it has an effect of promoting proliferation of cells rooting in mesoderm and exoderm. In order to solve this problem, we modified the construction of hbFGF.Ⅰ Construction of a novel m-hbFGFPurposeTo construct a novel hbFGF and test its biological activities. Method1. Clone and identification of mature peptide gene of m-hbFGF cDNA :replace 119th amino acid, perform PCR,synthesize a whole m-hbFGF cDNA sequence.Clone it into pUC18 carrier,forming m-hbFGF/ pUC18 carrier, measure DNA sequence after transforming DH5a.2. Expression of m-hbFGF in coliform and its identification:Clone mhbFGF gene into pET3C expressive carrier,transform the pET3C/m-hbFGF expressive carrier into coliform JM109 and then screen out high expressive pET3C/m-hbFGF/ JM109.3. Seperate and purify m-hbFGF recombining protein and identify it.Results1. m-hbFGFcDNA sequence was synthesized successfully, and was transfected into pET3C/m-hbFGF expression vector.2. pET3C/m-hbFGF/ JM109 had highly expressive ability,could express the 17KD m-hbFGF target protein.ConclusionWe constructed a novel m-hbFGF successfully,and got the high expressive m-hbFGF protein.Ⅱ Testing biological activities of m-hbFGFPurposeTo testify whether m-hbFGF possesses activities of promoting proliferation. Methods1. Prepare growth culture fluid of Balb/c 3T3 cells:DMEM + 0.4%FBS.2. Cell inoculation: Get human navel vein endothelial cells and Balb/c 3T3 cells ,plant the third generation cells into 96-hole plates at the density of 1×104/hole seperately. Plant human navel vein endothelial cells into culture bottle at the density of 5× 104/ cm2 .3. The identification of cells:use cruor factor Ⅷ correlated antigen as tested target,chemical dyeing the immune cells with SABC immunohistochemical reagent box.4. Group of test:m-hbFGF group, hbFGF group and contrast group, there are six holes per each group and each density.5. Change the culture fluids every 24hour, add growth culture fluids of different density(0,1,2.5,5,10,20,40 and 80ng/ml into m-hbFGF group and hbFGF group separately. Plant the 3rd generation human navel vein endothelial cells(diluted into 1 × 105/mL) in cultural bottle.6. Plant cells in the 96-hole plates for 48 hours,then test OD value which is consistent with proliferative rate.7. Statistical analysis:Data analysis was performed using ANOVA. the comparison betweenm-hbFGF and hbFGF is according to T test. Data are expressed as mean±SEM.Results1. Balb/c 3T3 cell1.1 In m-hbFGF group,OD values go up gradually along with the increase of the density(from lng/mL),peak at 20ng/mL, then go down gradually along with the increase of the density.1.2 There is no significance between m-hbFGF groups and hbFGF groups at specific density, (P>0.05)1.3 The OD value of m-hbFGF group is higher than contrast group at the densities from 5ng/mL to 80ng/mL (P<0.05) .2. Human navel vein endothelial cells2.1 Primary cultured human navel vein endothelial cells started to stick to wall,thereare some suspended blood cells visible;15% cells stick to wall at 7th day,80% at 11th day.the cells present polygon,roundness or oval,with much cell plasma. Offspring cells have clear boundary like road metal.2.2 The identification of cells:the vein endothelial cell plasma was brown,positive.2.3 In m-hbFGF group,OD values go up gradually along with the increase of the density(from lng/mL),peak at 20ng/mL, then go down gradually along with the increase of the density.2.4 The OD value of m-hbFGF group is higher than contrast group at the densities from 10ng/mL (P>0.05)2.5 The OD value of m-hbFGF group is lower than hbFGF group at the densities from 10ng/mL (P<0.05) .3. In hbFGF group:there are lots of ringy blood vessel like structures,vascularendothelial cells present flat, cell plasma syncretize together, cells connecting togetherformed ringy blood vessel like structures.In m-hbFGF group:only a few vein endothelial cells syncretize together,no wholeringy blood vessel like structures visible.In contrast group:no vein endothelial cells syncretize together and no whole ringyblood vessel like structures visibleConclusion1. Our study ,for the first time,tesitify that m-hbFGF recombing protein has biological activities.2. The activity of m-hbFGF promoting the proliferation of Balb/c 3T3 cells is dose-dependant, the peak density is 20ng/mL.3. m-hbFGF has the effect of promoting proliferation of human navel vein endothelial cells,and the effect is dose-dependant,the peak is 20ng/mL.4. The effect of m-hbFGF on promoting proliferation of human navel vein endothelial cells is obviously weaker than that of hbFGF.5. The effect of m-hbFGF on inducing blood vessel cavity like structure formation is obviously weaker than that of hbFGF.Part Ⅱ The effects of m-hbFGF on three kinds ofcorneal cellsCorneal wound healing is a very complicated processjncluding activation, proliferation and differentiation of cells.release of cytokines,synthesization and reconstruction of extracellular matrix and so on. We study the effects of m-hbFGF on three kinds of corneal cells in vitro and compare it with hbFGF.Ⅰ The effect of m-hbFGF on corneal epithelial cellsPurposeTo study the effect of m-hbFGF on cultured corneal epithelial cells,and compare it with hbFGF.Methods1. Prepare culture fluids containing corneal epithelial cells:DMEM/F12 + 0.4%FBS.2. Cell plantation:get corneal epithelial cells using trypsin digestion,plant the 2nd generation cells into 96-hole plates at the density of 1 × 104 /hole.3. Identification of cells:Immunohistochemical dying with keratin AE1/AE3 antibody and SABC Immunohistochemical reagent box.4. Group of test:m-hbFGF group, hbFGF group and contrast group.5. Change the culture fluids every 24hour, add growth culture fluids of different density(0,1,2.5,5,10,20,40 and 80ng/ml into m-hbFGF group and hbFGF group separately.6. Plant cells in the 96-hole plates for 48 hours,then test OD value which is consistent with proliferative rate7. Statistical analysis:Data analysis was performed using ANOVA. the comparison betweenm-hbFGF and hbFGF is according to T test. Data are expressed as mean±SEMResults1. Inverse microscope:primary cultured corneal epithelial cells started to stick to wall from 2nd day, joined together at 5th day, cells presented roundness or oval,containing much plasma. Offspring cells have very clear boundary like road metal.2. Identification of cellsxorneal epithelial cells were brown yellow,positive.3. In m-hbFGF group,OD values go up gradually along with the increase of the density(from lng/mL),peak at 20ng/mL, then go down gradually along with the increase of the density.4. The OD value of m-hbFGF group is higher than contrast group at the densities from 5 ng/mL (P>0.05) .5. The OD value of m-hbFGF group is lower than hbFGF group at each density,but there is no significance (P>0.05) .Conclusion1. m-hbFGF may promote the proliferation of corneal epithelial cells and the effect is dose-dependant,peak is 20ng/mL.2. The effect of m-hbFGF on promoting proliferation of corneal epithelial cells is not weaker than that of hbFGF.Ⅱ The effect of m-hbFGF on corneal stromal cellsm-hbFGFPurposeTo study the effect of m-hbFGF on cultured corneal stromal cells,and compare it with hbFGF.Methods1. Prepare culture fluids containing corneal stromal cells:DMEM/F 12 + 0.4%FBS.2. Cell plantation:get corneal stromal cells using collagenase digestion,plant the 3rd generation cells into 96-hole plates at the density of 1 × 104 /hole.3. Identification of cells:Immunohistochemical dying with vimentin monoclone antibody and SABC Immunohistochemical reagent box.4. Group of test:m-hbFGF group, hbFGF group and contrast group.5. Change the culture fluids every 24hour, add growth culture fluids of different density(0,1,2.5,5,10,20,40 and 80ng/ml into m-hbFGF group and hbFGF group separately.6. Plant cells in the 96-hole plates for 48 hours,then test OD value which is consistent with proliferative rate7. Statistical analysis:Data analysis was performed using ANOVA. the comparison between m-hbFGF and hbFGF is according to T test. Data are expressed asmean±SEM. Results1. Inverse microscope:primary cultured corneal stromal cells started to stick to wall from 2nd day, cells presented shuttlel,containing much plasma. Offspring cells have very clear boundary like vortex.2. Identification of cellsxorneal stromal cells were brown yellow,positive.3. In m-hbFGF group,OD values go up gradually along with the increase of the density(from lng/mL),peak at 20ng/mL, then go down gradually along with the increase of the density.4. The OD value of m-hbFGF group is higher than contrast group at the densities from 10ng/mL to 80 ng/mL (p<0.05) .5. There is no significance between OD value of m-hbFGF group and that of hbFGF group (P>0.05) .Conclusions1. m-hbFGF may promote the proliferation of corneal stromal cells and the effect is dose-dependant,peak is 20ng/mL.2. The effect of m-hbFGF on promoting proliferation of corneal epithelial cells is not weaker than that of hbFGF m-hbFGF.Ⅲ The effect of m-hbFGF on corneal endothelial cellsm-hbFGFPurposeTo study the effect of m-hbFGF on cultured corneal stromal cells,and compare it with hbFGF.Methods1. Prepare culture fluids containing corneal endothelial cells:DMEM/F12 + 0.4%FBS2. Cell plantation:get corneal stromal cells using collagenase digestion,plant the 3rd generation cells into 96-hole plates at the density of 1×10~4/ hole.3. Identification of cells:Immunohistochemical dying with neurofibreprotein and SABC Immunohistochemical reagent box4. Group of test :m-hbFGF group, hbFGF group and contrast group.5. Change the culture fluids every 24hour, add growth culture fluids of differentdensity(0,1,2.5,5,10,20,40 and 80ng/ml into m-hbFGF group and hbFGF group separately.6. Plant cells in the 96-hole plates for 48 hours,then test OD value which is consistent with proliferative rate.7. Statistical analysis:Data analysis was performed using ANOVA. the comparison betweenm-hbFGF and hbFGF is according to T test. Data are expressed as mean±SEMResults1. Inverse microscopeiprimary cultured corneal stromal cells started to stick to wall from 2nd day, cells presented polygon,three dimensional,monolayer,containing much plasma. Offspring cells have very clear boundary like road metal.2. Identification of cellsxorneal stromal cells were brown yellow,positive.3. In m-hbFGF group,OD values go up gradually along with the increase of the density(from lng/mL),peak at 20ng/mL, then go down gradually along with the increase of the density .4. The OD value of m-hbFGF group is higher than contrast group at the densities from 5ng/mL (p<0.05) .5. The OD value of m-hbFGF group is lower than hbFGF group at the densities from 10ng/mL (P<0.05) .Conclusions1. m-hbFGF may promote the proliferation of corneal endothelial cells and the effect is dose-dependant,peak is 20ng/mL.2. The effect of m-hbFGF on promoting proliferation of corneal endothelial cells is weaker than that of hbFGF.Part Ⅲ The role of m-hbFGF in corneal woundhealinghbFGF plays an important role in corneal wound healing. We made corneal penetrating wound model and corneal acid burn model in rabbits, compare the effects of m-hbFGF and hbFGF on corneal wound healing and their impacts on CNV.Ⅰ The effect of m-hbFGF on corneal wound healing in corneal penetrating wound modelPurposeWe use m-hbFGF and hbFGF to treat corneal penetrating wound separately, in order to study their effects on corneal wound healing in corneal penetrating wound model.Methods1. Corneal penetrating wound modehwe cut through the corneal center with shaver piece under microscope, then drop1000u/mL heparin at the wound immediately,then use some medicine to prevent inflammation.2. Make pressure meter with the windpipe of blood pressure meter and "T" pipe, pressure meter(250 kpa),transfision pipe.3. Group of test:m-hbFGF group, hbFGF group and contrast group, 12 rabbits in each group, and one eye of each rabbit as experimental eye.4. Treatment after wound:from 1st day after wound, 2000ng/mL hbFGF , m-hbFGF and PBS were dropped into the experimental eyes in hbFGF group, m-hbFGF group and contrast group seperately, two drops each time and four times each day.5. Observation after wound:observe ocular reaction after wound with handy slit- lampmicroscope.6. Test the limit value of corneal wound pressure: at the 8th day after wound ,select 6 rabbits from each group, test the limit value of corneal wound pressure after anaesthesia.7. Extirpate the corneas termly: extirpate two corneas at 7th ,14th and 28th day after wound separately,dehydrate,emhed inparaffin,slice up,dye and observe the healing condition under light microscope.Results1. Observation under slit-lampmicroscope:there is no obvious ciliary congestion, anterior chamber come back at 1st day after wound, the edge of wound had edema,turbidness, edema lightened at the 2nd day and fadeaway at 3rd day after wound.2. Test the limit value of corneal wound pressure: hbFGF group:114.67±9.03, m-hbFGF group:110.33±8.43, contrast group :75.33±13.66, there is significance between hbFGF group(m-hbFGF group) and contrast group (P<0.05 ) ,but nosignificance between hbFGF group and m-hbFGF group (P>0.05) . 3. Tissue pathology:3.1 hbFGF group: at 7th day,corneal epithelial cells at wounds arranged clearly(5~6 layers), there are lots of fibretissue and fibrablasts in corneal stroma,corneal endothelia did not heal. At 14th day, corneal epithelial cells had 5-7 layers, there are much more fibretissue and fibrablasts in corneal stroma, arranging in order,corneal endothelia healed.3.2 m-hbFGF group: at 7th day,corneal epithelial cells at wounds arranged clearly(5~6 layers), there are lots of fibretissue and fibrablasts in corneal stroma,corneal endothelia did not heal. At 14th day, corneal epithelial cells had 5~6 layers, there are much more fibretissue and fibrablasts in corneal stroma, arranging in order,corneal endothelia healed.3.3 contrast group: at 7th day,corneal epithelial cells at wounds arranged mussily(8~10 layers), there are less fibretissue and fibrablasts in corneal stroma,without corneal endothelia. At 14th day, corneal epithelial cells had 8-10 layers, there are more fibretissue and fibrablasts in corneal stroma, arranging mussily,corneal endothelia did not healed very well.Conclusion1. m-hbFGF can promote corneal penetrating wound healing.2. There is no significance between the effects of m-hbFGF and hbFGF on promoting corneal penetrating wound healing.Ⅱ The effect of m-hbFGF on corneal wound healing in corneal acid burn modelPurposeWe use m-hbFGF and hbFGF to treat corneal acid burn separately, in order to study their effects on corneal wound healing in corneal acid burn model.Methods1. Corneal acid burn modelrdip a piece of round filter paper(diameter 8mm) in 1mol/L H2SO4 for 10 seconds, then put it on the corneal center for 3 minutes,get it off and rinse conjunctival sac with 50ml PBS immediately, use some medicines to prevent inflammation.2. Group of test:m-hbFGF group, hbFGF group and contrast group, 8 rabbits in eachgroup, and one eye of each rabbit as experimental eye.3. Treatment after wound:from 1st day after wound, 2000ng/mL hbFGF , m-hbFGF and PBS were dropped into the experimental eyes in hbFGF group, m-hbFGF group and contrast group seperately, two drops each time and four times each day.4. Observation after wound:observe ocular reaction after wound with handy slit-lampmicroscope.5. Test the corneal epithelial healing condition and CNV: At 14th day,28th day,select 6 rabbits from each group,take pictures of corneas and scan them into computer,count the percentage of corneal epithelial healing and area of CNV.6. Extirpate the corneas termly; extirpate two corneas at 7th ,14th and 28th day after wound separately,dehydrate,embed inparaffin,slice up,dye and observe the healing condition under light microscope.Results1.corneal epithelial healing: At the 14th day,m-hbFGF group:healed 83.30%; hbFGF group: healed 85.17%; contrast group: healed 67.7%. there is significance between hbFGF group(m-hbFGF group) and contrast group (P<0.05) ,but no significance between hbFGF group and m-hbFGF group(P>0.05).At the 28th day, m-hbFGF group and hbFGF group both healed 100%,but there is a cornea not healed well in contrast group.2. CNV: At the 14th day, there are some CNVs growing out from limbus above in each group,but they did not reach the wound area. At the 28th day, the average area of CNV in m-hbFGF group:7.57mm2 ; the average area of CNV in hbFGF group :9.67 mm2 ; the average area of CNV in contrast group: 7.02mm2。 there is significance between hbFGF group and m-hbFGF group (P<0.05) ,but no significance between m-hbFGF group and contrast group (P>0.05) .Conclusions1. m-hbFGF can promote corneal acid burn wound healing.2. The ability of m-hbFGF to induce CNV is weaker than that of hbFGF.Part Ⅳ Transfection of m-hbFGF in corneal endothelial cells and its meaningHuman corneal endothelial cells are not reborn, its wound can only be filled by the movement and spreading of cells nearby. When the density of endothelia is lower than 400/mm2, corneas will be edema which can damage vision. We study the method of protecting corneal endothelia through transgenome in vitro.Ⅰ The construction of pSecTag/m-hbFGF, pSecTag/hbFGF eukaryotic expression vectorPurposeTo construct m-hbFGF and hbFGF eukaryotic expression vector (pSecTag/hbFGF and pSecTag/m-hbFGF) 。Methods1. Synthesization of m-hbFGF and hbFGF genesequence: according to m-hbFGF and hbFGF cDNA sequence, synthesize corresponding primer, then synthesize the two target gene by PCR reaction.2. Connect target gene and pSecTag/hygrocine ABC eukaryotic expression vector3. Identify eukaryotic expression vector plasmid.ResultshbFGF 和 m-hbFGF gene was transfected into eukaryotic expression vector and pSecTag/Hygrocine ABC vector, we got 438bp pSecTag/m-hbFGF (Figure 4) and pSecTag/hbFGF eukaryotic expression vector。Conclusion1. We constructed pSecTag/hbFGF eukaryotic expression vector successfully.2. pSecTag/m-hbFGF eukaryotic expression vector was constructed successfully for the first time.Ⅱ Establishment of corneal endothelial wound modelPurposeTo establish corneal endothelial wound model by saline, and observe its wound effect on corneal endothelia.Methods1. Culture of corneal endothelial cells: same to part Ⅱ, plant the 3 rd generation cells into 96-hole plates at the density of 1×104/hole,and into 24-hole plates at the density of 3×l04/hole.2. Wound model by saline:stimulate cells cultured for 24 hours by saline,observe the impacts on cellular shape at different time.3. Test livability of corneal endothelial cells wounded by saline with MTT, there is a direct ratio relationship between the livability and OD value.Results1. At 30 minutes after wounding with saline,the shape of corneal endothelial cells started to change,at 90 minutes,cellular conjunctions were destroyed,cells started to be atrophied,at 150 minutes, most cells were dehydrated,even died.2. From 90 minutes, the comparison with contract group is significant.Conclusion1. There is a direct ratio relationship between the corneal endothelial wound by saline and time.2. We successfully established corneal endothelial wound model:the corneal endothelial cells wounded by saline for 90 minutes are ideal for this model.Ⅲ pSecTag/hbFGF, pSecTag/m-hbFGF eukaryotic expression vector transfect corneal endothelial cellsPurposeTo study the protective effect of m-hbFGF on the corneal endothelial cells wounded by saline.Methods1. Culture of corneal endothelial cells: same to part Ⅱ.2. Group of test: divided into 4 group.- pSecTag/m-hbFGF group, pSecTag/hbFGF group, contrast group and saline group.3. Cellular transfection:Perform as illumination of LipofectamineTM2000.4. Test of transfection:Test secretion and expression of m-hbFGF and hbFGF using Western blotting method.5. Test cellular livability with MTT:add saline 0.1 ml into the each hole of pSecTag/m-hbFGF group, pSecTag/hbFGF group and saline group at 48h after transfection, continue culture for 90 minutes,then test cellular livability with MTT.6. Cellular shape changes under electron microscope: add saline 0.5 ml into the each hole of pSecTag/m-hbFGF group,pSecTag/hbFGF group and saline group at 48h after transfection, continue culture for 90 minutes,then get cells as sample for electron microscope .Results1. Western blotting test:: In pSecTag/hbFGF and pSecTag/m-hbFGF group, corneal endothelial cells expressed 17 KD protein.2. MTT test: OD value :pSecTag/hbFGF group: 0.448 ± 0.030;pSecTag/m-hbFGF group: 0.479 ± 0.019;contrast group: 0.496 ± 0.023;saline group:0.382 ± 0.025. There is a significance between pSecTag/m-hbFGF group(pSecTag/hbFGF group) and saline group (P<0.05) ;also pSecTag/m-hbFGF group and pSecTag/hbFGFgroup (P<0.05)。3. Electron microscope:3.1 pSecTag/hbFGF group:cells distort,shrink,presenting different sizes,faint boundary and structure,cellular conjunctions are loose,center of cells hollow,only a few microvillus,cellular membrane still keep integrity,structures in plasma are not clear.3.2 pSecTag/m-hbFGF groupxells present hexagon,connect with each other closely,rank in order like road metal,cellular nucleus is round, much more microvillus, structures in plasma are clear.3.3 Saline groupxells change their shapes,like triangle or roundness,there is no conjunction among cells,there are cracks on the surface of cells,no microvillus visible, cellular nucleus shrink,cellular membrane break, structures in plasma are turbulent.Conclusion1. For the first time, we transfect pSecTag/m-hbFGF and pSecTag/hbFGF into cultured corneal endothelial cells,and discover that their expression can protectcorneal endothelial cells wounded by saline.2. The protective effect of m-hbFGF on corneal endothelial cells is obviously strongerthan that of hbFGF.