TGF-β_1基因修饰后板层人工生物角膜的免疫学研究
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摘要
角膜病是主要的致盲性眼病之一,我国约有350万角膜病患者,居世界首位,同种异体角膜移植是角膜盲复明的主要手段。在无血管床进行角膜移植具有较高的成功率,排斥反应发生率仅为5～10%,但在高危患者(如:角膜化学伤和Stevens-Johnson综合征所致的角膜严重血管化,其他器官移植术后再行角膜移植,偏中心移植,大植片移植和二次移植等)其发生率可高达50%以上,此外供体材料来源匮乏也严重制约了该手术的开展和普及。因此,降低移植术后的排斥反应和寻找新的角膜供体材料成为眼科界研究的两大热点问题。
应用免疫抑制剂是目前预防和治疗角膜移植术后免疫排斥反应的主要手段,常用药物有皮质类固醇、环孢霉素A(Cyclosporin A,CsA)、FK506等,这些药物的联合使用能够有效地抑制排斥反应,但长期使用对机体有很大的毒副作用。故而,开发探索新型强效免疫抑制剂是提高异体或异种角膜移植手术成功率的有效途径。转化生长因子β_1(Transforming growth factor-β_1,TGF-β_1)作为一种比CsA活性更高的免疫抑制剂(Ruegemer等报道),越来越引起人们的关注。TGFβ_1是多肽类细胞因子,广泛参与免疫系统各细胞、因子间的相互调节,可抑制胸腺细胞、淋巴细胞、自然杀伤细胞等的增殖分化,干扰多种免疫球蛋白的产生和转换,并通过直接、间接影响IL-1、IL-2、IFN-γ及其受体而起以“负调节”为主的免疫平衡作用。近来,TG-β_1,作为一种强效免疫抑制因子己被用于器官移植对抗免疫排斥反应、诱导移植物局部的免疫耐受,在小鼠和大鼠同种异体心脏移植及胰岛细胞移植模型中应用TGF-β_1均能明显延长移植物存活时间,取
Keratonosus is the main eye disease that can lead to blindness. There are about 3.5 million blind patients caused by corneal diseases in China whose blindness incidence rate is the highest in the world. At present the most important treatment to reconstruct useful vision is corneal allogenic transplantation. Although keratoplasty has a high successful rate, the corneal allogeneic transplantation rejection rate is only 5-10%under the condition of vascular recipient beds, yet in the high-risk cases is higher than 65%. The high-risk factors include vascularized cornea, prolonged inflammation, severe injury of the cornea and conjunctiva, large size keratoplasty, and re-keratoplasty. Moreover, However, corneal grafts were limited to perform due to the shortage of cornea materials. Therefore it becomes a hot issue for ophthalmology researchers to find new cornea tissue resources and reduce allograft rejection.The application with the immunosuppressions is now the main methods to prevent and treat corneal allograft rejection after the keratoplasty, including hormone, Cyclosporin A, FK506, and so on. The associated application of these medicine can suppress immune rejection effectively, but side-effect is high if long term application. Therefore searching for high-effective and low-toxic immunosuppressive agent is effective method to increase the success rate of corneal allograft or heterograft. TGF-β1 (transforming growth factor- β1) is a multifunctional peptide with immunosuppressive property. These properties include inhibition of B and T cell proliferation, adherence of granulocytes, respiratory burst of macrophage, the synthesis of IL-1,
IL-2, IFN- γ, and HLA gene expression. It plays a central role in inhibition of inflammatory and alloreactive immune response. It was reported that the immunosuppressive property of TGF- β1 is more stronger than Cyclosporine A by Prof. Ruegemer and his colleagues. TGF- β1 has been used to antagonize allograft rejection as one of the most effective immunosuppressive cytokines recently. A study shows that TGF- β1 can induce the immunological tolerance, and the application to the allogenic heart transplantation and pancteas islet cell transplantation model in the mice and rats can significantly prolong the allograft survival. In addition, TGF- β1 can induce anterior chamber associated immune deviation(ACAID), inhibit antigen specific delayed-type hypersensitivity response (DTH) and protect eye from the impairment of immune reaction.But exogenous growth factor is diluted and metabolized quickly when used in part because of short half-life. As a result large quantity of growth factor have to be applied repeatedly. Therefore, it becomes a hot issue and key for tissue engineering research to find how to keep growth factor acting continually and effectually. Dr.GuoXiaodong and his colleagues transfer the TGF- β1 gene into MSCs (mesenchymal stem cell) for the first time in a study of articular cartilage repairing, which was the first attempt to combine the principles of tissue engineering with principles of molecular biology organically. The results showed that the repair was histologically and ultrastructurally better for TGF- β1 gene modified group than for control group, indicating that TGF- β1 as an immunosuppressive cytokines also has the potential to prolong the allograft survival. Artificial biologic cornea (also called tissue engineering cornea) is a normal cornea equivalent which is reconstructed by three-dimension culture in vitro with artificial materials or biologic materials as carrier and technology of tissue and cell engineering. With the development of life science and profounding research, artificial biologic cornea will be the substitute for human donor cornea. This will be quite beneficial for corneal disease treatment and of great clinical importance and practical value.On the base of researches in the past, this study will transfer TGF-β1MP ( Transforming growth factor-beta 1 Mature Peptide) gene into seed cell of tissue
engineering cornea, then planted to the frozen and preserved in glycerine porcine corneal stromal as carrier for constructing transfected artificial biologic cornea used for transplantation. The aim of this study is to resolve the shortage of corneal materials and search a new gene therapy method for reducing corneal graft rejection. A novel concept, molecular tissue engineering cornea was put forward for the fist time. As a rising branch it represents both a new domain and a significant trend of tissue engineering research. This study mentioned above has not been reported in the references read.Part One Construction of eukaryotic high expression vector containing TGF-β1MPObjectiveTo construct the eukaryotic expression vectors of the secreted TGF-β1 cDNA and to express high effectively the recombinant proteins in the corneal endothelial cells and corneal stromal cells cultured in vitro.MethodsExpression vectors pSecTag2-TGFβ1MP, based on the pSecTag2/HygroA, were constructed by way of molecular cloning.1. Vectors pSecTag2/ HygroA was digested to obtain linear vectors by restriction enzyme SfiI and HindIII.2. According to the gene sequence of hTGF-β1MP, design the primer used to amplification of PCR reactions to obtain TGF-β1MP gene fragment. The pET3C-TGFβ1MP plasmid vector was used as model.3. The resulting PCR products were separated by electrophoresis in agarose gels, then were digested by restriction enzyme SfiI and HindIII.4. Linear vector pSecTag2/ HygroA and TGF-β1MP gene fragment were linked by ligase.5. The recombination vector that TGF-β1MP gene fragment had correctly been
inserted into pSecTag2/HygroA was identified by digestant, PCR reaction and sequencing.Results1. Vectors pSecTag2/HygroA was digested by SfiI and HindIII to obtain two fragments whose length were 4.7kb and 1.0kb respectively, digested by Hindlll to obtain one fragment whose length was 5.7kb.2. Two of four recombination vectors were digested by SfiI and BgIII to obtain two fragments whose length were 4.7kb and 1350bp respectively. The second step, the recombination vectors that were maybe masculine can be amplificated to obtain about 366bp gene fragments. The third step, the masculine plasmid was certified that its sequences of vector segment , inserted segment and digested point were consistent with reports completely.ConclusionAcquired recombination vectors pSecTag2-TGFβ1MP lays a foundation for TGF-β1MP gene transfection into deep lamellar artificial biologic cornea.Part Two Effects on biological activity of cultured corneal cells in vitro produced by TGF- β1ObjectiveBefore transfection of TGF-β1MP gene, to explore effects on biological activity of cultured three kinds of corneal cells in vitro produced by TGF-β1, in order to prevent and reduce the other side-effects at the same time to make use of its immunosuppressive functionMethods1. Epithelial cells, stromal cells and endothelial cells were isolated from New Zealand rabbits cornea respectively then were put into the incubator at 37 ℃ in a humidified atmosphere containing 5% CO2. Conformation of cultured cells were
examined and recorded regularly.2. The cultured cells were HE stained, observed with optic microscope.3. Immunohistochemistry examination was made to identify the category of cells. Three peculiar antibodies were used which were monoclonal antibody Keratin AE1/AE3, monoclonal antibody Vimentin monoclonal antibody Neurofilament respectively.4. Effect on proliferation of three kinds of corneal cells produced by TGF- β1 was measured by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay in vitro.Results1. Inverted phase contrast microscope examination shows:1.1 After 2 days in culture containing rhTGF- β1, epithelial cells had successfully migrated from the limbal explant onto the floor of 6-well culture plate. After 3-4 days in culture formed a rim around the limbal fragment 4-5mm wide. After 6-7 days in culture covered the whole plate.1.2 After 6 hours in culture containing rhTGF- β1, stromal cells had began to explant onto the floor of the culture plate. After 24 hours cells extended gradually, after 3 days cells grew vigorously.1.3 After 2 days in culture containing rhTGF- β1, endothelial cells had successfully migrated from the Descemet's membrance(DM) onto the floor of 6-well culture plate. After 3 days in culture formed a rim around the limbal fragment 4-5mm wide. After 6-7 days in culture covered the whole plate.2. HE staining shows: The configuration of three kinds of corneal cells cultured in culture medium containing rhTGF- β1 were close to normal cells. Cells connected closely, stained densely in the karyon. There were 2-3 nucleolus seemed clearly.3.1mmunohistochenistry examination shows: The cytoplasm of Epithelial cells, stromal cells and endothelial cells cultured in culture medium containing rhTGF- β1 were stained to brown by peculiar monoclonal antibody respectively.4.MTT colorimetric assay shows:
4.1 The solution of 20ng/ml, 10ng/ml, 5ng/ml, 1ng/ml ATGF-β1 was able to inhibit the proliferation of the epithelial cells in vitro. The suppressive effect of 10ng/ml group is the most strong. The solution of 0.5ng/ml, 0.1ng/ml rhTGF- β1 had no toxicity to these cells. The solution of 0.05ng/ml rhTGF- β1 was able to accelerate the proliferation of these cells in vitro.4.2 The solution of rhTGF- β1 no matter what concentration was all able to promote the proliferation of corneal stromal cells in vitro. The stimulative effect 10ng/ml group is the most strongest, became a peak value.4.3 The solution of 20ng/ml, 10ng/ml, 5ng/ml rhTGF- β1 was able to inhibit the proliferation of the endothelial cells in vitro. The suppressive effect of 10ng/ml group is the most strongest. The solution of 1 ng/ml, 0.5ng/ml rhTGF- β1 had no toxicity to these cells. The solution of 0. 1 ng/ml, 0.05ng/ml rhTGF- β1 was able to accelerate the proliferation of these cells in vitro.Conclusion1. The three kinds of corneal cells cultured in vitro were resemble normal corneal cells in shape, histological structure and immuncyto- chemistry.2. The effect on the proliferation and growth period of corneal cells cultured in vitro depends on the concentration of exogenous rhTGF- β11:2.1 The effect on the epithelial cells: The higher concentration group suppressed proliferation, 10ng/ml group is the most strongest. The concentration became higher the suppressive effect became weaker. Medium concentration group had no effect on cells. The lower concentration group accelerated proliferation.2.2 The effect on the corneal stromal cells: All concentration group accelerated proliferation. 10ng/ml group is a peak value.2.3 The effect on the endothelial cells: The higher concentration group suppressed proliferation, 10ng/ml group is the most strongest. The concentration became higher the suppressive effect became weaker. Medium concentration group had no effect on cells. The lower concentration group accelerated proliferation.
Part Three Transfection and expression of exogenous TGF-β1MP gene in corneal cellsI TGF-β1MP gene transfected to corneal cells ObjectiveTo study the transfection efficiency of exogenous TGF-β1MP gene in corneal stromal and endothelial cells and to observe the effect on proliferation induced by transfection. To co-transfect pEGFP-N1 plasmid acted as indicator system in living corneal tissue.Methods1. Plasmid pEGFP-N1 were amplified and were detected by Vsp1 and EcoR I respectively.2. Cultured rabbit corneal stromal and endothelial cells in vitro.3. Confirm the concentration of G418 and Hygromycin B for filtrating.4. At the time of reaching 90-95% confluence, cells were co-transfected with pEGFP-N1 and the recombinant expression vectors pSecTag2-TGFβ1MP though Lipofectamine reagent.5. The resistant clones secreting the recombinant proteins were established with G418(200μg/ml) and Hygromycin B(150μg/ml) screening.6. Extending culture of the cell clones: When the cell clones had appeared, the solution was changed with DMEM-F12 without screening reagent. After inosculation, the cells were trypsinized with 0.25% trypsin/0.1% EDTA solution, then cultured extendly. The corneal stromal cell clone was named Fibro-cc, the endothelial cell clone was named Endo-CC. The endothelial cell clone that cultured with lOng/ml EGF was named Endo-CC-EGF.7. To observe the cell configuration with inverted phase contrast microscope.8. The transient expression of transgene with fluorescence microscope (FLM): Accomplishing the transfection, changed culture medium to DMEM-F12 without
screening reagent and cultured cells sequentially. At the time of 12 hours, 24 hours, 48hours, 96hours after gene transfer, the expression of GFP in corneal stromal cells and endothelial cells was detected with fluorescence microscope respectively.9. To observe the morphological change of transfected cells with HE staining.10. GFP-positive rate was detected by Flow cytometry examination. To collect the cells of normal control group and transient transfected groups (12 hours, 24 hours, 48hours, 96hours) and stably transfected groups (Fibro-cc, Endo-CC, Endo-CC-EGF), then examined by Flow cytometry. The GFP-positive rates were analyzed with cell-quest software.11. The change of cell cycle were examined by flow cytometry: To collect the cells cultured for 48 hours of normal control group, transfected empty plasmid group and stably transfected groups (Fibro-cc, Endo-CC, Endo-CC-EGF) with the density of 1 X 106cells/ml, then examined by Flow cytometry. Calculate the proliferation index(PI) according to the formula: PI= (S +G2M) / (GoG1 + S + G2M) ×10012. To detect cells apoptosis with DNA electrophoresis in agarose gels: The corneal endothelial cells were incubated with 160mM/L Sodiym arsenite(Ars) to form a endothelial cells apoptosis model. The corneal stromal cells were irradiated with 320nm Ultroviolet B(UVB) to form a stromal cells apoptosis model.Results1. Plasmid pEGFP-N1 were digested by VspI and EcoRI to produce two bands, about 4.6kb and 600bp respectively.2. The resistant clones secreting the recombinant proteins were obtained by cultured with G418(200μg/ml) and Hygromycin B (150μg/ml) for 14 days.3. GFP-positive cells were not detected in the normal control group and transfected empty plasmid group. GFP-positive expression of transient transfected groups(12 hours, 24 hours, 48hours, 96hours) were detected. GFP-positive rate of 48 hours groups were the highest, 31.0% and 24.5% respectively. GFP-positive rate of stably groups were all 100%.
4. After 2 hours in transfection, some cells fell off from plate. The configuration of most cells had no obvious difference with normal cells. Endo-CC-EGF group growth rapidly than Endo-CC group, magnitude and arrangement were more consistent.5. The GFP expression in endothelial cells and stromal cells could be detected in 12 hours after transfection by fluorescence microscope, 24 hours GFP expression increased, 48 hours green fluorescence were strongest, 96 hours could observed green colored cells was only 3-4cell/eyeshot. There were no green colored cells in the control group and transfected empty plasmid group. Dense green fluorescence could been detected in the stably transfected groups, Endo-CC-EGF group were stronger than Endo-CC group.6. Endo-CC-EGF of one time passage were multiangular, Fibro-cc of one time passage were shuttle shape. Cells grew flourishingly and connected tightly.7. Flow cytometry assay shows: There was no subdiploid peak appeared in the flow cytometry assay graph of transfected corneal cells. Compared with the control group, the cellular proportion of transfected group decreased in S phase and G2/M phase significantly(PI value decreased). Compared with the transfected group, the cellular proportion of transfected+EGF group increased in S phase and G2/M phase significantly(PI value increased). There was no distinct difference between control groups and transfected groups.8. No Ladder-bands(DNA bands of apoptosis cells) were detected in DNA electrophoresis in agarose gels of normal control group and transfected group without reference to corneal stromal cells or endothelial cells. Contrarily, emblematical Ladder-bands appeared in the positive control group.Conclusion1. The plasmids of pSecTaq2-TGFβ1MP and pEGFP — N1 have been successfully transfected into cultured corneal stromal cells and endothelial cells in vitro, can express transiently or stably.2. GFP-positive rate of 48 hours groups were the highest, 31.0% and 24.5%
respectively. GFP-positive rate of stably groups were all 100%.3. Lipofectamine 2000 reagent was safe and effective to transfer gene into corneal cells, had no effect on morphology and proliferation.4. The transfected TGF-β1 gene expressed in high level, decreased the proliferation of corneal endothelial cells, increased the proliferation of corneal stromal cells, had no apoptosis effect on the two kinds of cells.5.At first time, indicated that 10ng/ml EGF could counteract the suppressive effect of TGF- β1 expressed unduly, made the proliferation of endothelial cells become to normal.II Expression of exogenous TGF-β1MP gene in corneal cellsObjectiveTo detect the expression of exogenous TGF-β1MP gene in corneal stromal and endothelial cells.Methods1. Total RNA was isolated from the cells cultured for 48 hours of normal control group, transfected empty plasmid group and stably transfected groups (Fibro-cc, Endo-CC-EGF) with the density of 1 × 106cells/ml, and reverse-transcribed into cDNA. Amplification of PCR reactions was conduced. The resulting PCR products were separated by electrophoresis in 2% agarose gels.2. The expression of TGF-β1 protein within the corneal endothelial cells was detected by Western blotting and immunohistochemical SABC.3. The secreted TGF-β1 protein concentration in the corneal endothelial cell supernatant was assayed by ELISA. To obtain the standard curve by nonlinear regression analyse.Results1. TGF-β1MP gene was confirmed to have been integrated and expressed in transfected corneal stromal cells and endothelial cells on the level of mRNA through
the positive appearance in the agarose gels electrophoresis.2. The expression of TGF-β1 protein was deteced with Western blotting. Positive brown band of 25KD appeared in transfected group, not in control group and untransfected group. The staining became deeper and deeper with the extending of expressing time.3. The expression of TGF-β1 protein was deteced with immunohistochemical SABC examination. Positive cells were stained to brown. Control group and transfected empty plasmid group were negative.4. Linear regression equation was obtained: OD = 0. 001C-0. 0344 ( R = 0. 998) According to the equation, could calculate the concentration of the secreted TGF-β1 protein in the corneal endothelial cell supernatant. The concentration of transfected endothelial cells was (597 ±7)pg/ml, the concentration of transfected stromal cells was (995 ± 12)pg/ml, the value was zero in control group and transfected empty plasmid group.Conclusion1. TGF-β1MP gene was confirmed to have integrated and expressed in the transfected corneal endothelial cells and stromal cells on levels of mRNA and protein.2. The expressed amount of TGF-β1 in the transfected corneal stromal cells and endothelial cells supernatant were (995 ± 12)pg/ml and (597 ± 7)pg/ml after cells had been cultured for 48 hours in vitro respectively. Control group and untransfected group were negative.Ⅲ Immunological activity of the expressed TGF-β1 in corneal cell supernatantObjectiveTo study the immunological property of expressive production from transfected corneal endothelial cells with TGF-β1 MP gene.
Methods1. To prepared the splenic lymphocytes suspension of mice.2. To collect the supernatant of transfected endothelial cells, were activated by HCl and used to experiment.3. The effect of transfected cells supernatant on the splenic lymphocytes activated by ConA was measured by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazoliym bromide (MTT) colorimetric assay in vitro. First the most conformable concentration of ConA to activate lymphocytes was confirmed to be 5μg/ml. The splenic lymphocytes suspension was divided into four groups as follows: transfect gene group, transfect empty plasmid group, normal control group, and blank control group.4. The effect of transfected cells supernatant on the apoptosis of splenic lymphocyte was detected with Flow cytometry. The splenic lymphocytes suspension was divided into four groups as above. 10ng/ml TGF-β1 solution was used as positive control group.5. The effect of transfected cells supernatant on the apoptosis of splenic lymphocyte was detected with agarose gels electrophoresis. The splenic lymphocytes was divided into four groups as above, and was collected respectively used to extract the gene group DNA.6. The effect of transfected cells supernatant on the transformation of lymphocyte subsets was detected with Flow cytometry. The splenic lymphocytes was divided into four groups as above, and every group was stained by different fluorescent antibody at four time points. CD69-PE/CD3-FITC were used in 4h group; CD25-PE/CD3-FTTC were used in 24h group; CD25-PE/CD4-FITC, CD25-PE/ CD8-FITC、 CD71-PE/CD4-FTTC、 CD71-PE/CD8-FITC were used in 48h group; CD71-PE /CD3-FTTC was used in 72h group.7. The effect of transfected cells supernatant on the types of cytokine expressed by lymphocytes was detected by reverse reanscriptase-polymerse chain reaction (RT-PCR). Total RNA was isolated from the lymphocytes of normal control group and
transfected gene group, then reverse-transcribed into cDNA. Design the specific primer of Th1 cytokine (IL-2、 IFN- γ、 TNF-α ) 、Th2 cytokine (IL-4, IL-6), and β -actin. Amplification of PCR reactions was conducted.Results1. Comparing with the other groups, OD value of 5μg/ml ConA group was the highest, transfected groups was the lowest. OD value were no difference among the other groups.2.There was a obvious subdiploid peak in the transfected gene group and 10ng/ml rhTGF-β1 group, was not appeared in transfected empty plasmid group and normal control group. Apoptosis rate of transfected gene group and 10ng/ml rhTGF-β1 group were higher than that of normal control group.3. Emblematical DNA apoptosis band(Ladder band) appeared in agarose gels electrophoresis of transfected gene group, not in transfected empty plasmid group and normal control group.4. Comparing with control group, CD69+CD3+、 CD25+CD3+、 CD71+CD3+ lymphocytes rate of transfected gene group decreased. 10ng/ml rhTGF-pi decreased CD69+CD3+ 、 CD25+CD3+rate more seriously. Comparing with control group, CD25+CD4+、 CD71+CD4+ lymphocytes rate of transfected gene group decreased significantly, CD25+CD8+、 CD71+CD8+ lymphocytes rate had no varieties.5. Compared with negative control group and untransfected group, the expression of IL-2、 IFN- γ、 TNF-α was decreased, the expression of IL-4、 IL-6 was increased in mice spleen lymphocytes of transfected gene group.Conclusion1. The high expression of recombinant TGF-β1 from the transfected corneal cell possessed negative immunological activity which maybe benefit for suppressing immune rejection.2. TGF-β1 secreted from the transfected corneal cell could restrain the transformation of lymphocytes activated by ConA, induce apoptosis of mice spleen lymphocyte through suppressing the differentiation and expression of CD69+, CD25+,
CD71+ spleen T cells.3. TGF-β1 secreted from the transfected corneal cell could decreased the expression of Th1 cytokine, enhanced the expression of Th2 cytokine significantly. Therefore could induce lymphocytes immmue deviation from Thl to Th2.Part Four Study on constructing and transfecting TGF-β1MP gene in vitro deep lamellar endothelial keratoplastyObjectiveTo evaluate the subsistence of transfected corneal stromal cells and endothelial cells on the frozen and preserved in glycerin porcine corneal stroma. GFP gene transfection was used to detect the subsistence and distribution of transfected cells after transplantation in living cornea tissue to indicate whether the exogenous gene could express normally and inhibit graft rejection or not.Methods1. Cultured rabbit corneal stromal cells and endothelial cells. When reaching 60-70% confluence, cells were co-transfected with pEGFP-N1 and the recombinant expression vectors pSecTag2-TGFβ1MP through Lipofectamine reagent.2. The resistant clones expressing stably were established under the selection of G418 and Hygromycin B.3.Obtaining fresh porcine corneal stroma, 150μm thickness, 7.25mm diameter, preserving DM/posterior stroma and endothelial cell lay, stored at -80℃ for 3 days, dealt with dispase Ⅱ and was thawed and preserved in glycerine for 1-2 months. The carrier was washed with PBS, was sterilized by ultraviolet ray before using.4. The corneal transplantation model of New Zealand white rabbit was set up, and was divided into 3 groups as follows. Group A(control group, n=10): the seed cells were untransfected cells. Group B(pSecTag2+pEGFP-N1, n=10): the seed cells were cells transfected empty plasmid. Group C(pSecTag2-TGFβ1MP+pEGFP-N1, n= 10): the seed cells were transfected TGF-β1MP gene cells.
5. The positive expression corneal stromal cells were seeded onto stroma surface of the carrier, then the positive expression corneal endothelial cells were seeded onto DM/stroma surface of the carrier with cell density of more than 2× 106/ml and cultured in vitro. The carriers of control group were seeded with cells transfected empty plasmid, negative control group were seeded no cells.6. A centered, hinged flap 9mm diameter, two-thirds thickness of cornea was made, the flap was retracted superiorly and the central posterior lamellar were excised using a 7.25mm trephine. The donor button was placed in the recipient bed and sutured interruptedly using 10-0 suture which can be absorbed in eye tissue. The outer corneal flap was sutured back into place using 10-0 nylon.7. Postoperative observation: The operated eyes were observed with slit-lamp microscope everyday since the first day after operation, continuing 3 months. The rejection indexes(RI) that included the corneal clarity, edema and vascularization, and the rejection time of each group were compared with each other.8. Observing expression GFP of endothelial cells with fluorescence light microscope (FLM). The structure, ultrastructure and expression of TGF- β1 of corneal implant was detected by HE staining , immunohistochemical SABC, scanning electron microscopy (SEM) examination.Results1. Transfected rabbit corneal stromal cells could grow on the porcine stroma carrier, culturing 3-4 hours cells adhesion carrier, culturing 7 days cells can grow among carrier fiber. Transfected rabbit corneal endothelial cells could attach on the heterogenic DM/stroma carrier and formed a confluent of cuboidal- shaped cells after 7 days in culture.2. The GFP positive expression of transfeced stromal cells and endothelial cells radiating green fluorescence could been observed by FLM on the porcine DM/stroma carrier lasting 3 months.3. Slit-lamp microscope examination shows: Group A: The corneal graft rejection appeared on the 35th day after transplantation, reached a peak from the 38th
day to 44th day. The graft became opaque and cloudy. There were plentiful neovascular around the graft. Group B: The corneal graft rejection appeared on the 31th day after transplantation, reached a peak from the 33th to 39th day. The graft became opaque and cloudy badly. There were also plentiful neovascular around the graft. Group C: Two grafts rejected during observation period, the first one appeared on the 59th day, one eye was neovascularized, eight grafts maintained clear for 3 months.4. The survival time of group C was longest (average 85 days), group B was shortest (average 38 days). There were no distinct difference between group C (average 43days) and group B.5. The rejection index of group A and group B increased continually, had significant difference with group C.6. There was green fluorescence appeared in graft of group B and C, was not in group A. 1 month after grafting, the distributing of fluorescence was limited in the join of donor graft and receptor corneal bed, presenting bright green. 2 months after grafting, green fluorescence dispersed to around, became weak. 3 months after grafting, the join had came to mistiness.7. HE staining shows: 1 month after grafting, the grfts of group A and group B were thickened , the epithelial cells were absent partially, corneal stroma were edemaous and there were a lot of infiltrating lymphocytes in it. Contrarily, stroma edema was gentle, infiltrating lymphocytes were few in the group A. 2 months after grafting, the grafts of group A and group B were more thicker, edematous more hardly, the inflammatory cells were more, and neovascularization in stroma. In group C, grafts were edematous slightly, inflammatory cells spread around. 3 months after grafting, edema and inflammatory cells decreased in the group A and group B. The inflammatory cells of group C decreased significantly.8. Immunohistochemistry staining shows:In group A and B,the cells of TGF- β1 positive staining were few, increased gradually following the time prolonged after graftion until 2 month, then decreased gradually. In group C, positive brown cells was detected largely in the grafts within one month after grafting, then decreased gradually,
but could be observed in all lays of deep lamellar corneal graft at least 3 months.9. SEM examination shows: At 1st month, the endothelial cells connected tightly, appeared cuboidal shape could been found in every group. The cell amounts of the group A and group B decreased gradually, there was aperture appeared.The endothelial cells of group C changed unconspicuously.Conclusion1. GFP could express stably in vivo, indicate that a majority of transfected endothelial cells could be survival after transplantion keeping stable expression and secretion at least 3 months.2. The frozen and preserved in glycerin porcine corneal stroma carrier is benefit for culturing corneal endothelia cells and stromal cells to adhere and migrate. It is the excellent carrier material to reconstruct artificial biologic cornea.3. The transfergenic corneal cells could proliferate normally, expressing TGF-β1 effectively and continually. The superfluous expression of TGF-β1 had no effect on the histological structure and biologic function obviously.4. First indicate that high expression of TGF-β1 in transplanted corneal cells could suppress immune rejection and prolong the survival time of corneal plasty.
应用免疫抑制剂是目前预防和治疗角膜移植术后免疫排斥反应的主要手段,常用药物有皮质类固醇、环孢霉素A(Cyclosporin A,CsA)、FK506等,这些药物的联合使用能够有效地抑制排斥反应,但长期使用对机体有很大的毒副作用。故而,开发探索新型强效免疫抑制剂是提高异体或异种角膜移植手术成功率的有效途径。转化生长因子β_1(Transforming growth factor-β_1,TGF-β_1)作为一种比CsA活性更高的免疫抑制剂(Ruegemer等报道),越来越引起人们的关注。TGFβ_1是多肽类细胞因子,广泛参与免疫系统各细胞、因子间的相互调节,可抑制胸腺细胞、淋巴细胞、自然杀伤细胞等的增殖分化,干扰多种免疫球蛋白的产生和转换,并通过直接、间接影响IL-1、IL-2、IFN-γ及其受体而起以“负调节”为主的免疫平衡作用。近来,TG-β_1,作为一种强效免疫抑制因子己被用于器官移植对抗免疫排斥反应、诱导移植物局部的免疫耐受,在小鼠和大鼠同种异体心脏移植及胰岛细胞移植模型中应用TGF-β_1均能明显延长移植物存活时间,取
Keratonosus is the main eye disease that can lead to blindness. There are about 3.5 million blind patients caused by corneal diseases in China whose blindness incidence rate is the highest in the world. At present the most important treatment to reconstruct useful vision is corneal allogenic transplantation. Although keratoplasty has a high successful rate, the corneal allogeneic transplantation rejection rate is only 5-10%under the condition of vascular recipient beds, yet in the high-risk cases is higher than 65%. The high-risk factors include vascularized cornea, prolonged inflammation, severe injury of the cornea and conjunctiva, large size keratoplasty, and re-keratoplasty. Moreover, However, corneal grafts were limited to perform due to the shortage of cornea materials. Therefore it becomes a hot issue for ophthalmology researchers to find new cornea tissue resources and reduce allograft rejection.The application with the immunosuppressions is now the main methods to prevent and treat corneal allograft rejection after the keratoplasty, including hormone, Cyclosporin A, FK506, and so on. The associated application of these medicine can suppress immune rejection effectively, but side-effect is high if long term application. Therefore searching for high-effective and low-toxic immunosuppressive agent is effective method to increase the success rate of corneal allograft or heterograft. TGF-β1 (transforming growth factor- β1) is a multifunctional peptide with immunosuppressive property. These properties include inhibition of B and T cell proliferation, adherence of granulocytes, respiratory burst of macrophage, the synthesis of IL-1,
IL-2, IFN- γ, and HLA gene expression. It plays a central role in inhibition of inflammatory and alloreactive immune response. It was reported that the immunosuppressive property of TGF- β1 is more stronger than Cyclosporine A by Prof. Ruegemer and his colleagues. TGF- β1 has been used to antagonize allograft rejection as one of the most effective immunosuppressive cytokines recently. A study shows that TGF- β1 can induce the immunological tolerance, and the application to the allogenic heart transplantation and pancteas islet cell transplantation model in the mice and rats can significantly prolong the allograft survival. In addition, TGF- β1 can induce anterior chamber associated immune deviation(ACAID), inhibit antigen specific delayed-type hypersensitivity response (DTH) and protect eye from the impairment of immune reaction.But exogenous growth factor is diluted and metabolized quickly when used in part because of short half-life. As a result large quantity of growth factor have to be applied repeatedly. Therefore, it becomes a hot issue and key for tissue engineering research to find how to keep growth factor acting continually and effectually. Dr.GuoXiaodong and his colleagues transfer the TGF- β1 gene into MSCs (mesenchymal stem cell) for the first time in a study of articular cartilage repairing, which was the first attempt to combine the principles of tissue engineering with principles of molecular biology organically. The results showed that the repair was histologically and ultrastructurally better for TGF- β1 gene modified group than for control group, indicating that TGF- β1 as an immunosuppressive cytokines also has the potential to prolong the allograft survival. Artificial biologic cornea (also called tissue engineering cornea) is a normal cornea equivalent which is reconstructed by three-dimension culture in vitro with artificial materials or biologic materials as carrier and technology of tissue and cell engineering. With the development of life science and profounding research, artificial biologic cornea will be the substitute for human donor cornea. This will be quite beneficial for corneal disease treatment and of great clinical importance and practical value.On the base of researches in the past, this study will transfer TGF-β1MP ( Transforming growth factor-beta 1 Mature Peptide) gene into seed cell of tissue
engineering cornea, then planted to the frozen and preserved in glycerine porcine corneal stromal as carrier for constructing transfected artificial biologic cornea used for transplantation. The aim of this study is to resolve the shortage of corneal materials and search a new gene therapy method for reducing corneal graft rejection. A novel concept, molecular tissue engineering cornea was put forward for the fist time. As a rising branch it represents both a new domain and a significant trend of tissue engineering research. This study mentioned above has not been reported in the references read.Part One Construction of eukaryotic high expression vector containing TGF-β1MPObjectiveTo construct the eukaryotic expression vectors of the secreted TGF-β1 cDNA and to express high effectively the recombinant proteins in the corneal endothelial cells and corneal stromal cells cultured in vitro.MethodsExpression vectors pSecTag2-TGFβ1MP, based on the pSecTag2/HygroA, were constructed by way of molecular cloning.1. Vectors pSecTag2/ HygroA was digested to obtain linear vectors by restriction enzyme SfiI and HindIII.2. According to the gene sequence of hTGF-β1MP, design the primer used to amplification of PCR reactions to obtain TGF-β1MP gene fragment. The pET3C-TGFβ1MP plasmid vector was used as model.3. The resulting PCR products were separated by electrophoresis in agarose gels, then were digested by restriction enzyme SfiI and HindIII.4. Linear vector pSecTag2/ HygroA and TGF-β1MP gene fragment were linked by ligase.5. The recombination vector that TGF-β1MP gene fragment had correctly been
inserted into pSecTag2/HygroA was identified by digestant, PCR reaction and sequencing.Results1. Vectors pSecTag2/HygroA was digested by SfiI and HindIII to obtain two fragments whose length were 4.7kb and 1.0kb respectively, digested by Hindlll to obtain one fragment whose length was 5.7kb.2. Two of four recombination vectors were digested by SfiI and BgIII to obtain two fragments whose length were 4.7kb and 1350bp respectively. The second step, the recombination vectors that were maybe masculine can be amplificated to obtain about 366bp gene fragments. The third step, the masculine plasmid was certified that its sequences of vector segment , inserted segment and digested point were consistent with reports completely.ConclusionAcquired recombination vectors pSecTag2-TGFβ1MP lays a foundation for TGF-β1MP gene transfection into deep lamellar artificial biologic cornea.Part Two Effects on biological activity of cultured corneal cells in vitro produced by TGF- β1ObjectiveBefore transfection of TGF-β1MP gene, to explore effects on biological activity of cultured three kinds of corneal cells in vitro produced by TGF-β1, in order to prevent and reduce the other side-effects at the same time to make use of its immunosuppressive functionMethods1. Epithelial cells, stromal cells and endothelial cells were isolated from New Zealand rabbits cornea respectively then were put into the incubator at 37 ℃ in a humidified atmosphere containing 5% CO2. Conformation of cultured cells were
examined and recorded regularly.2. The cultured cells were HE stained, observed with optic microscope.3. Immunohistochemistry examination was made to identify the category of cells. Three peculiar antibodies were used which were monoclonal antibody Keratin AE1/AE3, monoclonal antibody Vimentin monoclonal antibody Neurofilament respectively.4. Effect on proliferation of three kinds of corneal cells produced by TGF- β1 was measured by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay in vitro.Results1. Inverted phase contrast microscope examination shows:1.1 After 2 days in culture containing rhTGF- β1, epithelial cells had successfully migrated from the limbal explant onto the floor of 6-well culture plate. After 3-4 days in culture formed a rim around the limbal fragment 4-5mm wide. After 6-7 days in culture covered the whole plate.1.2 After 6 hours in culture containing rhTGF- β1, stromal cells had began to explant onto the floor of the culture plate. After 24 hours cells extended gradually, after 3 days cells grew vigorously.1.3 After 2 days in culture containing rhTGF- β1, endothelial cells had successfully migrated from the Descemet's membrance(DM) onto the floor of 6-well culture plate. After 3 days in culture formed a rim around the limbal fragment 4-5mm wide. After 6-7 days in culture covered the whole plate.2. HE staining shows: The configuration of three kinds of corneal cells cultured in culture medium containing rhTGF- β1 were close to normal cells. Cells connected closely, stained densely in the karyon. There were 2-3 nucleolus seemed clearly.3.1mmunohistochenistry examination shows: The cytoplasm of Epithelial cells, stromal cells and endothelial cells cultured in culture medium containing rhTGF- β1 were stained to brown by peculiar monoclonal antibody respectively.4.MTT colorimetric assay shows:
4.1 The solution of 20ng/ml, 10ng/ml, 5ng/ml, 1ng/ml ATGF-β1 was able to inhibit the proliferation of the epithelial cells in vitro. The suppressive effect of 10ng/ml group is the most strong. The solution of 0.5ng/ml, 0.1ng/ml rhTGF- β1 had no toxicity to these cells. The solution of 0.05ng/ml rhTGF- β1 was able to accelerate the proliferation of these cells in vitro.4.2 The solution of rhTGF- β1 no matter what concentration was all able to promote the proliferation of corneal stromal cells in vitro. The stimulative effect 10ng/ml group is the most strongest, became a peak value.4.3 The solution of 20ng/ml, 10ng/ml, 5ng/ml rhTGF- β1 was able to inhibit the proliferation of the endothelial cells in vitro. The suppressive effect of 10ng/ml group is the most strongest. The solution of 1 ng/ml, 0.5ng/ml rhTGF- β1 had no toxicity to these cells. The solution of 0. 1 ng/ml, 0.05ng/ml rhTGF- β1 was able to accelerate the proliferation of these cells in vitro.Conclusion1. The three kinds of corneal cells cultured in vitro were resemble normal corneal cells in shape, histological structure and immuncyto- chemistry.2. The effect on the proliferation and growth period of corneal cells cultured in vitro depends on the concentration of exogenous rhTGF- β11:2.1 The effect on the epithelial cells: The higher concentration group suppressed proliferation, 10ng/ml group is the most strongest. The concentration became higher the suppressive effect became weaker. Medium concentration group had no effect on cells. The lower concentration group accelerated proliferation.2.2 The effect on the corneal stromal cells: All concentration group accelerated proliferation. 10ng/ml group is a peak value.2.3 The effect on the endothelial cells: The higher concentration group suppressed proliferation, 10ng/ml group is the most strongest. The concentration became higher the suppressive effect became weaker. Medium concentration group had no effect on cells. The lower concentration group accelerated proliferation.
Part Three Transfection and expression of exogenous TGF-β1MP gene in corneal cellsI TGF-β1MP gene transfected to corneal cells ObjectiveTo study the transfection efficiency of exogenous TGF-β1MP gene in corneal stromal and endothelial cells and to observe the effect on proliferation induced by transfection. To co-transfect pEGFP-N1 plasmid acted as indicator system in living corneal tissue.Methods1. Plasmid pEGFP-N1 were amplified and were detected by Vsp1 and EcoR I respectively.2. Cultured rabbit corneal stromal and endothelial cells in vitro.3. Confirm the concentration of G418 and Hygromycin B for filtrating.4. At the time of reaching 90-95% confluence, cells were co-transfected with pEGFP-N1 and the recombinant expression vectors pSecTag2-TGFβ1MP though Lipofectamine reagent.5. The resistant clones secreting the recombinant proteins were established with G418(200μg/ml) and Hygromycin B(150μg/ml) screening.6. Extending culture of the cell clones: When the cell clones had appeared, the solution was changed with DMEM-F12 without screening reagent. After inosculation, the cells were trypsinized with 0.25% trypsin/0.1% EDTA solution, then cultured extendly. The corneal stromal cell clone was named Fibro-cc, the endothelial cell clone was named Endo-CC. The endothelial cell clone that cultured with lOng/ml EGF was named Endo-CC-EGF.7. To observe the cell configuration with inverted phase contrast microscope.8. The transient expression of transgene with fluorescence microscope (FLM): Accomplishing the transfection, changed culture medium to DMEM-F12 without
screening reagent and cultured cells sequentially. At the time of 12 hours, 24 hours, 48hours, 96hours after gene transfer, the expression of GFP in corneal stromal cells and endothelial cells was detected with fluorescence microscope respectively.9. To observe the morphological change of transfected cells with HE staining.10. GFP-positive rate was detected by Flow cytometry examination. To collect the cells of normal control group and transient transfected groups (12 hours, 24 hours, 48hours, 96hours) and stably transfected groups (Fibro-cc, Endo-CC, Endo-CC-EGF), then examined by Flow cytometry. The GFP-positive rates were analyzed with cell-quest software.11. The change of cell cycle were examined by flow cytometry: To collect the cells cultured for 48 hours of normal control group, transfected empty plasmid group and stably transfected groups (Fibro-cc, Endo-CC, Endo-CC-EGF) with the density of 1 X 106cells/ml, then examined by Flow cytometry. Calculate the proliferation index(PI) according to the formula: PI= (S +G2M) / (GoG1 + S + G2M) ×10012. To detect cells apoptosis with DNA electrophoresis in agarose gels: The corneal endothelial cells were incubated with 160mM/L Sodiym arsenite(Ars) to form a endothelial cells apoptosis model. The corneal stromal cells were irradiated with 320nm Ultroviolet B(UVB) to form a stromal cells apoptosis model.Results1. Plasmid pEGFP-N1 were digested by VspI and EcoRI to produce two bands, about 4.6kb and 600bp respectively.2. The resistant clones secreting the recombinant proteins were obtained by cultured with G418(200μg/ml) and Hygromycin B (150μg/ml) for 14 days.3. GFP-positive cells were not detected in the normal control group and transfected empty plasmid group. GFP-positive expression of transient transfected groups(12 hours, 24 hours, 48hours, 96hours) were detected. GFP-positive rate of 48 hours groups were the highest, 31.0% and 24.5% respectively. GFP-positive rate of stably groups were all 100%.
4. After 2 hours in transfection, some cells fell off from plate. The configuration of most cells had no obvious difference with normal cells. Endo-CC-EGF group growth rapidly than Endo-CC group, magnitude and arrangement were more consistent.5. The GFP expression in endothelial cells and stromal cells could be detected in 12 hours after transfection by fluorescence microscope, 24 hours GFP expression increased, 48 hours green fluorescence were strongest, 96 hours could observed green colored cells was only 3-4cell/eyeshot. There were no green colored cells in the control group and transfected empty plasmid group. Dense green fluorescence could been detected in the stably transfected groups, Endo-CC-EGF group were stronger than Endo-CC group.6. Endo-CC-EGF of one time passage were multiangular, Fibro-cc of one time passage were shuttle shape. Cells grew flourishingly and connected tightly.7. Flow cytometry assay shows: There was no subdiploid peak appeared in the flow cytometry assay graph of transfected corneal cells. Compared with the control group, the cellular proportion of transfected group decreased in S phase and G2/M phase significantly(PI value decreased). Compared with the transfected group, the cellular proportion of transfected+EGF group increased in S phase and G2/M phase significantly(PI value increased). There was no distinct difference between control groups and transfected groups.8. No Ladder-bands(DNA bands of apoptosis cells) were detected in DNA electrophoresis in agarose gels of normal control group and transfected group without reference to corneal stromal cells or endothelial cells. Contrarily, emblematical Ladder-bands appeared in the positive control group.Conclusion1. The plasmids of pSecTaq2-TGFβ1MP and pEGFP — N1 have been successfully transfected into cultured corneal stromal cells and endothelial cells in vitro, can express transiently or stably.2. GFP-positive rate of 48 hours groups were the highest, 31.0% and 24.5%
respectively. GFP-positive rate of stably groups were all 100%.3. Lipofectamine 2000 reagent was safe and effective to transfer gene into corneal cells, had no effect on morphology and proliferation.4. The transfected TGF-β1 gene expressed in high level, decreased the proliferation of corneal endothelial cells, increased the proliferation of corneal stromal cells, had no apoptosis effect on the two kinds of cells.5.At first time, indicated that 10ng/ml EGF could counteract the suppressive effect of TGF- β1 expressed unduly, made the proliferation of endothelial cells become to normal.II Expression of exogenous TGF-β1MP gene in corneal cellsObjectiveTo detect the expression of exogenous TGF-β1MP gene in corneal stromal and endothelial cells.Methods1. Total RNA was isolated from the cells cultured for 48 hours of normal control group, transfected empty plasmid group and stably transfected groups (Fibro-cc, Endo-CC-EGF) with the density of 1 × 106cells/ml, and reverse-transcribed into cDNA. Amplification of PCR reactions was conduced. The resulting PCR products were separated by electrophoresis in 2% agarose gels.2. The expression of TGF-β1 protein within the corneal endothelial cells was detected by Western blotting and immunohistochemical SABC.3. The secreted TGF-β1 protein concentration in the corneal endothelial cell supernatant was assayed by ELISA. To obtain the standard curve by nonlinear regression analyse.Results1. TGF-β1MP gene was confirmed to have been integrated and expressed in transfected corneal stromal cells and endothelial cells on the level of mRNA through
the positive appearance in the agarose gels electrophoresis.2. The expression of TGF-β1 protein was deteced with Western blotting. Positive brown band of 25KD appeared in transfected group, not in control group and untransfected group. The staining became deeper and deeper with the extending of expressing time.3. The expression of TGF-β1 protein was deteced with immunohistochemical SABC examination. Positive cells were stained to brown. Control group and transfected empty plasmid group were negative.4. Linear regression equation was obtained: OD = 0. 001C-0. 0344 ( R = 0. 998) According to the equation, could calculate the concentration of the secreted TGF-β1 protein in the corneal endothelial cell supernatant. The concentration of transfected endothelial cells was (597 ±7)pg/ml, the concentration of transfected stromal cells was (995 ± 12)pg/ml, the value was zero in control group and transfected empty plasmid group.Conclusion1. TGF-β1MP gene was confirmed to have integrated and expressed in the transfected corneal endothelial cells and stromal cells on levels of mRNA and protein.2. The expressed amount of TGF-β1 in the transfected corneal stromal cells and endothelial cells supernatant were (995 ± 12)pg/ml and (597 ± 7)pg/ml after cells had been cultured for 48 hours in vitro respectively. Control group and untransfected group were negative.Ⅲ Immunological activity of the expressed TGF-β1 in corneal cell supernatantObjectiveTo study the immunological property of expressive production from transfected corneal endothelial cells with TGF-β1 MP gene.
Methods1. To prepared the splenic lymphocytes suspension of mice.2. To collect the supernatant of transfected endothelial cells, were activated by HCl and used to experiment.3. The effect of transfected cells supernatant on the splenic lymphocytes activated by ConA was measured by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazoliym bromide (MTT) colorimetric assay in vitro. First the most conformable concentration of ConA to activate lymphocytes was confirmed to be 5μg/ml. The splenic lymphocytes suspension was divided into four groups as follows: transfect gene group, transfect empty plasmid group, normal control group, and blank control group.4. The effect of transfected cells supernatant on the apoptosis of splenic lymphocyte was detected with Flow cytometry. The splenic lymphocytes suspension was divided into four groups as above. 10ng/ml TGF-β1 solution was used as positive control group.5. The effect of transfected cells supernatant on the apoptosis of splenic lymphocyte was detected with agarose gels electrophoresis. The splenic lymphocytes was divided into four groups as above, and was collected respectively used to extract the gene group DNA.6. The effect of transfected cells supernatant on the transformation of lymphocyte subsets was detected with Flow cytometry. The splenic lymphocytes was divided into four groups as above, and every group was stained by different fluorescent antibody at four time points. CD69-PE/CD3-FITC were used in 4h group; CD25-PE/CD3-FTTC were used in 24h group; CD25-PE/CD4-FITC, CD25-PE/ CD8-FITC、 CD71-PE/CD4-FTTC、 CD71-PE/CD8-FITC were used in 48h group; CD71-PE /CD3-FTTC was used in 72h group.7. The effect of transfected cells supernatant on the types of cytokine expressed by lymphocytes was detected by reverse reanscriptase-polymerse chain reaction (RT-PCR). Total RNA was isolated from the lymphocytes of normal control group and
transfected gene group, then reverse-transcribed into cDNA. Design the specific primer of Th1 cytokine (IL-2、 IFN- γ、 TNF-α ) 、Th2 cytokine (IL-4, IL-6), and β -actin. Amplification of PCR reactions was conducted.Results1. Comparing with the other groups, OD value of 5μg/ml ConA group was the highest, transfected groups was the lowest. OD value were no difference among the other groups.2.There was a obvious subdiploid peak in the transfected gene group and 10ng/ml rhTGF-β1 group, was not appeared in transfected empty plasmid group and normal control group. Apoptosis rate of transfected gene group and 10ng/ml rhTGF-β1 group were higher than that of normal control group.3. Emblematical DNA apoptosis band(Ladder band) appeared in agarose gels electrophoresis of transfected gene group, not in transfected empty plasmid group and normal control group.4. Comparing with control group, CD69+CD3+、 CD25+CD3+、 CD71+CD3+ lymphocytes rate of transfected gene group decreased. 10ng/ml rhTGF-pi decreased CD69+CD3+ 、 CD25+CD3+rate more seriously. Comparing with control group, CD25+CD4+、 CD71+CD4+ lymphocytes rate of transfected gene group decreased significantly, CD25+CD8+、 CD71+CD8+ lymphocytes rate had no varieties.5. Compared with negative control group and untransfected group, the expression of IL-2、 IFN- γ、 TNF-α was decreased, the expression of IL-4、 IL-6 was increased in mice spleen lymphocytes of transfected gene group.Conclusion1. The high expression of recombinant TGF-β1 from the transfected corneal cell possessed negative immunological activity which maybe benefit for suppressing immune rejection.2. TGF-β1 secreted from the transfected corneal cell could restrain the transformation of lymphocytes activated by ConA, induce apoptosis of mice spleen lymphocyte through suppressing the differentiation and expression of CD69+, CD25+,
CD71+ spleen T cells.3. TGF-β1 secreted from the transfected corneal cell could decreased the expression of Th1 cytokine, enhanced the expression of Th2 cytokine significantly. Therefore could induce lymphocytes immmue deviation from Thl to Th2.Part Four Study on constructing and transfecting TGF-β1MP gene in vitro deep lamellar endothelial keratoplastyObjectiveTo evaluate the subsistence of transfected corneal stromal cells and endothelial cells on the frozen and preserved in glycerin porcine corneal stroma. GFP gene transfection was used to detect the subsistence and distribution of transfected cells after transplantation in living cornea tissue to indicate whether the exogenous gene could express normally and inhibit graft rejection or not.Methods1. Cultured rabbit corneal stromal cells and endothelial cells. When reaching 60-70% confluence, cells were co-transfected with pEGFP-N1 and the recombinant expression vectors pSecTag2-TGFβ1MP through Lipofectamine reagent.2. The resistant clones expressing stably were established under the selection of G418 and Hygromycin B.3.Obtaining fresh porcine corneal stroma, 150μm thickness, 7.25mm diameter, preserving DM/posterior stroma and endothelial cell lay, stored at -80℃ for 3 days, dealt with dispase Ⅱ and was thawed and preserved in glycerine for 1-2 months. The carrier was washed with PBS, was sterilized by ultraviolet ray before using.4. The corneal transplantation model of New Zealand white rabbit was set up, and was divided into 3 groups as follows. Group A(control group, n=10): the seed cells were untransfected cells. Group B(pSecTag2+pEGFP-N1, n=10): the seed cells were cells transfected empty plasmid. Group C(pSecTag2-TGFβ1MP+pEGFP-N1, n= 10): the seed cells were transfected TGF-β1MP gene cells.
5. The positive expression corneal stromal cells were seeded onto stroma surface of the carrier, then the positive expression corneal endothelial cells were seeded onto DM/stroma surface of the carrier with cell density of more than 2× 106/ml and cultured in vitro. The carriers of control group were seeded with cells transfected empty plasmid, negative control group were seeded no cells.6. A centered, hinged flap 9mm diameter, two-thirds thickness of cornea was made, the flap was retracted superiorly and the central posterior lamellar were excised using a 7.25mm trephine. The donor button was placed in the recipient bed and sutured interruptedly using 10-0 suture which can be absorbed in eye tissue. The outer corneal flap was sutured back into place using 10-0 nylon.7. Postoperative observation: The operated eyes were observed with slit-lamp microscope everyday since the first day after operation, continuing 3 months. The rejection indexes(RI) that included the corneal clarity, edema and vascularization, and the rejection time of each group were compared with each other.8. Observing expression GFP of endothelial cells with fluorescence light microscope (FLM). The structure, ultrastructure and expression of TGF- β1 of corneal implant was detected by HE staining , immunohistochemical SABC, scanning electron microscopy (SEM) examination.Results1. Transfected rabbit corneal stromal cells could grow on the porcine stroma carrier, culturing 3-4 hours cells adhesion carrier, culturing 7 days cells can grow among carrier fiber. Transfected rabbit corneal endothelial cells could attach on the heterogenic DM/stroma carrier and formed a confluent of cuboidal- shaped cells after 7 days in culture.2. The GFP positive expression of transfeced stromal cells and endothelial cells radiating green fluorescence could been observed by FLM on the porcine DM/stroma carrier lasting 3 months.3. Slit-lamp microscope examination shows: Group A: The corneal graft rejection appeared on the 35th day after transplantation, reached a peak from the 38th
day to 44th day. The graft became opaque and cloudy. There were plentiful neovascular around the graft. Group B: The corneal graft rejection appeared on the 31th day after transplantation, reached a peak from the 33th to 39th day. The graft became opaque and cloudy badly. There were also plentiful neovascular around the graft. Group C: Two grafts rejected during observation period, the first one appeared on the 59th day, one eye was neovascularized, eight grafts maintained clear for 3 months.4. The survival time of group C was longest (average 85 days), group B was shortest (average 38 days). There were no distinct difference between group C (average 43days) and group B.5. The rejection index of group A and group B increased continually, had significant difference with group C.6. There was green fluorescence appeared in graft of group B and C, was not in group A. 1 month after grafting, the distributing of fluorescence was limited in the join of donor graft and receptor corneal bed, presenting bright green. 2 months after grafting, green fluorescence dispersed to around, became weak. 3 months after grafting, the join had came to mistiness.7. HE staining shows: 1 month after grafting, the grfts of group A and group B were thickened , the epithelial cells were absent partially, corneal stroma were edemaous and there were a lot of infiltrating lymphocytes in it. Contrarily, stroma edema was gentle, infiltrating lymphocytes were few in the group A. 2 months after grafting, the grafts of group A and group B were more thicker, edematous more hardly, the inflammatory cells were more, and neovascularization in stroma. In group C, grafts were edematous slightly, inflammatory cells spread around. 3 months after grafting, edema and inflammatory cells decreased in the group A and group B. The inflammatory cells of group C decreased significantly.8. Immunohistochemistry staining shows:In group A and B,the cells of TGF- β1 positive staining were few, increased gradually following the time prolonged after graftion until 2 month, then decreased gradually. In group C, positive brown cells was detected largely in the grafts within one month after grafting, then decreased gradually,
but could be observed in all lays of deep lamellar corneal graft at least 3 months.9. SEM examination shows: At 1st month, the endothelial cells connected tightly, appeared cuboidal shape could been found in every group. The cell amounts of the group A and group B decreased gradually, there was aperture appeared.The endothelial cells of group C changed unconspicuously.Conclusion1. GFP could express stably in vivo, indicate that a majority of transfected endothelial cells could be survival after transplantion keeping stable expression and secretion at least 3 months.2. The frozen and preserved in glycerin porcine corneal stroma carrier is benefit for culturing corneal endothelia cells and stromal cells to adhere and migrate. It is the excellent carrier material to reconstruct artificial biologic cornea.3. The transfergenic corneal cells could proliferate normally, expressing TGF-β1 effectively and continually. The superfluous expression of TGF-β1 had no effect on the histological structure and biologic function obviously.4. First indicate that high expression of TGF-β1 in transplanted corneal cells could suppress immune rejection and prolong the survival time of corneal plasty.
引文
1. Williams KA, Jessup CE Coster DJ. Gene therapy approaches to prolonging corneal allograft survival. Expert Opin Biol Ther. 2004, 4(7): 1059-1071.
2. Zhang EP, Franke J, Schroff M, Junghans C, Wittig B, Hoffmann F. Ballistic CTLA4 and IL-4 gene transfer into the lower lid prolongs orthotopic corneal graft survival in mice. Graefes Arch Clin Exp Ophthalmol, 2003, 241(11): 921-926.
3.郭萍.谢立信,史伟云,黎晓新.肿瘤坏死因子相关凋亡诱导配体转基因治疗鼠角膜移植免疫排斥的研究.中华眼科杂志,2003,39(1):19-23.
4. Klebe S, Sykes PJ, Coster DJ, Krishnan R, Williams KA. Prolongation of sheep corneal allograft survixal by ex vivo transfer of the gene encoding interleukin-10. Transplantation, 2001, 71(9): 12414-1220
5. Larkin DF, Oral HB, Ring CJ, Lemoine NR. George AJ. Adenovirus-mediated gene delivery to the corneal endothelium. Transplantation, 1996, 61(3): 363-370.
6. Comer RM, King WJ, Ardjemand N. Effect of Administration of CTLA4-Ig as protein or cDNA on corneal allograft. Invest Ophthalmol Vis Sci, 2002, 43(4): 1095-1103
7. Oral HB, Larkin DE Fehervari Z, Byrnes AP, Rankin AM, Haskard DO, Wood MJ, Dallman M J, George AJ. Ex vivo aderovirus-mediated gene transfer and immunomodulatory protein production in human cornea. Gene Ther, 1997, 4(7): 639-647.
8. Fehervari Z, Rayner SA, Ora HB, George AJ, Larkin DE Gene transfer to ex vivo stored corneas. Cornea, 1997, 16(4): 459-464.
9. Qin L. Chavin KD, Ding Y, Woodward JE, Favaro JP. Lin J, Bromberg JS. Gene transfer for transplantation. Prolongation of allograft survival with transforming growth factor-beta 1. Ann Surg, 1994, 220(4): 508-18, discussion: 518-519
10. Brauner R, Nonoyama M, Labs H, Drinkwater DC Jr, McCaffery S, Drake T, Berk AJ, Sen L, Wu L. Intracoronary adenovirus-mediated transfer of immuno-suppressive cytokine genes prolongs allograft survival. J Thorac Cardiovasc Surg, 1997, 114(6): 923-933
11. Holweg CT, Baan CC, Balk AH, Niesters HG, Maat AP, Mulder PM, Weimar W. The transforming growth factor-beta 1 codon 10 gene polymorphism and accelerated graft vascular disease after clinical heart trarsplantation, Transplantation, 2001, 71(10): 1463-1467
12. Hangai M, Kaneda Y. Tanihara H, Honda Y. In vivo gene transter into the retina mediated by a novel liposome system. Invest Ophthahnol Vis Sci, 1996, 37(13): 2678-2685
13. Hangai M, Tanihara H, Honca Y, Kaneda Y. Introduction of DNA into the rat and primate trabecular meshwork by fusogenic liposomes. Invest Ophthalmol Vis Sci, 1998, 39(3): 509-516
14. Thompson KR Hanna KD, Gipson IK, Gravagna P, Waring GO 3rd, Johnson-Wint B. Synthetic epikeratoplasty in rnesus monkeys with human type Ⅳ collagen. Cornea, 1993, 12 (1): 35-45
2. Zhang EP, Franke J, Schroff M, Junghans C, Wittig B, Hoffmann F. Ballistic CTLA4 and IL-4 gene transfer into the lower lid prolongs orthotopic corneal graft survival in mice. Graefes Arch Clin Exp Ophthalmol, 2003, 241(11): 921-926.
3.郭萍.谢立信,史伟云,黎晓新.肿瘤坏死因子相关凋亡诱导配体转基因治疗鼠角膜移植免疫排斥的研究.中华眼科杂志,2003,39(1):19-23.
4. Klebe S, Sykes PJ, Coster DJ, Krishnan R, Williams KA. Prolongation of sheep corneal allograft survixal by ex vivo transfer of the gene encoding interleukin-10. Transplantation, 2001, 71(9): 12414-1220
5. Larkin DF, Oral HB, Ring CJ, Lemoine NR. George AJ. Adenovirus-mediated gene delivery to the corneal endothelium. Transplantation, 1996, 61(3): 363-370.
6. Comer RM, King WJ, Ardjemand N. Effect of Administration of CTLA4-Ig as protein or cDNA on corneal allograft. Invest Ophthalmol Vis Sci, 2002, 43(4): 1095-1103
7. Oral HB, Larkin DE Fehervari Z, Byrnes AP, Rankin AM, Haskard DO, Wood MJ, Dallman M J, George AJ. Ex vivo aderovirus-mediated gene transfer and immunomodulatory protein production in human cornea. Gene Ther, 1997, 4(7): 639-647.
8. Fehervari Z, Rayner SA, Ora HB, George AJ, Larkin DE Gene transfer to ex vivo stored corneas. Cornea, 1997, 16(4): 459-464.
9. Qin L. Chavin KD, Ding Y, Woodward JE, Favaro JP. Lin J, Bromberg JS. Gene transfer for transplantation. Prolongation of allograft survival with transforming growth factor-beta 1. Ann Surg, 1994, 220(4): 508-18, discussion: 518-519
10. Brauner R, Nonoyama M, Labs H, Drinkwater DC Jr, McCaffery S, Drake T, Berk AJ, Sen L, Wu L. Intracoronary adenovirus-mediated transfer of immuno-suppressive cytokine genes prolongs allograft survival. J Thorac Cardiovasc Surg, 1997, 114(6): 923-933
11. Holweg CT, Baan CC, Balk AH, Niesters HG, Maat AP, Mulder PM, Weimar W. The transforming growth factor-beta 1 codon 10 gene polymorphism and accelerated graft vascular disease after clinical heart trarsplantation, Transplantation, 2001, 71(10): 1463-1467
12. Hangai M, Kaneda Y. Tanihara H, Honda Y. In vivo gene transter into the retina mediated by a novel liposome system. Invest Ophthahnol Vis Sci, 1996, 37(13): 2678-2685
13. Hangai M, Tanihara H, Honca Y, Kaneda Y. Introduction of DNA into the rat and primate trabecular meshwork by fusogenic liposomes. Invest Ophthalmol Vis Sci, 1998, 39(3): 509-516
14. Thompson KR Hanna KD, Gipson IK, Gravagna P, Waring GO 3rd, Johnson-Wint B. Synthetic epikeratoplasty in rnesus monkeys with human type Ⅳ collagen. Cornea, 1993, 12 (1): 35-45