Efficacy Of Recombinant Adenovirus-mediated Double Suicide Gene Therapy In Human Keloid

Background and objectiveKeloids are defined as an abnormal scar that grows beyond the boundary of the original site of a skin injury and keloids do not subside. Their biological behaviour is similar to that of tumor. The recurrence rate is high after operation. The current treatment methods include injections of cortisone, surgery, laser, cryosurgery, silicone sheeting and compression, etc. But there are no strategies available for keloid therapy. Inadequate treatment methods may result in another keloid and sometimes a larger one. At the same time, the mechanisms of the keloid's occurance are unknown. It haa been the clinical tough problem and researching focal point in plastic surgery.Gene therapy is one of the ways to tumor therapy now, and as one of the main way of gene therapy, Suicide gene therapy has shown up the value of potential clinical application. During the past decade, several prodrug activation and sensitization approaches have been tested in different tumor models in animals. Two of the most studied prodrug activation approaches involve transfection of tumors with herpes simplex virus type 1 thymidine kinase (HSV-TK) gene or Escbericbia coli cytosine deaminase (CD) genes followed by administration of ganciclovir (GCV) or 5-fluorocytosine (5-FC) respectively. CD gene is found in some bacteria and fungi, but it is absent in mammalian cells. The enzyme encoded by CD gene can convert 5-FC into the toxic anabolite 5-fluorouracil (5-FU), used in the treatment of many cancers. Actually, 5-FU itself is also a prodrug which must be converted intracellularly to cytostatic/cytotoxic 5-fluorouracil triphosphate or 5-fluoro-2'-deoxyuridine 5'-monophosphate, and then interferes with RNA processing, inhibits thymidylate synthase and thus interferes with the DNA synthesis. HSV-TK converts the nontoxic GCV to GCV-MP, which is then metabolized to the toxic triphosphate form by cellular kinase. GCV-triphosphate interacts with the cellular DNA polymerase, causing interference with DNA synthesis and thereby leading to the death of dividing cells. The CD and TK gene can be combined by a polyglycine spacer for a CDglyTK double suicide gene, which will enhance the effect significantly. One of the key aspects of CDglyTK suicide gene/prodrug system strategies is the reliance on the so-called bystander effect. With this effect, partial nonexpression cells will not survive drug therapy.In this study, CDglyTK double suicide gene was transferred into keloid fibroblasts by adenovirus vector. Apoptosis was induced by CDglyTK double suicide gene and prodrug in Keloid fibroblasts. The effect of CDglyTK double suicide gene to keloid, the expression of Bcl-2, Bax, Sphk1 and Sphk2 gene were detected to approach the mechanism of action of CDglyTK double suicide gene. The apoptosis of keloid fibroblasts and the change of cell cycle of keloid fibroblasts were detected. An ideal animal model for the human keloid in nude mice was established. The effect of CDglyTK double suicide gene to keloid in mice and the expression of Bcl-2, Bax, Sphk1 and Sphk2 gene were detected.Methods1. The CDglyTK gene was digested from PWZLneoCDglyTK and cloned into the pAdTrack-CMV vector, which contains a GFP expression cassette driven by a separate cytomegalovirus (CMV) promoter. The shuttle vector was linearized with Pme I and transformed into E.coli.BJ 5183 cells which contain the Adeasy-1 adenoviral backbone vector. Homologous recombinants are selected for kanamycin resistance, and recombinants were subsequently identified by restriction digest and PCR amplification.2. The keloid fibroblasts were originally derived from a spontaneous keloid. The fibroblasts from 4 to 8 generation were used. Lethal effect and bystander effect were measured by the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.3. 1×10~5 cells per well were seeded on object slides in 6-well plates. Four groups of subjects were studied. The four groups were as follows: group (a): blank, group (b): CDglyTK~- fibroblasts + prodrug, group (c): CDglyTK~+ fibroblasts and group (d): CDglyTK~+ fibroblasts + prodrug. Morphologic changes of keloid fibroblasts and apoptosis were detected by HE staining and TUNEL assay, respectively. The change of keloid cell cycle was detected by flow cytometry. Bcl-2/Bax and Sphk1/Sphk2 gene were detected by immunohistochemistry, Real-Time PCR and Western Blot.4. The modified animal model for the human keloid in nude mice were established and the keloid model was evaluated according to morphologic changes and histology.5. The CDglyTK suicide gene was injected into the keloid in mice and the prodrugs were injected into abdominal cavity. The four groups were as follows: group (a): normal sodium was injected into the keloid in mice, group (b): normal sodium was injected into the keloid in mice and prodrugs were injected into abdominal cavity, group (c): Ad-CDglyTK were injected into the keloid in mice, (d): Ad-CDglyTK were injected into the keloid in mice and prodrugs were injected into abdominal cavity. The therapy of CDglyTK suicide genes in nude mice was detected. The morphologic changes of fibroblasts and the Bcl-2/BAX and Sphk1/Sphk2 were detected by vernier caliper, HE staining and immunohistochemistry, respectively. The apoptosis of keloid fibroblasts were detected by TUNEL assay.Results1. The recombinant adenovirus-mediated CDglyTK double suicide gene could be constructed by modified AdEasy system. 30 kb and 4.5kb straps were found when the adenovirus was digested by Pac I. 8.2 kb strap was deleted when it was digested by Bstx I. CD gene and TK gene were identified by PCR amplification successfully. The adenovirus was identified by transmission electron microscope.2. The best MOI for keloid fibroblasts is 20. When the MOI was 20, the rate of GFP positive fibroblasts visualized by fluorescence microscope was more than 95%. It could be seen that when 50% of fibroblasts were infected, the lethal effect was powerful and complete. When 25% of fibroblasts were infected, the lethal effect was partly. The lethal effect and bystander effect of CDglyTK were shown significantly in keloid fibroblasts.3. Compared to the control groups, the quantity of keloid fibroblasts were reduced and their appearance was changed. Cell swelling, membrane blebbing and vacuolar nucleus were observed in the experiment group(group d) and none of them were observed in the control groups. The appearances of all the fibroblasts in the control groups were normal. In the test of apoptosis, almost all the fibroblasts in the group dwere apoptotic by the TUNEL method compared to the control groups. The keloid fibroblasts which were transferred by recombinant adenovirus were stopped in G1 phase. The number of keloid fibroblasts in G1 phase increased (P<0.05) . Both Bcl-2 and BAX were expressed in the cytoplasm of keloid fibroblasts. The expression of Bcl-2 and Sphk1 was decreased in group d but the expression of BAX and Sphk2 was increased in group d. The same trend was found in the repeated experiments.4. The volume of pieces of keloids with epidermis were more large and longer compared to the pieces of keloids without epidermis (p<0.05) in the period of 2-8 weeks. The pieces of keloids with epidermis were similar to the human keloids in histology at the 8 weeks.5. Compared to those before treatment the volume of the implanted keloid of experiment group (group d) began to decrease since 14 days after treatment time-dependently (all P<0.05), and the volumes of the other 3 groups continued to increase and peaked on days 21, 14, or 7 respectively (all P<0.05). Microscopy showed infiltration of a larger quantity of histiocyte in the keloid tissue, and more obvious collagen disorganization and apoptosis of fibroblasts in group d than in the other 3 groups. The protein expression of Bcl-2 and Sphk1 were more remarkable and the protein expression of BAX and Sphk2 were less remarkable in experiment group than in the other 3 groups.Conclusion1. The modified homologous recombination in bacteria by using AdEasy system is efficient, convenient and easy to be carried out. The revised methodology involves using the AdEasier cells that are E.coli strain BJ5183 bacteria containing adenoviral backbone vector. It can increase the efficiency of generating recombinant adenovirus vectors compared to the standard AdEasy system. The recombinant adenovirus can be constructed with relatively short time and conveniently.2. The keloid fibroblasts can be infected by the recombinant adenovirus expression CDglyTK suicide genes with high efficiency. It is the best MOI value to keloid fibroblasts when MOI is 20. CD gene and TK gene can both be expressed in keloid fibroblasts. Besides the lethal effect, the bystander effect plays an important role in keloid fibroblasts therapy.3. Apoptosis induced by lethal effect and bystander effect is one of the main manifestations leading keloid fibroblasts to death. The change of expression of Bcl-2/BAX and expression of Sphk1/Sphk2, especially the high lever expression of BAX and Sphk2, play an important role in the process of apoptosis. Stopping fibroblasts in G1 phase is the main form of the effect of CDglyTK suicide genes to keloid fibroblasts.4. The keoid model of human keloid with epidermis is an easily established model, are similar to human keloids both in gross appearance and histology. So this model is an ideal one for the studies of human keloids.5. The recombinant adenovirus-mediated double suicide gene therapy is effective on the implanted keloid tissue. The main mechanism may be induction of apoptosis in the keloid fibroblasts. The changes of expression of Bcl-2/BAX and expression of Sphk1/Sphk2 also play an important role in the process of apoptosis.