【作者】 徐斌；

【导师】 蔡景龙；

【作者基本信息】 山东大学 ， 外科学， 2009， 博士

【摘要】 背景与目的瘢痕疙瘩是临床上常见的病理性瘢痕,也是美容、整形外科最棘手的难题之一。瘢痕疙瘩生长行为有类似肿瘤的特性,表现为超出原病损界限的持续性增生,并渐向周围正常皮肤组织侵犯,单纯手术切除后复发率极高。目前国内外的研究尚未能很好的揭示瘢痕疙瘩呈浸润性过度生长的机制,因此临床上尚无理想的治疗方案,迫切需要研究出新的治疗方法解决这一医学难题,这已成为整形外科研究的重点和热点问题。自杀基因治疗作为肿瘤基因治疗的主要方法之一,已经显现出潜在的临床应用前景和价值。目前已发现的自杀基因有多种,其中研究应用最多的是单纯疱疹病毒胸苷激酶基因(herpes simplex virus-thymidine kinase,HSV-TK)和大肠杆菌胞嘧啶脱氨酶基因(E.coli-cytosine deaminase,CD)。TK基因编码胸苷激酶,该酶可将核苷类似物(NA)代谢为二磷酸化物,后者在细胞内酶的作用下成为有毒性的三磷酸化物,阻止DNA复制,导致细胞死亡,而发挥抗肿瘤作用。CD基因编码胞嘧啶脱氨酶,编码的胞嘧啶脱氨酶可将胞嘧啶代谢为尿嘧啶,亦可将无毒性的药物前体5-Fc代谢成抗DNA合成的化疗药5-Fu,使肿瘤局部药物浓度增加,达到杀死肿瘤细胞的作用。双自杀基因(CDglyTK基因)的产物同时具有两种酶的活性,即CD和TK两者的活性,可以产生互补的作用,可以大大提高其应用价值,而不需考虑自杀基因的肿瘤细胞类型依赖性。CDglyTK双自杀基因在肿瘤学的治疗研究中已经取得了较大的进展,但将其应用于瘢痕疙瘩治疗研究中在国内外尚未见报道。本研究拟通过腺病毒载体将CDglyTK双自杀基因在体外实验中导入到瘢痕疙瘩成纤维细胞中,加入前药后,观察其对瘢痕疙瘩成纤维细胞的杀伤效应和旁观者效应;观察瘢痕疙瘩成纤维细胞中促细胞凋亡基因Bax及Sphk2和促细胞增殖基因Bcl-2及Sphk1的mRNA和蛋白表达变化情况;观察瘢痕疙瘩成纤维细胞的细胞周期改变情况;观察瘢痕疙瘩成纤维细胞的凋亡和形态改变情况;分别建立保留表皮的瘢痕疙瘩裸鼠移植模型及不保留表皮的瘢痕疙瘩裸鼠移植模型并将两者作对比,确定保留表皮瘢痕疙瘩裸鼠移植模型更有优越性,并将其应用;观察腺病毒介导的CDglyTK双自杀基因系统对裸鼠皮下移植的瘢痕疙瘩组织块的治疗作用;观察组织块的形态学变化及组织病理改变、凋亡改变及促细胞凋亡基因Bax及Sphk2和促细胞增殖基因Bcl-2及Sphk1的蛋白表达变化情况,以探讨腺病毒介导的CDglyTK双自杀基因对瘢痕疙瘩的治疗作用及其作用机制。方法1.利用改良AdEasy系统构建并制备高滴度纯化的重组CDglyTK双自杀基因腺病毒液:将CDglyTK双自杀基因从PWZLneoCDglyTK质粒上双酶切切下通过中间载体pcDNA3转移到穿梭质粒track-CMV中,将track-CMV-CDglyTK通过PmeⅠ线性化后在5183菌中与骨架质粒pAdEasy-1重组,得到Ad-CDglyTK质粒;将Ad-CDglyTK质粒用PacⅠ酶切后转化到293细胞中,包装得到Ad-CDglyTK腺病毒;2.组织块法培养瘢痕疙瘩以成纤维细胞;取第4-8代细胞用于实验,分别以25%感染,50%感染,75%感染及100%感染的瘢痕疙瘩成纤维细胞加入前药后MTT法观察腺病毒介导的CDglyTK双自杀基因系统对瘢痕疙瘩成纤维细胞的杀伤效应和旁观者效应;3.分别培养转染CDglyTK基因(MOI=20)和未转染CDglyTK基因的成纤维细胞,共4组,分为a组(空白对照组)、b组(未转染CDglyTK基因加前药组)、c组(转染CDglyTK基因未加前药组)及d组(转染CDglyTK基因加前药组),利用HE染色检测细胞的形态变化;利用TUNEL凋亡检测法检测成纤维细胞的凋亡状态;利用流式细胞术检测细胞周期变化情况;利用免疫组化、Real-Time PCR及Western Blot检测Bcl-2/Bax及Sphk1/Sphk2基因的变化情况;4.分别使用保留表皮的瘢痕疙瘩组织块和不保留表皮的瘢痕疙瘩组织块制作瘢痕疙瘩裸鼠移植模型,根据瘢痕疙瘩大体形态及组织学观察对模型作评价;5.将裸鼠移植模型分为4组,分别为a组(瘢痕内注射生理盐水)、b组(瘢痕内注射生理盐水+腹腔注射5-Fc和GCV)、c组(瘢痕内注射CDglyTK双自杀基因腺病毒)及d组(瘢痕内注射CDglyTK双自杀基因腺病毒+腹腔注射5-Fc和GCV),观察瘢痕疙瘩体积块的变化情况;利用TUNEL凋亡检测法检测瘢痕疙瘩组织块成纤维细胞凋亡情况;利用免疫组化检测瘢痕疙瘩组织块中治疗前后Bcl-2/Bax及Sphk1/Sphk2基因的表达变化情况。结果1.利用改良AdEasy系统成功构建CDglyTK双自杀基因腺病毒质粒,PacⅠ酶切后产生2条DNA片段,分别为30kb和4.5kb;BstxⅠ酶切重组成功的质粒后,相对腺病毒骨架质粒可见其中8.24kb条带消失;PCR鉴定能扩增出特异性的CD和TK基因的目的条带;经293细胞包装得到腺病毒,透射电镜下证实腺病毒颗粒存在。2.CDglyTK双自杀基因腺病毒对瘢痕疙瘩成纤维细胞的最佳感染复数(MOI)为20,在MOI=20时感染效率达到＞95%;在前药作用下,当感染腺病毒的成纤维细胞的比例达到25%时可产生部分杀伤效应,当感染腺病毒的成纤维细胞的比例达到50%时能产生完全杀伤作用,证实腺病毒介导的CDglyTK双自杀基因系统对瘢痕疙瘩成纤维细胞具有强大的杀伤效应和旁观者效应;3.HE染色发现d组成纤维细胞数量明显减少,同时细胞形态学改变明显,表现为细胞膨胀呈球形改变,细胞膜及细胞核有空泡样改变,而在对照组中未出现异常变化;TUNEL凋亡检测发现d组中成纤维细胞几乎都出现凋亡改变,与a,b及c组有明显差别;流式细胞术检测发现d组中大量的成纤维细胞被阻滞于G1期,与a,b及c组间差异具有统计学意义(P＜0.05);Real-Time PCR及WesternBlot定量检测发现d组成纤维细胞中Bax表达增高及Bcl-2表达降低,Sphk1表达降低及Sphk2表达增高,免疫组化检测发现Bcl-2/Bax及Sphk1/Sphk2基因表达定位未出现明显异常;4.分别采用保留表皮组织块和不保留表皮组织块建立瘢痕疙瘩裸鼠移植模型,术后第2周至第8周瘢痕疙瘩组织块体积变化率的差异均有统计学意义(P＜0.05),保留表皮组瘢痕疙瘩裸鼠移植模型组织块维持时间较去表皮组更长;移植后瘢痕疙瘩组织内成纤维细胞数量明显增加,且随时间延长增加,胶原纤维结构随时间延长逐渐减少;5.形态学观察可见d组裸鼠瘢痕疙瘩组织块体积在治疗后随着治疗时间的延长逐渐缩小,治疗14天后与治疗前及同时间点a,b及c组比较差异均有统计学意义(均P＜0.05);a,b,c三组组织块体积最大值分别出现在用药后21,14,7天;HE染色可见d组瘢痕疙瘩组织正常形态被破坏,胶原结构被破坏,大量的细胞浸润,且将胶原分割包绕,胶原组织结构松散,仅残留部分胶原组织,可见较多血管形成;TUNEL法检测发现各组均有成纤维细胞的凋亡,但在d组中出现大量的成纤维细胞凋亡,较对照组明显增加;发现Bcl-2及Sphk1基因的表达在d组减弱,明显低于a组,b组和c组;Bax及Sphk2基因的表达在d组明显增强。结论1.利用改良AdEasy系统能够高效率的成功构建CDglyTK双自杀基因腺病毒;2.CDglyTK双自杀基因腺病毒能够有效的感染瘢痕疙瘩成纤维细胞并在成纤维细胞中成功表达双自杀基因,能够对瘢痕疙瘩成纤维细胞产生强大的杀伤效应和旁观者效应;3.腺病毒介导的CDglyTK双自杀基因系统通过诱导瘢痕疙瘩成纤维细胞出现凋亡实现其对瘢痕疙瘩成纤维细胞的杀伤作用;Bcl-2和Sphk1表达降低,Bax和Sphk2表达增高是导致成纤维细胞凋亡的重要因素之一;成纤维细胞被阻滞于G1期是腺病毒介导的CDglyTK双自杀基因系统诱导细胞凋亡的一个重要机制;4.保留表皮的瘢痕疙瘩裸鼠移植模型优于传统的瘢痕疙瘩裸鼠移植模型,组织块维持时间更长;5.在瘢痕疙瘩裸鼠移植模型中腺病毒介导的CDglyTK双自杀基因系统也有较强的治疗作用;成纤维细胞的凋亡是其主要的表现形式;Bcl-2和Sphk1表达降低,Bax和Sphk2表达增高同样在瘢痕疙瘩裸鼠移植模型的自杀基因能治疗中起重要作用。更多还原

【Abstract】 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.更多还原