【作者】 韩焱福；

【导师】 宋建星；

【作者基本信息】 第二军医大学 ， 外科学， 2008， 博士

【摘要】 皮肤缺损创面的修复一直是外科领域不断深入研究的课题,而理想的创面愈合应含有真皮层结构。目前已研究证实异种脱细胞真皮是较理想的真皮替代物,异种脱细胞真皮基质与自体薄皮片复合移植修复皮肤缺损创面在国内外已有报道。异种脱细胞真皮的来源广,成本低,有望在临床上广泛应用。但不具备血管结构的脱细胞真皮层血管化速度慢,这是导致其上方表皮细胞营养缺乏、局部感染和移植失败的主要原因。因而促进脱细胞真皮移植后的早期血管化是移植成功的关键。在目前实验研究中,为了提高移植皮片的存活能力,许多能诱导新生血管形成的生长因子应用于移植皮片的研究。其中血管内皮生长因子（vascular endothelial growthfactor,VEGF）是作用最强、特异性最高的因子,其促进血管生成作用已得到认可。但VEGF蛋白质的半衰期较短,限制了临床应用。目前尚无一个完善的释放传递系统以延长VEGF与组织的接触时间（即作用时间）,让其发挥最大的生物效应。VEGF基因治疗可克服单纯蛋白质治疗的缺点,因此人们考虑应用VEGF基因治疗。实践中遇到的问题是导入的基因难以在体内持续表达;有些难以获得有效基因转移;而且还存在安全性问题;体外细胞增殖困难,自体细胞来源少及再移植后存活率低等。1993年加拿大的Sun和Chang最早提出了异体体细胞基因治疗的策略,即将目的基因导入已建立的细胞系内,使其表达基因产物,再将其移入病人体内。这样大大扩展了细胞来源,但是免疫排斥反应又限制了其应用。微囊化细胞移植技术为解决这一问题提供了新方法,异体或异种细胞微囊后移植可避免宿主的免疫排斥反应,同时可发挥分泌等功能。20世纪80年代初,微囊化技术与组织细胞移植相结合,其特点在于选择性透过膜,可避免免疫系统对囊内细胞的攻击,起到免疫隔离作用,而小分子的营养物质和囊内生物活性物质及代谢产物可自由出入。之后,微囊化细胞技术广泛应用于神经内分泌疾病的基础和临床研究,并已取得可喜成绩。至90年代,随着基因重组技术的发展,人们尝试以微囊作为转基因细胞的免疫隔离和运载工具,利用基因修饰的异体或异种细胞的代谢产物调节机体生理功能,治疗相关疾病。微囊化基因工程细胞技术是将微囊化技术与基因工程细胞技术的有机结合,使基因工程细胞即基因修饰细胞借助微囊的免疫隔离作用在受体体内长期存活,并持续表达目的蛋白,有助于克服传统基因工程药物半衰期短,活性不高等缺点,相对于基因治疗则具有不改变受体基因组,更加安全,且可以批量生产,冻存后随时移植给所需患者,有降低工作量与成本的优点。所以理论上将微囊化技术与基因工程技术及组织移植技术相结合,通过转VEGF基因的方式使囊内细胞具有分泌VEGF的功能,可促进移植组织血管化。然而,微囊化VEGF基因修饰细胞能否在创面异种真皮移植中起到加快早期血管化作用,目前国内外未见相关报道。为此我们设计了本课题实验,进行了以下四个部分的实验研究。第一部分:目的:构建携带人血管内皮生长因子一165(hVEGF₁₆₅)基因的重组腺病毒载体（Ad.VEGF）,为后续基因转染、微囊化基因修饰细胞制备等研究提供实验基础。方法:采用脂质体转染法,将pAxCAwt.hVEGF₁₆₅与DNA-TPC共转染人胚肾293细胞,扩增后获得载hVEGF₁₆₅基因的复制缺陷型重组腺病毒。使用PCR、酶切证实重组腺病毒中的目的基因。并根据半数组织感染量(TCID₅₀)法计算病毒滴度。结果:采用脂质体法可使pAxCAwt.VEGF₁₆₅与DNA-TPC有效转染293细胞,扩增出载hVEGF₁₆₅基因的复制缺陷型重组腺病毒。PCR的产物进行NcoI酶切鉴定,可得到597bp、146bp两个片段,与GeneTool软件理论上计算的结果完全一致。计算病毒滴度为2.2×10¹²pfu/ml。结论:成功构建复制缺陷型重组腺病毒Ad.VEGF₁₆₅,其滴度高,毒性低,效率高,体外转染安全。第二部分:目的:探讨腺病毒介导人血管内皮细胞生长因子(hVEGF₁₆₅)基因转染NIH3T3细胞后目的基因表达情况及对NIH3T3细胞增殖分化的影响。方法:应用Ad.VEGF感染传代培养的NIH3T3细胞后,用荧光显微镜和流式细胞仪检测转染效果和转染率,免疫组化、RT-PCR和ELISA方法分别检测hVEGF₁₆₅基因转染NIH3T3细胞后VEGF的表达情况,MTT检测细胞增值活性。结果:腺病毒介导的hVEGF₁₆₅基因对于NIH3T3细胞具有较高的转染效率,转染效率与病毒感染复制数(multiplicities ofinfection,MOD具有量效关系。MOI为100倍时,转染效率达95%,转染VEGF后,RT-PCR、免疫组化显示NIH3T3细胞可有效表达VEGF,ELISA示7d时表达达到高峰（1052 pg/mL）,13d后仍可检测到VEGF的表达。两周内采用MTT动态检测OD值,转染组细胞与未转染组细胞相比无显著性差异（P＞0.05）。结论:腺病毒介导的hVEGF₁₆₅基因可以有效的转染NIH3T3细胞,NIH3T3细胞是一种较理想的基因载体细胞,其携带的VEGF基因可获得较高的表达水平。第三部分:目的:制备微囊化VEGF基因修饰NIH3T3细胞,并研究微囊化技术对VEGF基因修饰NIH3T3细胞增殖与活性及代谢分泌功能的影响。方法:应用纯化海藻酸钠-氯化钡技术制备微囊化VEGF基因修饰NIH3T3细胞,并与未微囊化细胞对照培养,在倒置相差显微镜下观察微囊及细胞形态,用MTT和PI染色流式细胞术检测细胞的增殖及活性情况,并每48h更换和收集微囊化和未微囊化基因修饰NIH3T3细胞培养液,-20℃保存,通过ELISA检测培养液VEGF的含量,两组进行对比分析。结果:微囊形态较圆整,细胞生长良好,两组细胞增殖与活性及培养液中VEGF的含量的差异无统计学意义（P＞0.05）。结论:微囊化并不影响基因修饰细胞的生长代谢功能,与对照组比较,在体外培养时生物学特性无明显差异,从而为研究微囊化基因修饰细胞体内移植打下实验基础。第四部分:目的:探讨微囊化VEGF基因修饰NIH3T3细胞移植对猪脱细胞真皮早期血管化及其复合皮存活的影响,从而探讨其应用于创面促进猪脱细胞真皮血管化的可行性。方法:以豚鼠背部急性皮肤全层缺损创面为模型,且对称性分为4个区,按移植物不同分为四组:微囊化VEGF-NIH3T3+复合皮移植组（A组）、未微囊化VEGF-NIH3T3+复合皮移植组（B组）、空囊+复合皮移植组（C组）、PBS空白对照+复合皮移植组（D组）。观察移植一周后微囊化细胞及未微囊化细胞在组织中病理形态改变;一周后ADM微血管化程度和两周后复合皮的存活率;术后3,7,14天取脱细胞真皮组织,采用免疫组化SP法检测hVEGF与CD34的表达,并计算微血管密度（microvessel density,MVD）,进行统计学分析。结果:微囊化基因修饰细胞移植一周后微囊形态较好,细胞有增值现象,囊周围无明显淋巴细胞浸润,而未微囊化基因修饰细胞周围有明显淋巴细胞浸润。一周后大体解剖及病理切片显示,A组血管化程度明显强于B组。两周后A组皮片存活率（91±7%）明显高于其他三组（B,C,D组分别为79±5%,76±2%,77±4%）（P＜0.01）,B、C、D三组之间无明显差异（P＞0.05）。移植后的3,7,14天A组hVEGF及CD34表达明显强于其他三组,且7,14天MVD明显高于其他三组（P＜0.01）,而其他三组之间无明显区别（P＞0.05）。结论:微囊化基因修饰细胞移植可促进创面异种脱细胞真皮早期血管化,提高复合皮片存活率,改善创面愈合质量。综上所述,本研究初步证实了微囊化基因修饰细胞制备的可行性,在体外培养微囊内细胞增值活性好,VEGF稳定表达。体内植入可促进创面脱细胞真皮早期快速血管化,改善创面愈合质量。可以期待,随着微囊化和基因工程技术的不断深入研究,必将产生一系列成果,为组织移植及难愈创面的血管化提供新的临床治疗策略。更多还原

【Abstract】 Skin defect wound repairing is always topic studied deeply in surgical field, and ideal wound healing should contain dermal structure. So far it has demonstrated that xenogeneic acellular dermis is satisfactory substitute of dermis, and there have been some reports about transplantation of xenogenic ADM and autologous split thickness skin graft on wound at home and aboard. Broad source and lower cost of porcine ADM make its application become promising in clinic. But ADM without vessel structure develops angiogenesis slowly, which could lead to insufficient nutrition of epidermic cells, regional infection and failure of transplantation. Therefore it is crucial for successful transplantation that early angiogenesis of ADM is accelerated.In experimental studies of plastic surgery, many angiogenic factors have been applied in study of skin graft to promote survival rate. Of which VEGF is a most effective and highly specific factor, and it is well known that its angiogenic effect is obvious. However, short half-life of VEGF protein limits clinical application. To make VEGF exert more bioactive effect, now there has been no perfect releasing and delivering system to lengthen effective time in tissue. VEGF gene therapy may overcome drawback of protein therapy, therefore we try to undertake gene therapy. We may adopt two pathways, namely in vivo and in vitro, but common problem encountered in reality include delivering gene hard to persistent express in vivo, some gene hard to efficaciously transfer, existing safety tissue, cells in vitro proliferating difficultly, short resource of autlogous cell and low survival rate after cell implantation. In 1993, Sun and Chang in Canada firstly raised strategy of allogenic cell gene therapy, namely making desired gene delivering into cell line, and making these cells express gene product, then making them implant into body. Thus we may greatly enlarge resource of cell, but immune rejection reactions limit their application. Microencapsulated cell transplantation technique provides a novel approach for resolving the problem. Microencapsulated allogenic or xenogenic cells transplantation may avoid immune rejection reaction, and simultaneously don’t influence their secreting function.In the early 20th century 80, microcapsule technique combined with tissue and cell transplantation, characteristic of which is its selective permeable membrane, and shelter cells from the recipient’s immune system, but allows free exchange of small molecular nutrient, bioactive agent and metabolites. Since then, microencapsulating technique has been widely used in experimental and clinical study on neuroendocrine disease, and it has achieved gratifying results. To the 1990s,with the development of gene recombination technology, people try to use microcapsule as immune isolation and their means of delivery, and to regulate physical function and treat associated disease by metabolic product of genetically modified allogenic or xenogenic cells.Mircroencapsulation of recombinant cell lines is viewed as a new delivery system for gene therapy. This approach would allow nonautologous genetically modified cells to be implanted into any host to deliver the desired gene product without triggering graft rejection. The advantages of nonautologous method of gene delivery are: it does not require modification of the host’s genome, thus providing additional measures of safety and cost saving; it provide ample material for quality assessment before implantation, a safety feature not available to most other forms of in vivo delivery. Therefore in theory by microencapsulation technique combining with genetically engineering and tissue transplantation technique, cells in microcapsule may secret VEGF through transferring gene, and promote angiogenesis of implanted tissue. However, so far there have been no reports about whether microencapsulated VEGF-secreting cells play a key role in accelerating angiogenesis for implanted ADM. So we designed this present experiment, and undertake following studies.Part one: Objective: To construct the recombinant adenovirus vector containing human vascular endothelial growth factor-165(hVEGF₁₆₅), so as to lay a foundation for study on genetically modified cells. Methods: pAxCAwt.VEGF₁₆₅ and DNA-TPC cotransfected into 293 cells by lipofection method. Being propagated in HEK293 cells and purified by cesium chloride gradient centrifugation, recombinant replication-deficient adenovirus named Ad.VEGF₁₆₅ was obtained. Then the titer of virus was detected by TCID₅₀ method. The Ad. VEGF₁₆₅ was identified by PCR restriction enzyme digestion and DNA sequencing methods. Results: An efficient and reliable method of constructing recombinant Ad vectors was established. Replication-deficient adenovirus vectors coding for VEGF₁₆₅ DNA were generated in high titer. 597bp and 146bp were obtained by NcoI restriction enzyme .the result was consistent with that of Gene Tool software calculating, virus titers was 2.2×10¹² pfu / ml. Conclusions: pAxCAwt.VEGF₁₆₅ and DNA-TPC can be used to construct replication-deficient recombinant Ad vectors with high titer and purity. It is proved to be efficacy and reliable.Part two: Objective: To investigate the expression of adenovirus transfected hVEGF165 in NIH3T3 cells in vitro and the effect of transfection on NIH3T3 cells proliferation. Methods: NIH3T3 cells were passaged and expanded, then infected by Ad. VEGF and Ad. GFP. The infection efficiency of adenovirus vector to NIH3T3 cells was tested by Ad.GFP infection procedure. Ad.VEGF expression in NIH3T3 cells was detected by immunohistochemical staining and RT-PCR, and its secretion in culture medium were measured with ELISA method. Proliferation of cells was determined by MTT. Results: NIH3T3 cells could be effectively transfected by adenovirus containing hVEGF165 gene in vitro, the transfection efficiency has the dose-effect relationship with multiplicities of infection （MOI）. When MOI was 100, the infection efficiency was more than 95% and stable. The expression of VEGF was traced both in cell lysate and in culture medium. A maximum production of VEGF was observed at 5～9 days after infection （1052 pg/mL at the 7th day）, and VEGF was found even in day 13. The result of MTT demonstrated there was no significant difference between infected cells and uninfected cells （P>0.05）. Conclusion: Gene transfer technology mediated by adenoviral vector can transfect VEGF gene into NIH3T3 cells with high efficiency. NIH3T3 cells transfected by hVEGF165 gene could efficiently express VEGF.Part three: Objective: To prepare microencapsulated VEGF-expressing NIH-3T3 cells , and study the effect of microencapsulated process on cellular metabolic functions and proliferation. Methods: Microencapsulated VEGF-expressing NIH-3T3 cell was made by Alginate-BaCl2 process. Morphological appearances of the microcapsule were observed under inverted phase microscope. The concentrations of VEGF in culture supernatant were measured by ELISA; The proliferation of microencapsulated VEGF-expressing NIH-3T3 cell was detected by MTT, and viability was tested by PI and flow cytometry. Unencapsulated VEGF-expressing NIH3T3 cells were used as control. Results: The morphological appearances of microcapsules were round and uniform, and the microencapsulated VEGF-expressing NIH-3T3 cells in vitro survived well. There was no statistical significance in concentration of VEGF, MTT value and viability of cells between two groups in vitro culture（P>0.05）. Conclusion: The physiologically metabolic functions of NIH3T3 cells within Alginate-BaCl2 microencapsule have not been impacted by microcapsule membrane. In vitro culture, there is no significant difference in biocharacteristic between microencapsulated VEGF-expressing NIH3T3 cells and unencapsulated cells. Then it lays experimental foundation for exploring further transplantation of microencapsulated VEGF-expressing NIH3T3 cells in vivo.Part four: Objective: To investigate the angiogenic effect of microencapsulated VEGF-expressing NIH3T3 cells transplantation on xenogenic acellular dermis on acute wound. Methods: Each guinea pig dorsal was divided into four symmetrical areas, cutting off the skin to fascia, causing the size of 2cm x 2cm skin defect region, then composite skin （porcine acellular dermis + autologous split thickness skin） were use to cover wound. According to different injected agent under ADM, experiment were further divided into four groups: Microencapsulated VEGF-NIH3T3 cells group （A group）, unencapsuled VEGF-NIH3T3 group （B group） ,empty microcapsules grafted group （C group ） , PBS blank control （D group ）. The morphological appearances of microcapsules and cells, the early angiogenesis of acellular ADM at one week and survival of composite skin at two week were observed, the expression of VEGF and CD34 were detected by IHC on the 3th, 7th and the 14th day. The microvessel density （MVD） was calculated. Results: The morphological appearances of microcapsules were smooth and round one week after implantation, of which the cells surviving well, and immersion of lymphocytes were not obvious surrounding microcapsule, but there were obvious lymphocytes surrounding unencapsuled cells, the extent of angiogenesis at one week in A group was obvious.the survived rate of composite skin at end of two weeks in A group（91±7%） was higher than that in other three groups（79±5%,76±2%,77±4%,respectively in B,C,D group） （P<0.01）, but there was no statistical difference among B,C,D groups（P>0.05）.Compared with other three groups, the expression of hVEGF and CD34 were marked in A group, and MVD at 7,14 day in A group was higher than other three groups （P<0.01）. Conclusion: Transplantation of microencapsulated VEGF-expressing NIH3T3 cells could augment angiogenesis of ADM in wound healing early stage, and elevate survive rate of composite skin.To sum up, the results of experiment demonstrate that preparation of microencapsulated genetically modified cells is feasible, which may accelerate early angiogenesis of implanted ADM, and improve quality of wound healing. With study deeply in this regard, a serial of achievement will be to bring about expectantly, providing a novel strategy of augmenting angiogenesis for tissue transplantation and refractory wound.更多还原