自体成纤维细胞注射移植的可行性研究
摘要
面部软组织的充填，对于整形外科学者来说，一直是一个极具挑战性的课题，而注射性软组织填充材料的开发则是的研究难点。自1957年Wegener首次报道使用液态硅胶注射以来，注射性软组织填充材料的开发经历了一个去伪存真，良好的愿望和客观的现实相矛盾的发展过程，至今没有很好的解决。
临床上使用的软组织填充材料主要分为人工合成品，生物性填充材料和具有生物活性的软组织填充材料。人工合成品主要有液态硅胶、硅胶微粒、Artecoll、Arteplast，这些材料进行面部填充的主要原理是利用机体对不降解的人工合成品的组织反应，包括巨噬细胞反应、纤维增生、胶原增多来达到充填面部细小凹陷的目的。生物性填充材料主要包括牛胶原、纤维蛋白(Fibral)、透明质酸(Hyaluronic Acid Derivatives)，这些材料因为没有生物活性，暂时驻留在机体内，都存在不同程度的吸收问题，因此长期的临床效果都不能达到令人满意。另外由于机体对上述两种材料都存在或多或少的排斥反应，因此容易出现皮肤红肿、破溃、或引发机体的免疫系统性疾病。因此，临床期待着有良好组织相容性的软组织填充材料的出现。
具有生物活性的软组织填充材料，是指既要参与体内正常的生理代谢，又具有分泌各种生长因子和其他功能性蛋白的功能，因此也就是具有生理活性细胞的移植。目前涉及整形外科领域的主要是脂肪细胞移植、成纤维细胞移植和软骨细胞移植。自1995年以来，美国组织工程中心Hackensack大学医学中心整形外科的William K．Boss等，采用特殊的培养方法，从病人耳后切取3mm的皮肤，培养出自体成纤维细胞(Isolagen自体胶原)，选择第4、5代的细胞用于充填面部细小凹陷，如鼻唇沟、眉间纹、唇周、痤疮、鱼尾纹及凹陷性瘢痕。在1450例病人4800个部位的治疗中，1年病人的满意率达92％，3—4年的满意率为70％。随后进行的研究表明，注射部位的真皮层增厚，Ⅰ型胶原增加。在裸鼠身上未发现明显的致瘤性。据目前公开发表的文献，未见其对机理和安全性进行深入的研究。本课题就是对自体成纤维细胞注射移植充填面部细小凹陷的可行性进行初步的探讨。本试验分5个方面进行研究。
成纤维细胞是结缔组织中最主要的细胞成分，在分泌细胞外基质成分及构建细胞外基质中扮演着重要的角色，在组织创伤修复中也发挥着重要的作用。整形外科学者对成纤维细胞的理解远远超过了其他学科。成纤维细胞具有较强的分裂增殖能力，适应性强，是较容易培养的细胞。常用的培养方法有植块培养法、消化法和消化—植块培养法。临床上，我们希望以最少的组织、最快的速度获得最大量的细胞，而这其中最为需要注意的最少量的组织这个条件，因而决定了植块法是最理想的培养方式。
为了研究成纤维细胞在体内的移植存活情况，我们以新西兰兔为研究对象，首次采用了~3H-TdR体外参入标记细胞，注射移植后体内示踪的技术，通过放射自显影客观的观察到标记的细胞在移植后5个月依然在体内存活，为其临床效果的持久性提供了严谨的科学证据。采用天狼猩红染色的方法，观察注射部位与对照部位Ⅰ、Ⅲ型胶原的变化，证实细胞移植部位Ⅲ型胶原的含量增加，P＜0.05，有明显的
For all the plastic surgeon, it is an highly challenge to fill the soft tissue on the face, which focus on the injectable soft tissue filler. Since Wegener firstly reported to use the liquid silicon gel inl957, it has gone through countless difficulties and obstacles to exploit the injectable soft tissue filler. Even those, it had not made the satisfactory and had not been solved in the day.
The injectable soft-tissue mainly include the manufacture, the organism filler and the active organism filler. The manufacture, such as the liquid silicon gel, the silicon particle, Artecoll and Arteplast, could cause the histology reaction to undegratable manufacture, to augment the soft-tissue. There were macrophage migration, fibroblast penetration and new collagen deposition. The organism filler, for example, Zyplast, Fibral, Hyaluronic Acid Derivatives, could not persist in the implant sites, and had been resorbed by the body, so the persistence of correction was not gratifying. Besides, adverse reaction to these above mentioned substance can occur on an allergic and nonallergic basis. These transient or durable reactions include bruising, swelling, ulcer, the local inflammatory responses, as well as the systemic immune disease. The plastic surgeon and patient are looking forward to the good biocompatibility materials to emerge.
The active organism soft-tissue augmention, which should not only take part in the normal physiological metabolism, but also play role in excreting many growth factors or other functional protein, had to be the living cell transplantation. In the present, there are fat cell transplantation, fibroblast transplantation and cartilage cell transplantation in the plastic surgery field. Since 1995, in the University of Medicine and Dentistry of New Jersey, Department of Plastic Surgery, Hackensack University Medical Center, NJ, autologous cultured fibroblast(Isolagen) have been utilized successfully as a living, dynamic protein repair system for dermal and subcutaneous deficiencies. The autologous fibroblast culture process was started with a 3-mm retroauricular punch biopsy, which were expanded by proprietary tissue culture techniques. Cells of early passages(4, 5, 6) were used to treat rhytids, depressed scars, subcutaneous atrophy, ache irregularities, and laser wounds. A total of 4800injections were given on 1450 cases. Long-term follow-up ranged from 36 to 48months. A subjective patient satisfaction survey showed 92% of the patients were satisfied with grade of correction. A long-term follow-up survey revealed continuing improvement beyond the initial correction in 70% of patients. The following study showed that cultured fibroblast were functionally active and produced large quantities of type I collagen. In vitro studies of scar formation potency of injectable fibroblasts showed that these cells possessed normal collagen gel contraction capacity. In vivo experiments showed that that cultured fibroblasts exhibited no oncogenic properties and induced no tumors in nude mice. The study is designed to discuss the possibility of the autologous fibroblast to augment the mini-deficiencies. The whole study include 5
临床上使用的软组织填充材料主要分为人工合成品，生物性填充材料和具有生物活性的软组织填充材料。人工合成品主要有液态硅胶、硅胶微粒、Artecoll、Arteplast，这些材料进行面部填充的主要原理是利用机体对不降解的人工合成品的组织反应，包括巨噬细胞反应、纤维增生、胶原增多来达到充填面部细小凹陷的目的。生物性填充材料主要包括牛胶原、纤维蛋白(Fibral)、透明质酸(Hyaluronic Acid Derivatives)，这些材料因为没有生物活性，暂时驻留在机体内，都存在不同程度的吸收问题，因此长期的临床效果都不能达到令人满意。另外由于机体对上述两种材料都存在或多或少的排斥反应，因此容易出现皮肤红肿、破溃、或引发机体的免疫系统性疾病。因此，临床期待着有良好组织相容性的软组织填充材料的出现。
具有生物活性的软组织填充材料，是指既要参与体内正常的生理代谢，又具有分泌各种生长因子和其他功能性蛋白的功能，因此也就是具有生理活性细胞的移植。目前涉及整形外科领域的主要是脂肪细胞移植、成纤维细胞移植和软骨细胞移植。自1995年以来，美国组织工程中心Hackensack大学医学中心整形外科的William K．Boss等，采用特殊的培养方法，从病人耳后切取3mm的皮肤，培养出自体成纤维细胞(Isolagen自体胶原)，选择第4、5代的细胞用于充填面部细小凹陷，如鼻唇沟、眉间纹、唇周、痤疮、鱼尾纹及凹陷性瘢痕。在1450例病人4800个部位的治疗中，1年病人的满意率达92％，3—4年的满意率为70％。随后进行的研究表明，注射部位的真皮层增厚，Ⅰ型胶原增加。在裸鼠身上未发现明显的致瘤性。据目前公开发表的文献，未见其对机理和安全性进行深入的研究。本课题就是对自体成纤维细胞注射移植充填面部细小凹陷的可行性进行初步的探讨。本试验分5个方面进行研究。
成纤维细胞是结缔组织中最主要的细胞成分，在分泌细胞外基质成分及构建细胞外基质中扮演着重要的角色，在组织创伤修复中也发挥着重要的作用。整形外科学者对成纤维细胞的理解远远超过了其他学科。成纤维细胞具有较强的分裂增殖能力，适应性强，是较容易培养的细胞。常用的培养方法有植块培养法、消化法和消化—植块培养法。临床上，我们希望以最少的组织、最快的速度获得最大量的细胞，而这其中最为需要注意的最少量的组织这个条件，因而决定了植块法是最理想的培养方式。
为了研究成纤维细胞在体内的移植存活情况，我们以新西兰兔为研究对象，首次采用了~3H-TdR体外参入标记细胞，注射移植后体内示踪的技术，通过放射自显影客观的观察到标记的细胞在移植后5个月依然在体内存活，为其临床效果的持久性提供了严谨的科学证据。采用天狼猩红染色的方法，观察注射部位与对照部位Ⅰ、Ⅲ型胶原的变化，证实细胞移植部位Ⅲ型胶原的含量增加，P＜0.05，有明显的
For all the plastic surgeon, it is an highly challenge to fill the soft tissue on the face, which focus on the injectable soft tissue filler. Since Wegener firstly reported to use the liquid silicon gel inl957, it has gone through countless difficulties and obstacles to exploit the injectable soft tissue filler. Even those, it had not made the satisfactory and had not been solved in the day.
The injectable soft-tissue mainly include the manufacture, the organism filler and the active organism filler. The manufacture, such as the liquid silicon gel, the silicon particle, Artecoll and Arteplast, could cause the histology reaction to undegratable manufacture, to augment the soft-tissue. There were macrophage migration, fibroblast penetration and new collagen deposition. The organism filler, for example, Zyplast, Fibral, Hyaluronic Acid Derivatives, could not persist in the implant sites, and had been resorbed by the body, so the persistence of correction was not gratifying. Besides, adverse reaction to these above mentioned substance can occur on an allergic and nonallergic basis. These transient or durable reactions include bruising, swelling, ulcer, the local inflammatory responses, as well as the systemic immune disease. The plastic surgeon and patient are looking forward to the good biocompatibility materials to emerge.
The active organism soft-tissue augmention, which should not only take part in the normal physiological metabolism, but also play role in excreting many growth factors or other functional protein, had to be the living cell transplantation. In the present, there are fat cell transplantation, fibroblast transplantation and cartilage cell transplantation in the plastic surgery field. Since 1995, in the University of Medicine and Dentistry of New Jersey, Department of Plastic Surgery, Hackensack University Medical Center, NJ, autologous cultured fibroblast(Isolagen) have been utilized successfully as a living, dynamic protein repair system for dermal and subcutaneous deficiencies. The autologous fibroblast culture process was started with a 3-mm retroauricular punch biopsy, which were expanded by proprietary tissue culture techniques. Cells of early passages(4, 5, 6) were used to treat rhytids, depressed scars, subcutaneous atrophy, ache irregularities, and laser wounds. A total of 4800injections were given on 1450 cases. Long-term follow-up ranged from 36 to 48months. A subjective patient satisfaction survey showed 92% of the patients were satisfied with grade of correction. A long-term follow-up survey revealed continuing improvement beyond the initial correction in 70% of patients. The following study showed that cultured fibroblast were functionally active and produced large quantities of type I collagen. In vitro studies of scar formation potency of injectable fibroblasts showed that these cells possessed normal collagen gel contraction capacity. In vivo experiments showed that that cultured fibroblasts exhibited no oncogenic properties and induced no tumors in nude mice. The study is designed to discuss the possibility of the autologous fibroblast to augment the mini-deficiencies. The whole study include 5
引文
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