胎猪皮肤前体组织异种移植的实验研究
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摘要
皮肤是身体最大的器官,它兼负着机械防护、免疫调节、物质代谢及体温调节等功能。局部皮肤的严重损伤可通过自体全厚皮、游离皮瓣及带蒂皮瓣移植重建其完整的结构与功能;但临床大面积烧、创伤患者由于自体皮源严重不足,大张异体皮加盖自体微粒皮虽然能够较快地封闭创面,挽救患者生命,但是往往遗留大量瘢痕,愈合后的皮肤组织机械强度和弹性远不及正常皮肤,严重影响患者的愈后生活质量。近年有研究发现特定胎龄的猪胚胎前体组织/器官(肝、肾、胰、脾等)异种移植后既能够生长发育成为相应的成熟组织(或器官)又不形成畸胎瘤,且能发挥一定的生理功能。胎猪皮肤前体组织是否也具有这种特性呢?
为解决大面积烧、创伤后创面覆盖材料来源及创面愈合质量等问题,本课题主要围绕胎猪皮肤前体组织移植裸鼠后的生长发育问题,应用简便有效的胎猪皮肤前体组织异种移植模型,观察了五个不同胎龄的胎猪皮肤前体组织移植裸鼠后的创面愈合特点及新生组织的形态结构,评价了不同胎龄的胎猪皮肤前体组织的移植后生长潜能及成瘤性,探讨胚胎皮肤前体组织移植后的生长发育及其可能的机理。全文主要结果及结论如下:
1、本课题采用精确胎龄为56天的胎猪皮肤前体组织对3种不同的移植模型进行了对比,观测移植后的生长特性。研究结果表明,裸鼠背部全层缺损创面加盖异种皮移植胎猪皮肤前体组织微粒的方式是简便有效的移植模型。
①背部创面移植模型:裸鼠背部正中开创至肌膜上,创面植入胎猪皮肤前体组织微粒悬液,用人尸体皮覆盖、缝合;②背部皮下移植模型:裸鼠背部正中横向切口至肌筋膜上,植入小块邮票皮,缝合切口;③耳廓皮下移植模型:裸鼠双耳廓皮下穿刺行胎猪皮肤前体组织微粒移植;背部创面模型及耳廓皮下模型新生组织块大小采用双样本t检验。3种模型中移植的胎猪皮肤前体组织均能继续生长发育成为具有表皮及真皮结构的双层皮肤组织,其中背部创面移植模型及背部皮下移植模型观察到了汗腺,皮脂腺及毛囊等附件,耳廓皮下移植模型未见汗腺;背部创面移植模型的生长潜能远高于耳廓皮下移植模型的生长潜能。
2、系统筛选了用于异种移植的胎猪皮肤前体组织的最佳“妊娠时间窗”(gestational window,以下简称时间窗)。选取了5个精确胎龄(E35、E42、E56、E70、E91)的胎猪皮肤前体组织,采用背部创面移植模型,观察移植后创面的愈合情况,于移植后6周及12周取材检测新生皮肤组织的结构形态,评价成瘤性。直接免疫荧光及黑色素染色鉴定新生组织的种属来源。移植后12周的新生组织块大小采用单因素方差分析,比较其移植后的生长潜能。研究结果认为胎猪皮肤前体组织异种移植后能够继续生长发育成为结构“完整”的成熟皮肤,且其最佳的取材时间窗在胎龄56天左右。
①各胎龄的胎猪皮肤前体组织移植均能生长发育为具有表皮、真皮及毛发、皮脂腺及汗腺等附件的“完整”成熟皮肤组织;②E42的胎猪皮肤前体组织具有最大的生长能力(新生组织平均大小为47.1 mm2,L×W),E56为31.1 mm2,E35为18.4 mm2,E70为20.1 mm2,E91为6.5 mm2;③抗鼠的H-2Dd(FITC)免疫荧光及Fortana法黑色素染色鉴定新生组织的种属来源。④新生的皮肤组织大部分胶原纤维排列规则,表皮钉突明显。⑤E56及以后的胎猪皮肤前体组织异种移植后未发现形成畸胎瘤。
[结论] E56的胎猪皮肤前体组织具有良好的移植后器官发育成力、生长能力及安全性;胎猪皮肤前体组织异种移植的最佳“妊娠时间窗”在胎龄56天左右。
Skin, the biggest organ of human body, assume the responsibility of protection, immunological regulation, metabolism and body temperature modulation, etc. The serious injury of local skin can be repaired by transplantation of autologous full-thickness skin, free skin flap and pedicle skin flap favoring restoration of integrate structure and function. Because of the lack of the autologous skin, autologous microskin grafting combined with large alloskin might be able to repair the wound quickly and save the patients’life but still leave many problems that can seriously influence the life quality not solved in the extensive deep burn and truma patients, in which the mechanical strength and elasticity of the neoregenerative skin tissue are far worse than the normal skin and the unavoidale scar emerge after convalescence. Recent research demonstrated that embryonic porcine precursor tissue or organ (i.e.liver, kidney, pancreas and spleen) at certain gestational age can develop into mature and functional tissue or organ without teratoma formation. What about the embryonic porcine skin precursor?
In order to overcome these obstacles in large scale burn and trauma, such as the source of wound covering material and wound healing quality, in our study, we focus on the growth and development of the embryonic porcine skin precursor graft from five different gestational ages. With the help of convenient and effective xenotransplantation model, we observe the characteristics of the convalescent wound and the histomorphous and the structure of the neoregenerative tissue after transplanting to nude mouse and evaluate the tumorigenesis of the graft and the growth potential after transplantation. The results and conclusions are summarized as follows:
1. In our research, we compared the following three transplantation models using the embryonic porcine skin precursor harvested at precise E(embryonic day)56. It is proved the model that the graft microgranules onto the dorsal full-thickness defect covering by xenogenous skin is the most convenient and effective xenotransplantation model.
a. dorsal wound model: The wound involving upper- tunica muscularis was made on the center of the nude mouse dorsum, then transplanted with the graft granule suspension and finally covered with human cadaver skin.
b. dorsal hypodermis model: The wound involving upper muscular fasciae was made horizontally on the center of the nude mouse dorsum, then transplanted with the stamp-like graft and finally sutured.
c. auricle hypodermis model: The graft granule suspension was injected into the ear auricle hypodermis via subcutaneous puncture.
The results of the first and the third model were evaluated using double-sample t test. The grafts in all the three models could develop into double-layer skin tissue with epidermis and dermis. Appendages such as hair follicles, sebaceous glands and sweat glands were seen in the first two models while sweat glands were not observed in the last model. Our data also demonstrated that growth potential of the grafts in the dorsal wound model was higher than that of the auricle hypodermis model.
2. We systematically studied the growth state and tumorigenesis posttransplantation of embryonic porcine skin precursors from different gestational ages (E35, E42, E56, E70, E91). Embryonic porcine skin precursors from five precise gestational ages, were prepared as microgranules and then implanted onto wound surfaces of full-thickness skin defect, followed by xenoskin covering for protection. The neoregenerative tissue was performed biopsy in the 6th and 12th week after transplantation to study histomorphous and tumorigenesis. Melanin staining and immunofluorescence were performed to identify the tissue origin. The size of the neoregenerative tissue recorded in the 12th week was analyzed by mono-factor variance analysis to evaluate the growth potential. Our data strongly indicated that skin precursors from five different gestational ages could grow and develop into the“integrate”skin and the optimal time for embryonic porcine skin precursor xenotransplantation is around E56.
a. The results showed that all skin precursors could grow and develop into the“integrate”skin, which consists of epidermis, dermis and appendages including hairs, hair follicles, sebaceous glands and sweat glands.
b. Skin precursors obtained at E42 exhibited the maximal growth potential—the average size of neoregenerative tissue from E42 was 47.1 mm2 (L×W), while the average size of neoregenerative tissue from E35, E56, E71 and E91 was 18.4, 31.7, 20.1 and 6.5 mm2 respectively.
c. Melanin staining and immunofluorescence were performed to identify the tissue origin.
d. Orderly arranged collagen fibers and rete ridges were observed obviously in the neoregenerative tissue.
e. No teratoma was detected in neoregenerative tissue from E56 and later gestational ages.
Conclusion: The embryonic porcine skin precursor from E56 showed high growth potential and could develop into the“integrate”skin including hair follicles, sebaceous glands and sweat glands without teratoma formation. The optimal time for embryonic porcine skin precursor xenotransplantation is around E56.
为解决大面积烧、创伤后创面覆盖材料来源及创面愈合质量等问题,本课题主要围绕胎猪皮肤前体组织移植裸鼠后的生长发育问题,应用简便有效的胎猪皮肤前体组织异种移植模型,观察了五个不同胎龄的胎猪皮肤前体组织移植裸鼠后的创面愈合特点及新生组织的形态结构,评价了不同胎龄的胎猪皮肤前体组织的移植后生长潜能及成瘤性,探讨胚胎皮肤前体组织移植后的生长发育及其可能的机理。全文主要结果及结论如下:
1、本课题采用精确胎龄为56天的胎猪皮肤前体组织对3种不同的移植模型进行了对比,观测移植后的生长特性。研究结果表明,裸鼠背部全层缺损创面加盖异种皮移植胎猪皮肤前体组织微粒的方式是简便有效的移植模型。
①背部创面移植模型:裸鼠背部正中开创至肌膜上,创面植入胎猪皮肤前体组织微粒悬液,用人尸体皮覆盖、缝合;②背部皮下移植模型:裸鼠背部正中横向切口至肌筋膜上,植入小块邮票皮,缝合切口;③耳廓皮下移植模型:裸鼠双耳廓皮下穿刺行胎猪皮肤前体组织微粒移植;背部创面模型及耳廓皮下模型新生组织块大小采用双样本t检验。3种模型中移植的胎猪皮肤前体组织均能继续生长发育成为具有表皮及真皮结构的双层皮肤组织,其中背部创面移植模型及背部皮下移植模型观察到了汗腺,皮脂腺及毛囊等附件,耳廓皮下移植模型未见汗腺;背部创面移植模型的生长潜能远高于耳廓皮下移植模型的生长潜能。
2、系统筛选了用于异种移植的胎猪皮肤前体组织的最佳“妊娠时间窗”(gestational window,以下简称时间窗)。选取了5个精确胎龄(E35、E42、E56、E70、E91)的胎猪皮肤前体组织,采用背部创面移植模型,观察移植后创面的愈合情况,于移植后6周及12周取材检测新生皮肤组织的结构形态,评价成瘤性。直接免疫荧光及黑色素染色鉴定新生组织的种属来源。移植后12周的新生组织块大小采用单因素方差分析,比较其移植后的生长潜能。研究结果认为胎猪皮肤前体组织异种移植后能够继续生长发育成为结构“完整”的成熟皮肤,且其最佳的取材时间窗在胎龄56天左右。
①各胎龄的胎猪皮肤前体组织移植均能生长发育为具有表皮、真皮及毛发、皮脂腺及汗腺等附件的“完整”成熟皮肤组织;②E42的胎猪皮肤前体组织具有最大的生长能力(新生组织平均大小为47.1 mm2,L×W),E56为31.1 mm2,E35为18.4 mm2,E70为20.1 mm2,E91为6.5 mm2;③抗鼠的H-2Dd(FITC)免疫荧光及Fortana法黑色素染色鉴定新生组织的种属来源。④新生的皮肤组织大部分胶原纤维排列规则,表皮钉突明显。⑤E56及以后的胎猪皮肤前体组织异种移植后未发现形成畸胎瘤。
[结论] E56的胎猪皮肤前体组织具有良好的移植后器官发育成力、生长能力及安全性;胎猪皮肤前体组织异种移植的最佳“妊娠时间窗”在胎龄56天左右。
Skin, the biggest organ of human body, assume the responsibility of protection, immunological regulation, metabolism and body temperature modulation, etc. The serious injury of local skin can be repaired by transplantation of autologous full-thickness skin, free skin flap and pedicle skin flap favoring restoration of integrate structure and function. Because of the lack of the autologous skin, autologous microskin grafting combined with large alloskin might be able to repair the wound quickly and save the patients’life but still leave many problems that can seriously influence the life quality not solved in the extensive deep burn and truma patients, in which the mechanical strength and elasticity of the neoregenerative skin tissue are far worse than the normal skin and the unavoidale scar emerge after convalescence. Recent research demonstrated that embryonic porcine precursor tissue or organ (i.e.liver, kidney, pancreas and spleen) at certain gestational age can develop into mature and functional tissue or organ without teratoma formation. What about the embryonic porcine skin precursor?
In order to overcome these obstacles in large scale burn and trauma, such as the source of wound covering material and wound healing quality, in our study, we focus on the growth and development of the embryonic porcine skin precursor graft from five different gestational ages. With the help of convenient and effective xenotransplantation model, we observe the characteristics of the convalescent wound and the histomorphous and the structure of the neoregenerative tissue after transplanting to nude mouse and evaluate the tumorigenesis of the graft and the growth potential after transplantation. The results and conclusions are summarized as follows:
1. In our research, we compared the following three transplantation models using the embryonic porcine skin precursor harvested at precise E(embryonic day)56. It is proved the model that the graft microgranules onto the dorsal full-thickness defect covering by xenogenous skin is the most convenient and effective xenotransplantation model.
a. dorsal wound model: The wound involving upper- tunica muscularis was made on the center of the nude mouse dorsum, then transplanted with the graft granule suspension and finally covered with human cadaver skin.
b. dorsal hypodermis model: The wound involving upper muscular fasciae was made horizontally on the center of the nude mouse dorsum, then transplanted with the stamp-like graft and finally sutured.
c. auricle hypodermis model: The graft granule suspension was injected into the ear auricle hypodermis via subcutaneous puncture.
The results of the first and the third model were evaluated using double-sample t test. The grafts in all the three models could develop into double-layer skin tissue with epidermis and dermis. Appendages such as hair follicles, sebaceous glands and sweat glands were seen in the first two models while sweat glands were not observed in the last model. Our data also demonstrated that growth potential of the grafts in the dorsal wound model was higher than that of the auricle hypodermis model.
2. We systematically studied the growth state and tumorigenesis posttransplantation of embryonic porcine skin precursors from different gestational ages (E35, E42, E56, E70, E91). Embryonic porcine skin precursors from five precise gestational ages, were prepared as microgranules and then implanted onto wound surfaces of full-thickness skin defect, followed by xenoskin covering for protection. The neoregenerative tissue was performed biopsy in the 6th and 12th week after transplantation to study histomorphous and tumorigenesis. Melanin staining and immunofluorescence were performed to identify the tissue origin. The size of the neoregenerative tissue recorded in the 12th week was analyzed by mono-factor variance analysis to evaluate the growth potential. Our data strongly indicated that skin precursors from five different gestational ages could grow and develop into the“integrate”skin and the optimal time for embryonic porcine skin precursor xenotransplantation is around E56.
a. The results showed that all skin precursors could grow and develop into the“integrate”skin, which consists of epidermis, dermis and appendages including hairs, hair follicles, sebaceous glands and sweat glands.
b. Skin precursors obtained at E42 exhibited the maximal growth potential—the average size of neoregenerative tissue from E42 was 47.1 mm2 (L×W), while the average size of neoregenerative tissue from E35, E56, E71 and E91 was 18.4, 31.7, 20.1 and 6.5 mm2 respectively.
c. Melanin staining and immunofluorescence were performed to identify the tissue origin.
d. Orderly arranged collagen fibers and rete ridges were observed obviously in the neoregenerative tissue.
e. No teratoma was detected in neoregenerative tissue from E56 and later gestational ages.
Conclusion: The embryonic porcine skin precursor from E56 showed high growth potential and could develop into the“integrate”skin including hair follicles, sebaceous glands and sweat glands without teratoma formation. The optimal time for embryonic porcine skin precursor xenotransplantation is around E56.
引文
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