【作者】 赵穆欣；

【导师】 李森恺； 李强； 周传德；

【作者基本信息】 中国协和医科大学 ， 外科学， 2009， 博士

【摘要】 第一部分自体口腔黏膜微粒移植修复创面的实验研究目的:观察自体口腔黏膜微粒移植后的成活生长情况,明确微粒黏膜移植于创面后的黏膜化过程及愈合速度,探讨不同的黏膜微粒形态和分布形式对创面愈合的影响,并探索提高创面上皮化速度的方法。材料与方法:1.在实验用猪的体表制备若干3×3cm~2全层皮肤缺损创面,将自体微粒黏膜扩张面积比例(即移植黏膜面积与创面面积比例)均设定为1:9,根据剪碎黏膜的时间不同,将创面分别随机分为5分钟组(Ⅰ组)、8分钟组(Ⅱ组)、10分钟组(Ⅲ组)、15分钟组(Ⅳ组)及空白对照组(Ⅴ组),分别将各组微粒黏膜移植于创面,并应用MTT法测量各组微粒黏膜的细胞活力。观察不同的微粒黏膜形态及在创面的分布形式,术后1周、2周、3周观察各组创面愈合情况,并行HE染色、免疫组织化学染色,第3周行扫描电镜和投射电镜观察。2.观察加速创面上皮化时,将剪碎黏膜的时间均设为8分钟,并将同样大小的体表创面随机分为空白对照组(A组)、微粒黏膜移植组(B组)、BMSCs+微粒黏膜移植组(C组)、BMSCs移植组(D组)、微粒黏膜-微粒皮混合移植组(E组)。术后1周、2周、3周比较各组创面上皮化情况,并行HE染色、免疫组织化学染色。结果:1.Ⅰ、Ⅱ、Ⅲ、Ⅳ组的黏膜微粒在数量、面积、周长、等效直径、形状因子等各项指标均有不同,对4组微粒黏膜的总周长进行统计学分析,发现各组之间均存在显著性差异(P＜0.05)。2.术后1周,Ⅱ组、Ⅲ组、Ⅳ组可见微粒黏膜成活并向外扩展生长,Ⅰ组成活的黏膜微粒较孤立,Ⅱ组的创面愈合率超过70%,Ⅴ组为肉芽创面。术后2周,Ⅱ组的新生黏膜融合成片,创面愈合率在85%以上;而Ⅰ组、Ⅲ组和Ⅳ组新生黏膜未完全成片,Ⅱ组优于Ⅰ组、Ⅲ组和Ⅳ组(P＜0.05)。术后3周,各实验组创面已经基本粘膜化,Ⅰ组、Ⅱ组和Ⅲ组的新生黏膜较光滑、质地厚,其中Ⅱ组的创面愈合率优于Ⅰ组和Ⅲ组(P＜0.05);而Ⅳ组的新生黏膜较薄,且Ⅰ组和Ⅲ组的创面愈合率优于Ⅳ组(P＜0.05)。3.术后1、2、3周,实验Ⅰ、Ⅱ、Ⅲ、Ⅳ各组的上皮细胞层数随时间逐渐增多,极性排列随时间变佳,Ⅱ组的细胞层数最多,极性排列最好;各组广谱角蛋白阳性染色也随时间逐渐增强,Ⅱ组的表达最强且完整连续。4.微粒黏膜细胞活力比较,Ⅰ组平均OD值最高,Ⅰ组与Ⅱ组无显著性差异(P＞0.05),其它各组之间比较P＜0.05。5.微粒黏膜与微粒皮复合移植组(E组)在术后1、2、3周的创面愈合率明显高于其它各组(P＜0.05)。HE染色发现皮肤与黏膜的交界处可以明显区分,黏膜处的上皮细胞层数明显多于皮肤处的上皮细胞,皮肤处存在角化层,黏膜处为不全角化,黏膜的钉突粗而深大。结论:1.微粒黏膜移植于创面后可以形成完整的片状黏膜,将面积扩增比为1:9的猪口腔黏膜微粒移植于创面,术后3周形成的黏膜上皮较完整,上皮质量较好。2.微粒黏膜的形态及在创面的分布形式对创面愈合有影响,制备的黏膜微粒直径平均约为0.7mm,面积平均约为0.46mm~2,单位创面上分布的黏膜微粒数约25个/cm~2时,其移植于创面后的愈合速度最快,形成的黏膜上皮质量最好。3.微粒黏膜与微粒皮复合移植可以加速创面的上皮化速度,但移植成活后仍保持其本身的组织特性。第二部分自体口腔黏膜微粒移植再造阴道的临床研究目的:探讨自体口腔黏膜微粒移植行阴道再造的手术方法及其疗效,并对再造的阴道分别通过形态学、组织学、微环境以及功能学来进行评价,进一步明确该方法的意义。材料与方法:自2006年7月至2008年9月,收治11例先天性无阴道及1例阴道再造术后7年继发阴道狭窄患者,年龄19～30岁,平均24.6岁。手术在患者两侧颊部及上下唇内侧黏膜区域点片状切取口腔黏膜,并剪碎成直径约0.7 mm的微粒,将微粒黏膜撒布于明胶海绵条上后移植于直肠与尿道之间的阴道再造腔穴。术后随访时测量再造阴道的深度及宽度,行阴道镜检查和阴道造影;取阴道壁组织行组织学检测;行人工阴道分泌物常规检查和PH值检测;对术后6个月以上的已有性生活的患者进行女性性功能的问卷调查并与正常女性性功能进行比较。结果:1.12例手术均获成功,手术时间平均约为112min(90～140min),术中出血约80～100 ml,平均住院时间为14天(10～19天),再造阴道完全上皮化的时间平均约为14天。所有患者均随访,平均随访时间约14个月(4～26个月)。口腔内供黏膜区域无继发畸形。2.所有患者术后外阴及阴道外口形态自然,会阴区无任何手术痕迹。再造阴道深约8cm,宽能容纳两横指。再造阴道壁的黏膜光滑、湿润,呈粉白色,弹性好,近阴道外口处阴道壁可见皱襞。组织学提示为复层鳞状上皮,未出现角化。阴道内可见浅黄色分泌物,平均PH值约为6.2(5.5～7.0)。3.12例患者中有8例患者已结婚,7例行女性性功能的问卷调查,对性生活均较满意。再造阴道组的性功能总评分和性高潮评分与正常对照组存在显著性差异(P＜0.05);而在性欲、性兴奋、阴道湿润度、性满意度及性舒适度评分无显著性差异(P＞0.05)。结论:1.应用自体口腔黏膜微粒游离移植可行阴道再造,手术操作简单,创伤小,供区及外阴形态无破坏。2.再造阴道具有足够的深度和宽敞度,阴道壁柔软、润滑、清洁,具有弹性和延展性。3.新阴道上皮为未角化的复层鳞状上皮,组织学表现与正常阴道组织相似。4.再造阴道功能接近于正常,性生活时不需要使用润滑剂。更多还原

【Abstract】 Part 1 Experimental study of autologous oral micro-mucosa graft on wound repairObjective:To investigate the survival and epithelization of the autologous oral micro-mucosa graft to the wound,and identify its mucosal process and healing pacing.We analyzed the morphology and distribution of the micro-mucosa on the wound and explored the method of accelerating wound epithetization.Materials and methods:1.We contive an animal model of full-thickness skin defect wound（3×3cm²） and use autologous micro-mucosa graft to repair the defect with area expansion ratio of 1;9. The wounds were randomly divided into 5-minute groups（groupⅠ）,8-minute groups （groupⅡ）,10-minute groups（groupⅢ）,15-minute groups（groupⅣ） and blank control groups（groupⅤ） depending on the different periods of mincing time.We applied MTT measurement in each group of mucosa cell viability.The morphology and distribution of the micro-mucosa were observed on the wound.Macroscopy and histological samples by HE and immunohistochemical staining were examined on the 7^th,14^th and 21^st day after grafting.SEM and TEM examination were taken on the 21^st day.2.The mincing time was set to 8 minutes when we observe the impact on the healing process.The Same size wounds were randomly divided into blank control group （group A）,micro-mucosa graft group（group B）,BMSCs + micro-mucosa transplantation group（group C）,BMSCs transplantation group（group D）,micro-mucosa combined with micro-skin transplantation group（group E）.Macroscopy and histological samples by HE and immunohistochemical staining were examined on the 7th,14th and 21st day after grafting.Results:1.Mucosal particles of all the experimental groups had different index in terms of quantity,area,perimeter,equivalent diameter and shape factor.All the total circumference of micro-mucosa in 4 groups were statistically analyzed and had significant difference（P＜0.05）.2.On the 7^th day postoperation,the micro-mucosa survived and grew outward in groupⅡ,ⅢandⅣ,however,the survived micro-mucoa in groupⅠseemed isolated.The wound healing rates of groupⅡwas over 70%while groupⅤwas granulation tissue. On the 14^th day postoperation,the neogenesis mucosa became to integrate into films in groupⅡand the wound healing rate was more than 85%and higher than the other three groups（P＜0.05）.The neogenesis mucosa was not seen to integrate in groupⅠ,ⅢandⅣ. On the 21^st day postoperation,the wounds of all experimental groups have covered by mucosa.The neogenesis mucosa showed smooth and thick texture in groupⅠ,ⅡandⅢwhile the neogenesis mucosa in groupⅣseemed thinner than them.The wound healing rate of groupⅠandⅢwas lower than groupⅡ（P＜0.05） but higher than groupⅣ（P＜0.05）.3.Epithelial cell layers and polarity of all the groups increased gradually over time postoperatively.GroupⅡhad the most epithelial cell layers and best polarity. Extended-spectrum keratin-positive staining of each group strengthened gradually with time.GroupⅡhad the strongest positive expression and complete consecutive.4.As for the comparison of mucosal cell viability,groupⅠachieved the highest average OD value but had no significance with groupⅡ（P＞0.05）.Comparison between other groups got P＜0.05.5.The wound healing rates of group E（micro-mucosa and micro-skin composite graft） were significantly higher than all the other groups postoperation（P＜0.05）.the junction between skin and mucous membrane can be clearly distinguished by HE staining.The mucous membrane had more epithelial cell layers,deep rete pegs and parakeratosis.The skin had obvious keratinized layer.Conclusions:1.Micro-mucosa graft to the wound can survive and grow to be a complete piece of flake.The epithelium becomes complete and achieves good quality on the 21st day postoperation.2.The morphology and distribution of the micro-mucosa on the wound have effects on the healing process after transplantation.The healing process goes fast and the neogenesis epithelium gets good quality when the average diameter,size and the density of the micro-mucosa is about 0.7mm,0.46mm² and 25/cm² respectively.3.The combination of micro-mucosa and micro-skin graft can accelerate the speed of wound epithelialization,however,both of them remain the histological characteristics of their own after transplantation. Part 2 Clinical research on vaginoplasty using autologous micro-mucosaObjective:The purpose of our study was to describe the procedure and outcome of creating neovagina lined with autologous micro-mucosa.To evaluate the neovagina by the terms of morphology,histology,microenvironment as well as the function and identify the significance of this method.Materials and methods:Between June 2006 and September 2008,12 patients underwent vaginaoplasty using autologous micro-mucosa graft from the oral cavity,whose age ranged from 19 to 30 years（mean 24.6 years）.11 of them were congenital absence of vagina,and the remaining one complained about vaginal stenosis after vaginal reconstruction 7 years ago. The oral mucosa graft was taken from the bilateral cheek and inner lips and minced into 0.7 mm in size and was transplanted to the cavity which is dissected between the bladder and the rectum.The follow-up includes measuring the depth and width of the neovagina, colposcopy,colpography,histological detection of the vaginal wall as well as secretion analysis.The function of the neovagina was assessed by female sexual function questionnaire in the patients having sexual life 6 months after operation and compared with normal female sexual function.Results:1.The operation was performed successfully in all twelve cases.The operative time was about 112 minutes（90～140 min） and operative blood loss was 80-100 mL.The average length of stay was about 14 days（10～19 days）.The time of epithelialization was about 14 days.All cases were followed up 4-26 months（average 14 months）.The donor sites healed uneventfully with no change in mouth opening.2.The perineal area was not disturbed,and a neovagina formed with a depth of 8cm （6-10 cm） and a width of two fingers.The lining is pink-colored and smooth and is confirmed as nonkeratizing squamous stratified mucosa.Vaginal plica was also visible from the orifice.The cream-colored viscous fluid was found in the neovaginal cavity and its PH value was about 6.2（5.5～7.0）.3.Of the twelve patients,eight were married.Seven of them answered the questionnaire and satisfied with their sexual life without pain and bleeding.There were significantly differences between the vaginoplasty group and normal control group on the total score and orgasm score（P＜0.05）,while no differences were found on desire, arousal,lubrication,relationship satisfaction and comfort score（P＞0.05）.Conclusions: 1.Vaginal reconstruction with autologous micro-mucosa graft is an easy,minimally invasive and useful method.The donor sites and perineal area were not disturbed.2.The neovagina lined with soft,smooth and pliable mucosa has adequate depth and width.3.Histologically,the neovaginal lining is confirmed as nonkeratinized squamous epithelium which is similar to the normal vagina.4.The neovagina achieves good function without any lubricants during sexual life.更多还原