乙状结肠阴道黏膜神经分布与性生活质量的关系及组织工程阴道动物模型构建的初步研究
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摘要
阴道成形术是解决先天性无子宫无阴道(Mayer-Rokitansky-Kuster-Hauser syndrome,MRKHS)患者性生活和性角色的主要方法。手术方式较多,临床上常采用自体或异体组织移植以及应用人工代用品植入尿道和直肠之间的人工腔穴做衬里进行阴道重建。乙状结肠代阴道成形术是较常用的一种术式,该术式成功率高,构建的阴道具有足够的长度、宽度和润滑,不易挛缩,多数文献报道患者有满意的性生活。我们前期研究已经观察到带蒂肠段形成新阴道后其组织形态、分泌功能和防御功能都发生了一系列的适应性改变,但与性活动关系密切的神经纤维在移位后的乙状结肠黏膜的分布和数量是否会发生变化?这种变化与患者的性生活质量有何关系?目前尚无相关研究,本研究第一部分以此为切入点,探讨乙状结肠阴道神经分布和功能之间的关系。
结合国内外对不同阴道成形术的临床效果以及手术近期和远期并发症的观察研究,发现尽管传统的阴道成形术解决了患者的痛苦,但同时也存在不同程度的缺陷。自体组织可造成机体的额外损伤,来源有限,异体组织存在免疫排斥和传播疾病的风险,而且利用这些非阴道组织再造的阴道在形态学和组织学方面与自然阴道组织差别较大,在功能上存在一定的局限性。因此,探索和改进阴道成形方法是必要而迫切的,组织工程学(tissue engineering)的兴起为其提供了空间和可能。本研究尝试构建组织工程阴道动物模型,期望建立形态和功能均接近正常的人工阴道,从而为在人体实现永久性替代、完美的形态修复和真正意义上的功能重建提供实验依据。
第一部分：乙状结肠代阴道成形术后阴道黏膜神经分布、雌激素受体表达及与性生活质量的关系
目的：检测乙状结肠代阴道成形术后阴道黏膜神经分布和雌激素受体(ER)表达的变化,探讨局部黏膜神经纤维密度和ER表达与患者性生活质量的关系,为乙状结肠阴道功能的评估和合理术式的选择提供依据。
方法：2006年1月-2010年12月,选择在河北医科大学第二医院妇产科接受乙状结肠代阴道成形术的MRKHS患者为研究对象。观察人工阴道解剖结构,对有性生活者,采用女性性功能量表(FSFI)中调查手术后患者近期的性生活质量。留取手术时患者乙状结肠断端黏膜(乙状结肠组,n=24),随访时取该患者的人工阴道黏膜组织(人工阴道组,n=24),取同期因非阴道疾患住院治疗患者的阴道黏膜(自然阴道组,n=10)。采用免疫组化法检测局部组织中的蛋白基因产物9.5(PGP9.5)的表达、血管活性肠肽(VIP)能神经和神经肽Y (NPY)能神经的分布和密度,以及ER的表达；实时荧光定量PCR (FQ-PCR)检测上述指标的mRNA表达；进一步分析人工阴道神经密度和ER表达与随访时限有无相关性；分析人工阴道神经分布和密度改变、ER表达和患者性功能指数评分之间的相关性。
结果：
1患者一般情况和FSFI评分
随访时限3个月-39个月。术后人工阴道外观与自然阴道相似,平均长度10.6±2.4cm,平均宽度2.9±0.6cm。随访时有性生活者17例(70.8%),性生活史从2个月-30个月不等。患者FSFI平均得分为24.11±1.33,23-29分之间有15例(88.2%),低于23分2例(11.8%)。其中性欲望评分为4.18±0.51,性兴奋4.044±0.55,阴道润滑3.57±0.49,性高潮4.00±0.48,性满意度5.06±0.65,疼痛3.25±0.90。
2神经分布特征
人工阴道黏膜组织的固有层、黏膜肌层、黏膜下层和肌层可见丰富的PGP9.5阳性表达的神经纤维,VIP和NPY表达阳性的神经纤维主要分布在血管周围和肌层；人工阴道黏膜组织中PGP9.5阳性表达的神经纤维密度分别高于VIP和NPY阳性表达的神经密度(38.22±8.93 vs7.45±2.01,6.13±2.38,P<0.05)。人工阴道的神经分布特征与乙状结肠相似。阳性神经纤维主要分布在自然阴道的固有层和肌层。
3人工阴道PGP9.5、VIP和NPY蛋白和mRNA的表达
本研究随访期限内,人工阴道黏膜PGP9.5、VIP、NPY阳性神经纤维的IOD均低于原位乙状结肠黏膜(38.22±8.93 vs 46.12±6.63,P1<0.05；7.45±2.01 vs 9.744±2.19,P2<0.05；6.13±2.38 vs 8.38±2.51,P3<0.05),但高于自然阴道黏膜(38.22±8.93 vs 22.76±6.61,Pj<0.05；7.45±2.01 vs4.59±1.76,P2<0.05；6.13±2.38 vs 3.58±1.23,P3<0.05)。
人工阴道PGP9.5、VIP和NPY mRNA表达均较原位乙状结肠降低(7.94±1.92 vs 10.17±2.24,Pj<0.05；2.37±0.87 vs 4.14±1.32,P2<0.05；2.68±0.75 vs 3.79±0.98,P3<0.05),较自然阴道黏膜高(7.94±1.92 vs 5.68±0.47,Pj<0.05；2.37±0.87 vs 1.04±0.36,P2<0.05；2.68±0.75 vs 1.15±0.33,P3<0.05)。
4人工阴道PGP9.5、VIP和NPY的表达与随访时限的相关性
人工阴道黏膜PGP9.5、VIP能阳性神经纤维表达与随访时限均呈正相关(r1=0.73,Pj=0.01；r2=0.16,P2=0.04),即随时间推移,移位的乙状结肠总的神经纤维密度和VIP能神经纤维密度降低后又有逐步回升趋势。人工阴道黏膜NPY能阳性神经纤维表达与随访时限无相关性(r=0.30,P=0.38)。
5人工阴道ER蛋白和mRNA表达及与随访时限的相关性
与原位乙状结肠比,人工阴道黏膜ER蛋白和mRNA表达明显升高(19.56±5.29 vs 10.49±2.84,Pj<0.05；4.36±1.41 vs 2.31±0.75,P2<0.05),但仍低于自然阴道黏膜的表达水平(19.56±5.29 vs 27.83±6.78,P1<0.05；4.36±1.41 vs 9.59±2.64,P2<0.05)。ER在人工阴道黏膜的表达与随访时限呈正相关(r=0.92,P=0.00),即随时间推移,ER表达逐步升高,但在本研究随访期限内,仍低于自然阴道。
6人工阴道神经分布、雌激素受体表达与患者性生活质量的相关性
PGP9.5和VIP mRNA表达与ER mRNA表达呈正相关(r1=0.66,P1=0.03；r2=0.65,P2=0.03)；NPY mRNA表达与ER mRNA表达无相关性(r=0.46,P=0.15)。FSFI与PGP9.5、VIP mRNA及NPY mRNA表达均无相关性(r1=0.12,Pj=0.72；r2=0.25,P2=0.43；r3=-0.47,P3=0.13)；FSFI与ER表达无相关性(r=0.50,P=0.12)。
结论：
1通过对乙状结肠代阴道成形术后患者的性功能指数调查,发现该术式可使患者获得较理想的性生活质量。
2人工阴道黏膜的神经分布特征与原位乙状结肠相似,其神经纤维密度介于乙状结肠和自然阴道之间,且随时间推移逐步回升,这种适应性改变可能是改善患者远期性生活质量的基础。
3人工阴道黏膜ER表达随时间的推移逐渐升高,提示移位后的乙状结肠黏膜对雌激素的敏感性增高,可能是移位后乙状结肠发生适应性改变及人工阴道pH值降低的基础。
4人工阴道黏膜PGP9.5和VIP mRNA表达与ER表达存在正相关,提示雌激素可能对局部神经有保护和促进再生作用。
5在对患者术后3年随访的研究期间,问卷调查显示患者FSFI评分与人工阴道的神经纤维密度和ER表达无明显相关性,提示影响性功能的因素复杂,需进一步深入广泛的研究。
第二部分：原代培养大鼠阴道黏膜上皮细胞的研究
目的：探讨大鼠阴道黏膜上皮细胞的体外培养和扩增技术,为构建组织工程化阴道动物模型提供种子细胞。
方法：(1)组织块法：将SD大鼠阴道全层组织剪切成0.5-1mm2小块,用解剖镊均匀地接种于一次性塑料培养皿中,在37。C 5%CO2饱和湿度细胞培养箱中静置培养3.0 h后,补加角化细胞无血清培养基继续培养。(2)酶消化法：取大鼠阴道全层组织,Dispase酶消化后分离上皮层,0.25%胰酶进一步消化成细胞悬液,接种于无血清角化细胞培养液中连续培养。观察两种方法细胞生长所需时间、细胞形态和体外生长特性,绘制生长曲线,免疫组化鉴定。
结果：
1组织块法于接种后3-5d有少量细胞自贴壁组织块周围爬出,3-4w后可传代。酶消化法24-36h后细胞开始贴壁,至7-1 Od细胞生长至80%融合,传代培养。
2倒置显微镜下见上皮细胞呈不规则圆形或多边形,连接成片后似铺路石状。扫描电镜下细胞表面可见微绒毛嵴。两种方法获得的细胞形态一致,传代后增殖特性和生长曲线基本一致。
3组织块法细胞培养成功率为37.5%,低于细胞法(50%),差异有显著性(P<0.05)。组织块法第一代细胞产量为2.67±0.23(×106/L),酶消化法为2.89±0.94(×106/L),差异无显著性(P>0.05)
4角蛋白染色阳性,组织块法细胞纯度为92%,酶消化法为98%。第四代细胞为正常二倍体核型。
结论：两种细胞培养方法均能获得不规则圆形或多边形的阴道上皮细胞,但酶消化法较组织块法细胞贴壁快,生长时间短,细胞增殖良好,成功率高,能较迅速获得较多细胞用于组织工程阴道的构建。
第三部分：聚乳酸-羟基乙酸/Ⅰ型胶原复合支架与阴道上皮细胞的相容性研究
目的：利用组织工程学原理,探讨聚乳酸-羟基乙酸(PLGA)/Ⅰ型胶原复合支架材料与阴道上皮细胞的生物相容性,验证该材料作为组织工程阴道支架的可行性。
方法：酶消化法培养SD大鼠阴道上皮细胞,制备PLGA/I型胶原支架材料。四甲基偶氮唑盐法测定支架材料浸提液对上皮细胞的毒性。上皮细胞接种于PLGA/I型胶原支架材料上体外培养,检测细胞与支架材料的黏附率,扫描电镜观察细胞在支架材料上的生长情况。将培养的细胞-支架材料移植到大鼠背部皮下,分别于2、4、8周取材,HE染色、免疫组化观察细胞在支架材料上的生长增殖。
结果：大鼠阴道上皮细胞在PLGA/I型胶原支架材料的浸提液中生长及增殖良好。PLGA/I型胶原支架的细胞黏附率为71.8%±9.2%,明显高于PLGA支架材料(63.4%±5.7%)。扫描电镜观察上皮细胞在复合支架材料上黏附、生长良好。皮下埋植2周,细胞在支架孔隙内生长增殖,成纤维细胞生长；4周材料表面形成1-3层上皮；8周后,支架材料部分降解,上皮层次增加,呈极性排列,广谱角蛋白免疫组化染色阳性。
结论：PLGA/I型胶原复合材料适合阴道上皮细胞的黏附生长,具有良好的相容性,可作为支架材料用于构建组织工程阴道。
第四部分：组织工程阴道动物模型的构建
目的：利用组织工程学原理,体外分离培养SD大鼠阴道上皮细胞,以PLGA/I型胶原为支架材料,构建人工阴道动物模型,以探讨更理想的阴道成形新术式的可行性,为进一步临床应用奠定实验基础。
方法：取10只SD大鼠阴道黏膜体外分离培养上皮细胞,接种于管状PLGA/I型胶原支架材料的内侧面,体外培养后原位移植到去除阴道的大鼠体内,分别于术后1、3、6个月处死动物,HE染色、Masson's染色和免疫组化动态观察组织学变化。
结果：
1大鼠的组织工程阴道重建手术成功完成。1只死于麻醉过量,观察过程中3只大鼠发生腹腔感染,其中2只死亡,1只阴道闭锁,其余6只大鼠阴道切口愈合良好,无阴道狭窄和塌陷。
2大体观察：1个月,阴道腔内可见岛状分布的黏膜,极薄,色红,支架材料少部分降解,阴道深度约1.4cm；3个月,阴道黏膜渐增厚,粉红色,有光泽,支架材料大部分降解,阴道深度约1.2cm.；术后6个月,组织工程阴道黏膜类似正常阴道黏膜,阴道深度约1.2cm.,无明显狭窄。
3组织学观察：术后1个月,仅有1-3层上皮细胞,未见平滑肌层,血管稀疏,胶原纤维较少,不均匀分布；术后3个月,上皮层增多,极性排列,可见少许平滑肌,间断存在,胶原纤维增多,欠整齐；术后6个月,上皮层与正常阴道无明显区别,基底层可见钉状突起,少于正常阴道,平滑肌束增多,胶原纤维致密,排列整齐。免疫组化证实阴道内腔面上皮组织的存在,与HE染色结果一致。
结论：以阴道上皮细胞和PLGA/I型胶原支架材料构建的组织工程阴道与大鼠正常阴道相似,可以成功修复大鼠阴道缺损。临床应用组织工程技术治疗先天性无阴道患者具有可行性。
Vaginoplasty is indicated in female with congenital absence or hypoplasia of the vagina, example for Mayer-Rokitansky-Kuster-Hauser syndrome (MRKHS). Many vaginal reconstruction methods have been described. The fundamental method is that a potential neovaginal space is created by blunt dissection of the space between the rectum and the bladder, and then the successive lining with autos or xenoma tissues were placed. Sigmoid colon vaginoplasty is particularly useful because it is anatomically close to the perineum, it is sufficiently long and the mobility of its vascular pedicle which allows it to be brought into the perineum. To date many studies have addressed that sigmoid vaginoplasty, which have shown the surgical procedure provides excellent results including adequate vaginal length, natural lubrication, a low incidence of shrinkage and early intercourse. Furthermore, over 80% studies had reported patients were satisfied with the psychosexual outcomes. After transplantation of sigmoid colon with vascular pedicle, its local mucosa have occurred some adaptative changes in morphology, secretion, and defense function with stimulation of intercourse and prolonged time from surgery. However, what change do the innervations and nerves density occur in the sigmoid vagina, and whether the changs correlate with sexual function of patients, which have not been examined to our knowledge. The contents will be explored in clinical part of our study.
Patients with MRKHS implement sexual intercourse using neovagina constructed by these traditional procedures. However, shortcomings of surgeries have been found, for example supernumerary trauma following autos tissues transplantation, rejection and propagation of diseases induced by xenoma tissues. Furthermore, the neovagina constructed by these ways show significantly difference in morphology and histology compared with the natural vagina. Therefore, it is necessary and emergent to explore and development the ideal vaginoplasty. With the development of tissue engineering, the ideal neovagina might be constructed with the technique. In fundament study part of the paper, we will attempt to reconstruction of tissue engineering vagina in rat, and expect to establish a new way for clinical vaginoplasty with more excellent outcomes.
Part one:Innervations, Expression of Estrogen Receptor in the Neovagina Mucosa and Correlation Analyses with Sexual Activity Quality after Sigmoid Vaginoplasty
Objective:To investigate the innervation and expression of estrogen receptor (ER) in the neovaginal wall of the patients undergoing sigmoid vaginoplasty for Mayer-Rokitansky-Kistner-Hauser Syndrome (MRKHS), and to assess whether the changs of nerve density and ER expression correlate with sexual function of patients
Methods:Twenty-four patients with MRKHS undergone sigmoid colon vaginoplasty were selected as research subjects from January 2006 to December 2010 in the Department. of OB & GYN of second hospital of HeBei Medical University. Surgical outcomes were observed during follow-up, and sexual activity quality were surveyed using female sexual function index (FSFI) to patients with intercourse. Biopsies of the intact sigmoid colon were obtained from these subjects during sigmoid vaginoplasty in the same period (sigmoid colon group, n=24). Tissue biopsies were taken out transvaginally from the posterior neovagina at the upper third position (neovagina group, n=24). Biopsies of natural vagina were obtained at the same position with neovagina from age-matched female without vaginal diseases (natural vagina group, n=10). The protein and mRNA expressions of protein gene product 9.5 (PGP9.5), and ER were detected by immunohistochemical method and real-time fluorescence quantitative PCR (FQ-PCR) in order to observed the distribution and density of nerve fibers containing particular neuropeptide. The expression of ER was studied with the same methods, and furtherly the correlation above parameters with interval time to surgery were analyzed. The relationship was also studied among nerves density, ER expression, and FSFI in neovagina.
Results:
1 The follow-up was 3 to 39 months. The appearances of neovagina were similar to that of the natural vagina, the mean length of the neovagina was 10.6±2.4cm, the mean width was 2.9±0.6cm, and no postoperative mortality and minimal morbidity occurred. Seventeen patients (70.8%) had regular vaginal intercourse. The range of sexual history was 2 to 30 months. The mean full FSFI score was 24.11±1.33, fifteen of these patients had a normal score whose range was 23 to 29, and two of them had lower score than 23. After creation of a sigmoid neovagina, patients with MRKHS can be considered normal in terms of desire (4.18±0.51), arousability (4.04±0.55), lubrification (3.57±0.49), orgasm (4.00±0.48), satisfaction (5.06±0.65), and comfort (3.25±0.90).
2 A larger number of PGP 9.5 nerve fibers were observed in lamina propria, muscle of the mucosa, submucosa, and muscle layer of the neovagina. The nerve fibers with VIP-and NPY-immunoreactivity (IR) mainly distributed around blood vessels and in the smooth muscles. In the neovagina, the expression intensity of nerve fibers was PGP 9.5> VIP and NPY (38.22±8.93 vs 7.45±2.01,6.13±2.38, P<0.05). These characters in neovagina were similar to the intact sigmoid colon in the study. In the natural vagina, the immunoreactivity nerve fibers distributed in the lamina propria and muscle layer.
3 An integral optical density (IOD) of PGP9.5, VIP, and NPY-IR was measured to evaluate total innervations in neovagina. PGP9.5, VIP, and NPY-positive expression were significantly lower in the neovagina than that in the intact sigmoid colon (38.22±8.93 vs 46.12±6.63, P1<0.05; 7.45±2.01 vs 9.74±2.19, P2<0.05; 6.13±2.38 vs 8.38±2.51, P3<0.05), but higher than that in the natural vagina (38.22±8.93 vs 22.76±6.61, P;<0.05; 7.45±2.01 vs 4.59±1.76,P2<0.05; 6.13±2.38 vs 3.58±1.23, P3<0.05)
These parameters mRNA expression displayed a similar tendency, which were the values in sigmoid colon> neovagina> vagina (PGP9.5:10.17±2.24 vs 7.94±1.92 vs 5.68±0.47, P1<0.05; VIP:4.14±1.32 vs 2.37±0.87 vs 1.04±0.36, P2<0.05; NPY:3.79±0.98 vs 2.68±0.75 vs 1.15±0.33, P3<0.05)
4 In the neovagina, PGP9.5 and VIP-IR nerve fibers and mRNA expression had a positive linear correlation with time interval to surgery r1=0.73, P1=0.01; r2=0.16, P2=0.04), which indicated the nerve density tended to decrease firstly and gradually upturn by time after surgery. Whereas, correlation between NPY expression and time interval to surgery wasn't found(r=0.30, P=0.38).
5 The expression ER protein and mRNA were significantly increase compared with the sigmoid colon (19.56±5.29 vs 10.49±2.84, P,<0.05; 4.36±1.41 vs 2.31±0.75, P2<0.05), but didn't reach the levels of that in the vagina (19.56±5.29 vs 27.83±6.78, P,<0.05; 4.36±1.41 vs 9.59±2.64, P2<0.05). ER expression had a positive linear correlation with time interval to surgery (r=0.92, P=0.00), which it gradually increased by time after surgery, but lower than that of in the natural vagina in our follow-up.
6 In the neovagina, the expression of PGP9.5 and VIP mRNA had a positive linear correlation with that of ER (n=0.66, P1=0.03; r2=0.65, P2=0.03), NPY mRNA expression hadn't correlation with ER (r=0.46, P=0.15). FSFI hadn't correlation with PGP9.5, NPY, VIP and ER mRNA expression (r1=0.12, P1=0.72; r2=0.25, P2=0.43; r3=-0.47, P3=0.13; r4=0.50, P4=0.12).
Conclusions:
1 Sigmoid neovagina allowed a normal sexual life and provided a good quality of sexual activity in patients with MRKHS who had sexual intercourse.
2 The distribution of sensory PGP9.5-, VIP-, and NPY- positive nerve fibers in the neovagina wall was similar to the pattern observed in the intact sigmoid colon wall. The density of nerve fibers in the neovagina was lower than that in the intact sigmoid but higher than that in the natural vagina, and gradually increased with time during our follow up. The adaptative change may be helpful in enhancing the sensitivity of the neovaginal mucosa and improving long-term quality of sexual activity.
3 The expression of ER protein and mRNA were increase by time, which was probably physiological foundation on the adaptative changes of histomorphology and decrease of pH value in neovagina.
4 The expression of PGP9.5 and VIP mRNA had a positive linear correlation with ER mRNA expression, which supported the protection of estrogen to nerves and maybe promoted incomplete reinnervation in neovagina by this way.
5 No correlation between FSFI of patients and innervations and ER expression in the neovagina suggested the factors of effecting sexual function were complicated and needed further study.
Part two:Primary Culture of Rat Vaginal Epithelial Cells in vitro
Objective:Vaginal epithelial cells from Sprague-Dawley rats were primary cultured by tissue explants and enzyme digestion methods in vitro respectively in order to explore the better culture technique and provide seed cells for vaginal tissue engineering.
Methods:
1) Tissue explants method:Vaginal tissues of rats were pruned to 0.5-1mm2, and then planted on the culture capsule. After 3 hours in incubator with 37℃and 5%CO2, the Keratinocyte Serum-Free Medium (K-SFM) were added and continuously cultured.
2) Enzyme digestion method:The rat vaginal tissues were digested by dispase enzyme to isolate the epithelial layer, and furtherly digested by trypsin. The cell suspension was cultured continuously in K-SFM.
3) Time of primary culture, the cell morphology, proliferation characteristics and ultrastructure were oberserved under light and electron microscope, and the growth curve was drawn. The cells were identified with Anti-pan-cytokeratin (P-CK) by immunohistochemistry stain.
Results:
1) The cells started to growth within 3-5 days by tissue explants, and confluent growth was noted for subculturing after 3-4 weeks. The culture cells with enzyme digestion method began to adhere within 24-36 hours, and reached 80% confluency after 7-10 days.
2) Vaginal epithelial cells were polygon-like or irregular globular and show typical appearances of "paving stone" after confluention under inversion microscope. Microvilli ridge were observed on cells surface with scanning electron microscope. The culture cells morphology and growth characteristics were essentially consistent by the two methods.
3) The success ratio of culture cells by tissue explants was significantly low compared with enzyme digestion method (37.5% vs 50%, P<0.05). The cell productivity by the two methods didn't show significant differences (2.67±0.23×106/L vs 2.89±0.94×106/L, P>0.05).
4) The expression of P-CK staining of the cultured cells was positive. Purity of cells using explants and enzyme digestion method were respectively 92% and 98%. The fourth generation cells pocess normal diploid karyotype.
Conclutions:Vaginal epithelial cells were obtained by the two methods. Compared with explants method, the culture cells using enzyme digestion method display faster adhesion, shorter growth time, and higher success rate. The larger numbers of epithelial cells were rapidly obtained to construct engineering of vaginal tissue by enzyme digestion method.
Part three:Biocompatibility Between PLGA/ⅠCollagen Scaffold and the Rat Vaginal Epithelial Cells
Objective:To explore the biocompatibility of the PLGA/Ⅰcollagen with rat vaginal epithelial cells, and verify the feasibility of using PLGA/Ⅰcollagen as scaffold to reconstruct vagina by the tissue engineering.
Methods:The rat vaginal epithelial cells were cultured by enzyme digestion method.Ⅰcollagen was combined with poly lactic-co-glycolic acid (PLGA) to form PLGA/Ⅰcollagen biodegradable polymer scaffold. The epithelial cells were culture in the leaching liquor of scaffold to detect its toxicity to cells by MTT. The epithelial cells were inoculated on the scaffold to calculate cell adhesion rate, and cells growth were observed in the epithelial cells-scaffold complexes with scanning electron microscope. Epithelial cells-scaffold complexes were implanted under cutis of rat back. At 2,4, and 8 weeks after implantation, the cells-scaffold were assessed by histological HE stain and immunohistochemical analysis
Results:The Epithelial cells grew and proliferated well in the leaching liquor of scaffold. On the PLGA/Ⅰcollagen scaffold, the cell adhesion rate was 71.8%±9.2%, which significantly higher than that on the PLGA scaffold. The epithelial cells were well distributed and adhered to the PLGA/Ⅰcollagen scaffolds under scanning electron microscope. At 2 weeks after implanted under cutis of rat back, the epithelial cells attached and proliferated in the space, and the fibroblasts were seen. At 4 weeks, the epithelium were seen about 1-3 layer. At 8 weeks, the epithelial cells increased and arranged regularitility, which formed the membrane-like layer on the scaffold. The expression of P-CK of the epithelium was positive.
Conclusions:The PLGA/Ⅰcollagen scaffolds are well-cytocompatible to the epithelial cells, and can be used as the biodegradable polymer scaffold of the vaginal tissue engineering.
Part four:Reconstruction of Tissue Engineering Vagina in Rat
Objective:To explore feasibility of reconstructing completely rat vagina by tissue engineering, and provide technical function on clinical application.
Methods:The epithelial cells were cultured from rat vaginal tissues, and innoculated on the medial surface of fistular PLGA/Ⅰcollagen biological material. The epithelial cells-scaffold complexes were transplanted to rat vagina in situ after culture in vitro to reconstruct vagina. After 1,3, and 6 months, we measured the vaginal length and observed the appearance at maero. The neovagina tissues were prepared for histological evaluation with HE, Masson's, and immunohistochemistry stain.
Results:
1) The experimental rat model of vaginal reconstruction surgery was performed sueeessfully.1 rat died to overdose of anesthesia after surgery, and during observation,3 rats subjected serious infection in abdominal cavity,2 of these died and 1 of these had ankylocolpos. and coleostenosis weren't be seen in terms of survival 6 rats.
2) At 1 month after operation, we observed thin and red epithelialization on the biological material in the vaginal canal, which distributed like islands. The little of biological material took place degradation, and the length of vagina was 1.4cm. Within 1-3 months, the vaginal mucosa get thick gradually and pink. The biological material had degradated more than a half, and the vagina was 1.2cm in length. After 6 months, the neovagina was hardly differentiated from the native one at either maero or miero appearance, and the vagina was 1.2cm in length and no shrink.
3) Light microscopy inspection showed the epithelium was 1-3 layers after 1 month, no smooth muscle, rare blood vessel and collagen fibers. At 3 months after surgery, the epithelium was thicker and multi-layere and the minor smooth muscle were seen occasionally. At 6 months, histology of neovagina was similar to the native one except less rete pegs in the epithelium basilar part, more smooth muscle and compact collagen fibers were observed by Masson's stain. Epithelial cells layer were identified by immunohistoch-emistry stain with P-CK since 1 month after surgery which was accordant to the result in HE stain.
Conclusins:The neovagina which was reconstructed using rat vaginal epithelial cells and PLGA/I collagen scaffold was hardly differentiated from the native one at either maero or miero appearance. A tissue engineering approach to clinical vaginal reconstruction in women is now a realistic possibility.
结合国内外对不同阴道成形术的临床效果以及手术近期和远期并发症的观察研究,发现尽管传统的阴道成形术解决了患者的痛苦,但同时也存在不同程度的缺陷。自体组织可造成机体的额外损伤,来源有限,异体组织存在免疫排斥和传播疾病的风险,而且利用这些非阴道组织再造的阴道在形态学和组织学方面与自然阴道组织差别较大,在功能上存在一定的局限性。因此,探索和改进阴道成形方法是必要而迫切的,组织工程学(tissue engineering)的兴起为其提供了空间和可能。本研究尝试构建组织工程阴道动物模型,期望建立形态和功能均接近正常的人工阴道,从而为在人体实现永久性替代、完美的形态修复和真正意义上的功能重建提供实验依据。
第一部分：乙状结肠代阴道成形术后阴道黏膜神经分布、雌激素受体表达及与性生活质量的关系
目的：检测乙状结肠代阴道成形术后阴道黏膜神经分布和雌激素受体(ER)表达的变化,探讨局部黏膜神经纤维密度和ER表达与患者性生活质量的关系,为乙状结肠阴道功能的评估和合理术式的选择提供依据。
方法：2006年1月-2010年12月,选择在河北医科大学第二医院妇产科接受乙状结肠代阴道成形术的MRKHS患者为研究对象。观察人工阴道解剖结构,对有性生活者,采用女性性功能量表(FSFI)中调查手术后患者近期的性生活质量。留取手术时患者乙状结肠断端黏膜(乙状结肠组,n=24),随访时取该患者的人工阴道黏膜组织(人工阴道组,n=24),取同期因非阴道疾患住院治疗患者的阴道黏膜(自然阴道组,n=10)。采用免疫组化法检测局部组织中的蛋白基因产物9.5(PGP9.5)的表达、血管活性肠肽(VIP)能神经和神经肽Y (NPY)能神经的分布和密度,以及ER的表达；实时荧光定量PCR (FQ-PCR)检测上述指标的mRNA表达；进一步分析人工阴道神经密度和ER表达与随访时限有无相关性；分析人工阴道神经分布和密度改变、ER表达和患者性功能指数评分之间的相关性。
结果：
1患者一般情况和FSFI评分
随访时限3个月-39个月。术后人工阴道外观与自然阴道相似,平均长度10.6±2.4cm,平均宽度2.9±0.6cm。随访时有性生活者17例(70.8%),性生活史从2个月-30个月不等。患者FSFI平均得分为24.11±1.33,23-29分之间有15例(88.2%),低于23分2例(11.8%)。其中性欲望评分为4.18±0.51,性兴奋4.044±0.55,阴道润滑3.57±0.49,性高潮4.00±0.48,性满意度5.06±0.65,疼痛3.25±0.90。
2神经分布特征
人工阴道黏膜组织的固有层、黏膜肌层、黏膜下层和肌层可见丰富的PGP9.5阳性表达的神经纤维,VIP和NPY表达阳性的神经纤维主要分布在血管周围和肌层；人工阴道黏膜组织中PGP9.5阳性表达的神经纤维密度分别高于VIP和NPY阳性表达的神经密度(38.22±8.93 vs7.45±2.01,6.13±2.38,P<0.05)。人工阴道的神经分布特征与乙状结肠相似。阳性神经纤维主要分布在自然阴道的固有层和肌层。
3人工阴道PGP9.5、VIP和NPY蛋白和mRNA的表达
本研究随访期限内,人工阴道黏膜PGP9.5、VIP、NPY阳性神经纤维的IOD均低于原位乙状结肠黏膜(38.22±8.93 vs 46.12±6.63,P1<0.05；7.45±2.01 vs 9.744±2.19,P2<0.05；6.13±2.38 vs 8.38±2.51,P3<0.05),但高于自然阴道黏膜(38.22±8.93 vs 22.76±6.61,Pj<0.05；7.45±2.01 vs4.59±1.76,P2<0.05；6.13±2.38 vs 3.58±1.23,P3<0.05)。
人工阴道PGP9.5、VIP和NPY mRNA表达均较原位乙状结肠降低(7.94±1.92 vs 10.17±2.24,Pj<0.05；2.37±0.87 vs 4.14±1.32,P2<0.05；2.68±0.75 vs 3.79±0.98,P3<0.05),较自然阴道黏膜高(7.94±1.92 vs 5.68±0.47,Pj<0.05；2.37±0.87 vs 1.04±0.36,P2<0.05；2.68±0.75 vs 1.15±0.33,P3<0.05)。
4人工阴道PGP9.5、VIP和NPY的表达与随访时限的相关性
人工阴道黏膜PGP9.5、VIP能阳性神经纤维表达与随访时限均呈正相关(r1=0.73,Pj=0.01；r2=0.16,P2=0.04),即随时间推移,移位的乙状结肠总的神经纤维密度和VIP能神经纤维密度降低后又有逐步回升趋势。人工阴道黏膜NPY能阳性神经纤维表达与随访时限无相关性(r=0.30,P=0.38)。
5人工阴道ER蛋白和mRNA表达及与随访时限的相关性
与原位乙状结肠比,人工阴道黏膜ER蛋白和mRNA表达明显升高(19.56±5.29 vs 10.49±2.84,Pj<0.05；4.36±1.41 vs 2.31±0.75,P2<0.05),但仍低于自然阴道黏膜的表达水平(19.56±5.29 vs 27.83±6.78,P1<0.05；4.36±1.41 vs 9.59±2.64,P2<0.05)。ER在人工阴道黏膜的表达与随访时限呈正相关(r=0.92,P=0.00),即随时间推移,ER表达逐步升高,但在本研究随访期限内,仍低于自然阴道。
6人工阴道神经分布、雌激素受体表达与患者性生活质量的相关性
PGP9.5和VIP mRNA表达与ER mRNA表达呈正相关(r1=0.66,P1=0.03；r2=0.65,P2=0.03)；NPY mRNA表达与ER mRNA表达无相关性(r=0.46,P=0.15)。FSFI与PGP9.5、VIP mRNA及NPY mRNA表达均无相关性(r1=0.12,Pj=0.72；r2=0.25,P2=0.43；r3=-0.47,P3=0.13)；FSFI与ER表达无相关性(r=0.50,P=0.12)。
结论：
1通过对乙状结肠代阴道成形术后患者的性功能指数调查,发现该术式可使患者获得较理想的性生活质量。
2人工阴道黏膜的神经分布特征与原位乙状结肠相似,其神经纤维密度介于乙状结肠和自然阴道之间,且随时间推移逐步回升,这种适应性改变可能是改善患者远期性生活质量的基础。
3人工阴道黏膜ER表达随时间的推移逐渐升高,提示移位后的乙状结肠黏膜对雌激素的敏感性增高,可能是移位后乙状结肠发生适应性改变及人工阴道pH值降低的基础。
4人工阴道黏膜PGP9.5和VIP mRNA表达与ER表达存在正相关,提示雌激素可能对局部神经有保护和促进再生作用。
5在对患者术后3年随访的研究期间,问卷调查显示患者FSFI评分与人工阴道的神经纤维密度和ER表达无明显相关性,提示影响性功能的因素复杂,需进一步深入广泛的研究。
第二部分：原代培养大鼠阴道黏膜上皮细胞的研究
目的：探讨大鼠阴道黏膜上皮细胞的体外培养和扩增技术,为构建组织工程化阴道动物模型提供种子细胞。
方法：(1)组织块法：将SD大鼠阴道全层组织剪切成0.5-1mm2小块,用解剖镊均匀地接种于一次性塑料培养皿中,在37。C 5%CO2饱和湿度细胞培养箱中静置培养3.0 h后,补加角化细胞无血清培养基继续培养。(2)酶消化法：取大鼠阴道全层组织,Dispase酶消化后分离上皮层,0.25%胰酶进一步消化成细胞悬液,接种于无血清角化细胞培养液中连续培养。观察两种方法细胞生长所需时间、细胞形态和体外生长特性,绘制生长曲线,免疫组化鉴定。
结果：
1组织块法于接种后3-5d有少量细胞自贴壁组织块周围爬出,3-4w后可传代。酶消化法24-36h后细胞开始贴壁,至7-1 Od细胞生长至80%融合,传代培养。
2倒置显微镜下见上皮细胞呈不规则圆形或多边形,连接成片后似铺路石状。扫描电镜下细胞表面可见微绒毛嵴。两种方法获得的细胞形态一致,传代后增殖特性和生长曲线基本一致。
3组织块法细胞培养成功率为37.5%,低于细胞法(50%),差异有显著性(P<0.05)。组织块法第一代细胞产量为2.67±0.23(×106/L),酶消化法为2.89±0.94(×106/L),差异无显著性(P>0.05)
4角蛋白染色阳性,组织块法细胞纯度为92%,酶消化法为98%。第四代细胞为正常二倍体核型。
结论：两种细胞培养方法均能获得不规则圆形或多边形的阴道上皮细胞,但酶消化法较组织块法细胞贴壁快,生长时间短,细胞增殖良好,成功率高,能较迅速获得较多细胞用于组织工程阴道的构建。
第三部分：聚乳酸-羟基乙酸/Ⅰ型胶原复合支架与阴道上皮细胞的相容性研究
目的：利用组织工程学原理,探讨聚乳酸-羟基乙酸(PLGA)/Ⅰ型胶原复合支架材料与阴道上皮细胞的生物相容性,验证该材料作为组织工程阴道支架的可行性。
方法：酶消化法培养SD大鼠阴道上皮细胞,制备PLGA/I型胶原支架材料。四甲基偶氮唑盐法测定支架材料浸提液对上皮细胞的毒性。上皮细胞接种于PLGA/I型胶原支架材料上体外培养,检测细胞与支架材料的黏附率,扫描电镜观察细胞在支架材料上的生长情况。将培养的细胞-支架材料移植到大鼠背部皮下,分别于2、4、8周取材,HE染色、免疫组化观察细胞在支架材料上的生长增殖。
结果：大鼠阴道上皮细胞在PLGA/I型胶原支架材料的浸提液中生长及增殖良好。PLGA/I型胶原支架的细胞黏附率为71.8%±9.2%,明显高于PLGA支架材料(63.4%±5.7%)。扫描电镜观察上皮细胞在复合支架材料上黏附、生长良好。皮下埋植2周,细胞在支架孔隙内生长增殖,成纤维细胞生长；4周材料表面形成1-3层上皮；8周后,支架材料部分降解,上皮层次增加,呈极性排列,广谱角蛋白免疫组化染色阳性。
结论：PLGA/I型胶原复合材料适合阴道上皮细胞的黏附生长,具有良好的相容性,可作为支架材料用于构建组织工程阴道。
第四部分：组织工程阴道动物模型的构建
目的：利用组织工程学原理,体外分离培养SD大鼠阴道上皮细胞,以PLGA/I型胶原为支架材料,构建人工阴道动物模型,以探讨更理想的阴道成形新术式的可行性,为进一步临床应用奠定实验基础。
方法：取10只SD大鼠阴道黏膜体外分离培养上皮细胞,接种于管状PLGA/I型胶原支架材料的内侧面,体外培养后原位移植到去除阴道的大鼠体内,分别于术后1、3、6个月处死动物,HE染色、Masson's染色和免疫组化动态观察组织学变化。
结果：
1大鼠的组织工程阴道重建手术成功完成。1只死于麻醉过量,观察过程中3只大鼠发生腹腔感染,其中2只死亡,1只阴道闭锁,其余6只大鼠阴道切口愈合良好,无阴道狭窄和塌陷。
2大体观察：1个月,阴道腔内可见岛状分布的黏膜,极薄,色红,支架材料少部分降解,阴道深度约1.4cm；3个月,阴道黏膜渐增厚,粉红色,有光泽,支架材料大部分降解,阴道深度约1.2cm.；术后6个月,组织工程阴道黏膜类似正常阴道黏膜,阴道深度约1.2cm.,无明显狭窄。
3组织学观察：术后1个月,仅有1-3层上皮细胞,未见平滑肌层,血管稀疏,胶原纤维较少,不均匀分布；术后3个月,上皮层增多,极性排列,可见少许平滑肌,间断存在,胶原纤维增多,欠整齐；术后6个月,上皮层与正常阴道无明显区别,基底层可见钉状突起,少于正常阴道,平滑肌束增多,胶原纤维致密,排列整齐。免疫组化证实阴道内腔面上皮组织的存在,与HE染色结果一致。
结论：以阴道上皮细胞和PLGA/I型胶原支架材料构建的组织工程阴道与大鼠正常阴道相似,可以成功修复大鼠阴道缺损。临床应用组织工程技术治疗先天性无阴道患者具有可行性。
Vaginoplasty is indicated in female with congenital absence or hypoplasia of the vagina, example for Mayer-Rokitansky-Kuster-Hauser syndrome (MRKHS). Many vaginal reconstruction methods have been described. The fundamental method is that a potential neovaginal space is created by blunt dissection of the space between the rectum and the bladder, and then the successive lining with autos or xenoma tissues were placed. Sigmoid colon vaginoplasty is particularly useful because it is anatomically close to the perineum, it is sufficiently long and the mobility of its vascular pedicle which allows it to be brought into the perineum. To date many studies have addressed that sigmoid vaginoplasty, which have shown the surgical procedure provides excellent results including adequate vaginal length, natural lubrication, a low incidence of shrinkage and early intercourse. Furthermore, over 80% studies had reported patients were satisfied with the psychosexual outcomes. After transplantation of sigmoid colon with vascular pedicle, its local mucosa have occurred some adaptative changes in morphology, secretion, and defense function with stimulation of intercourse and prolonged time from surgery. However, what change do the innervations and nerves density occur in the sigmoid vagina, and whether the changs correlate with sexual function of patients, which have not been examined to our knowledge. The contents will be explored in clinical part of our study.
Patients with MRKHS implement sexual intercourse using neovagina constructed by these traditional procedures. However, shortcomings of surgeries have been found, for example supernumerary trauma following autos tissues transplantation, rejection and propagation of diseases induced by xenoma tissues. Furthermore, the neovagina constructed by these ways show significantly difference in morphology and histology compared with the natural vagina. Therefore, it is necessary and emergent to explore and development the ideal vaginoplasty. With the development of tissue engineering, the ideal neovagina might be constructed with the technique. In fundament study part of the paper, we will attempt to reconstruction of tissue engineering vagina in rat, and expect to establish a new way for clinical vaginoplasty with more excellent outcomes.
Part one:Innervations, Expression of Estrogen Receptor in the Neovagina Mucosa and Correlation Analyses with Sexual Activity Quality after Sigmoid Vaginoplasty
Objective:To investigate the innervation and expression of estrogen receptor (ER) in the neovaginal wall of the patients undergoing sigmoid vaginoplasty for Mayer-Rokitansky-Kistner-Hauser Syndrome (MRKHS), and to assess whether the changs of nerve density and ER expression correlate with sexual function of patients
Methods:Twenty-four patients with MRKHS undergone sigmoid colon vaginoplasty were selected as research subjects from January 2006 to December 2010 in the Department. of OB & GYN of second hospital of HeBei Medical University. Surgical outcomes were observed during follow-up, and sexual activity quality were surveyed using female sexual function index (FSFI) to patients with intercourse. Biopsies of the intact sigmoid colon were obtained from these subjects during sigmoid vaginoplasty in the same period (sigmoid colon group, n=24). Tissue biopsies were taken out transvaginally from the posterior neovagina at the upper third position (neovagina group, n=24). Biopsies of natural vagina were obtained at the same position with neovagina from age-matched female without vaginal diseases (natural vagina group, n=10). The protein and mRNA expressions of protein gene product 9.5 (PGP9.5), and ER were detected by immunohistochemical method and real-time fluorescence quantitative PCR (FQ-PCR) in order to observed the distribution and density of nerve fibers containing particular neuropeptide. The expression of ER was studied with the same methods, and furtherly the correlation above parameters with interval time to surgery were analyzed. The relationship was also studied among nerves density, ER expression, and FSFI in neovagina.
Results:
1 The follow-up was 3 to 39 months. The appearances of neovagina were similar to that of the natural vagina, the mean length of the neovagina was 10.6±2.4cm, the mean width was 2.9±0.6cm, and no postoperative mortality and minimal morbidity occurred. Seventeen patients (70.8%) had regular vaginal intercourse. The range of sexual history was 2 to 30 months. The mean full FSFI score was 24.11±1.33, fifteen of these patients had a normal score whose range was 23 to 29, and two of them had lower score than 23. After creation of a sigmoid neovagina, patients with MRKHS can be considered normal in terms of desire (4.18±0.51), arousability (4.04±0.55), lubrification (3.57±0.49), orgasm (4.00±0.48), satisfaction (5.06±0.65), and comfort (3.25±0.90).
2 A larger number of PGP 9.5 nerve fibers were observed in lamina propria, muscle of the mucosa, submucosa, and muscle layer of the neovagina. The nerve fibers with VIP-and NPY-immunoreactivity (IR) mainly distributed around blood vessels and in the smooth muscles. In the neovagina, the expression intensity of nerve fibers was PGP 9.5> VIP and NPY (38.22±8.93 vs 7.45±2.01,6.13±2.38, P<0.05). These characters in neovagina were similar to the intact sigmoid colon in the study. In the natural vagina, the immunoreactivity nerve fibers distributed in the lamina propria and muscle layer.
3 An integral optical density (IOD) of PGP9.5, VIP, and NPY-IR was measured to evaluate total innervations in neovagina. PGP9.5, VIP, and NPY-positive expression were significantly lower in the neovagina than that in the intact sigmoid colon (38.22±8.93 vs 46.12±6.63, P1<0.05; 7.45±2.01 vs 9.74±2.19, P2<0.05; 6.13±2.38 vs 8.38±2.51, P3<0.05), but higher than that in the natural vagina (38.22±8.93 vs 22.76±6.61, P;<0.05; 7.45±2.01 vs 4.59±1.76,P2<0.05; 6.13±2.38 vs 3.58±1.23, P3<0.05)
These parameters mRNA expression displayed a similar tendency, which were the values in sigmoid colon> neovagina> vagina (PGP9.5:10.17±2.24 vs 7.94±1.92 vs 5.68±0.47, P1<0.05; VIP:4.14±1.32 vs 2.37±0.87 vs 1.04±0.36, P2<0.05; NPY:3.79±0.98 vs 2.68±0.75 vs 1.15±0.33, P3<0.05)
4 In the neovagina, PGP9.5 and VIP-IR nerve fibers and mRNA expression had a positive linear correlation with time interval to surgery r1=0.73, P1=0.01; r2=0.16, P2=0.04), which indicated the nerve density tended to decrease firstly and gradually upturn by time after surgery. Whereas, correlation between NPY expression and time interval to surgery wasn't found(r=0.30, P=0.38).
5 The expression ER protein and mRNA were significantly increase compared with the sigmoid colon (19.56±5.29 vs 10.49±2.84, P,<0.05; 4.36±1.41 vs 2.31±0.75, P2<0.05), but didn't reach the levels of that in the vagina (19.56±5.29 vs 27.83±6.78, P,<0.05; 4.36±1.41 vs 9.59±2.64, P2<0.05). ER expression had a positive linear correlation with time interval to surgery (r=0.92, P=0.00), which it gradually increased by time after surgery, but lower than that of in the natural vagina in our follow-up.
6 In the neovagina, the expression of PGP9.5 and VIP mRNA had a positive linear correlation with that of ER (n=0.66, P1=0.03; r2=0.65, P2=0.03), NPY mRNA expression hadn't correlation with ER (r=0.46, P=0.15). FSFI hadn't correlation with PGP9.5, NPY, VIP and ER mRNA expression (r1=0.12, P1=0.72; r2=0.25, P2=0.43; r3=-0.47, P3=0.13; r4=0.50, P4=0.12).
Conclusions:
1 Sigmoid neovagina allowed a normal sexual life and provided a good quality of sexual activity in patients with MRKHS who had sexual intercourse.
2 The distribution of sensory PGP9.5-, VIP-, and NPY- positive nerve fibers in the neovagina wall was similar to the pattern observed in the intact sigmoid colon wall. The density of nerve fibers in the neovagina was lower than that in the intact sigmoid but higher than that in the natural vagina, and gradually increased with time during our follow up. The adaptative change may be helpful in enhancing the sensitivity of the neovaginal mucosa and improving long-term quality of sexual activity.
3 The expression of ER protein and mRNA were increase by time, which was probably physiological foundation on the adaptative changes of histomorphology and decrease of pH value in neovagina.
4 The expression of PGP9.5 and VIP mRNA had a positive linear correlation with ER mRNA expression, which supported the protection of estrogen to nerves and maybe promoted incomplete reinnervation in neovagina by this way.
5 No correlation between FSFI of patients and innervations and ER expression in the neovagina suggested the factors of effecting sexual function were complicated and needed further study.
Part two:Primary Culture of Rat Vaginal Epithelial Cells in vitro
Objective:Vaginal epithelial cells from Sprague-Dawley rats were primary cultured by tissue explants and enzyme digestion methods in vitro respectively in order to explore the better culture technique and provide seed cells for vaginal tissue engineering.
Methods:
1) Tissue explants method:Vaginal tissues of rats were pruned to 0.5-1mm2, and then planted on the culture capsule. After 3 hours in incubator with 37℃and 5%CO2, the Keratinocyte Serum-Free Medium (K-SFM) were added and continuously cultured.
2) Enzyme digestion method:The rat vaginal tissues were digested by dispase enzyme to isolate the epithelial layer, and furtherly digested by trypsin. The cell suspension was cultured continuously in K-SFM.
3) Time of primary culture, the cell morphology, proliferation characteristics and ultrastructure were oberserved under light and electron microscope, and the growth curve was drawn. The cells were identified with Anti-pan-cytokeratin (P-CK) by immunohistochemistry stain.
Results:
1) The cells started to growth within 3-5 days by tissue explants, and confluent growth was noted for subculturing after 3-4 weeks. The culture cells with enzyme digestion method began to adhere within 24-36 hours, and reached 80% confluency after 7-10 days.
2) Vaginal epithelial cells were polygon-like or irregular globular and show typical appearances of "paving stone" after confluention under inversion microscope. Microvilli ridge were observed on cells surface with scanning electron microscope. The culture cells morphology and growth characteristics were essentially consistent by the two methods.
3) The success ratio of culture cells by tissue explants was significantly low compared with enzyme digestion method (37.5% vs 50%, P<0.05). The cell productivity by the two methods didn't show significant differences (2.67±0.23×106/L vs 2.89±0.94×106/L, P>0.05).
4) The expression of P-CK staining of the cultured cells was positive. Purity of cells using explants and enzyme digestion method were respectively 92% and 98%. The fourth generation cells pocess normal diploid karyotype.
Conclutions:Vaginal epithelial cells were obtained by the two methods. Compared with explants method, the culture cells using enzyme digestion method display faster adhesion, shorter growth time, and higher success rate. The larger numbers of epithelial cells were rapidly obtained to construct engineering of vaginal tissue by enzyme digestion method.
Part three:Biocompatibility Between PLGA/ⅠCollagen Scaffold and the Rat Vaginal Epithelial Cells
Objective:To explore the biocompatibility of the PLGA/Ⅰcollagen with rat vaginal epithelial cells, and verify the feasibility of using PLGA/Ⅰcollagen as scaffold to reconstruct vagina by the tissue engineering.
Methods:The rat vaginal epithelial cells were cultured by enzyme digestion method.Ⅰcollagen was combined with poly lactic-co-glycolic acid (PLGA) to form PLGA/Ⅰcollagen biodegradable polymer scaffold. The epithelial cells were culture in the leaching liquor of scaffold to detect its toxicity to cells by MTT. The epithelial cells were inoculated on the scaffold to calculate cell adhesion rate, and cells growth were observed in the epithelial cells-scaffold complexes with scanning electron microscope. Epithelial cells-scaffold complexes were implanted under cutis of rat back. At 2,4, and 8 weeks after implantation, the cells-scaffold were assessed by histological HE stain and immunohistochemical analysis
Results:The Epithelial cells grew and proliferated well in the leaching liquor of scaffold. On the PLGA/Ⅰcollagen scaffold, the cell adhesion rate was 71.8%±9.2%, which significantly higher than that on the PLGA scaffold. The epithelial cells were well distributed and adhered to the PLGA/Ⅰcollagen scaffolds under scanning electron microscope. At 2 weeks after implanted under cutis of rat back, the epithelial cells attached and proliferated in the space, and the fibroblasts were seen. At 4 weeks, the epithelium were seen about 1-3 layer. At 8 weeks, the epithelial cells increased and arranged regularitility, which formed the membrane-like layer on the scaffold. The expression of P-CK of the epithelium was positive.
Conclusions:The PLGA/Ⅰcollagen scaffolds are well-cytocompatible to the epithelial cells, and can be used as the biodegradable polymer scaffold of the vaginal tissue engineering.
Part four:Reconstruction of Tissue Engineering Vagina in Rat
Objective:To explore feasibility of reconstructing completely rat vagina by tissue engineering, and provide technical function on clinical application.
Methods:The epithelial cells were cultured from rat vaginal tissues, and innoculated on the medial surface of fistular PLGA/Ⅰcollagen biological material. The epithelial cells-scaffold complexes were transplanted to rat vagina in situ after culture in vitro to reconstruct vagina. After 1,3, and 6 months, we measured the vaginal length and observed the appearance at maero. The neovagina tissues were prepared for histological evaluation with HE, Masson's, and immunohistochemistry stain.
Results:
1) The experimental rat model of vaginal reconstruction surgery was performed sueeessfully.1 rat died to overdose of anesthesia after surgery, and during observation,3 rats subjected serious infection in abdominal cavity,2 of these died and 1 of these had ankylocolpos. and coleostenosis weren't be seen in terms of survival 6 rats.
2) At 1 month after operation, we observed thin and red epithelialization on the biological material in the vaginal canal, which distributed like islands. The little of biological material took place degradation, and the length of vagina was 1.4cm. Within 1-3 months, the vaginal mucosa get thick gradually and pink. The biological material had degradated more than a half, and the vagina was 1.2cm in length. After 6 months, the neovagina was hardly differentiated from the native one at either maero or miero appearance, and the vagina was 1.2cm in length and no shrink.
3) Light microscopy inspection showed the epithelium was 1-3 layers after 1 month, no smooth muscle, rare blood vessel and collagen fibers. At 3 months after surgery, the epithelium was thicker and multi-layere and the minor smooth muscle were seen occasionally. At 6 months, histology of neovagina was similar to the native one except less rete pegs in the epithelium basilar part, more smooth muscle and compact collagen fibers were observed by Masson's stain. Epithelial cells layer were identified by immunohistoch-emistry stain with P-CK since 1 month after surgery which was accordant to the result in HE stain.
Conclusins:The neovagina which was reconstructed using rat vaginal epithelial cells and PLGA/I collagen scaffold was hardly differentiated from the native one at either maero or miero appearance. A tissue engineering approach to clinical vaginal reconstruction in women is now a realistic possibility.
引文
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