Ho Matthew H, Heydarkhan Sanaz, Vernet Dolores, Kovanecz Istvan, Ferrini Monica G, Bhatia Narender N, Gonzalez-Cadavid Nestor F
From the Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Urology Research Laboratory, Torrance, California; the Division of Female Pelvic Medicine and Reconstructive Surgery, Department of Obstetrics and Gynecology, Harbor-UCLA Medical Center, David Geffen School of Medicine at UCLA, University of California, Los Angeles, California; the Division of Endocrinology and Molecular Medicine, Department of Internal Medicine, College of Medicine, Charles Drew University of Medicine and Science, Los Angeles, California; and the Department of Urology, David Geffen School of Medicine at UCLA, University of California, Los Angeles, California.
Obstet Gynecol. 2009 Aug;114(2 Pt 1):300-309. doi: 10.1097/AOG.0b013e3181af6abd.
Grafts are used for vaginal repair after prolapse, but their use to carry stem cells to regenerate vaginal tissue has not been reported. In this study, we investigated whether 1) muscle-derived stem cells (MDSC) grown on small intestinal submucosa (SIS) generate smooth-muscle cells (SMC) in vitro and upon implantation in a rat model of vaginal defects; 2) express markers applicable to the in-vivo detection of vaginal endogenous stem cells; and 3) stimulate the repair of the vagina.
Mouse MDSC grown on monolayer, SIS, or polymeric mesh, were tested for cell differentiation by immunocytochemistry, Western blot and real-time polymerase chain reaction (PCR). Stem cell markers were screened by DNA microarrays followed by real-time PCR, immunocytochemistry, and Western blot. Rats that underwent hysterectomy and partial vaginectomy were left as such or implanted in the vagina with 4',6-Diamidino-2-Phenylindole (DAPI)-labeled MDSC on SIS, or SIS without MDSC, immunosuppressed, and killed at 2-8 weeks. Immunofluorescence, hematoxylin-eosin, and Masson trichrome were applied to tissue sections.
Muscle-derived stem cell cultures on monolayer and on scaffolds differentiate into SMC, as shown by alpha-smooth muscle actin (ASMA), calponin, and smoothelin markers. Muscle-derived stem cells express embryonic stem cell markers Oct-4 and nanog. Dual DAPI/ASMA fluorescence indicated MDSC conversion to SMC. Muscle-derived stem cells/SIS stimulated vaginal tissue repair, including keratin-5 positive epithelium formation and prevented fibrosis at 4 and 8 weeks. Oct-4+ putative endogenous stem cells were identified.
Muscle-derived stem cells/SIS implants stimulate vaginal tissue repair in the rat, thus autologous MDSC on scaffolds may be a promising approach for the treatment of vaginal repair.
移植物用于脱垂后的阴道修复，但将其用于携带干细胞以再生阴道组织的情况尚未见报道。在本研究中，我们调查了：1）在小肠黏膜下层（SIS）上生长的肌肉衍生干细胞（MDSC）在体外以及植入阴道缺损大鼠模型后是否会生成平滑肌细胞（SMC）；2）是否表达适用于体内检测阴道内源性干细胞的标志物；3）是否能刺激阴道修复。
通过免疫细胞化学、蛋白质印迹法和实时聚合酶链反应（PCR）对在单层、SIS或聚合物网片上生长的小鼠MDSC进行细胞分化测试。通过DNA微阵列筛选干细胞标志物，随后进行实时PCR、免疫细胞化学和蛋白质印迹法。对接受子宫切除术和部分阴道切除术的大鼠不做处理，或在阴道内植入在SIS上培养的用4',6-二脒基-2-苯基吲哚（DAPI）标记的MDSC，或植入无MDSC的SIS，给予免疫抑制，在2 - 8周时处死。对组织切片应用免疫荧光、苏木精-伊红染色和Masson三色染色。
单层和支架上培养的肌肉衍生干细胞分化为SMC，α-平滑肌肌动蛋白（ASMA）、钙调蛋白和平滑肌蛋白标志物可证明这一点。肌肉衍生干细胞表达胚胎干细胞标志物Oct-4和nanog。双重DAPI/ASMA荧光表明MDSC转化为SMC。肌肉衍生干细胞/SIS刺激阴道组织修复，包括在4周和8周时形成角蛋白-5阳性上皮并防止纤维化。鉴定出Oct-4 +假定的内源性干细胞。
肌肉衍生干细胞/SIS植入物可刺激大鼠阴道组织修复，因此支架上的自体MDSC可能是治疗阴道修复的一种有前景的方法。
