膀胱平滑肌细胞与小肠黏膜下层体外复合培养的实验研究

目的探讨体外快速培养犬膀胱平滑肌细胞的方法及观察膀胱平滑肌细胞在脱细胞小肠黏膜下层(small intestinal submucosa,SIS上的生长状况,为构建组织工程膀胱平滑肌组织提供实验依据。方法分别采用酶消化法和组织块培养法分离、获取和原代培养犬膀胱平滑肌细胞,倒置相差显微镜下观察细胞的生长情况,透射电镜观察细胞的超微结构,免疫组织化学染色进行细胞鉴定。将犬膀胱平滑肌细胞接种到SIS支架材料上,于复合培养5、7及9d取材,行苏木素染色、石蜡切片HE染色和扫描电镜观察膀胱平滑肌细胞在SIS上的生长状况。以细胞-SIS复合培养组为实验组,以膀胱平滑肌细胞为对照组,每组各设9孔,分别于接种后3、5及7d取材,酶消化后收集细胞并计数。结果酶消化法原代培养获取犬膀胱平滑肌细胞数量多,细胞生长速度快,形态良好,培养5d细胞在培养瓶底生长汇合。组织块培养法接种3d见长梭形的膀胱平滑肌细胞从植块边缘萌出,获取的细胞数量较少。透射电镜下见膀胱平滑肌细胞胞质中有特征性细肌丝和细胞膜的密斑。抗α-肌动蛋白免疫组织化学染色胞浆呈棕黄色阳性反应。膀胱平滑肌细胞在SIS表面能黏附、生长和增殖。体外复合培养5d后,膀胱平滑肌细胞铺满SIS表面,呈单层细胞结构。7、9d细胞形态与5d相似。实验组3、5及7d的细胞计数分别为(16.85±0.79)×105、(39.74±2.16)×105及(37.15±2.02)×105个,对照组分别为(19.43±0.54)×105、(34.50±1.85)×105及(33.07±1.31)×105个。两组5d细胞计数差异有统计学意义(P<0.05)。结论酶消化法原代培养膀胱平滑肌细胞可提供大量活性良好的种子细胞。SIS支持膀胱平滑肌细胞黏附和生长,可为构建组织工程膀胱平滑肌组织提供良好支架。

EXPERIMENTAL STUDIES ON CANINE BLADDER SMOOTH MUSCLE CELLS CULTURED ON ACELLULAR SMALL INTESTINAL SUBMUCOSA IN VITRO

OBJECTIVE: To explore an effective method of culturing the canine bladder smooth muscle cells, observe the morphological characteristics of the bladder smooth muscle cells growing on acellular small intestinal submucosa(SIS) and offer an experimental basis for reconstruction of the bladder smooth muscle structure by the tissue engineering techniques. METHODS: The enzyme-treatment method and the explant method were respectively used to isolate and harvest the canine bladder smooth muscle cells, and then a primary culture of these cells was performed. The canine bladder smooth muscle cells were seeded on the SIS scaffold, and the composite of the bladder smooth muscle cells and the SIS scaffold were co-cultured for a further observation. At 5,7 and 9 days of the co-culture, the specimens were taken; the bladder smooth muscle cells growing on the SIS scaffold were observed by the hematoxylin staining, the HE staining, and the scanning electron microscopy. The composite of the bladder smooth muscle cells on the SIS scaffold was used as the experimental group, and the bladder smooth muscle cells with no SIS were used as the control group. In each group, 9 holes were chosen for the seeded bladder smooth muscle cells, and then the cells were collected at 3, 5 and 7 days for the cell counting after the enzyme treatment. Morphological characteristics of the cells were observed under the phase contrast microscope and the transmission electron microscope. Expression of the cell specific marker protein was assessed by the immunohistochemical examinaiton. The proliferation of the cells was assessed by the cell counting after the seeding on the SIS scaffold. RESULTS: The primary bladder smooth muscle cells that had been harvested by the enzyme-treatment method were rapidly proliferated, and the cells had good morphological characteristics. After the primary culture in vitro for 5 days, the bladder smooth muscle cells grew in confluence. When the bladder smooth muscle cells were seeded by the explant method, a small amount of the spindle-shaped bladder smooth muscle cells emigrated from the explant at 3 days. The cells were characterized by the well-developed actin filaments in the cytoplasm and the dense patches in the cell membrane under the transmission electron microscope. The immunohistochemical staining showed the canine bladder smooth muscle cells with positive reacting alpha-actin antibodies. The bladder smooth muscle cells adhered to the surface of the SIS scaffold, growing and proliferating there. After the culture in vitro for 5 days, the smooth muscle cells covered all the surface of the scaffold, showing a single-layer cellular structure. The cell counts at 3, 5 and 7 days in the experimental group were (16.85 +/- 0.79) x 10(5), (39.74 +/- 2.16) x 10(5) and (37.15 +/- 2.02) x 10(5), respectively. The cell counts in the control group were (19.43 +/- 0.54) x 10(5), (34.50 +/- 1.85) x 10(5) and (33.07 +/- 1.31) x 10(5), respectively. There was a significant difference between the two groups at 5 days (P < 0.05). CONCLUSION: With the enzyme-treatment method, the primarily cultured canine bladder smooth muscle cells can produce a great amount of good and active cells in vitro. The acellular SIS can offer an excellent bio-scaffold to support the bladder smooth muscle cells to adhere and grow, which has provided the technical foundation for a further experiment on the tissue engineered bladder reconstruction.