自体肌卫星细胞尿道内移植实验

Autologous transplantation of skeletal muscle satellite cells into rat urethra

目的:观察骨骼肌卫星细胞注射到尿道内存活的情况,探讨肌卫星细胞自体移植治疗压力性尿失禁的潜在可行性。方法:实验于2004-09/2005-09在福建医科大学附属协和医院内分泌学研究所和动物实验室完成。实验动物:清洁级3周龄雌性SD大鼠10只。实验分组:将大鼠分为实验组和对照组,每组5只。实验干预:①分别取其双下肢腓肠肌,采用1g/L胶原酶-2.4U/mLDispase和2.5g/L胰蛋白酶两步法消化分离、纯化细胞并培养。采用0.01%的活体荧光染料荧光金对骨骼肌卫星细胞进行标记。②将荧光金标记的骨骼肌卫星细胞自体注射于实验组大鼠近端尿道3点和9点处的黏膜下及肌层,对照组大鼠于相同位置注射等体积的DMEM/F12无血清培养液。实验评估:①荧光显微镜下观察荧光金的标记效果,计算标记后细胞的死亡率。②三四天后麻醉下处死大鼠,取出后尿道组织进行冰冻切片,荧光显微镜下观察是否有标记的细胞,并进行Desmin免疫组织化学染色观察。结果:10只大鼠均进入结果分析。①培养细胞的鉴定,荧光标记的效果:成功分离、培养出原代细胞,免疫细胞化学染色示90%左右的细胞特异性结蛋白染色阳性,证实所培养出的细胞为骨骼肌卫星细胞;0.01%的荧光金对细胞的标记效果好,细胞的存活率与未标记组无明显差异,适合用于骨骼肌卫星细胞自体移植标记。②后尿道组织冰冻切片观察:实验组切片于荧光显微镜下可见到有荧光标记的细胞,免疫组织化学染色有Desmin阳性的细胞;而对照组切片未见到荧光标记的细胞,免疫组织化学染色为阴性。结论:骨骼肌卫星细胞注射到尿道内后仍有存活,有可能成为压力性尿失禁注射疗法的一种新型注射材料。

AIM： To observe the survival of skeletal muscle satellite cells after transplanting into the urethra feasibility of autologous skeletal muscle satellite cell transplantation as a potential treatment incontinence. of stress urinary METHODS： The experiment was conducted at Endocrinology Institute and Animal Laboratory of Union Hospital of Fujian Medical University from September 2004 to September 2005. Experimental animals： Totally 10 female SD rats aged three weeks of clean grade. Experimental grouping： The rats were divided into experimental group and control group, with 5 in each group. Experimental intervention： （1）Gastroenemius of double lower extremities was obtained and gastroenemius cells were digested, isolated, purified and cultured by 1 g/L collagenase-2.4 U/mL Dispase and 2.5 g/L trypsin. Skeletal muscle satellite cells were labeled with 0.01% fluorogold. （2）The labeled cells were injected into point 3 and point 9 below mucosa and muscular layer of the autologous rats in the experimental group. Rats in the control group were injected with DMEM/F12 serum-free medium of the same volume. Experimental evaluation： （1）Fluorogold labeled effect was observed under fluorescence microscope and death rate of labeled cells were calculated. （2）Rats were killed after anesthesia 3 or 4 days later. Posterior urethral tissues were made into frozen section. Whether labeled cells appeared or not were observed under fluorescent microscope and then Desmin immunohistochemical staining. RESULTS： Totally 10 rats were involved in the result analysis. （1） Identification of cultured cells and effect of fluorescent-labeling： Primary cells were successively isolated and cultured. Immunocytochemical staining showed that about 90% cell specific desmin were positive, which verified that the cultured cells were skeletal muscle satellite cells. 0.01% fluorogold had good-labeled effect on cells. There was no significant difference in survival rate as compared with the non-labeled group. Fluorogold was fit for the marker of skeletal muscle satellite cells autografting. （2）Frozen section of posterior urethral tissues： Fluorescent-labeled cells appeared in frozen sections of the experimental groups under fluorescence microscopy. Tissue sections were positive for Desmin, whereas rats in the control group were negative. CONCLUSION： Skeletal muscle satellite cells are seen in tissue sections from urethra injection. It can be used as a new pattem agent to the injection treatment of stress urinary incontinence.