【作者】 姚润斯；

【导师】 罗新；

【作者基本信息】 暨南大学 ， 妇产科学， 2009， 硕士

【摘要】 研究目的:1.建立从SD大鼠骨髓和人足月脐带分离间充质干细胞(MSCs)、体外传代培养方法,研究其在体外的生物学特性及表型特征,并对两种MSCs进行标记,为后续实验提供理想的细胞源。2.模拟人类妊娠、分娩损伤、去势后低雌激素与压力性尿失禁(SUI)发生的关系,建立SUI动物模型,将获得的标记细胞注射于大鼠后尿道周围,依据尿流动力学指标、喷嚏实验阳性率、尿道及其周围组织形态学改变,观察间充质干细胞移植治疗压力性尿失禁大鼠是否有效可行。材料和方法:1.SUI模型的建立:①实验组:受孕SD雌鼠分娩后立即麻醉,将14F的弗莱氏导尿管插入阴道2～3cm并用丝线固定于阴道口,往气囊内注入5ml生理盐水,维持此状态4h;两周后行双侧卵巢切除术,常规饲养两月后SUI模型建成。②阳性对照组:受孕SD雌鼠分娩后立即麻醉,将14F的弗莱氏导尿管插入阴道2～3cm并用丝线固定于阴道口,维持此状态4h,常规饲养两月。所有实验大鼠均用BL-410生物机能实验系统检测尿流动力学指标,再加上喷嚏实验阳性率,检验模型建立的成功与否。2.在无菌条件下取出大鼠胫骨和股骨,采用贴壁法分离、纯化大鼠骨髓间充质干细胞(BMSCs);在无菌条件下取足月健康胎儿脐带标本,用双酶法分离、纯化脐带间充质干细胞(UCMSCs)。两种MSCs均用含10%胎牛血清(FBS)的DMEM/F12培养基培养。3.用流式细胞仪检测细胞表面标志,对培养的细胞进行鉴定;通过细胞形态学观察,MTT法检测细胞增值能力、描绘生长曲线,观察体外培养的MSCs的生物学特性。4.以脂质体2000为载体,用pEGFP-N1对两种MSCs进行标记,将获得的标记细胞分别注射于两组SUI模型大鼠尿道周围,常规饲养两周后用BL-410生物机能实验系统检测尿流动力学指标,并检测喷嚏实验阳性率。5.处死大鼠,进行尿道及其周围组织、膀胱取材,通过HE、Masson染色观察尿道及其周围组织和膀胱的病理形态学变化;在荧光显微镜下观察实验组标本切片有无绿色荧光,检验移植的间充质干细胞是否在体内成功生长增值。结果:1.建立SUI模型:模拟人类妊娠、难产产伤、去势后低雌激素能成功获得稳定的动物模型。实验组大鼠与阴性对照组、阳性对照组大鼠的最大膀胱容量分别为(1.93±0.49)ml、(3.21±0.58)ml、(3.14±0.52)ml;漏尿点压力值分别为(23.83±4.23)mmHg、(30.57±3.31)mmHg、(30.28±1.99)mmHg。这两个尿流动力学指标实验组均明显低于两对照组(p＜0.05),而两对照组之间无统计学差别(p＞0.05);喷嚏实验阳性率实验组(72.2%)明显高于两对照组(均为0)。2.体外培养MSCs和MSCs的生物学特性:原代BMSCs接种24h后部分贴壁,呈圆形,72h后绝大部分细胞贴壁,形态为梭形,呈集落样生长。10～12天后细胞近融合,可进行首次传代。传代后细胞增殖速度加快,传3～4代后细胞形态均一。培养的第3代细胞97.68%表达CD29,2.76%表达CD45,证实为间充质干细胞。原代培养的UCMSCs多于接种24～48h内贴壁,14～16天左右细胞80%融合,细胞梭形,呈平行排列样生长或漩涡样生长,传代后细胞增殖速度加快,传2～3代后细胞形态均一。培养的第3代细胞99%以上表达CD29、CD44,而CD34、CD45、CD31呈阴性,证实为间充质干细胞。两种MSCs大部分处于静止期及DNA合成前期(G0～G1期),少数处于有丝分裂期和DNA合成期。绿色荧光蛋白pEGFP-N1的标记率分别为(15.18±2.57)%和(15.68±1.03)%。3.SUI模型尿道周围MSCs移植:实验分3组:BMSCs注射组、UCMSCs注射组和PS注射组。(1)BMSCs移植组大鼠与UCMSCs移植组大鼠的最大膀胱容量(3.06±0.77&3.22±0.77)ml和漏尿点压力值(28.60±3.98&30.62±2.76)mmHg均明显高于治疗前(p＜0.05),并与阴性和阳性对照组无统计学差别(p＞0.05),而PS注射组治疗前后尿流动力学指标无统计学差别(p＞0.05);PS注射组最大膀胱容量和漏尿点压力均小于BMSCs注射组和UCMSCs注射组(p＜0.05),而两细胞移植组间的比较无统计学差别(p＞0.05)。两细胞移植组喷嚏实验阳性率(16.7%)明显低于PS注射组(83.3%)。4.尿道及其周围组织行HE、Masson染色后光镜形态学观察:PS注射组与阴性和阳性对照组比较,括约肌排列松散,部分肌肉有断裂现象,肌层变薄;筋膜间结缔组织所占比重增加,胶原纤维稀疏,排列紊乱;阴道粘膜上皮层萎缩。两细胞移植组大鼠尿道壁肌层较PS注射组增厚,但与对照组比较排列仍相对紊乱和松散;肌层和筋膜间结缔组织变紧密;阴道上皮萎缩未见明显改善。各组膀胱组织的光镜形态学无明显差别,平滑肌束清晰完整、连续,粘膜复层上皮未见明显异常。5.尿道及其周围组织切片后荧光显微镜下观察:移植2周后,两移植组可见到绿色荧光,而其余各对照组均未见到荧光的表达,证实移植细胞在大鼠体内存活,增殖情况良好。结论:1.依据SUI的发病机理为建模思路,根据尿流动力学指标、喷嚏实验阳性率的差异性可建立稳定的动物模型。2.应用贴壁法和双酶法可分别获得较纯的大鼠骨髓间充质干细胞和人脐带间充质干细胞。纯化的BMSCs不表达CD45,表达CD29;纯化的UCMSCs不表达CD34、CD45、CD31,表达CD29、CD44。两种MSCs的细胞周期主要是G0-G1期,并且在体外具有良好的扩增能力。原代培养的MSCs性状稳定,表型稳定均一,可用于进一步的组织工程研究。pEGFP-N1质粒可作为间充质干细胞的标记物,脂质体介导的质粒转染法适用于细胞的短期标记。3.PS注射组大鼠尿道括约肌和周围组织的不完整性,提示盆底组织结构破坏和生化改变是SUI的发生原因之一。SUI去势大鼠模型(PS注射组)的低雌激素和雌激素受体状态造成阴道上皮层萎缩是SUI发生的因素之一。4.大鼠骨髓间充质干细胞和人脐带间充质干细胞可以在损伤的大鼠尿道周围组织中存活、增殖,改善尿流动力学指标和喷嚏实验的阳性率;形态学上观察可修复尿道周围的支持结构,但未能改善大鼠体内的低雌激素水平状态。更多还原

【Abstract】 Objective:1.To set up the method of separating mesenchymal stem cells(MSCs) from the bone marrow of SD rat and full-term human umbilical cord,and the ways of serial subcultivation in vitro.To study that two MSCs’ biological properties and phenotypic characteristics in vitro,and tag two MSCs,provides an ideal cell sources for follow-up experiment.2.Simulation of human pregnancy,birth injury,low estrogen after castration and the relationship with stress urinary incontinence(SUI),set up animal models of SUI,labeled cells will be injected into the rats around the posterior urethral.Based on urodynamics、the positive rate of sneeze test、morphological changes of urethra and its surrounding tissue,to observe whether mesenchymal stem cells transplant in the treatment of SUI rats is effective and feasible or not.Materials and methods:1.Establishment of SUI model:①experimental group:pregnant female SD rats is anaesthetized immediately after birth.Fry’s 14F catheter will be inserted into the vagina 2～3 cm,and thread fixed in the Vagina mouth.Inject 5ml physiological saline into the balloon and maintain this status for 4 hours.Two weeks later with bilateral oophorectomy,the rats are routinely reared for two months.In this way,SUI model is built.②the positive control group:pregnant female SD rats is anaesthetized immediately after birth and thread fixed in the Vagina mouth,maintaining this status for 4 hours.The rats are routinely reared for two months.In order to test the success of the rat model,all rats are used BL-410 biological function experimental system to test urodynamics,in addition to the positive rate of sneeze experiment,.2.In sterile condition,remove the rat tibia and femur,and apply adherent method to separate and purify rat bone marrow mesenchymal stem cells(BMSCs).In sterile condition,obtain the umbilical cord samples from healthy full-term fetus and apply double enzymatic method to separate and purify umbilical cord mesenchymal stem cells(UCMSCs),Two MSCs are cultivated with 10%fetal bovine serum(FBS) in DMEM/F12 medium.3.Detect cell surface markers with flow cytometry and identify cultured cells.Through the observation of cell morphology,MTT method assays cells value-added、depicts the growth curve and observes the biological characteristics of MSCs in vitro.4.Take lipofectamine 2000 as carriers and tag the two MSCs with PEGFP-N1.The labeled cells will be injected into two groups of rats’ urethra separately.And then use BL-410 biological function experimental system to test urodynamics,and detect the positive rate of sneeze experiment after routinely reared for two weeks.5.Necropsied the rats,draw the materials from their bladder,urethra and the surrounding tissue,observe the pathological changes of their bladder,urethra and the surrounding tissue after HE and Masson staining.Observe the availability of green fluorescent of the experimental groups’ biopsy specimens under the fluorescence microscope,to test if the transplantation of mesenchymal stem cells successfully grow and add value in vivo.Results:1.Set up SUI modle:Set up a stable animal model successfully by simulation of human pregnancy,dystocia birth trauma,low estrogen after castration.The maximum bladder capacity of the experimental rats,the negative control group and positive control group of rats were(1.93±0.49) ml,(3.21±0.58 ) ml and(3.14±0.52) ml;leak point pressure values were(23.83±4.23) mmHg,(30.57±3.31) mmHg and(30.28±1.99) mmHg. The experimental group were significantly lower than the two control groups about the two Urodynamic index(p＜0.05),and there was no statistical difference between the two control groups(p＞0.05);the positive rate of Sneeze test(72.2%) was significantly higher than the two control groups(both 0).2.MSCs in vitro and biological characteristics of MSCs:Primary cultured BMSCs were adherent and round 24 hours after inoculation.Most of the cells were adherent and 72 hours later become fusiform-shaped after inoculation.The cells were passaged after 10～12 days.The proliferation became faster after passaging.The third and the fourth passage of BMSCs were morphologically homogeneous.97.68 percent of the cultured cells expressed CD29;2.76 percent expressed CD45,which were identified as BMSCs. Most of the primary cultured BMSCs were adherent for more than 24～48 hours after inoculation.With 80 percent of the cells fusion in about 14～16 days,cells were fusiform-shaped,parallel-like or whirlpool-like growth.The proliferation became faster after passaging.The third and the fourth passage of UCMSCs were morphologically homogeneous.99 percent of the cultured cells of the third passage expressed CD29 and CD44,but negative for markers of the hematopoietic lineage,including CD34 and CD45, which were identified as UCMSCs.Most of the two MSCs were in still and DNA presynthetic phase(G0/G1 phase),and only few were in mitotic phase and DNA synthetic phase.Labeling rate of green fluorescent protein marker pEGFP-N1 were (15.18±2.57)%and(15.68±1.03)%.3.MSCs transplantation around urethra of SUI model:Experiments were divided into 3 groups:BMSCs injection group、UCMSCs injection group and PS injection group.The maximum bladder capacity of the BMSCs injection group and the UCMSCs injection group were(3.06±0.77&3.22±0.77) ml;leak point pressure values were(28.60±3.98&30.62±2.76) mmHg.Both were significantly higher than pretherapy(p＜0.05), and had no statistical difference between the negative and positive control group(p＞0.05),but PS injection group had no statistical difference between before and after treatment(p＞0.05).The maximum bladder capacity and leak point pressure of the PS injection group were less than the BMSCs injection group and the UCMSCs injection group(p＜0.05),but two MSCs transplantation groups had no statistical difference(p＞0.05).Two MSCs transplantation groups(16.7%) were significantly lower than the PS injection group(83.3%) about the positive rate of sneeze test.4.Morphology by light microscopy of urethra and the surrounding tissues after HE、Masson stain:The PS injection group noted sphincter loose,some of the muscles collapse and decreased muscle layer.;vaginal epithelial atrophy,increased connective tissue content.Two MSCs transplantation groups showed muscle layer thicker,but still relatively disordered and loose.Compared with the control groups,normal connective tissue had no significant improvement in vaginal epithelial atrophy.The bladder tissue of each group had no significant difference in light microscopy of morphology.Smooth muscle bundles were intact and continuous,which showed no abnormal at stratified epithelium.5.Urethra and the surrounding tissue were observed under the fluorescence microscope:2 weeks after transplantation,the green fluorescence could be seen in two MSCs transplantion groups,and the rest of the control group was not showed the expression of fluorescence.It was confirmed that the transplanted cells can survive and increase in rats.Conclusions:1.Based on the pathogenesis of SUI,according to the difference of the urodynamic index and the positive rate of sneeze test,a stable animal model can be established.2.Purified BMSCs can be obtained by adherent method,and were negative for CD45, positive for CD29.Purified UCMSCs can be obtained by double enzymatic method, and negative for CD34、CD45、CD31,positive for CD29,CD44.Most of the two MSCs are in G0/G1 phase,and can proliferate in vitro.The cultured MSCs are uniform and stable,and can be used for tissue engineering,pEGFP-N1 plasmid can be used as the marker of mesenchymal stem cells,liposome-mediated transfection of plasmid is applicable to cells’ short-term tag.3.The imperfection of urethral sphincter and its surrounding tissue of PS injected rats, shows the change of morphology and biochemistry of pelvic framework is one of the cause of SUI.vaginal epithelial atrophy.Low estrogen and estrogen receptor status of ovariectomized rat model is one of the factors that caused SUI.4.BMSCs and UCMSCs can be survived and proliferated in the urethral and the surrounding tissue of injuried rats,and improve the urodynamic index and the positive rate of sneeze test.Morphology shows renovation of the support structures around the urethra,but fail to improve the low levels of estrogen status in rats.更多还原