-中国妇产科医院联盟
快速导航>>
 妊娠期糖尿病患者不同治疗时机不同对母婴预后的影响
 高龄经产妇围生期临床特点与妊娠结局的临床分析
 中西医结合治疗卵巢功能减退成功妊娠1例分析
 四价HPV疫苗不会增加多发性硬化症风险
 宫颈腺癌治疗进展
 他汀类药物的使用与子宫内膜癌生存率的相关性
 联合检查是子宫颈癌筛查的最优方法:三个中国大样本
 虚弱指数可预测妇科肿瘤患者严重并发症

更多内容...

大鼠骨髓及人脐带间充质干细胞对模型鼠尿失禁的修复研究  
 

大鼠骨髓及人脐带间充质干细胞对模型鼠尿失禁的修复研究

姚润斯  罗 新*  张 仙  范 瑾 帅翰林 蒋学风  李瑞满

(暨南大学附属第一医院妇产科,广东 广州,510630

【摘要】

目的:1. 探讨17β-雌二醇(17β-E2)骨髓间充质干细胞(bone marrow mesenchymal stem cells ,BMSCs)增殖﹑成肌分化的影响,为压力性尿失禁的细胞治疗提供理想的细胞源。

2.建立模拟人类分娩损伤、多产、密产、绝经后大鼠,并研究上述因素与压力性尿失禁发生的关系。3.观察SUI模型大鼠尿道周围组织、肛提肌组织形态学改变,探讨慢性损伤所致的盆底支持组织的改变是压力性尿失禁的病因之一。4.研究大鼠骨髓间充质干细胞和人足月分娩脐带中间充质干细胞的体外传代培养方法,并研究其生物学特性。通过对两种间充质干细胞标记,为细胞治疗的研究提供理想的识别方法。5.将获得的标记间充质干细胞注射于SUI大鼠后尿道周围组织,依据尿流动力学指标、喷嚏实验阳性率、尿道和尿道周围组织显微镜下形态学改变,验证移植间充质干细胞于模型大鼠后尿道来治疗压力性尿失禁大鼠的有效性。方法1.利用贴壁法体外分离、纯化BMSCs,连续传代3次;MTT法检测01nmol/L浓度范围内17β-E2BMSCs增殖活性的影响2.Hoechst33258染色测定BMSCsDNA含量;免疫荧光法检测添加不同浓度17β-E2诱导培养后BMSCs中骨骼肌分化标志肌球蛋白重链(MHC)myogenindesmin表达情况。3.建立SUI模型大鼠:妊娠SD大鼠分娩后立即麻醉,将14F导尿管插入阴道内23cm,往气囊注入5ml生理盐水撑开阴道,用1号手术丝线将导尿管固定于阴道口,并维持该状态4h;两周后再行上述操作一次;两周后麻醉下对其进行双侧卵巢切除术,术后连续7天注射抗生素,常规饲养两个月,获得模型大鼠。4.采用仪器BL-410生物机能实验系统检测模型大鼠最大膀胱容量、漏尿点压力等尿动力学指标,喷嚏实验阳性率,检验SUI模型大鼠建立的有效性。5.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;无菌条件下取足月妊娠健康胎儿脐带标本,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)DMEM/F12培养基培养。6.用流式细胞仪检测细胞表面标志,对体外传代培养的细胞进行鉴定并观察MSCs的生物学特性。7.以脂质体2000为载体,用pEGFP1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BL410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。8.处死大鼠,进行尿道及其周围组织、耻尾肌取材,通过HEMasson染色观察尿道及其周围组织和膀肤的病理形态学变化;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殖。结果1.17β-E2可增强BMSCs增殖活性,且呈浓度依赖关系,最佳促增殖浓度为1nmol/L;1nmol/L 17β-E2 诱导组与对照组BMSCsDNA含量随培养时间延长均呈增加趋势(  p<0.05);而两组细胞活性随培养时间的延长均呈下降趋势,17β-E2干预组下降缓慢。2.17β-E2可增加BMSCs三种骨骼肌特异标志myogeninMHCdesmin的表达。3.利用贴壁法和分离法可以获得高纯度的骨髓间充质干细胞,且细胞活性高。原代细胞生长相对较慢,BMSCs分离培养24h后开始少量贴壁,细胞形状呈短梭形,3d后贴壁细胞明显增多,呈集落样生长,10-14d细胞接近融合。传代后,细胞增殖、贴壁速度加快,10h后约90%以上细胞贴壁,细胞倍增时间大约为30h7-10d细胞接近融合。4.SUI模型建立情况:模拟人类妊娠、难产产伤、密产、去势后低雌激素能获得成功稳定的动物模型。实验组和对照组大鼠:漏尿点压力值分别为10.96±0.84mmHg28.51±0.95mmHg,(P<0.05);最大膀胱容量分别为1.33±0.20ml2.19±0.17ml,(P<0.05);喷嚏实验的阳性率分别为8/150/20P<0.055.实验组较对照组大鼠的尿道平滑肌、尿道括约肌及其周围筋膜层中胶原纤维结构松散、碎裂、呈灶性排列,染色不均。6.SUI模型尿道周围MSCs移植:实验分3组,分别为BMSCs注射组、UCMSCs注射组和PS注射组。(1)BMSCs注射组大鼠与UCMSCs注射组大鼠的最大膀胱容量(3.060.77&3.220.77)ml和漏尿点压力值(25.603.95&30.622.76)mmHg均明显高于治疗前(p<0.05),并与阴性和阳性对照组差异无统计学意义(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差异无统计学意义(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%)7.尿道及其周围组织行HEMasson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,部分肌纤维有断裂现象,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;阴道粘膜上皮萎缩。8.尿道及周围组织切片后在荧光显微镜下观察: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。1.17β-E2BMSCs的增殖具有促进作用,且能增强骨骼肌分化效率。2.模拟人类妊娠、难产产伤、密产、绝经建立的大鼠模型,表现可以诱发尿失禁。3.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。4.泌尿生殖道的形态学改变所致的盆底结构连接松散、张力减弱,与SUI的发生有关。5.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。6.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。

关键词:骨髓间充质干细胞 人脐带间充质干细胞细胞培养 移植治疗 尿道注射  雌激素  增殖  分化 压力性尿失禁  大鼠  胶原  免疫组化

 

基金项目:广东省医学科研基金立项课题(A2007338

广东省科技计划项目(2007B060401054)

广东省自然科学基博士科研启动项目 (8451063201000290)

国家自然科学基金面上项目 (81070459)

 

Experimental study on the repairing SUI rats by rat bone marrow and human umbilical cord mesenchymal stem cells

YAO Run-si,LUO Xin*, ZHANG XianFAN Jin, SHUAI Han-lin, JIANG Xue-feng, LI Rui-man

Department of gynecology and obstetrics, the First Affiliated Hospital of Jinan University, Guangzhou, Guangdong Province510630China.)

AbstractObjectives 1. To investigate the effects of 17 beta-estradiol(17β-E2) on bone marrow mesenchymal stem cells(BMSCs) myogenic differentiation and proliferation, so that to enhance skeletal differentiation ratio of BMSCs by 5-aza cytidine(5-aza) induction. 2. To investigate the establishing female rats of SUI can obtained by simulating human pregnancy, birth trauma, dense birth and menopause, and according to the index of urinary dynamics and the positive rate of sneeze tests to test the feasibility of model. This study provided the proof to treat stress urinary incontinence with injection. 3.To observe the alteration of histology and morphology in model rat of urethra and surrounding tissues, pubococcygeus. Change of these tissues showed that chronic injury leading to the change of function and structure of pelvic framework was one of the cause of SUI. 4. 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. 5. 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 urodynamicsthe 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. Methods 1.Bone mesenchymal stem cells were isolated from Sprague-Dawley rats and purified by the ability to adhere to the cultural plastic in vitro. Then BMSCs were expanded by subculture successively. All the third passage BMSCs were utilized for serial experiments as followes, respectively: Firstly, the cells were cultured respectively with medium containing different concentration of 17β-E2. Secondly, the proliferation effect of 17β-E2 on BMSCs was investigated by MTT method, so that to determine the optinal concentration of 17β-E2. Thirdly, Hoechst33258 staining was used to measure the effect of the optinal concentration of 17β-E2 on BMSCs DNA content, to explore the proliferative potential.of 17β-E2 on BMSCs. 2. The effects of 17β-E2 on BMSCs 5-aza-induced skeletal muscle differentiation were determined by Immunofluorescence staining (IF) and terminal deoxynucleotidyl transferase-mediated fluorescein-conjugated deoxyuridine triphosphate nick end-labeling(TUNEL) assay, to detect expression level of skeletal muscle differentiation markers. 3. The establishment of SUI models: Anesthetized immediately after delivery, female rats were taken 14F catheter inserting 2~3cm in the vagina, aerocyst was injected 5ml PS, and fixed the catheter to the vaginal orifice with silk suture. fixation the state 4h; do the operation again after 2 weeks; and do  bilateral ovariectomy after 2 weeks again. Rats were injected antibiotics 7 days continuously. At last, we gained the SUI model after ordinary breeding 2 months. 4. To use the instrument BL-410 biology and function experiment system to check the model. The indexes of urinary dynamics were the maximum bladder capacity, leak point pressure and the positive rate of sneezing test. 5. 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. 6. Detect cell surface markers with flow cytometry and identify cultured cells. Through the observation of cell morphology, MTT method assays cells value-addeddepicts the growth curve and observes the biological characteristics of MSCs in vitro. 7. 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. 8. 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.MTT method showed 17β-E2 concentation-dependently promoted BMSCs proliferation ability. The optimal concentration of 17β-E2 was 1nmol/L. Hoechst33258 staining suggested 17β-E2 could increase BMSCs DNA content with culture time dependency.  2. The modified leak point pressure of trial and control rats were 10.96±0.84mmHg28.51±0.95mmHgrespectively, P<0.05; the maximal  cystometry were 1.33±0.20ml and 2.19±0.17mlrespectively, P<0.05; the positive rate of sneeze test of model rats 8/15 and 0/20respectively, P<0.053. Morphologic images were showed that the smooth muscle fascicles, sphincter of urethra and the surrounding collagen fibril in fascia in model rat were disorder, sparse distribution, poorly organized and sparse in range compared with control group. 4.  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 1012 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 2448 hours after inoculation. With 80 percent of the cells fusion in about 1416 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% and15.68±1.03%. 5. MSCs transplantation around urethra of SUI model: Experiments were divided into 3 groupsBMSCs injection groupUCMSCs 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±0.77ml; leak point pressure values were28.60±3.98&30.62±2.76mmHg. Both were significantly higher than pretherapyp<0.05, and had no statistical difference between the negative and positive control groupp0.05, but PS injection group had no statistical difference between before and after treatmentp0.05. The maximum bladder capacity and leak point pressure of the PS injection group were less than the BMSCs injection group and the UCMSCs injection groupp<0.05, but two MSCs transplantation groups had no statistical differencep0.05. Two MSCs transplantation groups(16.7%) were significantly lower than the PS injection group (83.3%) about the positive rate of sneeze test. 6. 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. 7. 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.

Conclusion 1. 17β-E2 could promote BMSCs proliferation, enhance skeletal muscle differentiation of BMSCs induced by 5-aza. 2. The rat-models established by simulating human pregnancy, delivery trauma, multiparous birth, menopause may induce SUI. 3. The morphologic changes resulting in pelvic floor relaxation and tension decreasing was associated with SUI. 4. 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. 5. 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.

Key Words: Bone marrow mesenchymal stem cells; estrogen; proliferation; differentiation; Stress urinary incontinence; Rats; Collagen; Immunohistochemistry

·上一条:第一内含子DNA甲基化在子宫内膜异位症中对SF-1的上调作用
·下一条:关于宫颈癌手术相关问题的讨论

关于我们 | 活动影集 | 在线咨询 | 新闻动态 | 联系我们

Copyright 2011© 中国妇产科医院联盟 All Rights Reserved
咨询电话:电话:010-65735765  传真:010-65735695   地址:北京市朝阳区朝阳路8号朗廷大厦B座1007
Email:cpam2007@sohu.com  备案许可证 号:京ICP035号   技术支持:世瑞博网络