姚润斯 罗 新* 张 仙 范 瑾 帅翰林 蒋学风 李瑞满
目的：1. 探讨17β-雌二醇(17β-E2)对骨髓间充质干细胞(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。
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
Experimental study on the repairing SUI rats by rat bone marrow and human umbilical cord mesenchymal stem cells
YAO Run-si,LUO Xin*, ZHANG Xian，FAN Jin, SHUAI Han-lin, JIANG Xue-feng, LI Rui-man
（Department of gynecology and obstetrics, the First Affiliated Hospital of Jinan University, Guangzhou, Guangdong Province，510630，China.)
【Abstract】Objectives 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 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. 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-added、depicts 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±0.84mmHg和28.51±0.95mmHg，respectively, （P<0.05）; the maximal cystometry were 1.33±0.20ml and 2.19±0.17ml，respectively, （P<0.05）; the positive rate of sneeze test of model rats 8/15 and 0/20，respectively, P<0.05。3. 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 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）%. 5. 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. 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