Inhibitory effect of heat shock pretreated rat bone marrow mesenchymal stem cells on chemotherapy-induced ovarian granulocyte apoptosis / 医学研究生学报

ObjectiveBone marrow-derived mesenchymal stem cells (MSCs) can promote ovarian angiogenesis, improve ovarian insufficiency caused by chemotherapy, and repair ovarian function, while heat shock pretreatment can reduce the apoptosis rate of stem cells and improve the therapeutic effect of stem cells. This study aims to investigate the effect of heat shock pretreatment on MSCs, and further study the effect of heat shock pretreated mesenchymal stem cells on chemotherapy-induced apoptosis of ovarian granulosa cells.Methods1. The bone marrow-derived MSCs of rats were isolated, cultured and identified, and pretreated within a 42 °C water bath for one hour. 2. Cisplatin (5 mg/L) was added to MSCs to simulate the local microenvironment of chemotherapy. MSCs were divided into four groups: blank control group, heat shock control group, model group, and heat shock model group. The effects of heat shock pretreatment on the proliferation, apoptosis and survival rate of MSCs were investigated by CCK-8 method, Hoechst33342/PI, and flow cytometry. 3. We isolate and culture rat ovarian granulosa cells (GCs) to establish an in vitro model of GCs injury under the induction of cisplatin (5 mg/L). The experiment was carried out in four groups: a control group, model group, MSCs model group, HS-MSCs model group. The apoptosis and survival rate were detected by Hoechst33342/PI and flow cytometry, respectively.Results1. The proliferation level and survival rate of MSCs in the heat shock control group were significantly higher than those in the other three groups, and the apoptosis rate was significantly lower than the other three groups (P<0.05). Compared to the model group, the proliferation level of the heat shock model group was significantly increased, and the apoptosis rate was significantly decreased (P<0.05), and the cell survival rate increased; 2. The apoptosis rate of GCs in the HS-MSCs model group was significantly lower than that in the other three groups. Compared to the MSCs model group, the apoptosis rate of GCs in the HS-MSCs model group was significantly decreased (P<0.05).ConclusionHeat shock pretreatment can increase the proliferation level and survival rate of MSCs, and reduce its apoptosis rate. Heat shock pretreated stem cells can effectively inhibit chemotherapy-induced apoptosis of ovarian granulosa cells.

Therapeutic effect of lentivirus-mediated miR-21 on chemotherapy-induced premature ovarian failure in rats / 解放军医学杂志

Objective To explore the therapeutic potential of miR-21 in rat model of chemotherapy-induced premature ovarian failure (POF).Methods Lentivirus-mediated miR-21 (LV-miR-21) was constructed successfully in vitro with molecular biology methods.Rats were divided into 4 groups named control group,model group,blank vector group and miR-21 group.Rat models of chem0therapy-induced POF were established in the latter 3 groups by intraperitoneal injection ofcytoxan (CTX).Bilateral ovaries of rats in miR-21 group were injected with LV-miR-21,of rats in blank vector group were injected with lentivirus vector,and rats in model group received no treatment.At 1,15,30,45 and 60 days after the last injection,blood sample was collected,the rats were then sacrificed and the ovaries were removed.The estrous cycle was observed by vaginal smears.The E2 level was detected by chemiluminescent immunoassay,and the follicle stimulating hormone (FSH) level was detected by homologous double antibody radiation immunoassay.Ovary weights were measured,and the follicle count was conducted through observing paraffin section under microscope.The apoptosis of ovarian granulosa cells was analyzed by TUNEL assay.Results During 15-30 days,30-45 days and 45-60 days after the last injection,regular estrous cycle was recovered respectively in 8,5 and 3 rats in miR-21 group.At the 15th,30th,45th and 60th day after the last injection,the E2 level was higher in miR-21 group than in model group and blank vector group,but the FSH level showed the opposite trend (P=0.000).At the 45th and 60th day after the last injection,the follicle numbers at all stages increased markedly in miR-21 group than in model group and blank vector group (P=0.000).At the 30th,45th and 60th day after the last injection,the ovary weights were higher in miR-21 group than in model group and blank vector group.At the 15th,30th,45th and 60th day after the last injection,the apoptosis rate of ovarian granulosa cell were significantly lower in miR-21 group than in model group and blank vector group (P=0.000).Conclusion Up-regulation of miR-21 expression may partly recover the ovarian structure and function damaged by CTX.

Changes of serum miR-21 level in animal model and patients with chemotherapy-induced premature ovarian failure / 解放军医学杂志

Objective To investigate the correlation between miR-21 and chemotherapy-induced premature ovarian failure (CIPOF) by measuring the serum miR-21 level in animal models and patients. Methods CIPOF animal model was established by intraperitoneal injection of cyclophosphamide. Caudal vein blood samples were collected at the first and the 15th day after the models established. Serum miR-21 level was detected by QT-PCR. The correlations between miR-21 level with estradiol (E2) level, follicle-stimulating hormone (FSH) level and ovary structure were analyzed. From Jan. 2014 to Jun. 2015, 30 patients with CIPOF and 30 normal women of child-bearing age were enrolled in the study. Serum miR-21 levels of all cases were detected and the correlations between serum miR-21 levels and other laboratory indexes [luteinizing hormone (LH), E2, FSH and prolactin (PRL)] were analyzed. Results Compared to control group, the serum miR-21 levels in CIPOF animal models and patients were obviously lower with statistical significances (t=15.467, P=0.000; t=10.197, P=0.000). Positive correlations existed in animal models between miR-21 and E2, primordial, primary, secondary and antrum follicle numbers (r=0.750, P=0.000; r=0.403, P=0.006; r=0.704, P=0.000; r=0.783, P=0.000; r=0.849, P=0.000, respectively), and a negative correlation existed between miR-21 and FSH (r=–0.801, P=0.000). A positive correlation existed in patients between miR-21 and E2 (r=0.817, P=0.000), and negative correlations existed between miR-21 and FSH (r=–0.771, P=0.000) and miR-21 and LH (r=–0.784, P=0.000). No correlation existed between miR- 21 and PRL (r=0.204, P=0.207). Conclusion The evident decrease of serum miR-21 level in CIPOF animal models and patients suggests that miR-21 may play an important role in the process of CIPOF, and the miR-21 may be a potential prevention and therapeutic option for CIPOF.

Biological features and ultrastructure of human umbilical cord mesenchymal stem cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To isolate and culture human umbilical cord mesenchymal stem cells (MSCs) and explore their biological features and ultrastructure.</p><p><b>METHODS</b>After isolating MSCs from the human umbilical cord, the proliferation, cycle, and apoptosis were observed. The cell ultrastructure was observed under transmission electron microscope. The cytokines including vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1) were detected using enzyme-linked immunosorbent assay.</p><p><b>RESULTS</b>Human umbilical cord MSCs had fibroblast-like morphology and increased proliferation capability. Ultrastructural analysis showed that the MSCs had active cellular metabolism and strong migration and differentiation capabilities. Meanwhile, they could secrete anti-apoptotic cytokines such as VEGF, IGF-1, and HGF.</p><p><b>CONCLUSION</b>Human umbilical cord MSCs can secrete many anti-apoptotic cytokine and have good biological features.</p>

Green fluorescent protein gene transfection mediated by recombinant adenovirus vector to rat bone marrow mesenchymal stem cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the efficient transfection of green fluorescent protein gene (GFP) mediated by recombinant adenovirus vector(Ad-GFP) to rat bone marrow mesenchymal stem cells (MSCs) in vitro.</p><p><b>METHODS</b>Wistar rat bone marrow-derived MSCs were separated and purified in vitro by Percoll density gradient centrifugation combined with adherent cell culture followed by identification with flow cytometry. MSCs infected by Ad-GFP were observed and the transfection efficiency was assessed by fluorescence microscope. The proliferative ability of these cells was tested by CCK-8.</p><p><b>RESULTS</b>The transfection efficiency was as high as 90.0%. Expression of GFP gene of infected MSCs was stable for 1 month after infection. There was no statistically difference in proliferative ability between the infected MSCs and non-infected ones (P>0.05).</p><p><b>CONCLUSION</b>The infected MSCs with Ad-GFP expressed GFP with high efficiency and retain the ability of proliferation as non-infected MSCs. Transgection with Ad-GFP is a highly effective method for labeling MSCs.</p>