Objective To observe the effect of transplantation of embryonic stem cell(ES) on neurological functional recovery of injured spinal cord in adult mouse. Methods The ES cells were cultured and induced in vitro. Fifty C57/BL6J mice were made animal model of semicut mice of T9，10. The ES cellderived neural precursors cells were transplanted into the vertebral canalaround injured spinal cord semi-cut mice. Twenty-eight C57/BL6J mice were randomly divided into three groups: sham operation group(group A,n=9), operation／cell group (group B，n=10), and operation／DMEM group(group C,n=9). RT-PCR analysis, X-gal staining and immunofluorescence were used to observe the cells survival and differentiation in the spinal crod. BBB test was performed to study functional improvement. Results ES cells induced and cultured in vitro displayed clonal growth with circle or ovoid shape and had one or more nucleoli. RT-PCR result showed that the induced ES cells expressed mRNA of Nestin and microtubuleassociated protein, but did not express glial fibrillory acidic protein(GFAP). There was statistically significant difference in BBB scoring between group A and groups B, C after operation (P＜0.01). There was statistically significant difference in BBB scoring at 1, 2 and 4 weeks of operation(P＜0.01), but no statistically significant difference at 6 and 8 weeks of operation between groups B and C(P＞0.05). The X-gal staining results werepositive in group B and negative in groups A and C. The immunoflurescence resultshowed neurofilament green fluor and no expression of GFAP in injured spinal cord region. Conclusion After transplantation, ES cellderived cells can survive, transfer into the injury position, and differentiate into neurons, but spinal cord function has no obvious improvement.
ObjectiveTo investigate the mechanism of muscle-derived cells (MDCs) in repairing sciatic nerve defects in mice by observing the early growth of damaged peripheral nerves.MethodsThe hind limb skeletal muscles of mice carrying enhanced green fluorescent protein (EGFP) was collected to extract and culture EGFP-MDCs to P1 generation for later experiments. Five-mm-long nerve defects were created in the right sciatic nerves of C57BL/6 mice to establish a peripheral nerve defect model. The two stumps of sciatic nerve were bridged with 7-mm-long polyurethane (PUR) conduit. For the MDC group, EGFP-MDCs were injected into the PUR conduit. The PUR group without EGFP-MDCs was used as the negative control group. At 1 and 2 weeks after operation, the proximal and distal nerve stumps of the surgical side were collected to generally observe the early growth of nerve. Immunofluorescence staining of S100β, the marker of Schwann cells, was performed on longitudinal frozen sections of nerve tissues to calculate the maximum migration distance of Schwann cells, and observe the source of the Schwann cells expressing S100β. Immunofluorescence staining of phosphorylated erb-b2 receptor tyrosine kinase 2 (p-ErbB2) and phosphorylated focal adhesion kinase (p-FAK) in transverse frozen sections of nerve tissue was performed to calculate the positive rates of both proteins.ResultsThe general observation showed that the proximal and distal stumps of the surgical side in PUR group were not connected at 1 and 2 weeks after operation, while the bilateral nerve stumps in the MDC group were connected at 2 weeks after operation. Immunofluorescence staining showed that the Schwann cells expressing S100β in proximal and distal nerve stumps of PUR group and MDC group was not connected at 1 week after operation. At 2 weeks after operation, the Schwann cells expressing S100β in the two nerve stumps of the MDC group were connected, but not in the PUR group. At 2 weeks after operation, the sum of the maximum migration distance of Schwann cells in the regenerated nerve in both two groups was significantly increased when compared with that in each group at 1 week after operation, and that of MDC group was significantly higher than that in the PUR group at both 1 and 2 weeks after operation, the differences were all significant (P<0.05). At 1 week after operation, the positive rates of p-ErbB2 and p-FAK in the proximal nerve stump of MDC group were significantly higher than those in PUR group (P<0.05). There was no significant difference in the positive rate of p-ErbB2 of proximal stump between the two groups at 2 weeks after operation (t=0.327, P=0.747), while the positive rate of p-FAK of MDC group was significantly higher than that of PUR group (t=4.470, P=0.000). At 1 and 2 weeks after operation, the positive rates of p-ErbB2 and p-FAK in the distal stump of MDC group were significantly higher than those in PUR group (P<0.05). At 1 and 2 weeks after operation, part of Schwann cells expressing S100β, which were derived from EGFP-MDCs, could be observed in the regenerated nerves of MDC group.ConclusionMDCs can promote the phosphorylation of ErbB2 and FAK in the nerve stumps of mice, and promote the migration of Schwann cells. MDCs can be differentiated into cells expressing the Schwann cell marker S100β, or as other cellular components, to involve in the early repair of peripheral nerves.
ObjectiveTo approach the role of CD4+CD25+ regulatory T cells in the maintenance of immunotolerance in mouse liver allograft. MethodsThe mouse orthotopic liver transplantation was performed. After the liver transplantation immunotolerance induction, antiCD25 monoclonal antibody (PC61) was injected into the recipients with a delayed timing to remove the CD4+CD25+ T cells. The percentage of CD4+CD25+ T cells and the expression of forkhead/winged helix transcription factor (Foxp3) in the recipients were examined. Furthermore, the survival time of the recipient was observed. ResultsC3H/HeJ recipients receiving DBA/2 hepatic allografts survived over 70 d as in the syngeneic liver transplantation (C3H/HeJ recipients receiving C3H/HeJ hepatic grafts). With various protocols of the delayed PC61 treatment, the CD4+CD25+ T cell was completely disappeared as observed. However, the removal of CD4+CD25+ regulatory T cells after the induction of transplantation immunotolerance did not affect the survival of hepatic allografts. ConclusionCD4+CD25+ regulatory T cells are not essential for the maintenance of spontaneous mouse liver transplantation immunotolerance.
ObjectiveTo study the effect and mechanism of lipopolysaccharide (LPS) on osteoclasts formation and its bone resorption function.MethodsBone marrow-derived macrophages (BMMs) were extracted from the marrow of femur and tibia of 4-week-old male C57BL/6 mice. Flow cytometry was used to detect BMMs. The effect of different concentrations of LPS (0, 100, 200, 500, 1 000, 2 000 ng/mL) on BMMs activity was examined by cell counting kit 8 (CCK-8) activity test. In order to investigate the effect of LPS on osteoclastogenesis, BMMs were divided into macrophage colony-stimulating factor (M-CSF) group, M-CSF+receptor activator of nuclear factor κB ligand (RANKL) group, M-CSF+RANKL+50 ng/mL LPS group, M-CSF+RANKL+100 ng/mL LPS group. After the completion of culture, tartrate resistant acid phosphatase (TRAP) staining was used to observe the formation of osteoclasts. In order to investigate the effect of LPS on the expression of Connexin43, BMMs were divided into the control group (M-CSF+RANKL) and the LPS group (M-CSF+RANKL+100 ng/mL LPS); and the control group (M-CSF+RANKL), 50 ng/mL LPS group (M-CSF+RANKL+50 ng/mL LPS), and 100 ng/mL LPS group (M-CSF+RANKL+100 ng/mL LPS). The expressions of Connexin43 mRNA and protein were detected by Western blot and real-time fluorescent quantitative PCR, respectively. In order to investigate the effect of LPS on osteoclast bone resorption, BMMs were divided into M-CSF group, M-CSF+RANKL group, M-CSF+RANKL+50 ng/mL LPS group, and M-CSF+RANKL+100 ng/mL LPS group. Bone absorption test was used to detect the ratio of bone resorption area.ResultsThe flow cytometry test confirmed that the cultured cells were BMMs, and CCK-8 activity test proved that the 100 ng/mL LPS could promote the proliferation of BMMs, showing significant differences when compared with the 0, 200, 500, 1 000, and 2 000 ng/mL LPS (P<0.05). TRAP staining showed no osteoclast formation in M-CSF group. Compared with M-CSF+RANKL group, the osteoclasts in M-CSF+RANKL+50 ng/mL LPS group and M-CSF+RANKL+100 ng/mL LPS group were larger with more nuclei, while the osteoclasts in M-CSF+RANKL+100 ng/mL LPS group were more obvious, and the differences in the ratio of osteoclast area between groups were statistically significant (P<0.05). Western blot result showed that the relative expression of Connexin43 protein in LPS group was significantly higher than that in control group (P<0.05). Real-time fluorescent quantitative PCR showed that the relative expression of Connexin43 mRNA in control group, 50 ng/mL LPS group, and 100 ng/mL LPS group increased gradually, and the differences between groups were statistically significant (P<0.05). Bone resorption test showed that osteoclast bone resorption did not form in M-CSF group, but the ratio of bone resorption area increased gradually in M-CSF+RANKL group, M-CSF+RANKL+50 ng/mL LPS group, and M-CSF+RANKL+100 ng/mL LPS group, and the differences between groups were statistically significant (P<0.05).ConclusionLPS at concentration of 100 ng/mL can promote the expression of Connexin43, resulting in increased osteoclastogenesis and enhanced osteoclastic bone resorption.
Allogeneic mouse model of peritoneal heart transplant is a microscopic surgery on small animal with complex techniques. For a beginner, a learning curve of this surgical technique has to be experienced. The learning curve contains three stages:(1) to be familiar with the local anatomy of either donor or recipient mouse; (2) to be capable of collecting donor heart and well preparing the major peritoneal vessels of recipient; (3) to be skillful in the anastomosis of major vessels. The bottleneck of the learning curve is the valid skill of vascular anastomosis. The stepwise essentials are to "understand, be familiar, be accurate, and be quick" in the learning curve.
ObjectiveTo investigate the effects of adipose-derived stem cell released exosomes (ADSC-Exos) on wound healing in diabetic mice.MethodsThe ADSCs were isolated from the adipose tissue donated by the patients and cultured by enzymatic digestion. The supernatant of the 3rd generation ADSCs was used to extract Exos (ADSC-Exos). The morphology of ADSC-Exos was observed by transmission electron microscopy. The membrane-labeled proteins (Alix and CD63) were detected by Western blot, and the particle size distribution was detected by nanoparticle tracking analyzer. The fibroblasts were isolated from the skin tissue donated by the patients and cultured by enzymatic digestion. The 5th generation fibroblasts were cultured with PKH26-labeled ADSC-Exos, and observed by confocal fluorescence microscopy. The effects of ADSC-Exos on proliferation and migration of fibroblasts were observed with cell counting kit 8 (CCK-8) and scratch method. Twenty-four 8-week-old Balb/c male mice were used to prepare a diabetic model. A full-thickness skin defect of 8 mm in diameter was prepared on the back. And 0.2 mL of ADSC-Exos and PBS were injected into the dermis of the experimental group (n=12) and the control group (n=12), respectively. On the 1st, 4th, 7th, 11th, 16th, and 21st days, the wound healing was observed and the wound healing rate was calculated. On the 7th, 14th, and 21st days, the histology (HE and Masson) and CD31 immunohistochemical staining were performed to observe the wound structure, collagen fibers, and neovascularization.ResultsADSC-Exos were the membranous vesicles with clear edges and uniform size; the particle size was 40-200 nm with an average of 102.1 nm; the membrane-labeled proteins (Alix and CD63) were positive. The composite culture observation showed that ADSC-Exos could enter the fibroblasts and promote the proliferation and migration of fibroblasts. Animal experiments showed that the wound healing of the experimental group was significantly faster than that of the control group, and the wound healing rate was significantly different at each time point (P<0.05). Compared with the control group, the wound healing of the experimental group was better. There were more microvessels in the early healing stage, and more deposited collagen fibers in the late healing stage. There were significant differences in the length of wound on the 7th, 14th, and 21st days, the number of microvessels on the 7th and 14th days, and the rate of deposited collagen fibers on the 14th and 21st days between the two groups (P<0.05).ConclusionADSC-Exos can promote the wound healing in diabetic mice by promoting angiogenesis and proliferation and migration of fibroblasts and collagen synthesis.
ObjectiveTo investigate whether miR-93-5p suppresses osteogenic differentiation of mouse mesenchymal stem cells (C3H10T1/2) by targeting Smad5, a predicted target in silicon. MethodsSmad5 3'-UTRluciferase vector (pmiR-RB-REPORTTM) was constructed and dual-luciferase reporter gene assay was employed to examine the effect of miR-93-5p on Smad5 3'-UTR-luciferase activity to identify whether Smad5 was the target gene of miR-93-5p. miR-93-5p mimics (group M), miR-93-5p inhibitor (group In), miR-93-5p mimics negative control (group MC), and miR-93-5p inhibitor negative control (group InC) were transfected into the C3H10T1/2 cells, respectively, and followed by induction of osteogenic differentiation. After 48 hours, the real-time fluorescent quantitative PCR (qRTPCR) and Western blot assays were performed to detect the relative expressions of Smad5 mRNA and protein. At 14 days, to realize the regulation role of miR-93-5p in osteogenic differentiation, the extracellular calcium deposition during the osteogenesis of C3H10T1/2 cells was tested by Alizarin red staining. ResultsDual-luciferase reporter gene assay showed that miR-93-5p could combine with Smad5 mRNA 3'-UTR specificity, and inhibited its luciferase activity (P<0.05). After 48 hours, no significant difference was shown in the relative expression of Smad5 mRNA between group M and group MC as well as between group In and group InC by qRT-PCR assay (P>0.05); however, the results of Western blot assay showed that the relative expression of Smad5 protein was significantly decreased in group M and increased in group In when compared with groups MC and InC (P<0.05). At 14 days after osteogenic induction, Alizarin red staining showed that the extracellular calcium deposition of group M was obviously less than that of group MC, and it was obviously more in group In than in group InC. ConclusionSmad5 may be the target gene of miR-93-5p. And miR-93-5p can suppress osteogenic differentiation of C3H10T1/2 cells by directly targeting Smad5.
【摘要】 目的 研究肿瘤坏死因子相关凋亡诱导配体（TRAIL）蛋白对SKOV3移植瘤细胞半胱天冬氨酸蛋白酶-3（Caspase-3）表达的影响及其与肿瘤细胞凋亡的关系。 方法 建立雌性裸小鼠SKOV3移植瘤24只，随机分为4组，每组6只。TRAIL组单用重组人TRAIL蛋白（10 μg/kg），顺铂（DDP）组单用DDP（3 mg/kg），TRAIL+DDP组联合使用TRAIL蛋白（10 μg/kg）和DDP（3 mg/kg），空白对照组给予0.5 mL生理盐水。经处理后，各组的组织切片用免疫组织化学染色检测Caspase-3的表达和末端脱氧核苷酸转移酶介导核苷酸缺口标记技术（TUNEL）检测肿瘤细胞凋亡指数。 结果 Caspase-3的表达水平在TRAIL组（171.67±14.38）、DDP组（172.50±14.75）、联合组（230.00±40.99）中均明显高于对照组（135.83±16.25）（Plt;0.05）。SKOV3移植瘤细胞凋亡指数在空白对照组、TRAIL组、DDP组和联合组分别为16.67±5.43、33.17±8.42、24.33±4.59和40.50±6.16，TRAIL组和联合组细胞凋亡指数较空白对照组和DDP组明显增高（Plt;0.05）。 结论 TRAIL蛋白使卵巢癌移植瘤细胞的Caspase-3表达增强，TRAIL蛋白促进肿瘤细胞凋亡发生。【Abstract】 Objective To investigate the effects of Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on the expression of Cysteine/aspartic acid specific protease- 3 (Caspase-3) in SKOV3 ovarian tumor cells and its relationship with the apoptosis of the ovarian tumor xenografts in nude mice. Methods Twenty-four nude mice with SKOV3 cell line ovarian tumor were randomly divided into four groups with 6 in each group. TRAIL (10 μg/kg) was given to the mice in the TRAIL group; DDP (3 μg/kg) was given to the mice in the DDP group; TRAIL (10 μg/kg) and DDP (3 μg/kg) were given to the mice in the TRAIL+DDP group; and 0.5 mL of saline solution was give to the mice in the control group. The expression of Caspase-3 was detected with immunohistochemistry. The apoptosis index (AI) of cells was determined by Terminal deoxynucleotidyl transferase mediated-dUTP nick end labeling (TUNEL). Results The expression of Caspase-3 in the TRAIL group (171.67±14.38), DDP group (172.50±14.75), and TRAIL+DDP group (230.00±40.99) was significantly higher than that in the control group (135.83±16.25) (Plt;0.05). The apoptosis index for the control group, TRAIL group, DDP group and TRAIL+DDP group was 16.67±5.43, 33.17±8.42, 24.33±4.59, and 40.50±6.16, respectively. The apoptosis index for the TRAIL group and the TRAIL+DDP group was significantly higher than that in the control group and the DDP group (Plt;0.05). Conclusion Soluble TRAIL has an effect on enhancing the expression of Caspase-3 in implanted tumor in nude mice. TRAIL protein can inhibit the growth of SKOV3 cells in nude mice by inducing cell apoptosis.
目的 构建含小鼠血管内皮生长因子（mVEGF）的重组慢病毒表达载体，包装成病毒颗粒后感染NS-1小鼠骨髓瘤细胞株，以便进一步探索VEGF在骨髓瘤病理生理机制中的作用。 方法 聚合酶链反应法扩增mVEGF基因，克隆入含嘌呤霉素抗性的pCDH慢病毒表达载体，构建出表达mVEGF的慢病毒表达载体pCDH-mVEGF；采用磷酸钙法将慢病毒系统三质粒pCDH-mVEGF、psPAX2、pMD2.G共转染293FT细胞包装病毒，分别收集转染后48 h和72 h病毒上清并感染靶细胞NS-1，初次感染72 h后开始采用嘌呤霉素筛选稳定株，筛选2周后采用ELISA法检测稳定株细胞培养上清中mVEGF的表达，建立出稳定高表达mVEGF的NS-1小鼠骨髓瘤细胞株。 结果 成功构建重组慢病毒表达质粒pCDH-mVEGF，并包装成慢病毒颗粒，感染NS-1细胞株后获得靶基因的稳定高表达。 结论 成功构建出含mVEGF的慢病毒表达载体pCDH-mVEGF，慢病毒系统能有效介导目的基因在NS-1小鼠骨髓瘤细胞株中稳定表达，病毒包装成功并能有效感染NS-1细胞，为进一步探索VEGF在骨髓瘤病理生理机制中的作用奠定了基础。