Objective To investigate the effect of combined therapy of granulocyte colony stimulating factor (G-CSF) and bone marrow mesenchymal stem cells (BMSCs) carrying hepatocyte growth factor (HGF) gene on the angiogenesis of myocardial infarction (MI) in rats and the mechanisms of the synergistic effect. Methods BMSCs were aspirated from the femur and tibia of 3-week-old Sprague Dawley (SD) male rats. The third generation of BMSCs were harvested and transfectedwith Ad-HGF. The MI models were establ ished in 44 SD male rats (weighing 200-250 g) by l igating the left coronary artery. At 4 weeks after l igation, the shorting fraction (FS) of the left ventricle being below 30% was used as a criteria of model success. The BMSCs (5 × 107/ mL) transfected with Ad-HGF were transplanted into the infarct zone of 12 SD rats, and the expression of HGF protein was detected by Western blot method at 2, 7, and 14 days after transplantation. At 4 weeks, the other 32 SD rats were randomly divided into 4 groups (n=8). The 0.1 mL normal sal ine was injected into the infarct zone in control group; 0.1 mL normal sal ine was injected combined with intraperitoneal injection G-CSF [100 μg/ (kg•d)] for 5 days in G-CSF group; 0.1 mL BMSCs (5 × 107/ mL) transfected with Ad-HGF was injected into the infarct zone in HGF group; 0.1 mL BMSCs (5 × 107/ mL) transfected with Ad-HGF was injected combined with intraperitoneal injection G-CSF [100 μg/ (kg•d)] for 5 days in combined therapy group. At 2 weeks after transplantation, heart function was detected by cardiac ultrasound and hemodynamic analysis, and then myocardial tissue was harvested to analyse the angiogenesis of the infarct zone, and the expression of VEGF protein by immunofluorescence staining. Results The expression of HGF protein in vivo was detected at 2 days and 7 days of BMSCs transfected with Ad-HGF transplantation. There was no significant difference in left ventricular systol ic pressure (LVSP), left ventricular end-diastol ic pressure (LVEDP), dP/dtmax, and FS between G-CSF group and control group (P gt; 0.05). When compared with the control group, LVEDP decreased significantly; LVSP, FS, and dP/dtmax increased significantly (P lt; 0.05) in HGF group and combined therapy group. When compared with HGF group, FS and dP/dtmax increased significantly in combined therapy group (P lt; 0.05). Immunofluorescence staining showed that the vascular endothel ial cells were observed in myocardial infarction border zone. The vascular density and the expression of VEGF protein were significantly higher in combined therapygroup than in other 3 groups (P lt; 0.05). Conclusion The combined therapy of G-CSF and BMSCs carrying HGF gene has a synergistic effect and can enhance infarct zone angiogenesis through inducing the expression of VEGF protein.
Objective To evaluate the effectiveness and safety of autologous hemopoietic stem cell implantation for peripheral arterial disease (PAD). Methods Randomized controlled trials (RCTs) were identified from CBM (1978 to September 2010), CNKI (1979 to September 2010), MEDLINE (1950 to September 2010), Pubmed (1950 to September 2010), Embase (1970 to September 2010), and Cochrane l ibrary (issue 4, 2010). The papers of the RCTs of cl inical therapeutic studieson PAD treated by autologous hemopoietic stem cell implantation were included and analyzed according to the criteria of the Cochrane handbook. Results Eight RCTs involving 280 patients and 322 extremities were included, with majority of trials of low methodological qual ity. Meta-analysis indicated that autologous hemopoietic stem cell transplantation had an increased ulcer cure rate [RD=0.38, 95% CI= (0.25, 0.50)], a significant improvement in the ankle brachial index [MD=0.11, 95%CI= (0.04, 0.18)], transcutaneous oxygen tension [MD=7.33, 95%CI= (3.14, 11.51)], and pain-free walking distance [SMD=1.35, 95%CI= (0.90, 1.79)], a significant reduction in rest pain scores [MD= —1.70, 95%CI= (—2.15, —1.25)], and a significant benefit in terms of l imb salvage [RD= —0.19, 95%CI= (—0.31, —0.07)]. Only 2 trials reported the side effects of autologous hemopoietic stem cell transplantation, such as l imbs swell ing and concentrations of serum creatine phosphokinase increasing, and the long-term safety was not reported. Conclusion Based on the review, autologous hemopoietic stem cell transplantation may have positive effect on “no-option” patients with PAD. However, the evidence is not b enough due to the general low methodological qual ity, so we can not draw a rel iable conclusion about the effects of autologous stem cell transplantation for PAD at the moment. Further larger, randomized, double bl ind, placebo-controlled, and multicenter trials are needed.
OBJECTIVE Because of its special biological characteristics, myoblast might play a role in gene delivery and cell-to-biomaterial interactions. In this paper, the biological features of myoblast and its application on gene therapy and tissue engineering was discussed. METHODS Documents about proliferation and differentiation of myoblast were reviewed in details. The prospects of its application on gene therapy and tissue engineering were also presented. RESULTS Myoblast was important in muscle regeneration. The activation of myoblast to proliferate and differentiate was the very beginning of regeneration after injury. The cultured myoblast had high potential to proliferate, it was ready to fuse with each other and to form myotube (the special behavior of myoblast differentiation). Myoblast transplantation had been studied as a possible treatment for inherited myopathies, such as Duchenne muscular dystrophy. The transplanted myoblast could fuse with host myofibers, so the delivered target gene integrated into host. Several myoblast-mediated gene delivery system had been established, including the gene delivery of human factor IX (hFIX), erythropoietin (EPO) and clony stimulating factor-1 (CSF-1). Results from animal experiments demonstrated that myoblast-mediated gene delivery could be used as gene therapy for some inherited diseases. And recently, some authors have shown great interest in the interaction between myoblast and type I collagen gels. It was found that myoblast could keep on proliferating and differentiating in collagen gels and could form discoid, tubular materials. CONCLUSION Myoblast has great importance in gene therapy and tissue engineering. It is suggested that more efforts should be made in this field.
Objective To review the status and appl ication prospect in repair of spinal cord injury by stem cells. Methods The related articles in recent years were extensively reviewed, the biological characteristics of stem cells, the experimental and cl inical studies on repair of spinal cord injury by stem cells, the mechanism of the therapy and the problem were discussed and analyzed. Results The foundational and cl inical study indicated that the great advance was made in repair of spinal cord injury, the stem cells could immigrate in the spinal cord, and differentiate into neuron and secrete neurotrophic factors. So it could promote the repair effects. Conclusion Repair of spinal cord injury by stem cells is an effective therapystrategy, but many problems remain to be resolved.
Purpose To investigate the characteristics of intraocular growth of mice embryonic stem cells (ESC) in nude mice. Methods The undifferentiated murine ESC in vitro were transplanted into the eyes of nude mice.Mophological and immunohistochemical examinations were implemented. Results Two to three days after transplantation,yellowish-white granules and masses were seen inside the anterior chamber and vitreous cavity and enlarged gradually.Morphological examination showed that there were undifferentiated cells and differentiated cells in anterior chamber and vitreous cavity.The morphology and alignment of some differentiated cells were similar to those of the retina of nude mice.The cells were highly positive in NSE staining. Conclusion The transplanted ESC could grow in the eyes of nude mice and differentiate into neurons and retina-like structure. (Chin J Ocul Fundus Dis，2000，16：213-284)
Based on the pathogenic mechanisms of age-related macular degeneration (AMD), tremendous preclinical and clinical trials have demonstrated that cell transplantation which aim to replace impaired retinal pigment epithelium (RPE) with healthy RPE cells is a promising approach to treat AMD. So far, choices of cell sources mainly are autologous RPE, iris pigment epithelium, fetal RPE, human embryonic stem cell-derived RPE and human induced pluripotent stem cell-derived RPE, and some of them are undergoing clinical researches. Grafting manners in cell-based therapies are various including RPE sheet or RPE-choroid complex transplantation, RPE cell suspension injection, and RPE sheet transplantation with scaffolds. This review is limited to cell-based therapies for RPE that damaged first in the progress of AMD and focus on recent advances in cell sources, transplantation methods, preclinical and clinical trials, and the obstacles that must be overcome.
Abstract: Objective To observe the changes in morphology, structure, and ventricular function of infarct heart after bone marrow mononuclear cells (BMMNC) implantation. Methods Twenty-four dogs were divided into four groups with random number table, acute myocardial infarction (AM I) control group , AM I-BMMNC group , old myocardial infarct ion (OMI) control group and OM I-BMMNC group , 6 dogs each group. Autologous BMMNC were injected into infarct and peri-infarct myocardium fo r transplantation in AM I-BMMNC group and OM I-BMMNC group. The same volume of no-cells phosphate buffered solution (PBS) was injected into the myocardium in AM Icontrol group and OM I-control group. Before and at six weeks of cell t ransplantation, ult rasonic cardiography (UCG) were performed to observe the change of heart morphology and function, then the heart was harvested for morphological and histological study. Results U CG showed that left ventricular end diastolic dimension (LV EDD) , left ventricular end diastolic volume (LVEDV ) , the thickness of left ventricular postwall (LVPW ) in AM I-BMMNC group were significantly less than those in AM I-control group (32. 5±5. 1mm vs. 36. 6±3. 4mm , 46. 7±12. 1m l vs. 57. 5±10. 1m l, 6. 2±0. 6mm vs. 6. 9±0. 9mm; P lt; 0. 05). LVEDD, LVEDV , LVPW in OM I-BMMNC group were significantly less than those in OM I-control group (32. 8±4. 2 mm vs. 36. 8±4. 4mm , 48. 2±12. 9m l vs. 60.6±16.5m l, 7. 0±0. 4mm vs. 7. 3±0. 5mm; P lt; 0. 05). The value of eject fraction (EF) in OM I-BMMNC group were significantly higher than that in OM I-control group (53. 3% ±10. 3% vs. 44. 7%±10. 1% ). Compared with their control group in morphological measurement, the increase of infarct region thickness (7. 0 ± 1. 9mm vs. 5. 0 ±2.0mm , 6.0±0. 6mm vs. 4. 0±0. 5mm; P lt; 0. 05) and the reduction of infarct region length (25. 5±5. 2mm vs. 32. 1±612mm , 33. 6±5. 5mm vs. 39. 0±3. 2mm , P lt; 0. 05) were observed after transplantation in AM I-BMMNC group and OM I-BMMNC group, no ventricular aneurysm was found in AM I-BMMNC group, and the ratio between long axis and minor axis circumference of left ventricle increased in OM I-BMMNC group (0. 581±0. 013 vs. 0. 566±0.015; P lt; 0. 05). Both in AM I-BMMNC group and OM I-BMMNC group, fluorescence expressed in transplantation region was observed, the morphology of most nuclei with fluorescencew as irregular, and the differentiated cardiocyte with fluorescence was not found in myocardium after transplantation. The histological examination showed more neovascularization after transp lantation both in AMI and in OM I, and significant lymphocyte infiltration in AM I-BMMNC group. Conclusion BMMNC implantation into infarct myocardium both in AMI and OMI have a beneficial effect, which can attenuate deleterious ventricular remodeling in morphology and st ructure, and improve neovascularization in histology, and improve the heart function.
Objective To review the current status and problems in developing cardiac biological pacemaker(CBP) by cell transplantation. Methods The l iterature over the past decade concerning CBP constructed through celltransplantation was reviewed and summarized. Results Experiments in vivo testified that the cell transplantation was feasible for CBP construction, and the transplantation of sinus atrial node cell and stem cell was still the predominant method for constructing CBP. However, such problems as difficult ampl ification of transferred cardio muscle cell, low success rate of CBP construction as well as unstable function of CBP make it lag behind the tremendous cl inical demands. The gene transfection technology might be one of the approaches to resolve these issues. Conclusion As one feasible method for CBP construction, the cell transplantation has a bright future in the cl inical appl ication and is worthy of further study.
Objective To investigate the expression of micro-dystrophin gene in myoblast cultured in vitro, to explore the possibil ity of combining myoblast transplantation with gene transfer for Duchenne muscular dystrophy therapy. Methods Competent Escherichia coli JM109 was prepared, which transformed with plasmid pSL139, and positive clones were picked to cultivate. Plasmid was extracted with Alkal ine lysis method and cutted with both Pvu I and Cla I enzyme. Agarose gel electrophoresis was employed to take pictures. Ten healthy 5-7 days old male C57/BL10 mice were selected, weighing4-5 g, the primary and subcultured myoblasts were cultured with multi-step enzymatic digestion and differential adhesionmethod, and Desmin immunofluorescent method was used to identfy. The 3rd generation myoblasts that were transfected with plasmid pSL139 mediated by l iposome served as the experimental group, untransfected cells served as the control group. After 48 hours of transfection, the expressions of micro-dystrophin mRNA and protein in myoblasts were detected with RTPCR and cell immunofluorescent methods, and the transfection efficiency was caculated. Results After pSL139 plasmids being digested and for 40 minutes agarose gel of electrophoresis, 3.75 kb fragment of target gene and vector were observed. The cells were almost uniform, and triangular or diamond shape after 24-48 hours of culture; the cells turned to fusion manner and could be passaged after 4-6 days. Desmin immunofluorescent result showed that green fluorescence was seen in cytoplasm of most 2nd myoblasts, and the purity of the myoblasts was above 90%. At 48 hours after transfection of myoblasts with plasmid pSL139, RT- PCR results showed that about 300 bp fragment was seen in the experimental group and the control group, and the brightness was higher in experimental group. Immunofluorescent staining displayed that green fluorescence was seen in the cytoplasm of the myoblasts in the experimental group and no green fluorescence in the control group; the expression efficiency of positive cells for micro-dystrophin was 45%-55% in experimental group. Conclusion Micro-dystrophin gene can highly express at the levels of mRNA and protein respectively in myoblasts transfected with plasmid pSL139 mediated by l iposome.
Objective To investigate the cl inical effect of MSCs transplantation derived from human umbil ical cord on bone nonunion. Methods From December 2005 to December 2007, 72 patients with traumatic bone nonunion were treated. Auto-il iac bone transplantation was used in 36 patients (group A), including 27 males and 9 females, aging (34.0 ± 2.1) years; including 18 cases of femoral fracture and 18 cases of tibia fracture; and the time of bone nonunion being (9.1 ± 1.7)months. Percutaneous MSCs transplantation derived from human umbil ical cord was used in 36 patients (group B), including 28 males and 8 females, aging (36.0 ± 1.6) years; including 18 cases of femoral fracture and 18 cases of tibia fracture; and the time of bone nonunion being (6.4 ± 1.9) months. There were no statistically significant differences in general data between two groups (P gt; 0.05). In group A, the site of bone nonunion was filled with relevant auto-il iac bone. In group B, the mixture of 6-8 mL platelet-rich plasma prepared by centrifugal izing venous blood and 1 × (106-107) P5 MSCs extracted from human umbil ical cord denoted by volunteers was injected into the region of bone nonunion with 0.2 g demineral ized bone powder. Results Incision healed by first intention in group A. No puncture, deep infection, rejection and general fever reaction occurred in group B. All patients in two groups were followed up for (13.2 ± 4.6) months. No loosening and breakage of internal fixation were observed in two groups. The motil ity and function of hip, knee and ankle were good. The time of bone union was (10.3 ± 2.8) months in group A and (5.6 ± 0.8) months in groups B, showing significant difference between two groups (P lt; 0.05). Conclusion The percutaneous MSCs transplantation derived from human umbil ical cord is more effective on bone nonunion than the traditional treatment, it is easily-to-operate, safe, rel iable, and rapid for union. It is one of effective methods in treating bone nonunion.