Abstract： Objective To study the effect of different numbers of bone marrow mesenchymal stem cells（MSCs） transplanted into rats with pulmonary arterial hypertension （PAH）induced by monocrotaline（MCT）and their influence on the expression of endothelin-1（ET-1）. Methods Forty healthy male Wistar rats（weight，from 180 to 250 g） were divided into four groups by random number table（n=10）：group A：Wistar rats were intraperitoneally injected with MCT 60 mg/ kg， and then injected with 1×106 MSCs via the external jugular vein；group B：Wistar rats were intraperitoneally injected with MCT 60 mg/kg，and then injected with 5×105 MSCs via the external jugular vein；MCT group：Wistar rats were intraperitoneally injected with MCT 60 mg/kg， and then injected with equal amount of PBS via the external jugular vein； control group：Wistar rats were intraperitoneally injected with equal amount of saline and then injected with equal amount of PBS via the external jugular vein. Four weeks after MSCs transplantation，right ventricular systolic pressure（RVSP） and ventricular weight ratio of right ventricle/ （left ventricle＋ventricular septum）were measured. Histomorphology of lung tissue was observed. Genetic expression of ET-1 in lungs and serum peptide of ET-1 were also measured. Results Four weeks after MSCs transplantation，both RVSP and ventricular weight ratio decreased significantly in rats of group Acompared with those of MCT group（RVSP：35.8±4.2 mm Hg vs. 47.2±10.1 mm Hg，P＜ 0.01； ventricular weight ratio：0.357±0.032 vs. 0.452±0.056，P＜0.01）， but these two parameters didn’t decrease significantly in rats of group B（P＞ 0.05）. By histopathological staining， the percentage of medial wall thickness of the pulmonary arterioles was significantly less in rats of group A than that of MCT group（19.7%±3.0% vs. 26.8%±3.6%， P＜ 0.01）. There was no statistical difference in the percentage of medial wall thickness of the pulmonary arterioles between group B and MCT group. Reverse transcriptase-polymerase chain reaction （RTase-PCR）results showed that ET-1messenger ribonucleic acid（mRNA）expression was highest in MCT group and MSCs transplantation significantly decreasedits expression in group A， while its expression was similar between group B and MCT group. The expression ofET-1 in plasma was also significantly decreased in group A than that in MCT group. Conclusion Intravenous MSCs transplantation can significantly inhibit MCT-induced PAH，and reduce both ET-1 mRNA expression in lung and ET-1 peptide level in plasma. It’s a better choice to transplant 1×106 MSCs to inhibit PAH in rats.
Abstract： Objective To investigate the effects of hepatocyte growth factor（HGF）gene transfected bone marrow mesenchymal stem cells （MSCs）transplantation in pigs with chronic ischemic heart disease. Methods MSCs were isolated from pig bone marrow by density gradient centrifugation and adherent cell culture, purified, and determined by cellsurface antigens（CD34, CD44, CD71, Ⅷ factor and desmin）. MSCs were transfected by adenovirus expressing hepatocyte growth factor（AdHGF）, and the influence of HGF on the biological characteristics of MSCs was tested. The pig model of chronic myocardial ischemia was established by placing Ameroid ring inside the left circumflex coronary artery via leftthoracotomy. A total of 40 pigs were randomly divided into 5 groups （n=8） and were injected 5×106/ml MSCs＋ 4×109 pfu 200 μl AdHGF （MSCs＋ AdHGF group）, 4×109 pfu 200 μl AdHGF （AdHGF group）, 5×106/ml MSCs 200 μl（MSCs group），4×109 pfu 200 μl AdNull （AdNull group）and 1 ml saline（control group） into the ischemic myocardiumrespectively. Echocardiogram, digital subtraction angiography （DSA） of coronary artery, single photon emission computed tomography（SPECT） myocardial perfusion imaging and cardiomyocyte apoptosis were examined after 4 weeks. Results Positive CD44 and CD71 and negative CD34, Ⅷ factorand desmin were detected in MSCs by flow cytometer. HGF had a b influence on stimulating the proliferation and differentiation of MSCs. Echocardiogram examination showed that left ventricular end-diastolic volume（LVEDV），left ventricular ejection fraction（LVEF），fractional shortening（FS）of MSCs＋ AdHGF group were significantly increased after treatment （P＜ 0.05）. DSA detection showed that ischemic neovascularization of MSCs＋ AdHGF group was significantly higher than those of AdHGF group and MSCs group （P＜ 0.05）. SPECT showed that the left ventricular myocardium of MSCs＋ AdHGF group appeared thickened,myocardial perfusion was significantly improved and the myocardial motion was significantly increased （P＜ 0.05）. Vascular density of MSCs＋ AdHGF group was significantly higher than those of AdHGF group and MSCs group by HE stain of myocardium ［（39.4±1.2）/ HPF vs. （36.5±1.4）/ HPF and（34.5±1.7）/ HPF，P＜ 0.05］. Cardiomyocyte apoptosis rate of MSCs＋ AdHGF group was significantly lower than those of AdHGF group and MSCs group by TUNEL stain （P＜ 0.05）. Conclusion Combination transplantation can promote the angiogenesis of chronic ischemic myocardium, inhibit cardiomyocyte apoptosis and improve heart function in pigs with chronic ischemic heart disease. The effect of HGF gene transfected MSCs transplantation is better than that of MSCs or HGF transplantation alone.
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 investigate the feasibility of imaging of bone marrow mesenchymal stem cells (BMMSCs) labeled with superparamagnetic iron oxide(SPIO) transplanted into coronary artery in vivo using magnetic resonance imaging (MRI), and the redistribution of the cells into other organs. Methods BMMSCs were isolated, cultured from bone marrow of Chinese mini swine, and double labeled with SPIO and CMDiI(Cell TrackerTM C-7001). The labeled cells were injected into left anterior descending coronary artery through a catheter. The injected cells were detected by using MRI at 1 week,3weeks after transplantation. And different organs were harvested and evaluated the redistribution of transplanted cells through pathology. Results The SPIO labeled BMMSCs injected into coronary artery could be detected through MRI and confirmed by pathology and maintained more than 3 weeks. The SPIO labeled cells could be clearly imaged as signal void lesions in the related artery. The pathology showed that the injected cells could be distributed into the area of related artery, and the cells injected into coronary artery could be found in the lung, spleen, kidney, but scarcely in the liver, the structures of these organs remained normal. Conclusion The SPIO labeled BMMSCs injected into coronary artery can be detected by using MRI, the transplanted cells can be redistributed into the non-targeted organs.
Objective To investigate the dose-dependent relationship of bone marrow mesenchymal stem cells(MSCs) transplantation in improving ischemic myocardial dysfunction? in a rat ischemic heart model. Methods Myocardial infarction was induced in 32 inbred F344 rats by acute ligation of the left anterior descending(LAD) coronary artery. One week after ligation, the ratswere randomized? into four equal groups, with eight rats in each group. Equal volume Iscove’s modified Dulbecco’s medium was injected in the control group, 1×103(group 1), 1×105(group 2), and 1×107(group 3) 5-bromodeoxyuridine (BrdU) labeled bone marrow MSCs were injected into the infarcted myocardium. Cardiac function was evaluated by ultrasound before the ligation of the LAD, before the transplantation and the 4th week after transplantation. The expressions of BrdU,Connexin43,Myosin heavy chain β(MHC), and smooth muscle actin α(α-SMA) were detected by immunofluorescence and immunohistochemistry at the 4th week after transplantation. The amount of functional vessels stained by α-SMA was counted simultaneously. Results At the 4th week? after transplantation, the ejection fraction(EF) in goup 2 was more significantly improved than that in group1(0.54±0.20 vs. 0.34±0.16, P＝0.004) and EF in group 3 was more significantly improved than that in group 2(0.71±0.24 vs. 0.54±0.20,P＝0.018), whereas no significant difference between group 1 and control group was detected (0.34±0.16 vs. 0.36±0.15,Pgt;0.05). The BrdU labeled MSCs could be found in host myocardium. The number of cells in group 2 by double staining both for BrdU and for MHC observed in ischemic myocardium were significantly more than that in group 1? (323.20±91.62 n/HP vs. 51.75±27.58 n/HP,P＝0.049) and the same was true between group 3 and group 2(409.75±106.65 n/HP vs. 323.20±91.62 n/HP,Plt;0.001), whereas the result of control group was negative.The majority of transplanted cells were found positive staining both for MHC and for Connexin43 in all groups. There were lots of positive staining of α-SMA whose form were partly irregular in ischemic myocardium indicating that there was neovascularization in group1 and control group. More neovascularization in group2 was found than that in group 1 (28.38±12.79 n/HP vs. 22.75±9.07 n/HP, P＝0015) and more neovascularization in group 3 was found? than that in group 2 (35.63±13.27 n/HP vs. 28.38±12.79 n/HP, P＝0.002) . Conclusion Transplanted into infarcted myocardium, bone marrow MSCs may have significant and dose-dependent potential for cardiomyogenesis with functional recovery from myocardial ischemia.
Objective To study the influence of autologous bone mesenchymal stem cells (BMSCs) on myocardial structure and cardiac function after being implantated into acute infarcted myocardial site. Methods Bone marrow was aspirated from the posterosuperior iliac spine of Guizhou Xiang swine. After being isolated, cultured and co cultured with 5 azacytidine, either autologous BMSCs (total cells 2×10 6, experimental group, n =12), or a comparable volume of culture medium (control group, n =12), was injected into the left anterior descending(LAD) branch of coronary artery just distal to the ligation site of the LAD. The same volume of BMSCs or culture medium was injected into several spots in the infarcted myocardium. Echocardiographic measurements were performed three or six weeks after implantation to assess the myocardial structure and cardiac function. Results Left ventricular function, including eject fraction(EF), fractional shortening and wall thickening, were higher in experimental group when compared with control group. The thickness of the ventricular wall and septum was also found increased while the left ventricular chamber size was smaller in experimental group. Conclusion Implantation of BMSCs into the infarcted myocardium is believed to attenuate the remodeling process, inhibit the extent of wall thinning and dilatation of the ventricular chamber. BMSCs implantation may also improve the contractile ability of the myocardium and cardiac function.
Objective To investigate the influence of Nogo extracellular peptide residues 1-40 (NEP1-40) gene modification on the survival and differentiation of the neural stem cells (NSCs) after transplantation. Methods NSCs were isolated from the cortex tissue of rat embryo at the age of 18 days and identified by Nestin immunofluorescence. The lentiviruses were transduced to NSCs to construct NEP1-40 gene modified NSCs. The spinal cords of 30 Sprague Dawley rats were hemisected at T9 level. The rats were randomly assigned to 3 groups: group B (spinal cord injury, SCI), group C (NSCs), and group D (NEP1-40 gene modified NSCs). Cell culture medium, NSCs, and NEP1-40 gene modified NSCs were transplanted into the lesion site in groups B, C, and D, respectively at 7 days after injury. An additional 10 rats served as sham-operation group (group A), which only received laminectomy. At 8 weeks of transplantation, the survival and differentiation of transplanted cells were detected with counting neurofilament 200 (NF-200), glial fibrillary acidic portein (GFAP), and myelin basic protein (MBP) positive cells via immunohistochemical method; the quantity of horseradish peroxidase (HRP) positive nerve fiber was detected via HRP neural tracer technology. Results At 8 weeks after transplantation, HRP nerve trace showed the number of HRP-positive nerve fibers of group A (85.17 ± 6.97) was significantly more than that of group D (59.25 ± 7.75), group C (33.58 ± 5.47), and group B (12.17 ± 2.79) (P lt; 0.01); the number of groups C and D were significantly higher than that of group B, and the number of group D was significantly higher than that of group C (P lt; 0.01). Immunofluorescent staining for Nestin showed no obvious fluorescence signal in group A, a few scattered fluorescent signal in group B, and b fluorescence signal in groups C and D. The number of NF-200-positive cells and MBP integral absorbance value from high to low can be arranged as an order of group A, group D, group C, and group B (P lt; 0.05); the order of GFAP-positive cells from high to low was group B, group D, group C, and group A (P lt; 0.05); no significant difference was found in the percentage of NF-200, MBP, and GFAP-positive cells between group C and group D (P gt; 0.05). Conclusion NEP1-40 gene modification can significantly improve the survival and differentiation of NSCs after transplantation, but has no induction on cell differentiation. It can provide a new idea and reliable experimental base for the study of NSCs transplantation for SCI.
Objective To summarize the research situation of stem cells transplantation for intervertebral disc (IVD) degeneration. Methods The original articles about stem cells transplantation for repair of IVD degeneration were extensively reviewed; the clinical applications, the mechanisms, and related factors to influence repair effect were analyzed; and obstacles in stem cells transplantation for repair of IVD degeneration. Results Autogenic stem cells transplantation can repair IVD degeneration and effectively relieve the symptoms of low back and leg pain. Stem cells can differentiate into disc chondrocytes in the disc microenvironment, increase the production of various growth factors, and exert a trophic effect on disc cells. It is also evident that the transplanted stem cells can potentially protect disc cells from apoptosis and maintain an immune-privileged state in the IVD. Multiple factors such as tissue origin of stem cells, methods to pre-modulate the seeds, choice of injectable scaffolds, and even the severity of degeneration are closely related to the repair effects. To get a more efficient stem cell therapy, future researches are challenged to modulate the migration and distribution of stem cells in the IVD, avoid flow back, and better understand their ability to restore stemness properties within the degenerative disc niche. Conclusion Stem cells transplantation is proven to be a promising biological approach for repair of IVD degeneration.
【Abstract】 Objective To review the progress in the treatment of spinal cord injury (SCI) by graft of neuralstem cells (NSCs) or bone marrow mesenchymal stem cells (BMSCs) as well as immune characteristics of two stemcells. Methods Different kinds of documents were widely collected, and then immunologic characteristics of NSCs andBMSCs were summarized. The therapy of SCI by stem cell transplantation was reviewed. Additionally, some problems intreatment were analyzed. Results Experimental study showed that graft of NSCs and BMSCs can promote the functionalrecovery of the injured spinal cord in animals. Due to immunologic properties of two stem cells, rejection reaction oftransplantation could produce a harmful effect on SCI treatment. Conclusion Transplantation of NSCs or BMSCs might bean effective measure for SCI treatment, but immunologic rejection reaction must be considered.
Objective To compare single cell suspension of neural stem cells (NSCs) with neurospheres transplantation for spinal cord injury (SCI) so as to explore the therapeutic effectiveness of two NSCs transplantation methods for SCI. Methods The NSCs were isolated from the spinal cord of adult Sprague Dawley (SD) rats, purified and cultured. At passage 3, the cells were identified by Hoechst33342, Nestin staining, and gl ial fibrillary acidic protein staining for differentiated cells. Sixty adult SD rats (weighing 230-250 g) were made the SCI models at T10 level with modified Allen method and randomlydivided into 3 groups (20 rats in each). The injury sites were treated by injecting 5 μL sal ine (group A), 5 μL single cellssuspensions of NSCs at passage 3 (group B), and 5 μL neurospheres cell suspensions at passage 3 (group C). At preoperation and 3, 7, 14, 21, and 28 days after operation, the locomotor functions of each group were assessed using the Basso, Beattie, and Bresnahan (BBB) rating scale. HE staining was applied to observe the morphology of spinal cord. Subsequently immunofluorescence staining was used to observe microtubule-associated protein 2 (MAP-2). Results The cells cultured were NSCs by morphological observation and immunofluorescence staining. After 3 days of modeling surgery, BBB score significantly decreased when compared with preoperative score, and there was no significant difference among 3 groups at 3 and 7 days (P gt; 0.05). BBB score increased in different degrees with time; at 14, 21, and 28 days, BBB score of groups B and C was better than that of group A, and group C was better than group B, showing significant differences (P lt; 0.05). HE staining showed that spinal cord structure of group C was more clear than that of groups A and B, and had less scar. There was no significant difference in the number of MAP-2 positive cells among 3 groups at 3 and 7 days (P gt; 0.05). At 14, 21, and 28 days, the number of MAP-2 positive cells of groups B and C was significantly more than that of group A, and group C was more than group B, showing significant differences (P lt; 0.05). Conclusion Transplantation of neurospheres suspension compared with single cell can significantly promote NSCsto differentiate into neurons and is conducive to recover the lower extremity function after SCI.